Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are b elieved to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infe ct human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CT L occurring during natural infection, is mandatory to study CTL activity an d to define the hierarchy of immunodominance of CTL epitopes, To achieve th is goal, 5 different defective adenoviruses carrying structural and nonstru ctural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to in duce the endogenous synthesis of HCV proteins in human adherent mononuclear cells ill vitro and to allow their entry into the HLA class I cytosolic pa thway of antigen processing. The cytolytic activity of peripheral blood lym pho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulate d with recombinant adenovirus-infected cells was tested against target cell s either pulsed with a panel of synthetic peptides containing the HLA-A2 bi nding motif or infected with recombinant vaccinia viruses carrying HCV gene s. Our study defines a reproducible system to stimulate and expand HCV-spec ific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restrictcd epitopes tha t should correspond to immunodominant HCV sequences recognized by CTL durin g natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection .