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Journal of Virology, June 2005, p. 6976-6983, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6976-6983.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Strain-Specific T-Cell Suppression and Protective Immunity in Patients with Chronic Hepatitis C Virus Infection

Kazushi Sugimoto,1,{dagger} David E. Kaplan,1,{dagger} Fusao Ikeda,1 Jin Ding,1 Jonathan Schwartz,2 Frederick A. Nunes,3 Harvey J. Alter,4 and Kyong-Mi Chang1*

Division of Gastroenterology, Department of Medicine, University of Pennsylvania, and Philadelphia Veterans Affairs Medical Center,1 Pennsylvania Hospital, Philadelphia, Pennsylvania,3 Oregon Health Sciences University, Portland, Oregon,2 Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland4

Received 22 July 2004/ Accepted 26 January 2005


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ABSTRACT
 
Hepatitis C virus (HCV) frequently persists with an apparently ineffective antiviral T-cell response. We hypothesized that some patients may be exposed to multiple HCV subtypes and that strain-specific T cells could contribute to the viral dynamics in this setting. To test this hypothesis, CD4 T-cell responses to three genotype 1a-derived HCV antigens and HCV antibody serotype were examined in chronically HCV infected (genotypes 1a, 1b, 2, 3, and 4) and spontaneously HCV recovered subjects. Consistent with multiple HCV exposure, 63% of patients infected with genotypes 2 to 4 (genotypes 2-4) and 36% of those infected with genotype 1b displayed CD4 T-cell responses to 1a-derived HCV antigens, while 29% of genotype 2-4-infected patients showed serotype responses to genotype 1. Detection of 1a-specific T cells in patients without active 1a infection suggested prior self-limited 1a infection with T-cell-mediated protection from 1a but not from non-1a viruses. Remarkably, CD4 T-cell responses to 1a-derived HCV antigens were weakest in patients with homologous 1a infection and greater in non-1a-infected patients: proportions of patients responding were 19% (1a), 36% (1b), and 63% (2-4) (P = 0.0006). Increased 1a-specific CD4 T-cell responsiveness in non-1a-infected patients was not due to increased immunogenicity or cross-reactivity of non-1a viruses but directly related to sequence divergence. We conclude that the T-cell response to the circulating virus is either suppressed or not induced in a strain-specific manner in chronically HCV infected patients and that, despite their ability to clear one HCV strain, patients may be reinfected with a heterologous strain that can then persist. These findings provide new insights into host-virus interactions in HCV infection that have implications for vaccine development.


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INTRODUCTION
 
Hepatitis C virus (HCV) is a highly persistent virus associated with significant liver-related morbidity and mortality (2, 23, 30). It is also very heterogeneous, with 6 different molecular genotypes and over 50 subtypes (2, 23, 30). T cells play a key role in HCV clearance in both humans and chimpanzees (7, 9, 11, 14, 21, 33, 39, 42-44). A vigorous and broad antiviral T-cell response is associated with HCV clearance in acutely infected patients, while a weak or dysfunctional virus-specific T-cell response accompanies HCV persistence (9, 14, 27, 31, 33, 42-46). Following spontaneous viral clearance, an HCV-specific T-cell response is maintained for decades, whereas the antibody response may wane in the absence of viremia (9, 44). While a robust HCV-specific T-cell response generally correlates with prior HCV exposure and clearance, the significance of the HCV-specific T-cell response in established chronic infection is less clear. In chronic HCV infection, virus-specific T cells may contribute to HCV persistence by selecting for immune escape variants (8, 15, 21) and, conversely, may ameliorate clinical outcome even without viral clearance (4, 12, 24, 34).

The number and type of HCV exposures may also influence the outcome of HCV infection. For example, the likelihood of HCV transmission (and therefore persistent infection) may be increased with multiple rather than isolated exposures. However, individuals recovering from one HCV infection are more resistant to subsequent infection than previously uninfected persons (32). Conceivably, such individuals may maintain a memory T-cell response against the initial viral strain that protects against reinfection with that strain but not against heterologous strains. Interestingly, even among patients chronically infected with HCV, there is evidence for further host-virus and/or virus-virus interactions selecting one virus strain over another. For example, despite probable multiple HCV exposures (e.g., injection drug users), most patients are infected with one dominant HCV subtype (rather than multiple subtypes) (K. M. Chang, unpublished observation). It is interesting to consider the role of T cells in clearing one, but not another, HCV subtype. Along this line, T-cell responses to a virus may be influenced by prior exposures to other viruses eliciting cross-reactive T-cell responses (38, 47). Furthermore, heterologous viruses may be more prone to persist if the host immune response was fixed to the original antigen through "original antigenic sin," as suggested previously (22, 26).

Based on these considerations, we hypothesized that patients exposed to multiple HCV subtypes experience a dynamic interplay between viral strains and host T cells that determines the virological outcome. To this end, we examined 98 patients chronically infected with genotype 1 or non-genotype 1 viruses and 30 individuals who had recovered from HCV for immunological evidence of genotype 1 exposure. Interestingly, a significant proportion of patients infected with non-genotype 1 HCV displayed B- and T-cell responses to genotype 1-derived antigens, consistent with multiple viral exposures. Furthermore, the CD4 T-cell response to 1a-derived antigens was significantly greater in patients without 1a infection and weakest in patients with homologous 1a infection, in direct relationship to sequence divergence between the circulating viral genotype and the 1a-derived antigen tested. While these results suggested a strain-specific T cell-mediated protective immunity, they also suggested suppression of T cells specific for the circulating virus in HCV persistence.


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MATERIALS AND METHODS
 
Study subjects. Study subjects were enrolled through the clinics at the Philadelphia Veterans Affairs Medical Center (PVAMC), Temple University, the Department of Transfusion Medicine at the National Institutes of Health, and the Clinical Research Center at the University of Pennsylvania. All subjects gave written informed consent to participate in this study, approved by the respective institutional review board. All subjects were assessed for demographic and clinical parameters as well as serum anti-HCV (by second generation enzyme immunoassay) and HCV RNA by quantitative or qualitative Roche COBAS reverse transcriptase PCR (RT-PCR) (Roche Diagnostics, Branchburg, NJ). HCV RNA-positive patients were tested for HCV genotype by INNOLIPA (Innogenetics, Ghent, Belgium). We excluded subjects with prior antiviral or immunosuppressive therapy, acute hepatitis C within 1 year, human immunodeficiency virus (HIV) or hepatitis B virus coinfection, autoimmune diseases, or conditions precluding phlebotomy. Among genotype 1-infected patients, we included only patients with a definite 1a or 1b subtype so as to specifically compare 1a-specific T-cell responses in patients with 1a or 1b infection.

Ninety-eight patients with chronic (C) HCV infection were enrolled. Genotype 1-infected patients (C1; n = 71) were divided into the C1a (n = 36) and C1b(n = 35) subgroups based on 1a or 1b subtype. Group C2-4 (n = 27) included patients infected with genotype 2 (n = 20), 3 (n = 5), or 4 (n = 2). Most were males (96/98), due to high male predominance in our patient population. As shown in Table 1, the three groups had similar age distribution and liver function parameters, but there were fewer African-Americans in the C2-4 subgroup, as previously reported (36, 43). We also recruited 30 healthy HCV antibody-positive, RNA-negative persons without previous antiviral therapy (group R), who presumably had recovered spontaneously from HCV infection. Normal controls included 23 HCV antibody-negative, RNA-negative healthy volunteers (group N) without a history of liver disease or HCV exposure. Lack of HCV viremia in the R and N groups was confirmed by the qualitative Roche COBAS RT-PCR. Most chronic patients had prior injection drug and/or cocaine use, relevant for potential HCV exposure (74 to 80%), while 19 to 28% had a history of transfusion. HCV-recovered subjects had similar HCV risk factors (73% drugs, 17% transfusion).


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  		TABLE 1. Chronic patientsa

HCV-specific CD4 proliferative T-cell responses were assessed in all subjects. The HCV serotype was assessed in 88 chronic and 30 recovered subjects based on serum availability. HCV-specific Th1 responses were examined for 71 chronic, 18 recovered, and 13 normal controls by a gamma interferon (IFN-{gamma}) enzyme-linked immunospot (ELISPOT) assay based on lymphocyte availability.

PBMC. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Histopaque density gradient (Sigma Chemical Co., St Louis, MO) as previously described (43).

HCV proteins, NS4 peptides, and tetramers. Recombinant HCV core 2-120, NS4 1569-1931, NS3-4 1192-1931, NS5 2054-2995, and control superoxide dismutase (SOD) proteins based on an HCV type 1 (HCV-1) isolate (genotype 1a) were kindly provided by Michael Houghton (Chiron Corporation, Emeryville, CA). These proteins were immunogenic in patients with various HCV genotypes (9, 13, 17, 33, 34, 37).

HCV-derived 15-mers (serially offset by 6 and overlapped by 9 amino acid residues) synthesized by Mimotopes (Clayton, Victoria, Australia) (42) were used individually and in pools to map the NS4 epitope. NS4-specific T-cell responses between genotypes 1 and 2 were compared with three sets of 20 overlapping 15-mers spanning NS4 residues 1747 to 1875 derived from published genotypes 1a, 2a, and 2b.

NS3-1073/human leukocyte antigen (HLA)-A2 tetramers were synthesized by the tetramer facility of the National Institute of Allergy and Infectious Diseases and were used to stain PBMC in a 1:300 dilution. Tetramer specificity was confirmed using NS3-1073-specific T-cell lines (data not shown).

HCV serotype. Plasma HCV antibodies were serotyped for 30 recovered subjects and 88 chronic patients by using an HCV serotyping 1-6 assay (kindly performed by David Parker and Lara Sandler, Murex Diagnostic, London, United Kingdom) (43), based on immunodominant NS4 epitope peptides and with 80 to 90% concordance with molecular genotype (19, 29, 40).

HCV-specific CD4 proliferative T-cell response. Freshly isolated PBMC were stimulated in 5 replicates (0.2 million PBMC/well) for 7 days with recombinant HCV and control SOD proteins (10 µg/ml) as described elsewhere (43). Results were expressed as a stimulation index (SI), calculated as the mean cpm in stimulated wells divided by that in control wells. This response was mediated by CD4 T cells (9). A positive response was determined by cutoffs derived from 23 normal controls (>2 standard deviations + mean SI): 2.7 for core, 2.1 for NS3-4, 3.8 for NS5). For a positive control, PBMC from all patients were stimulated with/without 2 µg/ml phytohemagglutinin (PHA) in triplicate for 4 days. A total of 64 chronic and 23 recovered subjects were examined with 0.1 µg/ml tetanus toxoid (Connaught International Lab, Ontario, Canada) and 20 µg/ml Candida albicans (Greer Lab Inc, Lenoir, NC) in triplicate.

IFN-{gamma} ELISPOT assay. HCV-specific CD4 Th1 cell responses were examined ex vivo by an IFN-{gamma} ELISPOT assay with 0.2 million PBMC/well in triplicate stimulated with recombinant HCV and control SOD proteins (10 µg/ml) for 42 h as described elsewhere (42, 43). The HCV-specific IFN-{gamma} response was calculated by subtracting the mean IFN-{gamma} spot-forming units (SFU) in control wells from that in HCV-stimulated wells and was then expressed as HCV-specific IFN-{gamma} SFU/million PBMC, excluding assays with high background (>10 dots/well in the negative control) or no PHA responses. A positive HCV-specific ELISPOT response also required at least 2/3 wells with SFU 3 standard deviations above the mean in negative control wells (42). Additional control experiments were performed for 64 chronic and 23 recovered subjects with 0.1 µg/ml tetanus toxoid.

Sequence heterogeneity calculation. Amino acid sequence variance from the prototype HCV-1 sequence was calculated by aligning 13 full-length HCV sequences from GenBank with genotypes 1a (HCV-1, HCV-H), 1b (Con-1, HCV-N, HCV-MD2), 2 (JCH-1, VAT96, HC-J6CH, MA, MD2b-1), 3 (HPCHK6, NZL1), and 4 (ED43).

Mapping of a dominant CD4 T-cell epitope in the NS4 region from a genotype 2-infected patient, C2#10. By using PBMC from a genotype 2-infected patient with a dominant CD4 T-cell response to 1a-derived NS3-4, HCV-specific T cells were expanded in bulk (4 million PBMC/well) with 10 µg/ml recombinant HCV NS3-4 protein and biweekly interleukin 2 (IL-2) (30 U/ml) for 3 weeks. The resulting NS4-specific CD4 T-cell line was cloned at limiting dilutions (0.5, 1, and 5 cells/well) with 100,000 autologous irradiated PBMC, 1 µg/ml HCV NS3-4, PHA (1 µg/ml), and 30 U/ml recombinant IL-2 in 96-well U-bottom plates. Further mapping with overlapping NS4-derived 15-mers in an IFN-{gamma} ELISPOT assay (first with pools of 5 peptides, followed by individual peptides in duplicates) identified NS4 1825-1a (AATAFVGAGLAGAAI) as the dominant NS4 epitope.

Sequence analysis of NS4 1825 in patient C2#10. The HCV sequence corresponding to the NS4 epitope region (NS4 1825) was amplified by nested PCR from cDNA reverse transcribed from total plasma RNA and cloned into the TA vector (Invitrogen, Carlsbad, CA) as described elsewhere (8). Fourteen clones were sequenced with the automated sequence analyzer at the University of Pennsylvania Sequencing Core laboratory (Philadelphia, PA) and aligned with an HCV-1 (1a) sequence. The virus-encoded NS4 1825V sequence was a highly conserved genotype 2 sequence, GATGFVVSGLVGAAV, containing 6/15 (40%) residues different (underlined) from NS4 1825-1a.

T-cell immunogenicity and cross-reactivity of virus-encoded NS4 1825V. T-cell lines were established from PBMC by initial stimulation with genotype 1a-derived recombinant HCV NS4 protein (10 µg/ml) or the NS4 1825V peptide (10 µM) and twice-weekly IL-2 (30 U/ml). The T-cell lines were tested for responsiveness to recombinant NS4, NS4 1825-1a, and NS4 1825V in IFN-{gamma} ELISPOT assays (20,000 T cells with 100,000 autologous PBMC as antigen-presenting cells) at several antigen concentrations (0, 1, 3, and 10 µM).

Statistical analysis. The clinical and immunological parameters of patient subgroups were compared using a nonparametric Wilcoxon rank sum test or the Mann-Whitney U test. Positive responder frequency for each assay was compared using a chi-square test or Fisher's exact test. Increasing trends in T-cell responsiveness between patient subgroups were compared using the Mantel-Haenszel chi-square test for relative frequency and the Jonckheere-Terpstra test for median values.


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RESULTS
 
Patients without genotype 1 infection display HCV serotype evidence for prior genotype 1 exposure. We first looked for serologic evidence of genotype 1 exposure in patients with and without genotype 1 infection by using an antibody serotyping assay that distinguishes between six major HCV genotypes as described in Materials and Methods (43). Supporting its specificity in our population, the HCV serotype was concordant with the molecular genotype for 90% of chronically infected patients (Table 2). For example, 92% of the genotype 1-infected C1 patients were serotype 1 responders, while 91% of the genotype 2-infected patients were serotype 2 responders. Most HCV-recovered subjects were serotype 1 responders (80%) as well, suggesting that genotype 1 viruses are spontaneously cleared at a similar rate as non-1 genotypes without being overrepresented in chronic infection. Finally, among C2-4 patients, infected with genotype 2, 3, or 4, three were serotype 1 responders while two others responded to both 1 and non-1 serotypes. Thus, 5/17 (29%) C2-4 patients showed a serotype 1 response consistent with prior genotype 1 virus exposure. Conversely, two C1 and two C2-4 patients showed mixed-serotype responses to both genotype 1 and non-1 viral antigens. These results suggest that patients are exposed to multiple HCV isolates/subtypes with persistence of one viral isolate/subtype but not others.


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  		TABLE 2. HCV antibody serotypes in patient subgroupsa

A T-cell response to genotype 1a antigens is detected in HCV-seropositive patients without genotype 1a infection. We then looked for CD4 T-cell-based evidence for previous genotype 1a exposure of HCV-seropositive individuals with and without chronic genotype 1a infection by using genotype 1a-derived HCV core, NS3-4, and NS5 antigens in a standard CD4 proliferation assay. As with the HCV serotype assay, CD4 T-cell responses to the 1a-derived HCV antigens were detectable in HCV-seropositive patients without concurrent 1a infection. Remarkably, the CD4 T-cell response to the 1a-derived HCV antigens was weakest in genotype 1a-infected C1a patients, increasing significantly as the circulating virus diverged further from 1a (Fig. 1). For example, only 19% of C1a patients responded to at least one 1a-derived HCV antigen, compared to 36% of C1b and 63% of C2-4 patients (P < 0.0001) (Table 3). None of the C1a patients responded to two or more HCV antigens, while 17% of C1b and 37% of C2-4 patients did (P < 0.0001). Similarly, fewer C1a patients than C1b or C2-4 patients responded to the nonstructural (NS) antigens (P < 0.0001). Significant differences were observed only for the NS antigens, not for the core antigen. A similar trend was apparent for HCV-specific CD4 T-cell IFN-{gamma} responses, as shown in Fig. 1C. This was not due to different ethnic distributions in the C1 and C2 groups (Table 1), since the genotypic differences in T-cell responsiveness persisted for both Caucasians and African-Americans (Fig. 2). These results provide T-cell-based evidence for prior genotype 1a exposure of many patients without chronic 1a infection.



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  		FIG. 1. CD4 T-cell responses to genotype 1a-derived HCV antigens in HCV-seropositive persons with and without genotype 1a infection. (A) The percentage of HCV-specific CD4 proliferative T-cell responders to 1a-derived HCV antigens in chronically HCV-infected patients showed a significantly increasing trend from genotype 1a (C1a) to 1b (C1b) and genotypes 2-4 (C2-4) for NS3-4 and NS5 but not HCV core. Inclusion of the recovered group (R) in the comparison enhanced the statistical significance of the trend: core (P = 0.0850), NS3-4 (P < 0.0001), NS5 (P < 0.0001). (B) HCV-specific CD4 T-cell proliferation (expressed as SI) to the 1a-derived HCV antigens showed a similar trend for the C1a, C1b, and C2-4 groups. Inclusion of the recovered group (R) enhanced the statistical significance of the trend: core (P = 0.0805), NS3-4 (P < 0.0001), NS5 (P < 0.0001). (C) The HCV-specific Th1 response (expressed as the number of IFN-{gamma} SFU/106 PBMC) to the 1a-derived HCV antigens tended to increase from C1a or C1b to C2-4 based on an IFN-{gamma} ELISPOT assay for the NS antigens, although this trend was not statistically significant (core, P = 0.94; NS3-4, P = 0.157; NS5, P = 0.2209). Including the recovered group (R) resulted in statistically significant trends for NS3-4 (P < 0.0001) and NS5 (P = 0.0018) but not for HCV core (P = 0.55). Horizontal lines in panels B and C indicate mean values. The statistical significance of increasing trends in HCV-specific T-cell responses between patient subgroups was determined using the Mantel-Haenszel chi-square test for comparison of relative frequency (A) and the Jonckheere-Terpstra test for comparison of mean values (B and C), with P values less than 0.05 considered to be significant.


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  		TABLE 3. Pattern of CD4 T-cell responses to genotype 1a-derived HCV antigens



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  		FIG. 2. The HCV-specific CD4 proliferative T-cell response is independent of African-American or Caucasian ethnicity. Comparison of percentages of responders for each of the genotype 1a-derived HCV core, NS3-4, and NS5 proteins shows that the genotypic differences persist in African-Americans and Caucasians. As previously reported, African-Americans tended to display greater T-cell responsiveness than Caucasians to the NS antigens among genotype 1-infected patients (African-Americans versus Caucasians: NS3-4, 17% versus 0% [P = 0.049]).

The CD4 T-cell response to genotype 1a antigens is directly related to the sequence divergence of the infecting viral genotype from the 1a-derived HCV antigens. Since the genotypic difference in CD4 T-cell response was observed for the variable NS regions but not the conserved core region, we asked if this difference may be related to potential sequence variations between the circulating virus and the antigenic 1a-derived proteins based on the HCV-1 isolate. Therefore, potential sequence divergences between HCV-1 and genotypes 1a, 1b, and 2 to 4 within the antigenic core, NS3-4, and NS5 regions were estimated using 12 published full-length HCV sequences as described in Materials and Methods. As expected, HCV core was highly conserved, with only 0%, 3%, and 7% amino acid differences for genotypes 1a, 1b, and 2 to 4, respectively. By contrast, there were greater genotypic differences within the NS regions among genotypes 1a, 1b, and 2 to 4: 2%, 9%, and 19% for NS3-4 and 2%, 15%, and 31% for NS5, respectively. Thus, the T-cell response to the 1a-derived HCV antigens was weakest when the circulating viral subtype was most homologous to the antigenic 1a protein tested, greater with decreased homology, and greatest in the complete absence of any homologous virus, suggesting a sequence-specific suppression of HCV-specific T cells in HCV persistence. Furthermore, the increased frequency of a 1a-specific T-cell response in patients with chronic non-1a infection suggests that the 1a-specific T cells were protective against 1a but not non-1a infection.

Genotypic differences in the HCV-specific T-cell response are not due to poor immunogenicity for genotype 1 viruses. An alternative interpretation for our finding is that genotype 1 viruses are intrinsically poor immunogens compared to other genotypes. Therefore, we examined the T-cell responses to genotype 1a-derived HCV antigens in spontaneously HCV recovered patients with and without a serotype 1 response. As shown in Fig. 3A and B, HCV-specific T-cell responses were more frequent (P = 0.02) and broader (P = 0.011), with a tendency to target the NS antigens (P = 0.008), in serotype 1-positive subjects than in serotype 1-negative subjects. Thus, genotype 1 viruses were highly immunogenic in the setting of spontaneous HCV clearance (but not persistence), and a robust genotype 1a-specific T-cell response provided a marker for previous self-limited 1a infection. Further supporting this notion, we detected CD45RO+ memory CD8 T cells specific for the 1a-derived HLA-A2-restricted epitope NS3 1073 ex vivo in some of the genotype 2-infected C2 patients despite sequence differences between genotypes 1a and 2 (CINGVCWTV versus SISGVLWTV or TISGILWTV) (Fig. 3C).



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  		FIG. 3. T-cell responses to genotype 1a-derived HCV antigens in recovered subjects relative to serotype. (A) CD4 proliferative T-cell responses to genotype 1a-derived HCV antigens in recovered serotype 1-positive and -negative subjects show significantly greater SI scores among serotype 1-positive (n = 23) than serotype 1-negative (n = 6) subjects. Horizontal lines indicate median values (P values by the nonparametric Mann-Whitney U test). Percentages of T-cell responder frequency, shown below, are based on cutoff values derived from normal seronegative controls as described in Materials and Methods; corresponding P values by Fisher's exact test are given. (B) The pattern of HCV-specific CD4 T-cell response in recovered serotype 1-positive and -negative subjects showed that serotype 1-positive subjects react more frequently and broadly, with a tendency to target the NS antigens, than serotype 1-negative subjects. (C) Ex vivo tetramer detection of CD45RO+ memory CD8 T cells specific for the 1a-derived HCV NS3 1073 epitope in peripheral blood of HCV-recovered and genotype 2-infected subjects is demonstrated for four spontaneously HCV recovered and four chronically genotype 2 infected subjects in fluorescence-activated cell sorter density plots for CD8-gated lymphocytes. The x axis shows CD45RO staining. The y axis shows staining with the HCV NS3 1073 tetramer. The recovered subjects were all serotype 1 responders. As shown, varying numbers of NS3 1073-specific CD8 T cells with and without the memory marker CD45RO are detectable in some of the C2 patients as well as the recovered subjects. By contrast, A2-positive normal control subjects showed few or no NS3 1073 tetramer-positive CD8 T cells. Four representative fluorescence-activated cell sorter results out of seven different control subjects studied are shown (bottom graphs, gating on the CD8+ T cells, with the x axis showing CD8 staining and the y axis showing NS3 1073 tetramer staining). The genotype 1a-derived NS3 1073 sequence used for tetramer synthesis was CINGVCWTV, while published genotype 2 sequences encode SISGVLWTV or TISGILWTV containing three amino acid variations.

Patients with chronic genotype 2 infection display T-cell responses to HCV peptides derived from genotype 1 but not 2. Another possibility is that non-genotype 1 viruses are extremely immunogenic and activate T cells targeting highly cross-reactive or conserved epitopes. We examined this possibility for 10 C2 patients, 11 C1 patients, and 8 HCV-recovered subjects by comparing their T-cell IFN-{gamma} responses ex vivo to three sets of 20 overlapping 15-mers derived from genotypes 1a, 2a, and 2b and spanning the NS4 1747-1875 region, which is frequently immunogenic in HCV-recovered subjects (42). As shown in Fig. 4A, 5/8 recovered subjects responded to the 1a-derived peptides, while 4/8 also responded to the 2a- and/or 2b-derived peptides. Two of 11 C1 patients also responded to genotype 2-derived peptides. Interestingly, only 1/10 C2 patients responded to 2a peptides while 5/10 responded to 1a peptides. Thus, genotype 2-infected patients were similar to genotype 1-infected patients in their suppressed response to the persistent viral strain and their strong response to the previously encountered but cleared viral strain.



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  		FIG. 4. T-cell responses to genotype 1a- or 2-derived HCV peptides. (A) T-cell IFN-{gamma} responses to genotype 1a-, 2a-, and 2b-derived NS4 peptides ex vivo in 11 genotype 1-infected (C1), 10 genotype 2-infected (C2), and 8 recovered (R) subjects show that 5/10 C2 patients respond to 1a-derived NS4 peptides but only 1/10 to 2a-derived peptides and 0/10 to 2b-derived peptides in an ex vivo IFN-{gamma} ELISPOT assay (y axis shows the number of IFN-{gamma}+ SFU/106 PBMC). Thus, like genotype 1-infected patients, genotype 2-infected patients show a suppressed response to the circulating viral antigen. Among HCV-recovered subjects, 5/8 responded to 1a-derived peptides while 3/8 responded to 2a-derived peptides and 4/8 responded to 2b-derived peptides, suggesting prior exposure to both genotype 1 and genotype 2 viruses. (B) Cross-reactivity and immunogenicity of an HCV-specific CD4 T-cell epitope, NS4 1825V, encoded by the circulating virus, compared to the genotype 1a prototype. The NS4 1825-1a epitope was mapped with overlapping 15-mers by using a CD4 T-cell line derived from a C2 patient’s PBMC expanded in vitro by 1a-derived NS3-4 and NS4 proteins (see Materials and Methods). The patient's viral isolate encoded the NS4 1825V sequence, which differed in 6/15 residues from 1825-1a in 14/14 PCR clones (sequences homologous to 1a are indicated by dashes). The virus-encoded NS4 1825V was identical to that encoded by 6/6 published genotype 2 sequences (JCH-1, VAT96, HC-J6CH, MA, MD2b-1, BEBE1). A T-cell line expanded by 1a-derived NS4 recognized NS4 1825-1a and NS4 protein, but not NS4 1825V, in an IFN-{gamma} ELISPOT assay (top bar graph). Thus, the genotype 2-derived NS4 1825V in the infecting virus was not cross-recognized by the circulating 1a-specific T cells. NS4 1825V was also not immunogenic in itself (bottom bar graph).

To define the relationship between the viral sequence and T-cell response more directly, we mapped the CD4 T-cell epitope in a genotype 2-infected patient as described in Materials and Methods. The dominant CD4 T-cell epitope was located within NS4 (NS4 1825-1a). However, the circulating virus coded for a genotypic variant sequence with 6/15 amino acid substitutions in 14/14 clones (NS4 1825V) that were entirely conserved in 6/6 published genotype 2 sequences. As shown in Fig. 4B, the NS4 1825V sequence was neither cross-recognized by NS4 1825-1a-specific T cells nor immunogenic in itself, suggesting that the 1a-specific T cells in this patient were not primed by the existing genotype 2 virus and may have even selected for the existing virus. These results further support a dynamic host-virus interaction in HCV infection.


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DISCUSSION
 
HCV has a high propensity to persist in otherwise immune-competent individuals (7, 30). While the true HCV persistence rate is difficult to establish, studies of patients with documented single HCV exposure or experimentally infected chimpanzees suggest rates less than 50%, while cross-sectional analyses of HCV-seropositive individuals show higher rates, up to 90% (2, 5, 16, 23). In addition to the transmission route, the viral titer, inoculum size, various host factors, and the number of HCV exposures may influence the probability for persistent infection. Another major consideration is the antiviral T-cell response. For example, HCV-recovered subjects uniformly display stronger, broader, and more-effective HCV-specific T-cell responses than patients with chronic evolution (7, 9, 11, 14, 21, 33, 39, 42-44). Furthermore, there is evidence for memory T-cell-mediated protective immunity to HCV in chimpanzee rechallenge experiments (21, 39). Along this line, among injection drug users, individuals who cleared one HCV infection were more resistant to a second infection (32), although that study did not include concurrent T-cell analyses. Nonetheless, even after initial T-cell-mediated HCV clearance, repetitive exposures could ultimately lead to chronic infection, particularly with a heterologous virus not recognized by memory T cells directed to the original infecting strain. In this scenario, robust T-cell responses to one virus might be detected in patients chronically infected with a different strain.

In this study, T-cell responses to genotype 1a-derived HCV antigens were detected in patients without active genotype 1a infection. For example, 63% of patients infected with genotype 2, 3, or 4 displayed significant T-cell responses to 1a antigens, with a vigor, scope, and specificity resembling that of HCV-recovered subjects, suggesting prior genotype 1a exposure. Serotype evidence of genotype 1 exposure was detected for 29% of patients with genotypes 2 to 4 (genotypes 2-4), a lower frequency than for T-cell responses, perhaps reflecting the greater need for antigen to maintain humoral compared to cellular immune responses to HCV (44). The detection of both B- and T-cell responses to genotype 1a-derived HCV antigens in genotype 2-4 patients (as well as in 1b-infected patients) supported our hypothesis of multiple viral exposure of HCV-infected patients. Thus, the persistent virus may be, not the only virus to which the patient was exposed, but perhaps the only virus that could not be eliminated.

T-cell responses to the 1a-derived HCV antigens were significantly greater in patients with heterologous non-1a virus infection than in those with homologous 1a infection, resulting in an apparent genotypic difference in HCV-specific CD4 T-cell responses. We asked if genotype 1 viruses were less immunogenic than those of other genotypes and perhaps less readily cleared in natural infection, as suggested by their poor clearance rate during IFN-{alpha}-based antiviral therapy (4, 12, 24, 34). However, most HCV-recovered subjects were serotype 1 responders, with robust responses to genotype 1a-derived antigens. Thus, genotype 1 viruses can be highly immunogenic and readily cleared without being overrepresented in the chronic groups. Furthermore, a robust 1a-specific T-cell response was a marker of self-limited genotype 1a infection.

While 1a-specific T cells may contribute to clearing homologous 1a virus, they may also increase the likelihood of heterologous viral infection. For example, for a patient with an initial self-limited 1a infection, a subsequent encounter with a heterologous, non-1a virus may activate T- and B-cell responses against the original 1a strain, but not the heterologous strain, through original "antigenic sin," thus promoting persistence of the heterologous strain (3, 26, 41). Indeed, in an injection drug user with a persistent serotype 1 response, an initial acute 1a infection was spontaneously cleared while a second, 3a infection was not (35). Since genotype 1 viruses are more prevalent than non-genotype 1 viruses in North America, most of our patients were probably exposed to genotype 1 before non-genotype 1 viruses. Thus, many patients with chronic non-1a infection (especially those with 1a-specific T-cell responses) may have initially cleared a 1a infection before succumbing to infection by a non-1 subtype. The genotype-specific protective immunity in our study differs from the cross-genotype protection recently reported for chimpanzees (28), albeit without concurrent T-cell analysis. While differences in species, age, inoculum size, or other host/environmental factors could account for this discrepancy, a prospective analysis of HCV-specific T-cell response and outcome in chimpanzees sequentially challenged with different HCV genotypes (or rare patients acutely infected with different HCV subtypes) is needed to formally address the questions of protective immunity, immune selection, and antigenic sin.

Interestingly, most studies correlating HCV-specific T-cell responses with antiviral therapy were performed with genotype 1-derived HCV antigens irrespective of the infecting HCV genotype (4, 12, 24, 34). Based on our findings, the CD4 T-cell responses detected in these published studies may not be related to the circulating virus but directed to previously cleared viruses. While such T-cell responses could still correlate with clinical outcomes, some observations in HCV immune pathogenesis may need to be reexamined in consideration of the nature of circulating HCV subtypes and target antigens used to study the T-cell response. Further studies are also needed to examine if T cells homing to the liver are more specific for the circulating virus or not (20).

A notable finding in our study was the suppressed T-cell response specific to the circulating viral strain in patients who could respond to a heterologous strain. Since genotype classification is sequence based, we propose that this apparent T-cell suppression could also be sequence specific. A prospective study (e.g., in an animal model) is needed to address this question and to determine if the apparent lack of strain-specific T-cell response occurs at the level of induction, maintenance, or function. Nonetheless, there is increasing evidence for HCV-specific T-cell dysfunction in HCV persistence. For example, HCV persistence may be associated with T-cell escape variants that may inactivate or antagonize the circulating virus-specific T cells, as shown for several viruses including HCV (6, 8, 25). HCV persistence is also associated with immunoregulatory factors, including HCV-specific IL-10+ T cells (1, 31, 45) and CD4+ CD25+ regulatory T cells (42). Chronic antigenic exposure can further induce anergic and regulatory T-cell subsets (10) or even T-cell exhaustion (18), suggesting that the apparent T-cell dysfunction could also be a consequence of HCV persistence. It would be interesting to examine if therapeutic HCV clearance can restore the virus-specific T-cell effector function and to explore immunotherapeutic strategies to counter-regulate these suppressive factors.

In conclusion, we report that T-cell-mediated protective immunity to HCV is strain specific and readily evaded by heterologous viruses. We also find evidence for strain-specific T-cell suppression in HCV persistence that warrants further investigation. An effective HCV vaccine must target highly conserved regions or be sufficiently broad to avoid selection of resistant variants while overcoming the strain-specific T-cell suppression.


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ACKNOWLEDGMENTS
 
This study was supported by NIH grants AI47519 and AA12849 and the NIH/NIDDK Center of Molecular Studies in Digestive and Liver Diseases (P30DK50306) and its Molecular Biology and Cell Culture core facilities. D.E.K. was supported by NIH T32 07066. This study was also supported in part by Public Health Service research grant M01-RR00040 from the National Institutes of Health.

We thank Michael Houghton and Kevin Crawford at Chiron Corporation for generous provision of the recombinant HCV antigens; David Parker, Brian Rodgers, and Lara Sandler at Murex Diagnostic for performing the HCV serotyping assay (as well as for helpful information about HCV serotyping); and John Lippolis at the NIH Tetramer Facility for generous provision of the HLA-A2 tetramers. We gratefully acknowledge Mary Valiga, Marcia Johnson, and Barbara Rensman for patient recruitment, and we thank the individuals who participated in this study at the PVAMC clinics, the hospital of the University of Pennsylvania, and the NIH-funded Clinical Research Center within the University of Pennsylvania. Acknowledgment is also given to the PVAMC Research Facility, where much of the study was performed.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine, GI Division, University of Pennsylvania, and Philadelphia VAMC, A212 Medical Research, PVAMC, University and Woodland Avenues, Philadelphia, PA 19104. Phone: (215) 823-5893. Fax: (215) 823-4394. E-mail: kmchang{at}mail.med.upenn.edu. Back

{dagger} Both K.S. and D.E.K. contributed equally to this work and share first coauthorship. Back


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Journal of Virology, June 2005, p. 6976-6983, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6976-6983.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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