The study investigates the immunogenicity and protective efficacy of the Theileria parva CTL antigen tp1, delivered via had5/mva prime-boost vaccination, in cattle. Four groups of cattle were immunized with different formulations of the antigen, resulting in the generation of CTLs measurable by tetramer staining and other assays, with approximately 30% protection observed upon challenge. The results indicate the potential of the prime-boost regimen for inducing immune responses against the tp1 antigen, including the activation of CD8 and CD4 T cells.

1. N. Svitek1, R. Saya1, E. Awino1, S. Gilbert2, J. Poole1, V. Nene1, L. Steinaa1
1 Vaccine Biosciences, International Livestock Research Institute, Kenya; 2 The Jenner Institute, UK
Immunogenicity and protection of the Theileria parva CTL
antigen Tp1, with or without a leader sequence, using
HAd5/MVA prime-boost vaccination
FIG.1 Immunization groups: 5 cattle in each group, primed
with HAd5-Tp1 and boosted with MVA Tp1 with and without
leadersequence:
GROUP 1. Tp1 + tPA* leader sequence (HAd5/MVA)
GROUP 2. Tp1 without leader sequence (HAd5/MVA)
GROUP 3. Tp1 + native leader sequence (HAd5/MVA)
GROUP 4. GFP + tPA* leader sequence (HAd5/MVA)
0 14 28 42 56 63 70 84
HAd5
prime
MVA
boost
91
Challenge with
T. parva
FIG 2. Regimen and sample points:
Day
FIG 3. CTL are Tp1 specific (tetramer staining): Example of ex
vivo tetramer staining of 2 animals, 2 weeks post MVA boost.
Tp1-Tetramer
CD8
T
p
2
p
o
o
l
T
p
1
p
o
o
l
O
v
a
lb
u
m
in
T
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1
p
ro
te
in
0
5 0
1 0 0
1 5 0
C D 4
T
p
2
p
o
o
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T
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p
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0
5 0
1 0 0
1 5 0
C D 4
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p
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a
lb
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in
T
p
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p
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te
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0
2 0 0
4 0 0
6 0 0
C D 4
Group 1 Group 2 Group 3Foldincrease
0 2 4 6 8 10 12 14 16 18 20 22 24
0
50
100
D ays elapsed
Percentsurvival
C ontrol
H Ad5/M V A-Tp1
O ther experim ental vaccine group
H A d5/M V A -G FP
Animal 1 Animal 5
Tp1+
Tp1+
Perf+ Perf+
%Specifickilling
Tp1+
Perf+ Perf+
Tp1+Animal 2 Animal 4
17.3%70.3%4.15% 17.4%
2 4 8 1 6 3 2 6 4
0
1 0
2 0
3 0
4 0
E ffector/Target R atio
A nim al 5 TpM
Irr T p M 1
Irr T p M 2
P B M C
P B M C + T p1
0 .5 1 2 4 8 1 6
0
1 0
2 0
3 0
4 0
E ffector/Ta rg e t R a tio
A nim al 4 TpM
Irr T p M 1
Irr T p M 2
P B M C
P B M C + T p1
1 2 4 8 16 32
0
20
40
60
E ffector/Target R atio
A nim al 2 TpM
Irr T p M 1
Irr T p M 2
P B M C
P B M C + T p1
1 2 4 8 16 32
0
10
20
30
40
E ffector/Target R atio
A nim al 1 TpM
Irr T p M 1
Irr T p M 2
P B M C
P B M C + T p1
Animal 1 Animal 5Animal 2 Animal 4
G
ro
u
p
1
G
ro
u
p
2
G
ro
u
p
3
0
1 0 ,0 0 0
2 0 ,0 0 0
3 0 ,0 0 0
Spotformingunitsper
millionCD8+cells
0 1 4 2 1 2 8 5 6 6 3 8 4
0
1 0 ,0 0 0
2 0 ,0 0 0
C on tro l e pitop e-G rou p 1
T p1 e pito pe-G roup 1
C on tro l e pitop e-G rou p 2
T p1 e pito pe-G roup 2
C on tro l e pitop e-G rou p 3
T p1 e pito pe-G roup 3
FIG 4. CD8 cells respond to the Tp1 epitope (IFN-γ ELISpot ): SFU (per million
CD8 cells) 7 days after MVA boost. First diagram shows the group average, the
second shows the individual cattle.
Summary: 4 groups of 5 BoLA-typed animals were immunized with the T. parva Tp1
antigen with or without leader sequence in the HAd5 viral vector and boosted with the
same antigens in the MVA vector. Most animals generated CTL to the known epitope
measured using tetramer staining, ELISpot and Cr-51-release assay. The CTL expressed
perforin and lysed peptide pulsed PBMC. CD4 cells were shown to proliferate to the
antigen. Challenge of the animals resulted in about 30% protection.
FIG 1. Immunization groups: 5 cattle in each group, primed
with HAd5-Tp1 and boosted with MVA Tp1 with and without
leader sequence:
FIG 5. CD4 cells proliferate to the Tp1 antigen. 2x105 CD4 cells were cultured with
Tp1 full-length antigen, Tp1 pool (overlapping peptides), ovalbumin (control) and
Tp2 pool (control peptides), 3H-thymidine was added, cells were harvested.
FIG 6. Tp1 specific CTL are cytotoxic and they express perforin: Upper panel: PBMC
were restimulated 3 times using infected autologous cells, CD8 cells were purified
and cytotoxicity were measured by pulsing autologous PBMC with the peptide or
infected cell line (Cr-51 release). Lower panel: CD8 cells were costained with Tp1
tetramer and a perforin mAb.
FIG 7. Protection by prime-boost regimen with Tp1 in HAd5/MVA vectors. Groups of
cattle (Fig. 2) were challenged with a lethal dose of T. parva sporozoites. Kaplan-Meier
survival plot is shown.
Licensed for use under the Creative Commons Attribution 4.0 International Licence (May 2016)