Virological footprint of CD4+ T-cell responses during chronic hepatitis C virus infection

Abstract

Human and animal model evidence suggests that CD4+ T cells play a critical role in the control of chronic hepatitis C virus (HCV) infection. However, despite their importance, the mechanism behind the failure of such populations in chronic disease is not understood and the contribution of viral mutation is not known. To address this, this study defined the specificity and virological footprint of CD4+ T cells in chronic infection. CD8+ T-cell-depleted peripheral blood mononuclear cells from 61 HCV genotype 1-infected patients were analysed against a panel of peptides covering the HCV genotype 1 core--a region where CD4+ T-cell responses may be reproducibly obtained. In parallel, the core region and E2 protein were sequenced. Gamma interferon-secreting CD4+ T-cell responses directed against multiple epitopes were detected in 53% of individuals, targeting between one and four peptides in the HCV core. Viral sequence evaluation revealed that these CD4+ T-cell responses were associated with mutants in 2/21 individuals. In these two cases, the circulating sequence variant was poorly recognized by host CD4+ T cells. Bioinformatics analyses revealed no overall evidence of selection in the target epitopes and no differences between the groups with and without detectable CD4+ T-cell responses. It was concluded that sustained core peptide-specific CD4+ T-cell responses may be reproducibly measured during chronic HCV infection and that immune escape may occur in specific instances. However, overall the virological impact of such responses is limited and other causes for CD4+ T-cell failure in HCV must be sought.

Fig. 1.

IFN-γ ELISPOT response to core peptides. (a) The frequency of CD4⁺ T-cell responses against the panel of core peptides is indicated. Data were derived from Table 2. The sequences of the individual peptides are available in Supplementary Table S1. All donors were tested against all peptides, and the total numbers of donors positive from the 61 tested is shown. (b) Typical ELISPOT assay from patient 306, showing a positive IFN-γ response to HCV core peptide 61–80 (s.f.u. per 10⁶ CD8-depleted PBMCs=140). (c) Titration experiment using fresh ex vivo CD8-depleted PBMCs from donor 304. The peptide concentration used is displayed on the y-axis, with the background subtracted. Peptides: ▴, 31–50; ▪, 61–80; ▾, 151–170.

Fig. 2.

Relatedness of sequences amongst responders and non-responders. Phylogenetic tree of the CD4 responder (•) and non-responder (○) HCV core region based on the neighbour-joining method using 1000 bootstrap replicates (scores <30 are indicated by an asterisk). Bar, nucleotide substitutions per site.

Fig. 3.

Sequence mutants in targeted epitopes. (a) An alignment of the core region is shown. The upper line indicates the group consensus. The lower lines indicate donors 304 and 379 with mutations within targeted epitopes indicated. Dots indicate amino acids identical to the consensus sequence. (b) An alignment of the core region for cloned donor 304 is shown. Each clone was compared with the bulk sequencing product. The frequency of the variant within the epitope 61–80 is indicated by shading: A68V was observed in the majority of the sequenced population. (c) Peptide titrations using PBMCs from donors 304 and 379, using wild-type (▪) and mutant (□) peptide as indicated in Fig. 3(a). The assays were performed as in Fig. 1.