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Project ID: QLK2-CT-2001-01374
Finanziato nell'ambito di: FP5-LIFE QUALITY
Paese: Germany

Definition of CSFV-specific MHC class II epitopes

The identification of MHC class II epitopes in viral proteins is an essential point for the investigation of the humoral immuneresponse after a viral infection. In addition, the knowledge of defined T-cell epitopes is important for the development of novel viral vaccines. Overlapping 15mer peptides derived from the protein sequence of CSFV strain Glentorf were synthesized. For the generation of CSFV -specific T lymphocytes, three dfd-haplotype pigs were infected with CSFV-strain Glentorf .Plasma and blood samples were collected before infection and weekly post-infection. CSFV -specific T lymphocytes could be detected by lympho-proliferation assay using PBMC from CSFV-infected animals and CSFV strain Glentorf for the in vitro re-stimulation.

The experiments performed in this project have shown that both CD4+CD8- T-helper cells and CD4-CD8+ cytolytic T-cells were involved in the CSFV-specific response in vitro. The CSFV-specific T-cell response could be shown by the lymphoproliferation assays based on 3H-thymidine incorporation. In order to identify CSFV-specific T-cell epitopes, the overlapping 15mer synthetic peptides derived from CSFV strain Glentorf were applied in these proliferation assays. The 3H-thymidine incorporation assays identified 26 peptides distributed throughout the CSFV viral protein. Characterisation of the reactive epitope-specific T -cell fractions by flow cytometry (FCM) analyses using CFSE-staining, together with different combinations of swine- specific monoclonal antibodies showed that mainly CD4+ T-cells were involved.The phenotype of the main responding T -cell population was actually defined as CD4+CD8a+CD8a-CD45RC-MHCII+.

These analyses with CFSE-stained PBMC, together with identification of the surface antigen expression gave dear hints concerning the possible MHC-restriction of the responding T -cell subset. Therefore, these assays might replace blocking experiments employing monoclonal antibodies against CD4, CD8 MHCI and MHCII molecules. For the determination of the frequency of CSFV -specific T lymphocytes in PBMC from CSFV-infected animals, ELISPOT assays were established to detect interferon-a (IFN-alpha) and Interleukin 4 (IL-4) producing T cells. From these ELISPOT assays, the average frequency of IFN-alpha IL-4 secreting CSFV -specific T lymphocytes in PBMC preparations was calculated as between 1xl0-3 and 1xl0-4.

For a fine analysis the identified CSFV -specific T -cell epitopes were further investigated. They were synthesised for an identification of the amino acids involved in the binding to the MHC-II molecules as "Alanine-Scan-peptides". These "alanine-peptides" were further used in 3H-thymidine incorporation assays, CFSE-assays for the characterisation of the responding T-cell subpopulations and in IFN-y and IL-4 ELIPSOTS for the determination of their activation potency. It could be shown, that an exchange of the amino acids in position 1, 5, 6, 10 and 13 was combined with an reduced stimulation capacity in CSFE-assays, a diminished proliferative response of CSFV -specific T lymphocytes and a reduced frequency of IFN-gamma producing cells.

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Eberhard PFAFF, (DEPUTY HEAD OF INSTITUTE)
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