STSM Report
Helen Mokhtar
April-June 2011
COST Report: Identification of T cell epitopes within porcine reproductive and
respiratory syndrome virus-1 (Strain Olot91).
Introduction:
Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive problems
in sows, stillbirths and abortions. It also plays a major role in the porcine respiratory disease
complex, which leads to an enhanced susceptibility to secondary bacterial and viral
infections. An effective universal vaccine is yet to be developed due in part to the genetic
variation of the virus and as such, PRRSV has a significant economic impact worldwide.
There are two main strains of PRRSV; a European subtype (I) and a more virulent North
American subtype (II). The virus has an RNA genome which is subject to a high mutation
rate rapidly producing new variants.
There is limited information about how PRRSV affects the host immune system. It is known
that the target cells are differentiated macrophages, and that the virus is not cleared for a
number of weeks, indicating that the virus manipulates the cells of the immune system. There
is evidence that supports this; PRRSV does not illicit a strong IFN-α response and upon
infection immunosuppressive cytokines are also induced. In addition, data has shown that
PRRSV infection delays both neutralising antibody and T cell mediated responses and it has
been suggested that both of these responses are essential to provide protection from the virus.
While the exact mechanisms underlying protective immunity against PRRSV are not well
understood, vaccine induced IFN-γ secreting T cell responses have been associated with
protection. However little is known about their specificity, the exception being the
identification of epitopes on the surface glycoprotein GP5, which is the best-studied of
PRRSV antigens for its vaccine potential. The lack of knowledge about which other PRRSV
antigens contain the major T cell epitopes makes it difficult to construct effective vaccines
that stimulate both arms of the immune system.
Aim of the STSM:
To identify T cell epitopes within PRRSV (PRRSV-I strain Olot91) using a synthetic peptide
library spanning the entire proteome and a cohort of pigs rendered immune to PRRSV-1
Olot91 by repeated experimental infection. In addition to the identification of antigenic
peptides, the phenotype of responding T cells from immune pigs will be characterised.
Methods:
The synthetic peptide library used in this project comprised of 15mer peptides off-set by four
residues which is considered an optimal length and overlap for identification of both MHC
class-I and –II restricted epitopes. The peptide sequences were designed using the predicted
amino acid sequences of the structural proteins of PRRSV-1 Olot91 strain and the nonstructural proteins of the closely related PRRSV-1 Lelystad strain (non-structural protein
encoding open-reading frame sequences are not available for Olot91). The library consists of
1275 peptides which have additionally been combined into pools representing the 19 proteins
of PRRSV-1.
Peripheral blood mononuclear cells (PBMC) were isolated from the PRRSV-1 Olot91
immune pigs and stimulated with the peptide pools representing each PRRSV protein.
Reactivity was assessed by measuring IFN-γ release by ELISpot assay. Positive pools were
identified and peptides were screened individually again, using IFN-γ ELISpot. IFN-γ
inducing peptides were confirmed by titration. CD8+ and CD4+ cell depletions were then
carried out on PBMC and depleted cell subsets were stimulated with the IFN-γ inducing
peptides to determine the phenotype of the responding cells.
STSM Report
Helen Mokhtar
April-June 2011
Results:
Peptide pools were used to stimulate 1x106 PBMC per well (each individual peptide at a
concentration of 10μg/ml). Figure 1 shows the mean of 3 replicates and the error bars show
SEM. Cells and peptides were incubated at 37oC for 20hrs. Data was analysed using a twoway ANOVA statistical analysis, with Bonferroni post-test, to ascertain which pools
exhibited a significant positive response when compared to media. Positive pools identified
were NSP1b, NSP2, RdRp, GP3, GP4, GP5, and M.
800
Pig 52
Pig 53
Pig 54
SFC/106 PBMC
600
400
200
N
ed
ia
m
M
P4
P5
G
G
E
P3
G
P2
G
as
e
dR
p
SP
11
N
SP
12
N
R
SP
8
el
ic
H
N
SP
7
N
SP
5
SP
6
N
N
SP
3
SP
4
N
N
SP
2
N
N
SP
1
b
0
Peptide pool
Figure 1. Screening of peptide pools representative of PRRSV proteins with PBMC from 3
immune pigs.
Peptides making up GP3, GP4, GP5 and M were screened individually. Peptides making up
NSP1b, NSP2 and RdRp were screened in pools of 10 due to their length. Figure 2 shows an
example screen of individual peptides from the M protein.
300
SFC/10^6 cells
200
100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
0
Peptide ID
Figure 2. Representative data of screening individual peptides from protein pools. The 41
peptides that constituted the M-protein pool were screened individually with PBMC from pig
54.
STSM Report
Helen Mokhtar
April-June 2011
From these screens, 5 putative antigenic peptides identified in NSP1, 9 in NSP2, 7 in RdRp, 4
in GP3, 8 in GP4 8 in GP5 and 14 in M protein. Positive peptides were confirmed by titration
using a 10-fold dilution series starting at 1μg/ml. Figure 3 shows the assessment of
recognition of peptides 3, 4 and 5 from M protein by PBMC from pig 54.
300
SFC/106 cells
Peptides
3
4
5
200
100
0
1
0.1
0.01
0.001
peptide concentration (g/ml)
Figure 3. Representative data of screening individual peptide titrations. Putative antigenic and
control peptides from M-protein pool were screened with PBMC from pig 54.
Finally, PBMCs were depleted of either CD4+ or CD8+ T cells and then stimulated with
0.1μg/ml of each titration confirmed individual peptide or pool of 10 peptides or an irrelevant
peptide to test the phenotype of the responding cells. Figure 4 shows antigenic peptide 4 from
protein M assessed for recognition by CD4+ or CD8+ T cell depleted PBMC from pig 54.
SFC/106 cells
250
M4
Media
Irrelevent peptide
200
150
100
50
0
PBMC
CD4 depleted
CD8 depleted
Figure 4. Representative data of phenotyping of responder T cell populations. Reactivity
against antigenic and control peptides were assessed using intact PBMC, CD4+ and CD8+ T
cell depleted PBMC populations.
STSM Report
Helen Mokhtar
April-June 2011
A summary of the antigenic peptides identified and the phenotype of responder T cells is
shown below:
Protein
Antigenic Peptide ID Phenotype of responder cells
4
CD4
18
CD8
24
CD4
Non-structural protein1(NSP1)
33
CD8
38
CD8
52
CD8
88
CD4
164
Inconclusive
Non-structural protein 2 (NSP2)
175
Inconclusive
94
Inconclusive
Viral Polymerase (RdRp)
54
CD8
55
CD8
14
Inconclusive
Glycoprotein 5 (GP5)
16
CD4
4
CD4
5
Inconclusive
Matrix protein (M)
40
CD8
41
CD8
Conclusions and future work:
3 of the non-structural and 4 of the structural proteins of PRRSV induced a significant IFN-γ
response in PBMCs isolated from immune pigs.
A total of 18 antigenic peptides were identified across the PRRSV proteome and these
peptides stimulated a mixture of CD4+ and CD8+ responder T cells.
DNA from the Olot91 immune pigs used in the study is being obtained so as to determine the
SLA-I/II haplotypes of the pigs. This will allow further analysis of the antigenic regions
identified in this study.
The study is now being reproduced using a large number of pigs infected with a variety of
different strains of PRRSV; including the Lelystad virus.
Confirmation by the host institute of the successful execution of the mission:
The T-cell epitope screen with the PRRSV-1 peptide library on PBMC from PRRSV-1
immune pigs was performed in the BSL4 containment facility of the Institute of Virology and
Immunoprophylaxis (IVI) in Mittelhäusern, Switzerland, from April 25 to June 17 2011,
under the responsibility of Nicolas Ruggli and Artur Summerfield. The experiments were
carried out at the IVI to take advantage of three PRRSV-1 immune specific pathogen-free
blood donor pigs that had been generated by immunization with the cell culture-adapted
Olot91 strain. The pigs had received a total of three immunizations, at nine weeks and 12
months intervals respectively. The third immunization was administered two weeks before
the beginning of the stimulation experiments. The PBMC of all three pigs responded to the
STSM Report
Helen Mokhtar
April-June 2011
stimulation with PRRSV-1 virus with a strong proliferation of IFN-γ producing cells, which
allowed successful execution of the experiments.