1. A RECOMBINANT APHTHOVIRUS
CHIMERA OF THE GLYCOPROTEIN OF
VESICULAR STOMATITIS VIRUS AS DNA
AND PROTEIN-BASED VACCINE IN
CATTLE
Alejandra Capozzo
ICT MISLTEIN- CONICET. Buenos Aires, ARGENTINA
2. 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
3. .
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)
4. .
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
5. 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)
7. 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)
8. 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)
13. 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)
14. 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
15. 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
17. 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
18. 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
19. • 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)