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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Spontaneous recovery occurs in a minority of patients with acute hepatitis C but is associated with vigorous and long-lasting cellular immune responses. Treatment-induced recovery can be achieved in the majority of patients who are treated in the acute phase, but the kinetics and mechanisms of viral clearance and immune responsiveness are not known. Both direct antiviral effects and indirect immune-mediated effects, such as immune modulation of Th2 to Th1 responses and prevention of exhaustion of cellular responses by rapid reduction of viral titer, have been proposed. To investigate how early antiviral therapy affects hepatitis C virus (HCV)-specific T cell responses, we performed detailed prospective clinical, virological, and immunological studies on 7 patients with acute hepatitis C who received antiviral therapy and were followed at 2 to 4 week intervals for 1 to 2 years. The total CD4+ and CD8+ cell response was analyzed with 600 overlapping HCV peptides and 6 proteins by ex vivo enzyme-linked immunospot (ELISpot), intracellular cytokine staining, and proliferation assays. In contrast to earlier studies with selected HCV epitopes, this extended analysis detected multispecific interferon γ+ (IFN-γ+) responses in each patient, even in the absence of T-cell proliferation. After initiation of antiviral therapy (at a mean of 20 weeks after infection), all sustained responders demonstrated gradually decreasing, then nearly absent HCV-specific T-cell responses, whereas the sole patient who developed viral breakthrough after initial HCV control maintained cellular immune responses. In conclusion, a sustained response to antiviral therapy was not associated with a lasting enhancement of HCV-specific T-cell responsiveness in the blood. (HEPATOLOGY 2004;40:87–97.)

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis in Western nations. In the United States, approximately 2.7 million people are persistently infected with HCV, and 40,000 become newly infected each year.1 Progression to chronic hepatitis occurs in up to 70% to 80% of infected persons, and it is projected that the incidence of complications from chronic liver disease, such as cirrhosis and hepatocellular carcinoma, will dramatically increase over the next 20 years.2 Because of the high number of persistently infected patients and because of difficulties in developing effective vaccines and immunotherapies, emphasis has been placed on antiviral therapies.

Recent studies have shown that early antiviral therapy in acute hepatitis C can prevent the development of chronic hepatitis.3 In a multicenter study, interferon-α (IFN-α) monotherapy started within the first 4 months of onset of acute hepatitis C resulted in sustained biochemical and virological responses in 98% of patients.4 In a second study, in which the start of therapy was delayed to an average of 5.7 months after onset of acute hepatitis, the sustained virological response rate was 80%.5 These response rates are far higher than those reported in chronic hepatitis C. Once patients have been infected for several years, IFN-α monotherapy results in sustained response rates of only 5% to 15%,6 and even pegylated interferon/ribavirin combination therapy has sustained virological response rates of no more than 56%.7, 8

Elucidation of the reasons for the different response rates in acute and chronic HCV infection is important to understanding the causes of resistance to antiviral therapy and the underlying factors that lead to HCV persistence. Previous reports have shown that HCV-specific immune responses are vigorous in patients who spontaneously clear HCV during acute infection and weak in those who develop persistent infection.9–15 Such findings have led to the suggestion that early antiviral therapy may prevent down-regulation or exhaustion of HCV-specific T-cell responses. Specifically, early antiviral therapy may decrease the number of quasispecies and therefore prevent the emergence of HCV mutants that may escape or antagonize T-cell responses.16 Also, reduction of HCV antigen levels may prevent subversion of host immune responses by HCV proteins, such as E2 and NS5A, that have been shown to inhibit innate responses.17–21 Furthermore, HCV core may inhibit adaptive T-cell responses by binding to the complement receptor.22

In the present study, we prospectively examined HCV-specific immune responses in relation to viral levels and antiviral therapy in 7 patients with acute hepatitis C. We studied IFN-γ production and proliferation as T-cell effector functions because these are readily detectable in the blood of patients with spontaneous HCV clearance9–11, 13, 14 but weak or absent in chronic hepatitis C.15, 23 To assess the total CD4+ and CD8+ T-cell response against all potential HCV epitopes in the context of all given human leukocyte antigen (HLA) alleles, we used recombinant HCV proteins and 600 overlapping 15-mer peptides that span the entire HCV polyprotein. In contrast to earlier studies with selected HCV peptides,10, 11 this study analyzes the total immune response in acute HCV infection. Results provide insights into the effects of early antiviral therapy on the cellular immune response and the outcome of acute hepatitis C.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients.

Seven patients with acute hepatitis C (patients A1-A7) were assessed prospectively. For comparison, the immune response of 4 patients who cleared HCV RNA and recovered spontaneously more than 5 years (patients R1 and R2), 2 years (patient R4), and 0.26 years (patient R3) before this study, as well as 9 patients with chronic hepatitis C (C1-C9) and 29 healthy, anti-HCV negative blood donors without a history of hepatitis were studied at a single time point. Acute hepatitis C was diagnosed based on (1) exposure to HCV-infected blood, (2) detection of serum HCV RNA by reverse transcriptase-polymerase chain reaction (Amplicor; Roche Diagnostics, Branchburg, NJ), and (3) elevation of serum alanine aminotransferase (ALT) levels at least 4-fold above the upper limit of normal. All but 1 patient (A3), who was not tested until later in the acute phase, had documented seroconversion from anti-HCV–negative to –positive by enzyme-linked immunosorbent assay (Abbott Laboratories, Abbott Park, IL). In addition, all but 1 patient (A6) developed symptoms consistent with acute hepatitis (Table 1). One patient (A7) was infected with human immunodeficiency virus (HIV) and on highly active antiretroviral therapy but had normal CD4+ T-cell counts at enrollment and no detectable HIV viremia throughout the study. All subjects tested hepatitis B surface antigen-negative, gave written informed consent according to a protocol approved by the National Institute of Diabetes and Digestive and Kidney Diseases Institutional Review Board, and were treated according to standard of care.

Table 1. Patient Demographics and Treatment Regimen
Patient No.SexRaceAge (y)Route of InfectionSymptomsJaundiceHCV GenotypeMax. ALT (U/L)DiagnosisTreatment
Start (wk)*RegimenOutcome
  • Abbreviations: AA, African-American; PEG-IFN, peginterferon; RIBA, ribavirin; SR, sustained responder; C, Caucasian; A, Asian; B, breakthrough.

  • *

    Week from exposure.

  • Dose reduction of peginterferon after 12 weeks of treatment due to ear infection.

  • Time point estimated; presumed date of HCV infection calculated based on last potential exposure risk.

  • §

    HIV coinfected; under highly active antiretroviral therapy; CD4+ T-cell counts within normal limits.

  • Dose reduction after 16 weeks due to ear infection and neutropenia.

A1FAA45NeedlestickYesYes1b888anti-HCV–neg. to–pos. (wk 13)21PEG-IFN + RIBA × 21 wkSR
A2FAA52NeedlestickYesNo1b470anti-HCV–neg. to–pos. (wk 11)23PEG-IFN + RIBA × 24 wkSR
A3FC23RazorYesNo1a356anti-HCV–pos. (wk 8)16PEG-IFN + RIBA × 24 wkSR
A4MC39Occupational (skin cut)YesNo1a499anti-HCV–neg. to–pos. (wk 14)>18PEG-IFN + RIBA × 24 wkSR
A5FA39NeedlestickYesYes2b1351anti-HCV–neg. to–pos. (wk 6)37PEG-IFN + RIBA × 24 wkSR
A6MC26NeedlestickNoNo1a154anti-HCV–neg. to–pos. (wk 12)4IFN (3 × 106 3×/wk) + RIBA × 18 wkIFN (4.5 × 106 3×/wk)  + RIBA × 14 wkPEG-IFN + RIBA × 5 wkSR
A7§MAA35Razor/SexYesNo1b1383anti-HCV–neg. to–pos. (wk 8)24PEG-IFN × 22 wkB

Antiviral Therapy.

Patients A1-A5 were treated with peginterferon α-2b (1.5 μg/kg; Schering-Plough, Kenilworth, NJ) and ribavirin (1000 mg daily for < 75 kg body weight and 1200 mg daily for > 75 kg body weight; Schering-Plough). Patient A6 was treated before the availability of pegylated IFNs and received initially standard IFN-α2b (3 × 106 3 times/wk) and ribavirin (1200 mg daily). The IFN-α dose was subsequently increased and later replaced by peginterferon (Table 1). The HIV/HCV coinfected patient (A7) was treated with peginterferon α-2b monotherapy because of concern that ribavirin in combination with didanosine and zalcitabine (which he was taking for his HIV infection) placed him at a higher risk for lactic acidosis and because previous studies had successfully used interferon monotherapy.4 Serum HCV RNA was quantitated with the HCV-Monitor Amplification kit (Roche Diagnostics).

HCV Antigens.

Six hundred 15-mer peptides (Mimotopes; Clayton, Australia), overlapping by 10 amino acids and covering the complete HCV genotype 1 polyprotein sequence were resuspended at 20 mg/mL in dimethyl sulfoxide (DMSO), pooled, and further diluted with phosphate-buffered saline (PBS) solution to obtain 18 mixes with each peptide at 24 μg/mL. HCV core, NS3, helicase, NS4, NS5A, and NS5B proteins were purchased from Mikrogen (Munich, Germany).15

Enzyme-Linked Immunospot (ELISpot) Assay.

IFN-γ and, at selected study points, interleukin 5 (IL-5) ELISpot assays were performed as described15 with duplicate cultures of 3 × 105 freshly isolated peripheral blood mononuclear cells (PBMC) and with either (1) individual HCV peptide mixes containing each peptide at 1 μg/mL, (2) 1 μg/mL HCV protein, (3) 1 μg/mL phytohemagglutinin (PHA; Murex Biotech Limited, Dartford, England), (4) buffer control, or (5) DMSO control, respectively. Antigen-specific spot-forming cells (SFC in the presence of antigen minus SFC in buffer or DMSO controls) were quantitated with a KS ELISpot Reader (Zeiss, Thornwood, NY). Average background responses were 6 SFC/3 × 105 PBMC for the protein buffer control and 5 SFC/3 × 105 PBMC for the DMSO control. When peptides of selected, recognized mixes were retested individually, the sum of the individual peptide responses was comparable to the response against the peptide mix (data not shown).

To exclude the possibility of nonspecific stimulation by the peptide mix, patient PBMC were tested with a control mix of overlapping 15-mer hepatitis D virus (HDV) peptides. No significant responses were observed. In addition, PBMC of 29 healthy, anti-HCV–negative controls were tested with 5 HCV proteins and 18 HCV peptide mixes with the following results (mean number of SFC ± SD per 3 × 105 PBMC): HCV proteins—core, 1.19 ± 1.29; NS3, 1.24 ± 1.36; NS4, 1.5 ± 1.33; NS5A, 1.05 ± 0.65; NS5B, 1.18 ± 0.95— and HCV peptide mixes—core, 1.95 ± 4.83 (1 mix); E1, 3.24 ± 5.82; E2 (2 mixes), 1.05 ± 2.08; p7 (1 mix), 1.72 ± 2.55; NS2 (1 mix), 2.8 ± 4.92; NS3 (3 mixes), 1.81 ± 2.81; NS4A (1 mix), 2.22 ± 3.23; NS4B (2 mixes), 2.26 ± 2.64; NS5A (3 mixes), 7.29 ± 13.59; and NS5B (3 mixes), 5.68 ± 8.77. A response was scored as positive if it was greater than the mean response + 2 SD in healthy, anti-HCV–negative control subjects and more than 3-fold above the background response (buffer control for proteins or DMSO control for peptides) in HCV-infected patients.

Intracellular Cytokine Staining.

To differentiate between IFN-γ–producing CD4+ and CD8+ T cells, 2 × 106 PBMC were stimulated separately with combinations of the HCV peptide mixes previously described (final concentration of 1 μg/mL per peptide), the corresponding DMSO control, or 1 μg/mL PHA (Murex Biotech Limited) in 500 μL RPMI1640 (Cellgro, Herndon, VA), supplemented with 10% FBS, 2 mmol L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin. Anti-CD28 and -CD49d antibodies (BD Pharmingen, San Diego, CA) were added at 1 μg/mL each. After 2 hours, 2 μL monensin was added. After 4 additional hours, cells were treated with 2 mmol ethylenediaminetetraacetic acid for 20 minutes at room temperature and washed and stained with antibodies against CD4 and CD8 for 30 minutes at 4°C. Cells were washed again, fixed and permeabilized with the Cytofix/Cytoperm Kit (BD Pharmingen), stained with antibodies against IFN-γ for 30 minutes at 4°C, washed, resuspended in PBS, and immediately analyzed on a FACSCalibur (Becton Dickinson, Santa Ana, CA).

Proliferation Assays.

Triplicate cultures of 200,000 PBMC were stimulated with 1 μg/mL HCV protein, buffer control, or 1 μg/mL PHA as previously described.15 The cutoff for a significant response was set at a stimulation index (counts per minute [cpm] in the presence of antigen/cpm in the absence of antigen) of 3 for each protein, equivalent to the mean proliferative response of 29 healthy, anti-HCV–negative controls + 2 SDs.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

HCV-Specific Immune Response in Acute Hepatitis C Before Treatment.

Seven patients with acute hepatitis C were studied (patients A1-A7, Table 1). The group included 4 women and 3 men, ages 23 to 52 (3 African-American, 3 Caucasian, and 1 Asian-American). All but 1 patient (A6, who requested immediate treatment), were followed for at least 16 weeks before starting therapy to evaluate the possibility of spontaneous recovery. During this time, mild clinical symptoms such as fatigue and pain in the upper right quadrant of the abdomen were reported by all patients and 2 (A1 and A5) developed jaundice. Serum ALT levels increased to peak values of 154 to 1383 U/L and peak HCV RNA levels ranged between 103 and 107 copies/mL (Figs. 1A and B, Figs. 2A and B, Figs. 3A and B, Fig. 5A).

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Figure 1. Serological, virological, and immunological parameters during acute HCV-infection and antiviral therapy in sustained treatment responders (patients A1 and A2). (A and B) Serological and virological course of disease. Serum alanine aminotransferase (ALT) levels are expressed in U per liter [U/L] (blue diamonds); HCV RNA values are expressed as HCV RNA copies/mL (red squares). Details on seroconversion from anti-HCV–negative to –positive and antiviral therapy (gray area) are provided in Table 1. (C and D) Bars indicate the frequency of HCV-specific CD4+ and CD8+ T cells that produce IFN-γ in response to overlapping HCV peptides covering the complete HCV polyprotein (ELISpot analysis). Responses are shown as antigen-specific, IFN-γ spot-forming cells (SFC) per 3 × 105 peripheral blood lymphocytes (PBL). The line indicates the percentage of IFN-γ–producing CD8+ T cells within the total population of HCV-specific, IFN-γ–producing T cells as determined by intracellular cytokine analysis. (E and F) Frequency of HCV-specific CD4+ T cells that produce IFN-γ in response to HCV proteins (ELISpot analysis). (G and H) Proliferative T-cell response to HCV proteins. The stimulation index indicates proliferation in the presence of antigen relative to proliferation in the absence of antigen. Significant proliferative responses are indicated by colored bars. (I) Both CD4 and CD8 T cells contribute to the IFN-γ response to overlapping HCV peptides. Representative results are shown. The percentage of IFN-γ–producing CD4 and CD8 cells is indicated in each dot blot. n.t., not tested; FSC, forward scatter.

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Figure 2. Serological, virological and immunological parameters during acute HCV-infection and antiviral therapy in sustained treatment responders (patients A3 and A4). (A and B) Serological and virological course of disease. Gray area: time of antiviral therapy (see Table 1 for details). (C and D) Frequency of HCV-specific CD4+ and CD8+ T cells that produce IFN-γ in response to overlapping HCV peptides covering the complete HCV polyprotein (ELISpot analysis). (E and F) Frequency of HCV-specific CD4+ T cells that produce IFN-γ in response to HCV proteins (ELISpot analysis). (G and H) Proliferative T-cell response to HCV proteins, for details, see Fig. 1 legend. SFC, spot-forming cells; PBL, peripheral blood lymphocytes; n.t., not tested.

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Figure 3. Serological, virological, and immunological parameters during acute HCV infection and antiviral therapy in sustained treatment responders (patients A5 and A6). (A and B) Serological and virological course of disease. Gray area: time of antiviral therapy (see Table 1 for details). (C and D) Bars indicate the frequency of HCV-specific CD4+ and CD8+ T cells that produce IFN-γ in response to overlapping HCV peptides covering HCV core, NS3, NS4A, and NS4B proteins (ELISpot analysis). Responses are shown as antigen-specific, IFN-γ spot-forming cells (SFC) per 3 × 105 peripheral blood lymphocytes (PBL). The line indicates the percentage of IFN-γ–producing CD8+ T cells within the total population of HCV-specific, IFN-γ–producing T cells as determined by intracellular cytokine analysis. (E and F) Proliferative T-cell response to HCV proteins, for details, see Fig. 1 legend. (G) Both CD4 and CD8 T cells contribute to the IFN-γ response to overlapping HCV peptides. Representative results are shown. The percentage of IFN-γ–producing CD4 and CD8 cells is indicated in each dot blot. n.t., not tested; FSC, forward scatter.

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Figure 5. Serological, virological, and immunological parameters during acute HCV infection and antiviral therapy in patient A7, an initial treatment responder with a virological breakthrough toward the end of therapy. (A) Serological and virological course of disease. Gray area: time of antiviral therapy (see Table 1 for details). (B) Frequency of HCV-specific CD4+ and CD8+ T cells that produce IFN-γ in response to overlapping HCV peptides covering the complete HCV polyprotein (ELISpot analysis). (C) Frequency of HCV-specific CD4+ T cells that produce IFN-γ in response to HCV proteins (ELISpot analysis). (D) Proliferative T-cell response to HCV proteins for details, see Fig. 1 legend. SFC, spot-forming cells; PBL, peripheral blood lymphocytes; n.t., not tested.

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To evaluate the kinetics of the cellular immune response in relation to the clinical and virological course of acute HCV infection, the total CD4+ and CD8+ T-cell response that each subject mounted in the context of the complete HLA class haplotype was assessed with overlapping 15-mer peptides that covered the complete HCV polyprotein sequence. Multispecific IFN-γ responses were detectable in all patients (Figs. 1C and D, Figs. 2C and D, Figs. 3C and D, Fig. 5B) and mainly targeted against epitopes in the nonstructural HCV proteins (Figs. 1C and D, Figs. 2C and D, Fig. 5B). Both CD4+ and CD8+ T cells contributed to the overall IFN-γ response as demonstrated by intracellular cytokine staining (see Fig. 1I and Fig. 3G for representative dot plots and Fig. 1C, Fig. D, Fig. 3C for an assessment of the contribution of CD8+ T cells to the overall IFN-γ response).

Whereas overlapping HCV peptides were used to assess the total CD4+ and CD8+ T-cell response, recombinant HCV proteins were used to study CD4+ T-cell responses in ELISpot and proliferation assays. All patients mounted IFN-γ responses to recombinant proteins and, with the exception of patient A4 (Fig. 2F), nonstructural proteins were most frequently recognized (Figs. 1E and F, Fig. 2E, Fig. 5C). Despite the presence of IFN-γ+ T-cell responses in all patients, however, only 3 patients displayed significant proliferative T-cell responses: patient A1 (Fig. 1G), patient A2 (Fig. 1H), and patient A5 (Fig. 3E). In all 3 patients, this response targeted epitopes within nonstructural proteins, thereby confirming that proliferative T-cell responses, if present, were targeted against the same proteins as IFN-γ responses.

Jaundice and clinical symptoms as surrogate markers of vigorous immune responses are frequently discussed as indicators for a self-limited outcome of HCV infection.24, 25 Two recent studies report spontaneous HCV clearance in 12 of 14 patients26 and 24 of 46 patients5 with symptomatic hepatitis C, but in 0 of 2 and 0 of 9 patients with asymptomatic hepatitis C.5, 26 Because 2 patients (A1 and A5) in our study developed jaundice and clinical symptoms, we asked whether this was a correlate of stronger cellular immune responses. Indeed, these 2 patients had the strongest proliferative T-cell responses prior to therapy (stimulation indices of 70 to 80 against individual HCV proteins). Interestingly, these responses coincided with transient decreases in HCV RNA level. Patient A1, for example, displayed a decrease of HCV RNA levels at weeks 15 and 18 before therapy and at week 23 just after the start of antiviral therapy (Fig. 1A), and HCV-specific T-cell proliferation was significantly increased at precisely those time points (Fig. 1G). In contrast, when HCV RNA reached peak levels at weeks 17 and 21 after infection (Fig. 1A), HCV-specific T-cell proliferation was weak (Fig. 1G). Similarly, patient A5 displayed a transient decrease of HCV RNA levels at weeks 7, 9, and 18 after infection (Fig. 3A), and HCV-specific T-cell proliferation was significantly increased at those time points (Fig. 3E). When HCV RNA reached peak levels at weeks 5, 11, 15, and 35 after infection (Fig. 3A), HCV-specific T-cell proliferation was diminished (Fig. 3E). Thus, these 2 patients with clinically severe disease demonstrated periods of decreased HCV RNA levels that coincided with stronger cellular immune responses, followed by periods of weaker immune responses and increased HCV RNA and ALT levels. Notably, this pattern of virus/host interaction continued during the first 21 and 37 weeks of HCV-infection, but did not lead to spontaneous HCV clearance. Thus, as previously hypothesized,5, 24 a symptomatic onset of hepatitis was indeed associated with vigorous HCV-specific T-cell responses that coincided with a transient decline of HCV RNA levels. In contrast to previous reports,11, 26 however, rapid resolution of disease was not observed.

HCV-Specific Immune Responses of Patients With a Sustained Treatment Response.

Because no patient resolved HCV infection spontaneously, antiviral therapy was started at a mean of 20 weeks after infection (Table 1). Patients A1 to A6 (86% of all patients) had a sustained response to treatment. The following is a description of their their immune response during antiviral therapy.

Within the first month of treatment, HCV RNA levels decreased significantly by 2 (patient A3, Fig. 2A, and patient A6, Fig. 3B) to 6 logs (patient A1, Fig. 1A). One patient showed a 4-log decrease of HCV RNA just prior to therapy, and HCV RNA remained undetectable during treatment (patient A2, Fig. 1B). Reduction of HCV RNA levels during therapy was followed by a transient increase in HCV-specific T-cell responses in 3 of 6 patients. Patient A1, for example, started therapy at week 21 after infection and displayed an increased IFN-γ production (Fig. 1C) and proliferation (Fig. 1G) of HCV-specific T cells at week 23 after infection, when HCV RNA became undetectable. Patient A4 displayed an increased HCV-peptide and protein-specific IFN-γ response at weeks 19 and 21, respectively, when HCV RNA levels decreased (Figs. 2D and F). Likewise, patient A6 showed an increase of HCV protein-specific, IFN-γ production and T-cell proliferation at week 12, i.e., precisely when HCV RNA was undetectable (Figs. 3D and F). Surprisingly, however, continued antiviral therapy was associated with a gradual decrease of HCV-specific T-cell responses to levels below the detection limit of the assays. In all patients, the disappearance of HCV-specific IFN-γ–producing and proliferating T cells in the peripheral blood occurred at a slower pace than the decrease of HCV RNA levels. In patient A1, for example, HCV RNA levels were significantly decreased by week 23, but the HCV-specific IFN-γ+ T-cell response remained detectable until week 37 (Fig. 1B). Similar observations were made for patients A4 (Fig. 2D) and A5 (Fig. 3C). HCV-specific T-cell responses of patient A2 (Fig. 1D) that had already spontaneously decreased before treatment were not restored by antiviral therapy.

At the last bleed date, i.e., 21 to 24 weeks after cessation of therapy, sustained treatment responders displayed significantly weaker proliferative T-cell responses than a control group of spontaneously recovered patients (P = .027, Fig. 4). Indeed, proliferative T-cell responses of sustained treatment responders were comparable to those of untreated, persistently HCV-infected patients (Fig. 4). A similar trend, although not statistically significant, was observed for the HCV-specific IFN-γ response to recombinant proteins (P = .055; data not shown). Reduction of IFN-γ production was not due to a Th1 to Th2 shift, because IL-5 was not detected in IL-5 ELISpot assays (data not shown). Thus, HCV-specific immune responses remained relatively weak in the peripheral blood of sustained treatment responders for at least 24 weeks after cessation of therapy.

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Figure 4. Proliferative T-cell responses of sustained treatment responders in comparison to those of a group of spontaneously recovered patients and a group of persistently HCV-infected patients. At weeks 21 to 24 after cessation of antiviral therapy, the 6 sustained treatment responders (A1-A6) displayed significantly weaker HCV-specific proliferative T-cell responses than 4 spontaneously recovered patients (R1-R4). The proliferative T-cell response of 9 patients with persistent HCV infection (C1-C9) was assessed for comparison. The stimulation index indicates proliferation in the presence of antigen relative to proliferation in the absence of antigen.

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HCV-Specific Immune Responses of a Patient With Viral Breakthrough.

Patient A7 was the sole patient who did not show a sustained treatment response and, instead, experienced viral breakthrough after initial control of HCV (Fig. 5A). Because of reasons previously outlined, patient A7 received peginterferon-α-2b monotherapy. Compared to patients A1 to A6, he initially showed a significant decrease of HCV RNA levels (Fig. 5A). His HCV-specific T-cell response, however, did not decrease to undetectable levels, even though a virological response was maintained until week 40. Instead, HCV-specific T-cell responses showed some fluctuation with significant peaks of IFN-γ–producing T cells at week 28 to 36 (Figs. 5B and C). At the same time, ALT levels increased to 68 U/L (normal range 6-41 U/L). These results suggest that HCV-specific T-cell responses may have been stimulated and maintained by low levels of remaining antigen and that HCV may not have been completely cleared. Indeed, a viral breakthrough occurred several weeks later with an increase in HCV titer to 106 to 107 copies/mL (Fig. 5).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Whereas spontaneous HCV clearance occurs in about 30% to 50% of patients with acute hepatitis C,27 it is virtually absent once chronic infection is established. Treatment-induced HCV clearance is also more frequently achieved in patients with acute hepatitis C4 than in patients with chronic hepatitis C.6 The mechanisms responsible for the high rate of spontaneous and treatment-induced HCV clearance in acute hepatitis C are unknown.

One hypothesis that has been raised is based on the different immune responses of patients with acute and chronic hepatitis C. Patients who spontaneously recover display multispecific cellular immune responses in the peripheral blood,9–14 but HCV-specific immune responses are weak or completely undetectable during chronic hepatitis C, suggesting primary failure,11 secondary down-regulation, or exhaustion.10 It has therefore been proposed that early therapeutic reduction of HCV RNA levels may prevent down-regulation and exhaustion of HCV-specific immune responses and facilitate viral clearance.4, 24 This hypothesis is supported by data from lymphocytic choriomeningitis (LCMV) and simian immunodeficiency virus (SIV) models, in which early control of viral replication facilitates the development of virus-specific proliferative T-cell responses.28, 29

The current study addressed these issues in a prospective manner by the analysis of the cellular immune responses of 7 patients during acute HCV infection and subsequent antiviral therapy. In contrast to earlier studies with individual HCV peptides, the present study employed overlapping 15-mer peptides spanning the entire HCV polyprotein. Advantages of this method were the analysis of the CD4+ and CD8+ T-cell response in the context of the complete HLA haplotype of individual patients without the bias of preselecting a few epitopes that represent only a limited proportion of the response. Disadvantages were the necessity of performing additional assays to assess the differential contribution of CD4+ and CD8+ T cells to the overall response and the potential underestimation of responses because of the theoretical possibility of T cell receptor (TCR) and/or major histocompatibility complex (MHC) competition among peptides with similar binding motifs and affinities. Nevertheless, the present study clearly demonstrated that all patients mounted vigorous T-cell responses against HCV in the acute phase of hepatitis prior to treatment and that a substantial proportion of these responses was mediated by CD8+ T cells.

Peginterferon with and without ribavirin yielded a sustained response in 6 of 7 (86%) patients in this study. This is consistent with a previous report demonstrating a 98% response rate, in which patients with acute, symptomatic hepatitis C were treated with a 24-week course of IFN-α2b monotherapy.4 The sole patient whose initial response to antiviral therapy was not sustained and who eventually developed a viral breakthrough in this study did not receive ribavirin. This observation is consistent with the notion that ribavirin might facilitate the sustenance of the initial virological response.30 Apart from the omission of ribavirin, however, the patient's HIV status, African-American race and male sex may also have contributed to the lower likelihood of a sustained response.

Notably, interferon/ribavirin therapy was still effective when started at time points at which the peripheral blood T-cell responses—the hallmark of spontaneous viral clearance—had already decreased substantially (patient A2, Fig. 1D, and patient A3, Fig. 2C). Although, in these cases, interferon/ribavirin therapy was not associated with a significant, lasting increase of the HCV-specific T-cell responsiveness in the blood, it remains possible that it affected the intrahepatic T-cell response for a sufficient time to induce viral clearance. Intrahepatic HCV-specific T-cell responses have been detected during acute, self-limited hepatitis C in chimpanzees even in the absence of peripheral blood responses31–33; this is consistent with a compartmentalization at the site of inflammation. In addition, T–cell-independent, antiviral mechanisms of IFN action may have contributed to HCV clearance. In any case, HCV clearance was followed by a decrease of HCV-specific T-cell responses in the blood, similar to the natural contraction of T-cell effector populations after complete antigen clearance.34 Consistent with these findings, the maintenance of HCV-specific T-cell responses in patient A7 (Fig. 5) suggested that HCV antigens were not completely cleared in this patient; this was confirmed by the subsequent viral breakthrough.

How do HCV-specific T-cell responses of sustained treatment responders compare to those of spontaneously recovered patients? Because of the high rate of response to treatment, it was not possible to include a control group of untreated patients for this study. Interestingly, however, several additional investigators have prospectively analyzed the cellular immune response of patients with acute hepatitis C and described a decline of HCV-specific IFN-γ responses within 10 to 15 weeks of spontaneous HCV clearance and resolution of acute hepatitis C.10, 14, 35 In our study, a comparison of the proliferative T-cell responses of sustained treatment responders and spontaneously recovered patients revealed a significant difference in proliferative T-cell responses (P = .027; Fig. 4). A similar, although not statistically significant difference in IFN-γ production was also seen in HCV protein-specific IFN-γ production (P = .055; data not shown). These results are supported by several recent reports36–38 that uniformly describe significantly weaker HCV-specific immune responses in sustained treatment responders than in spontaneously recovered patients. Because HCV-specific T-cell proliferation has been described as an immunological hallmark of recovery from hepatitis C,9, 11, 13, 15, 39–41 these findings may have clinical implications for T cell-mediated protective immunity.32, 42, 43

Whether HCV-specific T-cell responses of sustained treatment responders remain weak during longer follow-up clearly requires further investigation. Because the difference between the HCV-specific T-cell responses of sustained treatment responders and spontaneously recovered patients was most pronounced in terms of HCV-specific T-cell proliferation, and because IFN-α is known for its antiproliferative, myelosuppressive, and proapoptotic effects,44, 45 it is possible that the impaired proliferative response represents a side effect of antiviral treatment. Only a small percentage of memory T cells divide at any given time point,46 and it may take years for the proliferative T-cell response of sustained treatment responders to recover completely. Because the current study included health care workers with an increased risk of reexposure to HCV, further prospective follow-up of the strength and quality of the memory T-cell response is warranted.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank all patients for participating; Dr. Elliott Aleskow for patient referral; Ms. Yoon Park and Ms. Rebecca McBurney for expert patient care; and Ms. Charma Zuber, Ms. Marina Chen, Ms. Zorayda Aben, Mr. Timothy Davis, Ms. Wan Hua Zhang, Ms. June Germain, and Ms. Tracy Peters for reverse transcriptase-polymerase chain reaction and antibody analysis.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References