Generation of Novel Chagas VaccinesEvolving Studies / Work in Progress

Christopher S. Eickhoff1, Matthew Ardito2, Eric Gustafson3,

William Martin2, and Daniel F. Hoft1

1 Saint Louis University

St. Louis, MO

2EpiVax, Inc.

Providence, RI

3 University of Rhode Island

Providence, RI

Presentation Summary

1) Chagas disease background

2) Study design & prototype vaccines

3) Future directions

Chagas DiseaseNeglected infection of poverty

• most common cause of heart disease deaths

in endemic areas (10-50,000 deaths/yr)

• drug treatment:•Benznidazole/Nifurtimox

-highly successful during acute infection

-promising in reducing disease progression

**- side effects: 30-40%=stop therapy

• available vaccines : NONE

Trypanosoma cruzi

• 11-18 million infected (300,000 in U.S.)

• 40-100 million at risk

• life-long chronic infection

T. cruzi mouse model

• develop acute disease & mortality

• models developed for studying mucosal and

systemic infection

• develop cardiac disease/inflammation (ECG/H&E)

• availability of ‘humanized’ mice (HLA-A2/DR1 tg)

Chagas Immunity

Mouse data:

• CD4+ and CD8+ T cells critical for protection

Human data:

• T- and B- cell responses against several T.

cruzi proteins

• CD8+ IFN- + T cells correlate with decreased

disease progression

GOAL Design a Chagas vaccine targeting

CD4+ and CD8+ T cell responses

Overall Study Design

Identify key CD4+ & CD8+ T cell epitopes & clusters (multiple HLA),

synthesize peptides

Validation using HLA binding assays &

in vitro assays with T. cruzi+ human T cells

Generate synthetic epitope-

based DNA vaccines

Vaccinate HLA A2+DR1+ Tg mice

Protection against acute T. cruzi

challenge

(prophylactic vaccine)

Protection against disease

progression

(therapeutic vaccine)

iVAX Tools

Conservatrix – identifies 9mer sequences

across sequence variants

iVAX Tools

EpiMatrix – Maps T cell epitopes across HLA

(8 common class II alleles, 6 class I

supertypes)

iVAX ToolsClustiMer – identifies promiscuous epitopes

(dense ‘clusters’ of T cell epitopes)

iVAX Tools

EpiAssembler – assembles overlapping

epitopes to encode immunogenic

consensus sequences (ICS)

T. cruzi genome/proteome

• Published 2005:

– genome of a single reference strain

– proteomes of all 4 T. cruzi life stages

• Excellent resource for many aspects of T.

cruzi research, including vaccine

development

T. cruzi genome/proteome

• Revealed the presence of several large

gene families– The trans-sialidase (TS) gene family is the largest:

>1,400 members

>5% of genome

Trans-sialidase (TS) • transfers sialic acid from

host to parasite

• necessary for parasite

infectivity

• virulence factor

• immunogenic (mice &

humans)

Functional and non-functional TS genes

Only 12 of the 1,400 TS gene family members

encode active enzyme.

Do T cell epitopes present in diverse non-functional

TS genes play a role in parasite persistence and

immunoevasion?

Identified 2,900

unique 9mers

392 conserved in

≥50% F-TS genes

Total of 11 class II ICS

clusters identified

* Top 5-6 sequences predicted by EpiMatrix for each of the

supertype class I alleles selected for further analysis

Functional

TS genes

(12)

Non-functional

TS genes

(723)

Identified 196,664

unique 9mers

750 conserved in

≥5% NF-TS genes

Total of 12 class II ICS

clusters identified

Epitope identification in F-TS and NF-TS genes

Conservatrix

EpiMatrix / EpiAssembler

F-TS Prototype vaccinesT cell responses induced by full length TS vaccination

HLA A2+DR1+ tg

Week 0 Week 2 Week 5 Week 8

DNA i.m DNA i.m. Adenovirus s.c. IFN- ELISPOT

CD4+ Spleen cells CD8+ Spleen cells

-Vaccination with native F-TS induces CD4+ and CD8+ T cell

responses against F-TS class II clusters (DRB*0101) &A0201 peptides predicted by EpiMatrix.

T. cruzi genome - 25,011 genes

T. cruzi proteome Mass spec data

(AMA/Tryp/BFT) - 2,185 genes

Potentially secreted proteins (SignalP,LipoP,Phobius)

204 genes

Class I 9mer EpiMatrix

Class II EpiMatrix/ClustiMer

BLAST analysis

HLA binding assays

IFN- ELISPOT with Chagasic T cells

Prepare ‘string-of-beads’ epitope based

vaccines & test in HLA A2+DR1+ Tg mice

Whole-genome-derived epitope identification

Validation of selected Class II clusters (Class II binding assays)

Whole genome-derived epitope identification

Seq-1 Surface protease gp63 KRDILTKEKRSIILNSLLPRAFGMH 4 25 40.27Seq-2 Surface protein GGIPLLLRAPLLMLAAVASFFGF 3 23 52.83Seq-3 Hypothetical conserved protein HIPFVFFFSITSSSKNSSQSR 2 21 57.35Seq-5 Dynein light chain GPGLRELKKLKILSLGRNVIRKIE 2 24 52.72Seq-6a Hypothetical conserved protein AEEVLKAAAPALFLSK 2 16 22.49Seq-6b Hypothetical conserved protein APALFLSKNKSAEEESV 2 17 25.06Seq-8 Hypothetical conserved protein RGLVLLLSFITSPLSLQQAFE 2 21 42.77Seq-9 Hypothetical conserved protein PTETLQLLTNILQNFPSLFKSV 2 22 42.1Seq-10 Hypothetical conserved protein HIAVKYVKLVYLLRANPSLSTPSL 1 24 89.66

Seq-15a Hypothetical conserved protein RNDVLIMESLLRQLRVS 1 17 21Seq-15b Hypothetical conserved protein ESLLRQLRVS ISNALRLASRT 1 21 64.16Seq-16 ATPdependent Clp protease ARPLKRLVQSVLLNRLALMLLDGR 1 24 59.32

Seq-17a Proteosome regulatory nonATPase ERQVDALVHLLSVIRSFFSL 1 20 46.08Seq-17b Proteosome regulatory nonATPase SVIRSFFSLLPKAKTTRM 1 18 36.61

10<IC50<100

IC50<10

Weak/Non-binder

Moderate binder

Strong binder

Cluster

Score

DRB*

0101

DRB*

0401

DRB*

0701

DRB*

1501

IC50>100

SEQUENCE

ID

TriTrypDB

gene description Sequence

#

genes

AA

length

• EpiMatrix predicted HLA binding with high success

(93% to HLA DRB*0101&1501; overall 70%)

Future Directions

• Continue synthesizing and validating

selected peptides– HLA binding assays

– In vitro studies with T. cruzi infected human

T cells

• Generate synthetic epitope vaccines

• Test in HLA A2+DR1+ tg animals as

prophylactic v. therapeutic vaccines

AcknowledgmentsSaint Louis University

St. Louis, MO

EpiVax, Inc.

Providence, RI

University of Rhode Island

Providence, RI

Daniel Hoft

Nicole Sullivan

Jenny Blase

Matthew Ardito

William Martin

Frances Terry

Annie De Groot

Eric Gustafson

Joseph Desrosiers

Sheba Meymandi – Olive View Chagas Clinic

NIH RO1-AI040196 to D.F.H.

3U19AI082642-02S1, Translational Immunology Research and Accelerated

[vaccine] Development Program