Susceptibility to chronic hepatitis C virus infection is influenced by sequence differences in immunodominant CD8+ T cell epitopes.

BACKGROUND & AIMS: The antiviral immune response against HCV by CD8+ T cells plays a central role in viral containment. In a large HCV genotype 1b outbreak in Ireland, HLA-B(∗)08 was identified as a risk allele for chronic infection and HLA-A(∗)03 and HLA-B(∗)27 were associated with higher clearance rates. Here we took advantage of a similar large common source HCV genotype 1b outbreak (East-German cohort) to determine the role of HLA class I alleles and the sequence of the infection source, in immunodominant CD8+ T cell epitopes for disease outcome. METHODS: HLA-type and IL28B genotype were determined in 216 patients with chronic and 95 with spontaneously resolved HCV infection. The viral sequence in immunodominant epitopes was determined in the infection source and in patients with chronic infection. RESULTS: In contrast to the Irish cohort, HLA-B(∗)08, HLA-A(∗)03 and HLA-B(∗)27 were neutral for disease outcome even when the cohort was stratified for the IL28B genotype. Sequence analysis of the immunodominant epitopes revealed that pre-existing substitutions in the infection source of both cohorts influenced the impact of the corresponding HLA-allele. The immunodominant epitopes presented by the "protective" alleles HLA-A(∗)03 and -B(∗)27 in the Irish cohort contained substitutions in the source virus of the East-German outbreak. Importantly, the pre-existing substitutions altered subsequent selection pressure and viral evolution in the East-German cohort. CONCLUSIONS: This study highlights that subtle sequence differences in the infection source may have profound effects on the ability to clear HCV infection in the presence of particular HLA class I alleles.

Escape from a dominant HLA-B*15-restricted CD8+ T cell response against hepatitis C virus requires compensatory mutations outside the epitope.

Antiviral CD8(+) T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8(+) T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8(+) epitopes in NS5B (LLRHHNMVY(2450-2458) and SQRQKKVTF(2466-2474)), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs.

CD8+ T-cell response promotes evolution of hepatitis C virus nonstructural proteins.

BACKGROUND & AIMS: Hepatitis C virus (HCV) acquires mutations that allow it to escape the CD8+ T-cell response, although the extent to which this process contributes to viral evolution at the population level is not clear. We studied viral adaptation using data from a large outbreak of HCV genotype 1b infection that occurred among women immunized with contaminated immunoglobulin from 1977 to 1978. METHODS: The HCV nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients, and mutations were mapped according to their location inside or outside previously described CD8+ T-cell epitopes. A statistical approach was developed to identify sites/regions under reproducible selection pressure associated with HLA class I. RESULTS: The frequency of nonsynonymous mutations was significantly higher inside previously described CD8+ T-cell epitopes than outside-particularly in NS3/4A and NS5B. We identified new regions that are under selection pressure, indicating that not all CD8+ T-cell epitopes have been identified; 6 new epitopes that interact with CD8+ T cells were identified and confirmed in vitro. In some CD8+ T-cell epitopes mutations were reproducibly identified in patients that shared the relevant HLA allele, indicating immune pressure at the population level. There was statistical support for selection of mutations in 18 individual epitopes. Interestingly, 14 of these were restricted by HLA-B allele. CONCLUSIONS: HLA class I-associated selection pressure on the nonstructural proteins and here predominantly on NS3/4A and NS5B promotes evolution of HCV. HLA-B alleles have a dominant effect in this selection process. Adaptation of HCV to the CD8+ T-cell response at the population level creates challenges for vaccine design.

Coexpression of PD-1, 2B4, CD160 and KLRG1 on exhausted HCV-specific CD8+ T cells is linked to antigen recognition and T cell differentiation.

Exhausted CD8+ T cell responses during chronic viral infections are defined by a complex expression pattern of inhibitory receptors. However, very little information is currently available about the coexpression patterns of these receptors on human virus-specific CD8+ T cells and their correlation with antiviral functions, T cell differentiation and antigen recognition. We addressed these important aspects in a cohort of 38 chronically HCV infected patients and found a coexpression of inhibitory receptors such as 2B4, CD160 and KLRG1 in association with PD-1 in about half of the HCV-specific CD8+ T cell responses. Importantly, this exhaustive phenotype was associated with low and intermediate levels of CD127 expression, an impaired proliferative capacity, an intermediate T cell differentiation stage and absence of sequence variations within the corresponding epitopes, indicating ongoing antigen triggering. In contrast, a low expression of inhibitory receptors by the remaining HCV-specific CD8+ T cells occurred in concert with a CD127hi phenotype, an early T cell differentiation stage and presence of viral sequence variations within the corresponding epitopes. In sum, these results suggest that T cell exhaustion contributes to the failure of about half of HCV-specific CD8+ T cell responses and that it is determined by a complex interplay of immunological (e.g. T cell differentiation) and virological (e.g. ongoing antigen triggering) factors.