Functional genomics as a tool to define a molecular signature of

Engineering Conferences International
ECI Digital Archives
Vaccine Technology IV
Proceedings
Spring 5-22-2012
Functional genomics as a tool to define a molecular
signature of effective vaccination against foot and
mouth disease virus
Jose Chabalgoity
Departamento de Desarrollo Biotecnologico, Facultad de Medicina, Universidad de la Republica, Uruguay
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Recommended Citation
Jose Chabalgoity, "Functional genomics as a tool to define a molecular signature of effective vaccination against foot and mouth disease
virus" in "Vaccine Technology IV", B. Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves , ITQB/
IBET; K. Jansen, Wyeth Vaccine Research Eds, ECI Symposium Series, (2013). http://dc.engconfintl.org/vaccine_iv/24
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Transcriptional biomarkers of
effective vaccination against
Foot and Mouth Disease
José A. Chabalgoity
Instituto de Higiene
http://www.higiene.edu.uy/ddbp
Laboratory for Vaccine Research
Salmonella
as vaccine
vector
Salmonella:
genetic
variation &
pathogenicity
Immunotherapies
for respiratory
infections
Biotech
industry
Veterinary
vaccines
Laboratory for Vaccine Research
Biotech
industry
Veterinary
vaccines
Foot and Mouth Disease (FMD)
• Highly contagious disease of mammals. Affects
cattle, pigs, sheep, goats, etc.
• Clinical signs can vary from mild to severe.
Fatalities may occur, especially in young animals
• Spreads rapidly
• Seven serotypes of FMD virus (FMDV): no cross
immunity between serotypes
• Severe economic losses
Outbreaks have major impact in
countries economy
S. Levy Vaccine Technology III - 2010
FMDV vaccines
• Inactivated virus vaccines
• No cross-protection between serotypes
• Multivalent vaccines to provide protection against
the different serotypes
• Challenge tests are needed to establish a PD50 (50%
protective dose) value or protection
• Challenge experiments requires appropriate
biosecurity containment (Group 4 pathogens)
• Slow and expensive procedure
O.I.E.- Manual of Diagnostic Tests and Vaccines for Terrestrial Animals
Potency Test
• PD50 test: The number of protective doses in a
vaccine is estimated from the resistance to live
virus challenge
• Cattle of at least 6 months of age, obtained from
areas free from FMD should be used.
• Unprotected animals show lesions at sites other
than the tongue
• Vaccines of high potency will prevent the
development of local tongue lesions at the site of
challenge
O.I.E.- Manual of Diagnostic Tests and Vaccines for Terrestrial Animals
FMDV vaccines
• The use of animals should be avoided where possible by
the use of in vitro alternatives.
• A serological test is considered to be satisfactory where a
valid correlation between the observed protection, and the
specific antibody response has been established.
• Time consuming assay
• Need to be delivered rapidly into the field for
outbreaks attack
• Short shelf life
O.I.E.- Manual of Diagnostic Tests and Vaccines for Terrestrial Animals
Cooperación Comunidad Europea - MERCOSUR
Cooperación Unión Europea - MERCOSUR
Genome-wide analysis of
transcriptional responses
against vaccination
Transcriptional signature
of protection as
surrogate of protection
New technologies for development & control of
Foot and Mouth vaccines
New vaccines
development
New analytical tools to
validate production
process
Functional genomics as a tool to
define a molecular signature of
effective vaccination against
FMDV
Work implementation
Analysis
Cell stimulation
Vaccination
Naive animals
Many parameters to be adjusted
•
•
•
•
•
•
Time before processing (samples transport)
Antigen preparation
Time of stimulation (kinetics of the response)
RNA conservation (QC for microarray)
Housekeeping gene
Initial qRT-PCR for sample selection
Genome-wide analysis of transcriptional
responses
B. Taurus (Bovine) Oligo Microarray v2 Design ID 023647 4 x 44K
(Agilent Technologies)
Genome-wide analysis of transcriptional
responses
1109 differentially expressed genes
344 genes upregulated
Naïve
Vaccinated
765 genes downregulated
Main altered biological process
Immunological Disease
Cell-To-Cell Signaling and Interaction – Activation of Leukocytes
Cell-To-Cell Signaling and Interaction – Activation of Blood cells
Antigen Presentation – Activation of phagocytes
Antigen Presentation – Activation of antigen presenting cells
Vaccine Potency Test
• PD50 test: estimated from the resistance
to live virus challenge
• Control animals must develop lesions on
at least three feet
• Unprotected animals show lesions at sites
other than the tongue
• Vaccine of high potency will prevent the
development of local tongue lesions at the
site of challenge.
O.I.E.- Manual of Diagnostic Tests and Vaccines for Terrestrial Animals
Genome-wide analysis of transcriptional
responses
341 differentially expressed genes
276 genes upregulated
Symptomatic
Highly
protected
65 genes downregulated
A molecular signature of
effective vaccination against
FMDV?
qRT-PCR for microarray validation
• TLDA not suitable due to
low numbers of inventored
genes
• Need to design appropriate set of
primers
Genes from
microarray
vaccinated vs naive
animals
Genes from
microarray
highly protected vs
symptomatic
animals
Gene Symbol
Gene Name
C5AR1
CCR1
CD14
CD180
CD36
CLEC7A
CSF1R
CXCL10
CXCL5
complement component 5a receptor 1
chemokine (C-C motif) receptor 1
CD14 molecule
CD180 molecule
CD36 molecule (thrombospondin receptor)
C-type lectin domain family 7, member A
colony stimulating factor 1 receptor
chemokine (C-X-C motif) ligand 10
chemokine (C-X-C motif) ligand 5
FCGR1A
Fc fragment of IgG, high affinity Ia, receptor (CD64)
FGF18
HCK
IL12A
IL12B
IL2RA
fibroblast growth factor 18
hemopoietic cell kinase
interleukin 12A (p35)
interleukin 12B (p40)
interleukin 2 receptor, alpha
LIF
leukemia inhibitory factor (cholinergic differentiation factor)
MMP9
PIGR
S100A9
matrix metallopeptidase 9
polymeric immunoglobulin receptor
S100 calcium binding protein A9
SLAMF1
signaling lymphocytic activation molecule family member 1
TAP1
transporter 1, ATP-binding cassette, sub-family B (MDR/TAP)
TG
THBS1
TNF
thyroglobulin
thrombospondin 1
tumor necrosis factor
TNFRSF1A
tumor necrosis factor receptor superfamily, member 1A
BLA-DQB
MHC class II antigen
BOLA-DQB
major histocompatibility complex, class II, DQ beta
BoLA-DRB3
major histocompatibility complex, class II, DRB3
CCNB1
cyclin B1
CD3G
CD3g molecule, gamma (CD3-TCR complex)
CDKN3
cyclin-dependent kinase inhibitor 3
EIF2B1
eukaryotic translation initiation factor 2B, subunit 1 alpha, 26kDa
EIF5A
GAPDH
GBP4
eukaryotic translation initiation factor 5A
glyceraldehyde-3-phosphate dehydrogenase
guanylate binding protein 4
IGJ
immunoglobulin J polypeptide, linker protein for Ig α and µ polypeptides
IRF8
KIR3DL2
RPL10
RPS24
THSD4
interferon regulatory factor 8
killer cell immunoglobulin-like receptor
ribosomal protein L10
ribosomal protein S24
thrombospondin, type I, domain containing 4
Gene Symbol
Gene Name
CLEC1A
C-type lectin domain family 1, member A
CCL4
chemokine (C-C motif) ligand 4
CREB3
cAMP responsive element binding protein 3
CXCR2
chemokine (C-X-C motif) receptor 2
CXCR5
chemokine (C-X-C motif) receptor 5
DNAJA1
DnaJ (Hsp40) homolog, subfamily A, member 1
FTSJ2
FtsJ homolog 2 (E. coli)
GADD45B
growth arrest and DNA-damage-inducible, beta
Genes present in both
microarrays
GPR128
G protein-coupled receptor 128
GPR137B
G protein-coupled receptor 137B
IFN-γ
interferon, gamma
IGF2
insulin-like growth factor 2 (somatomedin A)
IL23R
interleukin 23 receptor
P2RY6
pyrimidinergic receptor P2Y, G-protein coupled, 6
RPL9
ribosomal protein L9
SIX5
SIX homeobox 5
UPP1
uridine phosphorylase 1
qRT-PCR for microarray validation
GeneSymbol
Genes in
microarray
vaccinated vs
naive animals
Genes in
microarray
protected vs
symptomatic
animals
Genes shared by
both microarrays
C5AR1
CD180
CLEC7A
CXCL10
FCGR1A
FGF18
IL12A
IL12B
LIF
S100A9
SLAMF1
TG
THBS1
TNF
BLA-DQB
CCNB1
CD3G
EIF5A
GBP4
IGJ
IRF8
KIR3DL2
CLEC1A
CCL4
CREB3
CXCR5
DNAJA1
FTSJ2
GADD45B
GPR137B
IFNg
P2RY6
SIX5
UPP1
Array
Vaccinated vs
Naive
Array
Protected vs
Symptomatic
Transcriptional
biomarker of
vaccination ?
Symptomatic
Transcriptional
biomarker of
vaccination & high
protection ?
Symptomatic
Transcriptional
biomarker of high
protection ?
Symptomatic
Non-validated genes
Symptomatic
Symptomatic + 2
IFN-γγ a suitable
biomarker of
vaccination & high
protection ?
Symptomatic
Better than Neutralizing antibodies ?
Symptomatic
Molecular signature seems to better
differentiate between highly protected and
symptomatic animals
Animal
Symptoms
Neutralizing antibodies (UA)
Increase IFN-γγ mRNA
1
-
3,48
0,01034
2
-
2,26
0,01624
4
-
3,55
0,01155
11
-
2,99
0,00478
5
-
2,35
0,00463
10
-
3,29
0,00271
7
+
3,48
0,00123
8
+
2,26
0,00154
9
+
3,55
0,00177
13
+
2,99
0,00015
3
+
2,17
0,00728
12
+
1,79
0,00082
Naïve 1
+
0,00086
NaÏve 2
+
0,00058
Conclusions & future directions
• There is a need for proper subrogates of protection in
vaccines
• This is especially important when trying to translate
research from preclinical studies to clinical trials
• Genome-wide transcriptional profiling allowed the
discovery of biomarkers that seem more accurate
than neutralizing antibodies for FMD
• Need to conduct large screenings to validate the
process
• This shall accelerate the release of new batches of
vaccines into the field
Desarrollo Biotecnologico
Instituto de Higiene
Uruguay
Juan Martin Marques
Andrea Rossi
Sebastian Sasias
Gustavo Sarroca
Biogenesis Bago
Argentina
Susana Levy
Marcelo A. Spitteler
Eliana Smitsaart
Alejandro Ham
Jorge Tito Filippi
Institut des Hautes Etudes Scientifiques
France
Arndt Benecke
Instituto Leloir
Argentina
Osvaldo Podhajcer
Santiago Werbahj
Eduardo Cafferata
INTA Castelar
Argentina
Mariano Perez
Institut Pasteur of Montevideo
Uruguay
Carlos Robello
Gonzalo Greif
URUGUAY
Largest beef consumers in the
world: 53 kg per capita
The biggest “asado”: Guinnes record 2008
Beaches of Uruguay
Beaches of Uruguay
World cup South Africa 2010
American Cup Argentina 2011