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Immune function in healthy and African swine fever virus infected pigs - SE1509

Description

The current alarming increase in the frequency and severity of outbreaks of African Swine Fever virus in Africa, and continued incidence of sporadic cases throughout Western europe, coupled with a lack of effective vaccine, means that ASFV remains a major threat to the UK pig industry. This project aims to provide the knowledge of the porcine immune system necessary for the rational design of effective vaccines against ASFV. The work will also be applicable to the design of vaccines against other important pathogens representing threats to the British pig industry.

This work extends the previous project (SE1506) which characterized subpopulations of porcine lymphocytes, and demonstrated that the protective cellular immune response to ASFV was dependent on cytotoxic T-cells. This proposal builds on this information to identify the proteins encoded by the virus that are recognized by the immune system and assess whether these can be used as subunit vaccines. We will also further define the subpopulations of porcine T-cells that respond to ASFV during infection and vaccination. Ultimately we will use T-cell reponses to monitor the development of vaccines able to protect against disease.

This work will advance our understanding of mechanisms of cellular immunity in the pig, particularly in relation to viral infection. This will not only improve animal welfare by reducing the threat of widspread mortality in the commercial pig industry in the UK, but also maintain within the UK an internationally respected scientific program that can act on behalf of, and respond to, the requirements of MAFF.
Objective

1). Identification of ASFV encoded proteins recognized by porcine T-cells. Genes encoded by ASFV will be systematically expressed in fibroblasts expressing specific porcine MHC class 1 molecules. Initially, the work will focus on ASFV proteins which lack membrane targetting sequences since these are likely to be localized in the cytosol of infected cells, the site of antigen processing for presentation to CTLs. The search will be further resitricted by testing one member of each of the 5 multigene families encoded by ASFV. Taken together this will cut the number of candidate proteins to be tested by one third but still involve some 100 proteins. At present we have some 20 genes in expression vectors. It is here that our selection of proteins within the remaining genome will be guided by identification of MHC peptide anchor motifs (see objective 2). We have already identified two ASFV proteins recognized by CTLs isolated from infected animals. We will carry out deletion analysis to localize peptide determinants within these proteins.

2. Identification of MHC peptide anchor motifs . We will use DNA transfection to generate a source of cells expressing single porcine class one genes. The class 1 protein will be isolated by immunoaffinity chromatography from bulk tissue culture and the associated peptides will be analysed by HPLC followed by N-terminal sequencing. Anchor residues will be identified as amino acids conserved at particular positions within the mixed population of eluted peptides. The validity of this approach will be tested by assessing the ability of synthetic peptides containing anchor residues to bind porcine MHC class 1.

3). Phenotypic characterization of CD8 positive T-cell populations. In these experiments our unique panel of antibodies specific for T lymphocyte cell surface markers generated under the pevious MAFF project will be used to immunopurify 4 sub populations of CD8+ cells from inbred strains of NIH mini pig. These are CD4 /CD8 double positive cells expressing high or low levels of CD8, and CD4 negative cells expressing high or low levels of CD8. These will be added to irradiated virus infected fibroblasts expressing matched MHC haplotype to test for MHC restricted proliferation. These populations will be further analysed by three colour flow cytometry to detect the expression of proteins, eg: perforin or fas ligand, necessary to kill infected cells. They will also be analysed for binding of MHC tetramers (generated in 4 below), to determine the number of cells within each population that are specific for ASFV.

4) Generation of ASFV specific MHC class 1 tetramers. MHC class 1 tetramer methodology has been adapted for several human and mouse class 1 MHC proteins (1-4). Given the predicted overall structural similarity between porcine and human class 1 MHC proteins (4,11), there are no obvious reasons why this technology cannot be adapted to the porcine system. We have obtained cDNAs encoding the PD1 and PD14 MHC class 1 genes of the aa, cc and dd mini pig haplotypes (11), and these will be used to generate tetramer probes loaded with peptides shown above to be recognized by CTLs isolated from infected animals. The peptide/tetramer complexes will be used in flow cytometry as described in section 3 above.
Project Documents
• Final Report : Immune function in healthy and African swine fever virus infected pigs   (52k)
Time-Scale and Cost
From: 2000

To: 2003

Cost: £536,697
Contractor / Funded Organisations
Institute for Animal Health (BBSRC)
Keywords
African Swine Fever              
Animal Diseases              
Animal Health              
Biotechnology              
Plants and Animals              
Vaccines              
Fields of Study
Animal Health