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. 2005 Jun 6;201(11):1741-52.
doi: 10.1084/jem.20050121.

Cellular immune selection with hepatitis C virus persistence in humans

Andrea L Cox  1 , Timothy Mosbruger, Qing Mao, Zhi Liu, Xiao-Hong Wang, Hung-Chih Yang, John Sidney, Alessandro Sette, Drew Pardoll, David L Thomas, Stuart C Ray
Affiliations

Cellular immune selection with hepatitis C virus persistence in humans

Andrea L Cox et al. J Exp Med. .

Abstract

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific cellular immune responses. To determine if immunologically driven sequence variation occurs with HCV persistence, we coordinately analyzed sequence evolution and CD8+ T cell responses to epitopes covering the entire HCV polyprotein in subjects who were followed prospectively from before infection to beyond the first year. There were no substitutions in T cell epitopes for a year after infection in a subject who cleared viremia. In contrast, in subjects with persistent viremia and detectable T cell responses, we observed substitutions in 69% of T cell epitopes, and every subject had a substitution in at least one epitope. In addition, amino acid substitutions occurred 13-fold more often within than outside T cell epitopes (P < 0.001, range 5-38). T lymphocyte recognition of 8 of 10 mutant peptides was markedly reduced compared with the initial sequence, indicating viral escape. Of 16 nonenvelope substitutions that occurred outside of known T cell epitopes, 8 represented conversion to consensus (P = 0.015). These findings reveal two distinct mechanisms of sequence evolution involved in HCV persistence: viral escape from CD8+ T cell responses and optimization of replicative capacity.

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Figures

Figure 1.
Figure 1.
Fluctuating HCV RNA level during acute infection. Spontaneous clearance (SC) occurred in one subject, and persistence developed in seven others. A solid triangle indicates detectable HCV RNA. An open triangle indicates an HCV RNA level of less than 50 IU/ml. A gray inverted triangle indicates that IFN-γ ELISPOT analysis of T cell responses was performed at that time point. The number in the bottom right corner of each panel is the subject number. Subject 28 entered the study antibody negative and HCV RNA positive so that the time of infection is estimated using the average time from infection to seroconversion.
Figure 2.
Figure 2.
Amino acid substitutions and epitopes recognized during the first 6 mo of HCV infection for five subjects. The map at the top of the figure indicates the region of the HCV polyprotein sequenced. For each study subject, horizontal lines represent the sequences obtained at initial viremia (t0), 6 mo after viremia was first detected (t6), and 12 mo after viremia was first detected (t12). Thin vertical lines represent amino acid substitutions. Shorter, thicker vertical lines indicate recognized epitopes, below which the t0 and t6 sequences of the epitope are shown. Subject 29 had no detectable response to the t0 peptide HSKRKCDEL, but did respond to the t6 peptide; therefore, this was not counted as an escape mutation in subsequent analyses. *Subjects 11, 12, and 13 were not tested for IFN-γ responses using the entire panel of 524 peptides because of limited PBMC specimens; instead, they were screened using peptides overlapping sites of amino acid replacement.
Figure 3.
Figure 3.
Amino acid substitutions in epitopes reduced T cell responses. Peptide sequences that were observed to vary between t0 (circle, initial viremia) and t6 (triangle, 6 mo after onset of viremia) were used as antigens in IFN-γ ELISPOT, using PBMCs (filled symbols) or T cell lines generated from PBMCs (open symbols) obtained at t6 as effectors. (A) Loss of recognition: For 4 of 10 peptide pairs, recognition of the t6 variant peptide was reduced at least 20-fold at all concentrations. (B) Decreased recognition: For 4 of 10 peptide pairs, recognition of the t6 variant peptide was reduced more than 2-fold but less than 20-fold at at least two concentrations of peptide. (C) Comparable recognition: For 2 of 10 pairs of peptides, recognition of the t6 variant was not reduced more than twofold relative to the t0 peptide at more than one concentration tested.
Figure 4.
Figure 4.
Similar recognition patterns using lines and PBMCs. IFN-γ ELISPOT responses for T cell lines (open symbols) and PBMCs (closed symbols) from which the lines were generated using the t0 peptide are shown. Responses for PBMCs and T cell lines were consistently similar aside from the expected differences in the proportion of responding cells (note difference in axes).
Figure 5.
Figure 5.
Recognition patterns persist with time. To rule out the subsequent development of T cell responses to the t6 HCV sequence, IFN-γ ELISPOT testing for recognition of the t6 peptides demonstrating escape also was performed for subjects 17 and 28 using PBMCs that were obtained ∼12, 18, 24, and—for subject 17—36 mo after initial infection. The number in the bottom right corner of each panel is the subject number. Patterns of recognition persisted over time and recognition of the t6 peptides (▴) declined in parallel with the decline in recognition of the t0 peptides (•) that occurred with prolonged infection.
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