Interferon-$alpha; for hepatitis C: antiviral or immunotherapy?

Abstract

About 15–20% of patients with acute hepatitis C virus (HCV) infection achieve permanent viral clearance proving principally that HCV can be controlled by antiviral immune mechanisms. In the majority of patients, however, HCV is obviously able to escape the efficient antiviral immune responses and to establish chronic viral persistence. Several mechanisms have been implicated in the pathogenesis of chronic hepatitis C: first, HCV seems to interfere at several levels with the action of type I interferons [1Foy E Li K Wang C Sumpter Jr, R Ikeda M Lemon S.M et al.Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease.Science. 2003; 300: 1145-1148Crossref PubMed Scopus (700) Google Scholar, 2Gale Jr, M.J Korth M.J Tang N.M Tan S.L Hopkins D.A Dever T.E et al.Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein.Virology. 1997; 230: 217-227Crossref PubMed Scopus (719) Google Scholar, 3Duong F.H Filipowicz M Tripodi M La Monica N Heim M.H Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A.Gastroenterology. 2004; 126: 263-277Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar]; second, viral variability may lead to escape from specific humoral and cellular immune responses, and third, a general impairment of HCV-specific CD4+ and CD8+ T cells occurs, which is yet incompletely understood [4Ulsenheimer A Gerlach J.T Gruener N.H Jung M.C Schirren C.A Schraut W et al.Detection of functionally altered hepatitis C virus-specific CD4 T cells in acute and chronic hepatitis C.Hepatology. 2003; 37: 1189-1198Crossref PubMed Scopus (186) Google Scholar, 5Barnes E Lauer G Walker B Klenerman P T cell failure in hepatitis C virus infection.Viral Immunol. 2002; 15: 285-293Crossref PubMed Scopus (20) Google Scholar]. Whereas some studies suggest a dysfunction of dendritic cells leading to poor antigen presentation [[6]Bain C Fatmi A Zoulim F Zarski J.P Trepo C Inchauspe G Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.Gastroenterology. 2001; 120: 512-524Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar], animal models of other chronic viral infections suggest a mechanism of T cell exhaustion in the presence of high viral load [[7]Komarova N.L Barnes E Klenerman P Wodarz D Boosting immunity by antiviral drug therapy: a simple relationship among timing, efficacy, and success.Proc Natl Acad Sci USA. 2003; 100: 1855-1860Crossref PubMed Scopus (52) Google Scholar].Treatment of chronic hepatitis C with pegylated interferon-α/ribavirin combination therapy leads to sustained viral clearance in about 50% of patients with genotype 1 infection and more than 80% of patients with genotype 2 or 3 infection. In vitro, interferon-α has pleiotropic effects which can be separated into two major groups, inhibition of viral replication and immunomodulatory effects, respectively. During treatment of HCV infection, modelling of viral kinetics suggests that both mechanisms play a role for successful viral clearance [[8]Neumann A.U Lam N.P Dahari H Gretch D.R Wiley T.E Layden T.J et al.Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy.Science. 1998; 282: 103-107Crossref PubMed Scopus (1889) Google Scholar]. In a first phase, lasting 1 to 2 days, a rapid decline of viral load is observed, which has been attributed to direct antiviral effects of interferon-α. In the most recent models of HCV kinetics this is followed by a second phase with little change in the peripheral blood viral load, lasting for 2 to 28 days [[9]Herrmann E Lee J.H Marinos G Modi M Zeuzem S Effect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon.Hepatology. 2003; 37: 1351-1358Crossref PubMed Scopus (226) Google Scholar]. In a third phase, viral load declines again, although usually at a slower rate compared to the first phase. This phase is thought to reflect clearance of HCV infected hepatocytes, most likely by HCV-specific cellular immune responses. Several studies have tried to define this assumed HCV-specific cellular immune responses during interferon-α treatment. Indeed, a significant increase in the HCV-specific CD4+ T cell response was found to occur frequently during antiviral treatment [10Hoffmann R.M Diepolder H.M Zachoval R Zwiebel F.M Jung M.C Scholz S et al.Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection.Hepatology. 1995; 21: 632-638PubMed Google Scholar, 11Missale G Cariani E Lamonaca V Ravaggi A Rossini A Bertoni R et al.Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients.Hepatology. 1997; 26: 792-797Crossref PubMed Scopus (65) Google Scholar, 12Barnes E Harcourt G Brown D Lucas M Phillips R Dusheiko G et al.The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection.Hepatology. 2002; 36: 743-754Crossref PubMed Scopus (141) Google Scholar, 13Kamal S.M Fehr J Roesler B Peters T Rasenack J.W Peginterferon alone or with ribavirin enhances HCV-specific CD4 T-helper 1 responses in patients with chronic hepatitis C.Gastroenterology. 2002; 123: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar]. In contrast, little changes have been detected in HCV-specific CD8+ T cell responses [[12]Barnes E Harcourt G Brown D Lucas M Phillips R Dusheiko G et al.The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection.Hepatology. 2002; 36: 743-754Crossref PubMed Scopus (141) Google Scholar]. Some studies have also claimed an association between the recovery of HCV-specific CD4+ T cell responses and a sustained virological responses to treatment [10Hoffmann R.M Diepolder H.M Zachoval R Zwiebel F.M Jung M.C Scholz S et al.Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection.Hepatology. 1995; 21: 632-638PubMed Google Scholar, 13Kamal S.M Fehr J Roesler B Peters T Rasenack J.W Peginterferon alone or with ribavirin enhances HCV-specific CD4 T-helper 1 responses in patients with chronic hepatitis C.Gastroenterology. 2002; 123: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar]. Since patients infected with genotype 2 and 3 consistently show higher sustained viral response rates, not surprisingly also a higher frequency of HCV-specific T cell responses can be found in this group [[11]Missale G Cariani E Lamonaca V Ravaggi A Rossini A Bertoni R et al.Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients.Hepatology. 1997; 26: 792-797Crossref PubMed Scopus (65) Google Scholar]. From these studies, however, it has not become clear whether the recovery of the T cell response is due to suppression of viral replication or whether the HCV-specific T cells indeed contribute to viral clearance. In fact, recovery of virus specific CD4+ T cell responses during treatment with pure antiviral drugs has been reported in chronic hepatitis B [[14]Boni C Penna A Bertoletti A Lamonaca V Rapti I Missale G et al.Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.J Hepatol. 2003; 39: 595-605Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar] and HIV infection [[15]Rosenberg E.S Altfeld M Poon S.H Phillips M.N Wilkes B.M Eldridge R.L et al.Immune control of HIV-1 after early treatment of acute infection.Nature. 2000; 407: 523-526Crossref PubMed Scopus (907) Google Scholar] and could be due to the reduced T cell suppressive effect of a high viral load in addition to the redistribution of specific CD4+ T cells from the site of inflammation back into the circulation [[7]Komarova N.L Barnes E Klenerman P Wodarz D Boosting immunity by antiviral drug therapy: a simple relationship among timing, efficacy, and success.Proc Natl Acad Sci USA. 2003; 100: 1855-1860Crossref PubMed Scopus (52) Google Scholar].This subject has been addressed in the paper from Hultgren et al. in this issue of the Journal who initially studied a cohort of 71 treatment naı̈ve patients with chronic hepatitis C and subsequently looked at 32 patients undergoing antiviral therapy [[16]Hultgren C Desombere I Leroux-Roels G Quiroga J.A Carreno V Nilsson B et al.Evidence for a relation between the viral load and genotype and hepatitis C virus-specific T cell responses.J Hepatol. 2004; 40: 971-978Abstract Full Text Full Text PDF PubMed Google Scholar]. Frequent immunological and virological measurements were performed during the first 12 weeks of therapy allowing a detailed analysis of both viral and immunological kinetics. First, the authors could show in the group of treatment naı̈ve patients that a HCV-NS3 specific CD4+ T cell proliferative response is significantly more frequent in patients with genotype 3 infection. Next, during antiviral treatment, NS3-specific CD4+ T cell responses appeared more frequently in genotype 2 and 3 patients as compared to patients with genotype 1 infection, and an earlier appearance of the T cell response correlated with faster kinetics of viral elimination. Finally, there was a trend (P=0.07) for an association between the appearance of T cell responses and a sustained viral response. Interestingly, NS3 specific T cell responses occurred as early as on day 1 and in most patients within the first week after the start of treatment. This was after the initial one to two log drop in viral load (corresponding to phase 1) but well before viral clearance occurred. Accordingly, the authors conclude that during antiviral therapy NS3 specific CD4+ T cell responses are more frequently restored in patients with genotype 2 or 3 infection and that this immune response may contribute to a more rapid clearance of HCV RNA. Based on these findings, however, new questions arise: the frequent testing of T cell responses in this study came to the price of a limited analysis of the breadth of the cellular immune response. Future studies will certainly have to address the role of CD4+ T cell responses to other HCV antigens. In addition, the proliferation assay detects only a subset of HCV-specific CD4+ T cells, usually those with the ability to produce IL-2 and to proliferate, corresponding to the so-called central memory cells. Further differentiated effector cells (‘effector memory’), which may still produce interferon-γ but no IL-2 and which can also have important antiviral activity, may be missed in the proliferation assay. Nevertheless, recent evidence suggests that IL-2 production by CD4+ T cells is a critical factor for the differentiation of cytotoxic T cells into antiviral effector cells [[17]Francavilla V Accapezzato D De Salvo M Rawson P Cosimi O Lipp M et al.Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms.Eur J Immunol. 2004; 34: 427-437Crossref PubMed Scopus (60) Google Scholar], indicating that the proliferation assay measures a relevant function of CD4+ T cells in this scenario. Eventually, the frequent determinations of CD4+ T cell activity in the study of Hultgren et al. highlights another issue: CD4+ T cells responses during antiviral treatment of chronic hepatitis C are relatively weak and fluctuating compared to patients with acute self-limited disease and are usually not maintained. This is consistent with previous studies [10Hoffmann R.M Diepolder H.M Zachoval R Zwiebel F.M Jung M.C Scholz S et al.Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection.Hepatology. 1995; 21: 632-638PubMed Google Scholar, 12Barnes E Harcourt G Brown D Lucas M Phillips R Dusheiko G et al.The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection.Hepatology. 2002; 36: 743-754Crossref PubMed Scopus (141) Google Scholar] and leads to the important question whether these responses could be further augmented by therapeutic vaccination, potentially leading to a higher rate of sustained virological responses.In conclusion, the study of Hultgren et al. suggests that interferon-α based therapies hit HCV at two different sites, first by overriding the relative interferon resistance of the virus and second by partially restoring antiviral cellular immune responses, exemplified in this study by the NS3-specific CD4+ T cell response. HCV-specific CD4+ T cell responses seem to be more frequent in patients with genotype 2 or 3 infection, both before and during interferon-α treatment, which offers an attractive explanation for the significantly higher sustained virological response rates in these patients. About 15–20% of patients with acute hepatitis C virus (HCV) infection achieve permanent viral clearance proving principally that HCV can be controlled by antiviral immune mechanisms. In the majority of patients, however, HCV is obviously able to escape the efficient antiviral immune responses and to establish chronic viral persistence. Several mechanisms have been implicated in the pathogenesis of chronic hepatitis C: first, HCV seems to interfere at several levels with the action of type I interferons [1Foy E Li K Wang C Sumpter Jr, R Ikeda M Lemon S.M et al.Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease.Science. 2003; 300: 1145-1148Crossref PubMed Scopus (700) Google Scholar, 2Gale Jr, M.J Korth M.J Tang N.M Tan S.L Hopkins D.A Dever T.E et al.Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein.Virology. 1997; 230: 217-227Crossref PubMed Scopus (719) Google Scholar, 3Duong F.H Filipowicz M Tripodi M La Monica N Heim M.H Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A.Gastroenterology. 2004; 126: 263-277Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar]; second, viral variability may lead to escape from specific humoral and cellular immune responses, and third, a general impairment of HCV-specific CD4+ and CD8+ T cells occurs, which is yet incompletely understood [4Ulsenheimer A Gerlach J.T Gruener N.H Jung M.C Schirren C.A Schraut W et al.Detection of functionally altered hepatitis C virus-specific CD4 T cells in acute and chronic hepatitis C.Hepatology. 2003; 37: 1189-1198Crossref PubMed Scopus (186) Google Scholar, 5Barnes E Lauer G Walker B Klenerman P T cell failure in hepatitis C virus infection.Viral Immunol. 2002; 15: 285-293Crossref PubMed Scopus (20) Google Scholar]. Whereas some studies suggest a dysfunction of dendritic cells leading to poor antigen presentation [[6]Bain C Fatmi A Zoulim F Zarski J.P Trepo C Inchauspe G Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.Gastroenterology. 2001; 120: 512-524Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar], animal models of other chronic viral infections suggest a mechanism of T cell exhaustion in the presence of high viral load [[7]Komarova N.L Barnes E Klenerman P Wodarz D Boosting immunity by antiviral drug therapy: a simple relationship among timing, efficacy, and success.Proc Natl Acad Sci USA. 2003; 100: 1855-1860Crossref PubMed Scopus (52) Google Scholar]. Treatment of chronic hepatitis C with pegylated interferon-α/ribavirin combination therapy leads to sustained viral clearance in about 50% of patients with genotype 1 infection and more than 80% of patients with genotype 2 or 3 infection. In vitro, interferon-α has pleiotropic effects which can be separated into two major groups, inhibition of viral replication and immunomodulatory effects, respectively. During treatment of HCV infection, modelling of viral kinetics suggests that both mechanisms play a role for successful viral clearance [[8]Neumann A.U Lam N.P Dahari H Gretch D.R Wiley T.E Layden T.J et al.Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy.Science. 1998; 282: 103-107Crossref PubMed Scopus (1889) Google Scholar]. In a first phase, lasting 1 to 2 days, a rapid decline of viral load is observed, which has been attributed to direct antiviral effects of interferon-α. In the most recent models of HCV kinetics this is followed by a second phase with little change in the peripheral blood viral load, lasting for 2 to 28 days [[9]Herrmann E Lee J.H Marinos G Modi M Zeuzem S Effect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon.Hepatology. 2003; 37: 1351-1358Crossref PubMed Scopus (226) Google Scholar]. In a third phase, viral load declines again, although usually at a slower rate compared to the first phase. This phase is thought to reflect clearance of HCV infected hepatocytes, most likely by HCV-specific cellular immune responses. Several studies have tried to define this assumed HCV-specific cellular immune responses during interferon-α treatment. Indeed, a significant increase in the HCV-specific CD4+ T cell response was found to occur frequently during antiviral treatment [10Hoffmann R.M Diepolder H.M Zachoval R Zwiebel F.M Jung M.C Scholz S et al.Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection.Hepatology. 1995; 21: 632-638PubMed Google Scholar, 11Missale G Cariani E Lamonaca V Ravaggi A Rossini A Bertoni R et al.Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients.Hepatology. 1997; 26: 792-797Crossref PubMed Scopus (65) Google Scholar, 12Barnes E Harcourt G Brown D Lucas M Phillips R Dusheiko G et al.The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection.Hepatology. 2002; 36: 743-754Crossref PubMed Scopus (141) Google Scholar, 13Kamal S.M Fehr J Roesler B Peters T Rasenack J.W Peginterferon alone or with ribavirin enhances HCV-specific CD4 T-helper 1 responses in patients with chronic hepatitis C.Gastroenterology. 2002; 123: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar]. In contrast, little changes have been detected in HCV-specific CD8+ T cell responses [[12]Barnes E Harcourt G Brown D Lucas M Phillips R Dusheiko G et al.The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection.Hepatology. 2002; 36: 743-754Crossref PubMed Scopus (141) Google Scholar]. Some studies have also claimed an association between the recovery of HCV-specific CD4+ T cell responses and a sustained virological responses to treatment [10Hoffmann R.M Diepolder H.M Zachoval R Zwiebel F.M Jung M.C Scholz S et al.Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection.Hepatology. 1995; 21: 632-638PubMed Google Scholar, 13Kamal S.M Fehr J Roesler B Peters T Rasenack J.W Peginterferon alone or with ribavirin enhances HCV-specific CD4 T-helper 1 responses in patients with chronic hepatitis C.Gastroenterology. 2002; 123: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar]. Since patients infected with genotype 2 and 3 consistently show higher sustained viral response rates, not surprisingly also a higher frequency of HCV-specific T cell responses can be found in this group [[11]Missale G Cariani E Lamonaca V Ravaggi A Rossini A Bertoni R et al.Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients.Hepatology. 1997; 26: 792-797Crossref PubMed Scopus (65) Google Scholar]. From these studies, however, it has not become clear whether the recovery of the T cell response is due to suppression of viral replication or whether the HCV-specific T cells indeed contribute to viral clearance. In fact, recovery of virus specific CD4+ T cell responses during treatment with pure antiviral drugs has been reported in chronic hepatitis B [[14]Boni C Penna A Bertoletti A Lamonaca V Rapti I Missale G et al.Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.J Hepatol. 2003; 39: 595-605Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar] and HIV infection [[15]Rosenberg E.S Altfeld M Poon S.H Phillips M.N Wilkes B.M Eldridge R.L et al.Immune control of HIV-1 after early treatment of acute infection.Nature. 2000; 407: 523-526Crossref PubMed Scopus (907) Google Scholar] and could be due to the reduced T cell suppressive effect of a high viral load in addition to the redistribution of specific CD4+ T cells from the site of inflammation back into the circulation [[7]Komarova N.L Barnes E Klenerman P Wodarz D Boosting immunity by antiviral drug therapy: a simple relationship among timing, efficacy, and success.Proc Natl Acad Sci USA. 2003; 100: 1855-1860Crossref PubMed Scopus (52) Google Scholar]. This subject has been addressed in the paper from Hultgren et al. in this issue of the Journal who initially studied a cohort of 71 treatment naı̈ve patients with chronic hepatitis C and subsequently looked at 32 patients undergoing antiviral therapy [[16]Hultgren C Desombere I Leroux-Roels G Quiroga J.A Carreno V Nilsson B et al.Evidence for a relation between the viral load and genotype and hepatitis C virus-specific T cell responses.J Hepatol. 2004; 40: 971-978Abstract Full Text Full Text PDF PubMed Google Scholar]. Frequent immunological and virological measurements were performed during the first 12 weeks of therapy allowing a detailed analysis of both viral and immunological kinetics. First, the authors could show in the group of treatment naı̈ve patients that a HCV-NS3 specific CD4+ T cell proliferative response is significantly more frequent in patients with genotype 3 infection. Next, during antiviral treatment, NS3-specific CD4+ T cell responses appeared more frequently in genotype 2 and 3 patients as compared to patients with genotype 1 infection, and an earlier appearance of the T cell response correlated with faster kinetics of viral elimination. Finally, there was a trend (P=0.07) for an association between the appearance of T cell responses and a sustained viral response. Interestingly, NS3 specific T cell responses occurred as early as on day 1 and in most patients within the first week after the start of treatment. This was after the initial one to two log drop in viral load (corresponding to phase 1) but well before viral clearance occurred. Accordingly, the authors conclude that during antiviral therapy NS3 specific CD4+ T cell responses are more frequently restored in patients with genotype 2 or 3 infection and that this immune response may contribute to a more rapid clearance of HCV RNA. Based on these findings, however, new questions arise: the frequent testing of T cell responses in this study came to the price of a limited analysis of the breadth of the cellular immune response. Future studies will certainly have to address the role of CD4+ T cell responses to other HCV antigens. In addition, the proliferation assay detects only a subset of HCV-specific CD4+ T cells, usually those with the ability to produce IL-2 and to proliferate, corresponding to the so-called central memory cells. Further differentiated effector cells (‘effector memory’), which may still produce interferon-γ but no IL-2 and which can also have important antiviral activity, may be missed in the proliferation assay. Nevertheless, recent evidence suggests that IL-2 production by CD4+ T cells is a critical factor for the differentiation of cytotoxic T cells into antiviral effector cells [[17]Francavilla V Accapezzato D De Salvo M Rawson P Cosimi O Lipp M et al.Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms.Eur J Immunol. 2004; 34: 427-437Crossref PubMed Scopus (60) Google Scholar], indicating that the proliferation assay measures a relevant function of CD4+ T cells in this scenario. Eventually, the frequent determinations of CD4+ T cell activity in the study of Hultgren et al. highlights another issue: CD4+ T cells responses during antiviral treatment of chronic hepatitis C are relatively weak and fluctuating compared to patients with acute self-limited disease and are usually not maintained. This is consistent with previous studies [10Hoffmann R.M Diepolder H.M Zachoval R Zwiebel F.M Jung M.C Scholz S et al.Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection.Hepatology. 1995; 21: 632-638PubMed Google Scholar, 12Barnes E Harcourt G Brown D Lucas M Phillips R Dusheiko G et al.The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection.Hepatology. 2002; 36: 743-754Crossref PubMed Scopus (141) Google Scholar] and leads to the important question whether these responses could be further augmented by therapeutic vaccination, potentially leading to a higher rate of sustained virological responses. In conclusion, the study of Hultgren et al. suggests that interferon-α based therapies hit HCV at two different sites, first by overriding the relative interferon resistance of the virus and second by partially restoring antiviral cellular immune responses, exemplified in this study by the NS3-specific CD4+ T cell response. HCV-specific CD4+ T cell responses seem to be more frequent in patients with genotype 2 or 3 infection, both before and during interferon-α treatment, which offers an attractive explanation for the significantly higher sustained virological response rates in these patients.