April 14, 2013

Sex Hormones and HCV

Expert Review of Gastroenterology and Hepatology

An Unresolved Mystery

Radwa Y Mekky, Ahmed I Abdelaziz

Expert Rev Gastroenterol Hepatol. 2013;7(1):69-75.

Abstract and Introduction
Abstract

The biological differences between males and females advocate the ultimate need for gender-specific medicine. The variation in response to viral infection as well as therapy among different genders makes it very intriguing to reveal the responsible factors for causing this discrepancy. HCV is one of the most noxious infectious diseases, however the impact of gender on the response to HCV has received negligible attention in the literature. The controversial studies concerning the effect of gender on the outcome of interferon-based therapy urge a need to judge the gender discrepancy in host factors responsible for both interferon release and action. The main aim of this review is to disentangle the interplay between sex hormones and several viral and host factors responsible for viral clearance in an attempt to clarify the role of gender in modulating the response to HCV as well as interferon-based therapy.

Introduction

The numerous diversities among males and females make them respond variably to disease and therapy. This variation has stimulated an interest in the implementation of gender-specific medicine, wherein the genetic diversity between different genders is taken into consideration. Accordingly, the therapeutic dosage given to either males or females is adjusted in an attempt to reach the optimal therapeutic outcome with minimal adverse events.

Chronic hepatitis C infection is considered a major healthcare burden worldwide, with high prevalence in Africa and the Middle East, especially in Egypt.[1] The fact that HCV is characterized by high mutation rates makes it capable of escaping the host immunological response.[2] Consequently most HCV-infected patients suffer a chronic form of infection. Recently gender has emerged as a major factor affecting the innate response to HCV; the rate of spontaneous clearance of HCV was found to be higher among HCV-infected females when compared with male patients.[3–5] The exact factors responsible for this variability in the natural history of the disease are still not well known.

Gender has also been suspected to contribute to the variable response to standard HCV therapy (pegylated interferon (IFN) and ribavirin).[6] The goal of standard therapy of HCV infection is to achieve a sustained virologic response (SVR), which is defined as the absence of HCV RNA in the serum, 6 months after the end of treatment.[7] Unfortunately, the possible effects of gender on treatment response are still contested. On one hand, some recent studies have revealed that an SVR is more prominent among male patients.[8,9] On the other hand, female gender has been widely reported to be a good prognostic factor to IFN based therapy.[10–12] Alternatively, various studies have denied the association between gender and the outcome of therapy.[13–16] To help unravel the ambiguity surrounding this issue an important question should be investigated: could the actions of sex hormones on viral and host factors explain the disparate rates of viral clearance and response to therapy between males and females? Thus this review aims at analyzing the sex hormones' impact on viral and host factors that are important in viral clearance in an attempt to demystify the gender variation in self-limited infection to HCV and therapeutic response to IFN-based therapy.

Viral Behavior Among Different Genders

Although HCV infection is characterized by persistence, it is documented that 15–30% of individuals show spontaneous clearance of the virus.[17] Gender is one of the main factors that has been widely reported to influence the HCV clearance rate, where HCV-infected females have shown higher clearance rates when compared with their male counterparts.[3,4]

The disparity in self resolution of HCV infection among males and females suggests a role for sex hormones in influencing viral behavior. However, the impact of female sex hormones on HCV remains enigmatic; it is not known whether they hold a beneficial or detrimental role towards virological clearance. For example, 17β-estradiol was found to act directly on the virus itself by possessing an inhibitory effect on mature virion production in cultured Huh-7.5 cells transfected with viral replicon (Figure 1).[18] However, in the same study this inhibitory effect was not observed upon treatment with progesterone.[18] Moreover, the coumestans family of phytoestrogens, naturally occurring estrogen-like compounds derived from some plants and belonging to the flavonoids category of phytoestrogens, was reported as novel candidates for targeting viral replication by acting on HCV NS5B, a nonstructural viral protein essential for replication.[19] Pregnancy, a state of elevated estrogen and progesterone, was found to decrease the activity of chronic HCV. This effect was abolished after delivery.[20] In contrast to the aforementioned studies, Watashi et al. reported that estrogen receptor-α is functionally associated with HCV replication and its blockage with tamoxifen, a selective estrogen receptor modulator, may be a novel approach for targeting HCV infection (Figure 1).[21] The conflicting data concerning the influence of female sex hormones on HCV necessitate an in-depth look into their effects at various stages of the viral life cycle.

778968-fig1

Figure 1.

Sex hormone impact on the HCV life cycle. 1. HCV enters the host cells through cell surface receptors CD81, SR-B and LDLR. Tight junction proteins OCLN and CLDN are co-receptor molecules important for viral entry. 2. After viral entry, uncoating of the virus and cytoplasmic release takes place. 3. Translation occurs on the rough endoplamic reticulum. 4. Association of produced viral proteins with the ER results in production of a membranous web upon which replication takes place. 5. Assembly of the viral proteins occurs. 6. Mature virions exit host cell via exocytosis. Testosterone was found to enhance HCV entry by positively (+) regulating both SR-B as well as CLDN. Oppositely, estrogen decreased (-) the expression of SR-B. Estrogen receptor a was found to be a crucial host factor used by the virus to promote its own replication. However, estrogen was reported to inhibit (-) the release of mature virions from infected Huh-7 cell lines.

CLDN: Claudin; ER: Endoplasmic reticulum; LDLR: Low-density lipoprotein receptors; OCLN: Occludin; SR-B: Scavenger receptors class B.

The impact of testosterone on HCV behavior represents another piece of the puzzle. Surprisingly, to date, the effect of testosterone on HCV replication has not been studied. However, it was reported that testosterone enhanced the expression of scavenger receptors, which are critical for viral entry, on both HepG2 cell lines and human monocyte-derived macrophages in a dose-dependent manner.[22,23] Interestingly, estrogen suppressed the expression of hepatic scavenger receptors (Figure 1).[24] In the same context, testosterone enhanced the expression of the tight junction protein claudin, which also plays a central role in HCV cellular entry (Figure 1).[25,26] These aforementioned studies might elucidate the role of testosterone in enhancing viral entry to host cells thus highlighting a gender bias toward HCV response.

Discrepancy in IFN Release Among Different Genders

Variation in IFN induction among males and females may help to explain the differences in immune response to HCV. Toll-like receptor (TLR) 7 is a receptor which recognizes HCV RNA. The activation of this receptor leads to IFN-α induction, which consequently activates interferon-stimulated genes (ISGs) that allow viral clearance (Figure 2).[27,28] TLR7 showed a higher sensitivity to TLR7 agonists in healthy females when compared with healthy males, which consequently led to enhanced production of IFN-α.[29]

778968-fig2

Figure 2.

Impact of sex hormones on pre- and post-IFN release. (A) HCV attaches to cell surface receptors, and after uncoating the virus, binds to endosomal TLR7 which consequently leads to induction of IFN-α, -β, -λ and -γ via activation of several transcription factors. Among these transcription factors are IRF7 and NF-κB. Sex hormones regulate IFN release in several ways. For example, estrogen and progesterone were found to enhance expression of TLR7. However, progesterone was found to abrogate the expression of IRF7. Estrogen was also found to downregulate the expression of NF-κB. (B) After IFN is released they bind to their receptors, which activate the JAK/STAT pathway leading to production of ISGs. The JAK/STAT pathway is negatively regulated by certain transcription factors such as SOCs. Estrogen was found to attenuate the JAK/STAT pathway by enhancing the expression of SOCs and downregulating ISG expression.

+: increasing the expression; -: decreasing the expression; IFNAR: Interferon receptor; IRF7: Interferon regulatory factor 7; ISG: Interferon-stimulated gene; SOCS: Suppressors of cytokine release; TLR: Toll-like receptor.

Female sex hormones were reported to affect certain host factors that are important for IFN release. For example, estrogen was reported to suppress the maturation of dendritic cells, which are the main producers of IFN.[30] Estrogen was also found to negatively regulate NFκB, a transcription factor that is fundamental for production of IFN (Figure 2).[31] Progesterone was also reported to impair the antiviral immune response by decreasing the response of plasmacytoid dendritic cells through abrogating the activation of interferon regulatory factor 7, an important transcription factor for IFN production (Figure 2A).[32] However conflicting data by Meier et al. found a significant correlation between an increased production of IFN-α in healthy female plasmacytoid dendritic cells after being triggered with TLR7/8 ligands and plasma levels of progesterone (Figure 2A).[33]

The impact of testosterone on IFN release has received negligible attention in the literature. However, the discrepant rates of self-limited infection in males and females may be explained by the contrasting effects of estrogen and testosterone on the expression of the TLR family; where testosterone did not alter the expression of TLR on the surface of peripheral blood mononuclear cells (PBMCs) from healthy volunteers, while estrogen enhanced its expression (Figure 2A).[34]

Regulation of IFN Response Among Different Genders

Gender variation in response to endogenous and exogenous IFN might help decode the differences between the sexes with respect to response to standard IFN therapy. After IFN is released it binds to its receptor, which consequently activates the JAK/STAT pathway (Figure 2B). A line of evidence exists that posits that genetic variation among different genders may be responsible for the variability in IFN activity. Polymorphism in the promoter region of IL-10 was shown to induce high amounts of IL-10 in HCV-infected females.[35] This cytokine was reported to be associated with IFN resistance through inhibition of the expression of IFN-α inducible genes by prevention of tyrosine phosphorylation of STAT and upregulation of suppressor of cytokine 3 (Figure 2B).[36] It was also recently reported that females carrying the minor allele of single nucleotide polymorphism (rs8099917) in IL-28B, one of the strong predictors of response to IFN-based therapy, have the highest probability of null response to IFN therapy when compared with males. The same study found that males carrying the major allele have the lowest probability of null response.[37] Another recent study reported that females carrying the favorable genotype of IL-28B polymorphism showed higher chances of IFN response when compared with males carrying either the favorable or unfavorable genotype of IL-28B.[38] Another explanation for the enhanced IFN action in HCV-infected females is the higher rate of ISGs induction in PBMCs after IFN stimulation compared with their male counterparts.[39]

Young age and low BMI are two factors rendering premenopausal women better responders to IFN therapy.[40,41] Nevertheless, it is considered inevitable that female sex hormones play a major role in enhancing IFN action.[42,43] The exact mechanisms by which female sex hormones affect IFN response are not well characterized, however suppression of hepatic iron load by estrogen could offer an explanation for the decreased rate of IFN resistance among female HCV patients.[44,45] It was also found that estrogen represses the elevated levels of IL-6,[46] a cytokine found to be elevated during menopause and to play a major role in IFN resistance.[43,47] Among the beneficial effects of estrogen is the attenuation of IL-8 release by monocytes, which has been reported to inhibit IFN-induced antiviral response.[48,49] Recently, a cohort study suggested a potential synergism between female sex hormone and IL-28B polymorphism; where women carrying favorable CC genotype for rs12979860 have the greatest likelihood to resolve HCV infection.[38] Although several studies have underlined the beneficial role of estrogen in promoting SVR among females, others have still reached opposing results. For example, estrogen was also proven to attenuate the JAK/STAT pathway by downregulating ISGs in HCV-infected PBMCs and upregulating SOCS expression, a negative regulator of the JAK/STAT pathway, in hepatic cells both in vivo and in vitro (Figure 2B).[39,50] This effect was reversed by progesterone in ovariectomized hormonal treated mice.[51] This latter study highlights the role of estrogen–progesterone interaction on immune modulation. These contradicting studies urge the need to study the cross talk between estrogen and progesterone in modulating the response to HCV infection.

To underscore the sexual dimorphism in IFN action, it's worth mentioning the impact of testosterone on various factors affecting IFN resistance. In vitro stimulation of female monocytes with testosterone enhanced the production of IL-1b, a cytokine negatively associated with IFN outcome.[52,53] Additionally, a recent correlation was drawn between an undesired outcome of IFN treatment and a low baseline of adiponectin.[54] It was suggested that testosterone plays a role in decreasing adiponectin levels in males after puberty, which may propose an indirect role for male sex hormones in modulating the response to IFN-based therapy.[55]

Conclusion

The disparate rates of viral clearance and response to therapy between HCV-infected males and females is still considered an unresolved mystery. Thus in an attempt to unravel this ambiguity, the authors highlight a plausible impact of sex hormones on viral behavior and on host factors affecting both IFN release and action. The scarce scientific studies addressing this issue hampered the comprehensive understanding of gender bias to this noxious viral infection. Consequently, the implementation of gender-specific medicine on HCV-infected patients is challenged. Thus it is considered particularly important to deeply investigate the precise role sex hormones on each specific step of the HCV life cycle.

Expert Commentary

From our point of view, though female sex hormones are downregulators of endogenous release of IFN, they indirectly inhibit various host factors that cause IFN resistance, therefore indirectly render females better responders to interferon therapy. The exact role of sex hormones in the natural course of infection and treatment has not been critically investigated. This raises the question: 'can HCV-infected males and females undergo hormone replacement therapy either alone or during IFN treatment?' More investigation is needed to clarify this issue.

It is noteworthy that 20–25% of HCV patients progress to chronic liver cirrhosis, wherein sex hormone levels are altered.[56] Female sex hormone levels are elevated while testosterone levels are decreased in patients who exhibit liver cirrhosis.[57–59] This makes it crucially important to study the impact of hormonal imbalance after HCV liver disease progression on viral behavior.

Five-year View

In the upcoming 5 years, hopefully comprehensive understanding of genetic disparities between HCV-infected males and females will unravel the ambiguity concerning this issue. Moreover, after the emergence of a HCV cell-culture system it might be easy to study the role of sex hormones on each step of the HCV life cycle. This might open the gates toward novel investigation in HCV pathogenesis. To date, the impact of sex hormones on direct acting antiviral agents, such as the protease inhibitors telaprevir and boceprevir, has never been investigated except from a pharmacokinetic point of view. Co-administration of hormonal contraceptives such as ethinyl estradiol with these antiviral drugs lead to a decrease in the therapeutic concentration of oral contraceptive and treatment failure.[60] This mandates that in the next 5 years, extensive investigation of the role of gender and sex hormones on direct acting antiviral agents should take place.

Sidebar
Key Issues
  • Recently, gender has emerged as a major factor affecting the innate response to HCV; the rate of spontaneous clearance of HCV was found to be higher among HCV-infected females when compared with male patients. The exact factors responsible for this variability in the natural history of the disease are still not well known.

  • Sex hormones influence viral behavior. However, the impact of female sex hormones on HCV remains enigmatic; it is not known whether they hold a beneficial or detrimental role towards virological clearance. However, to date, nothing is known concerning the effect of testosterone on HCV replication.

  • Female sex hormones were reported to be downregulators of many host factors that are important for interferon (IFN) release while the role of testosterone on IFN release has received negligible attention in the literature.

  • A line of evidence exists that proposes that genetic variation among different genders may be responsible for the variability to IFN therapy. In particular, females carrying the favorable genotype of the IL-28Bpolymorphism showed higher chances of IFN response when compared with males carrying either the favorable or unfavorable genotype of IL-28B [38]. Moreover, peripheral blood mononuclear cells derived from HCV-infected females showed higher rates of interferon-stimulated gene induction after IFN stimulation compared with their male counterparts [39].

  • The higher chance of premenopausal women achieving sustained virologic response when compared with postmenopausal HCV patients suggests a key role for female sex hormones in enhancing IFN action. Female sex hormones indirectly promote IFN action by decreasing various factors that causes IFN resistance. On the other hand, testosterone enhances the production of various cytokines involved in IFN resistance.

References
  1. El-Zanaty FWA. Egypt Demographic and Health Survey 2008. Minstry of Health, Egypt 431 (2009).

  2. Farci P, Bukh J, Purcell RH. The quasispecies of hepatitis C virus and the host immune response. Springer Semin. Immunopathol. 19(1), 5–26 (1997).

  3. Fedorchenko SV, Martynovich TL, Liashok OV, Kariuk ZhA, Ianchenko VI. Spontaneous HCV clearance: an association with gender, age, viral genotypes, infection transmission routes, and markers of HBV and HIV. Ter. Arkh. 82(3), 52–56 (2010).

  4. Bakr I, Rekacewicz C, El Hosseiny M et al. Higher clearance of hepatitis C virus infection in females compared with males. Gut 55(8), 1183–1187(2006).
    ** A large retrospective study provides a strong evidence that females have a better chance of spontaneously clearing HCV compared with male counterparts.

  5. Spada E, Mele A, Berton A et al. Multispecific T cell response and negative HCV RNA tests during acute HCV infection are early prognostic factors of spontaneous clearance. Gut 53(11), 1673–1681 (2004).

  6. Conjeevaram HS, Fried MW, Jeffers LJ et al.; Virahep-C Study Group. Peginterferon and ribavirin treatment in African American and Caucasian American patients with hepatitis C genotype 1. Gastroenterology 131(2), 470–477 (2006).

  7. de Bruijne J, Buster EH, Gelderblom HC et al.; Netherlands Association of Gastroenterologists and Hepatologists. Treatment of chronic hepatitis C virus infection - Dutch national guidelines. Neth. J. Med. 66(7), 311–322 (2008).

  8. Akram M, Idrees M, Zafar S et al. Effects of host and virus related factors on interferon-α+ribavirin and pegylated-interferon+ribavirin treatment outcomes in chronic hepatitis C patients. Virol. J. 8, 234 (2011).

  9. Akuta N, Suzuki F, Kawamura Y et al. Predictive factors of early and sustained responses to peginterferon plus ribavirin combination therapy in Japanese patients infected with hepatitis C virus genotype 1b: amino acid substitutions in the core region and low-density lipoprotein cholesterol levels. J. Hepatol. 46(3), 403–410 (2007).

  10. Kryczka W, Zarebska-Michaluk D, Chrapek M. Assessment of selected clinical factors as predictors of response to combined interferon-α plus ribavirin therapy among patients with chronic hepatitis C. Med. Sci. Monit. 9(Suppl. 3), 32–35 (2003).

  11. Poynard T, McHutchison J, Goodman Z, Ling MH, Albrecht J. Is an "a la carte" combination interferon-α-2b plus ribavirin regimen possible for the first line treatment in patients with chronic hepatitis C? The ALGOVIRC Project Group. Hepatology 31(1), 211–218 (2000).

  12. Husa P, Slesinger P, Stroblová H, Svobodník A. The effect of patient's body weight, gender and baseline viral load on the efficacy of hepatitis C therapy. Vnitr. Lek. 52(6), 590–595 (2006).

  13. Qureshi S, Batool U, Iqbal M, Burki UF, Khan NU. Pre-treatment predictors of response for assessing outcomes to standard treatment in infection with HCV genotype 3. J. Coll. Physicians Surg. Pak. 21(2), 64–68 (2011).

  14. Di Bisceglie AM, Fan X, Chambers T, Strinko J. Pharmacokinetics, pharmacodynamics, and hepatitis C viral kinetics during antiviral therapy: the null responder. J. Med. Virol. 78(4), 446–451 (2006).

  15. Narciso-Schiavon JL, Schiavon Lde L, Carvalho-Filho RJ et al. Gender influence on treatment of chronic hepatitis C genotype 1. Rev. Soc. Bras. Med. Trop. 43(3), 217–223(2010).
    * Negates that gender plays a role in interferon treatment response. However, the authors conclude that females experience a high incidence of adverse events.

  16. Jiménez-Pérez M, Sáez-Gómez AB, Pérez-Daga JA, Lozano-Rey JM, de la Cruz-Lombardo J, Rodrigo-López JM. Hepatitis C virus recurrence after liver transplantation: analysis of factors related to sustained viral response. Transplant. Proc. 42(2), 666–668 (2010).

  17. Barrett S, Goh J, Coughlan B et al. The natural course of hepatitis C virus infection after 22 years in a unique homogenous cohort: spontaneous viral clearance and chronic HCV infection. Gut 49(3), 423–430 (2001).

  18. Hayashida K, Shoji I, Deng L, Jiang DP, Ide YH, Hotta H. 17β-estradiol inhibits the production of infectious particles of hepatitis C virus. Microbiol. Immunol. 54(11), 684–690(2010).
    * Concludes that 17β-estradiol has a beneficial effect on HCV infection by preventing the release of mature virions from hepatocellular carcinoma cell lines.

  19. Kaushik-Basu N, Bopda-Waffo A, Talele TT et al. Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors. Nucleic Acids Res. 36(5), 1482–1496 (2008).

  20. Latt NC, Spencer JD, Beeby PJ et al. Hepatitis C in injecting drug-using women during and after pregnancy. J. Gastroenterol. Hepatol. 15(2), 175–181 (2000).

  21. Watashi K, Inoue D, Hijikata M, Goto K, Aly HH, Shimotohno K. Anti-hepatitis C virus activity of tamoxifen reveals the functional association of estrogen receptor with viral RNA polymerase NS5B. J. Biol. Chem. 282(45), 32765–32772(2007).
    ** A study characterizing estrogen receptor-α as a host factor essential for HCV replication and identifying tamoxifen as an antihepatitis C agent.

  22. Langer C, Gansz B, Goepfert C et al. Testosterone up-regulates scavenger receptor BI and stimulates cholesterol efflux from macrophages. Biochem. Biophys. Res. Commun. 296(5), 1051–1057 (2002).

  23. Scarselli E, Ansuini H, Cerino R et al. The human scavenger receptor class B type 1 is a novel candidate receptor for the hepatitis C virus. EMBO J. 21(19), 5017–5025 (2002).

  24. Srivastava RA. Scavenger receptor class B type I expression in murine brain and regulation by estrogen and dietary cholesterol. J. Neurol. Sci. 210(1–2), 11–18 (2003).

  25. Meng J, Mostaghel EA, Vakar-Lopez F, Montgomery B, True L, Nelson PS. Testosterone regulates tight junction proteins and influences prostatic autoimmune responses. Horm. Cancer 2(3), 145–156 (2011).

  26. Evans MJ, von Hahn T, Tscherne DM et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446(7137), 801–805 (2007).

  27. Gibson SJ, Lindh JM, Riter TR et al. Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cell Immunol. 218(1–2), 74–86 (2002).

  28. Gorski KS, Waller EL, Bjornton-Severson J et al. Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists. Int. Immunol. 18(7), 1115–1126 (2006).

  29. Berghöfer B, Frommer T, Haley G, Fink L, Bein G, Hackstein H. TLR7 ligands induce higher IFN-α production in females. J. Immunol. 177(4), 2088–2096 (2006).

  30. Escribese MM, Kraus T, Rhee E, Fernandez-Sesma A, López CB, Moran TM. Estrogen inhibits dendritic cell maturation to RNA viruses. Blood 112(12), 4574–4584 (2008).

  31. Ghisletti S, Meda C, Maggi A, Vegeto E. 17β-estradiol inhibits inflammatory gene expression by controlling NF-κB intracellular localization. Mol. Cell. Biol. 25(8), 2957–2968 (2005).

  32. Hughes GC, Thomas S, Li C, Kaja MK, Clark EA. Cutting edge: progesterone regulates IFN-α production by plasmacytoid dendritic cells. J. Immunol. 180(4), 2029–2033 (2008).

  33. Meier A, Chang JJ, Chan ES et al. Sex differences in the Toll-like receptor-mediated response of plasmacytoid dendritic cells to HIV-1. Nat. Med. 15(8), 955–959 (2009).

  34. Young NA, Friedman A, Kaffenberger B, Jarjour WN. Estrogen stimulation of endosomal toll-like receptor expression lowers the threshold of activation in peripheral blood mononuclear cells and contributes to the gender bias of systemic lupus erythematosus. Arthritis Rheum. 63(Suppl. 10), 1425 (2011).

  35. Paladino N, Fainboim H, Theiler G et al. Gender susceptibility to chronic hepatitis C virus infection associated with interleukin 10 promoter polymorphism. J. Virol. 80(18), 9144–9150 (2006).

  36. Ito S, Ansari P, Sakatsume M et al. Interleukin-10 inhibits expression of both interferon α- and interferon γ-induced genes by suppressing tyrosine phosphorylation of STAT1. Blood 93(5), 1456–1463 (1999).

  37. Kurosaki M, Tanaka Y, Nishida N et al. Genetic polymorphism in IL28B predicts null virological response to pegylated-interferon plus ribavirin therapy for chronic hepatitis. J. Hepatol. 52, 451–452 (2010).

  38. van den Berg CH, Grady BP, Schinkel J et al. Female sex and IL28B, a synergism for spontaneous viral clearance in hepatitis C virus (HCV) seroconverters from a community-based cohort. PLoS ONE 6(11), e27555(2011).
    ** A study exploring the potential interaction between female sex and IL-28B polymorphism and identifying HCV-infected females as having a higher chance of spontaneously clearing HCV infection when compared with male patients.

  39. Mekky RY, Hamdi N, El-Akel W, Esmat G, Abdelaziz AI. Estrogen-related MxA transcriptional variation in hepatitis C virus-infected patients. Transl. Res. 159(3), 190–196(2012).
    ** The first study to conclude that estrogen attenuates the JAK/STAT pathway. This study showed that peripheral blood mononuclear cells derived from HCV premenopausal females showed higher induction rate of interferon-stimulated genes after interferon stimulation when compared with postmenopausal females or male patients.

  40. Hayashi J, Kishihara Y, Ueno K et al. Age-related response to interferon-α treatment in women vs men with chronic hepatitis C virus infection. Arch. Intern. Med. 158(2), 177–181 (1998).

  41. Bressler BL, Guindi M, Tomlinson G, Heathcote J. High body mass index is an independent risk factor for nonresponse to antiviral treatment in chronic hepatitis C. Hepatology 38(3), 639–644 (2003).

  42. Floreani A, Cazzagon N, Boemo DG et al. Female patients in fertile age with chronic hepatitis C, easy genotype, and persistently normal transaminases have a 100% chance to reach a sustained virological response. Eur. J. Gastroenterol. Hepatol. 23(11), 997–1003(2011).
    ** A recent study identifying premenopausal females infected with HCV genotype 2 or 3 as having a 100% chance of responding to interferon treatment.

  43. Villa E, Karampatou A, Cammà C et al. Early menopause is associated with lack of response to antiviral therapy in women with chronic hepatitis C. Gastroenterology 140(3), 818–829 (2011).

  44. Shimizu I, Omoya T, Kondo Y et al. Estrogen therapy in a male patient with chronic hepatitis C and irradiation-induced testicular dysfunction. Intern. Med. 40(2), 100–104 (2001).

  45. Fujita N, Sugimoto R, Urawa N et al. Hepatic iron accumulation is associated with disease progression and resistance to interferon/ribavirin combination therapy in chronic hepatitis C. J. Gastroenterol. Hepatol. 22(11), 1886–1893 (2007).

  46. Rachon D, Mysliwska J, Suchecka-Rachon K, Wieckiewicz J, Mysliwski A. Effects of oestrogen deprivation on interleukin-6 production by peripheral blood mononuclear cells of postmenopausal women. J. Endocrinol. 172(2), 387–395 (2002).

  47. Taliani G, Badolato MC, Nigro G et al. Serum concentration of gGT is a surrogate marker of hepatic TNF-α mRNA expression in chronic hepatitis C. Clin. Immunol. 105(3), 279–285 (2002).

  48. Mlisorn NMN, Sanvarinda Y, Wanikiat P. 17 β-estradiol attenuates LPS-induced interleukin-8 production by human peripheral blood monocytes through estrogen receptor-α activation. Afr. J. Pharm. Pharmacol. 4(11), 806–810 (2010).

  49. Polyak SJ, Khabar KS, Paschal DM et al. Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. J. Virol. 75(13), 6095–6106 (2001).

  50. Leong GM, Moverare S, Brce J et al. Estrogen up-regulates hepatic expression of suppressors of cytokine signaling-2 and -3 in vivo and in vitro. Endocrinology 145(12), 5525–5531 (2004).

  51. Steyn FJ, Anderson GM, Grattan DR. Hormonal regulation of suppressors of cytokine signaling (SOCS) messenger ribonucleic acid in the arcuate nucleus during late pregnancy. Endocrinology 149(6), 3206–3214 (2008).

  52. Posma E, Moes H, Heineman MJ, Faas MM. The effect of testosterone on cytokine production in the specific and non-specific immune response. Am. J. Reprod. Immunol. 52(4), 237–243 (2004).

  53. Kishihara Y, Hayashi J, Yoshimura E, Yamaji K, Nakashima K, Kashiwagi S. IL-1 β and TNF-α produced by peripheral blood mononuclear cells before and during interferon therapy in patients with chronic hepatitis C. Dig. Dis. Sci. 41(2), 315–321 (1996).

  54. Zografos TA, Liaskos C, Rigopoulou EI et al. Adiponectin: a new independent predictor of liver steatosis and response to IFN-α treatment in chronic hepatitis C. Am. J. Gastroenterol. 103(3), 605–614 (2008).

  55. Martos-Moreno GA, Barrios V, Argente J. Normative data for adiponectin, resistin, interleukin 6, and leptin/receptor ratio in a healthy Spanish pediatric population: relationship with sex steroids. Eur. J. Endocrinol. 155(3), 429–434 (2006).

  56. Rodger AJ, Roberts S, Lanigan A, Bowden S, Brown T, Crofts N. Assessment of long-term outcomes of community-acquired hepatitis C infection in a cohort with sera stored from 1971 to 1975. Hepatology 32(3), 582–587 (2000).

  57. Bandyopadhyay SK, Moulick A, Saha M, Dutta A, Bandyopadhyay R, Basu AK. A study on endocrine dysfunction in adult males with liver cirrhosis. J. Indian Med. Assoc. 107(12), 866, 868–869 (2009).

  58. Pignata S, Daniele B, Galati MG et al. Oestradiol and testosterone blood levels in patients with viral cirrhosis and hepatocellular carcinoma. Eur. J. Gastroenterol. Hepatol. 9(3), 283–286 (1997).

  59. Gordon GG, Olivo J, Rafil F, Southren AL. Conversion of androgens to estrogens in cirrhosis of the liver. J. Clin. Endocrinol. Metab. 40(6), 1018–1026 (1975).

  60. Garg V, van Heeswijk R, Yang Y, Kauffman R, Smith F, Adda N. The pharmacokinetic interaction between an oral contraceptive containing ethinyl estradiol and norethindrone and the HCV protease inhibitor telaprevir. J. Clin. Pharmacol. 52(10), 1574–1583(2012)

** A recent study highlighting a possible pharmacokinetic interaction between telaprevir and hormonal contraceptives advises that females should use nonhormonal contraceptives when administering telaprevir.

Papers of special note have been highlighted as:
* of interest
** of considerable interest

Expert Rev Gastroenterol Hepatol. 2013;7(1):69-75. © 2013 Expert Reviews Ltd.

Source

Expert Review of Gastroenterology and Hepatology

Chao Shi, Alexander Ploss

Expert Rev Gastroenterol Hepatol. 2013;7(2):171-185.

Abstract and Introduction
Abstract

Hepatitis C virus (HCV) infection is a major global health problem as it has a high propensity for establishing chronicity. Chronic HCV carriers are at risk of developing severe liver disease including fibrosis, cirrhosis and liver cancer. While treatment has considerably improved over the years, therapy is still only partially effective, and is plagued by side effects, which contribute to treatment failure and is expensive to manage. The drug development pipeline contains several compounds that hold promise to achieve the goal of a short and more tolerable therapy, and are also likely to improve treatment response rates. It remains to be seen, however, how potent antiviral drug cocktails will affect the hepatitis C burden worldwide. In resource-poor environments, considerable costs, inadequate infrastructure for medical supervision and distribution may diminish the impact of future therapies. Consequently, development of novel therapeutic and prophylactic strategies is imperative to contain HCV infection globally.

Prospects in HCV Treatment

An estimated 170 million people, or 3% of the world population, are chronically infected with hepatitis C virus (HCV) (Figure 1). Persistent HCV infection leads to liver cirrhosis and can culminate eventually in hepatocellular carcinomas. Since the discovery of HCV as a causative agent for non-A non-B hepatitis in 1989, constant efforts have been made to improve the outcome of hepatitis C patients. Before 1990, HCV was an incurable disease and monotherapy with IFN-α resulted in a sustained virologic response (SVR) in only 10% of the treated patients.[1] Combination therapies of pegylated interferon (peg-IFN) with ribavirin (RBV) were later applied and became the standard-of-care for HCV. This combination treatment improved SVR rates, but fell short of curing HCV infection in more than 50% of patients with HCV genotype 1 and had an even worse outcome or was contraindicated in patients with comorbidities such as HIV infection, cirrhosis, transplant recipients or in African–Americans,[2,3] thus creating a need for more effective therapies. The introduction of direct-acting antivirals (DAAs), which are inhibitors of virally encoded protein functions, to the market represents a milestone in HCV therapy. Incivek® (generic name: telaprevir; Vertex, MA, USA) and Victrelis™ (generic name: boceprevir; Merck, NJ, USA), two drugs that interfere with the virally encoded NS3/4A protease, were approved by the US FDA in 2011. Addition of telaprevir or boceprevir to the peg-IFN/RBV regimen increases SVR rates in certain clinical trial cohorts to 60–70%.[4–7] In the meantime, many candidates of HCV DAAs, including the next generation of protease inhibitors, NS5A inhibitors and polymerase inhibitors, are at the late stage of development. Recent clinical trials have demonstrated that combinations of orally administered DAAs with different mechanisms of action can cure chronic HCV infection with 90% rate,[8–10] although the optimal results remain to be confirmed in larger patient cohorts. The availability of these new, presumably more potent DAAs is expected to revolutionize the standard-of-care of HCV infection, with a promise to cure HCV with an all-oral, IFN-free cocktail regimen. In addition, drugs targeting host factors that are essential for HCV replication, such as cyclophilin A and miR122, are also in the pipeline. A drastic expansion of the ammunition for treating HCV infection is expected in the next few years.

779243-fig1

Figure 1.

Relative hepatitis C virus prevalence and distribution of common genotypes. The numbers in the figure indicate the most prevalent HCV genotype(s) in the respective regions. Data taken from WHO (2006) and [20,152].

Because HCV, unlike HIV and hepatitis B virus (HBV), does not integrate into the host genome, successful treatment with antiviral therapies is able to eradicate the virus from individuals. A 90% cure rate of new antiviral drugs suggests that the number of existing patients will shrink in the USA and other developed counties, where effective treatment can be applied. Moreover, as a consequence of implementation of rigorous blood supply screening for HCV since 1991, the number of new HCV infections in the USA fell from a peak of 180,000/year in the mid-1980s to 16,000/year in 2009 ([11] and CDC data). Currently, the most common cause of ongoing HCV transmission is the sharing of contaminated needles or syringes by injection drug users (IDUs; in the USA) and unsafe medical practices (globally). Some studies have projected the prevalence in the USA to decline from 3.2 million in 2005 to 2.5 million in the 2020s without considering the utilization of more effective antiviral regimens.[12,13] Using a similar but simplified approach, the authors predict that with the application of new DAAs, which can potentially improve the rate of SVR from 50 to 90%, the infected population in the USA will decline below 2 million in 2020s (Figure 2 & ). Furthermore, since the new regimens can be applied to patients who were previously ineligible for the standard-of-care treatment due to their insensitivity or intolerance to interferons, the percentage of patients receiving treatment is expected to increase. Currently, only 10–27% of people diagnosed with HCV infection are offered treatment.[14] If we assume the treatment rate increases to 50% with the application of new DAAs, the projection of the US prevalence will be below half a million in 2020s (Figure 2). However, this optimistic outlook comes with caveats.

Table 1. Variables and assumptions used in the projection.

Variable Base value Explanation Ref.
Prevalence of chronic infection 3,000,000 in 2012 2.7–3.9 million CDC data and [153]
Incidences of new infection 15,000/year Estimated 16,000 for 2009 by CDC, assuming remains stable
Spontaneous clearance rate 18%
[16,154]
Diagnosis rate of infected 20–70% of existing20% of newly infected Increases with ages as symptoms exacerbate; assume 70% in 2022
Treatment rate of diagnosed 10% with peg-IFN/RBV50% with DAAs Increased rate with DAAs due to improved tolerance and efficacy
SVR 50% with peg-IFN/RBV90% with DAAs
[8–10]
Mortality of infected Average: 21,000/year Mortality increases with ages, but decreases with application of DAAs

As new infections in the USA occurs mainly among injection drug user, the transmission rate within this population is assumed stable, given that many prevention programs have been in place, and that low penetration of treatment in this population will keep the number of the infected at a sustained level. DAA: Direct-acting antiviral; Peg-IFN: Pegylated interferon; RBV: Ribavirin; SVR: Sustained virologic response.

779243-fig2

Figure 2.

A projection of hepatitis C virus burdens in the USA for the next decade. Assuming 50% sustained virologic response rate for peg-IFN/ribavirin regimen and 90% for DAAs combo regimen. DAA: Direct-acting antiviral; HCV: Hepatitis C virus; peg-IFN: Pegylated interferon; RBV: Ribavirin.

Unmet Demands

First, access to the new drugs is limited due to their high costs. Depending on the duration of treatment, a regimen of telaprevir or boceprevir costs US$30,000–50,000.[15] According to the current paradigms, peg-IFN/RBV, which costs $35,000 per course, still needs to be added to the treatment with either DAAs. Meanwhile, additional expenses for managing the side effects, among which are anemia, rash and depression, must be considered. Because HCV infection is prevalent among marginalized groups with lower incomes, who usually lack adequate coverage by medical insurance, the penetration of new treatment will probably be low. Moreover, since most new incidences of HCV infection in the USA are acquired through sharing of contaminated needles or syringes by IDUs, a low penetrance of treatment in this population will keep the number of new infections at a sustained level. Even if a certain rate of treatment can be achieved in the high-risk population of IDUs, frequent re-exposure and reinfection can still pose a problem, but may be ameliorated if an at least partially protective vaccine were available. Therefore, hepatitis C may become more of a social problem than a medical one. This critical issue will be difficult to tackle without sufficient political support.

Second, although HCV drug development is moving forward rapidly, the efforts to identify chronic HCV carriers lag behind. As 80% of people do not exhibit any symptoms following initial infection, most people with HCV are unaware of their infection status.[16] This is especially a problem for the high-risk population, which do not have access to routine medical screening. The low rate of diagnosis not only leads to an underestimation of overall HCV burden, but also limits the utilization of effective treatments.

Third, HCV infection will remain a global epidemic in the foreseeable future. Even if it will be possible to treat all the infected individuals in developed countries with new DAA regimens, HCV infection is likely to persist in the vast majority who live in the developing world, where the medical infrastructure cannot support and afford the treatment. Those developing countries also face the additional challenge of a high transmission rate owing to inadequate screening of blood products and an increasing number of IDUs.[17] Given the constant human migration, it is impossible for the developed countries to insulate themselves from the epidemic elsewhere. Furthermore, as most new drugs were designed to treat HCV genotype 1, because it is difficult to cure with peg-IFN/RBV, and it is predominant in developed counties, the effectiveness of those drugs for controlling infection with other genotypes remains to be tested. This is particularly problematic as nongenotype 1 viruses are widely distributed around the globe (Figure 1).

HCV Vaccines

In general, vaccination has been considered the most effective approach for combatting infectious diseases. A lesson learned from fighting HIV is that emphasis on prevention as well as treatment is important for controlling the pandemic.[18] In addition to reducing the transmission with more efficient blood screening and prevention-focused programs for IDUs, a prophylactic vaccine for HCV will fill the gaps of treatment discussed earlier, and provide a long-term solution for the disease. The target population for the vaccine will be the high-risk groups in developed countries, including healthcare workers, IDUs and men who have sex with men, and the entire population of developing countries. Optimally, a putative HCV vaccine would be effective against all HCV genotypes, would have minimal side effects, administered in a single shot and could be produced and distributed at minimal cost. However, after more than two decades of research, no HCV vaccine is currently available.

The development of a vaccine for HCV is hampered by several challenges. The first is the tremendous genetic and antigenic diversity of the virus. Currently there are seven genotypes being recognized, with sequence dissimilarity of 30–35% across the genome.[19,20] Most genotypes contain genetic diversity within the group, and can be further categorized into viral quasispecies.[21] As a positive sense, single-stranded RNA virus, HCV replicates its genome rapidly through the highly error-prone polymerase. This feature, together with the remarkable capability of the virus to produce progeny during chronic infection, enables HCV to generate a vast reservoir of genetic diversity, which provides the basis for selecting a variant with higher replicative fitness and capability of immune evasion. This also suggests that HCV is more likely to escape the immune protection elicited by a vaccine designed for a limited number of epitopes. The new antiviral drugs are facing the same challenge, as HCV variants will emerge with mutated binding sites for inhibitors (reviewed in[22]). To avoid resistance, a combination of drugs acting with different mechanisms needs to be used. Similarly, a successful vaccine is likely to contain multiple conserved epitopes that minimize the risk of viral escape. Thus, many of the current vaccination approaches in clinical development, discussed in the following sections, include numerous viral proteins encompassing larger numbers of epitopes to alleviate this problem. Additional complexity stems from the putatively different sensitivities of usually linear T-cell epitopes and frequently conformational antibody epitopes to mutational sequence diversity. While amino acid mutations in critical residues can readily abrogate T-cell antigen recognition, sequence variations that do not alter shape or charge of a viral antigen may not necessarily affect antibody recognition. The second challenge for vaccine development is incomplete understanding in the immunology of HCV infection. This is largely due to a lag in the development of experimental systems. Researchers were not able to grow HCV and test the ability of antibodies to neutralize HCV in vitro until recently.[23–26] Because of a narrow species tropism of HCV, chimpanzees are the only in vivo experimental model with competent immune system available for HCV vaccine research, but those studies are limited due to ethical concerns, restricted availability and prohibitively high costs. As a result, much of our knowledge in HCV immunology relies on the studies of a very small number of animals. Clinical studies on infected patients have been informative and probably less biased by sample size, but are hampered by limited access to the relevant tissue compartment, that is, the liver and usually limited information on the dose, timing and antigenic composition of the transmitted viral genome. The lack of accessible animal models is also a hurdle for testing the efficacy of vaccine candidates.

Current HCV Vaccine Research
Immune Responses to HCV & Correlates of Protection

Despite the challenges in studying HCV, considerable progress has been in made in characterizing anti-HCV immune responses. It has been estimated that approximately 20% of individuals are able to clear the infection spontaneously following acute HCV infection, whereas the rest progresses to chronicity.[27] Longitudinal studies of the two cohorts of patients during and after acute infection have defined immunologic correlates that are associated with viral clearance.

A strong T-cell response, characterized by the production of effector cytokines including IFN-γ, and broad epitope specifically correlate with the resolution of acute infection.[28–34] After clearance of the acute infection, memory T cells are maintained, but whether they can provide protection against reinfection is incompletely understood.[35–39] While usually not resulting in sterilizing immunity, that is, prevention of acute infection after re-exposure especially to antigenically more divergent HCV strains, adaptive immunity protects against progression to chronic infection following repeated HCV exposure.[40,41] As chronic infection persists, the number of epitopes recognized decreases and T-cell responses are often lost.[42,43] Since HCV-associated morbidity and mortality are caused by chronic infection, a vaccine, even if it only prevents viral persistence, would greatly ameliorate the problem. Although neutralizing antibodies are present during the chronic phase of infection, these antibodies are not able to clear the virus.[44,45] Several mechanisms of viral escape from antibody-mediated neutralization have been postulated and tested (reviewed in [46]). Recently, several human monoclonal antibodies against HCV envelope protein E1 or E2, which show crossneutralizing capability, were identified.[47–50] These antibodies were able to prevent infection of heterologous HCV in the HCV pseudoparticle and HCV cell culture model system, suggesting passive prophylaxis with exogenous neutralizing antibodies or eliciting high-affinity antibodies with similar specificity representing a viable strategy to prevent or more efficiently control HCV infections.

Although resolution of the infection is dependent on adaptive immunity, innate responses are also observed early after HCV infection. Type I interferons and interferon response pathways are induced in the liver at early stages of infection regardless of the clinical outcomes.[29,51,52] The fact that various strategies have evolved in the HCV life cycle to interfere with the IFN response[53] indicates that the innate response exerts a significant pressure on HCV. Moreover, recent genome-wide association studies have identified single-nucleotide polymorphisms in the IL-28B gene locus that correlate with spontaneous clearance of an acute HCV infection and predict to a certain extent how likely patients with a given combination of IL28B alleles are to respond to peg-IFN/RBV therapy.[54–57] Genetic analysis has also revealed the important role of natural killer (NK) cells, which produce IFN-γ and are abundant in the liver. Genetic polymorphisms that affect the threshold of NK-cell activation influence the clinical outcomes of HCV infection.[58] Recent genetic data suggest that taking into account the combination of polymorphisms within the loci of IL28B, HLA-C and its ligands, the killer immunoglobulin-like receptors has greater predictive value for clearance of HCV infection.[59] These results not only highlight the importance of innate immunity during HCV infection, but also suggest the efficacy of a certain vaccine may depend on the genetic features of recipients.

Approaches of Vaccination

Along with the efforts in improving our understanding of the basic immunology of HCV infection, various approaches have been taken to develop vaccines. Specific approaches in the development of both prophylactic and especially therapeutic vaccines against HCV infection have been recently reviewed in great detail elsewhere.[60] In this article, the authors limit the discussion on general principals pertaining to the different vaccination approaches and highlight candidate vaccination approaches that are in active clinical development.

Prophylactic Vaccination

Prophylactic vaccinations aim at preventing infection often through the induction of a pathogen-specific humoral immune response. However, the role of neutralizing antibodies in protection against HCV infection remains controversial.[61] Although only few founder viruses appear to initiate the HCV infection during transmission,[62] antigenically diverse viral variants are readily produced once HCV starts to replicate. The antigenic diversity poses further challenges to the prophylactic vaccination approach. Early attempts focused on inducing the production of neutralizing antibodies against envelope proteins of HCV, E1 and E2. This was inspired by the success of HBV vaccines, which induce antibodies against HBV surface antigens, thereby preventing viral entry and infection. Induction of HCV envelope-specific antibodies in naive chimpanzees by vaccination with recombinant E1 and E2 or DNA yielded protection from virus challenge.[63,64] Similarly, immunization of healthy human volunteers with HCV envelope glycoproteins elicits antibodies that crossneutralize heterologous virus strains in vitro.[65,66,201] A major challenge remains in the identification of suitable immunogens that elicit broadly neutralizing antibody responses. The major antigen determinants within the viral envelope are in the hypervariable-region 1 of the E2 glycoprotein, which, as the name implies, is not necessarily suitable to confer broad protection against antigenically diverse viruses. It has been speculated that more broadly shared epitopes will become accessible when the HVR1 region is deleted from the viral envelope. However, the idea of engineering the immunogenicity of HCV by exposing better-conserved epitopes remains to be tested. Furthermore, analysis of the structural details of (conformational) epitopes recognized by antibodies with broad neutralizing activity may provide a starting point for the design of immunogens capable of eliciting antibodies with similar activity.[67,68]

Prophylactic vaccination approaches are not limited to those geared towards inducing neutralizing antibodies. Clinical trials are ongoing to assess the efficacy, safety and immunogenicity based on the sequential use of adeno- and/or modified vaccinia Ankara (MVA) vectors expressing HCV nonstructural proteins NS3-NS5B.[202,203] Conceivably, combining the approaches that prime both humoral and cellular immunity would protect more efficiently against HCV challenge, although the concept remains to be tested in suitable animal models and/or clinical trials.

Therapeutic Vaccination

The main rationale of therapeutic vaccination is to bolster new and/or restore ineffective previously primed antiviral adaptive immune responses to neutralize circulating virus and eliminate infected cells. Optimally, therapeutic vaccination, conceivably in combination with standard-of-care treatment, would eventually result in complete control of the previously established viral infection or at least significantly mitigates liver disease progression. Treatment of chronic HCV infection has considerably improved in recent years and numerous directly acting antiviral and host-targeting antiviral drug candidates have shown remarkable efficacy in clinical trials. These advances may ultimately reduce the need for therapeutic vaccines. However, as outlined earlier, the new treatment, while being expensive, is not effective in all patient populations, and is plagued with considerable side effects. Consequently, more cost-effective alternatives are required to improve treatment options, particularly in resource-poor environments.

Therapeutic vaccine trials have demonstrated that HCV-specific immune responses can be primed in chronically infected individuals, resulting in transient reductions in HCV RNA titers in subsets of patients.[69–71] However, to date, no therapeutic vaccine candidates have achieved sustained SVRs. Considering that immune exhaustion is frequently associated with chronic HCV infection, the fact that partially functional T-cell responses can be primed is still remarkable. These observations also argue that a better understanding of mechanisms of immune exhaustion is needed to pair therapeutic vaccinations with specific immunomodulatory regimens to bolster antiviral immunity. A plethora of approaches has been undertaken towards developing a therapeutic vaccine against HCV infection (reviewed in detail in [60,72]). These can be broadly divided into peptide- or protein-based vaccines, DNA vaccines, viral vector vaccines – including recombinant adenovirus, MVA, alphavirus or paramyxovirus vectors – recombinant yeast-based vaccines and vaccination approaches based on dendritic cells (DCs). Of those, some have advanced into early clinical development assessing their safety and immunogenicity (reviewed in detail in[72]), but only few are currently being actively pursued ( ). For example, it was previously demonstrated that HCV antigen expression from DNA can result in robust induction of HCV-specific humoral and T-cell immunity, depending on the antigen combination. Currently, administration with a plasmid expressing NS3/4a of HCV genotype 1 and subsequent in vivo electroporation is being tested in combination with peg-IFN and RBV in chronically infected HCV patients. In contrast to plasmid DNA vaccines, viral vectors are highly immunogenic and also allow for the expression of an antigen combination of choice. From a regulatory perspective of safety, insufficient or incomplete attenuation of replication of viral vector is a major concern. Replication incompetent adenoviral and MVA vectors have been extensively tested in this respect. To induce anti-HCV immunity, adenoviruses alone and/or with MVA expressing HCV nonstructural proteins in combination with standard-of-care therapy are currently being evaluated for their potential to restore dysfunctional T-cell response and to broaden HCV-specific T-cells' immunity. Saccharomyces cerevisiae, being nonpathogenic in humans but highly immunogenic, can be engineered to express heterologous proteins and thus present a desirable vaccine platform. The impact of vaccination with inactivated S. cerevisiae engineered to express a fusion protein of HCV core and NS3 in combination with peg-IFN and RBV is being investigated.

Table 2. Approaches for therapeutic vaccination in clinical development.

Strategy Approach Examples in clinical development ClinicalTrials.gov identifier Sponsor Outcome
Peptides Viral peptides coupled with adjuvants to induce humoral and cellular immunity
No active clinical trials
NA
DNA vaccines Expression of HCV protein(s) from a DNA plasmid In vivo electroporation of DNA plasmid expressing HCV NS3/4a NCT01335711 ChronTech Pharma AB Unpublished
Viral vector vaccines Use of viral vectors for delivering HCV RNA; produces a broader range of HCV epitopes and highly immunogenic AdV vector expressing HCV NS3-5B NCT01094873 Okairos Unpublished
MVA and AdV vectors expressing HCV NS3-5BMVA vector expressing NCT01296451 Okairos Unpublished
HCV NS3, NS4 and NS5B (+peg-IFN/RBV) NCT01055821 Transgene
Recombinant yeast-based vaccines Expression of HCV protein(s) in yeast to induce innate and adaptive immunity Inactivated Saccharomyces cerevisiae expressing NS3-core fusion protein (+peg-IFN/RBV) NCT00606086 GlobeImmune Unpublished
Vaccines based on DCs Infusion of ex vivo stimulated DCs loaded with HCV antigens
No active clinical trials
NA

AdV: Adenovirus; DC: Dendritic cell; HCV: Hepatitis C virus; MVA: Modified vaccinia Ankara; NA: Not applicable; NS: Nonstructural; Peg-IFN: Pegylated interferon; RBV: Ribavirin.

Clinical trial IDs obtained from [205].

Alternative Paths

Improved Design & Selection of Immunogens. Immunogen selection is a formidable challenge when thinking about a HCV vaccine due to the extreme diversity of the virus. Most of the currently licensed effective vaccines have been developed by inoculating attenuated or inactivated pathogens, or isolated antigenic components of a given pathogen. However, live-attenuated, or even inactivated, virus is not an easy approach for HCV. Safety issue is of course a concern, but more importantly, a robust replication system that allows large-scale production of HCV particles, and further purification for vaccine usage is not yet available. Selection of individual proteins and even combinations thereof does not cover adequately the genetic and antigenic complexity of the different HCV genotypes and existing quasispecies in a given patient. While some regions within the HCV open reading frame are more conserved across genotypes, they do not necessarily contain the most potent epitopes. Advances in computational and structural biology offer putative solutions to overcome this hurdle.

Reverse vaccinology starts with genomic sequences of the pathogen and uses bioinformatics tools to predict potentially antigenic protein products of the sequences. The protein candidate can then be tested with experimental systems.[73] Recent success in applying reverse vaccinology to develop vaccines against meningococus B has demonstrated the effectiveness and efficiency of this approach.[74] The selection of antigenic regions, especially putative antibody epitopes, can be further refined and verified using 3D structures of a given pathogen derived protein superimposed with the linear and/or conformational epitopes of known potent neutralizing antibodies pointing towards the 'Achilles heel' of a (viral) pathogen. Crystal structures of the HIV envelope have been solved and numerous very potent antibodies have been identified;[75–82] but this recent gain in knowledge has not yet translated into effective structure-based vaccine design for HIV.[83,84] In order to use structure-based approaches for designing vaccine candidates for HCV, some critical gaps need to be closed. Although, the HCV E2 proteins can now be purified under presumably native conditions[85] and based on biochemical data a model of E2 has been put forward,[86] high-resolution crystal structures for the viral envelope remain elusive. Considerable progress has been made in the identification of broadly crossreactive monoclonal antibodies.[47,49,50,87–92] The assumption that a reconstructed antigen designed to fit a neutralizing antibody will be an efficient immunogen to elicit protective antibodies in vivo remains to be proven. Nevertheless, as a supplemental approach, structure-based antigen design may help improve the efficacy of vaccine candidates identified by empirical immunogenicity trials.

To design polyvalent vaccine antigens for T-cell-based vaccines, a computational approach was developed for HIV[93,94] and has also been considered for HCV.[95] Such artificial antigens are comprised of sets of 'mosaic' proteins, which are computationally generated recombinants assembled from fragments of natural sequences using a genetic algorithm. Mosaic proteins are similar to natural proteins, but are optimized to maximize the coverage of common potential T-cell epitopes found in a population of natural sequences, and to minimize the inclusion of rare epitopes to avoid vaccine-specific responses. Sets of mosaic proteins provide coverage of the most common 9-mers in the circulating population, and enable the delivery of these variants in the form of intact proteins that could be processed naturally and delivered readily in a vaccine. Adenovirally expressed mosaic HIV-1 vaccines have been shown to expand the breadth and depth of cellular immune responses in rhesus monkeys;[96] however, it has yet to be proven that mosaic HIV vaccines are more immunogenic than conventional antigen combinations in clinical trials; theoretically, HCV-mosaic vaccines hold promise as more potent immunogens to elicit T-cell responses with pan-genotypic specificity.

For any of the aforementioned approaches to define potentially more potent immunogens, new or refined platforms are needed to elicit immunity in vivo. Expression of HCV protein antigen in various viral vectors, including replication incompetent MVA or adenoviral vectors, induces protective immune responses against diverse pathogens and cancer in various animal models, and can induce robust and sustained cellular immunity in humans. However, for the most commonly used serotype 5 adenoviruses, most humans have neutralizing antibodies, which can diminish the immunogenicity of such vaccines. Recently, more than 1000 adenoviruses from chimpanzees have been isolated and sequenced, which can induce potent cellular immunity across multiple species.[97] In clinical trials, these simian adenovirus vectors appear to be safe and highly immunogenic. Although there is so far no side-by-side comparison of vaccine efficacy to demonstrate its superiority to other nonhuman ones, these simian adenoviruses provide a viable alternative to their human counterparts as vectors for vaccine delivery.[98,99] Harnessing the inherent immunogenicity of activated DCs, the central orchestrator of adaptive immunity in vivo, may serve as one alternative to complement and boost viral vaccine vectors (reviewed in [100]). To avoid the need of isolation and expansion of autologous DCs in vitro, which would limit the utility of the approach for widespread use, direct targeting of antigens to DCs in vivo is currently being explored. It was previously demonstrated that antigens fused antibodies binding to cell-type-specific uptake receptors on the surface of DCs can induce antigen-specific immune responses in vivo (reviewed in [101]), and thus may be applied to induce in particular T-cell immunity to HCV with novel, tailored HCV antigens.

Passive Prophylaxis. Although neutralizing antibodies induced by natural infection or vaccines are not sufficient to prevent HCV infection,[102] strong and broadly reactive antibodies to HCV from exogenous sources can be used in postexposure prophylaxis or prevention of recurrent HCV infection after liver transplantation. Indeed, it has been shown that immunoglobulins prepared from chronic HCV patients can prevent hepatitis C in liver recipients, if antibodies are administered to patients without prior exposure to the virus.[103] However, those immunoglobulins have so far failed to prevent reinfection in HCV patients who have undergone liver transplantation.[104] Recent development of human monoclonal antibodies has enabled in vitro production of anti-HCV antibodies with defined specificity at a large scale. The observation that these monoclonal antibodies are able to neutralize diverse HCV quasispecies in human liver-chimeric mice[50] has raised the hope that potent antibodies at high dosages will be effective for postexposure prophylaxis in humans. To achieve a high, sustained dose of antibodies, vector-mediated gene delivery approaches are being explored. In the case of HIV, overexpression of broadly neutralizing antibodies using an adeno-associated virus vector was able to fully protect humanized mice from HIV infection.[105] Similar studies are sought to test whether this approach can produce effective prophylaxis against HCV.

Immunotherapeutic Approaches. As an immunotherapeutic approach, immune cells with antiviral activities are transferred to enhance the endogenous immune response in the recipient. In a study, HCV-infected patients who have undergone liver transplantation were infused with lymphocytes extracted from the liver allografts.[106] These lymphocytes include abundant NK and NK T cells, and were treated in vitro with IL-2/anti-CD3 mAbs before infusion. This treatment markedly lowered the HCV RNA titers in patients and completely prevented HCV infection in human liver-chimeric mice.[106] Furthermore, it has been shown that CD56+ cells obtained from the peripheral blood mononuclear cells show anti-HCV activity.[107] However, unlike T and B cells, NK cells lack an antigen-recognition receptor for distinguishing healthy and infected cells. Instead, their responses depend on the signals from inhibitory and activating receptors. A putative solution may be to guide NK cells to HCV-infected target cells using bispecific antibodies binding to an invariant domain of an activating receptor and viral antigens/antigen–MHC complexes on the target cell.

The efficacy of HCV vaccines may also be enhanced by immunomodulatory treatments. As overly activated T-cell response can cause excessive tissue damage, many regulatory mechanisms are in place to keep T-cell activity in check.[108] For instance, T cells express the inhibitory receptor programmed death-1 (PD-1)[109] and T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3).[110] Ligation of these regulatory receptors by their ligands induces negative signals to the responding cells and leads to reduction of cytokine production and cell proliferation.[111,112] Moreover, Foxp3+ Treg, which is a specialized CD4+ T cell, can suppress the function and proliferation of effector T cells.[113] These regulatory mechanisms together limit T-cell responses during chronic infection, and also affect the efficacy of a vaccine. It has been shown that blockade of PD-1 or Tim-3 can enhance the proliferation and cytotoxicity of HCV-specific cytotoxic T lymphocytes.[114] With this rationale, the effect of inhibitory receptor blockade or Treg depletion on the efficacy of a HCV vaccine needs to be examined.

Recent Progress in the Model Systems for Vaccine Studies

The development of an infectious cell culture system for HCV in 2005[23,115,116] marked a major milestone for hepatitis C research. It has not only enabled in-depth studies of the HCV life cycle but has also aided HCV vaccine research. The cell culture system for HCV has not only been applied to study the efficiency of neutralizing antibodies,[46,117,118] but also to understand the mechanisms of immune evasion of HCV.[47,119] However, the infectious cell culture system relies primarily on a single HCV molecular clone derived from a Japanese genotype 2a infected patient with fulminant hepatitis (JFH1), replicate efficiently both in tissue culture and in animal models. Subsequently, intergenotypic chimeras consisting of the core through NS2 regions of representative genomes of all seven HCV genotypes have been constructed.[120–123] These tools have become indispensable for in vitro and in vivo assays of HCV entry and neutralization. However, a broad spectrum of infectious clones of all HCV genotypes capable of replicating in cell culture is still missing. Despite these advances, a huge gap still exists between in vitro experimental systems and clinical studies. Although the efficacy of preventive vaccines can be assessed in individuals with high risks of exposure, such as IDUs and healthcare workers, the low incidence rates of infection in developed countries requires a large number of participants to be recruited in order to detect statistically significant differences. Consequently, it may be more feasible to conduct clinical trials in developing countries with high prevalence and incidence rates of new infections. Testing of preventative and especially therapeutic vaccines in human trials without adequate preclinical safety assessment in animal models can be problematic, since vaccine induced and/or exacerbated responses may lead to more severe hepatitis and in worst-case scenario to acute liver failure. Furthermore, vaccine dosing, formulations and vaccination schedules often need to be empirically tested to define regimens that result not only in maximal immunogenicity but also efficacy. However, animal models that can be infected with HCV and thus are suitable for testing of vaccine candidates are sparse ().

Table 3. Models for in vivo vaccine testing.

Models for in vivo tests Humans (clinical trials) Chimpanzees Mouse-adapted HCV Genetically humanized mice Xenotransplantation models
HCV life cycle in vivo
Entry Yes Yes ? (in vitro: yes) Yes Yes
Replication Yes Yes ? ? Yes
Assembly Yes Yes ? ? Yes
Immunity
Innate Yes Yes Impairment needed? Yes Yes
Adaptive – cellular Yes Yes Impairment needed? Yes Limited
Adaptive – humoral Yes Yes Impairment needed? Yes Impaired
Pathogenesis Hepatitis, fibrosis, cirrhosis, HCC Milder than in humans Not yet tested Not yet tested Evidence for fibrosis
Validated for vaccine testing Yes Yes No Limited No
Practical considerations
Costs High High Low Low Medium
Throughput Low Low High High Medium
Heterogeneity High High Low Low High

It is not known whether the respective parts of the HCV life cycle are supported.

It is unclear whether innate and/or adaptive responses need to be suppressed.

HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus.

Chimpanzees have been the central animal model to study HCV infection. Besides humans, they are the only species that is naturally susceptible to HCV infection. Chimpanzees partially recapitulate the natural course of infection in humans. While acute hepatitis is somewhat milder in chimpanzees than in humans, experimentally infected animals also frequently progress to chronicity.[74,124,125] Subclinical hepatitis, after many years of chronicity, however, is also not uncommon in humans without risk factors for rapid progression including, older age or alcohol intake. The chimpanzee model has been of critical importance for defining the nature of protective immunity following reinfection. Experiments in chimpanzees demonstrated that clearance of a primary infection with HCV does not provide sterilizing immunity against challenges with homologous or heterologous viruses.[83,84] However, despite their utility, the use of chimpanzees in biomedical research is intensely debated due to ethical concerns. In fact, strong public opposition against the use of large apes has led to the ban of chimpanzee research in many countries. Undoubtedly, an NIH moratorium on 'nonessential chimpanzee research'[204] is likely to constrain HCV vaccine development and HCV research in general in the future. Furthermore, experiments in chimpanzees are associated with very high cost, and usually only few animals, which are genetically heterogeneous, remain available. Consequently, the interpretation of data derived from experiments on a small number of chimpanzees is often statistically difficult.

Accessible animal models, in particularly immunocompetent small rodents and primates, are urgently needed. Significant progress has been recently made in attempts to adapt HCV to infect nonpermissive species and to engineer the host environment to provide an environment that is more conducive to viral infection. While many new tools are still in the early phases of development, they hold promise to dramatically improve our ability to preclinically assess vaccine candidates in the near future.

Adapting HCV to Infect Nonpermissive Species

The full life cycle of HCV can be divided into three critical steps: cell entry, replication and assembly/egress/release of viral particles. Host factors are involved in each step. For entering its target cell, the hepatocyte, HCV requires numerous cellular factors, including glycosaminoglycans,[126,127] low-density lipoprotein receptor, scavenger receptor class B type I (SCARB1),[128–131] CD81,[132] two tight junction proteins, claudin-1[133] and occludin (OCLN).[134,135] More recently, the cholesterol absorption receptor Niemann-Pick C1-like 1[136] and two receptor tyrosine kinases, EGF receptor and EphrinA2[137] have been implicated in the viral uptake pathway into human cells (reviewed in [138]). The difference in the ability of HCV to engage these host factors helps define the species tropism of HCV. In murine cells, HCV appears to less efficiently engage CD81 and OCLN, which precludes viral entry.[134] However, given the error-prone replication of HCV genome, it is conceivable to 'train' HCV to utilize murine orthologs of HCV entry factors. Critical proof-of-concept for this approach was recently provided in a study, in which a strain of HCV was selected to use murine CD81.[139] This virus acquired mutations in the E1 and E2 envelope proteins, which facilitated viral entry into murine cells in the absence of human entry factors. It remains to be tested whether the 'murine-tropic' HCV can actually infect mice in vivo. A mouse model would certainly be very attractive for HCV vaccine research; as multiple inbred and outbred lines are available, mice can be generated in large numbers at fairly low costs and numerous tools to analyze vaccine and virally induced immune responses are available. However, since the evolutionary divergence of mouse and man 65 million years ago, these two species have inhabited different ecological niches and have been challenged with minimally overlapping groups of pathogens. Therefore, the human and mouse immune systems, evolving to meet these challenges, have accumulated many differences,[140] making genes related to immunity, together with genes involved in reproduction and olfaction, the most divergent between the two species.[141] These differences are likely to affect the quality of an antiviral immune response and consequently may lessen the utility of the system for testing vaccines and therapeutics targeting human HCV.

Consequently, adaptation of HCV to other, more closely related species to humans needs to be pursued to ameliorate some of the caveats. Small nonhuman primates, such as Rhesus monkeys are more similar to humans but they are also resistant to HCV infection[142] possibly due to the inability of HCV to counteract antiviral innate immune responses.[143] Nonetheless, adaptation of HCV to small nonhuman primates offers several considerable advantages. Given the greater similarity to humans, it may potentially be easier to overcome putative incompatibilities between virally encoded proteins and host factors. Furthermore, rhesus monkeys have been extensively used in biomedical research, and a plethora of tools is available. Studies conducted in monkeys may translate more readily into clinical results. Macaques also offer a better platform for pharmacokinetic studies for HCV vaccines and drugs. In addition, the fact that a large fraction of HCV carriers is coinfected with HIV must be considered when assessing vaccine candidates. A macaque model for HIV/HCV coinfection is of interest and significant clinical relevance, especially when a simian-tropic HIV-1 is already available.[144,145]

Engineering the Hosts to Accommodate HCV

Another direction researchers have taken to create an animal model is to engineer the host environment to support HCV infection. Host adaptation can be achieved by actually transplanting human tissues to humanize relevant tissue compartments or by genetic adaptation of the host species. It has been demonstrated that engraftment of human hepatocytes into suitable xenorecipients can render mice susceptible to HCV infection.[146] In these xenotransplantation models, liver injury is induced in the recipients before transplantation to provide a growth stimulus to the engrafted human hepatocytes. Currently, the urokinase plasminogen activator transgenic mouse and fumary lacetoacetate hydrolase deficient mouse, both being susceptible to endogenous liver injury, were used in combination with severe immunodeficiency for liver xenotransplantation models.[147–149] Human liver-chimeric mice can be successfully infected with HCV.[146,147,150] However, a major drawback of these human liver-chimeric mice is the lack of a functional immune system. Although the efficacy of neutralizing antibodies and antiviral drugs can be assessed in those mice, it is difficult to study the immune responses induced by vaccines. Recently, an alternative xenorecipient strain has been constructed, in which FK506 binding protein fused to active caspase 8 is transgenically expressed.[125] Upon injection of the FK506 dimerizer, AP20187 apoptosis is induced in mouse hepatocytes. Remarkably, mice injected with a mixture of autologous human hepatoblasts, nonparenchymal cells and hematopoietic stem cells resulted in measurable human hepatic and hematopoietic chimerism. Dually engrafted mice not only supported HCV viremia at very low levels but also mounted antigen-specific viral immune response.[125] Although the functionality of the human immune system still requires substantial improvement, these and similar xenotransplant models may become a suitable platform to analyze vaccine induced, human anti-HCV immunity in a small animal model.

Alternatively, mice have been genetically modified to be more permissive for HCV infection. Based on the previous discovery that human CD81 and OCLN constitute the minimal set of entry factors required for viral uptake into rodent cells,[134] mice were engineered to express adenovirally delivered human entry factors.[151] These genetically humanized mice support HCV entry. Importantly, this mouse is fully immunocompetent, and it is thus suitable for immunization and challenge studies. However, in order for this model to gain additional utility, for example, to study virus-induced immunity or to evaluate therapeutic vaccine candidates, the recapitulation of the entire life cycle in an inbred, genetically humanized mouse would be critical. Nevertheless, the introduction of a small animal model with a competent immune system, would be an enabling tool in HCV vaccine research.

Expert Commentary

The field of HCV research has witnessed exciting breakthroughs in recent years. Two specific antiviral inhibitors have reached the market, and many more are at the late stage of development. Combination therapies with the new antivirals are projected to cure HCV infection efficiently in most patients who can afford the treatment. However, the socially disadvantaged, who face the highest risk of HCV infection in developed countries, along with the vast majority in the developing world, are less likely to gain immediate access to the current medical innovations. Thus, a vaccine administered to high-risk populations in both developed and, in particular, developing countries represents a cost-effective alternative in the era of new antivirals. Immunological control of HCV is possible, but the genetic and antigenic diversity of the virus poses a major challenge to vaccine development, yet substantial progress has been made. Various vaccination approaches are being exploited, having yielded several candidates for human trials. Vaccine research is prominently advanced by new methodologies adopted for in vitro and in vivo studies on HCV infection. The introduction of small animal models will enable a better understanding in the immunology of HCV infection and more rigorous preclinical tests on vaccine candidates. If we are experiencing a harvest season for DAAs, the coming one is for HCV vaccines.

Five-year View

Based on data from recently completed and ongoing clinical trials, it is foreseeable that potent combinations of directly acting and/or host-targeting antivirals that can effectively cure chronic HCV infection in most patient population will reach the market. However, predicted high costs and potential side effects requiring medical supervision may lessen the impact of pharmacological intervention in resource-poor environments. By contrast to the diverse portfolio of compounds, which currently fill the drug development pipelines, only few vaccination approaches are being evaluated in clinical trials, making it unlikely that a pan-genotypic vaccine will become accessible within the next half decade. Advances in the ability to grow HCV in vitro has provided critical insights in HCV biology but has also led to the construction of new tools to monitor the efficacy of vaccination approaches. The ban of the use of chimpanzees in many countries or the severely diminished resources allocated to chimpanzee research in others, such as the USA, will undoubtedly slow down the development of prophylactic and therapeutic vaccines. Considerable effort has been made in the development of alternative animal models yet falling short of a fully immunocompetent animal model that is readily susceptible to HCV infection and mimics closely HCV pathogenesis. Continued animal engineering may yield such an in vivo system, which is urgently needed to preclinically evaluate vaccine candidates.

Sidebar
Key Issues
  • Adequate resources need to be devoted to obtain more accurate data on the prevalence and rate of spreading of hepatitis C virus (HCV).

  • The tremendous activity in anti-HCV drug development needs to be extended to HCV vaccine development.

  • Continued research activities to provide further insights into the mechanisms of HCV clearance and persistence.

  • Additional virological tools including a broad array of infectious clones covering all HCV genotypes are needed to effectively test vaccine-induced immunity in vitroand in vivo.

  • Integration of novel computational and structure-based design of vaccine candidates to improve epitope selection. These attempts probably require 3D structures of the viral particle and high-resolution crystal structure of the viral envelope. Furthermore, alternative vaccine delivery vehicles are needed to efficiently induce immunity against HCV.

  • In the absence of chimpanzees, fully immunocompetent small animal models that recapitulate the entire HCV life cycle are urgently needed for testing vaccine candidates, particularly the ones for prophylactic purpose, prior to entering clinical trials.

References
  1. Hoofnagle JH, Mullen KD, Jones DB et al. Treatment of chronic non-A,non-B hepatitis with recombinant human alpha interferon. A preliminary report. N. Engl. J. Med. 315(25), 1575–1578 (1986).

  2. Fried MW, Shiffman ML, Reddy KR et al. Peginterferon α-2a plus ribavirin for chronic hepatitis C virus infection. N. Engl. J. Med. 347(13), 975–982 (2002).

  3. Manns MP, McHutchison JG, Gordon SC et al. Peginterferon α-2b plus ribavirin compared with interferon α-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 358(9286), 958–965 (2001).

  4. McHutchison JG, Everson GT, Gordon SC et al.; PROVE1 Study Team. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N. Engl. J. Med. 360(18), 1827–1838 (2009).

  5. Jacobson IM, McHutchison JG, Dusheiko G et al.; ADVANCE Study Team. Telaprevir for previously untreated chronic hepatitis C virus infection. N. Engl. J. Med. 364(25), 2405–2416 (2011).

  6. Poordad F, McCone J Jr, Bacon BR et al.; SPRINT-2 Investigators. Boceprevir for untreated chronic HCV genotype 1 infection. N. Engl. J. Med. 364(13), 1195–1206 (2011).

  7. Kwo PY, Lawitz EJ, McCone J et al.; SPRINT-1 investigators. Efficacy of boceprevir, an NS3 protease inhibitor, in combination with peginterferon α-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label, randomised, multicentre Phase 2 trial. Lancet 376(9742), 705–716 (2010).

  8. Lok AS, Gardiner DF, Lawitz E et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N. Engl. J. Med. 366(3), 216–224 (2012).

  9. Gane EJ, Roberts SK, Stedman CA et al. Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial. Lancet 376(9751), 1467–1475 (2010).

  10. Chayama K, Takahashi S, Toyota J et al. Dual therapy with the nonstructural protein 5A inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis C virus genotype 1b-infected null responders. Hepatology 55(3), 742–748 (2012).

  11. Alter MJ. Epidemiology of hepatitis C. Hepatology 26(3 Suppl. 1), 62S–65S (1997).

  12. Kershenobich D, Razavi HA, Cooper CL et al. Applying a system approach to forecast the total hepatitis C virus-infected population size: model validation using US data. Liver Int. 31(Suppl. 2), 4–17 (2011).

  13. Volk ML, Tocco R, Saini S, Lok AS. Public health impact of antiviral therapy for hepatitis C in the United States. Hepatology 50(6), 1750–1755 (2009).

  14. Morrill JA, Shrestha M, Grant RW. Barriers to the treatment of hepatitis C. Patient, provider, and system factors. J. Gen. Intern. Med. 20(8), 754–758 (2005).

  15. Liu S, Cipriano LE, Holodniy M, Owens DK, Goldhaber-Fiebert JD. New protease inhibitors for the treatment of chronic hepatitis C: a cost–effectiveness analysis. Ann. Intern. Med. 156(4), 279–290 (2012).

  16. Thomas DL, Seeff LB. Natural history of hepatitis C. Clin. Liver Dis. 9(3), 383–398, vi (2005).

  17. Ward JW, Averhoff FM, Koh HK. World Hepatitis Day: a new era for hepatitis Control. Lancet 378(9791), 552–553 (2011).

  18. Okie S. Fighting HIV – lessons from Brazil. N. Engl. J. Med. 354(19), 1977–1981 (2006).

  19. Bukh J, Miller RH, Purcell RH. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Semin. Liver Dis. 15(1), 41–63 (1995).

  20. Simmonds P. Genetic diversity and evolution of hepatitis C virus – 15 years on. J. Gen. Virol. 85(Pt 11), 3173–3188 (2004).

  21. Murray CL, Rice CM. Turning hepatitis C into a real virus. Annu. Rev. Microbiol. 65, 307–327 (2011).

  22. Welsch C, Jesudian A, Zeuzem S, Jacobson I. New direct-acting antiviral agents for the treatment of hepatitis C virus infection and perspectives. Gut 61(Suppl. 1), i36–i46 (2012).

  23. Lindenbach BD, Evans MJ, Syder AJ et al. Complete replication of hepatitis C virus in cell culture. Science 309(5734), 623–626 (2005).

  24. Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J. Exp. Med. 197(5), 633–642 (2003).

  25. Hsu M, Zhang J, Flint M et al. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc. Natl Acad. Sci. USA 100(12), 7271–7276 (2003).

  26. Yu MY, Bartosch B, Zhang P et al. Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma. Proc. Natl Acad. Sci. USA 101(20), 7705–7710 (2004).

  27. Afdhal NH. The natural history of hepatitis C. Semin. Liver Dis. 24(Suppl. 2), 3–8 (2004).

  28. Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J. Exp. Med. 194(10), 1395–1406 (2001).

  29. Thimme R, Bukh J, Spangenberg HC et al. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc. Natl Acad. Sci. USA 99(24), 15661–15668 (2002).

  30. Lechner F, Wong DK, Dunbar PR et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J. Exp. Med. 191(9), 1499–1512 (2000).

  31. Lauer GM, Barnes E, Lucas M et al. High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection. Gastroenterology 127(3), 924–936 (2004).

  32. Urbani S, Amadei B, Fisicaro P et al. Outcome of acute hepatitis C is related to virus-specific CD4 function and maturation of antiviral memory CD8 responses. Hepatology 44(1), 126–139 (2006).

  33. Shoukry NH, Grakoui A, Houghton M et al. Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection. J. Exp. Med. 197(12), 1645–1655 (2003).

  34. Grakoui A, Shoukry NH, Woollard DJ et al. HCV persistence and immune evasion in the absence of memory T cell help. Science 302(5645), 659–662 (2003).

  35. Farci P, Alter HJ, Govindarajan S et al. Lack of protective immunity against reinfection with hepatitis C virus. Science 258(5079), 135–140 (1992).

  36. Weiner AJ, Paliard X, Selby MJ et al. Intrahepatic genetic inoculation of hepatitis C virus RNA confers cross-protective immunity. J. Virol. 75(15), 7142–7148 (2001).

  37. Bassett SE, Guerra B, Brasky K et al. Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. Hepatology 33(6), 1479–1487 (2001).

  38. Major ME, Mihalik K, Puig M et al. Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge. J. Virol. 76(13), 6586–6595 (2002).

  39. Bukh J, Thimme R, Meunier JC et al. Previously infected chimpanzees are not consistently protected against reinfection or persistent infection after reexposure to the identical hepatitis C virus strain. J. Virol. 82(16), 8183–8195 (2008).

  40. Mehta SH, Cox A, Hoover DR et al. Protection against persistence of hepatitis C. Lancet 359(9316), 1478–1483 (2002).

  41. Osburn WO, Fisher BE, Dowd KA et al. Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection. Gastroenterology 138(1), 315–324 (2010).

  42. Wedemeyer H, He XS, Nascimbeni M et al. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J. Immunol. 169(6), 3447–3458 (2002).

  43. Gerlach JT, Diepolder HM, Jung MC et al. Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C. Gastroenterology 117(4), 933–941 (1999).

  44. Pestka JM, Zeisel MB, Bläser E et al. Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C. Proc. Natl Acad. Sci. USA 104(14), 6025–6030 (2007).

  45. von Hahn T, Yoon JC, Alter H et al. Hepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in vivo. Gastroenterology 132(2), 667–678 (2007).

  46. Zeisel MB, Cosset FL, Baumert TF. Host neutralizing responses and pathogenesis of hepatitis C virus infection. Hepatology 48(1), 299–307 (2008).

  47. Meunier JC, Russell RS, Goossens V et al. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus. J. Virol. 82(2), 966–973 (2008).

  48. Perotti M, Mancini N, Diotti RA et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J. Virol. 82(2), 1047–1052 (2008).

  49. Johansson DX, Voisset C, Tarr AW et al. Human combinatorial libraries yield rare antibodies that broadly neutralize hepatitis C virus. Proc. Natl Acad. Sci. USA 104(41), 16269–16274 (2007).

  50. Law M, Maruyama T, Lewis J et al. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat. Med. 14(1), 25–27 (2008).

  51. Bigger CB, Brasky KM, Lanford RE. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J. Virol. 75(15), 7059–7066 (2001).

  52. Su AI, Pezacki JP, Wodicka L et al. Genomic analysis of the host response to hepatitis C virus infection. Proc. Natl Acad. Sci. USA 99(24), 15669–15674 (2002).

  53. Dustin LB, Rice CM. Flying under the radar: the immunobiology of hepatitis C. Annu. Rev. Immunol. 25, 71–99 (2007).

  54. Suppiah V, Moldovan M, Ahlenstiel G et al. IL28B is associated with response to chronic hepatitis C interferon-α and ribavirin therapy. Nat. Genet. 41(10), 1100–1104 (2009).

  55. Tanaka Y, Nishida N, Sugiyama M et al. Genome-wide association of IL28B with response to pegylated interferon-α and ribavirin therapy for chronic hepatitis C. Nat. Genet. 41(10), 1105–1109 (2009).

  56. Thomas DL, Thio CL, Martin MP et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 461(7265), 798–801 (2009).

  57. Ge D, Fellay J, Thompson AJ et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461(7262), 399–401 (2009).

  58. Khakoo SI, Thio CL, Martin MP et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 305(5685), 872–874 (2004).

  59. Suppiah V, Gaudieri S, Armstrong NJ et al.; International Hepatitis C Genetics Consortium (IHCGC). IL28B, HLA-C, and KIR variants additively predict response to therapy in chronic hepatitis C virus infection in a European Cohort: a cross-sectional study. PLoS Med. 8(9), e1001092 (2011).

  60. Ip PP, Nijman HW, Wilschut J, Daemen T. Therapeutic vaccination against chronic hepatitis C virus infection. Antiviral Res. 96(1), 36–50 (2012).

  61. Bowen DG, Walker CM. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature 436(7053), 946–952 (2005).

  62. Li H, Stoddard MB, Wang S et al. Elucidation of hepatitis C virus transmission and early diversification by single genome sequencing. PLoS Pathog. 8(8), e1002880 (2012).

  63. Choo QL, Kuo G, Ralston R et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc. Natl Acad. Sci. USA 91(4), 1294–1298 (1994).

  64. Forns X, Payette PJ, Ma X 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 32(3), 618–625 (2000).

  65. Frey SE, Houghton M, Coates S et al. Safety and immunogenicity of HCV E1E2 vaccine adjuvanted with MF59 administered to healthy adults. Vaccine 28(38), 6367–6373 (2010).

  66. Stamataki Z, Coates S, Abrignani S, Houghton M, McKeating JA. Immunization of human volunteers with hepatitis C virus envelope glycoproteins elicits antibodies that cross-neutralize heterologous virus strains. J. Infect. Dis. 204(5), 811–813 (2011).

  67. Potter JA, Owsianka AM, Jeffery N et al. Toward a hepatitis C virus vaccine: the structural basis of hepatitis C virus neutralization by AP33, a broadly neutralizing antibody. J. Virol. 86(23), 12923–12932 (2012).

  68. Keck ZY, Xia J, Wang Y et al. Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate. PLoS Pathog. 8(4), e1002653 (2012).

  69. Yutani S, Yamada A, Yoshida K et al. Phase I clinical study of a personalized peptide vaccination for patients infected with hepatitis C virus (HCV) 1b who failed to respond to interferon-based therapy. Vaccine 25(42), 7429–7435 (2007).

  70. Klade CS, Wedemeyer H, Berg T et al. Therapeutic vaccination of chronic hepatitis C nonresponder patients with the peptide vaccine IC41. Gastroenterology 134(5), 1385–1395 (2008).

  71. Habersetzer F, Honnet G, Bain C et al. A poxvirus vaccine is safe, induces T-cell responses, and decreases viral load in patients with chronic hepatitis C. Gastroenterology 141(3), 890.e1–899.e1 (2011).

  72. Feinstone SM, Hu DJ, Major ME. Prospects for prophylactic and therapeutic vaccines against hepatitis C virus. Clin. Infect. Dis. 55(Suppl. 1), S25–S32 (2012).

  73. Sette A, Rappuoli R. Reverse vaccinology: developing vaccines in the era of genomics. Immunity 33(4), 530–541 (2010).

  74. Santolaya ME, O'Ryan ML, Valenzuela MT et al.; V72P10 Meningococcal B Adolescent Vaccine Study group. Immunogenicity and tolerability of a multicomponent meningococcal serogroup B (4CMenB) vaccine in healthy adolescents in Chile: a Phase 2b/3 randomised, observer-blind, placebo-controlled study. Lancet 379(9816), 617–624 (2012).

  75. Scheid JF, Mouquet H, Feldhahn N et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature 458(7238), 636–640 (2009).

  76. Walker LM, Huber M, Doores KJ et al.; Protocol G Principal Investigators. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477(7365), 466–470 (2011).

  77. Wu X, Yang ZY, Li Y et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329(5993), 856–861 (2010).

  78. Walker LM, Phogat SK, Chan-Hui PY et al.; Protocol G Principal Investigators. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326(5950), 285–289 (2009).

  79. Burton DR, Pyati J, Koduri R et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266(5187), 1024–1027 (1994).

  80. Muster T, Steindl F, Purtscher M et al. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67(11), 6642–6647 (1993).

  81. Buchacher A, Predl R, Strutzenberger K et al. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retroviruses 10(4), 359–369 (1994).

  82. Trkola A, Purtscher M, Muster T et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70(2), 1100–1108 (1996).

  83. Van Regenmortel MH. Requirements for empirical immunogenicity trials, rather than structure-based design, for developing an effective HIV vaccine. Arch. Virol. 157(1), 1–20 (2012).

  84. Virgin HW, Walker BD. Immunology and the elusive AIDS vaccine. Nature 464(7286), 224–231 (2010).

  85. Whidby J, Mateu G, Scarborough H, Demeler B, Grakoui A, Marcotrigiano J. Blocking hepatitis C virus infection with recombinant form of envelope protein 2 ectodomain. J. Virol. 83(21), 11078–11089 (2009).

  86. Krey T, d'Alayer J, Kikuti CM et al. The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathog. 6(2), e1000762 (2010).

  87. Giang E, Dorner M, Prentoe JC et al. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus. Proc. Natl Acad. Sci. USA 109(16), 6205–6210 (2012).

  88. Owsianka A, Tarr AW, Juttla VS et al. Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79(17), 11095–11104 (2005).

  89. Broering TJ, Garrity KA, Boatright NK et al. Identification and characterization of broadly neutralizing human monoclonal antibodies directed against the E2 envelope glycoprotein of hepatitis C virus. J. Virol. 83(23), 12473–12482 (2009).

  90. Schofield DJ, Bartosch B, Shimizu YK et al. Human monoclonal antibodies that react with the E2 glycoprotein of hepatitis C virus and possess neutralizing activity. Hepatology 42(5), 1055–1062 (2005).

  91. Keck ZY, Li TK, Xia J et al. Definition of a conserved immunodominant domain on hepatitis C virus E2 glycoprotein by neutralizing human monoclonal antibodies. J. Virol. 82(12), 6061–6066 (2008).

  92. Sabo MC, Luca VC, Prentoe J et al. Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step. J. Virol. 85(14), 7005–7019 (2011).

  93. Fischer W, Perkins S, Theiler J et al. Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nat. Med. 13(1), 100–106 (2007).

  94. Korber BT, Letvin NL, Haynes BF. T-cell vaccine strategies for human immunodeficiency virus, the virus with a thousand faces. J. Virol. 83(17), 8300–8314 (2009).

  95. Yusim K, Fischer W, Yoon H et al. Genotype 1 and global hepatitis C T-cell vaccines designed to optimize coverage of genetic diversity. J. Gen. Virol. 91(Pt 5), 1194–1206 (2010).

  96. Barouch DH, O'Brien KL, Simmons NL et al. Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat. Med. 16(3), 319–323 (2010).

  97. Colloca S, Barnes E, Folgori A et al. Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species. Sci. Transl. Med. 4(115), 115ra2 (2012).

  98. O'Hara GA, Duncan CJ, Ewer KJ et al. Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector. J. Infect. Dis. 205(5), 772–781 (2012).

  99. Sheehy SH, Duncan CJ, Elias SC et al. Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors. PLoS ONE 7(2), e31208 (2012).

  100. Zhou Y, Zhang Y, Yao Z, Moorman JP, Jia Z. Dendritic cell-based immunity and vaccination against hepatitis C virus infection. Immunology 136(4), 385–396 (2012).

  101. Trumpfheller C, Longhi MP, Caskey M et al. Dendritic cell-targeted protein vaccines: a novel approach to induce T-cell immunity. J. Intern. Med. 271(2), 183–192 (2012).

  102. Strickland GT, El-Kamary SS, Klenerman P, Nicosia A. Hepatitis C vaccine: supply and demand. Lancet Infect. Dis. 8(6), 379–386 (2008).

  103. Féray C, Gigou M, Samuel D et al. Incidence of hepatitis C in patients receiving different preparations of hepatitis B immunoglobulins after liver transplantation. Ann. Intern. Med. 128(10), 810–816 (1998).

  104. Davis GL. Hepatitis C immune globulin to prevent HCV recurrence after liver transplantation: chasing windmills? Liver Transpl. 12(9), 1317–1319 (2006).

  105. Balazs AB, Chen J, Hong CM, Rao DS, Yang L, Baltimore D. Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature 481(7379), 81–84 (2012).

  106. Ohira M, Ishiyama K, Tanaka Y et al. Adoptive immunotherapy with liver allograft-derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice. J. Clin. Invest. 119(11), 3226–3235 (2009).

  107. Doskali M, Tanaka Y, Ohira M et al. Possibility of adoptive immunotherapy with peripheral blood-derived CD3CD56+ and CD3+CD56+ cells for inducing antihepatocellular carcinoma and antihepatitis C virus activity. J. Immunother. 34(2), 129–138 (2011).

  108. Jameson SC. Maintaining the norm: T-cell homeostasis. Nat. Rev. Immunol. 2(8), 547–556 (2002).

  109. Yamazaki T, Akiba H, Iwai H et al. Expression of programmed death 1 ligands by murine T cells and APC. J. Immunol. 169(10), 5538–5545 (2002).

  110. Sánchez-Fueyo A, Tian J, Picarella D et al. Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance. Nat. Immunol. 4(11), 1093–1101 (2003).

  111. Freeman GJ, Long AJ, Iwai Y et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192(7), 1027–1034 (2000).

  112. Sabatos CA, Chakravarti S, Cha E et al. Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance. Nat. Immunol. 4(11), 1102–1110 (2003).

  113. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat. Rev. Immunol. 10(7), 490–500 (2010).

  114. McMahan RH, Golden-Mason L, Nishimura MI et al. Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity. J. Clin. Invest. 120(12), 4546–4557 (2010).

  115. Wakita T, Pietschmann T, Kato T et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11(7), 791–796 (2005).

  116. Zhong J, Gastaminza P, Cheng G et al. Robust hepatitis C virus infection in vitro. Proc. Natl Acad. Sci. USA 102(26), 9294–9299 (2005).

  117. Owsianka AM, Tarr AW, Keck ZY et al. Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein. J. Gen. Virol. 89(Pt 3), 653–659 (2008).

  118. Haberstroh A, Schnober EK, Zeisel MB et al. Neutralizing host responses in hepatitis C virus infection target viral entry at postbinding steps and membrane fusion. Gastroenterology 135(5), 1719.e1–1728.e1 (2008).

  119. Uebelhoer L, Han JH, Callendret B et al. Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness. PLoS Pathog. 4(9), e1000143 (2008).

  120. Pietschmann T, Kaul A, Koutsoudakis G et al. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc. Natl Acad. Sci. USA 103(19), 7408–7413 (2006).

  121. Gottwein JM, Scheel TK, Jensen TB et al. Development and characterization of hepatitis C virus genotype 1–7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 49(2), 364–377 (2009).

  122. Jensen TB, Gottwein JM, Scheel TK, Hoegh AM, Eugen-Olsen J, Bukh J. Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. J. Infect. Dis. 198(12), 1756–1765 (2008).

  123. Scheel TK, Gottwein JM, Jensen TB et al. Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc. Natl Acad. Sci. USA 105(3), 997–1002 (2008).

  124. Folgori A, Capone S, Ruggeri L et al. A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Nat. Med. 12(2), 190–197 (2006).

  125. Washburn ML, Bility MT, Zhang L et al. A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease. Gastroenterology 140(4), 1334–1344 (2011).

  126. Koutsoudakis G, Kaul A, Steinmann E et al. Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J. Virol. 80(11), 5308–5320 (2006).

  127. Barth H, Schafer C, Adah MI et al. Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate. J. Biol. Chem. 278(42), 41003–41012 (2003).

  128. Molina S, Castet V, Fournier-Wirth C et al. The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus. J. Hepatol. 46(3), 411–419 (2007).

  129. Agnello V, Abel G, Elfahal M, Knight GB, Zhang QX. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl Acad. Sci. USA 96(22), 12766–12771 (1999).

  130. Monazahian M, Böhme I, Bonk S et al. Low density lipoprotein receptor as a candidate receptor for hepatitis C virus. J. Med. Virol. 57(3), 223–229 (1999).

  131. OwenDM , HuangH , YeJ , GaleM Jr. Apolipoprotein E on hepatitis C virion facilitates infection through interaction with low-density lipoprotein receptor. Virology 394(1), 99–108 (2009).

  132. Pileri P, Uematsu Y, Campagnoli S et al. Binding of hepatitis C virus to CD81. Science 282(5390), 938–941 (1998).

  133. Evans MJ, von Hahn T, Tscherne DM et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446(7137), 801–805 (2007).

  134. Ploss A, Evans MJ, Gaysinskaya VA et al. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457(7231), 882–886 (2009).

  135. Liu S, Yang W, Shen L, Turner JR, Coyne CB, Wang T. Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J. Virol. 83(4), 2011–2014 (2009).

  136. Sainz B Jr, Barretto N, Martin DN et al. Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor. Nat. Med. 18(2), 281–285 (2012).

  137. Lupberger J, Zeisel MB, Xiao F et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat. Med. 17(5), 589–595 (2011).

  138. Ploss A, Evans MJ. Hepatitis C virus host cell entry. Curr. Opin. Virol. 2(1), 14–19 (2012).

  139. Bitzegeio J, Bankwitz D, Hueging K et al. Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors. PLoS Pathog. 6, e1000978 (2010).

  140. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J. Immunol. 172(5), 2731–2738 (2004).

  141. Waterston RH, Lander ES, Sulston JE. On the sequencing of the human genome. Proc. Natl Acad. Sci. USA 99(6), 3712–3716 (2002).

  142. Abe K, Kurata T, Teramoto Y, Shiga J, Shikata T. Lack of susceptibility of various primates and woodchucks to hepatitis C virus. J. Med. Primatol. 22(7–8), 433–434 (1993).

  143. Patel MR, Loo YM, Horner SM, Gale M Jr, Malik HS. Convergent evolution of escape from hepaciviral antagonism in primates. PLoS Biol. 10(3), e1001282 (2012).

  144. Hatziioannou T, Ambrose Z, Chung NP et al. A macaque model of HIV-1 infection. Proc. Natl Acad. Sci. USA 106(11), 4425–4429 (2009).

  145. HatziioannouT , PrinciottaM , PiatakM Jr et al. Generation of simian-tropic HIV-1 by restriction factor evasion. Science 314(5796), 95 (2006).

  146. Mercer DF, Schiller DE, Elliott JF et al. Hepatitis C virus replication in mice with chimeric human livers. Nat. Med. 7(8), 927–933 (2001).

  147. Meuleman P, Libbrecht L, De Vos R et al. Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera. Hepatology 41(4), 847–856 (2005).

  148. Azuma H, Paulk N, Ranade A et al. Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice. Nat. Biotechnol. 25(8), 903–910 (2007).

  149. Bissig KD, Le TT, Woods NB, Verma IM. Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model. Proc. Natl Acad. Sci. USA 104(51), 20507–20511 (2007).

  150. Bissig KD, Wieland SF, Tran P et al. Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J. Clin. Invest. 120(3), 924–930 (2010).

  151. Dorner M, Horwitz JA, Robbins JB et al. A genetically humanized mouse model for hepatitis C virus infection. Nature 474(7350), 208–211 (2011).

  152. Perz JF, Farrington LA, Pecoraro C, Hutin YJF, Armstrong GL. Estimated global prevalence of hepatitis C virus infection. Presented at: 42nd Annual Meeting of the Infectious Diseases Society of America. Boston, MA, USA, 30 September–3 October 2004.

  153. Armstrong GL, Wasley A, Simard EP, McQuillan GM, Kuhnert WL, Alter MJ. The prevalence of hepatitis C virus infection in the United States, 1999 through 2002. Ann. Intern. Med. 144(10), 705–714 (2006).

  154. Villano SA, Vlahov D, Nelson KE, Cohn S, Thomas DL. Persistence of viremia and the importance of long-term follow-up after acute hepatitis C infection. Hepatology 29(3), 908–914 (1999).
    Websites
    201. ClinicalTrials.gov Identifier: NCT00500747. www.clinicaltrials.gov
    202. ClinicalTrials.gov Identifier: NCT01436357. www.clinicaltrials.gov
    203. ClinicalTrials.gov Identifier: NCT01070407. www.clinicaltrials.gov
    204. NIH. Grants and funding. http://grants.nih.gov/grants/guide/notice-files/NOT-OD-12-025.html
    205. ClinicalTrials.gov. www.clinicaltrials.gov

Acknowledgements
The authors thank Naglaa Shoukry and Leonia Bozzacco for critical reading of the manuscript.

Expert Rev Gastroenterol Hepatol. 2013;7(2):171-185. © 2013 Expert Reviews Ltd.

Source