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Swimming against the current: Genetic vaccination against Trypanosoma cruzi infection in mice INCTV National Institute for Vaccine.

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Presentation on theme: "Swimming against the current: Genetic vaccination against Trypanosoma cruzi infection in mice INCTV National Institute for Vaccine."— Presentation transcript:

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

2 Prof. Dr. Carlos Chagas Filho

3 EpidemiologyPathogenesis ImmunologyChemotherapy Prevention

4 Attenuated live virus Inactivated virus Live attenuated bacteria Killed bacteria Toxoids Polysaccharides Recombinant proteins Chemical composition of the vaccines Hepatitis BHPV ~200 years Vaccines

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 Trypomastigotes Intracellular amastigotes

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

8 Experimental T cell-based genetic vaccination against experimental malaria 0 Days 2135 Rec. Influenza virus Rec. Vaccinia virus 56 Challenge P. yoelli Rodrigues et al., 1993 and 1994 Results: 9/15 - no malaria 4/15 - delayed malaria 2/15 - failure CD8 + T cell mediated Malaria antigen - CS protein Humans ?

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

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.HIVmalariabiotechnology Excess of regulations High risks Little financial return New vaccines Few or no products

11 Trans-sialidase of trypomastigotes of T. cruzi Catalytic Domain (aa ) GPI anchor Signal Peptide (aa 1-33) NH 2 COOH C-terminal repeats (CTR) (aa ) Genetic vaccination against Trypanosoma cruzi infection ?

12 Summary of the results using different plasmids BALB/c mice CD4 Th1 and CD8 Tc1 epitopes are important for efficient protective immunity CD4 Th1 CD8 Tc1 Protective Immunity / Fujimura et al., 2001 pcDNA3-TS CD4 Epitope(s) CD8 Epitope p154/13 p  154/13 -CD8 pCD8- Epitope

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

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

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

18 ASP-4(7015) RibpS PAR Tc MTP HSP RpL7a H2b EF TcG TcG TcG TcG ASP-3(5340) Other T. cruzi ORFs Da Silveira et al., 2008

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

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

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

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

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

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

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

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

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

28 Phenotypic characterization of specific CD8 + T cells C57BL/6 CD8 Naive pIgSPCl.9/ AdASP-2 Days H-2k b -VNHRFTLV de Alencar et al., submitted

29 CD11a % of Max % of Max Days Naive H-2k b -VNHRFTLV Purified CD8 + T cellls % of Max % of Max CD % of Max % of Max CD43 de Alencar et al., submitted

30 % of Max % of Max CD122 IL-2 rec % of Max CD127 IL-7 rec % of Max KLRG-1 Days Naive H-2k b -VNHRFTLV Purified CD8 + T cellls : % of Max CD62L de Alencar et al., submitted

31 Naive CD8 + T cells 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 Specific CD8 + T cells 14 days 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 Specific CD8 + T cells 98 days 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 Phenotype of the CD8 + T cells T effector memory T effector

32 4 h C57Bl/6 WT Perforin KO In vivo cytotoxicity C57Bl/6 WT Perforin KO 20 h Function of the immune CD8 + T cells CD107a IFN-  CD3 + CD8 + CD107a + IFN-  + CD107a and IFN-  expression ? de Alencar et al., submitted

33 CD3 + CD8 + Multifunctional cells ? No peptidePep. VNHRFTLV TNF IFN-  TNF AB CD pIgSPCl.9/AdASP-2 pcDNA3/Ad  -gal de Alencar et al., submitted

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

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

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

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 UFRJ - I. de Biofísica CCF Dr. Pedro M. Persechini IOC-FIOCRUZ Dr. Gabriel de Oliveira Dr. Joseli Lannes-Vieira PhD and Pos-Docs positions National Institute for Vaccine Technology Inst. Adolofo Lutz Dr. Vera Pereira-Chioccola


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