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)

6. 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

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)

9. NH
2
aa
110
Region IV
COOH
ASA dimer
aa 193
s –s-
Introduction (8/19)
VSV-G/ASA chimera

10. Results (9/19)
T CELL EPITOPES
IFN- ng/ml
Sample: whole blood
Method: 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
Naive
128 470,40 1,20 6,00 1,40
146 512,60 0.40 3.80 0.40
Recall-responses

11. Results (10/19)
VACCINE ANTIGENS
• BACULOVIRUS-expressed Protein: DEL BAC
• DNA VACCINE: pC DEL

12. 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

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

16. Vaccine DPV IgG1 titer IgG2 titer
RATIO
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)

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)

20. Thanks! (19/19)
MUCHAS GRACIAS
Instituto Milstein, CEVAN. CONICET