Determinants required for the recognition of the Tp9 CTL Epitope from Theileria parva
Transcript of Determinants required for the recognition of the Tp9 CTL Epitope from Theileria parva
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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