CD8+ T cell selection pressure is a major driving force for evolution of hepatitis C virus nonstructural proteins

Aims: Mutational escape from immune pressure exacted by CD8 T cells has been described during infection with hepatitis C virus (HCV). However, the extent to which this immune adaptation process contributes to viral evolution at the population level is less clear. Here, we took advantage of a large HCV genotype 1b outbreak after inoculation of contaminated immunoglobulins in 1977/78 to study this adaptation process.

Methods: Samples were collected since 2008 and HLA-typed. The nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients and mutations mapped according to their location inside or outside previously described CD8 epitopes. Moreover, a statistical approach was developed to identify sites/regions under reproducible HLA-class I-associated selection pressure.

Results: The frequency of nonsynonymous mutations was significantly higher inside previously described CD8 epitopes compared to the outside region. This difference was most prominent in NS3/4A and NS5B. In some previously described CD8 epitopes mutations were highly reproducible in patients sharing the relevant HLA-allele suggesting reproducible immune pressure at the population level. In 17 individual epitopes we find statistical support for selection of mutations. Interestingly, 13 of these 17 epitopes are restricted by HLA-B-alleles. Utilizing a statistical approach we were able to identify novel regions under selection pressure possibly pointing towards unknown CD8 epitopes. By this approach six novel CD8 epitopes were identified and experimentally confirmed in vitro.

Conclusion: HLA-class I-associated selection pressure is the major driving force for viral evolution of NS3/4A and NS5B. HLA-B-alleles have a dominant effect in this selection process. Adaptation of HCV to CD8 T cell immune pressure at the population level further complicates vaccine design.

CD8 T cell - anti-D cohort - immune escape - selection