[e-mail]

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Swimming against the current Genetic vaccination
against Trypanosoma cruzi infection in mice
INCTV National Institute for Vaccine Technology -
CNPq
mrodrigues_at_unifesp.br
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Prof. Dr. Carlos Chagas Filho
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Epidemiology
Pathogenesis
Prevention
Immunology
Chemotherapy
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Chemical composition of the vaccines
Attenuated live virus
Recombinant proteins
Inactivated virus
Live attenuated bacteria
Killed bacteria
Polysaccharides
200 years 20-25 Vaccines
Toxoids
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Protective mechanisms of current available
vaccines?
Antibodies ?
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Diseases with important antibody independent
mechanism of immunity
Leishmania sp. Mycobacterium HIV Plasmodium Tr
ypanosoma cruzi
Intracellular amastigotes
Intracellular Trypomastigotes
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Hypothesis
Drs. Fidel Zavala and Ruth Nussenzweig
1989-2009
Immunization
Specific T cells
Protective Immunity
Experimental malaria and T. cruzi infection
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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
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T cell-based genetic vaccination against
malaria The human challenge
2004 and 2005
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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
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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
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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
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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
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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 ____________________
_____________
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Amastigote Surface Protein-2 of T. cruzi (Group
II of TS)
(Pan McMahon-Pratt, 1989 and Low Tarleton,
1998)
VTV BOX (VTVxNVFLYNR)
ASP BOX (SxDxGxTW)
Signal Peptide
Amastigote Specific
Liver tissue
Boscardin et al., 2003
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pIgSPclone 9
aa 1-695 of ASP-2
Asp-2
- 3
5 -
Signal peptide of mouse immunoglobulin K chain
pcDNA3
Boscardin et al., 2003
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A/Sn mice Immunization with ts and asp-2 genes
4 doses
tsasp-2
asp-2
ts
pc DNA3
Dependent of CD4 and CD8 T cells
Vasconcelos et al., 2004
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Other T. cruzi ORFs

... 642
66...
ASP-3(5340)
Da Silveira et al., 2008
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pIgSP-ASP-4
pIgSP-ASP-3
Survival ()
Survival ()
ASP-3 p0.0005
ASP-4 p0.0061
pcDNA3
pcDNA3
Days after challenge
Days after challenge
Da Silveira et al., 2008
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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
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Heterologous prime-boost vaccination with ASP-2
of T. cruzi
Peak parasitemia



Adeno- ASP2 (1X)
1- pcDNA3/Adbgal 2- DNA-ASP2/DNA-ASP2 3-
None/Adeno-ASP2 4- Adeno-ASP2/Adeno-ASP2 5-
DNA-ASP2/Adeno-ASP2
13.6X
22.8X
de Alencar et al., submitted
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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
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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
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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
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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
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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
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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
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Phenotypic characterization of specific CD8 T
cells C57BL/6
de Alencar et al., submitted
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Purified CD8 T cellls
H-2kb-VNHRFTLV
CD11a
CD44
CD43
Days
14
98
de Alencar et al., submitted
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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
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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 memory
T effector
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?
Function of the immune CD8 T cells
In vivo cytotoxicity
4 h
C57Bl/6 WT
Perforin KO
de Alencar et al., submitted
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?
CD3CD8 Multifunctional cells
No peptide
Pep. VNHRFTLV
pcDNA3/Adb-gal
pIgSPCl.9/AdASP-2
de Alencar et al., submitted
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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
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Protective immunity after heterologous
prime-boost vaccination
?
Role for IFN-g
WT X IFN-g KO
de Alencar et al., submitted
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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
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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