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Swimming against the current: Genetic vaccination against
Trypanosoma cruzi infection in mice
INCTVNational Institute for Vaccine Technology - CNPq
Prof. Dr. Carlos Chagas Filho
Epidemiology
Pathogenesis
Immunology
Chemotherapy
Prevention
Attenuated live virus
Inactivated virus
Live attenuated
bacteria
Killed bacteria
Toxoids
Polysaccharides
Recombinant
proteins
Chemical composition of the vaccines
Hepatitis B HPV
~200 years
20-25 Vaccines
Protective mechanisms of current available vaccines?
Antibodies ?
Diseases with important antibody independent mechanism of immunity
Leishmania sp.
Mycobacterium
HIV
Plasmodium
Trypanosoma cruzi IntracellularTrypomastigotes
Intracellularamastigotes
Hypothesis
Specific T cells
Immunization
Protective Immunity
Experimentalmalaria
and T. cruzi infection
1989-2009
Drs. Fidel Zavala and Ruth Nussenzweig
Experimental T cell-based genetic vaccination against experimental malaria
0
Days
21 35
Rec. Influenza virus
Rec. Vaccinia virus
56
Challenge P. yoelli
Rodrigues et al., 1993 and 1994
Results:9/15 - no malaria4/15 - delayed malaria2/15 - failure
CD8+ T cell mediated
Malaria antigen - CS protein
Humans ?
2004 and 2005
T cell-based genetic vaccination against malaria:
The human challenge
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
New vaccinesFew or no products
Trans-sialidase of trypomastigotes of T. cruzi
CatalyticDomain
(aa 34-678)
GPIanchor
Signal Peptide(aa 1-33)
NH2 COOH
C-terminal repeats (CTR)(aa 679-1071)
Genetic vaccination against Trypanosoma cruzi infection ?
Summary of the results using different plasmidsBALB/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
CD4Epitope(s)
33-275
CD8 Epitope359-367
p154/13
p154/13
p154/13-CD8
pCD8-Epitope
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
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_________________________________
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 Epitope553-560
H-2Kb
CD8 Epitope320-327
H-2Kk
CD4 epitopes ?
(Pan & McMahon-Pratt, 1989 and Low & Tarleton, 1998)
pIgSPclone 9
Boscardin et al., 2003
aa 1-695 of ASP-2
- 3’Asp-25’ -
Signal peptide of mouse immunoglobulin
chain
pcDNA3
A/Sn mice Immunization with ts and asp-2 genes
Vasconcelos et al., 2004
4 doses
Dependent of CD4+ and CD8+ T cells
asp-2
ts
ts+asp-2
pcDNA3
66... ... 743ASP-4(7015)
1... ... 273RibpS4
1... ... 600PAR-2
1... ... 211Tc24
1... ... 656MTP70
1... ... 653HSP70
1... ... 319RpL7a
1... ... 112H2b
1... ... 776EF2
1... ... 218TcG2
1... ... 91TcG4
1... ... 449TcG5
1... ... 395TcG8
66... ... 642ASP-3(5340)
Other T. cruzi ORFs
Da Silveira et al., 2008
Days after challenge
Su
rviv
al (
%)
pcDNA3
pIgSP-ASP-3
66... ... 642ASP-3
Da Silveira et al., 2008
ASP-3 p=0.0005 S
urv
ival
(%
)
Days after challenge
pIgSP-ASP-4
pcDNA3
66... ... 743ASP-4
ASP-4 p=0.0061
Vaccination with ASP-2 of T. cruzi
Homologous X Heterologous 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 vaccination
Priming Boosting
Plasmid DNA Plasmid DNA
None Rec. Adenovirus
Rec. Adenovirus Rec. Adenovirus
Plasmid DNA Rec. Adenovirus
Days after infection
15 20 25 30 35 220 225S
urvi
val (
%)
0
20
40
60
80
100
1- pcDNA3/Adgal2- DNA-ASP2/DNA-ASP23- None/Adeno-ASP24- Adeno-ASP2/Adeno-ASP25- DNA-ASP2/Adeno-ASP2
1 2 3 4 5
Par
asite
mia
/mL
of b
lood
105
106
**
*
Heterologous prime-boost vaccination with ASP-2 of T. cruzi
Peak parasitemia
Adeno-ASP2 (1X)
22.8X
13.6X
de Alencar et al., submitted
ECG222 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
Days after infection
15 20 25 30S
urvi
val (
%)
0
20
40
60
80
100
Days after infection
8 9 10 11 12 13
Par
asite
mia
104
105
106
***
**
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
Days after infection15 20 25 30
Sur
viva
l (%
)0
20
40
60
80
100
pcDNA3 Ad-gal None pIgSPCl.9 AdASP-2 Rat igG pIgSPCl.9 AdASP-2 -CD8
Days after infection
8 9 10 11 12 13 14
Par
asite
mia
106
105
104
** *
**
Protective immunity is dependent on CD8+ T cells
Heterologous prime-boost vaccination with ASP-2 of T. cruzi
de Alencar et al., submitted
Longevity of protective T cells
Days after infection
15 20 25 30
Sur
viva
l (%
)
0
20
40
60
80
100
Days after infection
8 9 10 11 12 13
Par
asite
mia
104
105
106
* * *
** * *
*
98 days
14 days
98 days 14 days
de Alencar et al., submitted
Heterologous prime-boost vaccination with ASP-2 of T. cruzi
Strain-specificity of the protective immunity elicited by heterologous prime-boost vaccination
Days after challenge
0 20 40 60 80 100 120
Sur
viva
l (%
)
0
20
40
60
80
100
Col 1 v Col 4 Col 1 v Col 7
Colombian
Days after challenge
10 15 20 25 30 35 40 45 50
Sur
viva
l (%
)
0
20
40
60
80
100
pcDNA3/AdGalGr. IV - pIgSPCl.9 + p154/13 / AdASP-2 +AdTS
Failure
Success
COL
pcDNA3 – adeno-gal
pIgSP-Cl.9 – Adeno-ASP-2P154/13 - Adeno-TS
Haolla et al., submitted
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
Phenotypic characterization of specific CD8+ T cellsC57BL/6
CD8
NaivepIgSPCl.9/AdASP-2
Days
H-2
kb-V
NH
RFTLV
15.7
0.018
0.016 0.38
0.4
9.97
14
98
0.32
0.31
0.02
0.016
de Alencar et al., submitted
CD11a
0 102 103 104 105<FITC-A>
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105<FITC-A>
0
20
40
60
80
100
% o
f M
ax
Days
14
98
Naive H-2kb-VNHRFTLVPurified CD8+ T cellls
0 102 103 104 105<FITC-A>
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105<FITC-A>
0
20
40
60
80
100
% o
f M
ax
CD44
0 102 103 104 105<FITC-A>
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105<FITC-A>
0
20
40
60
80
100
% o
f M
ax
CD43
de Alencar et al., submitted
0 102 103 104 105<FITC-A>
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105
<FITC-A>
0
20
40
60
80
100
% o
f M
ax
CD122IL-2 rec.
0 102 103 104 105<PE-Cy7-A>
0
20
40
60
80
100
% o
f M
ax
CD127IL-7 rec.
0 102 103 104 105
<PE-Cy7-A>
0
20
40
60
80
100
% o
f Ma
x
0 102 103 104 105<APC-A>
0
20
40
60
80
100
% o
f M
ax
KLRG-1
0 102 103 104 105
<APC-A>
0
20
40
60
80
100
% o
f Ma
x
Days
14
98
Naive H-2kb-VNHRFTLVPurified CD8+ T cellls
0 102 103 104 105<FITC-A>: 6
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105
<APC-A>
0
20
40
60
80
100
% o
f Ma
x
CD62L
de Alencar et al., submitted
Naive CD8+ T cells
CD11a LowCD25 LowCD27 LowCD31 HighCD43 LowCD44 LowCD49d LowCD69 LowCD62L HighCD122 LowCD127 IntKLRG-1 Low
SpecificCD8+ T cells
14 days
CD11a HighCD25 HighCD27 HighCD31 LowCD43 HighCD44 HighCD49d LowCD69 HighCD62L LowCD122 HighCD127 LowKLRG-1 Low/High
SpecificCD8+ T cells
98 days
CD11a HighCD25 LowCD27 HighCD31 LowCD43 HighCD44 HighCD49d LowCD69 LowCD62L LowCD122 Intermed.CD127 Intermed.KLRG-1 Low/High
Phenotype of the CD8+ T cells
T effector memoryT effector
Cito
toxi
city
(%
)
0
20
40
60
80
100
pcDNA3+
Ad-gal
pIgSPCl.9 +
AdASP-2
pcDNA3+
Ad-gal
pIgSpCl.9 +
AdASP-2
4 h
C57Bl/6 WT Perforin KO
In vivo cytotoxicity
Cyt
otox
icity
(%
)
0
20
40
60
80
100
pcDNA3+
Ad-gal
pIgSPCl.9 +
AdASP-2
pcDNA3+
Ad-gal
pIgSpCl.9 +
AdASP-2
C57Bl/6 WT Perforin KO
20 h
Function of the immune CD8+ T cells
0 10 2 10 3 10 4 10 5
0
10 2
103
104
105 8.12 7.09
2.0882.7
CD
107a
IFN-
CD3+CD8+
CD107a+
IFN-+
CD107a and IFN- expression
?
de Alencar et al., submitted
CD3+CD8+ Multifunctional cells
?
No peptide Pep. VNHRFTLV
0 10 2 10 3 10 4 10 5
0
10 2
10 3
10 4
10 5 0.11 0.051
0.5799.3
0 10 2 10 3 10 4 10 5
0
10 2
10 3
10 4
10 5 0.082 0.065
0.5999.3
0 10 2 10 3 10 4 10 5
0
10 2
10 3
10 4
10 5 1.88 4.5
0.8492.8
0 10 2 10 3 10 4 10 5
0
10 2
10 3
10 4
10 5 0.068 0.032
0.4399.5
TN
F
IFN- IFN-
IFN- IFN-
TN
F
A B
C D
pIgSPCl.9/AdASP-2
pcDNA3/Ad-gal
de Alencar et al., submitted
Days after infection
20 30 40 50 60 70 80
Sur
viva
l (%
)
0
20
40
60
80
100
C57BL6/WT Perforin KOC57BL6/WT Perforin KO
pIgSPcl.9/AdASP-2
pIgSPcl.9/AdASP-2
pcDNA3/Adgal
pcDNA3/Adgal
Protective immunity after heterologous prime-boost vaccination
WT X Perforin KO
Role for Perforin ?
de Alencar et al., submitted
WT X IFN- KO
Protective immunity after heterologous prime-boost vaccination
?Role for IFN-
de Alencar et al., submitted
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 Teffector (14 days) or
Teffector 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 ?
Participants
UNIFESP-EPMBruna C. G. de Alencar Fanny TzelepisCarla Claser Filipe A. HaollaJosé Ronnie Vasconcelos
Dr. Sergio Schenkman
UFMG e CPRR-FIOCRUZ Dr. Ricardo T. Gazzinelli Dr. Oscar Bruna-RomeroDr. Marcus PenidoDr. Alexandre V. Machado
UFRJ - I. de Biofísica CCF Dr. Pedro M. Persechini
IOC-FIOCRUZ Dr. Gabriel de OliveiraDr. Joseli Lannes-Vieira
PhD and Pos-Docs positions National Institute for Vaccine
Technology
Inst. Adolofo Lutz Dr. Vera Pereira-Chioccola