Immune evasion versus recovery after acute hepatitis C virus infection from a shared source

Abstract

Acute infection with hepatitis C virus (HCV) rarely is identified, and hence, the determinants of spontaneous resolution versus chronicity remain incompletely understood. In particular, because of the retrospective nature and unknown source of infection in most human studies, direct evidence for emergence of escape mutations in immunodominant major histocompatibility complex class I-restricted epitopes leading to immune evasion is extremely limited. In two patients infected accidentally with an identical HCV strain but who developed divergent outcomes, the total lack of HCV-specific CD4+ T cells in conjunction with vigorous CD8+ T cells that targeted a single epitope in one patient was associated with mutational escape and viral persistence. Statistical evidence for positive Darwinian selective pressure against an immunodominant epitope is presented. Wild-type cytotoxic T lymphocytes persisted even after the cognate antigen was no longer present.

Figure 1.

HCV infection in two subjects who were exposed accidentally on the same day to HCV genotype 1a–infected patellar tendon that resulted in divergent outcomes. PD101 (top) developed elevation in serum alanine aminotransferase (ALT) and HCV RNA viremia that persisted until initiation of antiviral therapy with pegylated interferon and ribavirin. PD102 developed marked elevations in serum alanine aminotransferase and bilirubin; viremia became undetectable spontaneously without antiviral therapy.

Figure 2.

HCV–genome-wide analysis of CD4⁺ and CD8⁺ T cell responses in PD101 and PD102 at 6, 12, and 24 mo after infection. Overlapping 15 mer-peptides (n = 750) were synthesized to span the complete HCV polyprotein derived from HCV-1a (the sequence data are available from GenBank/EMBL/DDBJ under accession no. M62321) and divided into 32 peptide pools. IFN-γ production was detected using an established ELISPOT protocol. CD8⁺ cells were isolated from PBMCs by magnetic-activated cell sorting magnetic bead separation (Miltenyi Biotec) and the remaining CD4⁺ others were used. Negative controls in the ELISPOT assay were wells with purified T cells but no peptide (n = 8). After plates were dry, spots were quantified by a Zeiss microscope using KS ELISPOT software. Responses were considered positive if greater than the mean plus three SD of the control wells (DMSO only), if there were at least 10 spots above background, and/or were significant by Student's t test (P < 0.05). Shown are the mean ± SEM. (A and B) CD4⁺ and CD8⁺ T cell responses for PD101 and PD102 at 6 mo after infection. The difference in total immune response to the HCV peptides between patients was statistically significant (P = 0.0005, Fisher's two-tail test). (C and D) CD4⁺ and CD8⁺ T cell responses for PD101 and PD102 at 12 mo after infection. (E and F) T cell responses for PD101 and PD102 at 24 mo after infection.

Figure 3.

Proliferation of HCV-specific tetramer⁺ CD8⁺ T cells. On day 8 of culture in HCV peptide–stimulated T cell lines derived from PD101 and PD102, CFSE-labeled tetramer⁺ cells were detected. In brief, 10⁷ PBMCs were cultured in the presence of four HCV epitope peptides (NS3 1406–1415 [KLVALGINAV], NS3 1073–1081 [CINGVWCTV], and core 132–140 [DLMGYIPLV]) with IL-2 (0.5 ng/ml) added on days 0 and 3. Because the CFSE signal is diluted with each cell division, signals in the left upper quadrant represent viral-specific CD8⁺ T cells that have proliferated in culture. Those in right upper quadrant represent tetramer⁺ T cells that have not divided (reference 14). (A) In PD101, the NS3 1406–specific CD8⁺ T cells expanded after peptide stimulation (representing 29% of gated CD8⁺ T cells) and NS3 1073–specific CD8⁺ T cells demonstrated modest proliferation, whereas the other epitope tetramer⁺ cells were viable but failed to expand, as indicated by the tetramer⁺ cells in the upper right quadrants of the dot plots. Analysis from two other time points after infection revealed similar results. (B) For PD102, in vitro culture for 8 d led to proliferation of NS3 1406– and NS3 1073–specific CDD8⁺ T cell responses. In contrast, neither core-specific nor NS5B 2594–specific responses expanded in culture. Analysis from two other time points revealed similar results.

Figure 4.

Mutant viral sequence, recognition, and binding of mutant peptide. (A) Viral sequences corresponding to the single peptide that elicited CD8⁺ T cells from PD101 who developed viral persistence. The prototype HCV-1 peptide sequence is shown; dashes indicate identity with the prototype and substitutions are represented by standard letter codes at different time points after infection. The donor sequence is identical to the prototype sequence and the sequence derived from PD102 who spontaneously cleared HCV RNA. In contrast, PD101 developed a mutation within the NS3 1406–1415 epitope (A1409V) by 4.5 mo after infection that was propagated until antiviral therapy was initiated. (B) CD8⁺ T cells from PD101 (from 8 mo after infection) recognize wild-type NS3 1406 peptide, but not mutant 1406 peptide. Dose titrations of wild-type, mutant, and combination of both peptides reveal high frequency of IFN-γ–producing effector cells after pulsing of autologous DCs with wild-type peptide and culture with bead-purified CD8⁺ T cells without antagonism by the mutant peptide. Wild-type and nonwild-type peptides were screened at multiple concentrations. (C) MHC class I binding affinity. Transporter associtated with antigen-processing–deficient T2 cells (10⁵) were cultured for 16 h at 26°C to increase expression of peptide-receptive cell surface molecules, and then were incubated with various concentrations of individual peptides (wild-type NS3 1406, mutant NS3 1406, or DMSO) at 37°C for 1 h, washed, and stained with FITC-labeled anti–HLA-A2 antibody (BD Biosciences), as described previously (24). The variant peptide bound to the HLA-A2 with decreased affinity (P = 0.0014), comparable to DMSO (no peptide) control. Data are expressed as the mean fluorescence intensity.

Figure 5.

Comparison of viral genetic sequence of donor, PD101, and PD102. (A) Dendrogram showing genetic relatedness of donor, PD101, and PD102 relative to other HCV isolates. Donor, PD101 (the sequence data are available from GenBank/EMBL/DDBJ under accession no. AY695436), and PD102 PD102 (the sequence data are available from GenBank/EMBL/DDBJ under accession no. AY695437) amino acid sequences were aligned against the corresponding sequences from the reference 1a isolate from which the peptides were derived (M62321), and a genotype 1b isolate (J4). The length of the horizontal lines connecting two isolates by the shortest route (right to left and left to right) is proportional to the genetic distance between the isolates. (B) Viral sequences for the prototype HCV-1 peptide sequence, donor, PD101 (6 mo after infection), and PD102 (1.5 mo) are shown; dashes indicate identity with the prototype and substitutions are represented by standard letter codes at different time points after infection. Known class I restricted epitopes are indicated in brackets [HLA A2 and B35].