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Progress in the development of preventive and therapeutic vaccines for hepatitis C virus

  • Joseph Torresi
    Correspondence
    Corresponding author at: Department of Infectious Diseases, Austin Hospital, Heidelberg, Victoria 3084, Australia. Tel.: +61 3 9496 6676; fax: +61 3 9496 6677.
    Affiliations
    Austin Centre for Infection Research, Department of Infectious Diseases Austin Hospital, Heidelberg, Victoria 3084, Australia

    Department of Medicine, Austin Hospital, University of Melbourne, Heidelberg, Victoria 3084, Australia
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  • Doug Johnson
    Affiliations
    Austin Centre for Infection Research, Department of Infectious Diseases Austin Hospital, Heidelberg, Victoria 3084, Australia

    Department of Medicine, Austin Hospital, University of Melbourne, Heidelberg, Victoria 3084, Australia
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  • Heiner Wedemeyer
    Affiliations
    Department of Gastroenterology and Hepatology, Medizinische Hochschule, 30625 Hannover, Germany
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Open AccessPublished:January 12, 2011DOI:https://doi.org/10.1016/j.jhep.2010.09.040
      Hepatitis C virus (HCV) is a blood borne disease estimated to chronically infect 3% of the worlds’ population causing significant morbidity and mortality. Current medical therapy is curative in approximately 50% of patients. While recent treatment advances of genotype 1 infection using directly acting antiviral agents (DAAs) are encouraging, there is still a need to develop vaccine strategies capable of preventing infection. Moreover, vaccines may also be used in future in combination with DAAs enabling interferon-free treatment regimens.
      Viral and host specific factors contribute to viral evasion and present important impediments to vaccine development. Both, innate and adaptive immune responses are of major importance for the control of HCV infection. However, HCV has evolved ways of evading the host’s immune response in order to establish persistent infection. For example, HCV inhibits intracellular interferon signalling pathways, impairs the activation of dendritic cells, CD8+ and CD4+ T cell responses, induces a state of T-cell exhaustion and selects escape variants with mutations CD8+ T cell epitopes. An effective vaccine will need to produce strong and broadly cross-reactive CD4+, CD8+ T cell and neutralising antibody (NAb) responses to be successful in preventing or clearing HCV.
      Vaccines in clinical trials now include recombinant proteins, synthetic peptides, virosome based vaccines, tarmogens, modified vaccinia Ankara based vaccines, and DNA based vaccines. Several preclinical vaccine strategies are also under development and include recombinant adenoviral vaccines, virus like particles, and synthetic peptide vaccines. This paper will review the vaccines strategies employed, their success to date and future directions of vaccine design.

      Keywords

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      • Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: Perspectives and challenges
        Journal of HepatologyVol. 54Issue 6
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          The treatment of chronic hepatitis B virus (HBV) infection has greatly improved over the last 10 years, but alternative treatments are still needed. Therapeutic vaccination is a promising new strategy for controlling chronic infection. However, this approach has not been as successful as initially anticipated for chronic hepatitis B. General impairment of the immune responses generated during persistent HBV infection, with exhausted T cells not responding correctly to therapeutic vaccination, is probably responsible for the poor clinical responses observed to date.
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      Introduction

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      Figure thumbnail gr1
      Fig. 1Summary of the immune responses required to clear HCV and the major sites of action of different HCV preventive and therapeutic vaccines.

      Correlates of hepatitis C cell mediated immunity

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      ] responses to both structural and non-structural HCV proteins (Table 1)(Fig. 1). Clearance of HCV and protection from reinfection is determined not only by the magnitude and/or breadth of multifunctional CD8+ T cells but also by the quality, functional potency, and cytotoxic potential of HCV-specific CD8+ T cells [
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      ]. The ability to produce strong HCV specific CD8+ and CD4+ T cell responses are important considerations for effective HCV vaccine design. It may also be important to consider the differences that exist in CD8+ T cell specificities in peripheral compared to intrahepatic CD8+ T cells [
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      ].
      Table 1Summary of the correlates of protective HCV immunity and the mechanisms of viral evasion of immune responses.
      The relative contributions of individual viral proteins to the total magnitude of the HCV-specific CD4+ and CD8+ T cell responses also play an important role in determining the outcome of infection. Using various approaches it has been possible to determine a hierarchy of CD4+ and CD8+ T cell responses, particularly to the NS3 protein [
      • Lauer G.M.
      • Barnes E.
      • Lucas M.
      • Timm J.
      • Ouchi K.
      • Kim A.Y.
      • et al.
      High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection.
      ,
      • Schulze zur Wiesch J.
      • Lauer G.M.
      • Day C.L.
      • Kim A.Y.
      • Ouchi K.
      • Duncan J.E.
      • et al.
      Broad repertoire of the CD4+ Th cell response in spontaneously controlled hepatitis C virus infection includes dominant and highly promiscuous epitopes.
      ,
      • Thammanichanond D.
      • Moneer S.
      • Yotnda P.
      • Aitken C.
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      ,
      • Wertheimer A.M.
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      • Sasaki A.W.
      • Kaufman E.
      • Rosen H.R.
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      ]. It is also possible to elicit strong HCV core specific CD4+ and CD8+ T cell responses in naïve human lymphocytes [
      • Li W.
      • Krishnadas D.K.
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      • Tyrrell D.L.J.
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      ]. These studies argue that the inclusion of CD8+ T cell epitopes representing key viral proteins, like core and NS3, will be essential for the development of a cell mediated vaccine for HCV. However, a recent meta-analysis of the efficacy of HCV vaccines in chimpanzees has shown that the inclusion of structural proteins in vaccines was more significantly associated with protective immune responses compared to vaccines based on non-structural proteins of HCV [

      Dahari H, Feinstone S, Major ME. Meta-analysis of hepatitis C virus vaccine efficacy in chimpanzees indicates an importance for structural proteins. Gastrenterology 2010.

      ].

      Neutralising antibody (NAb) responses to HCV and neutralising epitope domains

      A strong line of evidence now exists demonstrating that NAb responses to epitopes in the viral E1 and E2 glycoproteins can be protective [
      • Yu M.
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      ] and is associated with resolution of hepatitis C infection [
      • Pestka J.M.
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      • Blaser E.
      • Schurmann P.
      • Bartosch B.
      • Cosset F.L.
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      Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.
      ]. Vaccination of chimpanzees with mammalian cell, but not yeast cell-derived recombinant HCV E1 and E2 glycoproteins has been shown to prevent the development of chronic infection with both homologous and heterologous viruses [
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      • Tanaka T.
      • Okamoto H.
      Influence of antibodies to the hypervariable region of E2/NS1 glycoprotein on the selective replication of hepatitis C virus in chimpanzees.
      ,
      • Rosa D.
      • Campagnoli S.
      • Moretto C.
      • Guenzi E.
      • Cousens L.
      • Chin M.
      • et al.
      A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.
      ]. Similarly, neutralisation of a viral inoculum with either rabbit hyperimmune serum directed to homologous virus [
      • 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.
      ] or human anti-HCV sera [
      • 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.
      ] and immunoglobulin [
      • Yu M.
      • Bartosch B.
      • Zhang P.
      • Guo Z.P.
      • Renzi P.M.
      • Shen L.
      • et al.
      Neutralising antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma.
      ] protected chimpanzees against viral challenge, thus providing strong evidence that NAb contributes to protection against HCV.

      E2 neutralising epitopes

      Distinct neutralizing antibody epitopes have been identified in hypervariable region 1 (HVR1) of the E2 protein [
      • 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.
      ,
      • 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.
      ,
      • Hsu M.
      • Zhang J.
      • Flint M.
      • Logvinoff C.
      • Cheng-Mayer C.
      • Rice C.M.
      • et al.
      Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.
      ,
      • Lee J.W.
      • Kim K.
      • Jung S.H.
      • Lee K.J.
      • Choi E.C.
      • Sung Y.C.
      • et al.
      Identification of a domain containing B-cell epitopes in hepatitis C virus E2 glycoprotein by using mouse monoclonal antibodies.
      ,
      • Rosa D.
      • Campagnoli S.
      • Moretto C.
      • Guenzi E.
      • Cousens L.
      • Chin M.
      • et al.
      A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.
      ] and in the region downstream of HVR1, including the binding sites for the putative HCV receptor, CD81 [
      • Bartosch B.
      • Bukh J.
      • Meunier J.C.
      • Granier C.
      • Engle R.E.
      • Blackwelder W.C.
      • et al.
      In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.
      ,
      • Grollo L.
      • Torresi J.
      • Drummer H.
      • Zeng W.
      • Williamson N.
      • Jackson D.C.
      Cross-reactive epitopes of hepatitis C virus induce antibodies that capture virions and inhibit pseudo virus particle cell entry.
      ,
      • Hsu M.
      • Zhang J.
      • Flint M.
      • Logvinoff C.
      • Cheng-Mayer C.
      • Rice C.M.
      • et al.
      Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.
      ,
      • Keck Z.
      • Op De Beeck A.
      • Hadlock K.G.
      • Xia J.
      • Li T.-K.
      • Dubuisson J.
      • et al.
      Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions.
      ,
      • Law M.
      • Maruyama T.
      • Lewis J.
      • Giang E.
      • Tarr A.W.
      • Stamataki Z.
      • et al.
      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ,
      • Torresi J.
      • Fischer A.
      • Grollo L.
      • Zeng W.
      • Drummer H.
      • Jackson D.C.
      Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T-helper epitopes.
      ,
      • Torresi J.
      • Stock O.M.
      • Fischer A.E.
      • Grollo L.
      • Drummer H.
      • Boo I.
      • et al.
      A self-adjuvanting multiepitope immunogen that induces a broadly cross-reactive antibody to hepatitis C virus.
      ]. Also, epitopes in the amino-terminus of HVR1 may induce neutralising antibody responses that are associated with self-limiting hepatitis C infection [
      • Zibert A.
      • Kraas W.
      • Meisel H.
      • Jung G.
      • Roggendorf M.
      Epitope mapping of antibodies directed against hypervariable region 1 in acute self-limiting and chronic infections due to hepatitis C virus.
      ]. In contrast, antibodies to the carboxy-terminal end of HVR1 may not be neutralising and may represent epitopes that interfere with the optimal development of neutralising antibody response [
      • Esumi M.
      • Zhou Y.
      • Tanoue T.
      • Tomoguri T.
      • Hayasaka I.
      In vivo and in vitro evidence that cross-reactive antibodies to C-terminus of hypervariable region 1 do not neutralize heterologous hepatitis C virus.
      ].
      It is now apparent that epitopes within E2 can be broadly cross neutralising [
      • Tarr A.W.
      • Owsianka A.M.
      • Timms J.M.
      • McClure P.C.
      • Brown R.J.P.
      • Hickling T.P.
      • et al.
      Characterization of the Hepatitis C Virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33.
      ], some of which do not overlap with the CD81 binding domain of the E2 protein [
      • Grollo L.
      • Torresi J.
      • Drummer H.
      • Zeng W.
      • Williamson N.
      • Jackson D.C.
      Cross-reactive epitopes of hepatitis C virus induce antibodies that capture virions and inhibit pseudo virus particle cell entry.
      ,
      • Torresi J.
      • Fischer A.
      • Grollo L.
      • Zeng W.
      • Drummer H.
      • Jackson D.C.
      Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T-helper epitopes.
      ]. Also, monoclonal antibodies directed to a conserved conformational region of the E2 protein that is known to contain a major neutralising antigenic region referred to as antigenic region 3 (AR3) [
      • Law M.
      • Maruyama T.
      • Lewis J.
      • Giang E.
      • Tarr A.W.
      • Stamataki Z.
      • et al.
      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ] of HCV genotype 1a virus are able to cross neutralise HCV of different genotypes. AR3 is located on the viral envelope and is formed by three discontinuous segments situated between amino acids 396–434, 436–447, and 523–540 of the E2 protein (Fig. 2). Importantly, passive immunization of human liver chimeric Alb-uPA/SCID mice with monoclonal antibodies directed to AR3 protect these mice against challenge with human serum derived HCV, further highlighting the importance of effective NAb and the significance of delivering HCV neutralising epitopes in the correct conformation [
      • Law M.
      • Maruyama T.
      • Lewis J.
      • Giang E.
      • Tarr A.W.
      • Stamataki Z.
      • et al.
      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ]. It should, therefore, be possible to develop an effective vaccine strategy that will include conserved neutralising epitopes outside of HVR1, and a HCV VLP based vaccine has the very real potential to achieve this goal.
      Figure thumbnail gr2
      Fig. 2Summary of neutralizing domains described in both the E1 and E2 proteins. AR3 is formed by three discontinuous epitopes on the viral envelope.

      E1 neutralising epitopes

      Less is known about neutralising immune responses to the E1 glycoprotein of HCV and until recently there have been relatively few reports describing neutralising epitopes contained within E1 [
      • Grollo L.
      • Torresi J.
      • Drummer H.
      • Zeng W.
      • Williamson N.
      • Jackson D.C.
      Cross-reactive epitopes of hepatitis C virus induce antibodies that capture virions and inhibit pseudo virus particle cell entry.
      ,
      • Meunier J.C.
      • Russell R.S.
      • Goossens V.
      • Priem S.
      • Walter H.
      • Depla E.
      • et al.
      Isolation and Characterization of Broadly Neutralizing Human Monoclonal Antibodies to the E1 Glycoprotein of Hepatitis C Virus.
      ,
      • Torresi J.
      • Fischer A.
      • Grollo L.
      • Zeng W.
      • Drummer H.
      • Jackson D.C.
      Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T-helper epitopes.
      ]. However, a broadly cross-neutralising epitope has been identified between amino acids 313–327 of a highly conserved region of the E1 protein [
      • Meunier J.C.
      • Russell R.S.
      • Goossens V.
      • Priem S.
      • Walter H.
      • Depla E.
      • et al.
      Isolation and Characterization of Broadly Neutralizing Human Monoclonal Antibodies to the E1 Glycoprotein of Hepatitis C Virus.
      ,
      • Torresi J.
      • Fischer A.
      • Grollo L.
      • Zeng W.
      • Drummer H.
      • Jackson D.C.
      Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T-helper epitopes.
      ] (Fig. 2). Monoclonal antibodies directed to this epitope strongly neutralise HCV/HIV pseudotypic particles bearing the envelope glycoproteins of HCV genotypes 1a, 1b, 4a, 5a, and 6a and less so against 2a and 2b. These monoclonal antibodies also neutralised cell culture derived HCV of genotypes 1a and 2a. In addition, it has also been shown that cross-neutralizing E1-specific antibodies can be produced following vaccination of mice with a retrovirus-based HCV virus like particle vaccine [
      • Dreux M.
      • Pietschmann T.
      • Granier C.
      • Voisset C.
      • Ricard-Blum S.
      • Mangeot P.-E.
      • et al.
      High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor B1.
      ,
      • Pietschmann T.
      • Kaul A.
      • Koutsoudakis G.
      • Shavinskaya A.
      • Kallis S.
      • Steinmann E.
      • et al.
      Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.
      ] highlighting the importance of including E1 in a preventive HCV vaccine.

      Evasion of the immune responses by HCV

      Hepatitis C virus has evolved several ways of evading the host’s immune response in order to establish persistent infection [
      • Bowen D.G.
      • Walker C.M.
      Adaptive immune responses in acute and chronic hepatitis C virus infection.
      ,
      • Bowen D.G.
      • Walker C.M.
      Mutational escape from CD8+ T cell immunity: HCV evolution, from chimpanzees to man.
      ,
      • Bowen D.G.
      • Walker C.M.
      The origin of quasispecies: cause or consequence of chronic hepatitis C viral infection?.
      ,
      • Gale Jr., M.
      • Foy E.M.
      Evasion of intracellular host defence by hepatitis C virus.
      ,
      • Grakoui A.
      • Shoukry N.H.
      • Woollard D.J.
      • Han J.H.
      • Hanson H.L.
      • Ghrayeb J.
      • et al.
      HCV persistence and immune evasion in the absence of memory T cell help.
      ,
      • Meylan E.
      • Curran J.
      • Hofmann K.
      • Moradpour D.
      • Binder M.
      • Bartenschlager R.
      • et al.
      Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.
      ,
      • Penna A.
      • Pilli M.
      • Zerbini A.
      • Orlandini A.
      • Mezzadri S.
      • Sacchelli L.
      • et al.
      Dysfunction and Functional Restoration of HCV-Specific CD8 Responses in Chronic Hepatitis C Virus Infection.
      ,
      • Wedemeyer H.
      • He X.S.
      • Nascimbeni M.
      • Davis A.R.
      • Greenberg H.B.
      • Hoofnagle J.H.
      • et al.
      Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection.
      ] (Table 1). HCV exists as a population of closely related viral quasi-species that are sufficiently distinct immunologically to enable emerging HCV variants to elude the host humoral and cellular immune responses [
      • Bowen D.G.
      • Walker C.M.
      The origin of quasispecies: cause or consequence of chronic hepatitis C viral infection?.
      ]. The virus is also able to modulate the host antiviral cytokine response by inhibiting intracellular antiviral interferon signalling pathways [
      • Bowen D.G.
      • Walker C.M.
      Adaptive immune responses in acute and chronic hepatitis C virus infection.
      ,
      • Meylan E.
      • Curran J.
      • Hofmann K.
      • Moradpour D.
      • Binder M.
      • Bartenschlager R.
      • et al.
      Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.
      ].
      HCV also impairs the activation of dendritic cells (DC) although this has not been a consistent finding [
      • Auffermann-Gretzinger S.
      • Keeffe E.B.
      • Levy S.
      Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection.
      ,
      • Bain C.
      • Fatmi A.
      • Zoulim F.
      • Zarski J.P.
      • Trepo C.
      • Inchauspe G.
      Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.
      ,
      • Bowie A.
      • Kiss-Toth E.
      • Symons J.A.
      • Smith G.L.
      • Dower S.K.
      • O’Neill L.A.
      A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling.
      ,
      • Ciesek S.
      • Liermann H.
      • Hadem J.
      • Greten T.
      • Tillmann H.L.
      • Cornberg M.
      • et al.
      Impaired TRAIL-dependent cytotoxicity of CD1c-positive dendritic cells in chronic hepatitis C virus infection.
      ,
      • Kanto T.
      • Hayashi N.
      • Takehara T.
      • Tatsumi T.
      • Kuzushita N.
      • Ito A.
      • et al.
      Impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis C virus-infected individuals.
      ,
      • Sarobe P.
      • Lasarte J.J.
      • Casares N.
      • Lopez-Diaz de Cerio A.
      • Baixeras E.
      • Labarga P.
      • et al.
      Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins.
      ]. DC isolated from patients chronically infected with HCV are able to mature normally in response to HCV lipopeptides [
      • Chua B.Y.
      • Eriksson E.M.
      • Brown L.E.
      • Zeng W.
      • Gowans E.J.
      • Torresi J.
      • et al.
      A self-adjuvanting lipopeptide-based vaccine candidate for the treatment of hepatitis C virus infection.
      ] and DC from chimpanzees chronically infected with HCV are not functionally impaired [
      • Larsson M.
      • Babcock E.
      • Grakoui A.
      • Shoukry N.
      • Lauer G.
      • Rice C.
      • et al.
      Lack of phenotypic and functional impairment in dendritic cells from chimpanzees chronically infected with hepatitis C virus.
      ]. In contrast, both CD8+ and CD4+ T cell responses are impaired by HCV. Hepatitis C virus-specific CD8+ T cells from patients with chronic hepatitis C have impaired effector function [
      • Wedemeyer H.
      • He X.S.
      • Nascimbeni M.
      • Davis A.R.
      • Greenberg H.B.
      • Hoofnagle J.H.
      • et al.
      Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection.
      ]. In addition, HCV upregulates the expression of PD-1 on peripheral and intrahepatic CD8+ T cells resulting in T-cell exhaustion [
      • Nakamoto N.
      • Kaplan D.E.
      • Coleclough J.
      • Li Y.
      • Valiga M.E.
      • Kaminski M.
      • et al.
      Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization.
      ,
      • Penna A.
      • Pilli M.
      • Zerbini A.
      • Orlandini A.
      • Mezzadri S.
      • Sacchelli L.
      • et al.
      Dysfunction and Functional Restoration of HCV-Specific CD8 Responses in Chronic Hepatitis C Virus Infection.
      ,
      • Radziewicz H.
      • Hanson H.L.
      • Ahmed R.
      • Grakoui A.
      Unraveling the role of PD-1/PD-L interactions in persistent hepatotropic infections: potential for therapeutic application?.
      ] while blockade of PD-1 is associated with a functional restoration of CD8+ T cell function in HCV [
      • Nakamoto N.
      • Kaplan D.E.
      • Coleclough J.
      • Li Y.
      • Valiga M.E.
      • Kaminski M.
      • et al.
      Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization.
      ]. Differential expression of several other co-stimulatory and co-inhibitory receptors on virus-specific CD8+ T cells may also further influence T cell responses. Examples include CTL-A4 [
      • Nakamoto N.
      • Kaplan D.E.
      • Coleclough J.
      • Li Y.
      • Valiga M.E.
      • Kaminski M.
      • et al.
      Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization.
      ] 2B4 (CD244) [
      • Schlaphoff V.
      • Jaroszewicz J.
      • Pothakamuri S.V.
      • Grabowski J.
      • Stegmann K.A.
      • Manns M.P.
      • et al.
      Expression of 2B4 (CD244) and its effect on virus-specific CD8+ T cells – another important costimulatory molecule for the control of viral hepatitis.
      ] or CD86 [
      • adziewicz H.
      • Ibegbu C.C.
      • Hon H.
      • Bedard N.
      • Bruneau J.
      • Workowski K.A.
      • et al.
      Transient CD86 Expression on Hepatitis C Virus-Specific CD8+ T Cells in Acute Infection Is Linked to Sufficient IL-2 Signaling.
      ]. These may all have important implications for the design of an effective therapeutic HCV vaccine as the primary vaccine recipients will be patients with chronic infection in whom significant abnormalities of T cell function already exist.
      Further mechanisms to evade the host immune response include the selection of escape variants that carry mutations in key CD8+ T cell epitopes [
      • Bowen D.G.
      • Walker C.M.
      Mutational escape from CD8+ T cell immunity: HCV evolution, from chimpanzees to man.
      ,
      • Neumann-Haefelin C.
      • Timm J.
      • Schmidt J.
      • Kersting N.
      • Fitzmaurice K.
      • Oniangue-Ndza C.
      • et al.
      Protective effect of human leukocyte antigen B27 in hepatitis C virus infection requires the presence of a genotype-specific immunodominant CD8+ T-cell epitope.
      ,
      • Urbani S.
      • Amadei B.
      • Cariani E.
      • Fisicaro P.
      • Orlandini A.
      • Missale G.
      • et al.
      The Impairment of CD8 Responses Limits the Selection of Escape Mutations in Acute Hepatitis C Virus Infection.
      ] or through the restriction of T-cell receptor repertoires [
      • Meyer-Olson D.
      • Shoukry N.H.
      • Brady K.W.
      • Kim H.
      • Olson D.P.
      • Hartman K.
      • et al.
      Limited T Cell Receptor Diversity of HCV-specific T Cell Responses Is Associated with CTL Escape.
      ] thereby impairing viral specific CD8 responses.
      In addition to escaping cellular immune responses HCV has also evolved several mechanisms to evade humoral responses. These include the association of HCV with high-density lipoprotein [
      • Dreux M.
      • Pietschmann T.
      • Granier C.
      • Voisset C.
      • Ricard-Blum S.
      • Mangeot P.-E.
      • et al.
      High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor B1.
      ,
      • Voisset C.
      • Op de Beeck A.
      • Horellou P.
      • Dreux M.
      • Gustot T.
      • Duverlie G.
      • et al.
      High-density lipoproteins reduce the neutralizing effect of hepatitis C virus (HCV)-infected patient antibodies by promoting HCV entry.
      ] leading to the interference of binding of neutralising antibody to HCV. The HDL mediated interference of neutralisation results from a complex interplay with the cell surface protein Scavenger Receptor-B1 (SR-B1) and enhanced viral entry into hepatocytes [
      • Bartosch 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.
      ]. The viral envelope is also heavily glycosylated and the presence of these glycans protects the virus against the development and binding of neutralising antibodies [
      • Helle F.
      • Goffard A.
      • Morel V.
      • Duverlie G.
      • McKeating J.
      • Keck Z.Y.
      • et al.
      The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein.
      ,
      • Liu M.
      • Chen H.
      • Luo F.
      • Li P.
      • Pan Q.
      • Xia B.
      • et al.
      Deletion of N-glycosylation sites of hepatitis C virus envelope protein E1 enhances specific cellular and humoral immune responses.
      ]. Finally, it has also been reported that some sequences in the C-terminal region of HVR1, (amino acids 434–446) may interfere with protective neutralizing antibody responses, providing the virus with another mechanism to evade protective antibody responses [
      • Zhang P.
      • Zhong L.
      • Struble E.B.
      • Watanabe H.
      • Kachkob A.
      • Mihalikb K.
      • et al.
      Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity.
      ].
      Finally, the ability to clear hepatitis C infection spontaneously and following antiviral treatment may be genetically predetermined, as shown by the high rate of spontaneous clearance of acute hepatitis C infection and the high sustained virological response rates following treatment with pegylated interferon plus ribavirin in association with specific polymorphisms of the IL28B gene [
      • Ge D.
      • Fellay J.
      • Thompson A.J.
      • Simon J.S.
      • Shianna K.V.
      • Urban T.J.
      • et al.
      Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance.
      ,
      • Rauch A.
      • Kutalik Z.
      • Descombes P.
      • Cai T.
      • Di Iulio J.
      • Mueller T.
      • et al.
      Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.
      ,
      • Suppiah V.
      • Moldovan M.
      • Ahlenstiel G.
      • Berg T.
      • Weltman M.
      • Abate M.L.
      • et al.
      IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy.
      ,
      • Thomas D.L.
      • Thio C.L.
      • Martin M.P.
      • Qi Y.
      • Ge D.
      • O’Huigin C.
      • et al.
      Genetic variation in IL28B and spontaneous clearance of hepatitis C virus.
      ]. The presence of such genetically predetermined antiviral responses may also have a significant impact on the host’s ability to develop protective immune responses following vaccination, adding yet another layer of complexity to the design of an effective vaccine for HCV.

      HCV vaccines in clinical trials

      Preventive HCV vaccines

      Recombinant proteins

      Recombinant envelope glycoprotein’s were amongst the earliest preventive vaccine candidates for HCV but relatively few have entered into clinical trials. One such vaccine candidate includes the envelope gpE1/gpE2 proteins in an oil water adjuvant MF59 together with a CpG oligonucleotide (Chiron Corp) [
      • Houghton M.
      • Abrignani S.
      Prospects for a vaccine against the hepatitis C virus.
      ,
      • Vajdy M.
      • Selby M.
      • Medina-Selby A.
      • Coit D.
      • Hall J.
      • Tandeske L.
      • et al.
      Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses.
      ]. This vaccine has entered into a Phase I randomized, observer-blinded, placebo-controlled study and was registered with clinicaltrials.gov on July 12, 2007. This trial will evaluate the safety, tolerability and immunogenicity of the HCV E1E2/MF59 vaccine in healthy HCV-negative adults. The study has been completed but not reported to date (Table 2).
      Table 2Summary clinical trials with preventive and therapeutic vaccines for HCV.
      Prophylactic and therapeutic trials registered with clinicaltrials.gov.
      In another approach a yeast derived recombinant HCV core protein adjuvanted with ISCOMATRIX has been studied in a Phase I placebo controlled, dose escalation clinical study of the safety of the vaccine in 30 human participants. Antibody responses were detected in all but one of the participants. In contrast, CD8+ T cell responses were only detected in two of the thirty participants and T cell cytokines were detected in 7 of the 8 participants in the highest dose group [
      • Drane D.
      • Maraskovsky E.
      • Gibson R.
      • Mitchell S.
      • Barnden M.
      • Moskwa A.
      • et al.
      Priming of CD4+ and CD8+ T cell responses using a HCV core ISCOMATRIXTM vaccine.
      ].

      Therapeutic HCV vaccines

      Approaches that aim to increase HCV specific cell-mediated responses may improve the likelihood of achieving a sustained virological response and therapeutic vaccines may provide a means of achieving this goal. The concepts underlying therapeutic vaccines can be divided into three major strategies; (i) combining a therapeutic vaccine with antiviral therapies to enhancing anti-HCV immunity with the major aim of preventing viral relapse after stopping antiviral therapy, (ii) treating first with a therapeutic vaccine to induce HCV-specific immune responses followed by antiviral treatment in order to maximise early viral suppression and thereby increase sustained virological response rates, (iii) using therapeutic vaccination to produce partial control of HCV infection without inducing HCV clearance. Several therapeutic vaccine strategies have been explored and although these have provided promising results to date they have had limited success in clearing infection. Consequently, studies to investigate their role as an adjunct to pegylated interferon/ribavirin therapy have now commenced.

      Synthetic peptide vaccines

      IC41 (Intercell)

      The IC41 vaccine (Intercell AG, Vienna, Austria) contains five synthetic peptides encoding for four HCV specific HLA-A2 restricted CTL epitopes (core35–44 and 132–140, NS3 1073–1081, NS4 1764–1772) and three highly promiscuous CD4+ T cell epitopes (core23–44, NS3 1248–1261, NS4 1767–1786) that have been adjuvanted with poly-l-arginine to augment Th1/Tc1 (IFN-gamma) responses (Fig. 1). The sequences contained within these epitopes are highly conserved in the most prevalent HCV genotypes 1a, 1b, and 2.
      The immunogenicity of IC41 with or without poly-l-arginine was initially investigated in a randomized, placebo controlled trial [
      • Firbas C.
      • Bernd J.
      • Tauber E.
      • Buerger V.
      • Jelovcan S.
      • Lingnau K.
      • et al.
      Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.
      ]. Although half of the recipients experienced local injection site reactions the vaccine was well tolerated. A dose dependent proliferative CD4+ and CD8+ T cell response was induced in the majority of vaccine recipients. Furthermore, the inclusion of poly-l-arginine was shown to be important for the production of functional interferon-γ secreting T cells [
      • Firbas C.
      • Bernd J.
      • Tauber E.
      • Buerger V.
      • Jelovcan S.
      • Lingnau K.
      • et al.
      Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.
      ].
      A subsequent randomized double-blind phase II study was performed in sixty HLA-A2-positive chronic HCV patients who had either relapsed or failed to respond to previous pegylated interferon/ribavirin therapy [
      • Klade C.S.
      • Wedemeyer H.
      • Berg T.
      • Hinrichsen H.
      • Cholewinska G.
      • Zeuzem S.
      • et al.
      Therapeutic vaccination of chronic hepatitis C nonresponder patients with the peptide vaccine IC41.
      ]. The vaccine was well tolerated with the most common adverse events in the vaccine recipients including local reactions and influenza-like illness. Over two thirds of patients developed HCV-specific Th1/Tc1 responses and one third developed sustained T cell responses lasting up to six months after the last vaccination. However, responses were generally weak with viremia persisting in all recipients and only one patient experiencing a one-log10 reduction in viral load (Table 2)[
      • Klade C.S.
      • Wedemeyer H.
      • Berg T.
      • Hinrichsen H.
      • Cholewinska G.
      • Zeuzem S.
      • et al.
      Therapeutic vaccination of chronic hepatitis C nonresponder patients with the peptide vaccine IC41.
      ].
      Based on the premise that IC41 is able to induce HCV specific T cell responses, the role of IC41 as an adjunctive add-on immunotherapy to treatment with pegylated interferon/ribavirin has also recently been investigated in a phase II trial [
      • Wedemeyer H.
      • Schuller E.
      • Schlaphoff V.
      • Stauber R.E.
      • Wiegand J.
      • Schiefke I.
      • et al.
      Therapeutic vaccine IC41 as late add-on to standard treatment in patients with chronic hepatitis C.
      ]. Thirty-five HLA A2 positive patients infected with HCV genotype 1 were given six doses of IC41 from weeks 24 to 48 of pegylated interferon/ribavirin treatment and followed for a further six months. Vaccination did not decrease the virological relapse rate although those who developed sustained virological responses also developed strong HCV-specific responses (Table 2) [
      • Wedemeyer H.
      • Schuller E.
      • Schlaphoff V.
      • Stauber R.E.
      • Wiegand J.
      • Schiefke I.
      • et al.
      Therapeutic vaccine IC41 as late add-on to standard treatment in patients with chronic hepatitis C.
      ].
      An optimized vaccine schedule was subsequently tested in healthy volunteers. Of note, bi-weekly intradermal injections of IC41 induced much stronger T cell responses than the initial monthly subcutaneous vaccination [

      Firbas C, Boehm T, Buerger V, Schuller E, Sabarth N, Jilma B, et al. Immunogenicity and safety of different injection routes and schedules of IC41, a Hepatitis C virus (HCV) peptide vaccine. Vaccine 2010.

      ]. This improved vaccine schedule was tested in 50 patients with chronic hepatitis C and resulted in a significant decline in viral load after 4 months of biweekly therapeutic vaccination [
      • Klade C.S.
      • von Gabain A.
      • Manns M.
      Significant continuous viral load decline in treatment-naive HCV genotype 1patients after therapeutic peptide vaccination with IC41.
      ]. This study provided proof of concept that therapeutic vaccination aimed at inducing HCV specific T cell responses is able contribute to the control of HCV infection.
      Recently, a group of investigators from Japan reported phase I dose-escalation study to assess the safety and immune responses to the HLA-A2-restricted HCV core peptide YLLPRRGPRL in 25 HCV-positive patients [
      • Yutani S.
      • Komatsu N.
      • Shichijo S.
      • Yoshida K.
      • Takedatsu H.
      • Itou M.
      • et al.
      Phase I clinical study of a peptide vaccination for hepatitis C virus-infected patients with different human leukocyte antigen-class I-A alleles.
      ]. The vaccine was well tolerated in all subjects and produced HCV core-specific CTL responses in peripheral blood mononuclear cells from 15 of 25 patients. Phase II studies to determine vaccine efficacy are expected to commence in the near future.

      Virosome-based HCV vaccines

      Pevion Biotech have announced the start of phase I clinical testing of its virosome-based hepatitis C virus (HCV) vaccine in December 2006. The vaccine is based on a combination of the PeviPRO and PeviTER platforms that utilize synthetic HCV peptide antigens. A Phase I single-blinded, randomised, placebo controlled, dose escalating study of one virosome formulated CD4 and two virosomes formulated CD8 HCV vaccine components (PEV2A and PEV2B) administered to healthy adult volunteers have been registered with clinicaltrials.gov in March 2007 (Table 2). However, to date the results of this trial have not yet been published.

      Tarmogens: globeImmune GI-5005

      Tarmogens are a novel development in immunotherapeutic vaccines that consist of whole heat-killed recombinant Saccharomyces cerevisiae yeast that have been genetically modified to express one or more protein targets, including viral proteins. Tarmogens are avidly taken up by dendritic cells and stimulate both innate and specific cellular immune responses [
      • Bernstein M.B.
      • Chakraborty M.
      • Wansley E.K.
      • Guo Z.
      • Franzusoff A.
      • Mostbock S.
      • et al.
      Recombinant Saccharomyces cerevisiae (yeast-CEA) as a potent activator of murine dendritic cells.
      ]. This technology has now been applied to HCV with the development of a vaccine candidate (GI-5005a) that encodes a core-NS3 fusion [
      • Haller A.A.
      • Lauer G.M.
      • King T.H.
      • Kemmler C.
      • Fiolkoski V.
      • Lu Y.
      • et al.
      Whole recombinant yeast-based immunotherapy induces potent T cell responses targeting HCV NS3 and Core proteins.
      ](Fig. 1). This vaccine induces potent HCV NS3 and core specific T cell responses in vaccinated mice [
      • Haller A.A.
      • Lauer G.M.
      • King T.H.
      • Kemmler C.
      • Fiolkoski V.
      • Lu Y.
      • et al.
      Whole recombinant yeast-based immunotherapy induces potent T cell responses targeting HCV NS3 and Core proteins.
      ].
      A Phase 1b double-blind, placebo-controlled, dose-escalation therapeutic trial study of GI-5005-01 evaluated the subcutaneous administration of 7 doses of GI-5005 as a monotherapy. There were no dose limiting serious adverse events (SAEs) reported. Patients receiving GI-5005 had viral load reductions of up to 1.4 log10 while subjects in the placebo group had HCV RNA reductions <0.75 log10. Normalisation of the ALT was observed in up to 50% of patients receiving the highest vaccine dose compared to only 11% in the lowest dosing group and 0% in the placebo group. HCV specific CD8+ T cell responses were also observed in GI-5005 treated subjects, but not placebo subjects [
      • Habersetzer F.
      • Baumert T.F.
      • Stoll-Keller F.
      GI-5005, a yeast vector vaccine expressing an NS3-core fusion protein for chronic HCV infection.
      ,
      • Schiff E.R.
      • Everson G.T.
      • Tsai N.
      • Bzowej N.H.
      • Gish R.G.
      • McHutchison J.G.
      • et al.
      HCV-specific cellular immunity, RNA reductions, and normalization of ALT in chronic HCV subjects after treatment with GI-5005, a yeast-based immunotherapy targeting NS3 and core: a randomized, double-blind, placebo controlled phase 1b study.
      ].
      A Phase 2 trial comparing the virological response rates of pegylated interferon/ribavirin (SOC) with and without G1-5005a in patients infected with HCV genotype 1 has commenced. Patients in the triple therapy arms were up to 12% more likely to achieve an early virological response (EVR) than those receiving SOC alone [
      • Lawitz E.
      • Jacobson I.
      • McHutchinson J.
      • Lee W.
      • Pockros P.
      • Shiffman M.
      • et al.
      GI-5005 Immunotherapy plus PEG-IFN/ribavirin versus PEG-IFN/ribavirin in genotype 1 chronic HCV subjects; preliminary phase 2 EVR analyses.
      ]. .A subsequent Phase 2b study in genotype 1 interferon-naïve patients GI-5005 administered with SOC increased the end of treatment response to 74% from 59% and SVR from 58% from 48% compared with SOC alone [
      • Jacobson I.M.
      • McHutchison J.G.
      • Boyer T.D.
      • Schiff E.R.
      • Everson G.T.
      • Pockros P.J.
      • et al.
      GI-5005 therapeutic vaccine plus PEG-IFN/ribavirin significantly improve virological response and ALT normalization at end-of-treatment and improves SVR24 compared to PEG-IFN/RIBAVIRIN in genotype 1 chronic HCV patients.
      ,
      • McHutchison J.G.
      • Jacobson I.M.
      • Boyer T.D.
      • Schiff E.R.
      • Everson G.T.
      • Lee W.M.
      • et al.
      GI-5005 therapeutic vaccine plus pegIFN/ ribavirin improves end of treatment response at 48 weeks versus pegIFN/ ribavirin in naive genotype 1 chronic HCV patients.
      ].

      Vaccination with HCV E1 protein (innogenetics/genImmune)

      In 2003, a pilot trial from Belgium was published suggesting that therapeutic vaccination of chronic hepatitis C patients with a 135aa C-terminally truncated recombinant form of the E1 protein, formulated on alum may slow down fibrosis progression although no changes in HCV RNA levels were seen in that trial [
      • Nevens F.
      • Roskams T.
      • Van Vlierberghe H.
      • Horsmans Y.
      • Sprengers D.
      • Elewaut A.
      • et al.
      A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C.
      ]. Subsequently, two placebo controlled multicentre trials were initiated in Europe exploring the effects of two different doses of HCV E1 on fibrosis progression. In the first study patients received 20 μg E1 and were biopsied after 15 months. The second trial (T2S-918 study) was a 3-year study in which 122 patients received 4 courses of 6 injections of 50 μg E1. In both trials, humoral and cellular immune responses against E1 were induced, however, the T2S-918 study failed to achieve its primary endpoint of an improvement in fibrosis scores [
      • Wedemeyer H.
      • Mazur W.
      • Nevens F.
      • Horsmans Y.
      • Adler M.
      • Blum H.
      • et al.
      Factors influencing progression of liver fibrosis in patients with chronic hepatitis C: results of the 3-year T2S-918-HCV study with HCVE1 therapeutic vaccine.
      ]. The programme was stopped in 2007.

      Vaccination HCV E1E2/MF59 (Chiron Corp)

      The HCV E1E2/MF59 vaccine has undergone further development in a phase Ib therapeutic trial in conjunction with pegylated interferon and ribavirin in patients who have previously not responded to standard therapy. The vaccine was shown to be safe and in those who developed a first phase antiviral effect the addition of the vaccine enhanced the second phase viral clearance [
      • Colombatto P.
      • Brunetto M.R.
      • Maina A.M.
      • Romagnoli V.
      • Craxı A.
      • Almasio P.
      • et al.
      The candidate HCV E1E2MF59 vaccine is safe in chronic hepatitis C patients and accelerates the second phase viral decline upon primary response to pegylated interferon-2A/ribavirin therapy.
      ]. This further highlights the potential utility of protein-based vaccines as adjunctive therapeutic agents.

      Modified vaccinia Ankara virus (MVA)-based HCV vaccines: TG4040

      TG4040 is a recombinant poly-antigenic T cell vaccine based on a modified vaccinia Ankara virus (MVA) that encodes for the HCV NS3, NS4, and NS5B proteins [
      • Fournillier A.
      • Gerossier E.
      • Evlashev A.
      • Schmitt D.
      • Simon B.
      • Chatel L.
      • et al.
      An accelerated vaccine schedule with a poly-antigenic hepatitis C virus MVA-based candidate vaccine induces potent, long lasting and in vivo cross-reactive T cell responses.
      ]. The safety and biological activity of TG4040 in 15 treatment-naïve HCV subjects have been evaluated in a phase I open-label, multicentre, dose escalation study (Table 2)[
      • Honnet G.
      • Veron L.
      • Olivier D.
      • Grellier B.
      • Marie-Bastien B.
      • Bonfils E.
      • et al.
      Phase 1 clinical trial with a novel HCV therapeutic vaccine TG4040: interim results of biomarker and immunomonitoring analyzes.
      ]. Six of 15 patients received 3 weekly injections of the vaccine while the remaining 9 patients received a 4th injection at 6 months. HCV-specific T cell responses were detected in all patients as early as one week after the first vaccination and were maintained during the 6-month follow-up. Vaccination reduced HCV viral loads by up to 1.5 log10 and the strongest vaccine specific T cell responses were observed in patients who achieved the greatest viral load reductions [
      • Honnet G.
      • Veron L.
      • Olivier D.
      • Grellier B.
      • Marie-Bastien B.
      • Bonfils E.
      • et al.
      Phase 1 clinical trial with a novel HCV therapeutic vaccine TG4040: interim results of biomarker and immunomonitoring analyzes.
      ].

      DNA based vaccines: ChronVac-C®, Tripep AB, Sweden

      Figure thumbnail fx4
      In a different approach, Alvarez-Lajonchere and coworkers vaccinated HCV-chronically infected individuals in a Phase I study with a therapeutic vaccine (CIGB-230) containing a combination of a DNA plasmid expressing HCV structural antigens and recombinant HCV core protein. The individuals, all non-responders to previous interferon plus ribavirin treatment, received 6 doses of vaccine by intramuscular injection at 4-week intervals. Neutralizing antibody and HCV core specific T cell responses developed in the majority of patients and although viremia persisted, almost half of the vaccinated individuals developed an improvement in liver histology with a reduction in fibrosis [
      • Alvarez-Lajonchere L.
      • Shoukry N.H.
      • Gra B.
      • Amador-Canizares Y.
      • Helle F.
      • Bedard N.
      • et al.
      Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial.
      ]. The CIGB-230 vaccine, therefore, may hold promise of a potential therapeutic vaccine for patients chronically infected with HCV.

      Preclinical vaccine strategies

      Recombinant adenoviral HCV vaccines

      Recombinant adenoviruses expressing structural and non-structural proteins of HCV have proven to be valuable tools to help define the hierarchy of HCV specific CD8+ T cell responses [
      • Bharadwaj M.
      • Thammanichanond D.
      • Aitken C.K.
      • Moneer S.
      • Drummer H.E.
      • Lilly S.L.
      • et al.
      TCD8 response in diverse outcomes of recurrent exposure to hepatitis C virus.
      ,
      • Thammanichanond D.
      • Moneer S.
      • Yotnda P.
      • Aitken C.
      • Earnest-Silveira L.
      • Jackson D.
      • et al.
      Fiber-modified recombinant adenoviral constructs encoding hepatitis C virus proteins induce potent HCV-specific T cell response.
      ](Fig. 1). Unlike synthetic peptides, which are able to deliver a limited number of epitopes adenoviral vectors contain complete viral genes, thereby delivering intact HCV proteins and are not limited by HLA class restriction. Adenoviral vectors encoding HCV core and NS3 proteins are able to stimulate protective HCV specific cellular immune responses [
      • Arribillaga L.
      • de Cerio A.L.
      • Sarobe P.
      • Casares N.
      • Gorraiz M.
      • Vales A.
      • et al.
      Vaccination with an adenoviral vector encoding hepatitis C virus (HCV) NS3 protein protects against infection with HCV-recombinant vaccinia virus.
      ,
      • Matsui M.
      • Moriya O.
      • Abdel-Aziz N.
      • Matsuura Y.
      • Miyamura T.
      • Akatsuka T.
      Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells transduced with replication-defective recombinant adenovirus.
      ] and when co-administered with an IL12 cytokine expression plasmid produces highly efficient cytotoxic CD8+ T cell responses [
      • Matsui M.
      • Moriya O.
      • Abdel-Aziz N.
      • Matsuura Y.
      • Miyamura T.
      • Akatsuka T.
      Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells transduced with replication-defective recombinant adenovirus.
      ]. These features make adenoviral-HCV vectors attractive T-cell based vaccine candidates.
      Recently, investigators have used an adenoviral serotype 6 vector to construct a recombinant adenoviral HCV vaccine expressing the viral NS3 to 5B proteins [
      • Capone S.
      • Meola A.
      • Ercole B.B.
      • Vitelli A.
      • Pezzanera M.
      • Ruggeri L.
      • et al.
      A novel adenovirus type 6 (Ad6)-based hepatitis C virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques.
      ,
      • Fattori E.
      • Zampaglione I.
      • Arcuri M.
      • Meola A.
      • Ercole B.B.
      • Cirillo A.
      • et al.
      Efficient immunization of rhesus macaques with an HCV candidate vaccine by heterologous priming-boosting with novel adenoviral vectors based on different serotypes.
      ]. This vaccine induced broad, cross reactive HCV specific CD8+ T cell responses in mice and rhesus macaques that are not suppressed by pre-existing adenovirus 5 immunity. In an extension of these initial studies, Folgori and coworkers investigated a prime boost strategy in five chimpanzees that were immunized with two doses, four weeks apart of a replication-deficient serotype 6 adenovirus encoding HCV NS3-NS5B. At week 25 the chimpanzees were vaccinated with a replication-deficient serotype 24 adenovirus encoding the same HCV antigens. This was followed by three intramuscular doses of a recombinant NS3-NS5B DNA vaccine before challenging the chimpanzees with a heterologous HCV inoculum. Potent HCV specific peripheral and intrahepatic CD8+ T cell responses were induced by vaccination and these were accompanied by the clearance of HCV in 4 of the 5 challenged chimpanzees. Vaccination also resulted in multi-specific T cell responses against several viral proteins, with the strongest responses directed against NS3 [
      • 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.
      ]. The success of these recombinant adenovirus vaccines provides promise for the development of effective therapeutic vaccines for HCV. The immunogenicity of the vaccine is now being tested further in healthy volunteers [
      • Barnes E.
      • Folgori A.
      • Aston S.
      • Smith K.
      • Brown A.
      • Capone S.
      • et al.
      Phase I trial of a highly immunogenic T-cell vaccine for hepatitis C virus based on novel adenoviral vectors from rare serotypes.
      ].

      Modified vaccinia Ankara virus (MVA)-based HCV vaccines

      Another approach to the development of HCV therapeutic vaccines has been the use of modified vaccinia Ankara (MVA) viruses engineered to encode HCV specific genes. One such vaccine encoding the HCV NS3-4-5B genes has been tested in HLA-class I transgenic mice and shown to produce strong long lasting cross-reactive HCV specific CD8+ and CD4+ T cells. These responses could be readily boosted by an additional dose of vaccine given after 6 months, suggesting that the vaccine is able to induce effective memory T cell responses [
      • Fournillier A.
      • Gerossier E.
      • Evlashev A.
      • Schmitt D.
      • Simon B.
      • Chatel L.
      • et al.
      An accelerated vaccine schedule with a poly-antigenic hepatitis C virus MVA-based candidate vaccine induces potent, long lasting and in vivo cross-reactive T cell responses.
      ].
      In a separate study chimpanzees were vaccinated with DNA plasmids encoding HCV core-E1-E2 and NS3 followed by booster immunizations with recombinant MVA encoding core-E1-E2 and NS3 genes [
      • Rollier C.S.
      • Paranhos-Baccala G.
      • Verschoor E.J.
      • Verstrepen B.E.
      • Drexhage J.A.
      • Fagrouch Z.
      • et al.
      Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity.
      ]. This DNA prime-MVA boost immunization induced strong Th1- and Th2-cytokine responses together with strong HCV specific CD8+ T cell responses. In addition, all animals achieved high HCV-specific antibody tires after the prime-boost combination, but not with the DNA prime alone. A subsequent challenge with a chimpanzee adapted heterologous virus demonstrated that the DNA prime-MVA boost was associated with the control of HCV viremia in the acute stage but unfortunately did not protect against chronic infection [
      • Rollier C.S.
      • Paranhos-Baccala G.
      • Verschoor E.J.
      • Verstrepen B.E.
      • Drexhage J.A.
      • Fagrouch Z.
      • et al.
      Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity.
      ]. This highlights the induction of T-cell responses alone may be insufficient for the prevention of chronic HCV persistence.

      HCV virus-like particles (VLP’s)

      A HCV VLP based vaccine would make it possible to deliver important neutralising antibody and core specific T cell epitopes in a single vaccine construct that will most closely resemble mature virions antigenically (Fig. 1). The development of an HCV VLP-based vaccine strategy is also supported by other examples of VLP based vaccines that have now been successfully licensed for persistent viral infections, such as hepatitis B virus [
      • Kao J.H.
      • Chen D.S.
      Global control of hepatitis B virus infection.
      ,
      • Papaevangelou G.
      • Dandolos E.
      • Roumeliotou-Karayannis A.
      • Richardson S.C.
      Immunogenicity of recombinant hepatitis B vaccine.
      ] and human papilloma virus [
      • Giannini S.
      • Hanon E.
      • Moris P.
      • Van Mechelen M.
      • Morel S.
      • Dessy F.
      • et al.
      Enhanced humoral and memory B cellular immunity using HPV16/18. L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only.
      ]. Although both vaccines induce cell-mediated immune responses the protective efficacy of these vaccines relies on their ability to induce long lasting neutralising antibody.
      Insect cell-derived HCV VLPs have produced encouraging results for HCV vaccine design [
      • Baumert T.F.
      • Ito S.
      • Wong D.T.
      • Liang T.J.
      Hepatitis C virus structural proteins assemble into virus like particles in insect cells.
      ,
      • Baumert T.F.
      • Vergalla J.
      • Satoi J.
      • Thomson M.
      • Lechmann M.
      • Herion D.
      • et al.
      Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate.
      ,
      • Murata K.
      • Lechmann M.
      • Qiao M.
      • Gunji T.
      • Alter H.J.
      • Liang T.J.
      Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection.
      ,
      • Steinmann D.
      • Barth H.
      • Gissler B.
      • Schurmann P.
      • Adah M.I.
      • Gerlach J.T.
      • et al.
      Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C.
      ]. However, insect cell-derived E2 antigens are not able to stimulate a protective antibody response in primates [
      • Choo Q.L.
      • Kuo G.
      • Ralston R.
      • Weiner A.
      • Chien D.
      • Van Nest G.
      • et al.
      Vaccination of chimpanzees against infection by the hepatitis C virus.
      ,
      • Rosa D.
      • Campagnoli S.
      • Moretto C.
      • Guenzi E.
      • Cousens L.
      • Chin M.
      • et al.
      A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.
      ] and methods to improve their immunogenicity will be necessary. HCV VLPs contain potentially neutralising epitopes that are present in the native protein sequence of both the E1 and E2 proteins [
      • Grollo L.
      • Torresi J.
      • Drummer H.
      • Zeng W.
      • Williamson N.
      • Jackson D.C.
      Cross-reactive epitopes of hepatitis C virus induce antibodies that capture virions and inhibit pseudo virus particle cell entry.
      ,
      • Law M.
      • Maruyama T.
      • Lewis J.
      • Giang E.
      • Tarr A.W.
      • Stamataki Z.
      • et al.
      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ,
      • Meunier J.C.
      • Russell R.S.
      • Goossens V.
      • Priem S.
      • Walter H.
      • Depla E.
      • et al.
      Isolation and Characterization of Broadly Neutralizing Human Monoclonal Antibodies to the E1 Glycoprotein of Hepatitis C Virus.
      ,
      • Torresi J.
      • Fischer A.
      • Grollo L.
      • Zeng W.
      • Drummer H.
      • Jackson D.C.
      Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T-helper epitopes.
      ,
      • Torresi J.
      • Stock O.M.
      • Fischer A.E.
      • Grollo L.
      • Drummer H.
      • Boo I.
      • et al.
      A self-adjuvanting multiepitope immunogen that induces a broadly cross-reactive antibody to hepatitis C virus.
      ]. An important feature of HCV VLPs is that cross protective neutralising epitopes will be present on the surface of the VLPs as part of AR3 [
      • Law M.
      • Maruyama T.
      • Lewis J.
      • Giang E.
      • Tarr A.W.
      • Stamataki Z.
      • et al.
      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ]. Immunisation with heterodimers of recombinant E1 and E2 proteins has been shown to protect chimpanzees against challenge with homologous virus [
      • Choo Q.L.
      • Kuo G.
      • Ralston R.
      • Weiner A.
      • Chien D.
      • Van Nest G.
      • et al.
      Vaccination of chimpanzees against infection by the hepatitis C virus.
      ]. However, it is unknown whether heterodimers of recombinant E1 and E2 will successfully reproduce important cross-neutralising regions like AR3. Unlike linear recombinant E1 and E2 proteins and synthetic peptides, HCV VLPs are the most likely vaccine candidate to faithfully reproduce this important antigenic region. Also, in both mice and non-human primates HCV VLPs are superior in immunogenicity compared to DNA or recombinant envelope protein vaccines [
      • Baumert T.F.
      • Vergalla J.
      • Satoi J.
      • Thomson M.
      • Lechmann M.
      • Herion D.
      • et al.
      Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate.
      ]. A HCV VLP vaccine based on a single genotype has been shown to induce cross-protective neutralisation of HCV [
      • Meunier J.C.
      • Engle R.E.
      • Faulk K.
      • Zhao M.
      • Bartosch B.
      • Alter H.
      • et al.
      Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1.
      ,
      • Murata K.
      • Lechmann M.
      • Qiao M.
      • Gunji T.
      • Alter H.J.
      • Liang T.J.
      Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection.
      ]. It may, therefore, be expected that a vaccine that includes VLPs of genotypes 1a, 1b, and 3a should induce broad cross protective immune responses.
      HCV VLPs provide a promising vaccine candidate because they induce humoral and cellular responses against HCV structural proteins [
      • Baumert T.F.
      • Vergalla J.
      • Satoi J.
      • Thomson M.
      • Lechmann M.
      • Herion D.
      • et al.
      Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate.
      ,
      • Murata K.
      • Lechmann M.
      • Qiao M.
      • Gunji T.
      • Alter H.J.
      • Liang T.J.
      Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection.
      ]; bind NAb against HCV [
      • Steinmann D.
      • Barth H.
      • Gissler B.
      • Schurmann P.
      • Adah M.I.
      • Gerlach J.T.
      • et al.
      Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C.
      ]; stimulate the maturation of human dendritic cells [
      • Barth H.
      • Ulsenheimer A.
      • Pape G.R.
      • Diepolder H.M.
      • Hoffman M.
      • Neumann-Haefelin C.
      • et al.
      Uptake and presentation of hepatitis C virus-like particles by human dendritic cells.
      ]; elicit protective CTL responses in mice against recombinant vaccinia viruses encoding structural proteins of HCV [
      • Barth H.
      • Ulsenheimer A.
      • Pape G.R.
      • Diepolder H.M.
      • Hoffman M.
      • Neumann-Haefelin C.
      • et al.
      Uptake and presentation of hepatitis C virus-like particles by human dendritic cells.
      ,
      • Murata K.
      • Lechmann M.
      • Qiao M.
      • Gunji T.
      • Alter H.J.
      • Liang T.J.
      Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection.
      ]; elicit protective CTL responses against HCV in chimpanzees [
      • Elmowalid G.A.
      • Qiao M.
      • Jeong S.-H.
      • Borg B.B.
      • Baumer T.F.
      • Sapp R.K.
      • et al.
      Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees.
      ] and have superior immunogenicity compared to recombinant proteins and DNA vaccines [
      • Jeong S.H.
      • Qiao M.
      • Nascimbeni M.
      • Hu Z.
      • Rehermann B.
      • Murthy K.
      • et al.
      Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates.
      ,
      • Lechmann M.
      • Murata K.
      • Satoi J.
      • Vergalla J.
      • Baumert T.F.
      • Liang T.J.
      Hepatitis C virus-like particles induce virus-specific humoral and cellular immune responses in mice.
      ,
      • Murata K.
      • Lechmann M.
      • Qiao M.
      • Gunji T.
      • Alter H.J.
      • Liang T.J.
      Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection.
      ]. With so many favourable immunological characteristics further development of a preventive HCV VLP based vaccine appears warranted.

      Recombinant proteins

      Recombinant envelope glycoproteins had shown early promise as vaccine candidates because of their ability to induce protective neutralising antibody responses in chimpanzees, although this was only directed against the homologous virus [
      • Choo Q.L.
      • Kuo G.
      • Ralston R.
      • Weiner A.
      • Chien D.
      • Van Nest G.
      • et al.
      Vaccination of chimpanzees against infection by the hepatitis C virus.
      ]. In addition, E1 and E2 glycoproteins expressed in insect, yeast, and bacteria are less effective at inducing protective antibody responses than glycoproteins produced in mammalian cells [
      • Rosa D.
      • Campagnoli S.
      • Moretto C.
      • Guenzi E.
      • Cousens L.
      • Chin M.
      • et al.
      A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.
      ]. Attempts to improve the immunogenicity of a HCV envelope gpE1/gpE2 vaccine have included the addition of an oil water adjuvant MF59 together with a CpG oligonucleotide (Chiron Corp). The vaccine prevented the development of chronic infection in chimpanzees after heterologous viral challenges [
      • Houghton M.
      • Abrignani S.
      Prospects for a vaccine against the hepatitis C virus.
      ,
      • Vajdy M.
      • Selby M.
      • Medina-Selby A.
      • Coit D.
      • Hall J.
      • Tandeske L.
      • et al.
      Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses.
      ].
      A recombinant core polyprotein vaccine produced in yeast formulated in ISCOMATRIX adjuvant [
      • Polakos N.K.
      • Drane D.
      • Cox J.
      • Ng P.
      • Selby M.J.
      • Chien D.
      • et al.
      Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine.
      ] has recently been tested in rhesus macaques and shown to induce strong long-lived CD4+ and CD8+ T-cell responses [
      • Polakos N.K.
      • Drane D.
      • Cox J.
      • Ng P.
      • Selby M.J.
      • Chien D.
      • et al.
      Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine.
      ]. A second vaccine consisting of a recombinant HCV core-NS3-NS4-NS5a-NS5b polyprotein adjuvanted in MF59 has also been shown to induce broad proliferative CD4+ and CD8+ T cell responses against the core and NS proteins in mice [
      • Vajdy M.
      • Selby M.
      • Medina-Selby A.
      • Coit D.
      • Hall J.
      • Tandeske L.
      • et al.
      Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses.
      ].
      In an attempt to improve the strength and breadth of antibody and cellular immune responses against HCV a recent study investigated a prime-boost vaccination strategy [
      • Lin Y.
      • Kwon T.
      • Polo J.
      • Zhu Y.F.
      • Coates S.
      • Crawford K.
      • et al.
      Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5.
      ]. Mice immunised with recombinant E1E2 glycoproteins adjuvanted with MF59 containing the CpG oligonucleotide 7909 or a recombinant NS3-4-5 polyprotein adjuvanted with ISCOMATRIX developed strong CD4+ T but not CD8+ T-cell responses. In contrast immunization of mice with defective chimeric Venezuelan equine encephalitis and Sindbis (VEE/SIN) viruses encoding HCV gpE1/gpE2 or NS3–NS4–NS5 produced strong CD8+ T-cell but low CD4+ T helper responses. However, by first priming with adjuvanted viral proteins followed by boosting with the chimeric alphavirus-HCV vaccines it was possible to induce strong CD8+ and CD4+ T cell responses against the respective viral proteins. First priming mice with MF59-CpG-adjuvanted E1E2 followed by a VEE/SIN-E1E2 boost induced strong cross-neutralising antibodies [
      • Lin Y.
      • Kwon T.
      • Polo J.
      • Zhu Y.F.
      • Coates S.
      • Crawford K.
      • et al.
      Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5.
      ]. These prime-boost vaccines may hold promise for the development of a preventive HCV vaccine.

      Synthetic peptide vaccines

      Studies using synthetic peptides have provided important insights into important protective epitopes. Both humoral and cellular immune responses to HCV have been produced with peptide vaccines [
      • 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.
      ,
      • Grollo L.
      • Torresi J.
      • Drummer H.
      • Zeng W.
      • Williamson N.
      • Jackson D.C.
      Cross-reactive epitopes of hepatitis C virus induce antibodies that capture virions and inhibit pseudo virus particle cell entry.
      ,
      • Khudyakov Yu E.
      • Khudyakova N.S.
      • Jue D.L.
      • Lambert S.B.
      • Fang S.
      • Fields H.A.
      Linear B-cell epitopes of the NS3-NS4-NS5 proteins of the hepatitis C virus as modeled with synthetic peptides.
      ,
      • Langhans B.
      • Braunschweiger I.
      • Schweitzer S.
      • Jung G.
      • Inchauspe G.
      • Sauerbruch T.
      • et al.
      Lipidation of T helper sequences from hepatitis C virus core significantly enhances T-cell activity in vitro.
      ,
      • Oseroff C.
      • Sette A.
      • Wentworth P.
      • Celis E.
      • Maewal A.
      • Dahlberg C.
      • et al.
      Pools of lipidated HTL-CTL constructs prime for multiple HBV and HCV CTL epitope responses.
      ,
      • Torresi J.
      • Fischer A.
      • Grollo L.
      • Zeng W.
      • Drummer H.
      • Jackson D.C.
      Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T-helper epitopes.
      ,
      • Torresi J.
      • Stock O.M.
      • Fischer A.E.
      • Grollo L.
      • Drummer H.
      • Boo I.
      • et al.
      A self-adjuvanting multiepitope immunogen that induces a broadly cross-reactive antibody to hepatitis C virus.
      ]. An approach aimed at delivering multiple neutralising epitopes involved the polymerisation of single vaccine peptides representing potentially cross-neutralising epitopes into synthetic polymer vaccines that also included a promiscuous T-helper epitope and a lipid moiety as an adjuvant [
      • Torresi J.
      • Stock O.M.
      • Fischer A.E.
      • Grollo L.
      • Drummer H.
      • Boo I.
      • et al.
      A self-adjuvanting multiepitope immunogen that induces a broadly cross-reactive antibody to hepatitis C virus.
      ]. This self-adjuvanting vaccine induced cross-neutralising antibodies in mice and although promising, the titres of neutralising antibody to individual viral epitopes were relatively low.
      Synthetic lipopeptides representing HCV specific MHC class I epitopes have also been shown to induce strong CD8+ T cell response in rodents, although this response is dependent on the presence of covalently linked helper-T cell epitopes [
      • Oseroff C.
      • Sette A.
      • Wentworth P.
      • Celis E.
      • Maewal A.
      • Dahlberg C.
      • et al.
      Pools of lipidated HTL-CTL constructs prime for multiple HBV and HCV CTL epitope responses.
      ]. The increased immunogenicity of lipopeptides containing both T helper and CD8+ epitopes may be related to their increased uptake by antigen presenting cells [
      • Zeng W.
      • Ghosh S.
      • Lau Y.F.
      • Brown L.E.
      • Jackson D.C.
      Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines.
      ]. However, peptide strategies based on delivering a limited number of epitopes are unlikely to be broadly cross protective for a virus like HCV.

      Conclusions

      HCV has a propensity to cause chronic infection, however, it is possible to develop robust cellular and humoral immune responses capable of resolving infection. An effective vaccine will need to reproduce such broad immune responses in order to ensure that viral clearance will occur. By better understanding the correlates of an effective immune response it will be possible to develop effective preventive and therapeutic vaccine strategies. However, HCV has evolved several mechanisms to evade the host immune response in order to sustain its own persistence. In addition to these well-developed viral escape adaptations, a vaccine that delivers relatively limited humoral and cellular immune responses could select neutralization escape variants or result in skewing T cell repertoires leading to viral escape, particularly following challenge with heterologous virus [
      • Cornberg M.
      • Chen A.T.
      • Wilkinson L.A.
      • Brehm M.A.
      • Kim S.K.
      • Calcagno C.
      • et al.
      Narrowed TCR repertoire and viral escape as a consequence of heterologous immunity.
      ].
      In addition to these concerns to date only a few vaccine candidates have progressed to phase I/II trials. Published data on both the efficacy and safety of these vaccines is limited. However, with several different vaccine approaches in various stages of development it appears inevitable that the most promising of these vaccines candidates will enter into clinical trials. For therapeutic vaccines novel approaches including combination therapy with pegylated interferon and ribavirin [
      • Jacobson I.M.
      • McHutchison J.G.
      • Boyer T.D.
      • Schiff E.R.
      • Everson G.T.
      • Pockros P.J.
      • et al.
      GI-5005 therapeutic vaccine plus PEG-IFN/ribavirin significantly improve virological response and ALT normalization at end-of-treatment and improves SVR24 compared to PEG-IFN/RIBAVIRIN in genotype 1 chronic HCV patients.
      ] or possibly with the newly developed DAAs [
      • Kwong A.D.
      • McNair L.
      • Jacobson I.
      • George S.
      Recent progress in the development of selected hepatitis C virus NS3.4A protease and NS5B polymerase inhibitors.
      ,
      • McHutchison J.G.
      • Everson G.T.
      • Gordon S.C.
      • Jacobson I.M.
      • Sulkowski M.
      • Kauffman R.
      • et al.
      Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection.
      ,
      • Thompson A.
      • Patel K.
      • Tillman H.
      • McHutchison J.G.
      Directly acting antivirals for the treatment of patients with hepatitis C infection: a clinical development update addressing key future challenges.
      ] may prove to be more effective approaches for the treatment of patients who fail to respond to currently available treatments. With the enormous global burden of disease posed by hepatitis C and the continuing transmission of HCV there is a genuine need for an effective vaccine for this disease. Success in developing effective vaccines for HCV will require a strongly committed effort by both research laboratories and industry partners alike.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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