Swimming against the current Genetic vaccination against Trypanosoma

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Swimming against the current: Genetic vaccination against Trypanosoma cruzi infection in mice INCTV National

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

Prof. Dr. Carlos Chagas Filho

Epidemiology Pathogenesis Prevention Immunology Chemotherapy

Epidemiology Pathogenesis Prevention Immunology Chemotherapy

Chemical composition of the vaccines Attenuated live virus Inactivated virus Recombinant proteins Live attenuated

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

Protective mechanisms of current available vaccines? Antibodies ?

Protective mechanisms of current available vaccines? Antibodies ?

Diseases with important antibody independent mechanism of immunity Leishmania sp. Mycobacterium HIV Intracellular amastigotes

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

Hypothesis 1989 -2009 Immunization Specific T cells Protective Immunity Experimental malaria and T. cruzi

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

Experimental T cell-based genetic vaccination against experimental malaria Rodrigues et al. , 1993 and

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

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

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

Wikipedia Economics of development One challenge in vaccine development is economic: many of the

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

Genetic vaccination against Trypanosoma cruzi infection ? Trans-sialidase of trypomastigotes of T. cruzi Signal

Genetic vaccination against Trypanosoma cruzi infection ? Trans-sialidase of trypomastigotes of T. cruzi Signal Peptide (aa 1 -33) NH 2 Catalytic Domain (aa 34 -678) C-terminal GPI repeats (CTR) anchor (aa 679 -1071) COOH

Summary of the results using different plasmids BALB/c mice CD 4 Epitope(s) 33 -275

Summary of the results using different plasmids BALB/c mice CD 4 Epitope(s) 33 -275 CD 8 Epitope 359 -367 CD 4 Th 1 CD 8 Tc 1 Protective Immunity pc. DNA 3 -TS + + + p 154/13 + + + p. D 154/13 + - - p. CD 8 -Epitope - -/+ - p. D 154/13 -CD 8 + + + CD 4 Th 1 and CD 8 Tc 1 epitopes are important for efficient protective immunity Fujimura et al. , 2001

2007 CD 4 Th 1 and CD 8 Tc 1 cells are important for

2007 CD 4 Th 1 and CD 8 Tc 1 cells are important for efficient protective immunity Rec. protein plus Cp. G – B cells are important for efficient priming CD 4 Th 1 and CD 8 Tc 1

Plasmids with TS gene fail to protect A/Sn mice against T. cruzi infection (Y

Plasmids with TS gene fail to protect A/Sn mice against T. cruzi infection (Y strain) Plasmid Protected / challenged _________________ pc. DNA 3 0/15 p. B 43 0/15 p 154/13 0/15 p. Vr 1012 0/10 p. Vr- Cl. 44 0/8 _________________

Amastigote Surface Protein-2 of T. cruzi (Group II of TS) (Pan & Mc. Mahon-Pratt,

Amastigote Surface Protein-2 of T. cruzi (Group II of TS) (Pan & Mc. Mahon-Pratt, 1989 and Low & Tarleton, 1998) Signal Peptide ASP BOX (Sx. Dx. Gx. TW) VTV BOX (VTVx. NVFLYNR) Clone 9 Amastigote Specific Liver tissue CD 8 Epitope 320 -327 H-2 Kk CD 8 Epitope 553 -560 H-2 Kb CD 4 epitopes ? Boscardin et al. , 2003

p. Ig. SPclone 9 aa 1 -695 of ASP-2 5’ - Asp-2 - 3’

p. Ig. SPclone 9 aa 1 -695 of ASP-2 5’ - Asp-2 - 3’ Signal peptide of mouse immunoglobulin K chain pc. DNA 3 Boscardin et al. , 2003

A/Sn mice Immunization with ts and asp-2 genes 4 doses ts+asp-2 pc DNA 3

A/Sn mice Immunization with ts and asp-2 genes 4 doses ts+asp-2 pc DNA 3 Dependent of CD 4+ and CD 8+ T cells ts Vasconcelos et al. , 2004

Other T. cruzi ORFs 1. . . Ribp. S 4 . . . 273

Other T. cruzi ORFs 1. . . Ribp. S 4 . . . 273 1. . . EF 2 . . . 776 1. . . PAR-2 . . . 600 1. . . Tc. G 2 . . . 218 1. . . Tc 24 . . . 211 1. . . Tc. G 4 . . . 91 1. . . MTP 70 . . . 656 1. . . Tc. G 5 . . . 449 1. . . HSP 70 . . . 653 1. . . Tc. G 8 . . . 395 1. . . Rp. L 7 a . . . 319 66. . . ASP-3(5340) . . . 642 1. . . H 2 b . . . 112 66. . . ASP-4(7015) . . . 743 Da Silveira et al. , 2008

. . . 642 ASP-3 66. . . ASP-4 . . . 743 p.

. . . 642 ASP-3 66. . . ASP-4 . . . 743 p. Ig. SP-ASP-4 p. Ig. SP-ASP-3 p=0. 0005 Survival (%) 66. . . ASP-4 p=0. 0061 pc. DNA 3 Days after challenge Da Silveira et al. , 2008

Vaccination with ASP-2 of T. cruzi Homologous vaccination Plasmid DNA - 4 doses -

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

Heterologous prime-boost vaccination with ASP-2 of T. cruzi Peak parasitemia * * * Adeno.

Heterologous prime-boost vaccination with ASP-2 of T. cruzi Peak parasitemia * * * Adeno. ASP 2 (1 X) 13. 6 X 22. 8 X 1 - pc. DNA 3/Adbgal 2 - DNA-ASP 2/DNA-ASP 2 3 - None/Adeno-ASP 2 4 - Adeno-ASP 2/Adeno-ASP 2 5 - DNA-ASP 2/Adeno-ASP 2 de Alencar et al. , submitted

Normal ECG in T. cruzi infected adenovirus-vaccinated mice Noninfected mice ECG 222 days after

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

Heterologous prime-boost vaccination with ASP-2 of T. cruzi pc. DNA 3 Adb-gal p. Ig.

Heterologous prime-boost vaccination with ASP-2 of T. cruzi pc. DNA 3 Adb-gal p. Ig. SPCl. 9 Ad. ASP-2 * None Rat ig. G a-CD 4 * * ü Protective immunity is dependent on CD 4+ T cells de Alencar et al. , submitted

Heterologous prime-boost vaccination with ASP-2 of T. cruzi pc. DNA 3 Adb-gal p. Ig.

Heterologous prime-boost vaccination with ASP-2 of T. cruzi pc. DNA 3 Adb-gal p. Ig. SPCl. 9 Ad. ASP-2 * * None Rat ig. G a-CD 8 * * * ü Protective immunity is dependent on CD 8+ T cells de Alencar et al. , submitted

Heterologous prime-boost vaccination with ASP-2 of T. cruzi Longevity of protective T cells 98

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

Strain-specificity of the protective immunity elicited by heterologous prime-boost vaccination COL Colombian Failure Success

Strain-specificity of the protective immunity elicited by heterologous prime-boost vaccination COL Colombian Failure Success pc. DNA 3 – adeno-bgal p. Ig. SP-Cl. 9 – Adeno-ASP-2 P 154/13 - Adeno-TS Haolla et al. , submitted

Heterologous prime-boost vaccination with ASP-2 of T. cruzi Highly Susceptible mouse strains Homologous and

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 CD 8+ T cell dependent Defined epitopes Epitope TEWETGQI Phenotype and functions of the protective CD 8+ T cells Monitoring the CD 8 T cells responses of vaccinated or immune individuals ?

Phenotypic characterization of specific CD 8+ T cells C 57 BL/6 p. Ig. SPCl.

Phenotypic characterization of specific CD 8+ T cells C 57 BL/6 p. Ig. SPCl. 9/ Ad. ASP-2 Naive Days 0. 018 0. 4 14 H-2 kb-VNHRFTLV 0. 016 0. 38 15. 7 9. 97 0. 02 0. 32 0. 016 0. 31 98 CD 8 de Alencar et al. , submitted

Purified CD 8+ T cellls CD 11 a Days CD 43 80 80 80

Purified CD 8+ T cellls CD 11 a Days CD 43 80 80 80 60 40 % of Max 100 60 40 20 0 102 103 104 <FITC-A> 0 105 100 80 80 80 60 40 20 0 % of Max 100 % of Max 98 CD 44 100 % of Max 14 H-2 kb-VNHRFTLV Naive 20 0 102 103 104 <FITC-A> 105 60 40 20 0 102 103 104 <FITC-A> 105 0 de Alencar et al. , submitted

Purified CD 8+ T cellls CD 62 L 100 80 80 40 20 20

Purified CD 8+ T cellls CD 62 L 100 80 80 40 20 20 105 0 0 102 103 104 <FITC-A> 105 100 100 80 80 80 % of Max 103 104 <FITC-A>: 6 KLRG-1 60 40 0 102 CD 127 IL-7 rec. 60 40 20 20 0 % of Max 60 0 98 % of Max 100 % of Max 14 CD 122 IL-2 rec. % of Max Days H-2 kb-VNHRFTLV Naive 0 102 103 104 <FITC-A> 105 0 60 40 20 0 102 103 104 <PE-Cy 7 -A> 105 0 0 102 103 104 <APC-A> de Alencar et al. , submitted 105

Phenotype of the CD 8+ T cells Naive CD 8+ T cells CD 11

Phenotype of the CD 8+ T cells Naive CD 8+ T cells CD 11 a Low CD 25 Low CD 27 Low CD 31 High CD 43 Low CD 44 Low CD 49 d Low CD 69 Low CD 62 L High CD 122 Low CD 127 Int KLRG-1 Low Specific CD 8+ T cells 14 days CD 11 a High CD 25 High CD 27 High CD 31 Low CD 43 High CD 44 High CD 49 d Low CD 69 High CD 62 L Low CD 122 High CD 127 Low KLRG-1 Low/High T effector Specific CD 8+ T cells 98 days CD 11 a High CD 25 Low CD 27 High CD 31 Low CD 43 High CD 44 High CD 49 d Low CD 69 Low CD 62 L Low CD 122 Intermed. CD 127 Intermed. KLRG-1 Low/High T effector memory

? Function of the immune CD 8+ T cells In vivo cytotoxicity 4 h

? Function of the immune CD 8+ T cells In vivo cytotoxicity 4 h CD 107 a and IFN-g expression 20 h CD 3+CD 8+ 10 CD 107 a 10 10 10 5 8. 12 7. 09 CD 107 a+ IFN-g+ 4 3 2 2. 08 0 82. 7 0 10 2 10 3 10 4 10 IFN-g C 57 Bl/6 WT Perforin KO de Alencar et al. , submitted 5

? CD 3+CD 8+ Multifunctional cells No peptide A pc. DNA 3/Adb-gal TNF 10

? CD 3+CD 8+ Multifunctional cells No peptide A pc. DNA 3/Adb-gal TNF 10 5 0. 082 0. 065 Pep. VNHRFTLV B 10 5 10 4 10 3 10 2 0 0. 59 99. 3 0 10 2 10 3 10 4 0 0. 57 99. 3 10 5 0 10 2 IFN-g C p. Ig. SPCl. 9/Ad. ASP-2 TNF 10 5 0. 068 0. 032 D 10 5 10 4 10 3 10 2 0 0. 43 99. 5 0 10 3 IFN-g 10 3 10 4 10 5 IFN-g 10 4 10 2 0. 051 0. 11 10 4 10 5 1. 88 4. 5 0. 84 0 92. 8 0 10 2 10 3 10 4 10 5 IFN-g de Alencar et al. , submitted

Protective immunity after heterologous prime-boost vaccination Role for Perforin ? WT X Perforin KO

Protective immunity after heterologous prime-boost vaccination Role for Perforin ? WT X Perforin KO p. Ig. SPcl. 9/ Ad. ASP-2 pc. DNA 3/ Adbgal de Alencar et al. , submitted

Protective immunity after heterologous prime-boost vaccination Role for IFN-g WT X IFN-g KO de

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

Conclusions from the mouse vaccination studies 1 - High degree of protective immunity 2

Conclusions from the mouse vaccination studies 1 - High degree of protective immunity 2 - Genetic vaccination (DNA/Ad. ASP-2) 3 - Specific protective CD 8+ T cells 4 - The cell surface markers A/Sn mice C 57 BL/6 mice 5 - Mechanisms mediated by CD 8+ T cells C 57 BL/6 mice Ad. ASP-2 (2 X) or DNA/Ad. ASP-2 Long lived, CD 4 and CD 8 dependent In vivo cytotoxicity and expression IFN-g, TNF-a and CD 107 a. T effector (14 days) or T effector memory (98 days). Perforin and IFN-g Humans ?

Participants UNIFESP-EPM Bruna C. G. de Alencar Fanny Tzelepis Carla Claser Filipe A. Haolla

Participants UNIFESP-EPM Bruna C. G. de Alencar Fanny Tzelepis Carla Claser Filipe A. Haolla José Ronnie Vasconcelos Dr. Sergio Schenkman Inst. Adolofo Lutz Dr. Vera Pereira-Chioccola 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 UFRJ - I. de Biofísica CCF Dr. Pedro M. Persechini Ph. D and Pos-Docs positions National Institute for Vaccine Technology