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Differential Antigenic Hierarchy Associated with Spontaneous Recovery from Hepatitis C Virus Infection: Implications for Vaccine Design

  1. Susan Smyk-Pearson1,
  2. Ian A. Tester1,
  3. Dennis Lezotte2,
  4. Anna W. Sasaki4,
  5. David M. Lewinsohn3 and
  6. Hugo R. Rosen1
  1. 1Division of Gastroenterology and Hepatology, Hepatitis C Center, and Integrated Program in Immunology, University of Colorado Health Sciences Center and National Jewish Hospital, and
  2. 2Department of Preventive Medicine, University of Colorado, Denver;
  3. 3Division of Pulmonary and Critical Care Medicine,
  4. 4Portland Veterans Affairs Medical Center and Oregon Health and Science University, Portland
  1. Reprints or correspondence: Dr. Hugo R. Rosen, UCHSC GI Div., 4200 E. Ninth Ave., No. B-158, Denver, CO 80262 (Hugo.Rosen{at}UCHSC.edu)
 
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Abstract

BackgroundCellular immune responses play a central role in the control of hepatitis C virus (HCV) infection, and in some individuals the adaptive immune response can spontaneously eradicate HCV infection. The development of vaccine candidates to prevent the spread of this infection remains a top priority; however, understanding the correlates of effective immunological containment is an important prerequisite

MethodsUsing 750 overlapping peptides, we directly characterized ex vivo total and subgenomic HCV-specific CD4+ and CD8+ T cell responses in a large cohort of participants with either chronic infection or spontaneously resolved infection

ResultsIn chronic infection, the frequency of total CD4+ T cells specific for HCV averaged 0.06%, compared with 0.38% in resolved infection. Total HCV-specific CD4+ and CD8+ T cell responses were strongly correlated in the setting of spontaneous resolution but not in the setting of viral persistence. NS3 protein–specific responses comprised a significantly greater proportion of the total response in resolved infection than in chronic infection, whereas responses to different regions comprised a larger proportion of responses in chronic infection

ConclusionBecause these data comprehensively define the breadth, specificity, and threshold of the T cell response associated with spontaneous recovery from HCV infection, they have important implications in the development of multigenic vaccine candidates for this common infection

Hepatitis C virus (HCV) is the major causative agent of chronic hepatitis and has an estimated global prevalence of 3%. In the United States alone, HCV infection affects an estimated 3 million persons, most of them <50 years of age [1, 2]. It is one of the leading causes of morbidity and mortality related to chronic liver disease and is the most common indication for liver transplantation throughout the world [3]. The development of vaccine candidates to prevent the spread of this infection and to improve treatment outcome remain a top priority, particularly in light of the fact that standard treatment is associated with suboptimal response rates and significant toxicity in a large proportion of patients [4]. Studies have shown that clearance of HCV infection in humans provides anti-HCV immunity that confers at least partial protection against new infections and reduces the severity of the chronic infections that do occur [5, 6]

Precisely why infection with HCV leads to persistence in a majority of exposed individuals and to spontaneous recovery in only a minority of them remains unclear, but abundant data demonstrate that the vigor and breadth of the cellular immune response is critical [7–10]. In particular, spontaneous resolution of infection has been associated with a vigorous and sustained CD4+ T cell response; indeed, recrudescence of viremia is temporally related to the loss of CD4+ T cell responses to recombinant HCV antigens in the setting of acute infection [11]. When combined with the spontaneous loss of CD4+ T cell help that precedes HCV persistence, CD8+ T cell function might be impaired to the point where cytotoxic T lymphocytes (CTLs) do not mediate viral clearance but still exert sufficient immune pressure to select for variant viruses. The appearance of functional, HCV-specific CD8+ T cells has been temporally associated with the control of viremia [12]. In chimpanzees, antibody-mediated depletion of CD4+ and CD8+ T cell subsets has provided compelling direct evidence of the importance of CD8+ T cells in the control of viremia during acute HIV infection [7] and of their absolute dependence on CD4+ T cell help [13]

In the present study, we comprehensively examined, using 750 overlapping peptides (15 aa in length, overlapping by 11 aa), HCV-specific CD4+ and CD8+ T cell responses in a large cohort of subjects with chronic infection or spontaneously resolved infection. This unbiased approach has allowed us to provide novel information on the relationship between HCV-specific CD4+ and CD8+ T cells and on the relative dominance of T cell determinants targeted across the entire HCV genome, including the relative contribution of individual gene products to the cumulative breadth of the HCV-specific immune response. These data represent important information for understanding the immunopathogenesis of this common disease, for defining the correlates of effective immunological containment, and for developing multigenic vaccine candidates

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Methods

SubjectsThe study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in the a priori approval by the human research committees and institutional review boards of the Oregon Health and Science University and the Portland Veterans Affairs Medical Center. The study was composed of 3 groups of consenting participants: subjects with risk factors for the acquisition of HCV infection who demonstrated antibody reactivity (by Chiron RIBA HCV 3.0 SIA) [14] but who had undetectable HCV RNA levels by a qualitative polymerase chain reaction–based assay (with a lower limit of detection of 100 copies/mL) and by a qualitative transcription-mediated amplification assay (Chiron Procleix) (n=25); HCV-seropositive patients infected with genotype 1 (n=25); and healthy, HCV-seronegative control subjects without risk factors for the acquisition of HCV infection (n=10). All of the control subjects were negative for viral markers of HCV, hepatitis A virus, hepatitis B virus, and HIV infection

Synthetic peptides for T cell analysisOverlapping peptides (n=750) were synthesized to span the complete amino acid sequence of the HCV polyprotein derived from HCV genotype 1a (GenBank accession number M62321) and were divided into subgenomic peptide pools (figure 1). The peptide compositions were confirmed by amino acid analysis using mass spectroscopy. These pentadecamers (15mer peptides), which overlapped by 11 aa, were resuspended with dimethyl sulfoxide (DMSO) at 20 mg/mL and then were concentrated so that the final volume of DMSO in the assay would not exceed 0.5%. The final concentration of peptides (either single or pooled) was 10 μg/mL

Figure 1
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Figure 1

Peptide pools used for stimulation in enzyme-linked immunospot assays. The hepatitis C virus polyprotein (genotype 1a; 3010 aa; 750 peptides in total) is composed of 4 structural proteins (Core–p7) and 6 nonstructural proteins (NS2–NS5B). This polyprotein was divided into 33 peptide pools (with 18–25 overlapping peptides in each pool), as described in Methods

Preparation of peripheral-blood mononuclear cells (PBMCs) PBMCs (obtained from a unit of blood or after leukophoresis) were separated using ficoll-histopaque density gradient (Amersham Pharmacia Biotech) and then were cryopreserved in medium containing 80% fetal calf serum, 10% DMSO, and 10% RPMI 1640 (GIBCO) with penicillin-streptomycin, in accordance with the manufacturer’s instructions

Generation of monocyte-derived dendritic cells (DCs)The generation of monocyte-derived DCs was based on the method previously described by Romani et al. [15] and previously used by us [16]. DCs used in enzyme-linked immunospot (ELISPOT) assays (see below) were resuspended in RPMI 1640 plus 10% human serum and plated at 10,000 cells/well in 96-well plates

Flow cytometryFlow-cytometric analysis was performed at the Oregon Health and Science University Flow Cytometry Core Facility by use of a FACScalibur flow cytometer and CellQuest Pro software (version 4.0.2; Becton Dickinson). Flow-cytometric analysis for each participant allowed precise enumeration of the proportion of CD4+ T cells comprising the CD4+/other cell population (PBMCs depleted of CD8+ T cells)

MACS bead separationCD8+ T cells were separated from PBMCs by use of a positive-selection strategy and MACS superparamagnetic beads (Miltenyi Biotec), in accordance with the manufacturer’s instructions

ELISPOT assaysInterferon (IFN)–γ production was detected using an established ELISPOT assay [16, 17]. Purified CD8+ T cells (2.5×105) were cultured with peptide-pulsed DCs, and 5.0×105 CD4+/other cells were incubated in plates for 40 h before development. Spots were visualized using a peroxidase substrate kit (Vector). After the plates were dry, spots were quantified using a Zeiss microscope and KS ELISPOT software (version 4.4). A qualitative response was defined as one that was at least 3 SDs above the mean response in control wells (n=8) containing DMSO but not peptide and that was at least 5 spots above background

Statistical analysesDescriptive statistics (including means, SDs, and frequencies) are tabled for the raw counts for the subjects with spontaneously resolved or chronic HCV infection, separately and across the entire set of regions considered. The simple t test and χ2 test were performed to assess differences in the results of ELISPOT assays for each subgenomic region. SAS (version 9.1.3; SAS Institute) and JMP (version 6.0; SAS Institute) were used for all statistical analyses. Statistical significance was assumed at P<.05

Logistic regression methodology [18] was used to assess single region contributions and to construct independent multivariate (i.e., multiregion) prediction models for the binary response variable “resolved or chronic HCV infection.” These are tabled along with odds ratios and P values [18]

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Results

Fifty HCV-seropositive participants were included in the present study—25 patients with persistent genotype 1 viremia, and 25 subjects with spontaneously resolved infection (positive for antibody but with undetectable HCV RNA levels). The demographic and clinical features of these individuals are shown in table 1

Differential magnitude of and relationship between CD4+ and CD8+ T cell responses according to virological outcome To comprehensively assess CD4+ and CD8+ T cell responses to the whole HCV polyprotein, 750 peptides (15 aa in length, overlapping by 11 aa) were pooled and used in an established ELISPOT assay [17]. Autologous DCs were used as antigen-presenting cells to stimulate bead-purified CD8+ T cells; we have previously shown that this approach increases the sensitivity of the detection of HCV-, Epstein-Barr virus–, and influenza virus–specific CTL responses without increasing nonspecific background responses [16]. The remaining CD4+/other cells (PBMCs depleted of CD8+ T cells; 5×105 cells/well) were stimulated with the same 33 peptide pools that spanned distinct subgenomic regions (figure 1). To enable comparison between our approach and a more conventional approach reported in the literature (use of unfractionated PBMCs or a CD4+ T cell–depleted population [CD8+/other cells]), in figure 2 we present results for a chronically infected patient (C16) and a subject with resolved infection (R7) side by side. Although the conventional approach with unfractionated PBMCs did not yield any significant responses (defined in Methods) for patient C16 (figure 2A), our approach detected responses to the core B, NS5A1, and NS5B2 pools (figure 2B). Use of CD8+/other cells yielded no significant responses (figure 2C), but use of autologous DCs and bead-purified CD8+ T cells detected significant responses to the NS36H pool (figure 2D). For subject R7, our approach with a higher concentration of CD4+/other cells yielded a >3-fold increase in the number of peptide pool responses (figure 2F), compared with that observed with unfractionated PBMCs (figure 2E). Similarly, for subject R7, our approach with autologous DCs and bead-purified CD8+ T cells identified significant responses to the core B, NS2A, and NS5B5 pools (figure 2H), which would have been missed with a less sensitive approach. Further breakdown of constituent peptides within these peptide pools confirmed the T cell determinants for both patient C16 and subject R7

Figure 2
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Figure 2

Comparison of a conventional approach (left panels) with the approach used in the present study with high input nos. (right panels) to enumerate hepatitis C virus (HCV)–specific responses in a chronically infected patient (C16) and in a subject with spontaneously resolved infection (R7). Stimulation of 3.0×105 unfractionated peripheral-blood mononuclear cells (PBMCs) revealed no significant responses in patient C16 (A) whereas stimulation of 5.0×105 CD8+ cell–depleted PBMCs (CD4+/other cells) revealed significant responses (indicated by asterisks) to 3 pools (B). For subject R7, the conventional approach with unfractionated PBMCs (E) yielded less than a third of the responses than did the present approach with CD4+/other cells (F). Both patient C16 and subject R7 had CD8+ T cell determinants within peptide pools (confirmed after further breakdown of constituent peptides) that were not detectable when a CD4+ cell–depleted population was stimulated (D vs. C and H vs. G)

In chronic infection, the frequency of total CD4+ T cells that were specific for HCV (the sum of the IFN-γ–producing CD4+ T cells identified in ELISPOT assays for all the peptide pools) averaged 0.06% (range, 0%–0.16%), compared with an average of 0.38% (range, 0.02%–3.2%) in resolved infection (P<.0001) (figure 3A). Accordingly, the total area of HCV-specific IFN-γ production was also significantly different (P<.0001)

Figure 3
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Figure 3

Total hepatitis C virus (HCV)–specific interferon (IFN)–γ-producing CD4+ and CD8+ T cells in patients with chronic infection, subjects with spontaneously resolved infection, and healthy control subjects. Horizontal black bars indicate means. A CD4+ T cells. The no. of spots for 33 peptide pools was totaled for each individual participant, and the frequency of HCV-specific CD4+ T cell responses (after enumeration by flow cytometry) is shown as a percentage of circulating CD4+ T cells. P<.0001, for chronic vs. resolved; P=.0002, for resolved vs. control; and no significant difference, for chronic vs. control (Wilcoxon&amp;rank sum test). B CD8+ T cells. Data were calculated as for CD4+ T cells. P=.07, for chronic vs. resolved (P=.03 when outlier patient C5 is excluded); P=.003, for resolved vs. control; and P=.02, for chronic vs. control (Wilcoxon&amp;rank sum test)

The frequency of HCV-specific CD8+ T cells in the circulating blood averaged 0.024% (range, 0%–0.39%) in chronic infection and 0.15% (range, 0%–2.4%) in resolved infection (figure 3B). This difference was statistically significant when outlier patient C5 (who, coincidentally, had the lowest HCV RNA level) was excluded. For CD8+ T cells, the total area of HCV-specific IFN-γ production was greater in resolved infection than in chronic infection (9.8×105 vs. 1.88×105 μm2; P=.03)

A statistically significant correlation between the frequency of circulating HCV-specific CD4+ T cells and that of circulating HCV-specific CD8+ T cells was observed in resolved infection (P=.004; nonparametric Spearman’s ρ=0.56) (figure 4A), but no such association was observed in chronic infection (P=.70; nonparametric Spearman’s ρ=-0.08) (figure 4B). Of interest, these data contrast with those from a previous study that was limited to an analysis of CTL responses to 5 epitopes [19], underscoring how a targeted screen will lead to underrepresentation of the breadth of immunity

Figure 4
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Figure 4

Correlation between hepatitis C virus (HCV)–specific CD4+ and CD8+ T cells. The total no. of HCV-specific interferon-γ–producing CD4+ and CD8+ T cells (per 2.5×105 cells) are shown log transformed and plotted against each other. There is a strong correlation between the 2 HCV-specific T cell subsets in spontaneously resolved infection (A) but not in chronic infection (B). Comparisons were made using Spearman’s&amp;rank correlation test

Once chronic infection is established, the magnitude of T cell recognition does not appear to contribute to the control of HCV infection. We found no correlation between serum HCV RNA levels and the magnitude and breadth of CD4+ and CD8+ T cell responses (for total and individual peptide pools and protein subunits; data not shown)

Variation in immunogenicity across the HCV genome: qualitative and quantitative differencesThe breadth of responses for each participant was determined by counting the number of pools for which there was a qualitative response (defined in Methods). We detected qualitative HCV-specific CD4+ T cell responses in all 25 subjects with resolved infection and in 19 of the 25 patients with chronic infection (P=.02). Qualitative HCV-specific CD8+ T cell responses were detected in 19 of the 25 subjects with resolved infection and in 16 of the 25 patients with chronic infection (difference not significant). On average, the subjects with resolved infection displayed CD4+ T cell responses to 11.4 pools (SE, 1.67 pools; median, 10 pools; range, 1–25 pools), compared with 5.24 pools for the chronically infected patients (SE, 1.22 pools; median, 5 pools; range, 0–25 pools) (P=.004, Wilcoxon&amp;rank sum test). Qualitative CD4+ T cell responses to >7 pools (i.e., 21% of the HCV polyprotein) correlated strongly with recovery (area under the receiver operating characteristic curve, 0.74; P=.0035)

Resolved infection was associated with CD8+ T cell responses to an average of 2.84 pools (SE, 0.98 pools; median, 2 pools; range, 0–25 pools), whereas in chronic infection an average of 1.28 pools (SE, 0.36 pools; median, 1 pool; range, 0–7 pools) were targeted per patient (P=.07). Among the 10 healthy control subjects, an average of 0.50 pools (SE, 0.27 pools) elicited CD8+ T cell responses (for the comparison with chronic infection, P=.040). The detection of low-level CD8+ T cell responses among healthy individuals with no exposure to HCV is in keeping with the findings of a recent analysis by Kennedy et al. [20] and may represent cross-reactivity to other pathogens

Figure 5A and 5B shows the percentages of patients in each group with qualitative response(s) to the individual peptide pools. These data indicate that all HCV proteins and protein subunits can serve as targets of CD4+ and CD8+ T cell responses. Some regions are highly immunogenic for CD4+ T cells, eliciting responses in more than half of the subjects with resolved infection, whereas other regions rarely contain antigenic determinants. There were major differences in responsiveness to individual peptide pools between chronic and resolved infection; in particular, nonstructural peptides frequently elicited responses from the subjects with resolved infection, confirming and extending the results of our previous analysis using recombinant proteins [21] as well as those of a recent analysis using overlapping peptides [22]. In contrast, the frequency of qualitative CD4+ T cell responses targeting the envelope peptides did not differ between chronic infection and resolved infection. Use of a more stringent criterion of a response being at least 10 spots above background (along with the criterion of a response being at least 3 SDs above the mean response in control wells) did not significantly change these results (figure 5 insets). In contradistinction to CD4+ T cell responses, CD8+ T cell responses were less prevalent, with only the NS35H pool eliciting responses in more than a quarter of the participants (figure 5B)

Figure 5
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Figure 5

Qualitative and quantitative responses to hepatitis C virus (HCV) peptide pools. A Percentages of patients with chronic infection and subjects with spontaneously resolved infection who had qualitative CD4+ T cell responses to 33 HCV peptide pools spanning the entire genome (as shown in figure 1). B Percentages of patients and subjects who had qualitative CD8+ T cell responses to the peptide pools. In contrast to the CD4+ T cell response, only 1 pool elicited CD8+ T cell responses in more than a quarter of the subjects with resolved infection. A qualitative response was defined as one that was at least 3 SDs above the mean response in control wells (n=8) containing dimethyl sulfoxide but not peptide and that was at least 5 spots above background; the insets show the results when the second criterion was changed to the response being at least 10 spots above background. C and D Mean ± SE HCV-specific CD4+ T cell responses (C) and CD8+ T cell responses (D) to each peptide pool for resolved and chronic infection, after subtraction of background (per 2.5×105 T cells). *P<.05 and **P<.005 (2-sided t test), for chronic vs. resolved

Quantitative analyses of the pools revealed a differential magnitude of responses across the HCV genome (figure 5C and 5D and table 2). Thus, in contrast to the findings of a recent, less comprehensive study that used a smaller sample size and bulk stimulation [23], we found statistically significant differences in the frequency and vigor of responses to individual peptide pools between resolved infection and chronic infection

Figure 6
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Figure 6

Relative contributions of individual hepatitis C virus (HCV) gene products to the cumulative breadth of the HCV-specific response. In brief, the sum of responses to pools spanning each region was divided by the total no. of spots for all pools. A CD4+ T cell responses. Responses to the E1–p7 peptides (20.6% vs. 12%; P=.02) and the NS2 peptides (3.7% vs. 1.23%; P=.02) comprised a significantly higher proportion in chronic infection than in spontaneously resolved infection. In contrast, NS3-specific responses were higher in resolved infection than in chronic infection (31% vs. 22%; P=.02). B CD8+ T cell responses. NS3-specific responses comprised 52% of the total HCV-specific response in resolved infection, compared with 23% in chronic infection (P=.02)

Table 1
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Table 1

Demographic and clinical features of the 50 hepatitis C virus (HCV)–seropositive study subjects

Table 2
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Table 2

Simple (univariate) logistic regression of qualitative and quantitative CD4+ T cell responses in subjects with spontaneously resolved infection

We next assessed the relative contributions of the individual proteins to the total magnitude of the HCV-specific CD4+ and CD8+ T cell responses. As is shown in figure 6A and 6B NS3-specific responses accounted for a significantly larger proportion of the total response in resolved infection than in chronic infection. For CD4+ T cell responses, NS3-specific responses comprised 31% of the cumulative magnitude in resolved infection, compared with 22% in chronic infection (P=.02). After normalization for protein length, NS3 remained the most immunogenic region per amino acid. The order of immunodominance was as follows: NS3 (mean, 0.27 CD4 ELISPOTs/aa), core (0.26), NS5 (0.19), NS4 (0.17), E1–p7 (0.09), and NS2 (0.04) (P=.006, for NS3 vs. all others). Moreover, NS3 contributed most importantly to the total magnitude of the HCV-specific CD8+ T cell response in resolved infection (52%, vs. 23% in chronic infection; P=.02)

The results of the simple (univariate) logistic regression analyses were used to help guide construction of the multiple logistic regression model. Forward model building was used, because there were a small number of outcome observations (recovery vs. persistence) and a large number of subgenomic regions to evaluate. Multivariate modeling identified qualitative CD4+ T cell responses to regions as being positively (P = .0066, for NS33H; P=.0033, for NS35H) or negatively (P = .03, for E1A) associated with recovery

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Discussion

The present study is the most comprehensive assessment of total HCV-specific immunity conducted to date and used the largest cohort of subjects. Recent studies using overlapping-peptide stimulation have focused on either CD4+ T cell responses [22, 24], CD8+ T cell responses [24–26], or unfractionated PBMC responses [23]. By providing data on the breadth, specificity, and strength of both CD4+ and CD8+ T cell responses, we believe the present results have significant implications for multipeptide vaccine design

Several methological aspects of our study merit particular attention. The IFN-γ ELISPOT assay we used to directly enumerate HCV-specific CD4+ and CD8+ T cells provides an unprecedented degree of analytical sensitivity that allows the detection of rare responder cells and represents one of the major strengths of the study. In the present study, each patient had at least 1 unit of blood collected or underwent leukophoresis, allowing a high number of cells to be cultured and IFN-γ secretion to be quantitated directly ex vivo without in vitro expansion. The likelihood that DCs in our assay induced in vitro priming of naive T cells is extremely low, because we used immature DCs [27] and did not use multiple rounds of stimulation [28]. Moreover, CD8+ T cell lines that were generated by sorting IFN-γ–producing CD8+ T cells after stimulation with NS3 demonstrated specificities that were identical to those of our ex vivo analyses (data not shown)

We screened responses to HCV genotype 1a–derived peptides, in part because of the high prevalence of this genotype and its association with injection drug use [29], the predominant risk factor among our study participants. Viral heterogeneity may have contributed to the different levels of immune responses detected (or not detected) by our assay [16]. The high genetic variability of HCV has several biological effects. First, it provides diversity for rapid viral evolution in response to selective pressures, such as an immune response or antiviral pressure. Second, many genomes contain mutations that are either lethal or reduce viral fitness, leading to error catastrophe and their loss from the viral population [30]

It was not possible to determine whether the infecting viral strain(s) coded for sequences that differed from our screening peptides, and it would be cost prohibitive to screen immune responses with autologous HCV peptides in the chronically infected patients. Moreover, the remote nature of exposure in the subjects with spontaneously resolved infection precluded knowledge of the precise infecting genotype, although 14 of the 25 subjects had serologic evidence of exposure to genotype 1 virus. The fact that a large proportion of the subjects with resolved infection might have been exposed to non–genotype 1 virus makes the finding of enhanced immunity in resolved infection (relative to chronic genotype 1 infection) even more compelling. For example, subjects R2, R14, R16, R18, R23, and R25 demonstrated T cell responses despite lacking evidence of serotype 1 infection. Indeed, there were no statistically significant differences in total HCV-specific CD4+ and CD8+ T cell responses according to serotype (data not shown)

Our analysis reveals a differential relationship between HCV-specific CD4+ and CD8+ T cells in resolved and chronic infection. CD4+ T cells are indispensable for the maintenance of functional CD8+ T cells that control chronic viral infections [31]. CD4+ T cells may help CD8+ T cells directly, via production of cytokines [32], or indirectly, by assisting professional antigen-presenting cells via CD40/CD40L-mediated activation [33]. The interdependence of viral-specific CD4+ and CD8+ T cell responses in disease resolution and the lack of such correlation in the setting of chronic viremia underscore the need to elucidate the mechanisms that underlie the failure to develop HCV-specific CD4+ T cells and their instructive signals during memory CD8+ T cell differentiation [17]. Accordingly, although it is well documented that CD4+ T cell help is required for the generation of stable and protective CD8+ T cell memory in a number of models [34], it is less crucial during the primary expansion phase of CD8+ T cells. Thus, it would be of considerable interest to characterize “helpless” CD8+ T cells that are primed in the absence of CD4+ T cells during the earliest phases of HCV infection [17] and then to determine their fate after recovery or chronicity is established. Data in this study suggest that, once chronic infection is established, the magnitude of T cell recognition does not contribute to the control of HCV infection, which is in accord with recent observations [35] but is in contrast to others [23]. Additional studies are needed to define functional (besides IFN-γ production) and phenotypic features that might mediate a viral set point or liver injury in chronic disease [36]. Moreover, it might be predicted that T cells that recognize antigen in multiple ways (on the basis of the genetics of the T cell receptor) and express high avidity for peptide (including naturally occurring variants) would lead to spontaneous recovery at a higher rate, but these possibilities remain undefined

Although the subjects in this study had been exposed to HCV in the remote past, it has been demonstrated in the chimpanzee model that the frequency of HCV-specific memory T cells becomes fixed immediately after the resolution of primary infection and that this memory cell set point does not decrease over a period of years [7]. Theoretically, boosting the responses that target the entire HCV polyprotein would be ideal for immunotherapy; however, the feasibility of doing so remains undetermined [4]. The data described here establish a much-needed framework by providing information on the strength, specificity, and relative immunodominance of T cell responses primed in vivo that correlate with the containment of HCV infection. In particular, CD4+ T cell responses to NS33H and NS35H are independently and positively associated with recovery; these regions might represent indispensable components of a prophylactic vaccine. Of interest, the NS33H pool contains NS31248–1261, which has been described by a number of groups as an immunodominant epitope that is presented by at least 7 different HLA class II molecules [22, 37, 38] and is highly conserved across genotypes. The promiscuous nature of this and other recently described class II epitopes (aa 1771–1790 within NS4B1; aa 2571–2590 within NS5B2; and aa 1531–1550 within NS36H [23]) suggests that recapitulation of these responses by vaccination might offer broad protection for a large proportion of HLA-diverse individuals at risk. Furthermore, because covalent linkage of CD4+ and CD8+ T cell epitopes on the same peptide vaccine construct has been shown to be important for induction of antigen-specific responses [39], the natural occurrence of nested CD4+ and CD8+T cell epitopes within NS35H provides promising potential to fulfill these requirements [40]

In summary, we have defined the correlates of effective immunological containment of HCV infection by analysis of all potential epitopes, and these findings have implications for novel immunotherapeutic approaches. Vaccines that contain only envelope peptides might induce neutralizing antibodies but not adequately prime CD4+ and CD8+ T cells to control HCV infection [12]

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Acknowledgments

We are grateful to the participants, for contributing their time to the study, and to Drs. Lucy Golden-Mason, Jay Burton, and Georg Lauer, for helpful review of the manuscript

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Footnotes

  • ↵Potential conflicts of interest: none reported

    Financial support: National Institutes of Health (grant RO1 DK060590 to H.R.R.)

  • Received February 1, 2006.
  • Accepted March 27, 2006.
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References

  1. ↵
    1. Alter MJ,
    2. Seeff LB,
    3. Bacon BR,
    4. Thomas DL,
    5. Rigsby MO
    . Testing for hepatitis C virus infection should be routine for persons at increased risk for infection. Ann Intern Med 2004;141:715-7.
    Abstract/FREE Full Text
  2. ↵
    1. Lauer GM,
    2. Walker BD
    . Hepatitis C virus infection. N Engl J Med 2001;345:41-52.
    CrossRefMedlineWeb of Science
  3. ↵
    1. Rosen HR,
    2. Martin P
    . Viral hepatitis in the liver transplant recipient. Infect Dis Clin North Am 2000;14:761-84.
    CrossRefMedlineWeb of Science
  4. ↵
    1. Houghton M,
    2. Abrignani S
    . Prospects for a vaccine against the hepatitis C virus. Nature 2005;436:961-6.
    CrossRefMedline
  5. ↵
    1. Mehta SH,
    2. Cox A,
    3. Hoover DR,
    4. et al
    . Protection against persistence of hepatitis C. Lancet 2002;359:1478-83.
    CrossRefMedlineWeb of Science
  6. ↵
    1. Grant M
    . Secondary persistent infection with hepatitis C virus: a challenge for adaptive immunity. Lancet 2002;359:1452.
    CrossRefMedlineWeb of Science
  7. ↵
    1. Shoukry NH,
    2. Grakoui A,
    3. Houghton M,
    4. et al
    . Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection. J Exp Med 2003;197:1645-55.
    Abstract/FREE Full Text
  8. ↵
    1. Thimme R,
    2. Oldach D,
    3. Chang KM,
    4. Steiger C,
    5. Ray SC
    . Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med 2001;194:1395-406.
    Abstract/FREE Full Text
  9. ↵
    1. Lechner F,
    2. Wong DK,
    3. Dunbar PR,
    4. et al
    . Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med 2000;191:1499-512.
    Abstract/FREE Full Text
  10. ↵
    1. Gruner NH,
    2. Gerlach TJ,
    3. Jung MC,
    4. et al
    . Association of hepatitis C virus–specific CD8+ T cells with viral clearance in acute hepatitis C. J Infect Dis 2000;181:1528-36.
    Abstract/FREE Full Text
  11. ↵
    1. Gerlach JT,
    2. Diepolder H,
    3. Jung MC
    . Recurrence of hepatitis C virus after loss of virus-specific CD4+ T-cell response in acute hepatitis C. Gastroenterology 1999;117:933-41.
    CrossRefMedlineWeb of Science
  12. ↵
    1. Shoukry NH,
    2. Cawthon AG,
    3. Walker CM
    . Cell-mediated immunity and the outcome of hepatitis C virus infection. Annu Rev Microbiol 2004;58:391-424.
    CrossRefMedlineWeb of Science
  13. ↵
    1. Grakoui A,
    2. Shoukry NH,
    3. Woollard DJ,
    4. et al
    . HCV persistence and immune evasion in the absence of memory T cell help. Science 2003;302:659-62.
    Abstract/FREE Full Text
  14. ↵
    1. Gretch DR,
    2. Lee W,
    3. Corey L
    . Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory. J Clin Microbiol 1992;30:2145-9.
    Abstract/FREE Full Text
  15. ↵
    1. Romani N,
    2. Gruner S,
    3. Brang D,
    4. et al
    . Proliferating dendritic cell progenitors in human blood. J Exp Med 1994;180:83-93.
    Abstract/FREE Full Text
  16. ↵
    1. Wertheimer AM,
    2. Miner C,
    3. Lewinsohn DM,
    4. Sasaki AW,
    5. Kaufman E
    . Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection. Hepatology 2003;37:577-89.
    CrossRefMedlineWeb of Science
  17. ↵
    1. Tester I,
    2. Smyk-Pearson S,
    3. Wang P,
    4. et al
    . Immune evasion versus recovery after acute hepatitis C virus infection from a shared source. J Exp Med 2005;201:1725-31.
    Abstract/FREE Full Text
  18. ↵
    1. Hamilton LC
    . A second course in applied statistics. Belmont, CA: Duxbury Press; 1992. Regression with graphics; p. 183-216.
  19. ↵
    1. Chang KM,
    2. Thimme R,
    3. Melpolder JJ,
    4. et al
    . Differential CD4+ and CD8+ T-cell responsiveness in hepatitis C virus infection. Hepatology 2001;33:267-76.
    CrossRefMedlineWeb of Science
  20. ↵
    1. Kennedy PT,
    2. Urbani S,
    3. Moses RA,
    4. et al
    . The influence of T cell cross-reactivity on HCV-peptide specific T cell response. Hepatology 2006;43:602-11.
    CrossRefMedlineWeb of Science
  21. ↵
    1. Rosen HR,
    2. Miner C,
    3. Sasaki AW,
    4. et al
    . Frequencies of HCV-specific effector CD4+ T cells by flow cytometry: correlation with clinical disease stages. Hepatology 2002;35:190-8.
    CrossRefMedlineWeb of Science
  22. ↵
    1. Schulze zur Wiesch J,
    2. Lauer GM,
    3. Day CL,
    4. et al
    . Broad repertoire of the CD4+ Th cell response in spontaneously controlled hepatitis C virus infection includes dominant and highly promiscuous epitopes. J Immunol 2005;175:3603-13.
    Abstract/FREE Full Text
  23. ↵
    1. Sugimoto K,
    2. Ikeda F,
    3. Stadanlick J,
    4. Nunes FA,
    5. Alter HJ
    . Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection. Hepatology 2003;38:1437-48.
    MedlineWeb of Science
  24. ↵
    1. Day CL,
    2. Lauer GM,
    3. Robbins GK,
    4. et al
    . Broad specificity of virus-specific CD4+ T-helper-cell responses in resolved hepatitis C virus infection. J Virol 2002;76:12584-95.
    Abstract/FREE Full Text
  25. ↵
    1. Lauer GM,
    2. Barnes E,
    3. Lucas M,
    4. et al
    . High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection. Gastroenterology 2004;127:924-36.
    CrossRefMedlineWeb of Science
  26. ↵
    1. Cox AL,
    2. Mosbruger T,
    3. Lauer GM,
    4. Pardoll D,
    5. Thomas DL
    . Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C. Hepatology 2005;42:104-12.
    CrossRefMedlineWeb of Science
  27. ↵
    1. Segura E,
    2. Nicco C,
    3. Lombard B,
    4. et al
    . ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming. Blood 2005;106:216-23.
    Abstract/FREE Full Text
  28. ↵
    1. Wilson CC,
    2. Olson WC,
    3. Tuting T,
    4. Rinaldo CR,
    5. Lotze MT
    . HIV-1-specific CTL responses primed in vitro by blood-derived dendritic cells and Th1-biasing cytokines. J Immunol 1999;162:3070-8.
    Abstract/FREE Full Text
  29. ↵
    1. Pybus OG,
    2. Charleston MA,
    3. Gupta S,
    4. Rambaut A,
    5. Holmes EC
    . The epidemic behavior of the hepatitis C virus. Science 2001;292:2323-5.
    Abstract/FREE Full Text
  30. ↵
    1. Gonzalez-Lopez C,
    2. Arias A,
    3. Pariente N,
    4. Gomez-Mariano G,
    5. Domingo E
    . Preextinction viral RNA can interfere with infectivity. J Virol 2004;78:3319-24.
    Abstract/FREE Full Text
  31. ↵
    1. Zajac AJ,
    2. Blattman JN,
    3. Murali-Krishna K,
    4. et al
    . Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med 1998;188:2205-13.
    Abstract/FREE Full Text
  32. ↵
    1. Lu Z,
    2. Yuan L,
    3. Zhou X,
    4. Sotomayor E,
    5. Levitsky HI
    . CD40-independent pathways of T cell help for priming of CD8+ cytotoxic T lymphocytes. J Exp Med 2000;191:541-50.
    Abstract/FREE Full Text
  33. ↵
    1. Bennett SR,
    2. Carbone FR,
    3. Karamalis F,
    4. Flavell RA,
    5. Miller JF
    . Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 1998;393:478-80.
    CrossRefMedline
  34. ↵
    1. Blattman JN,
    2. Cheng LE,
    3. Greenberg PD
    . CD8+ T cell responses: it’s all downhill after their prime. Nat Immunol 2002;3:601-2.
    CrossRefMedlineWeb of Science
  35. ↵
    1. Lauer GM,
    2. Lucas M,
    3. Timm J,
    4. et al
    . Full-breadth analysis of CD8+ T-cell responses in acute hepatitis C virus infection and early therapy. J Virol 2005;79:12979-88.
    Abstract/FREE Full Text
  36. ↵
    1. Wedemeyer H,
    2. He XS,
    3. Nascimbeni M,
    4. et al
    . Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J Immunol 2002;169:3447-58.
    Abstract/FREE Full Text
  37. ↵
    1. Diepolder HM,
    2. Gerlach JT,
    3. Zachoval R,
    4. et al
    . Immunodominant CD4+ T cell epitope within nonstructural protein 3 in acute hepatitis C virus infection. J Virology 1997;71:6011-9.
    Abstract/FREE Full Text
  38. ↵
    1. Lamonaca V,
    2. Missale G,
    3. Urbani S,
    4. et al
    . Conserved hepatitis C virus sequences are highly immunogenic for CD4+ T cells: implications for vaccine development. Hepatology 1999;30:1088-98.
    CrossRefMedlineWeb of Science
  39. ↵
    1. Shirai M,
    2. Pendleton CD,
    3. Ahlers J,
    4. Takeshita T,
    5. Newman M
    . Helper-cytotoxic T lymphocyte (CTL) determinant linkage required for priming of anti-HIV CD8+ CTL in vivo with peptide vaccine constructs. J Immunol 1994;152:549-56.
    Abstract
  40. ↵
    1. Mizukoshi E,
    2. Sidney J,
    3. Livingston B,
    4. et al
    . Cellular immune responses to the hepatitis B virus polymerase. J Immunol 2004;173:5863-71.
    Abstract/FREE Full Text
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  1. J Infect Dis. (2006) 194 (4): 454-463. doi: 10.1086/505714
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