Differential cytotoxic T-lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients.

Cytotoxic T lymphocytes (CTL) are thought to control hepatitis B virus (HBV) infection, since they are readily detectable in patients who clear the virus whereas they are hard to detect during chronic HBV infection. In chronic hepatitis C virus (HCV) infection, however, the virus persists in the face of a CTL response. Indeed, most infected patients respond to one or more HCV-1 (genotype 1a)-derived CTL epitopes in the core, NS3, and NS4 proteins, and the CTL response is equally strong in patients infected by different HCV genotypes, suggesting broad cross-reactivity. To examine the effect of the HCV-specific CTL response in patients with chronic hepatitis C on viral load and disease activity, we quantitated the strength of the multispecific CTL response against 10 independent epitopes within the HCV polyprotein. We could not detect a linear correlation between the CTL response and viral load or disease activity in these patients. However, the CTL response was stronger in the subgroup of patients whose HCV RNA was below the detection threshold of the HCV branched- chain DNA assay than in branched-chain-DNA-positive patients. These results suggest that the HCV-specific CTL response may be able to control viral load to some extent in chronically infected patients, and they indicate that prospective studies in acutely infected patients who successfully clear HCV should be performed to more precisely define the relationship between CTL responsiveness, viral clearance, and disease severity in this infection.     

Sequence heterogeneity of NS5A and core proteins of hepatitis C virus and virological responses to pegylated-interferon/ribavirin combination therapy

Both host and viral factors have been implicated in influencing the response to pegylated-interferon/ribavirin (PEG-IFN/RBV) therapy for hepatitis C virus (HCV) infection. Among the viral factors, sequence heterogeneity within NS5A and core regions has been proposed. This study aimed to clarify the relationship between virological responses to PEG-IFN/RBV therapy and sequence heterogeneity within NS5A, including the IFN/RBV resistance-determining region (IRRDR), the interferon sensitivity-determining region (ISDR) and the core region. Pretreatment sequences of NS5A and the core regions were analyzed in 57 HCV-1b-infected patients who were to be treated with PEG-IFN/RBV. Of 40 patients infected with HCV having an IRRDR with four or more mutations (IRRDR ≥ 4), 28 (70%) patients achieved a sustained virological response (SVR). On the other hand, only 4 (24%) of 17 patients infected with HCV having an IRRDR with three or fewer mutations (IRRDR ≤ 3) achieved a SVR (P = 0.001). Similarly, 22 (71%) of 31 patients infected with HCV and having an ISDR with one or more mutations (ISDR ≥ 1) achieved a SVR while 10 (38%) of 26 patients infected with HCV and having an ISDR without any mutations (ISDR = 0) achieved a SVR (P = 0.014). As for the core region, there was significant correlation between a single mutation at position 70 (Gln(70) ) and non-SVR (P = 0.02). Notably, Gln(70) was more prominently associated with the null response (P = 0.0007). In conclusion, sequence heterogeneity within the IRRDR and ISDR, and a single point mutation at position 70 of the core region of HCV-1b are likely to be correlated with virological responses to PEG-IFN/RBV therapy.     

Direct binding of a hepatitis C virus inhibitor to the viral capsid protein

Over 130 million people are infected chronically with hepatitis C virus (HCV), which, together with HBV, is the leading cause of liver disease. Novel small molecule inhibitors of Hepatitis C virus (HCV) are needed to complement or replace current treatments based on pegylated interferon and ribavirin, which are only partially successful and plagued with side-effects. Assembly of the virion is initiated by the oligomerization of core, the capsid protein, followed by the interaction with NS5A and other HCV proteins. By screening for inhibitors of core dimerization, we previously discovered peptides and drug-like compounds that disrupt interactions between core and other HCV proteins, NS3 and NS5A, and block HCV production. Here we report that a biotinylated derivative of SL209, a prototype small molecule inhibitor of core dimerization (IC(50) of 2.80 μM) that inhibits HCV production with an EC(50) of 3.20 μM, is capable of penetrating HCV-infected cells and tracking with core. Interaction between the inhibitors, core and other viral proteins was demonstrated by SL209-mediated affinity-isolation of HCV proteins from lysates of infected cells, or of the corresponding recombinant HCV proteins. SL209-like inhibitors of HCV core may form the basis of novel treatments of Hepatitis C in combination with other target-specific HCV drugs such as inhibitors of the NS3 protease, the NS5B polymerase, or the NS5A regulatory protein. More generally, our work supports the hypothesis that inhibitors of viral capsid formation might constitute a new class of potent antiviral agents, as was recently also shown for HIV capsid inhibitors.     

Genetic Clustering of Hepatitis C Virus Strains and Severity of Recurrent Hepatitis after Liver Transplantation

The influence of viral factors on the severity of hepatitis C virus (HCV)-related liver disease is controversial. We studied 68 liver transplant patients with recurrent hepatitis C, of whom 53 were infected by genotype 1 strains. Relationships between core sequences, serum HCV RNA levels, and fibrosis scores for each patient were analyzed in pairwise fashion 5 years after transplantation. We used Mantel's test, a matrix correlation method, to evaluate the correspondence between measured genetic distances and observed phenotypic differences. No clear relationship was found when all 68 patients were analyzed. In contrast, when the 53 patients infected by genotype 1 strains were analyzed, a strong positive relationship was found between genetic distance and differences in 5-year fibrosis scores (P = 0.001) and differences in virus load (P = 0.009). In other words, the smaller the genetic distance between two patients' viral core sequences, the smaller the difference between the two patients' fibrosis scores and viral replication levels. No relationship was found between genetic distance and differences in age, sex, or immunosuppression. In multivariate analysis, the degree of fibrosis was negatively related to the virus load (r = -0.68; P = 0.003). In the particular setting of liver transplantation, and among strains with closely related phylogenetic backgrounds (genotype 1), this study points to a correlation between the HCV genetic sequence and the variability of disease expression.     

Intrahepatic hepatitis C virus replication correlates with chronic hepatitis C disease severity in vivo

The role of viral factors in the pathogenesis of chronic hepatitis C is unknown. The objective of the present study was to characterize markers of hepatitis C virus (HCV) infection and replication in liver biopsy specimens obtained from 65 genotype 1-infected subjects, including 31 who were coinfected with human immunodeficiency virus (HIV), and to analyze associations between intrahepatic viral markers and hepatitis C disease severity. The percentages of liver cells harboring HCV genomes (%G) and replicative-intermediate RNAs (%RI) were evaluated using strand-specific in situ hybridization, while HCV core and NS3 antigens were assessed by immunocytochemistry. HIV-positive and HIV-negative subjects had similar mean grades and stages of liver disease and had similar indices of HCV infection and replication in liver, even though coinfected subjects had significantly shorter mean disease duration (P = 0.0003). Multivariate analysis showed that %G was not associated with grade or stage of liver disease (P = 0.5 and 0.4, respectively), while %RI was strongly associated with liver inflammation (P < 0.001), liver fibrosis (P < 0.001), and serum alanine aminotransferase levels (P = 0.01). NS3 antigen (but not core) was more frequently detected in HCV RI-positive versus RI-negative specimens (P = 0.028). These findings demonstrate a link between HCV proliferation and hepatitis C disease severity and suggest similar pathogenic mechanisms in HIV-positive and HIV-negative individuals.     

Viral sequence variation in chronic carriers of hepatitis C virus has a low impact on liver steatosis

Most clinical studies suggest that the prevalence and severity of liver steatosis are higher in patients infected with hepatitis C virus (HCV) genotype 3 than in patients infected with other genotypes. This may reflect the diversity and specific intrinsic properties of genotype 3 virus proteins. We analyzed the possible association of particular residues of the HCV core and NS5A proteins known to dysregulate lipid metabolism with steatosis severity in the livers of patients chronically infected with HCV. We used transmission electron microscopy to quantify liver steatosis precisely in a group of 27 patients, 12 of whom were infected with a genotype 3 virus, the other 15 being infected with viruses of other genotypes. We determined the area covered by lipid droplets in liver tissues and analyzed the diversity of the core and NS5A regions encoded by the viral variants circulating in these patients. The area covered by lipid droplets did not differ significantly between patients infected with genotype 3 viruses and those infected with other genotypes. The core and NS5A protein sequences of the viral variants circulating in patients with mild or severe steatosis were evenly distributed throughout the phylogenic trees established from all the collected sequences. Thus, individual host factors seem to play a much greater role than viral factors in the development of severe steatosis in patients chronically infected with HCV, including those infected with genotype 3 viruses.     

Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance

Hepatitis C virus (HCV) clearance has been associated with reduced viral evolution in targeted cytotoxic T-lymphocyte (CTL) epitopes, suggesting that HCV clearers may mount CTL responses with a superior ability to recognize epitope variants and prevent viral immune escape. Here, 40 HCV-infected subjects were tested with 406 10-mer peptides covering the vast majority of the sequence diversity spanning a 197-residue region of the NS3 protein. HCV clearers mounted significantly broader CTL responses of higher functional avidity and with wider variant cross-recognition capacity than nonclearers. These observations have important implications for vaccine approaches that may need to induce high-avidity responses in vivo.     

Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness

Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8(+) T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637), displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication.     

Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.     

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.     

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C.     

Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.     

Compensatory mutations restore the replication defects caused by cytotoxic T lymphocyte escape mutations in hepatitis C virus polymerase

While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.     

Frequent recovery and broad genotype 2 diversity characterize hepatitis C virus infection in Ghana,West Africa

Hepatitis C virus (HCV) infection is thought to mostly become chronic and rarely resolves. HCV infection was serologically screened in 4,984 samples from Ghanaian blood donors, and 1.3% prevalence was found. At least 53% of confirmed anti-HCV carriers had no detectable viral RNA and were considered to have cleared the virus and recovered from the infection. Confirmation was authenticated by the presence of antibodies specific to at least two viral antigens, mostly NS3 and E2. Reactivity to HCV core antigens was lower in Ghanaian than United Kingdom blood donors. The minority of chronically infected donors carried a viral load significantly lower than an unselected comparative group of United Kingdom blood donors (2.5 x 10(5) versus 2.9 x 10(6) IU/ml; P = 0.004). HCV genotype 2 was largely predominant (87%). Sequence clustering was similarly broad in the E1/E2 and NS5 regions. The phylogenetic diversity and the incapacity to distinguish subtypes within genotype 2 in our and others' West African strains suggested that West Africa may be the origin of HCV genotype 2. The genetic diversity extended to the identification of strains clearly separated from known subtypes of genotype 2 and genotype 1. One strain appears to be part of a new HCV genotype. HCV infection in Ghana is characterized by a high rate of recovery and the predominance of broadly divergent genotype 2 strains.     

Hepatitis C virus biology

Hepatitis C virus infection represents a major problem of public health with around 350 millions of chronically infected individuals worldwide. The frequent evolution towards severe liver disease and cancer are the main features of HCV chronic infection. Antiviral therapies, mainly based on the combination of IFN and ribavirin can only assure a long term eradication of the virus in less than half of treated patients. The mechanisms underlying HCV pathogenesis and persistence in the host are still largely unknown and the efforts made by researchers in the understanding the viral biology have been hampered by the absence of a reliable in vitro and in vivo system reproducing HCV infection. The present review will mainly focus on viral pathogenetic mechanisms based on the interaction of HCV proteins (especially core, NS3 and NS5A) with host cellular signaling transduction pathways regulating cell growth and viability and on the strategies developed by the virus to persist in the host and escape to antiviral therapy. Past and recent data obtained in this field with different experimental approaches will be discussed.     

Strain-specific T-cell suppression and protective immunity in patients with chronic hepatitis C virus infection

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.     

A concerted action of hepatitis C virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.     

Broad specificity of virus-specific CD4+ T-helper-cell responses in resolved hepatitis C virus infection

Vigorous virus-specific CD4+ T-helper-cell responses are associated with successful control of hepatitis C virus (HCV) and other human viral infections, but the breadth and specificity of responses associated with viral containment have not been defined. To address this we evaluated the HCV-specific CD4+ T-helper-cell response in HCV antibody-positive persons who lack detectable plasma viremia, and compared this response to that in persons with chronic HCV infection. Peripheral blood mononuclear cells were stimulated with HCV proteins, followed by measurement of HCV-specific CD4+ T-cell responses to a comprehensive set of overlapping HCV peptides by intracellular gamma interferon production. In three persons with resolved HCV infection studied in detail, 13 to 14 epitopes were targeted, but none was recognized by all three. The 37 defined epitopes were predominantly distributed among the HCV proteins core, NS3, NS4, and NS5. In an expanded analysis of responses to these proteins in persons with resolved infection, an average of 10 epitopes was targeted, whereas in persons with chronic viremia never was more than one epitope targeted (P < 0.001). This comprehensive analysis of the breadth and specificity of HCV-specific T-helper-cell responses indicates that up to 14 viral epitopes can be simultaneously targeted by circulating virus-specific CD4+ T helper cells in a controlled human viral infection. Moreover, these data provide important parameters for evaluation of candidate HCV vaccines, and provide rationale for immunotherapy in chronic HCV infection.