Anti-hepatitis C virus T-cell immunity in the context of multiple exposures to the virus

Abstract

Characterisation of Hepatitis C virus (HCV)-specific CD8+ T-cell responses in the context of multiple HCV exposures is critical to identify broadly protective immune responses necessary for an effective HCV vaccine against the different HCV genotypes. However, host and viral genetic diversity complicates vaccine development. To compensate for the observed variation in circulating autologous viruses and host molecules that restrict antigen presentation (human leucocyte antigens; HLA), this study used a reverse genomics approach that identified sites of viral adaptation to HLA-restricted T-cell immune pressure to predict genotype-specific HCV CD8+ T-cell targets. Peptides representing these putative HCV CD8+ T-cell targets, and their adapted form, were used in individualised IFN-γ ELISpot assays to screen for HCV-specific T-cell responses in 133 HCV-seropositive subjects with high-risk of multiple HCV exposures. The data obtained from this study i) confirmed that genetic studies of viral evolution is an effective approach to detect novel in vivo HCV T-cell targets, ii) showed that HCV-specific T-cell epitopes can be recognised in their adapted form and would not have been detected using wild-type peptides and iii) showed that HCV-specific T-cell (but not antibody) responses against alternate genotypes in chronic HCV-infected subjects are readily found, implying clearance of previous alternate genotype infection. In summary, HCV adaptation to HLA Class I-restricted T-cell responses plays a central role in anti-HCV immunity and multiple HCV genotype exposure is highly prevalent in at-risk exposure populations, which are important considerations for future vaccine design.

Fig 1. Breakdown of HLA-specific T-cell targets tested and number eliciting a T-cell response based on an IFN-γ ELISpot assay.

Predicted T-cell targets include published HCV T-cell targets that contain a site associated with a specific HLA allele with the same restriction. Number in bracket indicates number of subjects tested that carry the particular HLA type.

Fig 2

A. Contribution of HLA-A-, -B- and -C-restricted responses to overall anti-HCV immunity in the cohort. Response rates of predicted (circle), published with adaptation (square) and published (triangle) HCV CD8⁺ T-cell epitopes are shown in relation to the HLA loci. HLA-A-restricted T-cell epitopes have a significantly higher response rate compared to HLA-B- and -C-restricted T-cell epitopes (p = 0.008 and p = 0.0007, respectively). B. Response rate of T-cell epitopes within the HCV non-structural proteins. Predicted (circle) T-cell epitope, published T-cell epitope with adaptation (square) and published (triangle) T-cell epitopes that elicit an IFN-γ response. T-cell epitopes with the highest response rates are indicated. There was a greater response rate in NS3 relative to the other proteins (p = 0.02).

Fig 3. Chronic non-GT1-infected individuals are able to mount T-cell responses to GT1 epitopes.

The SFU/million PBMCs are shown for subjects with known GT1 infection and non-GT1 infection. Although subjects may have responded to more than one peptide covering an epitope, in this figure only the highest response was used.

Fig 4. Examples of likely GT-specific responses using ELISpot analysis.

The two panels (A) and (B) represent GT1-epitopes that elicited responses from GT3-infected subjects. Peptides representing GT1 peptides tested in the original ELISpot screen and in a subsequent ELISpot with the alternative GT3 peptide(s). Original screen result is shown on the front row (grey) and subsequent ELISpot assay on the second row (black; mean SFU/10⁶ PBMCs from duplicates). Each panel represents data from a single subject.