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SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 14

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code SE1511

2. Project title

Immune function in health and African swine fever virus infected pigs

3. Contractororganisation(s)

Institute for Animal Health                         

54. Total Defra project costs £ 587,293(agreed fixed price)

5. Project: start date................ 01 July 2003

end date................. 30 June 2006

SID 5 (Rev. 3/06) Page 2 of 14

6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.African swine fever (ASF) is a highly contagious fatal acute haemorrhagic disease of pigs. As the name suggests, ASF was first described in Kenya and during the outbreak of between 1909 and 1915, 98.9 % of pigs died as a result of this disease. ASF virus (ASFV) is endemic in Africa particularly in the east and south, but is not limited to the African continent. The virus was introduced to the Iberian peninsula in the 1960’s, to South America and Caribbean during 1970’s and between 1984 and 1997 there have been some 5,000 outbreaks on European farms including those in Italy, Belgium and France. More recently, the virus re-emerged in Sardinia in 2004, where it caused nearly 250 outbreaks. Coupled with a lack of vaccine, ASF remains a major threat to UK pig industry. Thus this project aims to provide the knowledge of the porcine immune system necessary for the rational design of effective vaccine against ASF. In order to achieve this aim, it is necessary to (A) identify which components of the immune response are important in protecting pigs from ASF and (B) identify which ASFV antigens are involved in the induction of protective immunity. In the previous project (SE1506) we demonstrated the importance of a subset of white blood cells that express the cell surface marker CD8 (which is usually associated with the function of “killing virus-infected cells” ) in protection against ASFV (Oura et al., 2005). In contrast to humans or mice, pigs have very complicated white blood cell subsets; the first objective of this project was, therefore, to characterise the phenotype and function of CD8+ lymphocytes sub-populations. We employed antibodies labelled with different fluorescent dyes to identify cell surface and intracellular molecules in order to classify porcine CD8+ lymphocytes. We identified at least four different CD8+ lymphocyte subsets that potentially have “killer” function as demonstrated by their expression of a protein called “perforin”, which lyses cells. Some subsets are unique to pigs and the findings are published in a scientific journal (Denyer et al., 2006). Two of the subsets have a specific killer function to ASFV-infected cells (thus identified as cytotoxic T-cell subsets) by functional “killing” assays. One is a typical cytotoxic T-cell phenotype (CD4-

CD8+ T-cell) and the other has the CD4hiCD8+ T-cell phenotype, which is unique to pigs. When using non-pathogenic, tick isolated ASFV strain OURT88/3 as an ASF vaccine model, some pigs were completely protected against virulent ASFV challenge and some pigs developed clinical symptoms (Oura et al., 2005). Interestingly, CD4hiCD8+ memory T cells were induced in pigs that were completely protected and these cells produced an immuno-modulating soluble factor called interferon gamma (IFN-). In contrast, CD4-

CD8+ memory T-cells were induced in pigs that were not protected and these animals had few CD4hiCD8+

T-cells. These results suggest that an effective ASF vaccine should induce CD4hiCD8+ memory T cells. Using NK (natural killer) cell specific markers we were able to define porcine NK cells in detail. We also identified porcine NKT cells, which possess both T-cell and NK cell markers, for the first time. Thus we are now in a position to dissect the role of these different lymphocyte subsets in health and disease, not just limited to ASF. Since an effective vaccine should contain those ASFV proteins recognised by CD4hiCD8+ memory T

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cells, it is essential to develop simple screening method to identify which of the 150 ASFV proteins are recognised by such cytotoxic T-cells. Since standard “killing” assays not only require the use of radio-isotopes but are also able to screen only a small number of proteins at time, we developed a method based upon the detection of IFN- which is produced by cytotoxic T-cells when they interact with specific ASFV antigens. In this method, up to 40 ASFV proteins can be screened in a single assay. This assay detected ASFV-specific cytotoxic T-cells very effectively, however none of the ASFV proteins screened so far to date were recognised by cytotoxic T-cells.An alternative strategy to identify ASFV proteins recognised by cytotoxic T cells was also proposed in original project. As viral antigens are recognised by cytotoxic T-cells as a complex of major histocompatibility complex class I (MHC class I) molecules and antigenic peptides, peptides that bind to MHC class I have unique motifs. Thus, identification of pig MHC class I peptide anchor motifs will enable the screening of ASFV proteins for the presence of these motifs and ASFV proteins identified in this way could be analysed for their ability to be recognised by cytotoxic T-cells. Identification of ASFV peptides recognised by cytotoxic T cells will allow the generation of recombinant pig MHC class 1 protein loaded with such ASFV peptide and labeled with a fluorescent dye (so called MHC class I tetramers), Such tetramers can be used to identify ASFV-specific cytotoxic T-cells since the MHC class I tetramer binds to the ASFV specific T-cell receptor on cytotoxic T-cells. To achieve this objective, we used three different inbred pig lines, cc, dd and bb haplotypes. We were able to clone and express all of the six MHC class I genes from these inbred pig lines and a common 2 microglobulin gene, which forms part of the MHC class I molecule, which would be refolded with antigenic peptides. However as a result of the IAH restructuring and loss of MHC tetramer expertise from the Institute, an amendment of this part of the objectives was proposed to focus on ELISPOT-based assays as described above. Nevertheless, the reagents developed from this part of the project are available for future use not only for ASFV but also other pig diseases such as Classical swine fever and Aujeszky’s disease.As this project identified potential immunological markers that correlated with protection (CD4hiCD8+ T-cell and IFN-production) against ASFV challenge, these markers will be used in the future to screen potential ASF vaccine candidates in a project funded by the Wellcome Trust. The role of different lymphocyte subsets, such as NK and NKT cell subsets, in ASFV infection will be analysed further in the next DEFRA project SE1512.

C.A.L. Oura, M.S. Denyer, H.-H. Takamatsu & R.M.E. Parkhouse (2005) The in vitro depletion of CD8+ T-lymphocytes abrogates protective immunity to African swine fever virus. Journal of General Virology, 86:2445-2450.

M.S. Denyer, T.E. Wileman, C.M.A. Stirling & H.-H. Takamatsu (2006) Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of Natural Killer, Cytotoxic T, Natural Killer T and MHC un-restricted cytotoxic T-cells. Veterinary Immunology and Immunopathology,110: 279-292.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

The rational design of an effective vaccine against ASF will be greatly facilitated by knowledge of (1) which components of the immune response are important in protection against ASF and (2) which ASFV antigens are involved in the induction of protective immune responses to ASF. In the previous project (SE1506) we demonstrated the importance of CD8+ lymphocytes in mediating protection against ASF infection in ASFV-immune pigs by depleting CD8+ lymphocytes in vivo. However as pig possess a variety of CD8+ lymphocyte

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subsets, the CD8+ lymphocyte subset(s) required for protection was not known. Thus one of the objectives of this project was to characterise pig CD8+ lymphocyte subsets.

1. Phenotypic characterisation of CD8 positive T-cell populations (Objective 3, milestones 3/01, 3/03, 3/04 and amended milestones 4/02, 4/03).

In order to characterise the phenotype and function of CD8+ lymphocytes sub-populations, different populations of CD8+ lymphocytes were examined for their expression of cytotoxic molecules, ability to kill virus-infected cells and their ability to produce IFN-. This part of the project was facilitated by the development of ELISPOT technology and the development of tools to identify porcine NK cells.

(a) Analysis of CD8+ lymphocytes in pigs. Using four colour FACS analysis and monoclonal antibodies (mAbs) to surface markers CD2, CD3, CD4, T-cell co-receptors CD5 and CD6, CD8 and CD8 (CD8 forms either CD8 heterodimer or CD8 homodimer), CD11b, CD16 and TCR, we investigated cytoplasmic expression of the cytotoxic molecule “perforin” in different lymphocyte subsets. The results are summarised in Fig.1.We identified two anti-human perforin mAbs that cross-reacted with porcine perforin and one of these detected a 60kDa molecule by Western blotting, which corresponds to the expected molecular mass of porcine perforin. Multicolour FACS analysis demonstrated that all perforin positive lymphocytes (which are therefore capable of lysing cells) co-expressed CD2 and CD8, but only a few of them expressed CD8. The majority of perforin positive lymphocytes were small dense lymphocytes (SDL) and up to 90 % were CD3 negative and therefore not T-cells. However, the number of perforin positive CD3+T-cells increased with the age of animals. The remaining perforin positive cells were large and granular and contained more CD3+CD5+CD6+ T-cells (≈ 40 %), of which a substantial proportion co-expressed CD4. The perforin positive, CD3 negative subset was phenotypically homogeneous and defined as CD5-CD6-CD8-. This subpopulation also expressed the NK markers CD16 and CD11b. When purified, this population had NK activity and expressed mRNA for the NK receptor NKG2D and adaptor proteins DAP10 and DAP12 (Fig 2). Thus, this subset was identified as NK cell. Unlike humans and mice, pig NK cells are not large granular lymphocyte (LGL) but SDL.

Perforin positive T-cells could be divided into at least four subsets (Fig.1):

(i) The first subset has the typical cytotoxic T lymphocyte (CTL) phenotype of CD4-CD5+CD6+CD11b-CD16- and was capable of lysing ASFV-infected cells in an MHC class I-restricted manner, but not all cells expressed CD8.

(ii) The second subset is CD4+CD5+CD6+CD8+CD16-CD11b- and also has ASFV-specific cytotoxic activity specific for ASFV-infected syngeneic target cells. This cytotoxic T-cell subset, which co-expresses CD4 and CD8 is phenotypically unique to pigs.

(iii)The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset also expressed NK markers CD11b and CD16, and possessed MHC-unrestricted cytotoxicity and LAK activity. As they co-expressed CD11b and CD16, which are usually observed on NK cells, this raises the possibility that this subset might be represent porcine NKT cells. Preliminary studies demonstrated that CD6- cells were selectively stimulated with -galactosylceramide. This compound specifically stimulates CD1d-restricted NKT cells in humans and mice. Furthermore, this subset expressed the NK adaptor proteins DAP10 and 12 suggesting that some of the T-cells in this subset are likely to be NKT-cells.

(iv)Two more very minor subsets were also identified. One subset expressed both T-cell co-receptors and NK markers (CD5+CD6+CD11b+CD16+), and the other subset lacked both T-cell co-receptor and NK markers (CD5-CD6-CD11b-CD16-).

(b) Analysis of CD8+ lymphocyte responses in ASFV-infected pigs. In order to determine which of the CD8 subsets are involved in ASF-specific immune responses, pigs previously infected with a non-haemadsorbing non-pathogenic isolate of ASFV (OURT88/3) were challenged with a related virulent isolate of ASFV (OURT88/1). When peripheral blood mononuclear cells (PBMC) from OURT88/3 immunised pigs were stimulated with ASFV in vitro, two subsets of CTL were detected. One was CD8 single positive CTL, which all expressed CD8, and the other was CD4+ CD8+ CTL, which expressed very high levels of CD4 (CD4hi). The proportion of CD4hiCD8+ CTL in peripheral blood was dependent upon the immune status of the pig. Thus, OUR T88/3-immunised pigs that did not develop clinical signs of disease or viraemia following challenge with virulent OUR T88/1 virus had a high proportion of CD4hiCD8+ CTL. In contrast, immunised pigs that developed clinical symptoms and viraemia following challenge with virulent virus, had a low proportion of CD4hiCD8+ CTL and a high proportion of CD8 single positive CTL in the peripheral blood. Following in vitro stimulation of CD4hiCD8+ lymphocytes with ASFV, these cells produced large amounts of IFN-

Figure 3 shows changes in circulating CD8+ lymphocytes following infection with ASFV OUR T88/3 and challenge with ASFV OUR T88/1. Three pigs (top right side) that developed clinical symptoms following challenge with virulent virus showed a transient increase in CD8+ lymphocytes following challenge with virulent virus, whereas three pigs (top left side) without clinical symptoms had no change in circulating CD8+ lymphocytes. Stimulation of PBMC from completely protected pigs with ASFV in vitro, induced a high proportion of CD4hi CTL

SID 5 (Rev. 3/06) Page 5 of 14

(bottom left) (shown as perforin positive CD4hi population, within red square). In contrast, in vitro stimulation of PBMC from diseased pigs induced mainly CD4- perforin+ CTL (bottom right).

A comparison of the antigen-specific re-call lymphocyte proliferation responses from protected and diseased pigs demonstrated that the majority of perforin positive, proliferating cells, which were identified as Ki67 positive cells, in completely protected pigs were CD4hi CD8+, whereas the majority of perforin positive, proliferating lymphocytes from diseased pigs were CD8 single positive (Fig. 4). The proportion of activated NK cells also appeared to be slightly higher in completely protected pigs than in diseased pigs. Taken together these results indicate a correlation between the induction of ASFV-specific CD4hi CD8+ T cells and protection against infection.

IFN-production by ASFV-specific pig lymphocytes was examined initially by FACS. FACS analysis demonstrated that following stimulation of lymphocytes from ASFV-immunised pigs with ASFV, in vitro, the majority (≈ 80 %) of IFN- producing cells expressed both CD4 and CD8. In addition, there was a small population of IFN- producing CD8 single positive and CD4 single positive lymphocytes. In order to analyse ASFV-specific IFN-producing lymphocytes further, we developed a porcine IFN- ELISPOT assay. Initial studies established the optimum concentrations and conditions for the assay. The IFN ELISPOT assay (Fig. 5) is simpler and more sensitive than FACS and can be used to analyse the development of IFN- producing, ASFV-specific memory T-cells after ASFV infection and /or vaccination. As shown in Fig. 6, non-pathogenic ASFV OURT88/3 infection induced IFN- producing memory T-cells 4 to 5 days post infection. In contrast, pigs infected with a virulent virus failed to generate such T-cells. We examined the phenotype of IFN- producing cells by depleting specific subsets from total lymphocyte and confirmed the observation obtained from FACS. Thus, IFN-producing cells express CD3+, CD4+ and CD8+ and IFN production was not affected by depleting T-cells. These observations indicate a correlation between the induction of IFN-producing cells and recovery from primary ASFV infection.

2. Identification of ASFV proteins recognised by porcine T-cells (Objective 1, milestones 1/01, 1/02, 3/04).

Studies to identify ASFV proteins recognised by porcine CTL is a continuation from the previous DEFRA project, but in this project more emphasis was placed on the development of a high through put assay for analysis of CTL activity. This objective and mile stones were achieved by adapting the ELISPOT assay described above. In initial studies, MHC class I-defined, ASFV-infected target cells (dd haplotype derived kidney cell line) stimulated MHC-matched ASFV immune pig (dd haplotype) lymphocytes to produce IFN- as detected by ELISPOT. To confirm that the IFN- producing cells were CTL and recognised ASFV-infected cells through the interaction of the T-cell receptor with the MHC class I/ASFV peptide complex, we performed a series of experiments. First we removed professional antigen-presenting cells (APC) which might take up ASFV-infected cells or ASFV proteins produced by infected cells and present antigen via MHC class II. We demonstrated that in contrast to ASFV-infected cells, free ASFV antigen failed to stimulate T-cells to produce IFN-. Secondly, using purified T-cells (i.e. in the absence of APC and NK cells), ASFV failed to stimulate T-cells whereas ASFV-infected cells activated IFN--producing T cells. Finally we used fixed ASFV-infected cells as antigen (therefore there was no free ASFV that could be presented via MHC class II) and the results demonstrate that direct interaction of ASFV-infected cells is required to induce IFN- production by immune T cells. Next we examined the ability of individual ASFV genes expressed in MHC class I dd cells to stimulate IFN-producing T cells. First we examined expression of ASFV genes in dd cells following transfection with individual ASFV protein coding genes. The results are summarised in Table 2. Not all of the genes tested expressed ASFV protein in pig dd cells (some of genes were able to express in Vero cells but not in pig dd cells). The genes showing a relatively high transfection rate (30-40%) were tested in the IFN ELISPOT assay for their ability to activate T cells. None of the genes tested so far induced IFN- from ASFV-immune T cells. However, as we tested only a few genes from at least 150 ASFV proteins and moreover some genes do not express effectively, further testing and improvement of gene expression are needed. Nevertheless, these studies demonstrated that the IFN ELISPOT assay can be used to screen up to 40 different gene transfectants in a single plate. Therefore, this system is far superior to the conventional CTL assay to screen individual antigens.

3. Identification of MHC peptide anchor motifs (Objective 2) and Generation of ASFV specific MHC class I tetramers (objective 4). In previous studies, MHC class I was purified by immunoaffinity chromatography from the spleens of inbred Babraham (bb) pigs. Peptides associated with the MHC class I molecules were analysed by HPLC and N-terminal sequencing. These studies identified a partial peptide anchor residue. In the present project, we proposed to express pig MHC class I molecules and identify MHC peptide binding motifs by using random peptides which have high affinity to the recombinant MHC molecules peptides. To this end, cDNA encoding porcine MHC class 1 genes from three different haplotypes of inbred pigs, dd, cc and Babraham pigs (therefore, PD1, PD14 from dd pigs, PC1 and PC14 from cc pigs and PB1 and PB14 from Babraham pigs) and porcine 2 microglobulin were engineered to generate vectors that express these proteins in bacteria. MHC class I genes carrying biotinylation motifs and His6 tags for affinity purification and labelling were prepared. Thus far, milligram quantities of MHC proteins (except PD1, PC1 and PC14 which need to be confirmed) and 2 microglobulin can be produced from 1 litre cultures. Vectors designed to express porcine MHC proteins in mammalian cells have also been produced. A panel of overlapping peptides covering a 9 mer motif recognised by CSFV immune dd pig CTL have been designed for use in peptide binding and refolding studies using recombinant PD1 and PD14. A Summary of the reagents generated from this part of the project is shown in Table

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1. Due to the restructuring of the IAH and loss of tetramer expertise, an amendment to this objective was proposed to give a greater emphasis to ELISPOT based assays. Nevertheless, the reagents developed from this part of the project are available for future use not only for ASFV but also other pig diseases such as Classical swine fever and Aujeszky’s disease.

Porcine CD8+ lymphocyte subsets (so far)

CD3- CD172a+ MHC I I hi DC subset?CD8-CD8+

TCR+

TCR-CD4-CD5+CD6+ naïve T

CD4+CD5+CD6+ memory Th

CD3+

Perforin -ve

Perforin +ve

CD3- CD4-CD5-CD6-CD8-CD11b+CD16+

CD3+

CD6+

CD6-

CD4-

CD4+CD5+CD6+CD11b-CD16-

CD5+CD8+CD11b-CD16-

CD5-CD8-

CD8 positive

CD11b-CD16-

CD11b+CD16+

CD11b+CD16+

CD11b-CD16-

T-cell subsets

Fig.1

Fig. 2.

Cloning of porcine NK receptors and adaptors:

NKG2D

DAP10

DAP12

Actin

1 2 3 4 5

• Expressed in bacteria and baculovirus either with His tag or as human Ig Fc fusion protein.• As DAP10 is small, a synthetic peptide was made for immunisation.

NK cellCD6+ T cells

Total T cells

Total lymphocytes

-+KIR--KIR3D--NKp46--p58++DAP12++DAP10++NKG2D

ExpressionPCRMolecule

T-cell

SID 5 (Rev. 3/06) Page 7 of 14

Fig. 3.

Fig.4.

0

20

40

60

80

0 5 14 28 41 56 70 84 98 112

96983

0

20

40

60

80

0 3 5 7 14 21 28 35 41 49 56 63 70 77 84 91 94

38

41

42

Challenge Challenge

Completely protected pigs Diseased pigs

Days post OURT88/3 infection Days post OURT88/3 infection

OURT88/3 OURT88/3

% C

D8

+ly

mph

ocyt

es

% C

D8

+ly

mph

ocyt

es

Perf

orin

CD4

Perf

orin

CD4

0 20 40 60 80 100

Perforin positive

42.1 %

Ki67+ cells*

CD8+

CD4+ CD4 hi B

NK

Protected pig

Without antigen

Re-call antigen stimulated

0 20 40 60 80 100

Perforin positive

49.8 %

Ki67+ cells*

CD8+

CD4+

CD4 hi

B

NK

Diseased pig

Without antigen

Re-call antigen stimulated

44% 56 % 86 % 14 %(5%) (1 %)

0.6 %0.8 %

: CTL phenotype of CD3+CD4- CD6+ CD8+

ASFV challenge protected pigs induce CD4hi perforin+ T cell subset

*Proliferating lymphocytes were identified as Ki67 positive

SID 5 (Rev. 3/06) Page 8 of 14

IFN-ELISPOT assay

Each spot corresponding the lymphocyte which produced IFN-by stimulation with specific antigen (ASFV).

Fig.5

-10

0

10

20

30

40

50

60

70

80

90

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Days post infection

ELIS

POT VF10

VF11

VF12

VF13

0

10

20

30

40

50

60

70

80

90

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Days post infection

ELIS

POT

VF18

VF19

VF20

VF21

Fig. 6. Early induction of IFN-producing memory T-cells by OURT88/3 infection, but not by virulent Malawi isolate of ASFV infection.

OURT88/3 infection Malawi infection

SID 5 (Rev. 3/06) Page 9 of 14

YESN/AYESYESYES2Mcommon

YES?YESYESYESYESPC14cc

YES?YESYESYESYESPC1cc

YESYESYESYESYESPB14bb

YESYESYESYESYESPB1bb

YESYESYESYESYESPD14dd

YES?YESYESYESYESPD1dd

ExpressionBiotin tagInserted into pet22b

Into shuttle vector

PCR products

MHC class I genes

Haplotype

Table 1. porcine MHC class 1 tetramer reagents generated

Table 2. ASFV genes tested for expression in MAX (pig dd) cells and ELISPOT

ASFV genes Expression in Vero cells Expression in MAX cells ELISPOT

1. B117L + + ND

2. B169L + - ND

3. C257L ND + ND

4. D117L + - ND

5. E120R + + -

6. E146L - - ND

7. E183l + + -

8. E248L + + ND

9. EP152R ND + ND

10. H223R - - ND

11. I226R - - ND

12. I329l + +/- ND

13. I77L + + ND

14. B318L + + ND

15. B438L + + ND

16. D339L + - ND

17. EP402R ND + ND

18. EP153R ND + ND

19. F334L - - ND

20. I1243L + + ND

21. I267L + - ND

22. K196R ND - ND

23. MGF110 ND + -

24. NP419L ND + ND

25. XP124L ND + -

26. XP178L ND + -

27. S273R + + ND

In summary, this project identified porcine CD8 subsets and the main findings from this objective were:1. Pigs posses four major CD8 cytotoxic subsets and two minor subsets.2. Unlike human and mice, pig NK cells are not LGL but SDL.3. Pigs have two distinct CTL subsets, one of which is unique to pigs.4. Unlike human and mice, pig T-cells do not express perforin.5. Pig NKT cells were identified for the first time.6. Induction of CD4hiCD8+ CTL was shown to correlate with complete protection against ASFV challenge.

Reagents and techniques developed were;1. Primers and probes for porcine NKG2D, DAP10 and DAP12, which will facilitate analysis of pig NK cells 2. Established ASFV-specific IFN- ELISPOT.3. Developed an IFN-ELISPOT screening assay to analyse recognition of ASFV proteins by pig CTL 4. Generated pig MHC class I tetramer reagents.

A number of findings from this project are extremely important for ASF vaccine development, for example identification of CD4hi phenotype of CTL and ability to analyse IFN- production from CTL are immediately applicable to screen potential ASF vaccine candidates in a project funded by the Wellcome Trust. The unique

SID 5 (Rev. 3/06) Page 10 of 14

phenotype of porcine cytotoxic lymphocyte subsets requires further analysis, especially functionally, and this forms part of the next DEFRA project. A number of publications were made from this project and the main publications are listed in section 9 with hyperlink. The publications generated relating to this project and abstracts published in conferences/ scientific meetings are listed below.

Heath, C. M., M. Windsor, T. Wileman (2003). Membrane association facilitates the correct processing of pp220 during production of the major matrix proteins of African swine fever virus. Journal of Virology, 77: 1682-90.

Netherton C, Rouiller I, Wileman T (2004). The subcellular distribution of multigene family 110 proteins of African swine fever virus is determined by differences in C-terminal KDEL endoplasmic reticulum retention motifs. Journal of Virology 78(7):3710-21.

Jouvenet N, Monaghan P, Way M, Wileman T (2004). Transport of African swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin. Journal of Virology 78(15):7990-8001.

Netherton CL, Parsley JC, Wileman T (2004). African swine fever virus inhibits induction of the stress-induced proapoptotic transcription factor CHOP/GADD153. Journal of Virology 78(19):10825-8.

Stefanovic S, Windsor M, Nagata KI, Inagaki M, Wileman T (2005). Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II. Journal of Virology . 79:11766-75.

Jouvenet N, Wileman T(2005). African swine fever virus infection disrupts centrosome assembly and function. Journal of General Virology 86:589-94.

M.S.Denyer, C.M.A. Stirling, T.E. Wileman & H.-H. Takamatsu (2003) Porcine lymphocyte subset(s) responding to recall antigens. Brithish Society for Immunology annual meeting, Harrogate. Immunology, 110, Supplement 1: pp109.

C.M.A. Stirling, M.S. Denyer, T.E. Wileman & H.-H. Takamatsu (2003) African swine fever virus (ASFV) infected macrophage cannot polarise the cytokine/chemokine production profile in vivo. British Society for Immunology annual meeting, Harrogate, Immunology, 110. Supplement 1: pp110.

M.S. Denyer, C.Stirling, T.E.Wileman & H.-H.Takamatsu (2004) Phenotypic and functional characterisation of porcine lymphocyte responding to re-call antigen. Seventh International Veterinary Immunology Symposium, Quebec, Canada. Abstract book pp302.

C.Stirling, M.S.DEnyer, T.E.Wileman & H.-H.Takamatsu (2004) Anti-inflammatory cytokine polarisation dose not occurs in African swine fever virus infected pigs. Seventh International Veterinary Immunology Symposium, Quebec, Canada. Abstract book pp140

SID 5 (Rev. 3/06) Page 11 of 14

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

SID 5 (Rev. 3/06) Page 12 of 14

Vet Immunol Immunopathol. 2006 Apr 15;110(3-4):279-92. Epub 2005 Dec 1.   Links

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells.

Denyer MS , Wileman TE , Stirling CM , Zuber B , Takamatsu HH .

Immunology. 2005 Jun;115(2):189-96.   Links

Monoclonal antibodies that identify the CD3 molecules expressed specifically at the surface of porcine gammadelta-T cells.

Yang H , Parkhouse RM , Wileman T .

Vet Immunol Immunopathol. 2006 Jul 15;112(1-2):49-61. Epub 2006 May 22.   Links

Porcine gammadelta T cells: possible roles on the innate and adaptive immune responses following virus infection.

Takamatsu HH , Denyer MS , Stirling C , Cox S , Aggarwal N , Dash P , Wileman TE , Barnett PV .

Immunogenetics. 2006 Jun;58(5-6):481-6. Epub 2006 Apr 29.   Links

Identification of a single killer immunoglobulin-like receptor (KIR) gene in the porcine leukocyte receptor complex on chromosome 6q.

Sambrook JG , Sehra H , Coggill P , Humphray S , Palmer S , Sims S , Takamatsu HH , Wileman T , Archibald AL , Beck S .

J Gen Virol. 2005 Sep;86(Pt 9):2445-50.   Links

In vivo depletion of CD8+ T lymphocytes abrogates protective immunity to African swine fever virus.

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