Strain-Specific T-Cell Suppression and Protective
http://www.100md.com
病菌学杂志 2005年第11期
Division of Gastroenterology, Department of Medicine, University of Pennsylvania, and Philadelphia Veterans Affairs Medical Center
Pennsylvania Hospital, Philadelphia, Pennsylvania
Oregon Health Sciences University, Portland, Oregon
Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland
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.
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.
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).
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-) 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- ELISPOT assay. HCV-specific CD4 Th1 cell responses were examined ex vivo by an IFN- 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- response was calculated by subtracting the mean IFN- spot-forming units (SFU) in control wells from that in HCV-stimulated wells and was then expressed as HCV-specific IFN- 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- 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- 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.
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.
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- 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.
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).
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- 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.
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.
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--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.
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.
Both K.S. and D.E.K. contributed equally to this work and share first coauthorship.
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Pennsylvania Hospital, Philadelphia, Pennsylvania
Oregon Health Sciences University, Portland, Oregon
Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland
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.
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.
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).
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-) 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- ELISPOT assay. HCV-specific CD4 Th1 cell responses were examined ex vivo by an IFN- 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- response was calculated by subtracting the mean IFN- spot-forming units (SFU) in control wells from that in HCV-stimulated wells and was then expressed as HCV-specific IFN- 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- 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- 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.
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.
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- 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.
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).
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- 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.
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.
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--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.
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.
Both K.S. and D.E.K. contributed equally to this work and share first coauthorship.
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