Association of cytotoxic T lymphocyte (CTL) escape mutations with persistent hepatitis C virus (HCV) infection

Mechanisms by which HCV evades the cellular immune response in persistently infected humans and chimpanzees are poorly defined, but could involve mutations in epitopes recognized by class I MHC restricted CTLs. To investigate this possibility, we identified an epitope in the NS3 protein of HCV that was recognized by intrahepatic CTLs from a chimpanzee that developed persistent HCV infection after experimental challenge with the virus. Fine mapping studies with truncated synthetic peptides revealed that the epitope was 9 amino acids in length, encompassing residues 1445 to 1454 (GDFDSVIDC) of NS3. This sequence was completely conserved in all full-length NS3 genomes described to date. In view of the fact that the major genotypes of HCV may differ by up to -30% in overall amino acid homology, it appears in contrast that this epitope is highly conserved. The role of CTL escape mutations in HCV persistence was assessed in the virus inoculum used to infect this chimpanzee and in post-inoculation plasma samples. Sequencing of 6-10 M13 clones containing a 232-nucleotide fragment amplified with NS3-specific primers revealed that the epitope in the challenge inoculum and a post-inoculum plasma sample obtained at week 4 were identical to the published sequence of HCV-I. In contrast, all molecular clones sequenced from week 16, 25 and 28 plasma samples contained a single Asp--> Glu (D-->E) amino acid substitution at residue 1449. Significantly, four independently derived CTL clones established from the liver of this chimpanzee at various times up to two years after infection recognized target cells pulsed with a nonameric peptide representing the wild-type HCV-I sequence, but not those pulsed with a peptide containing the D-->E mutation. These data suggest that CTL escape mutations may play a role in viral persistence.