mrodrigues@unifesp.br

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