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Recombinant aphtovirus ACapozzo

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Recombinant aphtovirus ACapozzo

  1. 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. 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. 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. 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. 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. 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. 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. 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. 9. NH 2 aa 110 Region IV COOH ASA dimer aa 193 s –s- Introduction (8/19) VSV-G/ASA chimera
  10. 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. 11. Results (10/19) VACCINE ANTIGENS • BACULOVIRUS-expressed Protein: DEL BAC • DNA VACCINE: pC DEL
  12. 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. 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. 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. 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. 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. 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. 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. 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. 20. Thanks! (19/19) MUCHAS GRACIAS Instituto Milstein, CEVAN. CONICET

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