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

This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Vaccine Technology IV by an authorized administrator of ECI Digital Archives. For more information, please contact [email protected]. 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: genetic Salmonella variation & as vaccine pathogenicity vector 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 signature transcriptional responses of protection as against vaccination surrogate of protection

New technologies for development & control of Foot and Mouth vaccines

New analytical tools to validate production New vaccines process development 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

Highly Symptomatic 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 Gene Symbol Gene Name C5AR1 complement component 5a receptor 1 CCR1 chemokine (C-C motif) receptor 1 CD14 CD14 molecule CD180 CD180 molecule CD36 CD36 molecule (thrombospondin receptor) CLEC7A C-type lectin domain family 7, member A CSF1R colony stimulating factor 1 receptor CXCL10 chemokine (C-X-C motif) ligand 10 CXCL5 chemokine (C-X-C motif) ligand 5 FCGR1A Fc fragment of IgG, high affinity Ia, receptor (CD64) FGF18 fibroblast growth factor 18 Genes from HCK hemopoietic cell kinase microarray IL12A interleukin 12A (p35) vaccinated vs naive IL12B interleukin 12B (p40) animals IL2RA interleukin 2 receptor, alpha LIF leukemia inhibitory factor (cholinergic differentiation factor) MMP9 matrix metallopeptidase 9 PIGR polymeric immunoglobulin receptor S100A9 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 thyroglobulin THBS1 thrombospondin 1 TNF 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) Genes from CDKN3 cyclin-dependent kinase inhibitor 3 microarray EIF2B1 eukaryotic translation initiation factor 2B, subunit 1 alpha, 26kDa highly protected vs EIF5A eukaryotic translation initiation factor 5A symptomatic GAPDH glyceraldehyde-3-phosphate dehydrogenase animals GBP4 guanylate binding protein 4 IGJ immunoglobulin J polypeptide, linker protein for Ig α and µ polypeptides IRF8 interferon regulatory factor 8 KIR3DL2 killer cell immunoglobulin-like receptor RPL10 ribosomal protein L10 RPS24 ribosomal protein S24 THSD4 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 FtsJhomolog2 (E. coli)

Genes present in both GADD45B growth arrest and DNA-damage-inducible, beta

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

Array Array GeneSymbol Vaccinated vs Protected vs Naive Symptomatic C5AR1 CD180 CLEC7A CXCL10 FCGR1A Genes in FGF18 microarray IL12A vaccinated vs IL12B naive animals LIF S100A9 SLAMF1 TG THBS1 TNF BLA-DQB Genes in CCNB1 microarray CD3G EIF5A protected vs GBP4 symptomatic IGJ animals IRF8 KIR3DL2 CLEC1A CCL4 CREB3 CXCR5 DNAJA1 Genes shared by FTSJ2 both microarrays GADD45B GPR137B IFNg P2RY6 SIX5 UPP1 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-γγγ 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-γγγ 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 Biogenesis Bago Instituto Leloir Argentina Argentina Juan Martin Marques Andrea Rossi Susana Levy Osvaldo Podhajcer Sebastian Sasias Marcelo A. Spitteler Santiago Werbahj Gustavo Sarroca Eliana Smitsaart Eduardo Cafferata Alejandro Ham Jorge Tito Filippi INTA Castelar Argentina Institut des Hautes Etudes Scientifiques Mariano Perez France Arndt Benecke 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