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Journal of Virology, December 2002, p. 12584-12595, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12584-12595.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Broad Specificity of Virus-Specific CD4+ T-Helper-Cell Responses in Resolved Hepatitis C Virus Infection

Cheryl L. Day,1,2 Georg M. Lauer,1,2 Gregory K. Robbins,1,2 Barbara McGovern,3,4 Alysse G. Wurcel,1,2,4 Rajesh T. Gandhi,1,2 Raymond T. Chung,5 and Bruce D. Walker1,2*

Partners AIDS Research Center,1 Infectious Disease Unit,2 Gastrointestinal Unit,5 Massachusetts General Hospital and Harvard Medical School, and Division of Infectious Diseases, New England Medical Center,3 Lemuel Shattuck Hospital Boston, Massachusetts4

Received 23 May 2002/ Accepted 3 September 2002


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ABSTRACT
 
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.


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INTRODUCTION
 
Chronic viremia and progressive disease related to persistent viral infections such as hepatitis C virus (HCV) represent a major global health problem. HCV infects an estimated 170 million persons worldwide and more than 10% of the population in some regions (9, 31). The majority of HCV-infected individuals develop chronic infection associated with persistent viremia, and infection with HCV has become the major indication for liver transplantation (1, 31). Although 20 to 50% of HCV-infected individuals are able to spontaneously resolve HCV infection, as indicated by the absence of detectable HCV RNA in plasma, the determinants of viral clearance following acute HCV infection versus establishment of chronic HCV infection are not clearly defined (reviewed in reference 31).

Increasing evidence indicates that the induction of a strong cellular immune response involving both cytotoxic T lymphocytes and CD4+ T helper cells is essential for spontaneous control of chronic viral infections (reviewed in reference 26). Previous studies of individuals with acute HCV infection who went on to resolve hepatitis and clear HCV viremia demonstrate strong proliferative responses by CD4+ T helper cells upon stimulation with HCV proteins (10, 12, 19, 32, 35, 47). Furthermore, recurrence of HCV viremia has been shown to occur following the loss of HCV-specific CD4+ T helper cells during acute HCV infection (19). Similar data regarding a key role of virus-specific T-helper-cell responses in controlling chronic viral infections have been reported for a number of other chronic murine or human viral infections, including lymphocytic choriomeningitis virus, gammaherpesviruses, and human immunodeficiency virus (2, 5, 34, 38, 39, 43, 44, 46, 48, 49, 53).

Despite increasing evidence that virus-specific CD4+ T-helper cells play an important role in HCV infection and other infections, the breadth and specificity of the virus-specific T-helper-cell responses associated with a good outcome are not well-defined. In HCV infection, numerous studies examining the ability of peripheral blood mononuclear cells (PBMC) to proliferate in response to stimulation with viral antigen indicate that a variety of viral proteins can serve as targets for this response and that responses are greater in magnitude in those who control infection spontaneously compared to those who develop chronic viremia (6, 12, 17, 19, 35, 41, 47). Of the few HCV CD4+ epitopes defined thus far, some have been defined by epitope prediction algorithms (20), and a few have been identified using selected peptides (11, 24, 25, 27, 30, 33). However, no studies have addressed all the potential targets within the expressed HCV proteins or the overall breadth of the HCV-specific T-helper-cell response within a single individual. Indeed, such comprehensive analysis of CD4 responses in human and murine chronic viral infections has yet to be reported.

Given the association between a strong CD4+ T-helper-cell response and spontaneous resolution of HCV viremia (19, 42, 47), dissection of the specific components of this response is important in order to gain insight into the mechanisms of immunological control of HCV infection. In this study, we performed a comprehensive analysis to determine the breadth and specificity of HCV-specific T-helper-cell responses associated with spontaneous control of HCV viremia in individuals who are persistently HCV antibody positive and HCV RNA negative. Gamma interferon (IFN-{gamma}) production in response to overlapping HCV peptides spanning all expressed proteins was measured using HCV-specific cell lines generated from three such individuals and then extended to a larger cohort of persons with different disease outcomes. This comprehensive study of the total virus-specific T-helper-cell response using sensitive and specific new techniques indicates that multiple viral epitopes are simultaneously targeted by the CD4+ T-helper-cell response in a controlled human viral infection and that these broad responses persist in the absence of detectable viremia.


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MATERIALS AND METHODS
 
Subjects. Six subjects with spontaneously controlled HCV infection and eight subjects with chronic HCV infection were randomly enrolled in this study that was approved by the Institutional Review Boards of the Massachusetts General Hospital and the Lemuel Shattuck Hospital; all subjects gave written informed consent. All subjects were anti-HCV positive as measured by enzyme immunoassay, and no subjects were treated with HCV antiviral therapy. HCV RNA was measured by the Roche Amplicor Monitor assay (detection limit of 300 HCV RNA copies/ml of plasma). All individuals with spontaneously controlled HCV viremia had documented undetectable plasma viral loads (<300 HCV RNA copies/ml of plasma) at multiple time points for more than 7 months (range, >7 months to >2.5 years). The HCV viral loads of the individuals with chronic HCV infection ranged from 2,240 to 850,000 HCV RNA copies/ml of plasma. Serum alanine transaminase (ALT) was normal (less than 28 U/liter) in the individuals with resolved HCV infection, with the exception of subject 1, who had intermittent mild elevation in ALT levels (range, 23 to 72 U/liter) attributed to chronic HBV infection. Serum ALT of the subjects with chronic HCV infection ranged from 11 to 207 U/liter (mean, 75 U/liter).

Synthesis of HCV-derived peptides. Peptides corresponding to the amino acid sequences of the HCV-1a strain were synthesized as free acids using the 9-fluorenylmethoxy carbonyl method. Peptides were 20 amino acids (aa) in length, overlapping adjacent peptides by 10 aa.

HLA typing. HLA typing was performed by the Massachusetts General Hospital Tissue Typing Laboratory by standard serological and molecular techniques (3, 4). The HLA class II alleles of three subjects studied in detail are as follows: subject 1: DRB1*11, DQB1*03; subject 2: DRB1*04, DRB1*10, DQB1*01, DQB1*03; and subject 3: DRB1*04, DRB1*08, DQB1*03.

Recombinant HCV proteins. The recombinant HCV proteins used in this study were expressed as carboxy-terminal fusion proteins with human superoxide dismutase in Saccharomyces cerevisiae or Escherichia coli and were kindly provided by Michael Houghton (Chiron Corporation, Emeryville, Calif.). These proteins were derived from the HCV-1 sequence and encoded core (C22-3 aa 2 to 120), NS3 (C33C aa 1192 to 1457), NS4 (C100 aa 1569 to 1931), NS3/NS4 (C200 aa 1192 to 1931), and NS5 (NS5 aa 2054 to 2995).

Proliferation assays. Lymphocyte proliferation assays were performed using the HCV proteins described above at concentrations of 10 µg/ml. PBMC were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation and plated at 100,000 cells/well in 96-well U-bottom plates (Costar) in 200 µl of R10/HAB medium (RPMI 1640 (Sigma-Aldrich), 10% human AB serum (Sigma-Aldrich), and 10 mM HEPES buffer (Sigma-Aldrich) with 2 mM glutamine and antibiotics (penicillin-streptomycin, 50 U/ml) and the designated proteins in quadruplicate wells. After a 6-day incubation at 37°C and 5% CO2, wells were pulsed for 6 h with 1 µCi of [3H]thymidine (NEN). Cells were then collected on filters, and the amount of incorporated radiolabel was measured with a beta counter. For the purposes of data interpretation, a stimulation index of 5 or more was considered significant.

Bulk stimulation of PBMC. Ten million fresh PBMC were stimulated with 1-µg/ml recombinant HCV antigens (either C22-3, C33C, C100, C200, or NS5) in 10 ml of R10 medium supplemented with recombinant interleukin-2 (IL-2) (50 U/ml). Alternatively, for the regions of the HCV genome for which recombinant antigens were not available (E1, E2, p7, and NS2), 10 x 106 PBMC were stimulated with overlapping 20-mer peptides (1 µg/ml each) in pools of 10 peptides. After 10 to 14 days the cells were assayed for IFN-{gamma} production by intracellular cytokine staining in response to stimulation with pools of overlapping 20-mer HCV peptides.

Establishment of HCV-specific CD4+ T-helper-cell clones. Fresh PBMC were stimulated with recombinant HCV protein (1 µg/ml) in 10 ml of R10 medium supplemented with recombinant IL-2 (50 U/ml) as described above. After 14 days, bulk stimulated PBMC were plated at limiting dilution (10, 5, and 1 PBMC per well) and cultured with allogeneic irradiated PBMC at 105 cells/well in a final volume of 200 µl of R10 medium per well with recombinant IL-2 (50 U/ml). Plates were incubated for 2 weeks in 5% CO2 at 37°C and fed twice weekly with medium exchanges. Contents of wells showing growth after 14 to 21 days were transferred to 24-well plates and restimulated with 106 irradiated allogeneic PBMC and the CD3-specific monoclonal antibody (MAb) 12F6 (0.1 µg/ml), followed by testing for proliferation using the same protocol as used for PBMC.

Intracellular IFN-{gamma} staining and flow cytometry. Intracellular cytokine staining was performed as previously described (21, 38). Briefly, 5 x 105 bulk expanded PBMC were added to 105 autologous B cells and incubated with 4 µM peptide at 37°C and 5% CO2 for 1 h before the addition of GolgiPlug (1 µl/ml; Becton Dickinson). The cells were incubated for an additional 5 h at 37°C and 5% CO2. PBMC were then washed and stained with surface antibodies, allophycocyanin-conjugated anti-CD3 and peridin-chlorophyll protein-conjugated anti-CD4 (Becton Dickinson) at room temperature for 20 min. Following washing, the PBMC were fixed and permeabilized (Caltag, Burlingame, Calif.), and the fluorescein isothiocyanate (FITC)-conjugated anti-IFN-{gamma} MAb (Becton Dickinson) was added. Cells were then washed and analyzed on a FACS-Calibur flow cytometer using CELLQuest software (Becton Dickinson). For ex vivo intracellular cytokine staining of fresh PBMC, 106 fresh PBMC were stimulated with 4 µM peptide and anti-CD28 and anti-CD49d MAbs (1 µg/ml each; Becton Dickinson) and processed as described above. Cells were stained for cytokine production with either FITC-conjugated anti-IFN-{gamma} MAb or FITC-conjugated anti-tumor necrosis factor alpha (anti-TNF-{alpha}) MAb (Becton Dickinson).

HLA restriction. Autologous and partially HLA-matched Epstein-Barr virus-transformed B-lymphoblastoid cell lines (BCL) were pulsed with 0.1 µM peptide for 1 h at 37°C and 5% CO2. Peptide-pulsed BCL were then washed twice and incubated with HCV-specific CD4+ T-helper-cell clones at a ratio of 105 BCL to 5 x 105 CD4+ clones in 1 ml of R10 medium. Intracellular cytokine staining for IFN-{gamma} production was then carried out as described above.

Statistical analysis. The difference between the numbers of CD4+ T-helper-cell epitopes recognized by subjects with spontaneously resolved HCV infection and subjects with chronic HCV infection was analyzed by an exact Wilcoxon signed-ranks test. Values of P that were <0.05 were considered statistically significant.


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RESULTS
 
HCV-specific T-helper-cell responses to recombinant HCV proteins in resolved HCV infection. In the initial phase of this study, the HCV-specific T-helper-cell responses in three individuals who had spontaneously resolved HCV infection were comprehensively analyzed. All three subjects consistently exhibited broadly directed T-helper-cell proliferative responses against multiple HCV proteins, as measured by tritiated thymidine incorporation (Fig. 1). Each subject responded to at least three of the four antigens tested, with stimulation indices ranging as high as 124. Longitudinal analysis indicated that these responses were persistent over time in all three persons (data not shown). These data using traditional lymphocyte proliferation assays in persons controlling HCV infection show the persistence of HCV-specific T-helper-cell responses to multiple regions of the virus despite absence of detectable viremia.



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  		FIG. 1. Lymphocyte proliferation by fresh PBMC in response to the recombinant antigens C22-3, C33C, C100, and NS5 for subjects 1, 2, and 3 with spontaneously resolved HCV infection. At the time of study, all three subjects had been anti-HCV positive and HCV RNA negative for more than 2.5 years.

Comprehensive analysis of HCV-specific T-helper-cell responses by bulk stimulation of PBMC and intracellular IFN-{gamma} staining. The above experiments indicate that the CD4+ T-helper-cell response is directed at multiple regions of the virus in individuals who spontaneously controlled HCV viremia. However, they do not address the number of epitopes targeted in each of the larger viral proteins tested, nor do they comprehensively test all expressed viral proteins. To address these issues, fresh PBMC were initially tested against recombinant HCV antigens and pools of overlapping 20-mer HCV peptides in a 6-h flow cytometric assay assessing intracellular IFN-{gamma} production, as described for other viruses (38). However, the low ex vivo frequency of HCV-specific CD4+ T-helper cells made it difficult to clearly identify specific peptides targeted (data not shown).

In an attempt to enhance the sensitivity of this assay, HCV-specific T-cell lines were generated by stimulating freshly isolated PBMC in separate cultures with the recombinant HCV antigens C22-3, C33C, C200, C100, or NS5, in the presence of IL-2. To assess responses to those regions for which recombinant antigens were not available (aa 121 to 1190, encoding E1, E2, p7, and NS2), PBMC were stimulated with pools of overlapping peptides spanning the respective regions. The expanded cell lines were then tested for HCV-specific activity by IFN-{gamma} production in an intracellular cytokine staining assay.

For all three subjects, separate pools of 10 to 30 peptides were tested initially to screen the in vitro-expanded cell lines for virus-specific IFN-{gamma} production, and representative data from subject 2 are shown in Fig. 2. Brief in vitro stimulation resulted in expansion of HCV-specific T-helper-cell populations with antigen-specific cells ranging from 8.7% CD3+ CD4+ IFN-{gamma}+ cells in the C33C-stimulated culture to 25.8% CD3+ CD4+ IFN-{gamma}+ cells in the C100-stimulated culture. Similar expansion was achieved in subjects 1 and 3 (data not shown). Stimulation of PBMC from four HCV seronegative controls with recombinant HCV antigens did not result in expansion of any HCV peptide-specific IFN-{gamma} producing cells (data not shown), indicating that stimulation of fresh PBMC with recombinant HCV proteins does not induce primary HCV-specific responses.



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  		FIG. 2. Expansion of HCV-specific CD4+ populations by stimulation of PBMC with recombinant HCV antigens in subject 2. HCV-specific activity of cell lines generated by separate stimulation with each of four antigens (core C22-3, NS3 C33C, NS4 C100, and NS5) were tested with pools of overlapping peptides corresponding to the sequence encoded by the whole antigen. The percentage of CD3+ CD4+ IFN-{gamma}+ cells is indicated in the upper right quadrant of each plot, and results are shown for those antigens resulting in positive responses. The recombinant antigen used to generate each line is indicated to the left of the dot plots.

Peptide pools yielding positive responses for HCV peptide-specific IFN-{gamma} production were further deconvoluted by individually testing for responses to each of the constituent peptides in the positive pools. Figure 3 indicates a representative deconvolution of the positive pool of peptides 150 to 169 (aa 1501 to 1710) in C100-stimulated cells from subject 2 for which peptide 162 (NS3 1621 to 1640) was identified to be targeted. Single peptides that elicited responses of more than three times background IFN-{gamma} production were considered positive. For every positive response to a pool of peptides, at least one peptide contained within the pool was identified to be targeted (data not shown).



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  		FIG. 3. Identification of individual 20-mer peptides by deconvolution of positive peptide pools in cell lines generated by stimulation of PBMC with recombinant HCV antigens. Deconvolution of peptide pool 150-169 (Fig. 2) in a C100-stimulated PBMC cell line from subject 2 indicates a positive response to the NS3 peptide aa 1621 to 1640. Peptides 150-169 were all negative (data not shown). The percentage of CD3+ CD4+ IFN-{gamma}+ cells is indicated in the upper right quadrant of each plot.

All positive peptide pools were systematically deconvoluted as described above to identify single 20-mer HCV peptides targeted by the CD4+ T-helper-cell response in each subject (Fig. 4 and Table 1). The majority of peptides identified were contained within nonstructural regions of the HCV genome and were identified in PBMC cultures stimulated with recombinant HCV proteins encoding NS3, NS4, and NS5. However, PBMC from each subject were also stimulated with pools of overlapping peptides covering the C-terminal end of core, E1, E2, p7, NS2, and the N-terminal end of NS3 regions of the virus, for which recombinant proteins were not available. When these cultures were tested after 10 to 14 days by intracellular IFN-{gamma} staining, positive responses were detected against the core peptide aa 141 to 160 in subject 1, NS2 peptide aa 1001 to 1020 in subject 2, and NS3 peptide aa 1131 to 1150 in subject 3 (Fig. 4 and data not shown). In all, 11 epitopes were identified for subject 1 (Fig. 4A), in whom the most responses were detected in the NS5 protein (four epitopes); 13 HCV peptides were identified that were targeted by the CD4+ T-helper-cell response in subject 2, with the most in NS3 (six epitopes); and 13 CD4+ T-helper-cell epitopes were identified in subject 3, with responses to NS5 (seven epitopes) predominating (Fig. 4C). The presence of HCV peptide-specific CD4+ T cells was confirmed a second time in each subject using the same assay from PBMC obtained 2 to 4 months later (data not shown). These results indicate that T-helper-cell responses to multiple epitopes contained within several HCV proteins are maintained in the absence of detectable plasma viremia. They also indicate that these cells are readily expandable in vitro following stimulation with appropriate antigens.



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  		FIG. 4. Percent CD3+ CD4+ IFN-{gamma}+ cells for discrete 20-mer peptides in HCV antigen-stimulated PBMC cultures in subjects 1 (A), 2 (B), and 3 (C). The background percent IFN-{gamma} production for each cell line in the absence of peptide has been subtracted. The percent IFN-{gamma} production for each peptide indicated was greater than three times background IFN-{gamma} production in all cases.


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  		TABLE 1. Summary of HCV epitopes targeted by the CD4+ T-helper-cell response

Identification of additional subdominant T-helper-cell epitopes by limiting dilution cloning. In order to further confirm the presence of HCV-specific CD4+ T helper responses and examine HLA restriction, HCV-specific clones were generated from subject 1. These studies confirmed the presence of HCV-specific T-helper cells specific for the peptides identified by bulk expansion of PBMC as described above, but also revealed additional epitopes not detected in the bulk-expanded cells. CD4+ T-helper-cell clones were isolated that were specific for all previously identified epitopes targeted by this subject (Fig. 4A) that are contained within the HCV proteins NS3, NS4, and NS5 (data not shown). Three additional clones were isolated from subject 1 that were specific for subdominant HCV epitopes that had not been detected in the expanded cell lines (Fig. 5 and data not shown). The specificity of these clones included NS4 aa 1671 to 1690, NS4 aa 1891 to 1910, and NS5 aa 2841 to 2860.



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  		FIG. 5. NS5-specific CD4+ T-helper cells are restricted by HLA DRB1*11 in subject 1. Peptide-pulsed autologous and partially HLA-matched B cells were incubated with the NS5 peptide-specific clone from subject 1 for 6 h and stained for CD3, CD4, and IFN-{gamma} and analyzed by flow cytometry. The percentage of CD3+ CD4+ IFN-{gamma}+ cells is indicated in the upper right quadrant of each plot.

The generation of CD4+ T-helper-cell clones in subject 1 also provided a means of assessing the HLA restriction of these HCV-specific CD4+ T helper cells. Clones were stimulated with peptide-pulsed autologous and partially HLA-matched B-cell lines, and IFN-{gamma} production was measured. Representative data demonstrating HLA class II restriction of a peptide 284-specific CD4+ clone from subject 1 are shown in Fig. 5. Peptide-pulsed DQB1*03-matched BCL were unable to stimulate IFN-{gamma} production by the clone, whereas DRB1*11-matched BCL induced IFN-{gamma} production comparable to autologous peptide-pulsed BCL, indicating that peptide 284 is DRB*11-restricted in this subject.

Comparison of the breadth of the HCV-specific CD4+ T-helper-cell response in spontaneously resolved and chronic HCV infection. The above studies suggest broadly directed HCV-specific CD4+ T-helper-cell responses are associated with spontaneous control of viremia. In order to determine whether there was a significant difference in the HCV-specific CD4+ T-helper-cell response in individuals with spontaneously controlled versus chronic HCV infection, the same assay as described above was used in an expanded cohort of persons with revolved viremia and persons with persistent viremia. This analysis focused on assessment of responses to those proteins for which recombinant proteins were available (core, NS3, NS4, and NS5), since these proteins had been shown to contain the dominant epitopes targeted (Fig. 4). Freshly isolated PBMC from three additional individuals with spontaneously controlled HCV infection and eight individuals with chronic HCV infection were stimulated with the recombinant HCV antigens and tested with pools of 10 overlapping 20-mer peptides as described above. Of the three additional subjects with spontaneously controlled HCV infection analyzed in this manner, multiple HCV-specific CD4+ T-helper-cell epitopes were identified (median, 10.5 epitopes; range, 4 to 11 epitopes). In contrast, when PBMC from eight individuals with chronic HCV infection were stimulated with recombinant antigens spanning core, NS3, NS4, and NS5 proteins, only one of eight had a positive response, and this was to a single peptide pool. This response was further deconvoluted and found to be directed against a single peptide (NS5 aa 2531 to 2550) with a maximum of 1.1% CD3+ CD4+ IFN-{gamma}+ cells in the NS5-stimulated culture from this subject (data not shown). The breadth of HCV-specific T-helper-cell epitopes identified following stimulation was then compared in the two groups. Statistical analysis revealed the difference in the breadth of CD4+ T-helper-cell epitopes between individuals with resolved HCV infection and individuals with chronic HCV infection to be highly significant (P < 0.001) (Fig. 6). These data indicate that expandable, broadly directed HCV-specific CD4 T-helper-cell responses are lacking in persons with chronic HCV infection.



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  		FIG. 6. Spontaneous resolution of HCV viremia is associated with multiple CD4+ T-helper-cell epitopes. PBMC from six individuals with resolved HCV infection and eight individuals with chronic HCV infection were stimulated with the recombinant antigens C22-3, C33C, C200, and NS5 covering the core, NS3, NS4, and NS5 proteins. After 10 to 14 days, expanded cell lines were tested for IFN-{gamma} production upon stimulation with pools of 10 to 20 overlapping peptides each. All peptide pools eliciting a positive response by IFN-{gamma} production were further deconvoluted to identify specific 20-mer epitopes targeted by the CD4+ T-helper-cell response.

Direct detection of circulating HCV-specific CD4 T-helper-cell responses. The above studies show that up to 14 different epitopes can be targeted by the CD4+ T-helper-cell response in persons who control HCV viremia, but do not provide a direct measure of the magnitude of these responses in the peripheral blood in resolved HCV infection. Sufficient fresh PBMC samples were available from subjects 1 and 2 to address this issue. Peptides that generated a positive response in bulk cultures from these two subjects were tested individually or pooled together in a 6-hour intracellular cytokine staining assay using fresh PBMC. Cells were stained separately for IFN-{gamma} and TNF-{alpha} production, both of which are implicated in effector function of these cells (14, 18, 22, 23). Figure 7 indicates the frequency of cytokine producing CD4+ T cells in fresh PBMC stimulated with peptide pools containing each of the HCV CD4+ peptides recognized by subjects 1 and 2 (Table 1). For both subjects, TNF-{alpha} production was more readily detectable in fresh PBMC in response to the HCV peptide pools than IFN-{gamma} production, consistent with previous findings (42). For subject 1, 0.19 and 0.88% of CD3+ CD4+ T cells are IFN-{gamma}+ and TNF-{alpha}+, respectively, when stimulated with a pool of all-positive peptides. Similarly for subject 2, 0.13 and 0.70% of CD3+ CD4+ T cells are IFN-{gamma}+ and TNF-{alpha}+, respectively, upon stimulation with the pool of peptides recognized by this subject. These results indicate the responses to the peptides identified by bulk stimulation of PBMC are detectable in circulating fresh PBMC by ex vivo intracellular cytokine staining. Responses were also tested by fresh PBMC to individual peptides recognized by subjects 1 and 2 as listed in Table 1 (data not shown). The epitope generating the highest frequency of IFN-{gamma}+ (0.09%) or TNF-{alpha}+ (0.31%) CD4+ T cells ex vivo in subject 1 was peptide 120 (NS3 aa 1201 to 1220), which also elicited the most dominant response in the expanded cell lines in this subject as indicated in Fig. 4. The peptide generating the highest frequency of CD3+ CD4+ IFN-{gamma}+ cells (0.06%) in subject 2 was NS3 aa 1621 to 1640 (data not shown), which was also the epitope that elicited the most dominant response in the NS3-expanded PBMC cultures from this subject (Fig. 4). Together these data indicate that HCV-specific CD4+ cells are present in significant numbers in the circulation during prolonged periods of undetectable viremia, and these cells can rapidly mediate effector function following encounter with the relevant viral protein.



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  		FIG. 7. Ex vivo intracellular cytokine staining with fresh PBMC for IFN-{gamma} (A) or TNF-{alpha} (B) in subjects 1 and 2. Positive peptide responses as identified by bulk stimulation of PBMC were pooled together and used to stimulate fresh PBMC for 6 h and then stained intracellularly for cytokine production. For each subject, the peptide pool tested consisted of the peptides identified as targeted by the CD4+ T-helper-cell response as listed in Table 1.


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DISCUSSION
 
Increasing evidence indicates that virus-specific T-helper-cell responses are critical for persistent immune control in HCV and other chronic viral infections (5, 26, 34, 40), but the breadth and specificity of responses associated with controlled viral infection has not been determined. In HCV infection, up to 1012 virions are produced per day (29) in the chronic phase of infection, but there is a subset of persons who are able to achieve and maintain undetectable levels of plasma viremia following infection (32, 47). In this detailed study of persons who control HCV viremia, a strikingly broadly directed HCV-specific T-helper-cell response targeting up to 14 HCV epitopes was detected in circulating peripheral blood lymphocytes. In contrast, responses to more than a single epitope were never detected in persons with persistent viremia. Although the absolute magnitude of responses in freshly isolated PBMC in the controllers was low, these cells were capable of rapid expansion upon in vitro stimulation. These results indicate that multiple epitopes can be simultaneously and persistently targeted by circulating T-helper cells in the absence of ongoing plasma HCV viremia, and indicate an unprecedented breadth of responses that remain in the circulation despite undetectable viremia.

Previous studies of the CD4+ T-helper-cell response in HCV infection have indicated that a majority of individuals with resolved HCV infection display proliferative responses to one or more HCV antigens, whereas individuals with chronic infection typically have few to no detectable responses (6, 17, 19, 35, 37, 41). Our results confirm the presence of strong CD4+ proliferative responses associated with the control of viremia but also extend those studies by indicating that most expressed proteins contain multiple simultaneously targeted epitopes that can be identified using panels of synthetic peptides. The multiple epitopes identified in each subject in this study likely represent the minimal breadth of the total HCV-specific CD4+ T-helper-cell response. Antigen-specific cells of a very low precursor frequency may be less readily expanded in the in vitro cultures, as evidenced by the identification of additional epitopes by limiting dilution cloning that were not detected in the expanded PBMC cultures. Furthermore, the greatest number of HCV-specific CD4+ T-helper-cell epitopes was seen in subject 1, who is homozygous for both the HLA class II DR and DQ alleles, indicating that numerous epitopes of a broad range of specificities can still be presented in the context of a limited number of HLA class II alleles within one person. It should be noted that the breadth of CD4 epitopes targeted in the subjects included in this study exceeds the greatest number of CD8-T-cell epitopes reported in human and chimpanzee infections with this virus, where a maximum of eight epitopes have been identified to be concurrently targeted (15, 32, 52). It should be noted as well that this study was necessarily limited to analysis of HCV-specific CD4+ T cells circulating in peripheral blood, due to lack of clinical indication for liver biopsy in persons with controlled infection. The lack of HCV-specific CD4+ T cells seen here in the majority of individuals with chronic HCV infection may be due to compartmentalization in the liver of HCV-specific CD4+ T cells.

A list of all epitopes identified, along with their amino acid coordinates, location in the HCV genome, and amino acid sequence are shown for the three subjects characterized in detail in Table 1. Of the 37 epitopes targeted by these three individuals, none was targeted by all three, despite the reported frequent promiscuous presentation of class II restricted epitopes (11, 13, 36, 45). Four epitopes were targeted by at least two individuals. The NS3 peptide aa 1241 to 1260 has been previously described, and was reported to be recognized by three out of four HLA-diverse persons with self-limited acute HCV infection (11), a study in which the breadth of responses was not defined. Our data indicate that spontaneous resolution and long-term control are not associated with narrow responses but rather with broadly directed responses that persist. Further studies are needed to determine whether a vigorous response to this particular epitope during the acute phase of HCV infection is a predictor of a favorable disease outcome associated with spontaneous resolution of viremia.

In addition to recognizing peptide NS3 aa 1241 to 1260, subjects 1 and 2 both recognized the NS4 peptide aa 1911 to 1930. In a previous study analyzing CD4+ T-helper-cell responses to selected conserved regions of the virus, highly immunogenic CD4+-T-cell epitopes were identified in the core, NS3, and NS4 proteins, including aa 1909 to 1929 (30), which overlap the peptide shown to be immunogenic in this study. Additionally, Lamonaca et al. identify core aa 21 to 40 and NS4 aa1767 to 1786 as highly immunogenic epitopes among a cohort of individuals with acute HCV infection, and each of these was shown to be a targeted epitope by at least one person in the present study (Table 1). The present study thus not only identifies previously reported epitopes but through comprehensive analysis of all expressed proteins also shows that T-helper-cell responses are broadly directed in persons who control HCV viremia.

A striking feature of the present study is the paucity of responses to the envelope proteins of the virus. Although this is a region heavily targeted by antibody responses, among the three individuals with resolved HCV infection analyzed most comprehensively in this study, HCV-specific CD4+ T-helper-cell responses were detected against epitopes contained within all HCV proteins except the E1, E2, and p7 proteins. The envelope glycoproteins E1 and E2 represent the most variable region of the HCV genome, particularly hypervariability regions within the E2 sequence (28, 51). The lack of detectable CD4+ T-helper-cell responses to the envelope region of the virus may be a reflection of a high degree of envelope sequence variation, such that the synthetic peptides used in this study do not have sufficient cross-reactivity, or might represent impaired epitope presentation (50). Studies of larger numbers of individuals will need to be conducted to determine regions of the HCV genome that are frequently targeted by the CD4+ T-helper-cell response. Additionally, sequencing of autologous virus during episodes of viremia and subsequent synthesis of peptides corresponding to autologous virus sequence may reveal additional epitopes targeted in particularly variable regions of the virus, and the extent to which escape from T-helper-cell responses contributes to disease progression (7, 8, 16).

In summary, we conclude that multiple epitopes are simultaneously targeted by the CD4+ T-helper-cell response in individuals with spontaneous resolution of HCV infection. This comprehensive analysis at an epitope level of the total breadth and specificity of virus-specific T-helper-cell responses also provides important parameters for consideration in development and testing of candidate vaccines for chronic human viral infections. Identification of immunodominant epitopes will provide important insights regarding HCV immunopathogenesis and vaccine design and evaluation, and the finding that persons with chronic infection lack diverse responses provides rationale for immunotherapeutic approaches to control HCV infection.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health to B.D.W. (AI31563), R.T.C. (DK57857), and C.L.D. (T332 AI07387-12); from the Doris Duke Charitable Foundation (B.D.W.); and from the Deutsche Forschungsgemeinschaft to G.M.L. (DFG LA 1241/1-1).


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FOOTNOTES
 
* Corresponding author. Mailing address: Massachusetts General Hospital, Partners AIDS Research Center, 149 13th St., Rm. 5212D, Charlestown, MA 02129. Phone: (617) 724-8332. Fax (617) 726-4691. E-mail: bwalker{at}partners.org. Back


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Journal of Virology, December 2002, p. 12584-12595, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12584-12595.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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