Background:  Co-infection with hepatitis C virus (HCV) is a major health problem among high-risk exposed HIV⁺ patients. However, the effect of these co-infections on their respective immune responses and viral evolution remains poorly understood. Viral escape from CD8 responses has been documented for many viral infections. In contrast, viral escape from CD8 T-cell responses in HCV has only been conclusively demonstrated in the chimpanzee model. To begin to address this issue we examined sequences from all non-structural HCV genes in 46 subjects with chronic HCV infection and identified associations between sequence polymorphisms and specific HLA class I molecules.

Methods:  Samples from 46 chronically HCV-infected (genotype 1a) treatment-naïve patients, as well as longitudinal samples from three patients with acute infection, were collected. PCR amplification and sequencing of all non-structural proteins was performed. CD8⁺ T-cell responses were defined using an interferon (IFN)-γ ELISpot assay with overlapping synthetic peptides and tetramers. HLA class I typing was derived on each subject.

Results:  Sequences derived from the 46 chronically HCV-infected patients revealed at least 3 strong associations between sequence polymorphisms in CD8 epitopes restricted by HLA-A1, -B8, and -B35 and expression of the corresponding HLA molecule. Examination of the HLA-B8 epitope in more detail revealed that development of mutations within this NS3 CD8 epitope in subjects with acute infection coinciding with loss of strong ex vivo responses. In vitro functional analysis indicated that recognition was altered not by affecting binding or TCR contact but rather through a rarely described phenomenon of impaired antigen processing. Transmission of the HLA-B8-associated escape mutation to an HLA-B8-negative subject resulted in rapid reversion of the mutation, suggesting that some CD8 escape mutations may be less well tolerated by the virus than others.

Conclusions:  These data provide significant evidence for viral escape from CD8⁺ T-cell responses during human HCV infection. That HLA-associated polymorphisms were observed in multiple CD8 epitopes reveals the reproducibility of this phenomenon and the extent to which the selective forces acting on HCV influence its evolution. These baseline studies will facilitate addressing more specific questions regarding how HIV co-infection affects the evolution of HCV and HCV-specific immune responses. 

Keywords: HCV; CTL escape; viral evolution