Plenary Lecture - Hepatitis Virus Research Group

Transcription

18th International
Symposium on Hepatitis C Virus
and Related Viruses
8 – 12 September, 2011
Seattle Sheraton Hotel | Seattle, Washington, USA
www.hcv2011.org
ABSTRACT BOOK
Abstract Book Sponsored By:
Thank You to Our 2011 Symposium Sponsors
Bristol-Myers Squibb
NIAID
Apath LLC
Kineta
The HCV 2011 Symposium has been endorsed by the
following organizations:
02
WELCOME MESSAGE FROM LOC
Dear Colleagues,
Table of Contents
On behalf of the Local Organizing Committee, I am honored to welcome
you to Seattle and the 18th International Symposium on Hepatitis C
Virus and Related Viruses.
Symposium Sponsors . . . . . . . . . . . . . Inside
Front Cover
Chronic infection with hepatitis C virus (HCV) afflicts nearly 200
million people worldwide. As such, HCV is a significant global health
problem. Since first initiated in Venice, Italy in 1992, the Symposium
has maintained the focus of advancing the science of hepatitis C virus
and related viruses. Since then, the significance of the Symposium has
grown in direct proportion to the maturation of this field of research.
The Symposium attracts the leading names in the hepatitis C field
and includes promising junior scientists, students, public health
experts, and clinicians from academia and industry. The Symposium
is the premier venue for the discipline of HCV research and provides
a world stage on which cutting edge research of the highest scientific
caliber focused on HCV and related viruses is presented and debated
among the international community.
This year’s Symposium is the first meeting to occur in a new era of
therapy for chronic hepatitis C: the approval of directly acting antiviral
(DAA) compounds. The new DAA compounds targeting the NS3/4A
protease have been added to pegylated IFN plus ribavirin therapy,
and will result in significant cure rates. While this landmark event
represents tremendous clinical advance, we need to balance the
celebration with the sober reminder that many patients remain undiagnosed of HCV infection, many patients remain to be treated, and
still others will not respond to the new therapy. As such, the struggle
to advance HCV research and knowledge must continue until we can
control HCV in all patients.
Seattle – the backdrop for this year’s Symposium – is a cosmopolitan
city comprised of unique neighborhoods that offer a stylish, artistic,
and small-town experience reflective of the Pacific Northwest. It sits
in the context of the breathtaking Cascade and Olympic mountain
ranges, forests, natural bodies of water, and wildlife. We hope that
you will find time to enjoy the city and surrounding area.
It is my hope that you will enjoy and benefit from the science
presented at HCV 2011, and that this experience will nurture existing
and establish new collaborations and friendships. Please enjoy your
stay in the “Emerald City”.
Sincerely,
Endorsing Organizations . . . . . . . . . . Inside
Front Cover
Committees . . . . . . . . . . . . . . . . . . . . . . . . ii
Venue Floor Plan . . . . . . . . . . . . . . . . . . . iv
Mayor of Seattle Welcome . . . . . . . . . . . . v
Program-at-a-Glance . . . . . . . . . . vi, Inside
Back Cover
HCV 2012 Save the Date . . . . . . . . . . . . . vii
Detailed Program . . . . . . . . . . . . . . . . . . . ix
HCV 2013 Save the Date . . . . . . . . . . . . xxii
General Information . . . . . . . . . . . . . . xxvi
Plenary Lecturers . . . . . . . . . . . . . . . . . xxxi
Oral Presentations . . . . . . . . . . . . . . . 1–79
Host Genetics and Host Responses . . . . 1
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . 7
Viral and Host Factors . . . . . . . . . . . . . 13
Innate Immunology . . . . . . . . . . . . . . . 19
Cellular Immunology . . . . . . . . . . . . . . 25
Epidemiology, Global Burden, and
Vaccine Development . . . . . . . . . . . . . . 31
Virology: Entry, Replication and
Assembly . . . . . . . . . . . . . . . . . . . . . . . . 37
Antiviral Therapy . . . . . . . . . . . . . . . . . . 53
Virus-Host Interactions . . . . . . . . . . . . . 63
Viral/Kinetics and Drug Resistance . . . 75
Poster Presentations . . . . . . . . . . . 81–340
Antiviral Therapy . . . . . . . . . . . . . . . . . . 81
Cellular Immunity . . . . . . . . . . . . . . . . 117
Vaccine Development . . . . . . . . . . . . . 133
Host Genetics . . . . . . . . . . . . . . . . . . . 155
Host Responses . . . . . . . . . . . . . . . . . . 165
Pathogenesis and Carcinogenesis . . . 183
Viral Kinetics and Drug Resistance . . 205
Virology: Entry and Translation . . . . . 217
Virology: Replication and Assembly . 239
Virus-Host Interactions . . . . . . . . . . . . 287
Author Index . . . . . . . . . . . . . . . . . . . . . 341
Stephen J. Polyak, Ph.D.
Chair
Certificate of Attendance . . . . . . . . . . . 359
Poster Layout . . . . . . . . . . . . . . . . . . . . . 361
18th International Symposium on Hepatitis C Virus and Related Viruses : i
COMMITTEES
Local Organizing Committee
COMMITTEES
Stephen J. Polyak
Symposium Chair, USA
Michael Gale, Jr.
Symposium Vice Chair, USA
Chihiro Morishima
John D. Scott
International Organizing Committee
Session Chairs
Sergio Abrignani, Italy
Ralf Bartenschlager, Germany
Michael Beard, Australia
Mark Harris, UK
Michael Houghton, Canada
Geneviève Inchauspé, France
Daniel Lamarre, Canada
Robert Lanford, USA
Stanley Lemon, USA
Jane McKeating, UK
John McLauchlan, UK
Tatsuo Miyamura, Japan
Jean-Michel Pawlotsky, France
Stephen J. Polyak, USA
Charles Rice, USA
Barbara Rehermann, USA
Heinz-Jüergen Thiel, Germany
Takaji Wakita, Japan
Christopher Walker, USA
Andrea Branch, USA
Andrea Cox, USA
Matt Evans, USA
Jordan J. Feld, Canada
Pablo Gastaminza, Spain
Jeffrey Glenn, USA
Lucy Golden-Mason, USA
Matthias Gotte, Canada
Young Hahn, USA
Masao Honda, Japan
Georg Lauer, USA
Kui Li, USA
Brett Lindenbach, USA
Volker Lohmann, Germany
Ming Loo, USA
Chihiro Morishima, USA
Thomas Pietschmann, Germany
Glenn Randall, USA
Ratna Ray, USA
Christopher Richardson, Canada
John D. Scott, USA
Evaldo Stanislau, Brazil
Susan Uprichard, USA
ii : HCV 2011
COMMITTEES
Sergio Abrignani, Italy
Ralf Bartenschlager, Germany
Thomas Baumert, France
Jens Bukh, Denmark
Andrea Cox, USA
Matt Evans, USA
Jordan J. Feld, Canada
Michael Gale, Jr., USA
Pablo Gastaminza, Spain
Jeffrey Glenn, USA
Lucy Golden-Mason, USA
Matthias Gotte, Canada
Young Hahn, USA
Mark Harris, UK
Markus Heim, Switzerland
Masao Honda, Japan
Robert Lanford, USA
Georg Lauer, USA
Stanley Lemon, USA
Kui Li, USA
Brett Lindenbach, USA
Volker Lohmann, Germany
Ming Loo, USA
George Luo, USA
Jane McKeating, UK
John McLauchlan, UK
Tatsuo Miyamura, Japan
Darius Moradpour, Switzerland
Chihiro Morishima, USA
Eve-Isabelle Pecheur, France
Francois Penin, France
Thomas Pietschmann, Germany
Stephen J. Polyak, USA
Ratna Ray, USA
Ranjit Ray, USA
Barbara Rehermann, USA
Charles Rice, USA
Christopher Richardson, Canada
Takeshi Saito, USA
John D. Scott, USA
Ken Sherman, USA
Evaldo Stanislau, Brazil
John Tavis, USA
Timothy Tellinghuisen, USA
Heinz-Jüergen Thiel, Germany
David Thomas, USA
D. Lorne Tyrrell, Canada
Susan Uprichard, USA
Takaji Wakita, Japan
Christopher Walker, USA
Gulam Waris, USA
Steven Wiersma, Switzerland
Past and Future Symposiums on Hepatitis C Virus and Related Viruses
1992 - Venice, Italy
1994 - San Diego, USA
1995 - Gold Coast, Australia
1997 - Kyoto, Japan
1999 - Bethesda, USA
2000 - Gold Coast, Australia
2001 - Paris, France
2002 - San Diego, USA
2003 - Kyoto, Japan
2004 - Heidelberg, Germany
2005 - Montreal, Canada
2006 - Cairns, Australia
2007 - Glasgow, UK
2008 - San Antonio, USA
2009 - Nice, France
2010 - Yokohama, Japan
2011 - Seattle, USA
2013 - Melbourne, Australia
Michael Houghton, Sergio Abrignani, Feruccio Bonino
Michael Houghton, Charles Rice
Eric Gowans, Graham Cooksley
Tatsuo Miyamura, Kunitada Shiomotohno
Feruccio Bonino, Heinz-Jüergen Thiel
Jay Hoofnagle, Jake Liang
Eric Gowans, Graham Cooksley
Jean-Michel Pawlotsky, Geneviève Inchauspé
Charles Rice, Sergio Abrignani, Michael Houghton
Tatsuo Miyamura, Kunitada Shiomotohno
Ralf Bartenschlager, Darius Moradpour, Heinz-Jüergen Thiel
Daniel Lamarre, Marc Bilodeau, Rafick Sekaly, D. Lorne Tyrrell
Eric Gowans, Michael Beard, Geoff McCaughan
John McLauchlan, Mark Harris, Arvind Patel, Elizabeth McCruden
Stanley Lemon, Robert Lanford
Jean-Michel Pawlotsky
Takaji Wakita
Stephen J. Polyak, Michael Gale, Jr., Chihiro Morishima, John D. Scott
Sergio Abrignani, Alfredo Alberti, Feruccio Bonino, Raffaele De Francesco
Michael Beard, Heidi Drummer, Jacob George, Rose French
18th International Symposium on Hepatitis C Virus and Related Viruses : iii
COMMITTEES
Thank You to Our Abstract Reviewers
VENUE FLOOR PLAN
Seattle Sheraton Hotel
Level 2
Registration, Speaker Check-In, Scientific Sessions, Symposium Meals
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iv : HCV 2011
GREENWOOD
18th International Symposium on Hepatitis C Virus and Related Viruses : v
PROGRAM-AT-A-GLANCE
8:00
Saturday
10 September
Plenary Lecture
Grand Ballroom CD
Session 3: Viral and
Host Factors
9:00
Grand Ballroom CD
10:00
Plenary Lectures
Grand Ballroom CD
Session 1: Host
Genecs and Host
Responses
15:00
Grand Ballroom CD
16:00
17:00
Refreshment Break
Pre-Funcon Area
Plenary Lecture
Grand Ballroom CD
Session 2:
Pathogenesis
Grand Ballroom CD
18:00
Welcome Recepon
19:00
Grand Ballroom AB
Plenary Lecture
Grand Ballroom CD
Session 7a: Virology:
Entry
Session 8a: Anviral
Therapy
Grand Ballroom CD
Grand Ballroom CD
Refreshment Break
Pre-Funcon Area
Refreshment Break
Pre-Funcon Area
Lunch
Lunchme
Symposium
Grand
Ballroom CD
Grand Ballroom CD
Grand Ballroom CD
7:00
8:00
Session 9b: Virus-Host
Interacons
Grand Ballroom CD
Refreshment Break
Pre-Funcon Area
9:00
10:00
11:00
Session 10: Viral Kinecs &
Drug Resistance
Grand Ballroom CD
12:00
Closing Remarks
13:00
Grand Ballroom AB
Grand Ballroom AB
Plenary Lecture
Grand Ballroom CD
Session 7c:
Virology: Assembly
Grand Ballroom CD
Monday
12 September
Grand Ballroom CD
Lunch
Session 9a: VirusHost Interacons
14:00
15:00
16:00
Grand Ballroom CD
Refreshment Break
Pre-Funcon Area
Session 6:
Epidemiology, Global
Burden & Vaccine
Development
Grand Ballroom CD
Session 8b: Anviral
Therapy
Lunch
Refreshment Break
Pre-Funcon Area
Session 5: Cellular
Immunology
Speaker Check-In - Aspen Room
Grand Ballroom CD
Session 7b: Virology:
Replicaon
Registraon - Spruce Room
Session 4: Innate
Immunology
Plenary Lecture
Grand Ballroom CD
Opening Remarks
PROGRAM-AT-A-GLANCE
14:00
Registraon - Grand Ballroom Pre-Funcon Area
13:00
Plenary Lecture
Grand Ballroom CD
Refreshment Break
Pre-Funcon Area
11:00
12:00
Sunday
11 September
Friday
9 September
Thursday
8 September
7:00
17:00
Poster Session 2
Metropolitan
Ballroom
18:00
Poster Session 1
19:00
Metropolitan
Ballroom
20:00
20:00
Symposium Gala
21:00
Museum of Flight
21:00
22:00
22:00
23:00
23:00
24:00
24:00
vi : HCV 2011
18th International Symposium on Hepatitis C Virus and Related Viruses : vii
HCV2011_abstract book_page_grayscale.indd 1
16/06/11 10.10
HCV 2011
viii : HCV 2011
DETAILED PROGRAM
Thursday, 8 September
12:45 – 13:00 Opening Remarks
Grand Ballroom CD
13:00 – 14:00 Plenary Lectures
Grand Ballroom CD
Systems biology, transformational technologies and the
emergence of proactive P4 medicine
Leroy Hood
Systems biology approaches to viral pathogenesis and
immunity: where are Google and IBM?
Michael Katze
14:00 – 15:30 Session 1: Host Genetics and Host Responses
Grand Ballroom CD
Session Chairs: Jordan J. Feld and Masao Honda
14:00
Liver cell-specific expression phenotypes and hepatitis C virus
treatment responses: phenotype outperforms IL28B genotype
O1.01
Jordan J. Feld, Limin Chen, Maha Guindi, Sandra Fischer, E. Jenny Heathcote,
Ivan Borozan, Xiangdong Liu, Aled Edwards, Nazia Selzner, Katherine Siminovitch,
Ian McGilvray
Association of TLR7 rs179008 gene variants with hepatic IL-29/IFN-lambda1 and IFN-lambda receptor expression
in chronic hepatitis C
O1.02
Sabine Mihm, Eva Askar, Giuliano Ramadori
14:30
NK inhibitory receptor expression associated with treatment
failure and IL-28B genotype in patients with chronic hepatitis C
O1.03
Hugo Rosen, Lucy Golden
14:45
High hepatic IFNλ-R1 receptor levels are associated with HCV RNA clearance in IL28B genotype CC HCV patients
O1.04
Jie Chen, Thomas Urban, Yuhong Zhang, Jeremie Guedj, Alan Perelson, Daryl Lau
15:00
Liver gene expression signature of mild fibrosis in chronic hepatitis C
O1.05
Aurélie Ducès, Martine Lapalus, Ivan Bièche, Bénédicte Jardin-Watelet, Eve Dupas,
Simon De Muynck, Isabelle Molina, Nadine Lambert, Bérénice Sallenave, Emilie Nicolas,
Emilie Estrabaud, Nathalie Julian, Michelle Martinot-Peignoux, Olivier Lada, Valérie Paradis,
Dominique Valla, Pierre Bedossa, Daniel Laune, Michel Vidaud, Patrick Marcellin, Tarik Asselah
15:15
Gene signatures associated with liver disease progression in HCV patients following liver transplantation
O1.06
Angela Rasmussen, Nathan Susnow, Alexei Krasnoselsky, Sujay Datta, Craig Magaret,
Matthew Yeh, Deborah Diamond, Sean Proll, Sharon Lederer, Kathie Walters,
Robert Carithers Jr., Steven Self, Michael Katze
18th International Symposium on Hepatitis C Virus and Related Viruses : ix
DETAILED PROGRAM
14:15
DETAILED PROGRAM
15:30 – 16:00 Refreshment Break
Grand Ballroom Pre-Function Area
16:00 – 16:30 Plenary Lecture
Grand Ballroom CD
The spectrum of HCV disease
Norah Terrault
16:30 – 18:00 Session 2: Pathogenesis
Grand Ballroom CD
Session Chairs: Chihiro Morishima and Christopher Richardson
16:30
Laser capture microdissection reveals that fibrosis in chronic
HCV infection is associated with a loss of immune, metabolic,
and redox functions
O2.01
Supriya Munshaw, Hyon S. Hwang, Michael Torbenson, David L. Thomas, Stuart C. Ray,
Ashwin Balagopal
16:45
HCV may trigger hepatic carcinogenesis through modulation
of c-Myc oncogene expression, enhancing oxidative stress
and DNA damage
O2.02
Martin Higgs, Herve Lerat, Jean-Michel Pawlotsky
17:00
TLR4-Nanog stemness pathway in HCV-induced cancer stem cells confers resistance to TGF-β-mediated tumor
suppression through YAP1 and IGF2BP3-AKT-mTOR
O2.03
Keigo Machida, Chia-Lin Chen, Jian-Chang Liu, Claudine Kashiwabara, Douglas Feldman,
Samuel French, Jeong Hyeongnam, Linda Sher, Hidekazu Tsukamoto
DETAILED PROGRAM
17:15
Infection of SCID/Alb-uPA mice by HCV leads to liver damage, apoptosis and fibrosis
O2.04
Michael Joyce, Kinola Williams, Gerald Lachance, Ran Chen, Lin-Fu Zhu, Sue-Ellen Lamb,
Deborah Burshtyn, D. Lorne Tyrrell
17:30
Persistent expression of the full genome of hepatitis C virus in
B cells induces spontaneous development of B-cell lymphomas
in mice
O2.05
Yuri Kasama, Satoshi Sekiguchi, Michinori Kohara, Kyoko Tsukiyama-Kohara
17:45
Hepatitis C virus stabilization of hypoxia inducible factor-1α promotes epithelial to mesenchymal transition
O2.06
Garrick Wilson, Gary Reynolds, Claire Brimacombe, Ke Hu, Maria Simoes, Margaret Ashcroft,
Peter Balfe, Stefan Hubscher, Jane McKeating
18:00 – 20:00 Welcome Reception
x : HCV 2011
Grand Ballroom AB
DETAILED PROGRAM
Friday, 9 September
Grand Ballroom CD
Host-based ribaviron resistence influences hepatitis C virus
replication and treatment response
Julie Pfeiffer
08:30 – 10:00 Session 3: Viral and Host Factors
Grand Ballroom CD
Sponsored by
Session Chairs: Andrea Branch and Glenn Randall
08:30
Identification of a host factor determining the anti-HCV
activity of ribavirin
O3.01
Kyoko Mori, Osamu Hiraoka, Masanori Ikeda, Yasuo Ariumi, Akiko Hiramoto,
Yusuke Wataya, Nobuyuki Kato
08:45
Ribavirin treatment reverses HCV NS3/4A-mediated interference with intrahepatic immunity in vivo
O3.02
Erwin Daniel Brenndörfer, Anette Brass, Matti Sällberg
09:00
HCV-infected hepatocytes produce CXCL10 in a RIG‐I and TLR3 dependent manner
O3.03
09:15
Activation of chemokine and inflammatory cytokine response
in HCV-infected hepatoma cells requires TLR3 sensing of
HCV dsRNA intermediates
O3.04
Qingming Dong, Nan Li, Dahai Wei, Susan Pfeffer, Meiyun Fan, Lawrence Pfeffer, Kui Li
09:30
Identification of activity profiles of novel hepatitis C virus
associated host factors
O3.05
David Blais, Neda Nasheri, Craig Mckay, Daniel Figeys, Shao Yao, Ragunath Singaravelu,
John Pezacki
09:45
Malnutrition impairs interferon signaling through mTOR and FoxO pathways in patients with chronic hepatitis C
O3.06
Masao Honda, Kenji Takehana, Akito Sakai, Takayoshi Shirasaki, Tetsuro Shimakami,
MinKyung Yi, Stanley M. Lemon, Shuichi Kaneko
18th International Symposium on Hepatitis C Virus and Related Viruses : xi
DETAILED PROGRAM
Jessica Brownell, Jessica Wagoner, Derek Thirstrup, Wesley Smith, Kui Li, Stephen Polyak
DETAILED PROGRAM
Friday, 9 September
Grand Ballroom CD
Natural killer cells in HCV infection
Hugo Rosen
11:00 – 12:30 Session 4: Innate Immunology
Grand Ballroom CD
Sponsored by
Session Chairs: Lucy Golden-Mason and Georg Lauer
11:00
Mitochondrial-associated ER membranes form an innate immune synapse that is targeted by hepatitis C virus
O4.01
Stacy M. Horner, Helene Liu, Jessica Briley, Michael Gale Jr.
11:15
Short-range exosomal transfer of viral RNA to plasmacytoid dendritic cells activates the innate host response
O4.02
Marlene Dreux, Urtzi Garaigorta, Bryan Boyd, Josan Chung, Francis V. Chisari
11:30
Type III interferons produced by hepatocytes play a significant
role in the induction of interferon stimulated genes during
acute HCV infection
O4.03
Heiyoung Park, Onyinyechi Eke, Stefania Capone, Antonella Folgori, Barbara Rehermann
11:45
Early natural killer cell responses in hepatitis C virus exposed
healthcare workers who do not develop acute infection
O4.04
Jens M. Werner, Theo Heller, Barbara Rehermann
12:00
Liver dendritic cell recruitment during chronic hepatitis C virus infection
O4.05
DETAILED PROGRAM
Victoria Best
12:15
Acute resolving infections with HCV and HAV differ in induction
of type 1 IFN responses and the prolonged persistence of
HAV RNA in the liver
O4.06
Robert Lanford, Zongdi Feng, Bernadette Guerra, Deborah Chavez, Kathleen Brasky,
Yan Zhou, Daisuke Yamane, Alan Perelson, Christopher Walker, Stanley Lemon
12:30 – 14:30 Lunch
13:00 – 14:30 Lunchtime Symposium: HCV Drug Development
Grand Ballroom CD
Please join us for lunch and exciting talks on current and future drug developments from leading scientists
from academia and industry. A panel discussion will follow the individual talks and will include a question and
answer period.
xii : HCV 2011
DETAILED PROGRAM
Friday, 9 September
13:00
Welcome and introductions
Jean-Michel Pawlotsky, MD, PhD, Director of the National Reference Center for Viral Hepatitis B, C and Delta,
Head of the Department of Virology, Hôpital Henri Mondor, Université Paris-Est, and INSERM U955, Créteil,
France
13:05
Mechanism of action and optimization of drug candidates for HCV
Han Ma, PhD, Associate Director, Head, Molecular Virology Group, Virology Discovery and Translation
Area, Roche, Nutley, NJ, USA
13:20
Cyclophilin inhibitors
Philippe Gallay, PhD, Professor, Department of Immunology & Microbial Science, The Scripps Research
Institute, La Jolla, CA, USA
13:40
Development of NS5B inhibitors
Michelle Berrey, MD, MPH, Chief Medical Officer, Pharmasset, Princeton, NJ, USA
14:00
HCV Drug Development: Where are we headed?
14:20
Panel Discussion
Panelists:
Han Ma
Philippe Gallay
Michelle Berrey
Ann Kwong, PhD, Vice President, HCV Franchise Lead, Vertex Pharmaceuticals, Inc, Cambridge, MA, USA
Raymond T. Chung, MD, Director of Hepatology, Vice Chief, Gastroenterology, Massachusetts General
Hospital, Associate Professor of Medicine, Harvard Medical School, Boston, MA, USA
15:00 – 16:15 Session 5: Cellular Immunology
Grand Ballroom CD
Session Chairs: Andrea Cox and Young Hahn
15:00
Modulation of HCV specific CD8 T cell exhaustion and survival
by IL-21 during acute hepatitis C
O5.01
Hassen Kared, Nathalie Bédard, Julie Bruneau, Naglaa H. Shoukry
15:15
IL-18 directly triggers innate functions of liver-homing CD4+ T cells
O5.02
Vicki Fleming, Joannah Fergusson, Yu-hoi Kang, Paul Klenerman, Cormac Cosgrove
15:30
Repeated exposure to trace amounts of HCV suppresses T-cell responses to subsequent high dose HCV challenge
via induction of regulatory T-cells
O5.03
Su-Hyung Park, Naga Suresh Veerapu, Barbara Rehermann
18th International Symposium on Hepatitis C Virus and Related Viruses : xiii
DETAILED PROGRAM
There will be a short break to pick up your boxed lunch in the foyer before the session begins and another
comfort break afterwards before reconvening for the regular agenda.
DETAILED PROGRAM
15:45
Friday, 9 September
Protection and immunodominance of an HLA-B27 restricted
HCV-specific CD8+ T cell epitope is linked to rapid antigen
processing and presentation
O5.04
Julia Schmidt, Astrid K. N. Iversen, Stefan Tenzer, David A. Price, Volker Lohmann, Paul Bowness,
Hansjörg Schild, Paul Klenerman, Hubert E. Blum, Christoph Neumann-Haefelin, Robert Thimme
16:00
Tissue-specific versus disease-specific expression of T-cell
inhibitory ligands and receptors
O5.05
Daniela Kroy, Donatella Ciuffreda, Michelle Tomlinson, Garrett Hauck, Joseph Misdraji, Georg M. Lauer
16:45 – 18:00 Session 6: Epidemiology, Global Burden, and Vaccine Development
Grand Ballroom CD
Session Chairs: Michael Houghton & Geneviève Inchauspé
16:45
Finding the 70%: cost-effectiveness and population-level impact
of general population screening for hepatitis C infection
O6.01
Phillip Coffin, John Scott, Matthew Golden, Sean Sullivan
17:00
HCV genotype 1a ancestral sequence elicits broad
CD8+ T cell responses
O6.02
Kelly P. Burke, Supriya Munshaw, William O. Osburn, Jordana Levine, Stuart C. Ray, Andrea L. Cox
17:15
DETAILED PROGRAM
Antibodies to the interfering epitope EP-II (aa434-446) of HCV E2 can mask neutralizing activity induced in
patients vaccinated with rE1E2 protein
O6.03
Alla Kachko, Sharon E. Frey, Frances Wells, Iryna Zubkova, Pei Zhang, Stephen M. Feinstone,
Michael Houghton, Marian Major
17:30
Enhancing T cell response and cross-neutralizing antibodies
against HCV envelope by combining adenovirus and protein
O6.04
Alicja Chmielewska, Mariarosaria Naddeo, Virginia Ammendola, Ke Hu, Lieven Verhoye,
Malgorzata Rychlowska, Rino Rappuoli, Stefano Colloca, Alfredo Nicosia, Riccardo Cortese,
Geert Leroux-Roels, Philip Meuleman, Krystyna Bienkowska-Szewczyk, Peter Balfe,
Jane A. McKeating, Antonella Folgori
17:45
Simian adenoviral and MVA vectors combined in a heterologous
prime boost strategy – a highly immunogenic novel vaccine
against HCV
Eleanor Barnes, Antonella Folgori, Richard Antrobus, John Halliday, Stefania Capone,
Ye Oo, Rachel Townsend, Leo Swadling, Steve Aston, Joel Myer, Katherine Gantlett,
Rachel Huddart, Anthony Brown, Ayako Kurioka, Christabel Kelly, Virginia Ammendola,
Stefano Colloca, Mariarosaria Naddeo, Loredana Siani, David Mutimer, Cinzia Traboni,
David Adams, Ricardo Cortese, Alfredo Nicosia, Paul Klenerman
xiv : HCV 2011
O6.05
DETAILED PROGRAM
Friday, 9 September
18:00 – 20:30 Poster Session 1
18:00 –19:00 & 19:00 – 20:00 (Two repeated sessions.)
Metropolitan Ballroom
Ballard
Overview and features of the freely available Virus Pathogen Resource (ViPR) Bioinformatics Resource Center
Are you looking for a one-stop shop for data and analysis tools for human pathogenic viruses? The Virus Pathogen
Resource (ViPR) is a solution for you. ViPR (http://www.viprbrc.org/) is a freely available, NIAID-sponsored one-stop
database and analysis resource to search, analyze, visualize, save and share data for NIAID Category A-C Priority
Pathogens and other human viruses. Stop by at 18:00 or 19:00 to see a short demonstration of ViPR’s major functionalities: sequence search and
retrieval, comparative genomics analysis, 3D protein structure visualization, Sequence Feature Variant Type
analysis, and saving and sharing your data and analyses. DETAILED PROGRAM
Presenter: Yun Zhang, ViPR Outreach Coordinator, University of Texas Southwestern Medical Center at Dallas
18th International Symposium on Hepatitis C Virus and Related Viruses : xv
DETAILED PROGRAM
Saturday, 10 September
Grand Ballroom CD
Liver disease in HCV infection: from cell death to
inflammation to fibrosis with a few things in between
Jacquelyn Maher
08:30 – 10:30 Session 7a: Virology: Entry
Grand Ballroom CD
Sponsored by
Session Chairs: Matt Evans and Thomas Pietschmann
08:30
Visualizing HCV-receptor dynamics at the cell surface
O7.01
Helen Harris, Caroline Clerté, Amy Barnes, Margaret Goodall, Ke Hu, Garrick Wilson,
Christine Thumann, Patrice Rassam, Ragai Mitry, Anil Dhawan, Thomas Baumert,
Pierre Milhiet, Jane McKeating
08:45
Viral and cellular determinants of HCV cell-to-cell spread
O7.02
Maria Teresa Catanese, Joana Loureiro, Christopher T. Jones, Timothy Sheahan,
Alfredo Nicosia, Charles M. Rice
09:00
High-resolution imaging studies of HCV entry pathways
O7.03
Janice Elaine Silverman, Zongyi Hu, T. Jake Liang
09:15
EGF promotes hepatitis C virus entry by activating EGFR phosphorylation at Y1086 and the Ras/MAP kinase signaling pathway
O7.04
DETAILED PROGRAM
Laetitia Zona, Joachim Lupberger, Christine Thumann, Benoit Fischer, Laure Froidevaux,
Jonathan Florentin, François Duong, Michelle J. Farquhar, Mirjam B. Zeisel, Jacques A. Nunès,
Ivan Hirsch, Laurent Brino, Jane A. McKeating, Thomas F. Baumert
09:30
The Niemann-Pick C1-Like 1 cholesterol absorption receptor:
a novel hepatitis C virus entry factor and therapeutic target
O7.05
Bruno Sainz, Naina Barretto, Danyelle Martin, Nobuhiko Hiraga, Michio Imamura,
Xuemei Yu, Kazuaki Chayama, Waddah Alrefai, Susan Uprichard
09:45
The SLAM family receptor CD229: a key role in hepatitis C virus
binding and infection
O7.06
Flora Cartier, Rémy Nyga, Christèle Ossart, Luciane Lamotte, Kaïss Lassoued, Hicham Bouhlal
10:00
HCV entry inhibitors arbidol and silibinin hinder early steps of
viral trafficking as revealed by single particle tracking analysis
O7.07
Julie Blaising, Pierre Levy, Elodie Teissier, Jean-Pierre Lavergne, Eve-Isabelle Pecheur
10:15
Genotype dependent usage of CLDN-family members as HCV
entry receptors
O7.08
Sibylle Haid, Christina Grethe, Thomas Pietschmann
xvi : HCV 2011
DETAILED PROGRAM
11:00 – 13:00 Session 7b: Virology: Replication
Grand Ballroom CD
Sponsored by
Session Chairs: Pablo Gastaminza and Volker Lohmann
11:00
High-resolution profiling HCV genome by combining random
insertion mutagenesis with next-generation sequencing
O7.09
Hangfei Qi, Sheng-Yao Su, Zugen Chen, Shu-hwa Chen, Vaithilingaraja Arumugaswami,
Stanley Nelson, Chung-yen Lin, Ren Sun
11:15
The SHAPE of RNA cis-acting replication elements of HCV
O7.10
Andrew Tuplin, Madeleine Struthers, Peter Simmonds, David Evans
11:30
CREB3L1: a host transcription factor that inhibits proliferation
of virus-infected cells
O7.11
Bray Denard, Joachim Seemann, Quiyue Chen, Austin Gay, Hua Huang, Jin Ye
11:45
Productive homologous and heterologous recombination of HCV RNA in vitro
O7.12
Troels K. H. Scheel, Judith M. Gottwein, Lotte S. Mikkelsen, Jens Bukh
12:00
Novel infectious clone of HCV 1a strain HCV-RMT efficiently replicate in vitro and in vivo using adaptive mutations
O7.13
Yuko Tokunaga, Masaaki Arai, Asako Nakaya, Yoshimi Tobita, Chise Tateno, Michinori Kohara
12:15
Mapping the nascent RNA channel of the hepatitis C virus RNA dependent RNA polymerase using mass spectrometry
O7.14
Robert Vaughan, Hui Cai, Jin-Sam You, C. Cheng Kao
NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional
HCV replication complexes
O7.15
David Paul, Inés Romero-Brey, Jérôme Gouttenoire, Savina Stoitsova,
Jacomine Krijnse-Locker, Darius Moradpour, Ralf Bartenschlager
12:45
Diacylglycerol acyltransferase 1 (DGAT1) functions as a cellular “hub” for HCV NS5A and core proteins
O7.16
Gregory Camus, Eva Herker, Charles Harris, Robert Farese, Melanie Ott
13:00 – 14:30 Lunch
Grand Ballroom AB
18th International Symposium on Hepatitis C Virus and Related Viruses : xvii
DETAILED PROGRAM
12:30
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xviii : HCV 2011
DETAILED PROGRAM
14:30 – 16:30 Session 7c: Virology: Assembly
Grand Ballroom CD
Sponsored by
Session Chairs: Brett Lindenbach and Susan Uprichard
14:30
A highly specific genetic interaction between core and NS3
is essential for the production of infectious hepatitis C virus
O7.17
Daniel Jones, Ali Atoom, Rodney Russell
14:45
Identification of lipid droplet-associated membrane proteins
that are involved in HCV production
O7.18
Hideki Aizaki, Yoshihiro Matsumoto, Koji Goto, Koichi Watashi, Ryosuke Suzuki,
Masayoshi Fukasawa, Kentaro Hanada, Shigeko Sato, Nobuhiro Takahashi,
Yoshiharu Matsuura, Kiyoto Motojima, Tatsuo Miyamura, Tetsuro Suzuki, Takaji Wakita
15:00
Dynamic trafficking of HCV core protein to and from cellular lipid droplets during virus particle assembly
O7.19
Natalie Counihan, Brett Lindenbach
15:15
A role for the trans-golgi network in HCV release
O7.20
Mark Harris, Jamel Mankouri, Stephen Griffin
15:30
Underlying molecular mechanisms of apolipoprotein E in
hepatitis C virus life cycle
O7.21
Wei Cun, Guangxiang Luo
15:45
HCV p7 and NS2 induce the early steps of virus assembly by
regulating core localization at the endoplasmic reticulum vs.
lipid droplets
O7.22
16:00
NS4B protein facilitates HCV assembly via genetic interaction
with p7 and NS5A proteins
O7.23
Kouacou Konan, David Manna, Qingxia Han
16:15
Two amino acid exchanges are sufficient to restore pestivirus
morphogenesis in the absence of uncleaved NS2-3
O7.24
Norbert Tautz, Oliver Klemens, Erik Lattwein
16:30 – 19:00 Poster Session 2
Metropolitan Ballroom
18th International Symposium on Hepatitis C Virus and Related Viruses : xix
DETAILED PROGRAM
Bertrand Boson, Ophelia Granio, Ralf Bartenschlager, F. L. Cosset
DETAILED PROGRAM
Sunday, 11 September
Grand Ballroom CD
A new era in HCV antiviral therapy
Donald Jensen
08:30 – 10:30 Session 8a: Antiviral Therapy
Sponsored by
Session Chairs: Jeffrey Glenn and Matthias Gotte
08:30
Targeting lipid droplet biogenesis for the discovery of novel anti-HCV drugs
Grand Ballroom CD
O8.01
Andrea Olmstead, Francois Jean
08:45
HCV NS5A: NMR analyses of its interactions with NS5B and
cyclophilin A and molecular characterization of a cyclophilin
inhibitor-resistance mutation
O8.02
Xavier Hanoulle, Isabelle Huvent, Claire Rosnoblet, Dries Verdegem, Marie Dujardin,
Aurélie Badillo, Lotte Coelmont, Bernd Fritzinger, Jean-Michel Wieruszeski, Isabelle Landrieu,
Johan Neyts, Ralf Bartenschlager, François Penin, Guy Lippens
09:00
Solution structure of monomeric hepatitis C virus (HCV) p7 in a native, drug binding, hairpin conformation
O8.03
Toshana Foster, Arnout Kalverda, Gary Thompson, Joseph Thompson, Arwen Pearson,
Richard Foster, Steven Homans, Mark Harris, Stephen Griffin
DETAILED PROGRAM
09:15
Structure and dynamics of membrane-associated p7 by
NMR spectroscopy
O8.04
Lindsay Dawson, Gabriel A. Cook, Stanley J. Opella
09:30
Deciphering conformational variability of the hepatitis C virus polymerase (genotype 1) in complex with inhibitors
O8.05
Célia Caillet-Saguy, Philip C. Simister, Stéphane Bressanelli
09:45
Elimination of HCVcc using recombinant AAV vector-mediated
delivery of anti-HCV miRNAs
O8.06
Xiao Yang, Shangzhen Zhou, Guangxiang Luo, Linda Couto
10:00
Phosphatidylinositol 4-kinase IIIα is targeted by 4-aminoquinazoline compounds with anti-HCV activity
O8.07
Raffaele De Francesco, Annalisa Bianco, Reinaldo Alvarez, Simone Fenu, Veronica Reghellin,
Massimiliano Pagani, Sergio Abrignani, Petra Neddermann
10:15
The non-nucleoside inhibitor tegobuvir utilizes a novel
mechanism of action to inhibit HCV NS5b polymerase function
Christy M. Hebner, Bin Han, Katherine M. Brendza, Michelle Nash, Maisoun Sulfab,
Yang Tian, Magdeleine Hung, Wanchi Fung, Randy Vivian, James Trenkle, Kyla Bjornson,
Steven Bondy, Xiaohong Liu, Swami Swaminathan, John Link, Johan Neyts,
Roman Sakowicz, Uli Schmitz, Weidong Zhong
xx : HCV 2011
O8.08
DETAILED PROGRAM
11:00 – 13:00 Session 8b: Antiviral Therapy
Sponsored by
Session Chairs: Daniel Lamarre and Jean-Michel Pawlotsky
11:00
NS5A dimerization: a potential drug target?
Grand Ballroom CD
O8.09
Precious Lim, Udayan Chatterji, Dan Cordek, Craig Cameron,
Paul Targett-Adams, Kai Lin, Philippe Gallay
11:15
The green tea polyphenol epigallocatechin-3-gallate (EGCG)
inhibits hepatitis C virus (HCV) entry
O8.10
Sandra Ciesek, Thomas von Hahn, Luis M. Schang, Martina Friesland, Michael P. Manns,
Michael Ott, Heiner Wedemeyer, Philip Meuleman, Thomas Pietschmann, Eike Steinmann
11:30
Marked synergy of entry inhibitors and interferon-alfa or direct
acting antivirals on the inhibition of hepatitis C virus infection
O8.11
Fei Xiao, Isabel Fofana, Joachim Lupberger, Pieter Leyssen, Johan Neyts, Mirjam B. Zeisel,
Thomas F. Baumert
11:45
The RAFIs aUY11 and dUY11 inhibit infectivity of HCV and other enveloped viruses by preventing fusion of viral
and cellular membranes
O8.12
Che C. Colpitts, Alexey V. Ustinov, Vladimir A. Korshun, Luis M. Schang
Identification and functional analysis of small molecules inhibiting the late step of hepatitis C virus life cycle
O8.13
Koichi Watashi, Nanako Uchida, Ryosuke Suzuki, Hideki Aizaki, Takaji Wakita
12:15
Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes
O8.14
Kyoko Tsukiyama-Kohara, Takashi Takano, Yuichi Hirata, Yuko Tokunaga, Chise Tateno,
Masayuki Sudo, Michinori Kohara
12:30
The impact of calcineurin inhibitors on HCV replication in a chimeric mouse model of HCV infection
O8.15
Norman Kneteman, Jamie Lewis, Donna Douglas, Mahra Nourbakhsh,
Lorne Tyrrell, Garry Lund
12:45
PPARα agonist WY14643 improves the antiviral effect
of interferon against hepatitis C virus
O8.16
Scott Read, Jacob George, Mark Douglas
13:00 – 14:30 Lunch
Grand Ballroom AB
18th International Symposium on Hepatitis C Virus and Related Viruses : xxi
DETAILED PROGRAM
12:00
xxii : HCV 2011
DETAILED PROGRAM
Grand Ballroom CD
Virus-host interactions: The role of type I, II, and III
interferons in acute and chronic hepatitis C
Markus Heim
15:00 – 17:00 Session 9a: Virus-Host Interactions
Grand Ballroom CD
Session Chairs: Michael Beard & Ratna Ray
15:00
A mouse model for hepatitis C virus infection and immunity
O9.01
Marcus Dorner, Joshua Horwitz, Alexander Vogt, Kathy Mu, Gisa Gerold,
Charles Rice, Alexander Ploss
15:15
Hepatitic C virus infection of differentiated human hepatocytes
derived from embryonic and induced pluripotent stem cells
O9.02
Xianfang Wu, Jason Robotham, Hongying Deng, Hengli Tang
15:30
Molecular determinants and dynamics of hepatitis C virus secretion
O9.03
Kelly Coller, Nicholas Heaton, Kristi Berger, Jacob Cooper, Jessica Saunders,
Glenn Randall
15:45
Impaired signaling and function induced in plasmacytoid
dendritic cells exposed to HCV
O9.04
Jonathan Florentin, Clélia Dental, Besma Aouar, Françoise Gondois-Rey,
David Durantel, Thomas F. Baumert, Daniel Olive, Ivan Hirsch, Ruzena Stranska
HCV infection leads to stimulation of LDL receptor expression
O9.05
Aleem Siddiqui, Huihui Tang, Gulam Syed, Tarek Hassanein
16:15
Inflammasome activation by hepatitis C virus
O9.06
Amina Negash, Hilario Ramos, Michael Gale Jr.
16:30
HCV directly stimulates the production of the inflammasome-
associated cytokine IL-18 in cells of the monocyte lineage
O9.07
Michael Chattergoon, Erik Orberg, Jordana Levine, Andrea Cox
16:45
miR-122 stabilizes hepatitis C virus RNA by an Ago2-dependent mechanism
O9.08
Tetsuro Shimakami, Daisuke Yamane, Rohit Jangra, Brian Kempf, Carolyn Spaniel,
David Barton, Stanley Lemon
18:00 – 23:30 Symposium Gala
Museum of Flight
Group transportation provided. Please plan to depart from the Union Street Entrance
promptly at 18:00.
18th International Symposium on Hepatitis C Virus and Related Viruses : xxiii
DETAILED PROGRAM
16:00
DETAILED PROGRAM
Monday, 12 September
08:30 – 10:30 Session 9b: Virus-Host Interactions
Grand Ballroom CD
Session Chairs: Kui Li and Ming Loo
08:30
A RIG-I translocon directs membrane-association of RIG-I
with IPS-1 and controls innate antiviral immunity
O9.09
Helene Minyi Liu, Michael Gale Jr.
08:45
Unraveling the complexity of the type I interferon antiviral response
O9.10
John Schoggins, Sam Wilson, Maryline Panis, Mary Murphy, Christoper Jones,
Paul Bieniasz, Charles Rice
09:00
In vivo suppression of interferon sensitive gene 15 (ISG15),
a proviral host factor in HCV replication, enhances the
endogenous interferon response
O9.11
Catherine I. Real, Ruth Broering, Kerstin Jahn-Hofmann, Ludger Ickenstein,
Matthias John, Kathrin Kleinehr, Guido Gerken, Joerg F. Schlaak
09:15
Robust induction of type III interferons and other cytokines
defines a unique pattern of innate immunity in response to
hepatitis C virus infection
O9.12
Emmanuel Thomas, Veronica Gonzalez, Qisheng Li, Ankit Modi, Weiping Chen,
Mazen Noureddin, Yaron Rotman, T. Jake Liang
09:30
DETAILED PROGRAM
Systems biology & computational network analysis identifies mitochondrial host targets of HCV infection
and liver disease progression
O9.13
Deborah L. Diamond, Jason E. McDermott, Alexei Krasnoselsky, Kristin E. Burnum,
Nathan Susnow, Bobbie-Jo Webb-Robertson, Courtney Courley, Matthew M. Yeh,
Angela L. Rasmussen, Jon M. Jacobs, Renuka Bhattacharya, James D. Perkins,
Robert L. Carithers Jr., Marina A. Gritsenko, Iris W. Liou, Susan Strom, Katrina M. Waters,
Richard D. Smith, Michael G. Katze
09:45
Overall structural model of non-structural protein 5A from hepatitis C virus and modulation by cyclophilin A
O9.14
Aurelie Badillo, Veronique Receveur-Brechot, Simona Miron, Xavier Hanoulle, Jennifer Molle,
Roland Montserret, Ralf Bartenschlager, Guy Lippens, Sylvie Ricard-Blum, Francois Penin
10:00
Hepatitis C virus infection induces autophagy and blocks innate immune response
O9.15
Shubham Shrivastava, Amit Raychoudhuri, Robert Steele, Ranjit Ray, Ratna Ray
10:15
Differential type I interferon-mediated autophagic trafficking
of hepatitis C virus proteins in mouse liver
O9.16
Mayura Desai, Bin Gong, Tehsheng Chan, Jiaren Sun
xxiv : HCV 2011
DETAILED PROGRAM
11:00 – 12:30 Session 10: Viral Kinetics and Drug Resistance
Session Chairs: John D. Scott and Evaldo Stanislau
11:00
Project ECHO: outcomes of hepatitis C treatment by
primary care providers
Grand Ballroom CD
O10.01
Sanjeev Arora, Karla Thornton, Summers Kalishman, Paulina Deming, Wesley Pak,
John Brown, Glen Murata
11:15
Molecular characterization of HCV resistance to telaprevir
by means of ultra-deep pyrosequencing
O10.02
Stephane Chevaliez, Christophe Rodriguez, Alexandre Soulier, Abdelhakim Ahmed-Belkacem,
Christophe Hezode, Jean-Michel Pawlotsky
11:30
New insights into the mechanism of action of interferon-alpha
and BMS-790052: a multi-scale mathematical modeling approach
O10.03
Jeremie Guedj, Harel Dahari, Libin Rong, Richard Nettles, Scott Cotler,
Thomas Layden, Alan Perelson
11:45
HCV persistence after sustained virological response to antiviral therapy in patients with or without past
exposure to hepatitis B virus
O10.04
Tram N. Q. Pham, Carla S. Coffin, Norma D. Churchill, Stefan J. Urbanski, Samuel S. Lee,
Thomas I. Michalak
12:00
Long-term follow-up of chronic HCV patients treated with
telaprevir: evaluation of persistence of resistant variants by
ultra-deep sequencing
O10.05
12:15
Selection of cyclophilin inhibitor resistance in transplant patient
O10.06
Rob Striker, Israr Ansari, Todd Allen, Andrew Berical
12:30 – 13:00 Closing Remarks
Grand Ballroom CD
18th International Symposium on Hepatitis C Virus and Related Viruses : xxv
DETAILED PROGRAM
Xiomara Thomas, Joep de Bruijne, Tara Kieffer, James Sullivan, Sjoerd Rebers,
Michel de Vries, Henk Reesink, Christine Weegink, Janke Schinkel, Richard Molenkamp
GENERAL INFORMATION
Symposium Information
Venue and Host Hotel Information
Sheraton Seattle Hotel
1400 6th Avenue
Seattle, Washington 98101
United States
Phone: 1.206.621.9000
Secure Fax: 1.206.389.5703
www.sheraton.com/seattle
Overflow Hotel
Renaissance Seattle Hotel
515 Madison St
Seattle, Washington 98104
United States
Phone: 1.206.694.4953
Fax: 1.206.624.8125
www.renaissanceseattle.com
Registration Desk Location and Hours
The Symposium Registration Desk will be open during the hours listed below:
Day
Time
Location at Sheraton
Thursday, 8 September:
Friday, 9 September:
Saturday, 10 September:
Sunday, 11 September:
Monday, 12 September:
08:00 – 18:00
07:00 – 17:00
07:00 – 17:00
07:00 – 17:00
07:00 – 14:00
Spruce Room
Spruce Room
Spruce Room
Spruce Room
Name Badges
Name badges issued at registration must be worn for admittance to all symposium sessions, meals and
social events.
Certificate of Attendance
A detachable Certificate of Attendance has been provided for you on page 361 of this book.
Speaker Check-In Information
GENERAL INFORMATION
Aspen Room, Level 2
All presenting authors must submit their final presentation at speaker check-in during the designated days and
times listed below. Computers will be provided by the Symposium and will be equipped with the following:
Mac computers with Mac OS 10.6 and Microsoft Office Mac Pro 2008, and PCs with Windows XP Pro and
Microsoft Office. Please note that personal laptops may not be used.
Presentation Date
Presentation Due at Speaker Check-In
Friday, 9 September
Saturday, 10 September:
Sunday, 11 September:
xxvi : HCV 2011
08:00 – 11:00
11:00 – 18:00
07:00 – 16:30
07:00 – 16:30
07:00 – 16:30
GENERAL INFORMATION
Poster Sessions
A detachable Poster Session Floor Plan has been provided for you on page 363 of this book.
Poster Information
Posters will be displayed in the Metropolitan Ballroom and Pre-Function Area on the 3rd floor of the Sheraton.
All posters should be set up on Thursday between 09:00 and 20:00. They will remain on display through
Saturday evening and will need to be removed by the author no later than Sunday at 14:30. Delegates can
view the posters at anytime during the Symposium; however, poster authors will be available for discussions
during the poster session hours designated below.
Event
DateTime
Poster Install: all posters
Poster Session 1: odd numbered posters
Poster Session 2: even numbered posters
Poster Dismantle: all posters
Saturday, 10 September: Sunday, 11 September:
09:00 – 20:00
18:00 – 20:30
16:30 – 19:00
07:00 – 14:30
Meals
The following meals are included in your symposium registration:
Thursday, 8 September: Friday, 9 September:
Saturday, 10 September: Sunday, 11 September: Monday, 12 September:
Refreshment Break and Welcome Reception
Refreshment Breaks and Lunch
Refreshment Break
The HCV 2011 Gala is an optional ticketed event on Sunday evening. Please visit the Registration Desk if you wish to
attend the Gala and haven’t already purchased a ticket. Onsite additions will be handled on a case-by-case basis.
Social Events
Networking Reception and Pacific Northwest Performing Arts Showcase
18:00 – 20:00
Grand Ballroom AB, Sheraton Seattle Hotel
Join us for the very first social mixer of the Symposium! The Local Organizing Committee has a special evening
planned that will highlight the strength and diversity of the Northwest’s local arts culture.
Be our guest for a few short, but exceptional performances that have been arranged exclusively for your
entertainment. Meanwhile, enjoy delicious hors d’oeuvres and regional wines and beers while you reconnect
with your colleagues and make new connections.
18:00 – 23:30
Museum of Flight (Group transportation provided). Please plan to depart from the Union Street Entrance
promptly at 18:00.
Dine among historic air and spacecraft artifacts while enjoying local Pacific Northwest fare and networking
with your colleagues at Seattle’s famous Museum of Flight. Browse impressive exhibits including a replica of
the International Space Station Laboratory, evoke your inner pilot in a flight simulator and boogie down to
classic Rock ‘n Roll with favorite local cover band, The Beatniks! Do you enjoy dancing? Then, this is the place
to be! This event is sure to create lasting memories.
18th International Symposium on Hepatitis C Virus and Related Viruses : xxvii
GENERAL INFORMATION
HCV 2011 Gala
GENERAL INFORMATION
General Symposium Policies
Language
English is the official language of the 18th International Symposium on Hepatitis C Virus and Related Viruses.
Mobile Phone Policy
Please ensure that your mobile phone is turned off during all sessions to avoid disrupting the presenters,
microphones and your fellow delegates.
Recording Policy
Photography, video recording and audio recording without permission from the Symposium Chair is
strictly prohibited.
Message Board
A message board is available near the Registration Desk for all delegates to use.
Hotel Information
Internet Access
Complimentary wireless internet access is available in the Sheraton Lobby and Lobby Bar area for all hotel
guests. For non-hotel guests, please visit the Front Desk for a complimentary 45 minute pass.
Or stay connected with the Sheraton’s Link@Sheraton, offering a secure and convenient area to work or relax.
Connect on the available computer terminals or bring your laptop for complimentary wireless broadband.
Fitness Center
Work out with comprehensive fitness equipment in the newly remodeled Fitness Center located on the 35th
floor, featuring a 40’ lap pool and jacuzzi. Enjoy sprawling city and mountain views from the bi-level exercise
room while working out. Access to the fitness center is complimentary for all overnight guests.
Hotel Parking
The Sheraton Seattle Hotel Garage will give priority valet parking (self-parking is not available) to overnight
guests at a rate of $43 inclusive per day. The Valet will direct other drivers and drivers of oversized vehicles to
alternate parking options.
Medical Assistance
In case of an emergency, please dial 911 from any hotel house phone.
GENERAL INFORMATION
Seattle Information
Currency
The U.S. dollar is the official currency of the United States and is normally abbreviated as the dollar sign, $, or
as USD or US$. It is divided into 100 cents.
To exchange foreign currency to USD, visit Travelex Currency Services at Sea-Tac airport or Westlake Center
in Seattle, just two (2) blocks away from the Sheraton. Most local banks are also able to exchange currencies.
ATMs will also dispense USD for a nominal fee.
Traveler’s checks are becoming relatively uncommon in Seattle, and they are accepted only by banks and
major hotels. VISA, MasterCard and American Express are widely accepted at hotels, department stores,
shops, restaurants and nightclubs.
xxviii : HCV 2011
GENERAL INFORMATION
ATM
There are two ATMs onsite at the Sheraton Seattle Hotel, located in the Business Center and in the Fireplace Lobby.
Sales Tax
Seattle has a sales tax of 9.5% on consumed goods, and a 15.6% tax on hotel rooms.
Electricity
United States operates 110 volts AC, 60 hertz electricity, which is half of the 220 volts that many other
countries use. Dual voltage appliances are recommended.
Smoking Policy
Washington has a very strict smoking ban in place. Seattle is a smoke free city, which means that smoking is
prohibited in all public places and workplaces, including all restaurants and bars. You may not smoke within 25
feet (7.6 m) of any door or window. Violating this ban can result in a $100 USD fine.
Tipping
At food stands or in restaurants that do not provide table service, it is at your discretion to tip 0-10%.
In a restaurant that provides table service, a 15% to 20% tip is customary. For large parties of 6 people or
more, restaurants will often include the tip or “gratuity”. If the tip is included, it will be clearly indicated on
your itemized bill and you do not need to leave an additional tip.
When ordering hotel room service, a tip will already be included on the bill and you do not need to add an
additional tip.
For carrying luggage to your room, $3-5 is average, unless you have excess luggage.
For taxis, a 10-20% tip is customary and is not included in the fee. For hotel shuttles, $2-5 for the driver is
average for luggage assistance, but is at your discretion.
Time Zone
Seattle lies within the Pacific Time Zone (PT) or Greenwich Mean Time (GMT) -7 hours.
Transportation to the Airport
Seattle-Tacoma International Airport (SEA) is located 20 minutes south of Seattle. Transportation options from
the airport to the Sheraton include the Link Light Rail, taxi and limousine service, bus and airport shuttles.
Guests are encouraged to consider Link Light Rail as a convenient and affordable transportation option. The
one-way fare from Westlake Center (approximately two (2) blocks from the Sheraton) to the Seattle-Tacoma
International Airport (SEA) to is $2.75 and takes approximately 38 minutes.
Typical Minimum Charge (one way)
Link Light Rail Taxi Shuttle Limo $2.75
$35
$19
$59
GENERAL INFORMATION
Type 18th International Symposium on Hepatitis C Virus and Related Viruses : xxix
xxx : HCV 2011
PLENARY LECTURERS
PLENARY LECTURERS
18th International Symposium on Hepatitis C Virus and Related Viruses : xxxi
PLENARY LECTURERS
xxxii : HCV 2011
PLENARY LECTURERS
Systems biology, transformational technologies and the emergence of proactive
P4 medicine
Leroy Hood, Institute of Systems Biology, Seattle, WA, USA
The challenge for biology in the 21st century is the need to deal with its incredible complexity. One powerful way to think
of biology is to view it as an informational science. This view leads to the conclusion that biological information is captured,
mined, integrated by biological networks and finally passed off to molecular machines for execution. Hence the challenge in
understanding biological complexity is that of deciphering the operation of dynamic biological networks across the three time
scales of life—evolution, development and physiological responses. Systems approaches to biology are focused on delineating
and deciphering dynamic biological networks and their interactions with simple and complex molecular machines. I will
discuss the principles and infrastructure needed for systems biology.
I will then focus on our efforts at a systems approach to disease—looking at a neurodegenerative (prion) disease and a brain
tumor (glioblastoma) in mice. We published a few years ago a study on prion disease that has taken more than five years
that integrates 6 different types of data and lays out the principles of a systems approach to disease including dealing with
the striking signal to noise problems of high throughput biological measurements and biology itself (e.g. polymorphisms).
One interesting question that I will discuss is how close the murine model diseases mimic their human counterparts. From
these studies comes a clear understanding of some of the principal opportunities systems biology brings to medicine and
the study of disease.
Then I will discuss the emerging technologies that will transform biology and medicine over the next 10 years—e.g., next
generation DNA sequencing, targeted mass spectrometry, microfluidic protein chips, new approaches to protein-capture
agents, single-cell analyses and the use of induced pluripotential cells to understand development, disease mechanisms and
stratify disease.
It appears that systems medicine, together with emerging technologies and the development of powerful new computational
and mathematical tools will transform medicine over the next 5-20 years from its currently reactive state to a mode that
is predictive, personalized, preventive and participatory (P4). I will describe the nature of P4 medicine and its societal
implications for healthcare.
Leroy Hood, MD, PhD, President and co-founder of the Institute for Systems Biology in Seattle, is
a pioneer in systems approaches to biology and medicine. Dr. Hood’s research has focused on the
study of molecular immunology, biotechnology and genomics. His professional career began at
Caltech, where he and his colleagues developed the DNA sequencer and synthesizer and the protein
synthesizer and sequencer––four instruments that paved the way for the successful mapping of
the human genome and lead to his receiving this year’s prestigious Russ Prize, awarded by the
Academy of Engineering. A pillar in the biotechnology field, Dr. Hood has played a role in founding
more than fourteen biotechnology companies, including Amgen, Applied Biosystems, Darwin, The
Accelerator and Integrated Diagnostics. He is a member of the National Academy of Sciences, the
National Academy of Engineering, and the Institute of Medicine, one of only 10 people in the
world to be elected to all three academies. In addition to having published more than 700 peer reviewed articles, he has
coauthored textbooks in biochemistry, immunology, molecular biology and genetics, as well as a popular book on the
human genome project, The Code of Codes. He is the recipient of numerous awards, including the Lasker Award, the Kyoto
Prize and the Heinz Award in Technology. Dr. Hood has also received 17 honorary degrees from prestigious universities in
the US and other countries.
18th International Symposium on Hepatitis C Virus and Related Viruses : xxxiii
PLENARY LECTURERS
Plenary Lecture: Thursday, 8 September, 13:00 -13:30
PLENARY LECTURERS
PLENARY LECTURERS
Systems biology approaches to viral pathogenesis and immunity: where are Google
and IBM?
Michael Katze, University of Washington, Seattle, WA, USA
After decades of research, vaccines against some of the greatest viral threats are still lacking and antiviral drugs remain few and
slow in coming. These shortcomings signal the need for new approaches that go beyond traditional virology methods. Highthroughput technologies and computational biology are poised to deliver a much-needed boost to the field. My laboratory
is using systems biology and computational approaches to understand and model integrated views of virus-host interactions,
viral evasion of host defense mechanisms, and viral pathogenesis. Much of our work is focused on the viruses responsible
for current worldwide pandemics, including hepatitis C virus and human immunodeficiency virus. We make extensive use of
serial liver biopsies from patients with recurrent HCV after liver transplantation, biopsies from patients co-infected with HCV
and HIV-1, and experimental systems for HCV infection such as the SCID-beige/Alb-uPA chimeric mouse model and the HCV
2a in vitro infection system. As new experimental systems and technologies continue to come online, such as mouse systems
genetics, metabolomics, lipidomics, and next-generation sequencing, our systems-level views have expanded to encompass
host genetic variation, metabolic pathways, microRNAs, and long noncoding RNAs. Because this amount of information
is beyond the capacity of human intuition to grasp and formulate predictions—such as for how to most effectively design
new therapeutics or vaccines—it is beyond question that mathematical frameworks and computational models must be
constructed. Such models are necessary to predict how molecular components work together to yield operational mechanisms
and phenotypic outcome. Models also facilitate a conceptual understanding of the coordination of the various facets of the
overall system. The combination of high-throughput datasets and computational methods provides best hope for speeding
vaccine and drug development.
Dr. Michael Katze is Professor of Microbiology at the University of Washington and Associate
Director and Core Staff Scientist at the Washington National Primate Research Center. He is also the
Program Director for a National Institute on Drug Abuse P30 Center on Functional Genomics and
HCV-Associated Liver Disease, Co-Director of the Pacific Northwest Regional Center of Excellence,
and Director of the Center for Systems and Translational Research on Infectious Disease (STRIDE). He
has studied virus-host interactions for more than 30 years and is a world leader in the use of systems
biology approaches, including high-throughput technologies and computational methods, to define
and model virus-host interactions and the varied strategies used by viruses to evade host defense
mechanisms. These approaches are applied a broad range of experimental systems, including those
focused on influenza virus, hepatitis C virus (HCV), Ebola virus, SARS-associated coronavirus, and
simian and human immunodeficiency viruses.
The spectrum of HCV disease
Norah Terrault, University of California San Francisco, San Francisco, CA, USA
Dr. Norah Terrault is the Professor of Medicine and Surgery as well as the Director of the Viral
Hepatitis Center at the University of California San Francisco.
She is recognized nationally and internationally for her work related to viral hepatitis, especially in
the setting of liver transplantation. She has authored over 100 original articles, reviews and book
chapters on viral hepatitis, and serves as the Deputy Editor for Liver Transplantation and Associate
Editor for Hepatology. She is an investigator on several NIH-funded clinical studies in hepatitis B
and C, and is an investigator on several ongoing clinical trials of antiviral therapies for patients with
chronic hepatitis B and C.
xxxiv : HCV 2011
PLENARY LECTURERS
Host-based ribavirin resistance influences hepatitis C virus replication and
treatment response
Julie Pfeiffer, University of Texas Southwestern Medical Center, Dallas, TX, USA
Kristie Ibarra, University of Texas Southwestern Medical Center, Dallas, TX, USA
Mamta Jain, University of Texas Southwestern Medical Center, Dallas, TX, USA
Many individuals infected with hepatitis C virus (HCV) develop a chronic infection, and of those who are treated with pegylated
interferon and ribavirin (RBV) many do not respond. While the nucleoside analog RBV improves treatment outcome, and will
likely be an important component of therapy with next-generation viral inhibitors, RBV’s mechanism is controversial. Most
of RBV’s proposed mechanisms require RBV import into cells. Therefore, we explored whether host-based RBV resistance
develops through reduced cellular uptake, akin to chemotherapy resistance in some cancers. We examined the effect of
host-based RBV resistance on HCV replication in cultured hepatoma Huh 7.5 liver cells, and whether RBV resistance develops
in HCV patients. When Huh 7.5 cells were exposed to RBV, resistance developed through reduced RBV uptake via the ENT1
nucleoside transporter and antiviral efficacy was reduced. Importantly, RBV uptake significantly declined in HCV patient
peripheral blood mononuclear cells (PBMCs) following four weeks of therapy. Furthermore, maintenance of RBV uptake
correlated with rapid treatment response. Our results uncovered a novel form of antiviral drug resistance, and suggest that
host-based RBV resistance develops in HCV patients undergoing therapy, and that maintenance of RBV uptake may contribute
to rapid viral clearance.
Dr. Julie K. Pfeiffer received her Ph.D. degree in microbiology in 2001 from the University of Michigan,
where she studied genetic recombination in retroviruses with Dr. Alice Telesnitsky. She was a Damon
Runyon postdoctoral fellow in the laboratory of Dr. Karla Kirkegaard at Stanford University, where
she studied RNA virus genetics, pathogenesis, and antiviral drug resistance. In 2006, she became an
Assistant Professor of Microbiology at the University of Texas Southwestern Medical Center in Dallas.
She was named a Pew Scholar in 2007. Her laboratory studies a variety of RNA viruses, including
poliovirus, hepatitis C virus, yellow fever virus, and reovirus, with a goal to understand viral evolution
mechanisms, antiviral drug resistance, and host barriers that limit viral trafficking. During the course
of these studies, her laboratory has found that 1) resistance to the antiviral drug ribavirin can occur
through host-based mechanisms, 2) several host barriers limit poliovirus trafficking in infected
mice, including inefficient transport in neurons, and 3) bacteria within the intestine promote enteric virus replication and
pathogenesis. Future work in her laboratory will dissect mechanisms of ribavirin resistance and inefficient retrograde axonal
transport of poliovirus and other viruses, and determine how intestinal bacteria enhance enteric virus infections.
18th International Symposium on Hepatitis C Virus and Related Viruses : xxxv
PLENARY LECTURERS
Plenary Lecture: Friday, 9 September, 08:00 - 08:30
PLENARY LECTURERS
Plenary Lecture: Friday, 9 September, 10:30 - 11:00
PLENARY LECTURERS
Natural killer cells in HCV Infection
Hugo Rosen, University of Colorado, Denver, CO, USA
This lecture will focus on the rapidly evolving role of natural killer cells in hepatitis C infection, including protection from acute
HCV and response to antiviral therapy.
Hugo R. Rosen, MD performed his fellowship training in Gastroenterology and Hepatology at UCLA
where he worked in the lab of Randy Wall, PhD at the Molecular Biology Institute to study genetic
regulation of immune cells. His first faculty position was at Oregon Health and Sciences University
and Portland VA where he developed one of the leading translational research programs studying
adaptive immunity to hepatitis C infection. He is the recipient of numerous Awards, including the
Senator Hatfield Award for Research; he became the first hepatologist awarded the prestigious
American Society of Transplantation Research Award for Clinical Science. In 2005, he became the
Waterman Endowed Chair in Liver Research and the Division Head of Gastroenterology & Hepatology
at University of Colorado. He is also the Program Director for an NIH-sponsored Hepatitis C Cooperative
Research Center (2005-2015), GI training grant director, PI on several RO1s and VA Merit Review
grants funded since 1998. His clinical and research interests include clinical hepatology with a special focus on viral hepatitis
and endstage liver disease; innate and adaptive immunity to HCV, transplantation mathematical modeling, transplantation
immunology, hepatic immunity in non-alcoholic steatohepatitis.
Plenary Lecture: Saturday, 10 September, 08:30 - 09:00
Liver disease in HCV infection: from cell death to inflammation to fibrosis with a few
things in between
Jacquelyn Maher, University of California San Francisco, San Francisco, CA, USA
Liver disease in the setting of hepatitis C infection follows a sequence similar to that in non-viral settings. The death of
hepatocytes represents a pivotal event; indeed, cell death is essential for triggering downstream consequences including
inflammation and fibrosis. Hepatocyte death promotes the release of cytokines that induce inflammatory and fibrotic
responses. Cell remnants can also be engulfed by leukocytes or myofibroblasts, with a similar outcome. Prolonged activation
of the cell-death-inflammation-fibrosis axis results in cirrhosis and portal hypertension. Steatosis of hepatocytes, which occurs
variably in HCV-infected patients, has been implicated as a risk factor for progressive liver disease but its precise contribution
to organ injury remains unclear. Although cirrhosis is often considered the “end-stage” of liver disease, this condition
is reversible if the underlying cause of liver injury can be successfully treated. Measures to treat fibrosis per se, without
simultaneous attention to the root cause of liver disease, have been explored but are less promising. Treatments aimed at
reducing hepatocyte death as the most proximal event in liver injury sound logical; ironically, these may be ineffective if they
also prevent the death of the cells that are contributing to hepatic fibrosis.
Jacquelyn Maher, M.D. is a Professor of Medicine at the University of California, San Francisco.
Her research focuses on basic mechanisms of liver injury, addressing specific questions about the
relationships between liver cell death, inflammation and fibrosis. She received her medical training
at the Duke University School of Medicine and completed an Internal Medicine residency at the
Duke University Medical Center. She trained in Gastroenterology UCSF, and joined the faculty at
UCSF in 1987. Dr. Maher currently serves as Program Director of the UCSF Liver Center and head
of the UCSF postdoctoral research training program in hepatology. She is an Associate Editor of
HEPATOLOGY, and recently completed a 3-year term as Secretary of the American Association for
the Study of Liver Diseases.
xxxvi : HCV 2011
PLENARY LECTURERS
A new era of HCV antiviral therapy
Donald Jensen, University of Chicago, Chicago, IL, USA
2011 marks the beginning of a new era in hepatitis C therapy. Understanding the reproductive machinery of the hepatitis C virus
was a first step in delivering new and improved therapies. Several potential targets for antiviral therapy were identified which
were considered “drug-able”, including the NS3/4A serine protease enzyme, the HCV helicase enzyme, the NS5A polymerase
enzyme and the NS5B polymerase enzyme. Of these, the NS3/4A protease enzyme seemed like the best initial target. The
first two successful compounds, VX950 ( Telaprevir) by Vertex and SCH503034 (Boceprevir) by Schering-Plough (now Merck)
were FDA approved in May 2011. A few key points about these compounds need emphasizing. First, they cannot be used as
monotherapy. The HCV virus rapidly develops resistance to these agents within days of exposure to monotherapy. For this
reason, they are always given in combination with PEG/RBV. Second, telaprevir and boceprevir are indicated only for genotype
1 infections. Third, they must be taken every eight hours, and telaprevir must be taken with a fatty meal – not TID. Fourth,
each agent has its own unique adverse events on top of those already associated with PEG/RBV. These include skin rash and
anemia with telaprevir and anemia and dysguesia with boceprevir. Finally, they have not been well-studied in certain key HCV
populations: decompensated cirrhosis, HIV coinfection, post-transplant and the elderly. In spite of all of these limitations and
difficulties, the results of treatment with telaprevir and boceprevir have been truly outstanding with a near doubling of SVR
rates to as high as 75% in genotype 1, treatment-naïve subjects. Response-guided therapy could shorten therapy to 24-28
weeks. Those with prior relapse responded extremely well to triple therapy, while those with null response to prior PEG/RBV
did no better than 30%. Future therapies are directed in two different approaches: quad therapy involving PEG/RBV/protease
inhibitor/and a fourth compound (either a nucleoside polymerase inhibitor or NS5A inhibitor). Interestingly, combination with
just two oral DAA agents (protease inhibitor plus NS5A inhibitor) in prior null responders could cure 37%. Clearly, the future
of HCV therapy is bright and the hope of an all oral, interferon-free therapy is possible….no, probable.
Dr. Donald Jensen, MD, FACP, is currently Professor of Medicine and Director of the Center for
Liver Diseases at the University of Chicago Hospitals. Dr. Jensen attended the University of Illinois
College of Medicine where he received his MD degree. He completed his internship and residency
in internal medicine at Rush University Medical Center and was also the Chief Medical Resident at
Rush. He was a Liver Research Fellow at King’s College Hospital in London, England. In 1992, he was
appointed Director of Clinical Hepatology at Rush University, and in 1999 was named the Richard B.
Capps Professor of Medicine. In July, 2005, Dr Jensen accepted his current position at the University
of Chicago Medical Center. Dr. Jensen is currently Treasurer of the AASLD and past President of the
Illinois chapter of the American Liver Foundation. He is an Associate Editor for Hepatology. He has
authored or co-authored over 150 research articles, reviews and book chapters. His research interest
is in hepatitis C new drug development.
18th International Symposium on Hepatitis C Virus and Related Viruses : xxxvii
PLENARY LECTURERS
Plenary Lecture: Sunday, 11 September, 08:30 - 09:00
PLENARY LECTURERS
Plenary Lecture: Sunday, 11 September, 14:30 - 15:00
PLENARY LECTURERS
Virus-Host Interactions: The role of type I, II and III interferons in acute and chronic
hepatitis C
Markus H. Heim, M.D., University Hospital Basel and University Basel, Switzerland
Experimental HCV infections in chimpanzees reveals three distinct phases during the acute hepatitis C: I) an initial phase with very
rapid increase of HCV RNA in the serum followed by II) a steady state lasting 4-8 weeks with viral titers between 105 to 107, and III)
the recruitment of HCV specific T cells that results in the elimination of HCV (acute self-limiting HCV) or in the reduction of the viral
titer by around 2 log10 in case of HCV persistence. The transition from phase I with its exponential increase of HCV titers to phase
II is accompanied by the upregulation of interferon stimulated genes such as OAS, MX1 and IFIT. Presumably, HCV infection triggers
a type I interferon (IFN) response that controls the initially unrestrained HCV replication. However, neither the precise type of IFN
(IFN beta, IFN alpha(s) or IFN lambda) nor the cellular source of IFN has been identified so far. Also, little is known about these early
phases of HCV infection in human liver.
In chimpanzees, the third phase of acute HCV is characterized by T cell infiltration and measurable IFN gamma mRNA in the liver,
an increase in serum ALT, and a rapid decline of viral titer. A recent analysis of 6 human liver biopsies obtained 3-4 months after
HCV infection found the same features. In all cases, microarray analysis showed a strong induction the mRNAs of hundreds of
ISGs, and immuno-histochemistry confirmed this induction for a limited number of ISGs on the protein level. Gene set enrichment
analysis of microarray data revealed a dominant role of IFN gamma in driving the ISG induction in the (late) acute phase of HCV. It is
unexplained how HCV can persist and establish a chronic infection in presence of such a strong induction of ISGs. Paradoxically, over
90% of patients can be cured with IFN alpha when treated during the acute phase of HCV infection. The efficacy of IFN alpha could
be due to purely quantitative difference of ISG induction. Alternatively, there could be a requirement for ISGs that are induced
specifically or preferentially by IFN alpha. Interestingly, the IL28B CC (rs860) genotype is associated with spontaneous elimination
of HCV. Since IFN alpha and IFN lambda induce vastly overlapping sets of ISGs, it is tempting to speculate that endogenous IFN
lambda stimulates important ISGs not induced by IFN gamma. However, there is no evidence that patients with the IL28B CC
genotype produce more IFN lambda in acute hepatitis C.
Whereas IFN alpha induced ISGs have strong antiviral efficacy in the acute phase of HCV, the same ISGs are little effective during
chronic hepatitis C. Hundreds of ISGs are persistently induced in the liver of chronically infected chimpanzees, but HCV persists.
The same is true for about 30-50% of humans with chronic hepatitis C. In these patients, hundreds of ISGs are strongly (comparable
to what can be achieved with therapeutically applied pegIFNalpha) induced in the liver during decades. However, mot only is
the persistent activation of the endogenous IFN system ineffective in clearing HCV, but it also strongly reduces the response to
treatments that contain (peg)IFN alpha. On the other side, patients without an induction of ISGs in the liver have a high response
rate to pegIFN alpha / ribavirin treatments. The reasons for the differential activation of the endogenous IFN system are unclear.
Genetically determined differences in IFN lambda production is one of the hypothesis presently being tested by several groups.
Indeed, there is a strong association of the IL28B rs860 T allele with hepatic ISG induction. However, experimental evidence for
increased IFN lambda production in the liver in patients with a T allele is lacking.
Refractoriness of IFN signal transduction pathways is a potential mechanisms responsible for non-response to (peg)IFN alpha in
presence of a persistently induced endogenous IFN system. Experiments in mice and chimpanzees have documented that activation
of STAT1 and ISG induction is transient. After an initial response, the IFN alpha signal transduction system remains unresponsive
to further stimulation for a prolonged period of time. Interestingly, IFN lambda signaling remains intact, most likely because the
IFN lambda receptor and the associated Jak kinases are not inhibited by USP18, the major mediator of refractoriness of IFN alpha.
Markus Heim, M.D. earned his medical degree at the University of Basel, Switzerland. He then trained
for 3 years in basic biomedical science at the Biozentrum in Basel with U.A.Meyer investigating genetic
polymorphisms of drug metabolism. Following two years in Internal Medicine at Basel University Hospital,
Heim joined the laboratory of James E. Darnell at Rockefeller University, New York, as a postdoctoral
fellow, where he worked on basic aspects of Jak-STAT signal transduction. He then completed his internal
medicine training and obtained a full training in gastroenterology and hepatology at the University
Hospitals in Freiburg, Germany, and Basel, Switzerland. Since 1999 he is Group Leader of the Hepatology
Laboratory, and since 2003 head of the hepatology service at the Basel University Hospital. Heim is
presently in a sabbatical in Frank Chisari’s Lab at the Scripps Research Institute in La Jolla, CA.
His research focuses on signal transduction in liver disease. He is specifically interested in the role of
interferons in acute and chronic viral hepatitis. For a complete list of his publications, please visit http://www.researcherid.com/
rid/A-7526-2008.
xxxviii : HCV 2011
ORAL ABSTRACTS
Host Genetics and Host Responses
O1.01
Liver cell-specific expression phenotypes and hepatitis C virus treatment
responses: phenotype outperforms IL28B genotype
O1.02
Association of TLR7 rs179008 gene variants with hepatic IL-29/IFN-lambda1
and IFN-lambda receptor expression in chronic hepatitis C
Sabine Mihm, Eva Askar, Giuliano Ramadori
O1.03
NK inhibitory receptor expression associated with treatment failure and
IL-28B genotype in patients with chronic hepatitis C
Hugo Rosen, Lucy Golden
O1.04
High hepatic IFNλ-R1 receptor levels are associated with HCV RNA clearance
in IL28B genotype CC HCV patients
Jie Chen, Thomas Urban, Yuhong Zhang, Jeremie Guedj, Alan Perelson, Daryl Lau
O1.05
Aurélie Ducès, Martine Lapalus, Ivan Bièche, Bénédicte Jardin-Watelet, Eve Dupas, Simon De Muynck, Isabelle Molina,
Nadine Lambert, Bérénice Sallenave, Emilie Nicolas, Emilie Estrabaud, Nathalie Julian, Michelle Martinot-Peignoux,
Olivier Lada, Valérie Paradis, Dominique Valla, Pierre Bedossa, Daniel Laune, Michel Vidaud, Patrick Marcellin,
Tarik Asselah
O1.06
Gene signatures associated with liver disease progression in HCV patients
following liver transplantation
Angela Rasmussen, Nathan Susnow, Alexei Krasnoselsky, Sujay Datta, Craig Magaret, Matthew Yeh, Deborah Diamond,
Sean Proll, Sharon Lederer, Kathie Walters, Robert Carithers Jr., Steven Self, Michael Katze
18th International Symposium on Hepatitis C Virus and Related Viruses : 1
ORAL ABSTRACTS
Jordan J. Feld, Limin Chen, Maha Guindi, Sandra Fischer, E. Jenny Heathcote, Ivan Borozan, Xiangdong Liu, Aled Edwards,
Nazia Selzner, Katherine Siminovitch, Ian McGilvray
ORAL ABSTRACTS
2 : HCV 2011
HOST GENETICS AND HOST RESPONSES
O1.01
Liver cell-specific expression phenotypes and hepatitis C virus treatment responses:
phenotype outperforms IL28B genotype
Background: Our previous data show that cell-type specific interferon stimulated gene (ISG) expression is associated with response to
interferon-based therapy. ISG staining in hepatocytes is associated with non-response while responders show increased staining in hepatic
macrophages at baseline. Aim: To compare the predictive ability and correlation between hepatic ISG staining and IL28B gt. Methods: The
IL28B SNP rs12979860 was sequenced. Hepatic gene expression was evaluated by MxA staining in hepatocytes and macrophages (0-3+) or
by microarray, using classifier analysis on a validated 6-gene set to predict response. Regression models were compared using the likelihood
ratio test. Results: 209 patients were evaluated: 60% M, 73% Caucasian, 69% HCV gt 1. Treatment responses to peginterferon/ribavirin were
47% SVR, 16% relapse (REL) and 37% non-response (NR). Both IL28B gt and mRNA gene expression were predictive of response (SVR/REL)
with high PPV (IL28B 0.94 and gene expression 0.94) but modest NPV (IL28B 0.51 and gene expression 0.58). Patients who subsequently
responded had stronger macrophage staining (2.29±0.1 vs 0.78±0.1, p<0.0001) but reduced hepatocyte staining (2.81±0.1 vs 1.64±0.2,
p<0.0001) compared to future NR. REL showed an intermediate pattern. Absence of macrophage staining was strongly predictive of NR (NPV
96%). Staining showed a gradient by IL28B gt with macrophage staining weakest in TT patients (absent 79%) and strongest in CC patients
(present 96%), with the opposite pattern in hepatocytes. By multivariable logistic regression, only ISG expression (mRNA or MxA staining),
but not IL28B or viral gt, remained significantly associated with treatment response. The best predictive model contained macrophage
staining and viral gt, but not IL28B or other factors. Conclusion: The presence of macrophage ISG staining appears to be a prerequisite
to respond to interferon-based therapy and is strongly associated with the IL28B gt. Although the host and viral genotype influence gene
expression, ultimately the phenotype ie cell-specific expression pattern, best predicts treatment outcome.
Contact: Jordan J. Feld
University of Toronto, Toronto, Canada
[email protected]
O1.02
Association of TLR7 rs179008 gene variants with hepatic IL-29/IFN-lambda1 and IFNlambda receptor expression in chronic hepatitis C
Sabine Mihm (1), Eva Askar (1), Giuliano Ramadori (1)
(1) University Medical Center Goettingen, D-37075 Goettingen, Germany
Toll-like receptor 7 (TLR7) is likely involved in early pathogen detection and host response to viral infections. It senses viral single-stranded
RNA within the endosomal compartment. The variant allele of a non-synonymous polymorphism within exon 3 of the X-linked TLR7 gene,
rs179008, encodes for a functionally impaired protein. This study compared hepatic expression of selected innate immunity genes in female
and male chronic hepatitis C patients being homo- or hemizygous for the wildtype allele A or the variant allele T, respectively. The A allele
carriers were not found to differ significantly from the T allele carriers with regard to the amount of hepatic viral load. They also do not differ
in the hepatic expression of genes that previously have been shown to be activated in chronic hepatitis C virus (HCV) infection as IP-10, p44,
MxA, or interferon (IFN)-γ. However, TLR7 rs179008 T homo- and hemizygotes were found to express significant lower amounts of IL-29/
IFN-lambda1, and of IL-10 receptor β (IL-10Rβ) and IL-28 receptor alpha (IL-28Ralpha) which constitute the two subunits of the IFN-lambda
heterodimeric receptor, as well as lower amounts of IFN-alpha and IFN-alpha receptor 2 (IFNAR2) transcripts. The finding of a significant
association between TLR7 variants and (distinct) hepatic gene expression of IFN-lambda1 and IFN-lambda receptor subunits might point to
a role of TLR7 in HCV pattern recognition linked to endogenous IFN-lambda1 and IFN-lambda receptor expression. Significant differences in
IL-29/IFN-lambda1 and IFN-lambda receptor gene expression between TLR7 rs179008 T and A allele patients might also have implications
for responsiveness to future IFN-lambda based therapeutic approaches.
Contact: Sabine Mihm
University Medical Center Goettingen, D-37075 Goettingen, Germany
[email protected]
ORAL ABSTRACTS
Jordan J. Feld (1), Limin Chen (1), Maha Guindi (1), Sandra Fischer (1), E. Jenny Heathcote (1), Ivan Borozan (1), Xiangdong Liu (1),
Aled Edwards (1), Nazia Selzner (1), Katherine Siminovitch (1), Ian McGilvray (1)
(1) University of Toronto, Toronto, Canada
ORAL ABSTRACTS
O1.03
NK inhibitory receptor expression associated with treatment failure and IL-28B
genotype in patients with chronic hepatitis C
Hugo Rosen (1), Lucy Golden (1)
(1) University of Colorado, Aurora, CO, USA
ORAL ABSTRACTS
Natural killer (NK) cells constitute a first line of defense against viral infections; their function is governed by the integration of signals
from a wide spectrum of activating and inhibitory cell surface receptors. Furthermore, in contrast to T cells, NK cells do not require
priming in order to recognize virus-infected cells. We studied 101 patients (56 African American and 45 Caucasian American patients)
who received PEG/RIB for 48 weeks. Multiparameter FACS analysis comprehensively examined relative expression of cell surface markers
on NK and natural T (NT) cells.
Pre-treatment levels of activating receptors NKp30 and NKp44 were lower on NK subsets, and inhibitory receptors CD158b and CD158e
were higher in patients who demonstrated poor viral decline within the first 28 days of therapy. Higher expression levels of inhibitory
receptors NKG2A, CD158e (KIR3DL1) and CD158b (KIR3DL2/DL3) were demonstrable in patients who failed to develop sustained virologic
response (SVR). HCV-infected patients carrying the IL-28B T allele had higher NKG2A expression on total lymphocytes and CD56+ cells.
Next, we created mathematical regression models that incorporated race, viral level, and two of the NK inhibitory receptors. The areaunder-the curve ROC was 0.863 which means it is highly predictive of SVR (p <.00001). Moreover, the model performed complementarily
with IL-28B across the CC, CT, and TT genotypes. Among TT patients with pre-treatment SVR model scores in the top 50th percentile, 88%
had a chance of virologic cure (versus 10% of patients with lower scores). Purified NK subpopulations treated with pegylated IFN-a for 4
hours demonstrated higher levels of OAS, IP-10, TRAIL, and CD107a if depleted of cells that expressed inhibitory receptors.
These results provide novel insights into the associations of NK phenotype with IL-28B genotype and gene expression patterns, as well as
the roles of NK cells in mediating IFN-induced viral clearance of chronic HCV infection.
Contact: Hugo Rosen
University of Colorado, Aurora, CO, USA
[email protected]
O1.04
High hepatic IFNλ-R1 receptor levels are associated with HCV RNA clearance in IL28B
genotype CC HCV patients
Jie Chen (1), Thomas Urban (2), Yuhong Zhang (1), Jeremie Guedj (3), Alan Perelson (3), Daryl Lau (1)
(1) Harvard Medical School, Newton, MA, USA
(2) Duke University, USA
(3) Los Alamos National Laboratory, USA
IL28B polymorphisms predict treatment response in hepatitis C. IL28B is an interferon lambda (IFN-λ) that signals through a receptor
complex comprised of IL-10R2 and IFNλ-R1. This study evaluated the roles of IL28B genotypes in viral clearance. We correlated IL28B
genotypes with acute phase 1 HCV RNA decline, hepatic interferon stimulated gene (ISG) expression by microarray profiling and hepatic
levels of IFNλ-R1 receptor using confocal methods in liver samples obtained before and 24 hours after a single dose of IFN-α(10 MU sc).
18 HCV genotype 1 patients (6 each of IL28B genotype CC, CT and TT) were enrolled. Results: Responder genotype (CC) was associated
with rapid HCV RNA decline (>2 log) in the first 24 hours as measured by Drug Effectiveness or Epsilon (ε) derived from mathematical
model. Mean ε for genotype CC, CT and TT were 0.96, 093 and 0.71 respectively. Most patients with the nonresponder genotype (TT) had
preactivation of hepatic ISGs including MX1, ISG15, ISG 56, Viperin and OAS before IFN-α. In contrast, ISGs were induced by IFN-α in most
genotype non-TT patients. The IFNλ-R1 levels were determined by mean immunostaining signal per cell from 10 images of each liver slide.
Genotype CC patients with high ε had significantly higher pretreatment IFNλ-R1 levels compared to genotype TT nonresponders (5.2 vs. 0.33
signal/cell, p<0.05). Furthermore, there was strong coexpression of IFNλ-R1 with HCV NS3 before treatment. The IFNλ-R1 levels between
pre and post IFN-α were not different. However, hepatocytes with IFNλ-R1 expression generally had no detectable HCV NS3 after treatment.
Conclusions: Patients with IL28B genotype CC had high IFNλ-R1 receptor level that was likely induced by HCV infection. The correlations
between high IFNλ-R1 expression and rapid HCV RNA decline acutely after IFN-α suggest the role of IL28B genotype CC in inducing IFN-λ
and ISGs that result in viral clearance.
Contact: Jie Chen
Harvard Medical School, Newton, MA, USA
[email protected]
4 : HCV 2011
HOST GENETICS AND HOST RESPONSES
O1.05
Background and aims: Fibrosis is a dynamic process mediated by factors that regulate matrix synthesis and degradation, resulting in an excessive
accumulation of a dense extracellular matrix. In chronic hepatitis C, the transition from mild (F1) to moderate fibrosis (F2) seems to be a major
prognostic step. The aim of this study was to identify molecular markers that could differentiate mild from moderate fibrosis in chronic hepatitis C.
Methods: Liver biopsies from 244 untreated patients (or prior to treatment) were studied. Among these patients 66% had mild fibrosis (F1, Metavir
score) and 34% septal fibrosis (F2). Patients were mainly infected with genotype 1 (55%), 2 (11%), 3 (11%), 4 (20%) and 5-6 (3%) respectively. Real-time
quantitative RT-PCR assays were used to analyse the mRNA expression of 51 genes selected from the literature to be involved in fibrogenesis. Results:
The expression profiles of mild fibrosis differ significantly from those of moderate fibrosis. We identified a 5-gene signature composed of A2M, CXCL10,
IL8, SPP1 and S100A4 able to discriminate F1 and F2 patients with 70% sensitivity, 75% specificity, 81% NPV and 60% PPV. These genes are mainly
involved in extracellular matrix production and remodelling (matrix proteases family), in cell-cell and cell-extracellular matrix interactions, in cell cycle,
or encode growth factors/cytokines families (CXC chemokines). Conclusion: We identified on a large independent cohort that mild and moderate
fibrosis have different liver gene expression. A 5-gene signature composed of A2M, CXCL10, IL8, SPP1 and S100A4 was able to discriminate F1 and F2
patients. The most notable changes occurred mainly in cell-matrix turn-over. Several genes included in this gene signature encode molecules secreted
in the serum and provide a rational functional approach for the development of serum markers of fibrosis progression.
Contact: Tarik Asselah
Service d’Hépatologie & INSERM U773-CRB3, Clichy, France
[email protected]
O1.06
Gene signatures associated with liver disease progression in HCV patients following
liver transplantation
Angela Rasmussen (1), Nathan Susnow (2), Alexei Krasnoselsky (1), Sujay Datta (3), Craig Magaret (3), Matthew Yeh (2), Deborah Diamond
(1), Sean Proll (1), Sharon Lederer (1), Kathie Walters (4), Robert Carithers Jr. (2), Steven Self (3), Michael Katze (1)
(1) University of Washington, Seattle, WA, USA
(2) University of Washington Medical Center, Seattle, WA, USA
(3) SCHARP, Fred Hutchinson Cancer Research Center, USA
(4) Institute for Systems Biology, USA
Liver failure due to chronic hepatitis C virus infection is a major cause for liver transplantation worldwide. Recurrent infection of the graft is universal
in HCV patients following transplant and results in rapid progression to severe fibrosis and end-stage liver disease in one-third of all patients.
Unfortunately, no single clinical variable, or combination thereof, has proven accurate in identifying patients at risk of clinical decompensation in
the transplant setting. We thus sought to identify gene expression signatures in HCV transplant patients associated with progression to severe liver
disease. Logistic regression modeling identified 609 genes significantly associated with eventual development of severe liver fibrosis post-transplant.
Because of the variable nature of this clinical patient population, we also identified donor age, donor cause of death, graft rejection, and history of
alcohol abuse as potentially confounding clinical covariates. Statistical adjustment for these variables produced a signature of 270 genes that predict
clinical outcome and provide insight into the pathogenesis of HCV-induced liver disease in a clinical context. Our data indicate that de-regulation of
genes linked to cell cycle control, including those functioning in tumor suppression and DNA damage repair, occurs concomitant with the up-regulation
of transcripts for numerous extracellular matrix components in those patients who eventually progress to severe liver disease. We hypothesize that
early during re-infection, HCV modulates the cell cycle of infected hepatocytes to create a favorable environment for viral replication, which in turn
favorably impacts on pro-fibrogenic processes involving hepatic stellate cell activation. Significantly, these perturbations in gene expression occur prior
to histologic evidence of liver disease progression, suggesting that events which occur during the very acute phase of infection may influence patient
outcome and implicating a role for cell cycle regulation in HCV pathogenesis.
Contact: Angela Rasmussen
University of Washington, Seattle, WA, USA
[email protected]
ORAL ABSTRACTS
Aurélie Ducès (1), Martine Lapalus (7), Ivan Bièche (2), Bénédicte Jardin-Watelet (3), Eve Dupas (3), Simon De Muynck (7), Isabelle Molina
(3), Nadine Lambert (4), Bérénice Sallenave (1), Emilie Nicolas (1), Emilie Estrabaud (7), Nathalie Julian (5), Michelle Martinot-Peignoux
(7), Olivier Lada (7), Valérie Paradis (6), Dominique Valla (7), Pierre Bedossa (6), Daniel Laune (3), Michel Vidaud (1), Patrick Marcellin (7),
Tarik Asselah (7)
(1) Service de Biochimie et Génétique Moléculaire, Beaujon Hospital, Clichy, France
(2) INSERM UMR745, University of Paris Descartes, Paris, France
(3) UMR 3145 CNRS/Bio-Rad, Montpellier, France
(4) Bio-Rad Laboratories, Marnes-La-Coquette, France
(5) Ariana Pharmaceuticals, Paris, France
(6) Service d’Anatomie Pathologique, Hôpital Beaujon, Clichy, France
(7) Service d’Hépatologie & INSERM U773-CRB3, Clichy, France
ORAL ABSTRACTS
6 : HCV 2011
ORAL ABSTRACTS
Pathogenesis
O2.01
Laser capture microdissection reveals that fibrosis in chronic HCV infection
is associated with a loss of immune, metabolic, and redox functions
O2.02
HCV may trigger hepatic carcinogenesis through modulation of c-Myc
oncogene expression, enhancing oxidative stress and DNA damage
Martin Higgs, Herve Lerat, Jean-Michel Pawlotsky
O2.03
TLR4-Nanog stemness pathway in HCV-induced cancer stem cells confers
resistance to TGF-β-mediated tumor suppression through YAP1 and
IGF2BP3-AKT-mTOR
Keigo Machida, Chia-Lin Chen, Jian-Chang Liu, Claudine Kashiwabara, Douglas Feldman, Samuel French,
Jeong Hyeongnam, Linda Sher, Hidekazu Tsukamoto
O2.04
Infection of SCID/Alb-uPA mice by HCV leads to liver damage, apoptosis
and fibrosis
Michael Joyce, Kinola Williams, Gerald Lachance, Ran Chen, Lin-Fu Zhu, Sue-Ellen Lamb, Deborah Burshtyn, D. Lorne Tyrrell
O2.05
Persistent expression of the full genome of hepatitis C virus in B cells
induces spontaneous development of B-cell lymphomas in mice
Yuri Kasama, Satoshi Sekiguchi, Michinori Kohara, Kyoko Tsukiyama-Kohara
O2.06
Hepatitis C virus stabilization of hypoxia inducible factor-1α promotes
epithelial to mesenchymal transition
Garrick Wilson, Gary Reynolds, Claire Brimacombe, Ke Hu, Maria Simoes, Margaret Ashcroft, Peter Balfe, Stefan Hubscher,
Jane McKeating
ORAL ABSTRACTS
Supriya Munshaw, Hyon S. Hwang, Michael Torbenson, David L. Thomas, Stuart C. Ray, Ashwin Balagopal
ORAL ABSTRACTS
8 : HCV 2011
PATHOGENESIS
O2.01
Laser capture microdissection reveals that fibrosis in chronic HCV infection is
associated with a loss of immune, metabolic, and redox functions
Chronic Hepatitis C Virus (HCV) Infection, occurring in 60-80% of infected persons, is complicated by hepatic fibrosis and cirrhosis, although
the biologic basis of fibrogenesis is poorly understood. Hypothesizing that particular cellular components of the liver contribute in distinct
pathways to produce fibrosis, laser capture microdissection (LCM) was used to isolate unique hepatic segments in persons with chronic
HCV at different stages of liver disease. Subjects from the ALIVE cohort with HCV-monoinfection and archived liver tissue were matched for
age, race, and gender. LCM was performed on liver biopsies from subjects with Metavir scores of ≤ 1 or ≥3, capturing hepatic parenchyma
(HP) and portal tracts (PT) separately from five persons with low fibrosis and four persons with high fibrosis. In an unbiased comparison
between all segments from persons with low and high fibrosis only two genes were differentially expressed. As expected, comparison of
HP with PT showed that HP segments were enriched for genes associated with hepatocyte function (e.g. albumin, FDR<10-12) while PT
segments were enriched for immune surveillance (e.g. MHC Class II, FDR<10-5). Partitioning the analysis by fibrosis stage demonstrated a
loss of immune surveillance enrichment in PT segments of persons with high fibrosis. A comparison of HP segments between fibrosis stages
yielded a set of 86 differentially expressed genes (FDR<0.05); most genes were asymmetrically upregulated in persons with low fibrosis. Low
fibrosis HP segments were enriched for genes involved in small molecule and drug metabolism (e.g. Butyrylcholinesterase, FDR<10-4) and
protection against reduction-oxidation processes (e.g. peroxiredoxin3, FDR<0.03). The heterogeneity of the liver prevents the distinction
of RNA expression patterns that arise from separate hepatic components. LCM of individual hepatic segments displays clear segregation of
pathways potentially involved in fibrogenesis. Fibrosis is associated with an overall loss of function, primarily of immune surveillance, small
molecule metabolism, and reduction-oxidation protection.
Contact: Supriya Munshaw
Johns Hopkins University School of Medicine, Baltimore, MD, USA
[email protected]
O2.02
HCV may trigger hepatic carcinogenesis through modulation of c-Myc oncogene
expression, enhancing oxidative stress and DNA damage
Martin Higgs (1), Herve Lerat (1), Jean-Michel Pawlotsky (1)
(1) INSERM U955, Hopital Henri Mondor, Universite Paris-Est, Creteil, AE, France
Chronic hepatitis C virus (HCV) infection is a major risk factor in the onset and progression of hepatocellular carcinoma (HCC). The HCV
proteins may play a direct role in triggering hepatocarcinogenesis, although the underlying molecular mechanisms are unclear. The cMyc
oncogene influences oxidative stress, cell cycle checkpoint function and DNA damage, and has been shown to be involved in a number of
human tumors, including HCC.
To determine whether cMyc plays a role in HCV-associated HCC, we first analyzed cMyc expression in HCC-positive patients by
immunoblotting and qRT-PCR. Augmented cMyc expression was observed in non-tumoral tissue from patients infected with HCV compared
to hepatitis B virus (HBV)-infected or non-infected patients. Analyses of liver biopsies from patients who did not develop HCC revealed
similarly augmented cMyc expression in HCV-infected patients when compared to non-infected individuals. To assess the role of the HCV
proteins in cMyc expression in the absence of local inflammation, we made use of FL-N/35 transgenic mice expressing the entire HCV ORF
in a liver-specific fashion at near-physiological levels, which display characteristic liver pathologies associated with chronic HCV infection,
especially HCC development. Increased c-Myc expression was observed in non-tumoral tissues of these mice when compared to wt mice,
mirroring data from infected patients. Using chromatin immunoprecipitation, RNA-silencing and reporter gene assays, we demonstrated
that activation of β-catenin, a transcriptional activator of cMyc, was responsible for increased cMyc transcription in the presence of the HCV
proteins. Furthermore, RNAi-mediated silencing of c-Myc or β-catenin in hepatocytes isolated from FL-N/35 mice led to decreased oxidative
stress, diminished DNA damage, and restored G1/S checkpoint function. Together, these data demonstrate that HCV protein expression is
associated with enhanced c-Myc expression through β-catenin-mediated activation of its promoter, contributing to oxidative stress and DNA
damage, a mechanism likely to play a role in triggering liver carcinogenesis.
Contact: Jean-Michel Pawlotsky
INSERM U955, Hopital Henri Mondor, Universite Paris-Est, Creteil, AE, France
[email protected]
ORAL ABSTRACTS
Supriya Munshaw (1), Hyon S. Hwang (1), Michael Torbenson (1), David L. Thomas (1), Stuart C. Ray (1), Ashwin Balagopal (1)
(1) Johns Hopkins University School of Medicine, Baltimore, MD, USA
ORAL ABSTRACTS
O2.03
TLR4-Nanog stemness pathway in HCV-induced cancer stem cells confers resistance to
TGF-β-mediated tumor suppression through YAP1 and IGF2BP3-AKT-mTOR
Keigo Machida (1), Chia-Lin Chen (1), Jian-Chang Liu (1), Claudine Kashiwabara (1), Douglas Feldman (1), Samuel French (2), Jeong
Hyeongnam (1), Linda Sher (1), Hidekazu Tsukamoto (1)
(1) University of Southern California, Los Angeles, CA, USA
(2) University of California Los Angeles, USA
ORAL ABSTRACTS
Tumor-initiating stem cells (TISC) confer resistance to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC)
commonly observed in hepatitis C virus (HCV)-infected patients with alcohol abuse. We recently identified the pluripotency marker Nanog
as a direct target of Toll-like receptor 4 (TLR4) signal activated by endotoxin in CD133+/CD49f+ TISC from liver tumor in HCV Ns5a transgenic
(Tg) mice fed alcohol. Here we report such TISC are also isolated from HCC in alcohol-fed HCV Core Tg mice and patients. We further
demonstrate that TLR4/Nanog-dependent TISC are defective in TGF-β signaling. Functional oncogene screening of a cDNA library identified
the organ size control pathway target Yap1 and AKT activator Igf2bp3 as Nanog-dependent genes that inhibit TGF-β signaling in TICS.
This inhibition is achieved by Yap1-mediated cytoplasmic retention of p-Smad3 and suppressed Smad3 phospho-activation by the AKTmTOR pathway. Silencing of both Yap1 and Igf2bp3 restores TGF-β signaling, inhibits pluripotency gene expression and tumorigenesis, and
sensitizes TISC to cell death by the conventional chemotherapy with Rapamycin and Sorafenib. Therefore, a combined therapy targeting
both the YAP1 and AKT-mTOR pathways to restore the TGF-β pathway should improve therapeutic efficacy for HCC.
Contact: Keigo Machida
University of Southern California, Los Angeles, CA, USA
[email protected]
O2.04
Infection of SCID/Alb-uPA mice by HCV leads to liver damage, apoptosis and fibrosis
Michael Joyce (1), Kinola Williams (1), Gerald Lachance (1), Ran Chen (1), Lin-Fu Zhu (1), Sue-Ellen Lamb (1), Deborah Burshtyn (1),
D. Lorne Tyrrell (1)
(1) University of Alberta, Edmonton, Canada
Hepatitis C virus is a blood borne pathogen which is a major cause of liver disease worldwide, with an estimated 200 million people infected.
It’s estimated that 30% of chronically infected patients eventually develop progressive liver disease including cirrhosis and end stage liver
disease. It has been shown that hepatocyte damage can lead to apoptosis, which plays a role in the recruitment and activation of stellate
cells and macrophages, and the subsequent development of fibrosis. We previously characterized the host transcriptional response to HCV
patient serum infection of chimeric SCID/Alb-uPA mice. Here, we describe HCV infection in SCID/Alb-uPA mice which develop fibrosis similar
to that seen in patients despite the lack of an adaptive immune system. Similar to mice infected with HCV H77c RNA, there was evidence
for ER stress and hepatocyte ballooning when mice were infected with HCV JFH-1 and increased levels of apoptosis. There are increased
numbers of macrophages in the livers of infected mice accompanied by increases in macrophage specific transcripts CD68 and iNOS. In
addition there are increases in TGF-β and TNF-α transcripts, which activate stellate cells, and as expected increased SMA transcription and
increase numbers of stellate cells. Fibrosis is evident both qualitatively and by quantitation of the amount of collagen I and III. Importantly
anti-NK antibodies decreased the amount of apoptosis seen, and exogenously added NK92 cells lead to increased levels of apoptosis. In
addition, infection of SCID/bg/Alb-uPA mice, which are incapable of degranulation, did not result in signs of liver damage.
Contact: Michael Joyce
Univertity of Alberta, Edmonton, Canada
[email protected]
10 : HCV 2011
PATHOGENESIS
O2.05
Persistent expression of the full genome of hepatitis C virus in B cells induces
spontaneous development of B-cell lymphomas in mice
Extrahepatic manifestations of hepatitis C virus (HCV) infection have been reported in 40–70% of HCV-infected patients. B-cell non-Hodgkin’s
lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection. Although infection of B cells by HCV has been
reported, the mechanism of disease onset remains unclear. Here we established HCV transgenic mice that express the full HCV genome in
B cells (RzCD19Cre mice) and observed a 25.0% incidence of non-Hodgkin’s diffuse large B-cell lymphomas (22.2 % in males and a 25.9%
in females) at 600 days after birth. Expression levels of aspartate aminotransferase, alanine aminotransferase, and 31 different cytokines,
chemokines and growth factors were examined. Among these, only the incidence of B-cell lymphoma and the level of soluble interleukin-2
receptor α subunit (sIL-2Rα) in RzCD19Cre mouse serum were significantly correlated. All RzCD19Cre mice with substantially elevated serum
sIL-2Rα levels developed B-cell lymphomas. Thus, expression of HCV in B cells promotes non-Hodgkin’s-type diffuse B-cell lymphoma,
and therefore the RzCD19Cre mouse represents a powerful model to study the mechanisms of HCV-associated development of B-cell
lymphoma. The further detailed mechanism of B cell lymphoma development in these mice is now under investigation.
Contact: Kyoko Tsukiyama-Kohara
Kumamoto University, Kumamoto, Japan
[email protected]
O2.06
Hepatitis C virus stabilization of hypoxia inducible factor-1α promotes epithelial to
mesenchymal transition
Garrick Wilson (2), Gary Reynolds (2), Claire Brimacombe (2), Ke Hu (2), Maria Simoes (1), Margaret Ashcroft (1), Peter Balfe (2), Stefan
Hubscher (2), Jane McKeating (2)
(1) Division of Medicine, University College London, United Kingdom
(2) The University of Birmingham, Birmingham, United Kingdom
Hepatitis C virus (HCV) infects hepatocytes of the liver causing progressive liver disease including hepatocellular carcinoma (HCC). However,
the precise mechanism(s) of HCV induced HCC are poorly understood. Hepatocytes are highly polarized with distinct apical and basal
membranes separated by tight junctions that are essential for the establishment and maintenance of cell polarity. Loss of tight junction
integrity is the hallmark of many malignant tumours. Recent data showed that the HCV encoded glycoproteins E1E2 induced a relocalization
of tight junction proteins in non-polarized hepatoma cells. To ascertain the functional effects of HCV E1E2 on hepatocellular polarity, we
stably expressed the viral glycoproteins in HepG2 cells that polarize and develop apical canalicular structures. We demonstrate that E1E2
alter tight junction Occludin and ZO-1 expression and localization, reduced HepG2 polarity, tight junction formation and integrity. This was
accompanied by increased migration, increased vascular endothelial growth factor production, enhanced sensitivity to the permeabilizing
effects of cytokines and the induction of epithelial to mesenchymal transition markers SNAIL and TWIST. Importantly, these observations
were confirmed following HCVcc infection of hepatoma cell lines or primary hepatocytes and HCV infected liver tissue. HCV infection stabilizes
hypoxia inducible factor-1α (HIF-1α), a transcription factor that regulates a variety of genes involved in angiogenesis, cell invasion and
metastasis. Inhibition of HIF-1α ablated the effects of E1E2 glycoproteins and virus infection on hepatocyte biology, demonstrating a HIF-1α
dependent perturbation of hepatocyte biology. These data provide new insights into the molecular biology of HCV induced carcinogenesis
of the liver and offer new approaches for treatment.
Contact: Jane McKeating
The University of Birmingham, Birmingham, United Kingdom
[email protected]
ORAL ABSTRACTS
Yuri Kasama (2), Satoshi Sekiguchi (1), Michinori Kohara (1), Kyoko Tsukiyama-Kohara (2)
(1) The Tokyo Metropolitan Institute, Japan
(2) Kumamoto University, Kumamoto, Japan
ORAL ABSTRACTS
12 : HCV 2011
ORAL ABSTRACTS
Viral and Host Factors
O3.01
Identification of a host factor determining the anti-HCV activity of ribavirin
O3.02
Ribavirin treatment reverses HCV NS3/4A-mediated interference with
intrahepatic immunity in vivo
Erwin Daniel Brenndörfer, Anette Brass, Matti Sällberg
O3.03
HCV-infected hepatocytes produce CXCL10 in a RIG‐I and TLR3
dependent manner
Jessica Brownell, Jessica Wagoner, Derek Thirstrup, Wesley Smith, Kui Li, Stephen Polyak
O3.04
Activation of chemokine and inflammatory cytokine response in HCV-infected
hepatoma cells requires TLR3 sensing of HCV dsRNA intermediates
Qingming Dong, Nan Li, Dahai Wei, Susan Pfeffer, Meiyun Fan, Lawrence Pfeffer, Kui Li
O3.05
Identification of activity profiles of novel hepatitis C virus associated
host factors
David Blais, Neda Nasheri, Craig Mckay, Daniel Figeys, Shao Yao, Ragunath Singaravelu, John Pezacki
O3.06
Malnutrition impairs interferon signaling through mTOR and FoxO
pathways in patients with chronic hepatitis C
Masao Honda, Kenji Takehana, Akito Sakai, Takayoshi Shirasaki, Tetsuro Shimakami, MinKyung Yi, Stanley M. Lemon,
Shuichi Kaneko
ORAL ABSTRACTS
Kyoko Mori, Osamu Hiraoka, Masanori Ikeda, Yasuo Ariumi, Akiko Hiramoto, Yusuke Wataya, Nobuyuki Kato
ORAL ABSTRACTS
14 : HCV 2011
VIRAL AND HOST FACTORS
O3.01
Identification of a host factor determining the anti-HCV activity of ribavirin
Recently we developed a new Li23 cell line-derived drug assay system (ORL8), in which genome-length HCV-RNA (O strain of genotype 1b)
encoding renilla luciferase efficiently replicates. In the last meeting, we reported the findings that ribavirin (RBV) at clinically achievable
concentrations (approximately 10 μM) efficiently inhibited HCV-RNA replication in ORL8, but not in OR6 (an HuH-7-derived drug assay
system), and clarified that its anti-HCV activity was mediated by the inhibition of inosine monophosphate dehydrogenase (IMPDH).
In this time, we report the finding of a host factor determining the anti-HCV activity of RBV. We first performed HPLC analysis of the
nucleotides extracted from RBV-treated ORL8 and OR6 cells, and showed that GTP concentration was reduced in ORL8 cells, but not in OR6
cells. Interestingly, we observed that IMP in ORL8 cells was accumulated approximately 30 times more than that in OR6 cells. In addition,
we demonstrated that IMPDH siRNA significantly suppressed HCV-RNA replication in ORL8 cells. These results support that anti-HCV activity
of RBV is mediated by the inhibition of IMPDH.
To identify the host factor determining the anti-HCV activity of RBV, we performed cDNA microarray analysis using Li23- and HuH-7derived cells. During the course of the study, we unexpectedly found that the ectopic expression of some host factor (RSG:RBV-sensitivitydetermining gene as a laboratory name) in OR6 cells converted the cells from a RBV-resistant phenotype (50% effective concentration
(EC50): >100 μM) to a RBV-sensitive phenotype (EC50: 2.6 μM). Furthermore, we demonstrated that the transfection of RSG siRNA to ORL8
cells possessing the RBV-sensitive phenotype led the cells to a moderately RBV-resistant phenotype. Taken together, these results suggest
that RSG is a determining factor for the anti-HCV activity of RBV. Further analysis on the mechanism underlying the differential expression
of RSG in ORL8 and OR6 cells is in progress.
Contact: Kyoko Mori
Department of Tumor Virology, Okayama University, Okayama, Japan
[email protected]
O3.02
Ribavirin treatment reverses HCV NS3/4A-mediated interference with intrahepatic
immunity in vivo
Erwin Daniel Brenndörfer (1), Anette Brass (1), Matti Sällberg (1)
(1) Karolinska Institutet, Stockholm, Sweden
Background: As a persistent virus, HCV has evolved mechanisms to evade elimination by both innate and adaptive immunity. The HCV
NS3/4A protease/helicase is known to modulate signaling pathways in the infected hepatocyte by cleaving Cardif, TC-PTP and TRIF. However,
the global effects exerted by NS3/4A in vivo still remain unclear. This is currently of a particular interest since new antiviral therapies are
being introduced based on inhibitors blocking the NS3/4A protease. Thus, these therapies may exert effects beyond the viral replication.
Objectives: We aimed to understand how NS3/4A affects intercellular signaling and immune cell function in vivo and to study the effects of
ribavirin treatment on the NS3/4A-mediated modulation of the host’s immune system.
Methods: The intrahepatic immune response of naive and lipopolysaccharide (LPS)/D-galactosamine (D-galN) treated NS3/4A-transgenic
(Tg) mice was determined by Western blot, ELISA, Real Time PCR, Flow cytometry, ALT levels, and survival. Ribavirin pretreatment was
performed to analyze the influence of ribavirin on the NS3/4A-mediated effects.
Results: Hepatic IL10 expression is enhanced in NS3/4A-Tg mice both basally and after LPS/D-galN treatment, while IFN-gamma secretion is
impaired. Furthermore, the chemokine profile (CCL2, CCL17, CXCL9) is altered towards an anti-inflammatory state. As result, the intrahepatic
number of Th1 (CXCR3+) cells in NS3/4A-Tg mice is decreased, while the amount of Th2 (CCR4+) cells is increased. Interestingly, the NS3/4Amediated effects could be reversed by ribavirin treatment. This is of particular importance since recent clinical trials suggest that ribavirin is
essential for a functional therapy with only directly acting antiviral compounds (DAAs).
Conclusions: NS3/4A induces a shift of the intrahepatic immune response towards a Th2-dominated immunity. This may impair the host
response to the infection and promote viral persistence. Importantly, ribavirin treatment can block the effects mediated by NS3/4A.
Contact: Erwin Daniel Brenndörfer
Karolinska Institutet, Stockholm, Sweden
[email protected]
ORAL ABSTRACTS
Kyoko Mori (1), Osamu Hiraoka (2), Masanori Ikeda (1), Yasuo Ariumi (1), Akiko Hiramoto (3), Yusuke Wataya (3), Nobuyuki Kato (1)
(1) Department of Tumor Virology, Okayama University, Okayama, Japan
(2) School of Pharmacy, Shujitsu University, Japan
(3) Faculty of Pharmaceutical Sciences, Okayama University, Japan
ORAL ABSTRACTS
O3.03
HCV-infected hepatocytes produce CXCL10 in a RIG‐I and TLR3 dependent manner
Jessica Brownell (1), Jessica Wagoner (1), Derek Thirstrup (1), Wesley Smith (1), Kui Li (2), Stephen J. Polyak (1)
(2) University of Tennessee-Memphis, Memphis, TN, USA
ORAL ABSTRACTS
Chronic hepatitis C affects 170 million people globally and causes liver damage by eliciting a persistent, local inflammatory response that
cannot eradicate hepatitis C virus (HCV) infection. Characterization of the role played by pro-inflammatory chemokines in establishing this
response within the liver during acute HCV infection may lead to development of more effective treatments for hepatitis C. To this end we
investigated how sensing of HCV infection by the pathogen recognition receptors (PRRs) TLR3 and RIG-I induced expression of the chemokine
CXCL10 (i.e. IP-10) in infected hepatocytes. Maximal CXCL10 mRNA and protein levels were detected 72 hours after infection with JFH-1 in
Huh7 cells expressing both TLR3 and RIG-I. CXCL10 mRNA and protein expression were also observed following JFH-1 infection of 4 different
primary human hepatocyte preparations, which were confirmed to endogenously express both PRRs. The mRNA response peaked at 48
hours post-infection in these cells and then declined over the subsequent 3 days in culture despite continued presence of viral genomes.
Conversely, CXCL10 protein expression remained elevated throughout the infection period. While a small dose-dependent increase in
CXCL10 mRNA was observed following treatment with several doses of interferon (IFN)-α, robust activation of CXCL10 transcription was
also observed by independently triggering RIG-I or TLR3 signaling using poly I:C. CXCL10 protein was further shown by deconvolution
fluorescence microscopy to be robustly expressed in HCV-infected cells, with lower levels of CXCL10 protein observed in neighboring noninfected cells. These observations suggest that CXCL10 induction during JFH-1 HCV infection is only partially attributable to the paracrine
effects of IFN-α. Current work is focusing on the relative contribution of RIG-I and TLR3 signaling as well as other soluble, paracrine factors
to CXCL10 production.
Contact: Jessica Brownell
[email protected]
O3.04
Activation of chemokine and inflammatory cytokine response in HCV-infected
hepatoma cells requires TLR3 sensing of HCV dsRNA intermediates
Qingming Dong (1), Nan Li (1), Dahai Wei (1), Susan Pfeffer (1), Meiyun Fan (1), Lawrence Pfeffer (1), Kui Li (1)
(1) University of Tennessee-Memphis, Memphis, TN, USA
Chemokines and inflammatory cytokines are key regulators of immunity and inflammation during viral infections. Hepatitis C virus (HCV) is
a hepatotropic RNA virus frequently associated with chronic liver inflammation and hepatocellular carcinoma. Intrahepatic and peripheral
blood levels of chemokines and cytokines are elevated in chronic HCV infections, but the underlying mechanisms remain unclear. We find
that Toll like receptor-3 (TLR3) senses HCV infection in cultured hepatoma cells, leading to NF-kB activation and the production of numerous
chemokines and inflammatory cytokines, such as RANTES, MIP-1a, MIP-1b, IP-10 and IL-6. The chemokine/cytokine induction occurred
late in HCV infection and was abrogated when HCV was UV-inactivated prior to infection, indicating a dependence on cellular recognition
of HCV replication products. Gel shift and chromatin immunoprecipitation assays revealed that NF-kB plays a pivotal role in HCV-induced
chemokine/cytokine transcription. Mutations specifically disrupting the dsRNA binding activity of TLR3 ablated the chemokine/cytokine
response to HCV infection, indicating that HCV dsRNA was the pathogen associated molecular pattern triggering TLR3 signaling. In vitro
synthesized HCV dsRNAs with a length of >100 bp activated TLR3-dependent chemokine expression, regardless of the genome position from
which they derive. In contrast, HCV ssRNAs, including those derived from 3’NTR highly potent for RIG-I activation, failed to do so. Moreover,
robust production of chemokines and inflammatory cytokines was also observed in primary human hepatocytes following stimulation with
extracellular poly-I:C, a TLR3 ligand. Taken together, our data suggest that TLR3-mediated chemokine and inflammatory cytokine responses
may play an important role in host immune responses to HCV and the pathogenesis of HCV-associated liver diseases.
Contact: Kui Li
University of Tennessee-Memphis, Memphis, TN, USA
[email protected]
16 : HCV 2011
VIRAL AND HOST FACTORS
O3.05
Identification of activity profiles of novel hepatitis C virus associated host factors
Activity-based proteome profiling (ABPP) is a novel proteomics tool used to investigate changes in the activity of enzyme classes and
identify host-pathogen interactions. The aim of the present study is to identify novel activity profiles for families of enzymes, as part of a
larger systems biology study during the transient expression of hepatitis C virus (HCV) proteins as well as during HCV infection. We have
performed ABPP of human hepatoma cells that express HCV proteins such as core and NS4B, as well as cells expressing HCV replicons and
JFH-1 infectious clones. We have obtained activity profiles using a combination of mechanism-based ABPP probes that target hydrolases,
substrate-based probes that target proteases and esterases, as well as non-directed probes, which allow for targeting enzyme classes lacking
cognate inhibitors. We have shown that carboxylesterase 1 (CES1) activity changes during HCV replication, and this activity correlated with
viral abundance in a number of models for HCV infection. We have also utilized probes to study fatty acid synthase (FAS) activity and found
that FAS activity changes rapidly upon expression of HCV proteins. ABPP gives new information about the dynamics and regulation of FAS
activity. Also we have used a wortmannin-based ABPP probe to examine the changes in kinase activity and found that kinases, including
PI3K, protein kinase C, and pyruvate kinase, were differentially active during HCV replication and infection. By combining SILAC with a
wortmannin-based ABPP probe, the precise quantification of changes in activity of several kinases was observed during HCV replication
and infection. The change in activity observed for PI3K is consistent with its predicted role in HCV propagation. We hypothesize that at least
some of these differentially active proteins facilitate host-virus interactions crucial to replication and infection in the human liver and could
serve as a targets for therapeutic intervention.
Contact: John Pezacki
NRC Steacie Institute for the Molecular Sciences, Ottawa, Canada
[email protected]
O3.06
Malnutrition impairs interferon signaling through mTOR and FoxO pathways in
patients with chronic hepatitis C
Masao Honda (1), Kenji Takehana (2), Akito Sakai (1), Takayoshi Shirasaki (1), Tetsuro Shimakami (1), MinKyung Yi (3), Stanley M. Lemon
(4), Shuichi Kaneko (1)
(1) Department of Gastroenterology, Kanazawa University, Kanazawa, Japan
(2) Ajinomoto Pharmaceuticals Co., Ltd, Japan
(3) University of Texas Medical Branch, Galveston, USA
(4) The University of North Carolina at Chapel Hill, USA
Background & Aims: Patients with advanced chronic hepatitis C (CH-C) are often malnourished, but the effects of malnutrition on interferon
(IFN) signaling and response to treatment have not been determined. We assessed the importance of the nutritional state of the liver on
IFN signaling and treatment response.
Methods: We studied data from 168 patients with CH-C who were treated with the combination of Peg-IFN and ribavirin. Plasma
concentrations of amino acids were measured by mass spectrometry. Liver gene expression profiles were obtained from 91 patients. Huh-7
cells were used to evaluate the IFN signaling pathway, mammalian target of rapamycin complex1 (mTORC1), and forkhead box O (Foxo).
Antiviral signaling induced by branched-chain amino acids (BCAAs) was determined using the in vitro HCV replication system.
Results: Multivariate logistic regression analysis showed that Fischer’s ratio was significantly associated with non-responders, independent
of IL28B polymorphisms or the histologic stage of the liver. Fischer’s ratio was inversely correlated with expression of BCAA transaminase 1,
and was affected by hepatic mTORC1 signaling. IFN stimulation was substantially impaired in Huh-7 cells grown in medium low in amino acid
concentration, through repressed mTORC1 signaling and increased Socs3 expression, which was regulated by Foxo3a. BCAA could restore
impaired IFN signaling and inhibit hepatitis C virus replication under conditions of malnutrition.
Conclusions: Malnutrition impaired IFN signaling, by inhibiting mTORC1 and activating Socs3 signaling through Foxo3a. Increasing BCAAs to
upregulate IFN signaling might be used as a new therapeutic approach for patients with advanced CH-C.
Contact: Masao Honda
Department of Gastroenterology, Kanazawa University, Kanazawa, Japan
[email protected]
ORAL ABSTRACTS
David Blais (3), Neda Nasheri (3), Craig Mckay (3), Daniel Figeys (1), Shao Yao (2), Ragunath Singaravelu (3), John Pezacki (3)
(1) Ottawa Institute of Systems Biology/University of Ottawa, Canada
(2) National University of Singapore, Singapore
(3) NRC Steacie Institute for the Molecular Sciences, Ottawa, Canada
ORAL ABSTRACTS
18 : HCV 2011
ORAL ABSTRACTS
Innate Immunology
O4.01
Mitochondrial-associated ER membranes form an innate immune synapse
that is targeted by hepatitis C virus
O4.02
Short-range exosomal transfer of viral RNA to plasmacytoid dendritic cells
activates the innate host response
Marlene Dreux, Urtzi Garaigorta, Bryan Boyd, Josan Chung, Francis V. Chisari
O4.03
Type III interferons produced by hepatocytes play a significant role in the
induction of interferon stimulated genes during acute HCV infection
Heiyoung Park, Onyinyechi Eke, Stefania Capone, Antonella Folgori, Barbara Rehermann
O4.04
Early natural killer cell responses in hepatitis C virus exposed healthcare
workers who do not develop acute infection
Jens M. Werner, Theo Heller, Barbara Rehermann
O4.05
Victoria Best
O4.06
Acute resolving infections with HCV and HAV differ in induction of type 1
IFN responses and the prolonged persistence of HAV RNA in the liver
Robert Lanford, Zongdi Feng, Bernadette Guerra, Deborah Chavez, Kathleen Brasky, Yan Zhou, Daisuke Yamane,
Alan Perelson, Christopher Walker, Stanley Lemon
ORAL ABSTRACTS
Stacy M. Horner, Helene Liu, Jessica Briley, Michael Gale Jr.
ORAL ABSTRACTS
20 : HCV 2011
INNATE IMMUNOLOGY
O4.01
Mitochondrial-associated ER membranes form an innate immune synapse that is
targeted by hepatitis C virus
Stacy M. Horner (1), Helene Liu (1), Jessica Briley (1), Michael Gale Jr. (1)
Contact: Stacy M. Horner
[email protected]
O4.02
Short-range exosomal transfer of viral RNA to plasmacytoid dendritic cells activates
the innate host response
Marlene Dreux (1), Urtzi Garaigorta (1), Bryan Boyd (1), Josan Chung (1), Francis V. Chisari (1)
(1) The Scripps Research Institute, La Jolla, CA, USA
Apart from virus secretion, infected cells can be sensed by bystander cells thereby modulating their activities. Here, we report that, in the
absence of virus production, RNAse A treatment-resistant HCV RNA that co-purifies with exosome markers (CD63, CD81) is specifically
released from subgenomic HCV replicon cells and is transferred in a short range cell-cell manner to cocultured plasmacytoid dendritic
cells (pDC) that then produce type 1 interferon (IFNα). Accordingly, inhibitors of neutral sphingomyelinase that are known to reduce
exosome release inhibit IFNα production by pDC, suggesting that exosomal vesicles transport HCV RNA across the intercellular space
between replicon cells and pDC. To examine the molecular basis of this novel cell-cell communication model, we knocked-down expression
of representative members of intracellular vesicular transport pathways in the replicon cells and monitored their ability to activate the
pDCs. Our results indicate that Annexin A2, and components of the ESCRT (e.g. CHMP4B, TSG101) and autophagy (e.g. Atg12) machinery
are required for the replicon cells to generate the pDC-activating signal. Moreover, deletion of domain III of NS5A (amino acid residues
2328-1435), which is known to be required for Annexin A2 recruitment to the HCV replicase, abrogates pDC activation, thereby confirming
the Annexin A2 requirement. In addition, we detected HCV RNA in ESCRT protein- and exosome marker-containing intracellular vesicles,
potentially representing exosome precursors prior to egress. Together, these results illustrate the existence of a cellular mechanism whereby
the intracellular vesicular transport machinery mediates the formation and transfer of immunostimulatory viral RNA-containing exosomes
across the interface between infected cells and pDC, thereby triggering the innate host response to HCV infection. They also suggest that
vesicle-mediated cell-cell communication may play an important role in other viral and nonviral systems as well.
Contact: Marlene Dreux
The Scripps Research Institute, La Jolla, CA, USA
[email protected]
ORAL ABSTRACTS
RIG-I is an intracellular protein that signals innate immune defenses against viruses and is essential for immunity against HCV. RIG-I engages
HCV RNA in infected cells to trigger innate immune defenses through its adaptor protein IPS-1. However, HCV blocks RIG-I signaling through
viral NS3/4A protease cleavage of IPS-1 to support persistent infection. While IPS-1 resides on mitochondria and peroxisomes, how its
signaling is coordinated among these organelles has been unknown. Here, we show that a major site of IPS-1 localization and signaling is
the mitochondrial-associated membrane (MAM), which links ER to mitochondria. During both acute RNA virus infection and persistent HCV
RNA replication, RIG-I is recruited to the MAM to bind IPS-1. Dynamic MAM tethering to mitochondria and peroxisomes then coordinates
IPS-1 localization to form a signaling synapse. The HCV NS3/4A protease localizes to the MAM at these junctions with peroxisomes and
mitochondria for IPS-1 targeting. Importantly, NS3/4A cleaves IPS-1 from MAM but not mitochondria to restrict RIG-I signaling. Thus, an
intracellular immune synapse anchored by the MAM coordinates innate immunity during RNA virus infection and is targeted by the HCV
NS3/4A protease.
ORAL ABSTRACTS
O4.03
Type III interferons produced by hepatocytes play a significant role in the induction of
interferon stimulated genes during acute HCV infection
Heiyoung Park (1), Onyinyechi Eke (1), Stefania Capone (2), Antonella Folgori (2), Barbara Rehermann (1)
(1) National Institute of Health, Liver Diseases Branch, NIDDK, Bethesda, MD, USA
(2) Okairos, Naples, Italy
ORAL ABSTRACTS
Strong induction of interferon-stimulated genes (ISGs) in the liver is a signature of acute hepatitis C virus (HCV) infection. Given that HCV
proteins have been shown to inhibit the induction of type I interferon (IFN-α and -β) in vitro, it remains unclear which cytokines and which
cells are responsible for the strong ISG induction in the HCV-infected liver in vivo.
To answer these questions, we studied the differential expression of type I and type III (IL28-AB, IL-29) IFNs in relation to ISG expression and
outcome of infection using plasma samples and serial liver biopsies of five chimpanzees throughout the course of acute HCV genotype 1a
infection. Three chimpanzees displayed a self-limited course and two chimpanzees displayed a chronically evolving course of HCV infection.
Despite vigorous and early induction of ISGs (Mx-1, IFIT1, CXCL11), IFN-α2 and IFN-β mRNA levels were minimally induced (<3.3-fold
compared to preinfection levels) in the liver and IFN-α and IFN-β proteins remained undetectable in the plasma. In contrast, type III IFN,
in particular IL-29 was strongly induced at the mRNA in the liver (>33-fold) and protein level in the plasma (>247 pg/ml) and its expression
kinetics correlated with ISG expression. However, there was no association between the expression level of type III IFNs and the outcome
of acute HCV infection.
To evaluate whether the IFN profile that we observed in vivo was derived from hepatocytes, we infected primary human hepatocytes with
HCV JFH-1. These experiments recapitulated the robust induction of type III IFNs followed by ISGs expression but almost no induction of
type I IFNs.
Collectively our data indicate a strong role of hepatocyte-derived type III IFNs in ISGs induction during acute HCV infection.
Contact: Heiyoung Park
National Institute of Health, Liver Diseases Branch, NIDDK, Bethesda, MD, USA
[email protected]
O4.04
Early natural killer cell responses in hepatitis C virus exposed healthcare workers who
do not develop acute infection
Jens M. Werner (1), Theo Heller (1), Barbara Rehermann (1)
(1) NIDDK, National Institutes of Health, Bethesda, MD, USA
Acute hepatitis C virus (HCV) infection typically results in chronic disease with HCV outpacing adaptive immune responses. Here we asked
whether innate immune responses from natural killer (NK) cells contribute to antiviral immunity using a unique cohort of prospectively
followed health care workers who were exposed to HCV but did not develop acute infection.
Twelve health care workers were studied 2, 4, 6, 12 and 24 weeks after exposure to HCV infected blood [needlestick (n=10), cut (n=2)].
All remained HCV RNA and HCV antibody negative at all study time points. Surprisingly, the mean fluorescence intensity of NK cells that
expressed the activation marker CD122 peaked 2 weeks after HCV exposure (p<0.05), and was followed by peak expression of the activating
receptors NKp44 and NKp46 at week 4 (p<0.05) and the inhibitory receptor NKG2A at week 6 (p<0.05). This change in NK cell phenotype
was accompanied by increased cytotoxicity (CD107a degranulation, TRAIL expression (p<0.05)) at week 4, and increased IFN-γ production
at week 6 (p<0.05). Importantly, the increase in NK cell cytotoxicity correlated to the magnitude of the subsequent HCV-specific T cell
response. CD8 T cells were primarily elicited against nonstructural HCV peptides, providing evidence that HCV had successfully entered
cells and initiated its life cycle with translation of nonstructural proteins. Consistent with this interpretation, we observed an early IFN-α
response, as evidenced by increased serum levels of CXCL10, an IFN-α-induced protein.
Collectively, these results demonstrate that an early NK cell response may contribute to the prevention of acute hepatitis and the successful
induction of adaptive immunity. The multifunctional NK cell response (cytotoxicity and IFN-γ production) in these HCV-exposed health care
workers differed from the typical NK cell response (increase in cytotoxicity and decrease in IFN-γ production) in persistent HCV infection.
Contact: Barbara Rehermann
NIDDK, National Institutes of Health, Bethesda, MD, USA
[email protected]
22 : HCV 2011
INNATE IMMUNOLOGY
O4.05
Hepatitis C virus (HCV) infection is a major global health problem with approximately 170 million people chronically infected worldwide.
80% of infected individuals fail to clear their infection, largely as the result of weak, narrowly targeting or waning antiviral T cell responses.
One possibility is that inadequate antigen presentation by dendritic cells (DCs) contributes to immune failure, yet the role of DCs at the
site of HCV replication in the liver is yet to be clearly undefined. Using a multicolor flow-based approach, we identified six phenotypically
distinct populations of professional antigen presenting cells among interstitial leukocytes isolated from human explanted liver specimens.
When the proportion of cells belonging to each of the six identified populations was compared between HCV-infected and non-infected
patients, HCV infection was characterized by a significant increase in the frequency of intrahepatic myeloid DCs (both CD1c+ mDC1s and
CD141+ mDC2s) and a corresponding decrease in the frequency of CD34+ DC progenitor cells. Phenotypic and functional analysis of mDC
populations isolated from HCV-infected livers further revealed increased expression of accessory molecules CD40, CD80 and CD83 in the
context of HCV infection, and that these cells were capable of secreting effector cytokine IL-12 in response to TLR stimulation in vitro.
Together these data demonstrate that i) chronic HCV infection may facilitate the “customized” recruitment and differentiation of liver DC
subsets with established functional roles in the presentation of endogenous viral antigens and ii) these DCs are characterized by a mature,
activated phenotype and respond to antigenic stimulation in vitro. Together these findings highlight a role for interstitial liver DCs in the
adaptive immune response to HCV, and may have implications for the strategic design of HCV therapies aimed towards the modulation of
antigen presentation and the assignment of immune context.
Contact: Victoria Best
Emory University, Atlanta, GA, USA
[email protected]
O4.06
Acute resolving infections with HCV and HAV differ in induction of type 1 IFN
responses and the prolonged persistence of HAV RNA in the liver
Robert Lanford (1), Zongdi Feng (2), Bernadette Guerra (1), Deborah Chavez (1), Kathleen Brasky (1), Yan Zhou (3), Daisuke Yamane (2),
Alan Perelson (4), Christopher Walker (3), Stanley Lemon (2)
(1) Texas Biomedical Research Institute, San Antonio, TX, USA
(3) The Research Institute at Nationwide Children’s Hospital, USA
HCV and HAV are both positive, single-stranded RNA viruses that cause acute hepatitis infections; however, only HCV is capable of causing
life-long persistent infections. We compared acute HCV and HAV infections in chimpanzees in search of factors that may contribute to the
different outcomes of the infections. Surprisingly, HAV-infected animals exhibited very limited induction of type I interferon-stimulated
genes in the liver compared to the robust response observed in HCV-infected chimpanzees, despite the observation that HAV infected
animals have similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Low level ISG responses peaked 1-2 weeks
after HAV challenge then subsided despite continuing high hepatic viral loads. Serum IFNα levels followed the same trend, suggesting that
HAV is capable of actively suppressing the type I IFN response. Similar to HCV, we have found that HAV has evolved mechanisms of blocking
IRF3 activation and IFN synthesis by infected cells through viral protease-mediated cleavage of MAVS/IPS-1 and TRIF. Ongoing studies are
evaluating whether HAV-infected cells induce IFN synthesis by plasmacytoid dendritic cells (pDCs), but high-titer HAV by itself does not
activate pDCs. An acute inflammatory response at 3-4 weeks correlated with the appearance of virus-specific antibodies and both apoptosis
and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months (35 to >48 weeks) after clearance from serum
and feces, revealing dramatic differences in the kinetics of clearance in the three compartments. In contrast, during acute resolving HCV
infection, virus is cleared from serum and liver with essentially the same kinetics and at much earlier times (10-20 weeks). Collectively, these
findings suggest that acute HAV infection is far stealthier than acute HCV infection and represents a distinctly different paradigm in viral-host
interactions within the liver.
Contact: Robert Lanford
Texas Biomedical Research Institute, San Antonio, TX, USA
[email protected]
ORAL ABSTRACTS
Victoria Best (1)
(1) Emory University, Atlanta, GA, USA
ORAL ABSTRACTS
24 : HCV 2011
ORAL ABSTRACTS
Cellular Immunology
O5.01
Modulation of HCV specific CD8 T cell exhaustion and survival by IL-21
during acute hepatitis C
O5.02
Vicki Fleming, Joannah Fergusson, Yu-hoi Kang, Paul Klenerman, Cormac Cosgrove
O5.03
Repeated exposure to trace amounts of HCV suppresses T-cell responses to
subsequent high dose HCV challenge via induction of regulatory T-cells
Su-Hyung Park, Naga Suresh Veerapu, Barbara Rehermann
O5.04
Protection and immunodominance of an HLA-B27 restricted HCV-specific
CD8+ T cell epitope is linked to rapid antigen processing and presentation
Julia Schmidt, Astrid K. N. Iversen, Stefan Tenzer, David A. Price, Volker Lohmann, Paul Bowness, Hansjörg Schild,
Paul Klenerman, Hubert E. Blum, Christoph Neumann-Haefelin, Robert Thimme
O5.05
Tissue-specific versus disease-specific expression of T-cell inhibitory ligands
and receptors
Daniela Kroy, Donatella Ciuffreda, Michelle Tomlinson, Garrett Hauck, Joseph Misdraji, Georg M. Lauer
ORAL ABSTRACTS
Hassen Kared, Nathalie Bédard, Julie Bruneau, Naglaa H. Shoukry
ORAL ABSTRACTS
26 : HCV 2011
CELLULAR IMMUNOLOGY
O5.01
Modulation of HCV specific CD8 T cell exhaustion and survival by IL-21 during acute
hepatitis C
Studies in humans and chimpanzees have demonstrated that CD4 helper T cells are essential for spontaneous clearance of Hepatitis C virus
(HCV) and long-term protection against viral persistence. However, the exact mechanisms underlying failure of CD4 helper T cells during the
early acute phase of HCV are not well understood. We hypothesized that early loss or exhaustion of CD8 T cells coupled with a defect in the
cytokine profile of helper CD4 T cells, is implicated in failure to clear HCV. We thus performed longitudinal analysis of the phenotype and
function of CD4 T cells in patients acutely infected with HCV who went on to achieve spontaneous resolution (n=7) or persistent infection
(n=13). Antigen specific proliferation, expression of inhibitory molecules like CTLA-4, PD1 or Tim-3 and the skewing of cytokines secretion
by polyclonal or HCV specific CD4 T cells were assessed. We observed a higher frequency of proliferating HCV specific CD4 T cells with a
Th1 (IFN-g and TNF-a) and Th17 (IL-17A, IL-21, IL-22) cytokine profile in patients who achieved spontaneous viral clearance as compared to
chronic evolution. This was coupled with higher concentration of IL-21 in serum during the late acute phase that correlated positively with
the frequency of HCV specific CTLs measured by MHC class I tetramers. In contrast, patients with chronic evolution displayed lower levels of
IL-21 and diminished proliferation associated with increased expression of exhaustion markers upon antigenic stimulation. This exhaustion
was reversible upon blockade of all three inhibitory receptors. Our results suggest that IL-21 may play a key role in enhancing proliferation
and survival of HCV specific memory CD8 T cells by limiting the up-regulation of exhaustion molecules. Such approach may prove promising
in boosting host immunity to enhance the rate of HCV clearance during acute infection and as an adjuvant during therapeutic vaccination.
Contact: Hassen Kared
University of Montréal -CR CHUM St Luc, Montréal, Canada
[email protected]
O5.02
Vicki Fleming (1), Joannah Fergusson (1), Yu-hoi Kang (1), Paul Klenerman (1), Cosmac Cosgrove (1)
(1) University of Oxford, Oxford, England
CD4+ and CD8+ T cells play a central role in host responses against HCV, although their function in chronic infection is still poorly defined.
CD161, a C-type lectin, which is significantly expressed on HCV-specific T cells, and especially on liver-homing populations (up to 50%), is
linked to both Type 17 lineage evolution and IL-18 receptor expression. IL-18 is a multifunctional proinflammatory cytokine that is elevated
in HCV infection and IL-18 genetic polymorphisms are linked to disease outcome. In this study we examined the impact of IL-18 cytokine
signaling on the function of CD4+ CD161+ T cells. We sorted CD4+CD161+/- T cells and analysed the full impact of stimulation using RNA
expression arrays and real time-PCR approaches, followed by functional confirmation. IL-18 stimulation directly and rapidly activates
CD4+CD161+ T cells without TCR ligation and upregulates expression of a distinct range of cytokines and chemokines. Amongst CD161+
T cells, IL-17F secretion was highly upregulated upon activation (2.94 log2-fold change) and confirmed by intracellular staining and ELISA.
In addition, matrix metallopeptidase 3 and 13 were among the most highly upregulated transcripts in CD4+CD161+ T cells upon IL-18
stimulation (5.1 & 5.06 log2-fold change respectively), as well as granulocyte-macrophage colony-stimulating factor (3.24 log2-fold change)
and IL-22 (3.27 log2-fold change). Our study, using a novel approach to functional profiling, reveals an important population of CD4+ T cells
in the liver, and a potent role for IL-18 in their activation. This includes rapid triggering of potent inflammatory mediators, which possess
protective and pathologic roles and would have potentially a very significant role in innate and adaptive immunity during HCV pathogenesis
& liver disease.
Contact: Cosmac Cosgrove
University of Oxford, Oxford, England
[email protected]
ORAL ABSTRACTS
Hassen Kared (1), Nathalie Bédard (1), Julie Bruneau (1), Naglaa H. Shoukry (1)
(1) University of Montréal -CR CHUM St Luc, Montréal, Canada
ORAL ABSTRACTS
O5.03
Repeated exposure to trace amounts of HCV suppresses T-cell responses to subsequent
high dose HCV challenge via induction of regulatory T-cells
Su-Hyung Park (1), Naga Suresh Veerapu (1), Barbara Rehermann (1)
(1) Immunology Section, Liver Diseases Branch, NIDDK, NIH, Bethesda, MD, USA
ORAL ABSTRACTS
Vigorous and sustained T-cell responses are important for hepatitis C virus (HCV) clearance. Low-level HCV-specific T-cell responses have
been observed in frequently exposed populations such as injection drug users, and it has been proposed that they confer immune protection.
To test this hypothesis we induced T-cell responses in 2 chimpanzees by four intravenous infusions at 9-week intervals of human plasma
containing trace amounts of HCV (<40 copies/ml). A control chimpanzee received HCV RNA-negative human plasma.
The two HCV-exposed chimpanzees remained HCV RNA-negative in blood and liver but developed HCV-specific T-cell responses. Such
responses were not detected in the control chimpanzee.
To assess the protective nature of these T cell responses all chimpanzees were challenged with high dose (100 CID50) HCV 15 weeks after
the last infusion. Unexpectedly, the pre-existing T-cell responses of the 2 exposed chimpanzees were not boosted but disappeared rapidly.
Even more surprisingly, new T-cell responses to the high dose HCV challenge were significantly suppressed, and intrahepatic CD8 and IFN-γ
mRNA levels remained significantly lower than in the control chimpanzee.
To study the mechanisms underlying this apparent immune suppression we evaluated the role of regulatory T-cells (Tregs). Whereas the
chimpanzees did not differ in the frequency of circulating CD4+Foxp3+ Tregs at the start of the study, low dose HCV exposure increased
the percentage of Tregs. Most importantly, high dose HCV challenge resulted in a significant increase in Treg frequency in the liver and the
blood of the two previously HCV-exposed chimpanzees compared to the control chimpanzee. This corresponded to dramatically higher
intrahepatic Foxp3, IL-10, TGF-β and B7-H4 mRNA levels.
Collectively, we show that repeated exposure to trace amounts of HCV expands functional Tregs that suppress HCV-specific memory response
upon reinfection. This may negatively impact the efficiency of candidate vaccines targeting high-risk populations with frequent exposure to HCV.
Contact: Su-Hyung Park
Immunology Section, Liver Diseases Branch, NIDDK, NIH, Bethesda, MD, USA
[email protected]
O5.04
Protection and immunodominance of an HLA-B27 restricted HCV-specific CD8+ T cell
epitope is linked to rapid antigen processing and presentation
Julia Schmidt (1), Astrid K. N. Iversen (2), Stefan Tenzer (3), David A. Price (4), Volker Lohmann (5), Paul Bowness (2), Hansjörg Schild (3),
Paul Klenerman (6), Hubert E. Blum (1), Christoph Neumann-Haefelin (1), Robert Thimme (1)
(1) Department of Medicine II, University of Freiburg, Freiburg, Germany
(2) Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, United Kingdom
(3) Institute of Immunology, University of Mainz, Germany
(4) Department of Infection, Immunity and Biochemistry, Cardiff University, United Kingdom
(5) Department of Virology, University of Heidelberg, Germany
(6) Nuffield Department of Clinical Medicine, Oxford, United Kingdom
HLA-B27 has been shown to have a protective role in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. The immunological
and virological features of HLA-B27 allele mediated protection are not fully understood. However, there is growing evidence that the protective
effect is linked to single dominant HIV- and HCV-specific epitopes that are presented by HLA-B27 to cytotoxic T lymphocytes. Indeed, in the case
of HCV, protection could be linked to constraints on virological escape within this conserved epitope region. In the present study, we assessed
the importance of several immunological factors that may account for protection mediated by HLA-B27 in human viral infections. Accordingly,
we analyzed the functional avidity, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the dominant HLA-B27 NS5B2841
epitope as well as the antigen processing and presentation of this epitope. The obtained findings were compared with results derived from
different HLA-A2 restricted HCV-specific epitopes. We could show that functional avidity, antiviral efficacy and the number of naïve precursors
do not contribute to the protective effect of the dominant HCV-specific HLA-B27 epitope. Importantly, however, the HLA-B27 restricted epitope
region was extraordinarily efficiently processed by immunoproteasomes, resulting in a faster presentation of the epitope on the cell surface
of antigen presenting cells. These results provide new insights into the immunological mechanisms of protection in HCV infection mediated by
HLA-B27. Specifically, they indicate that rapid and efficient antigen processing is a key immunological mechanism for the immunodominace of
the HLA-B27 restricted HCV-specific epitope and the protective effect of the HLA-B27 allele.
Contact: Julia Schmidt
Department of Medicine II, University of Freiburg, Freiburg, Germany
[email protected]
28 : HCV 2011
CELLULAR IMMUNOLOGY
O5.05
Tissue-specific versus disease-specific expression of T-cell inhibitory ligands and receptors
Background: Murine models of infection have demonstrated striking differences in the quantity and quality of immune responses in different
tissues, yet little is known about tissue specific immunity in humans. In HCV infection, increased intrahepatic expression of PD-1 and other
T-cell inhibitory receptors has been described, but important questions remain about the network of inhibitory receptors and ligands, the
activation status of inhibitory pathways and the degree to which observations are tissue- or disease-specific.
Specific aim: Our aim was to determine the intrahepatic expression patterns of T-cell inhibitory and stimulatory molecules in chronic
Hepatitis C infection compared to non-HCV disease and healthy liver tissue.
Methods: Intrahepatic lymphocytes (IHL) and PBMCs were extracted from 30 liver tissue specimens (5 healthy controls, 15 non-HCV disease
liver tissues, 10 HCV positive liver tissues) and blood. Flow cytometry was performed to determine the expression of PD-1, 2B4, LAG3,
CD160 on intrahepatic and peripheral CD4+ and CD8+ T-cells. Immunohistochemistry/immunofluorescence studies were done on paraffin
embedded liver sections to characterize inhibitory receptor and ligand expressing immune cells.
Results: Expression levels of PD-1, CD160, 2B4 and LAG3 were generally higher on IHL compared to PBMCs but did not differ between
the different disease groups on either CD4+ or CD8+ IHL. In contrast, we detected a significantly higher expression of PD-L1 and PD-L2 in
HCV positive liver compared to healthy controls and non-HCV disease tissue. PD-L1 was partially expressed by T-cells, whereas PD-L2 was
predominantly expressed by non-T-cell immune cells that we currently characterize in more detail.
Conclusion: Strong expression of inhibitory/stimulatory receptors is universally observed on IHL, even from healthy liver. In contrast,
expression of PD-1 ligands PD-L1 and PD-L2 is much more varied, with higher expression levels in HCV livers, indicating that expression of
inhibitory ligands might be the key event in determining the setpoint of inhibitory pathway activation and exhaustion.
Contact: Daniela Kroy
Massachusetts General Hospital, Boston, MA, USA
[email protected]
ORAL ABSTRACTS
Daniela Kroy (1), Donatella Ciuffreda (1), Michelle Tomlinson (1), Garrett Hauck (1), Joseph Misdraji (1), Georg M. Lauer (1)
(1) Massachusetts General Hospital, Boston, MA, USA
ORAL ABSTRACTS
30 : HCV 2011
ORAL ABSTRACTS
Vaccine Development
Finding the 70%: cost-effectiveness and population-level impact of general
population screening for hepatitis C infection
Phillip Coffin, John Scott, Matthew Golden, Sean Sullivan
O6.02
HCV genotype 1a ancestral sequence elicits broad CD8+ T cell responses
Kelly P. Burke, Supriya Munshaw, William O. Osburn, Jordana Levine, Stuart C. Ray, Andrea L. Cox
O6.03
Antibodies to the interfering epitope EP-II (aa434-446) of HCV E2 can mask
neutralizing activity induced in patients vaccinated with rE1E2 protein
Alla Kachko, Sharon E. Frey, Frances Wells, Iryna Zubkova, Pei Zhang, Stephen M. Feinstone, Michael Houghton,
Marian Major
O6.04
Enhancing T cell response and cross-neutralizing antibodies against HCV
envelope by combining adenovirus and protein
Alicja Chmielewska, Mariarosaria Naddeo, Virginia Ammendola, Ke Hu, Lieven Verhoye, Malgorzata Rychlowska,
Rino Rappuoli, Stefano Colloca, Alfredo Nicosia, Riccardo Cortese, Geert Leroux-Roels, Philip Meuleman,
Krystyna Bienkowska-Szewczyk, Peter Balfe, Jane A. McKeating, Antonella Folgori
O6.05
Simian adenoviral and MVA vectors combined in a heterologous prime
boost strategy – a highly immunogenic novel vaccine against HCV
Eleanor Barnes, Antonella Folgori, Richard Antrobus, John Halliday, Stefania Capone, Ye Oo, Rachel Townsend,
Leo Swadling, Steve Aston, Joel Myer, Katherine Gantlett, Rachel Huddart, Anthony Brown, Ayako Kurioka,
Christabel Kelly, Virginia Ammendola, Stefano Colloca, Mariarosaria Naddeo, Loredana Siani, David Mutimer,
Cinzia Traboni, David Adams, Ricardo Cortese, Alfredo Nicosia, Paul Klenerman
ORAL ABSTRACTS
O6.01
ORAL ABSTRACTS
32 : HCV 2011
EPIDEMIOLOGY, GLOBAL BURDEN, AND VACCINE DEVELOPEMENT
O6.01
Finding the 70%: cost-effectiveness and population-level impact of general population
screening for hepatitis C infection
Context: Current U.S. guidelines recommend limiting hepatitis C screening to high-risk individuals and approximately 70% of infected
persons remain unaware of their status.
Objective: To estimate the cost-effectiveness and population-level impact of adding one-time screening of the general adult U.S. population
to current hepatitis C testing guidelines.
Design and Setting: We used a decision analytic model for the screening intervention and developed a Markov model with annual transitions
to estimate natural history. The model reflects a societal perspective and lifetime horizon. We conducted one-way and probabilistic sensitivity
analyses on all modifiable inputs. Data sources for model parameters included clinical trials, reviews, meta-analyses, and expert opinion.
Main Outcome Measures: Incremental cost per quality-adjusted life year (QALY) gained for general population screening compared to
standard of care, calculated from lifetime costs and accumulated QALYs for each strategy, and hepatitis C-related health outcomes averted.
Results: Incremental cost for general population screening was $2,250/QALY compared to current guidelines and remained well below $50,000/
QALY in all one-way sensitivity analyses and Monte Carlo simulations. Incremental cost was lowest ($1,750/QALY) if screening was applied to a
population with a similar degree of liver fibrosis as estimated for 2010, but the absolute cost per QALY of managing hepatitis C increased with
degree of fibrosis at the time of detection, regardless of screening approach. Approximately 1% of hepatitis C-related health outcomes would
be averted per 7.5% of the population screened, although the impact would be greater with improved referral and treatment.
Conclusions: Broader screening for hepatitis C is likely to be cost-effective, but the absolute costs of managing hepatitis C will rise as fibrosis
advances among the undiagnosed population. Revisions of current guidelines to broaden screening criteria are needed, but realizing a
clinically significant impact on associated morbidity and mortality will require concomitant improvements in referral and treatment. Contact: Phillip Coffin
[email protected]
O6.02
HCV genotype 1a ancestral sequence elicits broad CD8+ T cell responses
Kelly P. Burke (1), Supriya Munshaw (1), William O. Osburn (1), Jordana Levine (1), Stuart C. Ray (1), Andrea L. Cox (1)
(1) Johns Hopkins School of Medicine, Baltimore, MD, USA
Background: Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross-reactivity against widely
divergent circulating strains. Computer-generated sequences minimize genetic distance when compared with circulating strains, and can
be generated as a consensus sequence (the most common amino acid at each position) or an ancestral sequence (derived using Bayesian
or maximum likelihood phylogenetic tools). To date, broader recognition of such sequences in HCV has not been demonstrated. We tested
the hypothesis that CD8 T cell responses to an ancestral HCV sequence (compared to those generated using circulating HCV strains) would
be more cross reactive.
Methods: A consensus HCV sequence and an ancestral representative sequence were generated from 390 full-length HCV genotype 1a
polypeptide sequences. Naturally occurring sequence mutations in known epitopes were identified by longitudinal sequencing of subjects
who generated CD8 T cell responses. Peptides encoding the ancestral, consensus, and natural sequence variants for each of eleven epitopes
were then tested for the capacity to expand CD8 T cell populations and to elicit cross-reactive responses by IFN-gamma ELISpot assay.
Results: The ancestral and the consensus sequence were identical for 8/11 epitopes examined. When the ancestral and consensus sequence
differed, expansion with ancestral sequence peptides produced significantly greater recognition of both ancestral and consensus peptides.
Lines generated against ancestral sequence peptides were generally cross-reactive to consensus sequence and natural sequence variants.
Furthermore, ancestral sequence peptides generated more robust T cell responses than did natural sequence variant peptides.
Conclusions: The broadest recognition of highly diverse circulating HCV strains was achieved using CD8+ T cells expanded with ancestral
sequence HCV. These data support the use of ancestral sequence in HCV vaccine design.
Contact: Kelly P. Burke
Johns Hopkins School of Medicine, Baltimore, MD, USA
[email protected]
ORAL ABSTRACTS
Phillip Coffin (1), John Scott (1), Matthew Golden (1), Sean Sullivan (1)
ORAL ABSTRACTS
O6.03
Antibodies to the interfering epitope EP-II (aa434-446) of HCV E2 can mask neutralizing
activity induced in patients vaccinated with rE1E2 protein
Alla Kachko (2), Sharon E. Frey (1), Frances Wells (2), Iryna Zubkova (2), Pei Zhang (2), Stephen M. Feinstone (2), Michael Houghton (3),
Marian Major (2)
(1) Division of Infectious Diseases and Immunology, Saint Louis University, USA
(2) CBER/FDA, Bethesda, MD, USA
(3) Dept. of Medical Microbiology and Immunology, University of Alberta, Canada
ORAL ABSTRACTS
We have previously demonstrated that depleting interfering antibodies to the HCV envelope protein E2 region aa434-446 (EP-II), increases
the neutralizing capacity of sera from persistently infected patients and vaccinated chimpanzees. We now demonstrate similar antibody
interference using serum from subjects vaccinated with recombinant HCV E1E2 protein adjuvanted with MF59C.1 (an oil-in-water emulsion).
The vaccine was given at 3 different dosages on Day 0 and Weeks 4, 24 and 48 in a phase 1, placebo-controlled, dose escalation trial with
healthy HCV negative adults (NIH/NIAID VTEU contract N01-A1-25464, Study number DMID 01-002). One hundred and twelve blinded serum
samples from the trial were tested in an ELISA for reactivity to peptides representing the E2 regions 412-426 (EP-I) and 434-446 (EP-II). Of these
112 samples, 8 samples tested positive for EP-I only; 4 tested positive for EP-II only and 18 samples were positive for both epitopes. Antibodies
to EP-II were depleted from 4 samples that tested positive for antibodies to both epitopes and the depletion of the antibodies was verified in
an ELISA. Untreated and depleted aliquots of these 4 samples were tested in a neutralization assay using an HCVcc 1a/2a chimera. In all cases,
the untreated samples were scored as non-neutralizing, with ID50 titers of <1:64 in the assay. However, following depletion of antibodies to the
EP II epitope, the samples were found to have detectable neutralizing activity with ID50 titers increasing up to 2.4 fold. These data show that
antibodies to the interfering region EP-II (aa434-446) are induced during immunization of patients with recombinant E1E2 proteins and that
these antibodies can mask effective neutralization of cell culture derived HCV. These findings further support the hypothesis that the efficacy of
HCV vaccines targeting the envelope proteins may be improved if the induction of antibodies to the interfering region EP-II can be prevented.
Contact: Marian Major
CBER/FDA, Bethesda, MD, USA
[email protected]
O6.04
Enhancing T cell response and cross-neutralizing antibodies against HCV envelope by
combining adenovirus and protein
Alicja Chmielewska (1), Mariarosaria Naddeo (2), Virginia Ammendola (2), Ke Hu (3), Lieven Verhoye (4), Malgorzata Rychlowska (1),
Rino Rappuoli (5), Stefano Colloca (2), Alfredo Nicosia (2), Riccardo Cortese (2), Geert Leroux-Roels (4), Philip Meuleman (4), Krystyna
Bienkowska-Szewczyk (1), Peter Balfe (3), Jane A. McKeating (3), Antonella Folgori (2)
(1) University of Gdansk, Gdansk, Poland
(3) Div. of Immunity and Infection, Univ. of Birmingham, Birmingham, United Kingdom
(4) University of Ghent, Ghent, Belgium
(5) Novartis Vaccines and Diagnostics, Cambridge, MA, USA, and Siena, Italy
Neutralizing antibodies (nAbs) may help control HCV viral replication in the early stages of infection as demonstrated by recent studies where
nAb responses during the acute phase were associated with viral clearance and fluctuating HCV RNA levels. Adenovirus (Ad) vectors expressing
the non-structural region of HCV genome elicited potent, durable and protective T cell response in chimpanzees and showed excellent safety and
immunogenicity in healthy volunteers. Chimpanzees vaccinated with purified HCV E1/E2 glycoprotein induced high-titer antibodies and displayed
a strong neutralizing activity. The same vaccine used in healthy volunteers was safe and immunogenic. To increase the probability of success of an
HCV vaccine for both prophylactic and therapeutic applications, the ‘ideal vaccine’ would be one that is capable of eliciting high-level neutralizing
antibody responses as well as potent T cell effector and memory populations. In this study, we constructed replication defective adenoviral
vectors encoding secreted and non-secreted HCV glycoproteins. Correct expression and post-translational maturation of HCV proteins was
confirmed upon infection of human cells. Heterologous prime-boost immunization regimens with Ad vectors expressing secreted E2 or the E1E2
heterodimer and recombinant E1E2 protein formulated with MF59 adjuvant were tested in mice and Guinea pigs. Strong and broad CD8+ and
CD4+ T cell responses and high-titer functional Th1-type IgG responses were induced by 4 sequential shots of Ad and protein vaccines. Immune
sera were capable of neutralizing HCVpp and HCVcc infection. Importantly, we demonstrated cross-reactive antibody responses that neutralized
genotypes 1a, 2, 3, 4 and 5. These animal studies showed that immunization regimens combining vectored vaccines with protein antigen in
adjuvant can induce strong antibody and T cell responses that surpass the best immune responses achieved by either vaccine technology alone.
Contact: Alicja Chmielewska
University of Gdansk, Gdansk, Poland
[email protected]
34 : HCV 2011
EPIDEMIOLOGY, GLOBAL BURDEN, AND VACCINE DEVELOPEMENT
O6.05
Simian adenoviral and MVA vectors combined in a heterologous prime boost strategy
– a highly immunogenic novel vaccine against HCV
Background and aims: A prophylactic HCV vaccine is urgently required. We have shown that adenoviral vaccine vectors encoding nonstructural (NS) HCV proteins induce potent T-cell responses that protect chimpanzees from chronic infection and that simian adenoviral
vectors encoding NS (AdCh3NS) administered to humans are highly immunogenic. In this study we evaluate the capacity of a rare human
adenovirus (Ad6NS) and an MVA-NS vector to boost functional HCV specific responses following AdCh3NS priming.
Methods: Replicative-defective AdCh3NS encoding 1985 amino acids derived from the NS3-5 region (genotype-1b) were administered (i.m.) as
a single or double-prime to 15 healthy volunteers (dose 2.5 x1010vp). A heterologous boosting vaccine was administered using either Ad6NS
(2.5 x1010vp) 8 or 24 weeks post prime, or MVA-NS 8 weeks post prime (2 x108pfu). Immunogenicity was assessed by ex-vivo IFNγ-ELISpot
(using 6 peptide pools spanning the HCV immunogen), HLA class-I tetramers, thymidine incorporation proliferation and ICCS assays.
Results: The AdCh3NS priming vaccination was highly immunogenic and induced multi-specific responses in all individuals; mean peak
1336 (range 642-4507) SFU/106 PBMC. HCV specific Ad6NS vaccination 8 or 24 weeks later effectively boosted responses; mean peak 1080
SFU/106 PBMC. These were maintained to at least a year post vaccination and could be tracked over this time using Class-I peptide tetramers.
However, boosting with MVA-NS 8 weeks post prime proved to be particularly potent; here the mean peak post-boost T-cell response was
3829 SFU/106 PBMC. ICCS showed that both polyfunctional CD4+ and CD8+ HCV specific T-cell responses are induced. Vaccination was very
well tolerated with mild/moderate, local/systemic reactions and no serious adverse advents.
Conclusions: We have generated a novel T-cell vaccine based on simian adenovirus and MVA vectors used in a prime boost regimen, that is
safe in man, induces polyfunctional CD4+ and CD8+ T-cells, and is highly immunogenic against multiple HCV antigenic targets.
Contact: Eleanor Barnes
Nuffiled Department of Medicine and NIHR BRC, University of Oxford, Oxford, United Kingdom
[email protected]
ORAL ABSTRACTS
Eleanor Barnes (1), Antonella Folgori (2), Richard Antrobus (3), John Halliday (4), Stefania Capone (2), Ye Oo (5), Rachel Townsend (4), Leo
Swadling (4), Steve Aston (3), Joel Myer (3), Katherine Gantlett (3), Rachel Huddart (4), Anthony Brown (4), Ayako Kurioka (4), Christabel
Kelly (4), Virginia Ammendola (2), Stefano Colloca (2), Mariarosaria Naddeo (2), Loredana Siani (2), David Mutimer (5), Cinzia Traboni (2),
David Adams (5), Ricardo Cortese (2), Alfredo Nicosia (2), Paul Klenerman (1)
(1) Nuffiled Department of Medicine and NIHR BRC, University of Oxford, Oxford, United Kingdom
(3) Centre for Clinical Vaccinology and Tropical Medicine, United Kingdom
(4) Nuffield Department of Medicine, University of Oxford, United Kingdom
(5) Queen Elizabeth Hospital Birmingham and WTCTF, Birmingham, United Kingdom
ORAL ABSTRACTS
36 : HCV 2011
ORAL ABSTRACTS
Virology: Entry, Replication and Assembly
O7.01
O7.02
Maria Teresa Catanese, Joana Loureiro, Christopher T. Jones, Timothy Sheahan, Alfredo Nicosia, Charles M. Rice
O7.03
Janice Elaine Silverman, Zongyi Hu, T. Jake Liang
O7.04
EGF promotes hepatitis C virus entry by activating EGFR phosphorylation
at Y1086 and the Ras/MAP kinase signaling pathway
Laetitia Zona, Joachim Lupberger, Christine Thumann, Benoit Fischer, Laure Froidevaux, Jonathan Florentin,
François Duong, Michelle J. Farquhar, Mirjam B. Zeisel, Jacques A. Nunès, Ivan Hirsch, Laurent Brino, Jane A. McKeating,
Thomas F. Baumert
O7.05
The Niemann-Pick C1-Like 1 cholesterol absorption receptor: a novel
hepatitis C virus entry factor and therapeutic target
Bruno Sainz, Naina Barretto, Danyelle Martin, Nobuhiko Hiraga, Michio Imamura, Xuemei Yu, Kazuaki Chayama,
Waddah Alrefai, Susan Uprichard
O7.06
The SLAM family receptor CD229: a key role in hepatitis C virus binding
and infection
Flora Cartier, Rémy Nyga, Christèle Ossart, Luciane Lamotte, Kaïss Lassoued, Hicham Bouhlal
O7.07
HCV entry inhibitors arbidol and silibinin hinder early steps of viral
trafficking as revealed by single particle tracking analysis
Julie Blaising, Pierre Levy, Elodie Teissier, Jean-Pierre Lavergne, Eve-Isabelle Pecheur
Continued on reverse.
ORAL ABSTRACTS
Helen Harris, Caroline Clerté, Amy Barnes, Margaret Goodall, Ke Hu, Garrick Wilson, Christine Thumann, Patrice Rassam,
Ragai Mitry, Anil Dhawan, Thomas Baumert, Pierre Milhiet, Jane McKeating
O7.08
Genotype dependent usage of CLDN-family members as HCV entry receptors
Sibylle Haid, Christina Grethe, Thomas Pietschmann
O7.09
High-resolution profiling HCV genome by combining random insertion
mutagenesis with next-generation sequencing
Hangfei Qi, Sheng-Yao Su, Zugen Chen, Shu-hwa Chen, Vaithilingaraja Arumugaswami, Stanley Nelson, Chung-yen Lin,
Ren Sun
O7.10
Andrew Tuplin, Madeleine Struthers, Peter Simmonds, David Evans
O7.11
CREB3L1: a host transcription factor that inhibits proliferation of virusinfected cells
Bray Denard, Joachim Seemann, Quiyue Chen, Austin Gay, Hua Huang, Jin Ye
ORAL ABSTRACTS
O7.12
Troels K. H. Scheel, Judith M. Gottwein, Lotte S. Mikkelsen, Jens Bukh
O7.13
Novel infectious clone of HCV 1a strain HCV-RMT efficiently replicate in
vitro and in vivo using adaptive mutations
Yuko Tokunaga, Masaaki Arai, Asako Nakaya, Yoshimi Tobita, Chise Tateno, Michinori Kohara
O7.14
Mapping the nascent RNA channel of the hepatitis C virus RNA dependent
RNA polymerase using mass spectrometry
Robert Vaughan, Hui Cai, Jin-Sam You, C. Cheng Kao
O7.15
NS4B self-interaction through conserved C-terminal elements is required for
the establishment of functional HCV replication complexes
David Paul, Inés Romero-Brey, Jérôme Gouttenoire, Savina Stoitsova, Jacomine Krijnse-Locker, Darius Moradpour,
Ralf Bartenschlager
O7.16
Diacylglycerol acyltransferase 1 (DGAT1) functions as a cellular “hub” for
HCV NS5A and core proteins
Gregory Camus, Eva Herker, Charles Harris, Robert Farese, Melanie Ott
O7.17
A highly specific genetic interaction between core and NS3 is essential for
the production of infectious hepatitis C virus
Daniel Jones, Ali Atoom, Rodney Russell
O7.18
Identification of lipid droplet-associated membrane proteins that are
involved in HCV production
Hideki Aizaki, Yoshihiro Matsumoto, Koji Goto, Koichi Watashi, Ryosuke Suzuki, Masayoshi Fukasawa, Kentaro Hanada,
Shigeko Sato, Nobuhiro Takahashi, Yoshiharu Matsuura, Kiyoto Motojima, Tatsuo Miyamura, Tetsuro Suzuki, Takaji Wakita
O7.19
Dynamic trafficking of HCV core protein to and from cellular lipid droplets
during virus particle assembly
Natalie Counihan, Brett Lindenbach
38 : HCV 2011
ORAL ABSTRACTS
O7.20
Mark Harris, Jamel Mankouri, Stephen Griffin
O7.21
Underlying molecular mechanisms of apolipoprotein E in hepatitis C virus
life cycle
Wei Cun, Guangxiang Luo
O7.22
HCV p7 and NS2 induce the early steps of virus assembly by regulating core
localization at the endoplasmic reticulum vs. lipid droplets
Bertrand Boson, Ophelia Granio, Ralf Bartenschlager, F. L. Cosset
O7.23
NS4B protein facilitates HCV assembly via genetic interaction with p7 and
NS5A proteins
O7.24
Two amino acid exchanges are sufficient to restore pestivirus
morphogenesis in the absence of uncleaved NS2-3
Norbert Tautz, Oliver Klemens, Erik Lattwein
ORAL ABSTRACTS
Kouacou Konan, David Manna, Qingxia Han
ORAL ABSTRACTS
40 : HCV 2011
VIROLOGY: ENTRY, REPLICATION AND ASSEMBLY
O7.01
Hepatitis C virus (HCV) entry is dependent on host cell expression of tetraspanin CD81, scavenger receptor BI (SR-BI) and tight junction
proteins Claudin-1 and Occludin. CD81 and SR-BI interact directly with HCV particles; however the exact role(s) played by each of the
‘entry’ factors in the multi-step entry process is unclear. Hepatocytes are the primary reservoir supporting HCV replication in the liver and
are highly polarized, with tight junctions separating their basolateral and apical domains. Tight junction proteins form homo- and heterodimeric complexes both within and between adjacent cells, raising questions as to their accessibility to HCV particles and their role in the
virus entry process. We have combined live cell imaging techniques such as FRET, FRAP, and single molecule tracking to characterise HCV
receptor complex association and motility in polarized HepG2 hepatoma cells and primary human hepatocytes.
Transduction of HepG2 and primary hepatocytes with monomeric fluorescent tagged HCV receptors enabled us to study protein-protein
interaction(s) and receptor motility at various cellular locations. All of the HCV receptors diffuse freely through the plasma membrane, with
the exception of Occludin that is retained at the tight junctions, reflecting the basolateral-apical trafficking of these molecules. We and
others reported that CD81 and Claudin-1 association is essential for viral entry, yet it is unknown whether this receptor complex is static
or dynamic. Utilising single molecule tracking techniques we have identified that CD81 and Claudin-1 diffuse through the membrane as
a complex. Small molecule entry inhibitors and siRNA receptor silencing allowed us to study the sequence of viral glycoprotein-receptor
engagement. HCV glycoproteins promoted the lateral diffusion and formation of receptor enriched domains. In summary, these data
support a model where HCV hijacks diffusing CD81/Claudin-1 receptors and re-localizes the complex to cholesterol enriched domains for
virus internalization.
[email protected]
O7.02
Maria Teresa Catanese (1), Joana Loureiro (1), Christopher T. Jones (1), Timothy Sheahan (1), Alfredo Nicosia (2), Charles M. Rice (1)
(1) Rockefeller University, New York, NY, USA
(2) CEINGE, Biotecnologie Avanzate, Naples, Italy
Hepatitis C virus (HCV) infection of hepatocytes occurs through interactions with at least four cellular entry factors. Since the co-receptors
are spatially segregated into different cellular domains, the current model of HCV entry involves initial virus binding to entry factors on the
basal side (CD81 and SR-BI) before organized transport towards tight junction associated molecules claudin 1 (CLDN1) and occludin (OCLN).
In addition, HCV is thought to propagate via cell-to-cell spread, in which virus is transported between neighboring cells without diffusion
through the extracellular environment. This mode of infection may shield the virus from the innate and adaptive immune responses
and promote chronic disease. The cellular and viral molecules guiding cell-to-cell spread of HCV remain poorly defined. Using live cell
imaging, a quantitative focus forming assay (qFFA), immunohistochemisty (IHC) and flow cytometry-based assays, we sought to elucidate
the molecular mechanisms of HCV cell-to-cell spread. An adapted version of J6/JFH, termed clone 2, forms much larger foci than the
parental virus, suggesting improved cell-to-cell transmission. We constructed chimeras between J6/JFH and clone 2 to identify the viral
determinants responsible for the large-focus phenotype. Our data suggest that mutations in the E1 and E2 glycoproteins are responsible for
enhancing cell-to-cell spread. On the cellular side, pretreatment with SR-BI antibodies blocked cell-free infection with both clone 2 and J6/
JFH. However, only J6/JFH cell-to-cell spread was blocked if the antibodies were added after infection. These results point towards a role for
SR-BI in the lateral transfer of J6/JFH between cells, and suggest that clone 2 has a reduced dependence on this molecule. By co-culturing
reporter primary human fetal hepatocytes with pre-infected Huh-7.5 cells, we are able to monitor the transfer of virus to primary cells in
real time. We are continuing to investigate the mechanisms of spread in this more biologically relevant system.
Contact: Maria Teresa Catanese
Rockefeller University, New York, NY, USA
[email protected]
ORAL ABSTRACTS
Helen Harris (5), Caroline Clerté (1), Amy Barnes (5), Margaret Goodall (5), Ke Hu (5), Garrick Wilson (5), Christine Thumann (2), Patrice
Rassam (1), Ragai Mitry (3), Anil Dhawan (4), Thomas Baumert (2), Pierre Milhiet (1), Jane McKeating (5)
(1) Inserm, Unité 1054, Université de Montpellier, France
(2) Inserm, U748 and Université de Strasbourg, , France
(3) King’s College Hospital, London, United Kingdom
(4) Institute of Liver Studies, King’s College Hospital, London, United Kingdom
ORAL ABSTRACTS
O7.03
Janice Elaine Silverman (1), Zongyi Hu (1), T. Jake Liang (1)
(1) NIH/NIDDK/LDB, Bethesda, MD, USA
ORAL ABSTRACTS
While clathrin-mediated endocytosis is the current accepted pathway for HCV entry, other viruses were recently shown to use alternative
pathways as immune evasion mechanism. We established a novel imaging technique to 1) study the host factors and pathways involved
in HCV entry and 2) identify alternative route used by HCV for productive infection. FlAsH labeling technique was developed to visualize
infectious HCV virion, in which a tetracysteine (TC) motif (CCXXCC) was genetically engineered to the N-terminus of HCV E2 protein of the
JFH1 strain. The TC sequence binds to fluorescein-derived compound FlAsH-EDT2 and allows visualization of the virus while bound to host
factors during the early stage of infection. Infectivity assays, Western blot and qPCR demonstrated that the tetracysteine-tagged virus
was active and stable for a few passages. Immunostaining of TC-labeled virus bound to the Huh7.5.1 cells with HCV E2 antibody showed
colocalization of FlAsH signal and HCV E2, validating the specific labeling of the virus by FlAsH. Confocal microscopy revealed specific
interactions of HCV with viral receptors including SR-B1, CD81, and occludin. Large foci of fluorescent virus were observed intracellularly 30
min to 1 h post-infection, and SR-B1, CD81 and occludin were shown to surround these virus-containing vesicles by high-resolution confocal
image analysis. These vesicles are much larger than and atypical of the endocytic vesicles. Antibodies against SR-B1, CD81, and Cldn1
reduced the formation of these virus foci. Using inhibitors of sodium/potassium exchange and actin rearrangement, we demonstrated that
HCV entry can occur via macropinocytosis, which likely represents an important entry pathway of HCV observed in this study.
Contact: Janice Elaine Silverman
NIH/NIDDK/LDB, Bethesda, MD, USA
[email protected]
O7.04
EGF promotes hepatitis C virus entry by activating EGFR phosphorylation at Y1086 and
the Ras/MAP kinase signaling pathway
Laetitia Zona (1), Joachim Lupberger (1), Christine Thumann (1), Benoit Fischer (2), Laure Froidevaux (2), Jonathan Florentin (3), François
Duong (4), Michelle J. Farquhar (5), Mirjam B. Zeisel (1), Jacques A. Nunès (3), Ivan Hirsch (3), Laurent Brino (2), Jane A. McKeating (5),
Thomas F. Baumert (1)
(1) Inserm, U748, Université de Strasbourg, Strasbourg, France
(2) High-Throughput Screening Platform, IGBMC, Illkirch, France
(3) Inserm U891, Centre de Recherche en Cancérologie de Marseille, France
(4) Hepatology Laboratory, University Hospital Basel, Switzerland
(5) Hepatitis C Research Group, University of Birmingham, United Kingdom
Hepatitis C virus (HCV) entry is the first step of infection and requires the cooperative interaction of several host cell factors including
CD81 and claudin-1. Using a functional RNAi kinase screen, we previously demonstrated that epidermal growth factor receptor (EGFR) is a
host co-factor for HCV entry that promotes CD81/claudin-1 interaction(s) and viral glycoprotein-dependent membrane fusion (Lupberger,
Zeisel et al. Nature Medicine 2011 in press). In this study, we aim to uncover the EGFR-triggered signaling events that mediate viral entry
in Huh7.5.1 hepatoma cells. Using inhibitory monoclonal antibodies specific for the extracellular domain of EGFR and antibodies specific
for EGFR tyrosine phosphorylation, we identified EGFR tyrosine 1086 (Y1086) as a phosphorylation site relevant for HCV entry. This site
is known to recruit adaptor protein Grb2 which provides a link between EGFR and Ras/MAP kinase signaling pathway. Indeed, a targeted
siRNA screen comprising cellular signaling pathways demonstrated that silencing of Grb2 or Ras-GTPase expression significantly inhibited
HCVpp and HCVcc infection of Huh7.5.1 cells with minimal effect on cell viability. Using dynamic phosphoflow cytometry, we demonstrate
that ligand-induced EGFR activation in Huh7.5.1 cells predominantly activates MAP kinase ERK but not other signaling pathways. Studies on
EGF signalling and CD81-Claudin-1 co-receptor associations and trafficking are ongoing. Taken together, our results support a model where
EGF-mediated HCV entry is mediated by EGFR activation through phosphorylation at Y1086 and the Ras/MAP kinase signaling pathway via
the Grb2 adaptor protein. These data contribute to the understanding of the molecular mechanisms of EGFR-mediated HCV entry and may
open new perspectives for the development of antivirals targeting receptor tyrosine kinases.
LZ and JL contributed equally.
Contact: Laetitia Zona
Inserm, U748, Université de Strasbourg, Strasbourg, France
[email protected]
42 : HCV 2011
O7.05
The Niemann-Pick C1-Like 1 cholesterol absorption receptor: a novel hepatitis C virus
entry factor and therapeutic target
Although Hepatitis C virus (HCV) protease and polymerase inhibitors are near FDA approval, analogous to HIV therapy, optimal HCV therapy
likely will require a combination of antivirals targeting multiple aspects of the viral lifecycle. HCV entry represents a promising multi-faceted
target for antiviral intervention; however, to date FDA-approved inhibitors of HCV entry are unavailable. Towards this end, we show that
the cellular Niemann-Pick C1 Like-1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor that is readily amenable to therapeutic
intervention. Specifically, targeting NPC1L1 cell-surface expression by RNAi silencing or antibody-mediated blocking in the Huh7 hepatoma
cell line prior to cell-cultured-derived HCV (HCVcc) infection resulted in reduced infection, suggesting that NPC1L1 may be involved in
HCV entry. To more directly test this hypothesis, we used the clinically-available FDA-approved NPC1L1 antagonist ezetimibe to show that
inhibition of cell-surface NPC1L1 potently blocks HCV uptake in vitro at a step prior to virion:cell membrane fusion. We next tested the
efficacy of ezetimibe to inhibit HCV infection in chronically infected cells. As expected of an entry inhibitor, ezetimibe alone had no effect
on chronic HCV infection; however, when used in combination with interferon, ezetimibe synergistically reduced intracellular HCV RNA
levels resulting in the elimination of HCV from the cultures. To assess the potential clinical utility of ezetimibe we used uPA-SCID mice
repopulated with human hepatocytes and show that in line with our in vitro observations, ezetimibe prevented the establishment of acute
HCV infection in mice when treatment was initiated prior to challenge and synergized with interferon-alpha to potently reduce HCV RNA
levels in chronically HCV-infected mice. Thus, we have not only identified NPC1L1 as an HCV entry factor, but also discovered a promising
new HCV antiviral target and available therapeutic agent.
Contact: Bruno Sainz
University of Illinois at Chicago, Chicago, IL, USA
[email protected]
O7.06
The SLAM family receptor CD229: a key role in hepatitis C virus binding and infection
Flora Cartier (1), Rémy Nyga (1), Christèle Ossart (1), Luciane Lamotte (1), Kaïss Lassoued (1), Hicham Bouhlal (1)
(1) UPJV-INSERM UMR925, Amiens, France
Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. HCV entry into hepatocytes is a complex multi-step process
involving several viral and cellular factors, mainly tetraspanin CD81, scavenger receptor type I (SR-B1), and tight junction proteins Claudin-1
(CLDN1) and Occludin (OCLDN). In addition, the virus may use glycosaminoglycans (GAGs) and low density receptors (LDL-R) as initial
attachment factors. However, the expression of these factors on HCV-resistant cells strongly indicates that the molecular mechanism of HCV
entry is not fully understood. We report here and for the first time the expression of CD229, a member of the SLAM family, on the surface of
human hepatocytes and its implication in HCV infection. The receptors of this family, initially described on the surface of T lymphocytes and
B lymphocytes are mostly activated by homotypic interactions to notably control the production of type 1 and 2 interferon. CD229, made
up of four extracellular domains called “immunoglobulin-like”, is the only member of the SLAM family able to self-internalize. We show
that SLAM-CD229 plays a critical role in HCV binding and infection. CD229 blockade by specific antibodies and down-modulation by specific
siRNA sharply decreases the susceptibility of hepatocytes to HCV. Conversely, CD229 over-expression in human hepatocarcinoma cells
Huh-7 significantly increases HCV attachment and replication. In addition, the induction of CD229 expression on HCV-resistant cells confers
them a susceptibility to this virus. Finally, the use of synthetic peptides mimicking each domain of CD229 shows that the first extracellular
domain of CD229 is required for HCV binding and infection. Our results point to CD229’s key role in the absorption and infection of HCV, thus
shedding new light on the understanding of the pathophysiology of HCV infection. The blockade of this interaction via various approaches
provides a breakthrough in the treatment of HCV infection.
Contact: Flora Cartier
UPJV-INSERM UMR925, Amiens, France
[email protected]
ORAL ABSTRACTS
Bruno Sainz (1), Naina Barretto (1), Danyelle Martin (1), Nobuhiko Hiraga (2), Michio Imamura (2), Xuemei Yu (1), Kazuaki Chayama (2),
Waddah Alrefai (1), Susan Uprichard (1)
(1) University of Illinois at Chicago, Chicago, IL, USA
(2) Hiroshima University, Japan
ORAL ABSTRACTS
O7.07
HCV entry inhibitors arbidol and silibinin hinder early steps of viral trafficking as
revealed by single particle tracking analysis
Julie Blaising (1), Pierre Levy (2), Elodie Teissier (1), Jean-Pierre Lavergne (1), Eve-Isabelle Pecheur (1)
(1) CNRS UMR 5086, IBCP, Université Lyon 1, Lyon, France
(2) UMR INSERM U1052 CNRS 5286, Université de Lyon, France
ORAL ABSTRACTS
Cell entry of the hepatitis C virus (HCV) is an attractive target for therapeutic intervention, with opportunities to prevent virus-receptor
interactions and inhibit viral fusion. Small molecule inhibitors targeting extracellular events are of great potential since they are expected to
exhibit good bioavailability and low toxicity.
Here we investigated in living cells anti-HCV activities of arbidol (Arb) and silibinin (SbN), two inhibitors of HCV entry we recently described.
Single particle tracking analyses in Huh-7.5.1 cells expressing clathrin-GFP displayed rapid HCV internalisation in early endosomes. Highly
mobile HCV particles coincided for ~30 s with clathrin-positive endosomes, and were observed after 15 min in structures positive for Lamp1,
a late endosomal marker. Conversely, in the presence of micromolar fusion-inhibitory concentrations of Arb or SbN, HCV stayed confined in
early endosomes ca. 2 min, while remaining virtually immobile. Arb and SbN therefore appear to act primarily at the HCV fusion stage with
endosomal membranes. Immunolabeling experiments on HCV-infected Huh-7.5.1 and primary human hepatocytes further revealed the
overall slowdown of HCV trafficking in Arb- or SbN-treated cells, but not in cells treated with DMSO, the vehicle of both drugs. Drastically
reduced HCV infectivity was observed at similar drug concentrations.
Biochemical analyses by surface plasmon resonance with liposomes were performed to further investigate Arb and SbN potential ability to
interact with membranes. SbN displayed an apparent membrane affinity of 15 µM at acidic pH, and only a 30 µM affinity at neutral pH. This
behaviour is reminiscent of what we recently described for Arb. This membrane affinity feature may therefore explain the inhibitory action
of Arb and SbN on HCV entry and membrane fusion.
Our ongoing investigations will help further define the cellular mode of action of these drugs, which open promising perspectives for the
implementation of HCV entry inhibitors in antiviral multidrug regimens.
Contact: Julie Blaising
CNRS UMR 5086, IBCP, Université Lyon 1, Lyon, France
[email protected]
O7.08
Genotype dependent usage of CLDN-family members as HCV entry receptors
Sibylle Haid (1), Christina Grethe (1), Thomas Pietschmann (1)
(1) TWINCORE-Centre for Experimental and Clinical Infection Research, 30625 Hannover, Germany
HCV entry is a complex process involving at least four cellular entry receptors: the tetraspanin CD81, the scavenger receptor SR-BI, the
tight junction protein Occludin (OCLN) and one of three Claudin-family members CLDN1, CLDN6 or CLDN9. To investigate the role of CLDN
proteins during HCV entry, we employed HuH6 cells which almost completely lack endogenous CLDN1 but express all other HCV entry
factors and efficiently replicate HCV. Due to low CLDN1 expression, HuH6 cells were not susceptible to infection with genotype 2a chimeric
virus Jc1. Surprisingly these cells were permissive to infection by HCV genotype 1, 2b and 3a pseudoparticles (HCVpp) as well as cell culture
derived HCV (HCVcc) particles. Protein expression analysis revealed high levels of CLDN6 in HuH6 cells suggesting that HCV genotype 1 can
use CLDN6 as HCV entry receptor whereas HCV genotype 2a preferentially/mainly uses CLDN1. To further analyze the genotype-dependent
usage of CLDN, CLDN1 and CLDN6 were expressed in 293T cells and challenged with HCVpp carrying envelope proteins from different
viral genotypes. These data confirmed efficient use of CLDN1 by all viral genotypes and of CLDN6 by genotype 1 but not by genotype
2. To map essential determinants of CLDN6 responsible for genotype-dependent receptor usage we constructed various CLDN1-CLDN6
chimeric proteins. Preliminary data indicate that determinants resident in the first extracellular loop of CLDN6 are responsible for genotypedependent utilization by HCV. Collectively, these findings point to viral genotype-dependent differences in CLDN entry factor usage which
may have implications for virus spread and pathogenesis.
Contact: Sibylle Haid
TWINCORE-Centre for Experimental and Clinical Infection Research, 30625 Hannover, Germany
[email protected]
44 : HCV 2011
O7.09
High-resolution profiling HCV genome by combining random insertion mutagenesis
with next-generation sequencing
We have developed a high-throughput, quantitative, genome-scale platform that allows us to systematically map the Hepatitis C virus (HCV)
sequence required for its replication at a variety of conditions. A highly complex library of mutant HCV was constructed by randomly inserting
15-nucleotides (nt) at almost every base pair position in the virus genome. The library of mutant viruses was then propagated and passaged
in cell culture. Quantitative genetic profiling using next-generation sequencing technology, Solexa from Ilumina, was performed to accurately
determine advantageous, neutral and deleterious mutations in the entire viral genome at high-resolution. By enriching for fragments containing
the insertion sites prior to sequencing, the average coverage for each mutant was about 1000 times, which enables statistical analysis. Of 8406
insertions identified in 9517 nucleotides of the genome, 595, 796, and 7015 were tolerated, attenuating, and lethal, respectively, for virus
replication. Individual mutant viruses were constructed accordingly and the phenotype was confirmed. We are currently carrying out selection
of the mutant virus library in the presence and absence of IFN-α treatment. Identification of any mutations that confer increased sensitivity to
IFN-α treatment will help to uncover the mechanisms of viral resistance and offer novel therapeutic strategies.
This method offers advantages over traditional forward and reverse genetics by enabling the identification of any cis-elements or protein
domains that are important for any particular phenotype without prior knowledge of the function of the region. It can also be applied
in identifying therapeutic target regions for drug development and mapping essential residues that could serve as potential epitopes for
novel vaccines.
Contact: Hangfei Qi
Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USA
[email protected]
O7.10
Andrew Tuplin (2), Madeleine Struthers (2), Peter Simmonds (1), David Evans (2)
(1) Centre for Infectious Diseases, University of Edinburgh, United Kingdom
(2) University of Warwick, Coventry, United Kingdom
RNA viruses, including hepatitis C virus, use higher-order RNA structures such as stem-loops and pseudoknots to recruit cellular and
viral proteins involved in the initiation and regulation of genome replication. These cis-acting replication elements (CRE) are usually
bioinformatically and functionally conserved within related viruses and are of particular interest as they may form therapeutic targets for
treatment. We have previously predicted phylogenetically conserved RNA structures in the NS5B-coding region of HCV which have been
subsequently confirmed as CRE’s by reverse genetic analysis using the Con1b sub-genomic replicon or JFH-1 HCVcc system. Specifically we
and others have defined a stem-loop (designated SL9266, or 5BSL3.2) which forms two long-range interactions, with sequences in the X-tail
of HCV and with a region located ~200nt. 5’ to SL9266. Using the Con1b sub-genomic replicon we have demonstrated that both interactions
are critical for replication.
We have extended these studies by completing the SHAPE (selective 2’-hydroxyl acylation analysed by primer extension) analysis of these
regions of both Con1b and JFH-1. We have analysed the structure of the native (replication competent) sequence of both genomes and of
an extensive range of modified genomes that are incapable of replicating. We have demonstrated that the long-range interactions of SL9266
occur simultaneously, and are not mutually exclusive. Additionally, we have confirmed that structural changes in the interactions of SL9266
with both the X-tail and the 5’ sequences correlate with the ability of the genome to replicate.
Intriguingly we also have evidence that the interactions observed in Con1b and JFH-1 are strikingly different, and that the consequences –
both on the RNA structures and on the phenotype of RNA replication – differ significantly.
These studies contribute to our understanding of HCV CRE structures and have highlighted unexpected differences between sequences
involved in the replication of genotypes 1b and 2a of HCV.
Contact: David Evans
University of Warwick, Coventry, United Kingdom
[email protected]
ORAL ABSTRACTS
Hangfei Qi (1), Sheng-Yao Su (2), Zugen Chen (3), Shu-hwa Chen (2), Vaithilingaraja Arumugaswami (1), Stanley Nelson (3), Chung-yen Lin
(2), Ren Sun (1)
(1) Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USA
(2) Institute of Information Science, Academia Sinica, Taipei, Taiwan
(3) Department of Human Genetics, UCLA, Los Angeles, CA, USA
ORAL ABSTRACTS
O7.11
CREB3L1: a host transcription factor that inhibits proliferation of virus-infected cells
Bray Denard (1), Joachim Seemann (1), Quiyue Chen (1), Austin Gay (1), Hua Huang (1), Jin Ye (1)
(1) UT Southwestern, Dallas, TX, USA
Hepatitis C virus (HCV) does not replicate efficiently in wild type human hepatoma Huh7 cells, but it replicates robustly in certain subclones
of Huh7 cells. Comparison of permissive subclones to the non-permissive parental Huh7 cells has previously been shown to be a powerful
way to determine host factors required for HCV replication. Here, we identify CREB3L1 as a transcription factor expressed in parental
Huh7 cells but not in two independent subclones of Huh7 cells that are highly permissive for HCV replication. CREB3L1 is synthesized as
a membrane-bound protein. Upon transfection into cells infected by HCV, CREB3L1 was proteolytically cleaved, allowing its NH2-terminal
fragment to enter the nucleus. Once in the nucleus, CREB3L1 induced multiple genes encoding inhibitors of the cell cycle to block cell
proliferation. As proliferation of host cells is known to be required for efficient replication of HCV, CREB3L1-mediated inhibition of the cell
cycle inhibits HCV replication. In addition to HCV, CREB3L1 also inhibits proliferation of cells infected by West Nile virus and Sendai virus. Our
data reveal that CREB3L1 is a novel innate antiviral protein that inhibits proliferation of virus-infected cells.
ORAL ABSTRACTS
Contact: Bray Denard
UT Southwestern, Dallas, TX, USA
[email protected]
O7.12
Troels K. H. Scheel (1), Judith M. Gottwein (1), Lotte S. Mikkelsen (1), Jens Bukh (1)
(1) Copenhagen Hepatitis C Program (CO-HEP), Hvidovre, Denmark
Epidemiologically important HCV recombinants were previously reported. In addition, recombination of pestiviruses was described in vitro.
Here we describe the first recombination of HCV RNA in vitro leading to production of infectious virus. Initially, a replicating JFH1dE1E2
(genotype 2a) genome lacking part of the envelope genes was co-transfected into Huh7.5 cells with J6CF (genotype 2a) or J6/JFH1-GND,
none of which replicate in cell culture. After a decrease in HCV positive cells, infection spread after 8-25 days. Sequencing of genomes
recovered after passage to naïve cells revealed heterologous recombination in all cases with the 5’UTR-(NS2) (parentheses indicate partial
gene) of J6 sequence followed by (Core)-3’UTR JFH1 sequence (including the E1/E2 deletion). These recombinants had two potentially
functional p7 genes and up to 1257 additional nucleotides. Reverse genetic studies verified the function of a representative recombinant,
which immediately spread in culture following transfection and yielded infectivity titers above 4 log10(FFU/mL). Similarly, JFH1dE1E2 was
co-transfected in triplicates with full-length genotype 1, 3 or 4 clones, which do not replicate in Huh7.5 cells. Recombination was identified
in only one case resulting from an S52 (genotype 3a) 5’UTR-(NS2) segment and an (E2)-3’UTR JFH1 segment. Recombination seemed to
occur independent of HCV replication, as recombination was always observed when a JFH1 genome lacking the 5’UTR was co-transfected
with J6CF or J6CF lacking the 3’UTR and part of NS5B. By co-transfection of truncated genomes we forced recombination to occur outside
the identified junction regions; recombinants with junctions in the Core-E1 region were isolated. In one case, homologous recombination
in the Core gene was observed. Thus, productive recombination of attenuated HCV genomes occurred in almost all intragenotypic but few
intergenotypic co-transfections. Most often, NS2 to Core-E2 heterologous recombination was observed, but junctions could be forced to
other genome regions. Recombination did apparently not depend on HCV replication.
Contact: Troels K. H. Scheel
Copenhagen Hepatitis C Program (CO-HEP), Hvidovre, Denmark
[email protected]
46 : HCV 2011
O7.13
Novel infectious clone of HCV 1a strain HCV-RMT efficiently replicate in vitro and in
vivo using adaptive mutations
Introduction: The recognition of genotype 2a strain, JFH1 allows us to study the whole lifecycles of hepatitis C virus (HCV). The genotype
1 strains are associated with liver diseases in the most region of the world. HCV-RMT strain, which we cloned from genotype 1a-infected
individual, replicates efficiently and shows high infectivity in the chimeric mice with humanized liver. We previously demonstrated that
HCV-RMT, with three cell culture-adaptive mutations (designated as RMTtri), could replicate efficiently in HuH7 cells as well as JFH1, as both
replicon and full-length virus. In this study, with analyzing three cell culture-adaptive mutations, we would like to discuss about relationship
between infecting and replicating ability in vitro and in vivo of individual mutants.
Results and Discussion: We demonstrated that transfection of in vitro-transcribed HCV-RMT RNA derivatives into HuH7 cells showed various
replicating abilities and HCV-RMTtri kept above 3.0 × 106 copies per μg total RNA for at least two months. HCV-RMTtri genome also supported
secretion of viral particles in cell culture. Secreted virus from cells transfected with HCV-RMTtri genome RNA was infectious for naïve HuH7
cells. The specific infectivity of RMTtri particles (1.5 × 103 RNA copies per focus-forming unit) was relatively lower than JFH1 virus (1.5 ×
102 RNA copies per focus-forming unit). However, HCV-RMTtri genome which directly injected into livers of the chimeric mice, showed no
replicating ability although the same injection of parental HCV-RMT genome resulted in viremia as the same level as original serum. Derivatives
carrying single mutation or combinations of them showed partial characters both in vitro and in vivo. Our data suggest that HCV-RMT strain is
capable of supporting all stages of authentic genotype 1a virus life cycle and would be useful tool for understanding of HCV.
Contact: Yuko Tokunaga
Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Japan
[email protected]
O7.14
Mapping the nascent RNA channel of the hepatitis C virus RNA dependent RNA
polymerase using mass spectrometry
Robert Vaughan (1), Hui Cai (1), Jin-Sam You (2), C. Cheng Kao (1)
(1) Indiana University, Bloomington, IN, USA
(2) IUPUI, USA
The HCV encoded RNA-dependent RNA polymerase (NS5B) is responsible for replicating the viral genome and is a key target for antiviral
development. A reversible crosslinking-peptide fingerprinting method had been used to map the regions of the polymerase that bind
RNA (Kim et al., 2005). In this work, we seek to map the regions that bind the nascent RNA. A major technical challenge was the low yield
of nascent RNA. This was overcome by the use of nascent RNAs that contain alkyns that could be captured by the highly efficiency click
chemistry. Combined with a template designed to allow the polymerase to synthesize different lengths of nascent RNA, we identified a
region adjacent to the Δ1 loop in the fingers domain of NS5B to contact the nascent RNA, specifically helices α2 and α3 (Bressanelli et al.,
1999). A modification protection assay to amidinate surface exposed lysines while walking NS5B down an RNA template was used to confirm
the assignment. Molecular docking showed that this region has features consistent with the ability to contact RNA. Several mutants within
the putative nascent RNA binding region were generated and analyzed for defects in RNA synthesis in vitro and in the HCV subgenomic
replicon. Additional analysis will be to examine the effects of polymerase inhibitors on these mutants. The results from this study will better
define the ternary complex of the HCV polymerase and the requirements for HCV RNA synthesis.
Contact: Robert Vaughan
Indiana University, Bloomington, IN, USA
[email protected]
ORAL ABSTRACTS
Yuko Tokunaga (1), Masaaki Arai (2), Asako Nakaya (2), Yoshimi Tobita (1), Chise Tateno (3), Michinori Kohara (1)
(1) Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Japan
(2) Mitsubishi Tanabe Pharma Corporation, Japan
(3) Phoenix Bio Co., Ltd., Japan
ORAL ABSTRACTS
O7.15
NS4B self-interaction through conserved C-terminal elements is required for the
establishment of functional HCV replication complexes
David Paul (1), Inés Romero-Brey (1), Jérôme Gouttenoire (2), Savina Stoitsova (1), Jacomine Krijnse-Locker (3), Darius Moradpour (2), Ralf
Bartenschlager (1)
(1) Molecular Virology, University Hospital Heidelberg, 69120 Heidelberg, Germany
(2) University of Lausanne, Switzerland
(3) Electron Microscopy Core Facility, Heidelberg University, Germany
ORAL ABSTRACTS
Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of
the HCV life cycle have become possible with the development of cell culture systems supporting replication of viral RNA and production
of infectious virus. However, the exact functions of individual proteins, especially of non-structural protein 4B (NS4B), remain poorly
understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, designated as membranous webs, which have
been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel
of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor
effect on replication but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and
used for selection of pseudo-reversions. Most of the pseudo-reversions also localized to the highly conserved NS4B C-terminal domain
and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudo-reversions
restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally,
electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which
were restored to wild type morphology by the corresponding pseudo-reversion. These findings demonstrate the important role of the
C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.
Contact: David Paul
Molecular Virology, University Hospital Heidelberg, 69120 Heidelberg, Germany
[email protected]
O7.16
Diacylglycerol acyltransferase 1 (DGAT1) functions as a cellular “hub” for HCV NS5A
and core proteins
Gregory Camus (1), Eva Herker (1), Charles Harris (1), Robert Farese (1), Melanie Ott (1)
(1) Gladstone Institutes, San Francisco, CA, USA
The association of HCV proteins core and NS5A to lipid droplets plays an essential role in the HCV life cycle. Triglycerides stored in lipid
droplets can be generated by two diacylglycerol acyltransferase enzymes, DGAT1 or DGAT2. We have previously shown that DGAT1 is critical
for the recruitment of the HCV core protein to lipid droplets and thereby regulates viral assembly. Here, we screened all viral proteins for
interactions with DGAT1 and identified NS5A as a novel DGAT1 interaction partner. DGAT1, and not DGAT2, forms a trimolecular complex
with core and NS5A as shown by sequential coimmunoprecipitation experiments and stabilizes the otherwise weak cellular interaction
between the two viral proteins. Trafficking of NS5A to lipid droplets is dependent on active DGAT1 as shown in knockdown and dominant
negative studies or after treatment with a specific DGAT1 inhibitor. Notably, expression of core does not alter NS5A’s localization to lipid
droplets as determined by fluorescent microscopy, but in biochemical fractionation studies enhances levels of NS5A detected in purified
lipid droplets fractions. These results support the model that NS5A specifically traffics to DGAT1-generated lipid droplets where it may get
more firmly anchored through the interaction with core. Our data underscore the central role of DGAT1 as a host factor for HCV infection
and identify DGAT1 as a novel link between core and NS5A proteins in cells. We propose that the newly identified interactions of DGAT1
with NS5A and core serve to guide trafficking of both viral proteins to the same subset of lipid droplets where viral assembly can take place.
Contact: Gregory Camus
Gladstone Institutes, San Francisco, CA, USA
[email protected]
48 : HCV 2011
O7.17
A highly specific genetic interaction between core and NS3 is essential for the
production of infectious hepatitis C virus
By analogy to other members of the flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the
viral nucleocapsid, which encloses the single-stranded RNA genome. Following cleavage by cellular signal peptide peptidase at the ER
membrane, core localizes to lipid droplets (LDs) and this process is critical for infectious virus production. The interaction between core
and LDs is mediated by two amphipathic α-helices harboured within domain 2 (D2) of the protein. Domain 1 (D1) of core is also important
for the formation of infectious particles, although its precise contribution is less well understood. In this study, we mutated amino acids
64-75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64-66 are critical
for generation of infectious progeny, whereas 67-75 were dispensable for this process. Further investigation of the defective 64-66 mutant
(termed JFH1T-64-66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly.
Iodixanol and sucrose gradient analyses revealed that JFH1T-64-66 assembled dense intracellular species of core, presumably representing
nucleopcapsids. Thus, amino acids 64-66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1T-64-66
led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to
produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together,
our results suggest that residues 64-66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation
of infectious HCV particles at a stage downstream of nucleocapsid assembly.
Contact: Daniel Jones
Memorial University, St. John’s, Canada
[email protected]
O7.18
Identification of lipid droplet-associated membrane proteins that are involved in HCV
production
Hideki Aizaki (1), Yoshihiro Matsumoto (1), Koji Goto (1), Koichi Watashi (1), Ryosuke Suzuki (1), Masayoshi Fukasawa (1), Kentaro
Hanada (1), Shigeko Sato (2), Nobuhiro Takahashi (2), Yoshiharu Matsuura (3), Kiyoto Motojima (4), Tatsuo Miyamura (1), Tetsuro Suzuki
(5), Takaji Wakita (1)
(1) National Institute of Infectious Diseases, Shinjuku-ku, Japan
(2) Tokyo University of Agriculture and Technology, Japan
(3) Research Institute for Microbial Diseases, Osaka University, Japan
(4) Meiji Pharmaceutical University, Japan
(5) Hamamatsu University School of Medicine, Japan
Lipid droplet (LD) is known to be important for the assembly of HCV. In this research, we analyzed LD-associated membrane, where infectious
HCV particles exist, and identified 45 proteins in the membrane fraction by proteome analysis. Among them, knocking-down of a subset
of genes such as 17 beta-hydroxysteroid dehydrogenase (HSD) 11 and apolipoprotein E (ApoE) in HCV-infected cells resulted in marked
impairment of infectious HCV production without reduction of the viral RNA replication in the subgenomic replicon system, suggesting their
role in formation of virus particles.
HSD11 is mainly localized on the endoplasmic reticulum but can also distribute to LD when the LD formation is induced in cells. A deletion
with putative LD-targeting sequence (HSDdel) abolished LD localization of HSD11. Expression of HSD11 led to increase in HCV production,
while that of HSDdel suppressed the production, suggesting that LD localization is important for the function of HSD11 in HCV particle
formation. In the cells transfected with HSD11 gene, lipid contents as well as size and numbers of LD were increased. By contrast, opposite
effects were found in HSDdel-transfected cells.
We further analyzed interactions between HSD11 and HCV proteins and found HSD11 specifically interacts with NS5A by coimmunoprecipitation. Combined with the evidence that interaction of NS5A with core protein on the peripheral area of LD is critical for the
process of virion assembly, our findings suggest that HSD11, whose function in hepatocytes has largely been unclear, is involved in assembly
of infectious HCV particles possibly through up-regulation of LD biogenesis and increase in local concentration of NS5A around LD.
Contact: Hideki Aizaki
National Institute of Infectious Diseases, Shinjuku-ku, Japan
[email protected]
ORAL ABSTRACTS
Daniel Jones (1), Ali Atoom (1), Rodney Russell (1)
(1) Memorial University, St. John’s, Canada
ORAL ABSTRACTS
O7.19
Dynamic trafficking of HCV core protein to and from cellular lipid droplets during virus
particle assembly
Natalie Counihan (1), Brett Lindenbach (1)
(1) Yale University, New Haven, CT, USA
ORAL ABSTRACTS
Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production.
However, it has not been directly shown that LD-associated core is recruited into virus particles, nor do we understand how this recruitment
process is regulated. To investigate the kinetics of core trafficking, we developed methods to fluorescently label and image functional core
protein in live, virus-producing cells. By pulse-chase labeling with contrasting fluorescent dyes, we were able to specifically image preexisting vs. newly synthesized core. During the peak of virus assembly, newly synthesized core formed polarized caps on large, immotile
LDs within two hours of viral translation, and could remain on LDs for up to 24 hours. Suprisingly, core trafficking to LDs was not affected
by inhibitors of the cellular DGAT1 enzyme. Putative sites of virus assembly were observed in close proximity to LD-associated core, while
motile puncta of core were seen to traffic on microtubules. Treatment with Brefeldin A, an inhibitor of ER-Golgi trafficking, showed that
these puncta represent a post-LD, post-endoplasmic reticulum form of core, most likely virus particles undergoing secretion. Importantly,
recruitment of core from LDs into these puncta required the interaction of NS2 and NS3 proteins. These data reveal new aspects of core
trafficking and identify an essential role for viral nonstructural proteins in virus particle assembly.
Contact: Brett Lindenbach
Yale University, New Haven, CT, USA
[email protected]
O7.20
Mark Harris (1), Jamel Mankouri (1), Stephen Griffin (1)
(1) University of Leeds, Leeds, United Kingdom
The release of infectious HCV particles from infected cells remains a poorly characterized aspect of the virus lifecycle. Whilst components
of very low density lipoprotein (VLDL) metabolism are clearly required for virus assembly, the route of secretion for assembled virions is
less clear. Our recent studies have demonstrated that virus production requires late-acting components of trans-Golgi network (TGN)endosomal trafficking. Here, we aimed to characterize the precise nature of the cellular compartments required for HCV egress.
Using a panel of dominant negative (DN) Rab GTPases selected to inhibit defined steps in intracytoplasmic membrane traffic, we demonstrate
that TGN to endosome trafficking is required for the secretion of infectious HCV virions. Importantly, DN Rabs that impair HCV release do
not have a concomitant effect on secretion of VLDL, apolipoproteins (Apo) E or ApoB. We further demonstrate that TGN markers are
redistributed to the surface of lipid droplets within infected cells, colocalising with HCV core protein. Interestingly, DN golgi adaptor proteins
also prevent virion release, confirming the importance of TGN-endosomal trafficking and suggesting that the droplets in HCV infected cells
may differ to naive cells, being TGN derived.
These findings support the notion that HCV infection perturbs TGN architecture and that TGN-endosome trafficking plays a critical role
during virion release. We conclude that the TGN-endosome trafficking pathway used by HCV is unique and appears distinct to the pathway
involved in secretion of VLDL.
Contact: Mark Harris
University of Leeds, Leeds, United Kingdom
[email protected]
50 : HCV 2011
O7.21
Underlying molecular mechanisms of apolipoprotein E in hepatitis C virus life cycle
Wei Cun (1), Guangxiang Luo (1)
(1) University of Kentucky College of Medicine, Lexington, KY, USA
Contact: Guangxiang Luo
University of Kentucky College of Medicine, Lexington, KY, USA
[email protected]
O7.22
HCV p7 and NS2 induce the early steps of virus assembly by regulating core localization
at the endoplasmic reticulum vs. lipid droplets
Bertrand Boson (2), Ophelia Granio (2), Ralf Bartenschlager (1), F. L. Cosset (2)
(1) University of Heidelberg, Germany
(2) INSERM, Lyon, France
HCV encodes a polyprotein that is translated and matured by cleavage at the endoplasmic reticulum (ER). The assembly of the viral structural
components, including core, E1/E2 glycoproteins, and vRNA is believed to occur at the ER in close proximity of lipid droplets (LDs) and
requires a coordinated action of cellular and viral components in which the HCV non-structural proteins play a major role. In cells infected
with the JFH1 HCV strain, core is predominantly detected in tight association with LDs. Conversely, despite its intrinsic capacity to localize
to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of
HCV-infected cells, but was mainly localized at ER membranes where it colocalized with the E1E2 glycoproteins. Furthermore, high-titer cell
culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1
virus strengthening the notion that core-ER co-localization is required for efficient HCV assembly. Our results further indicate that p7 and
NS2 are HCV strain-specific factors that govern the recruitment of core protein at ER assembly sites. Indeed, using expression constructs
and recombinant HCV genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity.
Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same subcellular localization of core detected in JFH1- vs.
Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between
the p7 and the first NS2 trans-membrane domains are required to induce core-ER localization and assembly of extra- and intra-cellular
infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing subcellular localization of HCV core to LDs
vs. ER and HCV assembly.
Contact: F. L. Cosset
INSERM, Lyon, France
[email protected]
ORAL ABSTRACTS
We have previously demonstrated that apolipoprotein E (apoE) has dual functions in the hepatitis C virus (HCV) infectious cycle (Chang et
al., J. Virol 81:13783-13793, 2007; Jiang and Luo, J. Virol 83:12680-12691, 2009; and Cun et al., J. Virol 84(21):11532-11541). To further
determine the underlying molecular mechanisms of apoE in HCV life cycle, we carried out an extensive mutagenesis analysis of apoE.
Progressive deletion mutagenesis analysis of apoE demonstrated that the C-terminal a-helical domain of apoE is important for interaction
with NS5A and determines HCV assembly/production. Mutations disrupting the specific apoE-NS5A interaction resulted in the blockade of
HCV assembly/production. The structural determinants for HCV infection lie in the receptor-binding region of apoE. Mutations within the
apoE receptor-binding domain affected both HCV secretion and infectivity. Some mutations had dominant negative effects on HCV infection
when expressed ectopically in Huh-7.5 cells. Additionally, we demonstrated that synthetic peptides derived from the apoE receptor-binding
region were able to block HCV infection. Findings derived from these studies demonstrate that the N-terminal receptor-binding domain of
apoE is important for HCV budding and infection. Collectively, our findings demonstrate that apoE play important roles in HCV assembly,
budding, and infection through distinct interactions with cellular and viral proteins, respectively.
ORAL ABSTRACTS
O7.23
NS4B protein facilitates HCV assembly via genetic interaction with p7 and NS5A proteins
Kouacou Konan (1), David Manna (1), Qingxia Han (1)
(1) Penn State University, University Park, PA, USA
ORAL ABSTRACTS
The C terminal domain (CTD) of Hepatitis C Virus (HCV) NS4B protein has many roles in HCV replication complex formation and NS4B
membrane association, as shown by extensive mutagenesis of this domain. Preliminary results from our lab and others (Jones et al. 2009.
JVI 83, 2163-77) also suggest a role for the NS4B CTD in virion production. Indeed, replacement of the NS4B CTD from HCV JFH1 (genotype
2a or G2a) virus with sequences from Con1 (G1b) or H77 (G1a) had negligible impact on JFH1 replication. However, the resulting JFH1
chimera displayed a significant defect in virion production. Our preliminary data suggest that the defect is at the level of virus assembly.
Subsequent experiments looking into the causes for lower virus titers revealed that the chimeric viruses have at least a three-fold decrease
in NS5A p58/p56 ratio relative to JFH1 virus. Interestingly, cell culture-adapted viruses, with titers similar to JFH1, displayed higher levels
of NS5A p58 but the p58/p56 ratio remained lower than in JFH1. Sequencing of the cell culture-adapted viruses revealed four mutations
in the virus genome. Two mutations were present in the chimeric NS4B CTD region and one such mutation specifically targeted residue
N216 that has been reported to increase JFH1 virus titers. Another mutation was identified in domain III of NS5A, a region important for
NS5A hyperphosphorylation and virion assembly. The fourth mutation targeted the cytosolic loop region of p7, another protein required
for virus assembly. Introduction of the second-site mutations, singly, into the parental chimera rescued virus production to various levels
suggesting a genetic interaction between NS4B, p7 and NS5A proteins. Thus, our studies reveal a role for NS4B in virion production, perhaps
via interaction and modification of other nonstructural proteins.
Contact: Kouacou Konan
Penn State University, University Park, PA, USA
[email protected]
O7.24
Two amino acid exchanges are sufficient to restore pestivirus morphogenesis in the
absence of uncleaved NS2-3
Norbert Tautz (1), Oliver Klemens (1), Erik Lattwein (1)
(1) Institute of Virology and Cell Biology, University of Lübeck, Lübeck, Germany
Pestiviruses, like bovine viral diarrhea virus, BVDV, are highly similar to HCV with respect to polyprotein organization and processing.
Cleavage at the NS2-3 junction is catalyzed by virus encoded proteases in NS2 and generates free NS3 which is an essential part of the
viral replication complex. However, with respect to cleavage efficiency at the NS2-3 site and the functional relevance of uncleaved NS2-3
pestiviruses and HCV differ significantly.
For HCV processing at the NS2-3 site is highly efficient and virion morphogensis occurs also in the absence of uncleaved NS2-3, at least
in cell culture. In contrast, uncleaved NS2-3 is detected in high amounts in BVDV infected cells and has been proven essential for virion
morphogenesis.
In recent work we observed, that a chimeric BVD virus which contains the NS2-3-4A coding region of BVDV strain Osloss and a cell culture
selected mutation in NS2 was able to produce infectious viral particles in the entire absence of uncleaved NS2-3. However, mainly due to its
chimeric nature this virus contained 52 amino acid exchanges compared to the parental strain.
To finally identify the mutations relevant for this phenotype we narrowed down the number of Osloss-derived exchanges required in the
NS3-4A part to exchange V1721A in NS3. Based on this finding, we started cell culture selection experiments and identified exchange
E1576V in NS2 as a second critical mutation. In the context of an infectious cDNA of BVDV strain NCP7 containing an EMCV-IRES between
the NS2 and NS3 genes exchanges E1576V (NS2) and V1721A (NS3) were sufficient to restore infectious virus production. Currently possible
effects of these mutations on protein-protein interactions possibly involved in virion morphogenesis are under investigation.
Taken together, the results of this study break with a previously accepted dogma for pestiviral replication and reveal interesting novel
parallels between pestiviruses and HCV.
Contact: Norbert Tautz
Institute of Virology and Cell Biology, University of Lübeck, Lübeck, Germany
[email protected]
52 : HCV 2011
ORAL ABSTRACTS
Antiviral Therapy
O8.01
O8.02
HCV NS5A: NMR analyses of its interactions with NS5B and cyclophilin
A and molecular characterization of a cyclophilin inhibitor-resistence
mutation
Xavier Hanoulle, Isabelle Huvent, Claire Rosnoblet, Dries Verdegem, Marie Dujardin, Aurélie Badillo, Lotte Coelmont,
Bernd Fritzinger, Jean-Michel Wieruszeski, Isabelle Landrieu, Johan Neyts, Ralf Bartenschlager, François Penin, Guy Lippens
O8.03
Solution structure of monomeric hepatitis C virus (HCV) p7 in a native, drug
binding, hairpin conformation
Toshana Foster, Arnout Kalverda, Gary Thompson, Joseph Thompson, Arwen Pearson, Richard Foster, Steven Homans,
Mark Harris, Stephen Griffin
O8.04
Structure and dynamics of membrane-associated p7 by NMR spectroscopy
Lindsay Dawson, Gabriel A. Cook, Stanley J. Opella
O8.05
Deciphering conformational variability of the hepatitis C virus polymerase
(genotype 1) in complex with inhibitors
Célia Caillet-Saguy, Philip C. Simister, Stéphane Bressanelli
O8.06
Elimination of HCVcc using recombinant AAV vector-mediated delivery of
anti-HCV miRNAs
Xiao Yang, Shangzhen Zhou, Guangxiang Luo, Linda Couto
ORAL ABSTRACTS
Andrea Olmstead, Francois Jean
ORAL ABSTRACTS
O8.07
Phosphatidylinositol 4-kinase IIIα is targeted by 4-aminoquinazoline
compounds with anti-HCV activity
Raffaele De Francesco, Annalisa Bianco, Reinaldo Alvarez, Simone Fenu, Veronica Reghellin, Massimiliano Pagani,
Sergio Abrignani, Petra Neddermann
O8.08
The non-nucleoside inhibitor tegobuvir utilizes a novel mechanism of action
to inhibit HCV NS5b polymerase function
Christy M. Hebner, Bin Han, Katherine M. Brendza, Michelle Nash, Maisoun Sulfab, Yang Tian, Magdeleine Hung,
Wanchi Fung, Randy Vivian, James Trenkle, Kyla Bjornson, Steven Bondy, Xiaohong Liu, Swami Swaminathan, John Link,
Johan Neyts, Roman Sakowicz, Uli Schmitz, Weidong Zhong
O8.09
Precious Lim, Udayan Chatterji, Dan Cordek, Craig Cameron, Paul Targett-Adams, Kai Lin, Philippe Gallay
O8.10
ORAL ABSTRACTS
The green tea polyphenol epigallocatechin-3-gallate (EGCG) inhibits
hepatitis C virus (HCV) entry
Sandra Ciesek, Thomas von Hahn, Luis M. Schang, Martina Friesland, Michael P. Manns, Michael Ott, Heiner Wedemeyer,
Philip Meuleman, Thomas Pietschmann, Eike Steinmann
O8.11
Marked synergy of entry inhibitors and interferon-alfa or direct acting
antivirals on the inhibition of hepatitis C virus infection
Fei Xiao, Isabel Fofana, Joachim Lupberger, Pieter Leyssen, Johan Neyts, Mirjam B. Zeisel, Thomas F. Baumert
O8.12
The RAFIs aUY11 and dUY11 inhibit infectivity of HCV and other enveloped
viruses by preventing fusion of viral and cellular membranes
Che C. Colpitts, Alexey V. Ustinov, Vladimir A. Korshun, Luis M. Schang
O8.13
Identification and functional analysis of small molecules inhibiting the late
step of hepatitis C virus life cycle
Koichi Watashi, Nanako Uchida, Ryosuke Suzuki, Hideki Aizaki, Takaji Wakita
O8.14
Augmentation of DHCR24 expression by hepatitis C virus infection facilitates
viral replication in hepatocytes
Kyoko Tsukiyama-Kohara, Takashi Takano, Yuichi Hirata, Yuko Tokunaga, Chise Tateno, Masayuki Sudo, Michinori Kohara
O8.15
The impact of calcineurin inhibitors on HCV replication in a chimeric mouse
model of HCV infection
Norman Kneteman, Jamie Lewis, Donna Douglas, Mahra Nourbakhsh, Lorne Tyrrell, Garry Lund
O8.16
PPARα agonist WY14643 improves the antiviral effect of interferon against
hepatitis C virus
Scott Read, Jacob George, Mark Douglas
54 : HCV 2011
ANTIVIRAL THERAPY
O8.01
It is now well established that the hijacking of host-cell biosynthetic pathways by human viruses is a shared molecular event that is essential
for the viral life cycle. The next frontier is identifying common critical host-cell pathways that are hijacked by pathogenic viruses, in order to
develop broad-spectrum host-directed anti-virals with novel mechanisms of action.
In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful
approach to developing a novel class of broad-spectrum anti-virals against infection associated with human Flaviviridae viruses such as
hepatitis C virus (HCV) and dengue virus, whose replication and pathogenesis depend on the interaction with lipid droplets (LDs). In the
case of HCV, overstimulation of host lipid metabolism in the liver during viral infection promotes cholesterol intracellular storage in host
LDs, a critical cellular event for HCV infection. One such master regulator of cholesterol metabolic pathways is the host subtilase S1P.
Also known as SKI-1, S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which
control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here, we report that strategic manipulation of S1P activity
levels by our novel S1P-directed protein-based inhibitors or S1P small-molecule inhibitors provides a means of effectively inhibiting the
S1P-dependent proteolytic cleavage of SREBPs in hepatoma cells, a critical step in the hijacking of the host cholesterol pathways by HCV.
Further, we demonstrate that S1P inhibition in hepatoma cells is associated with a dramatic reduction in LD biogenesis. As hypothesized,
inhibiting S1P activity with our inhibitors completely blocked HCV infection (JFH-1 strain) of Huh-7.5.1 cells in a dose-dependent manner.
Thus, targeting host LD biogenesis by inhibiting S1P may have far-reaching applications in the therapeutic treatment of many important
Flaviviridae viruses. Supported by CIHR to F. Jean.
Contact: Francois Jean
University of British Columbia, Vancouver, Canada
[email protected]
O8.02
HCV NS5A: NMR analyses of its interactions with NS5B and cyclophilin A and molecular
characterization of a cyclophilin inhibitor-resistance mutation
Xavier Hanoulle (1), Isabelle Huvent (1), Claire Rosnoblet (1), Dries Verdegem (1), Marie Dujardin (1), Aurélie Badillo (2), Lotte Coelmont
(3), Bernd Fritzinger (1), Jean-Michel Wieruszeski (1), Isabelle Landrieu (1), Johan Neyts (3), Ralf Bartenschlager (4), François Penin (2),
Guy Lippens (1)
(1) UMR 8576 CNRS - Univ Lille 1, Villeneuve d’Ascq, France
(2) UMR 5086 CNRS - Univ Lyon 1, France
(3) KU Leuven - Rega Insitute for Medical Resaerch, Belgium
(4) Department of Molecular Virology, University of Heidelberg, Germany
NS5A is essential for HCV replication and constitutes an attractive target for antiviral drug development. Structural and functional data are
required to decipher its exact role(s).
We show that NS5A-D2 and -D3 are mainly unfolded even if some structural elements have been identified. We report a comparative
molecular characterization of the NS5A-D3 from JFH-1 and Con1 strains. Combining several biophysical analyses, we show that both
NS5A-D3 are natively unfolded but exhibit propensity to partially fold into an alpha-helix. The amphipathic nature of the conserved first
helix and its conservation suggest that it might correspond to a molecular recognition element. We will report interaction studies with
potential partner such as apolipoprotein E.
Getting a detailed interaction map of NS5A is of utmost importance to decipher its precise function(s). We performed a molecular
characterization by NMR, at a per residue level, of the interactions between NS5A-D2 and -D3 with the HCV NS5B polymerase.
Because mutations in both NS5A-D2 and -D3 have been linked to Cyclosporin A (CsA) resistance, we used NMR to investigate potential
interactions between these domains and CypA. We observed direct molecular interactions of both NS5A-D2 and NS5A-D3 with CypA. We
identified sites that are substrates for the peptidyl cis/trans isomerase activity of CypA.
We previously showed that CypA interacts with the most conserved region of NS5A-D2 containing several prolines. In this region a mutation,
D320E (Con1) and D316E (JFH-1), has been shown to confer CsA or Alisporivir resistance. We performed a structural characterization of
the consequences induced by this mutation and investigated their functional consequences on the interaction with CypA. Based on these
results a molecular mechanism for the resistance of HCV replication to CsA analogues will be proposed.
Contact: Xavier Hanoulle
UMR 8576 CNRS - Univ Lille 1, Villeneuve d’Ascq, France
[email protected]
ORAL ABSTRACTS
Andrea Olmstead (1), Francois Jean (1)
(1) University of British Columbia, Vancouver, Canada
ORAL ABSTRACTS
O8.03
Solution structure of monomeric hepatitis C virus (HCV) p7 in a native, drug binding,
hairpin conformation
Toshana Foster (1), Arnout Kalverda (2), Gary Thompson (2), Joseph Thompson (3), Arwen Pearson (2), Richard Foster (3), Steven Homans
(2), Mark Harris (4), Stephen Griffin (1)
(1) Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom
(2) Astbury Centre for Structural Molecular Biology, University of Leeds, United Kingdom
(3) School of Chemistry, University of Leeds, United Kingdom
(4) Institute of Molecular & Cellular Biology, University of Leeds, United Kingdom
ORAL ABSTRACTS
Interferon-sparing treatments for hepatitis C virus (HCV) infection will require the development of novel inhibitors of multiple virus-specific
processes for use in combination. The p7 ion channel protein plays a critical role during the production of infectious virions and represents
an exciting new antiviral target. Prototype inhibitors block both ion channel function in vitro as well as particle production in culture,
providing proof of principle that they could contribute to future HCV therapies.
We present the first complete solution structure for monomeric p7 from HCV genotype 1b, calculated using both NOE and chemical
shift-based methods. p7 formed a distinctive hairpin structure with significant differences to previously reported models, including an
extended inter-helical loop with a distinctive kink. The hairpin is stabilised through multiple inter-helix contacts and supported biophysically
by paramagnetic relaxation enhancement measurements. The unusual top-down triangular symmetry from the arrangement of the two
helices and the loop region expedited the docking of p7 protomers into a symmetrical heptameric channel model, where the potential
proton sensor His17 projected into the channel lumen.
Interaction of monomeric p7 with rimantadine in solution illustrated the value of this structure for determining the binding site for prototype
inhibitors and permitted the development of novel active compounds. Specific chemical shift changes were observed in the presence of
drug which agreed with in silico binding predictions, yet these were absent when a recently identified resistance mutation was engineered
into the protein sequence. The rimantadine binding site resided on the membrane-exposed peripheral surface of the predicted channel
complex. This was used as a template to generate novel molecules with activity against both p7 channel function in vitro as well as infectious
HCV. Our structure therefore paves the way for rational drug design for an additional HCV target.
Contact: Toshana Foster
Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom
[email protected]
O8.04
Structure and dynamics of membrane-associated p7 by NMR spectroscopy
Lindsay Dawson (1), Gabriel A. Cook (1), Stanley J. Opella (1)
(1) University of California, San Diego, La Jolla, CA, USA
P7 is a 63-residue viroporin encoded by hepatitis C virus (HCV). It is essential for HCV proliferation. It consists of two transmembrane
spanning regions connected by a cytoplasmic loop, and has been shown to have ion channel activity in lipid bilayers. The NMR structural
data that has been acquired includes: hydrogen/deuterium exchange, paramagnetic relaxation enhancement, residual dipolar couplings,
and bicelle ‘q-titration.’ These data demonstrate that the protein has a range of dynamic properties, and surprisingly one helical segment
in each of the membrane spanning helices have independent dynamics, possibly related to function. Solid-state NMR spectra of p7 in
aligned phospholipid bilayers provided the tilt angles of the helical segments, 25° and 10° to the membrane normal. It been shown that
specific mutations to p7 result in a significant loss of channel activity, indicating that this function of p7 is sequence specific. It has also been
shown that known channel-blocking compounds such as amantadine, hexamethylene amiloride (HMA), and long alkyl-chain iminosugar
derivatives inhibit the ion channel activity of p7. There are six major genotypes and over 100 relevant subtypes. Our current studies aim to
investigate the structure and dynamic differences among the genotypes. In addition to studying different genotypes, specific mutations to
the HCV J4 genotype were made in order to modulate the dynamics of the protein and obtain additional secondary structural information.
Any differences will be detected using solution NMR of micelles or isotropic bicelles and solid state NMR of bilayers. These studies have the
potential to identify the reasons why different genotypes have varying activity and drug binding affinities.
Contact: Gabriel A. Cook
University of California, San Diego, La Jolla, CA, USA
[email protected]
56 : HCV 2011
ANTIVIRAL THERAPY
O8.05
Deciphering conformational variability of the hepatitis C virus polymerase (genotype
1) in complex with inhibitors
A major target for specifically targeted antiviral therapy against hepatitis C virus (HCV) is the HCV RNA-dependent RNA polymerase NS5B.
Huge efforts have been devoted to the development of nucleoside and non-nucleoside inhibitors (NNIs) of NS5B. An offshoot of these
efforts has been the structural characterization of the interaction of NS5B with NNIs by X-ray crystallography. These works have shown that
the conformation of recombinant NS5B is very similar across strains, constructs and complexes, making evaluation of the structural effects
of NNIs nontrivial.
Using procedures appropriate to the evaluation of just such minor but potentially important conformational differences, we recently
reported a breakthrough in the understanding of the special replicative properties of NS5B of the JFH1 strain (Simister et al. 2009, Schmitt
et al. 2011). In the present work we apply similar procedures to assess conformational differences associated with the binding of NNIs in the
ca 80 available genotype 1 NS5B structures.
We find that there are 20 significantly different available NS5B conformations, but all geometrically close to a closed, RNA synthesis initiationcompetent one (Harrus et al, 2010). Within this fairly restricted range, differences can be mapped to movements of NS5B domains and
subregions. Most of this information is actually defined by small but significant changes in complexes with NNIs. We thus establish rigorously
the moving parts of the NS5B molecular machine and the previously unrecognized hinge points that come into play upon NNI binding.
We propose that NNIs binding at several distinct sites all specifically inhibit the initiation step by the same mechanism: They prevent
NS5B’s ‘thumb’ from quite reaching the proper position. Furthermore, we suggest that a small number of critical hinges in the NS5B
structure may emerge as sites of resistance mutations during antiviral treatment, particularly for those NNIs whose binding sites are not
permissive to mutations.
Contact: Célia Caillet-Saguy
Laboratoire de Virologie Moléculaire et Structurale CNRS UPR3296, Gif-sur-Yvette, France
[email protected]
O8.06
Elimination of HCVcc using recombinant AAV vector-mediated delivery of anti-HCV
miRNAs
Xiao Yang (2), Shangzhen Zhou (2), Guangxiang Luo (1), Linda Couto (2)
(1) University of Kentucky College of Medicine, USA
(2) Children’s Hospital of Philadelphia, Philadelphia, PA, USA
The successful treatment of HCV requires a combination of multiple drugs, due to the genetic diversity of the virus and its propensity
to mutate. RNA interference (RNAi), the sequence-specific post‐transcriptional mechanism of gene silencing, provides a means for
achieving this. We are exploiting the endogenous miRNA‐17‐92 cluster as a scaffold for the expression of multiple exogenous miRNAs that
are complementary to the HCV genome. The miRNAs are delivered to cells using recombinant AAV (rAAV) vectors and are expressed in
hepatocytes, the site of HCV replication. Previously, we created an rAAV vector encoding four active miRNAs, and demonstrated up to 98%
inhibition of bona fide HCVcc replication in the Huh-7.5 cells (Yang et al; Hepatology 2010). We have now constructed a more potent rAAV
vector (rAAV-Cluster 5) that expresses five active anti-HCV miRNAs. Upon co-infection of Huh7.5 cells with rAAV-Cluster 5 and HCVcc, miRNA
levels of ~1000 copies/cell were produced, which resulted in 98% inhibition of HCVcc replication. Furthermore, when HCVcc was serially
propagated on AAV-Cluster 5-transduced cells, it was completely eliminated (> five log decrease) from the culture within four rounds of
propagation. This is in contrast to AAV vectors that expressed just a single miRNA, which inhibited HCVcc replication by two logs, but was
incapable of eradicating the infection. Following a single injection of mice with rAAV-Cluster 5, efficient gene transfer was observed, and
all five miRNAs were expressed at between 1500-3500 copies/cell, which resulted in up to 98% gene silencing of reporter plasmids. This
data strongly suggests that rAAV-Cluster 5 will be highly effective against an HCV infection in human hepatocytes, which harbor lower copy
numbers of HCV and where the AAV vector is predicted to be stably maintained. This vector represents a potential alternative to current
HCV therapies.
Contact: Linda Couto
Children’s Hospital of Philadelphia, Philadelphia, PA, USA
[email protected]
ORAL ABSTRACTS
Célia Caillet-Saguy (1), Philip C. Simister (1), Stéphane Bressanelli (1)
(1) Laboratoire de Virologie Moléculaire et Structurale CNRS UPR3296, Gif-sur-Yvette, France
ORAL ABSTRACTS
O8.07
Phosphatidylinositol 4-kinase IIIα is targeted by 4-aminoquinazoline compounds with
anti-HCV activity
Raffaele De Francesco (1), Annalisa Bianco (1), Reinaldo Alvarez (1), Simone Fenu (1), Veronica Reghellin (1), Massimiliano Pagani (1),
Sergio Abrignani (1), Petra Neddermann (1)
(1) INGM-Istituto Nazionale Genetica Molecolare, Milano, Italy
ORAL ABSTRACTS
4-aminoquinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be
the viral protein NS5A. However, an unequivocal proof of this has never been presented. A quinazoline moiety is found in many kinase
inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a
cellular kinase.
The lipid phosphatidylinositol kinase PI4KIIIα has recently been identified as a host factor crucial for HCV replication. We therefore evaluated
a compound prototypical of the 4-aminoquinazoline class (AL9) on a panel of selected phosphatidylinositol kinases. Interestingly, AL9
inhibited the activity of purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. Next, we assessed the inhibition of these kinases in cell culture. In
Huh-7.5 cells, PI4KIIIα is responsible for the synthesis of the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane.
Accordingly, we observed a gradually decrease of PI4P in the plasma membrane upon incubation with increasing concentration of AL9,
indicating that AL9 inhibits PI4KIIIα also in living cells. Conversely, AL9 did not prevent the accumulation of PIP4 in the Golgi membrane,
indicating that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions.
Interestingly, we observed that the increase of PI4P in HCV-specific membranous structures is accompanied by a decreased concentration
of PI4P in the plasma membrane. This observation might indicate that HCV – by recruiting PI4KIIIα in the RNA replication complex - redirects
the PI4P to the membranous web.
In light of our findings we propose that AL9 inhibits PI4KIIIα and interrupts the supply of PI4P to the HCV membranous web thus leading to
inhibition of HCV replication. We propose that the class of 4-aminoquinazolines, previously reported to act as NS5A inhibitors, needs to be
reevaluated as PI4KIIIα inhibitors.
Contact: Raffaele De Francesco
INGM-Istituto Nazionale Genetica Molecolare, Milano, Italy
[email protected]
O8.08
The non-nucleoside inhibitor tegobuvir utilizes a novel mechanism of action to inhibit
HCV NS5b polymerase function
Christy M. Hebner (1), Bin Han (1), Katherine M. Brendza (1), Michelle Nash (1), Maisoun Sulfab (1), Yang Tian (1), Magdeleine Hung (1),
Wanchi Fung (1), Randy Vivian (1), James Trenkle (1), Kyla Bjornson (1), Steven Bondy (1), Xiaohong Liu (1), Swami Swaminathan (1), John
Link (1), Johan Neyts (2), Roman Sakowicz (1), Uli Schmitz (1), Weidong Zhong (1)
(1) Gilead Sciences, Inc., Foster City, CA, USA
(2) Rega Institute, KU Leuven, Belgium
Tegobuvir (TGV) is a novel non-nucleoside (NNI) inhibitor of HCV RNA replication with demonstrated antiviral activity in patients with
genotype-1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance
mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting
that a cellular component is involved in its antiviral mechanism. Here we demonstrate that TGV exerts anti-HCV activity by a unique direct
interaction with the NS5B protein. Treatment of replicon cells with TGV results in a modified form of NS5B having a distinctly altered
mobility on an SDS-PAGE gel. Appearance of this modified species correlates with antiviral activity as it is delayed or completely absent
in TGV -resistant replicon cells. Further analysis revealed that the aberrantly migrating NS5B species contains the inhibitor molecule and
formation of this complex does not require the presence of any other HCV proteins. Furthermore, mass spectrometry analysis of NS5B
protein purified from a heterologous expression system treated with TGV revealed the physical nature of the complex. This analysis suggests
that TGV undergoes an intracellular activation step and the resulting metabolite directly and specifically interacts with NS5B. Taken together,
these data demonstrate that TGV utilizes a cellular factor(s) to specifically target the HCV NS5B polymerase and is mechanistically distinct
from other classes of the NNI family.
Contact: Christy M. Hebner
Gilead Sciences, Inc., Foster City, CA, USA
[email protected]
58 : HCV 2011
ANTIVIRAL THERAPY
O8.09
Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current
standard therapy of pegylated interferon-alpha and ribavirin not only has a low success rate, but is often associated with serious side
effects. Thus, there is a need for new anti-HCV agents with novel mechanisms of antiviral action. HCV non-structural 5A protein (NS5A)
is a pleiotropic protein with a key role in HCV replication and cellular signaling pathways. Here we demonstrate through recombinant
proteins pulldown assays and co-immunoprecipitations that NS5A dimerization occurs and domain I (amino acids 1-240) is essential for
NS5A-NS5A contacts. Nucleic acids do not serve as connectors since DNAse, RNAse and benzonase treatments do not prevent NS5A-NS5A
interactions. Importantly, we found that dithiothreitol (DTT) abrogates NS5A-NS5A interactions, but does not affect NS5A-cyclophilin A
interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) and 2-mercaptoethanol (2-ME) also abrogate NS5A-NS5A
interactions, whereas cyclophilin inhibitors such as cyclosporine A (CsA) and Alisporivir do not. Moreover, we found that NS5A inhibitors
such as BMS-790052 do not block NS5A dimerization, suggesting that their mechanism of antiviral effect does not involve the disruption of
NS5A-NS5A interactions. Since we observed that reducing agents inhibit NS5A dimerization, we asked whether disulfide bridges mediate
NS5A-NS5A contacts. To test this hypothesis, each cysteine in domain I of NS5A was replaced by an alanine and resulting NS5A mutants were
tested for dimerization. Importantly, C13A, C39A, C98A, C140A, C142A, C165A, C190A and C243A behave like wild-type NS5A, while C57A,
C59A and C80A fail to dimerize, suggesting that these cysteines are key residues in NS5A-NS5A interactions. We are currently investigating
whether they play a vital role in HCV replication. The ultimate goal is to determine whether NS5A-NS5A dimerization and/or multimerization
represent a novel target for the development of HCV therapies.
Contact: Precious Lim
[email protected]
O8.10
The green tea polyphenol epigallocatechin-3-gallate (EGCG) inhibits hepatitis C virus
(HCV) entry
Sandra Ciesek (4), Thomas von Hahn (1), Luis M. Schang (2), Martina Friesland (4), Michael P. Manns (1), Michael Ott (1), Heiner
Wedemeyer (1), Philip Meuleman (3), Thomas Pietschmann (4), Eike Steinmann (4)
(1) Department of Gastroenterology, Hepatology and Endocrinology, Hannover, Germany
(2) Department of Biochemistry, University of Alberta, Edmonton, Canada
(3) Center for Vaccinology, Ghent University Hospital, Ghent, Belgium
(4) Twincore, Hannover, USA
Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in
a substantial proportion of cases and drug development is focused on non-structural proteins required for RNA replication. Individuals
undergoing orthotopic liver transplantation face rapid and universal reinfection of the graft and antiviral strategies targeting the early stages
of infection are urgently needed for the prevention of HCV infection.
In this study, we identified polyphenol epigallocatechin-3-gallate (EGCG) as a potent inhibitor of the HCV entry pathway. Green tea catechins
such as EGCG and its derivatives epigallocatechin (EGC), epicatechingallate (ECG) and epicatechin (EC) have been previously found to exert
antiviral and anti-oncogenic properties. EGCG had no effect on HCV RNA replication or assembly and release of progeny virions. However,
it potently inhibited HCVcc entry. This inhibitory effect was also observed using pseudoparticles with non-HCV viral envelope proteins,
but we observed a particularly high activity against HCV. Furthermore, HCV entry into primary human hepatocytes was blocked by EGCG
independent of the HCV genotype. Application and removal of EGCG prior to inoculation with HCV did not reduce HCV infection while
treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV receptors were
unaltered by EGCG indicating that it does not act through a down regulation of the entry factors. Finally, viral spread in a cell-to-cell
transmission assay was blocked by the compound. Studies in the chimeric uPA-SCID mouse model system are currently ongoing.
In summary, the green tea molecule EGCG potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of
HCV reinfection after liver transplantation.
Contact: Eike Steinmann
Twincore, Hannover, Germany
[email protected]
ORAL ABSTRACTS
Precious Lim (1), Udayan Chatterji (1), Dan Cordek (2), Craig Cameron (2), Paul Targett-Adams (3), Kai Lin (4), Philippe Gallay (1)
(2) Pennsylvannia State University, USA
(3) Pfizer Global Research and Development, United Kingdom
(4) Novartis Institutes for Biomedical Research, Inc., USA
ORAL ABSTRACTS
O8.11
Marked synergy of entry inhibitors and interferon-alfa or direct acting antivirals on the
inhibition of hepatitis C virus infection
Fei Xiao (1), Isabel Fofana (1), Joachim Lupberger (1), Pieter Leyssen (2), Johan Neyts (2), Mirjam B. Zeisel (1), Thomas F. Baumert (1)
(1) INSERM, U748, Université de Strasbourg, Strasbourg, France
(2) Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium
ORAL ABSTRACTS
Hepatitis C virus (HCV) is a major cause of liver disease, but the current treatment is limited by resistance and adverse effect. Several small
molecule compounds targeting the HCV non-structural proteins including protease and polymerase are in late stage development. Clinical
trial data are promising but toxicity of the individual compounds and emergence of resistance remain major challenges. This suggests
that the combination of several drugs, ideally targeting different steps of the viral life cycle, is needed for efficient anti-HCV therapy. Viral
entry is required for initiation, dissemination and maintenance of infection and thus is a promising target for antiviral therapy. Indeed, we
have previously shown that monoclonal claudin-1-specific antibodies and kinase inhibitors targeting cell entry factors efficiently inhibit
infection of HCV strains which are resistant to neutralizing host immune responses. Aiming to uncover antiviral combinations that increase
the barrier to resistance, we investigated whether the combination of entry inhibitors (claudin-1-specific antibodies, kinase inhibitors) and
interferon-alfa or direct acting antivirals (DAAs) (Telaprevir, Danoprevir, R7128) act in a synergistic manner on the inhibition of HCV infection
in cell culture models. Combination of entry inhibitors and interferon-alfa or DAAs resulted in a marked synergistic effect in inhibition of
HCV infection as shown by a combination index (CI) between 0.14 and 0.83. Combinations of either anti-claudin-1 or kinase inhibitors with
interferon-alfa or Telaprevir decreased the IC50s of entry inhibitors up to 100 fold. Studies to uncover the molecular mechanisms underlying
these synergies are ongoing. Taken together, these results provide the rationale for antiviral strategies combining compounds targeting viral
and host cell factors and complementary steps of the viral life cycle.
Contact: Fei Xiao
INSERM, U748, Université de Strasbourg, Strasbourg, France
[email protected]
O8.12
The RAFIs aUY11 and dUY11 inhibit infectivity of HCV and other enveloped viruses by
preventing fusion of viral and cellular membranes
Che C. Colpitts (1), Alexey V. Ustinov (2), Vladimir A. Korshun (2), Luis M. Schang (3)
(1) Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Edmonton, Canada
(2) Russian Academy of Sciences, Moscow, Russia
(3) Biochemistry/Li Ka Shing Institute of Virology, University of Alberta, Canada
We have described a family of novel antiviral compounds, the rigid amphipathic fusion inhibitors (RAFIs). The RAFI dUY11 is active against
HCV and other otherwise unrelated enveloped viruses. RAFIs inhibit viral entry with no obvious effects on physiological cellular fusions. We
proposed that RAFIs target the lipids in virion envelopes to prevent the formation of the negative membrane curvature required for fusion.
We had reported that dUY11 inhibited the infectivity of HCV JFH-1 to Huh7.5 cells, as tested by NS3 expression at 4 days post-infection (IC50,
183 nM). We now used a focus-forming assay to directly test the effects of dUY11 and another RAFI, aUY11. HCV virions were exposed to
aUY11 or dUY11 prior to infection of Huh7.5 cells. Infected cells were fixed at 3 days post-infection and processed for immunocytochemistry
for HCV core protein. aUY11 and dUY11 inhibited HCV infectivity (IC50, 175 nM and 130 nM, respectively). We next tested the effect of
aUY11 on fusion, using fluorescence dequenching assays. Vesicular stomatitis virus (VSV) labeled at self-quenching concentrations with
the membrane dye octadecyl rhodamine chloride (R18) were exposed to aUY11, and then mixed with Vero cells on ice. Fusion, induced by
raising the temperature to 37°C and lowering the pH to 5.5, was monitored by fluorescence dequenching. Fluorescence was dequenched
by 15% when virions were exposed to vehicle, but by only 5% when they were exposed to 60 nM aUY11. Therefore, aUY11 also inhibits
the fusion of virion lipid envelopes to cell membranes. In conclusion, the RAFIs aUY11 and dUY11 inhibit HCV infectivity at low nanomolar
concentrations. The activity of aUY11 is most similar to that of dUY11, supporting our model for the antiviral mechanism of RAFIs, which
target virion envelope lipids to prevent fusion of viral and cellular membranes.
Contact: Che C. Colpitts
Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Edmonton, Canada
[email protected]
60 : HCV 2011
ANTIVIRAL THERAPY
O8.13
Identification and functional analysis of small molecules inhibiting the late step of
hepatitis C virus life cycle
There are no anti-hepatitis C virus (HCV) drugs developing clinically that target a late step of the life cycle including assembly, budding, and
release. To identify small molecules that inhibit its late step, we screened small molecules using the production system for infectious HCV
(HCVcc) with Huh7-25 cells. The cells, which are deficient for the expression of CD81, do not allow reinfection after the transfection of HCV
JFH1 RNA and thus are expected to select hit compounds that target the late steps with higher rate than a system using CD81-intact cells. By
screening a bioactive compound library on this cell-based assay system, we found 17 compounds reducing the infectious titer of the virus in
culture media without serious cytotoxicity. These included several compounds that have already been reported as HCV replication inhibitors
such as antagonists for peroxisome proliferator activated receptor-alpha and farnesoid X receptor. To narrow down the target step of hit
compounds among the life cycle, we used HCV pseudoparticle system (HCVpp) and replicon system. As a result, 1 and 4 compounds clearly
suppressed HCVpp and the replicon activity, respectively. 12 compounds (71% of the hit compounds) decreased the infectivity in culture
media on HCVcc system without affecting HCVpp nor replicon system. Among them, at least 2 compounds drastically reduced the infectivity
of both extracellular and intracellular HCV, suggesting that these compounds may target the assembly step. Thus, our assay system identified
late step inhibitors with higher rate compared with already reported screening system using HCVcc. Hit compounds include kinase inhibitors,
antagonists for nuclear hormone receptors, and lipid metabolism modulators. We will also present the characterization of hit compounds.
Contact: Koichi Watashi
National Institute of Infectious Diseases, Japan, Tokyo, Japan
[email protected]
O8.14
Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral
replication in hepatocytes
Kyoko Tsukiyama-Kohara (1), Takashi Takano (4), Yuichi Hirata (4), Yuko Tokunaga (4), Chise Tateno (2), Masayuki Sudo (3), Michinori
Kohara (4)
(1) Department of Experimental Phylaxiology, Faculty of Medical and Pharma, Japan
(2) Phoenix Bio Co., Ltd., Study Service Department, 3-4-1, Kagamiyama, Hi, Japan
(3) Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd. Kajiwa, Japan
(4) Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
Background & Aims: The purpose of this study was characterization of the role of 3b-hydroxysterol-D-24-reductase (DHCR24) in hepatitis
C virus infection (HCV), because DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which
is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV
therapeutic agents.
Methods: We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice
with human hepatocytes. We targeted DHCR24 with siRNA and cholesterol synthesis inhibitor U18666A into HCV replicon cell lines and HCV
infected cells, and measured the level of HCV replication. U18666A was administrated into humanized chimeric mice, and anti-viral effects
were assessed.
Results: Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse
liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with
cholesterol synthesis inhibitor, U18666A, suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of
U18666A in vivo, we administrated U18666A with or without pegylated interferon to humanized chimeric mice and observed an inhibitory
effect of U18666A on HCV infection and a synergistic effect with interferon.
Conclusions: DHCR24 is an essential host factor which is augmented its expression by HCV infection, and plays a significant role in HCV
replication. DHCR24 may serve as a novel anti-HCV drug target.
Contact: Michinori Kohara
Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
[email protected]
ORAL ABSTRACTS
Koichi Watashi (1), Nanako Uchida (1), Ryosuke Suzuki (1), Hideki Aizaki (1), Takaji Wakita (1)
(1) National Institute of Infectious Diseases, Japan, Tokyo, Japan
ORAL ABSTRACTS
O8.15
The impact of calcineurin inhibitors on HCV replication in a chimeric mouse model of
HCV infection
Norman Kneteman (1), Jamie Lewis (1), Donna Douglas (1), Mahra Nourbakhsh (1), Lorne Tyrrell (1), Garry Lund (2)
(2) KMT Hepatech, Inc., Canada
ORAL ABSTRACTS
The impact of immunosuppression on HCV replication results in early recurrence post liver transplant with accelerated disease and more
rapid progression of fibrosis and cirrhosis culminating with poorer 5 and 10 year graft and patient survival. Cyclosporine has demonstrated
potent anti-HCV impact in vitro while the potential for better outcomes in clinical application has been difficult to demonstrate conclusively.
We evaluated the impact on serum HCV titers of therapy with cyclosporine vs. tacrolimus, both with or without concomitant interferon (IFN)
therapy in our chimeric mouse model of HCV infection.
Six groups of HCV infected mice received vehicle only, IFN, cyclosporine, cyclosporine + IFN, tacrolimus, or tacrolimus + IFN for 4 weeks.
Cyclosporine and tacrolimus dosages were established by pilot studies to target clinically relevant whole blood levels of 800-1200ng/ml 2
hour peak, and 8-10 ng/ml 12 hr. troughs respectively. Quantitative HCV PCR levels were run after 1, 2 and 4 weeks of drug administration.
Achievement of target drug levels for both cyclosporine and tacrolimus was confirmed at 4 weeks in sampled mice. While Interferon had a
consistent impact in reducing HCV titers across groups, no significant impact of cyclosporine was seen on HCV levels alone or in combination
with IFN at any time point. No significant toxicity of any drug administration combination was noted. While these results do not rule out the
possibility for a benefit of cyclosporine in combination with IFN in achieving higher SVR rates in post transplant patients, we could find no
evidence to support routine use of cyclosporine after liver transplantation in HCV infected patients.
Contact: Norman Kneteman
University of Alberta, Edmonton, Canada
[email protected]
O8.16
PPARα agonist WY14643 improves the antiviral effect of interferon against hepatitis C virus
Scott Read (1), Jacob George (1), Mark Douglas (1)
(1) Storr Liver Unit, Westmead Millenium Institute, Westmead, Australia
Pegylated interferon (IFN) in combination with ribavirin is currently the gold standard treatment for HCV but achieves sustained viral response
(SVR) in only 50-60% of patients and is largely dependent on genotype. SVR is achieved in ~65% of genotype 2/3 patients, compared to less
than half of patients infected with genotypes 1/4. Interestingly, genotypes 1 and 4 are also more likely to cause insulin resistance, which is
independently associated with poor treatment response but improves following successful IFN treatment.
To examine the effects of insulin sensitivity on IFN response in a human hepatoma cell line (Huh7), several HCV models were used: The
JFH-1 strain of HCV; a modified JFH-1 construct incorporating a luciferase reporter; and a luciferase expressing subgenomic replicon (SGR)
expressing HCV non-structural proteins. PPARα agonist WY14643 demonstrated greater synergy with interferon compared to PPARγ agonist
pioglitazone and AMPK activator metformin, and was thus chosen for further study. The effects of WY14643 on responses to acute and
chronic IFN treatment were examined in JFH-1 infected Huh7 cells. Effects on interferon signalling, inflammatory responses, triglyceride and
cholesterol metabolism were also investigated.
The effectiveness of WY14643 as an insulin sensitiser was confirmed using p-AKT assay. Pre-treatment with WY14643 enhanced the IFNinduced reduction in HCV RNA in all models. Surprisingly no detectable increase in IFN signalling was observed using ISRE and NFkB reporters.
Select IFN-stimulated gene (ISG)s did show altered expression, especially following chronic IFN treatment. While there was no apparent
effect of WY14643 on intracellular triglyceride or cholesterol, viral particle density and infectivity were modified following treatment.
Our data indicates that PPARα agonist WY14643 synergises with interferon to reduce JFH-1 replication in vitro. This most likely involves
multiple mechanisms, including alterations of ISG expression and lipid metabolism.
Contact: Scott Read
Storr Liver Unit, Westmead Millenium Institute, Westmead, Australia
[email protected]
62 : HCV 2011
ORAL ABSTRACTS
Virus-Host Interactions
O9.01
O9.02
Hepatitic C virus infection of differentiated human hepatocytes derived
from embryonic and induced pluripotent stem cells
Xianfang Wu, Jason Robotham, Hongying Deng, Hengli Tang
O9.03
Kelly Coller, Nicholas Heaton, Kristi Berger, Jacob Cooper, Jessica Saunders, Glenn Randall
O9.04
Impaired signaling and function induced in plasmacytoid dendritic cells
exposed to HCV
Jonathan Florentin, Clélia Dental, Besma Aouar, Françoise Gondois-Rey, David Durantel, Thomas F. Baumert, Daniel
Olive, Ivan Hirsch, Ruzena Stranska
O9.05
Aleem Siddiqui, Huihui Tang, Gulam Syed, Tarek Hassanein
O9.06
Amina Negash, Hilario Ramos, Michael Gale Jr.
O9.07
HCV directly stimulates the production of the inflammasome-associated
cytokine IL-18 in cells of the monocyte lineage
Michael Chattergoon, Erik Orberg, Jordana Levine, Andrea Cox
O9.08
Tetsuro Shimakami, Daisuke Yamane, Rohit Jangra, Brian Kempf, Carolyn Spaniel, David Barton, Stanley Lemon
ORAL ABSTRACTS
Marcus Dorner, Joshua Horwitz, Alexander Vogt, Kathy Mu, Gisa Gerold, Charles Rice, Alexander Ploss
ORAL ABSTRACTS
O9.09
A RIG-I translocon directs membrane-association of RIG-I with IPS-1 and
controls innate antiviral immunity
Helene Minyi Liu, Michael Gale Jr.
O9.10
John Schoggins, Sam Wilson, Maryline Panis, Mary Murphy, Christoper Jones, Paul Bieniasz, Charles Rice
O9.11
In vivo suppression of interferon sensitive gene 15 (ISG15), a proviral host
factor in HCV replication, enhances the endogenous interferon response
Catherine I. Real, Ruth Broering, Kerstin Jahn-Hofmann, Ludger Ickenstein, Matthias John, Kathrin Kleinehr, Guido Gerken,
Joerg F. Schlaak
O9.12
Robust induction of type III interferons and other cytokines defines a
unique pattern of innate immunity in response to hepatitis C virus infection
ORAL ABSTRACTS
Emmanuel Thomas, Veronica Gonzalez, Qisheng Li, Ankit Modi, Weiping Chen, Mazen Noureddin, Yaron Rotman,
T. Jake Liang
O9.13
Systems biology & computational network analysis identifies mitochondrial
host targets of HCV infection and liver disease progression
Deborah L. Diamond, Jason E. McDermott, Alexei Krasnoselsky, Kristin E. Burnum, Nathan Susnow,
Bobbie-Jo Webb-Robertson, Courtney Courley, Matthew M. Yeh, Angela L. Rasmussen, Jon M. Jacobs,
Renuka Bhattacharya, James D. Perkins, Robert L. Carithers Jr., Marina A. Gritsenko, Iris W. Liou, Susan Strom,
Katrina M. Waters, Richard D. Smith, Michael G. Katze
O9.14
Overall structural model of non-structural protein 5A from hepatitis C virus
and modulation by cyclophilin A
Aurelie Badillo, Veronique Receveur-Brechot, Simona Miron, Xavier Hanoulle, Jennifer Molle,
Roland Montserret, Ralf Bartenschlager, Guy Lippens, Sylvie Ricard-Blum, Francois Penin
O9.15
Hepatitis C virus infection induces autophagy and blocks innate immune
response
Shubham Shrivastava, Amit Raychoudhuri, Robert Steele, Ranjit Ray, Ratna Ray
O9.16
Differential type I interferon-mediated autophagic trafficking of hepatitis C
virus proteins in mouse liver
Mayura Desai, Bin Gong, Tehsheng Chan, Jiaren Sun
64 : HCV 2011
VIRAL-HOST INTERACTIONS
O9.01
More than 130 million people world-wide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease.
Antiviral treatments are only partially effective and a vaccine is currently not available. Development of more efficient therapies has been
hampered by the lack of a small animal model. Murine cells are resistant to HCV entry, support only inefficiently RNA replication and may
also be blocked at later life steps of assembly and release. Previously, we demonstrated that human CD81 and occludin (OCLN) comprise
the minimal set of human entry factors required for rendering murine cells permissive to HCV entry. Adenoviral delivery of human CD81 and
OCLN facilitates HCV entry also into hepatocytes of immunocompetent mice. This transient approach allows for genetic dissection of HCV
entry and for testing HCV entry inhibitors in vivo.
However, to minimize intra- and inter-experimental variability and to prevent vector-mediated immune activation, we have generated
transgenic mice stably expressing human CD81, scavenger receptor class B type 1, claudin 1 and/or OCLN in the liver. Here, we confirm that
expression of human CD81 and OCLN is sufficient to allow HCV infection of fully immunocompetent inbred mice. Using intergenotypic virus
chimeras expressing CRE recombinase to activate a cellular reporter, in vivo viral uptake could be conveniently quantified by bioluminescence
imaging. In ongoing studies we aim to validate infection in mice engineered to express a cell-based fluorescent reporter system that can be
activated with unmodified HCV genomes. By characterizing HCV-induced innate and adaptive immune responses, we are also attempting
to identify a murine background that is highly permissive for HCV RNA replication. These transgenic models open up new opportunities
for studying HCV pathogenesis and immunity and comprise cost-effective, reproducible, and high-throughput platforms for testing HCV
vaccines in vivo.
Contact: Marcus Dorner
The Rockefeller University, Center for the Study of Hepatitis C, New York, NY, USA
[email protected]
O9.02
Hepatitic C virus infection of differentiated human hepatocytes derived from
embryonic and induced pluripotent stem cells
Xianfang Wu (1), Jason Robotham (1), Hongying Deng (1), Hengli Tang (1)
(1) Florida State University, Tallahassee, FL, USA
Infection of primary human hepatocytes (PHHs) ex vivo or post-transplantation in mice represents the most relevant infection model with
human cells outside of patients. Yet, PHHs are largely refractory to genetic manipulation and not readily available to most labs. Functional
hepatocytes differentiated from human embryonic stem cells (hESC) and induced pluripotent cells (iPS) provide an accessible alternative
that is also amenable to genetic modification. However, the permissiveness to HCV infection of these differentiated human hepatocytes
(DHHs) had not been studied. Here we report a novel approach to directly differentiate hESCs into functional hepatocytes that could be
infected by HCV particles generated in cell culture (HCVcc). H9 (hESC) cells were first differentiated into definitive endoderm and then into
hepatic progenitors, followed by a hepatocyte maturation stage. Viral proteins (Core, NS3, and NS5A) were detected by Western blotting
after infection of cells in the maturation stage. Continuous monitoring of reporter activity in the supernatant revealed persistent infection
of the DHHs for 10-12 days and the infection could be abolished by either entry blockers (an anti-CD81 antibody, an SR-BI antagonist, and
a neutralizing E2-antibody) or replication inhibitors (DEB-025/Cyclosporine A, and interferon). Taking advantage of the defined stages of
differentiation, we also identified a critical transition phase for the differentiating cell to become permissive for infection, which was not
correlated with the expression of the cell surface receptors. We next tested genetically modified hESCs harboring shRNA directed at cellular
factors such as CyPA and PI4K-IIIalpha in the DHH infection model. Suppressing CyPA or PI4K-IIIalpha expression in hESCs did not affect
hepatocyte differentiation but efficiently blocked HCV infection of the DHHs. Finally, we reprogrammed primary cells and used iPS cells
to produce differentiated hepatocytes (iDHH) for HCV infection. These studies have significance for both basic research and therapeutic
development of HCV infection.
Contact: Xianfang Wu
Florida State University, Tallahassee, FL, USA
[email protected]
ORAL ABSTRACTS
Marcus Dorner (1), Joshua Horwitz (1), Alexander Vogt (1), Kathy Mu (1), Gisa Gerold (1), Charles Rice (1), Alexander Ploss (1)
(1) The Rockefeller University, Center for the Study of Hepatitis C, New York, NY, USA
ORAL ABSTRACTS
O9.03
Kelly Coller (1), Nicholas Heaton (1), Kristi Berger (1), Jacob Cooper (1), Jessica Saunders (1), Glenn Randall (1)
(1) The University of Chicago, Chicago, IL, USA
The current model of HCV egress is that virions assemble at lipid droplets, envelope at the ER and then likely exit the hepatocyte via the
secretory pathway in association with apolipoproteins. To gain a more detailed insight into infectious HCV release, we combined an RNAi
analysis of host factors that are required for infectious HCV secretion with live cell imaging of HCV core trafficking. Using this approach, we
identified numerous components of the secretory pathway that are both required for infectious HCV release and co-traffic with HCV core.
The dynamics of HCV core trafficking, both in terms of frequency of transport, particle velocity, and the corresponding run lengths were
quantified. We observe that dynamic core movements in the periphery require NS2, a viral protein required for virion assembly. We found
using live cell microscopy, core co-traffics with multiple components of the secretory pathway identified in the RNAi screen. This study
identifies new molecular determinants of HCV secretion and describes the visualization of HCV core in real time.
Contact: Kelly Coller
The University of Chicago, Chicago, IL, USA
[email protected]
ORAL ABSTRACTS
O9.04
Impaired signaling and function induced in plasmacytoid dendritic cells exposed to HCV
Jonathan Florentin (1), Clélia Dental (1), Besma Aouar (1), Françoise Gondois-Rey (1), David Durantel (2), Thomas F. Baumert (3), Daniel
Olive (1), Ivan Hirsch (1), Ruzena Stranska (1)
(1) INSERM U891 CRCM, 13273 Marseille Cedex 09, France
(2) INSERM U1052, CNRS UMR5286, CRCL Lyon, France
(3) INSERM U748, Université de Strasbourg, Institut de Virologie, France
BACKGROUND & AIMS: The eradication of hepatitis C virus (HCV) in more than 50% of chronically infected patients by treatment with
interferon (IFN)-alpha suggests that production of endogenous IFN-alpha can play an important role in the control of HCV infection.
Plasmacytoid dendritic cells (pDCs) are responsible for the production of IFN-alpha during viral infection. pDCs exposed to HCV-infected
hepatoma cells, in contrast to cell-free HCV virions, produce large amounts of IFN-alpha. To further investigate the molecular mechanism of
HCV sensing, we studied whether exposure of pDCs to HCV-infected hepatoma cells induces additional pDC functions, namely production
of proinflammatory cytokines and cell differentiation, both related to phosphorylation of NF-kappa-B. METHODS: Flow cytometry was
used to analyze signaling and differentiation at one-pDC level. Cytokine secretion was measured by ELISA. RESULTS: Exposure of pDCs to
HCV-infected hepatoma cells, in contrast to influenza virus, or synthetic agonists of TLR7 (R848) and TLR9 (CpG-B), surprisingly did not
induce phosphorylation of NF-kappa-B or secretion of tumor necrosis factor (TNF)-alpha. In addition, HCV-infected hepatoma cells did not
induce expression of the differentiation markers CD40, CCR7, CD86, or of the TNF-related apoptosis-inducing ligand (TRAIL). Further analysis
revealed that the HCV-infected hepatoma cells did not actively block the NF-kappa-B phosphorylation. CONCLUSIONS: We propose that
cell-associated HCV signals in pDCs via IRF7 but not NF-kappa-B pathway, and that in spite of IFN-alpha induction, it does not induce a full
functional response of pDCs. Our results thus show a link between pDC signaling pathways and functions, and they are important for our
understanding of the mechanisms leading to chronic HCV infection.
Contact: Jonathan Florentin
INSERM U891 CRCM, 13273 Marseille Cedex 09, France
[email protected]
66 : HCV 2011
VIRAL-HOST INTERACTIONS
O9.05
Aleem Siddiqui (1), Huihui Tang (1), Gulam Syed (1), Tarek Hassanein (1)
(1) UCSD, San Diego, CA, USA
Contact: Gulam Syed
UCSD, San Diego, CA, USA
[email protected]
O9.06
Amina Negash (1), Hilario Ramos (1), Michael Gale Jr. (1)
Chronic HCV infection is marked by liver inflammation that progresses to cirrhosis. Here, we examined the molecular features of hepatic
inflammation through assessment of inflammasome signaling. The inflammasome is a cytoplasmic multiprotein complex that leads to
the production and maturation of key pro-inflammatory cytokines such as IL-1β and IL-18 that serve to recruit immune cells to the site
of infection. Inflammasome induction requires two signals to stimulate cytokine maturation; the first leads to gene expression via NF-κB
to produce proIL-1β and proIL-18. The second drives Nod-Like receptor (NLR) signaling, which activates caspase-1 to cleave proIL-1β and
proIL-18 thus producing active and mature IL-1β and IL-18, respectively. However, the role of the inflammasome in HCV infection and
immunity is not known. We found that while hepatocytes and hepatoma cells express intracellular IL-1β, they do not secrete it nor is the
production of active cytokine further induced by HCV infection. In contrast, we found that when exposed to HCV, primary human monocytederived macrophages and macrophage-like THP-1 cells mediate the rapid and robust maturation and secretion of IL-1β and IL-18. We found
macrophage uptake and internalization of HCV is mediated through phagocytosis, and that the internalized HCV RNA does not replicate but
serves as a potent trigger of IL-1β expression through RNA signaling by the RIG-I pathway. Furthermore, secretion of IL-1β by macrophages
required NALP3 and the expression and activation of caspase-1. Our results support a model of hepatic inflammatory signaling in which
HCV RNA initiates signaling in hepatic myeloid cells through RIG-I and NLRP3 pathways to drive inflammasome activation. In support of this
model, we found that IL-1β is expressed in Kupffer cells, the liver-resident macrophage, in HCV patient liver and in a mouse model of hepatic
HCV RNA signaling. Inflammasome triggering by HCV may provide a platform for chronic hepatic inflammation.
Contact: Amina Negash
[email protected]
ORAL ABSTRACTS
Hepatitis C virus (HCV) influences the host lipid metabolism to enrich cellular microenvironment with lipids to facilitate its proliferation.
In this context, we have observed that HCV stimulates the expression of low-density lipoprotein receptor (LDLr). The induction of LDLr
expression can increase the uptake of lipoproteins a source of lipid for the hepatocytes and also enhance the possibility of HCV infection
since LDLr has been shown to be involved in the initial process of HCV entry. We observed that the stimulation of LDLr expression by
HCV primarily involves enhanced transcription of LDLr gene. LDLr expression at the protein level is also regulated by two E3 ubiquitin
ligases, the proprotein convertase subtilisin/kexin type 9 (PCSK9) and the inducible degrader of LDLr ( IDOL). We observed that HCV did
not influence the levels of these two proteins and correlates with our observation that HCV gene expression increases LDLr expression at
transcriptional level via sterol response element binding protein (SREBP) and liver X receptor (LXR). The functional significance of increased
LDLr expression in HCV infection was also evaluated by analyzing the uptake of Dil labeled LDL (Dil-LDL). We observed that HCV infection
led to enhanced uptake of Dil-LDL in Huh7 cells. Furthermore, we observed that LDLr protein expression was elevated in liver tissues from
patients chronically infected with HCV.
ORAL ABSTRACTS
O9.07
HCV directly stimulates the production of the inflammasome-associated cytokine IL-18
in cells of the monocyte lineage
Michael Chattergoon (1), Erik Orberg (1), Jordana Levine (1), Andrea Cox (1)
(1) Johns Hopkins School of Medicine, Baltimore, MD, USA
ORAL ABSTRACTS
Background: Persistent HCV infection has been associated with cytokine and ISG dysregulation and a chronic inflammatory state in the liver.
The levels of IL-18, an inflammasome-associated and interferon-γ inducing cytokine, are uniformly high in the early stages of acute infection
and remain elevated in persons with persistent infection, while clearance is associated with normalization of IL-18 levels. In this study, we
begin to explore the mechanism by which IL-18 remains elevated during persistent infection by investigating the effect of exposure to HCVcontaining plasma on secretion of IL-18 by primary lymphoid and liver cells.
Methods: Primary lymphoid cells were cultured with plasma obtained from subjects in the BBAASH cohort, prior to and after documented
HCV infection. Primary lymphoid cells were enriched for or depleted of individual immune cell lineages by magnetic sorting and exposed to
heat-inactivated pre-infection and post-infection plasma. IL-18 concentration in the culture media was quantified by ELISA.
Results: Increased levels of IL-18 were detected in PBMC cultured with plasma containing high levels of HCV RNA but not seronegative
or pre-infection plasma. This secretion required the presence of CD14+ cells in the culture and was not affected by T-cell (CD3+) or B-cell
(CD19+) depletion. Release of IL-18 was enhanced by enrichment of CD14+ cells in the culture. Exposure of primary hepatocyte or hepatoma
cell lines to HCV RNA failed to elicit IL-18 secretion.
Conclusions: These data suggest that HCV containing plasma directly stimulates the release of IL-18 from PBMC, but not liver cells, in a CD14+
cell-dependent manner. Our findings suggest that HCV can activate the inflammasome in cells of the monocyte lineage. Further investigation
into the mechanism of this effect may provide insight into the chronic inflammatory state characteristic of persistent HCV infection.
Contact: Michael Chattergoon
Johns Hopkins School of Medicine, Baltimore, MD, USA
[email protected]
O9.08
Tetsuro Shimakami (1), Daisuke Yamane (1), Rohit Jangra (2), Brian Kempf (3), Carolyn Spaniel (1), David Barton (3), Stanley Lemon (1)
(1) The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
(2) Mount Sinai School of Medicine, USA
(3) University of Colorado School of Medicine, USA
miR-122 is a liver-specific microRNA (miRNA) that is an essential but poorly understood host factor required for replication of hepatitis C
virus (HCV). Prior studies show that the binding of miR-122 to two sites located near the 5’ end of the positive-strand RNA enhances its
translation and promotes both RNA replication and the production of infectious virus. This is an unusual action for a miRNA, as miRNAs
typically regulate gene expression by binding to regions of imperfect complementarity in the 3’ untranslated region (3’UTR) of mRNAs,
recruiting an Argonaute (Ago) protein complex that results in translational repression or destabilization of the target RNA. The translation
and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to
reduce translation or enhance decay of its target RNA is controversial. We show here that miR-122 binds the 5’ terminus of HCV RNA in
association with Ago2, and that this slows the decay of the viral genome in infected cells following the arrest of new viral RNA synthesis
with a potent nucleoside analog inhibitor of the NS5B RNA-dependent RNA polymerase. The stabilizing action of miR-122 is dependent on
Ago2 but does not require the viral RNA to be translationally active nor engaged in replication. The protective effect of the miR-122-Ago2
complex on the viral genome can be functionally substituted by a non-methylated 5’ cap. Our data reveal the surprising involvement of a
RISC-like complex in mediating the stability of HCV RNA, and suggest that Ago2 and miR-122 act coordinately to protect the viral genome
from 5’ exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and
slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.
Contact: Tetsuro Shimakami
The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
[email protected]
68 : HCV 2011
ORAL ABSTRACTS
O9.09
A RIG-I translocon directs membrane-association of RIG-I with IPS-1 and controls
innate antiviral immunity
Retinoic-acid-inducible gene-I (RIG-I) is a cytosolic RNA helicase that functions as a pathogen recognition receptor to sense and bind viral
RNA, including HCV RNA. RIG-I drives a response that controls HCV permissiveness of hepatocytes. Once bound to non-self RNA, RIG-I can
engage host protective innate immunity against viral infection through IPS-1-depndent signaling from mitochondria-associated membranes
(MAM). The interaction between RIG-I and IPS-1 at the MAM is essential for IPS-1-mediated signaling of interferon production and innate
immune response against acute RNA virus infection but it is unclear how RIG-I, a cytosolic protein, can identify and interact with MAM
localized IPS-1. Here we show that RIG-I, TRIM25, and 14-3-3ε trimerize to form a translocon during acute phase of RNA virus infection.
While TRIM25 is known as an E3 ubiquitin ligase of RIG-I, the chaperon 14-3-3ε is a virus-induced protein that serves a role in protein
targeting to mitochondria membranes. We found that hepatocytes with TRIM25 or 14-3-3ε knock-down showed higher permissiveness to
HCV infection in vitro. Moreover, both TRIM25 and 14-3-3ε were required for RIG-I translocon formation through interacting with the CARD
domains of RIG-I. RIG-I/TRIM25/14-3-3ε trimer formed during virus infection and was regulated by the conformational change of RIG-I
governed by its repressor domain, which is known as the off/on switch for immune signaling. We found that once the RIG-I translocon is
formed, RIG-I becomes relocalized from a diffused pattern in the cytoplasm to a peri-nuclear distribution. MAM targeting by RIG-I occurred
in a manner independent of IPS-1. Our results demonstrate that RIG-I association with the MAM/intracellular membrane compartment
is important for downstream innate immune signaling and is mediated by a trimeric complex of RIG-I/TRIM25/14-3-3ε as a functional
translocon. These findings define the critical steps of innate immune response initiating RIG-I signaling and the anti-HCV response.
Contact: Helene Minyi Liu
[email protected]
O9.10
John Schoggins (1), Sam Wilson (2), Maryline Panis (1), Mary Murphy (1), Christoper Jones (1), Paul Bieniasz (2), Charles Rice (1)
(1) The Rockefeller University, New York, NY, USA
(2) HHMI, Aaron Diamond AIDS Research Center, The Rockefeller University, USA
The cellular type I interferon (IFN) response protects cells from invading viral pathogens. Incoming viruses are sensed by cellular pattern
recognition receptors, leading to the transcriptional induction and secretion of IFN. Binding of IFN to the type I IFN receptor (IFNAR) transmit
signals via the JAK/STAT pathway, resulting in the increased transcription of interferon stimulated genes (ISGs). Although hundreds of ISGs
have been identified, only few have been characterized with respect to antiviral activity. For most, little is known about their antiviral
potential, their target specificity, and their mechanisms of action. Using an overexpression screening approach, we show that different viruses
are targeted by unique sets of ISGs, with each viral species susceptible to multiple antiviral genes with a range of inhibitory activities. To
conduct the screen, we inserted over 380 commonly upregulated ISGs into a lentiviral vector and tested their ability to inhibit the replication
of several medically important viruses including hepatitis C virus (HCV), yellow fever virus (YFV), West Nile virus (WNV), chikungunya virus
(CHIKV), Venezuelan equine encephalitis virus (VEEV), and human immunodeficiency virus (HIV-1). We identified and confirmed that more
than 25 genes had antiviral activity against one or more viruses, while at least four ISGs enhanced replication of certain viruses. A subset of
anti-HCV effectors were shown to have antiviral activity when expressed before or after infection with HCV. Combined expression of two-ISG
pairs showed additive antiviral effects that recapitulated those induced by moderate IFN doses. Life-cycle studies using HCV pseudoparticles
and subgenomic replicons revealed that select anti-HCV effectors have little impact on virus entry, but rather impair primary translation to
varying degrees. Current areas of investigation include evaluation of ISG activity against HCV in primary hepatocyte cultures and knockdown
studies to define the contribution of individual ISGs to the IFN response.
Contact: John Schoggins
The Rockefeller University, New York, NY, USA
[email protected]
ORAL ABSTRACTS
Helene Minyi Liu (1), Michael Gale Jr. (1)
ORAL ABSTRACTS
O9.11
In vivo suppression of interferon sensitive gene 15 (ISG15), a proviral host factor in
HCV replication, enhances the endogenous interferon response
Catherine I. Real (1), Ruth Broering (1), Kerstin Jahn-Hofmann (2), Ludger Ickenstein (2), Matthias John (2), Kathrin Kleinehr (1), Guido
Gerken (1), Joerg F. Schlaak (1)
(1) University Hospital of Essen, Essen, Germany
(2) Roche Kulmbach GmbH, Germany
ORAL ABSTRACTS
Introduction: Non-response of HCV patients to combination therapy has previously been associated with a hepatic up-regulation of selected
interferon stimulated genes (ISG) and in particular ISG15. This proviral host factor promotes HCV replication and negatively regulates
responsiveness to interferon in vitro. In this study we investigate the proviral function of ISG15 in vivo.
Methods: Nanolipid-formulated (LNP01) siRNAs preferentially targeting the liver were injected into C57Bl/6 and MyD88-/- mice. After 6h
and 48h liver tissue was prepared and expression of IFN-β, ISG15, IFI-T1, CIG5, MX2, TNF-α, IL-6 and IL-10 was determined by qtRT-PCR.
Furthermore, hepatocytes and a mix of NPC were isolated to analyze cell-specific knockdown efficiency and induction of immune responses.
Results: LNP01 carrying unmodified siRNA caused enhanced transient expression of ISGs and inflammatory genes (IFN-β, ISG15, IFI-T1,
TNF-α, IL-6, IL-10) in the liver of wildtype mice 6h after application. In MyD88-/- mice, the induction of these genes was not detectable.
LNP01-siRNAs, harbouring ribose-modified nucleotides, did not induce these off-target effects even in wildtype mice. LNP01-ISG15 led to
80% suppression in hepatocytes, while no knockdown was detectable in NPCs. Intravenous administration of the interferon-inducing Poly
I:C led to strong induction of all tested genes in NPCs, whereas in hepatocytes only ISGs were up-regulated, responding to endogenous
interferon. Poly I:C injection after ISG15 knockdown with modified siRNA further enhanced this up-regulation of ISGs in the liver. However,
Poly I:C had no additional stimulatory effect in combination with immune-activating LNP01-siRNA.
Conclusions: We demonstrated selective suppression of ISG15 expression in mouse hepatocytes by LNP01-siRNA in vivo. Under this
condition, Poly I:C triggered a very strong activation of other ISGs, while ISG15 levels remained low. This RNA interference-mediated change
from a proviral to a potentially antiviral state may lead to novel therapeutic options for the treatment of HCV patients non-responding to
IFN-based therapies.
Contact: Ruth Broering
University Hospital of Essen, Essen, Germany
[email protected]
O9.12
Robust induction of type III interferons and other cytokines defines a unique pattern of
innate immunity in response to hepatitis C virus infection
Emmanuel Thomas (1), Veronica Gonzalez (1), Qisheng Li (1), Ankit Modi (1), Weiping Chen (1), Mazen Noureddin (1), Yaron Rotman (1), T.
Jake Liang (1)
(1) Liver Diseases Branch/NIDDK/NIH, Bethesda, MD, USA
Human and chimpanzees infected with the hepatitis c virus (HCV) demonstrate evidence of a robust interferon (IFN) response to the virus
as demonstrated by upregulation of IFN-stimulated genes (ISGs) in the liver. Recent demonstration of polymorphisms near the IL-28B gene
(IFN-λ3) associated with spontaneous clearance and treatment response in HCV infected patients implicates a role for type III IFNs (IFN-λs)
in HCV infection. However, the function of type III IFNs in intrinsic innate immunity is poorly understood. HCV infection of primary human
hepatocytes, in addition to upregulating type III IFNs leading to IFN-stimulated gene (ISG) induction, results in a much broader range of
changes in gene expression. Type III IFN, in addition to upregulating ISGs with different kinetics, induces a distinct set of genes from type
I IFN, potentially explaining the functional difference between the two types of IFNs. Treatment of primary human hepatocytes (PHHs)
and hepatoma cell lines with poly(I:C) or an HCV pathogen-associated molecular pattern (PAMP) resulted in a robust induction of IL28 and
IP-10 that is mediated by the IPS-1/IRF3 whereas IFN-α or -β was only marginally induced. PHHs infected with HCV showed a significant
and rapid upregulation of IL28 and other cytokines including IP-10. Chimpanzees undergoing experimental HCV infection demonstrated a
prompt hepatic induction of IL28 but not type I IFNs, associating with ISG upregulation. Analysis of liver biopsies from HCV-infected patients
supported a close correlation among hepatic expression of IL28, ISGs, and treatment response. Our study shows that the type III IFN system
is activated predominantly in hepatocytes by HCV infection and plays a crucial role in host response to this virus.
Contact: Emmanuel Thomas
Liver Diseases Branch/NIDDK/NIH, Bethesda, MD, USA
[email protected]
70 : HCV 2011
ORAL ABSTRACTS
O9.13
Systems biology & computational network analysis identifies mitochondrial host
targets of HCV infection and liver disease progression
Systems biology has received a great deal of attention in the field of infectious disease research owing to the potential to provide a greater
understanding of the pathogen-host interactions that control infection phenotype and disease outcome. A key aspect of the approach is
the use of computational methods to collectively integrate high throughput “omics” and traditional (e.g. virological, histopathological)
data into a single systems-level view that allows the identification of functional processes involved in pathogen-associated disease and the
further illumination of host “targets” representing key points of control by pathogens. We previously described computational network
analyses of cell culture data (e.g. proteomics and lipidomics) to identify the mitochondrial fatty acid oxidation enzyme DCI as a key target
in HCV-associated metabolic reprogramming and, confirmed a requirement for DCI in the HCV life cycle by a series of in vitro perturbations
including gene silencing and pharmacologic inhibition. Here we describe comparative network-based analysis of proteomics data from
HCV infection experiments in cell culture with similar networks generated from liver biopsy samples to identify common targets that have
potential translational impact. We now demonstrate that DCI and several other proteins that localize to the mitochondria are common HCV
targets both in vitro and in vivo. These proteins are predicted to provide multiple control points for modulating metabolic reprogramming by
HCV, implicating a variety of mechanisms involving regulation of protein folding, mitochondrial morphology, and enzyme catalytic activity.
Significantly, clinical protein profiling studies of serial liver biopsies obtained from an independent cohort of HCV+ liver transplant patients
further revealed that a subset of these target proteins are differentially regulated prior to histologic evidence of significant liver injury. These
findings provide new insights into a potentially important, and diagnostically/therapeutically relevant, role for these target proteins in HCV
infection and liver disease progression.
Contact: Deborah L. Diamond
[email protected]
ORAL ABSTRACTS
Deborah L. Diamond (1), Jason E. McDermott (2), Alexei Krasnoselsky (1), Kristin E. Burnum (2), Nathan Susnow (1), Bobbie-Jo WebbRobertson (2), Courtney Courley (2), Matthew M. Yeh (1), Angela L. Rasmussen (1), Jon M. Jacobs (2), Renuka Bhattacharya (1), James D.
Perkins (1), Robert L. Carithers Jr. (1), Marina A. Gritsenko (2), Iris W. Liou (1), Susan Strom (1), Katrina M. Waters (2), Richard D. Smith (2),
Michael G. Katze (1)
(2) Pacific Northwest National Laboratory, USA
ORAL ABSTRACTS
O9.14
Overall structural model of non-structural protein 5A from hepatitis C virus and
modulation by cyclophilin A
Aurelie Badillo (1), Veronique Receveur-Brechot (2), Simona Miron (3), Xavier Hanoulle (4), Jennifer Molle (1), Roland Montserret (1),
Ralf Bartenschlager (5), Guy Lippens (4), Sylvie Ricard-Blum (1), Francois Penin (1)
(1) IBCP, UMR 5086 CNRS, University of Lyon, Lyon, France
(2) NRS / IBSM/ IMR Laboratory, Marseille, France
(3) INSERM U759, Institut Curie Centre de Recherche, Orsay., France
(4) UMR 8576 CNRS, University of Lille, France
(5) Department of Infectious Diseases, University of Heidelberg, Germany
ORAL ABSTRACTS
NS5A is a membrane-associated phosphoprotein exhibiting RNA binding features, and current evidences indicate that it likely functions as
a molecular switch between replication and assembly. NS5A is reported to have numerous cellular interactants (>100) and to be involved
in the resistance to Cyclosporin A (CsA), which inhibits HCV replication by inhibiting cyclophilin A (CypA). NS5A is anchored to membrane
via an amphipathic N-terminal alpha-helix and is composed of three domains denoted D1, D2 and D3. While D1 domain is well-structured,
our recent structural studies indicate that D2 and D3 are intrinsically unfolded and are substrates for the peptidyl-prolyl cis/trans isomerase
activity of cyclophilin A. Our structural analyses of NS5A domains by Small Angle X-ray scattering (SAXS) demonstrated that D2, D3 and D2D3
are intrinsically unfolded and behave as random coil polypeptides. These conformational analyses do not support the subdivision of D2D3
in 2 structural domains. These domains should be seen as a mixture of conformers in fast interconversion, but exhibiting some propensity
for alpha helix folding, which could take place upon interaction with host or viral partners. We propose an overall structural model of NS5A
where interactions of cyclophilin A with D2D3 have implications for the functional architecture of NS5A.
To measure the interactions of NS5A D2 and D3 domains with viral and host cell factors including cyclophilin A, reliable experimental
conditions have been set up by Isothermal titration microcalorimetry (ITC) and Surface Plasmon Resonance (SPR). ITC and SPR analyses of
NS5A D2 and D3 with CypA revealed these interactions with apparent affinities in the micromolar range. The study of D2 resistance mutants
by these approaches give essential interaction parameters to understand the role of CypA in NS5A structural and functional features as well
as the mechanism of resistance to CypA inhibitors.
Contact: Aurelie Badillo
IBCP, UMR 5086 CNRS, University of Lyon, Lyon, France
[email protected]
O9.15
Hepatitis C virus infection induces autophagy and blocks innate immune response
Shubham Shrivastava (1), Amit Raychoudhuri (1), Robert Steele (1), Ranjit Ray (1), Ratna Ray (1)
(1) Saint Louis University, St. Louis, MO, USA
We have previously reported that hepatitis C virus (HCV) infection induces autophagic vacuole formation in immortalized human
hepatocytes (IHH). Infected hepatocytes displayed localization of autophagic markers, such as LC3 and ATG5. We have recently observed
that knockdown of autophagy-related proteins, Beclin 1 (BCN1) or ATG7, in IHH inhibited HCV growth. Subsequent studies demonstrated
that HCV infection in autophagy impaired IHH displays caspase activation, poly (ADP-ribose) polymerase (PARP) cleavage, and apoptotic cell
death. We asked whether autophagy plays a role in modulation of innate immune response against HCV infection. For this, we examined
the modulation of the interferon (IFN) signaling pathway in autophagy impaired IHH. BCN1 or ATG7 knockdown IHH, when infected with
HCV, exhibited an increase expression of several IFN signaling molecules (interferon-β, 2’, 5’-oligoadenylate synthetase 1, interferon-α, and
interferon-α-inducible protein 27 messenger RNAs) as compared to virus infected control IHH. Our results indicated that the disruption of
the autophagy machinery in HCV infected hepatocytes activates IFN signaling pathway. Further in depth mechanisms of IFN induction in
autophagy impaired HCV infected cells will be discussed.
Contact: Shubham Shrivastava
Saint Louis University, St. Louis, MO, USA
[email protected]
72 : HCV 2011
ORAL ABSTRACTS
O9.16
Differential type I interferon-mediated autophagic trafficking of hepatitis C virus
proteins in mouse liver
HCV NS3/4A serine protease can cleave mitochondria-associated anti-viral signaling protein (MAVS) and block retinoic acid-inducible gene
I (RIG-I)-mediated IFN responses. While this mechanism is thought to play an important role in HCV-mediated innate immunosuppression,
its significance in viral persistence is not entirely clear. We generated a novel transgenic mouse with liver-specific expression of the HCV
NS3/4A proteins and challenged the animals with a recombinant VSV, synthetic HCV genome, IFN-a and IFN-b Then, we evaluated the effect
of HCV serine protease on the innate immune responses and the interaction between the two. Although HCV NS3/4A expression resulted
in efficient cleavage of intrahepatic MAVS, upon challenge with vesicular stomatitis virus or synthetic HCV genome, the transgenic mice
mounted robust IFN responses, which were not significantly lower than those in control animals. Additional experiments showed that
various challenge agents induced IFN-a and -b at different ratios, resulting in differential autophagic responses and vesicular trafficking
patterns of ER- and mitochondria-associated viral proteins. IFN-b promoted autolysosomal degradation of the viral proteins. Furthermore,
the presence of variant isoforms of MAVS in the disparate type I IFN-mediated autophagic responses suggests its possible involvement in
the trafficking of viral components to the TLR3-containing endosomal compartments. Our findings bring to light a distinctive IFN-b-mediated
mechanism of antiviral host defense and imply a role of MAVS in the IFN-induced autophagic trafficking of viral proteins.
Contact: Jiaren Sun
University of Texas Medical Branch, Galveston, TX, USA
[email protected]
ORAL ABSTRACTS
Mayura Desai (1), Bin Gong (1), Tehsheng Chan (1), Jiaren Sun (1)
(1) University of Texas Medical Branch, Galveston, TX, USA
ORAL ABSTRACTS
74 : HCV 2011
ORAL ABSTRACTS
Viral Kinetics and Drug Resistance
O10.01
Project ECHO: outcomes of hepatitis C treatment by primary care providers
O10.02
Molecular characterization of HCV resistance to telaprevir by means of
ultra-deep pyrosequencing
Stephane Chevaliez, Christophe Rodriguez, Alexandre Soulier, Abdelhakim Ahmed-Belkacem, Christophe Hezode,
O10.03
New insights into the mechanism of action of interferon-alpha and
BMS-790052: a multi-scale mathematical modeling approach
Jeremie Guedj, Harel Dahari, Libin Rong, Richard Nettles, Scott Cotler, Thomas Layden, Alan Perelson
O10.04
HCV persistence after sustained virological response to antiviral therapy in
patients with or without past exposure to hepatitis B virus
Tram N. Q. Pham, Carla S. Coffin, Norma D. Churchill, Stefan J. Urbanski, Samuel S. Lee, Thomas I. Michalak
O10.05
Long-term follow-up of chronic HCV patients treated with telaprevir:
evaluation of persistence of resistant variants by ultra-deep sequencing
Xiomara Thomas, Joep de Bruijne, Tara Kieffer, James Sullivan, Sjoerd Rebers, Michel de Vries, Henk Reesink,
Christine Weegink, Janke Schinkel, Richard Molenkamp
O10.06
Rob Striker, Israr Ansari, Todd Allen, Andrew Berical
ORAL ABSTRACTS
Sanjeev Arora, Karla Thornton, Summers Kalishman, Paulina Deming, Wesley Pak, John Brown, Glen Murata
ORAL ABSTRACTS
76 : HCV 2011
VIRAL KINETICS AND DRUG RESISTANCE
O10.01
Project ECHO: outcomes of hepatitis C treatment by primary care providers
Background: The Extension for Community Healthcare Outcomes (ECHO) model was developed to improve access to best practice care for
complex health problems such as hepatitis C virus (HCV) infection for underserved populations and minorities in rural areas and prisons.
Using videoconferencing technology, best practice protocols, and case based learning, ECHO trains and supports primary care providers to
develop knowledge and self-efficacy to deliver appropriate care for patients with complex diseases.
Methods: A prospective cohort study compared the efficacy of treatment of HCV at the University of New Mexico (UNM) HCV clinic to
treatment by primary care clinicians at 21 ECHO sites in New Mexico. A total of 407 treatment naive patients with chronic HCV were
enrolled. The primary end point was a sustained viral response (SVR) defined as undetectable virus 24 weeks after the end of therapy.
Results: The rate of SVR for UNM and ECHO sites was 57.5% (84 of 146) and 58.2% (152 of 261 patients), respectively. In genotype 1 infection
the SVR rate was 45.8% (38 of 83) at UNM and 49.7% (73 of 147) at ECHO (P=0.572). More ECHO patients were minorities, 67.8% (166 of
261) versus 49.3% (72 of 146) at UNM (P=0.001).
Conclusions: This study demonstrates that the ECHO model is an effective way to treat HCV in rural and underserved communities and
expands access to treatment for minorities. By implementing this model other states and nations can treat many more patients with HCV,
thereby preventing an enormous burden of illness and death.
Contact: Karla Thornton
University of New Mexico Health Sciences Center, Albuquerque, NM, USA
[email protected]
O10.02
Molecular characterization of HCV resistance to telaprevir by means of ultra-deep
pyrosequencing
Stephane Chevaliez (1), Christophe Rodriguez (1), Alexandre Soulier (1), Abdelhakim Ahmed-Belkacem (1), Christophe Hezode (1), JeanMichel Pawlotsky (1)
(1) INSERM U955, Hopital Henri Mondor, Universite Paris-Est, Creteil, France
Telaprevir is a potent inhibitor of HCV NS3 protease that will soon be approved in combination with pegylated interferon (IFN) and ribavirin
for the treatment of chronic genotype 1 hepatitis C. Telaprevir resistance plays an important role in treatment failures. We characterized the
dynamics of telaprevir-resistant variants during and after treatment with telaprevir and pegylated IFN with or without ribavirin in 18 patients
from the Phase II PROVE2 trial. For this, we used ultra-deep pyrosequencing (UDPS) by means of Titanium XL Read on a 454 device (Roche),
followed by analysis with our “in-house“ softwares PyroClass©, PyroMute©, PyroDyn© and PyroLink©. We demonstrated the preexistence
at baseline of HCV variants known to confer resistance to telaprevir in all patients, with the most frequently preexisting substitutions including
R155K/T/Q, A156S/T/V, D168A/V/T/H and I170A/T. Analysis of serial samples taken at baseline, during therapy and after its withdrawal showed
complex patterns of enrichment in telaprevir resistant viral populations during treatment administration. PyroLink© was used to establish
linkages between amino acid substitutions. The analysis showed stepwise increases in the number of amino acid substitutions present in
the dominant telaprevir-resistant populations, including substitutions not known to confer telaprevir resistance that were likely involved in
resistant variant fitness. A slow decline of telaprevir resistant populations started soon after telaprevir withdrawal and resulted in a return to
a dominant wild-type pattern in most cases. In conclusion, using UDPS and a set of original in-house software’s, we showed that: 1/ telaprevir
resistant variants preexist at detectable levels in virtually all treatment-naïve patients infected with HCV; 2/ the dynamics of telaprevir-resistant
variants are complex, characterized by stepwise enrichments in resistant viral populations during treatment, followed by an immediate but
slow decrease after telaprevir cessation that leads to return to a dominant wild-type pattern.
[email protected]
ORAL ABSTRACTS
Sanjeev Arora (1), Karla Thornton (1), Summers Kalishman (1), Paulina Deming (1), Wesley Pak (1), John Brown (1), Glen Murata (1)
(1) University of New Mexico Health Sciences Center, Albuquerque, NM, USA
ORAL ABSTRACTS
O10.03
New insights into the mechanism of action of interferon-alpha and BMS-790052: a
multi-scale mathematical modeling approach
Jeremie Guedj (1), Harel Dahari (1), Libin Rong (2), Richard Nettles (3), Scott Cotler (4), Thomas Layden (4), Alan Perelson (1)
(1) Los Alamos National Laboratory, Santa Fe, NM, USA
(2) Oakland University, Rochester, USA
(3) Bristol-Myers Squibb Research and Development, Princeton, New Jersey, USA
(4) Department of Medicine, University of Illinois at Chicago, USA
ORAL ABSTRACTS
Background: Using the standard HCV kinetics model (Science 1998 2;282(5386):103-7) we showed that HCV half life, t1/2, is shortened
during therapy with BMS-790052 (t1/2=0.7h), a NS5A inhibitor, compared to IFN therapy (t1/2=2.7h) (Hepatology 2010, 52S:718A-719A).
Since virion clearance is a physiological process, it should not depend on the treatment regimen and this striking discrepancy hints that
the determinants of HCV decline under treatment may not be fully understood. Here we sought to reconcile these contradictory findings
by using a new multi-scale model that includes intracellular HCV dynamics. Methods: Frequent HCV RNA data under IFN or BMS-790052
were obtained from the University of Illinois at Chicago and Bristol-Myers-Squibb, respectively. The model includes intracellular viral RNA
(vRNA) synthesis/production and its clearance due to degradation and virion secretion. Unlike the standard model, this model distinguishes
between drug effectiveness that slows vRNA production/synthesis (εp) and virion secretion (εs). Results: Best agreement between model
and data was achieved assuming a short t1/2=0.74h (CI95%[0.65;0.86]). While both high daily doses of IFN (10 or 15 MIU) and one dose
of BMS-790052 (10 or 100 mg) effectively blocked vRNA production, εp=0.97 ([0.92;1.0]) and εp=0.994 ([0.996;1.0]), respectively, IFN had
a considerably lower effect on virion secretion (εs=0.37 [0.09;0.65]) compared to BMS-790052 (εs=0.998 [0.996;1.00]). Conclusions: Our
results suggest that IFN mainly slows vRNA production rather than virion release, so that during the first phase of viral decline under IFN
therapy there is continued release of preformed virions, which masks the intrinsic plasma HCV clearance rate. This novel understanding of
HCV kinetics allows us to estimate that HCV clearance, and hence, daily virion production, may have been more than 4-fold underestimated
previously. Further, the high antiviral potency of BMS-790052 may be due to a capacity to block two distinct stages of viral replication, the
production of vRNA and virion assembly/secretion.
Contact: Jeremie Guedj
Los Alamos National Laboratory, Santa Fe, NM, USA
[email protected]
O10.04
HCV persistence after sustained virological response to antiviral therapy in patients
with or without past exposure to hepatitis B virus
Tram N. Q. Pham (2), Carla S. Coffin (1), Norma D. Churchill (2), Stefan J. Urbanski (1), Samuel S. Lee (1), Thomas I. Michalak (2)
(1) University of Calgary, Canada
(2) Memorial University, St. John’s, NL, St. John’s, Canada
Hepatitis C virus (HCV) and hepatitis B virus (HBV) frequently co-infect and persist long after resolution of symptomatic infection. We
assessed the incidence of low-level (occult) HCV infection (OCI) after sustained virological response (SVR), as evaluated by clinical laboratory
assays, to standard anti-HCV therapy with pegylated IFN alpha and ribavirin in individuals with or without past exposure to HBV. Our aim
was to determine whether prior HBV exposure alters OCI incidence, level of HCV persistence and hepatic histology. Up to three matched
pairs of plasma and peripheral blood mononuclear cells (PBMC) were collected from 24 individuals at maximum of 72 months after SVR.
Liver histology was available for 9/24 patients before and after therapy. HCV and HBV genomes were detected by nucleic acid amplification
assays with sensitivity <10 genome copies/mL. In individuals without HBV exposure (n=15), sequential samples, including RNA isolated from
up to 4 mL of plasma and from ex vivo stimulated PBMC, revealed HCV in all cases (75% plasma and 61% PBMC samples). In the group with
HBV exposure (n=9), evidenced by circulating anti-HBc and/or HBV DNA detection, HCV RNA were identified in all cases (83% plasma and
59% PBMC samples) at comparable levels to those in HBV non-exposed individuals. Liver biopsies acquired up to 5 years after SVR (n=10)
showed histological improvement, nonetheless the majority displayed a low grade inflammation (9/10) and different stages of fibrosis
(7/10). Clonal sequencing revealed polymorphisms in PBMC-derived HCV variants. In 3 individuals despite achieving a SVR, circulating HCV
was detected by immune electronmicroscopy with anti-HCV E2 antibodies. In conclusion, the prevalence of low-level HCV infection after
SVR is comparable in individuals either with or without past exposure to HBV. HCV loads and histological liver alterations in this form of HCV
persistence appear to be unaffected by low-level HBV infection.
Contact: Thomas I. Michalak
Memorial University, St. John’s, NL, St. John’s, Canada
[email protected]
78 : HCV 2011
O10.05
Long-term follow-up of chronic HCV patients treated with telaprevir: evaluation of
persistence of resistant variants by ultra-deep sequencing
Telaprevir, a NS3/4A protease inhibitor, in combination with peginterferon alfa-2a (Peg-IFN) and ribavirin (RBV), has demonstrated significant
improvement in sustained viral response (SVR) rates compared with Peg-IFN/RBV alone in HCV genotype 1 infected patients. In some
patients who do not achieve an SVR, drug-resistant variants have been reported. A better understanding of the persistence of these variants
is needed. The aim of this study was to assess the prevalence of variants in patients before a short-term Phase I monotherapy assessment
of telaprevir and at long term follow-up by using sensitive ultra-deep pyrosequencing (UDPS).
Fourteen patients were recruited from 2 phase 1 clinical studies (VX101 and 103) of telaprevir. In these trials patients received either
telaprevir monotherapy for 14 days or combination therapy with Peg-IFN. Previously well-described resistant variants at NS3 protease
positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by UDPS. Resistant variants were tabulated
at each time point and compared statistically between time points at each position.
Median number of sequence reads per position ranged from 4677-13326, resulting in a limit of detection of resistant variants of <0.1%.
Prevalence of variants associated with resistance to telaprevir at baseline was found to be very low. The highest baseline prevalence was
observed in a patient carrying a T54A variant (0.54%). No resistant variants were detected in 9 of 14 patients at baseline compared to 8
of 14 at long-term follow-up. In 13 of 14 patients, there was no indication of significant enrichment of resistant variants at the long-term
follow-up time point relative to baseline, despite the presence of resistant variants in the clinical trial in all patients. The remaining patient
had both V36M and T54S significantly enriched at long-term follow-up (4.5% and 0.35%, respectively) relative to baseline, where neither
variant was observed.
Contact: Richard Molenkamp
AMC, Amsterdam, Netherlands
[email protected]
O10.06
Rob Striker (1), Israr Ansari (2), Todd Allen (3), Andrew Berical (3)
(1) Veterans Administration, Madison, WI, USA
(2) UW-Madison, USA
(3) Massachusetts General Hospital, USA
Hepatitis C virus (HCV) is a leading cause of hepatocellular carcinoma and severe liver disease. For patients unresponsive to antiviral
therapy with severe liver disease, liver transplant is the only choice. After liver transplant some patients receive Cyclosporine (CsA) as an
immunosuppressant which has the added theoretical advantage of inhibiting the host protein cyclophilin required for the HCV replicase.
We determined the nucleotide sequences of NS5A and NS5B in a cohort of HIV/HCV infected transplant patients where CsA was used in
post-transplant cases to determine if CsA selected specific viral variants. The patient’s samples were sequenced before transplant and on
CsA treatment and nucleotide sequences were compared with reference strain H77. A total of three unique mutations were identified in a
patient post-transplant serum associated with breakthrough viremia on CsA compared to the pretransplant viral isolate. The pretransplant
isolate displayed high CsA susceptibility when tested in HCV 1b/1a chimeric replicon based transient replication assay. Further analysis
revealed a proline at codon 328 accounted for the atypical CsA susceptibility of this 1a isolate pretransplant. In addition, CsA susceptibility
was reduced when proline 328 was mutated to the serine that was posttransplant. This particular codon also increases CsA susceptibility
when proline 328 was tested in the context of HCV 1b replicon. Seven naturally occurring variants present at that particular location were
tested in context of HCV 1b/1a chimeric replicon and CsA susceptibility was highest with proline, intermediate with other hydrophobic
residues, while hydrophilic/charged residues at that position were CsA resistant in cell culture. In conclusion, variation in residue 328 of
NS5A is associated with CsA susceptibility/resistance in cell culture and in vivo.
Contact: Rob Striker
Veterans Administration, Madison, WI, USA
[email protected]
ORAL ABSTRACTS
Xiomara Thomas (2), Joep de Bruijne (2), Tara Kieffer (1), James Sullivan (1), Sjoerd Rebers (2), Michel de Vries (2), Henk Reesink (2),
Christine Weegink (2), Janke Schinkel (2), Richard Molenkamp (2)
(1) Vertex Pharmaceuticals Inc., USA
(2) AMC, Amsterdam, Netherlands
80 : HCV 2011
POSTERS
Antiviral Therapy
P1.01
The mode of action of a small peptide inhibitor of internal ribosome entry
site (IRES) for the hepatitis C virus propagation in human hepatoma cells
Santanu Raychaudhuri, Asim Dasgupta
P1.02
HCV envelope proteins E1 and E2 each contribute to susceptibility and
resistance to a small molecule viral entry inhibitor
Kevin Pokornowski, Ronald Rose, Guo Li, Zhiwei Yin, Annapurna Pendri, Tao Wang, Daniel Tenney, Stephen Mason,
Paul Scola, Nicholas Meanwell, Carl Baldick
P1.03
Small molecules that elicit strong anti-HCV activity through downmodulation of HCV entry receptors
Yutaka Takebe, Rie Uenishi, Hideki Tani, Ryosuke Suzuki, Motoki Takagi, Saiki Hase, Huanan Liao, Takayo Tsuchiura,
Kazuo Shinya, Takaji Wakita, Yoshiharu Matsuura, Arvind Patel
P1.04
The Efficacy of an NS4B Inhibitor in HCV Infected PXB-Mice
P1.05
Plural assay systems derived from different cell lines and HCV strains are
required for the objective evaluation of anti-HCV reagents
Youki Ueda, Kyoko Mori, Yasuo Ariumi, Masanori Ikeda, Nobuyuki Kato
P1.06
Nitazoxanide is an indirect inhibitor of HCV replication through modulation
of HCV NS5A hyperphosphorylation by up-regulation of casein kinase I alpha
Abigail Montero, Prasanth Viswanathan, Changsuek Yon, Brent Korba
POSTERS
Robert Hamatake, Jeff Pouliot, Mike Thomson, Mi Xie, John Johnson, Andy Peat, Chris Roberts, Amanda Mathis,
Ping Xiong, Rich Peterson, Yoshio Morikawa, Takashi Shimada, Zhi Hong, Andrew Spaltenstein
POSTERS
P1.07
MK-5172, a novel macrocyclic inhibitor of NS3/4a protease, exhibits efficacy
in in vitro long-term potency and combination studies
Stacia Kargman, Genevieve Lavallee, Christine Brideau, Helen Chan, Martine Hamel, Qian Huang, Steven Ludmerer,
Mark Stahlhut, Rino Stocco, Joseph Vacca, David Olsen
P1.08
Affinity maturation to improve a human monoclonal antibody
neutralization potency and breadth of reactivity against hepatitis C virus
Yong Wang, Anasuya Saha, Jinming Xia, Zhenyong Keck, Jianlong Lou, James D. Marks, Steven Foung
P1.09
Ribosome assembly on the HCV RNA: a novel target for developing antiviral
therapeutics
Prasanna Bhat, Sivakumar Vadivel Gnanasundram, Prashant Mani, Debi Prasad Sarkar, Saumitra Das
P1.10
Discovery of a novel series of 1,3,5-triazine HCV-entry inhibitors that block
virus spread and promote virus clearance in vitro
Jean-Marc de Muys, Glen Coburn, Danielle Fisch, Sameer Moorji, Jose Murga, Dorothy Paul, Yakov Rotshteyn, Amy Han,
Dapeng Qian, Paul Maddon, William Olson
P1.11
Naïve resistance mutations of protease (NS3) and polymerase (NS5B)
inhibitors in HCV genotypes sequences from Los Alamos databank
Rafael S. Alves, Artur T.L. Queiroz, Edvaldo F. da Silva, Flair J. Carrilho, Mário G. Pessoa, Isabel M. V. G. de Carvalho Mello
P1.12
Isolation of potent hepatitis C virus helicase inhibitors from the yellow dyes
thioflavine S and primulin
Craig A. Belon, Kelin Li, Kevin J. Frankowski, Ben Neuenswander, Jean Ndjoumou, Alicia Hanson, Frank Schoenen,
Brian S. J. Blagg, Jeffrey Aube, David N. Frick
P1.13
Effects of fluoroquinolones on the helicase activity of HCV NS3 protein
Irfan Khan, Sadiq Rehmani, Sammer Siddiqui, Shahana Kazmi, Syed Ali
POSTERS
P1.14
Bi-specific high affinity TCR antiCD3 fusions for redirected killing of HCV
infected cells
Annelise Vuidepot, Nikolai Lissin, Peter Molloy, Conor Hayes, Linda Hibbert, Bent Jakobsen
P1.15
Sorafenib impairs the antiviral effect of interferon alpha on hepatitis C virus
replication
Kiyoshi Himmelsbach, Eberhard Hildt
P1.16
Characterization of NS5A resistance for HCV replicon treated by
BMS-790052
Chunfu Wang, Haichang Huang, Lourdes Valera, Xin Huang, Aneela Altaf, Jin-Hua Sun, Donald O’Boyle, Peter Nower,
Min Gao, Robert Fridell
P1.17
In vitro comparative analysis between IFN-lambda and IFN-alpha during
combination treatment with direct-acting antivirals targeting hepatitis C virus
Jacques Friborg, Steven Levine, Chaoqun Chen, Amy Sheaffer, Susan Chaniewski, Julie Lemm, Stacey Voss, Min Gao,
Fiona McPhee
82 : HCV 2011
ANTIVIRAL THERAPY
P1.18
Hepatitis C viral kinetics with the nucleoside polymerase inhibitor RG7128
Jeremie Guedj, Harel Dahari, Emi Shudo, Patrick Smith, Alan Perelson
P1.19
The combination of proteasome inhibitors and interferons - a new and
promising approach for treatment of HCV infection
Artur Kaul, Daniel Lüftenegger, Danijela Cvijetic, Eva Godehardt, Jana Pforr, Vadim Bichko, Ralf Kircheis
P1.20
Phenotypic characterization of HCV NS3 protease from patient isolates in
phase 2 clinical trials of telaprevir
Min Jiang, Leen Vijgen, Jim Sullivan, Michelle Nelson, Judith Lippke, John Fulghum, Kenneth Bonanno, Kumkum Saxena,
Azin Nezami, William Taylor, Joyce Coll, Yu-Ping Luong, Sandra De Meyer, Inge Dierynck, Gaston Picchio, Tara Kieffer
P1.21
The Hsp90 inhibitor 17-AAG inhibits hepatitis C viral replication through
cytostasis
Rudolf Beran, Ruchi Sharma, Amoreena Corsa, Yang Tian, Justin Golde, Greta Lundgaard, William Delaney IV,
Weidong Zhong, Andrew Greenstein
P1.22
An improved amino acid covariance network approach to evaluate
sensitivity of HCV isolates to interferon-based therapy
Rajeev Aurora, Maureen Donlin, John Tavis
P1.23
Discovery of naturally occurring aurones that are potent allosteric inhibitors
of hepatitis C virus RNA-dependent RNA polymerase
Abdelhakim Ahmed-Belkacem, Romain Haudecoeur, Wei Yi, Antoine Fortune, Rozenn Brillet, Catherine Belle,
Edwige Nicolle, Coralie Pallier, Jean-Michel Pawlotsky, Ahcene Boumendjel
P1.24
Antiviral activity against hepatitis C virus by iCo-007, an antisense inhibitor
of expression of the host protein c-Raf (Raf-1) kinase
P1.25
Susceptibility of treatment-naïve clinical isolates to HCV polymerase inhibitors
Hadas Dvory-Sobol, Ross Martin, Siu-Chi Sun, Michael D. Miller, Hongmei Mo
P1.26
Ribavirin and interferon-alpha synergistically inhibits HCV replication in
cells with a functional Jak-STAT pathway
Srikanta Dash, Sruti Chandra, Sidhartha Hazari, Partha Chandra, Feiza Gunduz, Luis Balart
P1.27
A multiplexed cell-based platform to monitor the activity of Flaviviridae
proteases
Jason Machula, Ryan O’Hanlon, Plamena Silvieva, Roland Wolkowicz
P1.28
Analysis of IL28B rs8099917 and hepatic Stat1 activation HCV-positive
patients treated with pegylated-interferon plus ribavirin
Tatsuo Miyamura, Tatsuo Kanda, Shingo Nakamoto, Shuang Wu, Fumio Imazeki, Osamu Yokosuka
P1.29
E2 binding peptide identified by RAPID system inhibited HCV infection
Noriyuki Watanabe, Kazuki Futai, Hiroaki Suga, Takaji Wakita
POSTERS
Rineke Steenbergen, John Clement, D. Lorne Tyrrell
POSTERS
P1.30
Development of chimeric hepatitis C virus expressing NS5A from strains of
genotypes 1 and 2: virus production and susceptibility to NS5A inhibitor
Yuka Okamoto, Takahiro Masaki, Asako Murayama, Takaji Wakita, Takanobu Kato
P1.31
Phenotypic live cell screening with hepatitis C reporter viruses and
reporter cells
Hee Young Kim, Soo Hyun Kim, Myung Eun Lee, Michael A.E. Hansen, Christopher T. Jones, Charles M. Rice,
Marc P. Windisch
P1.32
Development of an automated tissue culture infectious dose 50
determination assay in 384-well plates
Myung Eun Lee, Michael A.E. Hansen, Soo Hyun Kim, Thierry Dorval, Hee Young Kim, Keumhyun Kim, Tae Kyu Kim,
Marc P. Windisch
P1.33
Discovery of the clinical candidate TMC055, a novel non-nucleoside
inhibitor of the hepatitis C virus NS5b polymerase
Sandrine Vendeville, Tse-I Lin, Lili Hu, Abdellah Tahri, David McGowan, Maxwell Cummings, Katie Amssoms, Stefaan Last,
Maxime Canard, Iris Van den Steen, Benoit Devogelaere, Marie-Claude Rouan, Leen Vijgen, Jan Martin Berke,
Gregory Fanning, Kristof Van Emelen, Kenny Simmen, Origene Nyanguile, Pierre Raboisson
P1.34
Genotype 5a response to combination therapy in a South African study
Nishi Prabdial-Sing, Deepak Ramauthar, Monalisa Kalimashe, Mark Goosen, Deidre Greyling, Ernest Song,
Adam Mohamed, Trevlyn Burger, Schalk Van der Merwe, A. Elnagar, F. Bassa, N. Rapiti, R. Ally, Wamda Abuelhassan,
Nazeer Chopdat, J. Van Zyl, V. G. Naidoo, K. Newton, K. Govender, B. Luke, D. Mushi, J. Zaldivar, A. R. J. Carrim,
Adrian J. Puren
P1.35
Using data mining techniques to explore baseline predictors of response to
peg-interferon ribavirin in 3720 chronic HCV Egyptian patients
Hadeel Gamal El-Deer, A. Awad, I. Farag, A. Kamal, W. El-Akel, W. Doss, N. Zayed, T. Awad, A. Radwan, M. Mabrouk
P1.36
POSTERS
Synergistic effect of calcitriol and interferon-α on hepatitis C virus
replication in vitro
Christian Lange, Kenichi Morikawa, Michael Dill, Markus Heim, Jerome Gouttenoire, Darius Moradpour
P1.37
The antimalarial drug Artemisinin and analogues inhibit in vitro HCV
replication
Susan Obeid, Jo Alen, Christophe Pannecouque, Wim Dehaen, Johan Neyts, Jan Paeshuyse
P1.38
Identification of new class inhibitors from a high-throughput screening
against hepatitis C virus
Shihyun You, Richard Hazen, Cindy Richards, Eric Burroughs, Laurie Kane-Carson, Laurie Overton, Brandy Pollard,
Shane Holmes, Sara Grab, Jesse Keicher, Robert Hamatake
P1.39
The picornavirus inhibitor enviroxime inhibits HCV RNA replication in vitro
Leen Delang, Pieter Leyssen, Johan Neyts
P1.40
HCV targets the JAK/STAT pathway for degradation to reduce anti-viral
responses to IFN-α
Nigel Stevenson, Nollaig Bourke, Catherine Keogh, Marco Binder, Elizabeth Ryan, Sinead Keating, Aideen Collins,
Andrew Bowie, James Johnston, John Hegarty, Cliona O’Farrelly
84 : HCV 2011
ANTIVIRAL THERAPY
P1.41
Quasispecies variability in NS5A region of Hepatitis C virus genotype 1 and
therapy response
Ana Carolina Jardim, Cíntia Bittar, Renata Matos, Paola Provazzi, Lílian Yamasaki, Rafael Silva, João Renato Pinho,
Claudia Márcia Carareto, Isabel Maria Carvalho-Mello, Paula Rahal
P1.42
The presence of hepatitis C virus deletion mutants in chronic hepatitis C
patients and their role during PEG-IFN/Ribavirin therapy in HCV-1 patients
Raffaele de Francesco, Cristina Cheroni, Lorena Donnici, Annalisa Bianco, Suwanna Noppornpanth, Vincenza Valveri,
Massimiliano Pagani, Roberta Soffredini, Gian Maria Prati, Alessio Aghemo, Maria Grazia Rumi, Massimo Colombo,
Petra Neddermann, Sergio Abrignani
P1.43
MicroRNA profiling identifies potential anti-viral targets induced by
interferon alpha in human hepatoma cells expressing hepatitis C virus
Marybeth Daucher, Xiaozhen Zhang, David Armistead, Rodney Russell, Shyamasundaran Kottilil
P1.44
Biliverdin restores type I interferon expression in cells expressing HCV
NS3/4a protease
Zhaowen Zhu, Meleah Mathahs, Warren Schmidt
P1.45
Transplacental transfer of hepatitis B immune globulin in an animal model
Evi Struble, Li Ma, Lilin Zhong, Pei Zhang
P1.46
Identification of hepatitis C virus inhibitors targeting different aspects of
virus life cycle
Xuemei Yu, Susan L. Uprichard
P1.47
Analysis of the inhibition of HCV replication with different RNAi mediators
P1.48
Epigallocatechin gallate inhibits infectivity of HCV by preventing attachment
of virions to cells
Che C. Colpitts, Sandra Ciesek, Eike Steinmann, Luis M. Schang
P1.49
MicroRNA signatures in chronic hepatitis C virus induced insulin resistance
as a potential therapeutic approach
Gokul Das, Chad Creighton, F. Blaine Hollinger
P1.50
Preclinical data of a new class of anti-HCV compounds acting as
conformational modulators and having different MOA than all known
molecules in clinic
Philippe Guedat, Amaya Berecibar, Angelina Bertrand, Luc Batard, Caroline Evain-Freslon, Céline Fernagut,
Mehdi Lahmar, Aurélie Martin, Annick Piessens, Isabelle Vallarche, Majid Mehtali
P1.51
Partial knockdown of cyclophilin A sensitizes HCV replicons to
cyclosporin A inhibition
Margaret Robinson, Katie Chan, Andrew Greenstein, William Delaney IV
POSTERS
Sandra Jovanna Gonzalez-Rojas, Abdullah Ely, Marta Garcia-Valdecasas, Imanol Fernandez-Perez, Elena Carnero,
Patrick Arbuthnot, Puri Fortes
POSTERS
P1.52
Differential reduction of hepatitis C viral load by viral entry and replication
inhibitors in a persistently-infected cell-culture system
Caroline Bush, Matthew Paulson, Weidong Zhong, Rudolf Beran
P1.53
Antiviral responses with nucleotide analogs PSI-7977 and PSI-938 are
independent of IL28B genotype
Melanie Cornpropst, Eric Lawitz, Maribel Rodriguez-Torres, Jill Denning, Desiree Clemons, Lindsay McNair, Lei Fang,
Michelle Berrey, William Symonds
P1.54
Approach for discovery of HCV NS2 protease inhibitors
Guangwei Yang, Yongsen Zhao, Joanne Fabrycki, Dharaben Patel, Akihiro Hashimoto, Godwin Pais, Atul Agarwal,
Avinash Phadke, Milind Deshpande, Mingjun Huang
P1.55
Antiviral and Preclinical Profiles of HCV NS5A Inhibitors IDX380 and IDX719
John Bilello, Cyril Dousson, Massimiliano La Colla, Christopher Chapron, Sanjeev Bhadresa, Michelle Camire, Ilaria Serra,
Joshua Gillum, Lisa Lallos, Joseph McCarville, Xin-Ru Pan-Zhou, Marita Larsson Cohen, David Standring
P1.56
Pegylated-interferon/ribavirin treatment causes an increase in vitamin D
and a decrease in serum calcium
Andrea D. Branch, Catherine Constable, Kian Bichoupan, Peter Benedict, Marie-Louise Vachon, Douglas Dieterich,
Amir Soumekh
P1.57
Characterization of a small molecule with potent virocidal activity against
HCV and HIV
Ana Maria Chamoun, Karuppiah Chockalingam, Rudo Simeon, Philippe Gallay, Zhilei Chen
POSTERS
86 : HCV 2011
ANTIVIRAL THERAPY
P1.01
The mode of action of a small peptide inhibitor of internal ribosome entry site (IRES)
for the hepatitis C virus propagation in human hepatoma cells
Santanu Raychaudhuri (1), Asim Dasgupta (1)
(1) UCLA, Los Angeles, CA, USA
Hepatitis C is the leading cause of hepatocellular carcinoma, liver fibrosis and cirrhosis representing a major public health problem with
greater than 170 million cases worldwide. Currently, there is no approved vaccine available and approximately 80% of individuals infected
by the virus develop chronic disease. We have been involved in developing a new group of next-generation viral inhibitors that target its
essential translation regulatory elements. The HCV IRES-mediated, cap-independent translation requires interaction of host cell transacting
factors along with the canonical initiation proteins that lead to 40S ribosome recruitment in the close proximity of the translation start site.
Some of these host factors are Lupus autoantigen (La), polypyrimidine tract binding protein (PTB) and poly(rC)-binding protein (PCBP2).
We discovered a specific small peptide inhibitor, called LAP, which inhibits drastically the HCV IRES mediated translation without affecting
the cellular cap-dependent protein synthesis. This small peptide proved to be highly non-toxic in the animal and cell-culture systems. In
this abstract, we are going to reveal its mode of action of how this small peptide inhibits the HCV propagation in the cell culture system.
This La-protein derived peptide LAP binds very significantly with the other transacting factors PTB and PCBP2 along with its efficient entry
into the liver cells. We also found out the dominant negative mutation in La (Y23Q, but not Y24Q) as opposed to the wild-type La that
binds with the LAP as strong as PTB and PCBP2. We are able to demonstrate that PTB and PCBP2 are both required for the restoration of
HCV translation inhibited by LAP. Thus LAP is extremely useful in restricting the HCV propagation by specific binding with PTB and PCBP2.
Currently, we are analyzing the mode of interactions in LAP-PTB and LAP-PCBP2 in X-ray crystallography for developing more efficient small
molecule inhibitors in combating HCV.
Contact: Santanu Raychaudhuri
UCLA, Los Angeles, CA, USA
[email protected]
P1.02
Kevin Pokornowski (1), Ronald Rose (1), Guo Li (1), Zhiwei Yin (1), Annapurna Pendri (1), Tao Wang (1), Daniel Tenney (1), Stephen Mason
(1), Paul Scola (1), Nicholas Meanwell (1), Carl Baldick (1)
(1) Bristol-Myers Squibb, Wallingford, CT, USA
Entry of HCV into hepatocytes involves a coordinated series of steps that includes initial attachment, specific binding of virus to cell
surface receptors, endocytosis and fusion of the viral and endosomal membranes. Thus, HCV entry presents a series of potential targets
for therapeutic intervention and small molecule entry inhibitors could complement antivirals targeting viral replication functions that are
currently in development. Attachment, receptor binding and fusion activities are mediated by the HCV envelope glycoproteins E1 and E2.
We have previously reported the use of HCV pseudoparticles (HCVpp) to identify a potent triazine class of HCV entry inhibitor specific for
genotype 1. Resistance selection using cell culture-adapted HCV expressing genotype 1 envelope proteins resulted in the substitutions
W716L or V719G/F within or adjacent to the C-terminal transmembrane domain of E2, implicating this region in the mechanism of inhibition
by these entry inhibitors. However, we observed that although HCVpp containing envelope proteins from genotypes 2-5 are intrinsically
resistant to these inhibitors, they contain residues W and V at E2 positions 716 and 719, respectively, implying that additional amino acids in
E1 or E2 could modulate susceptibility. In order to map these determinants we constructed HCVpp expressing inter- or intragenotypic E1E2
envelope protein chimeras derived from genotype 1a, 1b, or 2a isolates with disparate sensitivities to triazines. Surprisingly, we found that
the C-terminus of E2 from a triazine-resistant isolate (aa 689-746) did not confer resistance when expressed as a chimera with the remianing
sequences from a triazine-sensitive E1 and E2. Additional envelope chimeras allowed us to map a triazine susceptibility determinant to a
central region of E1. Site-directed mutagenesis identified several residues within this region that modulate triazine susceptibility. These
studies support a role for both E1 and E2 in the mechanism of action of this class of HCV entry inhibitor.
Contact: Carl Baldick
Bristol-Myers Squibb, Wallingford, CT, USA
[email protected]
POSTERS
HCV envelope proteins E1 and E2 each contribute to susceptibility and resistance to a
small molecule viral entry inhibitor
POSTERS
P1.03
Small molecules that elicit strong anti-HCV activity through down-modulation of HCV
entry receptors
Yutaka Takebe (1), Rie Uenishi (1), Hideki Tani (2), Ryosuke Suzuki (1), Motoki Takagi (3), Saiki Hase (1), Huanan Liao (1), Takayo Tsuchiura
(1), Kazuo Shinya (3), Takaji Wakita (1), Yoshiharu Matsuura (2), Arvind Patel (4)
(1) National Institute of Infectious Diseases, Tokyo, Japan
(3) National Institute of Advanced Industrial Science and Technology, Japan
(4) MRC Virology Unit, MRC-University of Glasgow Centre for Virus Research, United Kingdom
We screened for antiviral compounds with HCV JFH-1 and HIV-1 NL432-based full-replication assays done in parallel, using an in-house natural
product library of 800 compounds derived from various microbe cultures. We identified a series of small-molecules (6 hit compounds) that
elicited both anti-HCV and anti-HIV-1 activities. Compound Y is one of the representative hits that show strong anti-viral activities with a 50%
effective concentration (EC50) of sub nM (selective index~10,000) in both HCV and HIV-1 full replication assays.
Compound Y elicited anti-HCV activities against J6/JFH-1 and TH (genotype 1b)/JFH-1 chimera, while it did not show any appreciable antiHCV activities in replicon assay. On the other hand, compound Y inhibited the infection of pseudotype vesicular stomatitis virus (VSV)
bearing HCV E1 and E2 glycoproteins (HCVpv) at EC50 of low nM, suggesting that compound Y appears to act at entry. We found that
compound Y downregulates surface CD81 expression by 30-40% of control. This compound could be a new category of HCV inhibitors
targeting HCV entry receptor(s).
Contact: Yutaka Takebe
National Institute of Infectious Diseases, Tokyo, Japan
[email protected]
P1.04
The Efficacy of an NS4B Inhibitor in HCV Infected PXB-Mice
Robert Hamatake (1), Jeff Pouliot (1), Mike Thomson (1), Mi Xie (1), John Johnson (1), Andy Peat (1), Chris Roberts (1), Amanda Mathis (1),
Ping Xiong (1), Rich Peterson (1), Yoshio Morikawa (2), Takashi Shimada (2), Zhi Hong (1), Andrew Spaltenstein (1)
(1) GlaxoSmithKline, Research Triangle Park, NC, USA
(2) PhoenixBio, Japan
POSTERS
HCV NS4B protein is an integral membrane protein required for viral RNA replication. GSK has developed potent inhibitors of HCV replication
that target NS4B as shown by the emergence of resistance in NS4B and by direct binding assays to purified NS4B. Compounds targeting
NS3/4A protease, NS5A, and NS5B polymerase are inhibitory in the HCV replicon system and have shown clinical efficacy in HCV infected
patients. Because NS4B inhibitors have not been validated in vivo, we have undertaken a study with our NS4B inhibitor GSK8853 in PXBmice infected with genotype 1a HCV. GSK8853 has an EC50 of 0.74 nM against the genotype 1a replicon. PXB-mice are uPA/SCID mice with
70-90% of their livers repopulated with human hepatocytes. GSK9574, a prodrug that is rapidly converted to GSK8853 was used because
of its greater oral bioavailability. Oral doses given twice daily for 7 days of 10, 30, and 100 mg/kg GSK9574 were selected for the efficacy
study in PXB-mice infected with HCV. HCV-796 (NS5B inhibitor) at 100 mg/kg and BILN-2061(protease inhibitor) at 10 mg/kg were used as
comparators. GSK9574 was well tolerated at all doses and resulted in rapid and substantial decreases in HCV RNA levels. Mean log viral load
reductions at nadir were 4.15 log for the 10 mg/kg group, 4.22 log for the 30 mg/kg group, and 4.23 log for the 100 mg/kg group compared
to 2.26 log for HCV-796 and 2.41 for BILN-2061. The viral load increased after reaching nadir in most of the animals although one animal in
the 100 mg/kg GSK9574 group had undetectable HCV RNA on days 4 and 7. Sequence analysis of day 7 samples is currently being performed
to identify resistant mutants. These results show that NS4B is an attractive target for small molecule inhibition of HCV replication.
Contact: Robert Hamatake
GlaxoSmithKline, Research Triangle Park, NC, USA
[email protected]
88 : HCV 2011
ANTIVIRAL THERAPY
P1.05
Plural assay systems derived from different cell lines and HCV strains are required for
the objective evaluation of anti-HCV reagents
Youki Ueda (1), Kyoko Mori (1), Yasuo Ariumi (1), Masanori Ikeda (1), Nobuyuki Kato (1)
(1) Okayama University Graduate School, Okayama, Japan
Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. HuH-7 hepatoma-derived cells are
widely used as the only cell-based HCV replication system for HCV research, including drug assays. We have also developed a HuH-7-derived
drug assay system (OR6), in which genome-length HCV-RNA (O strain of genotype 1b) encoding renilla luciferase replicates efficiently.
Recently, we found a different human hepatoma cell line, Li23, that enables robust HCV-RNA replication, and using Li23-derived cells, we
developed an HCV drug assay system (ORL8), which is relevant to the HuH-7-derived OR6 assay system. During the process of evaluating
ORL8 assay system using anti-HCV reagents such as interferons and ribavirin, we noticed that ORL8 assay system was frequently more
sensitive to anti-HCV reagents than the OR6 assay system, and supposed that the anti-HCV reagents reported to date might show different
activities among the different drug assay systems. Using the OR6 and ORL8 assay systems, we tested this assumption regarding 26 anti-HCV
reagents, which other groups had reported as anti-HCV candidates using HuH-7-derived assay systems other than OR6. The results revealed
that more than half of the reagents showed different anti-HCV activities from those in the previous studies, and that anti-HCV activities
evaluated by OR6 and ORL8 assays were also frequently different. In further evaluation using the HuH-7-derived AH1R assay system, which
was developed using the AH1 strain (genotype 1b), several reagents showed different anti-HCV activities in comparison with those evaluated
by OR6 and ORL8 assays. These results suggest that the different activities of anti-HCV reagents are caused by the differences in cell lines
or HCV strains used for the development of assay systems. Therefore, we conclude that plural HCV assay systems developed using different
cell lines or HCV strains are required for the objective evaluation of anti-HCV reagents.
Contact: Youki Ueda
Okayama University Graduate School, Okayama, Japan
[email protected]
P1.06
Nitazoxanide is an indirect inhibitor of HCV replication through modulation of HCV
NS5A hyperphosphorylation by up-regulation of casein kinase I alpha
Nitazoxanide (NTZ, Alinia®, Romark Laboratories, LC) is a licensed thiazolide anti-infective currently in advanced stage clinical development
for treatment of chronic hepatitis C. Studies by ourselves and others previously demonstrated that NTZ and its active metabolite, tizoxanide
(TIZ), exhibit potent antiviral activity against HCV replication in cell culture, but the antiviral mechanism has remained undetermined.
The current investigations sought to determine the effect of TIZ on intracellular HCV proteins to reveal a mechanism of action. TIZ was
inactive against HCV polymerase, protease, and helicase in enzymatic assays. The rate of reduction of HCV proteins in TIZ-treated HCV
replicon cells was consistent with loss of viral RNA template as assessed by Western blot analysis. TIZ treatment induced a 4- to 6-fold
enhancement of hyperphosphorylated HCV NS5A (p58), and a similar reduction of basally phosphorylated NS5A (p56), in intracellular
membrane preparations (where HCV replication is localized). These changes were both time- and dose- dependent. The phosphorylation
state of NS5A is a regulator of the switch from active viral genome replication to packaging, and overproduction of p58 is known to reduce
HCV replication. Evaluations of the effect of TIZ on casein kinase I-alpha (CKIa), the cellular kinase reported to be responsible for conversion
of HCV NS5A p56 to p58, were performed. CKI kinase activity in intracellular membrane preparations from TIZ-treated HCV replicon cells
was approximately 2-fold higher than that from untreated cells (p <0.02). However, TIZ had no direct effect on purified CKIa activity in
enzymatic assays. Overproduction of hyperphosphorylated HCV NS5A appears to be the primary antiviral mode of action of NTZ against
HCV. A drug-associated intracellular enhancement of the cellular enzyme activity responsible for hyperphosphorylation of NS5A provides
a cell-based mechanism. We hypothesize the primary cellular target for TIZ is one of several proteins involved in the regulation of CK1a.
Contact: Abigail Montero
Georgetown University, Washington, DC, USA
[email protected]
POSTERS
Abigail Montero (1), Prasanth Viswanathan (1), Changsuek Yon (1), Brent Korba (1)
(1) Georgetown University, Washington, DC, USA
POSTERS
P1.07
MK-5172, a novel macrocyclic inhibitor of NS3/4a protease, exhibits efficacy in in vitro
long-term potency and combination studies
Stacia Kargman (1), Genevieve Lavallee (2), Christine Brideau (1), Helen Chan (2), Martine Hamel (2), Qian Huang (1), Steven Ludmerer
(1), Mark Stahlhut (1), Rino Stocco (2), Joseph Vacca (1), David Olsen (1)
(1) Merck & Co., Rahway, NJ, USA
(2) Merck Frosst Canada, Canada
MK-5172 is a potent macrocyclic inhibitor of NS3/4a protease enzyme (IC50 < 0.016 nM, replicon IC50 = 2 nM) optimized to have increased
potency against clinically important resistant virus while maintaining a favorable pharmacokinetic profile. We utilized Huh-7 cells harboring
a gt1b replicon (con1) to further analyze the in vitro efficacy of MK-5172 in long term potency experiments. Gt1b replicon cells were
incubated in the presence of varying concentrations of MK-5172 for two weeks, with samples collected periodically throughout the study.
Replicon RNA was extracted and quantified by real-time polymerase chain reaction (qrt-PCR). MK-5172 reduced replicon RNA by 2 to 3 logs
over the duration of drug treatment with no breakthrough. Sequencing of NS3/4a isolated at various time points confirmed the absence of
mutations arising during drug treatment. Combination studies were initiated to examine potential synergistic effects with an NS5A inhibitor.
Gt1b con1 cells were treated with MK-5172 and MK-4882, (a potent NS5A inhibitor). The effect of the combination in reducing replicon RNA
was analyzed using MacSynergy (based on the Bliss Independence Model) and CompuSyn (based on the Median-Effect Principle (Chou)
and the Combination Index-Isobologram Theorem (Chou-Talalay)) software. Analysis with MacSynergy resulted in a synergy value (uM2%)
of 50 – 100, and a log volume of 5-9, indicating moderate to strong synergy. Analysis with CompuSyn, resulted in a combination index of
0.39-0.75 and an isobologram indicative of moderate synergy. The data suggests a favorable potential for combinations of MK-5172 with
a potent NS5a inhibitor in clinical testing.
Contact: Stacia Kargman
Merck & Co., Rahway, NJ, USA
[email protected]
P1.08
Affinity maturation to improve a human monoclonal antibody neutralization potency
and breadth of reactivity against hepatitis C virus
POSTERS
Yong Wang (1), Anasuya Saha (1), Jinming Xia (1), Zhenyong Keck (1), Jianlong Lou (2), James D. Marks (2), Steven Foung (1)
(1) Department of Pathology, Stanford University, Palo Alto, CA, USA
(2) Department of Anesthesia, University of California, San Francisco, CA, USA
A potent neutralizing antibody to a highly conserved HCV epitope might overcome its extreme variability allowing immunotherapy. The
human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 envelope glycoprotein. Previous studies showed that
HC-1 has a broad neutralization profile against most HCV genotypes but has modest potency. Variation in potency was also observed against
a panel of HCV retroviral pseudotype particles (HCVpp) bearing E1E2 from multiple 1a and 1b isolates. Some were poorly neutralized and
some failed to be neutralized by HC-1. To improve neutralization, we affinity matured HC-1 by constructing a library of yeast displayed HC-1
single chain Fv (scFv) mutants and used for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by
parallel mutagenesis of the heavy chain variable (VH) and kappa chain variable (Vk) genes separately and then combining the optimized VH
and Vk mutants. This resulted in the generation of a panel of HC-1 scFv displaying improved affinities. The best scFv had a 92 fold improved
affinity. After conversion to IgG1, some of the antibodies exhibited a 30 fold improvement in neutralization activity. BIAcore analysis showed
the increase in affinity was largely due to a slowing of the dissocation rate constant (Koff). Neutralization against the panel of HCVpp isolates
and infectious 2a HCV virus (HCVcc) improved with the HC-1 affinity-matured IgG1 antibodies. Interestingly, some of the affinity matured
antibodies neutralized some viral isolates that were not neutralized by wildtype (wt) HC-1. Moreover, propagating 2a HCVcc under the
selective pressure of wt HC-1 or affinity matured HC-1 antibodies yielded no viral escape mutants with the affinity matured IgG1 needing 40
fold less antibody concentration to achieve complete virus kill. Taken together, these findings suggest that affinity matured HC-1 antibodies
are excellent candidates for therapeutic development.
Contact: Yong Wang
Department of Pathology, Stanford University, Palo Alto, , CA, USA
[email protected]
90 : HCV 2011
ANTIVIRAL THERAPY
P1.09
Ribosome assembly on the HCV RNA: a novel target for developing antiviral therapeutics
Prasanna Bhat (2), Sivakumar Vadivel Gnanasundram (2), Prashant Mani (1), Debi Prasad Sarkar (1), Saumitra Das (2)
(1) University of Delhi, South Campus, India
(2) Indian Institute of Science, Bangalore, Bangalore, India
Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome
Entry Site (IRES) present in the 5’-untranslated region (UTR). This mechanism is unique and fundamentally different from the translation
mechanism of host cell mRNAs and thus could be exploited for selective antiviral target. Translation initiation on the HCV IRES is mediated
by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. We have studied the effect of a small RNA
(SLR6, 34nt) that structurally mimics a part of the HCV-IRES on the ribosome assembly at the HCV RNA. The SLR6 RNA was found to bind
the ribosomal protein S5 and disrupt the IRES-40S interaction to inhibit internal initiation of translation. Interestingly, SLR6 RNA was also
found to bind and sequester human La protein, an important ITAF for the HCV IRES function. More importantly, SLR6 RNA showed significant
suppression of HCV RNA replication from a monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system.
Finally, using Sendai virus based virosome, the targeted delivery of SLR6 RNA into mice liver succeeded in selectively inhibiting HCV IRES
mediated translation in vivo. This study therefore demonstrates a strategy to inhibit HCV protein synthesis using a small RNA that disrupts
an essential interaction between the HCV RNA and host ribosomes, providing the promise of an effective anti-HCV therapeutic targeted
against an important aspect of host-virus interaction.
Contact: Saumitra Das
Indian Institute of Science, Bangalore, Bangalore, India
[email protected]
P1.10
Discovery of a novel series of 1,3,5-triazine HCV-entry inhibitors that block virus
spread and promote virus clearance in vitro
Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. The entry process of
HCV into hepatocytes presents a series of potential targets for therapeutic intervention. Combinations of specific HCV virus entry inhibitors
with specific anti-viral replication agents could be synergistic or generate different distinct profiles of drug resistance.
We have discovered and optimized a series of triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry.
The compounds potently inhibit cell-free virus and cell-to-cell spread in the HCV cell culture model and display favorable pharmacokinetic
properties in preclinical testing. A key finding from our work was the demonstration that a representative triazine inhibitor can fully suppress
viral infection and lead to viral clearance in culture. We also demonstrated that a single amino acid variant, V719G, in the transmembrane
domain of E2 is sufficient to confer resistance to several compounds from the triazine series. Localization of the determinants of resistance
to the transmembrane domain suggests HCV fusion as a potential mechanism of inhibition by this series of triazines. Natural polymorphisms
identified at position 719 also exhibited reduced susceptibility to the triazines, however, many of these variants also exhibited reduced
levels of infectivity in the HCVpp assay. An analysis of the HCV sequence database demonstrates that the frequency of occurrence of each
polymorphism is in substantial agreement with the observed levels of infectivity in the HCVpp assay suggesting that amino acid substitutions
at position 719 accompany a significant reduction in viral fitness in the genotype 1 background.
The preclinical properties of the lead triazine compounds support further investigation of triazine inhibitors as a potential therapy for
HCV infection.
Contact: Jean-Marc de Muys
Progenics Pharmaceuticals, Inc., Tarrytown, NY, USA
[email protected]
POSTERS
Jean-Marc de Muys (1), Glen Coburn (1), Danielle Fisch (1), Sameer Moorji (1), Jose Murga (1), Dorothy Paul (1), Yakov Rotshteyn (1),
Amy Han (1), Dapeng Qian (1), Paul Maddon (1), William Olson (1)
(1) Progenics Pharmaceuticals, Inc., Tarrytown, NY, USA
POSTERS
P1.11
Naïve resistance mutations of protease (NS3) and polymerase (NS5B) inhibitors in HCV
genotypes sequences from Los Alamos databank
Rafael S. Alves (4), Artur T.L. Queiroz (1), Edvaldo F. da Silva (2), Flair J. Carrilho (3), Mário G. Pessoa (3), Isabel M. V. G. de Carvalho Mello (4)
(1) Instituto de Biociências, USP, São Paulo, Brazil
(2) Department of Gastroenterology, University of S. Paulo School of Medici, Brazil
(3) Dep. of Gastroenterology, University of São Paulo School of Medicine, Brazil
(4) Instituto Butantan, São Paulo, Brazil
New STAT-C (specific targeted antiviral therapies for hepatitis C) drugs are in development to Hepatitis C Virus (HCV) infection. The inhibitors
of NS5B and NS3 proteins are the most promising drugs in clinical trials with a high rate of sustained virologic response when combine to
standard of care. The viral genome high mutation rate, during replication, causes a natural polymorphic population, called quasispecies.
Our objective was to demonstrate the frequency of amino acid substitutions associated with resistance in polymerase (NS5B) and protease
(NS3) regions of genotypes 1a, 1b and 3a sequences, from patients naïve to protease/polymerase inhibitors. We collected 810 HCV NS5B
polymerase sequences (genotypes 1a = 471, 1b = 333, 3a = 6) and 1009 sequences of NS3 protease (genotype 1a = 328, genotype 1b =
470 genotype 3a = 211) from Los Alamos databank. Sequences were aligned and the alignments were visually edited to calculate positions
entropy plot and amino acid frequency. The NS3 analyses characterized the signature position V36L in genotype 3a, knowing to cause a
low level treatment resistance. At the same position, one sequence of genotype 3a had the A156T causing the highest resistance already
described. All genotypes presented mutations on the position 155 of the NS3 protein, associated with an important epitope position. In the
polymerase analyses each genotype presented, at least, one position with resistance mutation. The genotype 3a showed five mutations at
positions L419I, I424V, I482L V499A and S556G. We observed two positions presenting high polymorphism: in the genotype 1a (V499A) and
in the genotype 1b (C316N). Our results show the occurrence of natural polymorphisms in resistance positions that can be found in all the
three genotypes, suggesting that naïve resistance mutations could influence in STAT-C therapy successful in the near future.
Contact: Isabel M. V. G. de Carvalho Mello
Instituto Butantan, São Paulo, Brazil
[email protected]
P1.12
Isolation of potent hepatitis C virus helicase inhibitors from the yellow dyes thioflavine
S and primulin
POSTERS
Craig A. Belon (1), Kelin Li (2), Kevin J. Frankowski (2), Ben Neuenswander (2), Jean Ndjoumou (3), Alicia Hanson (3), Frank Schoenen (2),
Brian S. J. Blagg (2), Jeffrey Aube (2), David N. Frick (3)
(1) New York Medical College, USA
(2) University of Kansas, USA
(3) University of Wisconsin-Milwaukee, Milwaukee, WI, USA
A molecular beacon based helicase assay (MBHA) was used to screen 827 compounds from the National Cancer Institute Mechanistic Set
for inhibitors of the hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3), which is a helicase, ATPase, protease, and target
of numerous HCV drugs in clinical development. Compounds that inhibited the MBHA that did not interfere with substrate fluorescence
were tested for their ability to bind the MBHA DNA substrate using a fluorescent intercalator displacement (FID) assay. All helicase inhibitors
identified in the primary MBHA bound DNA except three, one of which was the yellow dye Thioflavine S (direct yellow 7), which also inhibited
NS3-catalyzed RNA unwinding, ATP hydrolysis, and peptide cleavage. Two purified compounds were isolated from Thioflavine S (Sigma Cat.
#T1892). Only one of these was a potent NS3 inhibitor (IC50 < 20 µM), and it resembled the main component of the related yellow dye
Primulin (direct yellow 59). Six additional compounds (2 major and 4 minor components) were purified from Primulin (MP Biochemicals Cat
#195454), and four of these were potent NS3 inhibitors. All active compounds were polymers of substituted benzothiazoles, and inhibition
potency correlated with the number of benzothiazole ring systems linked together. The most potent compound (compound B4) was a
benzothiazole tetramer that inhibited MBHAs by more than 50% at 2±1 µM. Unlike other HCV helicase inhibitors this class of compounds also
inhibits NS3 catalyzed ATP hydrolysis and peptide cleavage. Although Thioflavine and Primulin had some antiviral effects in cells transiently
transfected with HCV RNA, they had no significant effects on cells containing stable HCV replicons. In contrast, compound B4 reduced HCV
replicon content in Huh7.5 cells more than 50% when administered at 10 µM.
Contact: David N. Frick
University of Wisconsin-Milwaukee, Milwaukee, WI, USA
[email protected]
92 : HCV 2011
ANTIVIRAL THERAPY
P1.13
Effects of fluoroquinolones on the helicase activity of HCV NS3 protein
Irfan Khan (1), Sadiq Rehmani (2), Sammer Siddiqui (2), Shahana Kazmi (1), Syed Ali (2)
(1) University of Karachi, Karachi, Pakistan
(2) The Aga Khan University, Pakistan
Background: Hepatitis C virus (HCV) has infected an estimated over 170 million individuals worldwide. Effective therapy against HCV is still
lacking. There is a desperate need therefore to develop new treatment against this virus. Current evolution in the field of HCV has come
from two major discoveries: construction of Con1, a genotype 1b replicon that is able to independently replicate in the cell line Huh 8, and
the development of a cell culture model, using Huh 7 cells transfected with synthetic HCV RNA, to produce virions that are also infectious.
Fluoroquinolones inhibit bacterial DNA replication by targeting the enzymes gyrase and topoispmerase lV. These drugs have also been
shown to have inhibitory activity against some viral helicases. In the present study, we have used the above-mentioned HCV culture models
to test the activity of Fluoroquinolones against HCV. Methodology: Huh 7 cells producing the HCV virion as well as actively dividing Huh 8
cells were grown in the presence or absence of over 10 different Fluoroquinolone antibiotics for either 72 or 96 hours. Afterwards, Both
Huh 7 and Huh 8 cells were lysed, and viral RNA was extracted. The extracted RNA was reverse transcribed and quantified by real-time qPCR
using primers against HCV gene NS3. Results: To varying degrees, all of the tested fluoroquinolones effectively inhibited HCV replication in
Both Huh 7 and Huh 8 models. Conclusions: The two HCV culture models can be effectively used for the development of novel therapeutics
and vaccines against HCV. Fluoroquinolones hold a great deal of promise for treatment against HCV infection.
Contact: Irfan Khan
University of Karachi, Karachi, Pakistan
[email protected]
P1.14
Bi-specific high affinity TCR antiCD3 fusions for redirected killing of HCV infected cells
Hepatitis C (HCV) is a viral infection whose outcome depends heavily upon the strength of anti-viral response mounted by the host’s
immune system.
HCV can only be cleared by a patient if all of the cells infected with viable virus are killed by the host’s immune system. Approximately 25% of
infected individuals do not mount an immune response capable of clearing HCV, even when treated with anti-viral therapies. These immune
deficiencies can be attributed to viral related factors such as active avoidance of the host immune system through HLA down-regulation.
Immunocore has developed a platform of biologics that couple a novel targeting system comprising a soluble, affinity-enhanced T Cell
Receptor (TCR) with a clinically validated anti-CD3 scFv T cell redirection effector function. These ImmTAC reagents are highly stable, easily
manufactured and we have demonstrated that they can specifically kill cells presenting as few as 5-20 epitopes per cell with picomolar
potency. Our lead candidate, specific for malignant melanoma, initiated Phase I clinical testing in October 2010.
ImmTACs can actively target virally infected cells and direct a potent polyclonal response against them, even in the face of severe HLA downregulation. Immunocore is currently conducting proof-of-concept pre-clinical studies with a HIV specific ImmTAC (supported by the Bill &
Melinda Gates Foundation). We have demonstrated in vitro that the ImmTAC can specifically kill HIV infected cells presenting the HLA-A2
presented target epitope, HIVgag77-85.
Contact: Annelise Vuidepot
Immunocore Ltd., Abingdon OX14 4RX, United Kingdom
[email protected]
POSTERS
Annelise Vuidepot (1), Nikolai Lissin (1), Peter Molloy (1), Conor Hayes (1), Linda Hibbert (1), Bent Jakobsen (1)
(1) Immunocore Ltd., Abingdon OX14 4RX, United Kingdom
POSTERS
P1.15
Sorafenib impairs the antiviral effect of interferon alpha on hepatitis C virus replication
Kiyoshi Himmelsbach (1), Eberhard Hildt (1)
(1) Paul Ehrlich Institut, Langen, Germany
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality worldwide.
Permanent inflammation of the liver due to HCV infection results in fibrosis progressing to cirrhosis and frequently causes formation of a
hepatocellular carinoma. Former work showed that NS5A is associated with c-Raf and that inhibition of c-Raf by the kinase inhibitor Sorafenib
strongly impairs HCV replication. Given that today’s standard therapy is the application of interferon alpha combined with ribavirin and also
given that the treatment is not well tolerated by the patients and its efficacy is poor, we tried to combine Sorafenib with interferon alpha to
elucidate if additive effects could be observed that would allow the application of reduced concentrations of these two compounds. Here we
show that a combined treatment of HCV replicating hepatoma cells resulted in an impaired antiviral effect of interferon alpha or Sorafenib,
respectively. Investigating this effect in more detail it turned out that interferon is capable to activate c-Raf and thereby counteracts the
inhibitory effect of Sorafenib. Vice versa the inhibition of cRaf by Sorafenib or by expression of a transdominant negative mutant of cRaf
(RafC4) leads to a decreased phosphorylation of PKR. Moreover, the inhibition of c-Raf impairs interferon dependent activation of STAT1
and its translocation into the nucleus. These data indicate that integrity of c-Raf plays a crucial role for interferon signaling in particular for
the activation of PKR and STAT1. Based on this, a combined application of Sorafenib and interferon aplpha for inhibition of HCV replication
cannot be recommended.
Contact: Kiyoshi Himmelsbach
Paul Ehrlich Institut, Langen, Germany
[email protected]
P1.16
Characterization of NS5A resistance for HCV replicon treated by BMS-790052
Chunfu Wang (1), Haichang Huang (1), Lourdes Valera (1), Xin Huang (2), Aneela Altaf (2), Jin-Hua Sun (1), Donald O’Boyle (1), Peter Nower (1),
Min Gao (1), Robert Fridell (1)
(1) Dept. of Virology, Bristol-Myers Squibb R&D, Wallingford, CT, USA
(2) Dept. of Applied Genomics, Bristol-Myers Squibb R&D, NJ, USA
POSTERS
BMS-790052, a first-in-class HCV replication complex inhibitor targeting non structural protein 5A (NS5A), displays picomolar potency
against genotype 1-5. Importantly, this exceptional potency translated into anti-HCV activity in early phase clinical studies. To characterize
the in vitro BMS-790052 resistant profile in genotype 1a and 1b and to uncover potentially clinically relevant resistant variants, HCV replicon
elimination and colony formation assays were performed. HCV replicon was treated with different concentrations of BMS-790052 for 3,
7, 10 and 14 days in the absence of G418. Cells were maintained in a sub-confluent state and samples were collected at each split for
genotypic and phenotypic analysis. In all instances, genotypic analysis revealed resistance mutations within the N-terminal 100 aa of NS5A.
In genotype 1b, L31 and Y93 were the predominant sites of resistance substitutions. A more complicated pattern of resistance was identified
in genotype 1a with M28, Q30 and H58 representing additional sites of resistance development. A progression of variants was observed
depending on treatment duration and dose of inhibitor. In general, single amino acid substitutions that conferred relatively low levels of
resistance were observed at early time-points and at low doses. Higher doses yielded single resistant variants conferring greater levels of
resistance, or linked mutations. Importantly, most single amino acid substitution variants were suppressed by BMS-790052 at clinically
achievable concentrations. Some single amino acid substitution and linked mutations were more difficult to suppress. However, these high
resistant variants retained full sensitivity to IFN and other HCV inhibitors. This study helps to characterize the NS5A resistant profile in vitro
and provides a valuable tool for predicting the emergence of clinically relevant resistant variants.
Contact: Chunfu Wang
Dept of Virology, Bristol-Myers Squibb R&D, Wallingford, CT, USA
[email protected]
94 : HCV 2011
ANTIVIRAL THERAPY
P1.17
In vitro comparative analysis between IFN-lambda and IFN-alpha during combination
treatment with direct-acting antivirals targeting hepatitis C virus
Jacques Friborg (1), Steven Levine (1), Chaoqun Chen (1), Amy Sheaffer (1), Susan Chaniewski (1), Julie Lemm (1), Stacey Voss (1), Min Gao (1),
Fiona McPhee (1)
(1) Bristol-Myers Squibb, Wallingford, CT, USA
Clinical efficacy of a pegylated form of interferon lambda-1 (pegIFNλ) has been recently demonstrated in subjects infected with HCV representing
genotypes 1 through 4. In these proof-of-concept studies, pegIFNλ showed an improved safety profile compared to the pegylated form of IFN
alpha-2a (pegIFNα), which may be a consequence of the limited cell distribution of the unique IFNλ receptor complex. We describe herein a
comprehensive analysis of the antiviral activity of IFNλ in combination with investigational direct-acting antivirals (DAA) using various HCV
replication cell-based models. Our findings indicate that the recombinant (r) form of IFNλ1 (rIFNλ1) exerts the most robust in vitro antiviral
activity of the three identified IFNλ family members, while rIFNλ3 exhibits greater activity than rIFNλ2. More importantly, cross-resistance is
not observed in various replicon cell lines constitutively expressing variants of NS3, NS5A or NS5B known to have reduced susceptibility to
DAA. Furthermore, in 2-drug combination studies, pegIFNλ acted in a synergistic and/or additive manner with pegIFNα or various DAA. In
line with these observations, the emergence of DAA-resistant HCV replicon colonies were equally suppressed in cell culture when exposed
to combination regimens of two DAA with either rIFNλ1 or rIFNα. The HCV resistance profile for these DAA was similar in both IFN-based
treatments. Overall, these findings support the future studies of pegIFNλ with novel DAA in patients infected with HCV.
Contact: Jacques Friborg
Bristol-Myers Squibb, Wallingford, CT, USA
[email protected]
P1.18
Hepatitis C viral kinetics with the nucleoside polymerase inhibitor RG7128
Background: Mathematical modeling of HCV RNA kinetics has provided important insights for characterizing viral decline and estimating in
vivo effectiveness of antiviral agents; however it has not been used to characterize nucleoside polymerase inhibitors. RG7128 (mericitabine)
is a first-in class nucleoside polymerase inhibitor (NPI) currently in Phase 2B development. RG7128 requires intracellular uptake and
phosphorylation to two active species, a cytidine and uridine-triphosphate, with intracellular half-lives of ~6h and 24h, respectively.
Methods: HCV RNA was frequently measured in N=32 treatment experienced patients infected with HCV genotype-1 during and after
RG7128 monotherapy for 14d with 750-mg or 1500-mg administered once (QD) or twice daily (BID).
Results: Initial kinetics of HCV RNA decline was slower than with IFN or protease inhibitors and 12 patients presented a novel pattern of HCV
RNA kinetics, characterized by a monophasic viral decline, particularly at lower doses. Viral load data could be well fitted by assuming that
the effectiveness in blocking viral production gradually increased over time to reach its final value, εfinal, consistent with accumulation of
intracellular triphosphates. εfinal was high with BID dosing (mean 750-mg and 1500-mg: 98% and 99.8%, P=0.018) and significantly higher
than in patients treated QD (mean QD vs BID: 90% vs 99%, P<10-7). Patients treated BID were predicted to reach 90% of εfinal after 2.8 days.
Virus rebounded rapidly upon dosing discontinuation, which was attributed to the elimination of active drug and the subsequent decline of
drug effectiveness with t1/2=14.5h in the BID regimens, which mirrors the plasma half-life of the parent compound.
Conclusion: RG7128 administered BID reached a high, dose-dependent, final effectiveness in blocking viral production. The observed
slower initial decline likely represents the time needed to accumulate intracellular triphosphates, and is consistent with in vitro data. Unlike
protease inhibitors, NPI’s do not appear to have a prolonged post-antiviral effect.
Contact: Jeremie Guedj
Los Alamos National Laboratory, Santa Fe, NM, USA
[email protected]
POSTERS
Jeremie Guedj (1), Harel Dahari (1), Emi Shudo (2), Patrick Smith (2), Alan Perelson (1)
(1) Los Alamos National Laboratory, Santa Fe, NM, USA
(2) Clinical Pharmacology, Pharma Research and Early Development, Roche, USA
POSTERS
P1.19
The combination of proteasome inhibitors and interferons - a new and promising
approach for treatment of HCV infection
Artur Kaul (1), Daniel Lüftenegger (1), Danijela Cvijetic (1), Eva Godehardt (1), Jana Pforr (1), Vadim Bichko (2), Ralf Kircheis (1)
(1) ViroLogik GmbH, Erlangen, Germany
(2) ChemDiv, USA
The combination of pegylated interferon-alpha in combination with ribavirin is the only currently approved therapy for hepatitis C infection.
However, this standard therapy is limited by side-effects and reveals limited efficacy.
Most antivirals currently in development target viral proteins and are prone to induce drug resistance of the rapidly mutating HCV. We are
following an alternative strategy to prevent the formation of drug resistance. Our approach aims at inhibition of the highly conserved host
cell proteasome which is essential for HCV replication. The novel peptide-based inhibitor VL-01 is highly specific for the chymotrypsin-like
activity of the human 26S proteasome and induces a dose-dependent inhibition of the proteasome in cell culture as well as in human blood
cells. VL-01 is free of boronic acid which is believed to account for some of the dose-limiting adverse effects of the clinically approved
proteasome inhibitor Velcade™. In HCV genotype 1b, 1a and 2a replicons as well as in cell culture infection systems, VL-01 inhibits HCV
replication in a dose-dependent manner. This effect is enhanced when VL-01 is combined either with Interferon-alpha or Interferon-gamma.
The antiviral effect is believed to be, at least partially, the result of increased and/or prolonged phosphorylation of STAT proteins.
Contact: Artur Kaul
ViroLogik GmbH, Erlangen, Germany
[email protected]
P1.20
Phenotypic characterization of HCV NS3 protease from patient isolates in phase 2
clinical trials of telaprevir
Min Jiang (1), Leen Vijgen (2), Jim Sullivan (1), Michelle Nelson (1), Judith Lippke (1), John Fulghum (1), Kenneth Bonanno (1),
Kumkum Saxena (1), Azin Nezami (1), William Taylor (1), Joyce Coll (1), Yu-Ping Luong (1), Sandra De Meyer (2), Inge Dierynck (2),
Gaston Picchio (2), Tara Kieffer (1)
(1) Vertex Pharmaceuticals Inc., Cambridge, MA, USA
(2) Tibotec BVBA, Beerse, Belgium
POSTERS
Background: Inhibition of HCV NS3•4A protease by telaprevir (TVR) led to rapid and significant decreases in HCV RNA levels in genotype
1 HCV-infected patients in clinical trials. TVR-selected variants have been observed at NS3 positions 36, 54, 155 and 156 in patients who
did not achieve SVR. These variants have shown varying degrees of resistance in sub-genomic replicons generated through site-directed
mutagenesis. The level of resistance conferred by these variants in the background of patient-specific protease sequence was studied.
Methods: The NS3 gene (Tyr6-Pro191) was reverse transcribed from clinical isolates (n=65), amplified and cloned into a Con1 replicon.
Clones were pooled to mimic the intrinsic genetic heterogeneity of the viral quasispecies in infected patients. The drug susceptibility (IC50
values) was determined after transfection of in vitro-transcribed RNA pools into Huh7-lunet cells. A subset (n=26) of the 65 isolates was
tested in a NS3 enzymatic assay for comparison with replicon data.
Results: Baseline isolates lacking resistant mutations (wild-type) were all sensitive to TVR in the HCV replicon assay, but showed a range
in sensitivities (IC50: 0.034-0.32 µM). This intrinsic variability in in vitro TVR-susceptibility did not correlate with early virologic responses
in Phase 2 trial patients. Post-baseline isolates containing V36A/M, T54A/S, R155K/T, A156T or V36M+R155K exhibited resistance levels
consistent with those obtained previously using site-directed mutant replicons. The resistance level conferred by TVR-resistant mutations
obtained in the enzymatic assay correlated with that obtained in the replicon assay.
Conclusions: Phenotypic characterization of telaprevir-resistant clinical isolates conducted in both HCV replicon and enzymatic assays
correlated with the degree of resistance observed with site-directed mutants. The level of resistance conferred by the predominant resistant
variants (V36A/M, T54A/S, R155K/T, A156S/T and V36M+R155K), as determined previously, was confirmed in the context of diverse patient
HCV NS3 quasispecies.
Contact: Min Jiang
Vertex Pharmaceuticals Inc., Cambridge, MA, USA
[email protected]
96 : HCV 2011
ANTIVIRAL THERAPY
P1.21
The Hsp90 inhibitor 17-AAG inhibits hepatitis C viral replication through cytostasis
Rudolf Beran (1), Ruchi Sharma (1), Amoreena Corsa (1), Yang Tian (1), Justin Golde (1), Greta Lundgaard (1), William Delaney IV (1),
Weidong Zhong (1), Andrew Greenstein (1)
Researchers screening chemical libraries for inhibitors of HCV replication need to be able to distinguish between compounds that directly
inhibit viral replication and those that inhibit viral replication non-specifically through slowing cellular growth. The HCV replicon is used
as a model in many screens and its replication is tied to the level of cellular replication. Thus, a compound that induces cytostasis would
indirectly inhibit HCV replicon replication.
Antivirals that target host factors such as chaperones are interesting in that they have a higher resistance barrier than direct antivirals.
Compounds that inhibit the cellular chaperones CypA and Hsp90 have recently been observed to inhibit HCV replication. However, because
CypA and Hsp90 are involved in cellular growth processes, it is possible that inhibitors of these chaperones might slow cellular growth at
their effective antiviral concentrations.
Here we compared the antiviral efficacies and toxicity of the Hsp90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) and the
CypA inhibitor, Cyclosporin A (CsA). Neither 17-AAG nor CsA were cytotoxic at their effective antiviral concentrations. However, when we
measured cell confluence over time, we observed that 17-AAG, but not CsA, induced cytostasis at the same concentrations that inhibited
HCV replication. This suggested that 17-AAG might inhibit HCV replication non-specifically by slowing cellular growth. Indeed, when we
induced cytostasis in HCV-infected cultures by prolonged treatment with 1% DMSO, subsequent 17-AAG treatment did not further reduce
the extracellular HCV titer over time. In contrast, CsA treatment reduced the extracellular HCV titer released by the cytostatic, infected
cultures by 2 logs over time. Our findings suggest that 17-AAG inhibits HCV replication through inducing cytostasis, while CsA has true
antiviral effects. Furthermore, our work suggests that newly-discovered inhibitors of HCV and other viruses should be carefully tested for
the ability to induce cytostasis before further characterization is pursued.
Contact: Rudolf Beran
[email protected]
P1.22
Rajeev Aurora (1), Maureen Donlin (1), John Tavis (1)
(1) Saint Louis University School of Medicine, Saint Louis, MO, USA
Success of interferon-based therapy for HCV is affected by differences in host interferon responses and by variable sensitivity of individual
HCV isolates to interferon. IL28B polymorphisms control much of the host effect, but evaluating the sensitivity of individual HCV isolates
to interferon is not yet possible. We previously found that differences in HCV genome-wide amino acid covariance networks are strongly
associated with outcome of therapy, and proposed that differences in the covariances between sensitive and insensitive HCV isolates
could help predict treatment outcome. Here, we used network descriptors as biomarkers to develop a network-based method to predict
sensitivity of HCV isolates to interferon-based therapy.
A reference covariance network was generated from an alignment of HCV subtype 1a non-responder sequences, and then 17 test sequences
were added individually to the alignment and 17 test networks were generated. The network metrics of the reference network were
compared with those of the test networks. If a test network’s metrics increased by more than the amount predicted by chance, then the
test sequence strengthened the network and hence was predicted to have low sensitivity to interferon. The method correctly predicted 88%
of the test sequences (15/17) as being non-responders. The algorithm required use of a small number of reference sequences to render it
sensitive to disruption by addition of the test sequence, and it was relatively independent of which reference sequences were employed.
Robust prediction required two independent network parameters, such as covariance strength and number of hydrophobic residue pairs.
This improved approach reveals the sensitivity of an HCV isolate to interferon-based therapy and eliminates the need for a comprehensive
set of reference covariances associated with treatment outcome that hindered our original approach. Extension of this approach to
prediction of SVR and non-SVR for subtypes 1a and 1b is ongoing.
Contact: John Tavis
Saint Louis University School of Medicine, Saint Louis, MO, USA
[email protected]
POSTERS
An improved amino acid covariance network approach to evaluate sensitivity of HCV
isolates to interferon-based therapy
POSTERS
P1.23
Discovery of naturally occurring aurones that are potent allosteric inhibitors of
hepatitis C virus RNA-dependent RNA polymerase
Abdelhakim Ahmed-Belkacem (3), Romain Haudecoeur (1), Wei Yi (1), Antoine Fortune (1), Rozenn Brillet (3), Catherine Belle (2),
Edwige Nicolle (1), Coralie Pallier (3), Jean-Michel Pawlotsky (3), Ahcene Boumendjel (1)
(1) CNRS UMR 5063, Universite Joseph Fourier, Grenoble, France
(2) CNRS UMR 5250, Universite Joseph Fourier, Grenoble, France
Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Approximately 170 million
individuals are infected worldwide and HCV infection causes approximately 300,000 deaths per year. The current standard treatment for
chronic hepatitis C is based on the use of pegylated interferon (IFN-α) in combination with ribavirin for up to 1 year. However, only up to
50% of patient with HCV genotype 1 infection can eradicate infection upon therapy. Moreover, both IFN-α and ribavirin are associated
with adverse effects. Therefore, more efficient and better tolerated therapies are needed for hepatitis C. We have identified naturally
occurring 2-benzylidenebenzofuran-3-ones (aurones) as new templates for non-nucleoside hepatitis C virus (HCV) RNA-dependent RNA
polymerase (RdRp) inhibitors. The aurone target site, identified by site-directed mutagenesis, is located in thumb pocket I of HCV RdRp. The
RdRp inhibitory activity of 42 aurones was rationally explored in an enzyme assay. Molecular docking studies were used to determine how
aurones bind to HCV RdRp and to predict their range of inhibitory activity. Seven aurone derivatives were found to have potent inhibitory
effects on HCV RdRp, with IC50s below 5 _M and excellent selectivity. The most active aurone analogue was (Z)-2-((1-butyl-1H-indol-3-yl)
methylene)-4,6-dihydroxybenzofuran-3(2H)-one, with an IC50 of 2.2 _M. The potent RdRp inhibitory activity of aurones, together with their
low toxicity, make these molecules attractive candidate direct-acting anti-HCV agents.
[email protected]
P1.24
Antiviral activity against hepatitis C virus by iCo-007, an antisense inhibitor of
expression of the host protein c-Raf (Raf-1) kinase
POSTERS
Rineke Steenbergen (1), John Clement (2), D. Lorne Tyrrell (1)
(1) Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada
(2) iCo Therapeutics Inc., Canada
iCo-007 is a 2nd generation antisense inhibitor targeting c-Raf mRNA. It has been reported previously that c-Raf associates with Hepatitis
C virus NS5A and regulates viral replication (1). Recently it was shown that high concentrations of the anticancer-drug Sorafenib, a multikinase inhibitor, inhibited HCV replication, likely through c-Raf inhibition (2). Here we tested if iCo-007, a highly specific antisense inhibitor
of c-Raf, has antiviral activity in Huh7.5 cell lines infected with HCV.
c-Raf protein levels in cells transfected with a single dose of iCo-007 (200-500 nM) were reduced by up to 95%, for up to 5 days post transfection.
c-Raf suppression did not result in reduced cell growth. We then studied its effect on HCV viral titers, as well as HCV structural and nonstructural protein levels. We showed that iCo-007 has a dose dependent reducing effect on viral titers, with a moderate reduction in viral titers
at 200 nM (60%) and viral titers that are reduced by 80-95% at higher concentrations (300-500 nM). The suppressing effect was short-lived at
lower concentrations, but sustained for up to 8 days post infection at higher concentrations, after a single dose of iCo-007. HCV core protein
showed a parallel response to the HCV titers. However, NS3 protein levels were considerably less affected by iCo-007 treatment.
We showed that iCo-007, an antisense inhibitor of c-Raf efficiently inhibits viral production in a tissue culture model of HCV infection. This
novel effect of iCo-007 will be further explored as a strategy to treat HCV infection in patients.
1. Burckstummer et al. FEBS letters 580: 575-580, 2006.
2. Himmelsbach et al. Gut 58: 1644-1653, 2009.
Contact: Rineke Steenbergen
Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada
[email protected]
98 : HCV 2011
ANTIVIRAL THERAPY
P1.25
Susceptibility of treatment-naïve clinical isolates to HCV polymerase inhibitors
Hadas Dvory-Sobol (1), Ross Martin (1), Siu-Chi Sun (1), Michael D. Miller (1), Hongmei Mo (1)
(1) Gilead Sciences, Foster City, CA, USA
Background: The high degree of natural sequence variation within the HCV genome may affect the susceptibility of patient isolates to HCV
NS5B inhibitors. The aim of this study was to assess the natural variation in drug susceptibility among treatment-naïve HCV patient isolates
to six HCV polymerase inhibitors.
Methods: HCV NS5B polymerase genes were amplified from 30 treatment-naïve HCV patients (GT1a and 1b, 15 each) and ligated into an
NS5B 1b-Con-1 replicon shuttle vector. Pooled viral RNAs from each patient were transfected into Huh-7-Lunet cells and tested for their
susceptibilities to six NS5B inhibitors including the nucleotide inhibitor (NI) GS-6620, and the non-nucleoside inhibitors (NNIs) GS-9669,
PF-868554 and GS-430319 (site II), GS-462581 (site III) and GS-9190 (Tegobuvir, site IV). Linear regression and correlation analyses were
performed.
Results: The natural variation in drug susceptibility in wild-type HCV patient isolates varies across drugs and drug classes. Minimal variation
was observed with the NI GS-6620 with <4-fold difference between the 95th and 5th percentiles of EC50 values. In contrast, greater variation
was observed among the five NNIs, with the order of GS-9669<GS-430319<GS-9190< PF-868554≤GS-462581. The EC50 values in the 95th
percentile were 8-, 11-, 32-, 40- and 42-fold higher than the 5th percentile EC50 values for GS-9669, GS-430319, GS-9190, PF-868554 and
GS-462581, respectively. GS-9190 and PF-868554 were slightly more potent against GT1b than GT1a patient isolates. Linear regression
analyses revealed high correlations between susceptibilities to GS-9669 and the other site II inhibitors GS-430319 and PF-868554, but little
or no correlation between GS-9669 and GS-6620 or the site III inhibitor GS-462581 or the site IV inhibitor GS-9190.
Conclusions: Natural variation in HCV susceptibility to NNIs may cause variable antiviral responses to HCV NS5B inhibitors depending on the
clinical dose. The NI inhibitor GS-6620 showed the least variation in NS5B susceptibility.
Contact: Hadas Dvory-Sobol
Gilead Sciences, Foster City, CA, USA
[email protected]
P1.26
Srikanta Dash (1), Sruti Chandra (1), Sidhartha Hazari (1), Partha Chandra (1), Feiza Gunduz (1), Luis Balart (1)
(1) Tulane University Health Science Center, New Orleans, LA, USA
Background: IFN-α plus ribavirin is the standard treatment for chronic HCV infection. The mechanism by which ribavirin and IFN-α
synergistically inhibit HCV replication in the infected liver is unknown. Aim: To investigate the synergy mechanism of ribavirin and IFN-α in
cell culture model using IFN-α sensitive and resistant R4-GFP cell lines. Methods: S3-GFP and R4-GFP cell lines were treated with ribavirin
and IFN-α singly and in combination. The antiviral effect of ribavirin and IFN-α was determined by G-418 cell colony assay, flow cytometry
and RPA. The synergy mechanism of ribavirin and IFN-α was examined at the level of IFN-β promoter activation and their ability to inhibit
IRES mediated translation of green fluorescence protein (GFP) and firefly luciferase. Western Blot served as the key in understanding
Jak-STAT signaling pathways and other mechanistic pathways. Results: Ribavirin inhibited HCV replication and GFP expression in both S3GFP and R4-GFP cell lines. The antiviral effect with IFN-α and ribavirin combination treatment was robust in interferon sensitive S3-GFP
cells as compared to interferon resistant R4-GFP cells. R4-GFP cell line shows more resistance to IFN/ribavirin combination treatment
than S3-GFP cell line. Ribavirin and IFN-α synergistically induced the activity of the IFN-β promoter in S3-GFP cells compared to R4-GFP.
Interferon and ribavirin each inhibit the HCV IRES-mediated translation through two separate mechanisms. IFN-α induced the PKR and eIF2α
phosphorylation in cured sensitive Huh-7 cells but not in the resistant Huh-7 cells. Ribavirin blocks HCV IRES-translation in both cell lines by
inhibiting ionosine monophosphate dehydrogenase (IMPDH) and eIF4E activity. Conclusions: These results suggest that ribavirin inhibited
HCV replication in both of the IFN sensitive and resistant cell lines. The synergistic effect was observed in the sensitive cell line because
IFN-α and ribavirin each inhibited IRES-mediated translation by different mechanisms. The work was supported by NIH grants CA127481
and CA129776.
Contact: Srikanta Dash
Tulane University Health Science Center, New Orleans, LA, USA
[email protected]
POSTERS
Ribavirin and interferon-alpha synergistically inhibits HCV replication in cells with a
functional Jak-STAT pathway
POSTERS
P1.27
A multiplexed cell-based platform to monitor the activity of Flaviviridae proteases
Jason Machula (1), Ryan O’Hanlon (1), Plamena Silvieva (1), Roland Wolkowicz (1)
(1) San Diego State University, San Diego, CA, USA
We have previously established a cell-based assay to monitor the catalytic activity of HIV-1 protease in T-cells. Here, we are establishing
a platform for drug discovery against proteases of the Flaviviridae family of viruses: Hepatitis C Virus (HCV), Dengue Virus (DenV), West
Nile Virus (WNV), and Yellow Fever Virus (YFV), which cause liver cancer, Dengue fever and other syndromes, meningitis/encephalitis, and
hemorrhagic fever, respectively. A processive RNA-dependent RNA polymerase prone to errors, the emergence of resistant strains, and lack
of vaccines, highlight the need for novel antivirals and innovative methods to facilitate their discovery.
Flaviviridae rely on the processing of their proteome, a process absolutely necessary for the viral life cycle. The assay is based on the
inducible expression of a Gal4-DNA-binding-Domain/protease/Gal4-Transactivation-Domain fusion, and the activation of the reporter
Green Fluorescence Protein (GFP). As active protease cleaves itself from the Gal4 fusion, GFP induction occurs only when protease is
inhibited. GFP thus acts as biosensor for protease activity. The assay has been adapted to hepatocytes, mimicking the natural milieu of HCV
infection. Hepatocytes and Hamster Vero cells are used as tissue-culture models for DenV, WNV and YFV infection.
We have engineered a set of hepatocytic cell lines genetically bar-coded with different fluorescent proteins. Bar-coded cell-lines were
further engineered to contain a reverse tetracycline transactivator for inducible expression of Gal4/Protease fusions, and a Gal4 promoterGFP reporter. We are engineering Gal4/NS3/NS4AHCV, Gal4/NS2B/NS3WNV Gal4/NS2B/NS3DenV, and Gal4/NS2B/NS3YFV, each of them
expressed in a distinct bar-coded cell-line. A mixed population thus includes different fluorescent backgrounds, but results in GFP expression
only when induced and inhibited. A multiplexed cell-based platform enables us to monitor the inhibition of each distinct protease
independently in the same sample, drastically enhancing high-throughput capabilities for drug discovery. Moreover, the assay facilitates the
study of the proteases and their dependence on co-factors.
Contact: Roland Wolkowicz
San Diego State University, San Diego, CA, USA
[email protected]
P1.28
Analysis of IL28B rs8099917 and hepatic Stat1 activation HCV-positive patients treated
with pegylated-interferon plus ribavirin
POSTERS
Tatsuo Miyamura (1), Tatsuo Kanda (1), Shingo Nakamoto (1), Shuang Wu (1), Fumio Imazeki (1), Osamu Yokosuka (1)
(1) Chiba University, Graduate School of Medicine, Chiba, Japan
It has been reported that IL28B rs8099917 is related to the antiviral treatment response in chronic hepatitis C patients. We analyzed
the relation between IL28B SNP and treatment response as well as the activation of Signal Transducer and Activator of Transcription 1
(Stat1) in the liver. We compared a clinical background and treatment response with IL28B rs8099917 status [major alleles (T/T) group or
minor alleles (T/G, G/G) groups]. We also examined Stat1 nuclear localization in pre-treatment liver biopsy samples in some patients by
immunohistochemically. We reconfirmed that IL28B rs8099917 is useful for predicting null responder. Stat1, one of ISG, tends to be stronger
activation in pre-treatment liver tissue of sustained virological responder although we did not recognize the significant difference. It is
possible that hepatic Stat1 activation before treatment might predict treatment response to pegylated-interferon and ribavirin combination
therapy. Further study is now going in progress.
Contact: Tatsuo Kanda
Chiba University, Graduate School of Medicine, Chiba, Japan
[email protected]
100 : HCV 2011
ANTIVIRAL THERAPY
P1.29
E2 binding peptide identified by RAPID system inhibited HCV infection
Noriyuki Watanabe (1), Kazuki Futai (2), Hiroaki Suga (2), Takaji Wakita (1)
(1) Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
(2) Graduate School of Engineering, The University of Tokyo, Japan
HCV envelope proteins, E1 and E2, play an important role during early stage of virus infection. Especially, E2 protein binds to receptors to
enter host cells. Entry step is a potential target of therapeutic treatment for chronic HCV infection and neutralization antibodies against to
E2 protein were established as an entry inhibitor. The effect of these antibodies was not significant to block HCV infection. Additional entry
inhibitor which binds to E2 protein with high affinity and exist stably in vivo is required for antiviral therapy. In this study we attempted to
search novel peptide capable to inhibit HCV infection by binding to E2 protein. We first purified E2 protein from drosophila S2 transfectant
and analyzed whether purified E2 protein forms native structure. Next, we searched E2 binding peptide by RAPID (random peptide integrated
discovery) system, which consists of high diversity cycle peptide library and in vitro selection method. As the result of screening by RAPID
system, four E2 binding peptides were identified. Among these four peptides, two inhibited HCV infection in a dose-dependent manner but
others did not. This result may indicate that the bound peptide masks an important region in E2 protein for HCV entry. This research may be
important for the analysis of HCV entry and also the anti-viral development of entry inhibitors.
Contact: Noriyuki Watanabe
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
[email protected]
P1.30
Development of chimeric hepatitis C virus expressing NS5A from strains of genotypes 1
and 2: virus production and susceptibility to NS5A inhibitor
NS5A of hepatitis C virus (HCV) is an essential component of the virus replication and plays critical roles in the HCV lifecycle. In addition,
NS5A has recently attracted much attention as an important target for development of HCV-specific inhibitors. However, robust HCV cell
culture systems allowing the analysis of an NS5A inhibitor are currently available only for genotype 2a. In the present study, to assess
genotype-dependent sensitivity of HCV to an NS5A inhibitor, we developed infectious cell culture systems using JFH-1-based chimeric
constructs bearing NS5A of genotype 1 (H77; 1a and Con1; 1b) and genotype 2 (J6CF; 2a and MA; 2b) strains. All these chimeric constructs
were capable of producing infectious viruses at varying degrees. Replacement of JFH-1 NS5A with that of genotype 1 strains, H77 and
Con1, resulted in similar or slightly lower virus production, whereas replacement with NS5A of genotype 2 strains, J6CF and MA, showed
an increase of infectious virus production as compared with the JFH-1 wild type. The single-cycle virus production assay demonstrated that
efficient virus production of the genotype 2 chimeric constructs depended on enhanced virus assembly and that substitutions at C terminus
of NS5A were responsible for this phenotype. Pulse chase experiments suggested that this enhanced virus assembly was mainly attributed
to accelerated cleavage kinetics at the NS5A-NS5B site. By use of these cell culture systems, we examined genotype-dependent effects of a
specific NS5A inhibitor and observed a more than 1,000-fold difference in susceptibility to the inhibitor between strains of genotypes 1 and
2. This system will be useful not only for better understanding of genotype-specific roles of NS5A in the HCV lifecycle but also for evaluation
of specific inhibitors against NS5A of different genotypes and strains.
Contact: Takanobu Kato
[email protected]
POSTERS
Yuka Okamoto (1), Takahiro Masaki (1), Asako Murayama (1), Takaji Wakita (1), Takanobu Kato (1)
POSTERS
P1.31
Phenotypic live cell screening with hepatitis C reporter viruses and reporter cells
Hee Young Kim (1), Soo Hyun Kim (1), Myung Eun Lee (1), Michael A.E. Hansen (1), Christopher T. Jones (2), Charles M. Rice (2),
Marc P. Windisch (1)
(1) Institut Pasteur Korea, Seongnam-si, Republic of Korea
(2) Rockefeller University, USA
Since the development of the infectious HCVcc system, enormous progress has been made in developing new tools to identify novel targets
for antiviral interventions. Recently, a reporter cell line enabling phenotypic analysis of HCVcc infection in live cells has been reported. The
reporter activity is based on NS3-4A mediated cleavage and subsequent nuclear translocation of a red fluorescent protein (RFP) tethered to
the mitochondrial targeting sequence of the interferon-β promoter stimulator protein 1 (RFP-NLS-IPS). Phenotypic assays have the advantage
over more traditional screening platforms in that multiple parameters including viral replication and spread as well as cytotoxicity and
cellular stress can be analyzed simultaneously. This encouraged us to develop an infection assay in live cells, to quickly identify compounds
acting on different stages in HCV life cycle. Briefly, HCV reporter cells were plated in 384-well plates followed by compound treatment. Cells
were then infected with an HCVcc reporter virus that expresses the green fluorescent protein (GFP). 72h post infection cells were imaged
using a high throughput confocal microscope and analyzed by software to examine infection based on RFP translocation into the nucleus
and GFP expression in the cytoplasm. We screened pharmacologically active compound libraries and selected molecules inhibiting RFP
translocation and GFP expression, with a high Selectivity Index (CC50/EC50) for further analysis. Surprisingly, compounds were identified
that prevented cleavage of RFP-NLS-IPS, but did not interfere with HCV replication. Furthermore, a late stage inhibitor phenotype was
identified in which virus could enter and replicate but was unable to spread. In summary, we developed a compound screen based on a
live cell phenotypic assay for HCVcc infection and were able to identify molecules with various mechanism of action. Since the screened
libraries encompass already well characterized molecules active in other disease areas, a re-positioning into the HCV therapeutic area could
be considered.
Contact: Hee Young Kim
Institut Pasteur Korea, Seongnam-si, Republic of Korea
[email protected]
P1.32
Development of an automated tissue culture infectious dose 50 determination assay in
384-well plates
POSTERS
Myung Eun Lee (1), Michael A.E. Hansen (1), Soo Hyun Kim (1), Thierry Dorval (1), Hee Young Kim (1), Keumhyun Kim (1), Tae Kyu Kim (1),
Marc P. Windisch (1)
(1) Institut Pasteur Korea, Seongnam-si, Republic of Korea
Background: Since the establishment of an infectious cell culture system for hepatitis C virus (HCV), the tissue culture infectious dose 50%
(TCID50) determination assay has been widely used to determine the number of infectious particle. Generally, Huh-7 cells are plated into 96well plates, inoculated with endpoint diluted HCV cell culture supernatants and analyzed at 72h post infection. The way of analysis is labour
intensive and time consuming due to visually read out by eye. Additionally, depending on scale of the primary experiments there might be
limitations in the total amount of viral supernatants to conduct a TCID50 determination assay in 96-well plates. Advanced image mining
technology and the need for a more convenient and efficient read out encouraged us to develop an automated TCID50 determination assay
for HCV in 384-well plates.
Material & Methods: Naïve Huh-7 cells were plated in 384-well plates followed by inoculation with endpoint diluted HCV cell culture
supernatants. At 72h post infection cell nuclei were stained and viral antigen was either detected by indirect immunofluorescence analysis,
using a secondary antibody conjugated to a fluorescent dye or even more conveniently by using HCV expressing a fluorescent protein.
Subsequently, experiments were analyzed with a high throughput/content confocal microscope (ImageXpress Ultra/Molecular Devices) by
acquisition of five pictures per well enabling to cover the well entirely.
Results: By using software which is capable to identify even a single infected cell, by firstly detecting cell nuclei and secondly measuring
the fluorescence intensity in the cytoplasm, we can very precisely and highly reproducible determine TCID50 values. Additionally, the viral
supernatant amounts to conduct this assay in 384- compared to 96-well plates are three-fold lower.
Conclusion: We have developed an automated TCID50 determination assay for HCV in 384-well plates enabling to analyse conveniently and
efficiently enormous amounts of samples in parallel.
Contact: Myung Eun Lee
Institut Pasteur Korea, Seongnam-si, Republic of Korea
[email protected]
102 : HCV 2011
ANTIVIRAL THERAPY
P1.33
Discovery of the clinical candidate TMC055, a novel non-nucleoside inhibitor of the
hepatitis C virus NS5b polymerase
Sandrine Vendeville (1), Tse-I Lin (1), Lili Hu (1), Abdellah Tahri (1), David McGowan (1), Maxwell Cummings (1), Katie Amssoms (1),
Stefaan Last (1), Maxime Canard (1), Iris Van den Steen (1), Benoit Devogelaere (1), Marie-Claude Rouan (1), Leen Vijgen (1),
Jan Martin Berke (1), Gregory Fanning (1), Kristof Van Emelen (1), Kenny Simmen (1), Origene Nyanguile (1), Pierre Raboisson (1)
(1) J&J, Beerse, Belgium
In the search for new direct acting antivirals for HCV, the NS5b polymerase is an attractive target, since it is responsible for the replication
of the HCV genome, and has been validated in clinical trials. Among the 4 different allosteric binding sites, the thumb domain 1 (or NNI1 binding site) is the most conserved across the 6 HCV genotypes. Recent clinical data suggest that the inhibition of HCV NS5b using
NNI-1 targeted polymerase inhibitors is associated with a higher barrier to resistance than NNIs binding in the other allosteric binding
sites. In order to find new potent and bioavailable NNI-1 inhibitors, we have applied a rationally designed macrocyclisation strategy to the
known indole-carboxylic acid chemotype, based on the introduction of a linker in a solvent-exposed region. Lead optimization allowed
the identification of potent (EC50 < 100 nM) and orally bioavailable macrocyclic NS5B inhibitors. Our discovery effort culminated with the
identification of the clinical candidate TMC055. The in vitro and in vivo profile of this 17-membered ring macrocyclic indole will be discussed,
together with preliminary results obtained in the clinics.
Contact: Sandrine Vendeville
J&J, Beerse, Belgium
[email protected]
P1.34
Nishi Prabdial-Sing (1), Deepak Ramauthar (1), Monalisa Kalimashe (1), Mark Goosen (1), Deidre Greyling (1), Ernest Song (2), Adam Mohamed
(2), Trevlyn Burger (2), Schalk Van der Merwe (3), A. Elnagar (3), F. Bassa (4), N. Rapiti (4), R. Ally (5), Wamda Abuelhassan (5), Nazeer Chopdat (5),
J. Van Zyl (6), V. G. Naidoo (7), K. Newton (7), K. Govender (7), B. Luke (8), D. Mushi (9), J. Zaldivar (10), A. R. J. Carrim (11), Adrian J. Puren (1)
(1) National Institute for Communicable Diseases, Sandringham, South Africa
(2) Charlotte Maxeke Hospital, South Africa
(3) Tshwane Academic Hospital, South Africa
(4) King Edward Hospital, South Africa
(5) Chris Hani Baragwanath Hospital, South Africa
(6) Universitas Hospital, South Africa
(7) Albert Luthuli Hospital, South Africa
(8) Tshepong Hospital, South Africa
(9) Dr. George Mukari Hospital, South Africa
(10) Polokwane State Hospital, South Africa
(11) 1 Military Hospital, South Africa
The standard of care for the treatment of chronic HCV hepatitis patients in South Africa is Pegylated-interferon and Ribavirin. HCV viral
genotype influences the type and period of response to therapy. Global data on responses of patients infected with genotype 5a, one of the
predominant genotypes found in South Africa (SA), is limited. The study described the response to treatment in single or mixed genotype
infections, particularly of genotype 5a, in SA. Patients (N=88) were prospectively enrolled from public hospitals in 4 provinces. The LiPA assay
(Siemens) was used to determine genotypes and the COBAS Ampliprep/TaqMan (Roche Diagnostics) for viral load at 0, 4, 12, 48 and 72 weeks.
Major HCV genotypes were: 5a,N=28(32%); 1b,N=18(21%); 3a,N=15(17%), 4,N=9(10%) and mixed infections N=3(3%). Seventy (80%)
individuals had started therapy, 4 were non-compliant, 4 were not eligible, 5 did not start and 5 had <4 weeks therapy. Twenty-four of
70 patients had genotype 5a. A sustained virological response (SVR) was observed in 40/70 (57%) individuals, 14/24 (58%) with genotype
5a; a rapid virological response (RVR) was observed in 8 (11%;5 genotype 5a and 3 genotype 4) and 1 (genotype 1a) had a complete early
virological response (cEVR). The SVR in patients with a RVR was higher (13/40, 32.5%) than in patients with cEVR (11/40, 27.5%, p=0.16).
There were 15 non-responders (21%, 6, genotype 1b), 1 patient (genotype 1b) had no RVR, 2 died (genotype 5a) due to complications
unrelated to therapy and 3 discontinued (adverse effects). One individual (genotypes 1b + 4) did not attain SVR while another (1b + 5a)
cleared virus at 32 weeks.
SVR was higher in patients with genotype 5a (58%) compared to those with genotypes 1b (53%) and 4(55%, p=0.12). Individuals with mixed
infections may require longer duration of therapy in mixtures of poor and good responder subtypes.
Contact: Nishi Prabdial-Sing
National Institute for Communicable Diseases, Sandringham, South Africa
[email protected]
POSTERS
Genotype 5a response to combination therapy in a South African study
POSTERS
P1.35
Using data mining techniques to explore baseline predictors of response to peginterferon ribavirin in 3720 chronic HCV Egyptian patients
Hadeel Gamal El-Deen (1), A. Awad (2), I. Farag, (2) A. Kamal (2), A. Kamel (2), W. El-Akel (1), W. Doss (1), N. Zayed (1), T. Awad (3),
A. Radwan ( 4), M. Mabrouk (1)
(1) Endemic Medicine and Hepatology Department, Faculty of Medicine, Cairo University
(2) Computer Science Department, Faculty of Computers and Information, Cairo University
(3) Cochrane Hepato-Biliary Group, Copenhagen Trial Unit, Rigshospitalet, Copenhagen, Denmark
(4) Academy of Scientific Research & Technology
Introduction: Because of the moderate efficacy of the treatment of interferon-ribavirin combination therapy in patients with HCV, there is
a need for an innovative technique to identify the characteristics of patients who will respond to treatment. Data mining analysis is used to
build predictive models and hence can predict the therapeutic outcome. Aim: Using decision tree learning algorithm to develop a model to
explore baseline predictors of response to PEG-IFN/RBV therapy in chronic HCV patients from routine clinical, laboratory, and histopathological
data. Patients and Methods: This retrospective study included pre-treatment data from 3720 Egyptian patients with chronic HCV treated by
PEG-IFN alpha and RBV. Decision tree learning algorithm (Weka implementation of C4.5) was applied for model building using 21 attributes.
Internal validation was performed with test mode of 10-fold cross-validation. External validation was done on set of 200 patients not included
in internal validation model. Results: End of treatment response (ETR) Responders were (54.2%), non-responders were (45.8%) including 757
patients (20.3%) who discontinued the treatment either due to side effect or non-compliance. At week 72 sustained virological response (SVR)
was (46%) with a relapse rate of (15%) of responders. Out of 20 attributes the decision tree showed 6 significant attributes (AFP, gender, age,
fibrosis, haemoglobin). At W48 the correctly classified patients were (60%) according to intention to treat protocol. After excluding patients
who discontinued the treatment (figure1) we get higher rates of prediction (70%) at W48 and (60%) at W72. The reproducibility of the model
was conﬁrmed by external validation set of 200 patients with correctly classified instances (65%). Conclusion: A pre-treatment decision tree
model with simple variables can be potentially useful for predicting the probability of response to anti-viral therapy in chronic HCV and has the
prospective to support clinical decisions regarding the proper selection of patients for therapy.
Contact: Hadeel Gamal El-Deen
Endemic Medicine and Hepatology Department, Faculty of Medicine, Cairo University
[email protected]
P1.36
Synergistic effect of calcitriol and interferon-α on hepatitis C virus replication in vitro
POSTERS
Christian Lange (1), Kenichi Morikawa (1), Michael Dill (2), Markus Heim (2), Jerome Gouttenoire (1), Darius Moradpour (1)
(1) University of Lausanne, Lausanne, Switzerland
(2) University of Basel, Switzerland
Background and aims: Vitamin D is an important modulator of numerous cellular processes, including innate and adaptive immune pathways.
We have recently established an association between the 1α-hydroxylase promoter single nucleotide polymorphism CYP27B1-1260 rs10877012
and sustained virologic response (SVR) after standard treatment of chronic hepatitis C in patients with a poor-response IL28B genotype. This
suggests an intrinsic role of vitamin D signalling in treatment response of chronic hepatitis C, especially in patients with limited sensitivity to
interferon-α (IFN-α). In the present study we investigated the effect of 1,25-(OH)2 vitamin D3 (calcitriol) alone or in combination with IFN-α
on the hepatitis C virus (HCV) life cycle in vitro. Methods: Huh-7.5 cells harboring HCV Con1- or JFH-1-derived replicons or cell culture-derived
HCV were exposed to 0.1-100 nM calcitriol +/- 1-100 IU/ml IFN-α. The effect on HCV replication and viral particle production was investigated
by quantitative real-time PCR, immunoblot analyses, and TCID50 determinations. The expression of interferon-stimulated genes (ISGs) and
of calcitriol target genes was assessed by quantitative real-time PCR. Results: Calcitriol had no relevant effect on the viability of Huh-7.5 cells.
Calcitriol strongly induced and repressed the expression of the calcitriol target genes CYP24A1 and CCNC, respectively, confirming that Huh-7.5
cells can respond to calcitriol signaling. Physiological doses of calcitriol did not significantly affect HCV RNA replication or infectious particle
production in vitro, and calcitriol alone had no significant effect on the expression of several ISGs. However, calcitriol in combination with
IFN-α substantially increased the expression of ISGs compared to IFN-α alone. In addition, calcitriol plus IFN-α synergistically inhibited HCV
RNA replication. Conclusions: Calcitriol at physiological concentrations and IFN-α act synergistically on the expression of ISGs and HCV RNA
replication in vitro. Experiments to address the underlying mechanisms are underway.
Contact: Christian Lange
University of Lausanne, Lausanne, Switzerland
[email protected]
104 : HCV 2011
ANTIVIRAL THERAPY
P1.37
The antimalarial drug Artemisinin and analogues inhibit in vitro HCV replication
Susan Obeid (2), Jo Alen (1), Christophe Pannecouque (2), Wim Dehaen (1), Johan Neyts (2), Jan Paeshuyse (2)
(1) Molecular Design and Synthesis Section, K.U.Leuven, Belgium
(2) Rega Institute for Medical Research, K.U.Leuven, 3000 Leuven, Belgium
We reported previously that Artemisinin (ART), a widely used anti-malarial drug, is able to inhibit in vitro HCV replicon replication (EC50= 78 ±
21 µM). We here demonstrate the anti-HCV activity of ART in hepatoma cells infected with HCVcc (JFH-1) (EC50= 167 ± 38 µM) and identified
an analogue, AJ-004, that it is at least 10-fold more potent and selective (EC50= 16 ± 4.0 µM). Remarkably, AJ-004 proved about 4-fold more
potent in inhibiting subgenomic HCV replication (EC50= 4.0 ± 0.1 µM) than HCVcc replication. We observed earlier that hemin results in a 14fold potentiation of the anti-HCV activity of ART in the HCV replicon assay. Hemin did only marginally potentiate the anti-HCV activity of ART in
HCVcc infected cultures (3-fold). Carbon-centred radicals have been shown to be critical for the anti-malaria activity of ART. We demonstrate
that none of the tested carbon-centred radical-trapping compounds (so-called TEMPO compounds) affected the anti-HCV activity of ART,
providing evidence that these radicals are not involved in the anti-HCV activity. A possible mechanism by which ART and analogues exert
activity against HCV may be by the induction of reactive oxygen species (ROS). Therefore, we studied the combined anti-HCV activity of either
ART or AJ-004 with L-N-Acetylcysteine (L-NAC) (the compound that inhibits ROS generation). L-NAC significantly reduced the anti-HCV activity
of ART and AJ-004. Taken together, we demonstrate that the in vitro anti-HCV activity of ART and analogue can, or at least in part, be explained
by induction of ROS and that carbon-centered radicals are not involved in the anti-HCV activity of these molecules.
Contact: Jan Paeshuyse
Rega Institute for Medical Research, K.U.Leuven, 3000 Leuven, Belgium
[email protected]
P1.38
Identification of new class inhibitors from a high-throughput screening against
hepatitis C virus
The current standard of care for the hepatitis C virus infected patients is a lengthy treatment with a combination of pegylated interferon-α
and ribavirin. Due to considerable side effects and limited efficacy, there is a need for new anti-viral inhibitors which can improve current
therapies. Since the development of the hepatitis C virus cell culture system (HCVcc) using the genotype 2a strain, JFH-1, potential new
target areas in addition to RNA replication are now accessible. In the quest for new antiviral inhibitors of entry or virus production, we
conducted a high-throughput luminescent screening of 2.2 million compounds using the J6JFH chimeric virus harboring a firefly luciferase
gene. The hit compounds scored as active from the primary screening at 2 µM were triaged by effects on cell viability and inhibition of
HCVcc and genotype 2a replicon activity. The compounds that were specifically inhibitory to the HCVcc assay were further investigated to
determine the mechanisms of action as entry or virus assembly. For instance, GSK1 blocks the entry process with a potency of a sub µM
EC50. It was specifically inhibitory to the 2a virus as it was less potent against the 1a/2a chimeric virus consisting of the H77 sequence from
core to NS2. A resistant mutation emerged in the transmembrane domain of E1 and was able to show resistance against GSK1. A series of
bona-fide viral assembly inhibitors were also identified and are currently being characterized to identify the targets and to evaluate their
suitability as drug candidates. In particular, the treatment of one series resulted in accumulation of core proteins around lipid droplets,
implicating its mechanism of action as an HCV assembly inhibitor.
Contact: Shihyun You
GlaxoSmithKline, RTP, NC, USA
[email protected]
POSTERS
Shihyun You (1), Richard Hazen (1), Cindy Richards (1), Eric Burroughs (1), Laurie Kane-Carson (1), Laurie Overton (1), Brandy Pollard (1),
Shane Holmes (1), Sara Grab (1), Jesse Keicher (1), Robert Hamatake (1)
(1) GlaxoSmithKline, RTP, NC, USA
POSTERS
P1.39
The picornavirus inhibitor enviroxime inhibits HCV RNA replication in vitro
Leen Delang (1), Pieter Leyssen (1), Johan Neyts (1)
(1) Rega Institute for Medical Research, K.U.Leuven, Leuven, Belgium
Enviroxime [2-amino-1-(isopropylsulfonyl)-6-benzimidazole phenyl ketone oxime] is since long known as an inhibitor of the in vitro
replication of rhinovirus and enteroviruses. Picornaviruses resistant to enviroxime carry mutations in protein 3A. It was recently shown that
enviroxime-like compounds inhibit RNA replication of picornaviruses by inhibiting phosphatidylinositolkinases (PIK) (Arita et al, 2011). The
activity of PI4KIIIβ regulates the synthesis of enteroviral RNA by creating a microenvironment of phosphatidyl-4 phosphate lipids at the viral
replication complexes. Interestingly, PI4KIIIα was recently shown to be an important host factor for HCV replication. Knockdown of PI4KIIIα
expression drastically changed the membranous web morphology and disturbed viral RNA replication (Reiss et al, 2011). We here report
that enviroxime selectively inhibits HCV genotype 1b subgenomic replicon replication. Viral RNA replication is inhibited in a dose-dependent
manner with an EC50 value of 0,26 ± 0,06 µM (CC50= 28 µM). Following 8 weeks of culturing, enviroxime-resistant replicons were generated
(EC50= 19 µM or 73x wild-type EC50). Enviroximeres replicons retain wild-type susceptibility to other HCV inhibitors [VX-950 (protease
inhibitor), 2’-C-methylcytidine (nucleoside polymerase inhibitor), VX-222 (non-nucleoside polymerase inhibitor) and BMS-790052 (NS5A
inhibitor)]. Genotyping of the resistant replicon population revealed the presence of several mutations in NS4B and in NS5A. Swapping of
NS5A of the enviroximeres replicon into a wild-type genome did not result in a transfer of the resistant phenotype. Therefore, mutations
in NS5A are probably not responsible for enviroxime resistance. We are currently exploring the role of NS4B mutants in the enviroximeres
phenotype and the precise mechanism by which inhibition of PI4KIII results in inhibition of HCV replication. Interestingly, both HCV NS4B
and picornavirus 3A are membrane associated proteins that are involved in the formation of the membranous web.
Contact: Leen Delang
Rega Institute for Medical Research, K.U.Leuven, Leuven, Belgium
[email protected]
P1.40
HCV targets the JAK/STAT pathway for degradation to reduce anti-viral responses
to IFN-α
POSTERS
Nigel Stevenson (1), Nollaig Bourke (1), Catherine Keogh (1), Marco Binder (2), Elizabeth Ryan (1), Sinead Keating (1), Aideen Collins (1),
Andrew Bowie (1), James Johnston (3), John Hegarty (4), Cliona O’Farrelly (1)
(1) Trinity College Dublin, Dublin, Ireland
(2) Heidelberg University, Germany
(3) Queen’s University of Belfast, United Kingdom
(4) St. Vincent’s University Hospital, Dublin, Ireland
The anti-viral cytokine interferon (IFN)-α signals through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway,
leading to upregulation of anti-viral genes. IFN-α is used to treat patients infected with Hepatitis C Virus (HCV). However, many HCV infected
patients do not clear the virus naturally and IFN-α therapy fails in a large proportion, for reasons that are poorly understood. We found that
STAT3 protein of the IFN-α pathway is absent in leukocytes and hepatocytes of HCV infected patients. Furthermore, we report that STAT3 could
be restored by proteasomal inhibition. These findings outline a mechanism by which HCV promotes STAT3 degradation via the host E3 ligase/
ubiquitin/proteasome system by mimicking a specific E3 ligase complex. The loss of STAT3 had significant functional consequences during the
anti-viral response to IFN-α, including reduced IFN stimulated gene (ISG) induction and vaccinia virus clearance in vitro. STAT3 recovery during
proteasomal inhibition led to a restoration of ISG induction in immune cells and hepatocytes expressing HCV, demonstrating an anti-viral
function for STAT3 in the IFN-α pathway, targeted by HCV to evade immunity. Our findings reveal a novel molecular mechanism used by HCV to
silence the innate immune response and highlight STAT3 as an essential anti-viral protein during IFN-α signalling.
Contact: Nigel Stevenson
Trinity College Dublin, Dublin, Ireland
[email protected]
106 : HCV 2011
ANTIVIRAL THERAPY
P1.41
Quasispecies variability in NS5A region of Hepatitis C virus genotype 1 and
therapy response
Ana Carolina Jardim (1), Cíntia Bittar (1), Renata Matos (1), Paola Provazzi (1), Lílian Yamasaki (1), Rafael Silva (2), João Renato Pinho (3),
Claudia Márcia Carareto (1), Isabel Maria Carvalho-Mello (2), Paula Rahal (1)
(1) São Paulo State University, São José do Rio Preto, Brazil
(2) Butantan Institute, Brazil
(3) University of São Paulo, Brazil
The quasispecies composition of Hepatitis C virus (HCV) could have important implications with regard to viral persistence and response to
interferon-based therapy. The complete NS5A was analyzed to evaluate whether the composition of NS5A quasispecies of HCV 1a/1b is related
to responsiveness to combined interferon pegylated (PEG-IFN) and ribavirin therapy. Six hundred and ninety full-length NS5A sequences were
generated from samples collected before, during and after treatment from virological sustained responder, non-responder and the end-oftreatment responder patients. This study provides evidence that homogeneity of quasispecies composition, low diversity and less complexity of
the NS5A region pre-therapy are associated with viral clearance. Therefore, higher diversity and complexity of quasispecies could offer the virus
a better opportunity of evading anti-viral therapy. Phylogenetic relationships concerning complete NS5A sequences obtained from patients
did not demonstrate clustering associated with specific response patterns. However, distinctive clustering of pre/post-therapy sequences was
observed, suggesting that an evolutionary process occurred during the time course examined. In addition, the evolution of quasispecies over
time was subjected to purifying or relaxed purifying selection. This could explain the initial diversified composition of quasispecies at baseline,
followed by an increase in the frequency of a predominant quasispecies in ‘after treatment’ samples of non-responders and end-of-treatment
responders, probably because it offers some advantage for the virus. These results suggest that quasispecies diversity of the NS5A region could
be important for elucidating the mechanism underlying treatment failure in patients infected with chronic hepatitis C.
Contact: Paola Provazzi
São Paulo State University, São José do Rio Preto, Brazil
[email protected]
P1.42
Raffaele de Francesco (1), Cristina Cheroni (1), Lorena Donnici (1), Annalisa Bianco (1), Suwanna Noppornpanth (1), Vincenza Valveri (1),
Massimiliano Pagani (1), Roberta Soffredini (3), Gian Maria Prati (3), Alessio Aghemo (3), Maria Grazia Rumi (3), Massimo Colombo (3),
Petra Neddermann (1), Sergio Abrignani (1)
(1) INGM-Istituto Nazionale Genetica Molecolare, Milano, Italy
(2) A.M. Migliavacca Center for Liver Disease, First Division of Gastroenterology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Milano,
Università degli Studi di Milano, Italy
(3) Division of Hepatology, Ospedale San Giuseppe, Universitá degli Studi di Milano, Italy
HCV particles with defective RNA genomes have been identified in the serum of some hepatitis C patients. The defective genomes are
characterized by deletions of up to 2 kb in size, encompassing the region encoding the viral envelope proteins. Such HCV defective genomes
are capable of autonomous RNA replication and they are packaged into infectious viral particles in cells co-infected with the wild-type virus.
In this study, we examined the relationship between the presence of deletion mutants in the serum of HCV-infected individuals and disease
severity or the outcome of PEG-IFN/RBV combination therapy. Study subjects were 132 individuals, chronically infected with genotype 1 HCV, who were treated with the PEG-IFN plus RBV combination
therapy at the Liver Center, Ospedale Maggiore, Milan (Italy). We identified HCV defective genomes in the serum of an unexpectedly high
fraction of genotype 1 hepatitis C chronic patients (19%). The presence of HCV deleted genomes was found to be associated with higher
viremic levels (P=0.04) and with the presence of hepatic necro-inflammatory activity (P=0.005) but not with the IL28B host genotype.
Although we could not detect a significant association between the presence of HCV defective genomes and treatment failure overall,
virological relapse was observed in 60% of the patients carrying HCV with defective genomes compared to 30% of the patients infected with
only the full-length virus (P<0.05). In conclusion, we found that, in chronic hepatitis C patients, the presence of HCV with defective genomes was associated with hepatic necroinflammatory lesions. Furthermore, the presence of defective HCV RNA may represent a novel predictor of relapse after initial virological
response following PEG-IFN/RBV combination therapy.
Contact: Raffaele de Francesco
INGM-Istituto Nazionale Genetica Molecolare, Milano, Italy
[email protected]
POSTERS
The presence of hepatitis C virus deletion mutants in chronic hepatitis C patients and
their role during PEG-IFN/Ribavirin therapy in HCV-1 patients
POSTERS
P1.43
MicroRNA profiling identifies potential anti-viral targets induced by interferon alpha in
human hepatoma cells expressing hepatitis C virus
Marybeth Daucher (1), Xiaozhen Zhang (1), David Armistead (2), Rodney Russell (3), Shyamasundaran Kottilil (1)
(1) NIH-NIAID-Laboratory of Immunoregulation, Bethesda, MD, USA
(2) Applied Biosystems, Inc., USA
(3) Health Sciences Center, Memorial University of Newfoundland, Canada
Background and Aims: MicroRNAs (miRNAs) are members of a class of small noncoding functional RNAs that modulate gene expression
at the post-transcriptional level by degrading or suppressing the translation of target mRNAs. Increasing evidence suggests that miRNAs
have a profound impact on host defense to HCV infection and clinical outcome of standard HCV therapy. The objective of this study was
to investigate global changes in miRNA expression profiles using an HCV continuous culture system and IFN-α in order to identify novel
biomarkers that may more efficiently predict therapeutic outcome for HCV infection.
Methods: Microarray profiling was performed on RNA derived from Huh-7.5 cells infected with HCV genotype 2a (J6JFH) and treated with
IFN-α. miRNAs associated with HCV infection and interferon response were validated by real-time RTPCR and miRNA mimics and inhibitors.
Results: Seven miRNAs (miR-324, -30c, -130a, -301, -30b, -192 and -565) were down-regulated in HCV-infected Huh-7.5 cells (p < 0.05) and
12 miRNAs were up-regulated in HCV-infected Huh-7.5 cells treated with IFN-α (p < 0.01). Interestingly, the same 7 miRNAs that were downregulated in HCV-infected Huh-7.5 cells without IFN-α were significantly up-regulated following IFN-α treatment. Silencing of miR-30b,
miR-30c, and miR-192 using miRNA inhibitors increased HCV replication in HCV-infected Huh-7.5 cells suggesting a role for these miRNAs in
the endogenous control of HCV replication.
Conclusion: The differential expression of a number of miRNAs is affected by the presence of HCV in vitro and can be altered by the addition
of IFN-α suggesting that miRNAs may be attractive biomarkers for predicting therapeutic outcome in HCV infection.
Contact: Marybeth Daucher
NIH-NIAID-Laboratory of Immunoregulation, Bethesda, MD, USA
[email protected]
P1.44
Biliverdin restores type I interferon expression in cells expressing HCV NS3/4a protease
Zhaowen Zhu (1), Meleah Mathahs (1), Warren Schmidt (1)
(1) Research Service, VA, Internal Medicine, University of Iowa, Iowa City, IA, USA
POSTERS
Introduction: BV, a tetrapyrrole product of heme oxidation, inhibits HCV replication through mixed competitive and non-competitive
inhibition of the HCV NS3/4a protease. BV also increases interferon expression and enhances the antiviral activity of α-interferon in vitro.
Herein, we hypothesized whether BV anti-protease activity would also influence type I interferon induction and enhance innate immune
system signaling.
Methods: The effect of BV and bilirubin (BR) on type I interferon induction was assessed in several replicon and control cell lines after
incubation with tetrapyrrole alone or following stimulation with double-stranded (ds) DNA or RNA. Interferon induction was quantified
using luciferase reporter constructs containing IFN-beta1 or ISRE promoters. NS3/4a inhibition of interferon induction was evaluated after
transfection of cells with expression vectors containing NS3/4a sequences.
Results: BV and to lesser extent, BR showed a mild increase in expression of IFN-alpha2 and IFN-beta1 in HEK293, Huh7 and Huh5-15
nonstructural replicons but not in Huh7.5 or Huh7.5 cells with replicons. The tetrapyrrole effect was dose-dependent with BV more potent
than BR. HEK293 or Huh7 cells stimulated with dsDNA or dsRNA showed robust induction of type I interferon that was severely attenuated
in cells co-transfected with NS3/4a. However, concomitant treatment of NS3/4a expressing cells with BV dramatically restored dsDNA or
dsRNA interferon induction. In contrast, BR showed only minimal ability to restore interferon induction in the presence of NS3/4a, indicating
that BV activity can not be attributed to reduction of BV to BR.
Conclusions: BV can modestly induce type I interferon, but dramatically restores dsDNA or dsRNA type I interferon signaling after inhibition
with HCV NS3/4a protease. These findings support our recent data demonstrating that BV is a potent NS3/4a protease inhibitor. Furthermore,
our data support the hypothesis that that BV and or related derivatives would be useful antiviral drugs for treatment of chronic HCV infection.
Contact: Zhaowen Zhu
Research Service, VA, Internal Medicine, University of Iowa, Iowa City, IA, USA
[email protected]
108 : HCV 2011
ANTIVIRAL THERAPY
P1.45
Transplacental transfer of hepatitis B immune globulin in an animal model
Evi Struble (1), Li Ma (1), Lilin Zhong (1), Pei Zhang (1)
(1) CBER, FDA, Bethesda, MD, USA
Hepatitis B virus (HBV) can be vertically transmitted from a pregnant woman to her child during delivery. Hepatitis B Immune Globulin
Intravenous (Human) (HBIGIV) is approved by the FDA for use in newborns of infected mothers as well as for immune prophylaxis following
HBV exposure. However, for unknown reasons, HBIGIV treatment of neonates is effective in only 50% of cases.
Our research aims to explore the use of HBIGIV during the last third of pregnancy. The overall goal is to determine if neutralizing antibody
levels in HBIGIV 1) can protect the chronically infected pregnant woman from HBV exacerbation and preterm delivery, and 2) block
transmission of HBV to her baby. To do this, a relevant animal model for transplacental HBIGIV transfer is sought.
To date, no clinical or preclinical studies have been performed during pregnancy with HBIGIV or other polyclonal preparations to characterize
maternal pharmacokinetics, clearance and the transplacental transfer of neutralizing antibody. In a pilot experiment, we tested the feasibility
of the guinea pig as an animal model to study transplacental transfer of human IgG in late pregnancy. Pregnant guinea pigs or age matched
non-pregnant controls received HBIGIV on day 65 of pregnancy at a dose of 50 IU/kg. Blood levels of human IgG, IgG subclasses and HBV
neutralizing antibodies in mothers and newborn animals were measured and compared to putative protective levels.
We found that human IgG does transfer from the pregnant sow to her offspring. Further, HBIG titers that are correlated with protection
from HBV infection in humans can be measured in the sow and the newborn guinea pigs. A follow-up study containing a larger sample size
is being performed.
Contact: Evi Struble
CBER, FDA, Bethesda, MD, USA
[email protected]
P1.46
Identification of hepatitis C virus inhibitors targeting different aspects of virus life cycle
Hepatitis C virus (HCV) infection is a major health problem that affects 2-3% of worldwide population. Because current treatment options
are not effective in all patients and are associated with significant toxic side effects, there is an ongoing need to identify novel anti-HCV
agents. As such, we previously developed a cell-based hepatitis C virus infection assay for high-throughput antiviral compound screening
(Yu et al, 2009 Antimicrob Agents Chemother. 53(10):4311-9). Here, we report the screening of the NCI Diversity Set library containing
1974 synthesized chemical compounds and the identification of potential anti-HCV compounds. Using our HCV infection screening assay
in combination with toxicity counter screening, we identified 30 hits from the 1974 compound library. Twenty of the hits subsequently
showed dose-dependent inhibition of intracellular HCV RNA when present during low moi infection which allows for multiple rounds of
viral replication and spread. Using HCV pseudotype and replicon systems of multiple HCV genotypes, as well as JFH-1 based assembly and
secretion analysis, we determined that different compounds within this group target these different steps of the viral life cycle. The utility of
these HCV inhibitors as probes to study the viral life cycle or for further HCV drug development are under investigation.
Contact: Xuemei Yu
Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA
[email protected]
POSTERS
Xuemei Yu (1), Susan L. Uprichard (1)
(1) Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA
POSTERS
P1.47
Analysis of the inhibition of HCV replication with different RNAi mediators
Sandra Jovanna Gonzalez-Rojas (1), Abdullah Ely (2), Marta Garcia-Valdecasas (1), Imanol Fernandez-Perez (1), Elena Carnero (1), Patrick
Arbuthnot (2), Puri Fortes (1)
(1) FIMA, Pamplona, Spain
(2) University of Witwatersrand Wits., South Africa
RNA interference (RNAi) has great potential for antiviral therapeutic application. Indeed, RNAi has proved to be efficient for the inhibition of
viral replication of many viruses such as hepatitis B or C viruses. Hepatitis C virus (HCV), as a positive-stranded RNA virus, is an attractive target
for RNAi therapy. However, HCV replicase lacks proof-reading activity and generates great HCV genome sequence variation that frequently
results in the emergence of viral mutants that evade suppression by a single RNAi inhibitor. Ways of overcoming this problem are (i) to target
conserved areas of HCV genome and (ii) to use several RNAi inhibitors simultaneously. Highly conserved areas have been located in the
5’UTR and the NS5B region of the HCV genome. Therefore, we have constructed expression cassettes that generate several RNAi inhibitors
and target these regions. The NS5B region was targeted with long double stranded hairpin RNAs (lhRNAs) of varying lengths that serve as
substrates for generating multiple small interference RNAs (siRNAs). Seven independent short hairpin RNAs (shRNAs) expressed from a
polymerase III promoter were used to target the 5’ UTR. These 7 shRNAs have been also been incorporated into pri-miR-31 derived shuttle
scaffolds to enable expression from liver-specific and inducible polymerase II promoters. The efficacy of these inhibitors will be compared in
cells expressing different HCV replicons after stable or transient electroporation or in HuH7 cells infected with HCV. Combinations of several
inhibitors will be used to evaluate improvement of efficacy and prevent emergence of HCV escape variants. Demonstration of improved
suppression of HCV replication using this approach will have potential therapeutic application.
Contact: Elena Carnero
FIMA, Pamplona, Spain
[email protected]
P1.48
Epigallocatechin gallate inhibits infectivity of HCV by preventing attachment of virions
to cells
POSTERS
Che C. Colpitts (1), Sandra Ciesek (2), Eike Steinmann (2), Luis M. Schang (3)
(1) Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada
(2) Experimental Virology, Twincore, Hannover, Germany
(3) Biochemistry/Li Ka Shing Institute of Virology, University of Alberta, Canada
Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea. The antiviral activity of EGCG against unrelated viruses, including
influenza and HIV-1, has been described, but the mechanisms of action remain unclear. As previously shown by Ciesek and Steinmann, EGCG
also inhibited the infectivity of HCV virions. We used a focus forming assay, in which HCV virions were exposed to EGCG prior to infection of
Huh7.5 cells. Infected cells were fixed at 3 days post-infection and processed for immunocytochemistry for HCV core protein. EGCG inhibited
HCV JFH-1 infectivity (IC50, 1.7 μM). EGCG also inhibited the infectivity of VSV and HSV-1 (IC50, 1 and 0.25 μM, respectively). Using VSV and
HSV-1 as models, we started to test the potential antiviral mechanisms. EGCG directly acted on the virions, not the cells. VSV and HSV-1
virions labeled at self-quenching concentrations with the membrane dye octadecyl rhodamine chloride (R18) were exposed to EGCG for 10
minutes at 37°C. There was no dequenching of R18 fluorescence under these conditions, indicating that EGCG does not lyse virions. We used
fluorescence dequenching assays to monitor fusion between EGCG-treated R18-labeled VSV and Vero cells. Fluorescence was dequenched
by 35% when virions were exposed to vehicle, and by 30% when they were exposed to 20 μM EGCG, which inhibits plaquing efficiency by
100%. Therefore, EGCG had minimal effects on viral fusion. In contrast, EGCG inhibited virion attachment. We inoculated cells at 4°C with
EGCG-treated R18-VSV and HSV-1 before removing the inoculum, washing the cells and measuring the fluorescence attached to cells. R18
fluorescence was decreased by 87% for HSV-1 virions treated with 20 μM EGCG. EGCG therefore inhibited virion binding. In conclusion, we
show that EGCG inhibits infectivity of enveloped viruses by preventing attachment of virions to cells.
Contact: Che C Colpitts
Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada
[email protected]
110 : HCV 2011
ANTIVIRAL THERAPY
P1.49
MicroRNA signatures in chronic hepatitis C virus induced insulin resistance as a
potential therapeutic approach
Gokul Das (1), Chad Creighton (1), F. Blaine Hollinger (1)
(1) Baylor College of Medicine, Houston, TX, USA
Chronic HCV infection induces insulin resistance (IR) and IR is associated with progression of liver disease and a reduced response to IFN
therapy, which again necessitates premature withdrawal due to various side effects. Our goals are to understand the molecular basis of IR
and to develop a better well-tolerated therapeutic strategy. We characterized a chronically HCV infected cell line that show evidence of IR by
a disruption of glucose homeostasis and insulin signaling by IRS-1 Ser 312 phosphorylation, which might interfere again with IFN signaling.
Identification of miRNAs and targets have therapeutic potential, but it is unknown how miRNA signature changes during IR development
and how it is associated with IFN sensitivity. We hypothesize that coordinate expression of multiple miRNAs target signaling pathways for
IR and IFN response pathways and understanding their interaction will facilitate therapeutic development singly or in conjunction with IFNbased therapy. We undertook a global approach to identify anticorrelated miRNAs/mRNA pairs in HCV infected and uninfected Huh 7 cells
to elucidate how various pathways are integrated to regulate IR and IFN response. We have identified 18 miRNAs differentially regulated by
>1.4 and <0.7 fold (p<0.01) in HCV infected cells and identified anticorrelated miRNA/mRNA pairs. In IFN treated infected cells, several of
these miRNAs are regulated oppositely, but several others are up regulated. These miRNAs potentially target genes with unknown or novel
function involved in glucose homeostasis, insulin signaling, IFN signaling, apoptosis and autophagy. MicroRNAs, miR 23a, miR 27a, miR 206,
miR 130 and miR 207 are of particular interest as they are involved in molecular pathways involved in insulin and IFN signaling. One or more
of these miRNAs may serve as target to reduce IR and improve IFN’s response, which will be validated in a humanized mouse model under
development in our laboratory.
Contact: Gokul Das
Baylor College of Medicine, Houston, TX, USA
[email protected]
P1.50
Philippe Guedat (1), Amaya Berecibar (1), Angelina Bertrand (1), Luc Batard (1), Caroline Evain-Freslon (1), Céline Fernagut (1), Mehdi
Lahmar (1), Aurélie Martin (1), Annick Piessens (1), Isabelle Vallarche (1), Majid Mehtali (1)
(1) VIVALIS, Saint-Herblain, France
Anti-hepatitis-C therapeutics targeting viral enzymes are being developed but tend to induce rapid resistance. Interest is growing for
candidate molecules affecting viral proteins functions indirectly or targeting host cell factors with the advantages of a higher genetic
barrier to resistance and broader activity on HCV genotypes. The 3D-SCREEN platform is an innovative human cell-based assay to identify
compounds that alter the tri-dimensional structure of target proteins. By screening for conformational rather than for functional alterations,
promising modulators acting through original mechanisms of action (MOA) have been identified.
A 3D-SCREEN platform was generated for genotype 1b native NS5B polymerase and validated with known allosteric inhibitors. Screening of
our small molecule library led to the identification of original chemical series. Interestingly, lead optimization through medicinal chemistry
enabled to improve by two logs the activity (<60nM) both on the 3D-SCREEN and replicon assays but not on the purified protein’s enzymatic
assay. Moreover, no significant changes in expression or recruitment of viral proteins to the replication complex were observed between
cells treated with our leads and non-treated cells. Several escape replicon mutants were identified and still under investigation. All these
results suggest an indirect MOA; fishing of the involved factors is being performed by affinity chromatography. In addition, the technology
was developed to profile compounds cross-resistance. Escape point mutations described in vitro and/or in vivo were introduced in NS5B in
order to generate as many 3D-SCREEN NS5B platforms. None of the tested variants was resistant to our candidate compound. Furthermore
our compound is active on genotype 1a and JFH1-based cell culture assay. ADME results will be also presented.
We will also present data with a new class of anti-NS3 molecule (non peptidomimetic with a MW<500) that we have identified with the
3D-SCREEN technology, and having an EC50 of 13nM on replicon assay.
Contact: Philippe Guedat
VIVALIS, Saint-Herblain, France
[email protected]
POSTERS
Preclinical data of a new class of anti-HCV compounds acting as conformational
modulators and having different MOA than all known molecules in clinic
POSTERS
P1.51
Partial knockdown of cyclophilin A sensitizes HCV replicons to cyclosporin A inhibition
Margaret Robinson (1), Katie Chan (1), Andrew Greenstein (1), William Delaney IV (1)
(1) Gilead Sciences, Foster City, CA, USA
Background & Aims: Cyclophilin A (CypA) is an essential cellular cofactor for Hepatitis C Virus (HCV) replication. While intracellular levels of
CypA have been estimated at 2 µM, CypA inhibitors, such as cyclosporin A (CsA) can inhibit HCV replication at concentrations below 200 nM.
To investigate the relationship between CypA concentration and HCV replication, we determined CsA potency in CypA knockdown cell lines.
Methods: CypA knockdown cell lines were generated via lentiviral shRNA. Western blot analysis was performed to confirm knock down and
quantify CypA levels. Replication of genotype 2a subgenomic replicon (JFH1), genotype 2a virus (JC-1 Rluc), or CsA-resistant genotype 1a
replicons were monitored with or without CsA in WT or CypA-knockdown cells.
Results: Using the JFH1 replicon, we observed a 92-fold increase in potency of CsA when CypA levels were reduced by more than 98%. A
modest 7-fold increase in CsA potency was demonstrated against the 2a virus (JC-1 Rluc) when CypA levels were reduced by 50%. Resistance
selection with CsA in genotype 1a replicon cells selected the D320E mutation in NS5A. When re-introduced into the wild-type 1a replicon, a
3-fold decrease in susceptibility to CsA was observed. Furthermore, the D320E replicon replicated efficiently in the CypA knockdown cells,
but the wild-type replicon did not.
Conclusions: CypA knockdown cell lines are useful tools for studying the effects of CypA level on HCV RNA replication and the antiviral
activity of CypA inhibitors. The NS5A D320E mutation restored replicon replication in CypA-knockdown cells but conferred only modest
resistance to CsA. Together these results suggest that cellular CypA concentrations limit the antiviral potency of CsA.
Contact: Margaret Robinson
Gilead Sciences, Foster City, CA, USA
[email protected]
P1.52
Differential reduction of hepatitis C viral load by viral entry and replication inhibitors in
a persistently-infected cell-culture system
Caroline Bush (1), Matthew Paulson (1), Weidong Zhong (1), Rudolf Beran (1)
POSTERS
Drug development of direct acting antivirals aganst HCV is focusing on antiviral combinations that can lead to sustained HCV suppression
and curing of HCV. Identification of the optimal combinations from various inhibitor classes of different antiviral mechanisms is essential to
this goal. Mathematical modeling predicts that inhibitors preventing HCV entry will reduce viral load in a monophasic manner, reflecting the
slow (t1/2~0.5-70 days) death rate of infected cells and assuming that virus is blocked from infecting naïve cells. In contrast, viral replication
inhibitors are predicted to reduce viral load in a biphasic manner, with the initial rapid reduction phase due to elimination of cell-free viruses
in plasma (t1/2~ 3 hours) followed by a slower reduction phase resulting from infected hepatocyte death.
To test the modeling projections in vitro, we established persistently-infected cell cultures to evaluate the effects of various classes of HCV
inhibitors in reducing viral load over time. Such a model bypasses the cytopathic effects observed when HCV-infected cells are passaged.
Huh-7 cells grown in the presence of DMSO can be infected with an cell culture adapted HCVcc. Subsequently, inhibitor compounds were
added to fully-infected cell-cultures, aliquots of the medium were then removed over time, and viral load was determined. High doses
of viral entry inhibitors did not greatly affect the steady-state viral load during a 7-day treatment. In contrast, addition of HCV replication
inhibitors significantly reduced viral load in a biphasic manner. These results confirm that HCV replication inhibitors can be adequately
assessed by measuring plasma viral RNA reduction in short-term monotherapy studies. However, entry inhibitors may require relatively
long-term studies in order to adequately assess their antiviral effect.
Contact: Caroline Bush
[email protected]
112 : HCV 2011
ANTIVIRAL THERAPY
P1.53
Antiviral responses with nucleotide analogs PSI-7977 and PSI-938 are independent of
IL28B genotype
Melanie Cornpropst (1), Eric Lawitz (2), Maribel Rodriguez-Torres (3), Jill Denning (1), Desiree Clemons (1), Lindsay McNair (1), Lei Fang
(4), Michelle Berrey (1), William Symonds (1)
(1) Pharmasset, Durham, NC, USA
(2) Alamo Medical Research, USA
(3) Fundacion de Investigacion de Diego, Puerto Rico
(4) Pharstat, Inc., USA
The rs12979860 SNP in the IL28B gene locus is an important predictor of antiviral response including SVR in subjects treated with the
current standard of care for HCV, PEG/RBV. Combinations of DAA with PEG/RBV have demonstrated varying impact of IL28B genotype on
response, potentially related to antiviral potency and/or barrier to resistance. It is unknown whether IL28B genotype will influence antiviral
response in IFN-free regimens of potent DAA, including nucleotide analogs PSI-7977 and PSI-938. Methods: The NUCLEAR study consisted
of 4 cohorts of treatment-naïve non-cirrhotic patients with HCV GT1: 1) PSI-938 d1-14; 2) PSI-938 d1-7 and PSI-938+PSI-7977 d8-14; 3)
PSI-7977 d1-7 and PSI-938+PSI-7977 d8-14; or 4) PSI-938+PSI-7977 d1-14. 10 patients/cohort (2 placebo) received PSI-938 300mg QD and/
or PSI-7977 400mg QD. Cohorts were not stratified by IL28B genotype. Results: Of 32 subjects receiving active therapy, 12 (38%) were CC.
Mean baseline HCV RNA levels were higher in patients with CC (6.93 log10 IU/mL), compared to non-CC (6.13 and 5.74 log10 IU/mL for CT
and TT genotypes, respectively). All 32 subjects experienced profound antiviral suppression (median 5.0-5.2 log10 decline over 14 days)
with no viral breakthrough. Half of subjects on PSI-938 monotherapy, and a combined 92% in combination cohorts achieved HCV RNA <
LOD. Across all 4 cohorts, there was a median time to <LOD of 9.3 days. Subjects with CC achieved HCV RNA <LOD at 9.0 days and non-CC
subjects achieved <LOD at 9.3 days (p=0.61). Conclusion: IL28B genotype did not influence antiviral responses during 14 days of PSI-938
monotherapy or PSI-7977/PSI-938 combinations. CC genotype was associated with higher baseline HCV RNA, which was a predictor of
time to HCV RNA < LOD. Nevertheless, profound viral reductions were observed in all cohorts and in all IL28B genotypes, with no viral
breakthrough during 14 days of potent nucleotide therapy.
Contact: Melanie Cornpropst
Pharmasset, Durham, NC, USA
[email protected]
P1.54
Guangwei Yang (1), Yongsen Zhao (1), Joanne Fabrycki (1), Dharaben Patel (1), Akihiro Hashimoto (1), Godwin Pais (1), Atul Agarwal (1),
Avinash Phadke (1), Milind Deshpande (1), Mingjun Huang (1)
(1) Achillion Pharmaceuticals, Inc, New Haven, CT, USA
Background: HCV NS2 is an autocatalytic cysteine protease that is responsible for autocleavage at the junction of NS2 and NS3. This
autocleavage is essential for HCV RNA replication in vitro and in vivo. Hence, NS2 protease is an attractive target for antiviral drug discovery.
Here we report an approach developed for discovery of NS2 protease inhibitors.
Methods: (i) A cell line was constructed that harbored a HCV subgenomic replicon encoding a luciferase in its 1st citron and an NS2-5B polyprotein
in its 2nd citron. Compounds were screened in this cell line for their antiviral activity and cellular toxicity. ii) A pCI-neo based plasmid carrying
the complete NS2-3 region was constructed for expression and processing of NS2-3 protein in the coupled transcription/translation system. Hits
discovered from aforementioned replicon screening were examined for their effect on the cleavage between NS2 and NS3.
Results: Compounds were screened in the replicon. These compounds include known cysteine protease inhibitors, HSP90 inhibitors and
new chemical entities synthesized based on NS2 protease cleavage substrate or product. Leads were identified from these compounds that
displayed EC50 values ranging from submicro-molar to micro-molar in concentration. The inhibitory effect of some of these leads on the
cleavage between NS2 and NS3 was observed in the coupled transcription/translation system.
Conclusion: An approach that combined an NS2 protease-dependent replicon assay with an in vitro NS2-protease cleavage assay was
described. Using this approach, promising lead compounds were identified as potential NS2 protease inhibitors.
Contact: Guangwei Yang
Achillion Pharmaceuticals, Inc, New Haven, CT, USA
[email protected]
POSTERS
Approach for discovery of HCV NS2 protease inhibitors
POSTERS
P1.55
Antiviral and Preclinical Profiles of HCV NS5A Inhibitors IDX380 and IDX719
John Bilello (1), Cyril Dousson (1), Massimiliano La Colla (1), Christopher Chapron (1), Sanjeev Bhadresa (1), Michelle Camire (1), Ilaria
Serra (1), Joshua Gillum (1), Lisa Lallos (1), Joseph McCarville (1), Xin-Ru Pan-Zhou (1), Marita Larsson Cohen (1), David Standring (1)
(1) Idenix Pharmaceuticals, Inc., Cambridge, MA, USA
Objectives: In this study, we describe the anti-HCV and early pharmacokinetic profiles of IDX380 and IDX719, NS5A-targeted compounds
that inhibit the replication of multiple HCV genotypes.
Methods: The in vitro activities of IDX380 and IDX719 were evaluated against an infectious HCV virus and replicons of multiple HCV genotypes.
The resistance profiles of these compounds were determined in replicon selection experiments as well as site-directed mutant replicons.
IDX380 and IDX719 were examined for cell permeability, effects of human serum proteins, CYP450 inhibition and PK characteristics in monkeys.
Results: IDX380 and IDX719 EC50 values were <50 pM and CC50 values >100 µM against an HCV infectious virus and replicons representing
HCV genotypes 1-5. The resistance profiles of IDX380 and IDX719 were comparable to other NS5A inhibitors and no cross-resistance was
observed between these compounds and other classes of direct acting HCV antivirals. Antiviral potency was reduced by <10- or <20-fold
in the presence of 45% or 100% (extrapolated) human serum, respectively, while AAG had no significant effect (<3-fold). The following
characteristics were observed in vitro: low potential for inhibition of 7 CYP450 isozymes; stability in whole blood; low to moderate
permeability in a Caco-2 assay with no P-glycoprotein efflux; and low clearance in human hepatocytes. Plasma drug levels remained far
above the EC50 values 24 h after administration of a single 10 mg/kg oral dose in the monkey.
Conclusions: IDX380 and IDX719 demonstrate low pM potency against HCV genotypes 1a, 1b, 2a, 3a, 4a and 5a with high selectivity indices
in vitro and favorable pharmacokinetic properties. The resistance profiles of these compounds are comparable to other NS5A inhibitors.
These favorable in vitro and in vivo characteristics warrant further preclinical investigation of IDX380 and IDX719 with the goal of choosing
one as the preferred clinical candidate for a regulatory filing this year.
Contact: John Bilello
Idenix Pharmaceuticals, Inc., Cambridge, MA, USA
[email protected]
P1.56
Pegylated-interferon/ribavirin treatment causes an increase in vitamin D and a
decrease in serum calcium
POSTERS
Andrea D. Branch (1), Catherine Constable (1), Kian Bichoupan (1), Peter Benedict (1), Marie-Louise Vachon (1), Douglas Dieterich (1),
Amir Soumekh (1)
(1) Mount Sinai School of Medicine, New York, NY, USA
Background: It is important to examine vitamin D and calcium levels in patients undergoing HCV treatment because low 25-hydroxyvitamin
D levels predict treatment failure and because many pharmaceutical agents alter bone and calcium metabolism. HIV/HCV positive patients
have an especially high prevalence of bone disease.
Methods: We retrospectively examined data from the Hepatitis C Research Network 004 study, a prospective, multi-center trial of HIV/HCV
co-infected patients undergoing peg-IFN/RBV retreatment. Subjects completed at least 24 weeks of therapy. The Diasorin assay was used
to measure 25(OH)D in baseline (N=88) and 24-week samples (N=70). Changes in 25(OH)D and calcium were analyzed using the Wilcoxon
Signed Ranks Test.
Results: Most subjects were male; 86% had genotype 1 HCV; 15% (N=13) achieved an SVR. During treatment, 25(OH)D levels increased
significantly, by a median of 2.20 ng/ml in a paired analysis of 70 patients (p=0.04). Baseline 25(OH)D >18 ng/ml was the only variable significantly
associated with SVR (OR, 5.08, p=0.04) in a multivariable logistic regression model. The baseline 25(OH)D level and genotype were the only
factors associated with cEVR (OR, 4.25, p=0.02; OR 12.10, p=0.005, respectively), while changes in 25(OH)D were significantly associated with
EVR (OR, 1.06, p=0.050). Despite the increase in 25(OH)D, serum calcium (corrected for albumin) decreased significantly during treatment, by a
median of -0.13 mg/dl (p=0.04) and fell below the lower-limit-of-normal in 33% of subjects who entered the trial with normal values.
Conclusions: We report two novel and important findings about IFN/RBV treatment: Vitamin D levels increase, and calcium levels fall.
Because treatment success is positively associated with vitamin D, the on-treatment increase in 25(OH)D may be a newly-discovered
component of drug action. The drop in serum calcium may have adverse consequences, e.g., on bone. The potential of vitamin D and
calcium supplements to improve outcome and to protect bone merits investigation (DA031095;DK090317;CA152514).
Contact: Andrea D. Branch
Mount Sinai School of Medicine, New York, NY, USA
[email protected]
114 : HCV 2011
ANTIVIRAL THERAPY
P1.57
Characterization of a small molecule with potent virocidal activity against HCV and HIV
Ana Maria Chamoun (1), Karuppiah Chockalingam (1), Rudo Simeon (1), Philippe Gallay (2), Zhilei Chen (1)
(1) Texas A&M University, College Station, TX, USA
(2) The Scripps Research Institute, USA
Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are both important human pathogens currently lacking effective vaccines. In
the United States, approximately 30% of HIV-positive patients are co-infected with HCV. Using a high-throughput screening assay developed
in our lab, we identified a small molecule with virocidal activity against cell-culture produced HCV. Further studies showed that this molecule
is virocidal against lentiviruses pseudotyped with envelope proteins from HCV, VSV, MLV and Sindbis virus (SINV). In vitro studies revealed
that this molecule lyses lentiviral particles pseudotyped with the VSV-G envelope and releases the viral RNA in a time-, temperature- and
virus-titer-dependent manner. However, when incubated with HCVcc, little to no viral lysis was observed, despite a clear inactivation of
HCVcc infectivity upon pre-incubation of the small molecule with the virus. This compound showed no antiviral activity against SINV and
DENV, and does not appear to lyse liposomes of various compositions and sizes. Most interestingly, this compound showed very strong
virocidal activity against primary HIV isolates, multidrug resistant HIV-1 isolates, HIV-2 and simian immunodeficiency virus (SIV), with an
IC50 of ~1 uM and a selectivity index (CC50/EC50) of ~100. Only a handful of examples exist of virocidal small molecules, and even fewer are
known to exert an action through membrane lysis. Our virocidal small molecule therefore allows for a powerful new approach to combat
the global health problems posed by HCV and HIV.
POSTERS
Contact: Ana Maria Chamoun
Texas A&M University, College Station, TX, USA
[email protected]
POSTERS
116 : HCV 2011
POSTERS
Cellular Immunity
P2.01
Coping with hyper mutable HCV
Mandvi Bharadwaj, Usha Nivarthi, John Miles, Lars Kjer-Nielsen, Margaret Hellard, Jamie Rossjohn, James McCluskey
P2.02
T cell receptor bias and CTL escape in hepatitis C virus infection
Usha Nivarthi, Stephanie Gras, Lars Kjer-Nielsen, Richard Berry, Margaret Hellard, Jamie Rossjohn, James McCluskey,
Mandvi Bharadwaj
P2.03
Transcriptional repression of complement components by hepatitis C virus
Hangeun Kim, Budhaditya Mazumdar, Keith Meyer, Adrian M. Di Bisceglie, Ratna B. Ray, Arup Banerjee, Ranjit Ray
P2.04
Liver infiltrating lymphocytes of in HCV and alcohol cirrhosis by protein profiling
Ramzan Muhammad, Nathalie Sturm, Christian Letoublon, Karim Arafah, Jean-Pierre Zarski, Evelyne Jouvin-Marche,
Francois Berger, Vincent Leroy, Philippe Bulet, Patrice N. Marche
P2.05
Lytic activity of NK cells from blood and liver in chronically HCV-infected and
non-infected patients
P2.06
HCV-NS4B targets STING and abrogates RIG-I-mediated type-I interferondependent innate immune response
Sayuri Nitta, Naoya Sakamoto, Megumi Tasaka-Fujita, Kei Kiyohashi, Akiko Kusano-Kitazume, Miyako Murakawa,
Kouhei Yoshino, Kako Mishima, Sei Kakinuma, Mina Nakagawa, Mamoru Watanabe
P2.07
The protective KIR/HLA compound genotype is associated with an
expansion of NKG2Aneg NK cells with reduced cytolytic and cytokine
secreting function
Christoph Berger, Qingquan Liu, Anna Kaliszewska, Patrick Cheney, Arthur Kim, Philip De Jager, Mary Carrington,
Georg Lauer, Galit Alter
POSTERS
Emilie Fugier, Marie Ange Thélu, Aurélie Dariz, Nicolas Van Campenhout, Vincent Leroy, Jean-Pierre Zarski,
Nathalie Sturm, Evelyne Jouvin-Marche, Patrice Marche
POSTERS
P2.08
Acute PARV4 infection in a high risk IDU cohort
Ruth Simmons, Colin Sharp, Jordana Levine, Andrea Cox, Peter Simmonds, Paul Bowness, Paul Klenerman
P2.09
Uncoordinated phenotype-5A and its ligand netrin-1 are modulated by HCV,
its NS3-4A protease / helicase complex, and type I and II interferons
Marie-Laure Plissonnier, Thomas Lahlali, Andrea Paradisi, Maud Michelet, David Durantel, Birke Bartosch, Patrick
Mehlen, Fabien Zoulim, Romain Parent
P2.10
Escape from a dominant HLA-B*15-restricted CD8 T cell response against
HCV requires compensatory mutations in the epitope flanking region
Marianne Ruhl, Patrick Chhatwal, Heiko Strathmann, Thomas Kuntzen, Falko Heinemann, Peter Horn, Todd M. Allen,
Daniel Hoffmann, Thomas Pietschmann, Joerg Timm
P2.11
Immune responses to a novel Chimigen® HCV prophylactic/therapeutic vaccine
Rajan George, Allan Ma, Dakun Wang, René Déry, Yun Xia, Klaus Gutfreund
P2.12
Analysis of natural killer cell modulation in a hepatitis C virus cell culture system
Kayla Harris, Michael Grant, Rodney Russell
P2.13
CD8+ T cell selection pressure is the dominant force driving hepatitis C virus
evolution during acute infection
Heather B. Eccleston, Jens Bukh, Benoit Callendret, Dana L. Hasselschwert, Robert H. Purcell, Austin L. Hughes,
Christopher M. Walker
P2.14
Race associated CD16+56- NK-cell IFN-alphaR expression regulates signaling
and is implicated in IFN-alpha-induced HCV decline
Sara Conry, Gareth Hardy, Qinglai Meng, Nicole Yonkers, Julia Sugalski, Amy Hirsch, Perika Davitkov, Anita Compan,
Yngve Falck-Ytter, Ronald Blanton, Benigno Rodriguez, Clifford Harding, Donald Anthony
P2.15
POSTERS
The Toll-like receptor mediated induction of cytokines and interferons is not
impaired in primary isolated liver cells from HCV-positive patients
Ruth Broering, Kathrin Kleinehr, Andreas Paul, Guido Gerken, Joerg F. Schlaak
P2.16
The percentage of hepatitis C virus-specific CD127+ memory T cells in the
acute phase T cell response predicts the outcome of HCV infection
Eui-Cheol Shin, Su-Hyung Park, Michelina Nascimbeni, Marian Major, Stephen Feinstone, Charles Rice,
Barbara Rehermann
P2.17
HCV alters DC activation through the production of thymic stromal
lympopoitein (TSLP) cytokine and promotes Th17 differentiation
HaiChon Lee, Young S. Hahn
P2.18
Natural killer cells and dendritic cells function during acute HCV
Sandy Pelletier, Elias Said, Petronela Ancuta, Julie Bruneau, Naglaa Shoukry
P2.19
Characterisation of interferon lambda(IFN λ)-producing dendritic cell (DC)
populations in healthy and hepatitis C virus (HCV)-infected liver perfusate
Aoife Kelly, Elizabeth Ryan, Nigel Stevenson, Justin Geoghegan, John Hegarty, Cliona O’Farrelly
118 : HCV 2011
HCV 2011
P2.20
CELLULAR IMMUNITY
Reversion of cytotoxic T-cell escape mutation during pregnancy permits
vertical transmission of most fit hepatitis C variant
Jonathan Honegger, Jennifer Kohout, Seungtaek Kim, Stanley Lemon, Arash Grakoui, Christopher Walker
P2.21
Comparison of T cell immunity to the hepatitis A and C viruses in acutely
infected chimpanzees.
Yan Zhou, Deborah Chavez, Kathleen Brasky, Heather Eccleston, Stanley Lemon, Robert Lanford, Christopher Walker
P2.22
In vivo assessment of the proliferative potential of Hepatitis C virus specific
CD8+ T cells
Benoît Callendret, Heather Eccleston, Jennifer Rutkiewicz, Dana Hasselschwert, David Bowen, Christopher Walker
B cell homeostasis in chronic hepatitis C virus-related mixed
cryoglobulinemia
Lauren Holz, Joo Chun Yoon, Sukanya Raghuraman, Susan Moir, Michael Sneller, Barbara Rehermann
POSTERS
P2.23
POSTERS
120 : HCV 2011
CELLULAR IMMUNITY
P2.01
Coping with hyper mutable HCV
Mandvi Bharadwaj (1), Usha Nivarthi (1), John Miles (2), Lars Kjer-Nielsen (1), Margaret Hellard (3), Jamie Rossjohn (4), James McCluskey (1)
(1) The University of Melbourne, Melbourne, Australia
(2) Queensland Institute of Medical Research, Australia
(3) Macfarlane Burnet Institute, Melbourne, Australia
(4) Department of Biochemistry and Molecular Biology, Monash University, Australia
HCV has prolific genetic variability as reflected in its 7 genotypes across the world. As well, the high replication and mutation rate result
in various viral quasispecies in chronically infected subjects. T cells play a central role in HCV clearance however there is currently little
understanding of the T cell receptors (TCR) targeted at key HCV antigens. To better understand the influence of TCR-repertoire in HCV
immunity, we examined the repertoire targeted at an immunodominant CTL epitope HLA-A*0101–restricted, 1435ATDALMTGY1443
(ATD) in clearly defined cohorts of HLA-matched, HCV-infected individuals with persistent infection and HCV clearance. Comprehensive
clonal analysis of antigen-specific T cells revealed widespread use of public TCRs displaying a high degree of predictability in TRBV/TRBJ
gene usage, CDR3 length, and amino acid composition. These public TCRs were shared across subjects with diverse disease outcomes.
Remarkably, we noted TCR sequence mosaicism wherein, many TCRs were seen to predictably switch TRBV and TRBJ genes around a
conserved, N nucleotide-encoded CDR3 core. Three TCR mosaic clusters were identified within and between HCV exposed individuals
including a) a TRBV7-2/TRBV7-3/TRBV7-8/TRBV11-2 group, b) a TRBV10-3/TRBV19/TRBV27 group and c) a TRBV2/TRBV9 group. All three
TCR mosaic clusters comprised a high degree of amino acid homology within their germline-encoded CDR1 and CDR2 loops however, these
CDR patterns were found to be wholly unique to each cluster and were not encoded by any other TRBV gene. The selection of different Vβ
families that share mosaic-like patterns of sequence motifs suggest a strategy for TCR diversification that still permits selection for preferred
CDR elements, structurally important for MHC-peptide ligation. Overall, TCR mosaicism reveals an inherent characteristic of the TCR system
and a potential host-mechanism to combat variant viruses such as HCV.
Contact: Mandvi Bharadwaj
The University of Melbourne, Melbourne, Australia
[email protected]
P2.02
Usha Nivarthi (3), Stephanie Gras (1), Lars Kjer-Nielsen (3), Richard Berry (1), Margaret Hellard (2), Jamie Rossjohn (1), James McCluskey
(3), Mandvi Bharadwaj (3)
(1) Department of Biochemistry and Molecular Biology, Monash University, Australia
(2) Macfarlane Burnet Institute, Melbourne, Australia
(3) The University of Melbourne, Melbourne, Australia
T cells play a central role in HCV clearance; however, there is currently little understanding of whether the disease-outcome in HCV infection
is influenced by the choice of T-cell receptor (TCR) repertoire. It is unclear, if repertoire diversity (clonotypic breadth) or particular Vb
segments, as shown in HIV, are associated with HCV-persistence/clearance in humans. We therefore analysed the TCR-repertoires utilised
against two immunodominant CTL epitopes: the highly polymorphic, HLA-B8-restricted 1395HSKKKCDEL1403 (HSK) and the comparatively
conserved, HLA-A1-restricted, 1435ATDALMTGY1443 (ATD), in clearly defined cohorts of HLA-matched, HCV-infected individuals with
persistent infection and HCV clearance. Based on this comparison we recently reported that the antigen-driven patterns of TCR bias are
shared across diverse outcomes of human HCV Infection. However we also noted that whilst a broad TCR repertoire was recruited against
HLAA1-ATD, the repertoire against HLAB8-HSK was narrowly focused, supporting reports of mutational escape in this epitope, in persistent
HCV-infection. To gain a better understanding of how the peptide-major histocompatibility complex (pMHC) landscape and viral variants
impact on the T-cell repertoire and to assess the extent of CTL-escape of the reported variants (quasispecies and inter-genotypic), we
performed a detailed immunological and crystallographic analysis of T cells targeted at this hypervariable epitope and its variants. Some of
our recent observations and their implication for vaccine design will be presented.
Contact: Mandvi Bharadwaj
The Universiy of Melbourne, Melbourne, Australia
[email protected]
POSTERS
T cell receptor bias and CTL escape in hepatitis C virus infection
POSTERS
P2.03
Transcriptional repression of complement components by hepatitis C virus
Hangeun Kim (1), Budhaditya Mazumdar (1), Keith Meyer (1), Adrian M. Di Bisceglie (1), Ratna B. Ray (1), Arup Banerjee (1), Ranjit Ray (1)
(1) Saint Louis University School of Medicine, Saint Louis, MO, USA
Human complement plays an important role in innate immune function. Liver biopsy specimens from chronically HCV infected patients
displayed significantly lower mRNA levels for a number of complement components (C3, C4, C9), and cellular factor H as compared to liver
tissues from unrelated disease. Further, mRNA levels of these components were significantly reduced in hepatocytes transfected with RNA
from HCV genotype 1a or 2a. We prioritized our immediate study on the C4 complement component. A significant C4 regulatory role of
HCV core or NS5A upon C4 promoter activity was observed. HCV core or NS5A transgenic mice displayed a reduction in C4 mRNA. IFN-γ
induced C4 promoter activation was also impaired in the presence of HCV proteins. We further demonstrated that HCV core reduced
the expression of upstream stimulating factor-1 (USF-1), a transcription factor important for basal C4 expression. On the other hand, the
expression of interferon regulatory factor-1 (IRF-1), important for IFN-γ induced C4 expression was inhibited by HCV NS5A expressing
hepatocytes. Studies are in progress to further understand the mechanism by which HCV proteins may modulate the expression of other
complement components. These results underscore the effect of HCV proteins on complement related genes via transcriptional regulatory
controls, implicating innate immune regulation as a factor in the establishment of chronic infection.
Contact: Hangeun Kim
Saint Louis University School of Medicine, Saint Louis, MO, USA
[email protected]
P2.04
Liver infiltrating lymphocytes of in HCV and alcohol cirrhosis by protein profiling
Ramzan Muhammad (3), Nathalie Sturm (3), Christian Letoublon (3), Karim Arafah (1), Jean-Pierre Zarski (3), Evelyne Jouvin-Marche (3),
Francois Berger (2), Vincent Leroy (3), Philippe Bulet (1), Patrice N. Marche (3)
(1) AGIM, FRE 3405 CNRS-UJF, USA
(2) INSERM-UJF U836, France
(3) INSERM U823, Grenoble, France
POSTERS
We determined the types and evaluated the numbers liver-infiltrating lymphocytes (LILs) in 40 cirrhotic patients: 20 infected by HCV
(HCV) and 20 with high alcohol consumption (OH) as non viral controls. LILs were analyzed in liver tissue sections, i.e. fibrous septa and
parenchymatous nodules of cirrhotic zones by 1) immuno histo chemistry (IHC) typing for CD3, CD4, CD8, FoxP3 (fro T lymphocytes), CD20
(for B lymphocytes) and CD56 (for NK cells) and 2) protein profiling achieved by SELDI-TOF-MS , µLC-MS and MS imaging methods. Protein
profiling was done by 1) SELDI-TOF: zones of interest were laid on 3 types of chips adsorbing hydrophobic, cationic and anionic proteins and
2) by µLC loaded with cell lysats of the zones of interest, 3) followed by mass identification (MALDI-TOF).
IHC showed that 1) most of LILs types in the portal tracts and fibrous septa, while less abundant in parenchyma, 2) LILs were significantly
more abundant among HCV patients than in OH ones, although the relative proportion of each type is conserved.
Protein profiling showed that >95% of protein peaks were identical in serial duplicates of the same cirrhotic samples, and that SELDI-TOFMS and µLC-MS yielded to reproducible data. Among all proteins detected by MS, 62 displayed different abundance, allowing a distinctive
classification by Partial Least Squares-Discrimination analysis statistical method of the two groups of patients. Variable Importance for the
Projection plot identified highly significant parameters (p<10-3): 12 higher and 4 lower in HCV. The presence of the proteins was further
confirmed in situ by imaging-MS of liver sections.
Conclusion: LILs display profiles of protein expression which are distinctive of the etiology of the cirrhosis, VHC infection versus alcohol abuse.
These findings open perspectives in the molecular identification of immune markers by direct in situ analysis of the protein expression.
Contact: Patrice N. Marche
INSERM U823, Grenoble , France
[email protected]
122 : HCV 2011
CELLULAR IMMUNITY
P2.05
Lytic activity of NK cells from blood and liver in chronically HCV-infected and noninfected patients
Emilie Fugier (1), Marie Ange Thélu (1), Aurélie Dariz (1), Nicolas Van Campenhout (1), Vincent Leroy (1), Jean-Pierre Zarski (1), Nathalie
Sturm (1), Evelyne Jouvin-Marche (1), Patrice Marche (1)
(1) INSERM U823, Grenoble, France
The effect of hepatitis C virus (HCV) on innate immune cells in chronically HCV-infected patients is a subject of debate. The data reported
in the literature were derived from studies using peripheral blood or in vitro assays. Here, we analyzed cytotoxic and cytolytic activities of
NK cells in HCV-infected liver and compared them to non-infected liver. Thirty-eight chronically HCV-infected patients (grade/stage A1/
F1-A2/F2) and 23 non alcoholic fatty liver disease (NAFLD) individuals (grade/stage A0/F0-A1/F1) were studied. Lymphocyte from blood
and liver biopsies were analyzed by flow cytometry to define, T, NK and NKT cells, to monitor their content in gINF and perforin, and their
secretory activity by CD107 expression. Furthermore, cytotoxic assays of target cells evaluated the killing function of blood cell samples and
of intra-hepatic lymphocytes within the liver biopsie samples. Analyses of activating or inhibitory NKG2 family receptors (frequencies and
mean fluorescent intensity) expression on the surface of intra-hepatic NK cells indicated no major differences in both cohorts of patients.
Nevertheless intra-hepatic NK cells from HCV patients displayed higher degranulation process intensity. Upon ex vivo stimulation by either
cytokines cocktail or K562 target cells, NK cells from both groups of patients reacted similarly. Taken together, these results indicate that
intra-hepatic NK cells from chronically infected patients are reactive. Moreover, they have same functional features than NK cells from NAFLD
non-infectious environment. Interestingly, the highest degranulation intensity of NK cells from HCV-patients was maintained. Conclusions.
Our study highlights that, within the human liver, persistent stimulation due to HCV presence coupled with commensurate inhibitions result
in a sustained NK cells responsiveness.
Contact: Patrice Marche
INSERM U823, Grenoble, France
[email protected]
P2.06
Sayuri Nitta (1), Naoya Sakamoto (2), Megumi Tasaka-Fujita (3), Kei Kiyohashi (1), Akiko Kusano-Kitazume (1), Miyako Murakawa (1),
Kouhei Yoshino (1), Kako Mishima (1), Sei Kakinuma (1), Mina Nakagawa (1), Mamoru Watanabe (1)
(1) Gastroenteroloy and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
(2) Hepatitis Control, Tokyo Medical and Dental University, Tokyo, Japan
(3) Virology II, National Institute of Infectious Diseases, Japan
HCV establishes persistent infection in immune competent hosts. It has been reported that HCV-NS34A cleaves Cardif (MAVS/IPS-1/VISA) and
blocks RIG-I-induced signaling of interferon(IFN)-alpha/beta expression. Recently, STING (MITA/ ERIS/ MPYS) was identified as an activator
of RIG-I pathway (Zhong, Immunity 2008; Ishikawa, 2008 Nature). Because the amino terminal domain of STING has structural homology
with flavivirus NS4B and NS4B inhibits RIG-I-mediated IFN-beta expression pathway (Tasaka, J.Gen.Virol.2005), it may be hypothesized
reasonably that HCV-NS4B suppresses RIG-I signaling through interaction with STING. Thus, we investigated the role of NS4B in RIG-I
induced, STING mediated IFN expression pathway. IFN-beta promoter reporter assay showed that RIG-I or Cardif induced activation of
IFN-beta promoter was significantly suppressed by both NS34A and NS4B, while STING-induced IFN-beta activation was suppressed only by
NS4B but not by NS34A, suggesting of a different point of action of NS4B on RIG-I pathway. Immunofluorescence microscopy revealed that
STING colocalized with NS4B in ER and that Cardif and NS4B colocalized partly at their boundary region. To assess direct interaction between
NS4B, Cardif and STING proteins, we performed immunoprecipitation and Bimolecular Fluorescence Complementation (BiFC) assays. In
both assays, NS4B specifically bound with STING but not with Cardif. These results suggested that NS4B directly interacts with STING and
suppress its function on IFN signaling. We finally assessed whether NS4B suppressed IFN-beta production in the presence of cleaved Cardif
by NS34A protease (Cardif1-508). Interestingly, NS4B completely blocked the residual function of the Cardif1-508 protein to activate IFNbeta expression, suggesting of an additive effect on RIG-I activating pathway by NS34A and NS4B. Taken these results together, we conclude
that NS4B and NS34A cooperatively suppress RIG-I mediated IFN-beta production pathway through interaction with their target proteins,
Cardif and STING, respectively, and contribute to the establishment of persistent HCV infection in the presence of intact immune system.
Contact: Sayuri Nitta
Gastroenteroloy and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
[email protected]
POSTERS
HCV-NS4B targets STING and abrogates RIG-I-mediated type-I interferon-dependent
innate immune response
POSTERS
P2.07
The protective KIR/HLA compound genotype is associated with an expansion of
NKG2Aneg NK cells with reduced cytolytic and cytokine secreting function
Christoph Berger (1), Qingquan Liu (1), Anna Kaliszewska (2), Patrick Cheney (1), Arthur Kim (3), Philip De Jager (2), Mary Carrington (4),
Georg Lauer (3), Galit Alter (1)
(1) Ragon Institute of MGH, MIT and Harvard, Charlestown/Boston, MA, USA
(2) Brigham and Women’s Hospital, Boston, MA, USA
(3) Massachusetts General Hospital, Boston, MA, USA
(4) National Cancer Institute at Frederick, USA
Background: A fraction of HCV infected individuals clear HCV spontaneously. This resolution occurs within the first few months of infection,
suggesting a critical role for innate immune functions including Natural killer (NK) cells in antiviral control. Epidemiologic studies have linked
the inhibitory NK receptor KIR2DL3, in the presence of its ligand (specific HLA-C alleles), to HCV clearance. However, the mechanism by which
this compound genotype is protective in HCV infection is unknown. Methods: We comprehensively assessed the impact of the KIR2DL3/HLAC1C1 compound genotype on NK phenotype and function by performing multicolor flow cytometry on a cohort of 183 healthy individuals. NK
cells were phenotyped for 16 NK receptors and activation markers. Functional assays were also performed to define differences in the capacity
of NK cells from these different donors to respond to a diverse set of target cells. Results: The homozygous 2DL3/C1 compound genotype was
associated with the expansion of an NKG2A- NK cell population (p=0.04) that expressed increased perforin (p=0.01), and produced less IFNg
in response to NKG2D-mediated NK activation (p=0.01). Conversely, NKG2A+ NK cells, previously associated with dendritic cell (DC) editing,
showed a significantly increased degranulation in response to all tested stimulation conditions and significantly increased IFNg production in
response to NKG2D and NCR induced activation. Conclusion: The HCV protective compound genotype, KIR2DL3+/+ HLA-C1/C1, is associated
with a reduced frequency of NKG2A expressing NK cells in healthy individuals, that are generally more cytolytic and secrete higher levels of
IFNg. Taken together, these data suggest that an accumulation of less cytolytic and IFNg secreting NK cells may promote a more effective
adaptive immune response, potentially via reduced deletion of DCs required for the induction of effective adaptive immune responses.
Contact: Christoph Berger
Ragon Institute of MGH, MIT and Harvard, Charlestown/Boston, MA, USA
[email protected]
P2.08
Acute PARV4 infection in a high risk IDU cohort
POSTERS
Ruth Simmons (1), Colin Sharp (2), Jordana Levine (3), Andrea Cox (3), Peter Simmonds (2), Paul Bowness (4), Paul Klenerman (1)
(1) University of Oxford, Oxford, United Kingdom
(2) University of Edinburgh, Centre for Infectious Diseases, United Kingdom
(3) Departments of Medicine, Johns Hopkins Medical Institutions, USA
(4) Weatherall Institute of Molecular Medicine, Oxford, United Kingdom
Background: PARV4 is a novel parvovirus which was initially identified in an intravenous drug user (IDU) at risk of HIV infection. Since its discovery
it has been associated principally with individuals at risk of blood-borne viral infections, with 30% of HCV-infected individuals and 85% of
HIV-infected individuals with AIDS being PARV4 seropositive. We have identified strong T cell responses to PARV4 in HCV-infected IDU populations.
We therefore analysed the acute acquisition of PARV4 and immune response in a longitudinal prospectively collected cohort of IDUs.
Methods: We first studied a cohort of 98 active IDUs at risk of HCV infection, but who remained HCV seronegative throughout the course
of the study. Longitudinal samples were tested using IFN-g ELISpots to detect PARV4-specific T cell responses. A PARV4 ELISA was used to
detect anti-PARV4 IgG and IgM, and nested PCR enabled us to determine the length of viremia.
Results: 36% of individuals were PARV4 seropositive at the last clinic appointment. 8 of these individuals PARV4 sero-converted during
the time they attended clinic. Acute acquisition was associated with transient viremia (32-109 days), seroconversion and emergence of
polyspecific T cell responses. Antibody titres waned rapidly in some patients.
Conclusions: PARV4 infection is common in individuals exposed to blood-borne viruses; this study shows that PARV4 infection can readily
occur independently of HCV infection. PARV4 acquisition is subclinical and associated with priming and maintenance of CD8+ T cell
responses. The rapid waning of antibody responses may explain the detection of individuals with PARV4-specific T cells but without antiPARV4 antibodies, observed in previous studies.
Contact: Ruth Simmons
University of Oxford, Oxford, United Kingdom
[email protected]
124 : HCV 2011
CELLULAR IMMUNITY
P2.09
Uncoordinated phenotype-5A and its ligand netrin-1 are modulated by HCV, its NS3-4A
protease / helicase complex, and type I and II interferons
Marie-Laure Plissonnier (1), Thomas Lahlali (1), Andrea Paradisi (1), Maud Michelet (1), David Durantel (1), Birke Bartosch (1), Patrick
Mehlen (1), Fabien Zoulim (1), Romain Parent (1)
(1) Lyon Cancer Research Center (Inserm Unit 1052), 69424 Lyon Cedex 03, France
Dependence receptors (DR) and their ligands are involved in development, inflammation, and tumorigenesis. HCV-induced alteration of (i) the
UNC5A DR involved in apoptosis induction, and (ii) its NTN1 ligand implicated in cell survival was investigated. Experiments were performed in
HCV JFH1 as well as genotypes 1a (H77) and 1b (N) HCV replicating Huh7.5 cells. The NTN1/UNC5A pathway was found functional in Huh7.5
cells. Recombinant NTN1 was able to antagonize both doxorubicin- and HCV-induced apoptosis. Next, we showed that HCV infection increases
NTN1 and UNC5A transcripts by 3 to 5-fold at days 1 to 5 p.i. In turn, NTN1 overexpression induced a 4-fold increase in HCV RNA levels,
suggesting an interplay between HCV infection and the DR pathway. HCV also induced UNC5A redistribution and aggregation in proliferative
and differentiated Huh7.5 cells. Extensive colocalization between the HCV E2 protein and UNC5A was observed by the presence of HCV-induced
aggregates composed of both proteins. These results were confirmed with electroporated H77 and N HCV genomes. The NS3-4A protein
complex was found responsible for UNC5A up-regulation, and colocalized also massively with the UNC5A protein upon lentiviral and plasmidbased delivery. Extremely tight correlation observed between UNC5A and HCV positivity at the unicellular level by immunofluorescence
suggest UNC5A’s relevance as a host marker for HCV replication. Interactions between the DR system and innate immunity were addressed. In
naive Huh7.5 cells, NTN1 was found decreased by 3-fold at the mRNA level by IFN gamma treatment, whereas UNC5A transcripts were found
elevated by 4-fold at the mRNA and protein levels by IFN alpha treatment. These results suggest that the DR system is functionally altered
by HCV. UNC5A-associated functions appear to locate at the crossroads of proviral (NTN1 upregulation) and innate immunity-related events
(UNC5A upregulation and redistribution) upon acute in vitro HCV infection.
Contact: Marie-Laure Plissonnier
Lyon Cancer Research Center (Inserm Unit 1052), 69424 Lyon Cedex 03, France
[email protected]
P2.10
Marianne Ruhl (1), Patrick Chhatwal (2), Heiko Strathmann (3), Thomas Kuntzen (4), Falko Heinemann (5), Peter Horn (5), Todd M. Allen
(4), Daniel Hoffmann (3), Thomas Pietschmann (2), Joerg Timm (1)
(1) Institute of Virology, University Duisburg-Essen, Essen, Germany
(2) Division of Experimental Virology, Twincore, Hannover, Germany
(3) Centre for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany
(4) Ragon Institute of MGH, MIT and Harvard, Boston, USA
(5) Institute for Transfusion Medicine, University of Duisburg-Essen, Essen, Germany
Antiviral CD8+ T cells are one of the key components of the adaptive immune system against HCV. For the development of immune therapies it
is essential to understand how CD8+ T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure
of HCV-specific CD8+ T cells is mutational escape of the virus. Some escape mutations in viral epitopes are highly reproducible in patients
sharing the same HLA class I-alleles suggesting reproducible selection pressure. Clearly, the space in which HCV can evolve is limited, indicated
by fitness costs associated with some escape mutations. We hypothesize that compensatory mutations may point towards epitopes under
particular strong selection pressure because of a higher genetic barrier to escape. We identified two novel HLA-B*15-restricted CD8 epitopes
in NS5B (LLRHHNMVY2450-2458 and SQRQKKVTF2466-2474) based on sequence analysis of 113 sequences of a large GT1b outbreak. Both
epitopes are targeted in >60% of HLA-B*1501-positive individuals exposed to HCV. Reproducible selection of escape mutations in both epitopes
in GT1b was confirmed in an independent multi-centre cohort. Interestingly in HLA-B*15-positive individuals, mutations were also selected
in the epitope flanking region suggesting that compensatory evolution may play a role. Co-variation analysis of all GT1b sequences from the
database confirmed a significant association between mutations in the epitope flanking region (S2439T and K2440Q) and escape mutations
inside one of the epitopes. To address whether mutations in the epitope flanking region truly represent compensatory mutations, we utilized
the HCV genotype 1b subgenomic replicon Con1 to determine the replication capacity. Indeed, escape mutations inside the epitope impaired
viral replication, however, fitness was restored after mutations in the epitope flanking region were added. Conclusion: Selection of escape
mutations inside a novel HLA-B*15 epitope requires secondary mutations in the epitope flanking region that compensate for fitness costs.
Contact: Marianne Ruhl
Institute of Virology, University Duisburg-Essen, Essen, Germany
[email protected]
POSTERS
Escape from a dominant HLA-B*15-restricted CD8 T cell response against HCV requires
compensatory mutations in the epitope flanking region
POSTERS
P2.11
Immune responses to a novel Chimigen® HCV prophylactic/therapeutic vaccine
Rajan George (1), Allan Ma (1), Dakun Wang (1), René Déry (1), Yun Xia (1), Klaus Gutfreund (2)
(1) Paladin Biosciences, a Division of Paladin Labs Inc., Edmonton, Canada
(2) University of Alberta, Alberta, Canada
Chimigen® Vaccines are a new class of chimeric molecules with functional attributes of both an antigen and a xenotypic monoclonal antibody.
These molecules target the host immune system using specific receptors on dendritic cells (DCs) and are processed for presentation to T cells
through both MHC class I and class II pathways to elicit cellular and humoral immune responses against specific viral antigens. Chimigen®
HCV Prophylactic/Therapeutic Vaccine, a fusion protein of HCV antigens (E1-E2-NS5A) and a murine Fc fragment, has been produced in
insect cells, purified and characterized.
The immune responses to the vaccine were evaluated using human peripheral blood mononuclear cell (PBMC)-derived DC/T cells, in an antigen
presentation assay (APA), ex vivo. The immune responses to the vaccine were assessed in T cells derived from patients with chronic HCV infection
and uninfected healthy donors. PBMCs were stimulated ex vivo with buffer, vaccine, HCV antigen, or the xenotypic Fc fragment and then restimulated with vaccine-loaded DCs or HCV antigen overlapping peptides. In cells derived from both healthy donors and chronically infected
patients, a single stimulation with the vaccine resulted in increased IFN-γ secretion as compared to treatment with vaccine components. Restimulation with vaccine or the overlapping peptides induced a marked increase in the percentage of CD8+ and CD4+ T cells producing IFN-γ
and TNF-α. The induction of The expansion of HCV-antigen specific CD4+ and CD8 +T cells from chronically infected patients shows the potential
of Chimigen® HCV Vaccine for the development as a prophylactic/ therapeutic vaccine for HCV infections.
Financial support from NRC-IRAP Canada and AITF is gratefully acknowledged.
Contact: Rajan George
Paladin Biosciences, a Division of Paladin Labs Inc., Edmonton, Canada
[email protected]
P2.12
Analysis of natural killer cell modulation in a hepatitis C virus cell culture system
Kayla Harris (1), Michael Grant (1), Rodney Russell (1)
(1) Memorial University of Newfoundland, St. John’s, Canada
POSTERS
Viruses employ numerous mechanisms to avoid host immune responses and establish chronic infection; hepatitis C virus (HCV) may elude
immune responses by modulating natural killer (NK) cell function. In the absence of a suitable animal model, the HCV cell culture (HCVcc)
system is the most effective and realistic system to determine whether HCV downregulates NK cell function, thereby reducing their ability
to eliminate HCV-infected cells. Previous studies demonstrated decreased NK cell function when exposed to immobilized HCV particles.
We hypothesize that NK modulation results from the interaction of cell surface-expressed HCV E2 with NK CD81 receptors. Titred virus
stocks of 2x105 ffu/ml were generated by transfecting Huh-7.5 cells with tissue culture adapted JFH-1 HCV. A multiplicity of infection
and duration of infection which maximizes the number of infected Huh-7.5 cells while maintaining cell viability was determined through
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) and immunofluorescence (IF) assays. A short-term coculture assay was
then developed to compare the ability of NK cells to lyse labelled target cells in the presence of uninfected and HCV-infected Huh-7.5 cells.
This assay was also used to assess direct NK cell-mediated lysis of HCV-infected Huh-7.5 cells and the effect free HCV particles have on NK
cell lysis. Preliminary data suggests free virus has no effect on NK cell function. Direct killing of uninfected Huh-7.5 cells by NK cells is limited,
ranging between four and fifteen percent at a 50:1 effector to target (E:T) ratio. In some assays, we observed a fifteen to thirty percent
decline in target cell lysis by NK cells at a 50:1 E:T ratio when cocultured with infected versus uninfected cells. This assay platform will be
useful to not only study this effect but also to examine the influence HCV-infected cells have on the function of other lymphocytes.
Contact: Kayla Harris
Memorial University of Newfoundland, St. John’s, Canada
[email protected]
126 : HCV 2011
CELLULAR IMMUNITY
P2.13
CD8+ T cell selection pressure is the dominant force driving hepatitis C virus evolution
during acute infection
Heather B. Eccleston (1), Jens Bukh (2), Benoit Callendret (1), Dana L. Hasselschwert (3), Robert H. Purcell (4), Austin L. Hughes (5),
Christopher M. Walker (1)
(1) The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
(2) NIAID NIH, Copenhagen University Hospital and University of Copenhagen, USA
(3) University of Louisiana at Lafayette, New Iberia Research Center, USA
(4) National Institute of Allergy and Infectious Diseases, NIH, USA
(5) Division of Biological Sciences, University of South Carolina, USA
Viral evolution is governed by the positive or negative selection of mutations and the accumulation of neutral substitutions (genetic drift).
The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection. However, the relative contribution of
the different selective forces that drive this process remain poorly defined. Here we examined the impact of Darwinian selection pressure
imposed by CD8+ T cells on HCV evolution during acute and chronic infection. This question was addressed in two chimpanzees followed
for 8-10 years after infection with a well defined inoculum comprised of a clonal genotype 1a (isolate H77C) HCV genome. Detailed
characterization of CD8+ T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute phase
non-synonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome.
Moreover the ratio of non-synonymous to synonymous substitutions, a measure of positive selection pressure, was increased 50-fold in
class I epitopes when compared with the rest of the HCV genome during the early phase of infection. No further amino acid changes could
be linked to CD8+ T cell positive selection pressure in the chronic phase of infection. Finally, some mutation of the clonal H77C genome
towards a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the
majority of these amino acid changes occurred slowly over several years of chronic infection. Together these observations indicate that
during acute hepatitis C infection, viral evolution is driven primarily by positive selection pressure exerted by CD8+ T cells. This immune
pressure appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.
Contact: Heather B. Eccleston
The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
[email protected]
Race associated CD16+56- NK-cell IFN-alphaR expression regulates signaling and is
implicated in IFN-alpha-induced HCV decline
Sara Conry (1), Gareth Hardy (1), Qinglai Meng (1), Nicole Yonkers (1), Julia Sugalski (1), Amy Hirsch (1), Perika Davitkov (1), Anita Compan
(1), Yngve Falck-Ytter (1), Ronald Blanton (1), Benigno Rodriguez (1), Clifford Harding (1), Donald Anthony (1)
(1) Case Western Reserve University, Cleveland, OH, USA
NK cells are thought to contribute to outcome during acute hepatitis C virus (HCV) infection, and newer evidence supports a role during
interferon (IFN) induced control of chronic HCV infection. We previously observed that IFN-alphaR and NKP30 expression were associated
with IFN-alpha dependent NK activity. Here we examined CD16+56-, CD16+56+, and CD16-56+ NK cell subset IFN-alpha R and NKP30
expression in relation to magnitude of HCV decline during IFN-alpha /ribavirin therapy for HCV genotype-1 infection. We observed greater
baseline IFN-alphaR and NKP30 expression on CD16+56+ and CD16-56+ NK subsets in HCV infected subjects than those of healthy controls.
Baseline CD16+56- NK IFN-alpha R expression was associated with IFN-alpha induced pSTAT1 and both were associated with magnitude
of HCV decline during IFN-alpha/ribavirin therapy. In addition, baseline CD16+56- NK IFN-alphaR expression was associated with race and
IL28B genotype, negatively associated with aspartate aminotransferase/platelet ratio index (APRI), and positively associated with increase
in NKP30 expression after in vivo IFN-alpha exposure. Finally, IFN-alpha activated NK cytolysis of HCV infected target cells was in part
dependent on NKP30, and CD16+56- NK cells contributed to cytolytic activity. These data indicate IFN-alpha R expression on CD16+56- NK
cells during chronic HCV infection may in part be genetically determined, and level of expression regulates IFN-alpha signaling, which in turn
may contribute to control of HCV infection.
Contact: Donald Anthony
Case Western Reserve University, Cleveland, OH, USA
[email protected]
POSTERS
P2.14
POSTERS
P2.15
The Toll-like receptor mediated induction of cytokines and interferons is not impaired
in primary isolated liver cells from HCV-positive patients
Ruth Broering (1), Kathrin Kleinehr (1), Andreas Paul (2), Guido Gerken (1), Joerg F. Schlaak (1)
(1) University Hospital of Essen, Essen, Germany
(2) Dep. of Visceral and Transplataion Surgery, Germany
Background: The function of the hepatic innate immune system and its role in the defence against HCV are not well understood. Recent data
from our lab suggested that murine hepatocytes and non-parenchymal liver cells (NPC) can elicit potent antiviral responses and immune
modulation. Aim of this study was to investigate the TLR signaling in human hepatocytes as well as non parenchymal liver cells from HCVpositive and HCV-negative individuals. Methods: Human liver samples were obtained after tumor resection (n=14) or liver transplantation
(n=24; HCV n=13), respectively. Human hepatocytes (n=25) were isolated after perfusion and digestion of the liver. In addition, endothelial
cells (LSEC, n=7), stellate cells (HSC, n=6) and remaining NPC (n=18) were purified by density centrifugation and MACS separation. Cells were
stimulated with TLR1-9 ligands for 6h, RNA was extracted and expression of TNF-α, IL-6, IL-10, a subset of interferons and interferon sensitive
genes (ISGs) was determined by qRT-PCR. Results: In human hepatocytes as well as in different NPCs stimulation with TLR1-9 ligands, except
TLR9, led to cell-type specific induction of pro-inflammatory (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10). In contrast, expression of
type-I and –II interferons (IFN-α, -β, -γ, IL-28A, IL-28B, IL-29) could only be induced after TLR3 stimulation, resulting in enhanced expression
of ISGs (ISG15, IFI-T1, RSAD2, MXA). These polyI:C-induced expression levels were partially enhanced in HCV infected patients compared to
uninfected controls. The different underlying diseases, the type of surgery, fibrosis stage as well as liver function tests (ALT, AST, GGT, AP)
were not correlated with TLR signaling in these cells. Conclusions: Liver cells isolated from HCV infected individuals respond to TLR ligands
in a similar fashion as compared to HCV-negative controls. In contrast to data generated in hepatoma cell lines, primary hepatocytes from
HCV-infected patients are able to produce a variety of IFNs after TLR3 stimulation.
Contact: Ruth Broering
University Hospital of Essen, Essen, Germany
[email protected]
P2.16
The percentage of hepatitis C virus-specific CD127+ memory T cells in the acute phase
T cell response predicts the outcome of HCV infection
POSTERS
Eui-Cheol Shin (1), Su-Hyung Park (2), Michelina Nascimbeni (2), Marian Major (3), Stephen Feinstone (3), Charles Rice (4), Barbara
Rehermann (2)
(1) KAIST, Graduate School of Medical Science & Engineering, Daejeon, Republic of Korea
(2) Immunology Section, Liver Diseases Branch, NIDDK, NIH, DHHS, USA
(3) Laboratory of Hepatitis Viruses, CBER, FDA, USA
(4) Center for the Study of Hepatitis C, The Rockefeller University, USA
It is well accepted that vigorous T cell responses contribute to clearance of acute HCV infection. In contrast, it remains unclear whether
the exhausted and inhibited phenotype of HCV-specific T cells observed in chronic HCV infection are the cause or consequence of HCV
persistence. Here we compared early activation, inhibition and memory markers of T cell responses in blood and liver throughout the course
of acute HCV infection in five chimpanzees. Chimpanzees with self-limited infection showed earlier and more vigorous HCV-specific T cell
responses in the blood and higher CD8 and FasL mRNA expression in the liver than chimpanzees with chronic progression of infection. In
addition, chimpanzees with self-limited infection displayed earlier and stronger activation of HCV-specific CD8 T cells in the blood as studied
by PD-1 and CD38 expression on tetramer+ T cells and confirmed by real-time PCR in the liver. The strong correlation between PD-1 and
CD38 levels suggests that PD-1 is an activation, not an exhaustion marker in acute HCV infection. Consistent with this interpretation, high
levels of PD-1 on HCV-specific T cells in acute hepatitis C did not interfere with the development of CD127+ memory T cells. The frequency of
CD127+ HCV-specific memory T cells at the first time point of their appearance in the blood predicted the outcome of infection. In contrast,
the intrahepatic expression level of T cell inhibitory factors such as CTLA-4, LAG-3, Tim-3, the Tim-3 ligand galectin-9, the regulatory T cell
marker Foxp3 and the immunosuppressive cytokines IL-10 and TGF-b during acute infection did not predict the outcome of HCV infection.
In conclusion we found that the strength and timing of the HCV-specific immune response and its capacity to generate CD127+ memory T
cells but not the expression of inhibitory molecules correlate with the outcome of acute HCV infection.
Contact: Eui-Cheol Shin
KAIST, Graduate School of Medical Science & Engineering, Daejeon, Republic of Korea
[email protected]
128 : HCV 2011
CELLULAR IMMUNITY
P2.17
HCV alters DC activation through the production of thymic stromal lympopoitein (TSLP)
cytokine and promotes Th17 differentiation
HaiChon Lee (1), Young S. Hahn (1)
(1) University of Virginia, Charlottesville, VA, USA
Hepatitis C Virus (HCV)-specific CD4 T cells are essential in the generation of a successful HCV-specific immune response. However, the
molecular and cellular mechanisms underlying the development of CD4 T cells during HCV infection remain unclear. We report that HCV
induces thymic stromal lymphopoietin (TSLP), an epithelial cell-derived IL-7-like cytokine, in human liver epithelial cells. This HCV-induced
production of TSLP is regulating in the NFκB-dependent manner, a crucial mediator in innate immune pathway. The TSLP secreted by
HCV-infected epithelial cells is capable of activating human monocyte-derived DC such that co-culture of HCV-infected liver epithelial with
monocyte cells significantly increases TGF-β, IL-6 and IL-21 production, a cytokine milieu that regulates and expands Th17 cells. In addition,
TSLP neutralizing antibody blocks TGF-β, IL-6 and IL-21 production by HCV-infected cells. Importantly, co-culture of naïve CD4 T cells with DC
exposed to HCV-infected cells results in the increased frequency of Th17 cells. These data suggest that HCV-mediated TSLP production plays
a critical role in DC activation and promote CD4 T cells to Th17 differentiation.
Contact: HaiChon Lee
University of Virginia, Charlottesville, VA, USA
[email protected]
P2.18
Natural killer cells and dendritic cells function during acute HCV
Natural killer (NK) cells provide an early defense line against viral infections by killing infected cells and producing cytokines that inhibit
viral replication. They also interact with dendritic cells (DCs) and this reciprocal interaction results in regulation of both innate and adaptive
immune responses. We hypothesized that NK cell activity during acute HCV infection will modulate DC function and the capacity to prime
an efficient T cell response resulting in viral clearance. This effect can be mediated through either modulating the maturation status and
cytokines produced by DCs or selective killing of DCs presenting HCV antigens. We have recently reported increased NK cell degranulation
during acute HCV irrespective of infectious outcome. Importantly, NK cell degranulation correlated positively with the magnitude of HCVspecific T cell responses, suggesting that NK cells might play an indirect role in priming HCV-specific T cells through interaction with DCs. To
examine the function of DCs during acute HCV infection with different outcomes in relation to NK cells, we have optimized a flow cytometry
based method to study the frequency, maturation status (CD83, 86, PDL1/2) and cytokine (IL-6,-12, TNF-a) production of DCs in response
to different Toll-like receptor (TLR) ligands. In addition, to measure the effect of NK cells on limiting antigen presentation by DCs we have
developed a co-culture system to study NK-DC interactions such as the capacity of NK cells to kill immature DCs or DCs presenting HCV
antigens and the reciprocal capacity of DCs to enhance the activity of NK cells. This analysis is ongoing and we expect to observe decreased
DC maturation and cytokine production as well as defects in the NK-DC cross-talk during acute HCV in patients who evolve to chronicity.
These defects would consequently reduce DC maturation or death of DC, and thus limit HCV Ag presentation to T cells.
Contact: Sandy Pelletier
Centre de Recherche du CHUM, Hôpital St-Luc, Montréal, Canada
[email protected]
POSTERS
Sandy Pelletier (1), Elias Said (1), Petronela Ancuta (1), Julie Bruneau (2), Naglaa Shoukry (1)
(1) Centre de Recherche du CHUM, Hôpital St-Luc, Montréal, Canada
(2) Département de Médecine Familiale Université de Montréal, Canada
POSTERS
P2.19
Characterisation of interferon lambda (IFN λ)-producing dendritic cell (DC) populations
in healthy and hepatitis C virus (HCV)-infected liver perfusate
Aoife Kelly (1), Elizabeth Ryan (2), Nigel Stevenson (1), Justin Geoghegan (3), John Hegarty (3), Cliona O’Farrelly (1)
(1) Trinity College Dublin, Dublin, Ireland
(2) Education and Research Centre, St. Vincent’s University Hospital, Ireland
(3) Liver Unit, St. Vincent’s University Hospital, Ireland
Plasmacytoid DCs (pDCs), the body’s major interferon-producing cells, are both responsive to, and produce IFN λ. A subpopulation of
myeloid DCs (mDCs), BDCA3+, has recently been shown to be a major producer of IFN λ in response to poly I:C. However, little is known
about DC populations, particularly BDCA3+, in the liver, the primary site of HCV replication. In this study, liver perfusate was collected
during transplantation. Hepatic mononuclear cells (HMNCs) were isolated from healthy and HCV+ explant liver perfusate. DC populations
and IFN λ receptor expression were then characterised by flow cytometry. Matched donor blood samples were also collected to directly
compare DCs in peripheral blood to liver. Significant populations of DCs are found in liver perfusate. The ratio of mDCs to pDCs was similar
in liver perfusate and blood (4.6:1, range 2.9-6.4:1). Within the liver mDCs, BDCA3+ DCs comprise a significant population (83.5%, 0.9%
of total cells) whereas they account for only 0.03% of total white cells in the blood. Liver perfusate from a HCV+ donor had an increased
proportion of pDCs compared to healthy perfusate (30.1%, mDC:pDC ratio 1.5:1) but the proportion of BDCA3+ was decreased (74.2%,
0.29% of total). Increased IFN λ receptor expression was also observed in HMNCs from healthy liver perfusate compared to peripheral blood
mononuclear cells indicating increased potential IFN λ responsiveness in the liver. These results indicate that the liver is an important site of
DC accumulation, particularly the IFN λ-secreting BDCA3+ DCs. Analysis of a single HCV-infected perfusate revealed increased proportion of
pDCs and depletion of BDCA3+ DCs compared to healthy perfusate. Further studies of DCs and cells that produce IFN λ in the liver should
provide insight into the relationship between IL28B genotype and the expression and biological function of IFN λ.
Contact: Aoife Kelly
Trinity College Dublin, Dublin, Ireland
[email protected]
P2.20
Reversion of cytotoxic T-cell escape mutation during pregnancy permits vertical
transmission of most fit hepatitis C variant
POSTERS
Jonathan Honegger (1), Jennifer Kohout (1), Seungtaek Kim (2), Stanley Lemon (2), Arash Grakoui (3), Christopher Walker (1)
(1) The Ohio State University, Columbus, OH, USA
(2) University of North Carolina, USA
(3) Emory University, USA
Background: Cytotoxic T-lymphocyte (CTL) escape mutations embedded in vertically transmitted hepatitis C genomes could limit the
number of CTL epitopes recognized by the infant but may be detrimental to viral replication. Here we assessed the transmission of CTL
escape mutants to an infant born to a genotype 1a infected mother and the fitness cost of a CTL escape mutation that reverted to wild-type
during pregnancy. Methods: Clonal sequencing of the HCV genome was performed on maternal plasma samples prior to, during, and after
pregnancy and on infant plasma 12 weeks after birth. Viral amino acid substitutions occurring in the mother were tested for CTL escape
potential using synthetic peptides with wild-type versus mutant sequences. The fitness costs of CTL escape mutations were tested in vitro
using the genotype 1a H77S.3 system. Results: Three of four CTL escape mutations persisted through pregnancy and were recovered from
infant plasma at week 12. One escape mutation (L1402F) located within overlapping B*0801 epitopes gradually reverted to >95% wild-type
sequence by late pregnancy and this wild-type sequence was found in infant plasma at week 12. The L1402F mutant reappeared in the
mother shortly after delivery but was eventually replaced by a double mutant K1398R/A1409T. Transfection of H77S.3 RNA bearing the
maternal wild-type sequence at 1402 resulted in nearly 5 fold greater RNA replication and infectious virus production than transfection of
RNA bearing any of the escape mutants. Conclusions: While three escape mutations persisted through pregnancy and were transmitted,
one escape mutation with substantially impaired replication in vitro reverted to wild-type during pregnancy resulting in transmission of the
more fit sequence. This finding suggests that the relatively immune-suppressed state of pregnancy may allow HCV to shed variants with the
most costly escape mutations, establishing neonatal infections with genomes optimized for viral replication.
Contact: Jonathan Honegger
The Ohio State University, Columbus, OH, USA
[email protected]
130 : HCV 2011
CELLULAR IMMUNITY
P2.21
Comparison of T cell immunity to the hepatitis A and C viruses in acutely infected
chimpanzees
Yan Zhou (1), Deborah Chavez (2), Kathleen Brasky (2), Heather Eccleston (1), Stanley Lemon (3), Robert Lanford (2), Christopher Walker (4)
(1) The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
(2) Texas Biomedical Research Institute, USA
(4) Research Institute at Nationwide Children’s Hospital, USA
Two chimpanzees were sequentially infected with HAV and HCV to compare the kinetic and function of T cell responses to these small
hepatotropic viruses. T cell immunity to HAV, which does not establish a persistent infection in humans or chimpanzees, was evident in
blood within 4 weeks of infection and was temporally associated with peak liver injury and initial control of viremia. After HCV challenge
of the same animals, virus-specific T cells were not detected in blood until week 8-9. At the peak of the cellular immune response after
HAV and HCV infection, circulating virus-specific CD4+ T cells targeted multiple proteins and produced IL-2, IL-21, IFN-γ and TNF-α. The
proportion of CD4+ T cells producing two or more of these cytokines was very similar after both infections. HAV and HCV-specific CD8+ T
cell responses were also similar. They were either absent from blood or peaked at lower frequencies after challenge with each virus. Fewer
epitopes were targeted when compared with CD4+ T cells and they primarily produced IFN-γ. HAV-specific CD8+ T cells expanded in blood
two weeks before acquiring function and contracted sharply before virus production was terminated. This stunned response during acute
resolving HAV infection was unexpected and reminiscent of acute phase CD8+ T cell responses in HCV infections that often persist. The longterm fate of multi-functional virus-specific CD4+ T cell responses detected at the peak of HCV and HAV infections will be described, and
should provide insight into differences in a key immune response that determines infection outcome.
Contact: Yan Zhou
The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
[email protected]
P2.22
In vivo assessment of the proliferative potential of hepatitis C virus specific CD8+ T cells
Progression to chronic hepatitis C is characterized by the accumulation of escape mutation in HCV epitopes targeted by the CD8+ T cell
response. However some epitopes remain intact throughout the chronic phase of infection. CD8+ T cells targeting these epitopes lack
effector functions and display an exhausted phenotype characterized by up-regulation of multiple inhibitory receptors and low expression
of the IL7Rα (CD127) molecule. It is assumed that this exhausted state is the consequence of continuous TCR triggering due to preservation
of the cognate epitopes. Consistent with this hypothesis, recent studies demonstrated that CD8+ T cells downregulate inhibitory receptors
and up-regulate CD127 following escape mutation of the targeted epitope. However, to our knowledge, whether the exhausted state is
completely overcome and these cells recover effector function is unknown. In this study, we enrolled two chimpanzees with a peripheral
CD127+ CD8+ T cell population to probe the in vivo function of cells targeting an escaped epitope. Autologous peripheral blood mononuclear
cells were loaded with a class I epitope from HCV or cytomegalovirus (CMV), and reinfused into the animals. Tetramer staining analysis revealed
a 10-fold expansion of CMV-specific CD8+ T cells in the peripheral blood of both animals. Surprisingly, HCV-specific CD8+ T cells did not expand,
despite phenotypical evidence of activation (e.g. up-regulation of CD38). These results contrast with previous studies describing potent in
vitro proliferation ability of virus specific CD8+ T cells expressing CD127. Rather, our data suggest (i) that CD127 expression is not a hallmark
of functional T cells or proliferation ability, and (ii) that some CD8+ T cells targeting escaped epitopes lack effector functions and have
impaired TCR signaling. These cells may require additional signals (such as CD4 help, costimulation or cytokines) to recover function.
Contact: Benoît Callendret
Nationwide Children’s Hospital Research Institute, Columbus, OH, USA
[email protected]
POSTERS
Benoît Callendret (1), Heather Eccleston (1), Jennifer Rutkiewicz (1), Dana Hasselschwert (2), David Bowen (1), Christopher Walker (1)
(1) Nationwide Children’s Hospital Research Institute, Columbus, OH, USA
(2) New Iberia Research Center, University of Lafayette, LA, USA
POSTERS
P2.23
B cell homeostasis in chronic hepatitis C virus-related mixed cryoglobulinemia
Lauren Holz (1), Joo Chun Yoon (1), Sukanya Raghuraman (1), Susan Moir (2), Michael Sneller (2), Barbara Rehermann (1)
(1) Immunology Section, LDB, NIDDK, National Institutes of Health, Bethesda, MD, USA
(2) Laboratory of Immunoregulation, NIAID, National Institutes of Health, USA
Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. Immune
complexes of HCV, anti-HCV IgG, rheumatoid factor IgM and complement trigger inflammation in small blood vessels. The formation of
immune complexes is driven by clonal expansions of autoreactive memory B cells but total B cell numbers are not increased.
To investigate mechanisms of B cell homeostasis, we studied HCV patients without MC (n=19), HCV patients with MC (n=17) and healthy
controls (n=50). HCV patients with MC displayed 4-fold expansions of activated and immature B cell subsets and an increased percentage of
immature T2 transitional B cells (lower T1/T2 ratio). Higher immature B cell percentages were also observed in HCV patients without MC,
but the T1/T2 ratio was not affected. The size of the naïve B cell subset was reduced in both HCV patient groups with an increased sensitivity
of naïve B cells to apoptosis in particular in the HCV group with MC. These results suggest that normal B cell numbers in HCV patients with
MC are maintained due to apoptosis of naïve B cells.
Nine HCV patients with MC underwent treatment with Rituximab, a B cell-depleting antibody. Immune reconstitution started 6 months post
treatment with almost half of all B cells being immature transitional. Six months later B cell subsets and cryoglobulin levels had normalized.
Newly generated B cells were more akin to B cells in healthy controls as evidenced by a normal T1/T2 ratio and reduced percentage of
activated B cells. The correlation between T2 proliferation and the percentage of cryoglobulins suggests a link between the skewing of the
T1/T2 ratio and the formation of immune complexes.
This study provides new insight into the complexity of B cell homeostasis in hepatitis C and cryoglobulinemia and shows that Rituximab
restores B cell homeostasis.
Contact: Lauren Holz
Immunology Section, LDB, NIDDK, National Institutes of Health, Bethesda, MD, USA
[email protected]
POSTERS
132 : HCV 2011
POSTERS
Vaccine Development
P3.01
Transmission of hepatitis C between spouses: an epidemiological study at
national liver institute hospital
Wesam Morad
P3.02
Nosocomial Infection in living donor liver transplantation and strategies
for prevention
Wesam Morad
P3.03
A novel 2b/1a recombinant virus in a US patient
Rob Striker, Dipankar Bhattacharya, Molly Accola, William Rehrauer, Israr Ansari
P3.04
Does a vaccine derived from a single HCV strain elicit broadly crossneutralising antibodies in humans?
John Law, Darren Hockman, Sharon Frey, Takaji Wakita, Jens Bukh, Christopher Jones, Charles Rice, Sergio Abrignani,
Michael Houghton
Modifications required to turn HCV core gene into potent DNA immunogen
Maria Isaguliants, Elizaveta Starodubova, Ekaterina Alekseeva, Maria Mikhailova, David Hallengärd, Oleg Latishev,
Andreas Bråve, Olga Smirnova, Alexander Ivanov, Irina Sominskaya
P3.06
HCV 6a prevalence in Guangdong province had the origin from Vietnam and
recent dissemination to other regions of China
Hongren Wang, Chunhua Li, Kenji Abe, Ling Lu, Yongshui Fu
P3.07
HCV seroprevalence among men-who-have-sex-with-men (MSM) in
New York City
Ethan M. Weinberg, Rositsa Dimova, Ann Drobnik, Eric Rude, Kristen M. Marks, Daniel S. Fierer, Andrew H. Talal
POSTERS
P3.05
POSTERS
P3.08
Chimeric HBV-HCV envelope proteins form subviral particles that induce a
strong immune response against HBV and HCV envelope proteins.
Elodie Beaumont, Romuald Patient, Christophe Hourioux, Isabelle Dimier-Poisson, Philippe Roingeard
P3.09
Clinical, biochemical, and pathological profiles of 5464 Egyptian chronic
hepatitis C infected patients
Tahany Awad, Mahasen Mabrouk, Maissa El-Raziky, Naglaa Zayed, Rabab Salama, Wafaa Al-Akel, Mohamed El-Beshlawy,
Hadeel Gamal, AbuBakr Awad, Gamal Esmat
P3.10
A new vaccine strategy for HCV: presentation of hepatitis C virus
hypervariable region 1 on HBV capsid-like particles
Milena Lange, Sergei Viazov, Olena Brovko, Hanaa M. Alam El-Din, Nehal Hussien, Yuri Khudyakov, Michael Nassal,
Paul Pumpens, Michael Roggendorf, Abdel-Rahman Zekri, Andreas Walker
P3.11
Immunological memory response to induce neutralizing immunoglobulin in
HCV particles-immunized mice
Masaki Moriyama, Hiroshi Yokokawa, Daisuke Akazawa, Kazumi Nishimura, Noriko Nakamura, Hidenori Mochizuki,
Takanobu Kato, Koji Ishii, Takaji Wakita
P3.12
Characterisation and imunogenicity of adenovector-expressed HCV
glycoproteins depleted of specific glycans
Alicja Chmielewska, Mariarosaria Naddeo, Virginia Ammendola, Stefano Colloca, Alfredo Nicosia, François Helle,
Jean Dubuisson, Krystyna Bienkowska-Szewczyk, Antonella Folgori
P3.13
Characterization of a canine homolog of hepatitis C virus
Amit Kapoor, Peter Simmonds, Gisa Gerold, Natasha Qaisar, Komal Jain, Jose A. Henriquez, Cadhla Firth,
David L. Hirschberg, Charles M. Rice, Shelly Shields, W. Ian Lipkin
P3.14
Expansion of functional CD8+ T cell response targeting the circulating virus
after therapeutic vaccination for hepatitis C virus
POSTERS
Benoît Callendret, Antonella Folgori, Heather Eccleston, Stefania Capone, Riccardo Cortese, Alfredo Nicosia,
Christopher Walker
P3.15
Considerations for expanding federal policies regarding viral hepatitis
screening
Bryce Smith, Rebecca Morgan, Geoff Beckett, John Ward
P3.16
Characterization of the full coding sequences of five HCV strains including
subtypes 2i and 2l as well as three undefined subtypes 2
Jean-Francois Cantaloube, Francois Jordier, Philippe de Micco
P3.17
HVR1, a decoy that interferes with the immunogenicity of conserved
epitopes in HCV E2 protein
Ping Zhao, Xiaoqing Liu, Shi Yan, Yimin Tong, Xiaoling Liao, Xian Hua, Yongzhe Zhu, Di Yin, Zhongtian Qi
P3.18
The neutralizing epitope in HVR1 inhibits the production of antibody to
HCV E2 protein
Ping Zhao, Xiaoqing Liu, Shi Yan, Yimin Tong, Xiaoling Liao, Xian Hua, Yongzhe Zhu, Di Yin, Zhongtian Qi
134 : HCV 2011
EPIDEMIOLOGY, GLOBAL BURDEN, AND VACCINE DEVELOPMENT
P3.19
Molecular analysis of hepatitis C virus genotypes in Croatia
Ivana Furcic, Ana Planinic, Snježana Židovec-Lepej, Juan Carlos Sáiz, Antonio Mas, Adriana Vince
P3.20
Immune responses by CD4+ and CD8+ T-cells in injecting drug user (IDU)
prison inmates with multiple genotype HCV infection
Son T. Pham, Rowena A. Bull, Elizabeth Keoshkerian, Barbara Cameron, Gregory J. Dore, Michaela Lucas, Katja Pfafferott,
Silvana Gaudieri, Andrew R. Lloyd, Peter A. White
P3.21
Development of purification method for HCV particles using
chromatographic technique
Hiroshi Yokokawa, Daisuke Akazawa, Masaki Moriyama, Noriko Nakamura, Takanobu Kato, Koji Ishii, Takaji Wakita
P3.22
Optimization of peptide arrays for dissecting antibody responses to HCV
infection and vaccination studies
Tinashe Ruwona, Ryan Mcbride, Steven Head, Phillip Ordoukhanian, Dennis Burton, Mansun Law
P3.23
A therapeutic vaccine for HCV based on novel, rare, adenoviral vectors
Christabel Kelly, Antonella Folgori, John Halliday, Ye Oo, Stefania Capone, Richard Antrobus, Katherine Gantlett,
Elizabeth Stafford, Denise O’Donnell, Jane Collier, Anthony Brown, Rachel Huddart, Ayako Kurioka, Rachel Townsend,
Leo Swadling, Virginia Ammendola, Stefano Colloca, Mariarosaria Naddeo, Loredana Siani, Cinzia Traboni, David Mutimer,
David H Adams, Ricardo Cortese, Alfredo Nicosia, Paul Klenerman, Eleanor Barnes
P3.24
Greatly enhanced immunogenicity using a heterologous prime-boost
strategy for activation of hepatitis C virus non-structural (NS)-specific T cells
G. Inchauspé, A. Fournillier, L. Frelin, E. Jacquier, G. Ahlém, E. Gérossier, A. Brass, F. Holmström, K. Broderick,
N. Y. Sardesai, M. Sälberg
P3.25
Convergence and tractability of HCV HVR1 cross-immunoreactivity
P3.26
Characterization of neutralizing and interfering epitopes on hepatitis C virus
E2 glycoprotein using monoclonal antibodies
Hongying Duan, Lilin Zhong, Evi Struble, Kathleen Mihalik, Alla Kachko, Marian Major, Pei Zhang, Stephen M. Feinstone
P3.27
All-cause, liver-, and non-liver, related mortality among Hepatitis C infected
individuals in the general US population
Samer El-Kamary, Ravi Jhaveri, Michelle Shardell
P3.28
A synthetic codon-optimized HCV non-structural 5A DNA vaccine primes
polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice
Fredrik Holmström, Sepideh Alahyari, Malte Kriegs, Eberhard Hildt, Matti Sällberg, Gustaf Ahlén, Lars Frelin
P3.29
High cure rate in patients with chronic HCV gt1 infection receiving SOC
therapy after participation in a clinical trial of therapeutic DNA vaccination
Matti Sällberg, Sepideh Alahyari, Lars Frelin, Ola Weiland
POSTERS
David S. Campo Rendon, Zoya Dimitrova, Jonny Yokosawa, Duc Hoang, Nestor Perez, Sumathi Ramachandran,
Yury Khudyakov
POSTERS
P3.30
Genomic sequence analysis tools and a genotype-phenotype association
platform in the virus pathogen resource
Yun Zhang, Brett Pickett, Jyothi Noronha, R. Burke Squires, Victoria Hunt, Mengya Liu, Liwei Zhou, Chris Larson,
Jonathan Dietrich, Edward Klem, Richard Scheuermann
P3.31
HCV spread in different intravenous drug user networks – a large
population assessment
Marianne Alanko-Blome, Maria Josephson, Vilma Molnegren, Per Björkman, Anders Widell
P3.32
Characterizing HCV full length genome sequences for 27 isolates
representing various subtypes with genotypes 2, 3, and 4
Chunhua Li, Ling Lu, Hongren Wang, Jie Yuan, Donald Murphy
P3.33
Properties of HCV particles produced in serum-free Huh7.5 cell culture
Christian Mathiesen, Tanja B. Jensen, Judith Gottwein, Jens Bukh
POSTERS
136 : HCV 2011
P3.01
Transmission of hepatitis C between spouses: an epidemiological study at national
liver institute hospital
Wesam Morad (1)
(1) National Liver Institute, Shebin El Kom, Egypt
Background/Aims: In Egypt, relatively higher rates of sexual transmission have been reported and reflect the higher background prevalence
in this country. In rural Egypt, sexual transmission between monogamous spouses ranged between 3 and 34%. This is a cross sectional
hospital based study evaluated the non-sexual and sexual risk factors of HCV transmission from index male and female cases to their
spouses, to assess proportionate morbidity rate of HCV disease and to assess the relationship between the degree of HCV viremia in
index cases and the transmission of HCV infection. Methods: One hundred index males and 100 index females with HCV and their spouses
provided informed consent to participate. Participants were interviewed to gather risk factor information using multiple question model
previously prepared questionnaire and serological tests (anti HCV Abs, Hbs Ag and HCV-RNA PCR). Results: Proportionate morbidity rate of
HCV disease in NLI was 80% and HCV transmission from wife to husband including sexual relationship occurred in 46% of cases while from
husband to wife was 25% and 35.5% overall HCV transmission between spouses. Conclusion: This study documented previously uncertain
life-style risk factors for HCV transmission between spouses, confirmed the high prevalence of HCV infection (17% to 26%) in Egypt which
is higher than other parts of the world, and provided evidence of sexual transmission of HCV among spouses but we can’t ignore the
significant role of non sexual risk factors of HCV among couples such as sharing clippers or nail cutters and others.
Contact: Wesam Morad
National Liver Institute, Shebin El Kom, Egypt
[email protected]
P3.02
Nosocomial Infection in living donor liver transplantation and strategies for prevention
Background/Aims: Living donor liver transplantation (LDLT) is becoming a widespread technique for patients with acute and chronic end
stage liver diseases with good results making liver transplantation a widely accepted treatment modality. Infection is the most frequent
cause of morbidity and mortality following liver transplantation in Egypt. This is a cross sectional hospital based study describe types,
characteristics and rate of infections occurring in the early postoperative period and the possible associated preoperative, operative, and
post operative risk factors in patients underwent living related liver transplantation aiming at designing strategy for infection control protocol
in liver transplant program applied at National Liver Institute. Methods: 50 living donor liver transplantation patients’ provided informed
consent to participate. Participants were interviewed to gather risk factors information using multiple question model previously prepared
questionnaire, biophysiologic measures and observation checklist. Results: Living donor liver transplantation complicated Infection was
76.0%. 68.8% of infection episodes occurred in the first month post transplantation and the incidence declined thereafter. Infection induced
mortality was 77.8%. Conclusion: This study confirmed that high infection rate was associated with prolonged operative time (14.8 ± 3.07)
and septic techniques used intra and post-operative. Designing a good healthcare educational program for the nurses, health care workers
and patient contacts’ focusing on the prevention of infection related risk factors and careful evaluation of donor and recipient prior to
liver transplantation prevents serious post transplantation infection, either by excluding risky donors or by defining the need for specific
antimicrobial therapy post liver transplantation.
Contact: Wesam Morad
National Liver Institute, Shebin El Kom, Egypt
[email protected]
POSTERS
Wesam Morad (1)
(1) National Liver Institute, Shebin El Kom, Egypt
POSTERS
P3.03
A novel 2b/1a recombinant virus in a US patient
Rob Striker (1), Dipankar Bhattacharya (2), Molly Accola (2), William Rehrauer (2), Israr Ansari (2)
(1) Veterans Administration, Madison, WI, USA
(2) UW-Madison School of Medicine, USA
Recombination between two different genotypes has only rarely been reported for Hepatitis C Virus (HCV) and never in the Western
Hemisphere. Recombination is frequently cited as a method of genetic diversification frequently patients with HCV have been exposed
multiple times. In order to improve the accuracy of the clinical genotyping offered at our hospital we routinely performed sequencing
reactions on both the 5’ Untranslated Region (UTR), and the NonStructural 5B (NS5B) polymerase and found only 1 discordant result out of
134 patients. Further sequencing of this viral isolate showed that it contains a genotype 2b lineage from the 5’ UTR to the end of NS2, at the
same time as a genotype 1a lineage from NS3-NS5B. At our hospital only one other viral isolate has had a nearly identical genotype 2b 5’
UTR and the entire lineage of that viral isolate was 2b. Multiple viral recombinants in Europe and southeast Asia all involve a 5’ genotype 2
genome recombining with a 3’ genome of either the 1b, 5, or 6 lineage. Some of these recombinants but not all also have a NS2/3 junction
suggesting that genotype 2 at least may have a hotspot for recombination. This is the first case report of a naturally occurring recombinant
HCV in the United States between two genotypes. Conclusions: A small percentage of patients may be infected with a recombinant virus in
the United States, and a 2b/1a recombinant should be considered as a reason for a genotype 2 infected HCV patient to have a slow response
to interferon.
Contact: Rob Striker
Veterans Administration, Madison, WI, USA
[email protected]
P3.04
Does a vaccine derived from a single HCV strain elicit broadly cross-neutralising
antibodies in humans?
POSTERS
John Law (1), Darren Hockman (1), Sharon Frey (2), Takaji Wakita (3), Jens Bukh (4), Christopher Jones (5), Charles Rice (5), Sergio
Abrignani (6), Michael Houghton (1)
(2) St Louis University School of Medicine, USA
(3) National Institutes of Infectious Diseases, Tokyo, Japan
(4) University of Copenhagen, Denmark
(5) Rockefeller University, USA
(6) Istituto Nazionale Genetica Molecolare, Milano, Italy
All successful vaccines developed to date protect largely through the induction of neutralising antibodies in the case of viral pathogens
and through the induction of bacteriocidal antibodies in the case of bacterial pathogens. A key challenge in the fields of HCV (and HIV)
vaccine research relates to the ability of vaccines to elicit antibodies that can cross-neutralise multiple clades of these highly heterogeneous
viruses since much data has been presented suggesting that neutralizing antibodies against HCV(and HIV) may be narrowly restricted or
only isolate-specific. We have addressed this question by assaying sera samples from a phase 1 clinical trial in which healthy volunteers
received recombinant gpE1/gpE2 derived from the original HCV1 strain along with the MF59 adjuvant. Sera samples have been assayed for
the presence of antibodies that can neutralize infectious HCVcc derived from Huh-7.5 human hepatoma cell cultures containing gpE1/gpE2
derived from heterologous 1a, 1b,2a and other HCV strains. These data will be discussed in the context of future efforts to develop regional
and global vaccines against this common pathogen which is a major cause of morbidity and mortality around the world.
This study performed under NIH/NIAID VTEU contract N01-A1-25464, Study number DMID 01-002 (Rajen Khoshy )
Contact: John Law
[email protected]
138 : HCV 2011
P3.05
Modifications required to turn HCV core gene into potent DNA immunogen
Maria Isaguliants (1), Elizaveta Starodubova (1), Ekaterina Alekseeva (2), Maria Mikhailova (2), David Hallengärd (1), Oleg Latishev (5),
Andreas Bråve (1), Olga Smirnova (4), Alexander Ivanov (5), Irina Sominskaya (2)
(2) Biomedical Research and Study Center, Latvia
(3) Ivanovsky Institute of Virology, Russia
(4) Engelgardt Institute of Molecular Biology, Russia
(5) Ivanovsky Institute of Virology & Institute of Molecular Biology, Russia
Hepatitis C is a serious healthcare problem. No vaccines are yet available, although many are in clinical trials. Human DNA vaccines are promising;
they are well-tolerated and induce an immune response that limit viral replication at least temporally (Sällberg, 2009; Alvarez-Lajonchere L,
2009). An attractive target of such vaccine is viral core, highly conserved protein that packages viral RNA and regulates its translation. It is
a strong immunogen with responses evolving early in infection. Despite this, core does not perform well as DNA-immunogen. This may be
due to its regulatory/immunoregulatory properties attributed to the C-terminal domain (regulation of NF-kB transcription, inhibition of cell
cycle progression, cytotoxicity). The C-terminal depletion may then yield an efficient and safe vaccine candidate. Plasmids were constructed
encoding core truncated at aa 60, 98, 152, 173 and 191 and core 1-152 encoded by nonviral codons destroying RNA-folding. BALB/C mice
were DNA-immunized by electroporation with intramuscular boost. In prime, core genes were co-administered with luciferase/Luc gene. On
days 4-9-15-21-26 mice were injected with luciferin and in vivo luminescence was monitored by 3D-optical imaging/IVIS. After that, mice were
sacrificed, and splenocytes were assessed for in vitro response to core/core-derived peptides (Fluorospot, Mabtech). The capacity to induce
core-specific IFN-g response was similar for constructs encoding 150-191 aminoacid-long variants, and low for severely truncated versions.
Only core 1-152 gene with nonviral codons induced strong IL-2 and dual IFN-g/IL-2 secretion and anti-HCV antibodies. The immunogenic
performance was predicted by IVIS monitoring: strong response coincided with strong luminescence/high expression by day 4 and expressing
cell clearance/quenching by day ≥15. Quenching correlated with IFN-g/IL-2 response to core. The study demonstrated that low immunogenicity
can be overruled by using synthetic/non-viral core gene encoding C-terminally truncated protein. Acknowledgements: Swedish Institute New
Visby Program; Swedish Research Council grant K2011-79X-21744-01-6; Russian Foundation for Basic Research grant 10-04-01402a.
Contact: Maria Isaguliants
[email protected]
HCV 6a prevalence in Guangdong province had the origin from Vietnam and recent
dissemination to other regions of China
Hongren Wang (1), Chunhua Li (1), Kenji Abe (2), Ling Lu (1), Yongshui Fu (3)
(1) University of Kansas Medical Center, Kansas City, KS, USA
(2) Department of Pathology, National Institute of Infectious Diseases, Japan
(3) Guangzhou Blood Center, China
Background: In varied regions of China, 6a has shown a consistent predominance among HCV infected IDUs. Recently in the Guangdong Province,
6a has dramatically increased among patients and blood donors. This may be due to IDU linked transmission. Methodology/Findings: Two
hundred and ten drug users were recruited from a mandatory detoxification center in Shanwei City, Guangdong Province, China. After assaying
for anti-HCV by EIA and HCV-RNA by real-time PCR, 94.5% and 71.4 % of the users were positive, respectively. From 136 (90.7%) HCV-RNA+ users,
both E1 and NS5B fragments were amplified and sequenced. This resulted in genotyping 6a in 70 users, 3a in 42, 1b in 13, 3b in 3, and multiple
infections in 8. Among the 6a isolates, most were grouped into three clusters while 23% formed two new clusters representing emerging strains.
For a detailed migration analysis, additional 6a sequences were determined from 21 blood donors in Vietnam, 22 blood donors in 12 provinces
of China, and 36 IDUs in Liuzhou City of the Guangxi Province. The phylogeography indicated that Vietnam was the origin of 6a in China. The
Guangxi Province, which borders Vietnam, was the first region to accept 6a for circulation. Migration from Yunnan, which also borders Vietnam,
might be equally important, but only caused limited circulation among IDUs and failed to merge in the torrent of 6a prevalence in the country.
From Guangxi, 6a was further spread to the vicinity provinces: Yunnan, Guangdong, Hunan, and Hubei. However, evidence only showed that in
Guangdong 6a has become epidemic, which has made Guangdong the second source region to disseminate 6a to other provinces.
Conclusions/Significance: With an origin from Vietnam, HCV 6a has become local epidemic in the Guangdong Province where the increasing
prevalence has resulted in 6a subsequently being disseminated to many other regions.
Contact: HONGREN WANG
University of Kansas Medical Center, Kansas City, KS, USA
[email protected]
POSTERS
P3.06
POSTERS
P3.07
HCV seroprevalence among men-who-have-sex-with-men (MSM) in New York City
Ethan M. Weinberg (1), Rositsa Dimova (1), Ann Drobnik (2), Eric Rude (2), Kristen M. Marks (1), Daniel S. Fierer (3), Andrew H. Talal (1)
(1) Weill Cornell Medical College/New York Presbyterian Hospital, New York, NY, USA
(2) New York City Department of Health and Mental Hygiene, USA
(3) Mount Sinai School of Medicine, USA
Background: An emerging international epidemic of acute HCV infection has recently been identified among HIV-infected MSM. Data are
limited on HCV prevalence, transmission routes, and risk factors among MSM in the United States.
Methods: New York City Department of Health (NYCDOH) tests HCV seroprevalence at seventeen different sites with high-risk patient
populations. Subjects complete a survey regarding demographics, sexual history, drug use, and communicable diseases. HCV antibodies are
assessed using a second-generation ELISA. We compared MSM to all other males (non-MSM) for HCV prevalence, injection drug use (IDU),
HIV/HCV coinfection, and other associated risk factors.
Results: From October 2009 to March 2011, 1233 men were screened for HCV seropositivity, of whom 283 (23.0%) were MSM. HCV
seroprevalence was lower among MSM (for all comparisons, MSM vs. non-MSM: 49/283 vs. 274/950, p=0.0001); this difference was mostly
attributable to less IDU among MSM (84/283 vs. 367/950, p=0.006). However, even among injection drug users, HCV seroprevalence was
lower in MSM (39/84 vs. 227/367, p=0.01). While HIV infection was more common among MSM compared to non-MSM, both groups had
similar rates of HIV/HCV coinfection, mostly secondary to IDU. MSM were more likely to receive their initial HCV diagnosis as a result of this
screening (40/49 vs. 175/273 p=0.014). HCV seroprevalence was similar between MSM and non-MSM who did not engage in IDU; a history
of incarceration was the most common risk factor for HCV among all who did not engage in IDU.
Conclusions: IDU accounts for most HCV acquisition at NYCDOH testing sites. Independent of IDU, no difference exists in HCV seroprevalence
between MSM and non-MSM in this population (regardless of HIV status). Lower HCV seroprevalence among MSM-IDU suggests that this
cohort employs harm reduction practices. Higher rates of new HCV diagnoses among MSM suggests for increased screening in this group.
Contact: Ethan M. Weinberg
Weill Cornell Medical College/New York Presbyterian Hospital, New York, NY, USA
[email protected]
P3.08
Chimeric HBV-HCV envelope proteins form subviral particles that induce a strong
immune response against HBV and HCV envelope proteins
POSTERS
Elodie Beaumont (1), Romuald Patient (1), Christophe Hourioux (1), Isabelle Dimier-Poisson (2), Philippe Roingeard (1)
(1) INSERM U966; Université François Rabelais, Tours, France
(2) UMR INRA 483, Tours, France
With more than 170 million people worldwide chronically infected by HCV, and 3 to 4 million new infections occurring each year, there is an
urgent need for an effective prophylactic vaccine.
The HCV envelope glycoproteins E1 and E2 are the potential targets of neutralizing antibodies and constitute appropriate immunogens for
the development of such a vaccine. However, these proteins harboring a transmembrane domain are retained in the cell, which renders
their purification extremely difficult.
The hepatitis B virus (HBV) is another important human pathogen whose infection can be prevented using a safe, efficient vaccine
established in the early 80’s. This vaccine is based on the remarkable property of the small (S) HBV envelope protein to self-assemble in
highly immunogenic subviral envelope particles.
We previously described the concept of a vaccine based on the construction of plasmids encoding chimeric HBV-HCV envelope proteins
(S-E1 and S-E2), in which the N-terminal transmembrane domain of S has been substituted by the transmembrane domain of E1 or E2.
Produced in stably transduced CHO cells, these chimeric proteins are efficiently coassembled with the wild-type HBV S protein into subviral
particles that incorporate the whole E1 and E2 proteins, and that can be easily purified. Preliminary investigations showed that these
chimeric particles displayed a strong specific antibody response against the HCV and HBV envelope proteins in female BALB/c mice and
New-Zealand rabbits. Moreover, our experiments showed that these particles elicit cell-mediated immunity to HCV and HBV, with a Th1/Th2
cytokine profile in mice. Experiments are in progress to evaluate the neutralizing properties of the mouse and rabbit sera.
This strategy could ultimately lead to the establishment of a bivalent HBV-HCV prophylactic vaccine candidate that could be produced with
the same procedures used for the HBV vaccine, reducing the time and cost of its industrial development.
Contact: Elodie Beaumont
INSERM U966; Université François Rabelais, Tours, France
[email protected]
140 : HCV 2011
P3.09
Clinical, biochemical, and pathological profiles of 5464 Egyptian chronic hepatitis C
infected patients
Tahany Awad (1), Mahasen Mabrouk (2), Maissa El-Raziky (2), Naglaa Zayed (2), Rabab Salama (2), Wafaa Al-Akel (2), Mohamed ElBeshlawy (2), Hadeel Gamal (2), AbuBakr Awad (3), Gamal Esmat (2)
(1) Cochrane Hepato-Biliary Group, Copenhagen, Denmark
(2) Endemic Medicine and Hepatology Department, Cairo University Hospital,, Egypt
(3) Faculty of Computer Science, Cairo University, Cairo, Egypt, Egypt
Profile
Age(years)
BMI(kg/m2)
Hb (g/dl)
WBC(x103/cmm)
PLT(x103/μL)
AST (U/L)
ALT (U/L)
Albumin (g/dl)
Glucose (mg/dl)
AFP (ng/dl)
HCV-RNA(IU/ml)
Mean ±SD
41.56±9.85 28.12±4.32
14.12±1.54
6.47±1.83
214.28±63.29
56.77± 38.79
63.25± 42.92
4.22±0.47
99.43±28.93
16.46±10.26
1,34± 1,27 (x106)
Median
42.00
28.03
14.2
6.20
208.00
46
52
4.20
93.00
3.30
97,5 (x103)
Contact: Tahany Awad
Cochrane Hepato-Biliary Group, Copenhagen, Denmark
[email protected]
POSTERS
Background: In Egypt, hepatitis C Virus (HCV) is highly endemic (10% of general population), about 91% of them are infected with HCV
genotype 4. However, data of HCV-specific burden disease for Egypt are scarce. Detailed baseline demographic, biochemical, and histological
features of HCV patients might have major implications in designing optimal strategies for antiviral therapy, and hence better qualities for
patient care.
Aim: To identify clinical, biochemical and pathological features of chronic HCV patients as a step to optimize and standardize the required
information about HCV population in Egypt.
Methods: We analyzed retrospective data of 5464 HCV-antibody and PCR positive patients attending pretreatment assessment at one of the
National Treatment Reference Centers, Cairo, Egypt.
Results: Demographic characteristics and laboratory profile of the chronic HCV patients are presented in table 1. Egyptian chronic HCV
patients are mostly males in their productive age. Most patients had mild elevation of liver enzymes, low viral load, and low AFP levels. Liver
biopsy revealed A1 and A2 in 60% and 32% of patients respectively. Almost 80% of the patients had ≤ F2 and only 7.3 % of the patients had
F4 according to the METAVIR score. Meta regression demonstrated that advanced stages of fibrosis were significantly correlated with the
platelet count, AST/ALT ratio, AFP levels and BMI (p ≤ 0.001). However there was no correlation with viral load.
Conclusion: To our knowledge, this study is the first to include this huge number of chronic HCV patients on a nationwide scale, providing us
with the demographic, biochemical, and histological characteristics in chronic hepatitis C (genotype 4) patients.
Table 1 Demographic characteristics and laboratory profile of the chronic HCV patients.
POSTERS
P3.10
A new vaccine strategy for HCV: presentation of hepatitis C virus hypervariable region 1
on HBV capsid-like particles
Milena Lange (1), Sergei Viazov (1), Olena Brovko (1), Hanaa M. Alam El-Din (2), Nehal Hussien (2), Yuri Khudyakov (3), Michael Nassal (4),
Paul Pumpens (5), Michael Roggendorf (1), Abdel-Rahman Zekri (2), Andreas Walker (1)
(1) Institute of Virology, University of Duisburg-Essen, Essen, Germany
(2) Virology and Immnunology Unit, National Cancer Institute, Cairo, Egypt
(3) Centers for Disease Control and Prevention, Atlanta, USA
(4) University Hospital Freiburg, Freiburg, Germany
(5) Latvian Biomedical Research and Study Centre, Riga, Latvia
Hepatitis C virus (HCV) remains one of the most important human pathogens worldwide and until now no HCV vaccine is in sight. Most
recent data have demonstrated that antibodies to hypervariable region I (HVRI) of HCV envelope protein 2 are able block both cell-free as
well as cell-to-cell transmission of HCV. Future vaccines against HCV should therefore also be able to induce anti-HVRI antibodies.
The immunogenicity of small peptides can considerably be enhanced by their presentation on large particulate antigen carriers, such as
capsid-like particles (CLPs) derived from the HBV nucleocapsid. In the current study four different highly cross-reactive HVRI variants were
displayed on the surface of such CLPs. Upon immunization of mice these particles induced high titres of anti-HVRI antibodies, which were
able to cross-react with large numbers of naturally occurring HVRI sequences. When used in an HCVpp neutralization assay, the antibodies
were able to neutralize pseudoparticles bearing homologous as well as heterologous HVRI sequences. Also HCVpp with patient derived
envelope proteins could be neutralized efficiently, demonstrating that HVR-CLPs are indeed able to induce cross-neutralizing HCV antibodies.
Contact: Milena Lange
Institute of Virology, University of Duisburg-Essen, Essen, Germany
[email protected]
P3.11
Immunological memory response to induce neutralizing immunoglobulin in HCV
particles-immunized mice
POSTERS
Masaki Moriyama (1), Hiroshi Yokokawa (1), Daisuke Akazawa (1), Kazumi Nishimura (1), Noriko Nakamura (1), Hidenori Mochizuki (1),
Takanobu Kato (2), Koji Ishii (2), Takaji Wakita (2)
(1) Toray Industries, Inc. Pharmaceutical Research Laboratories, Yokohama, Japan
(2) National Institute of Infectious Diseases, Virology II, Japan
Hepatitis C virus (HCV) is a major causative agent of liver cancer, and it is important to develop prophylactic strategy against HCV infection.
Recently, the cell culture system of infectious HCV particles has been established, which has raised the possibility of developing HCV vaccine.
In this study, we inoculated mice with HCV particles in combination with various adjuvants and examined their immunogenic capacity.
The cell culture derived J6/JFH-1 chimeric HCV particles were purified by 500 kDa cut off membrane and sucrose density gradient centrifugation.
Purified HCV particles were inactivated by UV irradiation, and conjugated with several promising adjuvants (Alum, CpG, polyI:C and their
combinations, or MPL+TDM), and then injected intraperitoneally (i.p.) into BALB/c mice. Vaccination was repeated 4 times at 2-week intervals.
Antibody titers in mouse sera were measured by EIA method. HCV neutralizing activity was examined by HCVcc infection assay.
All vaccinated mice developed anti-HCV neutralizing antibodies in the sera after four immunizations. In order to examine the persistence of
anti-HCV antibodies in mice, EIA and in vitro neutralization assay were performed at 5 months after the 4th immunization. Both antibody
titers and neutralizing activities were observed in the sera, however their levels were relatively lower.
Finally, to examine the potency of HCV vaccine for immunological memory response, we re-challenged the mice with HCV particles at 10
months after 4th immunization. As a result, quick increase in antibody titer was observed as compared to the previous immunizations.
Neutralizing antibodies were detected in all vaccinated mice. Robust neutralizing effects were observed with Alum+CpG or MPL+TDM
adjuvants, compared with others. Thus, these results suggest that the HCV vaccination develop the immunological memory effect in
immunized mice which is important for prophylactic HCV vaccine.
Contact: Masaki Moriyama
Toray Industries, Inc. Pharmaceutical Research Laboratories, Yokohama, Japan
[email protected]
142 : HCV 2011
P3.12
Characterisation and imunogenicity of adenovector-expressed HCV glycoproteins
depleted of specific glycans
Alicja Chmielewska (1), Mariarosaria Naddeo (2), Virginia Ammendola (2), Stefano Colloca (2), Alfredo Nicosia (2), François Helle (3), Jean
Dubuisson (3), Krystyna Bienkowska-Szewczyk (1), Antonella Folgori (2)
(1) University of Gdansk, Gdansk, Poland
(3) Center for Infection & Immunity of Lille, Pasteur Institute of Lille, France
HCV envelope glycoproteins E1 and E2 form a non-covalent heterodimer which play a major role in the virus entry into host cells. Being
the main target for neutralizing antibodies, E1 and E2 glycoproteins have been widely considered as antigens for vaccination against HCV.
E1E2 ectodomains are heavily glycosylated with 4 to 5 glycosylation sites in E1 and up to 11 glycosylation sites in E2 and it is hypothesized
that glycan molecules may play a protective role against antibody-dependent neutralization and contribute to the evasion of HCV from the
humoral immune response. Assuming that “glycan shield” can limit the immunogenicity of HCV envelope proteins, we aim to generate
glycan depleted glycoproteins as potential vaccine candidates. As vaccine carrier we selected adenovirus based vectors that were previously
shown to induce both humoral and cellular immune responses.
It has been demonstrated that at least three glycans on E2 reduced the sensitivity of HCV pseudoparticles to antibody neutralization and
that the removal of these three glycans from HCV E1E2 made the glycoproteins more sensitive to neutralisation by antibodies isolated from
sera of infected patients.
Therefore, we constructed and purified several adenovirus-based replication defective vectors encoding for E1E2 glycoproteins (gt 1a)
with mutations at specific glycosylation sites. Biochemical analysis of glycoproteins produced in infected cells showed strong expression
and correct processing of both E1 and E2, which was confirmed by Western blotting analysis and immunostaining assays. Furthermore,
we confirmed the ability of mutated glycoproteins to assemble into E1E2 heterodimer and to bind to CD81 LEL. Immunogenicity and
neutralization studies in animal models will be presented.
Contact: Alicja Chmielewska
University of Gdansk, Gdansk, Poland
[email protected]
P3.13
Amit Kapoor (1), Peter Simmonds (1), Gisa Gerold (1), Natasha Qaisar (1), Komal Jain (1), Jose A. Henriquez (1), Cadhla Firth (1), David L.
Hirschberg (1), Charles M. Rice (1), Shelly Shields (1), W. Ian Lipkin (1)
(1) Columbia University, New York, NY, USA
An estimated 3% of the world’s population is chronically infected with hepatitis C virus (HCV). Although it was discovered more than twenty
years ago the origin of HCV remains obscure largely because no closely related animal virus homolog has been identified; furthermore,
efforts to understand HCV pathogenesis have been hampered by the absence of animal models for human disease other than chimpanzees.
Here we report the identification in domestic dogs of the first non-primate hepacivirus. Comparative phylogenetic analysis of the canine
hepacivirus (CHV) confirmed it to be the most genetically similar animal virus homolog of HCV. The 5’UTR of CHV showed 66% and 57%
nt identity with HCV and GBV-B, respectively. The envelope protein E2 of HCV is among the most variable portions of its genome, yet has
remarkable sequence similarity with CHV. Moreover, the number and position of cysteine residues in E2 protein of CHV indicate that even
the tertiary structure of CHV is likely to be more similar to HCV than other genetically related viruses. However, there are notable differences
between CHV and HCV that may have biological significance. Most strikingly, the potential occlusion of the binding site of miR122 in the CHV
5’UTR suggests that the interaction, which enhances the replication of HCV in human liver may not be needed in CHV infections. Bayesian
Markov Chains Monte Carlo and associated TMRCA analyses suggest a mean recent divergence time of CHV and HCV clades within the past
500-1,000 years, well after the domestication of canines. The discovery of CHV may provide new insights into the origin and evolution of
HCV and a tractable model system with which to probe the pathogenesis, prevention and treatment of diseases due to hepacivirus infection.
Contact: Amit Kapoor
Columbia University, New York, NY, USA
[email protected]
POSTERS
Characterization of a canine homolog of hepatitis C virus
POSTERS
P3.14
Expansion of functional CD8+ T cell response targeting the circulating virus after
therapeutic vaccination for hepatitis C virus
Benoît Callendret (1), Antonella Folgori (2), Heather Eccleston (1), Stefania Capone (2), Riccardo Cortese (2), Alfredo Nicosia (2),
Christopher Walker (1)
(1) Nationwide Children’s Hospital Research Institute, Columbus, OH, USA
Chronic hepatitis C virus infection is associated with failure of the adaptive cellular immune response. We undertook studies to restore
virus-specific T cell activity by therapeutic vaccination. Briefly, two persistently infected chimpanzees were vaccinated with a recombinant
adenovirus expressing the HCV non-structural proteins while virus replication was transiently suppressed using an inhibitor of the viral
polymerase. Drug-resistant viral variants appeared within the first 21 days of treatment, and HCV replication returned to pre-treatment
levels despite additional boosts with the HCV non-structural genes.
Strong vaccine-induced T cell immunity was observed despite the apparent absence of viral control. Some of the circulating CD8+ T cells
targeted vaccine-specific epitopes that were not conserved in the virus used to infect the animals. Remarkably other circulating CD8+ T cells
targeted epitopes that were conserved in the virus inoculum and the vaccine. A fraction of these epitopes had acquired escape mutations
prior to initiation of treatment, and so vaccine-expanded CD8+ T cells could not exert any pressure on viral replication. Importantly, functional
CD8+ T cells targeting epitopes that were intact in the circulating virus expanded in blood upon vaccination, but they had no obvious impact
on HCV replication. Reasons for ongoing failure of this CD8+ T cell response to control the infection are unknown, but we are investigating
the possibility that they remained functionally exhausted in the liver or selected for new immune escape variants. Nevertheless, expansion
of CD8+ T cells targeting intact HCV epitopes upon vaccination suggests that successful restoration of immunity may be feasible if virus
replication is more effectively controlled during the prime and boost, or by interruption of negative signaling pathways like programmed cell
death 1 (PD-1) that contribute to HCV-specific CD8+ T cell exhaustion.
Contact: Benoît Callendret
Nationwide Children’s Hospital Research Institute, columbus, OH, USA
[email protected]
P3.15
Considerations for expanding federal policies regarding viral hepatitis screening
POSTERS
Bryce Smith (1), Rebecca Morgan (1), Geoff Beckett (1), John Ward (1)
(1) Centers for Disease Control and Prevention, Atlanta, GA, USA
In 1998, the Centers for Disease Control and Prevention (CDC) published Recommendations for Prevention and Control of Hepatitis C Virus
(HCV) Infection and HCV-Related Chronic Disease recommending anti-HCV testing for populations “most likely to be infected with HCV” such
as persons with a history of injection drug use, blood product exposure or persistently elevated alanine aminotransferase levels. However,
risk-based strategies for HCV testing require assessments to determine which patients should be tested. Barriers to risk assessments include
reluctance of providers to ask patients sensitive questions and patient reticence to disclose potentially stigmatizing behaviors.
CDC estimates that the prevalence of anti-HCV among persons born from 1945 to 1965 is 3.27%, 4.5 times more than persons born outside
that high prevalence cohort (0.73%). Studies have found that 45-85% of anti-HCV+ persons are unaware of their status, indicating they
would be unlikely to be referred for potentially life-saving treatment or to receive secondary prevention messages to decrease or eliminate
alcohol and other drug use. With the new therapies that are expected to be available this year, treatment duration may be shorter with
considerably improved outcomes.
CDC is expanding the 1998 recommendations by using an evidence-based methodology to consider a high HCV prevalence birth-cohort
based approach including routine one-time screening of all persons born from 1945 to 1965. This strategy circumvents the limitations of
risk-based approaches by not requiring the discussion of sensitive behaviors. The risk-based approach focuses primarily on persons born
after 1965 and targets specific high-risk populations such as persons who inject drugs. Combining high-prevalence and targeted risk-based
strategies could provide the best opportunity to increase the proportion of persons who are aware of their status so that they can make
informed treatment and prevention decisions.
Contact: Bryce Smith
Centers for Disease Control and Prevention, Atlanta, GA, USA
[email protected]
144 : HCV 2011
P3.16
Characterization of the full coding sequences of five HCV strains including subtypes 2i
and 2l as well as three undefined subtypes 2
Jean-Francois Cantaloube (1), Francois Jordier (1), Philippe de Micco (1)
(1) Etablissement Francais du Sang, Marseille, France
Infection with Hepatitis C virus genotype 2 is common in West Africa. A large number of HCV subtypes 2 is also observed in the West Indies
and France. It is important to know the full-length sequences of HCV-2 subtypes in order to classify them correctly and to obtain more
detailed knowledge about HCV epidemiology. In view of the lack of full length coding viral sequences currently available for this genotype,
we were interested in sequencing 5 additional genotypes 2 including one HCV-2i strain (MRS37), one HCV-2l strain (MRS89), as well as three
strains (MRS40, MRS41 and MRS117) belonging to undefined subtypes 2. In this study, the full coding region was amplified and sequenced
using overlapping amplifications. The resulting genomes contain a single ORF of 9042–9111 nt. The sequences of HCV-2i and HCV-2l strains
show p-distances of 18.6-23.6% and 25.6–26.4%, respectively, in comparison with subtypes 2a, 2b, 2c, and 2k (GenBank accession numbers
AF238481, D10988, D50409, and AB031663, respectively). In comparison with genotypes 1, 3 and 4, the five sequences show p-distances
of 29.0-34.3%. Four genomes (MRS37, MRS40, MRS41, and MRS117) contain a 60-nucleotide insertion in the NS5a region. We have found
strong conservation of potential N-linked glycosylation sites in the envelope protein as other HCV genotypes. The E1 and E2 proteins contain
four sites (positions 196, 209, 234 and 305; numbered as in strain D10988) and eleven sites (positions 417, 423, 430, 448, 477, 534, 542, 558,
578, 627 and 649), respectively. These sites are conserved among all sequences except for MRS117 that lacks site 477. Our new data could
be used as references not only for assigning new HCV-2 subtypes, but also for further molecular evolutionary studies.
Contact: Jean-Francois Cantaloube
Etablissement Francais du Sang, Marseille, France
[email protected]
P3.17
HVR1, a decoy that interferes with the immunogenicity of conserved epitopes in HCV
E2 protein
The hypervariable region 1 (HVR1) of hepatitis C virus (HCV), locating at the amino terminus of envelope protein 2 (E2), is the most variable
region in HCV genome. HVR1 contains dominant neutralizing epitopes, and plays an important role in HCV cell entry. Besides HVR1, other
regions in E2 protein have been demonstrated to contain neutralizing epitopes, and some of which were highly conserved across diverse
genotypes. Anti-HVR1 antibodies are the predominant antibodies against E2 protein in acute phase of HCV infection, and antibodies targeting
the conserved epitopes in E2 protein are typically delayed in appearance. In this study, DNA plasmids encoding soluble E2 protein (sE2,
364~661aa in HCV polyprotein) of H77 isolates and the truncated version deleted HVR1 (sE2Δ) were used to immune BABL/c mice (50 μg per
mouse) by direct intramuscular injection. After the first immunization, all the mice (n=8) received sE2-plasmid developed HVR1 antibodies,
and sE2Δ antibodies were detectable in only three mice. However, all the mice received sE2Δ-plasmid produced sE2Δ antibodies with
much higher titers. During the observation period of three months, sE2Δ antibody levels in sE2-plasmid immunized mice were much lower
compared with those in sE2Δ-plasmid immunized mice. The antibody levels of HVR1 in sE2-plasmid immunized mice were negatively related
with those of sE2Δ antibody. The sera of sE2Δ-plasmid immunized mice showed much higher cross-reactivity to heterologous E2 proteins
(Con1, J4, J6, JFH-1, UKN2B2.8, UKN3A1.28C, and UKN4.21.16 isolates), comparison with with the sera from sE2-plasmid immunized mice.
The serum IgG purified from sE2Δ-plasmid immunized mice showed a similar neutralizing activity to pseudoparticles of autologous isolate
compared with those from sE2-plasmid immunized mice, but demonstrated a much stronger neutralizing activity to heterologous HCVpp
and J6-JFH1 chimeric HCVcc. The similar results were obtained for immunization with sE2- and sE2Δ-plasmids of Con1 isolate.
Contact: Ping Zhao
Department of Microbiology, Second Military Medical University, Shanghai, China
[email protected]
POSTERS
Ping Zhao (1), Xiaoqing Liu (1), Shi Yan (1), Yimin Tong (1), Xiaoling Liao (1), Xian Hua (1), Yongzhe Zhu (1), Di Yin (1), Zhongtian Qi (1)
(1) Department of Microbiology, Second Military Medical University, Shanghai, China
POSTERS
P3.18
The neutralizing epitope in HVR1 inhibits the production of antibody to HCV E2 protein
Ping Zhao (1), Xiaoqing Liu (1), Shi Yan (1), Yimin Tong (1), Xiaoling Liao (1), Xian Hua (1), Yongzhe Zhu (1), Di Yin (1), Zhongtian Qi (1)
By using DNA immunization of mice and rabbits with plasmids containing cDNA of truncated soluble E2 (sE2) or deleted HVR1 encoding
sequence (sE2Δ) of H77 and Con1 isolates, we found that HVR1 strongly inhibits the humoral response to HCV E2 protein. To investigate the
underlying mechanisms, the following experiments were performed: 1, The HVR1 encoding sequence of H77 isolate was attached to the N or
C terminus of HBsAg cDNA, and the corresponding expression plasmids were then used to immunize Balb/c mice, followed by the detection
of the anti-HBs and anti-HVR1. 2, The recombinant sE2 and sE2Δ protein were utilized to test the reactivity with forty-five serum samples
from chronic hepatitis C patients, ten serum samples of sE2Δ-plasmid immunized mice, and two conformation dependent McAb H53 and
H48. 3, The expression plasmid containing cDNA of HVR1 partially deleted sE2 (only the neutralizing epitope aa 16~24 was preserved) ,
and the expression plasmid containing chimeric sE2, in which the neutralizing epitope in HVR1 was swapped with HA epitope of influenza
virus were constructed and used to immunize BALB/c mice. The results showed that: 1, HVR1 does not interfere with antibody response to
HBsAg either fused at its N or C terminus. 2, HVR1 does not inhibit the reactivity of sera of hepatitis C patients, sE2Δ immunized mice, or
two conformation dependent McAbs with E2 protein. 3, Both the neutralizing epitope (aa 16~24) in HVR1 and HA epitope in chimeric HVR1
inhibit antibody production to sE2Δ. Our results suggest that the neutralizing epitope in HVR1 should act as an immune decoy that skews
humoral response to this minimal domain and makes humoral immune system ignore other regions in E2 protein that containing conserved
neutralizing epitopes. These findings may be of significant implications for the development of protective vaccines against HCV infection.
Contact: Ping Zhao
Department of Microbiology, Second Military Medical University, Shanghai, China
[email protected]
P3.19
Molecular analysis of hepatitis C virus genotypes in Croatia
Ivana Furcic (1), Ana Planinic (2), Snježana Židovec-Lepej (2), Juan Carlos Sáiz (3), Antonio Mas (4), Adriana Vince (2)
(1) Zagreb, Croatia
(2) University Hospital for Infectious Diseases “Dr. Fran Mihaljevic”, Croatia
(3) INIA, Environmental Virology, Department of Biotechnology, Madrid, Spain
(4) CRIB-UCLM, Molecular Virology Group, Albacete, Spain
POSTERS
There is little information about the prevalence and genotype distribution of HCV in Croatia. Previous data have reported a prevalence of
1.4% (range 0.8-2.5%), being genotypes 1b, 3a and 1a the most common in Croatian population, but none of these studies have performed
detailed molecular characterization of HCV genotypes using sequence based analysis.
We have established in-house method for more accurate HCV genotyping and subtyping based on phylogenetic analysis of the HCV NS5B
region, and tested on 143 randomly selected samples from Croatian patients.
Amplification and sequencing of the NS5B region (340bp PCR fragments) was achieved in 134 out of 143 samples (93.7%), since nine
samples were not amplified or were not successfully sequenced (6.3%).
Our data show that the HCV genotype 3a is the most prevalent (31.5%), followed by genotypes 1b and 1a (28.7% and 23.8%, respectively).
We have also found genotype 4 in 6.3% and 2b in 3.5% of samples.
These results have been compared with the data obtained with standard diagnostic test (INNO-LiPA1), with an almost complete genotype
concordance. Only one sample which had been previously classified as genotype 3a, turned to be genotype 4d after sequencing.
Additionally, we have genotyped one previously unclassified sample, 26 samples were subtyped and we have detected 12 mismatches. One
sample classified differently (as 1a or 1b) depending on reference sequences.
We are currently amplifying and sequencing the HCV core-E1 region in this set of patients to confirm genotypes and subtypes, and to further
test for possible recombination events.
Our in-house method for more accurate HCV genotyping and subtyping is being implemented in the diagnostic laboratory at University
Hospital for Infectious Diseases (Zagreb), for improvement of the routine hospital practice in Croatia and for a better management of HCV
infected patients. Detailed analysis of HCV NS5B and core-E1 regions will be discussed and presented.
Contact: Ivana Furcic
Zagreb, Croatia
[email protected]
146 : HCV 2011
P3.20
Immune responses by CD4+ and CD8+ T-cells in injecting drug user (IDU) prison
inmates with multiple genotype HCV infection
Son T. Pham (1), Rowena A. Bull (1), Elizabeth Keoshkerian (1), Barbara Cameron (1), Gregory J. Dore (1), Michaela Lucas (2), Katja
Pfafferott (2), Silvana Gaudieri (3), Andrew R. Lloyd (1), Peter A. White (1)
(1) University of New South Wales, Sydney, Australia
(2) Royal Perth Hospital and Murdoch University, Australia
(3) University of Western Australia, Australia
A key question in vaccine development is whether cross-genotype immunoreactivity is elicited following primary hepatitis C virus (HCV)
infection. In the present study, we sought evidence for cross-genotype HCV-specific CD4+ and CD8+ T-cell responses in subjects with mixed
HCV infection.
Longitudinal peripheral blood mononuclear (PBMC) samples from five subjects previously identified with mixed HCV genotype 1a (G1a)/
G3a infection were collected as part of the Hepatitis C Incidence and Transmission Study (HITS), an Australian seronegative IDU inmate
prospective cohort. CD8+T cell responses to HCV G1 and G3 peptides were assessed by IFN-gamma ELISPOT. Relative proportions of HCVspecific CD4+T-effector and CD4+T-regulatory cells were assessed by a novel FACS assay based on CD134 and CD39, following 44-hour
culture with G1 and G3 HCV peptide pools (P), representing Core-p7 (P1), NS2-NS3 (P2), NS4A-NS5B (P3).
Two subjects showed CD8+T cell responses; one (ID303) to G1 peptides at month-12 post-infection (p-i) and one (ID304) to both G1/G3
peptides at month-18 p-i. In 3/5 subjects where clearance of one (n=2) or more viruses (n=1) was achieved within twelve months p-i, CD4+T
cell responses to one or more peptide pools (predominantly P1 and P2) of both G1 and G3 were observed over a period of 24 months p-i
(ID303, 374, 304). Of the remaining two cases where mixed infection was detected after twelve months p-i, one subject demonstrated no
CD4+ or CD8+ T-cells responses (ID203) and the other CD4+T cells responded to only one G3 peptide pool at month-12 p-i (ID081). The ratio
of HCV-specific CD4+ effector to regulatory T-cells during follow-up was also higher in subjects who cleared one or more viruses, than in
those who did not.
These results show evidence of cross-genotype immunoreactivity to G1 and G3 HCV antigens predominantly in CD4+T cells. Further
comprehensive investigation of cellular immunity in more mixed HCV infection cases is warranted.
Contact: Son T. Pham
University of New South Wales, Sydney, Australia
[email protected]
Development of purification method for HCV particles using chromatographic technique
Hiroshi Yokokawa (1), Daisuke Akazawa (1), Masaki Moriyama (1), Noriko Nakamura (1), Takanobu Kato (2), Koji Ishii (2), Takaji Wakita (2)
(1) Pharmaceutical Research Laboratory, Toray Industries, Inc., Kamakura, Japan
(2) Department of Virology II, National Institute of Infectious Diseases, Japan
Aim: In late years the cell culture system of the infectious HCV particle was established, and the inactivated particle has possibility for
vaccine development and production of neutralizing antibodies as an antigen. In this study, we aimed to construct highly efficient HCV
particle purification procedures by performing chromatography. Various types of column were tested, and the purification efficiency was
examined. Purification by chromatography method may take shorter time, be simpler, and can be automated.
Results: Infectious HCV particles were prepared from supernatant of Huh7 cells transfected J6/JFH-1 chimeric virus RNA. As first purification
step, ultrafiltration with a 500 kDa cut off membrane was performed. We obtained the virus-concentrated fluid with approximately 90%
recovery of HCV core protein and 90% exclusion of contaminated protein after this step. Next, size exclusion chromatography was carried
out using Sepharose6 Fast Flow. The recovery rate of HCV core protein in void fraction was approximately 80%, where contaminated protein
was reduced to less than 10%. Finally, ion-exchange chromatography was carried out using anion- and cation-exchange column. As a result,
contaminated nucleic acid was removed efficiently by anion-exchange column, and contaminated protein was removed efficiently by cationexchange column. In conclusion, HCV particles can be efficiently purified with ultrafiltration, size exclusion chromatography and ion-exchange
chromatography. Currently, we examined further analysis to optimize types of columns and other conditions for high purity and yield.
Contact: Hiroshi Yokokawa
Pharmaceutical Research Laboratory, Toray Industries, Inc., Kamakura, Japan
[email protected]
POSTERS
P3.21
POSTERS
P3.22
Optimization of peptide arrays for dissecting antibody responses to HCV infection and
vaccination studies
Tinashe Ruwona (1), Ryan Mcbride (1), Steven Head (1), Phillip Ordoukhanian (1), Dennis Burton (1), Mansun Law (1)
Therapeutic antibodies and vaccines have been successfully developed to protect at-risk populations against many viral diseases, but so far
have not been successful for HCV. A major roadblock is our lack of understanding of how antibodies neutralize and protect against HCV. Existing
methods for dissecting antibody responses to HCV vaccination rarely consider the extreme genetic diversity of HCV viral isolates and the quality
of antibody responses. We hereby have developed a low cost, high-throughput microarray-based assay for mapping antibody specificities
at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout and the
methods for data analysis were investigated. NHS-ester glass microscope Slide H was the preferred surface for conjugation of aminooxy-tagged
peptides which provide an orthogonal covalent linkage via the added amino group. The addition of poly vinyl alcohol to printing buffer gave
uniform spot morphology and improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering an entire HCV
proteome of the H77 isolate and the E1 and E2 polypeptides (15-mer, 10 amino acid overlap) of 6 representative HCV genotypic subtypes
were synthesized. Nanoliter volumes of the peptides were printed onto microscopic slides in quadruplicate for studying antibodies specific to
HCV linear epitopes. The utility of the peptide arrays were confirmed using HCV monoclonal antibodies (mAbs) specific to diverse continuous
epitopes. Interestingly a standard control mAb A4, specific to the E1 region 197 to 207, showed cross-reactivity with other E1 and E2 peptides
and the results are confirmed by site-directed mutagenesis analysis of E1E2. The peptide microarrays are an effective platform to rapidly detect
antibodies to HCV linear epitopes on a miniature scale, and are currently being used to dissect immune responses in vaccinated animal models
and infected individuals. Arrays for discontinuous epitopes are also currently under development.
Contact: Tinashe Ruwona
[email protected]
P3.23
A therapeutic vaccine for HCV based on novel, rare, adenoviral vectors
POSTERS
Christabel Kelly (1), Antonella Folgori (2), John Halliday (1), Ye Oo (3), Stefania Capone (2), Richard Antrobus (4), Katherine Gantlett (4),
Elizabeth Stafford (5), Denise O’Donnell (5), Jane Collier (5), Anthony Brown (1), Rachel Huddart (1), Ayako Kurioka (1), Rachel Townsend
(1), Leo Swadling (1), Virginia Ammendola (2), Stefano Colloca (2), Mariarosaria Naddeo (2), Loredana Siani (2), Cinzia Traboni (2), David
Mutimer (3), David H Adams (3), Ricardo Cortese (2), Alfredo Nicosia (2), Paul Klenerman (6), Eleanor Barnes (6)
(1) Nuffield Department of Medicine, University of Oxford, United Kingdom
(3) Queen Elizabeth Hospital Birmingham and WTCTF, Birmingham , United Kingdom
(4) Centre for Clinical Vaccinology and Tropical Medicine, United Kingdom
(5) John Radcliffe Hospital NHS trust, United Kingdom
(6) Nuffiled Department of Medicine and NIHR BRC, University of Oxford, Oxford, United Kingdom
Background and aims: We have recently shown that vaccination with adenoviral vectors based on rare human and simian serotypes encoding
the non-structural proteins of HCV induce highly potent, multi-specific and durable T-cell responses in healthy humans and that similar vectors
may prevent chronic infection in chimpanzees following heterologous viral challenge. We now assess the safety and immunogenicity of these
vectors for the first time in HCV infected patients. Methods: Patients with treatment-naïve chronic genotype-1 HCV infection are vaccinated
(i.m.) with novel replicative-defective adenoviral vectors encoding 1985 amino-acids derived from the NS3-5 region of a genotype 1b strain
in a dose escalation (5x108vp, 5x109vp, 2.5 x1010vp) strategy. Patients receive simian AdCh3 as a single or double prime and a heterologous
boost with a human Ad6 vector 12 weeks later. The first vaccination is administered 2 or 14 weeks into a 48-week course of PEG-IFNα2a/
ribavirin. Immunogenicity and antigenic mapping is assessed by ex-vivo IFNγ-ELISpot (using 6 peptide pools and single peptides spanning the
HCV immunogen) and ICS assays. A control cohort of patients treated with PEG-IFNα2a/ribavirin alone, are included. Results: Ten patients
have received both prime and boost vaccines at 2.5 x1010vp. Five patients responded to vaccination with the average peak HCV specific
T cell response 939 (range 62-2954) SFU/106 PBMC as assessed by ELISpot. Peak responses are detected 2-8 weeks post boost. ICS shows
that polyfunctional CD4+ and CD8+ HCV specific T cell responses are generated. Fine epitope mapping shows multiple antigenic targets may
be generated and are frequently those previously described in the setting of acute HCV infection. Vaccination is very well tolerated with no
evidence of liver immunopathology. Conclusions: We have generated a novel T-cell vaccine based on adenovirus vectors from rare serotype
expressing HCV NS proteins that is safe and highly immunogenic in patients with chronic HCV infection.
Contact: Eleanor Barnes
Nuffiled Department of Medicine and NIHR BRC, University of Oxford, Oxford, United Kingdom
[email protected]
148 : HCV 2011
P3.24
Greatly enhanced immunogenicity using a heterologous prime-boost strategy for
activation of hepatitis C virus non-structural (NS)-specific T cells
G. Inchauspé (1), A. Fournillier (1), L. Frelin (2), E. Jacquier (1), G. Ahlém (2), E. Gérossier (1), A. Brass (2), F. Holmström (2), K. Broderick (3),
N. Y. Sardesai (3), M. Sälberg (2)
(1) Transgene, Lyon, France
(2) Karolinska Institute, Sweden
(3) Inovio Pharmaceuticals Inc., USA
Studies adopting heterologous prime/boost strategies have induced enhanced and broader vaccine-specific immune responses as recently
shown in the clinical development of malaria and HIV vaccines. We deployed a prime-boost strategy based on two vaccines currently in phase
II clinical trials, both having shown safety, immunogenicity and early efficacy data when administered to HCV chronically infected patients. This
strategy started with two monthly intra-muscular injections followed by electroporation using Medpulser™-DDS (EP) of a plasmid expressing
a codon optimized genotype 1a NS3/4A gene (ChronVac-C), followed by three weekly sub-cutaneous booster doses 5 to 12 weeks later with
a Modified Vaccinia virus Ankara vector expressing genotype 1b NS3/4/5B genes (TG4040). Mice were submitted to this regimen to evaluate:
1/ immunogenicity 2 or 6 weeks post-vaccination by IFNγ ELISPOT, IFNγ/TNFα/IL2 ICS using a panel of NS3/4/5B 1b or 1a-specific stimulations;
2/ protective capacity of the induced responses in a surrogate challenge model. ChronVac-C/EP prime/TG4040 boost resulted in higher T cell
responses compared to vaccination by ChronVac-C/EP or TG4040 alone, no significant difference 5 or 12-week interval was seen. Increased
frequencies of IFNg and/or TNFa producing T cells, especially for CD8+ responses, were reported 2 weeks post-vaccination and both CD8+
and CD4+ responses were improved 6 weeks post-vaccination. In addition, the prime/boost enlarged the spectrum of epitopes recognized
as compared to ChronVac-C/EP or TG4040 alone due to cumulative responses against both genotype 1a and 1b peptides. Unexpectedly high
synergy was observed 6 weeks post-vaccination in CD4+ responses (10 fold). Finally the ChronVac-C/EP prime/TG4040 boost was able to confer
a significant protection against Listeria monocytogenes-based challenge. This study demonstrates the efficacy of the ChronVac-C/EP prime/
TG4040 boost strategy for improvement of polyfunctional CD4+ and CD8+ HCV-specific responses in terms of frequency, scope and longevity.
These data support further evaluation of this vaccine combination in the clinic.
Contact: G. Inchauspé
Transgene, Lyon, France
[email protected]
P3.25
David S. Campo Rendon (4), Zoya Dimitrova (4), Jonny Yokosawa (1), Duc Hoang (2), Nestor Perez (3), Sumathi Ramachandran (4),
Yury Khudyakov (4)
(1) Universidade Federal de Uberlândia, Brazil
(2) National Institute of Hygiene and Epidemiology, Vietnam
(3) Probiomed S.A., Mexico
(4) Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA, USA
Development of HCV vaccine is hindered by lack of understanding of the factors that define variability and immunological specificity of
neutralizing antigenic epitopes. One HCV neutralizing determinant has been mapped to the HVR1 of the structural protein E2. In order to study
factors affecting specificity of HVR1 cross-immunoreactivity, we tested a set of 262 synthetic peptides representing different HVR1 variants
in enzyme immunoassay with serum specimens obtained from mice immunized with 103 HVR1 peptides. Each peptide was conjugated to
BSA and used to immunize a group of 3 mice. In total, 26883 reactions were tested, of which 5039 were positive. The immense antigenic
diversity of HVR1 was found to be (i) effectively limited, with only 3 selected variants being needed to collectively cross-immunoreact with
all tested peptides; and (ii) characterized by extensive homoplasy, with immunological specificities shared by genetically distant variants. We
also found that cross-immunoreactivity can be accurately predicted using a decision tree model based on amino acid sequence alone. 10-fold
cross-validation showed that the average testing accuracy was 92.5%, with the average sensitivity and specificity being 99.7% and 85.3%,
respectively. In addition, we analyzed all sequences used in the cross-immunoreactivity experiment in the context of the HVR1 sequence
space, which was modeled using 11,319 HVR1 sequences obtained from 3,172 patients. Of all sequences, 65.4% were obtained from the Los
Alamos HCV Sequence Database and 34.6% were clonal variants generated in our laboratory. We discovered that the HVR1 sequence space is
homoplastic. Each HCV genotype and subgenotype explores this space entirely. Some intra-host viral populations traverse it on a scale similar to
genotypes. These findings indicate significant functional and structural HVR1 convergence, suggest tractability of its immunological specificity,
and offer a novel framework vaccine development against HCV, and other heterogeneous viruses.
Contact: Yury Khudyakov
Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA, USA
[email protected]
POSTERS
Convergence and tractability of HCV HVR1 cross-immunoreactivity
POSTERS
P3.26
Characterization of neutralizing and interfering epitopes on hepatitis C virus E2
glycoprotein using monoclonal antibodies
Hongying Duan (1), Lilin Zhong (1), Evi Struble (1), Kathleen Mihalik (1), Alla Kachko (1), Marian Major (1), Pei Zhang (1), Stephen M.
Feinstone (1)
Antibodies against different epitopes in the E2 glycoprotein of hepatitis C virus (HCV) have been reported to reduce infectivity in vivo and in
vitro. However, HCV can persist in patients despite high levels of neutralizing antibodies. We previously studied two epitopes termed EP I (aa
412-426) and EP II (aa434-446) located in the E2 glycoprotein. Antibodies specific for EPI have been shown by us and others to have broadly
neutralizing properties. We have shown that antibodies to EP II can interfere with the neutralization by anti-EP I antibodies. To further
understand the mechanism of the neutralization and interference, we made monoclonal antibodies by immunizing mice with a peptide
(aa 412-447) encompassed both the EP I and EP II regions of the HCV-H strain and monoclonal antibodies (mAb) that recognized either EPI
or EPII were selected. One antibody mAb 41 recognized specifically the EP II peptide and neutralized a chimeric 1a/JFH1 virus that has the
genotype 1a envelop glycoproteins on a genotype 2a virus backbone. However, the antibody was unable to neutralize the all 2a virus, J6/
JFH1. By contrast, mAb 12 that also binds to the EPII peptide specifically, did not neutralize either the 1a/JFH1 or the J6/JFH1 virus . However,
antibody 12 interfered with neutralizing function of a polyclonal antibody specific for EPI, while it did not block neutralization by mAb 41,
nor did it prevent binding of 41 to the EPII peptide in an ELISA assay. These results suggest that EPII region contains at least two epitopes
that can be recognized by the neutralizing antibodies and the interfering antibodies that block antibody-mediated neutralization at the EPI.
Contact: Marian Major
[email protected]
P3.27
All-cause, liver-, and non-liver, related mortality among Hepatitis C infected individuals
in the general US population
Samer El-Kamary (1), Ravi Jhaveri (2), Michelle Shardell (1)
(1) University of Maryland School of Medicine, Baltimore, MD, USA
(2) Duke University Medical Center, USA
POSTERS
Background: Liver-related mortality among those infected with hepatitis C virus (HCV) has been described, but little is known about non-liver
related mortality. Our objective was to determine HCV-associated all-cause, liver- and non-liver related mortality in the general US population.
Methods: A prospective cohort study of 9,378 nationally representative adults aged 17 to 59 years, from the Third National Health and
Nutrition Examination Survey (NHANES III) Linked Mortality File that was made publicly available in 2010. HCV status was assessed from
1988 to 1994, with mortality follow-up of the same individuals through 2006.
Results: There were 614 deaths over a median follow-up of 14.8 years. After adjusting for all covariate risk factors, HCV chronic infection
had a 2.37 times higher all-cause mortality rate ratio [MRR](95%CI: 1.28-4.38;P=0.008), a 26.46 times higher liver-related MRR (95%CI: 8.0087.48;P<0.001), and 1.79 times higher non-liver related MRR (95%CI: 0.77-4.19;P=0.18), compared to being HCV-negative. This represents an
estimated 2.46 million US adults aged 17-59 years with chronic HCV infection who had an estimated 31,163 deaths from all causes per year,
of which 57.8% (95%CI: 21.9%-77.2%) were attributable to HCV. Among those, there was an estimated 9,569 liver-related deaths per year, of
which 96.2% (95%CI: 87.5-98.9%) were attributable to HCV. Non-liver related deaths were not significantly associated with HCV status.
Conclusions: Chronic HCV all-cause mortality is more than twice as in HCV-negative individuals. This suggests that those with chronic HCV
infection are at a higher risk of death even after accounting for liver-related morbidity, and should be closely monitored.
Contact: Samer El-Kamary
University of Maryland School of Medicine, Baltimore, MD, USA
[email protected]
150 : HCV 2011
P3.28
A synthetic codon-optimized HCV non-structural 5A DNA vaccine primes polyfunctional
CD8+ T cell responses in wildtype and NS5A-transgenic mice
Fredrik Holmström (1), Sepideh Alahyari (1), Malte Kriegs (2), Eberhard Hildt (2), Matti Sällberg (1), Gustaf Ahlén (1), Lars Frelin (1)
(1) Karolinska Institute, Huddinge, Stockholm, Sweden
(2) Clinic of Radiotherapy and Radiooncology, Germany
The hepatitis C virus (HCV) is today one of the leading causes of severe liver disease. Approximately 170 million individuals are infected
globally. The function of the HCV non-structural 5A protein (NS5A) is not completely known, but it has been suggested to be an important
component of the HCV replication complex, to interfere with the interferon signaling pathways and to impair the function of HCV-specific T
cells. Recent studies have suggested NS5A as a new target for antiviral therapy. Several NS5A-based inhibitors are in clinical evaluation and
have shown potent antiviral activities in chronic HCV patients.
The aim of this study was to evaluate NS5A as a potential vaccine candidate for patients with chronic HCV infection.
A synthetic codon-optimized NS5A genotype 1b gene was cloned into a eucaryotic expression plasmid and the immunogenicity was
determined after intramuscular immunization in combination with in vivo electroporation. The coNS5A gene primed high antibody levels,
with IgG titres of >104 post immunization. With respect to CD8+ T cell responses did the coNS5A gene prime IFNγ-producing and lytic
cytotoxic T cells in both wildtype and NS5A-transgenic mice. In addition, high frequencies of NS5A-specific CD8+ T cells were found in mice
after one single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells
in vivo was analyzed after immunization. One single dose of coNS5A primed tumor-inhibiting responses. Finally, immunization with the
coNS5A gene primed polyfunctional NS5A-specific CD8+ T cells responses. Thus, the codon optimized NS5A gene is a promising therapeutic
vaccine candidate.
Contact: Fredrik Holmström
Karolinska Institute, Huddinge, Sweden
[email protected]
P3.29
Matti Sällberg (1), Sepideh Alahyari (1), Lars Frelin (1), Ola Weiland (2)
(2) Karolinska Institutet, Division of Infectious Diseases, Stockholm, Sweden
HCV effectively establishes chronic infection. A strong T cell response to HCV proteins can prevent chronic infection, whereas the HCV-specific
T cell response is severely impaired during a chronic HCV infection. We recently concluded a phase I/II clinical study with a therapeutic
NS3/4A-based DNA vaccine delivered by in vivo electroporation in 12 patients with chronic HCV genotype 1 infection. Seven of these
patients have entered standard of care (SOC) therapy consisting of pegylated interferon-alpha and ribavirin. Of these seven, six has now
completed SOC including a six-month follow-up. All patients were tested for the IL-28B genotypes associated with outcome of SOC therapy.
Of these six, five had a sustained viral response as determined by negativity of HCV RNA at six months after cessation of therapy. Of the five
cured patients, two had the CC IL-28B genotype, and three had non-CC genotypes. The patient who was not cured had a non-CC genotype.
This is an unexpectedly high cure rate and suggests that therapeutic vaccination may influence the outcome of SOC. We will therefore start
a controlled clinical trial of therapeutic vaccination given in combination with SOC therapy.
Contact: Matti Sällberg
[email protected]
POSTERS
High cure rate in patients with chronic HCV gt1 infection receiving SOC therapy after
participation in a clinical trial of therapeutic DNA vaccination
POSTERS
P3.30
Genomic sequence analysis tools and a genotype-phenotype association platform in
the virus pathogen resource
Yun Zhang (1), Brett Pickett (1), Jyothi Noronha (1), R. Burke Squires (1), Victoria Hunt (1), Mengya Liu (2), Liwei Zhou (3), Chris Larson (4),
Jonathan Dietrich (3), Edward Klem (3), Richard Scheuermann (1)
(1) UT Southwestern Medical Center, Dallas, TX, USA
(2) Southern Methodist University, USA
(3) Northrop Grumman Health Solutions, USA
(4) Vecna Technologies, USA
The NIAID-sponsored Virus Pathogen Resource (ViPR, www.viprbrc.org) serves as a single publicly-accessible repository of integrated datasets
and analysis tools for thirteen different virus families. ViPR includes information from: GenBank sequence records, gene annotations, strain
metadata, Gene Ontology classifications, UniProtKB protein annotations, relevant 3D protein structures from the Protein Databank (PDB),
immune epitopes from the Immune Epitope Database (IEDB), and others sources. ViPR data can be searched and analyzed through userfriendly web interfaces for calculating: BLAST, multiple sequence alignments, phylogenetic trees, single nucleotide polymorphisms, and
metadata-driven comparative genomics analyses. Tools also exist to: view phylogenetic trees, assign annotations to new genomes, and store
results from any search or analysis within a personal Workbench space on the ViPR server for future use. We have recently developed the
Sequence Feature Variant Type (SFVT) component, an enhanced community annotation system to define the precise location of published
characterized regions within each protein called ‘Sequence Features’ (SF), which are comprised of: structural (i.e. alpha helices) and
functional regions (i.e. active sites), host immune epitopes, and sequence alterations for virus families that we support. For each defined SF,
all sequence records within the same species are searched to identify all unique existing amino acid sequence variations, termed ‘Variant
Types’ (VT). The aim of the SFVT module is to provide researchers with a dynamic, community-based annotation platform that can be a
resource for rapidly identifying polymorphisms that correlate with a specific phenotype. We expect our current (and future) suite of tools
to further assist viral researchers in developing diagnostics, prophylactics, and/or therapeutics for viruses that are pathogenic to humans.
Contact: Yun Zhang
UT Southwestern Medical Center, Dallas, TX, USA
[email protected]
P3.31
HCV spread in different intravenous drug user networks – a large population assessment
POSTERS
Marianne Alanko-Blome (1), Maria Josephson (1), Vilma Molnegren (2), Per Björkman (1), Anders Widell (2)
(1) Department of Infectious Diseases, University Hospital Skåne, Malmö, Sweden
(2) Department of Laboratory Medicine, County of Skåne, Malmö, Sweden
Background: In most countries, HCV infection spreads via intravenous drug use (IDU) and those who survive drug use often become our
future HCV patients. Needle exchange programs (NEPs) were initiated in the mid 1980-ies to control the spread of HIV. NEPs have met
considerable success by limiting needle sharing. One key element has been identification of those infected by blood borne viruses by
conducting repeat blood testing. However, in contrast to HIV, HCV continues to spread among NEP users. One recent study conducted by
us thus showed a high incidence rate of >30 new HCV infections per 100 person years. Using repeat testing anti-HCV and PCR we have also
monitored viremia kinetics across seroconversion. In these viremic sera HCV strains in incident cases among IDUs could further be compared
with strains from already infected NEP participants from a midsize (280000inh) Swedish city.
Materials: Frozen sera from 158 viremic incident HCV cases and from 223 already HCV infected participants recruited into the NEP between
1997 and 2005 were available. RNA was extracted from these samples, amplified, sequenced across 320 nucleotides in the NS5B region,
and analyzed phylogenetically by Maximum Composite Likelihood using Mega 4 to generate a neighbor joining tree, supported by bootstrap
Results: For subtype 1a we found 73 incident cases and 84 established, for 1b 10 incident cases, and 14 established; for 2b 17 incident cases,
and 36 established, and for subtype 3a 56 incident and 89 established cases. There were rare other subtype strains. Interestingly, each
subtype contained several bootstrap supported clusters, each often dominated by either incident or established cases – indicating different
networks. Incident cases in clusters could spread out over years.
Conclusion: We have established a very large phylogenetic relationship among currently circulating HCV strains among IDUs in a Swedish city.
Contact: Anders Widell
Department of Laboratory Medicine, County of Skåne, Malmö, Sweden
[email protected]
152 : HCV 2011
P3.32
Characterizing HCV full length genome sequences for 27 isolates representing various
subtypes with genotypes 2, 3, and 4
Chunhua Li (1), Ling Lu (1), Hongren Wang (1), Jie Yuan (2), Donald Murphy (3)
(1) University of Kansas Medical Center, Kansas city, KS, USA
(2) Department of Biochemistry, Sun Yat-sen University, China
(3) Institut National De Santé Publique Du Québec, Laboratoire De Santé Pu, Canada
Although many subtypes have been classified, HCV genotypes 2, 3, and 4 need to be further defined at the complete genome level.
Taxonomically, they contain a total of 47 subtypes: 2a-2r, 3a-3l, and 4a-4q. However, among genotypes 2 and 3 only subtypes 2a, 2b, 2c,
2i, 2k, 3a, 3b and 3k have their full genomes described. Although we have recently reported 13 full-length genomes for genotype 4, many
unclassified variants are not characterized. In this study, serum samples were obtained from 27 patients in Canada designated QC114,
QC178, QC182, QC232, QC259, QC283, QC289, QC297, QC302, QC331, QC64, QC29, QC260, QC268, QC105, QC115, QC108, QC126, QC127,
QC132, QC147, QC215, QC253, QC352, QC361, QC429, and QC58. With these samples the full length HCV genomes were determined for
10 subtypes, 2d, 2e, 2j, 2m, 2r, 3g, 3i, 3h, 3k, 4q, and 17 subtype equivalents. Their genome lengths ranged from 9408 to 9825 nucleotides
and each contains a single open reading frame (ORF) of 9021 to 9318 nucleotides. Phylogenetic analysis demonstrated that these isolates
were subtypically distinct from each other and from the reference sequences. They represent the first complete genomes of the 27 HCV
subtypes/equivalents. This study provides sequence information necessary for improved strategies of HCV surveillance and diagnostic
testing and has potential applications for better design of HCV vaccines and antivirals.
Contact: Chunhua Li
University of Kansas Medical Center, Kansas city, KS, USA
[email protected]
P3.33
Properties of HCV particles produced in serum-free Huh7.5 cell culture
HCVcc particles could prove useful as vaccine candidates or as tools for the biophysical characterization of HCV. HCVcc is usually produced
in the presence of FBS, however, the safety issues regarding contamination of FBS with prions, as well as immunogenicity of serum
proteins makes FBS unwanted in vaccine production. HCVcc associates with lipids, making them difficult to purify by ultracentrifugation. To
circumvent this, we produced HCVcc from Huh7.5 cells cultured in serum-free AEM. Titers of serum-free HCVcc (sf-HCVcc) corresponded to
those of HCVcc harvested in conventional medium (CM) reaching titers of ~105.0FFU/mL for Core-NS2 intergenotypic recombinants J6/JFH1
and SA13/JFH1. We observed a shift in density profile of the sf-J6/JFH1 and sf-SA13/JFH1 particles, from a heterogeneous to a homogeneous
population with a density at ~1.10g/mL, suggesting decreased lipid association. To test whether FBS were essential for the infectivity of sfHCVcc we diluted sf-SA13/JFH1 in either AEM or CM and infected Huh7.5 cells for 3hrs. Surprisingly, cells incubated with sf-HCVcc diluted in
AEM expressed less NS5A than cells incubated with sf-HCVcc diluted in CM 48hrs post infection. This suggests that associations between sfHCVcc and FBS occurred in the supernatant, thus possibly promoting better entry. To determine whether sf-HCVcc and serum components
associated, we incubated sf-HCVcc with either FBS, DMEM/10% FBS, DMEM, human serum, stock cell culture supernatant or AEM for 6hrs
in the absence of cells and determined the density profile of the particles. We observed no changes in density profile in any of the reactions
suggesting that such associations did not take place.
We have produced and harvested sf-HCVcc particles of a single density. sf-HCVcc can be harvested at high titers and does not associate with
serum components in cell free solution. This could prove useful in applications where purity is pivotal such as vaccine studies or studies of
biophysical properties.
Contact: Christian Mathiesen
CO-HEP, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark
[email protected]
POSTERS
Christian Mathiesen (1), Tanja B. Jensen (1), Judith Gottwein (1), Jens Bukh (1)
(1) CO-HEP, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark
POSTERS
154 : HCV 2011
POSTERS
Host Genetics
P4.01
Rapid PCR-based IL28B genotyping assay
Jean-Francois Gelinas, Bernard Willems, Julie Bruneau, Naglaa H. Shoukry
P4.02
Hepatitis C virus homologous DNA in PBMC: Hints for an episomal DNA
structure containing parts of its 5’-non-coding region
Reinhard H. Dennin, James Wo
P4.03
Single nucleotide polymorphisms in the PPIA gene encoding cyclophilin A
modulate HCV replication in vitro and clinical severity of hepatitis C in vivo
Thomas von Hahn, Behya Karavul, Eike Steinmann, Andrej Potthoff, Christoph Sarrazin, Thomas Berg, Christian Strassburg,
Heiner Wedemeyer, Michael Manns, Thomas Pietschmann, Sandra Ciesek
P4.04
IL28B genotype of hepatocyte cell lines may impact the findings in hepatitis
C virus research
Paul Bensadoun, Christophe Rodriguez, Alexandre Soulier, Martin Higgs, Stephane Chevaliez, Jean-Michel Pawlotsky
P4.05
Pan-Asian variability of interleukin 28B (IL28B) gene polymorphism and
relationship to sustained viral response(SVR) in chronic hepatitis C genotype 1
P4.06
Interferon alfa antibody (IFN-α Ab) as a useful predictive marker of
sustained viral response (SVR) for therapy in chronic hepatitis C
P. Patrick Basu, Nithya Krishnaswamy, Niraj James Shah, Sakina Farhat, Sajith Joseph, Robert S. Brown
P4.07
IL28B rs8099917 is the SNP most strongly associated with spontaneous HCV
clearance and outcome of IFN-alfa therapy in HCV/HIV-1 coinfected patients
Floriana Barbera, Sara Caputo, Bruno Gridelli, Paolo Grossi, Pier Giulio Conaldi
POSTERS
P. Patrick Basu, Nithya Krishnaswamy, Niraj James Shah, Sakina Farhat, Robert S. Brown
POSTERS
P4.08
A genome-wide association study identified susceptibility loci for
HCV-induced hepatocellular carcinoma in MICA gene
Naoya Kato, Vinod Kumar, Ryosuke Muroyama, Norie Kowatari, Wenwen Li, Kaku Goto, Motoyuki Otsuka,
Ryosuke Tateishi, Haruhiko Yoshida, Masao Omata, Kazuhiko Koike, Yusuke Nakamura, Koichi Matsuda
P4.09
IL28B polymorphism is associated with treatment response in hepatitis C
virus genotype 4 patients
Tarik Asselah, Simon De Muynck, Philippe Broët, Julien Masliah-Planchon, Maud Blanluet, Ivan Bièche, Olivier Lada,
Emilie Estrabaud, Martine Lapalus, Michelle Martinot-Peignoux, Dominique Vidaud, Nathalie Boyer, Pierre Bedossa,
Dominique Valla, Michel Vidaud, Patrick Marcellin
P4.10
Robust antiviral activity of lambda-interferon (IL-29) against HCV in
interferon alpha resistant Huh-7 cells with a defective JAK-STAT signaling
Srikanta Dash, Sidhartha Hazari, Satyam Nayak, Partha Chandra, Deepak Kaushal, Luis Balart
P4.11
Polymorphisms in the interleukin 10 promoter and interleukin 28B gene in
chronic hepatitis C patients treated with interferon and ribavirin
Carlos Melo, Evaldo Araujo, Fatima Tengan, Antonio Barone
P4.12
Brazilian profile of IL28-B rs12979860 and rs8099917 single nucleotide
polymorphism: a retrospective analysis and possible consequences for therapy
Carlos Melo, Evaldo Araujo, Luciane Martins, Carla Almeida, Fatima Tengan, Antonio Barone
P4.13
PSI-7977 with PEG/RBV in HCV GT1, 2, or 3 results in consistent viral
suppression independent of IL28B genotype
Kris Kowdley, Jay Lalezari, Eric Lawitz, Robert Hindes, Rob Hyland, Lei Fang, Effie Albanis, William Symonds, Michelle Berrey
POSTERS
156 : HCV 2011
HOST GENETICS
P4.01
Rapid PCR-based IL28B genotyping assay
Jean-Francois Gelinas (1), Bernard Willems (2), Julie Bruneau (3), Naglaa H. Shoukry (2)
(1) Centre de Recherche, Centre Hospitalier de l’U de Montréal (CRCHUM), Montreal, Canada
(2) CRCHUM & Département de Médecine, Université de Montréal, Canada
(3) CRCHUM & Département de Médecine Familiale, Université de Montréal, Canada
Several recent reports demonstrated that single nucleotide polymorphisms (SNPs) in the proximity of the Interleukine-28B (IL28B) gene
could predict spontaneous resolution of Hepatitis C virus (HCV) infection or sustained viral response following interferon (IFN) therapy. This
test will soon be part of standard criteria considered in management of HCV infected patients. However, screening is currently done through
costly RT-PCR or sequencing techniques and a rapid, cost-effective technique is needed. In this study, we developed a rapid PCR-based test
to screen for two IL-28B SNPs (rs12979860 and rs8099917). This test can be performed on frozen peripheral blood mononuclear cells, but
also on as little as 100 µl of fresh or frozen blood allowing integration as a clinical diagnostic tool in hospitals or clinics. We used this test
to investigate the role of these IL28B SNPs in the Cohorte Saint-Luc, a cohort of intravenous drug users at high risk of exposure to HCV in
Montreal, Quebec, Canada. We measured the occurrence of the two IL28B SNPs and the relation with spontaneous or therapeutic viral. We
observed a genetic linkage between the two SNPs. Also, this cohort being primarily composed of participants of Caucasian French Canadian
descent, there was a very high prevalence of the favourable SNPs compared to other cohorts. Despite this prevalence, we observe a high
rate of chronic HCV in this population. This suggests that host genetics is not the primary determinant of HCV outcome, but that other
factors like viral genetics and immune response would tip the balance in these individuals.
Contact: Jean-Francois Gelinas
Centre de Recherche, Centre Hospitalier de l’U de Montréal (CRCHUM), Montreal, Canada
[email protected]
P4.02
Hepatitis C virus homologous DNA in PBMC: Hints for an episomal DNA structure
containing parts of its 5’-non-coding region
Background: The total DNA extract of the white blood cell fraction (PBMC) from HCV-negative individuals (> 120 individuals tested) contains
DNA that harbors sequences from 82 bp up to 320 bp with 92,5% up to 99% homology to the 5‘-NCR of HCV reference isolates. HCV-DNA
sequence sections of its 5’-NCR of this size are not contained in the human genome’s library. We suppose, the HCV DNA homologous
sequences are part of ‘extra chromosomal’ DNA molecules. This study aimed to characterize the structure of this extra chromosomal DNA.
Methods: A pre-PCR restriction enzyme digestion protocol was established making consistent use of four restriction endonucleases selected for
certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences.
We further applied the Linear amplification (LA) protocol, and the isothermal Rolling-circle amplification protocol (RCA).
Results: The pre-PCR digestion protocol revealed a diversity of individual pattern of methylation in these HCV-DNA sequences: this epigenetic
phenomenon corresponds to an individual Methylom. Using the LA, regular, repeated, and overlapping sequence sections of the 5’-NCR
revealed. With RCA, amplicons of about 20 kbp resulted dependent of the primer used. By regular PCR with HCV gsp primers using these
RCA amplicons HCV homologous amplicons resulted with various degrees of homologies < 100% compared to HCV reference strains. The
5’-NCR of HCV-DNA sequences present in CD34+ cells compared with those from total PBMC share the same as well as different pattern of
methylation.
Conclusion: These findings point at i. recombinant constructs resulting from short chromosomal DNA sections and ii. most probably
representing an episomal DNA. iii. This is to be seen with regard to clinical findings of HCV RNA in CD34+ cells, and iv. evolution.
Contact: Reinhard H. Dennin
University of Luebeck, Luebeck, Germany
[email protected]
POSTERS
Reinhard H. Dennin (1), James Wo (2)
(1) University of Luebeck, Luebeck, Germany
(2) State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, China
POSTERS
P4.03
Single nucleotide polymorphisms in the PPIA gene encoding cyclophilin A modulate
HCV replication in vitro and clinical severity of hepatitis C in vivo
Thomas von Hahn (1), Behya Karavul (1), Eike Steinmann (2), Andrej Potthoff (1), Christoph Sarrazin (3), Thomas Berg (4), Christian
Strassburg (1), Heiner Wedemeyer (1), Michael Manns (1), Thomas Pietschmann (2), Sandra Ciesek (1)
(1) Medizinische Hochschule Hannover, Hannover, Germany
(3) Johann Wolfgang Goethe-Universität Frankfurt, Germany
(4) Universitätsklinikum Leipzig, Germany
Background: Human cyclophilin A (CypA) is an essential cellular co-factor for Hepatitis C Virus (HCV). Numerous single nucleotide
polymorphisms (SNPs) in the PPIA gene encoding CypA have been described. These include SNPs that alter the amino acid sequence of the
CypA protein (coding non-synonymous SNPs) as well as SNPs in the gene’s promoter region some of which thought to modulate the clinical
course of HIV infection. Here we investigated if these SNPs have an influence on permissiveness for HCV in vitro and on the clinical course
of HCV infection in chronically HCV-infected patients.
Methods: Replication factor function of CypA variants was tested in Huh-7.5 cells where endogenous CypA had been down regulated by
RNA interference. The frequency of all SNPs in the human population was measured by melting curve-based genotyping. A limited number
of SNPs were selected for further study in larger cohorts of clinically characterized HCV patients and healthy controls.
Results: Three out of six SNPs in the coding region of CypA were associated with markedly reduced HCV RNA replication and virus production
in vitro. One of these SNPs was found to be present in different populations with an allele frequency of up to 3%. The clinical implications
of this SNP are currently under investigation.
A SNP in the non-coding region of CypA could be found in about 18% of individuals and may be associated with a lower fibrosis progression
rate in HCV positive patients. Further work to confirm this finding in a second cohort and cohorts of other ethnic origins is ongoing.
Conclusion: These results indicate that genetic variation in the PPIA gene coding for CypA may play an important role in HCV replication in
vitro and also in vivo.
Contact: Thomas von Hahn
Medizinische Hochschule Hannover, Hannover, Germany
[email protected]
P4.04
POSTERS
IL28B genotype of hepatocyte cell lines may impact the findings in hepatitis C virus
research
Paul Bensadoun (1), Christophe Rodriguez (1), Alexandre Soulier (1), Martin Higgs (1), Stephane Chevaliez (1), Jean-Michel Pawlotsky (1)
Most of the current knowledge on HCV biology and new HCV drug development is based on in vitro studies using hepatoma cell lines,
principally Huh7 cells and their derivatives. Nevertheless, their IL28B genotype remains uncharacterized, despite its likely effects on numerous
intracellular biological processes relevant to HCV infection. We genotyped the IL28B rs12979860 SNP of hepatoma cell lines used in HCV
research, including Huh7, Huh7.5, Huh7.5.1 and HepG2 cells, as well as the commonly used non-hepatoma HEK293 and Hela cell lines. We used
ultra-deep pyrosequencing to sequence 292 nucleotides flanking the rs12979860 locus, using a GS FLX Titanium Sequencing Kit in conjunction
with a Genome Sequencer FLX. Data were analyzed using two original in-house softwares: Pyroclass© and Pyromute©. The cell lines displayed
a “non-Mendelian” distribution of the polymorphisms, likely a consequence of the polyploidal nature of hepatoma cells, or an indication of
the presence of multiple clonal populations arising under different environmental pressures. Two Huh7 cell lines originating from different
laboratories exhibited 100% and 20% of C alleles at SNP position rs12979860, respectively; Huh7.5, Huh7.5.1 and HepG2 cells exhibited 56%, 0%
and 61% of C alleles, respectively; HEK293 and Hela cells, 17% and 0% of C alleles, respectively. Our results emphasize the need for the genetic
characterization of hepatoma cell lines used for HCV research, especially when these studies involve innate immunity, IFN responsiveness, the
assessment of antiviral compound efficacy, or experiments aimed at cell cure. This conclusion may be extended to a number of other in vitro
experiments in which the genetic background of the cell line has an influence on the properties studied.
[email protected]
158 : HCV 2011
HOST GENETICS
P4.05
Pan-Asian variability of interleukin 28B (IL28B) gene polymorphism and relationship to
sustained viral response (SVR) in chronic hepatitis C genotype 1
P. Patrick Basu (1), Nithya Krishnaswamy (2), Niraj James Shah (2), Sakina Farhat (2), Robert S. Brown (1)
(1) Columbia University College of Physicians and Surgeons, Forest Hills, NY, USA
(2) North Shore LIJ University-Forest Hills, USA
Background: Overall SVR achieved for chronic hepatitis C (CHC) genotype 1 with peginterferon plus ribavirin is 42% to 46% in pivotal trials.
The IL28B single nucleotide polymorphism (SNP) was recently discovered, that implicated SVR doubled in patients with the IL28B C/C
variant versus the C/T or T/T variants. The prevalence of the C/C allele predicts the SVR rate in different ethnic groups, with whites greater
than African-Americans, and Hispanics with intermediate frequency. Asians have nearly equal frequency of C/C allele, but the data on the
distribution of alleles amongst Pan-Asian population are not clear. This retrospective study investigated the Pan-Asian diversity of IL28B
gene and SVR rates in CHC G1 Asian patients.
Methods: Thirty-three patients, with CHC geno1, mean age (46 +/- 6 years), from Pan-Asian demographic were analyzed with IL28B SNP.
The ethnic diversity of Pan-Asians were classified into Middle Asian (MA) [11/33, 33.3%]; central Asian (CA) [7/33, 21.2%]; and East Asian
(EA) [15/33, 45.5%] groups. The overall SVR rate was 22/33(66.67%). The allele frequency was C/C in 24/33 (72.7%), C/T in 7/33 (21.2%),
and T/T in 2/33(6 %).
Results: Sub-analysis of the allele frequency in three groups:
Middle Asians: (Turkey Turkmenistan, Iraq) n=11
CC- 6; SVR 3(50%)
CT- 5; SVR 2(40%)
TT- 0; SVR –
Central Asian (Uzbekistan, Tajikistan, Kazakhstan, Afghanistan), n = 7
CC- 5; SVR 4(80%)
CT- 0; SVR –
TT- 2; SVR 0(0%)
East Asian (China, Tibet, Pakistan, India, Bangladesh, Philippines), n = 15
CC- 13; SVR 11(84.6%)
CT- 2; SVR 2(100%)
TT- 0; SVR —
POSTERS
% SVR attained:
CC- 18/24 (75%)
CT- 4/7 (57.1%)
TT- 0/2 (0%)
Conclusion: In this retrospective study, Pan-Asian population has a higher C/C allele predominance with a higher SVR in genotype 1 CHC. A
large population based analysis is required to confirm the hypothesis.
Contact: P. Patrick Basu
Columbia University College of Physicians and Surgeons, Forest Hills, NY, USA
[email protected]
POSTERS
P4.06
Interferon alfa antibody (IFN-α Ab) as a useful predictive marker of sustained viral
response (SVR) for therapy in chronic hepatitis C
P. Patrick Basu (1), Nithya Krishnaswamy (2), Niraj James Shah (2), Sakina Farhat (2), Sajith Joseph (2), Robert S. Brown (1)
Purpose: Interferon-α is the mainstay of therapy for hepatitis C infection. Sustained viral response(SVR) remains 46% in all genotypes. On
treatment, rapid viral response (RVR), early viral response(EVR) are predictive markers of SVR and treatment modification. We propose a
novel marker, IFN-α Ab, in predicting response of interferon-α therapy on chronic hepatitis C naïve genotype 1 patients.
Methods: Forty (n=40) patients (age:45.2±7.2 years, mean±SD; range:26-56 years) were recruited. Serum levels of IFN-α Ab were obtained
initially at 0 week, then 2nd, 4th, 12th, 24th, 48th, and 72nd week. Il28b allele frequency was measured. Exclusion: co-infection, hemolytic
syndromes, severe cardiac disease, severe depression, uncontrolled diabetes mellitus, decompensated cirrhosis, and patients actively using
illicit drugs and those with alcohol consumption >50 g per day.
Results: 25/40 (62.5%) achieved SVR. 31/40 (77.5%) developed IFN-α Ab during the treatment (14 at 12th week, 16 at 24th week, and 1 at
48th week);15/31(48.4%) had higher titers (≥1:150). 13/14 (92.9%) with Ab at 12th week had higher titers. The presence of high-titer Ab at
12th week predicted failure of SVR with 86.7% sensitivity, 100% specificity, 100% positive predictive value, and 92.6% negative predictive
value. 11 Afro-Americans developed high-titer Ab at 12th week and failed SVR. 22/40 (55%) with high viral load >400k failed SVR.
Conclusion: Majority of patients during interferon therapy developed IFN-α Ab at some period. This study postulates the early presence of
higher titer Ab at week 12 that predicts the failure of SVR with high sensitivity and specificity. Afro-Americans developed high-titer Ab early
and failed SVR. Ab titers do not correlate with BMI, age, sex, and fibrotic score but predict failure of SVR. Il28b imparts indirectly with IFN
AB. This study may modify the recent treatment paradigm with cost benefits. A larger prospective trial is needed.
[email protected]
P4.07
IL28B rs8099917 is the SNP most strongly associated with spontaneous HCV clearance
and outcome of IFN-alfa therapy in HCV/HIV-1 coinfected patients
POSTERS
Floriana Barbera (1), Sara Caputo (2), Bruno Gridelli (1), Paolo Grossi (2), Pier Giulio Conaldi (1)
(1) Unit of Regenerative Medicine and Biomedical Technologies, ISMETT, Palermo, Italy
(2) Institute of Infectious Diseases, University of Insubria, Varese, Italy
HCV and HIV coinfection is common and the HCV-related liver disease is accelerated in coinfected patients. Recent studies have demonstrated
that single nucleotide polymorphisms (SNPs) of interleukin 28B (IL28B) locus (mainly SNPs rs12979860, rs12980275 and rs8099917) are
strongly associated with the response to IFN-alfa based treatment and also with spontaneous virus clearance in HCV infected patients.
Herein, the importance of the three IL28B SNPs is comparatively analyzed in a cohort of HCV-HIV coinfected patients.
Of 121 patients in the study, 50 had spontaneous HCV clearance and 71 were treated with IFN therapy presenting a sustained viral response
(SVR: 30 patients), a relapsing infection (12), and a treatment failure (29). We found that the frequency of homozygosis for favorable alleles
of all the three SNPs was significantly higher in the patients who spontaneously cleared HCV (mean: 80%) than in those affected by chronic
HCV infection (mean: 51%). Particularly, in the former patients the prevalence of rs8099917 T/T genotype was 88%, while no unfavorable
G/G genotype was found. Concerning the achievement of SVR in HIV patients coinfected with HCV-1 or HCV-4, we found that homozygosis
for favorable alleles is a strong positive predictor particularly in case of SNP rs8099917 (100% of patients with SVR had T/T genotype).
Interestingly, while SNP rs12979860 and rs12980275 showed a negligible influence on the outcome of INF-alfa therapy in case of coinfection
with HCV-2 and HCV-3, rs8099917 T/T genotype was detected in 82% of these patients achieving SVR. Finally, in relapser patients the
prevalence of IL28B SNP alleles resulted similar to that of SVR patients, but the baseline titer of HCV-RNA was higher and/or CD4 cell number
lower in relapsers. In conclusion, rs8099917 T/T genotype is a strong positive predictor both of spontaneous virus clearance and anti-HCV
therapy response in HIV-HCV coinfected patients.
Contact: Floriana Barbera
Unit of Regenerative Medicine and Biomedical Technologies, ISMETT, Palermo, Italy
[email protected]
160 : HCV 2011
HOST GENETICS
P4.08
A genome-wide association study identified susceptibility loci for HCV-induced
hepatocellular carcinoma in MICA gene
Naoya Kato (1), Vinod Kumar (1), Ryosuke Muroyama (1), Norie Kowatari (1), Wenwen Li (1), Kaku Goto (1), Motoyuki Otsuka (2), Ryosuke
Tateishi (2), Haruhiko Yoshida (2), Masao Omata (3), Kazuhiko Koike (2), Yusuke Nakamura (1), Koichi Matsuda (1)
(1) Institute of Medical Science, University of Tokyo, Tokyo, Japan
(2) Department of Gastroenterology, University of Tokyo, Japan
(3) Yamanashi Prefectural Hospital Organization, Japan
Background & Aims: HCV infection is a major risk factor for developing hepatocellular carcinoma (HCC). Host genetic factors involved in the
development of HCC in patients with HCV infection remain to be investigated. Methods: To identify the genetic susceptibility factor(s) for
HCV-induced HCC (C-HCC), a genome-wide association study (GWAS) was conducted using 432,703 autosomal SNPs in 721 C-HCC cases and
2,890 HCV-negative controls of Japanese origin. Single nucleotide polymorphisms (SNPs) which showed possible association (P < 1 x 10-5) in
the GWAS were further genotyped in 673 cases and 2,596 controls. Results: We identified 8 independent loci showing possible association
in the GWAS. After replication, we found a novel SNP in the 5’UTR of MICA (MHC class I polypeptide-related sequence A) gene [Pcombined
= 4.21x10-13, odds ratio = 1.39] to be strongly associated with C-HCC, whereas the remaining 7 SNPs failed to replicate the association.
Subsequent analyses using patients with CHC indicated that this SNP is also significantly associated with progression from chronic hepatitis
C to C-HCC (P=3.13 x 10-8). We also found that risk allele of SNP was associated with lower soluble MICA levels in C-HCC patients (P = 1.38
x 10-13). Conclusions: Our results highlight the importance of MICA as not only a predictive biomarker for C-HCC but also a therapeutic
target against C-HCC.
Contact: Naoya Kato
Institute of Medical Science, University of Tokyo, Tokyo, Japan
[email protected]
P4.09
Tarik Asselah (1), Simon De Muynck (1), Philippe Broët (2), Julien Masliah-Planchon (3), Maud Blanluet (3), Ivan Bièche (4), Olivier Lada
(1), Emilie Estrabaud (1), Martine Lapalus (1), Michelle Martinot-Peignoux (1), Dominique Vidaud (3), Nathalie Boyer (1), Pierre Bedossa
(5), Dominique Valla (1), Michel Vidaud (3), Patrick Marcellin (1)
(1) INSERM U773-CRB3, Paris, France
(2) University Paris-Sud Inserm UMR669, Villejuif., France
(3) Biochemistry and Molecular Genetics Department, Beaujon Hospital, Clichy, France
(4) INSERM UMR745, University of Paris Descartes, Paris, France
(5) Pathological Anatomy Department, Beaujon Hospital, Clichy, France
Background: Polymorphisms in the region of the interleukin (IL)-28B gene on chromosome 19 have been associated with peginterferon-alfa–
induced clearance of genotype 1, 2 and 3 HCV infection; however there are no data for patients with genotype 4. We evaluated the effects of
IL-28B polymorphisms on response to treatment with peginterferon and ribavirin and on the severity in a well-characterized cohort of genotype
4 patients. Patients and Methods: Serum samples from 164 patients were analysed by direct sequencing of the SNP rs12979860 of IL-28B. This
study recruited patients from three different ethnic groups (as self-reported by the patient) with 70 (43%) Egyptian, 53 (32%) European and 37
(23%) Sub-Saharan African Among these patient, 82 were included in a response cohort and 160 in a severity cohort. Genetic and bio-clinical
features between patients having sustained virological response (43 responder patients) and those who did not respond to treatment nor had a
relapse after the end of the treatment (39 non-responder patients) were compared. Results: Our data show that IL28B rs12979860 CC genotype
is associated with a better treatment response rate for patients with chronic genotype 4 HCV infections (trend test: p-value = 0.0008). The
response rates were 81.8% [0.66-0.98], 46.5% [0.31-0.61] and 29.4% [0.07-0.50] for genotype CC, CT and TT respectively. Frequencies of the C
allele were 61.4%, 54.7% and 31.0% for patients of Egyptian, European and Sub-Saharan Africa origin respectively (p-value< 0.0002). We didn’t
see any significant relationship between IL28B rs12979860 and the severity of the disease (trend test: p-value = 0.37). Conclusion: The SNP
rs12979860 is strongly associated with sustained virological response (SVR) in patient infected with genotype 4 HCV, but not with severity of the
disease. Analysis of IL-28B genotype might be used to guide treatment for these patients but can’t substitute liver biopsy in fibrosis evaluation.
INSERM U773-CRB3, Paris, France
[email protected]
POSTERS
IL28B polymorphism is associated with treatment response in hepatitis C virus
genotype 4 patients
POSTERS
P4.10
Robust antiviral activity of lambda-interferon (IL-29) against HCV in interferon alpha
resistant Huh-7 cells with a defective JAK-STAT signaling
Srikanta Dash (1), Sidhartha Hazari (1), Satyam Nayak (1), Partha Chandra (1), Deepak Kaushal (1), Luis Balart (1)
Background: The polymorphism of IFN-λ gene is strongly associated with the natural clearance of HCV infection and treatment response
in chronic HCV patients. The mechanisms by which the lambda gene variants affect HCV clearance are unclear. Aim: To study the antiviral
properties, cell signaling and gene regulation kinetics of IFN-λ between the interferon alpha sensitive (S3-GFP) and resistant (R4-GFP) Huh-7
cells replicating HCV sub-genomic RNA. Methods: S3-GFP and R4-GFP cells were treated with IFN-λ and the antiviral effect was determined
by measuring the HCV replication by G-418 resistant cell colony, HCV GFP fusion protein expression by flow cytometry and viral RNA levels
were measured by RPA and real time RT-PCR. The activation of IFN-λ induced Jak-Stat signaling and the ISGs and microRNA expression
between the S3-GFP and R4-GFP cells were measured by western blot analysis and real time RT-PCR. Results: Cell colony assay, fluorescence
microscopy and flow cytometry analysis revealed that the antiviral effect of IFN-λ is very strong in R4-GFP replicon cell line that can inhibit
HCV replication to a completion as compared to S3-GFP cells. This was also confirmed by RPA and real time RT-PCR based measurement of
HCV mRNA. IFN-λ treatment induced phosphorylation of Tyk2, Stat1 and Stat2 proteins and IFN-β promoter activity in S3-GFP cells but not
in R4-GFP cells. IFN-λ specifically induced IFITM1 and ISG56 significantly (10 and 4 fold respectively) in R4-GFP cells as compared to S3-GFP
cells. Silencing of IFITM1 using small hairpin RNA prevented IFN-λ mediated antiviral activity in R4-GFP cells. Conclusions: IFN-λ inhibits
HCV replication in Jak-Stat defective IFN-α resistant cells by a unique mechanism. Our results suggest that IFN-λ can be effectively used to
treat chronic HCV infection that are resistant to interferon alpha due to a defective Jak-Stat signaling. Acknowledgements: This work was
supported by NIH grants CA127481
[email protected]
P4.11
Polymorphisms in the interleukin 10 promoter and interleukin 28B gene in chronic
hepatitis C patients treated with interferon and ribavirin
Carlos Melo (1), Evaldo Araujo (1), Fatima Tengan (1), Antonio Barone Alci (1)
(1) Hepatitis Laboratory - University of Sao Paulo, Sao Paulo, Brazil
POSTERS
In HCV infections, the persistence of the virus and the response to antiviral therapy has been shown to be associated with the production of
inappropriate cytokine levels in inflammatory and immune response. The interleukin-10 (IL10) is a potent, anti-inflammatory cytokine and
it seems to have an important role in the host outcome of HCV. Several polymorphic sites within the promoter region of the IL-10 gene have
been described, and a SNP at position –1082 (G/A), relative to the transcription start site, is associated with a differential IL-10 expression.
Recently, genome-wide association studies have linked response to PEG-IFN/RBV with common single nucleotide polymorphisms near
the IL28B gene (rs12979860), encoding for interferon-lambda-3 (IL28B), associated with an approximately twofold change in response
to treatment. The rs12979860 CC genotype is associated with a greater rate of sustained virological response (SVR) than the CT or TT
genotypes in different HCV patients. In this study, we have evaluated the frequency of this polymorphisms genes and their association with
the response to antiviral therapy.
Genomic DNA from 39 patients classified as responders and 61 nonresponders to a combination of PEG-IFN/RBV was molecular typing by
polymerase chain reaction (PCR) with allele-specific primers followed by electrophoresis on agarose gels (3%).
The distribution of the genotypes A/A, G/A and G/G for the IL-10 -1082 polymorphism in the studied individuals was for responders 35.9%,
41.0% and 23.1%, respectively, and among the nonresponders was 39.3%, 44.3% and 16.4%, respectively. The frequency of polymorphism
rs12979860 of IL28B gene for responders were as follows: C/C 33.3%, C/T 51.3%, T/T 15.4%. The distribution among the non-responders
was: C/C 19.7%, C/T 67.2%, T/T 13.1%.
Our data are still preliminary, but we found no correlation between the presence of two polymorphisms together and predicting response
to therapy. New data will be needed to answer this question.
Contact: Carlos Melo
Hepatitis Laboratory - University of Sao Paulo, Sao Paulo, Brazil
[email protected]
162 : HCV 2011
HOST GENETICS
P4.12
Brazilian profile of IL28-B rs12979860 and rs8099917 single nucleotide polymorphism:
a retrospective analysis and possible consequences for therapy
Carlos Melo (1), Evaldo Araujo (1), Luciane Martins (1), Carla Almeida (1), Fatima Tengan (1), Antonio Barone (1)
(1) Hepatitis Laboratory - University of Sao Paulo, Sao Paulo, Brazil
Recently, genome-wide association studies have linked response to PEG-IFN/RBV with common single nucleotide polymorphisms near
the IL28B gene. We describe prevalence and discuss implications of those SNPs among Brazilians. Frozen samples (-80ºC) from 411 HCV
infected Brazilian patients were retrospectively analyzed. The SNPs near the IL28B gene, rs12979860 and rs8099917, was examined using an
assay with allele specific PCR probes. Demographic data were collected. Race was defined as registered at original files: White (Caucasian),
Black (Afro-American) and Yellow (Asiatic). The mean age of patients was 46 years and genotype 1 was the most prevalent (99.7%). For the
rs12989760 we found the following distribution: 86 (20.9%) C/C, 243 (59.1%) T/C and 82 (20.0%) T/T. For the rs8099917 G/G were 30 (7.3%),
T/G 150 (36.5%) and T/T 231 (56.2%). These results show an unexpected disagreement in the distribution of genotypes maybe linked to
the technique used. Our preliminary result reveals a concerning low prevalence of the most favorable IL28b polymorphisms. This is very
relevant information once in our continental country with almost 4 million chronic HCV carriers we could have a large amount of them with
a poor baseline genetic predictor of SVR. Among distinct races there was not a significant difference on genotype distribution (p<0.05). This
could reflect that Brazilian genetic heritage is too much mixed to allow a clear racial distinction as it is possible in other north hemisphere
countries. Although recently DAAs data showed a distinguished susceptibility to the IL28b SNPs being less pronounced to Telaprevir than
Boceprevir meaning that maybe within a DAA context IL28b influence can be overcomed by DAAs, our results still are relevant to the
Brazilian patients once in an interferon based therapy scenario, due to low prevalence of favorable genotypes in our population, the risk of
failure might be high and the resistance consequences unknowned.
Contact: Carlos Melo
Hepatitis Laboratory - University of Sao Paulo, Sao Paulo, Brazil
[email protected]
P4.13
Kris Kowdley (1), Jay Lalezari (2), Eric Lawitz (3), Robert Hindes (4), Rob Hyland (4), Lei Fang (5), Effie Albanis (4), William Symonds (4),
Michelle Berrey (4)
(1) Virginia Mason Medical Center, Seattle, WA, USA
(2) Quest Clinical Research, USA
(3) Alamo Medical Research, USA
(4) Pharmasset, Inc, USA
(5) Pharmstat, USA
Polymorphisms in IL28B influence response to PEG/RBV in patients with HCV GT1, 2, or 3. Addition of first-generation DAA to PEG/RBV has
demonstrated improved responses in populations including IL28B CT or TT genotypes; however, risk of virologic breakthrough and relapse
remains higher in these patients. The antiviral potency and high barrier to resistance of the nucleotide analog PSI-7977 may be independent
of IFN-responsiveness, and thus independent of IL28B genotype. Methods: PSI-7977 demonstrated a 4.3 log10 HCV RNA decline during 7-day
monotherapy. PROTON is an ongoing Phase 2b dose-ranging study of PSI-7977/PEG/RBV in HCV GT1, 2, or 3. A planned week 12 on-therapy
interim analysis was performed. Results: IL28B CC genotype was present in 36/95 (38%) GT1 patients, 7/24 (28%) with GT2/3. All 95 GT1
patients receiving PSI-7977 200mg or 400mg experienced early and substantial declines in HCV RNA; all but one achieved RVR. 13 GT1 had
TT genotype, with no discernible differences vs CC patients in early viral kinetics; median time to <LOD = 14 d for both groups. No virologic
breakthroughs were observed through wk 12 (4 unrelated discontinuations); all 91 subjects evaluable at wk 12 remained <LOD (cEVR). 100%
of 24 evaluable GT2/3 patients achieved RVR, cEVR, and SVR12. Across HCV GT1, 2, or 3, >70% of patients achieved HCV RNA <LOD by d14,
with no effect of IL28B genotype on time to HCV RNA <LOD or risk of on-treatment breakthrough. Conclusion: PSI-7977 + PEG/RBV resulted
in profound and consistent antiviral suppression with no on-treatment breakthrough in patients with HCV GT 1, 2, or 3, independent of IL28B
genotype. Additional analyses of IL28B and other predictors of IFN non-response will continue as post-treatment (SVR) data are available.
Clinical studies exploring PSI-7977 with minimal IFN and IFN-free DAA are ongoing in patients with HCV genotypes 1-6.
Contact: Kris Kowdley
Virginia Mason Medical Center, Seattle, WA, USA
[email protected]
POSTERS
PSI-7977 with PEG/RBV in HCV GT1, 2, or 3 results in consistent viral suppression
independent of IL28B genotype
POSTERS
164 : HCV 2011
POSTERS
Host Responses
P5.01
A weak neutralizing antibody response to hepatitis C virus E2 glycoprotein
leads to enhancement of virus infection
Keith Meyer, Arup Banerjee, Michael Houghton, Sharon Frey, Robert Belshe, Ranjit Ray
P5.02
Intrahepatic gene expression profiling of hepatic samples obtained by fine
needle aspiration compared to core needle biopsies in HCV-infected patients
Sergey Lezhnin, I-Ming Wang, Matthew Marton, Jared Lunceford, Marija Zeremski, Melissa Drexel, Jessy Makeyeva,
David Stone, Christine Cervini, Ginevra Castagna, Mark Ferguson, Robert Iannone, Marcella Ruddy, Andrew Talal
P5.03
The IFN-induced effector molecule IFITM1 inhibits HCV entry through
interactions with cellular coreceptors
Courtney Wilkins, Daryl Lau, Jessica Woodward, Nanette Crochet, Michael Gale Jr.
P5.04
Emergence of a host cellular restriction mechanism on propagation of
hepatitis C virus
P5.05
HCV and ALT flares following ART initiation in HCV/HIV coinfected patients
are immune-mediated
Mohamed Tarek Shata, Soad Nady, Susan Rouster, Jason Blackard, Kenneth Sherman
P5.06
Evaluation of potentially protective immunity in highly exposed but
uninfected prison inmates in the HITS study
Barbara Cameron, Peter Sugden, Andrew Lloyd, HITS Investigators
P5.07
Center for the study of innate immunity to hepatitis C virus infection: a
Hepatitis C Cooperative Research Center
Melissa Petersen, Michael Gale Jr.
POSTERS
Po-Yuan Ke, Steve Chen
POSTERS
P5.08
Vitamin-D inhibits hepatitis C virus by enhancing interferon production and
activity in hepatocytes in vitro
Meital Gal-Tanamy, Larisa Bachmetov, Amiram Ravid, Ruth Koren, Arie Erman, Ran Tur-Kaspa, Romy Zemel
P5.09
sCD26 activity is associated with responsiveness to peginterferon-alpha/
ribavirin therapy in chronic hepatitis C virus genotype 1 infection
Galia Askarieh, Martin Lagging, Magnus Lindh, Kristoffer Hellstrand, Jonas Soderholm
P5.10
Distinct cytokine and chemokine profiles in acute HCV infection are
associated with infection outcome
Jordana Levine, William O. Osburn, Jacquie Astemborski, Michael Chattergoon, David Thomas, Andrea L. Cox
P5.11
Broad genotype 1 neutralizing antibodies are generated during HCV infection
with genotypes 1, 2, or 3 but fail to protect against persistent infection
William Osburn, Brian Fisher, Anna Snider, Andrea L. Cox, Stuart C. Ray
P5.12
Modulation of HCV RNA abundance by the micro-regulator XRN2
Cecilia Sedano, Cara Pager, Peter Sarnow
P5.13
A case of seronegative HCV infection in a child infected via
mother-to-child transmission
Ariane Larouche, Geneviève Gaëtan, Nabil El-Bilali, Mathieu Quesnel-Vallières, Steven R. Martin,
Fernando Alvarez, Naglaa Shoukry, Hugo Soudeyns
P5.14
Effects of interferon alpha on parathyroid gland in chronic hepatitis B and C:
An observational study
P. Patrick Basu, Nithya Krishnaswamy, Niraj James Shah, Sakina Farhat, Thankam Nair, Robert S. Brown
P5.15
POSTERS
Serum retinol binding protein 4 (RBP-4) is a unique surrogate marker of end
stage liver disease
P. Patrick Basu, Nithya Krishnaswamy, Niraj James Shah, Sakina Farhat, Thankam Nair, Robert S. Brown
P5.16
Lymphocyte-hepatocyte interactions: hepatitis C virus changes the rules
Zania Stamataki, Omar Qureshi, Gary Reynolds, Christopher Mee, Linda Hibbert, Jenny Waters, Graham Foster,
Joshua Rappoport, Stefan Hubscher, David Adams, Jane McKeating
P5.17
Hepatitis C virus infection is blocked by HMGB1 released from virus-infected cells
Jong Ha Jung, Sung Key Jang
P5.18
Attenuated activation of type I interferon pathways by cell culture-adapted
HCV core proteins
Byung-Yoon Ahn, Ju-Il Kang
P5.19
A rational approach identified a 4 gene molecular signature to predict
response to treatment in hepatitis C
Tarik Asselah, Ivan Bièche, Bénédicte Jardin-Watelet, Aurélie Ducès, Martine Lapalus, Simon De Muynck, Eve Dupas,
Nathalie Julian, Isabelle Molina, Nadine Lambert, Emilie Nicolas, Bérénice Sallenave, Maud Blanluet, Emilie Estrabaud,
Michelle Martinot-Peignoux, Olivier Lada, Dominique Valla, Pierre Bedossa, Daniel Laune, Patrick Marcellin, Michel Vidaud
166 : HCV 2011
HOST RESPONSES
P5.20
Interferon-induced formation of stress granules in hepatitis C virus
infected cells
Alessia Ruggieri, Eva Dazert, Philippe Metz, Sarah Hofmann, Jan-Philip Bergeest, Artur Kaul, Charles Samuel,
Michael Frese, Karl Rohr, Georg Stoecklin, Ralf Bartenschlager
P5.21
Characterisation of immune cell population changes during GBV-B infection
Simon Hood, Edward T. Mee, James Greenhow, Helen Bright, Neil Berry, Nicola J. Rose
P5.22
Regulation of HCV infection by the interferon-stimulated gene ISG15
Patricia Domingues, Chris Boutell, John McLauchlan
P5.23
Role of HCV NS3/4A in host cell transformation
Aileen Patterson, Xiaojie Hu, Keding Cheng, Garrett Westmacott, Michael Carpenter
P5.24
Novel innate immune signaling factor, TNK1 that regulates HCV infection
Takeshi Saito, Jessica Briley, Timothy Tellinghuisen, Michael Gale Jr.
P5.25
Transcriptional profiling of human hepatocytes recovered from livers of SCID/
Alb-UPA mice following interferon treatment for HCV genotype 1A and 3A in
Donna Douglas, Gordon Broderick, Norman Kneteman
P5.26
Two center, masked replication study of occult hepatitis C virus in peripheral
blood of high risk seronegative subjects who are apparently uninfected
Peter Sugden, Tram Pham, Shabnah Ratnarajah, Barbara Cameron, Rowena Bull, Peter White, Thomas Michalak,
Andrew Lloyd, HITS Investigators
P5.27
Pre-exposure to interferon-lambda or endogenous interferons impairs ISG
induction and antiviral responses to interferon-alpha
P5.28
Biomarkers of spontaneous acute hepatitis C virus resolution
Suganya Selvarajah, Sheila Keating, John Heitman, Graham Simmons, Philip Norris, Eva Operskalski, James Mosley,
Michael Busch
POSTERS
Zahra Alvandi, Vera Cherepanov, Alana Sherker, Nazia Selzner, Limin Chen, Ian McGilvray, Jordan J. Feld
POSTERS
168 : HCV 2011
HOST RESPONSES
P5.01
A weak neutralizing antibody response to hepatitis C virus E2 glycoprotein leads to
enhancement of virus infection
Keith Meyer (1), Arup Banerjee (1), Michael Houghton (2), Sharon Frey (1), Robert Belshe (1), Ranjit Ray (1)
(1) St Louis University, St Louis, MO, USA
(2) Novartis, USA
A recent phase 1 safety and immunogenicity trial with hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, revealed that a modest
level of neutralizing activity to HCV genotype 1a was present in approximately ~25% of the vaccinee sera. We evaluated vaccinee sera as
a potential source of antibody dependent enhancement (ADE) in those exhibiting a weak (≤1/10) neutralizing antibody response. Initially,
observations of those sera displaying a neutralizing activity (≥ 1/20) revealed no significant enhancement of VSV/HCV pseudotype infection
with increasing dilutions. However, vaccinee sera with a weak or undetectable neutralizing response enhanced cell culture grown HCV
genotype 1a or 2a infection; and surrogate VSV/HCV pseudotype infection, in a dilution dependant manner. The use of surrogate VSV
pseudotype generated from individual HCV glycoproteins suggested that antibody responses specific to the E2 glycoprotein; but not the
E1 glycoprotein, were the principle targets for enhancing antibodies. FcRII was observed to be expressed on hepatic cell (Huh-7.5 and IHH)
surface, and antibody specific to FcRII (or to the Fc portion of Ig) blocked enhancement of HCV exposed to ADE sera. Together, the results
from in vitro studies indicated that in the absence of a strong antibody response to HCV envelope glycoproteins, enhancement of viral
infectivity may occur.
Contact: Keith Meyer
St Louis University, St Louis, MO, USA
[email protected]
P5.02
Intrahepatic gene expression profiling of hepatic samples obtained by fine needle
aspiration compared to core needle biopsies in HCV-infected patients
Background and aims: Although core needle biopsy (CNB) remains the gold standard for hepatic histology assessment, safety concerns limit
use for serial sampling. A safe and efficient alternative, such as fine needle aspiration (FNA), would have wide applicability to liver diseases.
Limitations of FNA include low RNA quantity, RNA degradation, and blood contamination. We sought to validate FNA sampling for serial
evaluation of hepatic gene expression.
Methods: Liver CNBs and FNAs were performed on 16 patients with chronic HCV infection. Eight patients had mild/moderate (stage 1-2,
Scheuer), and eight had severe (stage 3-4) fibrosis. RNA quality was assessed by RNA Integrity Number>5. Preamplified Taqman qPCR
assays were developed and validated according to ABI specification. Liver-specific housekeeping genes were identified based on variation in
population and absence in co-expression networks. Gene expression profiling was performed using custom-designed Affymetrix microarrays.
Pathway analysis was performed using Ingenuity.
Results: The FNA technique was found to be safe, well-tolerated and produced high-quality RNA in 83% of cases using optimized protocol. In
cases of low-quantity RNA, amplification enabled assessment of 21 pre-selected genes from 5ng total RNA. Blood contamination, observed
in 67%+/-23% of samples was determined by assessing expression of six novel liver-specific housekeeping genes. We were able to obtain
almost identical gene expression results (R=0.99, P< 0.0001) comparing samples obtained by CNB or FNA for the 21 genes of interest.
Comparing patients with mild/moderate to those with severe fibrosis, we identified a potential liver fibrosis signature, highly correlated
(R=0.73, P < 0.01) between both techniques and consistent with recognized HCV-related fibrogenic processes. FNAs were well tolerated
without any bleeding episodes.
Conclusions: We overcame limitations of FNA including low sample quantity and blood contamination. Our approach validates the use of
FNA as a reasonably safe method for serial assessment of hepatic gene expression.
Contact: Andrew Talal
Weill Cornell Medical College, New York, NY, USA
[email protected]
POSTERS
Sergey Lezhnin (1), I-Ming Wang (1), Matthew Marton (1), Jared Lunceford (1), Marija Zeremski (2), Melissa Drexel (1), Jessy Makeyeva (2),
David Stone (1), Christine Cervini (2), Ginevra Castagna (2), Mark Ferguson (1), Robert Iannone (1), Marcella Ruddy (1), Andrew Talal (2)
(1) Merck Research Laboratories, USA
(2) Weill Cornell Medical College, New York, NY, USA
POSTERS
P5.03
The IFN-induced effector molecule IFITM1 inhibits HCV entry through interactions with
cellular coreceptors
Courtney Wilkins (1), Daryl Lau (2), Jessica Woodward (1), Nanette Crochet (1), Michael Gale Jr. (1)
(2) Beth Israel Deaconess Medical Center, USA
The antiviral activity of interferon (IFN) therapy against HCV is largely attributed to the effector functions of specific IFN-stimulated genes
(ISGs) expressed in the hepatocyte following systemic IFN treatment. Interferon-induced transmembrane protein 1 (IFITM1) was identified
among a bioset of ISGs whose expression associated with HCV suppression in a cell culture model. We found that IFITM1 inhibits HCV in
tissue culture, with an increase in HCV replication and reduction of IFN antiviral efficacy following IFITM1 knockdown. Cell analysis revealed
that IFITM1 is localized to hepatic tight junctions in complex with HCV coreceptors. During the IFN response, IFITM1 interaction with these
co-receptors alters their interactions, resulting in suppression of the HCV entry process. We found that IFITM1 is localized to tight junctions
within both patient liver biopsy tissue and humanized chimeric mouse liver, and high level hepatic expression of IFITM1 associated with
favorable IFN therapy outcome among a patient cohort undergoing acute IFN treatment. Together, these data define the molecular function
of IFITM1 as an IFN-induced tight junction protein that governs HCV entry to mediate IFN actions against HCV infection.
Contact: Courtney Wilkins
[email protected]
P5.04
Emergence of a host cellular restriction mechanism on propagation of hepatitis C virus
Po-Yuan Ke (1), Steve Chen (1)
(1) Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
POSTERS
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. So far the virological and cellular consequences of HCV infection
still remain largely unknown. Here we determine the impact of HCV replication status on the consequential cellular response and virus
growth by comparing normal and high levels of HCV expression using an engineered HCV genome containing a blasticidin resistant cassette.
Interestingly, maintenance of highly-replicating HCV cells, which was initially concomitant with the high levels of viral RNA as well as viral
proteins and the production of high-titered virus. However, long-term culture of the highly-replicating HCV cells rapidly led to attenuated
viral assembly/budding and amplification due to superinfection exclusion. The rapid emergence of superinfection exclusion in HCV highlyreplicating cells was associated with downregulated cell surface expressions of HCV (co)receptors, CD81, Claudin1, and Occludin through
sequestrating these (co)receptors within a unique endoplasmic reticulum (ER)-associated membranous structure. Likewise, HCV infection
also led to reduction in the cell permissiveness to HCV infection through retaining HCV (co)receptors within the ER-associated membranous
structure. Taken together, these findings not only indicate that the status of HCV replication plays a crucial determinant in HCV propagation
but also reveal a novel action of a cellular restrictive mechanism in counteracting productive viral replication by retaining HCV (co)receptors
in ER to maintain virus-host cell homeostasis.
Contact: Po-Yuan Ke
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
[email protected]
170 : HCV 2011
HOST RESPONSES
P5.05
HCV and ALT flares following ART initiation in HCV/HIV coinfected patients are
immune-mediated
Mohamed Tarek Shata (1), Soad Nady (1), Susan Rouster (1), Jason Blackard (1), Kenneth Sherman (1)
(1) University of Cincinnati, Cincinnati, OH, USA
Background: Initiation of antiretroviral therapy (ART) in HCV/HIV coinfected patients may be associated with paradoxical increases in
circulating HCV RNA levels and/or flares in serum transaminases (ALT). To identify the role of the Ag-specific immune responses in the
induction of flares, we evaluated and correlated specific and non-specific immune responses in HCV/HIV coinfected patients before and
during ART therapy with clinical and laboratory findings.
Method: 16 subjects with HCV/HIV coinfection were evaluated. Subjects were treated with efavirenz or boosted atazanavir with a tenofovir/
emtricitabine backbone. We quantified HIV-specific, HCV-specific and non-specific (CD3 and CEF) immune responses using IFN-gamma
ELISPOT assays before and after initiation of ART therapy. We examined the relationship between the magnitudes of the immune responses
with the presence of ALT/HCV flare. Additionally, correlations among the Ag-specific immune responses and HCV viral loads were evaluated.
Results: Nine of 16 (56.3%) HCV/HIV coinfected patients enrolled in the study experienced ALT /HCV flare during ART initiation. Prior to and
during ART therapy, the average magnitude of the HCV specific immune responses in the flared subjects were significantly higher compared
to non-flared subjects (p=0.006). No significant differences were seen in responses to CEF, anti-CD3, or HIV protein (p=0.5,p= 0.3 and p=0.1
respectively). Moreover, a significant positive correlation was found between the HCV viral load and HCV specific IFN-gamma_ immune
responses in the flared subjects (p=0.002).
Conclusion: Our findings suggest that increased HCV viral load after ART initiation is associated with strong HCV specific response. However,
these immune responses were ineffective in lowering HCV viral load. Additionally, during immune reconstitution following initiation of ART,
strong HCV specific response is associated with ALT elevation, suggesting heightened immune-mediated killing of infected hepatocytes.
Paradoxically, this injury pattern is accompanied by increased HCV RNA levels in a subset of subjects.
Contact: Mohamed Tarek Shata
University of Cincinnati, Cincinnati, OH, USA
[email protected]
P5.06
Barbara Cameron (1), Peter Sugden (1), Andrew Lloyd (1), HITS Investigators (1)
(1) University of New South Wales, Sydney, Australia
HCV seroprevalence surveys in injecting drug users (IDU) indicate that a minority remain seronegative despite decades of risk behaviours,
with a subset exhibiting HCV-specific cellular immunity. The aim of the present study is to determine whether this cellular immunity provides
protection against infection, and to correlate it’s occurrence with detailed behavioural data.
Adult IDU inmates were recruited into the prospective Hepatitis C Incidence and Transmission in Prisons Study (HITS), and followed up whilst
in prison at approximately 6 monthly intervals. At each visit inmates complete a structured interview, recording detailed risk factors for
transmission of HCV, and blood is collected to screen for HCV viraemia by PCR, and HCV antibodies by ELISA, and to store peripheral blood
mononuclear cells (PBMC).
PBMC from enrolment were screened for anti-HCV cellular immunity by IFN-gamma ELISpot (n=94: 78 seronegative, 16 had seroconverted).
Subsequent incident infections were investigated in a nested case-control study, where pre-incident PBMC from subjects who became
infected (cases, n=30), and the latest timepoint from those remaining uninfected (controls, n=30; matched by risk behaviour, age and sex),
were assayed using IFN-gamma ELISpot (whole genome GT1 and GT3 HCV peptides), in vitro culture followed by FACS for HCV-specific CD4
T cells, and NK cell subsets and function, and IL28B genotype.
HCV-specific immunity was present in 28% of uninfected subjects at enrolment and correlated with specific risk behaviours, but did
not predict infection outcome. Immunological testing of the pre-incident samples from the case-control series is under way and will be
presented. IL28B genotype was not different between cases and controls.
HCV-specific cellular immunity is evident in high-risk exposed-uninfected IDU and is associated with specific behaviours. Identification of
immunological correlates of protection against HCV infection will inform vaccine design.
Contact: Barbara Cameron
University of New South Wales, Sydney, Australia
[email protected]
POSTERS
Evaluation of potentially protective immunity in highly exposed but uninfected prison
inmates in the HITS study
POSTERS
P5.07
Center for the study of innate immunity to hepatitis C virus infection: a Hepatitis C
Cooperative Research Center
Melissa Petersen (1), Michael Gale Jr. (1)
The Center for the Study of Innate Immunity to Hepatitis C virus is a new Hepatitis Cooperative Research Center (U19) funded by the U.S.
National Institutes of Health (NIH). The multidisciplinary studies within this Center aim to understand how the innate immune response
governs HCV infection and immunity, with a goal to define novel virus and host targets for antiviral therapy. The Center is comprised of
three translational research projects served by a central Clinical Core and an Administrative Core. The Center features integrated research
programs, bringing together expertise in HCV immunobiology, virology, patient care and translational research to conduct studies of innate
immunity, hepatic immunology, and interferon anti-viral therapy to HCV infection. Specific research projects within the Center include: 1)
Defining how host cells discriminate HCV as non-self leading to antiviral innate immune signaling and hepatic immunity, and Determining HCV
genotype-specificity of innate immune response triggering and control; 2) Defining the function of plasmacytoid dendritic cells (pDC) in the
hepatic innate immune response to HCV infection, and Identifying the hepatocyte-pDC interactions that stimulate IFN and proinflammatory
cytokine production to modulate HCV infection and immunity; 3) Defining the spectrum of microRNAs (miRNA) and their target genes that
are regulated to control chronic HCV infection and the outcome of IFN therapy. The Center’s Clinical Core conducts patient research and
clinical trials, as well as in vivo validation assessment of Center research findings. The Center is directed by Dr. Michael Gale Jr. (University of
Washington School of Medicine) Dr. Gale also serves as Project 1 Leader. Dr. Francis Chisari (The Scripps Research Institute) serves as Project
2 Leader. Dr. Daryl Lau (Harvard Medical School) directs the Clinical Core. Center research and clinical activities offer specialized training
programs in HCV and innate immunity research, and in HCV patient clinical care and treatment.
Contact: Melissa Petersen
[email protected]
P5.08
Vitamin-D inhibits hepatitis C virus by enhancing interferon production and activity in
hepatocytes in vitro
POSTERS
Meital Gal-Tanamy (2), Larisa Bachmetov (2), Amiram Ravid (2), Ruth Koren (2), Arie Erman (1), Ran Tur-Kaspa (1), Romy Zemel (2)
(1) Rabin Medical Center, Beilinson Campus, Israel
(2) Tel-Aviv University, Rabin Medical Center, Petah Tikva, Israel
Recently, low serum vitamin-D levels were linked to low sustained-virological-response (SVR) to interferon-based therapy in chronic
HCV. Furthermore vitamin-D supplementation was reported to improve the probability of achieving a SVR when combined with antiviral
treatment. Our aim was to determine the in-vitro ability of vitamin-D to inhibit HCV infectious virus production and explore the mechanism(s)
of inhibition.
Here we demonstrate that vitamin-D inhibited HCV production in a dose dependent manner up to 70-80% as was observed by FFU reduction
assay, western blot analysis , and by qPCR for HCV RNA in Huh7.5 cells. Since vitamin-D activity is obtained through its active metabolite,
calcitriol, we tested whether calcitriol is produced in Huh7.5 cells. We found that these cells express 1-alpha-hydroxylase, the enzyme
responsible for vitamin-D conversion to calcitriol. Specific ELISA detected the presence of calcitriol in the supernatant of vitamin-D treated
cells. Vitamin-D induced the expression of calcitriol target gene 24-hydroxylase, indicating that vitamin-D receptor is activated. Thus, these
cells contain the full machinery for vitamin-D metabolism. Moreover, HCV increased vitamin-D metabolism resulting in higher calcitriol
levels compared to non-infected cells. Treatment with calcitriol resulted in HCV inhibition confirming that vitamin-D activity is mediated by
its active metabolite. Stimulation of Huh 7.5 cells with poly-(IC) and HCV indicated that the anti-viral mechanism of vitamin-D or calcitriol
inhibition involves the induction of interferon singling pathway resulting in expression of interferon-beta and the downstream interferonstimulated gene, MxA. Importantly, the combination of vitamin-D or calcitriol and interferon-alpha synergistically inhibited viral production.
This study is among the first to explore in vitro the anti-viral effect of vitamin-D. It reveals novel information concerning its metabolism in
hepatoma cells, its role in anti-viral treatment and its synergistic activity in combination with interferon-alpha. This may have important
implications for improving antiviral treatment of HCV infected patients.
Contact: Romy Zemel
Tel-Aviv University, Rabin Medical Center, Petah Tikva, Israel
[email protected]
172 : HCV 2011
HOST RESPONSES
P5.09
sCD26 activity is associated with responsiveness to peginterferon-alpha/ribavirin
therapy in chronic hepatitis C virus genotype 1 infection
Galia Askarieh (1), Martin Lagging (1), Magnus Lindh (1), Kristoffer Hellstrand (1), Jonas Soderholm (1)
(1) University of Gothenburg, Gothenburg, Sweden
Approximately 50% of patients chronically infected with genotype 1 hepatitis C virus (HCV) achieve a sustained virological response (SVR)
following treatment with pegylated interferon-alpha and ribavirin, and outcome is prognosticated by gender, age, baseline viral load, IL28B
polymorphisms, and baseline IP-10. The chemokine IP-10 attracts mononuclear immune cells through the CXCR3 receptor. The previous
finding that high baseline serum levels of IP-10 predict treatment failure may therefore appear counterintuitive. A recent study, however,
suggested that the presence of an antagonistic form of IP-10, truncated by soluble CD26 (sCD26 or DPPIV), hinders the IP-10-induced
recruitment of mononuclear cells. Studies on sCD26 in small cohorts of HCV patients have been inconclusive in that one study reported
lower concentrations of sCD26 and another showed higher enzymatic activity in HCV-infected patients compared to healthy volunteers.
Thus we aimed at further clarifying the interplay between IP-10 and various forms of sCD26 with a focus on prognostication of treatment
outcome in HCV genotype 1 infected patients (n=73). Our study confirms that high baseline serum levels of IP-10 (median 263 pg/mL versus
420 pg/mL for SVR and non-SVR patients respectively; P=0.007) and the presence of the unfavorable IL28B T allele at rs12979860 (81% SVR
versus 53% SVR for CC and CT/TT respectively; P=0.015) are associated with treatment failure. Furthermore, higher sCD26 levels (708 ng/mL
versus 872 ng/mL; P=0.01) were associated with failure to achieve SVR, and a similar non-significant trend toward reduced sCD26 activity
and unresponsiveness was also observed (P=0.13), with a significant correlation noted between sCD26 activity and sCD26 concentration
(rs=–0.4; P<0.01). Indeed the strongest association was noted between SVR and the ratio of CD26 activity to sCD26 concentration (median
0.32 versus 0.23 RLU/(ng/mL); P=0.0005). Hence, higher sCD26 activity per sCD26 molecule may predict SVR at least as accurately as
baseline IP-10 or IL28B polymorphisms.
Contact: Jonas Soderholm
University of Gothenburg, Gothenburg, Sweden
[email protected]
P5.10
Jordana Levine (1), William O. Osburn (1), Jacquie Astemborski (1), Michael Chattergoon (1), David Thomas (1), Andrea L. Cox (1)
(1) The Johns Hopkins University/School of Medicine, Baltimore, MD, USA
Objective: To determine if inflammatory chemokine and cytokine patterns during acute HCV infection correlate with outcome of infection.
Methods: Blood was obtained throughout acute infection from injection drug users with known outcomes of infection in the longitudinal
BBAASH cohort. Plasma levels of IFNgamma, TNFalpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, Eotaxin, IP-10, MCP-1, MCP4, MIP-1beta, and TARC were measured by MesoScale Discovery ELISA. HCV RNA levels were quantified using the Roche COBAS Taqman
Assay. Cytokine and chemokine levels measured at the time of initial viremia were compared between clearance and persistence groups.
Levels measured at the time of last viremia in the clearance group were compared to time-matched samples in the persistence group.
Results: TNFalpha levels were higher in the initial viremic sample in the clearance group compared to the persistence group. Increased levels
of IL-2, IL-10, and IL-13 were detected in the persistence group and were correlated with one another. Increased levels of Eotaxin, MCP-4,
IL-2 and IL-10 were detected in time-matched samples in the persistence group compared to the last viremic samples of subjects in the
clearance group. While HCV RNA and most chemokine and cytokine levels were not correlated during acute infection, IL-10 was positively
correlated and MCP-4 and Eotaxin were negatively correlated with HCV RNA. In both analyses, IP-10 levels did not differ between groups
and were positively correlated with HCV RNA.
Conclusion: These data suggest that persistence is associated with increased anti-inflammatory cytokine signaling throughout acute
infection and enhanced chemokine signaling late in acute infection while clearance is associated with increased pro-inflammatory cytokine
signaling early in acute infection. Despite correlations with IP-10 levels and response to HCV therapy, IP-10 levels were not correlated with
spontaneous clearance. Further investigation of these differences may identify cytokine and chemokine expression patterns predictive of
HCV clearance.
Contact: Jordana Levine
The Johns Hopkins University/School of Medicine, Baltimore, MD, USA
[email protected]
POSTERS
Distinct cytokine and chemokine profiles in acute HCV infection are associated with
infection outcome
POSTERS
P5.11
Broad genotype 1 neutralizing antibodies are generated during HCV infection with
genotypes 1, 2, or 3 but fail to protect against persistent infection
William Osburn (1), Brian Fisher (1), Anna Snider (1), Andrea L. Cox (1), Stuart C. Ray (1)
Objective: Previous experiments using a single HCV pseudoparticle (HCVpp) to screen for neutralization suggested that clearance is
associated with the presence of neutralizing antibodies (nAb). To determine the breadth of heterologous nAb responses, neutralization was
assessed using a novel genotype 1 HCVpp library.
Method: We generated a phylogenetically representative heterologous HCVpp library containing 13 genotype 1a HCVpp and 7 genotype
1b HCVpp. For each HCVpp in this library, we measured neutralization by plasma specimens obtained at 12 months of infection from
intravenous drug users in the BBAASH cohort with known outcomes (persistence versus clearance). Neutralization at the time of last viremia
was assessed in cleared infections with infection durations of less than 12 months.
Results: Neutralization of at least one HCVpp was observed in 58% of all samples and neutralization of at least one genotype 1a and 1b
HCVpp was observed in 36% of all samples. Neutralization of any construct (58%) was more frequent than neutralization of even the most
commonly-neutralized construct (42%). Neither neutralization detection (any positive) nor breadth (number of HCVpp neutralized) differed
according to outcome (clearance versus persistence). The breadth and frequency of nAb response was independent of infection genotype.
The most striking finding was that the neutralization resistance (proportion of subjects that did not neutralize) of an HCVpp increased with
the duration of viremia in the subject from which the HCVpp was derived, suggesting that envelope genes accumulate escape mutations
with transition from acute to chronic viremia.
Conclusions: These data suggest that 1) HCV infection results in the generation, even during non-genotype 1 infections, of broad antigenotype 1 nAb responses that do not provide protection against persistent infection 2) Using multiple HCVpp results in a more sensitive
assessment of nAb responses, and 3) HCV evades the nAb response during the transition from acute to chronic infection.
Contact: William Osburn
[email protected]
P5.12
Modulation of HCV RNA abundance by the micro-regulator XRN2
POSTERS
Cecilia Sedano (1), Cara Pager (1), Peter Sarnow (1)
(1) Stanford University, Stanford, CA, USA
MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs of 18-25 nucleotides in size that are involved in important biological
functions such as development, cancer, and viral infection. MiR-122 is specifically expressed in the human liver and constitutes 70%
of its total miRNA population. This miRNA is a key regulator of cholesterol and fatty-acid metabolism in the adult liver, and our lab
previously reported that the replication of hepatits C virus is dependent on its expression levels, because miR-122 upregulates HCV
mRNA abundance.
While miRNA biogenesis and miRNA-mediated gene regulation have been widely studied, however, detailed mechanisms of miRNA
turnover remain to be explored. In A. thaliana, the 5’-3’ exoribonucleases XRN2 and XRN3 are involved in miRNA precursor processing,
whereas in C. elegans, XRN2 has been implicated in miRNA turnover in vivo. In an effort to examine whether XRN2 is responsible for
the turnover of miRNAs in mammals, XRN2 was depleted by siRNAs in liver cells and in cells that had been infected with JFH1. It was
found that XRN2 depletion caused a significant increase in HCV RNA and HCV protein abundances. In contrast, depletion of XRN2
diminished the expression of luciferase mRNAs that contained binding sites for miR-122 in their 3’ noncoding regions. The cellular
localization of XRN2 did not change upon HCV infection, and XRN2 did not co-localize with viral proteins or viral RNA. Thus, these
findings suggest that XRN2 enhances HCV expression by modulating the turnover of cellular microRNAs. This notion is supported
by the discovery that compound 3, which stimulates miR-122 gene expression, enhanced HCV RNA abundance, as did the ectopic
expression of miR-122 mimetics.
Contact: Cecilia Sedano
Stanford University, Stanford, CA, USA
[email protected]
174 : HCV 2011
HOST RESPONSES
P5.13
A case of seronegative HCV infection in a child infected via mother-to-child transmission
Ariane Larouche (1), Geneviève Gaëtan (2), Nabil El-Bilali (3), Mathieu Quesnel-Vallières (1), Steven R. Martin (4), Fernando Alvarez (5),
Naglaa Shoukry (3), Hugo Soudeyns (1)
(1) Unité d’Immunnopathologie Virale, CHU Ste-Justine, Montréal, Canada
(2) Département de Médecine, Université de Montréal, Canada
(3) Centre De Recherche Du CHUM, Hopital St-Luc, Canada
(4) Département de Pédiatrie, Université De Montréal, Canada
(5) Gastroentérologie, Hépatologie et Nutrition, CHU Sainte-Justine, Canada
HCV infection typically leads to the development of antibody responses within weeks following primary infection. Here we describe the
case of a child who acquired HCV infection by mother-to-child transmission and who remained persistently HCV-seronegative despite the
presence of elevated levels of plasma HCV RNA. Born 03/1999 to an HCV-1a-infected mother who was not a carrier of HIV-1, this child was
diagnosed with HCV in 2000 on the basis of positive PCR assays. Testing for anti-HCV antibodies (Ab) using ELISA and RIBA was repeatedly
negative (11 times) despite the presence of Ab responses against childhood immunizations (HAV; HBV; tetanus; mumps; rubella) and no
apparent abnormalities in IgG profiles. High ALT and AST levels justified introduction of treatment with pegIFN-alpha + ribavirin at age
9.5 years. Viral load was 997,000 UI/ml, 576,000 UI/ml and 76,000 UI/ml at age 5.25, 9.33, and 9.67 years, respectively. Treatment was
stopped at age 10.0 because of absence of SVR and did not lead to HCV seroconversion. Liver histology showed mild fibrosis without any
treatment-related symptoms or side effects. HCV-specific cell-mediated immune responses, tested using ELISpot and a complete genomic
peptide panel, showed a complete absence of HCV peptide-driven IFN-gamma production. Finally, HCV quasispecies profile based on HVR1,
examined at 5 time points over a 10 year period (17-48 E2 recombinants per time point), showed little time-dependent evolution and was
characterized by the presence of a single predominant E2 variant that represented more than 80% of the total number of clones analyzed in
subject 1. This variant persisted through time in subject 1 and remained largely predominant post-treatment. Interestingly, it also comprised
80% of sequences analyzed in the mother. These results are consistent with absence of both humoral and cell-mediated HCV-specific
immune responses in the child, suggestive of neonatal tolerance potentially associated with in utero transmission.
Contact: Ariane Larouche
Unité d’Immunnopathologie Virale, CHU Ste-Justine, Montréal, Canada
[email protected]
Effects of interferon alpha on parathyroid gland in chronic hepatitis B and C:
An observational study
P. Patrick Basu (1), Nithya Krishnaswamy (2), Niraj James Shah (2), Sakina Farhat (2), Thankam Nair (2), Robert S. Brown (1)
Objectives: Interferon alpha (IFN-α) has been the mainstay of therapy for chronic hepatitis C. Side events of IFN-based therapy include
autoimmune thyroiditis (10.7%). The effects of IFN gamma on calcium homeostasis have been well documented. Impacts of IFN-α on
osteoclasts have been postulated. This study evaluated IFN-α induced autoimmune hyperparathyroidism, with or without eucalcemic state.
Methods: 96 patients were evaluated, 45/90 patients treated with Pegylated IFN-α for chronic hepatitis C (group A- 75% alfa-2a, and 25%
on alfa-2b), 36 patients with chronic hepatitis B (group B- 100% alfa-2a). 15 patients with multiple sclerosis (MS) (group C) treated with IFN
beta for MS were recruited. Baseline and 24-week ionized calcium, serum parathyroid hormone intact (PTH intact), 24-hour urine calcium,
bone densitometry, and parathyroid antibody (PTH-Ab) were measured in all subjects. Exclusion criteria: Parathyroid dysfunction, multiple
endocrine neoplasia, hypercalcemic syndromes, hypervitaminosis D, renal failure, osteoporosis and sarcoidosis
Results: 23/96 patients (24%) had high normal serum calcium (>9.6 mg/dL). The 24-hour urine and total ionized calcium levels were
elevated in 2/36 (5.6%) patients in Group B. Baseline serum PTH-intact and PTH Ab were normal in all groups. Group A, 9/45 patients (20%,
alfa -2a 44%, 2b, 55%) in and Group-B, 6/36 patients (16.7%) developed elevated PTH levels and none in group C. PTH radionucleotide
scan identified 1/36 (2.8%) patient with adenoma in group B. PTH Ab was found 9/45 (20%) in group A, 5/36(13.9%) in group B, and none
in Group C. Conclusion: Interferon alpha has autoimmune effects on parathyroid gland expressing elevated PTH level with PTH Ab without
overt clinical manifestation of hyperparathyroidism. A larger sample size is required to confirm the potential effect of IFN alpha on calcium
homeostasis, which might explain the symptoms of fatigue in chronic hepatitis C associated with IFN therapy.
[email protected]
POSTERS
P5.14
POSTERS
P5.15
Serum retinol binding protein 4 (RBP-4) is a unique surrogate marker of end stage liver
disease
P. Patrick Basu (1), Nithya Krishnaswamy (2), Niraj James Shah (2), Sakina Farhat (2), Thankam Nair (2), Robert S. Brown (1)
Background: Retinol binding protein 4 (RBP-4), secreted from liver and adipose tissue, transports retinol in the circulation. RBP-4 is inversely
proportional to disease progression and decompensation. This study evaluates the role of serum RBP-4 in severity of liver disease.
Methods: 85 patients (n=85) (age=49.6±11.4, mean±SD) with moderate to -severe liver disease (mean MELD of 14) were recruited: Group
A- Chronic liver disease (n=30):20/30 HCV (67%) 8/30 HBV (27%) 2/30 Misc (6.6%) with mean MELD 20; Group B- (n=30) mean 10, 18/30
HCV (60%), 5/30 HBV (17%), 4/30 Alcoholics (13%), and 3/30 misc (10%) with mean MELD 18. Group C- controls (n=25) mean MELD 4. Liver
biopsy, HOMA-score, Adiponectin, Leptin, serum RBP-4, APRI, Hyaluronic acid, and Hepascore for liver fibrosis were analyzed. Exclusion
includes: NASH, NAFLD, metabolic, vascular liver diseases.
Results: RBP-4 has significant differences between all groups (F(2, 82)=47.9, p<0.001) over controls (n=25, 4.21+/1.60; mean+/-SD). RBP4 is
decreased in Cirrhotics (p<0.001; n=30, 1.46+/0.51) over non-Cirrhotics (p=0.015; n=30, 3.37+/0.97) and controls. Cirrhotics have decrease
levels of HOMA (1.09±0.09 vs. 1.51±0.60; p=0.001), Adiponectin (0.93±0.40 vs. 1.26±0.53; p=0.01), but increased TNF-alpha (11.23±3.81 vs.
3.72±3.37; p<0.001) compared to non-cirrhotic. RBP 4 has negative correlation with TNF-alpha (r=-0.62, p<0.001), and MELD score (r=-0.57,
p=0.001), but positive correlation with leptin (r=0.12, p=0.38) and hepatic steatometry (BURNT score<4)-(r=0.12, p=0.41). There is poor
correlation between RBP4 and APRI (r = -0.158, p = 0.228). RBP4 independently (R-square = 0.475, p = 0.005) correlates with Adiponectin
(p = 0.001) and HOMA (p = 0.038)
Conclusions: This study proposed the role of RBP-4 as a biomarker for endstage liver disease, with inverse relationship with severity of
fibrosis, hepatic decompensation, MELD score and positive correlation with insulin resistance and Adipokines, without hepatic steatometry.
A larger prospective clinical trial needs to validate the role of RBP-4 in fibrosis and decompensated liver disease.
[email protected]
P5.16
Lymphocyte-hepatocyte interactions: hepatitis C virus changes the rules
POSTERS
Zania Stamataki (2), Omar Qureshi (2), Gary Reynolds (2), Christopher Mee (2), Linda Hibbert (1), Jenny Waters (1), Graham Foster (1),
Joshua Rappoport (2), Stefan Hubscher (2), David Adams (2), Jane McKeating (2)
(1) Queen Mary University of London, United Kingdom
Hepatitis C Virus (HCV) is a major cause of liver disease worldwide. Innate and adaptive cellular immune responses play a role in resolving
acute HCV infection. However, the majority of infections are not cleared, resulting in a progressive chronic liver disease, consistent with
inadequate immune control. Evidence from both human and animal models suggest that CD4+ T cells play a critical role in controlling acute
HCV infection. However, the mechanism(s) behind their failure to control chronic HCV replication are unknown. The major site of HCV
replication is the liver and virus specific immune cells need to target infected hepatocytes to control virus replication. HCV specific effector
cells have been reported to home to the liver, however, little is known on their subsequent trafficking and fate within the organ. Our aim is
to investigate the role of HCV infection on lymphocyte-hepatocyte interactions, migration and immune cell effector function.
We used in vitro and ex vivo models to study the effect of HCV infection on lymphocyte-hepatocyte interactions. Primary lymphocytes and
hepatocytes were used in combination with hepatoma cell lines and replication competent HCV clones. Ex vivo lymphocyte migration assays
were performed using biopsy material and tissue from explanted liver. Results were confirmed by in vivo observations using tissue sections
from patients with end stage liver disease of viral and non-viral origin.
Our studies demonstrate: i. A role for hepatocyte ICAM-1 in mediating T-lymphocyte adhesion and migration; ii T-lymphocytes migrate
through hepatocytes (trans-cellular) and via cell-cell junctions (para-cellular); iii. HCV perturbs CD4 T-cell trans-cellular migration and proinflammatory cytokine expression. Our data highlight the existence of para- and trans-cellular routes for lymphocyte trafficking through
hepatocytes that impact on T-cell effector function. Furthermore, interaction with HCV-infected hepatocytes alters CD4+ T-cell trafficking
and cytokine expression, providing a mechanism for HCV to persist in the inflamed liver.
[email protected]
176 : HCV 2011
HOST RESPONSES
P5.17
Hepatitis C virus infection is blocked by HMGB1 released from virus-infected cells
Jong Ha Jung (1), Sung Key Jang (1)
(1) Department of Life Science, Pohang University of Science and Technology, Pohang, Republic of Korea
High mobility group box 1 (HMGB1), an abundant nuclear protein that triggers host immune responses, is an endogenous danger signal
involved in the pathogeneses of various infectious agents. However, its role in hepatitis C virus (HCV) infection is not known. Here, we show
that HMGB1 protein is translocated from the nucleus to cytoplasm, and subsequently released into the extracellular milieu by HCV infection.
Secreted HMGB1 triggers anti-viral responses and blocks HCV infection, a mechanism that may limit HCV propagation in HCV patients.
Secreted HMGB1 may also have a potential role in liver cirrhosis, which is a common comorbidity in HCV patients. Further investigations
into the roles of HMGB1 in the diseases caused by HCV infection will shed light on and potentially help prevent these serious and prevalent
HCV-related diseases.
Contact: Jong Ha Jung
Department of Life Science, Pohang University of Science and Technology, Pohang, Republic of Korea
[email protected]
P5.18
Attenuated activation of type I interferon pathways by cell culture-adapted HCV core
proteins
Hepatitis C virus (HCV) establishes persistent infection that frequently leads to chronic liver diseases. HCV core protein is believed to
modulate various cellular activities in these processes. In this study, we found two missense mutations (K23E and V31A) in the core protein
of the viral population prevalent in Huh-7 cells which had been persistently infected with JFH-1 virus for 158 days. These mutations caused
the viral population to escape from immunochemical detection by a core-specific antibody but apparently did not affect viral protein
expression (including that of core) or capsid assembly in the cell and in vitro. Reporter assay indicated that the activation of interferon-beta
promoter (IFN-b) and an IFN-stimulated response element (ISRE) was significantly attenuated by the expression of mutant core (K23E/
V31A) compared to that by wild-type core in cells either transfected with core alone or co-transfected with IKK kinases. We also found that
the mutant core binds less efficiently than wild-type core to DDX3, the cellular RNA helicase known to stimulate IFN pathways and bind at
the amino-terminal region of core. With the two mutations introduced the mutant virus showed enhanced RNA replication and prolonged
viral protein expression compared to the parental virus. These results indicate that HCV core activates type I IFN pathways and suggest a
mechanism by which HCV establishes persistent infection.
Contact: Byung-Yoon Ahn
Korea University, Seoul, Republic of Korea
[email protected]
POSTERS
Byung-Yoon Ahn (1), Ju-Il Kang (1)
(1) Korea University, Seoul, Republic of Korea
POSTERS
P5.19
A rational approach identified a 4 gene molecular signature to predict response to
treatment in hepatitis C
Tarik Asselah (1), Ivan Bièche (2), Bénédicte Jardin-Watelet (5), Aurélie Ducès (4), Martine Lapalus (1), Simon De Muynck (1),
Eve Dupas (5), Nathalie Julian (6), Isabelle Molina (5), Nadine Lambert (7), Emilie Nicolas (4), Bérénice Sallenave (4), Maud Blanluet (2),
Emilie Estrabaud (1), Michelle Martinot-Peignoux (1), Olivier Lada (1), Dominique Valla (1), Pierre Bedossa (8), Daniel Laune (5),
Patrick Marcellin (1), Michel Vidaud (4)
(1) INSERM U773, CRB3, Clichy, France
(2) INSERM UMR 745, University of Paris Descartes, Paris, France
(4) Service de Biochimie et Génétique Moléculaire, AP-HP, Beaujon Hospital, France
(6) Ariana Pharmaceuticals, Paris, France
(7) Bio-Rad Laboratories, Marnes-La-Coquette, France
(8) Service d’Anatomie Pathologique, AP-HP, Hôpital Beaujon, Clichy, France
Background and Aims: The treatment of chronic hepatitis C (CHC) is combined pegylated-interferon and ribavirin. Considering side effects and
treatment cost, prediction of treatment response is mandatory. The aim of this study was to identify a liver gene signature to predict sustained
virological response (SVR) in patients with CHC. Methods: Pretreatment liver biopsies from 140 patients treated with pegylated-interferon plus
ribavirin were studied. Patients were mainly infected with genotype 1, 3, 4 and 2. Among these patients 49% had SVRs, 36% non response (NR)
and 15% relapse (RR). Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of 54 genes involved in various cellular
and molecular mechanisms associated with liver gene expression during hepatitis C infection. Results: The expression profiles of responders differ
significantly from those of NRs. The most notable changes were mainly observed in the IFN-stimulated genes, and in growth factors/cytokines genes.
Interestingly, the basal liver levels of expression of IFN-stimulated genes were higher in NRs in comparison with RR and SVRs. We identified a 4-gene
signature composed of HERC5, IFI44, IL8 and MDK able to discriminate NRs and SVRs with 84% sensitivity, 86% specificity, 80% NPV and 89% PPV. In
NRs, the failure to respond to exogenous PEG-IFN could indicate a blunted response to IFN. This raises the possibility that, in NRs, the IFN-stimulated
genes are already maximally induced. Conclusion: NRs and SVRs have different liver gene expression profiles before treatment. We identified a 4
gene signature to predict response to treatment. The most notable changes occurred mainly in interferon-stimulated genes. Treatment response
could be predicted with a molecular gene signature including mainly interferon-stimulated genes. The genes included in the signature encode
molecules secreted in the serum and provide a logical functional approach for the development of serum markers to predict response to treatment.
INSERM U773, CRB3, Clichy, France
[email protected]
POSTERS
P5.20
Interferon-induced formation of stress granules in hepatitis C virus infected cells
Alessia Ruggieri (1), Eva Dazert (1), Philippe Metz (1), Sarah Hofmann (2), Jan-Philip Bergeest (3), Artur Kaul (1), Charles Samuel (4),
Michael Frese (1), Karl Rohr (3), Georg Stoecklin (2), Ralf Bartenschlager (1)
(1) Department of Infectious Diseases, Molecular Virology, University Heidelberg, Heidelberg, Germany
(2) German Cancer Research Center, DKFZ-ZMBH-Alliance, Heidelberg, Germany
(3) Biomedical Computer Vision Group, BioQuant Center, Heidelberg, Germany
(4) Department of Molecular, Cellular, and Developmental Biology, UCSB, CA, USA
Infection by many viruses induces a stress response leading to, amongst others, the formation of cytoplasmic stress granules (SGs), which
are thought to be sites of transient RNA storage or RNA degradation. This stress response allows cell recovery, but sustained stress may lead
to apoptosis. Although previously associated with ER- and oxidative stresses, HCV infection induces a very limited stress response in human
hepatoma (Huh7) cells. By using time-resolved immunofluorescence analyses we found that less than 10% of HCV-infected cells contain SGs.
Surprisingly however, their abundance is greatly increased upon interferon treatment with 80% of infected cells containing SGs. HCV-induced
SGs contain canonical markers such as TIA1, G3BP and PCBP2. Interestingly, the adenosine deaminase ADAR1 was also found in HCV-induced
SGs whereas none of the viral proteins localized to these structures. We established a real-time and quantitative live cell imaging system
based on confocal microscopy allowing repeated Z-stack capture of HCV-infected cells for more than 72h. We found that HCV-induced SGs are
motile, highly dynamic and that their appearance oscillates over time. Moreover, we elucidated the key stress kinase activating this pathway in
response to HCV infection. In summary, HCV infection appears to trigger a long-term stress response, which in contrast to other types of stress
does not induce apoptosis. We therefore hypothesize that this oscillating stress response contributes to cell survival and thus HCV persistence.
Contact: Alessia Ruggieri
Department of Infectious Diseases, Molecular Virology, University Heidelberg, Heidelberg, Germany
[email protected]
178 : HCV 2011
HOST RESPONSES
P5.21
Characterisation of immune cell population changes during GBV-B infection
Simon Hood (2), Edward T. Mee (2), James Greenhow (2), Helen Bright (1), Neil Berry (2), Nicola J. Rose (2)
(1) Pfizer Research and Development, United Kingdom
(2) NIBSC, Potters Bar, United Kingdom
Infection of red-bellied tamarins (Saguinus labiatus) with GBV-B is a recognised surrogate model of HCV infection in man. Detailed
characterisation of immune cell populations in GBV-B-infected tamarins has hitherto not been reported. We have identified crossreactive antibodies for flow cytometric analysis of CD3+, CD4+, CD8+ and CD20+ cells, as well as Programmed Death 1 (PD-1), a marker
of T-cell exhaustion. We have analysed peripheral and intrahepatic lymphocytes from uninfected tamarins and at time of termination,
either 6 or 24 weeks post-infection.
In the periphery during infection the proportion of CD3+ and CD20+ lymphocytes was unchanged whereas CD20+ lymphocytes were
more prominent following clearance of virus. The CD4:CD8 ratio increased during infection and returned to pre-infection levels following
clearance of peripheral viraemia. The PD-1 expression level fell following GBV-B infection. Analysis of intra-hepatic lymphocytes showed
a significant increase in the number of PD1+/CD4+ cells during infection compared to convalescent and uninfected tamarins and the
levels of PD1 expression on these cells was also significantly higher.
The data generated provide the first insight into the dynamics of immune cell populations in this model of HCV infection. The developed
antibody panels will be applicable to a range of studies in red-bellied tamarins. Further work will include the identification of CD4+ and
CD8+ T cell epitopes targeted during GBV-B infection in order to determine those responses associated with viral clearance.
Contact: Nicola J. Rose
NIBSC, Potters Bar, United Kingdom
[email protected]
P5.22
Regulation of HCV infection by the interferon-stimulated gene ISG15
HCV typically establishes a chronic infection that can be successfully eradicated by IFN-a based therapies. However, the underlying
mechanisms leading to virus clearance by IFN-a are poorly understood. From a siRNA library screen, we identified ISG15 as an interferonstimulated gene which modulates HCV RNA replication. In this study, we wished to understand the contribution of ISG15 to clearance
of HCV infection.
To examine directly the role of ISG15 in lowering the abundance of viral RNA, its gene expression was reduced using ISG15-specific
siRNAs, followed by treatment with and without IFN-a. In the absence of IFN-a, knock-down of ISG15 promoted HCV replication in Huh-7
and U2OS cells harbouring sub-genomic replicons by up to 150%. Abrogating expression of ISG15 in the presence of IFN-a also alleviated
repression of HCV replication by IFN-a. To further validate repression by ISG15, cell lines that constitutively expressed ISG15-specific
shRNAs were infected with a J6/JFH1 HCV chimera (JC-1) in the presence and absence of IFN-a. Data from these experiments mirrored
those from the sub-genomic replicon cells and together implicate ISG15 as a repressor of HCV RNA replication.
The mechanism used by ISG15 to modulate HCV infection is not known. Since it is an ubiquitin-like protein, ISG15 forms conjugates with
an array of other proteins; the majority of these conjugates are dependent on HERC5 E3 ligase activity. Interestingly, our results show
that conjugated ISG15 is produced to a much lesser extent in U2OS as compared to Huh-7 cells yet the IFN-mediated decrease in HCV
RNA replication is similar in both cell types. Expression of HERC5 is barely detected in U2OS cells even in the presence of IFN. Hence,
production of ISG15 conjugates may not be necessary for its anti-HCV properties. Experiments are in progress to determine directly the
need for ISG15 conjugation to mediate its anti-viral activity.
Contact: Patricia Domingues
MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
[email protected]
POSTERS
Patricia Domingues (1), Chris Boutell (1), John McLauchlan (1)
(1) MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
POSTERS
P5.23
Role of HCV NS3/4A in host cell transformation
Aileen Patterson (1), Xiaojie Hu (1), Keding Cheng (1), Garrett Westmacott (1), Michael Carpenter (1)
(1) Public Health Agency of Canada/Medical Microbiol., University Manitoba, Winnipeg, Canada
Hepatitis C virus NS3/4A is a multifunctional enzyme encoding a serine protease within its N-terminus and an NTPase/helicase activity in its
C-terminus. The C-terminal activities likely play roles in virus genome replication while the serine protease activity is essential for cleaving
the viral polyprotein into individual components. In addition, the protease has the capacity to cleave host proteins involved in innate
immunity such as MAVS and TRIF. Expression of NS3 in mouse (NIH3T3), rat (RF1) or human (QSG7701) cells also induces advanced cellular
transformation with characteristics including anchorage independent growth, loss of contact inhibition and the ability to induce tumors
when injected into nude mice. A functional protease is essential for this phenotype supporting the hypothesis that host proteins involved
in the event are being cleaved. Recently, a host protein T-cell protein tyrosine phosphatase protein was also shown to be targeted. In order
to more completely understand the mechanism by which NS3/4A transforms host cells, we have generated a series of NIH 3T3 cell lines
that stably express: 1) wt NS3/4A, 2) a protease deficient NS3/4A or 3) control (GFP or vector only) proteins. Quantitative proteomics was
performed by growing cells in SILAC (stable isotope labeling of amino acids in cell culture) media. Equal amounts of wt NS3/4A, and mutant
NS3/4A or wt NS3/4A and GFP expressing cell lysates were mixed prior to prefractionation at both the protein (SDS-PAGE separation)
or peptide (isoelectric focusing) levels. Samples were further fractionated online by reverse phase nano-HPLC prior to injection into a
high resolution mass spectrometer. Identification and relative ratios were determined for more than 2500 proteins (min 2 peptides, false
discovery rate <0.1). Network analysis of the proteins significant altered in their abundance (p<0.05) will be presented.
Contact: Aileen Patterson
Public Health Agency of Canada/Medical Microbiol., University Manitoba, Winnipeg, Canada
[email protected]
P5.24
Novel innate immune signaling factor, TNK1 that regulates HCV infection
Takeshi Saito (1), Jessica Briley (2), Timothy Tellinghuisen (3), Michael Gale Jr. (2)
(1) University of Southern California Keck School of Medicine, Los Angeles, CA, USA
(2) University of Washington Department of Immunology, USA
(3) Scripps Florida, USA
POSTERS
HCV infection is treated with interferon (IFN)-based therapy. IFN suppresses HCV replication through the induction of Interferon Stimulated
Genes (ISGs). Cellular engagement of IFN initiates the JAK-STAT signaling pathway driven by the type-1 interferon (IFN) receptor and results
in the ISG expression. However, it has been known that HCV attenuates JAK-STAT signaling pathway, leading HCV infected hepatocyte
refractory to ISGs expression by therapeutic IFN. In addition, systemic expression of IFN receptors limits the dose of IFN due to medication
related toxicities. Therefore, the identification of hepatocyte-specific intracellular ISGs induction pathway would be therapeutic target that
relive the patient from medication related side effect. Here, we conducted genome-wide high throughput cDNA screening to identify novel
cellular factors ISGs. A hepatoma cell line stably expressing the IFIT1 promoter fused to a firefly luciferase reporter gene was applied to
a high throughput cDNA screen, resulting in identification of more than forty genes as potential ISG inducers. Among these genes we
identified the non-receptor tyrosine kinase 1 (TNK1) as a potent inducer of ISG expression that activates JAK-STAT signaling in the absence
of IFN. TNK1 was sufficient to induce the ISGs through a process that involves its direct phosphorylation of STAT1 and STAT2. Moreover, we
found that TNK1-/- mice exhibit a severe deficit of hepatic ISG expression in response to type 1 IFN, indicating that TNK1 serves to potentiate
of IFN action. Therefore we conclude that TNK1 plays a critical role in IFN mediated innate antiviral immunity. The regulation of hepatic TNK1
activity represents a potential therapeutic target for HCV infection.
Contact: Takeshi Saito
University of Southern California Keck School of Medicine, Los Angeles, CA, USA
[email protected]
180 : HCV 2011
HOST RESPONSES
P5.25
Transcriptional profiling of human hepatocytes recovered from livers of SCID/Alb-UPA
mice following interferon treatment for HCV genotype 1A and 3A in
Donna Douglas (1), Gordon Broderick (1), Norman Kneteman (1)
We have previously shown that SCID/Alb-UPA mice infected with genotype 3A HCV respond more favorably to interferon alpha 2b (IFNα−2b)
treatment than those infected with HCV genotype 1A. In this study, we applied transcriptional profiling to human hepatocytes recovered
from the chimeric livers of a series of SCID/Alb-UPA mice that had been transplanted with hepatocytes isolated from a single human donor.
The treatment groups were: naïve mice (n=3 mice); HCV-1A (n=4 mice) and HCV-3A (n=3 mice) infected mice; naïve mice that received
IFNα−2b monotherapy (n=4 mice); and HCV-1A (n=3 mice) and HCV-3A (n=3 mice) infected mice that received IFNα−2b for the treatment of
infection. Principal component analysis (PCA) was used to provide a single optimal description for each microarray and this analysis depicted
agreement between the expression profiles within treatment groups and distinct profiles across treatment groups. Significantly changed
genes were selected by Significance Analysis of Microarrays (SAM). This method assigns a relative difference score and a false discovery
rate (FDR) to each gene on the basis of changes in expression levels and the standard deviation of repeated measurements. IFNα−2b
treatment enhanced the overall abundance of cellular mRNA transcripts with increased hybridization to approximately 3.9% of the 54,675
probe sets interrogated (FDR=0%); the most highly ranked were host defense interferon stimulated genes (ISGs) that have roles in antigen
presentation/processing, immune modulation, and ISGs of unknown function. HCV infection had a negative impact on the stimulation
of genes by IFNα−2b since hybridizations occurred to under 0.3% (FDR=0%). Genotype distinct ISG profiles were observed with human
hepatocytes recovered from mice that had undergone IFNα−2b therapy for the treatment of infection. The possibility that these distinct
profiles contribute IFN responsiveness in the clinical setting is the focus of future studies.
Contact: Donna Douglas
[email protected]
P5.26
Peter Sugden (1), Tram Pham (2), Shabnah Ratnarajah (1), Barbara Cameron (1), Rowena Bull (1), Peter White (1), Thomas Michalak (2),
Andrew Lloyd (1), HITS Investigators (3)
(1) University of New South Wales, Randwick, Australia
(2) Memorial University, St Johns, Canada
(3) HITS Investigators, Australia
Occult HCV infection is designated when subjects who may have been previously infected, do not have detectable HCV RNA using sensitive
commercial assays, but do have virus detected by ultra-sensitive techniques. Occult infection has been identified in individuals who have
cleared HCV, although there are several negative reports. A two center, masked, case-control study examining occult infection in highrisk, apparently uninfected subjects was undertaken. Coded plasma and peripheral blood mononuclear cells (PBMC) were studied:
20 antibody negative/PCR negative Australian prison inmates who reported injecting drug use (IDU); 5 chronic cases; 5 with cleared
infection; and 5 low-risk, uninfected Red Cross blood donors as a negative control. RNA extracted from plasma and PBMC were tested
in the Canadian laboratory by ultra-sensitive nested RT-PCR targeting the 5’UTR and Southern hybridization; the Australian laboratory
used nested RT-PCR for two genomic fragments (5’UTR and Core), followed by sequencing. Decoded analysis revealed that HCV RNA was
consistently detected (i.e. in both laboratories, with sequence identity, and both genomic locations) in one of 20 uninfected subjects (5%).
Evidence of probable contamination was also found with an identical sequence from a chronic case found in a Red Cross sample, highlighting
the need for stringent safeguards. As this result supports the likelihood that occult infection does occur in high-risk, apparently uninfected
subjects, a new study of 32 seronegative IDU prisoners with longitudinally-collected samples has been initiated - no subjects have evidence
of occult infection in one genomic location in the initial screening collection, with further analyses on later collections is underway. High-risk
subjects may harbor occult HCV. Further studies are needed to better understand whether these sequences are consistently present and
whether they are associated with immune recognition.
Contact: Peter Sugden
University of New South Wales, Randwick, Australia
[email protected]
POSTERS
Two center, masked replication study of occult hepatitis C virus in peripheral blood of
high risk seronegative subjects who are apparently uninfected
POSTERS
P5.27
Pre-exposure to interferon-lambda or endogenous interferons impairs ISG induction
and antiviral responses to interferon-alpha
Zahra Alvandi (1), Vera Cherepanov (1), Alana Sherker (1), Nazia Selzner (1), Limin Chen (1), Ian McGilvray (1), Jordan J. Feld (1)
(1) University of Toronto, Toronto, Canada
Background: The strong association between IL-28B gene polymorphisms and clearance of HCV suggest a role for IFN-λ3 in responses to
HCV infection. Studies have also shown IFN-signaling pathways are pre-activated and refractory to further stimulation in patients who fail
to respond to IFN therapy. Aims: To clarify how exposure to different interferons affects responses to subsequent antiviral treatment with
IFN-α. Methods: Huh7.5.1 cells were infected with HCVcc and treated with different interferons: mock, IFN-α (100 U/ml), IFN-λ (10 ng/ml),
or supernatant from poly:IC-treated Huh7.5.1 cells to mimic endogenous IFNs. Cells were treated one-time and left to recover or received
daily treatment for 7 days. At day 7, cells were given a final dose of IFN-α (100 U/ml) or mock-treated. Cells were harvested and interferonstimulated gene (ISG) induction and HCV RNA titer were assessed using qPCR. Results: Pre-treatment with IFN-α, IFN-λ or endogenous IFNs
led to a significant reduction in ISG induction with the final dose of IFN-α. Chronic exposure (daily treatment) made cells more refractory to
the final dose of IFN-α than a single dose. Exposure to IFN-λ or endogenous IFNs resulted in a prolonged and more potent refractory state
than pre-treatment with IFN-α. One-time or daily doses of all 3 IFNs led to a reduction in HCV RNA titer, however the antiviral effect of the
final dose of IFN-α (1.05 log reduction control) was significantly reduced by one-time or chronic pre-exposure to IFN-λ (-0.31 log 1-time and
0.32 log daily, p<0.03) or endogenous IFNs (0.34 log 1-time and 0.16 log daily, p<0.05) but not to IFN-α. Conclusion: Pre-exposure of cells to
IFN-λ or endogenous IFNs led to a prolonged refractory state that prevented ISG induction and antiviral activity upon treatment with IFN-α.
Pre-exposure to endogenous IFNs may lead to ISG pre-activation and prevent subsequent responses to therapeutic IFN-α.
Contact: Jordan J. Feld
University of Toronto, Toronto, Canada
[email protected]
P5.28
Biomarkers of spontaneous acute hepatitis C virus resolution
Suganya Selvarajah (1), Sheila Keating (1), John Heitman (1), Graham Simmons (1), Philip Norris (1), Eva Operskalski (2), James Mosley (2),
Michael Busch (1)
(1) Blood Systems Research Institute, University of California San Francisco, San Francisco, CA, USA
(2) University of Southern California, USA
POSTERS
We previously reported detailed characterization of the demographic, serological and clinical course of acute HCV infection in 94 recipients
who acquired HCV in Transfusion-Transmitted Viruses Study (TTVS). Our previous work also established the rate of viral resolution and
classified individuals as HCV resolvers and chronic HCV. In the current study we investigated soluble immunologic factors present in serial
serum samples of sixteen individuals during acute infection in order to identify biomarkers of spontaneous HCV resolution and chronic HCV.
Thirty-four of thirty-nine soluble cytokines/chemokines and growth factors using multi-analyte bead technology did not show significant
difference in expression levels in HCV resolvers or chronic HCV compared to uninfected controls. However, we observed increased expression
of IP-10, IL-10 and TNF-alpha in HCV resolvers and chronic HCV. The cytokine/chemokine expression kinetics was aligned with ALT and viral
load changes. For example, IL-10 expression was significantly higher in the early-post ALT elevation phase and waned during late-post
ALT elevation phase in HCV resolvers. However, higher IL-10 expression was observed during early- and late-post-ALT elevation phase in
individuals who progressed to chronic HCV. Interestingly, increased expression of sIL-2Ra was observed only in spontaneous HCV resolvers
and not in chronic HCV or uninfected controls.
We describe here a unique study to determine serum biomarkers of HCV infection and resolution, using a rare cohort (TTVS) with a large
number of serial serum samples spanning a year following transfusion transmission of HCV. We detected significant changes in expression
kinetics of IP-10, TNF-alpha, IL-10, sIL-2Ra and TGF-alpha as a result of HCV infection. Based on our interesting results we hypothesize that
elevation in sIL-2Ra expression during acute HCV infection is a serum biomarker of immune-stimulation and resolution in spontaneous HCV
resolvers. We believe, further studies for elevation of sIL-2Ra as a biomarker of spontaneous acute HCV resolution is warranted.
Contact: Suganya Selvarajah
Blood Systems Research Institute, University of California San Francisco, San Francisco, CA, USA
[email protected]
182 : HCV 2011
POSTERS
Pathogenesis
P6.01
Hepatitis C Virus activates mTOR/S6K1 signaling pathway in inhibiting IRS-1
function for insulin resistance
Sandip K. Bose, Shubham Shrivastava, Keith Meyer, Ratna B. Ray, Ranjit Ray
P6.02
CXCL9 and CXCL10 chemokines as predictors of fibrosis in a cohort of
primarily African-American injection drug users with chronic hepatitis C
Marija Zeremski, Rositsa Dimova, Jacquie Astemborski, David Thomas, Andrew Talal
P6.03
Induction of the antioxidant Nrf2/ARE defense pathway by HCV core is
controlled by amino acids 1-36
Alexander Ivanov, Olga Smirnova, Ekaterina Alekseeva, Olga Ivanova, Irina Somninskaya, Sergey Kochetkov,
Maria Isaguliants
P6.04
Hepatitis C virus infection promotes hepatic gluconeogenesis through an
NS5A-mediated, FoxO1-dependent pathway
P6.05
HCV-induced suppression of glucose transporter 2 gene expression via
downregulation of hepatocyte nuclear factor 1α
Chieko Matsui, Ikuo Shoji, Shusaku Kaneda, Lin Deng, Da-Peng Jiang, Yoshi-Hiro Ide, Hak Hotta
P6.06
Development of hepatocellular carcinoma in NS3 transgenic mice
Yoshi-Hiro Ide, Tatsuya Maebo, Chunying An, Dapeng Jiang, Lin Deng, Ikuo Shoji, Hak Hotta
P6.07
Network generation based on quantitative proteomic data reveals cellular
factors involved in the HCV-induced inhibition of VLDL production
Carmine Mancone, Claudia Montaldo, Laura Santangelo, Cristina Di Giacomo, Marco Vignetti, Laura Amicone,
Leopoldo Paolo Pucillo, Tonino Alonzi, Marco Tripodi
POSTERS
Lin Deng, Ikuo Shoji, Wataru Ogawa, Shusaku Kaneda, Tomoyoshi Soga, Da-peng Jiang, Yoshi-Hiro Ide, Hak Hotta
POSTERS
P6.08
Evidence for protective role of HCV-specific T cell transforming growth
factor beta in liver pathogenesis
Li Shaoyong, Imad Nasser, Nezam Afdhal, Margaret Koziel, Yury Popov, Detlef Schuppan, Mark A. Exley, Nadia Alatrakchi
P6.09
Effect of hepatitis C virus (HCV) on activation of the Human
Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR)
Satarupa Sengupta, Christina Martin, Ling Kong, Jason T. Blackard
P6.10
Hepatitis C virus NS5A protein impairs cytokine production through toll-like
receptor signaling pathway in mouse liver
Takeya Tsutsumi, Seiko Shinzawa, Koji Goto, Hidetaka Fujinaga, Hideyuki Miyoshi, Yoshizumi Shintani, Kyoji Moriya,
Hiroshi Yotsuyanagi, Tetsuro Suzuki, Yoshiharu Matsuura, Kazuhiko Koike
P6.11
Expression of the newly-discovered HCV minicore proteins is regulated by
viral mutations that predict treatment failure and liver cancer development
Francis Eng, Andrea Branch
P6.12
Hepatitis C viral protein NS5A induces EMT and participates in oncogenic
transformation of primary hepatocyte precursors
Damien Gregoire, Leila Akkari, Nicolas Floc’h, Yannick Simonin, Patrice Lassus, Urszula Hibner
P6.13
Detection of hepatitis C virus antigens in liver biopsies of patients with
hepatitis C recurrence after liver transplantation
Laura Mensa, Sofía Pérez-del-Pulgar, Gonzalo Crespo, Rosa Miquel, Xavier Forns
P6.14
HCV infection suppresses mitochondrial tri-functional protein transport to
impair lipid β-oxidation
Yutaka Amako, Mark Dallas, Aleem Siddiqui, Chris Peers, Mark Harris
POSTERS
P6.15
HCV suppresses a pro-apoptotic K+ current by Kv2.1 channel protein: a possible
mechanism of viral interference with stress-response signalling kinases
Yutaka Amako, Jamel Mankouri, Zsofia Igloi, Kalle Saksela, Arunas Kaslauskas, Mark Dallas, Chris Peers, Mark Harris
P6.16
During chronic HCV infection NK cells are increased in Huh 7.5 directed
cytolytic activity though are decreased in HCV infected target specificity
Qinglai Meng, Donald Anthony
P6.17
HCV-induced changes in gene expression in non-infected ‘bystander’ Huh-7 cells
Kate Muller, Nicholas Eyre, Michael Beard
P6.18
Hepatitis C virus core and NS5A proteins modulate expression of ODC and
SSAT thus affecting polyamine metabolism
Olga Smirnova, Maria Isaguliants, Olga Ivanova, Alexey Khomutov, Sergey Kochetkov, Alexander Ivanov
P6.19
Hepatitis C virus replication induces expression of vascular endothelial
growth factor and accelerates vascularisation of human hepatoma
Eileen Winkler, Hendryk Aurich, Rene Geissler, Alexander Mensch, Nadine Stöhr, Susann Friedrich, Stefan Hüttelmaier,
Sven-Erik Behrens
184 : HCV 2011
PATHOGENESIS
P6.20
Supplementation with branched-chain amino acids reduces hepatic iron
accumulation induced by hepatitis C virus proteins and iron overload in mice
Masaaki Korenaga, Keiko Korenaga, Shiho Tanaka, Sohji Nishina, Yasuyuki Tomiyama, Ryohei Ugaji, Noriko Dohi,
Yamato Tada, Kyo Sasaki, Yoshiharu Nakashima, Tomoya Kawase, Naoko Yoshioka, Yuhichi Hara, Kouji Yoshida,
Keisuke Hino
P6.21
PARV4 infection is associated with intravenous drug use, HCV and HIV
disease progression
Ruth Simmons, Colin Sharp, Patrick Mcclure, Janine Rohrbach, Helen Kovari, Will Irving, Andri Rauch, Peter Simmonds,
Paul Bowness, Paul Klenerman
P6.22
Hepatitis C virus induces type 2 diabetes through deregulation of insulin
secretion and glucose transport
Herve Lerat, Martin Higgs, Aurore Gaudin, Camille Cohen, Emilie Merour, Raphael Boisgard, Bertrand Tavitian,
Helene Kammoun, Isabelle Hainaut, Bertrand Blondeau, Fabienne Foufelle, Jean-Michel Pawlotsky
P6.23
Hepatic miRNAs expression in chronic hepatitis C patients at early stages of
liver fibrosis
Emilie Estrabaud, Ivan Bièche, Martine Lapalus, Simon De Muynck, Olivier Lada, Michelle Martinot-Peignoux,
Aurelie Duces, Dominique Valla, Pierre Bedossa, Patrick Marcellin, Michel Vidaud, Tarik Asselah
P6.24
Liver-specific targeting of viral transgenes in zebrafish: application to the
study of hepatitis C virus alternate reading frame protein
Amélie Pagliuzza, Mathieu Quesnel-Vallières, Zen-Kuei Chang, Shin-Jie Huang, Wangta Liu, Pierre Drapeau, Jen-Leih Wu,
Hugo Soudeyns
P6.25
Hepatitis C virus enhances tumor necrosis factor-alpha induced cell death
via suppression of nuclear factor-kappaB activation
P6.26
Hepatitis C virus infection down regulates IFNAR1 through ER stress
response leading defective JAK-STAT signaling and impaired IFN-α response
Srikanta Dash, Partha Chandra, Sidhartha Hazari, Feiza Gunduz, Sruti Chandra, Robert Garry, Luis Balart
P6.27
Molecular identification of transmitted hepatitis C viruses by single genome
amplification and sequencing
Hui Li, Mark Stoddard, Ruy Ribeiro, Shuyi Wang, Erica Parrish, Truman Grayson, Chuanxi Sun, Raoul Louzao,
Paul Goepfert, Thomas Denny, Barton Haynes, Alan Perelson, Beatrice Hahn, George Shaw
P6.28
Maintenance of miR-122 expression in hepatitis C virus-associated
hepatocellular carcinoma
Carolyn Spaniel, Tetsuro Shimakami, Masao Honda, Shuichi Kaneko, Stanley M. Lemon
P6.29
Detection of HCV-specific CD4+ T-cell proliferation and interferon-γ release
in seronegative spouses of chronically infected HCV patients
Bijan Raziorrouh, Dominique Tomlinson, Peter Kurktschiev, Winfried Schraut, Norbert Grüner, Reinhart Zachoval,
Maria-Christina Jung, Hans M. Nitschko, Martin Wächtler, Michael Spannagl, Helmut M. Diepolder, Axel Ulsenheimer
POSTERS
Junseong Park, Wonseok Kang, Seung-Wook Ryu, Woo-Il Kim, Dong-Yeop Chang, Eui-Cheol Shin, Chulhee Choi
POSTERS
P6.30
HCV infection causes cell cycle arrest at the G2/M-phase boundary
David McGivern, Rathi Kannan, Lucinda Hensley, Lauren Evers, Stanley Lemon
P6.31
Gene expression profiling in formalin fixed paraffin embedded (FFPE)
samples from hepatitis C virus infected patients with hepatocellular
carcinoma
Trina Das, James D. Perkins, Sean C. Proll, Jeffrey J. Delrow, Deborah L. Diamond, Michael G. Katze
P6.32
In vitro modeling of the HCV/HIV-1 co-infected liver environment
Brian Doehle, Amina Negash, Kristina Chang, Michael Gale Jr.
P6.33
Ethanol increases the morphogenesis of infectious HCV and the unfolded
protein response in primary human hepatocytes
Céline Hernandez, Béatrice Legrand, Arnaud Carpentier, Robert Barouki, Philippe Podevin, Hélène Rouach,
Arielle R. Rosenberg, Michèle Garlatti
POSTERS
186 : HCV 2011
PATHOGENESIS
P6.01
Hepatitis C virus activates mTOR/S6K1 signaling pathway in inhibiting IRS-1 function
for insulin resistance
Sandip K. Bose (1), Shubham Shrivastava (1), Keith Meyer (1), Ratna B. Ray (1), Ranjit Ray (1)
(1) Saint Louis University, Saint Louis, MO, USA
Insulin receptor substrates (IRS) are involved in activating numerous signaling pathways. IRS-1 plays a key role in the insulin signaling, thus
enabling metabolic regulation in mammalian cells. We examined the status of IRS-1 in HCV genotype 1a (clone H77) and genotype 2a (clone
JFH1) infected immortalized human hepatocytes (IHH). A detectable total and phosphorylated IRS-1 expression in hepatocytes infected
with HCV genotype 1a was observed. However, IRS-1 in HCV genotype 2a infected cells could not be detected. The status of Akt, which is
regulated by PI3K, was analyzed to determine its downstream signaling pathway. Ser473 phosphorylation status in Akt was increased in
HCV (genotype 1a and 2a) infected hepatocytes, while Thr308 phosphorylation status was not significantly altered. Further study indicated
an increase in total and phosphorylated mTOR expression, and this increase was more pronounced in IHH infected with HCV genotype 2a
as compared to genotype 1a. Interestingly, phosphoS6K level was higher in HCV genotype 2a infected hepatocytes suggesting a feed-back
mechanism for IRS-1 inhibition. Together, our observations suggest that HCV genotypes 1a and 2a act differently in activating mTOR/S6K1
signaling and IRS-1 inhibition for insulin resistance.
Contact: Sandip K. Bose
Saint Louis University, Saint Louis, MO, USA
[email protected]
P6.02
CXCL9 and CXCL10 chemokines as predictors of fibrosis in a cohort of primarily AfricanAmerican injection drug users with chronic hepatitis C
Background: CXCR3-associated chemokines CXCL9-11 promote hepatic inflammation in chronic HCV infection through the recruitment of
lymphocytes to the liver parenchyma. Peripheral CXCL10 and CXCL9 levels have been associated with hepatic fibrosis in predominantly
Caucasian HCV-infected patients, making them promising candidate-markers of liver fibrosis in this patient population. As fibrosis
progression as well as chemokine expression differs depending upon race, we investigated associations between CXCL9-10 and hepatic
fibrosis in African-American patients. Additionally, we analyzed potential role of these chemokines in predicting fibrosis progression.
Methods: Peripheral chemokine levels were measured using ELISA in 115 HCV-infected ALIVE cohort patients within 4 months of a liver
biopsy. All patients underwent a second biopsy 3-5 years following the initial one. Fibrosis was assessed using the Ishak fibrosis index by the
same hepatopathologist. Median age of study participants was 41.8 years, 84.4% were male, 93.9% African-American, 96% were infected
with HCV genotype 1, and 26% were HIV/HCV coinfected.
Results: CXCL10 levels appeared to be higher in patients with advanced fibrosis on the contemporaneous biopsy and were significantly higher
in patients with advanced compared to those with minimal fibrosis on the future biopsy (p = 0.0045). Moreover, CXCL10 had reasonable
ability to identify patients who are unlikely to develop advanced fibrosis in a 3-5 year interval; the odds ratio of having advanced fibrosis at
the second biopsy per unit increase in ln(IP10) was 2.368 (95% CI [1.15, 4.877]), adjusting for stage of fibrosis at the first biopsy. Performance
characteristic of CXCL10 in discriminating patients with advanced versus minimal fibrosis on the second biopsy did not differ significantly
compared to that obtained for Fibrosure and AST to platelet ratio (APRI). CXCL9 levels were not associated with fibrosis stage on present or
future biopsy.
Conclusion: CXCL10 has potential as a marker of fibrosis progression in African-American HCV-infected patients.
Contact: Marija Zeremski
Weill Medical College of Cornell University, New York, NY, USA
[email protected]
POSTERS
Marija Zeremski (1), Rositsa Dimova (1), Jacquie Astemborski (2), David Thomas (2), Andrew Talal (1)
(1) Weill Medical College of Cornell University, New York, NY, USA
(2) Johns Hopkins Medical Institutions, USA
POSTERS
P6.03
Induction of the antioxidant Nrf2/ARE defense pathway by HCV core is controlled by
amino acids 1-36
Alexander Ivanov (1), Olga Smirnova (1), Ekaterina Alekseeva (2), Olga Ivanova (1), Irina Somninskaya (2), Sergey Kochetkov (1),
Maria Isaguliants (3)
(1) Engelhardt Institute of Molecular Biology, Moscow, Russia
(2) Biomedical Research and Study Center, Latvia
(3) Karolinska Institute, Stockholm, Sweden; Institute of Virology, Moscow, Russia
Hepatitis C virus is the etiological agent for a wide-spread chronic liver disease which often leads to the development of liver cirrhosis,
steatosis, and cancer, as well as many extrahepatic manifestations. The development of liver fibrosis and hepatocellular carcinoma has
been tightly associated with HCV-induced oxidative stress (OS). Induction of OS by replicating HCV has been observed both in cell culture
systems and in blood of chronic hepatitis C patients. We and others have shown that HCV proteins induce oxidative stress induces and
activate Nrf2/ARE antioxidant defense pathway. The main inducers of both are Core and NS5A proteins. Here we aimed to study the
mechanism of oxidative stress induction and Nrf2/ARE pathway activation by HCV Core. We found that production of reactive oxygen
species is stimulated in response to expression of both N- and C-terminally truncated Core aa 37-191, and 1-36, respectively. However, only
Core1-36 activated the transcription factor Nrf2 and induced the Nrf2-ARE dependent antioxidant genes. This was mediated by protein
kinase C in ROS-dependent and by phosphoinositide-3-kinase (PI3K) and casein kinase 2 in ROS-independent manner (as for the full-length
Core). Core1-36 triggered Ca2+ efflux from ER to the cytoplasm and mitochondria, Ca2+ release suggestively responsible for signalling
through the antioxidant pathway. A detailed molecular mechanism underlying calcium homeostasis alterations and regulation of the cellular
redox state by the N-terminal Core domain is currently elucidated. Acknowledgements: supported by following grants: Russian Foundation
for Basic Research grants 10-04-00047 and 10-04-01402a, the President of Russian Federation МК-5035.2011.4, Swedish Research Council
K2009-66X-21053-01-3 and Swedish Institute New Visby program 00747/2010.
Contact: Alexander Ivanov
Engelhardt Institute of Molecular Biology, Moscow, Russia
[email protected]
P6.04
Hepatitis C virus infection promotes hepatic gluconeogenesis through an NS5A-mediated,
FoxO1-dependent pathway
POSTERS
Lin Deng (1), Ikuo Shoji (1), Wataru Ogawa (2), Shusaku Kaneda (1), Tomoyoshi Soga (3), Da-peng Jiang (1), Yoshi-Hiro Ide (1), Hak Hotta (1)
(1) Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan
(2) Division of Diabetes, Metabolism and Endocrinology, Kobe University Gr, Japan
(3) Institute for Advanced Biosciences, Keio University, Japan
Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association
is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally
upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for
hepatic gluconeogenesis, while downregulating the gene for glucokinase (GK), which catalyzes glycolysis. Accordingly, HCV enhanced the
cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor
forkhead box O1 (FoxO1). The phosphorylated form of FoxO1 is transported from the nucleus to the cytoplasm so that the transcriptional
activity of FoxO1 is downregulated. We observed that, although neither the mRNA levels nor the protein levels of FoxO1 expression were
affected by HCV, phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting
in increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It is well known that the FoxO1
phosphorylation is regulated by Akt/protein kinase B (PKB) through the insulin signaling pathway. However, it was unlikely that the decreased
FoxO1 phosphorylation was mediated through Akt inactivation as we observed increased phosphorylation of Akt at Ser473 in HCV-infected
cells compared to the control. Using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated
that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in the suppressed FoxO1 phosphorylation,
increased nuclear accumulation of FoxO1, and elevated glucose production. Furthermore, HCV NS5A is directly linked with the production of
ROS, reduction of FoxO1 phosphorylation, upregulation of PEPCK and G6Pase gene expression, and glucose production. Taken together, these
observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.
Contact: Lin Deng
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan
[email protected]
188 : HCV 2011
PATHOGENESIS
P6.05
HCV-induced suppression of glucose transporter 2 gene expression via downregulation
of hepatocyte nuclear factor 1α
Chieko Matsui (1), Ikuo Shoji (1), Shusaku Kaneda (1), Lin Deng (1), Da-Peng Jiang (1), Yoshi-Hiro Ide (1), Hak Hotta (1)
(1) Kobe University Graduate School of Medicine, Kobe, Japan
Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic diseases, including metabolic disorders and
autoimmune diseases. A number of studies have shown that HCV infection often predisposes the host towards type 2 diabetes. We previously
reported that HCV replication suppresses cellular glucose uptake by downregulation of cell surface expression of glucose transporter (GLUT)
2 (Kasai et al., J Hepatol, 2009). The detailed mechanism, however, remains to be elucidated. In this study, we aimed to clarify the molecular
mechanism of HCV-induced suppression of GLUT2 expression. We quantified the GLUT2 mRNA levels in HCV RNA replicon cells and in HCV J6/
JFH1-infected Huh-7.5 cells by RT-qPCR. Expression levels of GLUT2 mRNA were decreased in both cells. To determine a transcription factor
involved in downregulation of GLUT2 gene transcription, we analyzed GLUT2 promoter activities by luciferase assay using a series of GLUT2
promoter reporter plasmids. Our results suggested that hepatocyte nuclear factor (HNF)-1α is important for HCV-induced suppression of
GLUT2 gene transcription. We examined the levels of HNF-1α mRNA in HCV RNA replicon cells and in HCV J6/JFH1-infected cells by RT-qPCR.
HNF-1α mRNA levels were decreased in both cells, although HNF-1β mRNA levels were unchanged. Moreover, HCV infection dramatically
decreased the HNF-1α protein levels. Treatment of HCV-infected cells with IFN-α recovered the HNF-1α protein levels, indicating that HCV
specifically induced down-regulation of HNF-1α protein. To determine whether the HNF-1α protein is degraded through the proteasome
pathway or the lysosomal pathway, we assessed the effects of proteasome inhibitor and lysosomal inhibitor on the HNF-1α protein levels.
Treatment of the HCV-infected cells with lysosomal inhibitor, but not with proteasome inhibitor, increased HNF-1α protein levels, suggesting
that HCV infection promotes lysosomal degradation of HNF-1α protein. Taken together, our data suggest that HCV infection suppresses
GLUT2 gene expression by downregulation of HNF-1α expression.
Contact: Chieko Matsui
Kobe University Graduate School of Medicine, Kobe, Japan
[email protected]
P6.06
Yoshi-Hiro Ide (1), Tatsuya Maebo (1), Chunying An (2), Dapeng Jiang (1), Lin Deng (1), Ikuo Shoji (1), Hak Hotta (1)
(1) Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan
(2) Department of Oral Anatomy, Osaka Dental University, Japan
Hepatitis C virus (HCV) non-structural protein 3 (NS3) has emerged as an important target for new therapeutic agents, since it has a serine
protease domain and an RNA helicase domain, both of which are important for viral replication. We have previously reported that NS3
interacts with p53 and inhibits its tumor suppressor functions, and that N-terminal region of NS3 has anti-apoptotic activity. Correlation
between secondary structure of NS3 and development of hepatocellular carcinoma (HCC) was also reported in our epidemiological study.
To clarify the tumorigenic roles of NS3 in vivo, a transgenic (Tg) mouse model was established. Full length or protease domain of NS3 gene
derived from HCV genotype 1b were inserted into the pBEP vector under the control of HBx promoter. The purified vectors were injected in
oocytes of C57BL/6N mouse. Insertion of NS3 transgene was detected by PCR, and Tg mouse lines were established. Expression level of NS3
was analyzed by real-time PCR. Two Tg mouse lines expressing NS3 were successfully generated. The L189 line has the full length, and the
L218 line has a protease domain. NS3 mRNA expression was detected in both Tg mouse lines. After the age of 20 months, a solitary hepatic
tumor was developed in 12.2% of L189 (5/41, p<0.05) and 17.6% of L218 (3/17, p<0.01), whereas no hepatic tumor was seen in normal
control mice (0/37). Histopathological analysis revealed that all these tumors were well to moderately differentiated HCC without apparent
inflammation. These results suggest that HCV NS3 itself has specific carcinogenic activities in the liver. This NS3 Tg mouse model provides a
useful tool to investigate the molecular events for hepatocarcinogenesis.
Contact: Yoshi-Hiro Ide
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan
[email protected]
POSTERS
Development of hepatocellular carcinoma in NS3 transgenic mice
POSTERS
P6.07
Network generation based on quantitative proteomic data reveals cellular factors
involved in the HCV-induced inhibition of VLDL production
Carmine Mancone (1), Claudia Montaldo (1), Laura Santangelo (2), Cristina Di Giacomo (1), Marco Vignetti (2), Laura Amicone (2),
Leopoldo Paolo Pucillo (1), Tonino Alonzi (1), Marco Tripodi (2)
(1) INMI L. Spallanzani IRCCS, Rome, Italy
(2) Inst. Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy
Hypobetalipoproteinemia (HBL) is a HCV-associated pathological feature characterized by the reduced secretion of hepatic fat trough the
very low-density lipoprotein (VLDL) production. This represents a risk factor for the onset of hepatic steatosis and for the progression of
disease to cirrhosis. HBL is mainly caused by the impaired secretion of apolipoprotein B-100 (apoB-100), the main constituent of VLDL.
However, beside a correlation between the sole expression of HCV nonstructural (NS) proteins and the inhibition of apoB-100 secretion has
been described, the molecular mechanism of HCV-induced HBL is yet unknown. Our study aims to address this issue.
To investigate on the entire multistep process spanning from apoB-100 synthesis to VLDL secretion, we isolated apoB-100 containing subcellular fractions of both the RepBlast cell line, which express the sub-genome HCV-1b replicon, and the control counterpart repliconfree cell line (Huh7). A quantitative SILAC-based proteomic strategy allowed to unveil differences in the apoB-100 containing sub-cellular
fractions induced by HCV NS proteins. Twenty-three proteins, whose expression was significantly affected in RepBlast cells, were identified
by nanoHPLC and MALDI-TOF/TOF mass spectrometry approach. Among them, many are involved in pathways or biological functions
perturbed during HCV infection including lipid metabolism, cytoskeleton remodeling, protein synthesis and folding. Our proteomic data were
analyzed by protein-protein interaction software to gain insight on a possible functional significance for VLDL production: three proteins are
currently investigated as cellular determinants for the HCV-induced inhibition of VLDL production. The results obtained will be presented.
Contact: Carmine Mancone
INMI L. Spallanzani IRCCS, Rome, Italy
[email protected]
P6.08
Evidence for protective role of HCV-specific T cell transforming growth factor beta in
liver pathogenesis
POSTERS
Li Shaoyong (1), Imad Nasser (1), Nezam Afdhal (1), Margaret Koziel (1), Yury Popov (1), Detlef Schuppan (1), Mark A. Exley (1), Nadia
Alatrakchi (1)
(1) BIDMC and Harvard Medical School, Boston, MA, USA
HCV-specific immune effector responses can cause liver tissue damage in the setting of chronic infection. We previously identified TGF-beta,
produced by HCV-specific CD8+ T cells, as key regulatory cytokine involved in potentially controlling HCV-specific effector responses. Here
we investigated, in both liver and periphery, the role of HCV-specific TGF-beta in liver disease pathogenesis.
Methods: Cross sectional study of 2 well-defined groups of HCV-infected subjects: 13 with slow liver fibrosis progression (‘SP’)(< 0.1 Metavir/
year) and 6 with rapid progression (‘RP’). HCV-specific T cell responses were studied in peripheral blood (PBMC) and matched expanded
intra-hepatic lymphocytes (IHL) using IFN-gamma ELISpot ± blocking regulatory cyokines IL-10 and TGF-beta mAbs, along with multiplex and
conventional ELISA (for PBMC). Effects of IHL products of the response to HCV on human stellate cells (HSC) were studied by measuring HSC
expression of pro-fibrotic and anti-fibrotic genes.
Results: In both periphery (Wilcoxon; p=0.003) and liver (p=0.01), blocking regulatory cytokines significantly raised detection of HCV-specific
effector (IFN-gamma) responses only in SP group. HCV-specific IFN-gamma response, revealed upon regulatory cytokine blockade, strongly
correlated with HCV-specific TGF-beta measured without blockade (Spearman; R=0.70, p=0.005), but not with IL-10. HCV-specific TGF-beta,
not IL-10, inversely correlated with liver inflammation grade (R=-0.63, p=0.008) and fibrosis stage (R=-0.46, p=0.05). With SP only, with
whom blocking TGF-beta increased IFN-gamma response, HCV-stimulated IHL supernatants had an antifibrotic effect on HSC and prior
treatment of supernatants with TGF-beta mAbs abrogated this effect.
Conclusion: While TGF-beta can be pro-fibrotic, its role in HCV is contradictory. Our results support a protective role for selectively HCVspecific T cell TGF-beta, in ameliorating HCV liver disease progression.
Contact: Nadia Alatrakchi
BIDMC and Harvard Medical School, Boston, MA, USA
[email protected]
190 : HCV 2011
PATHOGENESIS
P6.09
Effect of hepatitis C virus (HCV) on activation of the Human Immunodeficiency Virus
(HIV) Long Terminal Repeat (LTR)
Satarupa Sengupta (1), Christina Martin (1), Ling Kong (1), Jason T. Blackard (1)
(1) University of Cincinnati College of Medicine, Cincinnati, OH, USA
Background and Purpose: HCV co-infection is common in HIV seropositive persons in the US. While a significant body of research suggests
an adverse effect of HIV on HCV, the impact of HCV on HIV infection is less clear. We evaluated the effects of the HCV proteins and infectious
virus on basal and Tat-induced activation of the HIV long terminal repeat (LTR).
Methods: Hepatocyte-derived cell lines were transfected with HCV Core, NS3/4A or infected with HCV and then transfected with a
luciferase construct containing the HIV LTR, in the presence or absence of the HIV-1 Tat protein. Luminescence was quantified as a
measurement of LTR activity.
Results: HIV LTR activation in hepatocytes was higher in the presence of Tat compared to basal expression levels. A Tat-activated subtype-C
LTR showed increased levels of expression compared to subtype-B which has only two NFkB sites. In the presence of Tat, HCV Core protein
partially suppressed LTR activation, while NS3/4A up-regulated LTR activation. Huh7.5 cells showed higher levels of LTR activation compared
to HepG2 cells. HCV infected hepatocytes had higher levels of Tat-induced HIV LTR activation in a dose- and time-dependent manner.
Conclusions: Recent data suggest that HIV can result in low-level infection of hepatocytes. Consistent with those findings, we found that
the HIV LTR was activated in hepatocytes in the presence of Tat. Moreover, the HCV core and NS3/4A proteins, as well as infectious HCV,
were able to alter tat-induced LTR activation. These studies may provide additional insight into the mechanisms by which HCV impacts HIV
replication and disease progression.
Contact: Jason T. Blackard
University of Cincinnati College of Medicine, Cincinnati, OH, USA
[email protected]
P6.10
Takeya Tsutsumi (1), Seiko Shinzawa (1), Koji Goto (1), Hidetaka Fujinaga (1), Hideyuki Miyoshi (1), Yoshizumi Shintani (1), Kyoji Moriya (1),
Hiroshi Yotsuyanagi (1), Tetsuro Suzuki (2), Yoshiharu Matsuura (3), Kazuhiko Koike (1)
(1) University of Tokyo, Tokyo, Japan
(3) Osaka University, Japan
Nonstructural (NS) 5A protein of hepatitis C virus (HCV) is shown to be associated with not only viral replication but also host immune
response and IFN sensitivity. We previously reported that NS5A protein interrupts toll-like receptor (TLR) signaling and represses cytokine
production in macrophage cell lines. Here, we established transgenic mouse lines expressing NS5A protein in the liver and explored
whether or not NS5A protein behaves similarly in vivo. NS5A protein in the liver was detected by western blotting although its expression
level was very low. Intrahepatic NS5A mRNA expression levels determined by real-time PCR were similar between transgenic mice and
HCV-infected patients. There were no significant histopathologic changes in the liver of transgenic mice until the age of 18 months. To
assess the immune response, we injected lipopolysaccaride (LPS) (1 µg/g body weight) intraperitoneally, and sacrificed mice after 6 hours.
Intrahepatic expression levels of cytokines such as IL-6 and IFN-γ were increased by LPS injection but there was a significant suppression of
the cytokine induction in transgenic mice compared with nontransgenic mice. Phosphorylation and activation of STAT3 and expression levels
of IFN-inducible genes, PKR and 2-5OAS, were also suppressed in transgenic mice. Phosphorylation of IRF7 and ERK, which comprise an
intracellular TLR signaling pathway, was similarly suppressed. In addition, the interaction of TRAF6 with IRAK1, molecules just downstream
of MyD88 in the TLR signaling, was suppressed in transgenic mice, suggesting that NS5A protein inhibits the TLR signaling at the level of
TLR-MyD88-IRAKs interaction. These results suggest that HCV NS5A protein interferes with innate immune response and impairs cytokine
production in vivo, which may contribute to persistent infection of HCV.
Contact: Takeya Tsutsumi
University of Tokyo, Tokyo, Japan
[email protected]
POSTERS
Hepatitis C virus NS5A protein impairs cytokine production through toll-like receptor
signaling pathway in mouse liver
POSTERS
P6.11
Expression of the newly-discovered HCV minicore proteins is regulated by viral
mutations that predict treatment failure and liver cancer development
Francis Eng (1), Andrea Branch (1)
Background: Mutations in codons 70 and 91 of the core gene are determinants of peg-IFN/RBV treatment failure and liver cancer
development. These mutations are valuable tools for investigating the molecular basis of IFN resistance and carcinogenesis. Intriguingly, the
newly-discovered 70 and 91 minicore proteins have N-termini at codons 70 and 91 (Eng et al, J Virol, 2009).
Aims: To determine whether mutations in codons 70 and 91 alter expression of minicore proteins; to explore minicore synthesis, and to
determine if the mutations confer IFN resistance to HCV in culture.
Methods: Infectious Con1/JFH chimeric viruses carrying wild-type or disease-associated mutations at codons 70 and 91 were constructed
and transfected into Huh-7.5 cells. In addition, mutations at codons 70 and 91 were introduced into FLAG-tagged core genes and expressed
from plasmids in 293T cells.
Results: The disease-associated R70N mutation produced less 70 minicore than wild-type when expressed from either infectious virus or
from core genes expressed in 293T cells. Interestingly, the R70N mutation in virus enhanced expression of a larger minicore protein that
may be a 70 minicore precursor. In 293T cells, core genes with the L91M mutation produced more 91 minicore than wild-type. 70 and 91
minicore proteins were produced independent of full-length p21 core protein. Viruses with disease-associated mutations in codons 70 and
91 did not have enhanced IFN resistance in Huh-7.5 hepatoma cells, as shown by real-time PCR.
Conclusions: The molecular basis of IFN resistance and increased liver cancer risk associated with mutations in codons 70 and 91 may be
due to alterations in viral RNA translation and minicore protein expression. Huh-7.5 cells may not be the optimal system to examine the
mechanism of IFN resistance mediated by minicores. Minicore proteins may be intra- and extra-cellular virulence factors promoting IFN
resistance, persistence, and increased risk of liver cancer (NIH:DK090317;DA031097;CA152514).
Contact: Francis Eng
[email protected]
P6.12
Hepatitis C viral protein NS5A induces EMT and participates in oncogenic
transformation of primary hepatocyte precursors
POSTERS
Damien Gregoire (1), Leila Akkari (1), Nicolas Floc’h (1), Yannick Simonin (1), Patrice Lassus (1), Urszula Hibner (1)
(1) CNRS IGMM Montpellier, Montpellier, France
Chronic hepatitis C is associated with strong risk of hepatocellular carcinoma. In addition to profound alterations of the liver microenvironment
caused by the long-term chronic infection, direct effects of the viral proteins on hepatocyte physiology are likely to participate in the
initiation of tumorigenesis.
We found that the HCV nonstructural protein NS5A gives rise to alterations of epithelial polarity of primary hepatic precursors and of
immortalized hepatocyte cell lines. Modifications of morphology are associated with the loss or displacement of epithelial cell markers, the
acquisition of mesenchymal ones and increased cell motility and invasiveness. Overall, NS5A expressing cells show features of epithelialto-mesenchymal transition (EMT). These virus-driven effects are cooperative with those of TGFβ, a well-known inducer of EMT. Molecular
characterization of signal transduction pathways initiated by the two stimuli revealed that they activate distinct transcriptional EMT
regulators, providing a molecular explanation of their cooperative effects.
We show that NS5A cooperates with activated Ras in oncogenic transformation of primary hepatocyte precursors, both in vitro and in vivo.
Our results suggest that the EMT triggered by the viral protein forms the basis of this oncogenic activity.
It would thus appear that in the context of chronic HCV infection, direct effects of NS5A on hepatocyte polarity and function participate
in the process of carcinogenesis. In addition to the well-described effect of EMT on tumor progression, our data suggest that virus-linked
loss of epithelial polarity participates in the formation of preneoplastic lesions. In conjunction with further oncogenic events, and in the
presence of inflammation-linked extrinsic signalling, it impacts on the acquisition of motile and invasive tumour phenotype.
Contact: Damien Gregoire
CNRS IGMM Montpellier, Montpellier, France
[email protected]
192 : HCV 2011
PATHOGENESIS
P6.13
Detection of hepatitis C virus antigens in liver biopsies of patients with hepatitis C
recurrence after liver transplantation
Laura Mensa (1), Sofía Pérez-del-Pulgar (1), Gonzalo Crespo (1), Rosa Miquel (1), Xavier Forns (1)
(1) IDIBAPS, Hospital Clínic, CIBERehd, Barcelona, Spain
Background and aim: Hepatitis C virus (HCV) infection recurs universally after LT. The detection of native HCV antigens may be relevant
not only for the understanding of the pathogenesis HCV infection, but also for the diagnosis and early treatment of HCV infection. For this
reason, the aim of our study was to detect HCV antigens in liver biopsies of patients with HCV infection after LT.
Methods: We selected 9 LT recipients with hepatitis C recurrence that had experienced an HCV-RNA peak higher than 7 Log IU/mL early after
LT (range 1 to 6 months; viral load range 7.7 to 9.0 Log IU/mL). Core and NS5A protein antigens were detected in formalin-fixed paraffinembedded (FFPE) liver biopsies obtained during reperfusion of the liver graft (negative control) and at the time of peak viral load using a
highly sensitive immunohistochemical detection system. In addition, immunofluorescence images were acquired by confocal microscopy
and a detailed colocalization study was performed using the Imaris software.
Results: Liver biopsies obtained at the peak of viremia after LT were all positive for both core and NS5A protein antigens. No staining of core
or NS5A was detected in reperfusion biopsies. Single infected hepatocytes were found throughout the liver section, with tendency to localize
in the periportal areas. The immunostaining was mainly hepatocellular with a granular cytoplasmatic pattern, showing a wide spectrum of
intensity. In some hepatocytes an intense ring-like pattern mainly located at the sinusoidal pole of the cells was observed. Colocalization
analyses strongly suggested the specificity of our staining, with strong colocalization (>80%) of both viral antigens in hepatocyte cytoplasm.
Conclusions: Viral antigens can be readily detected in FFPE liver biopsies by immunohistochemistry. The distribution of single infected
hepatocytes disseminated over the liver tissue is in agreement with the histological evidence of acute hepatitis C in these patients.
Contact: Laura Mensa
IDIBAPS, Hospital Clínic, CIBERehd, Barcelona, Spain
[email protected]
P6.14
Yutaka Amako (1), Mark Dallas (2), Aleem Siddiqui (3), Chris Peers (2), Mark Harris (1)
(1) Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom
(2) Faculty of Medicine and Health, University of Leeds, United Kingdom
(3) Department of Medicine, Division of Infectious Diseases, UCSD, USA
The course of HCV infection and disease progression is now known to involve metabolic alteration of lipid biogenesis and homeostasis
within the host cell. Since HCV infection targets the liver, the most important organ for lipid transportation and storage, an understanding
of why the viral life cycle is so tightly associated with lipid biogenesis and transport will be key in understanding the mechanism of
viral pathogenesis including steatosis and steatohepatitis. Lipid accumulation in the liver can be caused by either up-regulation of lipid
biosynthesis or down-regulation of lipid β-oxidation. Although many studies have focused on up-regulation of lipogenesis, little is known
about the effects of HCV on mitochondrial lipid β-oxidation. In this study, we found that NS5A interacted with both the α and β-subunits
of mitochondrial trifunctional protein (MTP), a complex which possesses hydroxyl CoA dehydrogenase, 3-ketoacyl CoA thiolase and enoyl
CoA hydratase enzymatic activities, and catalyses the last 3 steps of mitochondrial lipid β-oxidation. Through these interactions, NS5A
interferes with MTP trafficking to mitochondria, resulting in severe down-regulation of their expression and functions in mitochondria. The
presentation will discuss the molecular mechanisms of HCV perturbation of lipid β-oxidation and possible contribution to virulence by this
virus/host interaction.
Contact: Yutaka Amako
Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom
[email protected]
POSTERS
HCV infection suppresses mitochondrial tri-functional protein transport to impair
lipid β-oxidation
POSTERS
P6.15
HCV suppresses a pro-apoptotic K+ current by Kv2.1 channel protein: a possible
mechanism of viral interference with stress-response signalling kinases
Yutaka Amako (1), Jamel Mankouri (1), Zsofia Igloi (1), Kalle Saksela (2), Arunas Kaslauskas (2), Mark Dallas (3), Chris Peers (3), Mark Harris (1)
(1) IMCB, University of Leeds, Leeds, United Kingdom
(2) Haartman Institute, Department of Virology, University of Helsinki, Finland
(3) Faculty of Medicine and Health, University of Leeds, United Kingdom
HCV infection triggers a number of stress responses within the host cell including oxidative stress, with the potential for induction of a proapoptotic state. Previously we showed that HCV attenuated p38/MAPK activity in a manner that was dependent on a proline rich motif
(PP2.2) in NS5A. This resulted in impaired p38/MAPK activity in turn reducing Kv2.1 phosphorylation on serine 800 and channel activation.
To identify potential effectors of this PP2.2 mediated response, we screened a phage-display library expressing the entire complement of
human SH3 domains – this analysis identified mixed lineage kinase 3 (MLK3), a known upstream effecter of p38/MAPK. The interaction
between NS5A and MLK3 was verified by an immunoprecipitation / Western blotting study. The role of MLK3 in modulation of Kv2.1 activity
by NS5A was investigated by whole cell patch clamp analysis of HEK293 cells expressing Kv2.1 from a tetracycline regulated promoter.
Contact: Yutaka Amako
IMCB, University of Leeds, Leeds, United Kingdom
[email protected]
P6.16
During chronic HCV infection NK cells are increased in Huh 7.5 directed cytolytic
activity though are decreased in HCV infected target specificity
Qinglai Meng (1), Donald Anthony (1)
(1) Case Western Reserve University, Cleveland, OH, USA
POSTERS
NK cells have been implicated in contributing to resolution of acute HCV infection as well as the pathogenesis of chronic HCV infection.
Recent data indicate that K562 directed NK cytolytic activity of NK cells from chronic HCV infected subjects is comparable or greater than
that of NK cells from healthy controls. Whether this cytolytic function is extended to HCV infected target cells has been unclear. Here, HCV
(JFH-1) infected or uninfected hepatoma cells (Huh7.5) were used as target cells, and cytolytic activity to each target was analyzed using
negatively selected peripheral blood healthy control and untreated chronic HCV infected subject NK cells treated with media or IFN-alpha.
HCV subject media treated NK cell cytolytic activity directed at HCV+ and HCV- targets was greater than that of healthy control samples (P =
0.017 and 0.0038, respectively). However, the ratio of activity targeting HCV+ to HCV- targets was significantly lower in chronic HCV infection
compared to controls (P < 0.0001). IFN-alpha treatment of NK cells didn’t improve selectivity of HCV infected target killing in either HCV
infected or control groups. HCV subject CD16+CD56+ NK cell NKp46 expression correlated with cytolytic activity directed at HCV- targets
but not HCV infected targets. In addition, antibody blockade assays showed that in chronic HCV infection, NKp46, NKp30 and TRAIL directly
contributed to NK mediated killing, and NKp46 blockade most effectively enhanced selectivity of cytolytic activity towards HCV infected
targets. These data indicate that NK cells from chronic HCV infection had increased overall Huh 7.5 directed cytolytic activity, but decreased
specificity of activity directed towards HCV infected Huh cells. This implies NK cells mediated cellkilling not only increases in quantity but
also changes in quality during chronic HCV infection.
Contact: Qinglai Meng
Case Western Reserve University, Cleveland , OH, USA
[email protected]
194 : HCV 2011
PATHOGENESIS
P6.17
HCV-induced changes in gene expression in non-infected ‘bystander’ Huh-7 cells
Kate Muller (1), Nicholas Eyre (1), Michael Beard (1)
(1) University of Adelaide/SA Pathology, Adelaide, Australia
The mechanisms underlying the progression to fibrosis and hence advanced liver disease in HCV infection are complex. It is recognized that
not every hepatocyte within the HCV-infected liver is infected by virus, however, the effect of HCV infection on non-infected ‘bystander’ cells
is poorly understood. Nevertheless, damage to non-infected hepatocytes is postulated to expand the tissue injury and thus contribute to
fibrosis. Furthermore, non-infected ‘bystander’ cells may be relatively resistant to infection as result of the paracrine action of interferon. To
examine the bystander effect exerted on non-infected hepatocytes by HCV-infected hepatocytes, we generated a Huh-7 cell line that displays
stable shRNA knockdown of the entry receptor CD81. This CD81 knockdown cell line, which is GFP-positive and resistant to HCVcc infection,
was co-cultured with GFP-negative HCVcc (Jc1) infected hepatocytes. The same cells were co-cultured in an HCV-negative system as a
control. The cells were then separated on the basis of GFP positivity using FACS. Microarray analysis (Affymetrix GeneChip) was performed
on RNA extracted from these ‘bystander’ cells. Similar experiments using GFP-positive Huh-7 cells that were co-cultured with a replicon
cell line were performed to assess changes in their gene expression in the absence of extracellular virus. Transcriptome analysis is currently
underway with preliminary analysis revealing that non-infected bystander cells display a unique expression profile compared to controls.
These studies will reveal the changes in hepatocyte gene expression that are not exclusively the direct result of active HCV replication and
will inform us about the pathogenesis of HCV related liver disease.
Contact: Kate Muller
University of Adelaide/SA Pathology, Adelaide, Australia
[email protected]
P6.18
Hepatitis C virus core and NS5A proteins modulate expression of ODC and SSAT thus
affecting polyamine metabolism
Hepatitis C virus belongs to the wide-spread viruses which cause chronic and dangerous human diseases. HCV infection is often accompanied
by liver damages and metabolic disorders including fibrosis, steatosis, cancer and iron overload. Treatment of chronic hepatitis C is based
on ineffective combination of interferon alfa and ribavirin, with two HCV protease inhibitors to enter the market this year. So, a study of the
virus-associated liver and metabolic diseases and search for alternative treatment strategies remains an important goal for modern virology.
Natural polyamines spermine and spermidine play a crucial role in various cellular processes including replication, transcription, and
protection against oxidative stress. Our goal was to investigate the effect of HCV proteins on polyamine metabolism. We showed that
expression of Core and NS5A proteins results in up-regulation of transcription of ornithine decarboxylase (ODC) and spermidine/spermine-N1acetyltranferase (SSAT) genes which catalyze the rate-limiting steps of polyamine biosynthesis and degradation, respectively. This activation
was a result of HCV-induced oxidative stress. In case of ODC, transcription up-regulation was mediated solely by Nrf2 transcription factor,
the key regulator of the antioxidant defense system. Activation of SSAT gene transcription was achieved via Nrf2 and NF-kB transcription
factors. According to the preliminary results, activation of ODC and SSAT gene transcription was not accompanied by an increase in the
enzymes’ activity. In contrast, Core- and NS5A-expressing cells exhibited significantly lower ODC and SSAT activity and decreased polyamine
levels. Such discrepancy could be attributed to enhanced degradation of both enzymes, previously described for ODC. Role of polyamines
in HCV replication, and potential of their synthetic analogues as antiviral agents is currently elucidated.
Acknowledgements: The work was supported by following grants: Russian Foundation for Basic Research grants 10-04-00047 and 10-0401402a, the President of Russian Federation МК-5035.2011.4, Swedish Research Council K2009-66X-21053-01-3 and Swedish Institute New
Visby program 00747/2010.
Contact: Olga Smirnova
Engelhardt Institute of Molecular Biology Russian Academy of Sciences, Moscow, Russia
[email protected]
POSTERS
Olga Smirnova (1), Maria Isaguliants (2), Olga Ivanova (1), Alexey Khomutov (1), Sergey Kochetkov (1), Alexander Ivanov (1)
(1) Engelhardt Institute of Molecular Biology Russian Academy of Sciences, Moscow, Russia
(2) Karolinska Institutet, Stockholm, Sweden; Institute of Virology, Moscow, Russia
POSTERS
P6.19
Hepatitis C virus replication induces expression of vascular endothelial growth factor
and accelerates vascularisation of human hepatoma
Eileen Winkler (1), Hendryk Aurich (1), Rene Geissler (1), Alexander Mensch (1), Nadine Stöhr (1), Susann Friedrich (1), Stefan Hüttelmaier
(1), Sven-Erik Behrens (1)
(1) Martin-Luther-University Halle-Wittenberg, 06120 Halle/Saale, Germany
Hepatitis C virus (HCV) induced hepatocellular carcinoma (HCC) coincides with hepatic angiogenesis; however, the underlying mechanisms
are uncertain. In this study we examined the expression of the angiogenic vascular endothelial growth factor (VEGF) in human hepatoma
(Huh-7) cells that were stressed or transfected with different viral RNAs. Interestingly, solely cells that contained replicating HCV RNA
revealed a significantly increased expression level of VEGF as well as a lowered turnover of the VEGF mRNA. Correspondingly, these cells
secreted increased quantities of VEGF into the culture medium with respect to control cells. To correlate these observations with tumordevelopment, we established a mouse Xenograft model to monitor the vascularisation of human hepatoma. Xenotumors that derived from
HCV RNA-transfected Huh-7 cells revealed a considerably higher degree of neovascularisation versus control-tumors. Further analysis of the
molecular mechanism of VEGF mRNA stabilization in HCV containing cells indicated an essential role of a single RNA-binding protein. Thus,
HCV mediated hepatic angiogenesis relies on transcriptional as well as post-transcriptional effects.
Contact: Eileen Winkler
Martin-Luther-University Halle-Wittenberg, 06120 Halle/Saale, Germany
[email protected]
P6.20
Supplementation with branched-chain amino acids reduces hepatic iron accumulation
induced by hepatitis C virus proteins and iron overload in mice
Masaaki Korenaga (1), Keiko Korenaga (1), Shiho Tanaka (1), Sohji Nishina (1), Yasuyuki Tomiyama (1), Ryohei Ugaji (1), Noriko Dohi (1),
Yamato Tada (1), Kyo Sasaki (1), Yoshiharu Nakashima (1), Tomoya Kawase (1), Naoko Yoshioka (1), Yuhichi Hara (1), Kouji Yoshida (1),
Keisuke Hino (1)
(1) Kawasaki Medical College, Kurashiki, Japan
POSTERS
Background: Obesity and related metabolic abnormalities, including insulin resistance (IR) and iron metabolic disorder, are risk factors for
hepatocellular carcinoma in patients with hepatitis C virus (HCV). Branched-chain amino acids (BCAA)have been reported to inhibit the
incidence of hepatocellular carcinoma(HCC)in obese patients with liver cirrhosis. But its underlying mechanisms remain elusive. We found
that oxidative stress and hepcidin disorder play a critical role in the development of HCC in iron overloaded male transgenic mice expressing
HCV polyprotein(TgM). The aim of this study was to investigate whether the BCAA supplementation improves hepatic iron accumulation
and oxidative stress. Methods: Male TgM aged 2 months were divided into two groups. These were fed an excess-iron diet (carbonyl iron
225 mg/kg diet) with containing 3.0% of BCAA or casein, which served as a nitrogen content-matched control of BCAA. Serum levels of
ALT, glucose, insulin, hepcidin25, hydroperoxide measured by the derivatives of reactive oxygen metabolites(dROM) and reduced iron
measured by the biochemical antioxidant potential(BAP), and hepatic steatosis, triglyceride(Tg) and iron content were assessed at 6 months
after initiation of BCAA supplementation. Results: There were no differences in the ratio of liver to body weight, hepatic steatosis and
Tg, and insulin levels in serum between BCAA group and control group. Hepatic iron content were significantly lower in TgM treated with
BCAA than in control(218±15vs338±42μg/wet g). The hepcidin25 levels in serum were significantly higher in BCAA group than in control
group (348±27vs241±35). The ratio of BAP to dROM in the serum, for a marker of antioxidant status was significantly higher in BCAA group
than in control (27.6±2.1vs23.4±1.1). The levels of ALT and FBS tended to be lower in BCAA group. Conclusions BCAA supplementation
improves iron metabolism and oxidative stress in iron-overloaded TgM, suggesting a potential mechanism underlying the inhibitory effect
on hepatocarcinogenesis in HCV infected cirrhotic patients.
Contact: Masaaki Korenaga
Kawasaki Medical College, Kurashiki, Japan
[email protected]
196 : HCV 2011
PATHOGENESIS
P6.21
PARV4 infection is associated with intravenous drug use, HCV and HIV disease progression
Ruth Simmons (1), Colin Sharp (2), Patrick Mcclure (3), Janine Rohrbach (4), Helen Kovari (5), Will Irving (3), Andri Rauch (4),
Peter Simmonds (2), Paul Bowness (6), Paul Klenerman (1)
(1) University of Oxford, Nuffield Department of Medicine, Oxford, United Kingdom
(2) University of Edinburgh, Centre of Infectious Diseases, United Kingdom
(3) School of Molecular Medical Sciences, NIHR BRU, Univ. of Nottingham, United Kingdom
(4) University Clinic of Infectious Diseases, University Hospital Bern, Switzerland
(5) Division of ID and Hospital Epidemiology, University Hospital of Zurich, Switzerland
(6) WIMM and NIHR BRU, John Radcliffe Hospital, Oxford, United Kingdom
Background: Novel virus PARV4 is predominantly found in HCV- or HIV-infected individuals in Western countries. The pathogenic potential of
PARV4 is unknown. We present a study of PARV4 serostatus in several cohorts exposed to blood-borne viruses and their progression of HCVor HIV-related illness. Methods: Three cohorts were tested for PARV4 IgG, totalling 554 individuals. The first was a cohort of chronic HCV+
outpatients. The second, from the Nottingham Trent HCV cohort, was divided into individuals who cleared HCV versus those who became
chronically infected, and individuals who had mild liver fibrosis versus those who progressed to severe fibrosis. The third cohort, from the
Swiss HIV cohort, was made up of HIV+ HCV- men who have sex with men (MSM) and HIV-HCV coinfected IDUs. ALT, PCR status, genotype,
HCV treatment outcome, CD4 slopes and symptoms of HIV progression, were analysed in relation to PARV4 serostatus. Results: 26% of the
outpatient cohort was PARV4 IgG+ but this did not correlate with genotype, ALT or treatment outcome. An average of 31% of all groups studied
in the Nottingham Trent cohort was PARV4 seropositive, but with no significant difference observed between clinical groups (PCR+ vs PCR- or
mild vs severe liver disease). Strikingly, 95% of HIV-HCV coinfected IDUs were PARV4 seropositive versus 11% of HIV+ MSMs, however this was
unrelated to CD4 slopes or progression of disease. Overall though, there was a strong correlation between PARV4 serostatus and HIV disease
progression (p=0.02). Conclusions: PARV4 infection is common in individuals exposed to blood-borne viruses and approaches 100% in HCV-HIV
coinfected IDUs. There were no apparent correlations between PARV4 status and HCV outcome; but there was a clear association with HIV
progression, although since this was tightly linked to both HCV status and clinical group (IDU), the specific role of PARV4 is not yet clear.
Contact: Ruth Simmons
University of Oxford, Nuffield Department of Medicine, Oxford, United Kingdom
[email protected]
P6.22
Herve Lerat (3), Martin Higgs (3), Aurore Gaudin (3), Camille Cohen (3), Emilie Merour (3), Raphael Boisgard (1), Bertrand Tavitian (1),
Helene Kammoun (2), Isabelle Hainaut (2), Bertrand Blondeau (2), Fabienne Foufelle (2), Jean-Michel Pawlotsky (3)
(1) INSERM U1023, CEA, France
(2) UMRS 872, Université Paris 6, France
HCV infection is an independent risk factor of type 2 diabetes in humans. The underlying molecular mechanisms remain unknown. In this study, we
assessed whether FL-N/35 transgenic mice expressing the full HCV ORF develop the same pathology, and we analyzed the mechanisms related to
HCV-related type 2 diabetes and aberrant glucose homeostasis in this model. HCV transgenic mice displayed similar baseline glycemia, but higher
insulinemia and a higher HOMA-IR than control littermates. Intraperitoneal glucose tolerance tests (IPGTT) resulted in higher glycemia levels in
transgenic mice, demonstrating that HCV protein expression is associated with glucose intolerance. During IPGTT, the early peak of insulin secretion
observed in non-transgenic animals was absent in HCV transgenic mice, whilst the pancreatic β-cell mass and total pancreatic insulin pools were
identical in both groups, suggesting altered insulin secretion in HCV transgenic mice. At the hepatocyte level, insulin receptor substrate 1 (IRS-1)
levels were significantly lower in transgenic animals, as a result of higher SOCS-3 and PKC-theta levels. This resulted in downregulation of pyruvate
dehydrogenase kinase isozyme 1 (PKD1) phosphorylation and, ultimately, lower levels of T308-phosphorylated AKT. However, downstream events,
such as glycogen synthesis and insulin-regulated gene expression, remained unperturbed, due to AKT activation via insulin-independent pathways.
Dynamic whole-body positron emission tomography after 2-deoxy-2[18F]fluoro glucose injection showed no changes in liver glucose uptake in
HCV transgenic mice, despite higher baseline insulinemia. Finally, we observed significantly lower amounts of GLUT2, a glucose transporter-sensor,
at the plasma membrane of HCV transgenic mouse hepatocytes. In conclusion, 1) we show for the first time that, like HCV-infected humans, mice
expressing the full HCV ORF develop type 2 diabetes; 2) we demonstrate that this pathology is the result of perturbations of glucose transport and
insulin secretion as a consequence of reduced GLUT2 membrane localization in the absence of hepatic insulin resistance.
[email protected]
POSTERS
Hepatitis C virus induces type 2 diabetes through deregulation of insulin secretion and
glucose transport
POSTERS
P6.23
Hepatic miRNAs expression in chronic hepatitis C patients at early stages of liver fibrosis
Emilie Estrabaud (1), Ivan Bièche (2), Martine Lapalus (1), Simon De Muynck (1), Olivier Lada (1), Michelle Martinot-Peignoux (1), Aurelie
Duces (2), Dominique Valla (3), Pierre Bedossa (4), Patrick Marcellin (1), Michel Vidaud (5), Tarik Asselah (1)
(1) INSERM U773-CRB3 Bichat- Service d’Hépatologie Beaujon, Paris, France
(2) Université Paris Descartes, France
(3) Service d’Hépatologie, Beaujon Hospital, France
(4) Service d’Anatomie Pathologique, Beaujon Hospital, France
(5) Service de Biochimie et Génétique Moléculaire, Beaujon Hospital, France
Background and Aims: In chronic Hepatitis C (CHC), the fibrosis stage is correlated with the risk of development of cirrhosis and hepatocellular
carcinoma. Although some circulating markers can help to distinguish high from low fibrosis score, analysis of the liver biopsy remains the
only way to differentiate mild (F1) and moderate (F2) fibrosis. Micro RNAs (miRNAs) have been implicated in the regulation of many cellular
pathways. The aim of this study was to identify miRNAs differentially expressed at early stages of fibrosis F1 and F2. Methods: Liver biopsies
from 106 untreated CHC patients were studied. Male gender represents 67% of the patients, the mean age was 47.1 ± 8.9 and the BMI was
25.38± 4.1 kg/m2. Before treatment, the mean viral load was 2.97.106 copies/ml and the ALT mean was 105.1 ± 72.5. Among these patients, 34
presented a Metavir score F1, 40 F2, 16 F3 and 16 F4. Patients were mainly infected with genotype 1 and 4 (68% and 14.5%). The expression of
851 human miRNAs was assessed by RT-qPCR. Results: The miRNAs expression profiles are significantly different in patients with low (F1) and
high (F2-F4) fibrosis score. Among the 851 miRNAs, 7 miRNAs presented differential expression in moderate fibrosis (F2) in comparison with
mild fibrosis (F1). In particular, mir-675b has been validated up-regulated in F2 compared to F1; mir-675b has been described to stimulate the
expression of COL2A1. Moreover, mir-224 was gradually expressed upon fibrosis and has been previously described up-regulated in cancers.
Interestingly, mir-122 expression presents 50% reduction in HCV infected patients but is not modify upon liver fibrosis. Conclusion: Patients with
metavir score F1 and F2 present different miRNAs hepatic expression. Mir-675b was up-regulated in F2 compared to F1 in the two independent
groups of patients. Thus, miRNAs profile expression could help to identify the liver fibrosis score.
Contact: Emilie Estrabaud
INSERM U773-CRB3 Bichat- Service d’Hépatologie Beaujon, Paris, France
[email protected]
P6.24
Liver-specific targeting of viral transgenes in zebrafish: application to the study of
hepatitis C virus alternate reading frame protein
POSTERS
Amélie Pagliuzza (1), Mathieu Quesnel-Vallières (1), Zen-Kuei Chang (2), Shin-Jie Huang (2), Wangta Liu (2), Pierre Drapeau (3), Jen-Leih
Wu (2), Hugo Soudeyns (1)
(1) Unité d’Immunopathologie Virale, CHU Sainte Justine, Montreal, Canada
(2) Academia Sinica, Taipei, Taiwan
(3) Département de Pathologie et Biologie Cellulaire, University de Montréal, Canada
The nucleocapsid or core protein is thought to be responsible for the major pathogenic effects of HCV, including the development of fibrosis,
steatosis, cirrhosis and hepatocellular carcinoma. An alternate translational open reading frame exists in the core gene that allows the synthesis of
another protein termed «F protein» or «alternate reading frame protein» (ARFP), the role of which remains poorly understood. Since we cannot
exclude the presence of this protein in most studies of core biological functions, it is possible that the roles attributed to core reflect the activity of
ARFP. Our objective is to determine the biological functions of ARFP in hepatocytes and their influence on HCV-associated pathogenesis through the
study of transgenic lines of zebrafish (Danio rerio) in which the liver fatty acid binding protein (L-FABP) was used to direct liver-specific expression of
various forms of ARFP, including AUG26 (initiation at codon 26[+1]), AUG26opti (initiation at codon 26[+1], codon-optimized), AF11opti (frameshift
at codon 11, codon-optimized), and CoreMutI (core control). Founders (F0) were obtained with all constructs by microinjection of transgenic
vectors in one-cell stage embryos and screening for liver-specific GFP expression. F1 fishes were obtained by crossing F0 with wild-type, and F2
fishes were obtained by inbreeding F1 families. Germ line integration of transgenes was confirmed by PCR in all lines except those generated with
the AUG26 construct. Upon reaching maturity, the phenotype of F2 transgenic zebrafishes will be analyzed for morphological, histological and
microscopic signs of liver-associated pathology. Transgene expression will be verified by RT-PCR, western blotting and immunohistochemistry, and
compared to the results obtained with non-transgenic lines or lines expressing core protein. Results from these experiments will clarify the role of
ARFP in HCV-associated pathology, which could lead to the development of novel antiviral strategies.
Contact: Amélie Pagliuzza
Unité d’Immunopathologie Virale, CHU Sainte Justine, Montreal, Canada
[email protected]
198 : HCV 2011
PATHOGENESIS
P6.25
Hepatitis C virus enhances tumor necrosis factor-alpha induced cell death via
suppression of nuclear factor-kappaB activation
Junseong Park (1), Wonseok Kang (2), Seung-Wook Ryu (1), Woo-Il Kim (2), Dong-Yeop Chang (2), Eui-Cheol Shin (2), Chulhee Choi (1)
(1) KAIST, Department of Brain and Bioengineering, Daejeon, Republic of Korea
(2) KAIST, Graduate School of Medical Science and Engineering, Daejeon, Republic of Korea
Background & Aims: Hepatitis C virus (HCV) infection results in liver injury and long-term complications such as cirrhosis and hepatocellular
carcinoma. Liver injury in HCV infection is known to be caused by immune responses, not by viral cytopathic effects. Tumor necrosis
factor-α (TNF-α), an inflammatory cytokine, induces cell death that is prevented by nuclear factor kappaB (NF-κB) activation. In the
present study, we investigated the regulation of TNF-α signal transduction by HCV infection and identified HCV proteins responsible
for the regulation. Methods: We studied the effect of HCV infection on TNF-α signal transduction by using in vitro HCV infection model
(JFH-1, genotype 2a) of Huh-7 and Huh-7.5 cells. TNF-α-induced cell death was evaluated by WST-1 assay and annexin V staining. TNF-α
signal transduction was investigated by immunoblot and immunocytochemistry. To identify HCV proteins responsible for TNF-α signal
regulation, transfection was performed by using plasmids encoding each HCV protein. Results: TNF-α-induced cell death was significantly
enhanced in HCV-infected cells. NF-κB and c-Jun N-terminal kinase (JNK) pathways were examined as the balance between the both signal
pathways which regulate TNF-α-induced cell death. HCV infection diminished TNF-α-induced phosphorylation of IKK and IκB, which are
upstream components of NF-κB activation. HCV infection also inhibited nuclear translocation of NF-κB and subsequent expression of
NF-κB-dependent genes such as XIAP and c-FLIPL. Transfection study with plasmids encoding each HCV protein revealed that core, NS4B,
and NS5B attenuated TNF-α-induced NF-κB activation and enhanced TNF-α-induced cell death. Conclusions: HCV infection enhances
TNF-α-induced cell death via suppression of NF-κB activation, by the action of core, NS4B, and NS5B. This mechanism may contribute to
immune-mediated liver injury in HCV infection.
Contact: Wonseok Kang
KAIST, Graduate School of Medical Science and Engineering, Daejeon, Republic of Korea
[email protected]
P6.26
Srikanta Dash (1), Partha Chandra (1), Sidhartha Hazari (1), Feiza Gunduz (1), Sruti Chandra (1), Robert Garry (1), Luis Balart (1)
Background: The mechanism by which HCV induces a high rate of chronic infection and escapes the IFN-treatment is not understood.
Aim: To investigate the mechanism of chronic infection and impaired antiviral action of IFN-α in the infectious cell culture model of HCV.
Methods: Huh-7.5 cells were infected with cell culture produced virus particles for multiple cycles to remove the minute amount of plasmid
DNA carryover form the transfection. Replication of HCV in the infected Huh-7.5 cells was confirmed by the core protein expression by
Western blot analysis and increase in HCV-RNA levels by real-time RT-PCR. The complete clearance of HCV genome replication in infected
culture after IFN-α treatment was investigated. The effect of HCV replication on ER stress response of Huh-7.5 cells was measured by using
ATF6 firefly luciferase activity in a kinetic study. IFNAR1 expression, pSTAT1 and pSTAT2 levels were measured by Western blot and IFN-β
promoter activity. Results: Measuring the core protein expression and HCV-RNA levels indicated that HCV replication in the persistent
infected Huh-7.5 cells was resistant to IFN-α. The highly sensitive RT-nested PCR followed by Southern blot also confirmed the abovementioned findings. Our results show that HCV replication induces ER stress response and ATF6 promoter activity in the infected cells. We
observed that HCV infection itself down regulates the IFNAR1 expression, which prevents the phosphorylation of Stat1 and Stat2 proteins
and the IFN-β promoter activity thus creating the defective Jak-Stat signaling of the host cell. Conclusions: HCV infection down regulation
of IFNAR1 by induced ER stress leading to suppression of IFN-α signaling and impaired response to IFN-α. The work was supported by NIH
grants CA127481 and CA129776 and the Louisiana Cancer Research Consortium, New Orleans, LA.
[email protected]
POSTERS
Hepatitis C virus infection down regulates IFNAR1 through ER stress response leading
defective JAK-STAT signaling and impaired IFN-α response
POSTERS
P6.27
Molecular identification of transmitted hepatitis C viruses by single genome
amplification and sequencing
Hui Li (1), Mark Stoddard (1), Ruy Ribeiro (2), Shuyi Wang (1), Erica Parrish (1), Truman Grayson (1), Chuanxi Sun (1), Raoul Louzao (3), Paul
Goepfert (1), Thomas Denny (3), Barton Haynes (3), Alan Perelson (2), Beatrice Hahn (1), George Shaw (1)
(1) University of Alabama at Birmingham, Birmingham, AL, USA
(3) Duke Human Vaccine Institute, USA
Background: Extreme genetic diversity of HCV, even within an individual, has made molecular structure-function studies of HCV challenging.
We hypothesized that the exact nucleotide sequences of transmitted viral genomes that result in productive human infection can be inferred
from plasma viral RNA during the ramp-up and early plateau phases of acute infection, analogous to recent findings with HIV-1 (J Exp Med
206:1273, 2009). Methods: Twice weekly plasma specimens from 16 human source plasma donors who became acutely infected by HCV and
from 12 chronic controls were analyzed by single genome amplification and sequencing. 2,700 overlapping 5’ and 3’ viral half-genomes were
sequenced and evaluated by phylogenetic inference and mathematical modeling. Results: Sequences formed patient-specific monophyletic
lineages of genotypes 1a, 1b, 2b and 3a. Viral sequences from chronic subjects showed expected diversity (1 - 5%); sequences from acutely
infected subjects exhibited one or more discrete sublineages of low maximum diversity (0.04-0.19%). Sequences within each sublineage
exhibited a star-like phylogeny that coalesced to an unambiguous transmitted/founder genome. Transmitted genomes evolved at a rate of
<3x10-5/site/week. Viral recombination was not evident and immune selection was suggested in only a single subject at a single (potential)
HLA-restricted epitope. The median number of transmitted viruses was 3 (range 1-13). Conclusions: A novel molecular strategy for identifying
full-length and subgenomic transmitted HCV genomes responsible for productive clinical infection is described. This approach provided unique
molecular insights into HCV transmission and early diversification. Transmitted/founder HCV genomes, by definition, contain all sequence
elements necessary and sufficient for virus transmission and replication in vivo and may thus be uniquely suitable for molecular structurefunction studies, including analyses of virus susceptibility to antiviral drugs, immune modulators, natural immune responses, and immune
responses elicited by candidate vaccines.
Contact: Hui Li
University of Alabama at Birmingham, Birmingham, AL, USA
[email protected]
P6.28
POSTERS
Maintenance of miR-122 expression in hepatitis C virus-associated hepatocellular
carcinoma
Carolyn Spaniel (1), Tetsuro Shimakami (2), Masao Honda (2), Shuichi Kaneko (2), Stanley M. Lemon (1)
(1) University of North Carolina-Chapel Hill, Chapel Hill, NC, USA
(2) Kanazawa University, Japan
miR-122, an abundant, liver-specific microRNA, is an essential host factor for HCV replication. It promotes translation and amplification of HCV
RNA through a direct interaction with the viral 5’ UTR. In the host, miR-122 regulates numerous aspects of hepatocyte physiology, including
cholesterol metabolism and amino acid transport, through conventional miRNA interactions with the 3’UTRs of cellular mRNAs. Prior studies
suggest that miR-122 expression is often lost during development of hepatocellular carcinoma (HCC), and that miR-122 levels are typically
reduced in tumor tissue. Reconstitution of miR-122 expression in hepatoma cells lacking endogenous miR-122 also reduces the malignant
phenotype of these cells in nude mice. However, the role of miR-122 in HCV-associated HCC is uncertain, as some prior studies suggest that
miR-122 expression is maintained in tumor tissue. To determine whether miR-122 expression is lost or maintained during progression to HCC
in patients with HCV-associated cirrhosis, we used a sensitive and quantitative RT-PCR assay for miR-122 (Exiqon) to compare expression
levels in 16 HCV-associated HCC tissue samples (mostly moderate- to well-differentiated HCC) and paired, non-malignant cirrhotic tissues from
the same patients. Similar assays were run in parallel for U6 snRNA, a ubiquitously expressed spliceosome component, and let-7a, a miRNA
reduced in abundance in several cancer types. When normalized to U6 snRNA abundance, we observed an approximate 2-fold increase in miR122 abundance in tumor vs. nontumor tissue, while miR-122 levels were unchanged relative to let7-a. Thus, miR-122 expression is preserved
and possibly even increased in HCV-associated HCC, in contrast to what has been reported for HCC due to other causes. We postulate that a
requirement for continued miR-122 expression to support ongoing HCV replication results in unique oncogenic pathways that distinguish HCVassociated HCC, an hypothesis that suggests cancer arises in HCV-infected hepatocytes rather than uninfected bystander cells.
Contact: Carolyn Spaniel
University of North Carolina-Chapel Hill, Chapel Hill, NC, USA
[email protected]
200 : HCV 2011
PATHOGENESIS
P6.29
Detection of HCV-specific CD4+ T-cell proliferation and interferon-γ release in
seronegative spouses of chronically infected HCV patients
Bijan Raziorrouh (1), Dominique Tomlinson (1), Peter Kurktschiev (1), Winfried Schraut (1), Norbert Grüner (1), Reinhart Zachoval (1),
Maria-Christina Jung (2), Hans M. Nitschko (3), Martin Wächtler (4), Michael Spannagl (5), Helmut M. Diepolder (1), Axel Ulsenheimer (1)
(1) Medical Department II, University of Munich, Munich, Germany
(2) Leberzentrum, Munich, Germany
(3) Max von Pettenkofer Institute, University of Munich, Germany
(4) Medical Department I, Klinikum Schwabing, Munich, Germany
(5) Laboratory of Immunogenetics, University of Munich, Germany
Background: Despite continuous exposure, spouses of hepatitis C virus (HCV)-infected patients rarely develop apparent HCV- infection.
HCV-specific CD8+ T-cell response in HCV exposed seronegative subjects has been described as a potential correlate of antiviral protection.
Therefore we asked for the presence and the functional properties of HCV-specific CD4+ T cells in HCV seronegative spouses of chronically
infected patients. Methods: Virus-specific CD4+ T-cell response from 14 HCV seronegative spouses was characterized by CD154 activation
marker assay, CFSE proliferation assay, three MHC class II tetramers targeting epitopes from the NS3/4 region and intracellular cytokine
staining after in vitro expansion of CD4+ T-cells. Samples from the peripheral blood were stimulated with peptides or proteins from the
structural and non-structural HCV region and obtained data were compared to healthy donors (n=13), chronically infected patients (n=9)
and patients with spontaneous clearance of HCV infection(n=15). Results: After 14 days of in vitro expansion, high frequencies of MHC
class II tetramer+ CD4+ T-cells (p=0.04) as well as a significant proportion of IFN-γ producing CD4+ T-cells (p=0.008) were measurable in
HCV seronegative spouses compared to healthy donors. In addition, HCV-specific CD4+ T-cell proliferation was detected in 7 of 12 (58.3%)
spouses, while no proliferative response occurred in healthy individuals (p=0.01). Stimulation with HCV proteins from the NS3/4/5 region
resulted in significantly higher frequencies of CD154 coexpressing HCV-specific CD4+ T-cells in seronegative spouses compared to healthy
controls (p=0.004). Conclusion: We detected a virus-specific CD4+ T-cell response in HCV seronegative spouses of chronically infected
patients. Upon antigen stimulation, HCV-specific CD4+ T-cells proliferate, produce IFN-γ and upregulate CD154. As spouses of infected
patients are rarely infected with HCV despite persistent viral exposure, the antigen-specific CD4+ T-cell response we described might be
a further correlate of antiviral protection.
Contact: Peter Kurktschiev
Medical Department II, University of Munich, Munich, Germany
[email protected]
HCV infection causes cell cycle arrest at the G2/M-phase boundary
David McGivern (1), Rathi Kannan (2), Lucinda Hensley (1), Lauren Evers (2), Stanley Lemon (1)
(1) University of North Carolina, Chapel Hill, NC, USA
(2) University of Texas Medical Branch, USA
Chronic infection with hepatitis C virus (HCV) is associated with increased risk for hepatocellular carcinoma (HCC). Cancer develops typically
after several decades of infection, and while chronic immune-mediated inflammation is likely to be an important factor, HCV-specific virologic
mechanisms of carcinogenesis may also be involved. In vitro studies have revealed multiple interactions between HCV proteins and host-cell
cycle regulators and tumor suppressor proteins. Some prior studies suggest a pro-apoptotic role for these proteins while others suggest an antiapoptotic role. We set out to determine the net effect of these interactions on cell proliferation and cell cycle regulation in the context of virus
replication in cultured cells. Flow cytometric analyses using CFSE labeling indicated a slow-down in cellular proliferation in infected Huh-7.5
cells that correlated with levels of viral antigen. A decrease in the proportion of cells in G1- and S-phases with an accumulation of cells in G2/Mphase was observed in cells supporting replication of either genotype 1a (H77S) or 2a (JFH-1) RNA. However, an additional G1-phase arrest was
observed only in cells replicating JFH-1 RNA. Dramatic decreases in markers of mitosis such as phospho-histone H3 in infected cells suggested
a block to mitotic entry. In common with published literature, we observed caspase 3 activation, suggesting that cell cycle arrest is associated
with apoptosis. Important differences were observed in patterns of cell cycle disturbance and levels of apoptosis with different strains of HCV.
However, the data suggest that cell cycle arrest at the interface of G2 and mitosis is a common feature of HCV infection.
Contact: David McGivern
University of North Carolina, Chapel Hill, NC, USA
[email protected]
POSTERS
P6.30
POSTERS
P6.31
Gene expression profiling in formalin fixed paraffin embedded (FFPE) samples from
hepatitis C virus infected patients with hepatocellular carcinoma
Trina Das (1), James D. Perkins (1), Sean C. Proll (1), Jeffrey J. Delrow (2), Deborah L. Diamond (1), Michael G. Katze (1)
(1) University Of Washington, Seattle, WA, USA
(2) Fred Hutchinson Cancer Research Center, USA
Chronic infection with hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC), one of the most
common cancers worldwide and the third most common cancer mortality. We are studying hepatic gene expression signatures associated
with HCC development in HCV patients undergoing liver transplantation in order to better understand the molecular pathogenesis of
HCC and to identify potential HCC markers. In the present study we utilized the Illumina Whole Genome-DASL assay, which includes all
24,000 well-annotated RefSeq genes, for gene expression studies in formalin-fixed paraffin embedded (FFPE) tumor and non-tumor tissue.
We initially investigated the impact of sample characteristics on various RNA quality-control metrics and performance in the WG-DASL
assay. Our findings demonstrate that time from surgical explant to formalin fixation, a significant variable in our transplant cohort, did
not adversely affect gene detection or replicate reproducibility. Principal component analysis further demonstrated that segregation was
primarily based on sample source (e.g. tumor vs non-tumor). Functional analyses identified tumorigenesis as the top ranked function
represented by genes significantly differentially regulated in tumor versus non-tumor tissue. These include several genes previously linked
to hepatocellular carcinoma, pointing to the validity of FFPE-based gene expression profiling approaches to identify physiologically relevant
gene expression changes of HCV-associated hepatocarcinogenesis. We further describe de-regulation of genes implicated in numerous
other processes including for example, endothelial cell migration/angiogenesis and the trafficking/activation of immune cell populations.
Finally, we discuss these findings relative to subsequent analyses identifying differential gene expression patterns in non-tumor tissue from
HCV infected patients in the presence and absence of HCC.
Contact: Trina Das
University Of Washington, Seattle, WA, USA
[email protected]
P6.32
In vitro modeling of the HCV/HIV-1 co-infected liver environment
Brian Doehle (1), Amina Negash (1), Kristina Chang (1), Michael Gale Jr. (1)
POSTERS
Co-infection of individuals with HIV and HCV is a major global health problem, with HCV-related liver disease becoming a leading cause of
non-AIDS death within the population. The consequences of HCV infection are much more severe in HIV infected individuals, with increased
rates of liver fibrosis, an increased progression to cirrhosis and higher levels of complications. These effects have been attributed to both
direct viral effects as well as the dramatic dysregulation of the innate and adaptive immune responses during infection. Understanding
the mechanisms underlying these phenomenons is severely hampered by both the lack of appropriate model systems and the reliance
on human liver biopsies. Cross-talk between hepatocytes and the resident immune cells of the liver is a potential interface where HIV-1
infection of immune cells and HCV infection of hepatocytes may lead to exasperated dysregulation and increased pathogenesis of disease
in co-infected individuals. Recently, we have developed a number of HIV-1 specific in vitro and ex vivo model systems for the study of cell
intrinsic recognition of virus infection. We have found that HIV-1 specifically targets the transcription factor IRF3, which facilitates a global
disruption of effective recognition and signaling in response to a wide variety of pathogen associated molecular patterns (PAMPs). These
HIV-1 innate immune evasion strategies are potent in CD4+ T cells and macrophages, and potentially even dendritic cells. In conjunction
with our in vitro models for HCV inflammation and immune evasion, we have examined both immune cells and hepatocytes in HCV/HIV-1
mono and co-infections, and have demonstrated cell contact independent signaling cross-talk in response to infection. Our studies reveal
the potential to model a variety of direct viral and innate immune effects of co-infection in vitro. These studies will allow for more detailed
understanding of the dynamics of HCV/HIV-1 co-infection, and may inform future therapeutics design.
Contact: Brian Doehle
[email protected]
202 : HCV 2011
PATHOGENESIS
P6.33
Ethanol increases the morphogenesis of infectious HCV and the unfolded protein
response in primary human hepatocytes
Céline Hernandez (1), Béatrice Legrand (2), Arnaud Carpentier (1), Robert Barouki (2), Philippe Podevin (1), Hélène Rouach (2), Arielle R.
Rosenberg (1), Michèle Garlatti (3)
(1) Univeristé Paris Descartes, EA 4474, Paris, France
(2) INSERM, UMRS 747, France
(3) INSERM UMRS 872, France
Excessive alcohol consumption by HCV-infected patients increases the viral load and aggravates the liver disease, but the mechanisms
underlying this co-morbidity remain elusive. HCV replication and exposure to ethanol have both been reported to cause an endoplasmic
reticulum (ER) stress. Cells may respond to ER stress by activating the unfolded protein response (UPR), which aims at maintaining ER
homeostasis and cell survival unless it exceeds a threshold leading to cell death. Here we took advantage of a recently described model of
productive HCV infection in primary human adult hepatocytes (PHH) to assess the independent or combined effects of HCV and ethanol on
the UPR and the impact of ethanol on the production of infectious virus. PHH were inoculated with the JFH1-derived chimera Con1/C3, and
monitored during two weeks for HCV infection, expression of UPR-associated genes, and cytotoxic effects. HCV infection and exposure to
ethanol independently induced a complete, moderate UPR response that did not reach the threshold leading to cell death. Combination of
HCV and ethanol showed additive effects on the expression of genes allowing ER homeostasis recovery (XBP1, BiP, EDEM, Herp) and genes
critical for maintenance of cell survival (TRB3, ATF4). By contrast, no additive effect was seen on the expression of pro-apoptotic genes
(CHOP, ATF3), and no cytotoxic effect was observed. Addition of ethanol increased the production of infectious virus in a dose-dependent
manner. Interestingly, this effect was related to an increase in the specific infectivity of produced viral particles, but not in HCV genome
replication. Collectively, these results show that ethanol increases both the morphogenesis of infectious HCV and the UPR of hepatocytes,
suggesting that the viral load increase caused by alcohol may result from the improvement of ER functionality due to upregulation of the
adaptive program of the UPR.
POSTERS
Contact: Céline Hernandez
Univeristé Paris Descartes, EA 4474, Paris, France
[email protected]
POSTERS
POSTERS
204 : HCV 2011
POSTERS
Viral Kinetics and Drug Resistance
P7.01
Cross resistance of hepatitis C virus to ribavirin and other nucleoside analogs
Dino Feigelstock, Kathleen Mihalik, Stephen Feinstone
P7.02
Deep sequencing of hepatitis C virus mixtures allows sensitive detection of
minority species
Reinhold Pollner, Shyun Shyun Lee, Tyson Clark, Jason Chin, Jonas Korlach, Mark Reynolds, Matthew Friedenberg
P7.03
Discovery of full-length HCV genome quasispecies by deep sequencing
Tomomi Ando, Hideki Aizaki, Masaya Sugiyama, Masashi Mizokami, Tsuyoshi Sekizuka, Makoto Kuroda, Takaji Wakita
P7.04
Hepatitis C virus envelope glycoprotein signatures and resistance to
antiviral treatment
Rémy Moenne-Loccoz, Aurélie Velay, John Murray, Anne-Claire Erba, Mirjam B. Zeisel, Marine Turek, Isabel Fofana,
Samira Fafi-Kremer, François Habersetzer, Michel Doffoël, Jean-Pierre Gut, Françoise Stoll-Keller, Thomas F. Baumert,
Evelyne Schvoerer
P7.05
Analysis of interferon signaling by infectious hepatitis C virus clones with
substitutions of core amino acids 70 and 91
P7.06
Cell culture analysis of the effect of HCV drug resistance mutations on
viral fitness
Heidi Morris, Ali Atoom, Matthias Götte, Rodney Russell
P7.07
Enrichment of the NS5B polymerase variant F415Y following failure of
ribavirin containing regimens in patients with subtype 1a HCV
Ann Tigges, Doug Bartels, James Sullivan, Joshua Henshaw, Min Jiang, Eileen Zhang, Jennifer Dorrian, Ann Kwong,
Tara Kieffer
POSTERS
Naoya Sakamoto, Yusuke Funaoka, Goki Suda, Mina Nakagawa, Sei Kakinuma, Mamoru Watanabe
POSTERS
P7.08
Signature resistance mutations to directly acting antiviral agents occur at
low prevalence in treatment naïve subjects with recently acquired HCV
Silvana Gaudieri, Tanya Applegate, Monika Tschochner, Anne Plauzolles, Katja Pfafferott, Michaela Lucas, Helmut
Diepolder, Axel Ulsenheimer, Simon Mallal, Jason Grebely, Margaret Hellard, Andrew Lloyd, John Kaldor, Gregory Dore,
Gail Matthews
P7.09
Sequence and replication capacity of NS3 protease genes of individual
clones from patients treated with the HCV protease inhibitor GS-9256
Angela Worth, Kelly A. Wong, Michael D. Miller, Hongmei Mo
P7.10
Footprinting reveals simultaneous binding of NS3 to single-stranded and
double-stranded regions of nucleic acid substrates
Melody Harrison, Veronica Raney, Kim Reynolds, David Harrison, Craig Cameron, Kevin Raney
P7.11
HCV NS3 catalyzes the first step for DNA strand exchange
David Harrison, Melody Harrison, Bartek Sikora, Craig Cameron, Kevin Raney
P7.12
N236T-HBV mutants Isolated from patients with primary non response to
adefovir show a reduction of susceptibility to tenofovir in vitro
Olivier Lada, Qian Zhang, Emilie Estrabaud, Roberto Carvalho-Filho, Rami Moucari, Simon De Muynck, Marie-Pierre
Ripault, Corrine Castelnau, Michelle Martinot-Peignoux, Nathalie Boyer, Tarik Asselah, Patrick Marcellin
P7.13
HBsAg level and HBV related liver disease
Michelle Martinot-Peignoux, Roberto Carvalho-Filho, Ana Carolina Ferreira Netto Cardoso, Martine Lapalus, Olivier Lada,
Tarik Asselah, Patrick Marcellin
P7.14
Quantitative HBsAg: a new specific marker for the diagnosis of HBsAg
inactive carriage
Michelle Martinot-Peignoux, Martine Lapalus, Marie-Pierre Ripault, Nathalie Boyer, Tarik Asselah, Patrick Marcellin
POSTERS
P7.15
In vitro selection and characterization of genotype 2a J6/JFH-1 replicon
variants resistant to PSI-7977
Christine Espiritu, Shalini Bansal, Michael Otto, Phillip Furman, Angela Lam
P7.16
Modeling inhibition kinetics of HCV sg1b RNA in non-growing Huh7 cells
Harel Dahari, Naina Barretto, Natasha Sansone, Jeremie Guedj, Alan Perelson, Susan Uprichard
P7.17
Envelope mutations found after IFN-alpha treatment of HCV 1a and 3a coreNS2 JFH1-based recombinants led to increased viral fitness and IFN resistance
Stéphanie Serre, Jens Bukh, Judith Gottwein
206 : HCV 2011
P7.01
Cross resistance of hepatitis C virus to ribavirin and other nucleoside analogs
Dino Feigelstock (1), Kathleen Mihalik (1), Stephen Feinstone (1)
The standard therapy for the treatment of chronically HCV infected patients consists of a combination of pegylated interferon alpha and
ribavirin. Given the side effects associated with injections of interferon, an interferon-free regimen for the treatment of HCV infections is
highly desirable. Recent studies have shown that ribavirin in combination with other antiviral drugs, without interferon, can be efficacious,
suggesting that an interferon-free therapy could be adopted in the near future. However, generation of drug resistant mutants and cross
resistance to different drugs could impair the efficacy of the treatment. In order to study the cross-resistance of HCV to ribavirin and other
nucleoside analogs, we selected a J6/JFH1 HCV mutant resistant to ribavirin (HCV-RR) and tested its ability to grow in the presence of two other
nucleoside analogs, 2’-C-methylcytidine (previously tested in HCV clinical trials) and 5-Fluorouracil (a mutagenic agent) in Huh7D cells (a Huh7
cell derivative more permissive to HCV replication). We found that J6/JFH1 HCV-RR was more resistant than wild type J6/JFH1 to 5-Fluorouracil,
but was actually more sensitive to 2’-C-methylcytidine than wild type J6/JFH1. These results indicate that ribavirin resistant viruses could have
elevated resistance to other nucleoside analogs and highlight the importance of combination drug selection for HCV treatment.
Contact: Dino Feigelstock
[email protected]
P7.02
Deep sequencing of hepatitis C virus mixtures allows sensitive detection of minority
species
Background: Hepatitis C virus (HCV) is the causative agent of chronic and acute hepatitis in approximately 3% of the human population. The
virus exhibits extreme genetic heterogeneity and is classified into six major genotypes and more than 70 different subtypes.
HCV genotyping provides important information regarding the response to and the duration of current antiviral therapy. Promising nextgeneration antiviral drugs are currently under development, which will inevitably exert pressure on the already heterogeneous viral
population resulting in new sequence variants. Next-generation sequencing methods are ideally suited to study the viral heterogeneity
prior and after administration of those drugs.
Goal: Our objective was to develop a prototype assay for the sensitive detection and differentiation of a HCV minority species in the
presence of a HCV majority species by combining Gen-Probe’s hybridization-based Target Capture method with Pacific Biosciences’ single
molecule, real-time (SMRTTM) DNA sequencing technology.
Results: We demonstrated that the combination of Target Capture, RT-PCR, and SMRTTM sequencing allows the detection and assignment
of different HCV subtypes in clinical samples, including novel variants not listed in the HCV database. Mixtures of different subtypes were
quantitated by sequencing to allow detection of at least 1 % of a minority species in the presence of a majority species. Targeted sequencing
was extended to include the 5’-UTR, NS3, NS4 and NS5 regions.
Conclusion: Viral deep sequencing assays are becoming increasingly important in the clinical laboratory allowing a better understanding of
the quasispecies complexity and a better description of the entire mutation spectrum in response to antiviral drugs.
Contact: Reinhold Pollner
Gen-Probe, San Diego, CA, USA
[email protected]
POSTERS
Reinhold Pollner (1), Shyun Shyun Lee (1), Tyson Clark (2), Jason Chin (2), Jonas Korlach (2), Mark Reynolds (1), Matthew Friedenberg (1)
(1) Gen-Probe, San Diego, CA, USA
(2) Pacific Biosciences, USA
POSTERS
P7.03
Discovery of full-length HCV genome quasispecies by deep sequencing
Tomomi Ando (1), Hideki Aizaki (1), Masaya Sugiyama (2), Masashi Mizokami (2), Tsuyoshi Sekizuka (3), Makoto Kuroda (3), Takaji Wakita (1)
(1) Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
(2) National Center for Global Health and Medicine, Japan
(3) Center for Patho. Genom., National Institute of Infectious Diseases, Japan
Hepatitis C Virus (HCV) genome accumulates nucleotide substitutions during its persistent infection because of low viral RNA polymerase
fidelity and high viral replication ratio. This intra-host variability, i.e. “quasispecies”, has been believed to contribute for viral escape from
the selective pressure exerted by the host immune system or anti-viral drug exposure. For the virus genome analysis, we use “consensus
sequence” to represent the viral genome containing “quasispecies”. “Consensus sequence” is the combination of dominant nucleotides in
each position. However, it has been unclear if the virus having “consensus sequence” exists. Furthermore, significance of “quasispecies” in
the viral life cycle is also not clear. Here, we performed the next-generation sequencing (NGS) to detect “quasispecies” on the full-length
viral genome RNA. Combined the date sets from the illumina Genome Analyzer II, 454 GS FLX, and capillary sequencing, we determined
at least three independent sequences existed in one patient serum. The single mutation at position 70 of the core (core70) seems to be
associated with response to combination therapy of the peg-interferon and ribavirin. First sequence had core70(Arg:R) related to the
favorable response, and other two had core70(Gln:Q) associated with the null response. The numbers of nucleotide difference between the
sequence having core70(R) and the sequence having core70(Q) was 139 bp although the numbers of difference between two sequences
having core70(Q) was only 44 bp. Moreover, each sequence contained further minor variations. These findings indicate that the persistently
infected HCV genome may be composed of some independent major sequences, and each sequence has each “quasispecies”. Surprisingly,
we could not detect the viral genome with “consensus sequence”. These sequence variations in the persistently infected viral genome may
be important for the viral persistence, pathogenesis, and susceptibility.
Contact: Tomomi Ando
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
[email protected]
P7.04
Hepatitis C virus envelope glycoprotein signatures and resistance to antiviral treatment
POSTERS
Rémy Moenne-Loccoz (1), Aurélie Velay (1), John Murray (2), Anne-Claire Erba (1), Mirjam B. Zeisel (1), Marine Turek (1), Isabel Fofana (1),
Samira Fafi-Kremer (1), François Habersetzer (1), Michel Doffoël (1), Jean-Pierre Gut (1), Françoise Stoll-Keller (1), Thomas F. Baumert (1),
Evelyne Schvoerer (1)
(1) INSERM U748, Strasbourg, France
(2) UNSW, School of Mathematics and Statistics, Australia
A major challenge for antiviral treatment of hepatitis C virus (HCV) infection is viral resistance. The mechanisms of resistance are only
incompletely understood. The high variability of HCV envelope glycoproteins may contribute to resistance by selection of strains with
enhanced infectivity and/or escape from host immune responses. To evaluate this hypothesis, we investigated the association of envelope
glycoprotein-specific molecular signatures with treatment failure and characterized their functional relevance using a HCV pseudoparticle
system. HCV RNA was amplified, sequenced and cloned from 92 patients infected with genotype 1 (43 for 1a, 49 for 1b) and treated with
pegylated interferon-alfa and ribavirin. Patients were classified into 46 responders (R; 27 1b, 19 1a) and 46 non responders (NR; 22 1b,
24 1a). Envelope glycoprotein sequences, molecular signatures and minimal networks of covariant amino acid pairs were correlated with
treatment response using bioinformatics. Bioinformatics identified four NR-related molecular signatures in E1 and E2: 219A (E1; 1a), 391V
(E2-HVR1;1b), 431A (E2-HVR3;1a) and 642V (E2;1a). These positions also appeared in minimal networks separating NR from R. Functional
analyses showed that introduction of residue 431A into nonrelated HCV strains resulted (i) in a decrease in antibody-mediated HCVpp
neutralization by pre-treatment sera (mean titer 1/100 for 431A versus 1/1066 for 431D, p < 0.0001) and (ii) in a 431A-dependent increase
of HCVpp entry into Huh7.5 cells overexpressing CD81 and SR-BI (p < 0.0005). The functional analysis of additional signatures is ongoing.
Conclusions: Bioinformatics retained four envelope glycoprotein signatures associated with treatment failure. Functional analyses
demonstrate that the identified signatures result in an alteration of host cell entry factor usage with concomitant escape from neutralizing
antibodies. Taken together, these data demonstrate that virus-host interactions during viral entry may contribute to treatment failure and
provide a rationale for the development of HCV entry inhibitors as part of combination therapies.
Contact: Rémy Moenne-Loccoz
INSERM U748, Strasbourg, France
[email protected]
208 : HCV 2011
P7.05
Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions
of core amino acids 70 and 91
Naoya Sakamoto (1), Yusuke Funaoka (1), Goki Suda (1), Mina Nakagawa (1), Sei Kakinuma (1), Mamoru Watanabe (1)
(1) Tokyo Medical and Dental University, Tokyo, Japan
Substitution of amino acids 70 and 91 in HCV core region is a significant predictor of poor responses to peginterferon plus ribavirin therapy,
while their molecular mechanisms remain unclear. Here, we investigated these differences in the response to interferon-alpha (IFN) by using
HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with wild type or the mutant HCV
clones showed that the core mutants, R70Q, R70H, and L91M, were significantly more resistant than the wild type. Among HCV-transfected
cells, intracellular HCV-RNA levels were significantly higher in the core mutants as compared with the wild type, while HCV-RNA in culture
supernatant was significantly lower in these mutants than the wild type. IFN-induced phosphorylation of STAT1 and STAT2, and expression
of the interferon-inducible genes were significantly lower in the core mutants than the wild type, suggesting of cellular unresponsiveness
to IFN. Expression level of an interferon-signal attenuator, SOCS3, was significantly higher in R70Q, R70H and L91M than the wild type. IL-6,
which upregulates SOCS3, was significantly higher in R70Q, R70H and L91M than the wild type, suggesting of interferon resistance possibly
through IL-6 induced, SOCS3-mediated suppression of interferon-signaling. Expression level of ER stress proteins were significantly higher in
cells transfected with core mutant than the wild type. In conclusion, HCV core mutants of R70 and L91 were resistant to interferon in vitro
and that the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance
of HCV core mutants.
Contact: Naoya Sakamoto
Tokyo Medical and Dental University, Tokyo, Japan
[email protected]
P7.06
Cell culture analysis of the effect of HCV drug resistance mutations on viral fitness
Treatment of HCV is currently transitioning to the use of direct-acting antiviral therapies (DAAs), which specifically target viral proteins
such as the NS5B polymerase and the NS3/4A protease. However, amino acid changes that confer drug resistance against these DAAs have
already been identified in vitro and in clinical trials. Some of these mutations emerge rapidly, while others appear to be associated with a
high barrier to the development of resistance. A prominent example is the S282T signature mutation associated with nucleoside analogue
NS5B inhibitors. The goal of this study is to utilize the HCV cell culture (HCVcc) system for in vitro analysis of the effects of resistanceconferring mutations on HCV fitness. The S282T mutation was inserted into the wild-type JFH-1 strain of HCV, as well as a highly infectious
cell cultured-adapted strain of JFH-1. Novel cell-based assays will be used to assess the kinetics of the selection process and determine the
primary and secondary mutations that emerge under drug pressure. We observed that the S282T mutation, located near the active site of
NS5B, reduced the levels of infectious virus produced compared to the wild-type JFH-1. The S282T mutation also reduced virus production
by ten-fold in the context of the more infectious adapted JFH-1. However, despite this decrease in virus production, the adapted JFH-1 was
still able to replicate at significant levels. Competition assays at various ratios showed the virus containing the S282T mutation to have a 10to 100-fold lower level of fitness than the adapted virus containing wild-type NS5B. Together these data demonstrate that S282T strongly
impacts virus replication capacity; however, the possible co-emergence of secondary mutations throughout the genome may at least in part
repair such deficits.
Contact: Heidi Morris
Memorial University of Newfoundland, St. John’s, Canada
[email protected]
POSTERS
Heidi Morris (1), Ali Atoom (1), Matthias Götte (2), Rodney Russell (1)
(1) Memorial University of Newfoundland, St. John’s, Canada
(2) McGill University, Canada
POSTERS
P7.07
Enrichment of the NS5B polymerase variant F415Y following failure of ribavirin
containing regimens in patients with subtype 1a HCV
Ann Tigges (1), Doug Bartels (1), James Sullivan (1), Joshua Henshaw (1), Min Jiang (1), Eileen Zhang (1), Jennifer Dorrian (1), Ann Kwong (1),
Tara Kieffer (1)
(1) Vertex Pharmaceuticals Inc., Cambridge, MA, USA
Introduction: Data obtained from clinical studies of limited sample size has lead to the suggestion that the HCV NS5B variant F415Y may
be enriched after ribavirin (RBV) monotherapy in patients with chronic subtype 1a HCV infection. Methods: To determine if F415Y or other
changes in the NS5B polymerase are selected during treatment with RBV-containing regimens in subtype 1a patients, population sequencing
was performed on 319 PROVE3 study patients who had previously failed a Peg-IFN/RBV (PR) regimen and 231 treatment-naïve patients from
studies PROVE1 and PROVE2. Patient isolates were analyzed before, during and after receiving either PR or regimens containing telaprevir (T)
with (TPR) or without RBV (TP). Results: NS5B F415Y is rare in subtype 1a and was observed in only 0.9% of treatment-naïve patients (2 of 231)
at baseline. F415Y was observed in 32% of the subtype 1a PR-experienced patients (n=319), greatly exceeding the treatment-naïve prevalence
(p<0.0001). The F415Y variant appears quite fit as it was present at baseline in PROVE3, a median of 2.4 years after PR failure. Amongst
treatment-naïve patients (PROVE1) significant (p<0.0001) enrichment of F415Y was observed during or after a RBV-containing treatment, with
the on-treatment mutation occurring in 7 of 65 patients that failed to achieve SVR. Enrichment of F415Y occurred after a median time of 48
weeks (range: 6-60) in patients with on-going replication in the presence of RBV; 5 of these 7 patients had detectable HCV RNA (LOQ 30 IU/
mL) throughout the RBV dosing period. By contrast, F415Y was not enriched in patients that did not receive RBV (i.e., TP). Conclusion: Multiple
mechanisms of action have been proposed for ribavirin in HCV therapy. The clinically observed enrichment of NS5B F415Y in subtype 1a
following regimens containing ribavirin suggests that at least one mechanism may be the specific targeting of the NS5B polymerase.
Contact: Ann Tigges
Vertex Pharmaceuticals Inc., Cambridge, MA, USA
[email protected]
P7.08
Signature resistance mutations to directly acting antiviral agents occur at low
prevalence in treatment naïve subjects with recently acquired HCV
POSTERS
Silvana Gaudieri (1), Tanya Applegate (2), Monika Tschochner (1), Anne Plauzolles (3), Katja Pfafferott (1), Michaela Lucas (1), Helmut
Diepolder (4), Axel Ulsenheimer (4), Simon Mallal (1), Jason Grebely (2), Margaret Hellard (5), Andrew Lloyd (6), John Kaldor (2), Gregory
Dore (2), Gail Matthews (2)
(1) Institute for Immunology and Infectious Diseases, Murdoch University, Australia
(2) The Kirby Institute for Infection and Immunity in Science, UNSW, Sydney, Australia
(3) University of Western Australia, Australia
(4) Institute for Immunology, University of Munich, Germany
(5) Macfarlane Burnett Institute for Population Health, Australia
(6) Centre for Infection and Inflammation Research, UNSW, Australia
Background and Aims: The identification of pre-existing signature DAA mutations in HCV infected patients is important for therapeutic options.
This projects aims to assess the prevalence of resistance mutations to DAA in treatment naïve individuals with recent hepatitis C infection.
Methods: Signature DAA resistance mutations were identified by bulk sequencing of the HCV NS3 protease and NS5 polymerase genes from 95
treatment naïve subjects within the Munich AHC and Australian Trial in Acute Hepatitis C (ATAHC) cohorts. Subjects with genotype (GT) 1a, 1b
or 3a, within 3-4 months (MUNICH cohort) or up to 2 years (ATAHC cohort) after HCV infection were sequenced. DAA resistance prevalence was
compared to 405 treatment naïve chronic HCV (CHC) subjects. These regions were also sequenced in a subset of subjects by 454 FLX. Results:
Protease and polymerase mutations in recent HCV subjects were found at a range of 0–22.6% of GT 1a patients (coverage 26-49 subjects at
each site), 0-23.5% of GT 1b (6-17 subjects) and 0-6.2% of GT 3a (13-21 subjects). The prevalence of resistance mutations in both regions were
similar to that found in CHC subjects. The Protease Q80K/R and Polymerase C316Y/N mutations were found at the highest rates (22.6 and 23.5 %
respectively) Longitudinal analysis by bulk sequencing of 8 patients indicated the mutation profile was maintained up to 2 years. Deep sequencing
in patients with recent HCV indicated <1.5 % of minor variants existing as quasispecies. Conclusions: Analysis of DAA resistance mutations by bulk
sequencing in treatment naïve recent HCV subjects indicates the prevalence to be similar to that in CHC and is particularly concerning for GT1a
infections. Preliminary deep sequencing results suggest that virus at the resistant sites is relatively homogenous.
Contact: Tanya Applegate
The Kirby Institute for Infection and Immunity in Science, UNSW, Sydney, Australia
[email protected]
210 : HCV 2011
P7.09
Sequence and replication capacity of NS3 protease genes of individual clones from
patients treated with the HCV protease inhibitor GS-9256
Angela Worth (1), Kelly A. Wong (1), Michael D. Miller (1), Hongmei Mo (1)
Background: Results from population-based sequencing and replication assays reflect predominant, but not minor, viral quasispecies. The
objectives of this study were to investigate the emergence of minor resistance quasispecies not detected by population sequencing and
to determine the replication capacity of drug resistant mutants (DRMs) on patient-derived-NS3 protease backbones. Methods: Protease
domains from baseline and on-treatment samples from seven genotype 1 HCV-infected patients treated with the NS3 protease inhibitor
GS-9256 were amplified and cloned into a 1b-Con-1-Rluc NS3 replicon. Up to twenty individual clones per sample were sequenced. The
replication capacity was determined by transfecting the RNA from individual patient-derived wild-type (no DRM) and on-treatment clones
carrying representative NS3 mutations into Huh-7 Lunet cells. Results: The NS3 mutations R155K, D168E, D168G, and D168V were detected
by both population and clonal sequencing in on-treatment samples. In addition, the NS3 mutations A156V, R155S, R155T, R155G, D168A,
D168H, and the double mutation R155K/D168G were detected only by clonal sequencing. These minor resistance quasispecies represented
5-21% of the clones. No replication was observed with either wild-type or mutant clones from 3/7 patients. In contrast, wild-type clones
from three patients replicated at a level comparable to the 1b-Con-1, and one replicated at approximately 8% of 1b-Con-1. The various
individual clones of the mutants from these four patients displayed reduced replication capacity (<10%) compared to corresponding
wild-type clones. The order of replication capacity was similar among mutants derived from the different patients and ranked as follows:
D168E>D168H>D168G≥ D168V and R155K>A156V>R155S>R155G. Conclusions: We identified the presence of multiple minor HCV drugresistant quasispecies in on-treatment samples with substitutions at R155, A156, and D168. These were detectable at lower frequency than
predominant DRMs, likely due to their comparatively reduced replication capacity.
Contact: Angela Worth
[email protected]
P7.10
Melody Harrison (1), Veronica Raney (1), Kim Reynolds (1), David Harrison (1), Craig Cameron (2), Kevin Raney (1)
(1) University of Arkansas for Medical Sciences, Little Rock, AR, USA
(2) The Pennsylvania State University, USA
NS3 is a dual-action viral protein encoded by HCV that consists of an N-terminal protease domain and a C-terminal helicase domain and is
required for viral replication. NS3 is a DExH motor protein with common helicase motifs including the Walker A and Walker B regions that
participate in binding and hydrolysis of ATP. The natural substrate of NS3 is RNA, but this helicase also unwinds DNA substrates at similar
rates in vitro. Reports have indicated the presence of NS3 in the nuclei of patients infected with HCV. Binding of DNA to NS3 was investigated
by DNA footprinting with KMnO4, which reacts preferentially with thymidine residues in ssDNA compared to those in dsDNA. When NS3
was bound to ssDNA, a distinct pattern of reactivity was observed, which repeated every 8 nt and is consistent with the known binding site
size of NS3. Binding was examined using a DNA substrate with a 15 nt ssDNA overhang made entirely of thymidine residues adjacent to a
22 bp duplex that contained thymidine at every other residue. The pattern of reactivity with this substrate in the presence of NS3 mirrored
that which was observed with ssDNA, indicating that the ssDNA portion was saturated. Surprisingly, the reactivity pattern extended from the
ssDNA into the dsDNA region of the substrate. Control experiments indicated that the duplex was not fully separated under the conditions
for DNA footprinting, indicating that NS3 was bound to the duplex as well as the ssDNA. The enhanced reactivity within the duplex region
is likely due to some degree of melting or untwisting of the duplex, thereby enhancing the reactivity of thymidine residues in the duplex
region. A model for binding of a partial duplex DNA to NS3 is proposed and the relationship of binding to DNA unwinding is discussed.
Contact: Melody Harrison
University of Arkansas for Medical Sciences, Little Rock, AR, USA
[email protected]
POSTERS
Footprinting reveals simultaneous binding of NS3 to single-stranded and doublestranded regions of nucleic acid substrates
POSTERS
P7.11
HCV NS3 catalyzes the first step for DNA strand exchange
David Harrison (1), Melody Harrison (1), Bartek Sikora (1), Craig Cameron (2), Kevin Raney (1)
(1) University of Arkansas for Medical Sciences, Little Rock, AR, USA
(2) The Pennsylvania State University, USA
Despite the well known functions of NS3 in the cytoplasm, recent data suggest that it may play a role in the nucleus as well. We have
observed that small amounts of NS3 can be isolated from the nuclei of transfected En5-3 cells, and chromatin immunoprecipitation reveals
DNA bound to nuclear NS3. In addition, electron microscopy has shown that in vitro NS3 can form large aggregates. The presence of NS3 in
the nucleus, coupled with its propensity to aggregate, led us to examine the binding of NS3 to DNA. Binding experiments indicate moderate
affinity of NS3 for duplex DNA, while KMnO4 footprinting reveals that NS3 binds to ssDNA in a repeating pattern, suggestive of a “filamentlike” structure. The DNA binding and protein aggregation properties of NS3 are also reminiscent of recombinase proteins. HCV NS3 protein is
known to contain two RecA-like domains that couple the binding and hydrolysis of ATP to nucleic acid unwinding. Further, our studies using
DNA substrates suggest that NS3 can catalyze the formation of joint molecules, the first step in strand exchange. The relationship between
joint molecule formation and nucleic acid unwinding will be discussed.
Contact: David Harrison
University of Arkansas for Medical Sciences, Little Rock, AR, USA
[email protected]
P7.12
N236T-HBV mutants Isolated from patients with primary non response to adefovir
show a reduction of susceptibility to tenofovir in vitro
Olivier Lada (1), Qian Zhang (1), Emilie Estrabaud (1), Roberto Carvalho-Filho (1), Rami Moucari (1), Simon De Muynck (1), Marie-Pierre
Ripault (1), Corrine Castelnau (1), Michelle Martinot-Peignoux (1), Nathalie Boyer (1), Tarik Asselah (1), Patrick Marcellin (1)
(1) Service d’Hépatologie, Beaujon and INSERM CRB3, Bichat, Paris, France
POSTERS
Background/Aims: Tenofovir disoproxil fumarate (TDF) and Entecavir (ETV) have demonstrated high antiviral efficacy in treatment-naive
patients with chronic hepatitis B virus (HBV) infection. However, few data are available on patients with primary nonresponse (PNR) (HBVDNA viral decrease <2 log IU/mL at month 6 of therapy) to nucleotide or nucleoside analogues. Among 138 patients treated with adefovir
dipivoxil (ADV) and followed from 2004 to 2008 at Beaujon hospital, we observed 10 patients with PNR to ADV. The aim of this study was to
assess the in vitro drug susceptibility of HBV strains isolated from these patients.
Methods: Whole HBV genome from each patient’s serum at baseline and M6 ADV therapy was PCR-amplified and cloned. HBV quasispecies
populations was sequenced and analysed. The main HBV mutants were tested after transfection into HepG2 for their resistance profile to
nucleoside analog. A wild type (wt) strain isolated from an untreated patient served as control.
Results: Baseline characteristics of the 10 patient s were: median age 46 (range, 37-63) years, 8 males, 8 HBeAg-positive, median baseline
HBV DNA level 7.1 log UI/mL (4.5-8.6). Six out 10 patients was lamivudine-experienced. Clonal analysis showed the co-localization on
the same HBV genome of mixture of lamivudine (3TC) plus ADV resistance mutations. In vitro data of the main HBV mutants detected in
patient’s serum are summarized in table below.
Conclusion: We showed in our in vitro system that presence of rtA181V mutation may induce cross-resistance to 3TC and ADV. TDF is effective
on all ADV-resistant HBV strains except HBV strain carrying N236T mutation alone. ETV is effective on all ADV-resistant HBV. By this clonal
approach, we showed that genotypic and phenotypic analysis could be clinically relevant in the management of antiviral drug resistance.
Contact: Emilie Estrabaud
Service d’Hépatologie, Beaujon and INSERM CRB3, Bichat, Paris, France
[email protected]
212 : HCV 2011
P7.13
HBsAg level and HBV related liver disease
Michelle Martinot-Peignoux (1), Roberto Carvalho-Filho (1), Ana Carolina Ferreira Netto Cardoso (1), Martine Lapalus (1), Olivier Lada (1),
Tarik Asselah (1), Patrick Marcellin (1)
(1) INSERM U773-CRB3 Bichat- Service d’Hépatologie Beaujon, Clichy, France
Discrepancies between histological lesions, serum ALT and HBV-DNA are frequent. We aimed to evaluate in chronic hepatitis B treatmentnaïve patients (CHB): the relation between HBsAg level and liver histological lesions, HBV DNA, genotype, HBeAg status and ALT.
Methods-Patients. Inclusion criteria were presence HBsAg and a liver biopsy, HBeAg positive or negative. HBsAg and HBV-DNA quantification,
genotype determination and ALT measurements were performed the day of the liver biopsy. Histological lesions assessed with Metavir
scoring system.
Results. 406 CHB patients were included (101 HBeAg positive). Genotypes distribution was: A: 27%/23%; B: 9%/12%; C: 29%/2%; D: 15%/28%;
E: 19%/33% in HBeAg positive and negative patients, respectively. 61%; 23%; 8% and 8% of the patients had a fibrosis stage ≤ F1, F2, F3 and
F4, respectively. Serum ALT measurements were above the normal in 65% of the patients. HBsAg levels were: 4.24±0.90 and 3.53±0.92 log
IU/ml in HBeAg positive and negative patients, respectively (p<0.0001). The ration HBsAg/HBV-DNA levels were 0.63±0.21versus 0.99±0.56
in HBeAg positive and negative patients, respectively (p<0.0001). In HBeAg positive patients there were strong correlations between HBsAg
and HBV-DNA levels (r=0.438: p<0.0001) and HBsAg and fibrosis stages (r=0.43; p<0.0001) (showing a decrease from F1 to F4), no correlation
was observed in HBeAg negative patients. No association was found between HBsAg level and gender, age, ALT or activity grade. HBsAg
levels were 3.84±0.90; 3.23±1.12; 3.93±0.91; 3.74±0.84; 3.97±0.60; in genotypes A; B; C; D; E, respectively (p<0.001).
Conclusions. Our study performed in a large cohort of patients (genotype A to E) show that HBsAg levels: are lower in HBeAg negative
patients than in HBeAg positive patients, are correlated with HBV DNA and fibrosis stages in HBeAg positive patients.
Contact: Michelle Martinot-Peignoux
INSERM U773-CRB3 Bichat- Service d’Hépatologie Beaujon, Clichy, France
[email protected]
P7.14
Quantitative HBsAg: a new specific marker for the diagnosis of HBsAg inactive carriage
Accurate identification of patients with HBeAg-negative chronic hepatitis B (CHB) from inactive hepatitis B virus carriers (IC) is a major
medical need since they could benefit from therapy with increased survival.
Our aim was to evaluate the role of hepatitis B surface antigen (HBsAg) as a new marker for identification of IC.
Methods: Inclusion criteria were: normal ALT, presence of HBsAg and anti-HBe without HBeAg at the first visit. IC was defined as persistently
normal ALT (3 measurements during one year). HBsAg was quantified with Elecsys HBsAg II assay (Roche Diagnostics).
Results: 135 untreated patients included; 101 had persistently normal ALT were classified IC and 34 with fluctuating activity were classified
CHB. Genotypes distribution was A: 40%; B: 10%; C: 3%; D: 31%; E: 16%. Mean baseline HBsAg titers were 3.36±0.92 and 3.70±0.68 log IU/ml
in IC and CHB, respectively (p<0.05); mean HBV-DNA levels were 2.54±0.91 and 3.74±0.90 log IU/ml in IC and CHB, respectively (p<0.001). At
baseline 54 patients had a HBsAg level ≤2000IU/l , 46 were IC PPV 85%; 40 had a HBsAg level ≤1000 IU/l , 36 were IC PPV 90%. Combination
of HBsAg level ≤ 2000 IU/ HBV-DNA ≤1000 IU/ml showed a PPV of 94%. The ratio HBsAg/HBV DNA was 1.38±0.50 and 1.04±0.32 in IC and
CHB, respectively (p<0.001) A ratio HBsAg/HBV DNA >1 allowed identification of IC with a PPV 84%.
Fibrosis stage ≤1 was observed in 84% IC and 61% CHB, (p<0.02).
Conclusion: Quantitative HBsAg is a specific marker for diagnosis of IC. HBsAg ≤2000 IU/mL and HBV DNA ≤1000 IU/ml allow identification
of IC with VPP 94%. HBsAg monitoring might be discussed in future guidelines as a new tool for HBV management.
Contact: Michelle Martinot-Peignoux
INSERM U773-CRB3 Bichat- Service d’Hépatologie Beaujon, Clichy, France
[email protected]
POSTERS
Michelle Martinot-Peignoux (1), Martine Lapalus (1), Marie-Pierre Ripault (1), Nathalie Boyer (1), Tarik Asselah (1), Patrick Marcellin (1)
(1) INSERM U773-CRB3 Bichat- Service d’Hépatologie Beaujon, Clichy, France
POSTERS
P7.15
In vitro selection and characterization of genotype 2a J6/JFH-1 replicon variants
resistant to PSI-7977
Christine Espiritu (1), Shalini Bansal (1), Michael Otto (1), Phillip Furman (1), Angela Lam (1)
(1) Pharmasset Inc., Princeton, NJ, USA
Background: A prodrug of beta-D-2′-F-2′-C-methyluridine-5′-monophosphate, PSI-7851 selects for the S282T resistant mutation in GT 1a
and 1b replicons. PSI-7977 is the purified diastereoisomer of PSI-7851 that has been selected for further clinical investigation. In a recent
Phase 2b study, a 96% SVR12 (sustained virological response for 12 weeks after treatment) was achieved in GT 2/3 patients treated for 12
weeks with PSI-7977 and SOC. Herein we present the resistance profile of PSI-7977 in J6/JFH-1 derived GT 2a replicon cells. Methods: GT 2a
replicon cells were cultured in the presence of G418 and increasing concentrations of PSI-7977. Cells were tested for sensitivity to PSI-7977
and replicons were analyzed for mutations within the NS5B polymerase. Replicon variants were constructed by site directed mutagenesis
and examined for resistance to PSI-7977. Results: GT 2a replicon cells showed reduced sensitivity for PSI-7977 after 137 days of selection.
In addition to the NS5B S282T mutation, sequence analysis at days 149 and 158 showed PSI-7977 also selected the following amino acid
changes: T179A, M289L, I293L, M434T (day 149 only), and H479P (day 158 only). Residues 179, 289 and 293 are located along two parallel
helices within the Finger and Palm domains. While S282T alone showed only a modest increase in EC50 value (~2-fold), a 5-fold shift was
observed when S282T was combined with M289L. Adding I293L to S282T/M289L caused a 7-fold increase in EC50 value. Fitness analysis
showed T179A and M289L compensated for the poor fitness of the S282T mutant. Conclusion: Resistance selection using HCV replicon
has been shown to predict resistance profiles of antiviral compounds in HCV infected patients. Our results showed S282T alone was not
sufficient to confer resistance to PSI-7977 in J6/JFH-1 GT 2a replicon. Only when S282T was combined with M289L or M289L/I293L did we
observe a significant decrease in susceptibility to PSI-7977.
Contact: Angela Lam
Pharmasset Inc., Princeton, NJ, USA
[email protected]
P7.16
Modeling inhibition kinetics of HCV sg1b RNA in non-growing Huh7 cells
Harel Dahari (1), Naina Barretto (2), Natasha Sansone (2), Jeremie Guedj (1), Alan Perelson (1), Susan Uprichard (2)
(2) University of Illinois, Chicago, IL, USA
POSTERS
Background: Modeling HCV sg1b-RNA inhibition kinetics in response to IFNa in growing cells has shown IFNa mainly blocks RNA replication,
but also enhances HCV RNA degradation. Here we use direct-acting agents, such as protease and polymerase inhibitors, to more effectively
estimate the intrinsic HCV-RNA degradation rate. We also use the non-growing DMSO-Huh7 system because it avoids issues of cells reaching
confluence and requiring splitting, reduces well-to-well variability, and reduces changes in intracellular drug concentration that may result
from cell growth. Methods: Non-growing sg1b replicon cells were treated with 100, 250 or 500U/ml IFN_, 10 or 80nM BILN2061 (protease
inhibitor), or 18.5uM NM107 (polymerase inhibitor). Levels of sg1b-RNA were frequently measured by RT-qPCR and the effect on blocking
sg1b-RNA production, ein, and sg1b-RNA degradation, mu, were calculated using a mathematical model (Eq.6; J.Virol 2009;83(13):63836390). Results: NM107 treatment led to ein=0.996 and sg1b-RNA degradation of mu=0.83/day similar to that observed in growing cells.
Since NM107 directly inhibits HCV RNA production and the mu_calculated in growing and non-growing cells was the same, we consider
mu=0.83/day as the intrinsic rate of sg1b-RNA degradation. Both doses of BILN2061 had ein=~0.9999, but there may have also been slight
enhancement of sg1b-RNA degradation (~2-fold) relative to that found with NM107. Similar to growing cells, in non-growing cells IFNa
primarily reduced sg1b-RNA production while higher doses also enhanced sg1b-RNA degradation ~3-fold. Conclusions: Non-growing Huh7
cells represent a more convenient and reproducible system for assessing HCV drug inhibition kinetics and drug mode of action. Using this
system, we confirmed that IFNa has a dose-dependent effect on blocking sg1b-RNA production and enhancing sg1b-RNA degradation.
Likewise, we show that in addition to blocking sg1b-RNA production, BILN2061 might also slightly enhance sg1b-RNA degradation, which
may be related to the effect protease inhibitors have on blocking NS3-mediated inhibition of host innate antiviral pathways.
Contact: Susan Uprichard
University of Illinois, Chicago, Chicago, IL, USA
[email protected]
214 : HCV 2011
P7.17
Envelope mutations found after IFN-alpha treatment of HCV 1a and 3a core-NS2 JFH1based recombinants led to increased viral fitness and IFN resistance
Stéphanie Serre (1), Jens Bukh (1), Judith Gottwein (1)
(1) Copenhagen Hepatitis C Program (CO-HEP), Hvidovre, Denmark
To identify HCV genomic correlates of resistance to interferon-alpha (IFN), basis of current treatment regimens, we promoted in vitro
escape of previously developed HCV genotype 1a (strain H77) and 3a (strain S52) Core-NS2 JFH1-based recombinants. At viral breakthrough,
in presence of IFN, H77 had acquired 5 coding mutations in E1, NS2, NS5A (2 mutations) and NS5B. The effect of these mutations was
investigated in reverse genetic studies. In transfection and viral passage kinetic experiments in Huh7.5 cells, with IFN, H77 with the single
E1 mutation or with all 5 mutations showed efficient viral spread with peak infectivity titers ≥3.5 log FFU/ml, while spread of the original
H77 was inhibited with titers below the limit of detection. Without IFN, these two recombinants showed accelerated spread and ~0.5
log increased titers. Thus, the E1 mutation was mainly responsible for increased viral fitness and IFN resistance. Reverse genetic studies
showed that this E1 mutation conferred partial IFN resistance to another 1a Core-NS2 recombinant (strain TN). As observed for H77, for
S52, studies of the 8 coding mutations identified in E1, E2, p7, NS2, NS5A (3 mutations) and NS5B suggested that combination of E1/E2
mutations increased viral fitness and conferred IFN resistance. To initiate mechanistic studies, we determined intracellular and extracellular
infectivity titers, following transfection of viral RNA transcripts in CD81-deficient S29 cells, precluding HCV entry. For H77, the E1 mutation
led to a ~0.5 log increase of intracellular and extracellular titers compared to the original virus. For S52 with E1/E2 mutations, intracellular
titers were similar to the original virus, while extracellular titers were increased by ~0.5 log. These preliminary data suggest that envelope
mutations identified in H77 or S52 affect different steps of the viral life cycle. Our data indicate that HCV envelope mutations might play an
important role in IFN resistance.
POSTERS
Contact: Stéphanie Serre
Copenhagen Hepatitis C Program (CO-HEP), Hvidovre, Denmark
[email protected]
POSTERS
216 : HCV 2011
POSTERS
Virology: Entry and Translation
P8.01
Low pH promotes reversible conformational changes in hepatitis C virus E1E2
Nishi Sharma, Gregory Melikian
P8.02
The association of CD81 with lipid rafts is a key determinant for hepatitis C
virus entry
Yongzhe Zhu, Ping Zhao, Yimin Tong, Yuan Liu, Xiaoqing Liu, Wen Wang, Lanjuan Zhao, Mingmei Cao, Zhongtian Qi
P8.03
Comparative expression of HCV candidate receptors in JFH1-susceptible
hepatoma cells and human T lymphocytes prone to wild-type HCV
Mohammed A. Sarhan, Thomas I. Michalak
P8.04
Post-transcriptional regulation of HCV gene expression by miR-122 in vitro
Shelton Bradrick
P8.05
Monoclonal antibodies specific for the SR-BI N-terminal ectodomain block
hepatitis C virus entry at postbinding steps and cell-cell transmission
P8.06
Lipoprotein lipase inhibits hepatitis C virus infection by blocking virus cell entry
Patrick Maillard, Marine Walic, Philip Meuleman, Farzin Roohvand, Thierry Huby, Willfried Le Goff, Geert Leroux-Roels,
Eve-Isabelle Pécheur, Agata Budkowska
P8.07
Mechanism of stimulation of hepatitis C virus RNA translation by
microRNA-122
Dagmar Goergen, Dominik Conrad, Florian Giering, Michael Niepmann
POSTERS
Mirjam B. Zeisel, Muhammad N. Zahid, Fei Xiao, Viet Loan Dao Thi, François-Loïc Cosset, Isabel Fofana, John Thompson,
Eva K. Schnober, Daniel Da Costa, Marine Turek, Fritz Grunert, Thomas F. Baumert
POSTERS
P8.08
Normalization of translation readouts after transfection of cells: the urgent
need for an independent method monitoring transfection efficiency
Christiane Bung, Carmen Fehr, Dagmar Goergen, Juliane Hirnet, Michael Niepmann
P8.09
Hepatitis C virus RNA translation stimulation coincides with expression of
microRNA-122 and argonaute protein during cell cycle
Carmen Fehr, Dominik Conrad, Michael Niepmann
P8.10
Hypervariable region 2 and the intergenotypic variable region modulate
neutralization resistance of HCV strains and virus entry properties
Kevin Tewierek, Irene Boo, Yousef Alhammad, Patricia Vietheer, Pantelis Poumbourios, Heidi Drummer
P8.11
Isolation of a highly infectious hepatitis C virus with adaptive mutations
Masayoshi Fukasawa, Yoshitaka Shirasago, Kyoko Saito, Yuko Murakami, Hidesuke Fukazawa, Tetsuro Suzuki,
Ryosuke Suzuki, Takaji Wakita, Kentaro Hanada, Jo Chiba
P8.12
Effect of genetic variation in the occludin gene on its co-receptor function
for HCVcc
Sandra Ciesek, Sandra Westhaus, Christoph Sarrazin, Nabila Hamdi, Ahmed Abdelaziz, Christian Strassburg,
Heiner Wedemeyer, Michael Manns, Thomas Pietschmann, Thomas von Hahn
P8.13
Panning for gold: a screen for Hepatitis C virus entry inhibitors
Vanessa Cowton, Sarah Beattie, Allison Marty, Ania Owisianka, Arvind Patel
P8.14
The role of HCV non-structural protein 5A (NS5A) in modulating viral
protein translation
Brett Hoffman, Qiang Liu
P8.15
Primary human hepatocytes to investigate HCV infection in vitro:
optimization of a relevant cell-culture model
POSTERS
Claire Gondeau, Philippe Briolotti, Francia Razafy, Dominique Larrey, Francis Navarro, Antonio Sa-Cunha, Sylvain Laporte,
Patrick Maurel, Martine Daujat-Chavanieu
P8.16
Uncovering host and viral genetic factors regulating HCV dissemination
Luke Meredith, Garrick Wilson, Helen Harris, Michelle Farquhar, Peter Balfe, Jane McKeating
P8.17
Modeling viral kinetics in vitro reconciles disparate observations of
claudin-1 expression on cells following hepatitis C virus (HCV) infection
Pranesh Padmanabhan, Narendra Dixit
P8.18
The NS5A/HSP70 Complex facilitates hepatitis C viral IRES-mediated translation
Samuel French, Ronik Khachatoorian, Edna Maio, Edna Maloney, Vaithilingaraja Arumugaswami, Ren Sun, Asim Dasgupta
P8.19
Receptor endocytosis defines hepatitis C virus entry
Michelle Farquhar, Ke Hu, Helen Harris, Christopher Davis, Claire Brimacombe, Joshua Rappoport, Peter Balfe, Jane McKeating
P8.20
Identification of a cysteine residue in E1 glycoprotein that determines particle
assembly and alters CD81 and heparan sulphate receptors dependency
George Koutsoudakis, Sofía Pérez-del-Pulgar, Jakub Dragun, Mairene Coto-Llerena, Laura Mensa, Xavier Forns
218 : HCV 2011
VIROLOGY: ENTRY AND TRANSLATION
P8.21
Variation of HCV infectivity and HVR1 quasispecies selection are affected by
HCV E1E2 synonymous mutation
Kazunori Kawaguchi, Masao Honda, Takayoshi Shirasaki, Suzanne Emerson, Purcell Robert, Shuichi Kaneko
P8.22
Dynamic of HCV entry inhibition by EWI-2wint
Julie Potel, Patrice Rassam, Claire Montpellier, Cyril Couturier, Birke Andrea Tews, Ioan Costin Popescu, Jean Dubuisson,
Pierre-Emmanuel Milhiet, Laurence Cocquerel
P8.23
Identification of (-)-epigallocatechin gallate as a new inhibitor of hepatitis C
virus entry
Noémie Calland, Anna Albecka, Sandrine Belouzard, Czeslaw Wychowski, Véronique Descamps, Gilles Duverlie,
Jean Dubuisson, Yves Rouillé, Karin Séron
P8.24
Active role of E1 in the conformation of E1E2 and in the exposition of
receptor binding domains
Florian Douam, Guillemette Maurin, Judith Fresquet, François-Loïc Cosset, Dimitri Lavillette
P8.25
Impact of primary human hepatocytes polarity on hepatitis C virus
internalization: an electron microscopy study
Mihayl Varbanov, Marie Perrault, Claire Gondeau, Patrick Maurel, Beatrice Burdin, François-Loic Cosset, Florence Ruggiero,
Eve-Isabelle Pecheur
P8.26
Expression of the HCV core+1/ARF protein in the context of the HCV
replicon system
Ioly Kotta-Loizou, Niki Vassilaki, Penelope Mavromara
P8.27
HCV quasispecies complexity and implications for the detection of
viral infection
P8.28
HIV fusion inhibitors derived from a flaviviridae glycoprotein
Kristin Eissmann, Yvonne Ködel, Holger Wend, Bernhard Fleckenstein, Heide Reil
P8.29
Hepatitis C virus has a genetically determined lymphotropism through a
coreceptor B7.2
Keigo Machida, Jeffrey Huang, Chun-Hsiang Wang, Jian-Chung Liu, Yasuteru Kondo, Joel Schechter, Steven Foung,
Takaji Wakita, Jae Jung, Michael Lai
P8.30
Characterization of new hepatocellular carcinoma cell lines for the study of
hepatitis C virus infection
Mairene Coto-Llerena, Loreto Boix, George Koutsoudakis, Juan Manuel López-Oliva, Jordi Bruix, Xavier Forns,
Sofía Pérez-del-Pulgar
P8.31
Molecular Analysis of the Interaction between Hepatitis C Virus E2 Protein
and Host Entry Factor CD81
Peter Liu, Lilin Zhong, Evi Struble, Christine Harman, Maria Luisa Virata-Theimer, Li Ma, Pei Zhang
POSTERS
Brendan A. Palmer, John Menton, Mairead Finn, Jamie O’Sullivan, Liam J. Fanning
POSTERS
P8.32
SR-BI mediates different steps of HCV entry into cells, involving ApoE
associated to HCV particles and direct or indirect interactions with HCV E2
Viet Loan Dao This, Christelle Granier, Mirjam Zeisel, Jimmy Mancip, Ophélia Granio, Francois Pénin, Dimitri Lavillette,
Thomas Baumert, Marlène Dreux, F. L. Cosset
P8.33
Identification of single amino acids in the E2 stem region vital for hepatitis C
virus entry
Thomas H.R. Carlsen, Troels K.H. Scheel, Santseharay Ramirez, Jens Bukh
P8.34
Occludin is required for a late step in hepatitis C virus cell entry, after
binding and CD81 requirement, but before viral fusion
Marion Sourisseau, Maria Michta, Lum Chati Zoni, Benjamin Israelow, Sharon Hopcraft, Matthew Evans
P8.35
The adaptor protein Disabled-2 mediates hepatitis C virus entry and is a
potential target for modulating viral infectivity
Ju-Chien Cheng, Yung-Ju Yeh, Meng-Hong Lu, Ching-Ping Tseng
P8.36
Antibody and lipid correlates of outcome of acute HCV infection
Maria Isaguliants, Natalia Petrakova, Olga Znoiko, Kristina Dudina, Sergey Domogatsky, Juris Jansons, Anders Widell
POSTERS
220 : HCV 2011
P8.01
Low pH promotes reversible conformational changes in hepatitis C virus E1E2
Nishi Sharma (1), Gregory Melikian (1)
At least four receptors, CD81, SR-BI, claudin-1 and occludin, are required for productive entry of the Hepatitis C Virus (HCV) that culminates
in low pH-dependent fusion with endosomes. Previous studies have shown that cell-free HCV is not inactivated at mildly acidic conditions,
implying that E1E2 do not irreversibly refold into a post-fusion conformation in the absence of cellular receptors. On the other hand,
retroviral particles pseudotyped with E1E2 (HCVpp) were reported to fuse with liposomes at low pH. We therefore examined the pHsensitivity of HCVpp bearing a luciferase reporter gene. To prevent possible virus aggregation at low pH, which could reduce the infectivity
without inactivating E1E2, we immobilized HCVpp on multi-well plates and pre-treated the virus with acidic or neutral buffers. The effect
of this treatment on HCVpp was assessed by overlaying viruses with Huh-7.5 cells and measuring the resulting infection. Pretreatment of
HCVpp with a pH 5.2 buffer did not compromise their infectivity. Moreover, we observed a modest but significant enhancement of infectivity
compared to control viruses pre-treated with a neutral buffer. This enhanced infectivity returned to a basal level when cells were added after
a 30 min-chase at neutral pH, but was increased again upon application of a second low pH pulse. Importantly, low pH pretreatment altered
the reactivity of conformation-specific antibodies against E1 and E2, suggesting that HCV glycoproteins undergo reversible conformational
changes that augment virus fusion with permissive cells. Acid-mediated conformational changes were not sufficient to bypass an endocytic
entry route and induce fusion with the plasma membrane of Huh-7.5 cells in the presence of drugs elevating the endosomal pH. This work
was supported by ARRA R21 AI079714 grant to G.M.
Contact: Gregory Melikian
[email protected]
P8.02
The association of CD81 with lipid rafts is a key determinant for hepatitis C virus entry
Hepatitis C virus (HCV) entry is a coordinated multistep process mediated by specific factors, involving tetraspanin CD81, scavenger receptor
class B member I (SR-BI), and the tight junction proteins claudin-1 (CLDN1) and occludin. Previous studies have suggested the interactions
between the viral envelope and host cell receptors may depend on cholesterol-enriched membrane microdomains, termed lipid rafts, an
important microdomain in cytosol membrane for many viruses’ cell entry. We found that HCV receptors CD81, SR-BI, CLDN1 and occludin
all partition in lipid rafts, co-localizing with HCV envelope glycoprotein E2 during viral entry. To investigate whether the raft localization of
CD81 affects HCV cell entry, we generated CD81 partitioning mutants by substituting the CD81 transmembrane and cytoplasmic domains
with those of raft proteins (SR-BI or CD9) or that of non-raft protein (CD71), while preserving the native CD81 large extracellular loop (LEL)
in chimeric molecule. All CD81 chimeras that retain raft partitioning mediate HCV entry and CD81-induced Rho-GTPase activation which
allows lateral movement of the virus and its delivery to cell-cell contact areas. Conversely, CD81 LEL targeting to a non-raft membrane
fraction results in a CD81 receptor with severely diminished capacity to mediate Rho-GTPase activation and HCV entry, although this mutant
binds HCV E2 as well as wild type CD81. Moreover, we show that the non-raft CD81 mutant fails to relocalize HCV E2 to the areas of cellcell contact, thus disrupting the formation of E2-CD81-CLDN1 complexes which was recently described as a determinant for viral entry.
Altogether, our results not only demonstrate that the association of CD81 with lipid rafts plays an essential role in HCV entry, but also
suggest new strategies for blocking HCV infection.
Contact: Yongzhe Zhu
Department of microbiology, Second Military Medical University, Shanghai, China
[email protected]
POSTERS
Yongzhe Zhu (1), Ping Zhao (1), Yimin Tong (1), Yuan Liu (1), Xiaoqing Liu (1), Wen Wang (1), Lanjuan Zhao (1), Mingmei Cao (1),
Zhongtian Qi (1)
POSTERS
P8.03
Comparative expression of HCV candidate receptors in JFH1-susceptible hepatoma
cells and human T lymphocytes prone to wild-type HCV
Mohammed A. Sarhan (1), Thomas I. Michalak (1)
(1) Molecular Virology and Hepatology Research Group, Memorial University, St. John’s, A1B 3V6, Canada
HCV candidate receptors were identified using surrogate HCV models and hepatoma cells as targets. However, molecules mediating HCV
attachment and entry to immune cells, which are extrahepatic sites of virus occurrence, have not yet been defined. The aim of this study was to
assess expression of different HCV candidate receptors in human PBMC, primary T cells and T cell lines known to be susceptible or not to wildtype HCV and compare their expression to that in HCV JFH1-susceptible (Huh7.5) and nonsusceptible (HepG2) hepatoma cells, and primary
human hepatocytes (PHH). All cell lines and primary cells were examined for the expression of CD81, SR-B1, CD5, claudin (CLDN)-1, 4, and 6,
and occludin (OCLN) by real-time RT-PCR and Western blotting. HCV RNA positive and negative strands and HCV NS5a and core proteins in
infected lymphocytes were assessed by RT-PCR and confocal microscopy, respectively. Although CD81 and CLDN-6 expression was essentially
comparable in all cell types investigated (except HepG2), SR-B1, CLDN-1 and CLDN-4 expression was significantly higher (P=0.001) in Huh7.5
cells and PHH. In contrast, OCLN was transcribed at significantly greater levels (P=0.0001) in hepatoma cell lines, PHH, and HCV-susceptible
Molt4, Jurkat and primary T cells compared to HCV-resistant PM1 and CEM T cells. In addition, CD5 was expressed by primary T cells, PBMC,
Molt4 and Jurkat cells at levels significantly higher (P=0.0001) than in HCV-resistant PM1 and CEM T cell lines, and was absent in hepatoma cells
and PHH. In conclusion, T cells prone to wild-type HCV infection, such as primary T cells, PBMC, Molt4 and Jurkat, express significantly higher
levels of CD5 and OCLN than HCV-resistant T cell lines, suggesting that these molecules, particularly CD5, might be directly involved in HCV
lymphotropism. The results from this study also imply that HCV utilizes distinct receptors to enter different cell types.
Contact: Mohammed A. Sarhan
Molecular Virology and Hepatology Research Group, Memorial University, St. John’s, A1B 3V6, Canada
[email protected]
P8.04
Post-transcriptional regulation of HCV gene expression by miR-122 in vitro
Shelton Bradrick (1)
(1) Duke University Medical Center, Durham, NC, USA
POSTERS
Functional interactions have been well documented between a liver-specific microRNA (miRNA), miR-122, and tandem target sites located
at the 5’ end of the HCV genome. miR-122 considerably stimulates HCV replication in both cultured cells and primates. However, the
precise mechanism(s) and associated host factors required for miR-122 enhancement of HCV replication have not been fully elucidated.
Development of relevant cell-free assays designed to probe the underlying molecular details of miR-122-HCV interaction may enhance our
understanding of both HCV and miRNA biology.
I have employed a biochemical approach in an effort to recapitulate miR-122-HCV interaction in cell-free, translation-competent cytoplasmic
lysates derived from cells that express miR-122. Specifically, lysates produced from stable 293 cells that inducibly transcribe and process
pri-miR-122 have been characterized alongside those from isogenic cells lacking miR-122 expression. Huh7.5 hepatoma cell lysates that
harbor endogenous miR-122 have also been utilized. These extracts support translation of sub-genomic, non-replicating HCV reporter RNAs
that depend on a functional internal ribosome entry site (IRES) for translation initiation. Dual mutation of miR-122-binding sites significantly
reduced reporter protein accumulation in transfected Huh7.5 cells, in agreement with published data, suggesting that miR-122 stimulates
viral replication at least partly through promotion of HCV RNA translation and/or stability. A stimulatory effect was correspondingly
observed in cell-free assays in a manner that depended on both the presence of endogenous miR-122 and wild-type target sites within
the HCV 5’ untranslated region. This reductionist in vitro approach allows direct analysis of RNA turnover, as well as formation of HCVribosome complexes during translation initiation, in the presence or absence of active miR-122. Moreover, this strategy may facilitate
affinity purification of operative HCV ribonucleoprotein complexes that coincide with miR-122-HCV interaction, and enable identification of
novel host factors involved in HCV replication.
Contact: Shelton Bradrick
Duke University Medical Center, Durham, NC, USA
[email protected]
222 : HCV 2011
P8.05
Monoclonal antibodies specific for the SR-BI N-terminal ectodomain block hepatitis C
virus entry at postbinding steps and cell-cell transmission
Mirjam B. Zeisel (1), Muhammad N. Zahid (1), Fei Xiao (1), Viet Loan Dao Thi (2), François-Loïc Cosset (2), Isabel Fofana (1), John
Thompson (3), Eva K. Schnober (4), Daniel Da Costa (1), Marine Turek (1), Fritz Grunert (3), Thomas F. Baumert (1)
(1) INSERM, U748, Université de Strasbourg, Strasbourg, France
(2) INSERM, U758, ENS de Lyon, IFR128 BioSciences Lyon-Gerland, France
(3) Genovac GmbH, Freiburg, Germany
(4) Department of Medicine II, University of Freiburg, Germany
Scavenger receptor class B type I (SR-BI) had been originally proposed as a hepatitis C virus (HCV) receptor because it binds HCV envelope
glycoprotein E2. Further studies then demonstrated that the molecular mechanisms underlying HCV-SR-BI interactions are complex and involve
an orchestrated interplay between HCV, SR-BI and HDL to promote efficient HCV entry and infection. We have previously generated polyclonal
anti-SR-BI antibodies and demonstrated that SR-BI is required for an entry step closely-linked to HCVcc interaction with CD81 and CLDN1. Here
we report novel rat and mouse monoclonal anti-SR-BI antibodies that, in contrast to previous monoclonal anti-SR-BI antibodies, do not inhibit
recombinant HCV E2 binding to SR-BI but inhibit HCVcc infection predominantly at post-binding steps. Combination of anti-HCV envelope and
anti-SR-BI antibodies resulted in synergistic effect on inhibition of both HCVpp entry and HCVcc infection without directly interfering with E2CD81 binding. Moreover, these monoclonal anti-SR-BI antibodies potently inhibit HCV cell-to-cell transmission and viral dissemination. Mapping
studies using chimeric mouse and human SR-BI expression constructs suggest that the antibodies target epitope(s) within the N-terminal part
of the extracellular loop of SR-BI. Taken together, our results suggest that distinct regions of SR-BI are responsible for its dual role in HCV entry
including a key role for the N-terminal ectodomain of SR-BI for mediating post-binding steps of HCV entry. Further studies aiming to fine-map
the epitope(s) for entry and cell-cell transmission and to investigate whether these monoclonal anti-SR-BI antibodies interfere with HDL binding
or SR-BI-mediated cholesterol transfer are ongoing. These antibodies will be useful for the investigation of the mechanistic role of SR-BI during
viral entry as well as the development of entry inhibitors targeting HCV-SR-BI interactions.
Contact: Mirjam B. Zeisel
INSERM, U748, Université de Strasbourg, Strasbourg, France
[email protected]
P8.06
Patrick Maillard (5), Marine Walic (5), Philip Meuleman (1), Farzin Roohvand (2), Thierry Huby (3), Willfried Le Goff (3), Geert Leroux-Roels
(1), Eve-Isabelle Pécheur (4), Agata Budkowska (5)
(1) Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium
(2) Hepatitis and AIDS Department, Pasteur Institute of Iran, Teheran, Iran
(3) INSERM UMR-S939, Hopital de la Pitié, Paris, France
(4) University of Lyon 1, CNRS UMR 5086, IBCP, Lyon, France
(5) Hepacivirus and Innate Immunity, Pasteur Institute, Paris, France
Lipoprotein lipase (LPL) hydrolyzes triglycerid rich lipoproteins within chylomicrons and VLDL, but independently of its catalytic activity, it
has a bridging activity, mediating hepatic uptake of chylomicrons and VLDL remnants. We have previously shown that exogenously added
LPL increases binding of serum HCV to hepatoma cells by acting as a bridge between viral-associated lipoproteins and cell surface heparan
sulfate, while at the same time decreasing infection levels (Andreo et al. 2007). We report here that LPL efficiently inhibits cell infection
with two HCV strains produced either in hepatoma cells, or in primary human hepatocytes grafted onto chimeric uPA-SCID mice. Viruses
produced either in vitro or in vivo were separated in iodixanol gradients into low and higher density populations, and cell infection with both
virus populations was inhibited by LPL. We determined the mechanism of action of LPL on HCV infection, which involves both the catalytic and
structural functions of the enzyme. The effect of LPL depended on its enzymatic activity, however HCV infectivity was only partially restored in
the presence of the lipase inhibitor, tetrahydrolipstatin, suggesting a major role of LPL “bridging” activity. For the first time we followed virus
cell entry by immunoelectron microscopy using anti-envelope and anti-core antibodies. These analyses demonstrated rapid internalization of
virus particles into hepatoma cells and their presence in vesicles and in association with lipid droplets. In the presence of LPL, HCV was retained
at the cell surface. In conclusion, LPL acts as natural inhibitor of HCV infectivity by dual mechanisms: lipolytic modification of triglycerid
rich lipoproteins associated with virus particles but mainly by the bridging function, which may act in concert or in parallel with lipolysis.
Together, these two functions of LPL lead to the retention of the virus at the cell surface, resulting in a significant decrease in infection levels.
Contact: Agata Budkowska
Hepacivirus and Innate Immunity, Pasteur Institute, Paris, France
[email protected]
POSTERS
Lipoprotein lipase inhibits hepatitis C virus infection by blocking virus cell entry
POSTERS
P8.07
Mechanism of stimulation of hepatitis C virus RNA translation by microRNA-122
Dagmar Goergen (1), Dominik Conrad (1), Florian Giering (1), Michael Niepmann (1)
(1) University of Giessen, Giessen, Germany
Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5´-untranslated region (5´-UTR). HCV
propagates preferentially in the liver, and its translation is stimulated by the liver-specific microRNA-122 (miR-122). This stimulation is
effective in living cells as well as in rabbit reticulocyte lysate (RRL). Here we show that the modes of miR-122 action on HCV translation are
fundamentally different in living cells compared to RRL. In cells, a duplex miR-122 precursor is required for HCV translation stimulation.
In contrast, in RRL only the single-stranded mature miR-122 guide strand is effective in translation stimulation. Also RNA oligonucleotides
shorter than a typical microRNA stimulate HCV translation in RRL. Binding of these single-stranded RNA oligonucleotides displaces an
inhibitory long-range HCV RNA-RNA interaction that can form between the region containing the two miR-122 target sites and a RNA
sequence located in the Core protein coding sequence. In contrast, in transfected cells this interference of miR-122 with the inhibitory longrange RNA-RNA interaction plays a negligible role. In cells, slight increases in the distance between miR-122 target sites and the IRES impair
miR-122 mediated translation stimulation, and only duplex miR-122 precursors of the correct length stimulate HCV translation, suggesting
that in cells microRNA-protein complexes are involved in HCV translation stimulation by miR-122.
Contact: Michael Niepmann
University of Giessen, Giessen, Germany
[email protected]
P8.08
Normalization of translation readouts after transfection of cells: the urgent need for an
independent method monitoring transfection efficiency
Christiane Bung (2), Carmen Fehr (2), Dagmar Goergen (2), Juliane Hirnet (1), Michael Niepmann (2)
(1) Boston University School of Medicine, USA
POSTERS
In studies investigating the efficiency of cis-elements involved in translation regulation, there should be an internal control that can be used
for correcting and normalizing well-to-well variations in cell density and transfection efficiency. However, any RNA used as cotransfected
control also interacts with the translation machinery and thus may interfere with translation of the RNA actually under investigation.
We used RNAs containing the 5´- and 3´-untranslated regions (UTRs) of Hepatitis C Virus (HCV) RNA coupled to a firefly luciferase (Fluc)
reporter gene as RNAs of interest, and a capped and polyadenylated Renilla luciferase (Rluc) RNA as cotransfected normalization reporter.
We found that both RNAs mutually interfere in translation in trans. The nature of the HCV Fluc reporter RNA could have an influence on
the expression of the Rluc reporter RNA. In turn, also the presence of the Rluc RNA had an influence on HCV reporter RNA expression.
Moreover, the expression of both RNAs varied strikingly during different cell cycle phases. Thus, care must be taken when normalizing the
results of translation experiments to the expression of a cotransfected reporter RNA. These results point to an urgent need for a reagent
that measures transfection efficiency independently.
[email protected]
224 : HCV 2011
P8.09
Hepatitis C virus RNA translation stimulation coincides with expression of
microRNA-122 and argonaute protein during cell cycle
Carmen Fehr (1), Dominik Conrad (1), Michael Niepmann (1)
Hepatitis C Virus (HCV) replicates preferentially in the liver, and in most cases the HCV infection becomes chronic and often results in
hepatocellular carcinoma. When the HCV plus-strand RNA genome has been delivered to the cytosol of the infected cell, its translation
is directed by the Internal Ribosome Entry Site (IRES) in the 5´-untranslated region (5´-UTR) of the viral RNA. Thereby, IRES activity is
modulated by several host factors. In particular, the liver-specific microRNA-122 (miR-122) interacts with two target sites in the HCV 5´UTR and stimulates HCV translation, a process that most likely contributes to HCV liver tropism. Here we show that HCV IRES-dependent
translation efficiency in the hepatoma cell line Huh7 is highest during the G0 and G1 phases of the cell cycle but significantly drops during
the S phase and even more in the G2/M phase. The additional stimulation of HCV translation by miR-122 works best during the G0, G1 and
G2/M phases but is lower during the S phase. Moreover, the levels of miR-122 and the levels of Argonaute-2 (Ago-2) protein are lowest
in the S phase. Our results suggest that miR-122 stimulation of HCV translation coincides with the availability of miRNP key components
during the cell cycle and facilitates a high expression of the HCV genome in the regular G0/G1 state of non-dividing hepatocytes in the liver.
[email protected]
P8.10
Hypervariable region 2 and the intergenotypic variable region modulate neutralization
resistance of HCV strains and virus entry properties
The HCV glycoprotein E2 interacts with the cell surface receptor CD81 and is a target of neutralizing antibodies. Glycoprotein E2 comprises
an N-terminal receptor binding domain (RBD; residues 384-661) that is connected to the transmembrane domain via a heptad repeat region
(residues 675-699). Within the RBD are three variable regions, hypervariable region 1 (HVR1; residues 384-410), HVR2 (residues 460-485)
and the intergenotypic variable region (igVR; residues 570-580). HVR1 elicits type specific neutralizing antibodies that drive hypermutation
and antibody escape. The evolutionary pressures driving mutation in HVR2 and the igVR are unknown. In this study we investigated the roles
of HVR1, HVR2 and the igVR in HCV entry and neutralization by monoclonal (MAb) and polyclonal (PAb) antibodies using a domain swapping
approach. Replacement of the H77c (genotype 1a) igVR with that of Con1 (genotype 1b) enhances virus entry by approximately 10-fold,
with any one of the four amino acids distinguishing H77c and Con1 igVR sequences sufficient to enhance entry. We examined the ability of
wild-type and chimeric viruses to be neutralized by MAbs specific to defined epitopes present on the core domain of E2. Replacement of
the H77c HVR2 with that of Con1 conferred resistance to neutralization by MAb24, directed to residues 407-424. By contrast replacement of
the H77c igVR with that of Con1 conferred resistance to neutralization by MAb44, directed to residues 512-529. Interestingly, examination
of patient H sequences isolated after 1990 revealed a single amino acid substitution within the igVR (L580R) that conferred resistance to
neutralization by MAb44 but not MAb24. The Con1 HVR1 and HVR2 sequences also conferred neutralization resistance to PAb. These results
assign new roles for HVR2 and the igVR in regulating the ability of HCV strains to be neutralized by antibody and in the case of the igVR, in
modulating virus entry properties.
Contact: Heidi Drummer
Burnet Institute, Melbourne, Australia
[email protected]
POSTERS
Kevin Tewierek (1), Irene Boo (1), Yousef Alhammad (1), Patricia Vietheer (1), Pantelis Poumbourios (1), Heidi Drummer (1)
(1) Burnet Institute, Melbourne, Australia
POSTERS
P8.11
Isolation of a highly infectious hepatitis C virus with adaptive mutations
Masayoshi Fukasawa (1), Yoshitaka Shirasago (1), Kyoko Saito (1), Yuko Murakami (1), Hidesuke Fukazawa (1), Tetsuro Suzuki (2), Ryosuke
Suzuki (1), Takaji Wakita (1), Kentaro Hanada (1), Jo Chiba (3)
(3) Tokyo University of Science, Japan
In this study, we have isolated and characterized a highly infectious hepatitis C virus (HCV) strain with adaptive mutations. By repeated
infections of naïve Huh7.5.1 cells with a parental HCV-JFH1 strain, we had an HCV-JFH1 variant with enhanced cytotoxicity against the host
cells. This variant showed ~1,000-fold higher infectivity than the wild type HCV-JFH1. By sequencing of the virus genome, two mutations
were found in the variant genome: K74T in the core protein and I414T in the N-terminal region of E2 protein. K74T/I414T mutations in the
variant were very stable after further repeated infections, and the higher infectivity of the variant was also preserved. Thus, this highly
infectious HCV-JFH1 variant with K74T/I414T adaptive mutations would be a useful tool for studying the molecular mechanism of HCV life
cycle. Recombinant HCV-JFH1 having I414T or K74T/I414T mutations showed ~100- and ~1,000-fold higher infectivity than wild-type HCVJFH1, respectively. However, there was no significant difference in the replication activity of HCV replicon RNA among the wild-type, K74T,
I414T and K74T/I414T replicon-harboring cells. The entry assay with pseudotyped HCV particles carrying a luciferase reporter gene showed
that I414T pseudoparticles had ~6-times higher luciferase activity than wild-type ones. These results suggested that the entry step, not the
replication step, of HCV life cycle was at least promoted in HCV-JFH1 strains having I414T or K74T/I414T mutations.
When the wild-type or K74T core protein was transiently expressed in Huh7.5.1 cells, K74T-core protein showed prolonged accumulation
in cells, compared with the wild-type core protein. K74T-core protein was less sensitive to the proteasome inhibitor MG-132. These result
suggested that K74T-core protein is more resistant to the proteasome-mediated protein degradation.
Contact: Masayoshi Fukasawa
[email protected]
P8.12
Effect of genetic variation in the occludin gene on its co-receptor function for HCVcc
POSTERS
Sandra Ciesek (1), Sandra Westhaus (3), Christoph Sarrazin (3), Nabila Hamdi (2), Ahmed Abdelaziz (2), Christian Strassburg (3), Heiner
Wedemeyer (3), Michael Manns (3), Thomas Pietschmann (1), Thomas von Hahn (3)
(2) The German University, Cairo, Egypt
(3) Medizinische Hochschule Hannover, Hannover, Germany
Hepatitis C Virus (HCV) is characterized by a narrow host range and high inter-individual variability in the clinical course of infection. Both
traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component
occludin (OCLN) is essential for HCV entry into host cells and differences between human and murine OCLN are thought to account in part
for the inability of HCV to infect mice and hence preclude their use as a convenient small animal model. This study assesses the impact of
genetic variation in OCLN on cell culture grown HCV (HCVcc) using a newly generated and characterized subclone of the Huh-7.5 cell line
harboring an shRNA targeting all known occludin transcripts and hence being highly resistant to HCV entry while retaining the ability to
support HCV replication and assembly. This cell line was used to functionally characterize OCLN variants. We report the frequency of coding
non-synonymous single nucleotide polymorphisms, i.e. polymorphisms resulting in amino acid exchanges, present in the human population
and found that the L233S variant had an allele frequency of 2.5% in Caucasians while two other variants were very rare. However, all variants
retain the ability to function as HCVcc (co-)receptors in vitro. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with
about 5-fold reduced efficiency compared to human OCLN. This reduction in efficiency is due solely to two amino acid residues previously
identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLNlow cell line to show that HCV spread between
neighboring cells is strictly dependent on OCLN.
Contact: Thomas von Hahn
Medizinische Hochschule Hannover, Hannover, Germany
[email protected]
226 : HCV 2011
P8.13
Panning for gold: a screen for hepatitis C virus entry inhibitors
Vanessa Cowton (1), Sarah Beattie (1), Allison Marty (1), Ania Owisianka (1), Arvind Patel (1)
The entry step of HCV infection is a multi-stage process. Virus entry involves cell attachment, receptor interaction, cellular uptake by clathrinmediated endocytosis and fusion of the viral and endosomal membranes to release the viral genome. This process is co-ordinated through
cellular signalling events. The entry phase of the viral life cycle is an attractive target for antiviral therapy.
We have developed a cell-based assay using a novel reporter cell line for screening small-molecule compounds to identify inhibitors of HCV
entry. We will present the findings of a pilot screening study using a kinase inhibitor library. Cellular kinases are key mediators of signal
transduction and this group of molecules has proven to be very druggable. We have identified a number of compounds of interest with IC50
values below 1uM. In addition, the screening process has been expanded to assess a library of known bioactive compounds. Our studies to
elucidate further the underlying mechanisms of inhibition are ongoing. These data not only identify compounds with antiviral potential but
also inform our understanding of the signalling events involved in the HCV entry process.
Contact: Vanessa Cowton
[email protected]
P8.14
The role of HCV non-structural protein 5A (NS5A) in modulating viral protein translation
HCV NS5A protein, which is essential for viral replication, has been implicated in the modulation of viral translation. However, the role NS5A
plays in viral translation remains controversial as contradictory studies have been published suggesting that NS5A either stimulates, inhibits
or has no effect on viral translation. Possible reasons for these discrepancies include the use of reporter constructs that lack the HCV 3’UTR,
where NS5A binds to the poly-U/UC region, as well as use of plasmid encoded reporters that do not accurately reflect the role of the 3’UTR
in HCV translation.
In this study, we investigated the role of NS5A in the modulation of HCV translation and the function of the 3’UTR poly-U/UC region in this
modulation. We used monocistronic RNA reporter constructs containing the 5’ and 3’UTRs of HCV and an internal Renilla luciferase gene
in combination with an NS5A expression plasmid. The HCV 5’UTR contains the internal ribosome entry site (IRES) that drives expression of
the internal Renilla luciferase gene, which can be quantified as a measurement of HCV-IRES mediated translation. A ∆poly-U/UC construct
is used to investigate the role of this region.
Our results showed that NS5A down-regulates viral translation in a dose-dependent manner that requires the presence of the poly-U/
UC region of the viral 3’UTR. Furthermore this modulation appears to compete with the cellular factor IGF2BP1, which enhances viral
translation, for the effect on HCV translation. In addition, the viral NS5B protein appears to reduce NS5A down-regulation of viral translation,
possibly by binding to it and preventing NS5A from binding to the 3’UTR.
In conclusion, HCV NS5A plays an important role in the modulation of viral translation and the poly-U/UC region of the viral 3’UTR is
necessary for this modulation, suggesting that NS5A binding to this region leads to down-regulation of viral translation
Contact: Brett Hoffman
Vaccine and Infectious Disease Organization, Saskatoon, Canada
[email protected]
POSTERS
Brett Hoffman (1), Qiang Liu (1)
(1) Vaccine and Infectious Disease Organization, Saskatoon, Canada
POSTERS
P8.15
Primary human hepatocytes to investigate HCV infection in vitro: optimization of a
relevant cell-culture model
Claire Gondeau (1), Philippe Briolotti (1), Francia Razafy (1), Dominique Larrey (2), Francis Navarro (3), Antonio Sa-Cunha (4), Sylvain
Laporte (5), Patrick Maurel (1), Martine Daujat-Chavanieu (1)
(1) INSERM U1040 - Institut de Recherche en Biotherapie, Montpellier, France
(2) Hepato-Gastroenteroly, Hopital Saint Eloi, Montpellier, France
(3) Digestive Surgery and Liver Transplant., Hopital St Eloi, Montpellier, France
(4) Digestive Surgery, Hopital Haut-Leveque, Pessac, France
(5) Visceral and Digestive Surgery - CHU Nimes, France
Several recent studies point out the importance of primary human hepatocytes (PHH) in culture as a model for hepatitis C virus (HCV)
studies: despite of different experimental conditions and varying performances from a laboratory to another, this in vitro system remains
clearly the one that most closely mimics the physiological situation. We previously showed that PHH isolated from specimens of surgical
liver resections can be successfully infected by HCV-positive sera (HCVser) and cell-culture particles produced in hepatoma cells (HCVcc/
JFH1). For both cell/virion combinations, we demonstrated that a complete viral cycle is achieved and that the interferon signaling pathways
are active. Unlike hepatoma cell lines, PHH do not proliferate in our cell-culture conditions: kinetic studies allowed us to show that the cell
monolayer organization varies from day 1 to day 7 post-seeding, especially with the appearance of functional biliary canaliculi. We analyzed
the link between HCV infection, cell polarization, the host receptors expression level and their spatial distribution. We then defined optimal
culture conditions used to evaluate the infectivity of more than 80 HCV-positive sera. Some of the highly infectious strain identified (mainly
genotype 3a) could allow long-term studies in culture (more than 15 days post-infection). Moreover, the HCVser/PHH system could be an
interesting tool to select new HCV clones able to replicate in hepatoma cell lines.
Contact: Claire Gondeau
INSERM U1040 - Institut de Recherche en Biotherapie, MONTPELLIER, France
[email protected]
P8.16
Uncovering host and viral genetic factors regulating HCV dissemination
Luke Meredith (1), Garrick Wilson (1), Helen Harris (1), Michelle Farquhar (1), Peter Balfe (1), Jane McKeating (1)
(1) University of Birmingham, Birmingham, United Kingdom
POSTERS
HCV poses a global health problem, with over 170 million chronically infected individuals worldwide at risk of developing progressive liver
disease, including cirrhosis and hepatocellular carcinoma. The major reservoir supporting HCV replication is hepatocytes and the ability of a
virus to spread within a host is a key determinant of its virulence. We recently demonstrated that HCV can disseminate by cell-free particle
diffusion or via direct contacts between infected and naïve hepatocytes. Cell-to-cell infection provides an efficient route for the virus to
disseminate and to evade host immune responses. We have developed our previously published co-culture assay to quantify single-cycle
early infection events to study the host cell receptors and pathways defining cell-to-cell infection. Filopodia expressing the major attachment
factors CD81 and scavenger receptor BI (SR-BI) were detected between the basal surface(s) of cultured hepatocytes in vitro, suggesting a
possible route for cell-contact dependent HCV spread. However, infection was only observed after a 2h co-culture of HCV infected producer
and target cells coincident with the localization of E-cadherin at cell-cell contacts, arguing for an adherens junction-dependent process.
Chimeric JFH-1 viruses expressing patient derived glycoproteins show different dissemination kinetics and dependence on co-receptor
SR-BI, highlighting a role for both the viral glycoproteins and host SR-BI expression levels in defining particle dissemination. Furthermore,
in contrast to cell-free particle infection, cell-to-cell infection occurs independent of intravesicular pH, suggesting different maturation
processes for cell-free and cell-to-cell infection. Importantly, cell-free and cell-to-cell routes of HCV infection show different sensitivities
to immune modulators such as interferon alpha, supporting the importance of cell-to-cell infection in immune evasion. In summary, these
studies show different maturation processes required for the genesis of infectious virus that disseminate via cell-free and cell-to-cell routes
and highlight the heterogeneity of infectious particles.
Contact: Luke Meredith
University of Birmingham, Birmingham, United Kingdom
[email protected]
228 : HCV 2011
P8.17
Modeling viral kinetics in vitro reconciles disparate observations of claudin-1
expression on cells following hepatitis C virus (HCV) infection
Pranesh Padmanabhan (1), Narendra Dixit (1)
(1) Indian Institute of Science, Bangalore, India
Recent studies made disparate observations of claudin-1 (CLDN1) expression on cells after HCV infection in vitro. Reynolds et al. (Hepatology
47:418-27, 2008) reported a significant increase in CLDN1 expression on infected cells compared to uninfected cells, suggesting upregulation
of CLDN1 after infection. In contrast, Liu et al. (J Virol. 83:2011-14, 2009) found that the mean CLDN1 expression decreased following
infection, suggesting downregulation of CLDN1 after infection. We hypothesized that viral kinetics and not HCV-induced regulation of CLDN1
expression may underlie these disparate observations: CLDN1 expression varies across cells in culture. Cells with higher CLDN1 expression
may be more susceptible to infection. Consequently, following exposure to HCV, cells with higher CLDN1 expression are more likely to
be infected, so that random sampling of uninfected and infected cells is likely to yield higher CLDN1 expression on the latter cells. At the
same time, the population of infected cells may grow slower than uninfected cells, due in part to virus-induced cytopathicity, which would
manifest as a shift of the overall CLDN1 expression to lower levels. To test this hypothesis, we constructed a mathematical model of HCV
kinetics in vitro mimicking experiments where cells with a known distribution of the CLDN1 expression level are exposed to HCVcc. We
divided the cells into subpopulations, each with a distinct expression level of CLDN1, and constructed dynamical equations to describe the
time-evolution of each of these subpopulations, recognizing that susceptibility to infection increases with CLDN1 expression. Indeed, we
found that random samples of infected cells had significantly higher CLDN1 expression than uninfected cells at any time post-exposure and
that the mean CLDN1 expression decreased with time. Our model thus reconciles the above conflicting observations of CLDN1 expression
following HCV infection and suggests that an HCV superinfection block in vitro may not be due to HCV-induced CLDN1 downregulation.
Contact: Pranesh Padmanabhan
Indian Institute of Science, Bangalore, India
[email protected]
P8.18
Samuel French (1), Ronik Khachatoorian (1), Edna Maio (1), Edna Maloney (1), Vaithilingaraja Arumugaswami (2), Ren Sun (3), Asim
Dasgupta (4)
(1) Department of Pathology and Laboratory Medicine at UCLA, Los Angeles, CA, USA
(2) Department of Surgery, Cedars-Sinai, Los Angeles, USA
(3) Department of Molecular and Medical Pharmacology at UCLA, Los Angeles, USA
(4) Department of Microbiology, Immunology, and Molecular Genetics at UCLA, Los Angeles, USA
NS5A is thought to be a key regulator of the hepatitis C virus life cycle including RNA replication, assembly and translation. We and others
have previously shown NS5A to augment HCV IRES-mediated translation. Further, treatment with the heat shock protein synthesis (HSP)
inhibitor Quercetin and knockdown of HSP70 inhibit IRES-mediated translation as measured by a bicistronic reporter construct implicating
HSP70 in this process. Viral infection is also inhibited in these conditions. We have also coimmunoprecipitated NS5A with HSP70 and
demonstrated cellular colocalization leading to the hypothesis that NS5A/HSP70 complex formation is important in HCV IRES mediated
translation. Here we identify the region in NS5A responsible for complex formation through in vitro deletion mutant analysis. Deletion of
domains II and III failed to reduce binding, whereas deletion of domain I eliminated complex formation. Domain I alone also bound HSP70.
Fine deletion mapping of domain I identified a 34 aa element (C34) responsible for the interaction. Further, addition of this element to
domains II and III restored complex formation. Introduction of C34 into the HCVcc viral cell culture system reduced cellular viral protein levels
in contrast to control peptides of the same size from other areas of NS5A. C34 also inhibited NS5A augmented IRES-mediated translation
and controls did not. Scanning alanine mutagenesis identified an exposed beta-sheet in C34 to be primarily responsible for NS5A driven
IRES-mediated translation. These results support the NS5A/HSP70 complex mediated IRES translation hypothesis and indentify a sequence
specific element in NS5A responsible for complex formation. Identification of this element will allow for further interrogation of NS5A
mediated IRES activity, sequence specific heat shock protein recognition and rational drug design.
Contact: Samuel French
Department of Pathology and Laboratory Medicine at UCLA, Los Angeles, CA, USA
[email protected]
POSTERS
The NS5A/HSP70 Complex facilitates hepatitis C viral IRES-mediated translation
POSTERS
P8.19
Receptor endocytosis defines hepatitis C virus entry
Michelle Farquhar (1), Ke Hu (1), Helen Harris (1), Christopher Davis (1), Claire Brimacombe (1), Joshua Rappoport (1), Peter Balfe (1), Jane
McKeating (1)
(1) The University of Birmingham, United Kingdom
Hepatitis C virus (HCV) entry is dependent on host cell expression of tetraspanin CD81, scavenger receptor BI and the tight junction proteins
Claudin-1 and Occludin. HCV infects hepatocytes via a low pH dependent endocytic process and fusion occurs within early endosomes.
Many viruses cooperatively endocytose with their cognate receptors, HCV binds CD81, however, subsequent trafficking events are currently
unknown. We demonstrate that HCV or anti-CD81 antibody ligation promotes CD81 endocytosis in a dynamin and clathrin-dependent
manner. CD81 does not contain a recognized GYxxØ endocytic motif and deletion of its C-terminal domain has minimal impact on protein
endocytosis or viral receptor-activity, suggesting that associating proteins define or regulate CD81 and virus endocytosis. CD81 associates
with Claudin-1 and we previously demonstrated that receptor complexes play a role in HCV infection. Current experiments demonstrate
that ligation of either receptor with infectious HCV particles or specific antibodies promotes CD81 and Claudin-1 co-internalization. Live cell
confocal microscopy demonstrates the fusion of CD81-Claudin-1 containing intracellular vesicles with Rab5 expressing early endosomes,
supporting a role for virus-receptor complexes in the fusion of viral and endosomal membranes.
[email protected]
P8.20
Identification of a cysteine residue in E1 glycoprotein that determines particle
assembly and alters CD81 and heparan sulphate receptors dependency
George Koutsoudakis (1), Sofía Pérez-del-Pulgar (1), Jakub Dragun (1), Mairene Coto-Llerena (1), Laura Mensa (1), Xavier Forns (1)
(1) IDIBAPS, Hospital Clínic, CIBERehd, Barcelona, Spain
POSTERS
Hepatitis C virus (HCV) JFH1-intergenotypic chimeras need cell culture adaptive mutations in order to increase viral particle assembly and
release. We have created a genotype 1b/2a chimera between the Barcelona HCV1 (BHCV1, genotype 1b) and the JFH1 (genotype 2a)
isolates (BHCV1/JFH1). Upon adaptation, this virus presented 1 adaptive mutation at a cysteine residue of the E1 glycoprotein at position
306: C306R. E1 possesses 8 cysteine residues critical for its structure due to disulphide bridges. This mutation was accompanied by E2
compensatory mutations, most of them clustered at E2 hypervariable region 2. After 10 passages, Huh7.5 cells were producing BHCV1/
JFH1 viruses harbouring all the adaptive mutations. Adaptation to cell culture led to increased virus production compared to wild-type
chimeric viruses and resistance to anti-CD81 and heparin neutralization. For further examination, we created by reverse genetics, HCV
pseudoparticles (HCVpp) harbouring the C306R mutation for the BHCV1 and the J6 (genotype 2a) isolates. These HCVpp were less infectious
than wild-type ones, nevertheless still resistant to anti-CD81 and heparin neutralization. Furthermore, we created BHCV1/JFH1 and J6/JFH1
chimeric viruses (HCVcc) encompassing the E1 mutation and we subjected these viruses to replication and infection studies. C306R mutation
did not affect replication neither of both chimeric viruses nor to a luciferase-carrying reporter variant of the J6/JFH1 virus. However, particle
production was completely abrogated intra – and extracellularly. In summary, these results suggest that the assembly mechanism between
HCVpp and HCVcc may differ. Additionally, a highly conserved cysteine residue at E1 glycoprotein is critical for virus assembly which disrupts
the dependency of the virus to the CD81 and heparan sulphate receptors. These data shed more light to the adaptation flexibility of the
virus in cell culture and to the complex interplay between HCV assembly and viral receptors.
Contact: George Koutsoudakis
IDIBAPS, Hospital Clínic, CIBERehd, Barcelona, Spain
[email protected]
230 : HCV 2011
P8.21
Variation of HCV infectivity and HVR1 quasispecies selection are affected by HCV E1E2
synonymous mutation
Kazunori Kawaguchi (1), Masao Honda (1), Takayoshi Shirasaki (1), Suzanne Emerson (2), Purcell Robert (2), Shuichi Kaneko (1)
(1) Gastroenterology / Kanazawa University, Kanazawa, Japan
(2) LID/NIAID/NIH, USA
Aims: We previously found that HCV virion dominancy between hyper-variable region 1 (HVR1) quasispecies were affected by E1E2 amino
acid changes outside of HVR1 rather than inside HVR1. However sequence analysis showed infectivity discrepancy between HCV clones with
different synonymous mutations even if these clones were same amino acid sequences. Since synonymous mutations were discussed only
for genomic diversity, we analyzed the effect of these mutations between two dominant HVR1 quasispecies.
Methods: The entire E1E2 region of the two major HVR1 quasispecies from H77 patient plasma using H77/JFH1 chimera clone that dominated
at various times during serial passage were sequenced to identify new amino acid mutations. We observed synonymous mutations in E1E2
region between two dominant species and analyzed distribution and frequency in picked HCV clones construct.
Results: We could observe infectivity discrepancy between two same amino acid constructs with one E1 adaptive mutation of H77/JFH1
chimera clones. One is mutagenized this adaptive mutation and the other is acquiring adaptive mutation after serial passage by ligating
E1E2 qusispecies of H77 plasma samples into H77/JFH1 chimera. The latter infectivity was around double compared with mutagenized
one. Inoculated dominant species of E1E2 sample from H77 plasma which disappeared after long term culture showed that synonymous
mutations were broadly distributed but these area was limited in 5’ end of E1 and HVR1 to 3 regions in E2 after several passages, while in
the second dominant quasispecies synonymous mutations were distributed entire E1E2 region even after several passages.
Conclusion: Synonymous mutation act not only just showing HCV genomic diversity but influencing HCV infectivity and dominancy
between quasispecies.
Contact: Kazunori Kawaguchi
Gastroenterology / Kanazawa University, Kanazawa, Japan
[email protected]
P8.22
Julie Potel (3), Patrice Rassam (1), Claire Montpellier (3), Cyril Couturier (2), Birke Andrea Tews (3), Costin-Ioan Popescu (4), Jean
Dubuisson (3), Pierre-Emmanuel Milhiet (1), Laurence Cocquerel (3)
(1) CBS, CNRS-UMR5048, INSERM-U1054, Université Sud de France, France
(2) INSERM-U761, Université Lille 2, France
(3) Center for Infection & Immunity of Lille / CNRS-8204 / INSERM-U1019, Lille, France
(4) Institute of Biochemistry of the Romanian Academy, Romania
CD81 is a major receptor for Hepatitis C Virus (HCV). This protein belongs to the tetraspanin family whose members form dynamic clusters
with numerous partner proteins and with one another, forming tetraspanin-enriched microdomains (TEM). The direct interaction between
tetraspanins and their partner proteins results in the modulation of their functions. Indeed, TEM-associated CD81 is not used by HCV for its
entry. In most cell lines, CD81 is associated with EWI-F and EWI-2, two members of the EWI family. Moreover, we have identified EWI-2wint,
a cleavage product of EWI-2 that still interacts with CD81 and CD9. In contrast to full-length EWI-2, EWI-2wint inhibits the binding of HCV
envelope glycoproteins to CD81, thus inhibiting viral entry by a mechanism that remained to be elucidated.
In our study, we generated HCV permissive cell lines expressing murine CD81 (mCD81) in combination with EWI-2wint or control proteins.
Thanks to antibodies, which preferentially recognize TEM-associated mCD81, we found that EWI-2wint might inhibit HCV entry by promoting
CD81 association with TEM. To strengthen this hypothesis, we analyzed the behavior of human CD81 molecules by single-molecule tracking
experiments. We defined the diffusion rate and the type of motion of single CD81 molecules in presence or in absence of EWI-2wint.
Interestingly, we found that EWI-2wint induces a decrease in the global diffusion of CD81 molecules due to an increase in the proportion
of confined trajectories. Our results indicate that a change in membrane partitioning of CD81 occurs in presence of EWI-2wint, suggesting
that HCV entry is likely a highly dynamic process. We are currently testing the effect of EWI-2wint on CD81-Claudin-1 complexes that are
essential for viral entry. Together, our results may open the way to the understanding of the mechanism by which HCV enters into target
cells and how EWI-2wint inhibits this step.
Contact: Birke Andrea Tews
Center for Infection & Immunity of Lille / CNRS-8204 / INSERM-U1019, Lille, France
[email protected]
POSTERS
Dynamic of HCV entry inhibition by EWI-2wint
POSTERS
P8.23
Identification of (-)-epigallocatechin gallate as a new inhibitor of hepatitis C virus entry
Noémie Calland (1), Anna Albecka (1), Sandrine Belouzard (1), Czeslaw Wychowski (1), Véronique Descamps (2), Gilles Duverlie (2), Jean
Dubuisson (1), Yves Rouillé (1), Karin Séron (1)
(1) Center for Infection and Immunity of Lille, Lille, France
(2) Université de Picardie Jules Verne and CHU, Amiens, France
With the current clinical development of direct acting antivirals (DAAs) targeting HCV replication, it remains essential to identify drugs
targeting other steps of the viral life cycle, like virus entry or assembly. Combinations of DAAs should indeed improve viral response rates
and therapeutic success in the absence of interferon alpha.
Here, we identified (-)-epigallocatechin gallate (EGCG) as a new inhibitor of HCV entry. EGCG is a flavonoid from the subclass of catechins,
which is present in green tea extract. This molecule has been described among other properties to impair cellular lipid metabolism. By
time addition assays, we demonstrate that EGCG at 50 µM inhibits HCV infectivity by more than 90% at an early step of the viral life cycle.
This inhibition is specific to HCV and was not observed with other Flaviviridae tested. The antiviral activity of EGCG on HCV was confirmed
with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, we demonstrated that
EGCG acts directly on HCV virion probably by targeting E1 and/or E2 and that it affects a very early step in virus entry. Importantly, a HCV
mutant resistant to EGCG was isolated and mutations in E1 and E2 were identified. Furthermore, we showed that EGCG has no effect on viral
replication and virion secretion. Finally, we also showed that EGCG inhibits HCV cell-to-cell spread and is able to clear HCV from cell culture
supernatant. In conclusion, EGCG is a new interesting anti-HCV molecule that could be used in combination with other DAAs. Furthermore,
it will be a major tool to further dissect the mechanisms of HCV entry into the hepatocyte.
Contact: Noémie Calland
Center for Infection and Immunity of Lille, Lille, France
[email protected]
P8.24
Active role of E1 in the conformation of E1E2 and in the exposition of receptor
binding domains
Florian Douam (1), Guillemette Maurin (1), Judith Fresquet (1), François-Loïc Cosset (1), Dimitri Lavillette (1)
(1) INSERM U758, 69007 Lyon, France
POSTERS
The precise functions of HCV E1 and E2 envelope glycoproteins during cell entry process remain poorly defined. Although many efforts have
focused on the role of conserved domains, we aimed to identify the role of less conserved domains that are involved in “cross-talks” within
the E1E2 heterodimer through its conformational change during entry. Using E1E2 complexes harboring E1 and E2 from different genotypes,
which induced suboptimal cell entry, we previously identified E1 N-terminal and trans-membrane domains motifs crucial for HCV entry. In
this study, we characterized alternative sub-optimal E1E2 heterodimers but carrying heterologous E1 and E2 proteins constructed from
two strains belonging to the same genotype (1a) and harboring no differences in the previously identified E1 motifs. We generated several
chimeras or point mutants aiming to restore their functionality in HCVpp and HCVcc assays. Through correlation of assays to characterize
E1E2 heterodimerization, receptor binding and cell entry, we show that a motif consisting of three residues in E1 ectodomain and four
residues in E2 domain III is involved in cross-talks between the two glycoproteins during entry and thus, is important for the functionality
and global conformation of the E1E2 heterodimer. Then, using inhibition assays with soluble CD81, receptor-specific antibodies and E2specific antibodies targeting CD81-binding site, we found that the sub-optimal heterogeneous E1E2 complexes have increased exposure
of the CD81-binding site. Moreover, we found that restoring the heterodimer functionality through mutations in E1 reduced the exposure
of the CD81-binding site. Altogether, the functional characterization of the interaction between these domains and the inhibition assays
highlighted an active role of E1 in modulating the interaction of E1E2 with CD81 and entry. We are currently investigating the involvement
of the identified “cross-talks” between specific E1E2 residues in other steps of cell entry.
Contact: Florian Douam
INSERM U758, 69007 Lyon, France
[email protected]
232 : HCV 2011
P8.25
Impact of primary human hepatocytes polarity on hepatitis C virus internalization: an
electron microscopy study
Mihayl Varbanov (1), Marie Perrault (1), Claire Gondeau (2), Patrick Maurel (2), Beatrice Burdin (3), François-Loic Cosset (4), Florence
Ruggiero (5), Eve-Isabelle Pecheur (1)
(1) CNRS, IBCP, Lyon, France
(2) INSERM, France
(3) UCBL, France
(4) ENS, France
(5) IGFL CNRS, France
Hepatitis C virus (HCV) mainly infects hepatocytes, which are polarized cells. The discovery of claudin-1 and occludin, components of
tight junctions (TJ), as new HCV receptors raises the question of the relationship between cell polarity and HCV entry. However the
nature of molecular actors and kinetics of HCV entry into hepatocytes remain poorly characterized. Here we used transmission electron
microscopy (TEM), first to characterize primary human hepatocytes (PHH) freshly isolated from liver biopsies. After two days in culture, TJ
appeared, defining bile canaliculi. At day three a profound reorganisation of cell-cell contacts occurred, with appearance of desmosomes
and zonula adherens forming junctional complexes. Bile canaliculi diameter increased, and luminal microvilli and secretions appeared.
Junctional complexes, crucial to maintain hepatocyte polarity, persisted at least eleven days, with apical and junctional proteins detectable
by immunofluorescence.
Second we studied HCV early entry steps into PHH and Huh-7.5.1 cells. In both cell types, virions adhered to plasma membrane via electrondense molecular bridges, internalized with virions by clathrin-dependent endocytosis. Virions were observed mainly outside hepatocytes
treated with chlorpromazine, which causes cell surface loss of coated pits and assembly of clathrin lattices on endosomal membranes.
Interestingly, mutant virions bearing mutated or devoid of any envelope glycoproteins were similarly internalized, but accumulated
intracellularly, leading to non-productive infection. This suggests that productive hepatocyte infection could be driven by late post-endocytic
events relying on HCV glycoproteins. In hepatoma cells, numerous internalization events were observed within 20 min. Fewer and delayed
events were seen in PHH. Our ongoing investigations will help define whether this could be due to the migration of HCV from basolateral
to apical membranes.
In conclusion, our TEM approach proved powerful to directly assess PHH morphology and function. Differences in HCV internalization
kinetics in hepatoma cells and PHH highlight the impact of cell polarization on virus entry.
P8.26
Expression of the HCV core+1/ARF protein in the context of the HCV replicon system
Ioly Kotta-Loizou (1), Niki Vassilaki (1), Penelope Mavromara (1)
(1) Hellenic Pasteur Institute, Athens, Greece
Previous studies from our laboratory and others have shown that HCV possesses a second functional open reading frame within the core
gene encoding the core+1/ARF protein (also known as ARFP, F, core+1), by internal translation initiation. Up to date expression of core+1/ARF
protein has been shown in transfected hepatoma cells or in an in vitro translation system. The aim of the present study was to investigate
the expression of core+1/ARF protein in the HCV JFH1 replicon system. For this purpose, luciferase-tagging experiments combined with sitedirected mutagenesis studies were carried out. Bicistronic reporter JFH1-based replicons with Firefly luciferase cloned in frame with core or
core+1/ARF sequence were constructed and used to transfect Huh7-Lunet and HepG2 cell lines. Expression of core+1/ARF-LUC protein was
observed, 48h p.t. in the replication-permissive Huh7-Lunet cells and 8h p.t. in HepG2 cells. A series of non-sense insertion mutations within
the core+1/ARF ORF have been constructed and are currently analysed. In order to investigate the molecular mechanism that controls
core+1/ARF protein synthesis, a dual luciferase reporter construct was used: nucleotides 345-591 of the core-coding sequence were cloned
between the Renilla and the Firefly luciferase genes. Huh7 cells were transfected with in vitro transcribed RNA derived from the plasmid.
Luciferase expression was assessed at 4, 8 and 24h p.t. and the results suggest the presence of a functional regulatory element within the
core-coding sequence that could direct internal translation initiation of the core+1/ARF.
Contact: Niki Vassilaki
Hellenic Pasteur Institute, Athens, Greece
[email protected]
POSTERS
Contact: Mihayl Varbanov
CNRS, IBCP, Lyon, France
[email protected]
POSTERS
P8.27
HCV quasispecies complexity and implications for the detection of viral infection
Brendan A. Palmer (1), John Menton (1), Mairead Finn (1), Jamie O’Sullivan (1), Liam J. Fanning (1)
(1) University College Cork, Cork, Ireland
The quasispecies nature of the viral population is a significant contributor to the development of chronic hepatitis C infection in humans. The
use of serum-derived HCV (sdHCV) and replicon based systems with its complex mixture of variants has served as a useful tool in the study
the natural infection of hepatocytes. The quasispecies complexity of sdHCV is reflected in both the viral genome and proteome. Mutations,
even in conserved domains, that are used as antibody epitopes in the assessment of infection and viral permissiveness may interfere with
in vitro detection of these same viral proteins. This natural phenomenon has the potential to negatively impact on the detection of sdHCV
in vitro and replicon encoded viral proteins.
Immunofluorescence detection of intracellular core protein, analysed at 24 h post-infection of Huh7 cells with sdHCV (genotype 2-5), was
achieved using the commercially available monoclonal anti-core antibody C7-50. However, the detection of core protein expressed from
the genotype 3a construct was only observed at a frequency of 5% of that noted for genotypes 2b, 4a and 5a, at 24, 48 and 72 h posttransfection. Comparative genetic analysis of the cloned core genes revealed 3 amino acid substitutions within the conserved antibody
epitope of the genotype 3a core gene clone. We reconstructed the antibody binding by serial site-directed mutagenesis to reveal that one
critical site (Val36) was the foremost contributor to the lack of antibody recognition. In summary, reverse mutagenesis of the Val36 site to
the consensus amino acid Leu36 restored the detection of core protein to 70% that of wild type levels observed for genotypes 2b, 4a and
5a. Dominance of an individual variant within a quasispecies population will lead to possible false negative observations with respect to
antibody binding. This phenomenon will likely apply to studies involving all hepatitis C virus genotypes.
Contact: Brendan A. Palmer
University College Cork, Cork, Ireland
[email protected]
P8.28
HIV fusion inhibitors derived from a flaviviridae glycoprotein
Kristin Eissmann (1), Yvonne Ködel (1), Holger Wend (1), Bernhard Fleckenstein (1), Heide Reil (1)
(1) Institute for Virology, University of Erlangen-Nuremberg, Erlangen, Germany
POSTERS
The identification of innovative drugs inhibiting viral replication is a pressing need for the treatment of chronic virus infections. For the
Human Immunodeficiency Virus (HIV), the entry process is a target that provides multiple options for therapeutic interventions. Therefore,
broad efforts are being made to develop new peptide-based or small molecule inhibitors to intervene the multi-stage entry process involving
attachment, receptor and coreceptor binding, and membrane fusion.
We studied viral interference mechanisms between HIV and a lymphotropic member of the flaviviridae, the GB Virus C (GBV-C) that uses the
same ecological niche in coinfected humans. GBV-C is a non-pathogenic ancient human virus that can replicate in T-cells and interfere with
HIV replication in vitro. HIV patients with long-term GBV-C viremia show better survival rates than HIV positive individuals without persisting
GBV-C. There are different possibilities of suppression described, but the underlying mechanisms are still undefined.
In earlier work we could demonstrate that recombinant glycoprotein E2 of GBV-C can interfere with HIV entry. An E2-peptide scanning
revealed a distinct region of the N-terminus of E2 being involved in HIV suppression. IC50 values of respective peptides are between 0.1 and
2µM with a variety of primary HIV viruses. Using an enzyme-based virus-cell fusion assay and temperature-arrested HIV-infection kinetics,
we can demonstrate that the E2-peptides do not prevent initial HIV binding but subsequent membrane fusion. Furthermore we provide
evidence that the E2-peptides directly target HIV-1 virions. Performing ELISA and FACS analyses, we identified the surface glycoprotein
complex gp120/gp41 of HIV as the binding partner.
To our knowledge, this is the first example of a flaviviral glycoprotein inhibiting the function of a retroviral glycoprotein.
Contact: Heide Reil
Institute for Virology, University of Erlangen-Nuremberg, Erlangen, Germany
[email protected]
234 : HCV 2011
P8.29
Hepatitis C virus has a genetically determined lymphotropism through a coreceptor B7.2
Keigo Machida (1), Jeffrey Huang (1), Chun-Hsiang Wang (1), Jian-Chung Liu (1), Yasuteru Kondo (1), Joel Schechter (1), Steven Foung (2),
Takaji Wakita (3), Jae Jung (1), Michael Lai (1)
(1) University of Southern California, Los Angeles, CA, USA
(2) Stanford University, USA
(3) National Institute of Infectious Diseases, Japan
HCV replicates not only in the liver but also in the B cells; however, the nature of B-cell infection by HCV has been controversial. Here, we
constructed an infectious full-length cDNA of a novel HCV (SB strain) derived from an HCV (+) B-cell lymphoma. The RNA transcript could
replicate in Raji cells, but not Huh7.5.1 cells, while the opposite was true for the hepatotropic JFH1 strain. Various chimera RNA studies
between SB and JFH1 cDNA demonstrated that the viral envelope region is primarily responsible for this preferential cell tropism. Pseudotype
retroviruses containing the lymphotropic viral envelope can infect B cells but not hepatocytes. Humanized PBMCs in immunocompromised
mice are chronically infected with SB strain. The lymphotropic strain requires CD81 and a novel immune cell–specific receptor B7.2 (CD86)
for infection. In addition, the presence of a unique 5’-UTR sequence enhances viral replication in B cells. Taken together, we have identified
a lymphotropic HCV strain with unique properties of the viral envelope, which enable HCV to infect lymphoid cells.
Contact: Keigo Machida
University of Southern California, Los Angeles, CA, USA
[email protected]
P8.30
Characterization of new hepatocellular carcinoma cell lines for the study of hepatitis C
virus infection
Background and aim: Hepatitis C virus (HCV) cell culture model is virtually restricted to the JFH-1 isolate and the human hepatoma cell line
Huh7. Since hepatocytes are the main site of viral replication in vivo and in order to expand the host cell lines to study HCV infection in vitro,
we have screened a series of cell lines derived from human malignant hepatocytes for their ability to support HCV entry.
Methods: We have developed and characterised six human cell lines (BCLC-1, 2, 3, 4, 5 and 6) derived from resected hepatocellular carcinoma
(HCC). All but one cell line (BCLC-6) were obtained from HCV-related HCC. The BCLC-6 cell line was obtained from an alcohol-related HCC,
with no serological markers of HCV infection. HCV receptor expression was determined by Western Blot and immunofluorescence. HCV
entry was assessed using HCV pseudoparticles (HCVpp).
Results: Tumor tissue fragments obtained at the time of surgical resection or liver transplantation were implanted in nude mice. After
several passages, xenografts were collected and tumoral hepatocytes isolated and cultured in vitro. BCLC cell lines present an epithelialshape and they grow as a monolayer or as colonies. Karyotype analysis confirmed the human origin, and hepatocyte lineage was confirmed
by cytokeratin profile. Western Blot analysis revealed that all of the BCLC cells expressed all HCV entry receptors CD81, SRB1 and the tightjunction proteins, claudin-1 and occludin. Immunofluorescence studies showed that HCV receptors were localized in the plasma membrane,
with minimal or none intracellular staining. In addition, all BCLC cells were highly permissive for HCVpp entry at comparable levels to Huh7.5
cells. In particular, BCLC-5 cells showed 2-fold higher infectivity than Huh7.5.
Conclusions: The BCLC cell lines express all of the essential HCV receptors and support HCVpp entry, thus becoming a useful tool to study
HCV entry in vitro.
Contact: Mairene Coto-Llerena
IDIBAPS, Hospital Clinic, Ciberehd, Barcelona, Spain
[email protected]
POSTERS
Mairene Coto-Llerena (1), Loreto Boix (1), George Koutsoudakis (1), Juan Manuel López-Oliva (1), Jordi Bruix (1), Xavier Forns (1), Sofía
Pérez-del-Pulgar (1)
(1) IDIBAPS, Hospital Clinic, Ciberehd, Barcelona, Spain
POSTERS
P8.31
Molecular Analysis of the Interaction between Hepatitis C Virus E2 Protein and Host
Entry Factor CD81
Peter Liu (1), Lilin Zhong (1), Evi Struble (1), Christine Harman (1), Maria Luisa Virata-Theimer (1), Li Ma (1), Pei Zhang (1)
(1) CBER, FDA, Bethesda, MD, USA
The interaction of hepatitis C virus (HCV) E2 glycoprotein with cellular receptors plays an important role in viral entry. Blocking HCV E2
protein-mediated viral entry can be achieved by means of an antibody binding to the host entry factor CD81 or to specific residues of HCV
E2 protein required for interaction with CD81. Understanding the mechanism for this blockage at a molecular level may provide insights
on ways to generate neutralizing immune globulins and design effective HCV vaccines. In this study, we transiently expressed soluble
truncated forms of the E2 protein of HCV H77 strain in Huh7 cells to test for their structural integrity and ability to bind to CD81. By using
immunoprecipitation assays, we observed that two truncated forms of E2 (a.a. 384-661 and a.a. 384-570) co-precipitated with the CD81
large extracellular loop (LEL). In contrast, a shorter E2 fragment (a.a. 384-509) failed to co-precipitate with CD81 under similar experimental
conditions. Consistent with the findings by others, our studies support the hypothesis that the residues 384-570 of the E2 protein are
sufficient for facilitating CD81 binding. Further site-directed mutagenesis studies will address the determinants within the E2 fragment for
the viral entry.
Contact: Pei Zhang
CBER, FDA, Bethesda, MD, USA
[email protected]
P8.32
SR-BI mediates different steps of HCV entry into cells, involving ApoE associated to
HCV particles and direct or indirect interactions with HCV E2
Viet Loan Dao This (1), Christelle Granier (1), Mirjam Zeisel (2), Jimmy Mancip (1), Ophélia Granio (1), Francois Pénin (3), Dimitri Lavillette
(1), Thomas Baumert (2), Marlène Dreux (1), F. L. Cosset (1)
(1) INSERM, Lyon, France
(2) INSERM, U748, Université de Strasbourg, France
(3) CNRS UMR 5086, Université de Lyon, France
POSTERS
HCV entry is a multi-step process. The nature of the primary interaction between HCV and the cell surface remains unclear, although
interactions with glycosaminoglycans were proposed to contribute to initial capture of virus particles to host cells. The scavenger receptor BI
(SR-BI) mediates a direct interaction with soluble forms of HCV-E2 glycoprotein, involving its amino-terminal HVR1 region; yet, no evidence
for an interaction between SR-BI and the E1E2 heterodimer is available. Ex vivo, ApoB-containing lipoproteins associated with natural serumderived HCV may play a key role in the initial interaction with SR-BI. Furthermore, we found that high-density lipoprotein (HDL), which does
not associate to HCV particles, increases entry into cells at a post-binding stage through interplay with SR-BI. Using cells expressing human
SR-BI or rodent orthologs, which do not mediate sE2 binding, and infection assays with HCVpp or HCVcc particles harbouring wild-type or
HVR1-mutated E2, we characterized the determinants of HCV-cell primary interaction in vitro. We found that SR-BI induces HCVpp entry
independent of E2-SR-BI direct interaction, through a pathway that involves its lipid transfer activity. In contrast, as shown using E2 point
mutants unable to bind SR-BI as well as SR-BI point mutants impairing selective lipid uptake and efflux, we found that HDL-mediated entryenhancement depends on E2 binding to SR-BI. Intriguingly, despite the loss of E2-SR-BI interaction, we show that human or mouse SR-BI
still binds HCVcc. Through studies of the HCVcc populations separated by density gradients and using SR-BI, ApoE, ApoB, E2 and HVR1
antibodies, and recombinant apolipoproteins, we demonstrate that ApoE, present at the surface of lipoprotein-associated HCV particles, is
a primary determinant of HCV interaction with SR-BI. Altogether, our data indicate that while they may not act as ligands determining the
initial HCV-cell interaction, the HCV glycoproteins induce crucial, SR-BI-dependent entry events required at post-binding levels.
Contact: F. L. Cosset
INSERM, Lyon, France
[email protected]
236 : HCV 2011
P8.33
Identification of single amino acids in the E2 stem region vital for hepatitis C virus entry
Thomas H.R. Carlsen (1), Troels K.H. Scheel (1), Santseharay Ramirez (1), Jens Bukh (1)
(1) Copenhagen Hepatitis C Program, Copenhagen University Hospital, Hvidovre, Denmark
The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in mediating entry through interaction with host cell receptors.
Previous studies have reported the transmembrane domain and parts of the E2 ectodomain to be involved in this process. In this study we
further characterized the E1/E2 glycoproteins function. Using a JFH1-based recombinant expressing Core-NS2 from genotype 1a isolate
H77, the E2 gene was substituted by corresponding functional sequences from genotype 1b isolates (DH1, DH5 and J4). All E2-exchanged
recombinants showed extensive eclipse phases before spread of infection after transfection of Huh7.5 cells. Sequence analyses of recovered
viruses from five independent transfections with each 1b E2 H77/JFH1 recombinant revealed, that the majority of recombinants contained
single isolate specific mutations within a five amino acid stretch in the stem region of E2. Introduction of the identified single putative
adaptive mutations into each of the corresponding 1b E2 H77/JFH1 recombinants fully restored virus viability with peak titers comparable
to controls. In addition, introduction of the mutations into the other E2-exchanged recombinants accelerated growth kinetics significantly,
although additional changes were required in the majority of recombinants for efficient production of infectious viral particles.
We did not identify any effect of the E2 mutations on replication, assembly or release, when measuring intra- and extracellular HCV Core,
infectivity titers, and particle densities. To investigate entry, HCVpp, with genotype 1a (H77) E1 and 1b (DH1, DH5 or J4) E2 genes, were
developed. The identified isolate-specific E2 mutations significantly increased entry for all chimeric E1/E2 pseudoparticles, demonstrating
the importance of the adaptive mutations for viral entry.
This study demonstrates a short 5 amino acid region of the E2 stem region to be highly important for HCV entry. Single adaptive mutations
fully restored viral entry, in most cases at an isolate specific level.
Contact: Thomas H.R. Carlsen
Copenhagen Hepatitis C Program, Copenhagen University Hospital, Hvidovre, Denmark
[email protected]
P8.34
Occludin is required for a late step in hepatitis C virus cell entry, after binding and
CD81 requirement, but before viral fusion
Hepatitis C virus (HCV) is the major cause of liver cancer worldwide. A better understanding of all the HCV life cycle steps, including entry
into a host cell, is of crucial importance to the development of HCV therapies and model systems. Although the tight junction protein
Occludin (OCLN) has been shown to be essential for HCV cell entry, little is known about how this protein mediates this event. To develop
an approach to conditionally regulate OCLN functions during HCV cell entry, we identified sites in extracellular regions of the protein where
Flag epitope sequences could be inserted without impairing HCV pseudoparticle (HCVpp) entry in naturally OCLN-deficient human renal
carcinoma 786-O cells. Flag antibodies blocked HCVpp infection of 786-O cells expressing these mutants in a specific and dose-dependant
manner. Using these antibodies in kinetic studies, we demonstrated that OCLN acts at a post-binding stage of HCV entry. Furthermore, OCLN
usage occurred soon after another HCV cell entry factor, CD81, and prior to endosomal acidification. This result supports the model that HCV
cell entry is a step-wise process where the virion sequentially uses an array of cellular proteins to gain access to the cell. We are currently
using these OCLN mutants and a variety of other HCV cell entry factor inhibitors to further define the timing of HCV cell entry events. We
are also using these mutants to explore whether OCLN interactions with viral or cellular factors are required during this process.
Contact: Marion Sourisseau
[email protected]
POSTERS
Marion Sourisseau (1), Maria Michta (1), Lum Chati Zoni (1), Benjamin Israelow (1), Sharon Hopcraft (1), Matthew Evans (1)
POSTERS
P8.35
The adaptor protein Disabled-2 mediates hepatitis C virus entry and is a potential
target for modulating viral infectivity
Ju-Chien Cheng (1), Yung-Ju Yeh (1), Meng-Hong Lu (1), Ching-Ping Tseng (2)
(1) China Medical University, Taichung, Taiwan
(2) Chang Gung University, Taiwan
The entry of hepatitis C virus (HCV) into host cells is a multi-step process that involves receptor binding and internalization. Despite
that clathrin-dependent endocytosis has been demonstrated to play a pivotal role in mediating HCV entry, little is known regarding the
component of the cargo-specific adaptor protein that modulates the assembly of clathrin-coated pits at plasma membrane upon HCV entry.
In this study, we investigated whether the cargo-specific adaptor protein Disabled-2 (DAB2) that is involved in various receptor-mediated
and clathrin-dependent endocytosis modulates HCV entry. Accordingly, the Huh7.5 cells were stably transfected with a expressing construct
encoding the short hairpin RNA of DAB2 (shDAB2) followed by infection with the HCV pseudotype virus (HCVpv). Our data revealed that
the HCVpv infectivity for DAB2 knockdown cells was approximate 50% of the vector control cells. Consistent with these observations, the
decrease in the HCV infectivity was reversed by overexpression of DAB2 but not another cargo-specific adaptor protein autosomal recessive
hypercholesterolemia (ARH). Notably, the deletion of DAB2 N-terminal phosphotyrosine binding (PTB) domain but not the C-terminal
proline-rich domain served as a dominant negative mutant and diminished further the entry of HCVpv in the DAB2 knockdown cells. Taken
together, DAB2 mediates HCV enter into host cells and could be a molecular target for modulating HCV infectivity.
Contact: Ju-Chien Cheng
China Medical University, Taichung, Taiwan
[email protected]
P8.36
Antibody and lipid correlates of outcome of acute HCV infection
Maria Isaguliants (1), Natalia Petrakova (2), Olga Znoiko (3), Kristina Dudina (2), Sergey Domogatsky (4), Juris Jansons (5), Anders Widell (6)
(1) Department of Microbiology, Tumoer, Cell Biology, Karolinska Institutet, Stockholm, Sweden
(2) Ivanovsky Institute of Virology, Moscow, Russia
(3) Moscow State University of Medicine and Dentistry, Russia
(4) IMTEK, Russia
(5) Malmö University Hospital; Biomedical Research & Study Center, Riga, Sweden
(6) Department of Medical Microbiology, Malmö University Hospital, Sweden
POSTERS
BACKGROUND HCV circulates in lipid particles and uses lipid pathway for entry; lipids play an important role in infection inhibition. We
studied these serum components in the acute and late phase of infection in patients who resolved or turned chronic. Experimental Sera
of 19 pts, 7 spontaneously resolving, were collected at 1 and ≥9 months post jaundice. Genotypes were 1b (12), 3a (4), 2 were untypable.
These and healthy control sera diluted 60, 240, 960 were tested for ability to inhibit infection with HCV 1b and 3a pseudoparticles (pp). Sera
were used intact, or were deprived of total Ig, HDL, LDL, or Ig+LDL using affinity columns. Serum lipid content and lipid ratios were assessed
by biochemical assays. Antilipid and anti-HCV envelope antibodies (Ab) were determined by ELISA.
RESULTS Acute phase sera of resolving patients diluted 1/60 inhibited 40-90% of HCVpp infection, while patients turning chronic inhibit
infection to <30% (p<0.05). There was no difference in inhibition at ≥ 9 months. Acute hepatitis C patients had increased TG/HDL, TC/
HDL and LDL/HDL that normalized in recovering patients. In multivariate regression analysis, the inhibition of HCVpp infection correlated
with high serum content of TC and TG, and high TC/HDL ratio (p<0.01) but not with serum HDL levels. Anti-lipid/lipoprotein Ab were more
prevalent in HCV patients than in controls, particularly in patients turning chronic (p<0.05). Antilipid Ab levels did not correlate with serum
lipid/lipoprotein content, or infection inhibition. Nor did HCVpp infection inhibition correlate to the levels of anti-E1, E2 or HVR1 Ab. Serum
deprivation of Ig+LDL decreased inhibition, while removal of Ig or LDL alone decreased it only moderately.
CONCLUSION Our data supports a complex role of LDL and antibodies in HCV entry, and points at metabolic correlates of HCV
infection outcome.
ACKNOWLEDGENTS: Russian Foundation for Basic Research #10-04-01402a; Swedish Institute #00747/2010.
Contact: Maria Isaguliants
Department of Microbiology, Tumoer, Cell Biology, Karolinska Institutet, Stockholm, Sweden
[email protected]
238 : HCV 2011
POSTERS
Virology: Replication and Assembly
P9.01
Development of an RNA polymerase I-driven adenoviral vector and its
application in an HCV replication assay
Takeshi Yoshida, Fumi Satoh, Watari Akihiro, Masuo Kondoh, Hiroyuki Mizuguchi, Naoya Sakamoto, Kiyohito Yagi
P9.02
Hepatitis C virus hijacks P-Body and stress granule components around
lipid droplets
Yasuo Ariumi, Misao Kuroki, Yukihiro Kushima, Kanae Osugi, Makoto Hijikata, Masatoshi Maki, Masanori Ikeda,
Takaji Wakita, Nobuyuki Kato
P9.03
miR122 facilitates replication of hepatitis C virus in non-hepatic cells
Takasuke Fukuhara, Mai Shiokawa, Chikako Ono, Hiroto Kambara, Eiji Morita, Wataru Kamitani, Yoshiharu Matsuura
P9.04
Annexins participate in the HCV RNA replication
Takayuki Abe, Takasuke Fukuhara, Eiji Morita, Yoshiharu Matsuura
P9.05
Replication of genotype 3a hepatitis C virus subgenomic replicon in cell culture
Mohsan Saeed, SuSu Hmwe, Claire Gondeau, Patrick Maurel, Takaji Wakita
HCV production requires the PML tumor suppressor protein
Misao Kuroki, Yasuo Ariumi, Masanori Ikeda, Hiromichi Dansako, Takaji Wakita, Nobuyuki Kato
P9.07
Genetic evidence for dual functions of NS5A in RNA replication
Robert Fridell, Lourdes Valera, Chunfu Wang, Dike Qiu, Melissa Kirk, Min Gao
P9.08
Regulation of HCV replication by FKBP8-dependent or -independent
Hsp90 activity
Kunihiro Kawakami, Hirotaka Kasai, Atsuya Yamashita, Nobuyuki Enomoto, Yoshiharu Matsuura, Masami Kusunoki,
Kohji Moriishi
POSTERS
P9.06
POSTERS
P9.09
Hepatitis C virus replication enhancement at low oxygen tension is
associated with higher anaerobic energy metabolism in hepatoma cells
Niki Vassilaki, Katerina I. Kalliampakou, Ioly Kotta-Loizou, Christina Befani, Panagiotis Liakos, Georgios Simos, Despina Smirlis,
Oliver Bauhofer, Marion Poenisch, Marc P. Windisch, Myungeun Lee, Ralf Bartenschlager, Penelope Mavromara
P9.10
Contrary to published models of HCV RNA replication, heat denaturation
reveals that much of the HCV in natural infections is in double-stranded form
Arielle Klepper, Francis Eng, Jose Walewski, Myron Schwartz, Thomas Schiano, Andrea Branch
P9.11
Highly sensitive and specific visualization of HCV plus- and minus-strand
RNA in vitro and in vivo
Urtzi Garaigorta, Stefan Wieland, Francis V. Chisari
P9.12
Engineering genetic suppressor elements against hepatits C virus
Zhilei Chen, Rudo Simeon
P9.13
Molecular mechanism and function of the HCV NS3 helicase
Meigang Gu, Charles Rice
P9.14
Occult HCV infections in hemodialysis patients
Ilkay Bozkurt, Bilgehan Aygen, Selma Gokahmetoglu, Orhan Yildiz
P9.15
Adaptative mutations in non structural genes that increase transcomplementation of HCV JFH1 replicon lacking E1E2 glycoproteins
Carole Fournier, Francois Helle, Véronique Descamps, Virginie Morel, Catherine Francois, Sarah Dedeurwaerder,
Czeslaw Wychowski, Gilles Duverlie, Sandrine Castelain
P9.16
Investigating the role of the cellular RNA helicase DDX3 in HCV replication
Seamus Stack, Allan Angus, Arvind Patel
POSTERS
P9.17
Disruption of a conserved disulfide bond in HCV E2 protein impacts CD81
binding and abrogates infectivity
Guaniri Mateu, Luke Uebelhoer, Jillian Whidby Freund, Daniel Claiborne, Joseph Marcotrigiano, Arash Grakoui
P9.18
Synthetic genotype 1a hepatitis C virus: reconstructing a
representative sequence
Supriya Munshaw, Lin Liu, Justin R. Bailey, Kelly Burke, Andrea Cox, Stuart C. Ray
P9.19
Geranylgeranyl transferase II is essential for HCV RNA replication
Masanori Ikeda, Midori Takeda, Yasuo Ariumi, Takaji Wakita, Nobuyuki Kato
P9.20
Hepatitis C Virus p7 is crucial for capsid assembly and envelopment
Juliane Gentzsch, Christiane Brohm, Gabrielle Vieyres, Martina Friesland, Eike Steinmann, Thomas Pietschmann
P9.21
The p7 protein of hepatitis C virus forms structurally plastic, rudimentary
ion channels
Francois Penin, Danielle E. Chandler, Roland Montserret, Eike Steinmann, Thomas Pietschmann, Klaus Schulten,
Christopher Chipot
240 : HCV 2011
VIROLOGY: REPLICATION AND ASSEMBLY
P9.22
Robust replication of primary or cultured hepatitis C virus isolates in human
liver slices: a relevant ex vivo model of liver infection
Sylvie Lagaye, Hong Shen, Jesintha Gaston, Pierre Bourdoncle, Laurent Hannoun, Pierre-Philippe Massault,
Anaïs Vallet-Pichard, Vincent Mallet, Stanislas Pol
P9.23
Replication efficiency of the non-structural 5B (NS5B) protein is increased
after initiation of antiretroviral therapy in HIV/HCV co-infected patients
Jason Blackard, Gang Ma, Christina Martin, Yupeng He, M. Tarek Shata, Kenneth Sherman
P9.24
A genetic bottleneck in HCV diversity occurs during mitosis
Brian Webster, Silke Wissing, Eva Herker, Melanie Ott, Warner Greene
P9.25
Infectious hepatitis C virus bearing genotype-1b subgenomic replicon by
trans-encapsidation with the structural regions of genotype 1b
Kazuo Sugiyama, Yuko Murakami, Takayuki Hishiki, Yuko Shimizu, Kenji Funami, Saneyuki Ujino, Hidetsugu Saito, Satoshi
Saito, Nobuyuki Kato, Toshifumi Hibi, Hiroshi Takaku
P9.26
Identification of a novel cellular RNA helicase-like protein as a target for
cyclosporin A that is involved in hepatitis C virus genome replication
Koichi Watashi, Hiroeki Sahara, Kengo Morohashi, Kazuki Iwabata, Takashi Sunoki, Kouji Kuramochi, Kaori Takakusagi,
Yoichi Takakusagi, Hiroki Miyashita, Noriyuki Sato, Atsushi Tanabe, Junpei Konno, Kunitada Shimotohno, Susumu Kobayashi,
Kengo Sakaguchi, Fumio Sugawara
P9.27
Identification of signal pathway involved in infectious hepatitis C virus (HCV)
particle production
Yuichi Abe, Takaji Wakita, Kunitada Shimotohno, Makoto Hijikata
P9.28
Genotype-specific function of hepatitis C virus glycoproteins in virus assembly
P9.29
Hepatitis C virus NS5A and the Rab1-GAP TBC1D20 colocalize on the rims of
lipid droplets
Inbar Nevo, Meital Asher, Yaakov Nahmias, Koret Hirschberg, Ella Sklan
P9.30
Reverse genetic characterization of Bovine Viral Diarrhea Virus (BVDV) NS5A
Norbert Tautz, Lars Krüger, Olaf Isken
P9.31
Biochemical analysis of NS2 topology provides evidence for three transmembrane domains
Barnabas King, Philip Tedbury, Stephen Griffin, Mark Harris
P9.32
Expression and purification of hepatitis C virus NS3 protease from
virological sustained responder and non-responder patients
Paola Provazzi, Vinícius Santana, Hamilton Cabral, Fernanda Canduri, Maurício Nogueira, Paula Rahal
P9.33
Evolution of NS5A genetic variability before, during, and after treatment in
patients infected with hepatitis C virus genotype 3a
Cintia Bittar, Ana Carolina Gomes Jardim, Paola Provazzi, Lilian Hiromi Tomonari Yamasaki, Claudia Marcia Aparecida Carareto,
João Renato Rebello Pinho, Isabel Maria Vicente Guedes de-Carvalho-Mello, Paula Rahal
POSTERS
Eike Steinmann, Juliane Dörrbecker, Martina Friesland, Sandra Ciesek, Heiner Wedemeyer, Christoph Sarrazin,
Thomas Pietschmann
POSTERS
P9.34
Biochemical and functional studies of MOBKL1B in HCV replication
Hyo-young Chung, Meigang Gu, Charles M. Rice
P9.35
Evidence for a role of p7 protein after hepatitis C virus nucleocapsid formation
Ali M. Atoom, Daniel M. Jones, Rodney S. Russell
P9.36
Identification of novel NS5A-associated proteins in the host-cell membrane
fraction and their role in HCV life cycle
Koji Goto, Toshiro Kimura, Koichi Watashi, Ryosuke Suzuki, Satoshi Yamagoe, Tatsuo Miyamura, Kyoji Moriya,
Hiroshi Yotsuyanagi, Kazuhiko Koike, Tetsuro Suzuki, Takaji Wakita, Hideki Aizaki
P9.37
Acylation of HCV core C184 residue is essential for protein maturation
Audrey Filion, Nathalie Majeau, Denis Leclerc
P9.38
A novel NS5A-core interaction: implications in HCV life cycle
Katarzyna Gawlik, Philippe Gallay
P9.39
Lactoferrin inhibits the HCV NS3 ATPase/Helicase protein which impairs
HCV replicon replication
Frederic Picard-Jean, Sabrina Bouchard, Angelique Lanthier, Martin Bisaillon
P9.40
Hepatitis C virus infection activates a novel NF-κB-independent function of
IKKα in lipid droplet biogenesis and viral assembly
Qisheng Li, Véronique Pène, Siddharth Krishnamurthy, Keng-Hsin Lan, Yun-Ping Wu, T. Jake Liang
P9.41
The hepatitis C virus RNA polymerase elongation complex: its assembly and
pre-steady-state kinetic analysis
Zhinan Jin, Vincent Leveque, Han Ma, Kenneth Johnson, Klaus Klumpp
P9.42
POSTERS
Development of a hepatocytic cell-based assay for monitoring the activity
of HCV NS3 protease
Plamena Silvieva, Tobin Dennis, Roland Wolkowicz
P9.43
Analysis of hepatitis C virus cultured in vitro indicates that culturing HCV in
different cell types affects selection of variants
Dennis Revie, Ryan Begley, Peter Henderson, John G. Prichard, Ann S. Kelley, S. Zaki Salahuddin
P9.44
Efficient HCV production system using HuH-7 subclone with high virus
assembly efficiency
Asako Murayama, Nao Sugiyama, Seiko Yoshimura, Mitsuko Ishihara-Sugano, Takaji Wakita, Takanobu Kato
P9.45
TIP47 interacts with hepatitis C virus nonstructural protein 5A and
participates in the assembly of infectious particles
Daniela Ploen, Mohamed Hafirassou, Kiyoshi Himmelsbach, Daniel Sauter, Martin Biniossek, Thomas F. Baumert,
Catherine Schuster, Eberhard Hildt
P9.46
Decreased levels of the the syntaxin-4 binding protein alpha-taxilin in
hepatitis C virus replicating cells
Daniela Ploen, Markus Heilmann, Jasmin Guhl, Kiyoshi Himmelsbach, Eberhard Hildt
242 : HCV 2011
P9.47
Infection and replication of serum-derived HCV in NS3/4A-protease
cleavage-dependent reporter cell lines
George Koutsoudakis, Sofía Pérez-del-Pulgar, Patricia Gonzalez, Gonzalo Crespo, Miguel Navasa, Xavier Forns
P9.48
Mapping the membrane topology of hepatitis C virus nonstructural protein 4B
Jérôme Gouttenoire, Pascal Roulin, Audrey Kennel, François Penin, Darius Moradpour
P9.49
The association of hedgehog pathway activity and hepatitis C virus replication
Steve Choi, Shelton Bradrick, Guan Qiang, Anahita Mostafavi, Anna Mae Diehl, Ravi Jhaveri
P9.50
Defining the hedgehog effect: probing the relationship to hepatitis C virus
Guan Qiang, Anahita Mostafavi, Mark Drinker, M. Anthony Moody, Karen Soldano, Allison Ashley-Koch, Brianna Norton,
Susanna Naggie, Ravi Jhaveri
P9.51
A role of microRNA-27a in the regulation of lipid metabolism and HCV
replication
Takayoshi Shirasaki, Masao Honda, Norihiro Tokumaru, Tetsuro Shimakami, Kazunori Kawaguchi, MinKyung Yi,
Stanley Lemon, Seishi Murakami, Shuichi Kaneko
P9.52
Impaired IFN signaling in chronic hepatitis C with advanced fibrosis stage
through TGF-β signaling pathway
Norihiro Tokumaru, Masao Honda, Takayoshi Shirasaki, Tetsuro Shimakami, Kazunori Kawaguchi, Seishi Murakami,
MinKyung Yi, Stanley Lemon, Shuichi Kaneko
P9.53
Multiple adaptive roles of the E1/E2 junction in HCV biology
Brendan A. Palmer, Isabelle Moreau, John Levis, Orla Crosbie, Elizabeth Kenny-Walsh, Liam J. Fanning
P9.54
A novel cell-based assay for RNA synthesis by the HCV polymerase reveals new
insights on mechanism of polymerase inhibitors and modulation by NS5A
P9.55
Identification of essential residues in the NS5A hinge region
Timothy Tellinghuisen, Katie LeMay, Jason Treadaway, Ivan Angulo
P9.56
Hepatitis C virus induces ultrastructural modifications in differentiated
Huh7.5 cells
Baptiste Jammart, Eve-Isabelle Pécheur, Philippe Roingeard, Fabien Zoulim, David Durantel
P9.57
Lipoprotein secretion profiles and VLDL production in hepatocytes and
hepatoma cell lines
Baptiste Jammart, Fabien Zoulim, David Durantel
P9.58
HCV-replicating HepG2 cells produce infectious ApoE positive, but ApoB
negative hybrid particles
Baptiste Jammart, Maud Michelet, Clarisse Benoit, Birke Bartosch, Romain Parent, Fabien Zoulim, David Durantel
P9.59
3’ supplementary base-pair interactions of miR-122 with hepatitis C virus RNA
Tetsuro Shimakami, Christoph Welsch, Masao Honda, Shuichi Kaneko, Stanley Lemon
POSTERS
C.T. Ranjith-Kumar, Yahong Wen, Nielson Baxter, Cheng Kao
POSTERS
P9.60
Propagation of HCV in human hepatoma Hep3B cells
Patricia Thibault, Joyce Wilson
P9.61
Differential regulation of HCV core protein throughout the viral lifecycle
Katherine Marsh, Susan Uprichard
P9.62
Regulation of infectious genotype 1a hepatitis C virus production by domain III
of NS5A
Seungtaek Kim, Christoph Welsch, MinKyung Yi, Stanley Lemon
P9.63
Comparison of replication and spread by HCV genotype 1 H77Sv3 and the
genotype 1/2 chimera HJ3-5
David McGivern, MinKyung Yi, Stanley Lemon
P9.64
Analysis of the topology and membrane association of GB virus B NS2 protein
Célia Boukadida, Caroline Marnata, Lisette Cohen, Jérôme Gouttenoire, Darius Moradpour, François Penin, Annette Martin
P9.65
NS3 protease-mediated and NS4A-dependent NS2 internal cleavage
Yongsen Zhao, Guangwei Yang, Joanne Fabrycki, Dharaben Patel, Atul Agarwal, Milind Deshpande, Mingjun Huang
P9.66
Emergence of chimeric interspecies pestiviruses by nonhomologous RNA
recombination
Paul Becher, Andreas Gallei
P9.67
Comparison of HCV amino acid covariance networks to covariance networks
other viruses
Maureen Donlin, Brandon Szeto, John Tavis
P9.68
POSTERS
Conformation-dependent phosphorylation of hepatitis C virus RNA
polymerase by the cellular protein kinase C-related kinase 2
Guanghui Yi, Wei Cun, Ziqing Liu, Johnny J. He, Guangxiang Luo, C. Cheng Kao
P9.69
Non-genotype-specific role of the hepatitis C virus 5’ untranslated region in
virus production and in inhibition by interferon and 5’UTR-based siRNAs
Yiping Li, Judith Gottwein, Jens Bukh
P9.70
Recruitment of nuclear pore complex proteins to sites of HCV assembly
Christopher Neufeldt, Michael A. Joyce, Aviad Levin, Rineke H. Steenbergen, D. Lorne J. Tyrrell, Richard W. Wozniak
P9.71
HCV secretion through the trans-Golgi network requires a PI4P-binding
protein, GOLPH3, and the unusual myosin MYO18A
Bryan Bishe, Aleem Siddiqui
P9.72
Characterizing the role of Rab1A in HCV infection
Christopher M. Williams, Angela L. Rasmussen, Deborah L. Diamond, Michael G. Katze
244 : HCV 2011
P9.73
Alanine scanning of residues 300 to 350 of HCV NS5A does not reveal the
cyclophilin-binding site
Udayan Chatterji, Precious Lim, Sam Hopkins, Daniel Cordek, Craig Cameron, Paul Targett-Adams, Kai Lin, Philippe Gallay
P9.74
Understanding the role of nonstructural protein 5A (NS5A) in the HCV life cycle
Anita Nag, Jason Robotham, Hengli Tang
P9.75
Coupling between the NS3 protease and helicase domains plays a critical
role in replication and infectivity of hepatitis C virus
Andrew Kohlway, Steve Ding, Brett Lindenbach, Anna Pyle
P9.76
Analysis of functional differences between hepatitis C virus NS5A of
genotypes 1-7 in infectious cell culture systems
Troels K. H. Scheel, Lotte S. Mikkelsen, Judith M. Gottwein, Jens Bukh
NS2 topology change as a negative feedback control mechanism to limit
infectious HCV production
Manu Anantpadma, Saravanabalaji Shanmugam, MinKyung Yi
POSTERS
P9.77
POSTERS
246 : HCV 2011
P9.01
Development of an RNA polymerase I-driven adenoviral vector and its application in
an HCV replication assay
Takeshi Yoshida (1), Fumi Satoh (1), Watari Akihiro (1), Masuo Kondoh (1), Hiroyuki Mizuguchi (2), Naoya Sakamoto (3), Kiyohito Yagi (1)
(1) Laboratory of Bio-Functional Molecular Chemistry, Osaka Univ., Suita, Japan
(2) Department of Biochemistry and Molecular Biology, Osaka Univ., Japan
(3) Dept. of Gastroenterol. & Hepatol., Tokyo Medical and Dental Univ., Japan
Objective: Approximately 170 million people are infected with HCV, and chronic infection with HCV is a major cause of hepatocellular
carcinoma. Interferon therapy is the sole effective therapy for HCV patients, but this therapy is ineffective in 30%–50% of patients. Thus,
novel therapeutic strategies for HCV are required. The transfer of complete or subgenomic HCV RNA into cells is useful for pharmaceutical
studies. Adenoviral (Ad) vectors are convenient and efficient tools for transducing foreign genes into cells, but Ad vector expressing HCV
genome has not yet been developed. In the present study, we developed a tetracycline-regulated RNA polymerase (pol) I cassette-driven
Ad vector system expressing HCV genome.
Methods: First, we attempted to prepare an RNA pol I-driven Ad vector expressing HCV subgenome that contains the luciferase gene
and HCV non-structural genes, but without success. Because HCV protease can cleave some components of Ad vector particles, we next
constructed a hybrid promoter consisting of the tetracycline-responsive element and the RNA pol I promoter in which HCV protease
expression was suppressed by the addition of tetracycline during the amplification of Ad vector particles. Ad vectors with HCV genome
expression controlled by the chimeric RNA pol I promoter were prepared. Cells were transfected with the Ad vectors, and HCV replication
was assessed by measuring luciferase activity and HCV RNA expression.
Results: Ad vector particles expressing the tetracycline-regulated RNA pol I-driven HCV genome could be amplified, and luciferase activity
was observed in cells transduced with the Ad vectors. HCV RNA was also detected, and the Ad vectors with mutated HCV RNA polymerase
did not exhibit HCV RNA replication. HCV replication in cells was attenuated by interferon treatment.
Conclusion: The Ad vector-mediated HCV replicon system will contribute to understanding the mechanism of HCV replication as well as the
development of anti-HCV drugs and vaccines.
Contact: Takeshi Yoshida
Laboratory of Bio-functional Molecular Chemistry, Osaka Univ., Suita, Japan
[email protected]
P9.02
Yasuo Ariumi (1), Misao Kuroki (2), Yukihiro Kushima (3), Kanae Osugi (4), Makoto Hijikata (3), Masatoshi Maki (4), Masanori Ikeda (2),
Takaji Wakita (5), Nobuyuki Kato (2)
(1) Center for AIDS Research Kumamoto University, Kumamoto, Japan
(2) Okayama University, Japan
(3) Institute for Virus Research, Kyoto University, Japan
(4) Nagoya University, Japan
(5) National Institute of Infectious Diseases, Japan
MicroRNA miR-122 and DDX6/Rck/p54, a microRNA effector, have been implicated in hepatitis C virus (HCV) replication. In this study, we
demonstrated for the first time that HCV-JFH1 infection disrupted processing (P)-body formation of microRNA effectors DDX6, Lsm1, Xrn1,
PATL1, or Ago2, except decapping enzyme DCP2 and dynamically redistributed these microRNA effectors to HCV production factory around
lipid droplets in HuH-7-derived RSc cells. Notably, HCV-JFH1 infection also redistributed stress granule component, GTPase-activating
protein (SH3 domain) binding protein 1 (G3BP1), ataxin-2 (ATX2), or poly(A)-binding protein 1 (PABP1) to the HCV production factory. In
this regard, we found that P-body formation of DDX6 began to be disrupted at 36 hrs post-infection. Consistently, G3BP1 formed stress
granules at 36 hrs post-infection. Then, we have observed the ring-like formation of DDX6 or G3BP1 and colocalization with HCV Core after
48 hrs post-infection, suggesting that disruption of P-body formation and hijacking of P-body and stress granule components occur in the
late step of HCV infection. Furthermore, HCV infection could suppress the stress granule formation in response to heat shock or treatment
with arsenite. Importantly, we demonstrated that the accumulation of HCV RNA was significantly suppressed in DDX6, Lsm1, ATX2, or PABP1
knockdown cells after inoculation of HCV-JFH1, suggesting that the P-body and the stress granule components are required for HCV life
cycle. Altogether, HCV seems to hijack the P-body and the stress granule components for the HCV replication.
Contact: Yasuo Ariumi
Center for AIDS Research Kumamoto University, Kumamoto, Japan
[email protected]
POSTERS
Hepatitis C virus hijacks P-Body and stress granule components around lipid droplets
POSTERS
P9.03
miR122 facilitates replication of hepatitis C virus in non-hepatic cells
Takasuke Fukuhara (1), Mai Shiokawa (1), Chikako Ono (1), Hiroto Kambara (1), Eiji Morita (1), Wataru Kamitani (1), Yoshiharu Matsuura (1)
(1) Department of Molecular Virology, Osaka University, Suita-shi, Japan
The liver-specific microRNA, miR122 has been shown to enhance translation of hepatitis C virus (HCV) RNA through a direct interaction with
two sites in the 5’ untranslated region (UTR). In vitro propagation of HCV (HCVcc) has been established by the combination of viral RNA from
genotype 2a JFH1 strain and cells derived from human hepatoma cell line Huh7. However, Huh7 cell lines are not suitable to investigate the
impact of miR122 on the HCV replication, due to an abundant expression of miR122. To determine the biological significance of miR122 on
the propagation of HCV, we examined various cell lines facilitate replication of HCV RNA by an exogenous expression of miR122. Among the
cell lines we examined, overexpression of miR122 facilitated HCV RNA replication in not only hepatic cell lines, but also several non-hepatic
cell lines. Although only a small amount of HCV RNA was detected in the uterus-derived cell line upon infection with HCVcc, 100-times
increase of viral RNA replication was achieved by the expression of miR122. In addition, HCVcc mutants carrying base substitutions in the 5’
UTR replicated efficiently by the expression of complementary miR122 mutants but not of a wild-type miR122, suggesting that expression of
miR122 specific to the 5’UTR of HCV is crucial for an efficient replication of HCV. Furthermore, subgenomic HCV replicon RNAs of genotype
1b Con-1 and genotype 2a JFH1 strains were shown to stably replicate in the uterus-derived cell line by the expression of miR122. These
results suggest that expression of miR122 facilitates an efficient replication of HCV not only in hepatic cells but also in non-hepatic cells
through a specific interaction with HCV RNA.
Contact: Takasuke Fukuhara
Department of Molecular Virology, Osaka University, Suita-shi, Japan
[email protected]
P9.04
Annexins participate in the HCV RNA replication
Takayuki Abe (1), Takasuke Fukuhara (1), Eiji Morita (1), Yoshiharu Matsuura (1)
(1) Department of Molecular Virology, RIMD, Osaka University, Suita-shi, Japan
POSTERS
Annexin is a calcium- and phospholipid-binding protein and forms a heterotetrameric complex with p11 light chain (also called S100A10).
The annexin family proteins are implicated in many cellular processes, including channel formation, membrane fusion, vesicle transport,
and regulation of the tight junction (TJ) formation. Recently, a cytosolic annexin A2 has been shown to be involved in the HCV particle
formation through an interaction with domain III of HCV NS5A. A quantitative PCR analyses revealed that expression of annexin A2, A4,
and A5 was upregulated in the HCV replicon cells and knockdown of A2 and A5 but not of A4 enhanced expression of NS5A in the replicon
and HCV infected cells, in contrast to the previous report. Knockdown of annexin A2 or A5 exhibited no effect on the HCV IRES-dependent
translation and on the particle formation, suggesting that annexins participate in the RNA replication of HCV. Although knockdown of A2 or
A5 exhibited no effect on the infectivity of the HCV pseudotype viruses, the distribution of TJ proteins including ZO-1, OCLN and CLDN-1 was
changed. In addition, dislocation of the TJ proteins was also observed in the RNA replicon cells of HCV but not in those of other flavivirus,
and was restored by the elimination of HCV RNA by the treatment with IFN or an HCV protease inhibitor. These results suggest that annexins
participate in the HCV RNA replication through the modification of TJ formation.
Contact: Takayuki Abe
Department of Molecular Virology, RIMD, Osaka University, Suita-shi, Japan
[email protected]
248 : HCV 2011
P9.05
Replication of genotype 3a hepatitis C virus subgenomic replicon in cell culture
Mohsan Saeed (1), SuSu Hmwe (1), Claire Gondeau (2), Patrick Maurel (2), Takaji Wakita (1)
(1) National Institute of Infectious Diseases, Tokyo, Japan, Tokyo, Japan
(2) INSERM U1040, Montpellier, France, France
Hepatitis C virus is a leading cause of viral hepatitis worldwide and its genotype 3a is predominant in vast areas of world. No efficient cell
culture system for studying the replication of this genotype has been reported so far. In this study, we cloned a full-length consensus genome
from viral RNA isolated from the serum of an infected person and used it to construct a bicistronic subgenomic replicon carrying neomycin
phospho transferase gene as a selectable reporter. This replicon was transfected into a human hepatoma cell line to estimate the replication
efficiency. Upon transfection into the cells and selection with G418, small number of colonies was observed. RTD-PCR, using genotype 3a
specific primers and probe, showed that the copy number of subgenomic replicon in cellular RNA was in the range of 3.96×107 to 2.08×108
copies/µg cellular RNA. Treatment of replicon cells with interferon attenuated the replication of viral genomes. Furthermore, viral replicon
RNA was transmissible to naïve HuH-7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of ten colonies at
two time points revealed the presence of at least one non-synonymous mutation in all the replicons, one of them being common among
five clones. All the non-synonymous mutations, when introduced back into wild type replicons, caused multiple fold increase in the colony
formation. An adaptive mutation S2204I, reported to increase the replication of genotype 1a replicon, also increased the colony formation
efficiency of genotype 3a replicon. In conclusion, we have established a genotype 3a replicon system in HuH-7 cells, which could provide a
powerful tool for researching this genotype.
Contact: Mohsan Saeed
National Institute of Infectious Diseases, Tokyo, Japan, Tokyo, Japan
[email protected]
P9.06
HCV production requires the PML tumor suppressor protein
Hepatitis C virus (HCV) is a causative agent of hepatocellular carcinoma. It has been suggested that interaction of HCV protein with tumor
suppressor protein is involved in the hepatocellular carcinogenesis. PML tumor suppressor protein, which forms discrete nuclear structures
termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense.
Recently, Herzer et al. reported the interaction of PML with HCV core protein. In this study, we investigated the further potential role of
PML in HCV life cycle. We examined the RNA levels of HCV-JFH1 in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently
replicate, stably expressing short hairpin RNA targeted to PML. The level of HCV-JFH1 RNA in PML knockdown cells was unaffected at 4h or
12h post-infection, suggesting that PML is unrelated to the early step of HCV replication. In contrast, the level of secreted HCV core in the
supernatants of PML knockdown cells was remarkably reduced at 96h post-infection, whereas the level of HCV-JFH1 RNA was somewhat
decreased in PML knockdown cells. To determine whether or not PML is involved in HCV RNA replication, we examined the level of HCV-JFH1
RNA in PML knockdown JRN/35B cells harboring a replicative subgenomic HCV-JFH1 RNA. Consequently, the level of HCV-JFH1 RNA was
similar to that of the control cells, suggesting that PML is unrelated to HCV RNA replication. Furthermore, we found that the infectivity of
the supernatants of PML knockdown JRN/35B cells overexpressing HCV-JFH1 core-NS2 was significantly reduced in comparison with that of
control cells, even though the level of HCV-JFH1 RNA was similar between these cells in the trans-packaging system. Taken together, these
findings suggest that PML is required for HCV production.
Contact: Misao Kuroki
Department of Tumor Virology, Okayama University, Okayama, Japan
[email protected]
POSTERS
Misao Kuroki (1), Yasuo Ariumi (2), Masanori Ikeda (1), Hiromichi Dansako (1), Takaji Wakita (3), Nobuyuki Kato (1)
(1) Department of Tumor Virology, Okayama University, Okayama, Japan
(2) Center for AIDS Research, Kumamoto University, Japan
(3) Department of Virology II, National Institute of Infectious Diseases, Japan
POSTERS
P9.07
Genetic evidence for dual functions of NS5A in RNA replication
Robert Fridell (1), Lourdes Valera (1), Chunfu Wang (1), Dike Qiu (1), Melissa Kirk (1), Min Gao (1)
(1) Bristol-Myers Squibb Research and Development, Wallingford, CT, USA
NS5A is an essential component of the HCV RNA replication complex but its precise roles in RNA replication remain obscure. BMS-790052
targets NS5A and is a potent and selective inhibitor of HCV RNA replication. In addition to its promise as an effective anti-HCV therapeutic
agent, BMS-790052 also provides a unique tool to probe NS5A functions in viral replication. Similar to other positive strand RNA viruses, HCV
RNA replication occurs in highly sequestered, specialized intracellular membrane compartments. Many of the HCV non-structural proteins
display a strong preference for cis-acting RNA replication functions. NS5A is an exception in that multiple studies have demonstrated a transacting function of NS5A in RNA replication. It has been suggested that, since NS5A is monotopically tethered to intracellular membranes by an
amphipathic alpha-helix, rather than being integrally anchored by a trans-membrane domain, there may be latitude for movement of NS5A
between replication complexes. By incorporating replicons with different sensitivities to BMS-790052 in a NS5A trans-complementation
assay, we demonstrate that, in addition to a trans-acting function, NS5A also possesses a distinct cis-acting function in RNA replication.
Specifically, we observed that while a JFH1 replicon with a replication defect (S2204I mutation in the hyperphosphorylation region of NS5A)
could be efficiently complemented by co-expression of a functional helper replicon, the inhibitor sensitivity phenotype of the rescued
replicon was not trans-complemented. These results imply that trans-complementation, in this instance, did not occur by exchange of
functional NS5A into the replication complex of the defective replicon. Instead, they support a model wherein hyperphosphorylation
deficient NS5A encoded by the defective replicon still performed an essential role as a component of the HCV RNA replication complex,
while hyperphosphorylated NS5A, encoded by the helper replicon, provided a separate trans-acting function. BMS-790052 is predicted to
inhibit both of these functions.
Contact: Robert Fridell
Bristol-Myers Squibb Research and Development, Wallingford, CT, USA
[email protected]
P9.08
Regulation of HCV replication by FKBP8-dependent or -independent Hsp90 activity
POSTERS
Kunihiro Kawakami (1), Hirotaka Kasai (4), Atsuya Yamashita (4), Nobuyuki Enomoto (2), Yoshiharu Matsuura (3), Masami Kusunoki (1),
Kohji Moriishi (4)
(1) Dpt. Struct. Biochem., Faculty of Engineering, University of Yamanashi, Japan
(2) 1st Dpt. Medicine, Faculty of Medicine, University of Yamanashi, Japan
(4) Dept. Microbiol., Faculty of Medicine, University of Yamanashi, Chuo-shi, Japan
We have reported previously that the host protein FKBP8 interacts with Hsp90 and HCV NS5A of Con1 or JFH1 strain and then regulates
HCV replication by sequestration of Hsp90 in the replication complex. In this study we investigated involvement of FKBP8 in HCV replication
of other strains of HCV genotype 1b. The immunoprecipitation test showed that FKBP8 interacted with NS5A proteins of all three cell lines
but FKBP51, FKBP52 and Cyp40 did not. Val121, which is critical for HCV replication and FKBP8 binding, was conserved among NS5A genes
amplified from these replicon cell lines. Endogenous FKBP8 was efficiently reduced by transfection with the siRNA targeting to FKBP8 in
all cell lines. Although treatment with 17-AAG, an inhibitor of Hsp90, impaired the HCV replication of all cell lines at the similar level, HCV
replication was impaired by the FKBP8 knockdown in two out of three cell lines. These results suggest that HCV replication is capable
of being supported by FKBP8-dependent or -independent Hsp90 activity. Other regulatory molecule(s) might be involved in the Hsp90dependent regulation of HCV replication.
Contact: Kohji Moriishi
Dept. Microbiol., Faculty of Medicine, University of Yamanashi, Chuo-shi, Japan
[email protected]
250 : HCV 2011
P9.09
Hepatitis C virus replication enhancement at low oxygen tension is associated with
higher anaerobic energy metabolism in hepatoma cells
Niki Vassilaki (1), Katerina I. Kalliampakou (1), Ioly Kotta-Loizou (1), Christina Befani (2), Panagiotis Liakos (2), Georgios Simos (2), Despina
Smirlis (3), Oliver Bauhofer (4), Marion Poenisch (4), Marc P. Windisch (5), Myungeun Lee (5), Ralf Bartenschlager (4), Penelope Mavromara (1)
(1) Hellenic Pasteur Institute, Athens, Greece
(2) Laboratory of Biochemistry, School of Medicine, Univ. of Thessaly, Greece
(3) Molecular Parasitology, Hellenic Pasteur Institute, Athens, Greece
(4) Department of Infectious Diseases, Mol. Virology, Univ. of Heidelberg, Germany
(5) Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, Republic of Korea
Liver oxygen tension ranges from 12% to 1% (v/v) O2 (median value 3% v/v), resulting in a metabolic activity zonation. In tissue culture, low
oxygen was shown to preserve cellular metabolism and to increase replication efficiency of several viruses. In this study, we examined the effect
of oxygen tension on HCV RNA replication and virus production in cultured cells. Huh7 cells non- or DMSO-differentiated (dif.) were infected
with cell culture-grown HCV or transfected with JFH1-derived HCV genomes under low (3% v/v) or atmospheric (20% v/v) O2. We found
that low oxygen tension positively regulated HCV production, viral protein expression and RNA replication, in both infected and transfected
Huh7 and Huh7 dif. cells, as determined by infectivity assay, immunofluorescence/western blot and qRT-PCR analyses. This enhancement was
affected by cell density, was not related to virus entry and occurred at an early stage of viral RNA replication. Hypoxia signaling pathway-focused
DNA microarray analysis revealed an up-regulation of genes related to hypoxic stress, glycolytic metabolism, cell growth and proliferation at 3%
O2. qRT-PCR combined with intracellular ATP measurement confirmed the reprogramming of cells to favour anaerobic energetic metabolism at
3% O2. HCV replication enhancement at 3% O2 correlated with increased glycolysis- and creatine kinase B-dependent ATP synthesis. Silencing
of hypoxia-inducible factors HIF-1α and HIF-2α as well as treatment with chemicals increasing the activity of HIFs neither affected HCV RNA
replication nor intracellular ATP amounts, arguing for alternative mechanisms for modulation of cellular metabolism at 3% O2. Interestingly,
selected oncogenes known to control glycolysis were up-regulated. In summary, low oxygen conditions occurring in the liver (3% v/v O2)
enhance HCV RNA replication and virus production in cultured hepatoma cells dependent on higher anaerobic metabolic activity.
Contact: Niki Vassilaki
Hellenic Pasteur Institute, Athens, Greece
[email protected]
Contrary to published models of HCV RNA replication, heat denaturation reveals that
much of the HCV in natural infections is in double-stranded form
Arielle Klepper (1), Francis Eng (1), Jose Walewski (2), Myron Schwartz (1), Thomas Schiano (1), Andrea Branch (1)
(2) Columbia University, New York, USA
Background: The viral factors promoting HCV persistence are incompletely understood and may include use of a stable double-stranded
duplex. Objective: To test the hypothesis that the majority of HCV RNA in the liver of HCV-infected patients is double-stranded. Methods:
With IRB approval, RNA was Trizol extracted from human serum and liver tissues of HCV-infected patients and controls, and from Huh7.5 cells replicating a Con1/JFH chimeric virus (Apath). Quantitative RT/PCR using Superscript III and a Lightcycler 480 SYBR green assay
was performed on purified RNA with or without prior heating to 106˚C for 2.5 min. Results: Heat treatment of single-stranded HCV RNA
extracted from serum reduced the HCV RNA signal to zero. When these conditions were applied to RNA from human liver (N=4), the
HCV RNA signal increased by a factor of 2.0. Intriguingly, heating increased the signal by a similar amount (1.4-fold) when applied to RNA
extracted from confluent HCV-infected Huh-7.5 cells, but did not change the signal from sub-confluent cells. Conclusions: We developed
heating conditions that allow HCV RNA to be released from dsRNA duplexes and to be quantified. These same conditions destroy ssHCV RNA
present in the original (untreated) samples. Heating RNA extracted from liver specimens of HCV-infected patients increased the HCV RNA
signal 2-fold, whereas heating ssHCV RNA extracted from serum eliminated the HCV RNA signal. We propose that heating to temperatures
over 99˚C leads to denaturation of HCV dsRNA duplexes, liberating (+) and (-) strands that can then be detected by RT/PCR. Based on our
results, we calculate that in human liver, the overall ratio of (+) to (-) strands is 1:1, or higher. Thus, half or more of the HCV RNA is present
in double-stranded form. The stability of this highly prevalent dsRNA may have important implications for viral persistence.
Contact: Arielle Klepper
[email protected]
POSTERS
P9.10
POSTERS
P9.11
Highly sensitive and specific visualization of HCV plus- and minus-strand RNA in vitro
and in vivo
Urtzi Garaigorta (1), Stefan Wieland (1), Francis V. Chisari (1)
Determining the frequency and identity of HCV infected cells during HCV infection has proven to be difficult, presumably due to the absence
of sufficiently sensitive detection systems. Here we describe a highly sensitive fluorescence in situ hybridization (FISH) system that can detect
HCV-infected hepatocytes in vitro and in vivo thanks to exceptionally strong signal amplification and high signal to noise ratio (QuantiGene
ViewRNA, Panomics/Affymetrix). Multiplex capability of the system allows simultaneous HCV positive- and negative-strand RNA detection
at an apparent sensitivity of <10 RNA molecules per JFH-1-infected Huh-7 cell. Combined FISH and antibody-based immunofluorescence
analysis reveals the presence of HCV positive- and negative-strand RNA and NS5A protein in the perinuclear region in JFH-1-infected Huh-7
cells, consistent with sites of HCV RNA replication. Positive-strand RNA is also detected peripherally in the cytoplasm, perhaps revealing
distinct sites of RNA translation, assembly and/or egress. A chromogenic version of the same system was used to specifically detect positiveand negative-strand JFH-1 RNA in formalin fixed/paraffin embedded liver sections from JFH-1 infected chimeric mice but not in uninfected
control mice. Experiments using HCV isolate specific probes to detect HCV RNA in the livers of HCV-infected humans, chimpanzees, and
chimeric mice infected with patient-derived HCV isolates are ongoing and will be discussed. In conclusion, this system’s exceptionally high
sensitivity and signal to noise ratio make it ideal to study the intracellular localization of HCV RNA in vitro and to determine the identity,
frequency and tissue distribution of infected cells during natural HCV infection in vivo.
Contact: Urtzi Garaigorta
[email protected]
P9.12
Engineering genetic suppressor elements against hepatits C virus
Zhilei Chen (1), Rudo Simeon (1)
(1) Texas A&M University, College Station, TX, USA
POSTERS
We describe the efficient selection of anti-HCV genetic suppressor elements (GSEs). GSEs are sequences derived from a gene or genome
of interest that act as transdominant inhibitors of a particular biological function. Using an engineered hepatoma cell line – n4mBid – that
undergoes significant HCV-induced cytopathic effect by cell culture-derived HCV (HCVcc), we developed an efficient selection system for
isolating anti-HCV GSEs from a library comprising a fragmented HCV genome. Huh-7.5 cells transduced with genetic fragments obtained
from the 5th round of selection showed significant resistance to HCV-induced cytotoxicity and lower levels of permissivity to HCV replication/
infection. These studies represent the first reported unbiased selection for anti-HCV genetic elements. We are currently isolating individual
GSEs from the library and characterizing their specific effects on the HCV life cycle.
Contact: Zhilei Chen
Texas A&M University, College Station, TX, USA
[email protected]
252 : HCV 2011
P9.13
Molecular mechanism and function of the HCV NS3 helicase
Meigang Gu (1), Charles Rice (1)
The hepatitis C virus (HCV) NS3 helicase (NS3h) is essential for the function of the membrane-associated replicase complex. Despite extensive
characterization of NS3h and similar helicases from other RNA viruses, the exact function(s) of these viral proteins remain to be defined.
We determined a series of structures of NS3h complexes. The structures representing three conformational states along translocation
reveal transitions within motif V and a “spring” α-helix, which couple the changes of ATP-mimic coordination and single-stranded (ss)DNA
association. These conformations suggest that NS3h steps one base per ATP hydrolysis, in a “ratchet” mode. The associated ssDNA strands
in the complexes show a series of structural changes, especially variations of the backbone sugar puckers. This suggests that efficient
translocation along ssRNA, which should have little pucker flexibility within a limited space, must require modulation in the nucleic-acid
binding site for RNA association. Comparing the binary NS3h-ADP·AlF4- and ternary NS3h-ADP·AlF4--ssDNA complexes further suggests
that ssDNA promotes an overall conformational change and extension of the spring α-helix, which collectively stabilize the transitionstate ATP for catalysis. This is supported by an assay, in which crosslinking engineered cysteines within the spring α-helix suppressed ATP
hydrolysis and DNA translocation. The binary complex likely approximates an intermediate state, which limits (d)NTP utilization in the
absence of proper nucleic-acid targets. Although it’s unclear how a NS3h-like viral helicase is involved in the replicase function, this highly
regulated protein probably acts in several aspects of virus replication. It may be involved in viral RNA trafficking, recruiting templates from
translational machinery to replication compartments and/or exporting synthesized genomes to assembly complexes. It may also act as a
chaperone to facilitate proper viral RNA folding and functions. We are using X-ray crystallography in combination with biochemistry and cell
culture studies to further understand the actions of this important viral enzyme.
Contact: Meigang Gu
[email protected]
P9.14
Occult HCV infections in hemodialysis patients
Hepatitis C virus (HCV) infection is a significant public health problem in patients receiving hemodialysis. Detection of extrahepatic HCV
replication is extremely important to break the chain of transmission and to treat the chronic liver disease. In this study, we aimed to detect
HCV RNA status in peripheral blood mononuclear cells (PBMCs) and to investigate the prevalence of occult HCV infection by real-time PCR
(Taqman 48, Roche, analytical sensitivity of PCR method was 25 IU/mL). HCV RNA in plasma and PBMCs, HBsAg, anti-HCV and liver function
tests were measured in 100 patients on maintenance hemodialysis (58.5 ±13.9 years). After the isolation of RNA from both of the plasma and
PBMCs, we have used the PCR method. Three of the 85 (3.5%) patients whose anti-HCV and plasma HCV RNA negative, PBMCs HCV RNA were
positive. Liver function tests were normal and HBsAg results were negative in patients with occult HCV infection. PCR in plasma was positive in
5 (5.6%) out of the 90 patients whose serum anti-HCV were negative. The levels of RNA were between 75-338 IU/mL by these patients. None
of these patients had positive results for RNA in PBMCs. Five of the 10 patients whose anti HCV positive were also plasma RNA positive. Four of
these patients were also PBMCs positive. Five of the 10 patients were RNA negative in both plasma and PBMCs. This is the most comprehensive
prevalence study in the population of hemodialysis patients. As a result of this study we suggest that the investigation of HCV RNA in both
plasma and PBMCs for the diagnosis of occult HCV infection but we need more studies to support this hypothesis.
Contact: Bilgehan Aygen
Erciyes University Medical Faculty, Department of Infectious Diseases, Kayseri, Turkey
[email protected]
POSTERS
Ilkay Bozkurt (1), Bilgehan Aygen (1), Selma Gokahmetoglu (2), Orhan Yildiz (1)
(1) Erciyes University Medical Faculty, Department of Infectious Diseases, Kayseri, Turkey
(2) Erciyes University Medical Faculty, Department of Medical Microbiology, Kayseri, Turkey
POSTERS
P9.15
Adaptative mutations in non structural genes that increase trans-complementation of
HCV JFH1 replicon lacking E1E2 glycoproteins
Carole Fournier (1), Francois Helle (1), Véronique Descamps (1), Virginie Morel (1), Catherine Francois (1), Sarah Dedeurwaerder (1),
Czeslaw Wychowski (1), Gilles Duverlie (1), Sandrine Castelain (1)
(1) University Hospital Center, Amiens, France
The recent development of the JFH1 hepatitis C virus infectious system (HCVcc) by Wakita and colleagues enabled to envisage the study of
trans-complementation of both nonstructural and structural HCV proteins. In the present study we wanted to investigate a trans-packaging
system of HCV replicon lacking the envelope glycoproteins.
An HCV replicon deleted for envelope genes was efficiently encapsidated by homologous HCV envelope proteins that were expressed under
control of adenoviral vector in trans. Expression in trans of envelope proteins associated with Core, p7 and NS2 did not enhance transcomplementation. In contrast, expression of heterologous envelope proteins with or without heterologous Core, p7, NS2 did not rescue
virus production.
To increase production of homologous particles with our system, successive cycle of trans-complementations and infections were performed.
After four successive cycles, the production of particles was improved one hundred fold. Sequence analysis of adapted genomes revealed
seven adaptive mutations that differed in two independent experiments. Except one core mutation in an experiment, adaptive mutations
were observed in the non-structural region with the majority in NS5A. These mutations do not affect genomic replication but have a
positive effect on virion produced by trans-complementation assembly and/or secretion. These adapted variants clearly promote transcomplementation mechanism and will be an interesting tool for the development of HCV replicon- based vaccines.
Contact: Carole Fournier
University Hospital Center, Amiens, France
[email protected]
P9.16
Investigating the role of the cellular RNA helicase DDX3 in HCV replication
Seamus Stack (1), Allan Angus (1), Arvind Patel (1)
POSTERS
DDX3 is a member of the DEAD-box family of RNA helicases. It is a ubiquitous cellular protein possessing ATPase and helicase activities. The
exact cellular function of DDX3 is as yet undefined, but there is evidence for its involvement in biological processes as diverse as splicing,
translation initiation and repression, cell cycle regulation, nucleo-cytoplasmic RNA shuttling, RNA transport, interferon induction and
apoptosis. Recent studies have shown that DDX3 may be one of the primary host targets for manipulation by a number of different viruses,
including vaccinia virus, HIV and HCV. The exact stage of the HCV lifecycle at which DDX3 functions is as yet unknown. To investigate this, we
analysed various parts of the viral lifecycle using a number of model systems, including HCV psuedoparticles (HCVpp), subgenomic replicons
(SGR) and the HCV cell culture system (HCVcc). The effect of DDX3 knockdown on these systems was determined by transducing target
cells with lentivirus encoding short hairpin RNA against the C-terminus of DDX3. DDX3-knockdown cells did not alter HCVpp infectivity,
indicating that DDX3 does not affect virus entry. Knockdown of DDX3 from SGR-containing cells caused no disruption to RNA replication,
suggesting DDX3 is not involved in RNA replication. However, HCVcc infectivity was severely impaired in cells transduced with shDDX3 RNA
prior to HCVcc infection. Other experiments performed included introducing HCVcc in vitro transcribed RNA into DDX3 depleted cells by
electroporation, which did not significantly reduce virus production in comparison to control cell lines. This result showed that DDX3 is not
essential for infectious virus assembly. Collectively, these results indicate that DDX3 functions at an early step in the viral lifecycle, acting
after entry but before RNA replication begins.
Contact: Seamus Stack
[email protected]
254 : HCV 2011
P9.17
Disruption of a conserved disulfide bond in HCV E2 protein impacts CD81 binding and
abrogates infectivity
Guaniri Mateu (1), Luke Uebelhoer (1), Jillian Whidby Freund (2), Daniel Claiborne (1), Joseph Marcotrigiano (2), Arash Grakoui (1)
(2) Rutgers University, USA
The high sequence variability in the hepatitis C virus genome is a major contributor to the development of inefficient humoral and adaptive
immune responses in chronically infected patients. While the ectodomain of the HCV envelope glycoprotein 2 (eE2) contains some of
the most variable regions of the entire genome, its encoded eighteen cysteine residues are absolutely conserved across all genotypes. In
this study we assessed the importance of these cysteine residues with respect to HCV viral replication, virion production, and infectivity.
Using a mutagenesis approach, all eighteen cysteines were dispensable for HCV replication in cell culture. Each E2 cysteine mutant was
characterized for virion production and classified into one of three categories based on phenotype: (i) absent to low production of noninfectious particles, (ii) low production of particles with limited infectivity, and (iii) high production of non-infectious particles. The lack of
infectivity for the latter mutant corresponded with its inability to bind HCV co-receptor, CD81. Further mutagenesis in the highly conserved
sequence adjacent to this specific cysteine revealed a novel region involved in CD81 binding within HCV eE2. Interestingly, our preliminary
data suggest that the oligomeric state of the E2 protein on the surface of these virions containing this cysteine mutation differs from the
wild type HCVcc. Our results highlight the importance of conserved cysteines found within this region for proper folding, virion assembly,
receptor binding, and infectivity.
Contact: Guaniri Mateu
[email protected]
P9.18
Synthetic genotype 1a hepatitis C virus: reconstructing a representative sequence
Hepatitis C Virus (HCV) research is hampered by the use of arbitrary representative isolates in cell culture. The most commonly used
isolate in vitro is a genotype 2a virus (JFH-1); however, genotype 1 is more prevalent worldwide and represents about 70% of infections
in the United States, and genotypes differ from one another by 31% to 33% at the nucleotide level. For phylogenetic and immunologic
analyses, H77 (subtype 1a) and HCV-J (subtype 1b) are commonly used based on their historic importance. In an effort to rationally design a
representative genotype 1a virus (bole1a), we used Bayesian phylogenetics, ancestral sequence reconstruction, and covariance analysis on a
curated set of 390 full-length human HCV 1a sequences from Genbank. We found that bole1a branches from the center of any phylogenetic
tree constructed on a given set of sequences suggesting that it contains variations present in widely circulating strains. Additionally, bole1a
provided epitope coverage of 78% on a full-genome comparison suggesting its use as a potential vaccine candidate for genotype 1a.
Preliminary analyses (Burke et al., abstract submitted) confirm that selected epitopes from the bole1a genome were able to elicit a robust
T cell response. As a proof of concept for infectivity, we have synthesized the envelope genes (E1 and E2) of bole1a and expressed them
in a HIV pseudoparticle system, which contains HCV envelope genes and HIV non-envelope genes with luciferase detection. Initial results
demonstrate that the bole1a pseudoparticle robustly infected Hep3B cells, whereas many individual variants from chronically-infected
people are noninfectious. In this proof-of-concept study, we demonstrate that these rationally designed fully synthetic HCV envelope genes,
which contain representative immunogens, assemble properly and mediate entry into target cells.
Contact: Supriya Munshaw
[email protected]
POSTERS
Supriya Munshaw (1), Lin Liu (1), Justin R. Bailey (1), Kelly Burke (1), Andrea Cox (1), Stuart C. Ray (1)
POSTERS
P9.19
Geranylgeranyl transferase II is essential for HCV RNA replication
Masanori Ikeda (1), Midori Takeda (1), Yasuo Ariumi (1), Takaji Wakita (2), Nobuyuki Kato (1)
(1) Okayama University, Department of Tumor Virology, Okayama, Japan
(2) Virology II, National Institute of Infectious Diseases, Japan
Mevalonate pathway produces cholesterol and geranylgeranyl pyrophosphate (GGPP). Some of the host proteins essential for HCV RNA
replication (e.g. FBL2) are subjected to geranylgeranylation with GGPP as a substrate. Therefore, inhibition of the GGPP production may be
a target in the development of anti-HCV reagents. To date, HMG-CoA reductase inhibitors, statins, have been reported that they inhibit HCV
RNA replication both in cell culture and in clinical studies. It has been reported that inhibition of geranylgeranyl transferase (GGTase) I caused
suppression of HCV RNA replication. However, the role of another GGTase, GGTase II, in HCV RNA replication has remained unclear. GGTaseII
is a Rab specific GGTase and needs Rab escort protein (REP)-1 and/or REP-2 for its enzyme activity and translocation of geranylgeranylated
Rab to the target membranes. In this study, we examined the role of GGTase II including REP-1 and REP-2 in HCV RNA replication.
We first examined the expression levels of REP-1 and REP-2 in the competent HuH-7 and Li23 cells for HCV replication. Li23 cells highly
expressed both REP-1 and REP-2. However, interestingly, HuH-7 cells expressed only REP-2 but not REP-1. The silencing of GGTaseII, REP1, or REP-2 inhibited HCV RNA replication in HCV RNA harboring Li23 cell line (ORL8). In the clinical study, it has been reported that the
deficiency of REP-1 causes visual disorder by the accumulation of unprenylated Rab27, because REP-2 is not able to compensate for REP-1 in
the prenylation of Rab27. Therefore, our findings suggest that HuH-7 cells possess the failure of Rab system. By the comparison with HuH-7
and Li23 cells, we are now under investigation for the role of REP-1 and REP-2 in HCV life cycle including viral entry, assembly, and release.
Contact: Masanori Ikeda
Okayama University, Department of Tumor Virology, Okayama, Japan
[email protected]
P9.20
Hepatitis C virus p7 is crucial for capsid assembly and envelopment
Juliane Gentzsch (1), Christiane Brohm (1), Gabrielle Vieyres (1), Martina Friesland (1), Eike Steinmann (1), Thomas Pietschmann (1)
(1) TWINCORE - Centre for Experimental and Clinical Infection Research, Hannover, Germany
POSTERS
Hepatitis C virus (HCV) p7 is a small membrane-associated ion channel protein which is important for virus production. To analyze how
p7 contributes to virus assembly, we constructed assembly-deficient p7 mutants and phenotypically compared these with HCV genomes
with mutations in alternative viral proteins known to contribute to virus production. We noted increased susceptibility of intracellular core
protein to proteinase K in cells transfected with p7, core, E1, NS2 or NS5A mutants compared to the wild type genome, indicating that
inactivation of p7 (core, E1, NS2 and NS5A) arrests virus assembly prior to membrane envelopment of core protein to varying degrees. In
case of p7 mutants, density gradients showed a second, slower sedimenting peak of core protein compared to wild type virus which was not
protected by a membrane and was not infectious. Similar core species accumulated when core was inactivated, and to a minor extent also
when NS2 or NS5A proteins were mutated. Fluorescence microscopy and biochemical preparation of lipid droplets from cells transfected
with the p7 mutant revealed an accumulation of core and NS5A at those organelles. Furthermore, rate zonal centrifugation revealed a
disturbance in capsid assembly with elevated levels of monomers and capsid-assembly intermediates for the p7, core and NS2 mutants
compared to wild type. Taken together these results indicate that p7, and to a minor extent NS2 and NS5A, are crucial for membrane
envelopment of HCV core protein. Moreover, p7 and NS2, or a complex consisting of both proteins, are essential for proper capsid assembly.
Contact: Juliane Gentzsch
TWINCORE - Centre for Experimental and Clinical Infection Research, Hannover, Germany
[email protected]
256 : HCV 2011
P9.21
The p7 protein of hepatitis C virus forms structurally plastic, rudimentary ion channels
Francois Penin (1), Danielle E. Chandler (2), Roland Montserret (1), Eike Steinmann (3), Thomas Pietschmann (3), Klaus Schulten (2),
Christophe Chipot (4)
(1) IBCP, UMR 5086, CNRS, University of Lyon, Lyon, France
(2) Beckman Institute, University of Illinois at Urbana-Champaign, USA
(3) Division Experimental Virology, TWINCORE, Hannover, Germany
(4) UMR 7565, CNRS, University of Nancy, France
Hepatitis C virus (HCV) p7 is a membrane-associated oligomeric protein possessing ion channel activity. It is essential for effective assembly
and release of infectious HCV particles and an attractive target for antiviral intervention. Yet, the self-assembly and molecular mechanism of
p7 ion channelling are currently only partially understood. The secondary structure elements of the monomeric form of p7 were determined
by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically
with structural data obtained from NMR experiments to propose a three-dimensional model of the monomeric form of p7 associated with
the phospholipid bilayer. Using molecular dynamics simulations in phospholipid bilayer, we show that p7 may form stable oligomers of four
to seven subunits, with an energetic bias towards six or seven monomeric subunits, and that p7 oligomers are capable of flexibly rearranging
to adapt to the membrane phospholipid thickness. We describe a model of a hexameric p7 complex, which forms a transiently-open pore.
This pore was blocked in two places by a ring of adjacent hydrophobic residue side chains forming an energetic barrier to water permeation.
The gating mechanism seems to involve minimal conformational changes. Notably, none of the p7 hydrophilic pore-lining residues studied
by mutagenesis appears to be essential for p7 functioning. This suggests that hydrophilic pore-lining residues would be all that is required
to attract and conduct ions across the channel. These results allowed us to propose a view of p7 oligomerization, wherein hexameric and
heptameric complexes may coexist, forming rudimentary, yet robust functional ion channels. This model has implications for the elucidation
of p7 functions, as well as for the development of drugs aimed at blocking p7 activity.
Contact: Francois PENIN
IBCP, UMR 5086, CNRS, University of Lyon, Lyon, France
[email protected]
P9.22
Sylvie Lagaye (1), Hong Shen (1), Jesintha Gaston (1), Pierre Bourdoncle (1), Laurent Hannoun (2), Pierre-Philippe Massault (3), Anaïs
Vallet-Pichard (3), Vincent Mallet (3), Stanislas Pol (3)
(1) Institut Cochin, INSERM U1016, Paris, France
(2) AP-HP, Groupe Hospitalier La Pitié Salpétrière, Paris, France
(3) AP-HP, Groupe Hospitalier Cochin-Saint Vincent de Paul, Paris, France
The model of human cultured HCV-replication-permissive hepatocarcinoma cell lines(HCVcc) is an important virological tool to study the
mechanisms of HCV infection but it remains distantly related to physiological and pathological conditions. It is why we developed an ex
vivo model of human adult liver slices. Our aim was to culture and infect liver slices with HCV. Non-infected liver 350 µm-thick slices,
obtained from human liver resection, were infected either with the HCVcc supernatant(Con1b/JFH-1)(MOI=0.1) or primary patients isolates
(genotype 1b) and cultivated up to ten days. The liver phenotypic markers expression was checked by RT-qPCR. Replication of HCV genome
was evaluated by strand-specific RT-qPCR. The HCV core and NS3 proteins were revealed by western blotting analysis. The infectious titers
were evaluated by focus formation upon inoculation into naïve Huh-7.5.1 cells. We validated the model by the neutralization assay with
CD81 mAb and neutralizing human serum, and by the interferon/ribavirin treatment on human liver slices infection by Con1b/HCVcc.
Human liver slices replicated, expressed efficiently as well HCVcc as primary patients isolates and produced high titers of progeny virions
(HCVpc). HCV infection was ascertained by: 1- the increasing amounts of intracellular viral RNA (up to 106 copies/µg RNA at day 10),
intracellular core and NS3 proteins and infectious HCV particles released into the culture supernatants (titer~105 focus-forming units/mL
by day 10), evidencing de novo viral particles production; 2- the presence of specific stained foci by in situ immunofluorescence with mAb,
3- the dose-dependent neutralization of HCV infection by CD81 mAbs or by convalescent serum from a recovered HCV genotype 1 patient
and 4- the dose-dependent inhibition by interferon/ribavirin treatment of HCV infection. This new ex vivo model provides a powerful tool
for studying the complete viral life cycle and dymanic spreading in the liver, and evaluating the potency of new antiviral therapies.
Contact: Sylvie Lagaye
Institut Cochin, INSERM U1016, Paris, France
[email protected]
POSTERS
Robust replication of primary or cultured hepatitis C virus isolates in human liver
slices: a relevant ex vivo model of liver infection
POSTERS
P9.23
Replication efficiency of the non-structural 5B (NS5B) protein is increased after
initiation of antiretroviral therapy in HIV/HCV co-infected patients
Jason Blackard (1), Gang Ma (1), Christina Martin (1), Yupeng He (2), M. Tarek Shata (1), Kenneth Sherman (1)
(1) University of Cincinnati, Cincinnati, OH, USA
(2) Abbott Laboratories, USA
Background: It is well established that HIV co-infection is associated with elevated hepatitis C virus (HCV) RNA levels, accelerated liver
disease, and lower HCV treatment response. Several studies have also reported that initiation of highly active antiretroviral therapy (HAART)
is associated with an increase in HCV RNA levels and/or elevated ALT levels. However, the mechanisms that contribute to these flares have
not been defined. Methods: 15 subjects with HIV/HCV co-infection were treated with efavirenz or boosted atazanavir with a tenofovir/
emtricitabine backbone. Full-length genotype 1 NS5B sequences were amplified from patient. A replicon-based shuttle vector system was
utilized to assess NS5B replication efficiency before (week 0) and 8 weeks after HAART initiation. The relationship between NS5B replication
efficiency and HIV viral load, HCV viral load, CD4 cell count, ALT, and AST was assessed by Spearman Rank Correlation. Results: 3 subjects
experienced an ALT flare post-HAART initiation, 2 experienced an HCV flare, and 3 experienced both an ALT and an HCV flare. The median
NS5B replication efficiency increased from 66.1% at week 0 to 140.8% at week 8. 8 subjects had >10% increase in replication efficiency from
week 0 to week 8. In contrast, only 2 subjects had >10% decrease, while 5 had <10% increase/decrease in replication efficiency between
weeks 0 and 8. Replication efficiency was positively associated with CD4 cell count (p = 0.03), but was not significantly associated with HIV
viral load, HCV viral load, ALT, or AST. Conclusions: The NS5B polymerase plays a critical role in the HCV replication cycle. Our in vitro data
suggest that NS5B fitness may be increased shortly after initiating HAART in the majority of HIV/HCV co-infected subjects; however, this was
not strongly associated with increased HCV RNA or ALT elevations observed in vivo after initiating HAART.
Contact: Jason Blackard
University of Cincinnati, Cincinnati, OH, USA
[email protected]
P9.24
A genetic bottleneck in HCV diversity occurs during mitosis
Brian Webster (1), Silke Wissing (1), Eva Herker (1), Melanie Ott (1), Warner Greene (1)
(1) Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
POSTERS
The hepatitis C virus (HCV) has been recently demonstrated to induce superinfection exclusion, a state where cells infected by HCV
become refractory to further infection by HCV. This phenomenon was shown to act at a post-entry step of infection, largely at the level
of RNA replication. HCV replicons are also capable of competition with each other, even when introduced into cells simultaneously. We
demonstrate, using congenic Jc1 reporter strains, that dual infection with HCV is inherently unstable in Huh7.5 hepatoma cells, as isolated
productively dually-infected cells assort out into singly-infected cells within ~10 days. This assortment is not due to higher cytotoxicity
or growth impairment in dually-infected cells. As there is no bias during assortment toward one or the other congenic Jc1 strains, this
appears to be a stochastic phenomenon, not dependent on viral fitness. Furthermore, the assortment represents an explicit loss of one of
the two viral genomes, as measured by quantitative PCR following assortment of dually-infected cells into singly-infected cells. Our data
indicates that this instability of the dually-infected state is due to a genetic bottleneck in viral genomic diversity at or shortly following
mitosis. When isolated premitotic dually-infected cells proceed through cell division, there is a significant enrichment in singly-infected cells
within ~48 h. We further observe, via time-lapse microscopy, that cells dually-infected with fluorophore-tagged Jc1 reporter strains tend to
lose fluorescence from one of the two strains within ~48 h following mitosis, in an apparently random manner. Along with superinfection
exclusion, this may be an additional mechanism that limits HCV genetic diversity and ability to recombine in vivo, and may further explain
the paucity of natural HCV recombinants.
Contact: Brian Webster
Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
[email protected]
258 : HCV 2011
P9.25
Infectious hepatitis C virus bearing genotype-1b subgenomic replicon by transencapsidation with the structural regions of genotype 1b
Kazuo Sugiyama (1), Yuko Murakami (2), Takayuki Hishiki (3), Yuko Shimizu (3), Kenji Funami (3), Saneyuki Ujino (3), Hidetsugu Saito (4),
Satoshi Saito (5), Nobuyuki Kato (6), Toshifumi Hibi (4), Hiroshi Takaku (2)
(1) Center for the Study of Chronic Liver Diseases, Keio University, Tokyo, Japan
(2) Dept. of Life and Environment Sciences, Chiba Institute of Technology, Japan
(3) Research Institute, Chiba Institute of Technology, Japan
(4) Internal Medicine, Keio University, Japan
(5) Internal Medicine, Yokohama City University, Japan
(6) Department of Molecular Biology, Okayama University, Japan
The infectious HCV strain, JFH-1, has brought about a great advance in the HCV research revealing almost a whole infectious cycle of HCV. However,
genotype of JFH-1 is 2a, which is generally sensitive to interferon treatment, whereas a major genotype in Japan is 1b, which is less sensitive to
the treatment and more frequently gives rise to hepatocellular carcinoma. Therefore, establishment of an efficiently infectious HCV strain of
genotype 1b is indispensable for further HCV research and improvement in the treatment. Here, we attempted to isolate subgenomic replicons
(NS3 to NS5B) of genotype 1b that can be preferentially packed into an infectious virion by trans-complementation of the structure regions (core
to NS2) of genotype 1b. At first, to obtain diverse repertoire of replicon-replicating cell lines, we employed a replicon library method reflecting
the genetic diversity of HCV within patient serum. We actually generated replicon libraries from 8 patient sera and established dozens of repliconreplicating cell lines in total, confirming replication of the replicon by RT-PCR, Western blotting and immunostaining for HCV. Of them, only 2 cell
lines produced infectious virion bearing subgenomic replicon by trans-complementation of the structure regions of 1b; the culture supernatant
of both cell lines formed infectious foci on naïve cells 2 days after inoculation, and formed neomycin-resistant colonies 3 weeks after, where the
authentic size of subgenomic RNA was detected by RT-PCR. Taken together, not all but only limited numbers (2 lines) of the subgenomic replicon
of genotype 1b was capable of trans-encapsidation with the structure regions of genotype 1b, suggesting adaptive mutations for an appropriate
interaction with the structural proteins upon assembly. Genetic analysis will be needed to identify such mutations. And we are attempting to
combine the structure region with the non-structure region from such replicons expecting an infectious full-genome HCV of genotype 1b.
Contact: Kazuo Sugiyama
Center for the Study of Chronic Liver Diseases, Keio University, Tokyo, Japan
[email protected]
Identification of a novel cellular RNA helicase-like protein as a target for cyclosporin A
that is involved in hepatitis C virus genome replication
Koichi Watashi (1), Hiroeki Sahara (2), Kengo Morohashi (3), Kazuki Iwabata (3), Takashi Sunoki (3), Kouji Kuramochi (3), Kaori Takakusagi
(3), Yoichi Takakusagi (3), Hiroki Miyashita (4), Noriyuki Sato (4), Atsushi Tanabe (2), Junpei Konno (2), Kunitada Shimotohno (5), Susumu
Kobayashi (3), Kengo Sakaguchi (3), Fumio Sugawara (3)
(1) Dept. Virology II, National Institute of Infectious Diseases, Tokyo, Japan
(2) Lab. Biology, Azabu University School of Veterinary Medicine, Japan
(3) Genome and Drug Research Center, Tokyo University of Science, Japan
(4) Dept. Pathology, Sapporo Medical University School of Medicine, Japan
(5) Research Institute, Chiba Institute of Technology, Japan
Cyclosporin A (CsA) suppresses the replication of hepatitis C virus (HCV) genome. It has been demonstrated that cyclophilins (CyPs) play a critical
role in HCV genome replication and one of the probable mechanisms for the anti-HCV activity of CsA is mediated by the inactivation of CyPs. To
get further insights into the interaction of CsA with HCV genome replication, we screened for a novel target factor of CsA. We developed a phage
display screening system to efficiently investigate the compound-peptide interaction. Using a random peptide library, we screened for interacting
peptides with CsA, and found a protein in the database that shows a significant homology to hit peptides. This protein, named CsA-associated
helicase like protein (CAHL), showed a RNA-dependent ATPase activity. CAHL interacted with CsA, and its ATPase activity was negated by treatment
with CsA but not FK506 in vitro. CAHL was distributed around the endoplasmic reticulum in Huh-7 cells where it colocalized with HCV NS proteins.
A downregulation of endogenous CAHL reduced the HCV genome replication in the subgenomic replicon system. Overexpression of this protein
enhanced the genome replication. It was also suggested that CAHL formed a complex with HCV NS5B as well as RNA. Thus, an approach directed
by a chemical biology method using CsA as a bio-probe presented another aspect of the mechanism underlying HCV genome replication.
Contact: Koichi Watashi
Dept. Virology II, National Institute of Infectious Diseases, Japan, Tokyo, Japan
[email protected]
POSTERS
P9.26
POSTERS
P9.27
Identification of signal pathway involved in infectious hepatitis C virus (HCV)
particle production
Yuichi Abe (1), Takaji Wakita (2), Kunitada Shimotohno (3), Makoto Hijikata (1)
(1) Institute for Virus Research, Kyoto University, Kyoto-shi, Japan
(2) Department of Virology 2, Natural Institute of Infectious Diseases, Japan
(3) Chiba Institute of Technology, Japan
Previously, we reported the development of three dimensional (3D) cell culture system that reproduces the lifecycle of HCV from patients.
Then we found that HCV infection, replication and virus particle production were enhanced in 3D condition compared with two dimensional
(2D) culture condition of immortalized hepatocytes, HuS-E/2 cells. In this study, we compared the difference of gene expression profiles
between 2D-, and 3D-cultured HuS-E/2 cells, to identify the signal pathways related with HCV lifecycle. Microarray analysis between 2D-,
and 3D-cultured HuS-E/2 cells was performed to identify signal pathways that are modulated in 3D culture condition. Then, the relationship
between those signal pathways and HCV lifecycle were analyzed by treatment of recombinant HCV (JFH1) producing culture with several
inhibitors, agonists, and antagonists. The amount of HCV RNA in medium and cells, infectivity of HCV particles, and localization of viral
proteins was analyzed. Microarray analysis showed differential mRNA expression levels of prostaglandin (PG) synthases in 3D-cultured
cells. Inhibitors, agonists, and antagonist did not affect HCV replication, secretion, and localization of viral proteins markedly. However,
cyclooxygenase1 inhibitor and some PGI2 receptor (IP) agonists decreased infectivity of HCV particles. These findings will provide new
insight about the mechanism of infectious particle production as well as a new target for anti-HCV drugs.
Contact: Yuichi Abe
Institute for Virus Research, Kyoto University, Kyoto-shi, Japan
[email protected]
P9.28
Genotype-specific function of hepatitis C virus glycoproteins in virus assembly
Eike Steinmann (1), Juliane Dörrbecker (1), Martina Friesland (1), Sandra Ciesek (1), Heiner Wedemeyer (2), Christoph Sarrazin (3),
Thomas Pietschmann (1)
(1) Twincore, Hannover, Germany
(2) Department of Gastroenterology, Hepatology and Endocrinology, MHH, Germany
(3) Medizinische Klinik 1, Johann Wolfgang Goethe-Universität, Germany
POSTERS
Development of the JFH1-based HCV infection system has permitted analysis of the complete replication cycle in tissue culture. However,
lack of tissue culture systems for primary, patient-derived isolates continues to limit systematic functional studies of viral quasiespecies.
This impediment has been partially overcome by construction of chimeric viruses. At the same time, these studies highlighted that viral
determinants in the core to NS2 proteins modulate the efficiency of virus production.
To permit studies of glycoprotein function from patient isolates in the JFH1-based cell culture system, we analyzed the importance of genetic
compatibility between the HCV glycoproteins E1 and E2 and remaining viral factors in the course of virus production. To this end, glycoprotein
genes from all seven genotypes and from patients infected with different viral genotypes were cloned. Samples included 7 isolates from
genotype 1, 11 from genotype 2 and one isolate of genotypes 3 to 7, respectively. Subsequently, the ability of these glycoproteins to restore
virus production was assessed by trans-complementation in Huh7.5 packaging cell lines constitutively expressing Jc1 core, p7 and NS2.
Among all patient-derived glycoproteins tested, genotype 2 and 7 isolates restored virus production more efficient than genotype 1, 3, 4, 5, 6
patient-derived variants. When introduced into full-length HCV genomes, GT1a E1/E2 proteins also did not support production of infectious
virus in the context of a GT2a virus genome. These results highlight that HCV glycoproteins support virus production in a genotype-specific
fashion due to specific interactions with additional viral factors. These studies should increase our understanding of HCV morphogenesis
and ultimately permit incorporation of patient-derived HCV glycoproteins in authentic infectious particles produced in tissue culture.
Contact: Eike Steinmann
Twincore, Hannover, AE, Germany
[email protected]
260 : HCV 2011
P9.29
Hepatitis C virus NS5A and the Rab1-GAP TBC1D20 colocalize on the rims of lipid droplets
Inbar Nevo (2), Meital Asher (2), Yaakov Nahmias (1), Koret Hirschberg (2), Ella Sklan (2)
(1) The Hebrew University of Jerusalem, Israel
(2) Tel-Aviv University, Tel-Aviv, Israel
HCV interacts with the host’s secretory and lipid-biosynthetic machinery to facilitate its replication and assembly. Lipid droplets (LDs), an
endoplasmic reticulum (ER) derived lipid-storage organelles, play a key role in HCV particle production. The viral core protein is known as the
major LD-associated viral protein that recruits the replication complex via interactions with the non-structural protein 5A (NS5A) and other
NS proteins. Although typically reported to be ER-associated, NS5A was shown to interact with LDs as well. Thus, the exact nature of NS5A
association with LDs and its role in HCV production are still unclear. Here, we show that NS5A is independently and tightly bound to the LD
surface and recruits TBC1D20, a Rab1- GAP, to ER-LD contact zones. Interestingly, the NS5A-TBC1D20 interaction was previously shown to
be essential for the viral life cycle. Using live cell imaging we analyzed the membrane dynamics of NS5A-GFP and found its binding to LDs
to be apparently irreversible, both in HCV-infected cells and when ectopically expressed. In HCV infected cells NS5A fluoresce was observed
around LDs and in perinuclear structures that were incorporated into a highly immobile platform positioned on the ER membrane surface.
Our results confirm the independent localization of NS5A to LDs and support a model where the LD localization of both core and NS5A is
pivotal for viral assembly. Our data further confirms a role of the host factors TBC1D20 and Rab1 in HCV production.
Contact: Ella Sklan
Tel-Aviv University, Tel-Aviv, Israel
[email protected]
P9.30
Reverse genetic characterization of Bovine Viral Diarrhea Virus (BVDV) NS5A
NS5A of HCV and BVDV are Zn-binding phosphoproteins associated with cellular membranes which play crucial roles in viral RNA replication.
Genetic mapping of HCV NS5A revealed that domain I and domain II are critical for viral RNA replication whereas domain III was shown to
be essential for virion production, but dispensable for RNA synthesis.
In silico analyses also proposed a three-domain structure for NS5A of BVDV; domain I has been shown to be essential for viral RNA replication.
Here we conducted a mutagenesis analysis of BVDV NS5A domains II and III including interdomain regions I and II. A series of 10 aa deletions
in NS5A was generated and tested for effects on RNA replication in the context of a BVDV replicon (DI9). All deletions in a colinear region of
about 100 aa, covering most of domain II and interdomain region II, displayed a lethal phenotype. However, 10 mutants with deletions in
interdomain region I and domain III showed RNA replication competence which was further quantified by the use of a luciferase expressing
bicistronic BVDV replicon.
Insertion of a DsRed-coding sequence into interdomain region I of NS5A did not abrogate viral RNA replication. Thus, formation of viral RNA
replication complexes could be monitored by live cell imaging in cells transfected with the NS5A/dsRed fusion protein coding replicons.
Furthermore, NS5A deletions which allowed for RNA replication were introduced into the cDNA of BVDV strain CP7. In this context only
virus mutants carrying deletions in interdomain region I and the N-terminal part of domain II of NS5A were capable of RNA replication and
infectious virus production.
In conclusion, our results suggest a critical role of large parts of BVDV NS5A domains II and III for virion morphogenesis. Moreover, the
replicon expressing a NS5A/DsRed fusion protein represents a novel tool to study the pestiviral life cycle.
Contact: Norbert Tautz
Institute of Virology and Cell Biology, University of Lübeck, Lübeck, Germany
[email protected]
POSTERS
Norbert Tautz (1), Lars Krüger (1), Olaf Isken (1)
(1) Institute of Virology and Cell Biology, University of Lübeck, Lübeck, Germany
POSTERS
P9.31
Biochemical analysis of NS2 topology provides evidence for three trans-membrane domains
Barnabas King (1), Philip Tedbury (2), Stephen Griffin (1), Mark Harris (1)
(1) University of Leeds, Leeds, United Kingdom
(2) National Cancer Institute at Frederick, USA
Non-structural protein 2 (NS2) is an integral membrane protein that has recently been shown to interact with other viral proteins, potentially
via intra-membrane contacts. The vital role of NS2 has not yet been characterised, however it is thought to be dependent upon these
interactions. Although NS2 has long been assumed to form a three transmembrane domain (TMD) topology; the biochemical evidence for
this remains ambiguous. Determining the topology of NS2 is key to our understanding of how NS2 interacts with the other viral proteins
and functions within the virus lifecycle. Here we provide the first biochemical evidence that NS2 forms three TMDs and define the regions
essential for its interactions with membranes and the viral proteins E2 and NS3. To accomplish this we compiled a consensus topology
prediction which was scrutinised using a series of C terminal truncations of NS2 expressed in Huh7 cells as fusions to a glycoprotein reporter.
Glycosylation analysis demonstrated that truncation of NS2 at aa70 orientated the reporter to the ER lumen, consistent with a 3 TMD
topology. From the truncation data discrete regions of NS2 were used to characterise the membrane targeting of NS2 and its ability to
integrally insert into membranes by membrane fractionation combined with high salt and alkaline carbonate treatment. These constructs
were also used to define the regions of NS2 responsible for interacting with the viral proteins E2 and NS3 by co-immunoprecipitation. This
work has provided the first unambiguous biochemical evidence for the membrane topology of NS2 and further elucidated its interactions
with membranes and other viral proteins. These data provide a framework for dissecting the role of NS2 in the virus life cycle.
Contact: Barnabas King
University of Leeds, Leeds, United Kingdom
[email protected]
P9.32
Expression and purification of hepatitis C virus NS3 protease from virological sustained
responder and non-responder patients
POSTERS
Paola Provazzi (1), Vinícius Santana (2), Hamilton Cabral (3), Fernanda Canduri (4), Maurício Nogueira (5), Paula Rahal (1)
(1) Department of Biology, São Paulo State University, São José do Rio Preto, Brazil
(2) Department of Physics, São Paulo State University, São José do Rio Preto, Brazil
(3) University of São Paulo, Ribeirão Preto, Brazil
(4) University of São Paulo, São Carlos, Brazil
(5) Faculty of Medicine, São José do Rio Preto, Brazil
The hepatitis C virus (HCV) infects 130 million people worldwide and is responsible for acute and chronic hepatitis, cirrhosis and hepatocelular
carcinoma. Nevertheless most effective treatment options are not yet available. The HCV Genotype 3 has a high frequency in Brazil and in
clinical evaluations it is associated with a mild illness manifestation and a better response to the antiviral therapy. Since the processing of
the viral polyprotein is essential for HCV replication, the NS3 protease has been considered to be a primary target for the development of
anti-HCV drugs. In previous work we identified amino acids substitutions on active site and zinc ion binding site of NS3 protease genotype
3 of virological sustained responder patient and non-responder patients. With that the objective of this work is to evaluate the enzymatic
activity of NS3 Protease genotype 3 variants sequences from virological sustained responder and non-responder patients. The Protease NS3
variants sequence was cloned, expressed in E. coli cells to assess their level of expression, and purified from soluble fraction. We believe
that evaluation of the NS3 Protease’s enzymatic activity from sensible and resistant to treatment patients will provide key information about
the NS3 and consequently the replication viral and the Hepatitis C establishment. Additional studies will provide more conclusive results.
Financial support: FAPESP, CAPES and CNPq.
Contact: Paola Provazzi
Department of Biology, São Paulo State University, São José do Rio Preto, Brazil
[email protected]
262 : HCV 2011
P9.33
Evolution of NS5A genetic variability before, during, and after treatment in patients
infected with hepatitis C virus genotype 3a
Cintia Bittar (1), Ana Carolina Gomes Jardim (1), Paola Provazzi (1), Lilian Hiromi Tomonari Yamasaki (1), Claudia Marcia Aparecida
Carareto (1), João Renato Rebello Pinho (2), Isabel Maria Vicente Guedes de-Carvalho-Mello (3), Paula Rahal (1)
(1) São Paulo State University, São José do Rio Preto, Brazil
(2) USP, São Paulo University, Department of Gastroenterology, Brazil
(3) Butantan Institute, Viral Immunology Laboratory, Brazil
Hepatitis C is a global health issue, with a prevalence of 130 million seropositive persons worldwide. The high mutation rates of the virus
RNA polymerase leads to a vast genetic variability presented in multiple levels, the genotypes, subtypes and the quasispecies. The variability
of one of the viral proteins, NS5A, is believed to be related to the response to the IFN therapy, the adopted treatment for the infection. This
study aimed to analyze the quasispecies composition of NS5A protein in patients infected with HCV genotype 3a before, during and after
treatment in patients with Sustained-virological-responder (SVR), Non-responder (NR) and End-of-treatment responder (ETR). Samples
from 12 patients (4 SVR, 4NR, 4ETR) were collected. Viral RNA was extracted, cDNA synthesized, the NS5A region was amplified and cloned.
Until now 540 sequences from 12 patients were generated. The analysis of the sequences revealed a tendency in ETR patients of presenting
less variable quasispecies population when compared to NR. We hypothesize that this has to do with a quest for the most suitable genetic
composition to survive and evade the treatment leading to the rebound. In 2 NR patients we identified one quasispecies that was found
before the treatment also in after treatment samples suggesting that it must have an advantage in the virus survival. The reconstruction of
the phylogeny shows the grouping of all sequences of each patient in a monophyletic branch. Sequences from before treatment samples
grouped in a monophyletic branch apart from the after treatment samples in 3 patients (2 ETR and 1 NR). Also the sequences of two patients
formed a monophyletic branch with hLRT branch support value of 99.2% suggesting an epidemiological link between these two patients.
Our data can help to understand the role of NS5A protein genetic variability on therapy outcome. Financial Support: FAPESP
Contact: Cintia Bittar
São Paulo State University, São José do Rio Preto, Brazil
[email protected]
P9.34
Hyo-young Chung (1), Meigang Gu (1), Charles M. Rice (1)
The hepatitis C virus (HCV) genome encodes a single polyprotein, which is cleaved by viral and cellular proteases producing three structural
and seven nonstructural proteins, core-E1-E2 and p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B, respectively. Nonstructural protein NS5A is a multifunctional protein that facilitates viral RNA replication and virion assembly/budding via interacting with other viral proteins and cellular
factors. However, the detailed mechanism of NS5A function in HCV replication is not completely understood yet.
In this study, we generated a J6/JFH-based infectious clone in which NS5A bears a One-STrEP (OST) tag. The OST-tagged viral genome was
introduced to Huh-7.5 cells and a pull-down assay was performed to detect NS5A interacting proteins. We found that a cellular protein,
Mps one binder kinase activator-like 1B (MOBKL1B) bound to NS5A. MOBKL1B is a component of the Salvador/Warts/Hippo (SWH) tumor
suppressor pathway. The SWH pathway is also called the mitotic exit network (MEN) that regulates mitosis and cytokinesis. A direct
interaction between MOBKL1B and NS5A was confirmed by yeast two-hybrid and surface plasmon resonance assays. MOBKL1B specifically
interacted with a highly conserved stretch of 28 amino acid residues in NS5A domain 2 with a binding affinity (KD) of ~18µM. In addition,
RNAi mediated depletion of MOBKL1B in Huh-7.5 cells significantly inhibited the spread of HCVcc. Further analyses indicated that early step
in viral RNA genome replication was blocked by MOBKL1B depletion. Continuing studies of MOBKL1B should reveal new insights into the
molecular details of HCV replication and spread, and may also be relevant to the link between HCV and hepatocellular carcinoma.
Contact: Hyo-young Chung
[email protected]
POSTERS
Biochemical and functional studies of MOBKL1B in HCV replication
POSTERS
P9.35
Evidence for a role of p7 protein after hepatitis C virus nucleocapsid formation
Ali M. Atoom (1), Daniel M. Jones (1), Rodney S. Russell (1)
(1) Memorial University, St.John’s, Canada
The HCV genome encodes a 63aa protein, p7, which is located between the structural and non-structural proteins. p7 localizes to endoplasmic
reticulum membranes and is composed of two transmembrane domains and a cytoplasmic loop. The role of p7 is relatively unknown and
is currently classified as neither a structural nor non-structural protein. Several studies have shown that p7 acts as an ion channel and is
crucial for infectious virus production in cell culture as well as infectivity in chimpanzees. Recently, it was demonstrated that p7 may prevent
acidification of intracellular compartments that may otherwise render assembled HCV particles non-infectious. The aim of this study was
to investigate whether p7 has additional roles in virus assembly and/or release. Accordingly, we created multiple alanine triplet mutants
along one third of the p7 coding region. We observed that mutations in these regions decreased infectious virus production with variable
efficiencies and these results were confirmed using a single-cycle virus production assay. Analysis of intra- and extracellular virus titres
indicated that p7 functions at a stage prior to generation of infectious particles. These effects were not due to altered RNA replication,
since no affect on NS3 or NS5A protein expression was observed. Confocal microscopy analyses revealed that the p7 mutations did not
affect recruitment of core protein to lipid droplets. Additionally, these mutants could still form nucleocapsids as determined by density
gradient fractionation, suggesting that p7 acts after nucleocapsid formation. Next, we intend to determine whether these mutations have
an effect on additional stages of the HCV life cycle. Ongoing genomic sequencing of adapted viruses that restored virus production after
forced evolution will provide genetic clues of potential p7 binding partners. The results of this study will provide a better understanding of
the function of p7 in virion morphogenesis.
Contact: Ali M. Atoom
Memorial University, St.John’s, Canada
[email protected]
P9.36
Identification of novel NS5A-associated proteins in the host-cell membrane fraction
and their role in HCV life cycle
POSTERS
Koji Goto (1), Toshiro Kimura (2), Koichi Watashi (2), Ryosuke Suzuki (2), Satoshi Yamagoe (3), Tatsuo Miyamura (2), Kyoji Moriya (4),
Hiroshi Yotsuyanagi (4), Kazuhiko Koike (4), Tetsuro Suzuki (5), Takaji Wakita (2), Hideki Aizaki (2)
(1) National Institute of Infectious Diseases, The University of Tokyo, Tokyo, Japan
(2) National Institute of Infectious Diseases, Department of Virology II, Japan
(3) National Institute of Infectious Diseases, Department of Chemotherapy, Japan
(4) The University of Tokyo, Department of Internal Medicine, Japan
(5) Hamamatsu University School of Medicine, Department of Infectious Diseases, Japan
Hepatitis C virus (HCV) NS5A protein is a membrane-associated, essential component of the viral replication complex. In addition, NS5A
is now known to play an important role in assembly of the infectious particles, for which the surface of lipid droplets (LDs) and/or the
LD-associated membranes function as a site. Although several host cellular proteins have been identified as NS5A-binding partners and
their significance in the viral replication has been reported, the molecular basis of NS5A in HCV life cycle has not been fully understood
yet. In this study, to identify novel cellular factors interacting with NS5A, we performed a co-purification, pull-down approach using the
cytoplasmic membrane fraction of cells expressing an epitope-tagged NS5A. We identified twenty host factors, which include previouslypublished ones such as vesicle-associated membrane protein-associated protein, protein-tyrosine kinase fyn, and FK506-binding protein 8.
By subsequent siRNA screening, six of novel NS5A-interacting factors were found to be involved in HCV life cycle; knocking-down the gene
of each factor significantly decreased production of infectious virus in HCV JFH-1-infected cells. Results from further investigation using
a subgenomic replication system and internal ribosomal entry site-assay suggest that these factors can be classified as those affecting 1)
viral RNA replication, 2) viral protein translation, and 3) both viral RNA replication and protein translation. We are going to elucidate the
molecular mechanisms that modulate HCV life cycle through interaction between NS5A and host factors identified in our screening system.
Contact: Koji Goto
National Institute of Infectious Diseases, The University of Tokyo, Tokyo, Japan
[email protected]
264 : HCV 2011
P9.37
Acylation of HCV core C184 residue is essential for protein maturation
Audrey Filion (1), Nathalie Majeau (1), Denis Leclerc (1)
(1) Université Laval, Ste-Marie Bce, Canada
Hepatitis C virus (HCV) core protein is a key factor in the production of infectious viral particles. Core protein also plays a significant role in
HCV pathogenesis especially through modification of the host lipid metabolism. A series of post-translational modifications enables core to
fulfill its different functions. Successive cleavages by the signal peptidase (sp) and signal peptide peptidase (spp) lead to the maturation of
core protein. Subsequently, the addition of a palmitate group near the C-terminus of the mature protein alters its affinity for membranes, a
critical step in particle formation. Herein, we identified by biotin-switch a second acylation site at cysteine 184 in the ER targeting domain of
the immature protein. We demonstrated that cleavage by sp was not dependent on acylation. However, acylation at this site was necessary
for the complete maturation of core by spp cleavage. A better understanding of post-translational mechanisms would be of great interest in
the design of new drugs against HCV infection.
Contact: Audrey Filion
Université Laval, Ste-Marie Bce, Canada
[email protected]
P9.38
A novel NS5A-core interaction: implications in HCV life cycle
Despite recent progress, the molecular machinery of packaging, assembly and particle release of hepatitis C virus (HCV) is still not fully
understood, but of a great importance for new therapeutic approaches. Both NS5A and core play critical roles in the HCV life cycle by acting
at multiple steps including the RNA replication, the recruitment of replication complexes (RC) to lipid droplets (LD), the formation of virions
and the release of infectious particles into the extracellular milieu. A recent study suggested that NS5A and core proteins interacted, we
thus examined the possibility that NS5A-core interactions control one or several steps of the HCV life cycle. We first confirmed by binding
assay using recombinant proteins and by co-immunoprecipitation in cell lysates that NS5A-core interactions occur. We considered the
binding a direct protein-protein interaction since DNase and RNase treatments do not prevent it. In contrast to previous reports, we found
that the domain I of NS5A, but not domain II or III, is responsible for direct binding to the mature form of core (residues 1-170). We further
narrowed the interaction region down to the C-terminal acidic subdomain IB (residues 101-213) of NS5A. Interestingly, we found that the
domain I of core contains the NS5A-binding site, suggesting NS5A-core interactions occur via their respective domains I. We mapped the
major NS5A-binding site in the basic P38 to K74 strech of the HCV core. We are currently mapping the exact residues in both NS5A and core
that mediate NS5A-core interactions. Identified residues will then be analyzed for their significance in HCV replication, assembly and egress
of infectious virus particles. The ultimate goal is to determine whether the NS5A-core interaction, by governing specific steps of the HCV life
cycle, represents a novel target for the development of HCV therapies.
Contact: Katarzyna Gawlik
[email protected]
POSTERS
Katarzyna Gawlik (1), Philippe Gallay (1)
POSTERS
P9.39
Lactoferrin inhibits the HCV NS3 ATPase/Helicase protein which impairs HCV replicon
replication
Frederic Picard-Jean (1), Sabrina Bouchard (1), Angelique Lanthier (1), Martin Bisaillon (1)
(1) Universite de Sherbrooke, Sherbrooke, Canada
More than 170 million individuals are infected by the hepatitis C virus (HCV) worldwide. Current state of therapy fail for 50% of the patients
and despite novel promising therapy, reliable sustained viral response or viral cure are yet to be achieved. About 15-20% of HCV infected
individuals undergo a successful viral eradication underlying the potency of the host immune system to fight HCV. Lactoferrin is part of
the innate immune system and has been reported to harbor antiviral effect. We investigated the effect of human lactoferrin (HLf) on the
replicative HCV non-structural (NS) proteins activity and identified NS3 ATPase/Helicase as a potential target for HLf. NS3 ATPase activity is
inhibited in a dose dependant matter by HLf where 50% inhibition is achieved at HLf concentration of 1 µM. This inhibition is independent
from the Fe status of HLf and is not mediated through a sequestration of the Mg2+ cofactor, but requires a native HLf conformation.
Quantitative analysis

Plenary Lecture - Hepatitis Virus Research Group

Transcription

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