A RECOMBINANT APHTHOVIRUS CHIMERA OF THE GLYCOPROTEIN OF

VESICULAR STOMATITIS VIRUS AS DNA AND PROTEIN-BASED VACCINE IN

CATTLE

Alejandra CapozzoICT MISLTEIN- CONICET. Buenos Aires, ARGENTINA

SUMMARY (1/19)

A chimeric construct conformed by:-an in tandem-dimer insertion of the antigenic site A (ASA) of VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3, aa 139-149)-within aa160 and 161 of the vesicular stomatitis virus G protein (VSV-G) was able to display the appropriate ASA conformation/s to elicit anti FMDV-specific neutralizing immune responses in calves.

SUMMARY

.

PEPTIDE-based FMDV VACCINES -Overview

- FMDV derived antigens administered as peptide vaccines were unable to induce significant humoral responses and protection in cattle even associated with foreign and FMDV-derived T cell epitope/s (Taboga et al. 1997; Rodriguez et al. 2003)

- However, a dendrimeric peptide containing VP1-ASA and FMDV T-cell epitopes conferred protection in swine (Cubillos et al. 2008)

Introduction (2/19)

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Introduction (3/19)

PEPTIDE-based FMDV VACCINES –issues…

1- Several aligned T cell epitopes might be required to induce humoral responses in cattle

2- ASA must be correctly exposed to preserve conformation

3- ASA must be presented as a repetitive motif

HYPOTHESIS

The correct display of epitopes that bind conformational-dependent-neutralizing antibodies aligned with bovine T cell epitopes can circumvent immunological limitations of peptide-based vaccines in cattle:

• difficulty to correctly expose conformational epitopes

• low T-cell induction due to highly polymorphic bovine MHC

Conclusions (4/19)

Conclusions (5/19)

CARRIER

SEQUENCE

TARGET

SEQUENCE

Long sequence

Must expose Target

sequence correctly

Provide T cell epitopes

Short sequence

B-cell Epitope/s

Antigenic Site A – “ASA”, VP1

FMDV-C3, Tandem - dimer

RECOMBINANT

IMMUNOGEN

STRATEGY

VSV-NJ Glycoprotein

slide title (1/20)

FMDV - VP1, Antigenic Site A: ASA

NH2

- helix

Loop

Binding of Neutralizing antibodies requires one or several conformations of ASA

Oligopeptide 11 aa long corresponding to VP1 sequence aa 139 y 149: ARRGDLAHLAT (Serotipe C).

Antigenic site composed of several overlapping conformational epitopes reactive to neutralizing antibodies.

Introduction (6/19)

CARRIER SEQUENCE : VSV-NJ/G• Potent immunogen in bovines, rabbit, swine, mice; even without adjuvants

• Bovine T cell epitopes have been mapped along its sequence

• Deletion of c-terminal end brings more stability and facilitates refolding

aa 80-193

EPITOPE IV

Re-naturalizes correctly in vitro

Very stable- loops stabilized by disulfur bonds

Not target of neutralizing antibodies

NH2 COOH

Aa 110-111-Sensible to V8 protease

BINDING OF NEUTALIZING ABs

aa 193-267

Transmembrane

Endo-domainEcto-domain

VSV-NJ GLYCOPROTEIN G (VSV-G; 514 aa)Introduction (7/19)

NH2

aa 110

Region IV

COOH

ASA dimer

aa 193

s –s-

Introduction (8/19)

VSV-G/ASA chimera

Results (9/19)

T CELL EPITOPES

IFN- ng/ml

Sample: whole bloodMethod: BOVIGAM®

Status

Stimulating antigen

Bovine #PWD FMDV

C3

DEL

BAC

NIL

FMDV Vaccinated commercial vaccine

44 512,60 216,80 68,20 1,60

47 421,60 315,40 30,60 0,60

50 418,60 366,20 54,40 1,00

Naive128 470,40 1,20 6,00 1,40

146 512,60 0.40 3.80 0.40

Recall-responses

Results (10/19)

VACCINE ANTIGENS

• BACULOVIRUS-expressed Protein: DEL BAC

• DNA VACCINE: pC DEL

Results (11/19)

IMMUNIZATION STUDIES

Route: i.m (Neck).

Vol/ dose: 3 ml.

Adyuvant: Marcol Montanide (w/o)

Schedule: 2 doses, 30 days.

DEL BAC 30µg

•Commercial Vaccine (Frenkel-Multivalent)

•Empty plasmid

•Heterologous Baculovirus antigen +Ady

Controls

Route: i.m (Neck).

Vol/ dose: 3 ml.

Adyuvant: none

Schedule: 2 doses, 15 days.

5 month-old calves

pC DEL 150µg

Reactivity with conformational ASA

Mab Mab Commercial DEL BAC pCDEL

VP1 G Sera

29 kDa

45 kDa

60 kDa

WESTERN BLOT IMMUNOPRECIPITATION

SERA FROM CHIMERA-VACCINATED ANIMALS STRONGLY REACT WITH WHOLE FMDV PARTICLES

Whole virus

IP + WB (Mab anti VP1)

Results (12/19)

Reactivity with conformational VP1 epitopes

SERA FROM CHIMERA-VACCINATED ANIMALS STRONGLY REACT WITH NATIVE FMDV

ELISA AGAINST WHOLE AND DENATURED VIRUS

WHOLE particles

Results (13/19)

Denatured particles

FMDV-SPECIFIC ANTIBODIES MEASURED BY VNT and Liquid Phase Blocking ELISA

INDIVIDUAL ANIMALS SURPASS PROTECTIVE LEVELS OF ABs (EPP >80%)

Results (14/19)

VNT LP ELISA

Vaccine DPV IgG1 titer IgG2 titerRATIO

IgG1/IgG2

pC DEL

15 1.62 +/- 0.32 1.54 +/- 0.42 1.05

30 1.60 +/- 0.43 1.72 +/- 0.41 0.93

45 1.64 +/- 0.14 2.08+/-0.16 0.78

60 1.63+/-0.18 2.20+/-0.17 0.74

DEL BAC

15 ND ND ND

45 1.54+/-0.38 1.35+/-0.31 1.14

60 1.74+/-0.36 1.33+/-0.28 1.31

90 1.90+/-0.37 1.34+/-0.29 1.42

Commercial

FMDVi

(Frenkel)

15 ND ND ND

45 2.30+/-0.40 1.36+/-0.18 1.69

90 2.20+/-0.23 1.38+/-0.19 1.59

FMDV-specific IgG subtypes

Results (15/19)

Conclusions (16/19)

T-cell epitopes in the chimeric construct could induce T-cell

activation in whole blood samples from commercially-

vaccinated animals.

DNA-coded and baculovirus expressed forms of G-ASA

induced strong humoral responses in cattle

Serum from all G-ASA vaccinated animals recognized

conformational ASA in the native FMDV-140S particles

3 out of 5 animals had EPP% values (LP ELISA) above 80%

and all DEL-BAC immunized calves showed high serum IgG1

titers, with values comparable to those recorded for protection

with inactivated vaccines

CONCLUSIONS

Conclusions (17/19)

CONCLUSIONS

The association of the FMDV-C3/85 ASA as a tandem dimer to VSV-G N terminal sequence could circumvent limitations of FMDV peptide-based antigens as effective vaccines for bovines

• Pablo Grigera• José La Torre• María de los Ángeles Lavoria CEVAN- ICT MISLTEIN• Olga Franco Mahecha• Florencia Mansilla• Danilo Bucafusco CICVyA – INTA

MANY-SPECIAL THANKS TO Mariano Pérez Filgueira and Marina Marzocca

Credits and acknowledgements…

Participants (18/19)

Thanks! (19/19)

MUCHAS GRACIAS

Instituto Milstein, CEVAN. CONICET