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Background:  We hypothesized that immune selection is a dominant force driving hepatitis C virus (HCV) variation during acute infection.

Methods:  To test this hypothesis, we prospectively collected serum, plasma, and peripheral blood mononuclear cells (PBMC) from HCV antibody-negative injection drug users in a protocol designed for monthly follow-up. ELISpot assay was used to detect interferon-γ production using a combination of previously identified optimal CD8⁺ T-cell epitopes and overlapping peptides covering the HCV polyprotein as potential antigens. For 8 subjects screened for cellular immune responses, RNA was extracted from serum or plasma at the initial point of viremia and 6 months later. Long-template RT-PCR was used to amplify and clone the region from the 5' UTR to the NS3/NS4A junction. Of 40 cDNA clones assigned to clonotypes, 2 clones representing the modal clonotype were sequenced.

Results:  In subjects with chronic viremia and T-cell responses, there were substitutions in 69% of T-cell epitopes and every subject had a substitution in at least 1 epitope. In contrast, in a subject who cleared infection, there were no substitutions in T-cell epitopes at 6 or at 12 months after infection. For the 5 subjects with persistent viremia and T-cell responses, amino acid substitutions were a median of 16.7-fold more likely to occur within T-cell epitopes than outside epitopes (range 11.4 to 24.9). For 8 of the 10 mutations in recognized epitopes tested, recognition by T lymphocytes was lost or significantly reduced compared with recognition of the sequence present at initial viremia, indicating escape (both measured using PBMC or lines). Of the 46 amino acid substitutions observed in those 5 subjects, only 10 were observed outside of the envelope proteins or recognized T-cell epitopes and 7 of those represented conversion to the consensus sequence (convergence).

Conclusions:  During acute HCV infection, amino acid substitutions are nonrandom, and may be explained by escape from CD8⁺ T-cell recognition and convergence, possibly because of less fit substitutions selected in a previous host. There is early commitment of the T-cell repertoire since neither lines nor PBMC recognized escape mutants.

Keywords: hepatitis C virus; immunology; T lymphocyte