New perspectives for T-cell-based HCV vaccines

Abstract

A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Folgori A, Capone S, Ruggeri L, Meola A, Sporeno E, Ercole BB, Pezzanera M, Tafi R, Arcuri M, Fattori E, Lahm A, Luzzago A, Vitelli A, Colloca S, Cortese R, Nicosia A.Three percent of the world’s population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here, we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.[Abstract reproduced by permission of Nat Med 2006;12(2):190–197] A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Folgori A, Capone S, Ruggeri L, Meola A, Sporeno E, Ercole BB, Pezzanera M, Tafi R, Arcuri M, Fattori E, Lahm A, Luzzago A, Vitelli A, Colloca S, Cortese R, Nicosia A. Three percent of the world’s population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here, we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses. [Abstract reproduced by permission of Nat Med 2006;12(2):190–197] HCV infection is a major health problem because more than 170 million people are infected by HCV worldwide. HCV infection is followed by persistence of the virus and chronic liver disease in 60–80% of infected individuals.The cell-mediated immune response to HCV is believed to play a central role in the pathogenesis of HCV infection and quality, strength and breadth of the HCV-specific T-cell response seem to deeply influence the evolution of infection towards control or chronic viral persistence [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. This concept is further supported by a recent report by Folgori et al. published in the February issue of Nature Medicine [[4]Folgori A. Capone S. Ruggeri L. Meola A. Sporeno E. Ercole B.B. et al.A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.Nat Med. 2006; 12: 190-197Crossref PubMed Scopus (270) Google Scholar] showing that immunization of chimpanzees with a T-cell-based genetic vaccine can elicit protective immunity against heterologous HCV strains by stimulation of the cellular arm of the immune system.Many recent reports indicate that priming of the adaptive responses by HCV antigens leads to the development of vigorous and multi-specific T-cell responses only in those individuals who eventually succeed in controlling the infection [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. The type of T-cell response associated with control of infection contrasts with the weaker and more narrowly focused T-cell response mounted by patients who develop a chronic infection [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. Irrespective of the final outcome, development of a functional HCV-specific T-cell response seems to proceed slowly after infection, with an initial generation of dysfunctional HCV-specific CD8 cells [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. Only in the presence of an efficient help provided by the CD4 T-cell response, which is known to be significantly stronger in self-limited than in chronically evolving infections, CD8 cells can complete their maturation program, acquiring a full antiviral activity which is essential for final and persistent control of infection [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar].Less clear at present is the role of the antibody response in HCV pathogenesis because the analysis of neutralizing anti-HCV antibodies has been hampered until recently by the lack of suitable in vitro systems to support HCV infection of human cells and productive virus replication in vitro. The recent development of infectious retrovirus-HCV pseudo-particles requiring co-expression of both E1 and E2 HCV glycoproteins for target cell infection in vitro [5Bartosch B. Dubuisson J. Cosset F.L. J Exp Med. 2003; 197: 633-642Crossref PubMed Scopus (942) Google Scholar, 6Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C.M. et al.Proc Natl Acad Sci USA. 2003; 100: 7271-7276Crossref PubMed Scopus (687) Google Scholar, 7Logvinoff C. Major M.E. Oldach D. Heyward S. Talal A. Balfe P. et al.Proc Natl Acad Sci USA. 2004; 101: 10149-10154Crossref PubMed Scopus (348) Google Scholar, 8Lavillette D. Morice Y. Germanidis G. Donot P. Soulier A. Pagkalos E. et al.Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection.J Virol. 2005; 79: 6023-6034Crossref PubMed Scopus (236) Google Scholar, 9Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. et al.An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies.J Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (245) Google Scholar] has allowed investigation of the neutralizing effect of serum anti-HCV antibodies from patients at different stages of infection. Most persistently infected patients show high titers of pseudotype-particle neutralizing antibodies, while neutralizing antibody responses are slow to develop after infection and seem to be of relatively low titers in acute HCV infection, irrespective of the outcome to control or persistence of the virus. Therefore, efficient and long-term control of HCV infection seems to require effective cellular immune responses, while evidence for a role of humoral responses in protection against HCV is much weaker. Based on this, priming HCV-specific T-cell responses before exposure to HCV may be an efficient strategy to protect from acute hepatitis C.Although clear evidence of protective immunity was not provided by previous T-cell-based vaccines in the chimpanzee model of infection [10Forns X. Payette P.J. Ma X. Satterfield W. Eder G. Mushahwar I.K. et al.Vaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV.Hepatology. 2000; 32: 618-625Crossref PubMed Scopus (143) Google Scholar, 11Rollier C. Depla E. Drexhage J.A. Verschoor E.J. Verstrepen B.E. Fatmi A. et al.Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.J Virol. 2004; 78: 187-196Crossref PubMed Scopus (84) Google Scholar, 12Puig M. Major M.E. Mihalik K. Feinstone S.M. Immunization of chimpanzees with an envelope protein-based vaccine enhances specific humoral and cellular immune responses that delay hepatitis C virus infection.Vaccine. 2004; 22: 991-1000Crossref PubMed Scopus (73) Google Scholar], a potent and efficient induction of protective T-cell responses able to suppress acute viremia in vaccinated chimpanzees is now reported by Alfredo Nicosia’s group [[4]Folgori A. Capone S. Ruggeri L. Meola A. Sporeno E. Ercole B.B. et al.A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.Nat Med. 2006; 12: 190-197Crossref PubMed Scopus (270) Google Scholar]. In this study, particular attention was paid to design carefully the immunogen and the delivery platform, in order to induce a vigorous and multi-specific T-cell reactivity able to mimic the type of response detectable in natural self-limited infections. HCV non-structural proteins were selected as immunogens because they contain most of the previously mapped T-cell epitopes. The sequence of the immunizing vaccine corresponded to the HCV-BK strain of genotype 1b. Adenoviral vectors and plasmid DNA were constructed and used as delivery systems because of their high potency in the induction of cellular responses. Moreover, adenoviruses of serotypes 6 and 24 were selected because these serotypes are less prevalent in the human population, thereby allowing to minimize the risk of inducing anti-adenovirus immune responses, which could limit the efficacy of the vaccination.The adenovirus 6/non-structural HCV proteins construct was used for the initial priming of the T-cell responses in five chimpanzees by two consecutive administrations 4 weeks apart (week 0 and week 4). Responses were subsequently boosted at week 25 with the adenovirus 24/non-structural HCV proteins construct and with three consecutive injections at weeks 35, 37 and 39 of electroporated DNA plasmid encoding non-structural HCV proteins. The five vaccinated chimpanzees were then infected by intravenous administration of a heterologous H77 inoculum (genotype 1a; 100 CID50). Five non-vaccinated chimpanzees were similarly infected and served as controls.A multi-specific CD4 and CD8-mediated T-cell response was induced by vaccination, with recognition of at least two different epitopes in all immunized animals, except one. All non-structural proteins were targeted, with most responses directed against NS3. T-cell responses were fully functional and detectable not only in the peripheral blood but also within the liver of vaccinated animals. T-cell responses in vaccinated animals were mostly sustained by CD8 cells and protected chimpanzees from acute hepatitis following infection with a heterologous HCV strain because no elevation of serum transaminase was observed. Detection of viremia was delayed and HCV-RNA levels were significantly lower in vaccine recipients than in control animals. Viremia was transient in four of the five vaccinated chimpanzees who eventually cleared the virus. Cross-reactive CD8 responses able to recognize not only the genotype 1b sequences of the immunizing vaccine but also the heterologous sequences of the HCV genotype 1a strain used for infection were detected in the four of the five vaccinated animals who resolved infection. Finally, escape mutations were found within the immunodominant CD8 epitope of the only vaccinated chimpanzee that did not control infection. Since non-structural regions of the infecting H77 virus (genotype 1a) differ by more than 13% from those of the genotype 1b immunizing vaccine, induction of protective cross-reactive T-cell responses is promising for the development of an effective T-cell-based vaccine, because published HCV isolates of genotype 1 show levels of amino acid variation similar to those present between virus sequences of vaccine and challenge viruses.Thus, the study provides the important demonstration that a strong, multi-specific and functional T-cell response can be successfully induced by vaccination. Importantly, this response can protect effectively against heterologous HCV strains, reproducing the situation which will occur when a vaccine will be available for humans, i.e., infection by a heterologous virus different from the immunizing vaccine. These results provide also additional evidence for a crucial role played by the cell-mediated immune response in the control of HCV infection. While this approach may be of practical efficacy to prevent chronic HCV persistence when vaccination is given before infection, results cannot be directly translated to therapy of long-lasting chronic infections because the requirements for successful stimulation of persistently dysfunctional T cells are expected to be totally different. HCV infection is a major health problem because more than 170 million people are infected by HCV worldwide. HCV infection is followed by persistence of the virus and chronic liver disease in 60–80% of infected individuals. The cell-mediated immune response to HCV is believed to play a central role in the pathogenesis of HCV infection and quality, strength and breadth of the HCV-specific T-cell response seem to deeply influence the evolution of infection towards control or chronic viral persistence [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. This concept is further supported by a recent report by Folgori et al. published in the February issue of Nature Medicine [[4]Folgori A. Capone S. Ruggeri L. Meola A. Sporeno E. Ercole B.B. et al.A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.Nat Med. 2006; 12: 190-197Crossref PubMed Scopus (270) Google Scholar] showing that immunization of chimpanzees with a T-cell-based genetic vaccine can elicit protective immunity against heterologous HCV strains by stimulation of the cellular arm of the immune system. Many recent reports indicate that priming of the adaptive responses by HCV antigens leads to the development of vigorous and multi-specific T-cell responses only in those individuals who eventually succeed in controlling the infection [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. The type of T-cell response associated with control of infection contrasts with the weaker and more narrowly focused T-cell response mounted by patients who develop a chronic infection [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. Irrespective of the final outcome, development of a functional HCV-specific T-cell response seems to proceed slowly after infection, with an initial generation of dysfunctional HCV-specific CD8 cells [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. Only in the presence of an efficient help provided by the CD4 T-cell response, which is known to be significantly stronger in self-limited than in chronically evolving infections, CD8 cells can complete their maturation program, acquiring a full antiviral activity which is essential for final and persistent control of infection [1Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1353) Google Scholar, 2Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (231) Google Scholar, 3Wieland S.F. Chisari F.V. Stealth and Cunning: hepatitis B and hepatitis C viruses.J Virol. 2005; 79: 9369-9380Crossref PubMed Scopus (374) Google Scholar]. Less clear at present is the role of the antibody response in HCV pathogenesis because the analysis of neutralizing anti-HCV antibodies has been hampered until recently by the lack of suitable in vitro systems to support HCV infection of human cells and productive virus replication in vitro. The recent development of infectious retrovirus-HCV pseudo-particles requiring co-expression of both E1 and E2 HCV glycoproteins for target cell infection in vitro [5Bartosch B. Dubuisson J. Cosset F.L. J Exp Med. 2003; 197: 633-642Crossref PubMed Scopus (942) Google Scholar, 6Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C.M. et al.Proc Natl Acad Sci USA. 2003; 100: 7271-7276Crossref PubMed Scopus (687) Google Scholar, 7Logvinoff C. Major M.E. Oldach D. Heyward S. Talal A. Balfe P. et al.Proc Natl Acad Sci USA. 2004; 101: 10149-10154Crossref PubMed Scopus (348) Google Scholar, 8Lavillette D. Morice Y. Germanidis G. Donot P. Soulier A. Pagkalos E. et al.Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection.J Virol. 2005; 79: 6023-6034Crossref PubMed Scopus (236) Google Scholar, 9Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. et al.An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies.J Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (245) Google Scholar] has allowed investigation of the neutralizing effect of serum anti-HCV antibodies from patients at different stages of infection. Most persistently infected patients show high titers of pseudotype-particle neutralizing antibodies, while neutralizing antibody responses are slow to develop after infection and seem to be of relatively low titers in acute HCV infection, irrespective of the outcome to control or persistence of the virus. Therefore, efficient and long-term control of HCV infection seems to require effective cellular immune responses, while evidence for a role of humoral responses in protection against HCV is much weaker. Based on this, priming HCV-specific T-cell responses before exposure to HCV may be an efficient strategy to protect from acute hepatitis C. Although clear evidence of protective immunity was not provided by previous T-cell-based vaccines in the chimpanzee model of infection [10Forns X. Payette P.J. Ma X. Satterfield W. Eder G. Mushahwar I.K. et al.Vaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV.Hepatology. 2000; 32: 618-625Crossref PubMed Scopus (143) Google Scholar, 11Rollier C. Depla E. Drexhage J.A. Verschoor E.J. Verstrepen B.E. Fatmi A. et al.Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.J Virol. 2004; 78: 187-196Crossref PubMed Scopus (84) Google Scholar, 12Puig M. Major M.E. Mihalik K. Feinstone S.M. Immunization of chimpanzees with an envelope protein-based vaccine enhances specific humoral and cellular immune responses that delay hepatitis C virus infection.Vaccine. 2004; 22: 991-1000Crossref PubMed Scopus (73) Google Scholar], a potent and efficient induction of protective T-cell responses able to suppress acute viremia in vaccinated chimpanzees is now reported by Alfredo Nicosia’s group [[4]Folgori A. Capone S. Ruggeri L. Meola A. Sporeno E. Ercole B.B. et al.A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.Nat Med. 2006; 12: 190-197Crossref PubMed Scopus (270) Google Scholar]. In this study, particular attention was paid to design carefully the immunogen and the delivery platform, in order to induce a vigorous and multi-specific T-cell reactivity able to mimic the type of response detectable in natural self-limited infections. HCV non-structural proteins were selected as immunogens because they contain most of the previously mapped T-cell epitopes. The sequence of the immunizing vaccine corresponded to the HCV-BK strain of genotype 1b. Adenoviral vectors and plasmid DNA were constructed and used as delivery systems because of their high potency in the induction of cellular responses. Moreover, adenoviruses of serotypes 6 and 24 were selected because these serotypes are less prevalent in the human population, thereby allowing to minimize the risk of inducing anti-adenovirus immune responses, which could limit the efficacy of the vaccination. The adenovirus 6/non-structural HCV proteins construct was used for the initial priming of the T-cell responses in five chimpanzees by two consecutive administrations 4 weeks apart (week 0 and week 4). Responses were subsequently boosted at week 25 with the adenovirus 24/non-structural HCV proteins construct and with three consecutive injections at weeks 35, 37 and 39 of electroporated DNA plasmid encoding non-structural HCV proteins. The five vaccinated chimpanzees were then infected by intravenous administration of a heterologous H77 inoculum (genotype 1a; 100 CID50). Five non-vaccinated chimpanzees were similarly infected and served as controls. A multi-specific CD4 and CD8-mediated T-cell response was induced by vaccination, with recognition of at least two different epitopes in all immunized animals, except one. All non-structural proteins were targeted, with most responses directed against NS3. T-cell responses were fully functional and detectable not only in the peripheral blood but also within the liver of vaccinated animals. T-cell responses in vaccinated animals were mostly sustained by CD8 cells and protected chimpanzees from acute hepatitis following infection with a heterologous HCV strain because no elevation of serum transaminase was observed. Detection of viremia was delayed and HCV-RNA levels were significantly lower in vaccine recipients than in control animals. Viremia was transient in four of the five vaccinated chimpanzees who eventually cleared the virus. Cross-reactive CD8 responses able to recognize not only the genotype 1b sequences of the immunizing vaccine but also the heterologous sequences of the HCV genotype 1a strain used for infection were detected in the four of the five vaccinated animals who resolved infection. Finally, escape mutations were found within the immunodominant CD8 epitope of the only vaccinated chimpanzee that did not control infection. Since non-structural regions of the infecting H77 virus (genotype 1a) differ by more than 13% from those of the genotype 1b immunizing vaccine, induction of protective cross-reactive T-cell responses is promising for the development of an effective T-cell-based vaccine, because published HCV isolates of genotype 1 show levels of amino acid variation similar to those present between virus sequences of vaccine and challenge viruses. Thus, the study provides the important demonstration that a strong, multi-specific and functional T-cell response can be successfully induced by vaccination. Importantly, this response can protect effectively against heterologous HCV strains, reproducing the situation which will occur when a vaccine will be available for humans, i.e., infection by a heterologous virus different from the immunizing vaccine. These results provide also additional evidence for a crucial role played by the cell-mediated immune response in the control of HCV infection. While this approach may be of practical efficacy to prevent chronic HCV persistence when vaccination is given before infection, results cannot be directly translated to therapy of long-lasting chronic infections because the requirements for successful stimulation of persistently dysfunctional T cells are expected to be totally different.