Determinants Required for the Recognition of the
Tp9 CTL Epitope from Theileria parva
Nicholas Svitek
8 International Congress on Ticks and Tick-Borne Pathogens (TTP8) and 12 Biennial
Conference, South Africa, 24-29 August 2014

T. parva Infection Induces Cytotoxic T
Lymphocyte (CTL) Responses
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
Infection & Treatment Method (ITM)
+
Muguga
cocktail
Oxytetracycline
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
• 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].
[BoLA-6*04101] Tp2 27SHEELKKLGML37

The Tp9 Epitope is Polymorphic in
Different Field Isolates
Tp9Mug AKFPGMKKSK (T. parva Muguga)
Tp9V2 EKFKHMGIGK
Tp9V3 EKFQRMGIGK
Tp9V4 AKFPGMKKGK
Tp9V5 EKFKHMRIGK
Tp9V6 EKFKRMGMKK
Tp9V7 NKFPGMKKGK
Tp9V8 NKFKHMRIGK
Tp9V9 AKFPNLKARR
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?

Alanine Scanning of the Tp9Mug Epitope
Tp9Mug AKFPGMKKSK (T. parva Muguga)
Tp9G1 GKFPGMKKSK
Tp9A2 AAFPGMKKSK
Tp9A3 AKAPGMKKSK
Tp9A4 AKFAGMKKSK
Tp9A5 AKFPAMKKSK
Tp9A6 AKFPGAKKSK
Tp9A7 AKFPGMAKSK
Tp9A8 AKFPGMKASK
Tp9A9 AKFPGMKKAK
Figure by Anne Mølgaard
Tp9A10 AKFPGMKKSA

Amino Acids at Positions 2 & 3 are
Important for Binding to BoLA-1*02301
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
ELISA peptide-BoLA binding assay
1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
3
2
1
0
[P e p t id e ] nM
O .D . [4 5 0 nm ]
Tp9
Tp9 G 1
Tp9 A 2
Tp9 A 3
Tp9 A 4
Tp9 A 5
Tp9 A 6
Tp9 A 7
Tp9 A 8
Tp9 A 9
Tp9 A 1 0
Svitek N., et al., In preparation.
A lower Kd value is
indicative of a higher
binding peptide

Positions 1, 4, 5 & 7 are Required for
Recognition by the CTL
****p < 0.0001
*p < 0.0153 NS p = 0.0883
Spot Forming Units (SFU) per 106 cells
IFN- Enzyme Linked Immunospot (ELISpot) Assay
with 495 bulk Tp9Muguga-specific CTL
6 0 0 0 0
4 0 0 0 0
2 0 0 0 0
M e d iu m
T p 9 G 1
T p 9 A 2
T p 9 A 3
T p 9 A 4
T p 9 A 5
T p 9 A 6
T p 9 A 7
T p 9 A 8
T p 9 A 9
T p 9
T p 9 A 1 0
0
SFU are indicative of
IFN- synthesis.
More SFU = better response
by the CTL.
Important for
binding

Summary 1 [Alanine Scanning]
Tp9Mug AKFPGMKKSK (T. parva Muguga)
Tp9G1 GKFPGMKKSK
Tp9A2 AAFPGMKKSK
Tp9A3 AKAPGMKKSK
Tp9A4 AKFAGMKKSK
Tp9A5 AKFPAMKKSK
Tp9A6 AKFPGAKKSK
Tp9A7 AKFPGMAKSK
Tp9A8 AKFPGMKASK
Tp9A9 AKFPGMKKAK
Tp9A10 AKFPGMKKSA
Tp9 AKFPGMKKSK
BoLA class I binding
TCR recognition
TCR recognition
BoLA class I
binding
TCR
recognition
TCR recognition

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

Tp9 Variants 4 and 9 Bind to
BoLA-1*02301
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
A lower Kd value is
indicative of a higher
binding peptide
1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
2
1
0
[p e p t id e ] (nM )
O .D . [4 5 0 nm ]
T p 9 M u g
T p 9 V 2
T p 9 V 3
T p 9 V 4
T p 9 V 5
T p 9 V 6
T p 9 V 7
T p 9 V 8
T p 9 V 9

Tp9Mug-Specific CTL Cross-Reacts
Only with Tp9V4
Spot Forming Units (SFU) per 106 cells
IFN- Enzyme Linked Immunospot (ELISpot) Assay
with 495 bulk Tp9Muguga-specific CTL
1 0 0 0 0 0
5 0 0 0 0
M e d iu m
T p 9
Tp9Mug AKFPGMKKSK
Tp9V2 EKFKHMGIGK
Tp9V3 EKFQRMGIGK
Tp9V4 AKFPGMKKGK
Tp9V5 EKFKHMRIGK
Tp9V6 EKFKRMGMKK
Tp9V7 NKFPGMKKGK
Tp9V8 NKFKHMRIGK
Tp9V9 AKFPNLKARR
T p 9 V 2
T p 9 V 3
T p 9 V 4
T p 9 V 5
T p 9 V 6
T p 9 V 7
T p 9 V 8
T p 9 V 9
0
SFU are indicative of
IFN- synthesis.
More SFU = better response
by the CTL.

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 protect
against 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 Nene
Roger Pellé
Rosemary Saya
Elias Awino
John Wasilwa
Stephen Munyao
Søren Buus
Expression of rBoLA class I
Ivan Morrison
Niall McHugh
Hanneke Hemink
Tim Connelley
Generation of the Tp9 CTL & sequencing of
the Tp9 gene from field isolates
Funding:

Better lives through livestock
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