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Abstract

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

Pegylated interferon α (PEG IFN-α) improves sustained virological response rates in chronic hepatitis C, but neither its role in acute hepatitis C nor the biologic basis for its action has been defined. This prospective study assessed the efficacy of PEG IFN-α treatment in acute hepatitis C in relation to the kinetics of hepatitis C virus (HCV)-specific CD4+ T cell responses during therapy and follow-up. Forty subjects with proven acute hepatitis C who received either PEG IFN-α plus ribavirin (n = 20) or PEG IFN-α monotherapy (n = 20) for 24 weeks in addition to 14 untreated subjects with acute hepatitis C were prospectively followed. Serum HCV RNA, HCV-specific CD4+ T cell responses, and cytokine production were measured before and during therapy and at follow-up and correlated to the outcome. The sustained virological response rate was 85% with PEG IFN-α/ribavirin combination and 80% with PEG IFN-α monotherapy. Five untreated subjects had spontaneous recovery. The frequency, magnitude, and breadth of HCV-specific CD4+ T helper 1 responses were significantly higher in treated subjects compared with untreated subjects with self-limited disease or subjects with chronic evolution. The CD4+ T cell responses were maintained in subjects with sustained virological responses and self-limited disease but fluctuated in those who developed chronic infection. In conclusion, PEG IFN-α therapy in acute hepatitis induces high rates of sustained virological response and prevents choronicity, probably through efficient early stimulation of multispecific HCV-specific CD4+ T helper 1 responses. (HEPATOLOGY 2004;39:1721–1731.)

Hepatitis C virus (HCV) is an important public health issue that affects 170–200 million people worldwide.1, 2 Acute HCV is usually asymptomatic and therefore rarely recognized. Approximately 70% of infected persons develop chronic hepatitis, and between 20%–30% of infected persons may develop cirrhosis.2, 3 Several controlled and uncontrolled trials4–10 using different doses and durations of conventional interferon α (IFN-α) monotherapy in acute HCV reported viral clearance rates ranging between 37% and 100% in IFN-α–treated patients compared with only 12%–20% of untreated subjects. Most of these studies were problematic because of the small number of subjects, the diverse enrollment criteria, the different types and doses of IFN-α, the differences in the definition of response, and the duration of follow-up.11 The lack of controls in some studies renders the results of such trials difficult to interpret given that 14%–40% of patients with acute hepatitis C may resolve spontaneously.12, 13

Pegylated IFN-α (PEG IFN-α) has recently replaced conventional IFN-α formulations in treatment of chronic hepatitis C because of the higher rate of virological response induced by PEG IFN-α.14–19 The overall sustained virological response (SVR) rate in patients with chronic hepatitis due to genotype 2 and 3 reaches 85%; however, SVR in genotype 1 and genotype 4 ranges between 42% and 50%, thus leaving half of patients with chronic hepatitis.14, 15 Assessment of the efficacy and safety of PEG IFN-α monotherapy or PEG IFN-α/ribavirin combination therapy in acute hepatitis C has not been assessed to date.

The host immune response plays an important role in controlling HCV replication and determining the outcome of infection. Several studies in individuals who experienced complete virological recovery either spontaneously20–22 or after IFN-α therapy23–25 showed that vigorous, polyclonal, multispecific CD4+ T helper 1 responses correlate with HCV clearance, while absent or narrowly focused CD4+ T cell responses are typical of chronic hepatitis C.21, 22, 26, 27 We have previously shown that PEG IFN-α therapy enhances restoration of HCV-specific CD4+ T cell responses in subjects with chronic hepatitis25; however, the patterns and kinetics of these responses during PEG IFN-α therapy in a setting of acute hepatitis C have not been described. Therefore, this prospective longitudinal study characterized the kinetics of HCV-specific CD4+ responses and cytokine patterns in patients with acute hepatitis C receiving PEG IFN-α/ribavirin combination therapy, PEG IFNα-monotherapy, or no therapy, and correlated these parameters with the outcome of therapy and acute hepatitis.

Patients and Methods

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

Study Population.

Fifty-four patients (male/female ratio 31:23; Table 1) with proven acute hepatitis C were recruited from cohorts participating in open-label, parallel-dose controlled trials performed at four Egyptian and German centers. The mean time between infection and referral was 4.2 ± 2.5 weeks (range 2–7 weeks). The diagnosis of acute HCV infection was based on the following criteria: elevated serum alanine aminotransferase (ALT) more than 10 times the upper limit of normal; seroconversion from negative to anti–HCV-positive antibody status by second-generation enzyme-linked immunosorbent assay (Roche Diagnostics, Branchburg, NJ); and positive polymerase chain reaction for HCV RNA (Cobas Amplicor HCV version 2.0, with a lower limit of detection of 100 copies/mL, Roche Diagnostics). Patients were matched for age, gender, duration of HCV infection, baseline ALT, and viral load to improve uniformity and exclude other factors that might affect the outcome of acute HCV infection. Twenty subjects had genotype 1 and 34 subjects had genotype 4 as determined by a second-generation, reverse hybridization line-probe assay (Inno-LiPA HCV II, Innogenetics, Zwijndrecht, Belgium). The subjects were health care workers with archived pre-exposure HCV-negative serum specimens and reported occupational HCV exposure, which allowed us to detect seroconversion and monitor the clinical and immunological events very close to the date of infection. Eight subjects had symptoms and jaundice, while 46 subjects had mild nonspecific symptoms. The mean time from exposure to onset of symptoms in symptomatic subjects was 7.8 weeks (range 6–10 weeks). Other causes of hepatitis were ruled out by history and appropriate serologic studies. None of the study cohort had decompensated liver disease, coinfection with HIV, Schistosoma mansoni, neutropenia (<1,500/mm3), thrombocytopenia (<90,000/mm3), creatinine concentration more than 1.5 times the upper limit of normal, serum α feto-protein above 25 ng/mL, organ transplant, neoplastic disease, severe cardiac or pulmonary disease, unstable thyroid dysfunction, psychiatric disorder, current pregnancy or breast feeding, or therapy with immunomodulatory agents within the last 6 months.

Table 1. Demographic Baseline Characteristics of Subjects and Controls
ParameterGroup A: Acute HCV Treated With PEG IFN-α/RibavirinGroup B: Acute HCV Treated With PEG IFN-α MonotherapyControl: Acute Hepatitis, No Treatment
  • NOTE: Group A received PEG IFN-α plus ribavirin for 24 weeks. Group B received PEG IFN-α monotherapy for 24 weeks. The control group included 14 untreated subjects with acute hepatitis C.

  • Abbreviation: AST, aspartate transaminase.

  • *

    P values were calculated by Mann-Whitney U test. Statistically significant difference is defined as a P value > .05.

Number202014
Male/Female12/811/98/6
Age (y): mean ± SD36.6 ± 8.134.2 ± 7.631.7 ± 2.9
Caucasian n (%)20 (100)20 (100)20 (100)
Body weight (kg)   
 <85 n (%)15 (75)17 (85)9 (64)
 >85 n (%)5 (25)3 (15)5 (36)
Risk factors for HCV transmission   
 Occupational exposure201913
 Blood transfusion000
 Intravenous drug use000
 Sexual011
Symptoms/jaundice n (%)01 (5)7 (50)
Total bilirubin (mg/dL) mean ± SD1.9 ± 0.7*2.1 ± 0.653.8 ± 1.2*
ALT (U/L) mean ± SD544.3 ± 162.7608.4 ± 124.8613 ± 131.7
AST (U/L) mean ± SD688.6 ± 195.3712.5 ± 137.6720 ± 89.6
Genotype   
 Genotype 11082
 Genotype 4101212
RNA titer (cop × 106/mL) mean ± SD1.7 ± 0.351.8 ± 0.461.8 ± 0.23
Pegylated interferon alfa-2a1212NA
Pegylated interferon alfa-2b88NA

Study Design.

All patients were screened after the first positive HCV RNA or the onset of symptoms in symptomatic patients for 12 weeks with serum ALT and HCV RNA. The protocol was designed to treat subjects who did not recover spontaneously by 12 weeks. Of the 54 enrolled subjects, four had spontaneous resolution (7.4%) with persistently normal levels of aminotransferases and no detectable serum HCV RNA during further follow-up. The remaining 50 subjects had elevated transaminases and detectable HCV RNA after 12 weeks (Fig. 1). Forty subjects (male/female ratio 23:17; mean age 36.7 ± 8.5 years) provided informed consent for therapy and were categorized into two groups (Table 1): group A included 20 subjects who received PEG IFN-α/ribavirin combination therapy, and group B included 20 subjects who received PEG IFN-α monotherapy for 24 weeks. According to the type of pegylated interferon available in each center, 24 subjects received pegylated interferon alfa-2a (40 kd) (Pegasys Hoffman-La Roche, Basel, Switzerland) subcutaneously once weekly at a dose of 180 μg/wk alone (n = 12) or plus ribavirin 800 mg/d (n = 12), and 16 received pegylated interferon alfa-2b (PEG-Intron, Schering Corporation, Kenilworth, NJ) at a dose of 1.5 μg/kg each week alone (n = 8) plus ribavirin (Rebetol; Schering Corporation) (n = 8) at a dose of 10.6 mg/kg/d for 24 weeks. All treated subjects were followed for 48 weeks after cessation of therapy. Ten subjects who did not achieve spontaneous viral clearance refused therapy; one of the 10 subjects who was positive at week 12 cleared HCV RNA at study week 14. For this study, T cell responses in this and the four patients who resolved prior to week 12 are considered to represent self-limited disease. The 10 subjects who did not achieve spontaneous viral clearance at week 12 as well as the five subjects with self-limited disease were prospectively followed. Peripheral blood mononuclear cells (PBMCs) from 20 healthy donors served as negative controls for HCV-specific proliferative responses and cytokine enzyme-linked immunosorbent spot (ELISpot) assays.

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Figure 1. Study design and study flow chart. All patients with acute hepatitis C were screened until 12 weeks after infection. Four subjects had self-limited disease during screening, and a fifth untreated subject cleared HCV shortly after the end of screening. Forty subjects received either PEG IFN-α plus ribavirin (group A; n = 20) or PEG IFN-α monotherapy (group B, n = 20) as mentioned in Patients and Methods. Abbreviations: HCV, hepatitis C virus; PCR, polymerase chain reaction; PEG IFN-α, pegylated interferon α.

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The study protocol was reviewed and approved by the ethics committee of each of the participating centers, and all study procedures were conducted in conformity with the ethical guidelines of the Declaration of Helsinki. All patients participating in the study presented a written informed consent before enrollment and before any study related procedures.

Assessment and End Points.

Clinical biochemical laboratory values (serum ALT, aspartate aminotransferase, bilirubin) and HCV RNA were assessed before therapy and weekly during the first treatment month, then at 4-week intervals throughout the 24-week treatment period and 48-week posttreatment follow-up period. The primary efficacy end point was defined as undetectable serum HCV RNA levels (Cobas Amplicor HCV version 2.0 with a lower limit of detection of 100 copies/mL) 48 weeks after treatment (SVR). Secondary end points included biochemical response (normalization of serum ALT and aspartate aminotransferase at week 72) and virological and biochemical response at treatment week 24.

Antigens.

Genotype 1a–derived recombinant HCV protein coding for parts of core, nonstructural proteins NS3–4 and NS5,25 and control superoxide dismutase (SOD) protein were purchased from Chiron Corporation (Emeryville, CA). Yeast and SOD were used as controls for nonspecific stimulation in proliferation assays and ELISpot assays. As a positive control, phytohemoagglutinin (4 μg/mL at 1:200 dilution, Murex Diagnostics, Chatillon, France) and tetanus toxoid (1 μg/mL, Wyeth Laboratories, St. Davids, PA) were used.

Cell Preparation.

PBMCs were isolated as previously described22, 25 and used whole and after CD4 depletion in complete media. CD4 depletion was performed by CD4-dynabeads (Dynal Biotech, NY) per the manufacturer's instructions, with greater than 90% CD4 depletion confirmed by flow cytometry in all cases. Cryopreserved PBMCs were tested using proliferation assay and ELISpot assay.

Proliferation Assays.

Proliferation assays were performed at entry, before initiation of therapy, and at weeks 0, 4, 8, 12, 24, 48, and 72 following therapy induction as described previously21, 22, 25 using HCV proteins described above at concentrations of 2 μg/mL and control antigens. All proliferation assays were performed after 5 days of culture with HCV antigens or control antigens. All values were obtained in triplicate. A stimulation index (SI) of 3 or more (3 SD above the mean SI of normal control subjects) was considered significant. Healthy controls mounted no (0/20) significant response to any HCV-specific antigen (mean SIcore: 1.0; SINS3: 0.8; SINS4: 0.9; SINS5: 0.8; data not shown). Depletion assays performed as described previously22, 25 showed that proliferative responses are mediated by CD4+ cells (data not shown).

ELISpot Assay.

ELISpot assays were performed as described previously.21, 25 Briefly, 96-well nitrocellulose plates (Millipore, MA) were coated with 10 μg/mL of appropriate capture monoclonal antibody (human anti–interferon-γ [IFN-γ], anti–interleukin 4 [IL-4] antibody, Endogen, Woburn, MA) and incubated overnight at 4°C. PBMCs (200,000 cells/well) were added in triplicate together with HCV proteins (2 μg/mL), positive control (phytohemoagglutinin: 5 μg/mL; tetanus toxoid: 1 μg/mL), SOD, yeast stimulation, and medium as negative control. The plates were incubated at 37°C, 5% CO2 for 24 hours for the IFN-γ assays and 40 hours for the IL-4 assays, then antigen stimulation was stopped. The plates were developed and analyzed for spot-forming cells (SFCs) counted on an automated ELISpot reader (AID, Strassberg, Germany) as described previously.21 The background was always fewer than 15 × 106 SFCs. A cytokine response is considered positive if the mean SFCs/well in at least two of three wells is greater than 4 SD above the mean negative control response (at least 30 × 106 SFCs). In all experiments, stimulation with phytohemoagglutinin and tetanus toxoid induced similar numbers of IFN-γ and IL-4 SFCs in both patient groups, indicating the capacity of T cells of all subjects to produce these cytokines (data not shown). Healthy controls had no significant production of the tested cytokines in response to any HCV-specific antigens (data not shown). Depletion experiments confirmed that cytokine (IFN-γ, IL-4) secretion was due to CD4+T cells (data not shown).

Fluorescence Activated Cell Sorter Analysis.

Differentiation of CD4+ and CD8+ lymphocytes was performed by triple immunofluorescence staining with a FACscan (Becton Dickinson, Heidelberg, Germany) as previously described.22, 25

Statistical Analysis.

Results were expressed as mean ± SD. The mean values for clinical and immunologic parameters were compared using the nonparametric Mann-Whitney U test. The frequency of positive responses was compared using the χ2 or Fisher exact test based on sample size. Correlations between parameters were tested for statistical significance by Spearman rank correlation. All statistical procedures were performed using an SSPS for windows version 11 package (SPSS Inc., Chicago, IL).

Results

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

Baseline Characteristics of Subjects.

Study and control subjects did not show significant differences with respect to age, gender, route or duration of HCV infection, serum ALT level, or HCV RNA titers (Table 1). Forty-six subjects were either asymptomatic or had minimal nonspecific symptoms despite elevated serum ALT levels and positive HCV RNA after needle-stick injury, while eight patients presented with acute illness and jaundice. The time from exposure to onset of symptoms in symptomatic patients ranged from 5–8 weeks. ALT levels were slightly higher in subjects who achieved spontaneous clearance; however, the difference was not statistically significant (P = .06). Twenty subjects were infected with genotype 1 (10/20 of group A, 8/20 of group B, and 2/14 of the control group), while 34 patients were infected with HCV genotype 4 (10/20 of group A, 12/20 of group B, and 12/14 of the control group).

Outcome of Acute Infection With and Without Therapy.

Besides the four subjects who spontaneously recovered during screening (before week 12), one untreated subject recovered spontaneously at week 14, thus raising the number of subjects with self-limited disease in our cohort to five subjects. The five subjects with self-limited disease were infected with genotype 4 and were symptomatic and jaundiced at entry. The time from exposure to onset of symptoms in these patients ranged from 5–8 weeks. ALT levels were slightly higher in subjects who achieved spontaneous clearance; however, the difference was not statistically significant (P = .06).

At the end of treatment (week 24), viral clearance was achieved in 19/20 subjects treated with PEG IFN-α plus ribavirin (95%) versus 18/20 patients (90%) treated with PEG IFN-α monotherapy (P = .3; Fig. 2A). PEG IFN-α/ribavirin combination therapy induced earlier normalization of ALT and undetectable HCV RNA (mean 3.5 ± 1.9 weeks) compared with PEG IFN-α monotherapy (5.1 ± 1 week). An SVR was achieved in 17 subjects with PEG IFN-α/ribavirin combination therapy (85%) and 16/20 subjects treated with PEG IFN-α monotherapy (80%) (P = .27). The SVR rate achieved in either treated group was significantly higher than the rate of spontaneous viral clearance in untreated subjects (P < .0001 between group A and untreated control subjects, P = .0009 between group B and untreated control subjects; Fig. 2A). No statistically significant difference in the outcome of therapy was detected between pegylated interferon alfa-2a– and pegylated interferon alfa-2b–based regimen (data not shown). Of the 33 treated patients who achieved an SVR, 19 had genotype 4 (nine in group A and 10 in group B), and 14 subjects had genotype 1 (eight in group A and six in group B). The SVR was 80% and 75% in genotype 1 subjects treated with PEG IFN-α/ribavirin combination therapy and PEG IFN-α monotherapy, respectively (Fig. 2B). In genotype 4 subjects, an SVR was achieved in 90% and 83% subjects treated with PEG IFN-α/ribavirin combination therapy and PEG IFN-α monotherapy, respectively (Fig. 2C). Taken together, our data suggest the efficacy of pegylated interferon α treatment in preventing chronicity in acute hepatitis C, even for genotypes 1 and 4. The addition of ribavirin slightly increased the rate of viral clearance, particularly in genotype 1; however, the difference was not statistically significant. Therapy was well tolerated in all patients, with no serious adverse effects leading to discontinuation of therapy.

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Figure 2. End of treatment virological responses (week 24 after therapy, which corresponds to study week 36) and sustained virological response (SVR: week 72 after therapy, which corresponds to study week 84). (A) Comparison of the outcome of treatment in the two patient groups versus untreated control subjects who did not clear viremia at week 12 (n = 1). Group A (shaded columns): PEG IFN-α plus ribavirin (n = 20). Group B: PEG IFN-α monotherapy (n = 20) (black columns). The control group is indicated by the white columns. End of treatment response was 95% and 90% in groups A and B, respectively. (B) End of treatment response and sustained response to the different treatment regimens versus spontaneous recovery in genotype 1. (C) End of treatment response and sustained response to the different treatment regimens versus spontaneous recovery in genotype 4. Abbreviation: PEG IFN-α, pegylated interferon α.

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PEG IFN-α Therapy Is Associated With Enhanced HCV-Specific CD4+ T Cell Responses.

Our results revealed differences in the evolution, frequency, magnitude, and specificity of HCV-specific CD4+ T cell responses between treated and untreated subjects during treatment and follow-up. Regardless of therapeutic regimen or therapy outcome, treatment induced a statistically significant increase in the frequency and strength of CD4+ T cell responses to HCV antigens—particularly nonstructural antigens—in all treated patients at study week 24 (SIcore: 32.6 ± 8.5; SINS3: 38.2 ± 4.1; SINS4: 28 ± 0.5, SINS5: 25.6 ± 2.5) compared with untreated subjects (SIcore: 16.5 ± 0.9; SINS3: 18.5 ± 1.3; SI NS4: 12 ± 0.8; SINS5: 15.6 ± 1.2; P < .001) (Fig. 3).

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Figure 3. Time course of ALT, HCV RNA, CD4+ proliferative response, and IFN-γ in (A) patients treated with PEG IFN-α plus ribavirin (n = 20), (B) patients treated with PEG IFN-α monotherapy (n = 20), and (C) untreated control subjects who did not achieve spontaneous recovery (n = 9). The upper panels show the mean ALT levels (upper limit of normal: 40 U/L) (•) and the mean serum HCV RNA levels (♦). The middle panel shows CD4+ proliferative T cell responses to four HCV proteins (core, NS3, NS4, and NS5) expressed as the stimulation index (Y axis). The cut-off for a positive response is 3. The bars represent the mean response to a given antigen and the lines represent SD. The lower panel shows the number of spots representing IFN-γ–secreting cells in the ELISpot assay after stimulation with core, NS3, NS4, and NS5. The bars represent the mean response and the lines represent SD. Horizontal lines represent cut-off values. The X axis represents weeks elapsed since the first positive HCV RNA result (0) until end of follow-up (week 84). The interval of therapy is indicated by arrows. Abbreviations: PEG IFN-α, pegylated interferon α; ALT, alanine aminotransferase; HCV, hepatitis C virus; NS, nonstructural protein; SFCs, spot-forming cells; PBMCs, peripheral blood mononuclear cells.

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PEG IFN-α Therapy During Acute HCV Infection Is Associated With Increased HCV-Specific Type 1 T Cell Responses.

We confirmed these findings using ELISpot assay, which also provided measurement of the qualitative nature of the immune response. Responses against HCV antigens were predominately T helper 1 type (IFN-γ production, no IL-4) in treated subjects and untreated controls irrespective of the treatment type or outcome (Figs. 3–5). No significant HCV-specific IL-4 production was detected at any time point. Pretreatment HCV specific IFN-γ production was comparable in all study subjects with a response to at least 1 HCV antigen. PEG IFN-α therapy alone or in combination with ribavirin induced a more vigorous T helper 1 response to the nonstructural antigens (>200 SFCs/million) and HCV core (mean 145 SFCs/million,) compared with pretreatment levels (P = .02) and untreated control subjects (P = .01) (Figs. 3–5). Experiments depleting PBMCs of CD4+ T cells confirmed these responses to be mediated primarily by CD4+ T cells (data not shown).

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Figure 4. Time course of ALT, HCV RNA, CD4+ proliferative response, and IFN-γ production in (A) subjects with SVR (n = 33), (B) subjects who recovered spontaneously (n = 5), (C) subjects who relapsed or failed to respond (n = 7:), and (D) untreated subjects with chronic evolution (n = 9). Upper panels: The Y1 axis shows CD4+ T cell responses to four HCV proteins (core, NS3, NS4, and NS5) expressed as the stimulation index. The cut-off for a positive response is 3 (3 SD above the mean stimulation index of normal control subjects), which is shown by the horizontal line. The Y2 axis shows the mean serum HCV RNA levels (▪) before, during, and after therapy. Lower panels: The Y1 axis shows the mean number of spots representing IFN-γ–secreting cells in the ELISpot assay after stimulation with core, NS3, NS4, and NS5. The cut-off for a positive ELISpot response is shown by the horizontal line. The Y2 axis shows the ALT level (upper limit of normal: 40 U/L) (•). The X axis represents the weeks between the first positive HCV RNA result (week 0) and the end of follow-up (week 84). The interval of therapy is indicated by arrows. Bars represent the mean response to a given antigen; lines represent SD. SVR, sustained virological response; NS, nonstructural protein; HCV, hepatitis C virus; PEG IFN-α, pegylated interferon α; ALT, alanine aminotransferase; SFCs, spot-forming cells; PBMCs, peripheral blood mononuclear cells.

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Figure 5. (A) HCV-specific IFN-γ kinetics in treated and untreated patients according to the outcome of HCV infection.The change in IFN-γ production between study week 4 (after the first positive HCV RNA result, left) and end of treatment (right) are shown. The X axis indicates the type of patients: sustained responders (•), subjects who spontaneously recovered (▴), and nonresponders and subjects with chronic evolution (solid diamonds). The Y axis indicates the number of IFN-γ–producing T cells per 106. Each panel represents IFN-γ production after exposure to a given antigen. A significant increase in HCV-specific IFN-γ production at study week 36 (end of treatment) was detected in sustained responders and subjects with spontaneous recovery compared with IFN-γ production at study week 4. Horizontal lines in each graph indicate cut-off levels for a positive ELISpot response based on control wells. (B) IFN-γ production in response to tetanus toxoid at baseline, end of treatment, and end of follow-up. In all patients, irrespective of study group, responses elicited by HCV-unrelated control antigens, such as tetanus toxoid, did not vary at any time point. The Y axis indicates the number of IFN-γ–producing T cells per 106. Abbreviations: IFN-α, interferon α; PBMCs, peripheral blood mononuclear cells; NS, nonstructural protein; SVR, sustained virological response; SFCs, spot-forming cells.

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The Kinetics of the HCV-Specific CD4+ T Cell Response Correlates With the Outcome of Acute Hepatitis and Antiviral Therapy.

We then asked if the outcome of therapy was related to the pattern of HCV-specific CD4+ T cell response. Patients with SVR, self-limited disease, nonresponders, and chronic evolution had comparable baseline clinical, virological, and immunological parameters (Table 2). HCV clearance either spontaneously or after therapy was associated strongly with induction of a quantitatively strong and persistent type 1 CD4+ T cell response. Sustained responders in group A and B and untreated subjects with self-limited disease maintained significant virus-specific T cell responses to at least three antigens throughout follow-up (group A, SIcore: 23.9 ± 1.3; SINS3: 26.7 ± 2.1; SINS4: 15 ± 0.8; SINS5: 15.5 ± 4.5; group B, SIcore: 21.9 ± 3.1; SINS3: 24.1 ± 1.6; SINS4: 19.6 ± 4.2; SINS5: 15.6 ± 2.3; untreated control subjects, SIcore: 11 ± 0.4; SINS3: 9.6 ± 0.75; SINS4: 10.2 ± 0.4; SINS5: 12.6 ± 0.5) (Figs. 4 and 6). Although HCV-specific IFN-γ production tended to be higher and more sustained in subjects treated with PEG IFN-α/ribavirin combination compared with PEG IFN-α monotherapy, the difference was not statistically significant (P = .06; see Fig. 3). The HCV-specific T helper 1 responses in patients with an SVR were greater than those detected in untreated patients with self-limited disease (n = 5; P < .05) and significantly higher than the responses detected in subjects with chronic evolution (P < .001) who showed rapid decline of IFN-γ production to nonstructural antigens (Figs. 4 and 5A). Subjects who relapsed during follow-up benefited from therapy because they had significantly lower ALT and HCV RNA levels through follow-up compared with untreated subjects with chronic evolution (n = 9). The T cell responses in the patients who relapsed after an end of treatment response increased gradually with treatment but started to wane 4–6 months after completion of treatment. Interestingly, the decline in T cell responses was shortly followed by reappearance of viremia within 1–2 months (Fig. 4C). This suggests that early enhancement and maintenance of HCV-specific CD4+ T cell responses, either spontanously or induced by PEG IFN-α therapy, is crucial to viral clearance and in preventing evolution to chronic hepatitis.

Table 2. Baseline Clinical and Virological Parameters in Sustained Responders, Spontaneously Recovered Nonresponders, and Untreated Patients With Chronic Evolution
Clinical ParametersSustained Responders (n = 33) and Spontaneously Recovered (n = 5)Nonresponders/relapse (n = 7) and Chronic Evolution (n = 9)P Value*
  • NOTE: All subjects had comparable baseline clinical and virological characteristics.

  • *

    P values were calculated by Mann-Whitney U test for all. Statistically significant difference was considered with a P value > .05.

Males2011.052
Females185.002*
Age (y): mean ± SD 34.2 ± 7.6.94
Risk factors for HCV transmission n (%)   
 Occupational36 (94.7)16 (100) 
 Blood transfusion00 
 Intravenous drug use00 
 Sexual20 
Symptoms n (%)8 (21)0 
Total bilirubin (mg/dL)3.9 ± 2.61.8 ± 0.4.04*
ALT (IU/L)625.3 ± 204.6592.5 ± 183.4.95
Genotype   
 Genotype 1146 
 Genotype 42410 
Viral load, × 106 IU/mL1.5 ± 1.71.8 ± 1.9.300
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Figure 6. HCV-specific CD4+ T cell proliferation associated with HCV clearance (therapy-induced or spontaneous) or chronic evolution. CD4+ T cell responses to each HCV protein (core, NS3, NS4, and NS5) are shown as the stimulation index (S.I., Y axis) in subjects with a treatment-induced SVR (n = 33) (○), subjects who recovered spontaneously (n = 5) (•), and subjects with chronic evolution (n = 16) (▵) (X axis). The three columns represent different time points. (A) HCV-specific CD4+ T cell proliferation before therapy induction (week 8). (B) HCV-specific CD4+ T cell proliferation at week 12 after therapy induction (corresponding to study week 24). (C) HCV-specific CD4+ T cell proliferation at the end of follow-up (week 72 after therapy, corresponding to study week 84). Horizontal lines in each graph indicate cut-off levels for a positive response based on normal controls (mean ± 3 SD). CD4+ proliferative responses to tetanus toxoid are shown in the lowest scatter plot, in which the Y axis represents the SI and the X axis represents patients with treatment-induced SVR (n = 33) (○), subjects who recovered spontaneously (n = 5) (•), and subjects with chronic evolution (n = 16, including 7 relapser/nonresponders and 9 untreated subjects) (⋄). Statistical comparisons were performed by χ2 test, and P values pertain to the mean SI between the different groups as assessed by Mann-Whitney U test. P values > .05 are considered not to be significant. SI, stimulation index; NS, nonstructural protein; TT, tetanus toxoid; SVR, sustained virological response; SLD, self-limited disease.

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In all patients, irrespective of group, only HCV-specific immune responses significantly changed over time during treatment, while responses elicited by HCV-unrelated control antigens such as tetanus toxoid did not vary at any time point. This indicates that the observed differences in T cell responses are accounted for by HCV-specific immune effects rather than general ones (Figs. 5B and 6).

Discussion

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

The current prospective study included a large cohort of health care workers with proven acute HCV who were enrolled shortly after acquiring the infection. We included both symptomatic and asymptomatic patients infected with difficult-to-treat genotypes, namely genotypes 1 and 4, to assess whether or not long-term viral eradication and prevention of chronic evolution is likely with PEG IFN-α therapy in acute hepatitis. Therapy was initiated after 12 weeks of acute infection to avoid unnecessary treatment of cases that might recover spontaneously.10, 12, 13 In accordance with these reports, four of the five subjects (80%) with self-limited disease in our study achieved spontaneous viral clearance within 12 weeks after infection, while the fifth was HCV RNA–negative after 14 weeks. These findings may suggest that delaying therapy by 3 or 4 months after onset may be a reasonable strategy to target only those who fail to clear spontaneously without significant loss of treatment efficacy.

To date, the optimal regimen and duration of therapy in acute hepatitis C have not been defined. The overall SVR detected in our treated subjects (85% and 75% with PEG IFN-α/ribavirin or PEG IFN-α monotherapy, respectively) is significantly higher than the rate of spontaneous viral clearance detected in the control group. This is consistent with previous reports showing significant improvement in the rate of SVR with conventional IFN-α monotherapy compared with no treatment.4–9 According to our findings, the addition of ribavirin to pegylated interferon therapy did not provide a significant increase in the SVR rate. Given the additional costs and side effects of ribavirin, PEG IFN-α monotherapy might be an appropriate approach in the setting of acute hepatitis; however, this needs to be further investigated in larger cohorts. The SVR rate in our treated subjects was higher than that reported in studies using 3 million units of IFN-α three times a week for 6–24 weeks but comparable to studies using high daily doses of conventional IFN-α monotherapy for acute hepatitis, although some groups have reported a higher SVR.4, 6 The differences in SVR rate could be explained by the fact that our study included only patients infected with genotypes 1 and 4, which were associated in previous studies on subjects with chronic hepatitis with lower rates of virological response compared with genotypes 2 and 3. Moreover, therapy was delayed until week 12 following infection, while in other studies therapy was initiated in an earlier phase of acute infection in all cases, which might have resulted in treatment of some patients who would recover spontaneously as shown in our control group. In most previous reports, follow-up ranged from 6–24 weeks, while our study included a long-term follow-up evaluation 48 weeks after therapy to ensure that the HCV infection resolved after the 24-week regimen allocated in the current study. Our results thus suggest that a PEG IFN-α–based regimen provides benefit for patients with genotypes 1 and 4, especially given that the highest reported SVR rate achieved when treating chronic hepatitis C subjects infected with genotype 1 does not exceed 50%.13–15 Moreover, PEG IFN-α therapy was well tolerated in our study with a similar spectrum of side effects to that reported in previous trials with PEG IFN-α in chronic hepatitis patients14–19 or in trials of IFN-α monotherapy of acute hepatitis.5–10

To understand the immunological correlates of spontaneous and treatment-related recovery, we monitored the HCV-specific CD4+ T cell kinetics during screening, therapy, and follow-up in treated subjects and the control group. Our results provide additional support to previous studies demonstrating that resolution of acute HCV is marked by the development of vigorous polyclonal-persistent CD4+ T helper 1 cell responses during the acute phase, while absence or waning of CD4+ T helper 1 responses (as detected in untreated patients) favors chronic evolution.21–25 Our results suggest that PEG IFN-α monotherapy or PEG IFN-α/ribavirin therapy is associated with more efficient responses, particularly to nonstructural antigens, and helps to maintain these responses, thus decreasing the possibility of relapse. However, we cannot exclude the possibility that a decrease in viral load led to enhancement of the T cell response, due to some inhibition of T cell responses by HCV. At present, it is not clear if increased CD4+ responsiveness might be the result rather than the cause of viral clearance. Our findings in those who relapsed after cessation of therapy showed that the decline in T cell responses was detected at follow-up, followed by the reappearance of viremia. Similar observations were reported by Gerlach et al.21 However, these observations need to be further investigated, because the current study is limited to monitoring HCV-specific CD4+ responses. We are currently analyzing the kinetics of cytotoxic CD8+ T cell responses given the importance of these responses in the setting of acute HCV infection26, 27 and during interferon therapy.23

The biologic basis for the association of a higher SVR rate with more vigorous T cell responses remains to be explained. Our findings are in accordance with the findings of Schirren et al.,28 who detected that SVR following treatment of acute HCV infection after orthopedic liver transplantation was associated with a strong, multispecific CD4+ T cell response directed at nonstructural proteins. Our findings are also consistent with a report from Farci et al.29 that showed that acute resolving hepatitis was associated with homogeneity of the HCV quasispecies, whereas progressing hepatitis correlated with genetic diversity, presumably reflecting greater immune pressure during acute spontaneous clearance.29 Similarly, a reduction in genetic diversity leading to an increasingly homogeneous viral population was a consistent feature associated with viral clearance in sustained responders.30 Taken together, our data support the hypothesis that early treatment with a potent agent stimulates polyclonal T cell responses, both of which act to limit emergence of viral mutations and sequence heterogeneity.

In conclusion, our data indicate that treatment with PEG IFN-α alone or in combination with ribavirin in acute hepatitis C genotype 1 and 4 prevents chronic evolution and is associated with more vigorous, multispecific, sustained HCV-specific CD4+ T helper 1 cells. To date, it is not clear why few individuals with chronic infection generate sufficiently strong T helper 1 responses to achieve clearance, while others fail to mount or maintain these responses; therefore, finding a means to sustain efficient HCV-specific T cell responses might be crucial for development of a more effective therapeutic approach.

References

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