Selection-driven immune escape is not a significant factor in the failure of CD4 T cell responses in persistent hepatitis C virus infection

Abstract

Immune escape driven by selection pressure from virus-specific CD8 T cells has been demonstrated in both chimpanzees and humans infected with the hepatitis C virus (HCV). Although escape mutations have also been characterized in major histocompatibility complex (MHC) class II-restricted HCV epitopes, it is unknown whether selection-driven immune escape by CD4 T cell epitopes is a significant factor in the failure of these responses or contributes to persistent infection. To address this issue, evolution of MHC class I- and class II-restricted HCV epitopes was compared in four chimpanzees persistently infected with the virus for more than 10 years. We identified an amino acid change in a CD4 epitope of the HCV NS3 protein in one of the chimpanzees 3 years after infection. This mutation resulted in diminished activation, cytokine production (interferon-gamma and interleukin-2), and proliferation by an epitope-specific CD4 T cell line. We expanded our analysis to determine if mutations were common in multiple CD4 versus CD8 T cell epitopes in the four chronically infected animals. Whereas we observed mutations in over 75% of CD8 T cell epitopes analyzed in this study, only 18% of CD4 T cell epitopes analyzed showed amino acid changes. The frequency of changes in class II epitopes was not different from flanking regions, so CD4 T cells rarely exert selection pressure against the HCV genome.

Conclusion: Apparent mutational escape can occur in MHC class II-restricted epitopes, but this is uncommon when compared with class I-restricted epitopes in the same individual. This indicates that other mechanisms for silencing CD4 T cells are dominant in persistent HCV infections.

Fig. 1

P8 V to A mutation in the NS3₁₃₇₆ epitope impairs IL-2 production in a cognate CD4 T cell clone. (A) Using an overnight intracellular cytokine staining assay, the E to A and V to A amino acid changes at positions P8 and P9, respectively, of the NS3₁₃₇₆ (YGKAIPLEVI) epitope were analyzed both separately and in combination for their ability to stimulate IFN-γ and IL-2 production in a CD4 T cell clone specific for the epitope. Representative flow cytometry data are shown, and values in the lower left, upper left, and upper right quadrants represent percentages of gated CD3⁺CD4⁺ cells that are IFN-γ⁻/IL-2⁻, IFN-γ⁺/IL-2⁻, or IFN-γ⁺/IL-2⁺, respectively. (B) Titrated concentrations of the NS3₁₃₇₆ WT and double mutant NS3₁₃₇₆ M1 peptides were analyzed for their ability to stimulate the CD4 T cell clone 3D to produce IFN-γ and IL-2 in an overnight intracellular cytokine staining assay. Representative results are shown. Values in the lower left, upper left, and upper right quadrants represent percentages of gated CD3⁺CD4⁺ cells that are IFN-γ⁻/IL-2⁻, IFN-γ⁺/IL-2⁻, or IFN-γ⁺/IL-2⁺, respectively.

Fig. 2

Mutation in the NS3₁₃₇₆ epitope stimulates impaired activation and proliferation in a cognate CD4 T cell clone. (A) Activation of the NS3₁₃₇₆-specific CD4 T cell clone 3D was assessed by intracellular staining for the phosphorylated form of the mitogen-activated protein kinase ERK 1/2 following a 15-minute stimulation with various concentrations of the NS3₁₃₇₆ WT or NS3₁₃₇₆ M1 peptides. Representative histograms are shown. White histograms show phosphorylated ERK 1/2 staining levels in unstimulated control cells; levels in stimulated test cells are depicted by gray histograms. (B) CFSE-labeled CD4 T cell clone 3D cells were stimulated with either the NS3₁₃₇₆ WT or NS3₁₃₇₆ M1 peptides (0.01 μg/mL) and proliferation, measured as CFSE dilution by way of flow cytometric analysis, was determined on day 3, 4, or 5. Representative plots show gated live CD3⁺CD4⁺ cells with frequencies of cells that either remained undivided (CFSE^hi) or had undergone up to four rounds of division as indicated above each plot.

Fig. 3

Comparison of the NS3₁₃₇₆ epitope in animals with and without the restricting MHC class II allele. Viral RNA was isolated from the serum of four persistently infected chimpanzees (CB0603, CB0609, CH-503, and NM1238) and amplified by PCR. Subsequent molecular cloning and sequencing were then performed to assess the sequence of the NS3₁₃₇₆ epitope in two DRB5*0310-positive chimpanzees (CB0609 and CH-503; highlighted) and two chimpanzees (NM1238 and CB0603) that did not express this restricting MHC class II allele. The frequencies of each identified sequence are indicated on the right.

Fig. 4

Infrequent sequence variation in MHC class II–restricted HCV epitopes in persistently infected chimpanzees. HCV RNA isolated from the serum of four persistently infected animals (CBO603, CBO609, CH-503, and NM1238) was amplified by way of PCR, and multiple molecular clones were sequenced to identify amino acid changes in 10 CD4 T cell epitopes compared with a consensus sequence of the infecting genotype 1a 1/910 strain. The frequency of each sequence is indicated to the right in each box. Viral sequences from animals expressing the restricting class II MHC allele are highlighted.

Fig. 5

Amino acid changes in CD8 T cell–targeted epitopes of HCV proteins in persistently infected chimpanzees. Multiple clonal sequences of class I MHC-restricted epitopes in HCV structural (A) and nonstructural (B) genes were compared from the four persistently infected chimpanzees to the consensus HCV-1/910 sequence. Epitopes in animals that express the appropriate restricting MHC class I allele are highlighted, and frequencies for the different sequences are shown on the right side of each box.

Fig. 6

HCV-specific CD8 T cells, but not CD4 T cells, exert significant pressure to drive increased nonsynonymous mutation rates. Median rates of synonymous substitutions per synonymous site (dS) and of nonsynonymous substitutions per nonsynonymous site (dN) were computed separately for nonepitope regions, and for CD4 and CD8 T cell epitopes both in animals that express the specific restricting MHC alleles and in animals that lack the necessary MHC allele to present the epitope to specific CD4 or CD8 T cells (data not shown). Tests of the hypotheses that (1) the median dS is equal to the median dN and (2) an individual dS or dN value in presented epitopes equals that in nonpresented epitopes were performed using the Wilcoxon signed-rank test. Tests of the hypothesis that an individual dS or dN value in epitopes equals that in nonepitope regions were performed using the Mann-Whitney test.