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HCV Acute Infection: immune and clinical responses
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Treatment-Induced vs. Spontaneous Clearance of Acute HCV Infection
Hepatology
Volume 40, Issue 1, July 2004
Three studies, published in the June and July issues of HEPATOLOGY, examined hepatitis C virus (HCV)-specific immune responses during and after antiviral treatment of acute hepatitis C. Rahman et al. (see abstract below) studied 7 acutely infected patients and found that all mounted HCV-specific T cell responses prior to therapy. However, after initiation of therapy, HCV-specific cellular immune responses waned in the 6 patients who achieved a sustained virological response. At a follow-up of 21-24 weeks, sustained treatment responders showed significantly weaker proliferative T cell responses than a control group of spontaneously recovered patients. Indeed, responses were comparable to those of patients persistently infected with HCV (Figure). This is a surprising finding, since cellular immune responses have been shown to persist for decades after spontaneous viral clearance. Indeed, the results suggest that treatment-induced viral clearance is different from spontaneous viral clearance. Is it possible that viral elimination was achieved by the direct antiviral effects of interferon (IFN)-a and that cellular immune responses are less important in this setting? Does rapid and complete suppression of viral antigens abrogate the development and maintenance of immunological memory? Would one have to search for HCV-specific T cells in the liver instead of the peripheral blood? Or are we looking at an IFN-a-mediated inhibition of T cell effector functions? Further studies and an extended follow-up will be required to address these questions.
A second study, by Wiegand et al., may provide some answers. These investigators report the long-term outcome of 31 patients successfully treated for acute hepatitis C. The good news is that viral elimination is complete and durable in these patients. Interestingly, at their latest follow-up (i.e., 52-224 weeks after completion of therapy), these authors found that while antibody and CD4+ T cell responses had vanished in the majority of patients, robust CD8+ T cell responses were detectable in 4 out of 5 patients analyzed in detail. Thus, although the number of patients examined with respect to CD8+ T cell responses is small, it is possible that some cellular immune responses recover after longer follow-up. (See HEPATOLOGY 2004;40:98-107; see abstract below)
A third study, by Kamal et al., provides quite a different view. These investigators prospectively followed 40 patients with acute hepatitis C treated with pegylated IFN- with or without ribavirin. In contrast to the other two studies, a clear increase in the frequency and strength of HCV-specific CD4+ T cell responses was observed in these patients both during therapy and at follow-up (i.e., 48 weeks following cessation of therapy). (See HEPATOLOGY 2004;39:1721-1731.)
How do we explain these different results? A clear explanation is not forthcoming. Subtle differences in the timing and schedule of treatment, the viral genotype, and the techniques used to monitor T cell responses may, among other factors, explain the differences. In any case, further investigation of these issues may not only contribute to optimal care of patients with acute hepatitis C, but may shed light on basic aspects of immunological memory and the pathogenesis of hepatitis C.
Outcome of HCV Infection: Does Timing and Homing Make the Difference?
Not tired of studies on acute hepatitis C? Here's another elegant one. Previous studies in acutely HCV-infected chimpanzees have clearly shown that innate and adaptive immune responses contribute significantly to viral clearance and liver disease. However, these studies have been limited by small numbers of animals, the fact that some chimpanzees were previously exposed or immunized, and the heterogeneity of the inocula used. Major et al. have now analyzed 10 naïve chimpanzees that were inoculated with virus or RNA encoding the same clonal HCV sequence. Four animals cleared the virus and 6 developed chronic infection. As has been shown previously, all animals displayed an intrahepatic induction of IFN-a/b-related genes during the first 2 weeks of infection, irrespective of the outcome of infection. This correlated with the first significant slowing of virus accumulation, suggesting that early partial control of viral replication may be achieved by type I IFNs. Eight to 10 weeks after infection, liver disease and intrahepatic induction of IFN-g were observed in all animals. This coincided with decreases of viral titers by at least 2 log10, and this effect was observed in all animals. Importantly, however, it occurred approximately 2 weeks later in the chronic group. In addition, the inability of the immune response to sustain viral clearance was associated with reduced intrahepatic CD3 mRNA, reflecting a reduced accumulation of presumably HCV-specific T cells at the site of disease. Thus, the most important finding of this study is that the difference between clearance and persistence of HCV may be governed by the timing of the host immune response and the accumulation of the right cells at the right place. But the big questions remain: What causes these differences? Can we influence them? Can the virus influence them? Looking more closely at viral evolution may yield additional insight into the complex mechanisms of viral persistence. (See HEPATOLOGY 2004;39:1709-1720.)
Effects of antiviral therapy on the cellular immune response in acute hepatitis C
Hepatology
Volume 40, Issue 1, July 2004
Fareed Rahman 1, Theo Heller 1, Yuji Sobao 1, Eishiro Mizukoshi 1, Michelina Nascimbeni 1, Harvey Alter 2, Steven Herrine 3, Jay Hoofnagle 1, T. Jake Liang 1, Barbara Rehermann 1 *
1Liver Diseases Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD
2Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Department of Health and Human Services, Bethesda, MD
3Thomas Jefferson University, Philadelphia, PA
ABSTRACT
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 g+ (IFN-g+) 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.
BACKGROUND
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. 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. 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. In a multicenter study, interferon-a (IFN-a) monotherapy started within the first 4 months of onset of acute hepatitis C resulted in sustained biochemical and virological responses in 98% of patients. 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%. These response rates are far higher than those reported in chronic hepatitis C. Once patients have been infected for several years, IFN-a monotherapy results in sustained response rates of only 5% to 15%, and even pegylated interferon/ribavirin combination therapy has sustained virological response rates of no more than 56%.
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. 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. 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. Furthermore, HCV core may inhibit adaptive T-cell responses by binding to the complement receptor.
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-g production and proliferation as T-cell effector functions because these are readily detectable in the blood of patients with spontaneous HCV clearance but weak or absent in chronic hepatitis C. 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, 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.
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. 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.
Routes of infection for the 7 patients were reported as: 4 needlesticks, 1 razor/sex, 1 razor, 1 occupational (skin cut). 6/7 reported symptoms. 2 reported jaundice. 6/7 were genotype 1. Maximum ALT was 154-1400. Antibody conversion from neg to pos was reported as 8-14 weeks for all patients. 1 patient started therapy at week 4, the others at 16-24 weeks. All pts received Peg-2b plus RBV, one pt started with IFN/rbv and switched I think to Peg/RBV. 6/7 had SVR & 1 had breakthrough. The one coinfected patient used PegRBV monotherapy due to concern for lactic acidosis since they were taking ddI and I think ddC. Of note, 3 pts were African-American.
DISCUSSION
Whereas spontaneous HCV clearance occurs in about 30% to 50% of patients with acute hepatitis C, 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. 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, but HCV-specific immune responses are weak or completely undetectable during chronic hepatitis C, suggesting primary failure, secondary down-regulation, or exhaustion. 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. 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.
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-a2b monotherapy. 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. 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, and patient A3,). 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 responses; 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 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-g responses within 10 to 15 weeks of spontaneous HCV clearance and resolution of acute hepatitis C. 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). A similar, although not statistically significant difference in IFN-g production was also seen in HCV protein-specific IFN- production (P = .055; data not shown). These results are supported by several recent reports 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, these findings may have clinical implications for T cell-mediated protective immunity.
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-a is known for its antiproliferative, myelosuppressive, and proapoptotic effects, 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, 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.
Long-term follow-up after successful interferon therapy of acute hepatitis C
Johannes Wiegand 1, Elmar Jäckel 1, Markus Cornberg 1, Holger Hinrichsen 2, Manfred Dietrich 3, Julian Kroeger 4, Wolfgang P. Fritsch 5, Anne Kubitschke 1, Nuray Aslan 1, Hans L. Tillmann 1, Michael Peter Manns 1, Heiner Wedemeyer 1
*1Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
2Medizinische Klinik, Universität Kiel (Health Clinic, University of Kiel), Kiel, Germany
3Bernhard-Nocht Institut Hamburg (Bernhard-Nocht Institute Hamburg), Hamburg, Germany
4Medzinische Klinik II, Universität des Saarlandes (University of Saarlandes), Hamburg/Saar, Germany
5Städtisches Krankenhaus Hildesheim (State Hospital), Hildesheim, Germany
ABSTRACT
Early treatment of acute hepatitis C infection with interferon alfa-2b (IFN--2b) prevents chronicity in almost all patients. So far, no data are available on the long-term outcome after interferon (IFN) therapy of acute hepatitis C.
The aim of this study was to assess the clinical, virological, and immunological long-term outcome of 31 successfully treated patients with acute hepatitis C infection who were followed for a median of 135 weeks (52-224 weeks) after end of therapy.
None of the individuals had clinical evidence of liver disease. Alanine aminotransferase (ALT) levels were normal in all but 1 patient. Serum hepatitis C virus (HCV) RNA was negative throughout follow-up, even when investigated with the highly sensitive transcription-mediated amplification (TMA) assay (cutoff 5-10 IU/mL). In addition, no HCV RNA was detected in peripheral blood mononuclear cells (PBMC) of 15 cases tested. The patients' overall quality-of-life scores as determined by the SF-36 questionnaire did not differ from the German reference control cohort.
Ex vivo interferon gamma (IFN-) ELISPOT analysis detected HCV-specific CD4+ T-helper cell reactivity in only 35% of cases, whereas HCV-specific CD8+ T-cell responses were found in 4 of 5 HLA-A2-positive individuals. Anti-HCV antibody levels decreased significantly during and after therapy in all individuals.
In conclusion, early treatment of symptomatic acute hepatitis C with IFN--2b leads to a long-term virological, biochemical, and clinical response. Waning of anti-HCV humoral immunity and presence of HCV-specific CD8+ (but not CD4+) T cells highlights the complexity of T-cell and B-cell memory to HCV, which might be significantly altered by IFN treatment.
BACKGROUND
Chronic hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease, affecting about 170 million people worldwide. Acute infection with HCV leads to a chronic course in 55% to 85% of cases. Although chronic HCV infection can be cured in 54% to 56% of cases with pegylated interferon alfa (IFN-a) and ribavirin, there is still no successful treatment option available for the majority of patients infected with HCV genotype 1. Thus, prevention of HCV chronicity seems to be desirable. Early treatment of acute symptomatic HCV infection with interferon alfa-2b (IFN-a-2b) monotherapy has been shown to be very successful: 98% of cases achieved a sustained virological response after a treatment period of 24 weeks. So far, there are no data available concerning the long-term outcome after treatment of acute hepatitis C with IFN-a. This might be of special interest because late relapses more than 6 months after spontaneous clearance of acute HCV infection have been described. Therefore, the aim of this study was to assess the clinical, virological, and immunological outcome of 31 patients who were followed for 52 to 224 weeks after end of monotherapy with IFN--2b.
We also investigated HCV-specific humoral and cellular immune responses in these patients. Immunological memory to HCV has been discussed controversially in recent years. While anti-HCV antibodies are thought to decline after clearance, there are conflicting data on the strength and specificity of HCV-specific CD4+ and CD8+ T-cell responses. In addition, it is not known whether antiviral therapy with IFN-a might influence the evolution of virus-specific cellular immune responses. However, detailed knowledge of T-cell memory might be of importance for the development of vaccines against HCV.
Patients and Methods
Thirty-one patients were included in this analysis. All patients had a history of acute HCV infection and were successfully treated with IFN-a monotherapy. Twenty-four patients were treated within the study reported by Jaeckel et al. In addition, 7 patients were included who were treated with the same regimen but outside the trial. Treatment success was defined as sustained virological response (HCV-RNA [polymerase chain reaction]-negative 24 weeks after end of therapy, measured by COBAS Amplicor, version 2.0; Roche Diagnostics, Mannheim, Germany; detection limit 600 copies HCV RNA/mL). The exact definition of acute hepatitis C and criteria for indication for therapy have been described in detail.
Eighteen patients were examined at Hannover Medical School; 13 patients were seen by their local physician and asked for their history using a questionnaire created for this study. This questionnaire covered the following topics: current health status, change of health status since IFN therapy, alcohol consumption, smoking habits or illicit drug use, concomitant diseases, body weight and height, current medication, and design and duration of IFN therapy. In Hannover, abdominal ultrasound examination and evaluation of autoantibodies (ANA, AMA, SMA, LKM, and SLA) were offered to the patients. Serum levels of HCV RNA were centrally tested by quantitative polymerase chain reaction (PCR) using COBAS Amplicor, version 2.0 (Roche Diagnostics; detection limit 600 IU HCV RNA/mL) at Hannover Medical School. Biochemical and hematological tests were performed locally.
The control group for T-cell analysis consisted of 13 individuals who had cleared chronic HCV infection after interferon (IFN)-based therapies. Patients were studied after a median of 56 weeks after the end of therapy (range, 24-262 weeks). All control patients were treated at Hannover Medical School and were constantly HCV RNA-negative in all regular follow-up visits. Control patients had no evidence of liver disease or other concomitant illnesses.
Optical density (OD) of anti-HCV antibodies was centrally analyzed with microparticle enzyme immunoassay AxSYM HCV version 3.0 (Abbott Laboratories, Abbott Park, IL) at weeks 0, 2, 4, 12, 24, and 48 and at the most recent time point after initiation of therapy. Antibody reactivity against HCV-specific antigens was evaluated using the immunoblot assay INNO-LIA HCV III Update (Innogenetics, Ghent, Belgium) at weeks 0 and 48 and at the most recent time point after the initiation of therapy.
Clinical Outcome
Thirty-one patients with a history of acute hepatitis C were studied. All of them were HCV RNA-negative 24 weeks after cessation of therapy (sustained response). Patients were followed thereafter for a median of 135 weeks (range, 52-224 weeks) after the end of therapy.
None of the patients had clinical evidence of liver disease at their most recent visit. Complete biochemical response (aminotransferases within normal limits) was achieved in all but 1 patient, who had only minimally increased alanine aminotransferase (ALT) levels (25 U/L: upper limit of normal 22 U/L). This patient had a body mass index (BMI) of 28 kg/m2 and showed signs of steatosis on ultrasound examination. Liver function as determined by bilirubin, albumin levels, and prothrombin time was normal in all individuals.
At their most recent follow-up visit, 18 of 28 patients (64%) reported being completely healthy without any evidence of symptoms potentially associated with hepatitis or IFN therapy. Two patients presented with mild intermittent abdominal pain in the right upper quadrant; one of these patients had cholecystolithiasis as documented by ultrasound. Another 3 patients suffered from fatigue, associated with an impaired ability to concentrate in 2 cases. The third patient additionally had thalassemia. Four of the patients (14%) reported persistence of symptoms that might be related to the previous IFN therapy: thyroid dysfunction, n = 1; dermatologic symptoms, n = 3; neurological/psychiatric symptoms, n = 2.
Seventeen patients were examined by high-resolution abdominal ultrasound. None of them displayed sonographic signs of chronic liver disease. Portal-vein flow and the resistance index of the hepatic artery were normal in all individuals. Patients were also screened for lymph nodes in the hepatoduodenal ligament because in the absence of liver biopsies, enlarged lymph nodes in the hepatoduodenal ligament have been shown to reflect intrahepatic inflammatory processes. Importantly, lymph nodes larger than 15 mm were detected in only 3 patients (18%). In addition, there was no serological or clinical evidence of autoimmune liver disease; all patients tested negative for ANA, AMA, SMA, LKM, and SLA autoantibodies.
The quality-of-life analysis was assessed using the SF-36 questionnaire. The study population showed similar scores compared to those of the general German control group. The results were within the standard deviation of the controls in each of 8 categories.
Virological Outcome
All patients included in this study had been HCV RNA-negative at the end of IFN--2b therapy and after 24 weeks of follow-up.
Thereafter, all 31 individuals were serum HCV RNA-negative by COBAS Amplicor (Roche Diagnostics, Mannheim, Germany) assay (detection limit 600 IU/mL) for the entire follow-up and at their most recent visit (range, 52-224 weeks).
For 20 patients, serum was available to investigate low levels of HCV RNA with the more sensitive TMA assay (detection limit 5-10 IU/mL). All samples tested were HCV RNA-negative. Finally, RNA could be isolated from PBMC of 15 patients. HCV RNA could not be detected in the PBMC of any of these individuals.
DISCUSSION
Early treatment of symptomatic acute hepatitis C infection with IFN--2b for 24 weeks leads to a virological response in 98% of cases. The aim of this study was to assess the clinical, virological, and immunological outcome of patients after a median follow-up period of 2.6 years after the end of IFN-a-2b monotherapy.
The most important finding of this study was that none of the patients had evidence of a late virological relapse. All patients remained serum HCV RNA-negative as determined by standard HCV PCR. Also important, there was no evidence for low levels of persisting virus using the more sensitive TMA assay. In addition, all patients tested were HCV-RNA-negative in PBMC. However, we cannot exclude persistence of low levels of HCV in the liver because there was no indication to perform liver biopsies in patients who were serum HCV -RNA-negative for 2 to 4 years. The virological response was accompanied by a biochemical response. ALT values were normal in all but 1 patient. This patients minimal increased ALT might reflect steatohepatitis in the presence of obesity (BMI 28 kg/m2); there was no evidence of diabetes mellitus in this case.
Analysis of the SF-36 questionnaire revealed that the study populations quality of life did not differ from that of the German reference population in all 8 categories. However, it is not possible to judge whether quality-of-life status changed after the successful IFN therapy because there was no evaluation before, during, or right after treatment. It is also not known whether the patients will develop a more impaired health condition in further follow-up. Patients who had spontaneously cleared HCV from serum and who were followed for 15 years still demonstrated impaired quality-of-life scores, thereby supporting potential long-term effects of HCV on mental health status.
Although the clinical and virological long-term outcome after IFN-a-2b monotherapy seems to be excellent, it is important to note that there were possible IFN-related long-term side effects in 14% of the patients. About 15% to 30% of patients with acute hepatitis C show a self-limited course of the disease. These patients may be unnecessarily at risk to develop IFN-related long-term side effects when being treated. However, there are no sufficient predictive parameters available yet to identify patients with self-limited acute hepatitis C prior to treatment. The kinetics of HCV RNA decline during the first weeks of symptomatic infection may indicate self-limited disease, but it must be pointed out that the mean time from exposure to HCV RNA negativity was 77 days in the study of Hofer et al. whereas the average time from infection until the start of therapy was 89 days in our previous trial. Thus, Hofer's patients would not have been included in our study. However, our data imply that patients should be informed extensively about potential risks associated with IFN-a therapy before treatment is initiated. An alternative approach to managing symptomatic acute hepatitis C infection is to treat only those patients who are HCV-RNA-positive 12 weeks after the onset of symptoms to avoid unnecessary therapy. This concept may also lead to sustained virological response rates of up to 90%. However, there are no data available concerning the long-term results of the wait-and-see strategy. A more comprehensive overview on different strategies for the management of acute hepatitis C has been recently published.
We also analyzed the HCV-specific humoral and cellular immune response after long-term recovery from acute hepatitis C infection. Interestingly, after an early increase of the OD 405 of the anti-HCV-ELISA, we observed a biphasic decline of the HCV-specific humoral immune response. The rapid decrease of the anti-HCV antibodies until the end of the IFN therapy could be caused by the fast clearance of HCV-RNA, leading to an absence of viral antigen required for stimulation of B-cells. Alternatively, IFN-a could also have inhibited B-cell proliferation. After the end of therapy, OD 405 levels further declined, potentially leading to an earlier disappearance of anti-HCV antibodies compared to previous reports on patients who had recovered spontaneously from acute HCV infection without antiviral treatment.
Kinetics of humoral responses during antiviral therapy did differ between patients with acute HCV infection and patients being treated for chronic hepatitis C. First, pretreatment antibody levels were higher in chronic infection; this is in line with previous reports. Second, the early OD increase during treatment was more pronounced in patients with acute HCV, although pretreatment viral load and HCV-RNA decline during therapy were similar in both groups. Thus, the OD rise may be explained not only by a simple titration phenomenon due to drop in antigen load but also by differences in responsiveness of B-cells to type I IFN stimulation. Third, during therapy, antibody levels did not decline in patients being treated successfully for chronic hepatitis C but remained at high levels even after the end of therapy. The reasons for these observations are not clarified yet and remain to be investigated.
The cellular immune response has been shown to play a crucial role in the outcome of acute HCV infection. Strong CTL and CD4+ T-cell responses are associated with spontaneous clearance of the virus, while T-cell responses are very weak or even absent in chronic hepatitis C. A maintained CD4+ T-cell response is a prerequisite for prolonged HCV-RNA negativity and protection from secondary infection, while a loss of CD4+ T-cell reactivity in the acute phase of infection is accompanied with a reoccurrence of HCV RNA after initial viral clearance. Using assay techniques identical to those applied in the present study, we usually detect HCV-specific CD4+ and CD8+ T cells in less than 10% of chronic HCV patients (data not shown). However, in IFN-recovered patients, responses were now detected only slightly more often, because HCV-specific CD4+ T cells were absent in about two-thirds of patients at their most recent follow-up. These data imply that there may be no longlasting CD4+ T-cell immune response against HCV after IFN-induced clearance of acute hepatitis C. The results are supported by our findings of weak CD4+ T-cell responses in the control patients recovered from chronic hepatitis C. Although some studies have suggested a restoration of T-cell responses in chronic hepatitis C by successful antiviral therapy, no data are available for the investigation of HCV-specific cellular immunity later than 24 weeks after the end of therapy.
The lack of low levels of persisting viral RNA could be an explanation for the observed vanishing of CD4+ T-cell responses because it has been reported that CD4+ T-cell activity depends on restimulation with low levels of persisting antigen.[43] The degree of CD4+ memory T-cell persistence also depends on the initial burst size of immune responses during early infection[44]; however, this is not known for our patients. CD4+ T-cell responses observed in our study were multispecific and showed a broader spectrum than those in earlier studies of patients with self-limited acute hepatitis C.
In contrast to the CD4+ T-cell immune response, an HCV-specific CD8+ T-cell immunity could be observed in 4 of 5 HLA-A2-positive patients. HCV-specific CD8+ T-cell memory has recently been proven to be essential for protection against persistent HCV infection on reexposure to HCV. The positive CD8+ T-cell response in the present study does not necessarily seem to be dependent on CD4+ help, although more patients are needed to draw final conclusions. However, the presence of CD8+ responses in our control group was also not accompanied by strong HCV-specific CD4+ responses. The rapid decline of the viral load at the beginning of therapy may have prevented an initial dysfunction and stunning of CD8+ T -cells; this can be observed in the initial phase of acute hepatitis C with high levels of antigen. The level of CD8+ T cells correlates with viral clearance and outcome of acute hepatitis C infection, but the high level of CD8+ T-cell immunity is not preserved after the first months in untreated patients with acute hepatitis C. Interestingly, patients without CD8+ T-cell response in the first 6 months after the onset of disease start to produce a specific immunity in the following months after the initiation of specific antiviral therapy. Overall, these data support the hypothesis that IFN- stimulates the induction of virus-specific CD8+ T cells.
In summary, the data from the present study prove the long-term efficacy of early IFN--2b therapy in patients with acute hepatitis C. However, the excellent treatment success may be accompanied by IFN-related side effects in some patients. In addition, this study gives evidence that IFN-a treatment may significantly influence the evolution of both cellular and humoral memory immune responses; this could have consequences if reexposure to HCV occurs.
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