Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity

AIM:To understand how interactions between hepatitis C virus(HCV) and the host's immune system might lead to viral persistence or effective elimination of HCV.METHODS:Nucleotides 3519-3935 of the non-structural 3(NS3) region were amplified by using reverse transcription polymerase chain reaction(PCR).PCR products of the HCV NS3 regions were integrated into a PCR T7TOPO TA vector and then sequenced in both directions using an automated DNA sequencer.Relative major histocompatibility complex binding levels of wild-type and variant peptides were performed by fluorescence polarization-based peptide competition assays.Peptides with wild type and variant sequences of NS3 were synthesized locally using F-moc chemistry and purified by high-performance liquid chromatography.Specific cytotoxic T lymphocytes(CTLs) clones toward HCV NS3 wild-type peptides were generated through limiting dilution cloning.The CTL clones specifically recognizing HCV NS3 wild-type peptides were tested by tetramer staining and flow cytometry.Cytolytic activity of CTL clones was measured using target cells labeled with the fluorescence enhancing ligand,DELFIA EuTDA.RESULTS:The pattern of natural variants within three human leukocyte antigen(HLA)-A2-restricted NS3 epitopes has been examined in one patient with chronic HCV infection at 12,28 and 63 mo post-infection.Results obtained may provide convincing evidence of immune selection pressure for all epitopes investigated.Statistical analysis of the extensive sequence variation found within these NS3 epitopes favors a Darwinian selection model of variant viruses.Mutations within the epitopes coincided with the decline of CTL responses,and peptide-binding studies suggested a signif icant impact of the mutation on T cell recognition rather than peptide presentation by HLA molecules.While most variants were either not recognized or elicited low responses,such could antagonize CTL responses to target cells pulsed with wild-type peptides.CONCLUSION:Cross-recognition of CTL epitopes from wild-type and naturally-occurring HCV variants may lead to impaired immune responses and ultimately contribute to viral persistence.

AIM:To understand how interactions between hepatitis C virus(HCV) and the host s immune system might lead to viral persistence or effective elimination of HCV.METHODS:Nucleotides 3519-3935 of the non-structural 3(NS3) region were amplified by using reverse transcription polymerase chain reaction(PCR).PCR products of the HCV NS3 regions were integrated into a PCR T7TOPO TA vector and then sequenced in both directions using an automated DNA sequencer.Relative major histocompatibility complex binding levels ...