The challenge of developing a vaccine against hepatitis C virus

Abstract

1. IntroductionHepatitis C virus (HCV) is a single stranded positive-sense RNA virus belonging to the Flaviviridae family [[1]Rice C.M Fields B.N Knippe D.M Howley P.M Fields Virology. Lippnicot-Raven, Philadelphia1996: 931-960Google Scholar]. Members of this family are small enveloped viruses that have been classified into three different genera: Pestivirus, which contains animal pathogens such as bovine viral diarrhea virus and hog cholera virus; Flavirirus, which contains mostly arthropod-transmitted human pathogens such as dengue fever and yellow fever viruses; and Hepacivirus, whose only member is HCV [[1]Rice C.M Fields B.N Knippe D.M Howley P.M Fields Virology. Lippnicot-Raven, Philadelphia1996: 931-960Google Scholar]. The recently discovered GB virus-B (GBV-B), which causes hepatitis in experimentally infected tamarins, will probably be classified with HCV [2Bukh J Apgar C.L Yanagi M Toward a surrogate model for hepatitis C virus: an infectious molecular clone of the GB virus-B hepatitis agent.Virology. 1999; 262: 470-478Crossref PubMed Scopus (119) Google Scholar, 3Simons J.N Leary T.P Dawson G.J Pilot-Matias T.J Muerhoff A.S Schlauder G.G et al.Isolation of novel virus-like sequences associated with human hepatitis.Nat Med. 1995; 1: 564-569Crossref PubMed Scopus (1160) Google Scholar].HCV is an important human pathogen, but the scope of its impact on human health has only recently been truly appreciated. The prevalence of HCV infection in the general population varies depending on the geographical area and ranges from less than 1% in Northern Europe to as high as 20% in some developing countries such as Egypt. It has been estimated that approximately 170 million people are chronically infected with HCV worldwide [[4]Global surveillance and control of hepatitis CReport of a WHO Consultation organized in collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium.J Viral Hepat. 1999; 6: 35-47Crossref PubMed Scopus (751) Google Scholar]. However, despite effective screening of blood and blood products, and use of sterile techniques, acute HCV is still a problem in industrialized countries. For example, around 40 000 new HCV infections occur each year in the US [[5]Williams I Epidemiology of hepatitis C in the United States.Am J Med. 1999; 107: 2S-9SAbstract Full Text Full Text PDF PubMed Google Scholar] and the majority of individuals with acute infection become persistently infected [[6]Houghton M The hepatitis C viruses.in: Fields B.N Knippe D.M Howley P.M Fields Virology. Lippnicot-Raven, Philadelphia1996: 1035-1058Google Scholar]. The source of these infections is principally the illicit use of parenteral drugs. Exposure to contaminated blood, especially via contaminated needles, syringes and surgical instruments, also accounts for the spread of HCV in developing countries. Chronic HCV infection is an important cause of liver cirrhosis and hepatocellular carcinoma in the Western World and Japan and, furthermore, represents the most frequent indication for liver transplantation in developed countries.Treatment of chronic HCV infection has improved considerably during the last few years: the combination of interferon and the nucleoside analogue ribavirin achieves a sustained virological response in approximately 40% of patients with chronic hepatitis C [7Manns M.P McHutchison J.G Gordon S.C Rustgi V.K Shiffman M Reindollar R et al.Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial.Lancet. 2001; 358: 958-965Abstract Full Text Full Text PDF PubMed Scopus (5873) Google Scholar, 8McHutchison J.G Gordon S.C Schiff E.R Shiffman M.L Lee W.M Rustgi V.K et al.Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group.N Engl J Med. 1998; 339: 1485-1492Crossref PubMed Scopus (3343) Google Scholar, 9Poynard T Marcellin P Lee S.S Niederau C Minuk G.S Ideo G et al.Randomized trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT).Lancet. 1998; 352: 1426-1432Abstract Full Text Full Text PDF PubMed Scopus (2393) Google Scholar]. It is possible that during the next few years, new antiviral agents such as inhibitors of the viral protease, helicase or polymerase will further improve the response rate of the current therapeutic agents. However, antiviral therapy is not affordable in most developing countries, where the prevalence of HCV is generally the highest. Thus, given the huge reservoir of HCV worldwide, the development of an effective vaccine will be the only way to control disease associated with HCV infection.2. The prospect of success for the development of HCV vaccinesProphylactic vaccines against some other members of the Flaviviridae family already exist or are under development [[1]Rice C.M Fields B.N Knippe D.M Howley P.M Fields Virology. Lippnicot-Raven, Philadelphia1996: 931-960Google Scholar]. For example, a live-attenuated vaccine against yellow fever virus is available and has proven to be highly effective. Efforts have been made since the 1940s to produce dengue vaccines. Immunity acquired from natural infection is specific for each dengue serotype and infection of an individual with three different serotypes has been reported. For this reason, tetravalent vaccines are being developed. However, the development of an effective vaccine against HCV will probably be more difficult. Viruses such as dengue or yellow fever cause acute self-limited infections in humans, which means that the host immune responses are capable of controlling the disease, and also provide immunity against subsequent exposure. Despite the generation of humoral and cellular immune responses against HCV, only 15–30% of the individuals infected with HCV resolve their infection [[10]Alberti A Chemello L Benvegnu L Natural history of hepatitis C.J Hepatol. 1999; 31: 17-24Abstract Full Text PDF PubMed Google Scholar]. Additionally, it has been reported that HCV can cause more than one episode of acute hepatitis in the same individual [[11]Lai M.E Mazzoleni A.P Argiolu F De Virgilis S Balestrieri A Purcell R.H et al.Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children.Lancet. 1994; 343: 388-390Abstract PubMed Scopus (275) Google Scholar]. Similarly, in the chimpanzee model, animals re-challenged with the same strain or with a closely related HCV strain were not protected against reinfection following acute resolving infection [12Bukh J Thimme R Forns X Chang K.M Yanagi M Emerson S.U et al.Acute resolving monoclonal hepatitis C virus (HCV) infection in a chimpanzee modifies subsequent infections with the homologous monoclonal virus.Hepatology (Abstract). 1999; 30: 452AGoogle Scholar, 13Farci P Alter H.J Govindarajan S Wong D.C Engle R Lesniewski R.R et al.Lack of protective immunity against reinfection with hepatitis C virus.Science. 1992; 258: 135-140Crossref PubMed Scopus (693) Google Scholar, 14Prince A.M Brotman B Huima T Pascual D Jaffery M Inchauspe G Immunity in hepatitis C infection.J Infect Dis. 1992; 165: 438-443Crossref PubMed Scopus (200) Google Scholar]. Thus infection with HCV apparently does not provide complete protection against subsequent exposures.2.1 The genetic heterogeneity of HCV contributes to its ability to elude preexisting immunityOne of the possible explanations for the lack of protective immunity after HCV infection is the genetic heterogeneity of HCV [15Bukh J Miller R.H Purcell R.H Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes.Semin Liver Dis. 1995; 15: 41-63Crossref PubMed Scopus (745) Google Scholar, 16Forns X Purcell R.H Bukh J Quasispecies in viral persistence and pathogenesis of hepatitis C virus.Trends Microbiol. 1999; 7: 402-410Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar]. HCV can be classified into six major genotypes and over 100 subtypes. The polyprotein sequences of the most diverse isolates differ by approximately 30% and their envelope proteins (E1 and E2) differ by up to 50% [[15]Bukh J Miller R.H Purcell R.H Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes.Semin Liver Dis. 1995; 15: 41-63Crossref PubMed Scopus (745) Google Scholar]. Within infected individuals, HCV circulates as a quasispecies, which is a mixture of closely related but distinct genomes [[17]Martell M Esteban J.I Quer J Genesca J Weiner A Esteban R et al.Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution.J Virol. 1992; 66: 3225-3229Crossref PubMed Google Scholar]. Genetic variability is particularly high at the amino terminus of E2, the hypervariable region 1 (HVR1). Genetic evolution of the virus might permit HCV to escape the host immune surveillance. In fact, genomic changes in the virus can emerge in a pattern consistent with repeated selection by escape from the host humoral or cellular immune responses [18Weiner A.J Geysen H.M Christopherson C Hall J.E Mason T.J Saracco G et al.Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections.Proc Natl Acad Sci USA. 1992; 89: 3468-3472Crossref PubMed Scopus (652) Google Scholar, 19Weiner A Erickson A.L Kansopon J Crawford K Muchmore E Hughes A.L et al.Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant.Proc Natl Acad Sci. 1995; 92: 2755-2759Crossref PubMed Scopus (311) Google Scholar, 20Sitia G Cella D De Mitri M.S Novati R Foppa C.U Perackis K et al.Evolution of the E2 region of hepatitis C virus in an infant infected by mother-to-infant transmission.J Med Virol. 2001; 64: 476-481Crossref PubMed Scopus (11) Google Scholar, 21Erickson A.L Kimura Y Igarashi S Eichelberger J Houghton M Sidney J et al.The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes.Immunity. 2001; 15: 883-895Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar]. Farci et al. [[22]Farci P Shimoda A Wong D Cabezon T De Gioannis D Strazzera A et al.Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.Proc Natl Acad Sci USA. 1996; 93: 15394-15399Crossref PubMed Scopus (529) Google Scholar] demonstrated that antibodies raised against the dominant HVR1 sequence of an HCV strain could neutralize in vitro that strain but not minor species present in the inoculum when tested in the chimpanzee model (Fig. 1) . Also, it has been observed that even a chimpanzee with effective immunity against homologous genotype 1a challenge was not protected against challenge with isolates of genotype 1b or 2a [[23]Bukh J Thimme R Saterfield W Forns X Chang K Yanagi M et al.Sterilizing immunity against hepatitis C virus is subtype specific, is apparently not mediated by neutralization antibodies, but is correlated with anamnestic cellular immune responses.Hepatology (Abstract). 2001; 34: 253AGoogle Scholar]. Therefore, the possibility that immunity against HCV is strain- or isolate-specific will have to be taken into consideration when developing an HCV vaccine. In addition, the apparent inherent ability of HCV to escape even specific immunity by developing escape mutants could result in vaccine failure as was recently described for simian immunodeficiency virus [[24]Barouch D.H Kunstmann J Kuroda M Schmitz J Santra S Peyerl F et al.Eventual AIDS vaccine failurein a rhesus monkey by viral escape from cytotoxic T lymphocytes.Nature. 2002; 415: 335-339Crossref PubMed Scopus (621) Google Scholar].2.2 The reason for the high chronicity rate of HCV is poorly understoodHCV infection becomes chronic in most cases. However, the mechanisms by which HCV escapes the host immune responses and establishes a chronic infection are not well defined, but clearly a better understanding of the components involved would be valuable in designing vaccine candidates. As stated above, the development of immune escape mutants has been postulated as one of the main mechanisms of HCV persistence [25Major M.E Mihalik K Fernandez J Seidman J Kleiner D Kolykhalov A.A et al.Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus.J Virol. 1999; 73: 3317-3325PubMed Google Scholar, 26Erickson A.L Kimura Y Igarashi S Eichelberger J Houghton M Sidney J et al.The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes.Immunity. 2001; 15: 883-895Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar]. However, this does not appear to be the only mechanism involved in the establishment of a persistent infection. Differing quality of the host responses are most likely involved.The recent development of infectious complementary DNA (cDNA) clones of HCV [27Kolykhalov A.A Agapov E Blight K.J Mihalik K Feinstone S Rice C.M Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.Science. 1997; 277: 570-574Crossref PubMed Scopus (621) Google Scholar, 28Yanagi M Purcell R Emerson S.U Bukh J Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.Proc Natl Acad Sci USA. 1997; 94: 8738-8743Crossref PubMed Scopus (457) Google Scholar, 29Yanagi M St Claire M Shapiro M Emerson S.U Purcell R.H Bukh J Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo.Virology. 1998; 244: 161-172Crossref PubMed Scopus (199) Google Scholar, 30Yanagi M Purcell R.H Emerson S.U Bukh J Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.Virology. 1999; 262: 250-263Crossref PubMed Scopus (162) Google Scholar] has permitted more controlled studies of the role of viral evolution in the persistence of HCV in chimpanzees. Since the RNA that is used for transfection is generated from a single HCV sequence, the initial infection in the chimpanzee is monoclonal. Interestingly, despite the absence of a quasispecies during the early acute phase of the infection the majority of transfected animals developed a chronic infection [[25]Major M.E Mihalik K Fernandez J Seidman J Kleiner D Kolykhalov A.A et al.Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus.J Virol. 1999; 73: 3317-3325PubMed Google Scholar]. Also the virus could persist without evolution in the envelope proteins, indicating that escape from neutralizing antibodies did not play a role, whereas mutations in the non-structural proteins could have represented cytotoxic T lymphocyte escape mutants. Furthermore, of two chimpanzees infected with recombinant HCV lacking the HVR1, one became chronically infected and the other cleared the infection, demonstrating that the most variable region of HCV was not the determinant of clearance of infection or progression to chronicity [[31]Forns X Thimme R Govindarajan S Emerson S.U Purcell R.H Chisari F.V et al.Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees.Proc Natl Acad Sci USA. 2000; 97: 13318-13323Crossref PubMed Scopus (132) Google Scholar]. Although these experiments provide extremely important information, they do not reproduce a natural infection, where multiple viral strains (quasispecies) infect the host. In fact, the potential relevance of the quasispecies evolution in the outcome of HCV infection was recently demonstrated in a study of patients with acute hepatitis C following blood transfusions [[32]Farci P Shimoda A Coiana A Diaz G Peddis G Melpolder J.C et al.The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.Science. 2000; 288: 339-344Crossref PubMed Scopus (775) Google Scholar], which showed that an increase in viral population diversity during the acute phase of HCV infection was associated with evolution to chronicity.As relates to the quality of the host responses, quantitatively inadequate cellular immunity might permit HCV persistence and contribute to the development of chronic liver disease [33Nelson D.R Marousis C.G Davis G.L Rice C.M Wong J Houghton M et al.The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C.J Immunol. 1997; 158: 1473-1481PubMed Google Scholar, 34Rehermann B Chang K.M McHutchinson J Kokka R Houghton M Rice C.M et al.Differential cytotoxic T-lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients.J Virol. 1996; 70: 7092-7102Crossref PubMed Google Scholar] (see below). It is also possible that the virus itself might contribute to diversion or inhibition of the host immune responses to viral antigens by employing decoy antigens [[35]Ray S.C Wang Y.M Laeyendecker O Ticehurst J.R Villano S.A Thomas D.L Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy.J Virol. 1999; 73: 2938-2946PubMed Google Scholar] or by inhibition of the host's interferon-inducible antiviral response [36Gale 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 non-structural 5A protein.Virology. 1997; 230: 217-227Crossref PubMed Scopus (719) Google Scholar, 37Taylor D.R Shi S.T Romano P.R Barber G.N Lai M.M Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.Science. 1999; 285: 107-110Crossref PubMed Scopus (660) Google Scholar].2.3 Limited understanding of the immunologic correlates of HCV clearanceThe existence of neutralizing antibodies has been shown in several experiments performed in chimpanzees. Farci et al. [[38]Farci P Alter H.J Wong D.C Miller R.H Govindarajan S Engle R et al.Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization.Proc Natl Acad Sci USA. 1994; 91: 7792-7796Crossref PubMed Scopus (438) Google Scholar] demonstrated that serum from a patient chronically infected with HCV was able to neutralize HCV strains present in this patient 2 years before. Yu et al. [[39]Yu M.W Shen L Major M.E Feinstone S.M Jong J.S Protective antibodies in immune globulins prepared from anti-hepatitis C virus-positive plasma units: update of a chimpanzee study.in: Margolis H.S Alter M.J Liang T.J Dienstag J.L Viral Hepatitis and Liver Disease (Proceedings of the 2000 International Symposium on Viral Hepatitis and Liver Disease). International Medical Press, Atlanta, Georgia2002: 374-377Google Scholar] reported that immune globulin pools containing antibodies against the envelope HCV proteins and generated from approximately 200 anti-HCV positive blood donors could neutralize HCV: a chimpanzee inoculated with a mixture of anti-HCV positive immune globulin and virus did not become infected, whereas a control animal inoculated only with the virus developed HCV infection. Krawzynski et al. [[40]Krawczynski K Fattom A Culver D Kamili S Spelbring J Basham I et al.Passive transfer of anti-HCV in chronic and acute HCV infection in chimpanzees -trials of experimental immune treatment.Hepatology (Abstract). 1999; 30: 423AGoogle Scholar] investigated the utility of post-exposure prophylaxis in chimpanzees, using an immune globulin preparation made from plasma units collected from 460 anti-HCV positive patients. The treated chimpanzees had early clearance of viremia and did not develop acute hepatitis. A study performed in patients undergoing liver transplantation for HCV- and HBV-related liver cirrhosis showed that infusion of anti-HBs hyperimmune globulin manufactured before 1990 was associated with a low incidence of HCV infection of the graft [[41]Feray C Gigou M Samuel D Ducot B Maisonneuve P Reynes M et al.Incidence of hepatitis C in patients receiving different preparations of hepatitis B immunoglobulins after liver transplantation.Ann Intern Med. 1998; 128: 810-816Crossref PubMed Scopus (129) Google Scholar]. These data strongly suggest that immune globulin produced before 1990 and containing anti-HCV was capable of neutralizing HCV. It is important to note, however, that the immune globulin preparations used in these studies, though containing neutralizing antibodies, came from plasma of chronically infected patients. The role of neutralizing antibodies in viral clearance during acute infection is still unknown. Although some data suggest that the early appearance of antibodies against HVR1 after HCV infection might facilitate HCV clearance [42Zibert A Meisel H Kraas W Schulz A Jung G Roggendorf M Early antibody response against hypervariable region 1 is associated with acute self-limiting infections of hepatitis C virus.Hepatology. 1997; 25: 1245-1249Crossref PubMed Scopus (106) Google Scholar, 43Allander T Beyene A Jacobson S.H Grillner L Persson M.A Patients infected with the same hepatitis C virus strain display different kinetics of the isolate-specific antibody response.J Infect Dis. 1997; 175: 26-31Crossref PubMed Scopus (41) Google Scholar], there are no definitive data to support this hypothesis. Recent data obtained in the chimpanzee model seem to indicate that the presence of neutralizing antibodies is not necessary to obtain protective immunity [[23]Bukh J Thimme R Saterfield W Forns X Chang K Yanagi M et al.Sterilizing immunity against hepatitis C virus is subtype specific, is apparently not mediated by neutralization antibodies, but is correlated with anamnestic cellular immune responses.Hepatology (Abstract). 2001; 34: 253AGoogle Scholar].There are data supporting a more relevant role of the cellular immune response for HCV clearance. One of the first studies that suggested the significance of cell-mediated immunity for HCV clearance was a report by Bjoro et al. [[44]Bjoro K Skaug K Haaland T Froland S.S Long-term outcome of chronic hepatitis C virus infection in primary hypogammaglobulinaemia.QJM. 1999; 92: 433-441Crossref PubMed Scopus (46) Google Scholar] of patients with hypogammaglobulinemia who became infected with HCV. In this study it became clear that some of the patients were able to clear HCV and therefore to eradicate the virus in the absence of measurable antibodies against the virus.Correlates of effective cellular immunity are difficult to establish and are mainly based on the analysis of CD4+ proliferative and CD8+ CTL responses during the acute phase of HCV infection in patients or chimpanzees [45Lechmann M Liang T.J Vaccine development for hepatitis C.Semin Liver Dis. 2000; 20: 211-226Crossref PubMed Scopus (73) Google Scholar, 46Abrignani S Houghton M Hsu H.H Perspectives for a vaccine against hepatitis C virus.J Hepatol. 1999; 31: 259-263Abstract Full Text PDF PubMed Google Scholar, 47Thimme R Oldach D Chang K.M Steiger C Ray S.C Chisari F.V Determinants of viral clearance and persistence during acute hepatitis C virus infection.J Exp Med. 2001; 194: 1395-1406Crossref PubMed Scopus (1011) Google Scholar]. In general, studies analyzing the cell-mediated immunity against HCV have described the polyclonal nature of a host immune response as well as its low vigor during chronic HCV infection [48Wong D.K Dudley D.D Afdhal N.H Dienstag J Rice C.M Wang L et al.Liver-derived CTL in hepatitis C virus infection: breadth and specificity of responses in a cohort of persons with chronic infection.J Immunol. 1998; 160: 1479-1488PubMed Google Scholar, 49Koziel M.J Dudley D Wong J.T Dienstag J Houghton M Ralston R et al.Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis.J Immunol. 1992; 149: 3339-3344PubMed Google Scholar, 50Chang K.M Gruener N.H Southwood S Sidney J Pape G.R Chisari F.V et al.Identification of HLA-A3 and -B7-restricted CTL response to hepatitis C virus in patients with acute and chronic hepatitis C.J Immunol. 1999; 162: 1156-1164PubMed Google Scholar]. CD4+ T-cell proliferative responses are stronger in patients resolving an infection compared with those developing a chronic infection: multi-specific T-cell responses directed against both structural and non-structural antigens are more frequent and are significantly stronger in patients who clear viremia than in those who do not [47Thimme R Oldach D Chang K.M Steiger C Ray S.C Chisari F.V Determinants of viral clearance and persistence during acute hepatitis C virus infection.J Exp Med. 2001; 194: 1395-1406Crossref PubMed Scopus (1011) Google Scholar, 51Missale G Bertoni R Lamonaca V Valli A Massari M Mori C et al.Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response.J Clin Invest. 1996; 98: 706-714Crossref PubMed Scopus (602) Google Scholar, 52Botarelli P Brunetto M.R Minutello M.A Calvo P Unutmaz D Weiner A.J et al.T-lymphocyte response to hepatitis C virus in different clinical courses of infection.Gastroenterology. 1993; 104: 580-587PubMed Google Scholar, 53Ferrari C Valli A Galati L Penna A Scaccaglia P Giuberti T et al.T-cell response to structural and non-structural hepatitis C virus antigens in persistent and self-limited hepatitis C virus infections.Hepatology. 1994; 19: 286-295Crossref PubMed Scopus (239) Google Scholar, 54Lechmann M Ihlenfeldt H.G Braunschweiger I Giers G Jung G Matz B et al.T- and B-cell responses to different hepatitis C virus antigens in patients with chronic hepatitis C infection and in healthy anti-hepatitis C virus – positive blood donors without viremia.Hepatology. 1996; 24: 790-795PubMed Google Scholar]. Furthermore, T-cells obtained from individuals with self-limiting infection display a strong profile of Th1 cytokines, such as interleukin 2 (IL-2) and interferon (IFN)-gamma, after stimulation with HCV antigen [51Missale G Bertoni R Lamonaca V Valli A Massari M Mori C et al.Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response.J Clin Invest. 1996; 98: 706-714Crossref PubMed Scopus (602) Google Scholar, 55Diepolder H.M Zachoval R Hoffmann R.M Wierenga E.A Santantonio T Jung M.C et al.Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection.Lancet. 1995; 346: 1006-1007Abstract PubMed Google Scholar, 56Takaki A Wiese M Maertens G Depla E Seifert U Liebetrau A et al.Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C.Nat Med. 2000; 6: 578-582Crossref PubMed Scopus (668) Google Scholar]. Further evidence supporting a role of Th1-type responses in contributing to viral clearance comes from the finding that strong CTL responses correlate with successful clearance of HCV infection in chimpanzees [[57]Cooper S Erickson A.L Adams E.J Kansopon J Weiner A.J Chien D.Y et al.Analysis of a successful immune response against hepatitis C virus.Immunity. 1999; 10: 439-449Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar]. Cooper et al. [[57]Cooper S Erickson A.L Adams E.J Kansopon J Weiner A.J Chien D.Y et al.Analysis of a successful immune response against hepatitis C virus.Immunity. 1999; 10: 439-449Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar] found that two chimpanzees with acute resolving infection had an early and strong intrahepatic CTL response to several HCV epitopes, whereas four animals that became chronically infected had much weaker CTL responses. Thimme et al. [[58]Thimme R Bukh J Chang K.M Pemberton J Purcell R.H Chisari F.V HCV persists despite a multispecific peripheral and intrahepatic T cell response in acutely infected chimpanzees.Hepatology (Abstract). 1999; 30: 422ACrossref PubMed Scopus (25) Google Scholar] have recently reported that in naive chimpanzees experimentally infected with HCV, the animals that controlled the virus at low levels or had viral clearance showed strong intrahepatic CD4+ and CD8+ responses, whereas animals without control did not.New data obtained in the chimpanzee model suggest that protective immunity against HCV can be elicited [[59]Bassett S.E Guerra B Brasky K Miskovsky E Houghton M Klimpel G.R et al.Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.Hepatology. 2001; 33: 1479-1487Crossref PubMed Scopus (203) Google Scholar]. In fact, chimpanzees that had previously cleared HCV infection were able to clear HCV rapidly following re-challenge with homologous or heterologous HCV [60Weiner A.J Paliard X Selby M.J Medina-Selby A Coit D Nguyen S et al.Intrahepatic genetic inoculation of hepatitis C virus RNA confers cross-protective immunity.J Virol. 2001; 75: 7142-7148Crossref PubMed Scopus (88) Google Scholar, 61Major M.E Mihalik K Puig M Rehermann B Nascimbeni M Rice C.M et al.Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge.J Virol. 2002; 76: 6586-6595Crossref PubMed Scopus (158) Google Scholar] (Fig. 2) . Strong peripheral and intrahepatic CD4+ responses appear to be associated with this protective immunity. However, in at least one case, HCV was able to persist following homologous re-challenge in an animal that had previously cleared the exact same virus, despite a st