virology afternoons 2015

Department of Virology
VIROLOGY AFTERNOONS 2015
July 1st and 2nd, 2015
Institut Pasteur – Amphithéâtre Emile Duclaux
Abstract Book
Organizing committee:
C. Pereira-Bittencourt, G. Fournier, C. Barbezange,
B. Di Duca, M. Lafon
Wednesday, July 1st, 2015
13:15 Welcome: Monique Lafon
13:30 Keynote session: Marie-Louise Michel
14:10 Session 1 – Virus-Host Interactions
Chairs: Nadia Naffakh and Philippe Afonso
Jun Jin – CCR5 dimerization and export
Elise Biquand – Delineating the interplay between the PB2 protein of influenza A virus
and the host Ubiquitin Proteasome System
14:50 Jose Carlos Valle-Casuso – HIV restriction in mature dendritic cells is associated with
p21-mediated decrease if pSAMHD1
15:10 Daniel Donahue – The inner nuclear membrane protein SUN2 inhibits HIV replication
14:10
14:30
15:30 Coffee break
16:00 Session 2 – Arboviruses: vectors and beyond
Chairs: Nathalie Pardigon and Anavaj Sakuntabhai
16:00
16:20
16:40
17:00
17:20
Faustine Louis – Risk evaluation of the Rift Valley fever emergence in Europe:
Competence of the European mosquitoes and adaptation of the virus
Vanesa Mongelli – Determining the impact of innate immune pathway on viral
diversity and evolution
Vincent Legros – Proteomic profiling of Aedes albopictus infected with Chikungunya
virus or Dengue virus provides new insights into vector/arbovirus interactions
Marine Petit – Do piRNAs contribute to the RNAi-mediated antiviral response in
Drosophila melanogaster?
Pei-Shi Yen – Transgenic mosquitoes for controlling transmission of arboviruses
17:40 Social hour
Thursday, July 2nd, 2015
13:30 Keynote session: Noël Tordo
14:10 Session 3 – New insights on arbovirus pathogenesis
Chairs: Marie Flamand and Bertsy Goic
Essia Belarbi – Bio-imaging of alphavirus disease in mice: A study using Ross River
virus
14:30 Enzo Poirier – A Sindbis mutator generates enhanced levels of defective interfering
particles
14:50 Cécile Khou – Pathogenesis of two molecularly-clones strains of West nile virus and
effect of E protein glycosylation
14:10
15:10 Coffee break
15:40 Session 4 – Weapons against viruses: immunity, vaccine and therapies
Chairs: Nolwenn Jouvenet and Marie-Isabel Thoulouze
15:40
16:00
16:20
16:40
17:00
17:20
Timothée Bruel – Broadly neutralizing antibodies that kill HIV-1 infected cells
Moran Galperin – Public TCR clonotypes preferentially expressed by HIV controllers
confer high sensitivity CD4 responses against Gag
Samira Khiar – Chemical stimulations of the innate immune response with ChX710:
toward a new class of antiviral molecules
Bryan Mounce – Inhibition of polyamine biosynthesis is a broad-spectrum strategy
against RNA viruses
Daria Jacob – A new subunit vaccine against enterovirus-71 based on VP1-carrying
nanoparticles delivered in Pichia pastoris yeast
Kirill Nemirov – Reverse genetics for hantaviruses
17:40 Meeting Wrap-up & Virology Afternoons 2015 Awards
18:00 Social hour
CCR5 dimerization and export
Jun Jin1, Gaelle Boncompain2, Floriane Herit3, Florence Niedergang3, Franck Perez2, Esther
Kellenberger4, Fernando Arenzana-Seisdedos1, Bernard Lagane1, Anne Brelot1
1
Unité Pathologie virale, Département de Virologie, Institut Pasteur, Paris, France
Institut Curie, Paris, France
3
Institut Cochin, Paris, France
4
Université de Strasbourg, Illkirch, France
2
The HIV-1 coreceptors CCR5 and CXCR4 are chemokine receptors that belong to
class A of the family of G-protein coupled receptors (GPCR). Data in the literature showed
that GPCR form dimers or larger oligomers, a process that plays role in numerous receptor
functions including ligand binding, cell signaling, and cell surface expression. In the last
years, the X-ray crystal structures of several GPCR have confirmed that they exist as dimers
and characterized the regions within the receptors involved in receptor oligomerization,
thereby paving the way for new studies on the functional significance of this process.
Our laboratory and others demonstrated that CCR5 can exist as homodimers and also
forms heterodimers with CCR2, but whether these processes influence the HIV-1 coreceptor
function of CCR5 and the antiviral activity of CCR5 ligands is poorly known. To assess these
questions, we plan to identify potential dimerization interfaces in the CCR5 homodimers and
the CCR5/CCR2 heterodimers by bioinformatic analysis and molecular modeling by
homology to the recently published structure of CXCR4. We validate the existence of CCR5
dimerization interfaces by covalent intermolecular cross-linking experiments and we study the
consequences of altering the integrity of dimer interfaces on receptor dimerization. By using
different experimental approaches including directed mutagenesis, energy transfer approaches
on living cells and a new export assay, we showed the importance of receptor dimerization in
the regulation of CCR5 export to the cell surface.
Session 1 Virus-­‐host interactions
Delineating the interplay between the PB2 protein of influenza A virus and
the host Ubiquitin Proteasome System
Elise Biquand, Sylvie van der Werf, Yves Jacob, Caroline Demeret
Unité Génétique moléculaire des virus à ARN, Département de Virologie, Institut Pasteur, Paris, France
The Ubiquitin-Proteasome system regulates diverse cell functions, by inducing protein
degradation, mediating protein activation or shaping their sub-cellular localization.
Accordingly, UPS hijacking is a recurrent theme in viral life cycles. Indeed, eukaryotic
viruses manipulate protein ubiquitination in various ways. Recent evidence indicates that an
intricate regulatory network involving viral proteins and the cellular UPS is likely to
contribute to viral replication and immune evasion of influenza A viruses. However,
usurpation of the host UPS by influenza A viruses is far from being comprehensively
deciphered. To progress in this understanding, we undertook to assess the interplay between
the UPS and the PB2 sub-unit of the influenza A virus polymerase through a global proteomic
profiling approach. For that purpose, a human UPS-dedicated library was constituted and
fully characterized. It consists in 623 ORFs coding for all factors of the human ORFeome
collection with acknowledged or potential functions related to the UPS system. This library
has an estimated global coverage of 63% of the human UPS. In an initial screen, all factors of
the UPS-library were challenged for interaction with PB2 from H1N1wsn using a highthroughput split luciferase assay. A total of 103 UPS factors emerged as potential partners of
PB2. Upon further validation of their interaction with PB2, 46 UPS factors were validated as
high confidence PB2 partners. Importantly, the same UPS-library has been simultaneously
screened with the human papillomavirus E6 and E7 proteins, followed by the same validation
strategy. Such combined screening enabled to detect probable non-specific interactions. It
also pointed to UPS targets common to divergent viruses, and by contrast to factors
specifically targeted by the influenza virus PB2 protein.
An interaction network was constructed by plugging the PB2 UPS targets against the
human interactome network. It appeared that PB2 preferentially targets highest connected
UPS factors, mostly consisting in factors dedicated to substrate recognition by E3 ligase
complexes. In addition, several DUBs emerged as partners of PB2. Our interactomic strategy
will be described, and we will present the resuting PB2/UPS interactome.
Session 1 Virus-­‐host interactions
HIV restriction in mature dendritic cells is associated with p21-mediated
decrease of pSAMHD1
Jose Carlos Valle-Casuso1, Awatef Allouch2, Annie David1, Michaela Müller-Trutwin1,
Monsef Benkirane3, Gianfranco Pancino2 and Asier Sáez-Cirión1
1
Unité HIV inflammation et persistance, Département de Virologie, Institut Pasteur, Paris, France
Unité Régulation des infections rétrovirales, Département de Virologie, Institut Pasteur, Paris, France
3
Laboratory of Molecular Virology, Institute of Human Genetics, CNRS UPR1142, Montpellier, France
2
Dendritic cells (DCs) play a key role in the induction of immune responses against HIV.
However, HIV has evolved ways to exploit them, facilitating immune evasion and viral
dissemination. Immature myeloid DCs (mDC) can sustain HIV-1 replication but are poor
inducers of adaptive immunity. In contrast mature mDC have a higher potential to efficiently
present antigens but are strongly resistant to HIV infection. Better understanding the interplay
between HIV and DCs could inform the design of therapeutic approaches to decrease viral
dissemination and to optimize immune responses. Our group has recently shown that the
cellular factor p21cip/waf potently blocks HIV infection in macrophages by reducing the pool of
dNTPs through the inhibition of RNR2. p21 through its cyclin-dependent kinase inhibitory
activity may also modulate the phosphorylation state of SAMHD1 and its antiviral activity.
We wondered whether p21 could be involved in the strong resistance of mature mDCs to HIV
infection. We found that the maturation of mDCs with IFNγ and CD40L, which strongly
blocked HIV-1 replication, was associated with a strong increase in the expression of p21.
While HIV-1 inhibition in mDCs was not associated with changes in RNR2 or total SAMHD1
expression, maturation of mDCs was accompanied by a decrease in the levels of pSAMHD1.
Knockdown of p21 by siRNA in immature and mature mDCs induced an increase in the
levels of pSAMHD1 and rescued HIV-1 replication. The inhibition of HIV replication in
mature mDCs was completely abrogated by the degradation of SAMHD1 in presence of VLPVPX particles. Although our study is still in progress, our results suggest that the block of
HIV replication in mature mDCs is due to an increase in non-phosphorylated forms of
SAMHD1 resulting from the induction of p21 expression during the maturation of the cells.
Overall, p21 appears as a master regulator of HIV infection in myeloid cells.
Session 1 Virus-­‐host interactions
The inner nuclear membrane protein SUN2 inhibits HIV replication
Daniel Aaron Donahue, Timothée Bruel, Nicoletta Casartelli, Olivier Schwartz
Unité Virus et immunité, Département de Virologie, Institut Pasteur, Paris, France
Background: In a screen of genes reported to be induced by interferon (IFN), SUN2 was
previously shown to inhibit HIV-1 infection through mechanisms that were not characterized.
SUN2 is an inner nuclear membrane protein belonging to the LINC complex. LINC
complexes form a bridge between the cytoskeleton and the nucleus, and play a
mechanostructural role important for cytoskeletal and nuclear function. The aim of our study
is to uncover the role of SUN2 in HIV replication.
Results: SUN2 was only weakly induced by IFNs and other cytokines in cell lines and
in monocyte-derived dendritic cells, and not in PBMCs or CD4 T cells. However, SUN2
overexpression blocked the infection of several cell lines with various HIV-1 and HIV-2
strains, with peaks of infection delayed by up to 2 weeks in CHME microglial cells. SUN2
silencing, in contrast, did not enhance infection. Using qPCR we show that total reverse
transcribed DNA levels were unchanged when SUN2 was overexpressed, while levels of 2LTR circles and integrated DNA were reduced, suggesting a nuclear import block. We also
identified HIV-1 and HIV-2 strains which were naturally resistant to SUN2, indicating that
viral components may regulate the sensitivity to this nuclear protein. In serial passaging
experiments we selected for SUN2-resistant HIV-1, which could lead to the identification of
the viral target of SUN2. By using SUN2 mutants and deletants we characterized the SUN2
domains required for the antiviral activity of the protein.
Conclusions: SUN2 is a novel cellular protein that inhibits HIV nuclear import. Current
work will help determine which cellular proteins and nuclear import pathways are involved in
this antiviral activity.
Session 1 Virus-­‐host interactions
Risk evaluation of the Rift Valley fever emergence in Europe: Competence
of the European mosquitoes and adaptation of the virus
Faustine Louis, Ilaria Castelli, Marie Vazeille, Anna-Bella Failloux
Unité Arbovirus et insectes vecteurs, Département de Virologie, Institut Pasteur, Paris, France
The Rift Valley Fever virus (RVFV) is an arbovirus capable to infect numerous species
of mammals and be transmitted in majority by mosquitoes but not only, this virus could also
be transmitted directly via fluids like raw milk, blood and semen, providing to this virus a
larger capacity to disseminate. It circulates among wild mammals at a low prevalence but
when environmental conditions are favorable for mosquito proliferation, an epidemic can
occur causing mass abortions in cattle and death of young animals. Humans are mainly
contaminated by direct contacts with tissues and blood when manipulating infected animals,
impacting the industry of breeders with dramatic economic and social consequences. RVFV
was first detected in Kenya in 1930 but recently, it has expanded its natural range of
distribution outside the Sub-Saharan Africa, in Saudi Arabia, Yemen, Madagascar, the
Comoros and Mayotte islands. Its current expansion questions on the risk of a RVFV
emergence in Europe. With a world trade in expansion, climate change and political
instabilities in some Africa countries, the risk for RVFV emergence outside its natural range
of distribution remains high. To initiate an epidemic, local mosquitoes should be capable to
transmit the virus and in other words, be competent for transmission. Our study will be
focused on the risk of RVFV emergence in Europe, and more particularly in the south of
France. The wetlands in Camargue facing North Africa offer a favorable environment for
mosquito proliferation and RVFV establishment from southern regions. Our study develops
two objectives: (i) determine the distribution and the competence to RVFV of French
mosquito vectors and (ii) determine if molecular changes in the viral genome can be
associated to an increased transmission by European mosquitoes.
Session 2 Arboviruses: vectors and beyond
Determining the impact of innate immune pathway on viral diversity and
evolution.
Vanesa Mongelli, Valerie Dorey, Marine Petit, Herve Blanc, Lionel Frangel, Maria Carla
Saleh
Unité Virus et interférence ARN, Département de Virologie, Institut Pasteur, Paris, France
Host-pathogen interactions trigger selective pressures on both partners. Hosts face a
vast array of parasites to which they must adapt, and it is widely believed that pathogens are a
major selection pressure in all natural populations. The fact that immunity genes evolve
faster, and often much faster, than other genes indicates that adaptive evolution is occurring.
On the other hand, RNA viruses accumulate mutations at very high rates. Consequently, viral
populations are very diverse and contain several genomes that diverge from the parental
consensus sequence. Such diversity is critical for efficient transmission and full pathogenicity
of the virus. It is thus likely that innate immunity, through an array of antiviral responses,
shapes the diversity of viral populations. Our goal is to conduct a comprehensive study, under
laboratory-controlled conditions, of the link between insect innate immunity and viral
evolution using D. melanogaster as model insect and its natural pathogen Drosophila C virus
(DCV). In order to do so, we generated innate immunity deficient mutant flies and serial
passaged DCV through those fly mutants. Viral population diversity was assessed by deep
sequencing after serial passages in the different innate immunity deficient flies.
Session 2 Arboviruses: vectors and beyond
Proteomic profiling of Aedes albopictus infected with Chikungunya virus or
Dengue virus provides new insights into vector/arbovirus interactions
Vincent Legros1, Thibault Chaze2, Mariette Matondo2, Dorothée Missée3, Valérie Choumet1
1
Unité Environnement et risques infectieux, groupe Interactions Moléculaires Flavivirus-Hôtes, Département
d’Infection et Epidémiologie, Institut Pasteur, Paris, France
2
Plate-forme de protéomique, Institut Pasteur, Paris, France
3
Institut de Recherche pour le Développement, Montpellier, France
Diseases caused by arbovirus are a major public health challenge in many parts of the
world. Despite intense research, vaccination or specific treatments are still needed for some
arbovirus such as chikungunya virus (CHIKV) and dengue virus (DENV). In order to control
those diseases, a better understanding of host-vector-virus interactions is required. In
particular, vector-virus interaction remains poorly understood although it could provide key
information about the virus epidemiology, physiopathology or virulence.
Our study focused on the interaction between Aedes albopictus and two of the main
arbovirus transmitted by this mosquito: chikungunya virus and dengue virus. For this purpose,
we performed experimental infections of adults Aedes albopictus by DENV and CHIKV in
our laboratory. Then, salivary glands and midguts were removed; the viral loads in those
organs were analyzed by RT-qPCR. Thus, we were allowed to determine the dissemination of
the arbovirus in the vector’s organism during the first two weeks of the infection. The second
step was to perform a label-free LC-MS proteomic analysis of these organs in the presence
and absence of the virus. We found that infected proteomes organs are significantly different
from those of uninfected organs, which suggests a modulation of proteins synthesis during the
infection of mosquito’s organs with the arbovirus. Quantitative analysis revealed 211 and 114
differentially expressed proteins in chikungunya infected midguts and salivary glands,
respectively. For organs infected with dengue virus, we found 100 and 103 proteins. In the
midgut, most of the proteins are upregulated, which is different from the salivary glands
where the proportion of downregulated proteins is higher. Using online libraries such as
UniProt, we were able to map the cellular pathways that are mostly affected during the
infection. We demonstrated that protein synthesis pathway and energy metabolism are
activated in salivary organs associated with an intense replication while apoptosis and stress
response are not stimulated. Additionally, this innovating approach allowed the identification
of potential restriction factors that we could target in order to inhibit virus transmission during
the bite.
Session 2 Arboviruses: vectors and beyond
Do piRNAs contribute to the RNAi-mediated antiviral response in
Drosophila melanogaster?
Marine Petit, Vanessa Mongelli, Lionel Frangel, Hervé Blanc, Carla Saleh
Unité Virus et interférence ARN, Département de Virologie, Institut Pasteur, Paris, France
The small interfering RNA (siRNA) pathway was shown to be the major antiviral
defense mechanism in insects. However, recent work describing the production of viral
derived piwi-interacting RNAs (vi-piRNAs) in mosquitoes during arbovirus infection,
suggested the involvement of the piRNA pathway in antiviral defense. In this work we
propose to address the involvement of the piRNA pathway on antiviral immunity in D.
melanogaster.
To study the impact of piRNA pathway on fly immunity, we studied the effect of viral
acute infections on piRNA pathway mutants. We inoculated wild type and Piwi, Aub, Ago-3
and Zuc mutant flies with Drosophila C virus (DCV), Drosophila X virus (DXV) or Sindbis
virus (SINV). No significant differences were observed between the fly genotypes neither in
survival nor in viral titer. We also analyzed the production of vi-piRNAs during acute
infections on wild type and RNAi Dcr-2 and Ago-2 mutant flies infected with DCV, DXV or
SINV. The siRNA pathway mutants were used to test if the absence of this antiviral
mechanism could put in evidence a contribution of piRNA pathway. Interestingly, no vipiRNAs were found by deep-sequencing analyses in none of the genotypes or viral infections.
Finally, we analyzed the presence of vi-piRNAs in fly stocks persistently infected with
Drosophila A virus (DAV), Nora virus and DCV. Recent work from our lab showed that the
establishment of persistent infections in insects depends on the production of a viral DNA
form, which could constitute the source of vi-piRNAs. Also in this case, no vi-piRNAs were
found.
Taken together, our results suggest that the piRNA pathway does not play a role in
antiviral defense in D. melanogaster, which might highlight an evolutionary divergence in the
speciation of the antiviral response between flies and mosquitoes.
Session 2 Arboviruses: vectors and beyond
Transgenic mosquitoes for controlling transmission of arboviruses
Pei-Shi Yen1, Chun-Hong Chen2, Anna-Bella Failloux1
1
2
Unité Arbovirus et insectes vecteurs, Département de Virologie, Institut Pasteur, Paris, France
National Health Research Institutes, Division of Molecular and Genomic Medicine, Miaoli, Taiwan
Dengue (DENV) and chikungunya (CHIKV) share both the same geographical
distribution and the mosquito vectors: Aedes aegypti and Aedes albopictus. These arboviruses
affect over 100 million people annually, and more than one-third of the world’s population
lives in regions at risk of infection. Currently no licensed vaccines or specific antiviral
treatments are available. The most effective intervention is the control of mosquito
populations through the use of insecticides. However control strategies based on insecticides
are threatened by the emergence of resistance in mosquito populations. Therefore, alternative
strategies for mosquito vector control are needed, including the development of new geneticsbased approaches. In our project, we try to activate the mosquito antiviral responses using two
different induction systems. The first approach utilizes the miRNA-based control strategy,
which corresponds to a genetic construct expressing copies of anti-DENV and -CHIKV
microRNAs under the control of Ae. aegypti ubiquitin and carboxypeptidase A promoter.
Upon infection, the transgenic mosquito mounts a potent RNAi response, limiting viral
infection of the host. Because continuous RNAi induction is likely to impose a fitness cost on
the mosquito, another approach is being explored using transcriptional elements of the
CHIKV genome. An exogenous immune gene will be activated at the very early stage of
infection, improving the antiviral efficiency and also reducing the fitness impact on transgenic
mosquitoes. To date, we have already obtained the basic information of susceptibility to
CHIKV and DENV of the mosquito strain that we use for transgenesis, and we are now
screening for the best performance line for miRNA-based control strategies. Meanwhile, we
have already completed the construction of some expression cassettes for the alternative
alphavirus induction system, and are performing mosquito transgenesis studies.
Session 2 Arboviruses: vectors and beyond
Bio-imaging of alphaviral disease in mice: A study using Ross River virus
Essia Belarbi1,2, Philippe Desprès2,3, Dorothée Missé4, Pierre Roques2, Valérie Choumet1
1
Unité Environnement et risques infectieux, groupe Arbovirus, Département d’Infection et Epidémiologie,
Institut Pasteur, Paris, France
2
Service d’immuno-virologie, Commissariat à l’Energie Atomique, Fontenay aux Roses, France
3
Université de la Réunion, Ile de la Réunion, France
4
Institut de Recherche pour le Développement, Montpellier, France
Due to their epidemic potential, alphaviruses, especially arthritogenic ones, are a serious
threat for the public health worldwide.
Arthritis and myalgia due to the alphaviruses Chikunguya (CHIKV) and Ross River
(RRV) are remarkable for their prevalence and duration of the disease. These arboviruses are
transmitted by mosquito bite. CHIKV causes millions of cases worldwide with 10 to 30%
chronicity resulting in a significant loss of life-quality and an important socio-economical
burden. RRV is found in the south pacific and causes around 8000 cases per year. Currently,
there is no effective treatment and no vaccine against these viruses.
The patients suffering from these viral arthralgia/myalgia are given symptomatic
treatments such as non-steroidal anti-inflammatory drugs or corticosteroids with
unsatisfactory results. Moreover, recent data suggest that the chronic phase of the disease
might be related to the persistence of the virus at low level in the macrophages, emphasizing
the need for new antiviral molecules efficient against alphaviruses and able to target these
compartments: macrophages or other reservoir cells embedded in tissues.
Using a molecular clone of RRV, we designed recombinant viruses expressing Renilla
luciferase, a bioluminescent protein. We characterized these viruses in vitro and in vivo and
performed a longitudinal study on an albino mice strain. The bioluminescent virus allowed us
to monitor the acute phases of the disease, and to detect replicating virus during the chronic
phase.
This novel non-invasive bio-imaging model of alphaviral arthritogenic disease will help
us deciphering the complex mechanisms tuning the switch to chronicity and evaluating
vaccines and new antiviral molecules.
Session 3 New insights on arbovirus pathogenesis
A Sindbis mutator generates enhanced levels of defective interfering
particles
Enzo Z. Poirier1, Kathryn Rozen-Gagnon1,2, Kenneth A. Stapleford1, Bryan C. Mounce1,
Peter Jan Hooikaas1, Gonzalo Moratorio1, Marco Vignuzzi1
1
Unité Populations virales et pathogenèse, Département de Virologie, Institut Pasteur, Paris, France
Present address, Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The
Rockefeller University, New York, NY 10065, USA
2
Alphaviruses, such as chikungunya virus, are medically important positive-stranded
RNA viruses that can infect both mammals and insects. Sindbis virus (SINV), a prototypic
alphavirus, can be genetically altered to generate more mutations during viral RNA
replication. These so-called mutators have been demonstrated to be attenuated in several
model organisms such as mice, mosquitoes and fruit flies. However, the precise mechanism(s)
as to why these mutants are attenuated has yet to be elucidated. A common feature of RNA
viruses is the production of defective interfering genomes, i.e. genomes that are truncated in
one or several coding sequences and that interfere with full-length genome replication.
Interestingly, recent studies have shown the high capacity of these defective genomes to
induce innate immunity. Here we demonstrate that defective genomes are produced in a larger
amount by SINV mutator compared to wildtype. When mixed with wildtype virus, these
defective genomes interfere with replication of the full-length virus. We identify a putative
RNA hairpin structure that may be the origin of these defective genomes. Our results suggest
that in addition to accumulation of lethal mutations on full-length genomes, mutator strains
may be further attenuated in vivo by the more rapid accumulation of defective interfering
particles.
Session 3 New insights on arbovirus pathogenesis
Pathogenesis of two molecularly-cloned strains of West Nile Virus and
effect of E protein glycosylation
Cécile Khou1, Marie-Pascale Frenkiel1, Khaled Alsaleh1, Sylvie Paulous2, Gamou Fall3,
Amadou Alpha Sall3, Nathalie Pardigon1
1
Unité Environnement et risques infectieux, groupe Arbovirus, Départment Infection et Epidémiologie, Institut
Pasteur, Paris, France
2
Unité Hépacivirus et immunité innée, Département de Virologie, Institut Pasteur, Paris, France
3
Unité Arbovirus et virus de fièvres hémorragiques, Institut Pasteur de Dakar, Dakar, Sénégal
West Nile virus (WNV) is an emerging neuroinvasive flavivirus that causes significant
morbidity and mortality worldwide. There are currently seven lineages identified, but only
lineages 1 and 2 viruses are responsible for West Nile diseases. In Africa, lineage 1 (L1)
seems to be more virulent than lineage 2 (L2). Current hypothesis is that difference in E
protein glycosylation may have an effect on virus pathogenesis. In this work, we constructed
molecular clones from both lineages isolated in Senegal, and produced infectious viruses in
cell culture. Glycosylation site at position 154 of L1 virus E protein was removed by sitedirected mutagenesis. Gene synthesis was used to add the glycosylation site at position 154 of
L2 virus E protein. Pathogenesis in a mouse model of WNV infection was investigated for all
these viruses.
Session 3 New insights on arbovirus pathogenesis
Session 4 Weapons against viruses: immunity, vaccine and therapies
Broadly neutralizing antibodies that kill HIV-1 infected cells
Timothée Bruel1,2,3, Florence Guivel1,2, Léa Richard1,2, Francoise Porrot1,2, Marine Malbec4,5,
Daniel A Donahue1,2, Nicoletta Casartelli1,2, Hugo Mouquet4,5, Olivier Schwartz1,2,3
1
Unité Virus et immunité, Département de Virologie, Institut Pasteur, Paris, France
CNRS URA 3015, Paris, France
3
Vaccine Research Institute, Creteil, France
4
G5 Réponse humorale aux pathogènes, Département d’Immunologie, Institut Pasteur, Paris, France
5
CNRS URA 1961, Paris, France
2
HIV-1 envelope (Env)-specific broadly neutralizing monoclonal antibodies (bNAbs)
with high potency have been described. A clinical trial recently demonstrated the antiviral
activity of one bNAb (3BNC117) in HIV-1-infected humans. In animal models, the Fc
domain of bNAbs is required for suppressing viremia and targeting the viral reservoir,
through mechanisms only partially understood. Here, we identify a subset of bNAbs that exert
antibody-dependent cellular cytotoxicity (ADCC) and kill HIV-1 infected lymphocytes in cell
culture. These antibodies target the CD4 binding site (NIH45-46, VRC01, and 3BNC117), the
glycan/V3 loop (10-1074 and PGT121), the V1/V2 loop (PG16) on gp120, or the gp41 moiety
(10E8). Efficient ADCC requires stable binding of bNAbs to Env proteins at the surface of
infected cells and engagement of NK cells in a FcγR-dependent manner. There is a strong
variability in sensitivity to ADCC depending on the viral strain and the individual bNAb used.
However, a combination of bNabs generally achieved better efficacy. Our study delineates the
parameters controlling the ADCC activity of the most potent bNAbs.
Daniela Benati*1, Moran Galperin*1, Olivier Lambotte2,3,4, Stéphanie Gras5,6, Annick Lim7,
Madhura Mukhopadhyay1, Alexandre Nouël1, Kristy-Anne Campbell5, Brigitte Lemercier7,
Mathieu Claireaux1, Samia Hendou8, Pierre Lechat9, Pierre De Truchis10, Faroudy Boufassa8,
Jamie Rossjohn5,6,11, Jean-François Delfraissy2,3,4, Fernando Arenzana-Seisdedos1,12, Lisa
Chakrabarti1
1
Unité de Pathogénie virale, Département de Virologie, Institut Pasteur, Paris, France
INSERM U802, Bicêtre Hospital, France
3
AP-HP, Department of Internal Medicine and Infectious Diseases, Bicêtre Hospital, France
4
Université Paris-Sud, Le Kremlin-Bicêtre, France
5
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University
6
Australian Research Council Centre of Excellence for Advanced Molecular Imaging, Monash University,
Clayton, Victoria 3800, Australia
7
Département d’Immunologie, Institut Pasteur, Paris, France
8
Center for Reseach in Epidemiology and Population Health (CESP), INSERM U1018, Le Kremlin-Bicêtre,
France
9
Genomic Bioanalysis Group, Institut Pasteur, Paris, France
10
Raymond Poincaré Hospital, AP-HP, Infectious and Tropical Diseases Department, Garches, France
11
Institute of Infection and Immunity, School of Medicine, Cardiff University, Heath Park, Cardiff, UK
12
INSERM U1108, Paris, France
2
The rare patients who spontaneously control HIV replication in the absence of therapy
show signs of a particularly efficient cellular immune response. To identify the molecular
determinants underlying this response, we characterized the TCR repertoire directed at the
most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers showed a
highly skewed repertoire characterized by a predominance of the TRAV24 and TRBV2
variable gene families, the presence of conserved motifs in both CDR3 regions, and a high
prevalence of public clonotypes (n=18 for each TCR chain). The most prevalent public
clonotypes generated TCR with affinities in the micromolar range, at the high end of values
reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs conferred
broad HLA II cross-restriction, with up to 5 HLA-DR alleles recognized, high antigen
sensitivity, and polyfunctionality to primary CD4+ T cells. In addition, CD8+ T cells could be
redirected to target the conserved capsid major homology region by expressing a highaffinity
Gag293-specific TCR. These findings indicate that TCR clonotypes with superior functions
are associated with HIV control. Amplifying or transferring such clonotypes may contribute
to immunotherapeutic approaches that aim at a functional HIV cure.
Session 4 Weapons against viruses: immunity, vaccine and therapies
Public TCR clonotypes preferentially expressed by HIV Controllers confer
high sensitivity CD4 responses against Gag
* contributed equally
Session 4 Weapons against viruses: immunity, vaccine and therapies
Chemical stimulation of the innate immune response with ChX710: toward
a new class of antiviral molecules
Samira Khiar1, Marianne Lucas-Hourani1, Hélène Munier-Lehmann2, Nikaïa Smith4, Olivier
Helynck2, Sébastien Nisole3, Jean-Philippe Herbeuval4, Frédéric Tangy1, Pierre-Olivier
Vidalain4
1
Unité Génomique virale et vaccination, Département de Virologie, Institut Pasteur, Paris, France
Unité Chimie et biocatalyse, Département Biologie Structurale et Chimie, Institut Pasteur, Paris, France
3
Unité Mechanism of Interferon action and bio-therapeutic pathways, Université Paris Descartes, Paris, France
4
Equipe Chimie & Biologie, Nucléos(t)ides et Immunologie pour la Thérapie (CBNIT), Université Paris
Descartes, Paris, France
2
Although viral infections represent a major burden for public health, our therapeutic
arsenal is quite limited. Current antiviral therapies mostly rely on nucleoside analogs, few
virus-specific drugs (oseltamivir; palivizumab), and recombinant IFN-α/β of limited efficacy
in most cases. Despite the success of direct acting antivirals (DAA) against HIV or HCV,
viruses often escape drugs targeting components of their replication machinery through
mutations. Added to the fact that new pathogenic viruses are emerging permanently, there is a
critical need for innovative approaches to fight seasonal epidemics and unexpected outbreaks
like Ebola and chikungunya crises. In this perspective, we are exploring a new concept:
broad-spectrum antivirals that, instead of targeting the virus itself, block viral growth by
modulating cellular defense mechanisms. To identify new chemical entities that stimulate the
expression of the antiviral cluster of interferon-stimulated genes (ISGs), we developed a
stable cell-line expressing luciferase under the control of an interferon-stimulated response
element that is both activated by IRF1/3/7 transcription factors and type I interferons. This
cellular assay was used to screen an unbiased chemical library of 10.000 compounds, and
only one referred as ChX710 was found to activate the ISRE promoter in a range similar to
IFN-β. Further analyses by qRT-PCR showed that ChX710 efficiently stimulates the
expression of interferon genes and ISGs in different human cell types, in particular primary
monocytes and plasmacytoid dendritic cells. Such immuno-stimulatory properties were never
reported before for this chemical series, and its mode of action is totally unknown. To
decipher the signaling pathways activated by ChX710, we knocked-down cellular factors in
the induction of ISGs by pathogen recognition receptors from RLR family (MAVS) or type I
interferon signaling (JAK1). None of these different factors, except IRF1, seems to be
involved in the activation of the ISRE promoter by ChX710. Added to the fact that our ISREluciferase reporter cell line does not express TLR7/8/9 or STING, our data suggest that
ChX710 is targeting a novel signaling pathway to induce ISGs and the innate antiviral
response. Furthermore, this molecule increased the antibody response to ovalbumin in mice,
establishing its immunostimulatory properties in vivo. Finally, and despite the induction of
some stress-related apoptosis in vitro that probably relates to the innate immune response
induced, ChX710 showed neither local nor systemic toxicity when injected to animals.
Altogether, our data suggest that ChX710 is a potent stimulator of the innate antiviral
response that targets some yet uncharacterized cellular pathway.
Session 4 Weapons against viruses: immunity, vaccine and therapies
Inhibition of polyamine biosynthesis is a broad-spectrum strategy against
RNA viruses
Bryan C. Mounce1, Enzo Z. Poirier1,2, Gonzalo Moratorio1, Everett Clinton Smith3, Carole
Tamietti4, Sandrine Vitry5, Etienne Simon-Loriere6, Romain Volle7,8, Cécile Khou9, Matthieu
Prot6, Marie-Pascale Frenkiel9, Kenneth A. Stapleford1, Anavaj Sakuntabhai6, Francis
Delpeyroux7,8, Nathalie Pardigon9, Marie Flamand4, Giovanna Barba-Spaeth4, Monique
Lafon5, Mark R. Denison3,10, Marco Vignuzzi1
1
Unité Populations virales et pathogenèse, Institut Pasteur, Paris, France
University Paris Diderot, Sorbonne Paris Cite, Cellule Pasteur, Paris, France
3
Department of Pediatrics, the Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical
Center, Nashville, USA
4
Unité Virologie structurale, Institut Pasteur, Paris, France
5
Unité Neuroimmunologie virale, Institut Pasteur, Paris, France
6
Unité Génétique fonctionnelle des maladies infectieuses, Institut Pasteur, Paris, France
7
Unité Biologie des virus entériques, Institut Pasteur, Paris, France
8
INSERM, Unité 994, France
9
Unité Environnement et risques infectieux, Institut Pasteur, Paris, France
10
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville,
USA
2
Emerging viruses present an extraordinary threat to human health, given their sudden
and unpredictable appearance and the potential for rapid spread among the human population.
Recent emergence of chikungunya virus (CHIKV) in the Americas, Middle East respiratory
syndrome coronavirus (MCoV) in the Arabian peninsula and Ebola virus in Western Africa
highlight the struggles to contain outbreaks. A significant hurdle is the availability and
implementation of antiviral therapies to treat the infected or protect at-risk populations, such
as family members and healthcare workers. While several compounds show promise in vitro
and in vivo, these recent outbreaks underscore the need to accelerate drug discovery, as well
as to explore therapeutic avenues of broad antiviral activity. In this report, we describe the
antiviral effects of difluoromethylornithine (DFMO, eflornithine), a potent suicide inhibitor of
ornithine decarboxylase (ODC1), a critical enzyme in polyamine synthesis. We show that
DFMO is active against diverse families of RNA viruses both in vitro and in vivo. Our data
show that polyamines are a general requirement for viral RNA synthesis. DFMO is
bioavailable and currently used in treating trypanosomiasis and hirsutism, and given its
tolerance in humans, may be an immediately available and viable option for controlling
infection during outbreaks of significant concern.
Daria Jacob1, Claude Ruffie1, Thibault Rosazza1,3, Mégane Babiak1, Luisa Mandorli1, Chantal
Combredet1, Romain Volle2, Jean Balanant2, Francis Delpeyroux2, Frederic Tangy1, Monica
Sala1.
1
Unité Génomique virale et vaccination, Département de Virologie, Institut Pasteur, Paris, France
Unité Biologie des virus entériques, Département de Virologie, Institut Pasteur, Paris, France
3
Unité Parasitologie moléculaire et signalisation, Département de Parasitologie, Institut Pasteur, Paris, France
2
Enterovirus 71 (EV71) is the cause of hand-foot-and-mouth disease (HFMD), which
may have neurological complications and lead to fatalities in young children. Its prevalence is
constantly growing with large epidemics in South-Eastern Asia and outbreaks in many other
parts of the world. Therefore, an efficient vaccine is urgently required.
We developed a subunit vaccine candidate against EV71, based on its VP1 capsid
protein known to induce neutralizing antibodies, which correlate with protection. The VP1
protein was fused to the measles virus nucleoprotein N capable of assembly into rod-shaped
multimeric ribonucleoparticles (RNPs) in various expression systems, particularly in yeast.
We used whole Pichia pastoris yeast as both production and delivery system of N-VP1 RNPs,
rendering vaccine preparation technologically and economically advantageous.
The possibility of fusing EV-71 VP1 to N and maintaining RNP formation was
validated by electron microcopy analysis following yeast lysis and ultracentrifugation.
Several homologous prime-boost protocols were screened for immunizing C57Bl/6 mice to
select the most efficient dose and protocol regimen. In the absence of accessory adjuvants,
30YU (yeast unit, 107 yeast cells) of heat-inactivated P. pastoris carrying N-VP1 RNPs
induced anti-VP1 IgG antibodies, which were capable of neutralizing the homologous EV71
strain. Substitution of two amino acids within the N multimerization site know to hamper
multimerization capacity (NQD mutant, Karlin et al., 2002 Virology) hampered the formation
of VP1-carrying RNPs and significantly decreased their ability to induce anti-VP1 antibodies,
indicating the intrinsic adjuvancy of the subunit platform provided through VP1
multimerization.
We suggest that a subunit platform based on P. pastoris yeast expressing VP1-carrying
RNPs is an attractive approach for developing an efficient and low-cost vaccine against
Session 4 Weapons against viruses: immunity, vaccine and therapies
A new subunit vaccine against enterovirus-71 based on VP1-carrying
nanoparticles delivered in Pichia pastoris yeast
EV71.
Session 4 Weapons against viruses: immunity, vaccine and therapies
Reverse genetics for hantaviruses
Kirill Nemirov1, Alexander Plyusnin2, Åke Lundkvist3, Noël Tordo1
1
Unité Stratégies antivirales, Département de Virologie, Institut Pasteur, Paris, France
Department of Virology, University of Helsinki, Haartman Institute, Helsinki, Finland
3
Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
2
Hantaviruses (genus Hantavirus, family Bunyaviridae) are rodent-borne viruses that
cause two severe and often fatal human zoonoses: hemorrhagic fever with renal syndrome
(HFRS), and hantavirus cardiopulmonary syndrome (HCPS). For many years research of
these pathogens was hampered by the lack of a reverse genetics system (RG). Such systems,
developed for other RNA viruses, may be extremely useful for dissecting the basis of viral
pathogenesis, generation of live-attenuated vaccines, and testing of anti-virals. Although
several reports claiming the development of minigenome (MG) for hantaviruses were
published, these MGs appeared to have very limited capacity and have not been developed for
practical use. We are currently exploring different approaches in order to develop functional
MG and RG system for non-pathogenic Tula hantavirus, which could be later adopted for
pathogenic hantaviruses, such as Dobrava and Puumala. Current progress of the work as well
as specific issues related to adaptation of RG approaches to hantaviruses will be disscussed.