Determinants Required for the Recognition of the

Tp9 CTL Epitope from Theileria parva

Nicholas Svitek8 International Congress on Ticks and Tick-Borne Pathogens (TTP8) and 12 Biennial

Conference, South Africa, 24-29 August 2014

Restricting BoLA [MHC]class I molecule

T. parva antigen CTL Epitope

1) 6*01301 Tp1 214VGYPKVKEEML224

2) 6*04101 Tp2 27SHEELKKLGML37

3) 2*01201 Tp2 49KSSHGMGKVGK59

4) BoLA-T2c Tp2 96FAQSLVCVL104

5) BoLA-T5 Tp5 87SKADVIAKY95

6) 3*00101 Tp8 379CGAELNHFL387

7) 1*02301 Tp9 67AKFPGMKKSK76

T. parva Infection Induces Cytotoxic T Lymphocyte (CTL) Responses

+Mugugacocktail

Oxytetracycline

Infection & Treatment Method (ITM)

Graham et al., 2006, 2008

• Can induce a carrier state.• Expensive to produce.• Necessitates a liquid nitrogen

cold chain to be delivered.

Developing Reagents to Assay CTL Responses in Cattle

• Immunoinformatics: prediction, using NetMHCpan, of peptide binding to MHC class I [antigen identification].

• Peptide-MHC class I binding using recombinant bovine MHC (BoLA) class I molecules [peptide specificity].

• Bovine peptide-MHC class I tetramers to measure T-cell responses in cattle [characterize cellular immune response].

Developing Reagents to Assay CTL Responses in Cattle

[BoLA-6*04101] Tp2 27SHEELKKLGML37

• Immunoinformatics: prediction, using NetMHCpan, of peptide binding to MHC class I [antigen identification].

• Peptide-MHC class I binding using recombinant bovine MHC (BoLA) class I molecules [peptide specificity].

• Bovine peptide-MHC class I tetramers to measure T-cell responses in cattle [characterize cellular immune response].

Tp9Mug AKFPGMKKSK (T. parva Muguga)Tp9V2 EKFKHMGIGK Tp9V3 EKFQRMGIGK Tp9V4 AKFPGMKKGK Tp9V5 EKFKHMRIGK Tp9V6 EKFKRMGMKK Tp9V7 NKFPGMKKGK Tp9V8 NKFKHMRIGK Tp9V9 AKFPNLKARR

The Tp9 Epitope is Polymorphic in Different Field Isolates

Pellé R., et al., In preparation.

How well does CTL raised against the Tp9 epitope from the Muguga strain cross-react towards Tp9 from other isolates?

WHICH AMINO ACIDS IN TP9MUG ARE IMPORTANT FOR BOLA BINDING AND RECOGNITION BY CTL?

Tp9Mug AKFPGMKKSK (T. parva Muguga)Tp9G1 GKFPGMKKSKTp9A2 AAFPGMKKSKTp9A3 AKAPGMKKSKTp9A4 AKFAGMKKSKTp9A5 AKFPAMKKSKTp9A6 AKFPGAKKSKTp9A7 AKFPGMAKSKTp9A8 AKFPGMKASKTp9A9 AKFPGMKKAKTp9A10 AKFPGMKKSA

Alanine Scanning of the Tp9Mug Epitope

Figure by Anne Mølgaard

Peptide Kd (nM)

Tp9 111

Tp9G1 131.5

Tp9A2 1935

Tp9A3 2804

Tp9A4 26.6

Tp9A5 203.8

Tp9A6 50.2

Tp9A7 16.6

Tp9A8 20.8

Tp9A9 54

Tp9A10 3.7

Amino Acids at Positions 2 & 3 are Important for Binding to BoLA-1*02301

1 10 100 1000 10000 1000000

1

2

3

[Peptide] nM

O.D

. [4

50

nm

]

Tp9Tp9G1

Tp9A2

Tp9A3

Tp9A4

Tp9A5

Tp9A6

Tp9A7

Tp9A8

Tp9A9

Tp9A10

A lower Kd value isindicative of a higher binding peptide

Svitek N., et al., In preparation.

ELISA peptide-BoLA binding assay

****p < 0.0001

NS p = 0.0883*p < 0.0153

Positions 1, 4, 5 & 7 are Required for Recognition by the CTL

Spot

For

min

g U

nits

(SFU

) per

106 c

ells

IFN- Enzyme Linked Immunospot (ELISpot) Assaywith 495 bulk Tp9Muguga-specific CTL

Med

ium

Tp9

Tp9G1

Tp9A2

Tp9A3

Tp9A4

Tp9A5

Tp9A6

Tp9A7

Tp9A8

Tp9A9

Tp9A10

0

20000

40000

60000

SFU are indicative ofIFN- synthesis. More SFU = better responseby the CTL.

Important for binding

Tp9Mug AKFPGMKKSK (T. parva Muguga)Tp9G1 GKFPGMKKSKTp9A2 AAFPGMKKSKTp9A3 AKAPGMKKSKTp9A4 AKFAGMKKSKTp9A5 AKFPAMKKSKTp9A6 AKFPGAKKSKTp9A7 AKFPGMAKSKTp9A8 AKFPGMKASKTp9A9 AKFPGMKKAKTp9A10 AKFPGMKKSA Tp9 AKFPGMKKSK

BoLA class I binding

TCR recognition

TCR recognition

BoLA class I binding

TCR recognition

Summary 1 [Alanine Scanning]

TCR recognition

WHAT ARE THE CONSEQUENCES OF THE Tp9 POLYMORPHISMS FOR BOLA-1*02301 CLASS I BINDING & RECOGNITION BY A TP9MUGUGA-SPECIFIC CTL?

Peptide Kd (nM) Sequence

Tp9 111 AKFPGMKKSK

Tp9V2 2609 EKFKHMGIGK

Tp9V3 988.5 EKFQRMGIGK

Tp9V4 271.6 AKFPGMKKGK

Tp9V5 2865 EKFKHMRIGK

Tp9V6 1584 EKFKRMGMKK

Tp9V7 2245 NKFPGMKKGK

Tp9V8 1318 NKFKHMRIGK

Tp9V9 485.9 AKFPNLKARR

Tp9 Variants 4 and 9 Bind to BoLA-1*02301

A lower Kd value isindicative of a higher binding peptide

1 10 100 1000 10000 1000000

1

2

[peptide] (nM)O

.D. [

45

0 n

m]

Tp9Mug

Tp9V2

Tp9V3

Tp9V4

Tp9V5

Tp9V6

Tp9V7

Tp9V8

Tp9V9

Tp9Mug-Specific CTL Cross-Reacts Only with Tp9V4

Spot

For

min

g U

nits

(SFU

) per

106 c

ells

IFN- Enzyme Linked Immunospot (ELISpot) Assaywith 495 bulk Tp9Muguga-specific CTL

Med

ium

Tp9

Tp9V2

Tp9V3

Tp9V4

Tp9V5

Tp9V6

Tp9V7

Tp9V8

Tp9V9

0

50000

100000

SFU are indicative ofIFN- synthesis. More SFU = better responseby the CTL.

Tp9Mug AKFPGMKKSKTp9V2 EKFKHMGIGK Tp9V3 EKFQRMGIGK Tp9V4 AKFPGMKKGK Tp9V5 EKFKHMRIGK Tp9V6 EKFKRMGMKK Tp9V7 NKFPGMKKGK Tp9V8 NKFKHMRIGK Tp9V9 AKFPNLKARR

Summary 2 [Field Isolates]

Tp9Mug AKFPGMKKSK (T. parva Muguga)Tp9V2 EKFKHMGIGK Tp9V3 EKFQRMGIGK Tp9V4 AKFPGMKKGK Tp9V5 EKFKHMRIGK Tp9V6 EKFKRMGMKK Tp9V7 NKFPGMKKGK Tp9V8 NKFKHMRIGK Tp9V9 AKFPNLKARR

Binding to BoLA-1*02301

Cross-reaction with Tp9Mug-specific CTL

Vaccinating with Muguga strain can potentially protectagainst variant 4 in the context of the BoLA-1*02301 class I molecule.

The other peptides can not be considered as potential CTL epitopes in the context of the BoLA-1*02301 class I molecule since they are non-binders.

Perspectives

Similar studies with the other polymorphic T. parva CTL epitopes. Tp1 is polymorphic but a CTL raised against Tp1Mug cross-

react with Tp1 from other field isolates.

Identification of antigens that are not polymorphic and would induce protection against all strains in the context of a particular BoLA (MHC) class I molecule.

Acknowledgments

Lucilla Steinaa & Vish NeneRoger PelléRosemary Saya Elias Awino John Wasilwa Stephen Munyao

Søren Buus Expression of rBoLA class I

Ivan MorrisonNiall McHughHanneke HeminkTim Connelley

Funding:

Generation of the Tp9 CTL & sequencing of the Tp9 gene from field isolates

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