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 of
approximately 1 million cattle every year. Immune animals to the
parasite develop a lifelong immunity based on a cytotoxic T
lymphocyte (CTL) response with a strong immunodominance
restricted by the bovine leukocyte antigen (BoLA) class I
molecules. In our goal of developing a next-generation vaccine
against T. parva, we have undertaken to identify new CTL
inducing antigens that can be included in a recombinant vaccine.
A peptide library of 18-mer peptides overlapping by 12 amino
acids and covering 500 genes of the whole parasite genome was
synthesized; giving approximately 40,000 peptides aliquoted in
pools of 50 peptides. Genes were selected based on the presence
of a signal peptide, abundance, and divergence to the related
parasite T. annulata, as well as by a selection using
immunoinformatic tools predicting, by artificial neural network,
peptides binding with strong affinity to the BoLA class I molecules
present in cattle of Africa. A bank of ten CTL lines from cattle
immunized with the live parasite expressing different BoLA class I
specificities were screened by IFN- ELISpot assay. Among these
cells, four secreted IFN- in the presence of a different peptide
library pool. Peptides from these pools were synthesized and
positive individual 18-mer peptides were identified. Dissection of
the minimal epitope is underway using IFN- ELISpot, cytotoxicity
as well as peptide-BoLA class I flow cytometry assays. These
newly identified antigens will hopefully allow us to develop a
vaccine towards T. parva giving wide coverage in cattle
population 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 via
a “zippering” mechanism
Sporozoite attachment
to host lymphocyte
Mature schizont in
“transformed” lymphocyte
Merogony
Bos indicus
Syncerus caffer
Piroplasms in
red blood cells
Larvae & nymphs
acquire infection
Rhipicephalus appendiculatus
(a three-host tick)
Merozoites released by
host cell rupture
Zygote
Kinete
Gametes
Daughter host-cells
remain infected
Sporogony in
type III acinus
Bos taurus
Proliferation of
schizont-infected cells
Sporozoites released
during tick feeding
Infected
nymphal & adult ticks
Rb
Rb
Rb
Mz
Sz
Sz
Rb
CTL LINES FROM T. PARVAMuguga
IMMUNIZED ANIMALS
Theileria
parvaMuguga
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. Positive
BX64 (B) A10/KN104 N.A. Positive
BF091 A18/A11/A19 Tp1214-224 Negative
BB007 A18/? Tp1214-224 Negative
BF092 A18/A12 Tp1214-224 Positive
BK173 A14 Not reacting to Tp9 Negative
BA219 A18 Tp1214-224 Negative
BH055 A11/A15v Tp5214-224 Negative
BG042 A10/A12 Not reacting to Tp2 Negative
BW012(B) T7/? Tp7206-214 Positive
PARTNERS FUNDING
SUMMARY & PERSPECTIVE
1
2 3
A10
12%
A11
10%
A12
11%
A14/A15
31%A17
4%
A18
9%
A19
11%
A20
6%
A31
6%
Haplotype Frequency in Cattle
N=127
4
4 chromosomes
≈ 4,000 genes
Theileria
parvaMuguga
- 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.[450nm]
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 TLTLQGHRMKVT
F100.2 LTLQGHRMKVTN
F100.3 TLQGHRMKVTNL
F100.4 LQGHRMKVTNLK
F100.5 QGHRMKVTNLKW
F100.6 GHRMKVTNLKWN
F100.7 HRMKVTNLKWNP
F100.8 RMKVTNLKWNPT
F100.9 MKVTNLKWNPTV
F100.10 KVTNLKWNPTVD
F100.11 VTNLKWNPTVDY
F100.12 TNLKWNPTVDYA
F100.13 NLKWNPTVDYAL
MHC CLASS I HAPLOTYPE FREQUENCIES
IN CATTLE
SELECTION OF PEPTIDES
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NEW ANTIGEN IDENTIFIED WITH F100 CTL
6
POSITIVE PEPTIDES IDENTIFIED WITH BF092 CTL
IFN ELISpot Assay
IFN ELISpot Assay
7
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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+
CD8+
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)