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mrodrigues@unifesp.br

Swimming against the current: Genetic vaccination against Trypanosoma cruzi infection in mice. INCTV National Institute for Vaccine Technology - CNPq. mrodrigues@unifesp.br. Prof. Dr. Carlos Chagas Filho. Epidemiology. Pathogenesis. Prevention. Immunology. Chemotherapy.

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mrodrigues@unifesp.br

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  1. Swimming against the current: Genetic vaccination against Trypanosoma cruzi infection in mice INCTV National Institute for Vaccine Technology - CNPq mrodrigues@unifesp.br

  2. Prof. Dr. Carlos Chagas Filho

  3. Epidemiology Pathogenesis Prevention Immunology Chemotherapy

  4. Chemical composition of the vaccines Hepatitis B HPV Attenuated live virus Recombinant proteins Inactivated virus Live attenuated bacteria Killed bacteria Polysaccharides ~200 years 20-25 Vaccines Toxoids

  5. Protective mechanisms of current available vaccines? Antibodies ?

  6. Diseases with important antibody independent mechanism of immunity Leishmania sp. Mycobacterium HIV Plasmodium Trypanosoma cruzi Intracellular amastigotes Intracellular Trypomastigotes

  7. Hypothesis Drs. Fidel Zavala and Ruth Nussenzweig 1989-2009 Immunization Specific T cells Protective Immunity Experimental malaria and T. cruzi infection

  8. Humans ? Experimental T cell-based genetic vaccination against experimental malaria Rodrigues et al., 1993 and 1994 Rec. Influenza virus Rec. Vaccinia virus Challenge P. yoelli Results: 9/15 - no malaria 4/15 - delayed malaria 2/15 - failure 56 0 21 35 Days Malaria antigen - CS protein CD8+ T cell mediated

  9. T cell-based genetic vaccination against malaria: The human challenge 2004 and 2005

  10. Wikipedia Economics of development One challenge in vaccine development is economic: many of the diseases most demanding a vaccine, including HIV, malaria and tuberculosis, exist principally in poor countries. Pharmaceutical firms and biotechnology companies have little incentive to develop vaccines for these diseases, because there is little revenue potential. Even in more affluent countries, financial returns are usually minimal and the financial and other risks are great. Excess of regulations High risks Little financial return Few or no products New vaccines

  11. Genetic vaccination against Trypanosoma cruzi infection ? Trans-sialidase of trypomastigotes of T. cruzi Catalytic Domain (aa 34-678) C-terminal repeats (CTR) (aa 679-1071) Signal Peptide (aa 1-33) GPI anchor NH2 COOH

  12. CD4 Epitope(s) 33-275 CD8 Epitope 359-367 CD8 Tc1 CD4 Th1 Protective Immunity + + + pcDNA3-TS + + + p154/13 + - - pD154/13 pCD8-Epitope - -/+ - pD154/13 -CD8 + + + Summary of the results using different plasmids BALB/c mice CD4 Th1 and CD8 Tc1 epitopes are important for efficient protective immunity Fujimura et al., 2001

  13. 2007 CD4 Th1 and CD8 Tc1 cells are important for efficient protective immunity Rec. protein plus CpG – B cells are important for efficient priming CD4 Th1 and CD8 Tc1

  14. Plasmids with TS gene fail to protect A/Sn mice against T. cruzi infection (Y strain) Plasmid Protected / challenged _________________________________ pcDNA3 0/15 pB43 0/15 p154/13 0/15 pVr1012 0/10 pVr-Cl. 44 0/8 _________________________________

  15. Clone 9 CD8 Epitope 320-327 H-2Kk CD8 Epitope 553-560 H-2Kb CD4 epitopes ? Amastigote Surface Protein-2 of T. cruzi(Group II of TS) (Pan & McMahon-Pratt, 1989 and Low & Tarleton, 1998) ASP BOX (SxDxGxTW) VTV BOX (VTVxNVFLYNR) Signal Peptide Amastigote Specific Liver tissue Boscardin et al., 2003

  16. pIgSPclone 9 aa 1-695 of ASP-2 Asp-2 5’ - - 3’ Signal peptide of mouse immunoglobulin K chain pcDNA3 Boscardin et al., 2003

  17. A/Sn mice Immunization with ts and asp-2 genes 4 doses ts+asp-2 asp-2 ts pc DNA3 Dependent of CD4+ and CD8+ T cells Vasconcelos et al., 2004

  18. ... 273 1... RibpS4 ... 656 ... 776 ... 211 ... 319 ... 218 ... 112 ... 449 ... 395 ... 600 ... 91 1... 1... 1... 1... 1... 1... 1... 1... 1... 1... PAR-2 MTP70 RpL7a TcG5 TcG4 Tc24 TcG2 TcG8 H2b EF2 ... 653 1... HSP70 ASP-4(7015) ... 743 66... Other T. cruzi ORFs ... 642 66... ASP-3(5340) Da Silveira et al., 2008

  19. ASP-4 ... 642 ... 743 66... ASP-3 66... pIgSP-ASP-4 pIgSP-ASP-3 Survival (%) Survival (%) ASP-3 p=0.0005 ASP-4 p=0.0061 pcDNA3 pcDNA3 Days after challenge Days after challenge Da Silveira et al., 2008

  20. Vaccination with ASP-2 of T. cruzi Homologous vaccination Plasmid DNA - 4 doses - A/Sn mice - Vasconcelos et al. 2004 Rec. Protein + CpG - 3 doses - A/Sn mice - Araújo et al. 2005 Homologous X Heterologous vaccination Priming Boosting Plasmid DNA Plasmid DNA None Rec. Adenovirus Rec. Adenovirus Rec. Adenovirus Plasmid DNA Rec. Adenovirus

  21. Heterologous prime-boost vaccination with ASP-2 of T. cruzi Peak parasitemia * * * Adeno- ASP2 (1X) 13.6X 1- pcDNA3/Adbgal 2- DNA-ASP2/DNA-ASP2 3- None/Adeno-ASP2 4- Adeno-ASP2/Adeno-ASP2 5- DNA-ASP2/Adeno-ASP2 22.8X de Alencar et al., submitted

  22. Normal ECG in T. cruzi infected adenovirus-vaccinated mice Non- infected mice pIgSPcl.9 + Ad-ASP-2 ECG 222 days after challenge Ad-ASP-2 + Ad-ASP-2 None + Ad-ASP-2 Hemocultures = negative de Alencar et al., submitted

  23. * * * * * Heterologous prime-boost vaccination with ASP-2 of T. cruzi pcDNA3 Adb-gal None pIgSPCl.9 AdASP-2 Rat igG pIgSPCl.9 AdASP-2 a-CD4 • Protective immunity is dependent on CD4+ T cells de Alencar et al., submitted

  24. Heterologous prime-boost vaccination with ASP-2 of T. cruzi pcDNA3 Adb-gal None pIgSPCl.9 AdASP-2 Rat igG pIgSPCl.9 AdASP-2 a-CD8 * * * * * • Protective immunity is dependent on CD8+ T cells de Alencar et al., submitted

  25. * * * * * * * * Heterologous prime-boost vaccination with ASP-2 of T. cruzi Longevity of protective T cells 98 days 14 days 98 days 14 days de Alencar et al., submitted

  26. Colombian Failure Strain-specificity of the protective immunity elicited by heterologous prime-boost vaccination COL Success pcDNA3 – adeno-bgal pIgSP-Cl.9 – Adeno-ASP-2 P154/13 - Adeno-TS Haolla et al., submitted

  27. Heterologous prime-boost vaccination with ASP-2 of T. cruzi Highly Susceptible mouse strains Homologous and heterologous challenge 2 strains Few doses (1 or 2) Short and Long term Defined mechanisms CD8+ T cell dependent Epitope TEWETGQI Defined epitopes Phenotype and functions of the protective CD8+ T cells ? Monitoring the CD8 T cells responses of vaccinated or immune individuals

  28. 0.4 0.016 0.02 0.32 15.7 9.97 0.31 0.016 Phenotypic characterization of specific CD8+ T cells C57BL/6 pIgSPCl.9/ AdASP-2 Naive Days 0.018 0.38 14 H-2kb-VNHRFTLV 98 CD8 de Alencar et al., submitted

  29. Naive 100 100 100 80 80 80 60 60 % of Max % of Max 100 100 100 60 % of Max 40 40 80 80 80 40 20 20 60 % of Max 60 60 20 % of Max % of Max 0 0 40 2 3 4 5 2 3 4 5 10 0 10 10 10 0 10 10 10 10 40 40 0 <FITC-A> <FITC-A> 2 3 4 5 0 10 10 10 10 <FITC-A> 20 20 20 0 0 0 2 3 4 5 10 0 10 10 10 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 <FITC-A> <FITC-A> <FITC-A> Purified CD8+ T cellls H-2kb-VNHRFTLV CD11a CD43 CD44 Days 14 98 de Alencar et al., submitted

  30. Naive 100 80 100 60 % of Max 100 80 100 100 40 80 60 80 80 % of Max 20 60 % of Max 40 60 60 % of Max 0 % of Max 40 2 3 4 5 0 10 10 10 10 <FITC-A>: 6 20 40 40 20 0 20 20 2 3 4 5 0 0 10 10 10 10 <FITC-A> 2 3 4 5 0 10 10 10 10 0 0 <FITC-A> 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 <PE-Cy7-A> <APC-A> Purified CD8+ T cellls H-2kb-VNHRFTLV CD122 IL-2 rec. CD127 IL-7 rec. Days KLRG-1 CD62L 14 98 de Alencar et al., submitted

  31. Phenotype of the CD8+ T cells Specific CD8+ T cells 14 days Specific CD8+ T cells 98 days Naive CD8+ T cells CD11a High CD25 Low CD27 High CD31 Low CD43 High CD44 High CD49d Low CD69 Low CD62L Low CD122 Intermed. CD127 Intermed. KLRG-1 Low/High CD11a Low CD25 Low CD27 Low CD31 High CD43 Low CD44 Low CD49d Low CD69 Low CD62L High CD122 Low CD127 Int KLRG-1 Low CD11a High CD25 High CD27 High CD31 Low CD43 High CD44 High CD49d Low CD69 High CD62L Low CD122 High CD127 Low KLRG-1 Low/High T effector T effector memory

  32. CD107a and IFN-g expression CD3+CD8+ 8.12 7.09 CD107a+ IFN-g+ 5 10 4 10 2.08 CD107a 82.7 3 10 2 10 0 2 3 4 5 0 10 10 10 10 IFN-g ? Function of the immune CD8+ T cells In vivo cytotoxicity 20 h 4 h C57Bl/6 WT Perforin KO C57Bl/6 WT Perforin KO de Alencar et al., submitted

  33. A B 5 5 10 10 0.051 0.082 0.065 0.11 4 4 10 10 3 3 10 10 TNF 2 2 10 10 0 0 99.3 99.3 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0.59 0.57 IFN-g IFN-g C D 1.88 4.5 0.068 0.032 5 5 10 10 4 4 10 10 TNF 3 3 10 10 2 2 10 10 0 0 0.84 0.43 92.8 99.5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 IFN-g IFN-g ? CD3+CD8+ Multifunctional cells No peptide Pep. VNHRFTLV pcDNA3/Adb-gal pIgSPCl.9/AdASP-2 de Alencar et al., submitted

  34. Protective immunity after heterologous prime-boost vaccination ? Role for Perforin WT X Perforin KO pIgSPcl.9/ AdASP-2 pcDNA3/ Adbgal pIgSPcl.9/ AdASP-2 pcDNA3/ Adbgal de Alencar et al., submitted

  35. Protective immunity after heterologous prime-boost vaccination ? Role for IFN-g WT X IFN-g KO de Alencar et al., submitted

  36. Humans ? Conclusions from the mouse vaccination studies 1- High degree of protective immunity A/Sn mice AdASP-2 (2X) or DNA/AdASP-2 2- Genetic vaccination (DNA/AdASP-2) A/Sn mice Long lived, CD4 and CD8 dependent 3- Specific protective CD8+ T cells C57BL/6 mice In vivo cytotoxicity and expression IFN-g, TNF-a and CD107a. 4- The cell surface markers C57BL/6 mice Teffector (14 days) or Teffector memory (98 days). 5- Mechanisms mediated by CD8+ T cells C57BL/6 mice Perforin and IFN-g

  37. Participants UNIFESP-EPM Bruna C. G. de Alencar Fanny Tzelepis Carla Claser Filipe A. Haolla José Ronnie Vasconcelos Dr. Sergio Schenkman UFMG e CPRR-FIOCRUZ Dr. Ricardo T. Gazzinelli Dr. Oscar Bruna-Romero Dr. Marcus Penido Dr. Alexandre V. Machado IOC-FIOCRUZ Dr. Gabriel de Oliveira Dr. Joseli Lannes-Vieira Inst. Adolofo Lutz Dr. Vera Pereira-Chioccola UFRJ - I. de Biofísica CCF Dr. Pedro M. Persechini PhD and Pos-Docs positions National Institute for Vaccine Technology

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