Broad specificity of virus-specific CD4+ T-helper-cell responses in resolved hepatitis C virus infection

Abstract

Vigorous virus-specific CD4+ T-helper-cell responses are associated with successful control of hepatitis C virus (HCV) and other human viral infections, but the breadth and specificity of responses associated with viral containment have not been defined. To address this we evaluated the HCV-specific CD4+ T-helper-cell response in HCV antibody-positive persons who lack detectable plasma viremia, and compared this response to that in persons with chronic HCV infection. Peripheral blood mononuclear cells were stimulated with HCV proteins, followed by measurement of HCV-specific CD4+ T-cell responses to a comprehensive set of overlapping HCV peptides by intracellular gamma interferon production. In three persons with resolved HCV infection studied in detail, 13 to 14 epitopes were targeted, but none was recognized by all three. The 37 defined epitopes were predominantly distributed among the HCV proteins core, NS3, NS4, and NS5. In an expanded analysis of responses to these proteins in persons with resolved infection, an average of 10 epitopes was targeted, whereas in persons with chronic viremia never was more than one epitope targeted (P < 0.001). This comprehensive analysis of the breadth and specificity of HCV-specific T-helper-cell responses indicates that up to 14 viral epitopes can be simultaneously targeted by circulating virus-specific CD4+ T helper cells in a controlled human viral infection. Moreover, these data provide important parameters for evaluation of candidate HCV vaccines, and provide rationale for immunotherapy in chronic HCV infection.

FIG. 1.

Lymphocyte proliferation by fresh PBMC in response to the recombinant antigens C22-3, C33C, C100, and NS5 for subjects 1, 2, and 3 with spontaneously resolved HCV infection. At the time of study, all three subjects had been anti-HCV positive and HCV RNA negative for more than 2.5 years.

FIG. 2.

Expansion of HCV-specific CD4⁺ populations by stimulation of PBMC with recombinant HCV antigens in subject 2. HCV-specific activity of cell lines generated by separate stimulation with each of four antigens (core C22-3, NS3 C33C, NS4 C100, and NS5) were tested with pools of overlapping peptides corresponding to the sequence encoded by the whole antigen. The percentage of CD3⁺ CD4⁺ IFN-γ⁺ cells is indicated in the upper right quadrant of each plot, and results are shown for those antigens resulting in positive responses. The recombinant antigen used to generate each line is indicated to the left of the dot plots.

FIG. 3.

Identification of individual 20-mer peptides by deconvolution of positive peptide pools in cell lines generated by stimulation of PBMC with recombinant HCV antigens. Deconvolution of peptide pool 150-169 (Fig. 2) in a C100-stimulated PBMC cell line from subject 2 indicates a positive response to the NS3 peptide aa 1621 to 1640. Peptides 150-169 were all negative (data not shown). The percentage of CD3⁺ CD4⁺ IFN-γ⁺ cells is indicated in the upper right quadrant of each plot.

FIG. 4.

Percent CD3⁺ CD4⁺ IFN-γ⁺ cells for discrete 20-mer peptides in HCV antigen-stimulated PBMC cultures in subjects 1 (A), 2 (B), and 3 (C). The background percent IFN-γ production for each cell line in the absence of peptide has been subtracted. The percent IFN-γ production for each peptide indicated was greater than three times background IFN-γ production in all cases.

FIG. 5.

NS5-specific CD4⁺ T-helper cells are restricted by HLA DRB1*11 in subject 1. Peptide-pulsed autologous and partially HLA-matched B cells were incubated with the NS5 peptide-specific clone from subject 1 for 6 h and stained for CD3, CD4, and IFN-γ and analyzed by flow cytometry. The percentage of CD3⁺ CD4⁺ IFN-γ⁺ cells is indicated in the upper right quadrant of each plot.

FIG. 6.

Spontaneous resolution of HCV viremia is associated with multiple CD4⁺ T-helper-cell epitopes. PBMC from six individuals with resolved HCV infection and eight individuals with chronic HCV infection were stimulated with the recombinant antigens C22-3, C33C, C200, and NS5 covering the core, NS3, NS4, and NS5 proteins. After 10 to 14 days, expanded cell lines were tested for IFN-γ production upon stimulation with pools of 10 to 20 overlapping peptides each. All peptide pools eliciting a positive response by IFN-γ production were further deconvoluted to identify specific 20-mer epitopes targeted by the CD4⁺ T-helper-cell response.

FIG. 7.

Ex vivo intracellular cytokine staining with fresh PBMC for IFN-γ (A) or TNF-α (B) in subjects 1 and 2. Positive peptide responses as identified by bulk stimulation of PBMC were pooled together and used to stimulate fresh PBMC for 6 h and then stained intracellularly for cytokine production. For each subject, the peptide pool tested consisted of the peptides identified as targeted by the CD4⁺ T-helper-cell response as listed in Table 1.