This proposal has two major specific aims: 1) To demonstrate the existence HCV-specific liver-infiltrating cytotoxic T lymphocytes in chronic HCV infection. The applicant has already determined the HCV genotype of many patients and will complete this. To detect the presence of HCV-specific CTLs, he will employ a target cell consisting EBV immortalized B-lymphoblastoid cells from each patient with chronic HCV. These cells will be infected with vaccinia virus carrying HCV gene inserts coding for specific candidate regions that are suspected of serving as epitopes in the CTL recognition process. CTLs will be obtained by bulk expansion of lymphocytes harvested from liver biopsies. chromium release assay of target cell lysis will be used to detect cells and quantitate the cytotoxic activity. 2) The second specific aim is to define the actual epitopes involved in the CTL- mediated recognition. CTLs identified in specific aim 1 will be cloned for use in screening small synthetically produced peptides overlapping in regions identified in specific aim 1. Sensitization of target cells and the cytolysis assay will be used.
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