Discovery of novel CTL epitopes by peptide library screening of CTL lines from Theileria parva immune animals

ABSTRACT

The parasite Theileria parva claims the life ofapproximately 1 million cattle every year. Immune animals to theparasite develop a lifelong immunity based on a cytotoxic Tlymphocyte (CTL) response with a strong immunodominancerestricted by the bovine leukocyte antigen (BoLA) class Imolecules. In our goal of developing a next-generation vaccineagainst T. parva, we have undertaken to identify new CTLinducing antigens that can be included in a recombinant vaccine.A peptide library of 18-mer peptides overlapping by 12 aminoacids and covering 500 genes of the whole parasite genome wassynthesized; giving approximately 40,000 peptides aliquoted inpools of 50 peptides. Genes were selected based on the presenceof a signal peptide, abundance, and divergence to the relatedparasite T. annulata, as well as by a selection usingimmunoinformatic tools predicting, by artificial neural network,peptides binding with strong affinity to the BoLA class I moleculespresent in cattle of Africa. A bank of ten CTL lines from cattleimmunized with the live parasite expressing different BoLA class Ispecificities were screened by IFN- ELISpot assay. Among thesecells, four secreted IFN- in the presence of a different peptidelibrary pool. Peptides from these pools were synthesized andpositive individual 18-mer peptides were identified. Dissection ofthe minimal epitope is underway using IFN- ELISpot, cytotoxicityas well as peptide-BoLA class I flow cytometry assays. Thesenewly identified antigens will hopefully allow us to develop avaccine towards T. parva giving wide coverage in cattlepopulation with diverse BoLA class I molecules.

N. Svitek1, R. Saya1, E. Awino1, M. Nielsen2, N. MacHugh3, J.C. da Silva4, V. Nene1, and L. Steinaa1

1Vaccines Biosciences, International Livestock Research Institute, Nairobi, Kenya 2Centre for Biological Sequence Analysis, Technical University of Denmark, Copenhagen, Denmark 3The Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom 4The Institute for Genome Sciences, University of Maryland, Baltimore, USA

LIFE CYCLE OF T. PARVA

Sporozoite entry viaa “zippering” mechanism

Sporozoite attachment to host lymphocyte

Mature schizont in“transformed” lymphocyte

Merogony

Bos indicus

Syncerus cafferPiroplasms in red blood cells

Larvae & nymphsacquire infection

Rhipicephalus appendiculatus(a three-host tick)

Merozoites released byhost cell rupture

Zygote

Kinete

Gametes

Daughter host-cellsremain infected

Sporogony in type III acinus

Bos taurusProliferation of

schizont-infected cells

Sporozoites releasedduring tick feeding

Infectednymphal & adult ticks

Rb

Rb

Rb

Mz

Sz

Sz

Rb

CTL LINES FROM T. PARVAMuguga

IMMUNIZED ANIMALS

TheileriaparvaMuguga

Oxytetracycline

INFECTION & TREATMENT METHOD (ITM) OF VACCINATION

Positive peptides (TpMuguga_04g00772):

TLTLQGHRMKVTNLKWNPG (D5.31)

HRMKVTNLKWNPTVDYALG (lD5.32)

Positive peptides (TpMuguga_01g01091 [Tp12]):

DTLDSNREQLLNQFYQLFG (E4.34)

REQLLNQFYQLFNTPVDQG (E4.35)

Cell line MHC Haplotype Epitope Screening

F100 (B) A10/KN104 N.A. PositiveBX64 (B) A10/KN104 N.A. PositiveBF091 A18/A11/A19 Tp1214-224 NegativeBB007 A18/? Tp1214-224 NegativeBF092 A18/A12 Tp1214-224 PositiveBK173 A14 Not reacting to Tp9 NegativeBA219 A18 Tp1214-224 NegativeBH055 A11/A15v Tp5214-224 NegativeBG042 A10/A12 Not reacting to Tp2 NegativeBW012(B) T7/? Tp7206-214 Positive

PARTNERS FUNDING

SUMMARY & PERSPECTIVE

1

2 3

A1012%

A1110%

A1211%

A14/A1531%A17

4%

A189%

A1911%

A206%

A316%

Haplotype Frequency in Cattle

N=1274

4 chromosomes≈ 4,000 genes

TheileriaparvaMuguga

- Abundance- Signal peptide- Divergence to T. annulata- Promiscuity to

BoLA class I molecules (NetMHCpan)

Selection of 500 genes 40,000 peptides

5

1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0

0

1

2

3

4

[p e p tid e ] (n M )

O.D

. [4

50

nm

]

B in d e r 1

B in d e r 2

N o n -b in d e r 1

N o n -b in d e r 2

F100.1 TLTLQGHRMKVTF100.2 LTLQGHRMKVTNF100.3 TLQGHRMKVTNLF100.4 LQGHRMKVTNLKF100.5 QGHRMKVTNLKWF100.6 GHRMKVTNLKWNF100.7 HRMKVTNLKWNPF100.8 RMKVTNLKWNPTF100.9 MKVTNLKWNPTVF100.10 KVTNLKWNPTVDF100.11 VTNLKWNPTVDYF100.12 TNLKWNPTVDYAF100.13 NLKWNPTVDYAL

MHC CLASS I HAPLOTYPE FREQUENCIES IN CATTLE

SELECTION OF PEPTIDES

Med

ium

CT

L+T

pM

Tp

M o

nly

F100.1

F100.2

F100.3

F100.4

F100.5

F100.6

F100.7

F100.8

F100.9

F100.1

0

F100.1

1

F100.1

2

F100.1

3

Tp

1 e

pit

op

e

Po

sit

ive p

oo

l

D5.3

1

D5.3

2

0

1 0 0

2 0 0

3 0 0

Sp

ot

Fo

rm

ing

Un

it (

SF

U)

Med

ium

Co

nA

Po

ol D

5, p

late

8

D5.3

1

D5.3

2

0

1 0 0

2 0 0

3 0 0

Sp

ot

Fo

rm

ing

Un

it (

SF

U)

NEW ANTIGEN IDENTIFIED WITH F100 CTL

6

POSITIVE PEPTIDES IDENTIFIED WITH BF092 CTL

IFN ELISpot Assay

IFN ELISpot Assay

7

8M

ed

ium

Co

nA

BF

092 T

pM

Tp

M a

lon

e

Tp

1 e

pit

op

e

Po

ol E

4, p

late

2

E4.3

3

E4.3

4

E4.3

5

E4.3

6

0

2 0 0

4 0 0

6 0 0

Sp

ot

Fo

rm

ing

Un

it (

SF

U)

IFN ELISpot Assay

- 4 new epitopes under identification;- Restricting BoLA class I molecule need to be

identified;- Minimal epitope sequence will be confirmed

by ELISpot, cytotoxicity assay as well as by:

A) BoLA class I monomer binding assay

B) peptide-BoLA class I tetramer staining of positive cells

Tetramer+

CD

8+

The figure illustrates the different life cycle stages of the parasite as it cycles through the mammalian and tick host.

Induction of a strong CTL response

negative positive

Looking for the minimal epitope:

A20

Licensed for use under the Creative Commons Attribution 4.0 International Licence (May 2016)