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Host-targeting agents for prevention and treatment of chronic hepatitis C – Perspectives and challenges

Open AccessPublished:October 08, 2012DOI:https://doi.org/10.1016/j.jhep.2012.09.022

      Summary

      Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. Furthermore, HCV-induced liver disease is a major indication of liver transplantation. In the past years, direct-acting antivirals (DAAs) targeting HCV enzymes have been developed. DAAs increase the virologic response to anti-HCV therapy but may lead to selection of drug-resistant variants and treatment failure. To date, strategies to prevent HCV infection are still lacking and antiviral therapy in immunocompromised patients, patients with advanced liver disease and HIV/HCV-co-infection remains limited. Alternative or complementary approaches addressing the limitations of current antiviral therapies are to boost the host’s innate immunity or interfere with host factors required for pathogenesis. Host-targeting agents (HTAs) provide an interesting perspective for novel antiviral strategies against viral hepatitis since they have (i) a high genetic barrier to resistance, (ii) a pan-genotypic antiviral activity, and (iii) complementary mechanisms of action to DAAs and might therefore act in a synergistic manner with current standard of care or DAAs in clinical development. This review highlights HTAs against HCV infection that have potential as novel antivirals and are in preclinical or clinical development.

      Abbreviations:

      DAA (direct-acting antiviral), HBV (hepatitis B virus), HCV (hepatitis C virus), HIV (human immunodeficiency virus), HTA (host-targeting agent), IFN (interferon), miR (microRNA), RBV (ribavirin)

      Keywords

      Introduction

      With approximately 130 million chronically infected individuals worldwide, hepatitis C virus (HCV) infection is a major cause of chronic liver disease including liver cirrhosis, liver failure and hepatocellular carcinoma (HCC) [
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      ]. HCV-induced liver cirrhosis and HCC are major indications for liver transplantation (LT) [
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      Management of HBV- and HCV-induced end stage liver disease.
      ]. Thus, HCV-induced liver disease is a major challenge for public health [
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      HCV is a single-stranded RNA virus of positive polarity belonging to the Flaviviridae family and the hepacivirus genus (reviewed in [
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      ]). While six major genotypes and several different subtypes have been described worldwide, the virus also circulates as a quasispecies within a given infected individual. This high variability represents a challenge for preventive and therapeutic antiviral strategies as the virus may rapidly evade the host immune responses and antivirals [
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      ]. The current standard of care (SOC) of chronic HCV infection consists of pegylated interferon-α (PegIFN-α) and ribavirin (RBV). Moreover, since 2011, the new SOC for HCV genotype 1 infected patients is a triple combination of PegIFN-α/RBV and a HCV protease inhibitor (telaprevir or boceprevir). Although the addition of these direct-acting antivirals (DAAs) improves outcome, an important limitation of these DAAs that may contribute to therapy failure is their low genetic barrier for resistance resulting in drug-escape mutants during long-term treatment due to their general mechanism of action [
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      ] and without imposing a large viral fitness cost. DAAs are not approved for LT [
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      ] and IFN-α-based antiviral therapies have limited efficacy and tolerability in LT recipients. In addition to licensed DAAs, other DAAs are at various stages of clinical development in combination with PegIFN-α or in IFN-free regimens, including second-generation protease inhibitors, polymerase and non-structural protein 5A (NS5A) inhibitors. Although a rapid decline in HCV RNA levels and/or eradication of HCV in IFN-free regimens have been demonstrated in clinical trials, viral breakthroughs due to the selection of HCV resistant variants as well as differences in virological outcomes for different genotypes and subtypes have been reported. Furthermore, many of these drugs were associated with side effects and raised issues related to drug–drug interactions [
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      ]. Finally, it is not yet clear whether DAA-based therapies will be effective in difficult-to-treat patients, such as null responders to prior PegIFN-α/RBV therapy, patients with advanced liver disease, LT recipients, HIV/HCV-co-infected individuals, hemodialysis patients, or immunosuppressed patients [
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      ].
      Another challenge in the management of chronically infected patients is the absence of strategies for prevention of liver graft infection. Development of preventive strategies based on anti-HCV envelope antibodies has been challenged by the high variability of HCV, resulting in rapid viral escape [
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      Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation.
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      • Fofana I.
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      • Fauvelle C.
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      Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.
      ]. Proof-of-concept of broadly cross-neutralizing antibodies in humans remains to be demonstrated. Thus, there is an unmet medical need for efficient and safe antiviral strategies for difficult-to-treat patients and for prevention of HCV graft infection during LT.
      Recent proof-of-concept studies in preclinical models and clinical trials have highlighted that host-targeting agents (HTAs) provide a novel and promising strategy to address current unmet medical needs and limitations of SOC. Two main concepts for HTAs are explored: the first strategy aims at interfering with host factors required for pathogenesis, i.e., to target host factors indispensible for the viral life cycle. These include host cell entry, replication and assembly factors. The second strategy is to target the host by boosting the host’s innate immunity, e.g., through the administration of IFN-λ [
      • Zeuzem S.
      • Arora S.
      • Bacon B.
      • Box T.
      • Charlton M.
      • Diago M.
      • et al.
      Pegylated interferon-lambda (PEGIFN-λ) shows superior viral response with improved safety and tolerability versus PEGIFNα-2A in HCV patients (Gl/2/3/4): EMERGE Phase IIB through week 12.
      ] or Toll-like receptor (TLR) agonists [
      • Boonstra A.
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      • Bergmann J.F.
      • de Bruijne J.
      • Hotho D.M.
      • et al.
      Potent immune activation in chronic hepatitis C patients upon administration of an oral inducer of endogenous interferons that acts via Toll-like receptor 7.
      ,
      • Rodriguez-Torres M.
      • Ghalib R.H.
      • Gordon S.C.
      • Lawitz E.
      • Patel K.
      • Pruitt R.
      • et al.
      IMO-2125, a TLR9 agonist, induces immune responses which correlate with reductions in viral load in null responder HCV patients.
      ,
      • Zhang X.
      • Kraft A.
      • Broering R.
      • Schlaak J.F.
      • Dittmer U.
      • Lu M.
      Preclinical development of TLR ligands as drugs for the treatment of chronic viral infections.
      ].
      HTAs offer a promising perspective due to the following features distinguishing them from DAAs: compared to the viral variability, genetic variability of the host is low. Thus, HTAs impose a very high genetic barrier to resistance [
      • Fafi-Kremer S.
      • Fofana I.
      • Soulier E.
      • Carolla P.
      • Meuleman P.
      • Leroux-Roels G.
      • et al.
      Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation.
      ,
      • Fofana I.
      • Fafi-Kremer S.
      • Carolla P.
      • Fauvelle C.
      • Zahid M.N.
      • Turek M.
      • et al.
      Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.
      ,
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ,
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Li B.
      • Snoeck J.
      • Tang Y.
      • Jones C.T.
      • Tiongyip C.
      • Bao W.
      • et al.
      Alisporivir – a host-targeting antiviral, provides low viral breakthrough rate and high barrier to resistance in HCV genotype 1 treatment-naïve patients in the Phase IIb ESSENTIAL study.
      ,
      • Janssen H.L.
      • Reesink H.W.
      • Zeuzem S.
      • Lawitz E.
      • Rodriguez-Torres M.
      • Chen A.
      • et al.
      A randomized, double-blind, placebo (plb) controlled safety and anti-viral proof-of-concept study of miravirsen (mir), an oligonucleotide targeting miR122, in treatment naive patients with genotype 1 (gt1) chronic HCV infection.
      ]. As HTAs are essential for the viral life cycle, HTAs are characterized by a broad pan-genotypic activity while first generation DAAs targeting HCV are characterized by a very narrow antiviral activity limited to genotype 1. Indeed, HTAs have been shown to inhibit infection by HCV of all major genotypes, highly variable quasispecies isolated from individual patients and highly infectious escape variants that are resistant to host neutralizing antibodies [
      • Fafi-Kremer S.
      • Fofana I.
      • Soulier E.
      • Carolla P.
      • Meuleman P.
      • Leroux-Roels G.
      • et al.
      Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation.
      ,
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ,
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Jopling C.L.
      • Yi M.
      • Lancaster A.M.
      • Lemon S.M.
      • Sarnow P.
      Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA.
      ,
      • Li Y.P.
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Bukh J.
      MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.
      ,
      • Syder A.J.
      • Lee H.
      • Zeisel M.B.
      • Grove J.
      • Soulier E.
      • Macdonald J.
      • et al.
      Small molecule scavenger receptor BI antagonists are potent HCV entry inhibitors.
      ,
      • Zahid M.N.
      • Turek M.
      • Xiao F.
      • Dao Thi V.L.
      • Guérin M.
      • Fofana I.
      • et al.
      The post-binding activity of scavenger receptor BI mediates initiation of hepatitis C virus infection and viral dissemination.
      ]. Finally, by acting through a complementary mechanism of action, HTAs may synergistically act with current anti-HCV SOC [
      • Fofana I.
      • Xiao F.
      • Thumann C.
      • Lupberger J.
      • Leyssen P.
      • Neyts J.H.
      • et al.
      Synergy of entry inhibitors and direct acting antivirals or interferon-alfa identifies novel antiviral combinations for hepatitis C virus infection.
      ,
      • Zhu H.
      • Wong-Staal F.
      • Lee H.
      • Syder A.
      • McKelvy J.
      • Schooley R.T.
      • et al.
      Evaluation of ITX 5061, a scavenger receptor B1 antagonist: resistance selection and activity in combination with other hepatitis C virus antivirals.
      ]. It is expected that this synergy will increase the genetic barrier for resistance, shorten treatment schedules and ameliorate adverse effects by reducing the doses of the individual compounds.
      This review will highlight recent progress in the development of HTAs targeting HCV infection that have the potential to clear chronic HCV infection or prevent HCV infection of the liver graft.

      Host-targeting agents against hepatitis C virus infection

      The HCV life cycle may be divided into three main steps: viral entry into the target cell, viral replication as well as assembly and release of new infectious virions (Fig. 1). Each step of the HCV life cycle is dependent on host cell factors [
      • Da Costa D.
      • Turek M.
      • Felmlee D.J.
      • Girardi E.
      • Pfeffer S.
      • Long G.
      • et al.
      Reconstitution of the entire hepatitis C virus life cycle in non-hepatic cells.
      ], thereby offering numerous targets for HTAs (Fig. 1, Fig. 2, Fig. 3 and Table 1).
      Figure thumbnail gr1
      Fig. 1Host factors required for the hepatitis C virus life cycle as antiviral targets. Outline of the hepatitis C virus (HCV) life cycle in polarized hepatocytes. Host-targeting agents (HTAs) and biological response modifiers (BRMs) are indicated in the figure according to their presumable point of interference with the viral life cycle. ER, endoplasmic reticulum; HS, heparan sulfate proteoglycans; RTKs, receptor tyrosine kinases; SR-BI, scavenger receptor BI; CD81, cluster of differentiation 81; CLDN1, claudin-1; OCLN, occludin; NPC1L1, Niemann-Pick C1-like 1 cholesterol absorption receptor; apo, apolipoprotein; BC, bile canaliculus; TJ, tight junction; Ab, antibody; miR, microRNA; HMGCoA, 3-hydroxy-3-methyglutaryl CoA reductase; MTP, microsomal triglyceride transfer protein; TLR, Toll-like receptor; IFN, interferon.
      Figure thumbnail gr2
      Fig. 2Host-targeting entry inhibitors for prevention of HCV liver graft infection. During liver transplantation, highly infectious variants of the HCV quasispecies escaping from the host neutralizing antibodies (nAbs) infect the liver graft. This “bottleneck” effect is related to the implantation of a new graft and the lack of selective pressure due to the strong immunosuppression (inset). The inset shows the mechanism of re-infection of naïve hepatocytes and viral spread in the liver graft. HCV variants may spread (i) by cell-free transmission and (ii) by cell–cell transmission. As a consequence, highly infectious HCV variants escaping the host neutralizing immune response are selected during re-infection of the new liver graft through a “bottleneck” effect [
      • Fafi-Kremer S.
      • Fofana I.
      • Soulier E.
      • Carolla P.
      • Meuleman P.
      • Leroux-Roels G.
      • et al.
      Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation.
      ,
      • Fofana I.
      • Fafi-Kremer S.
      • Carolla P.
      • Fauvelle C.
      • Zahid M.N.
      • Turek M.
      • et al.
      Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.
      ]. HCV entry factors are required for both ways of transmission and are targets of HTAs. Entry HTAs targeting HCV entry factors inhibit HCV entry and spread of all major genotype as well as of HCV escape variants that re-infect the liver graft [
      • Fafi-Kremer S.
      • Fofana I.
      • Soulier E.
      • Carolla P.
      • Meuleman P.
      • Leroux-Roels G.
      • et al.
      Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation.
      ,
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ,
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Syder A.J.
      • Lee H.
      • Zeisel M.B.
      • Grove J.
      • Soulier E.
      • Macdonald J.
      • et al.
      Small molecule scavenger receptor BI antagonists are potent HCV entry inhibitors.
      ,
      • Zeisel M.B.
      • Zahid M.N.
      • Xiao F.
      • Dao Thi V.L.
      • Cosset F.-L.
      • Fofana I.
      • et al.
      Monoclonal antibodies specific for the SR-BI N-terminal ectodomain block hepatitis C virus entry into human hepatocytes at postbinding steps and cell–cell transmission.
      ].
      Figure thumbnail gr3
      Fig. 3Host-targeting agents exhibit a high genetic barrier of resistance. HCV lipoviral particles circulate as quasispecies of viral variants that infect and replicate in hepatocytes. The mechanism of viral escape to drug therapy differs between direct-acting antivirals (DAAs) and host targeting agents (HTAs). (Left panel) DAAs efficiently inhibit the replication of DAA-sensitive HCV variants. An HCV variant that is resistant to DAA treatment becomes the predominant HCV variant escaping the antiviral treatment. (Right panel) Targeting host factors required for HCV entry and infection inhibits a broader spectrum of variants and genotypes since the host factor usage is usually highly conserved for all viral variants. As a consequence, the genetic barrier of viral resistance to HTAs can be higher compared to many DAAs.
      Table 1Host-targeting agents against hepatitis C virus infection.

      Entry inhibitors

      Viral entry is the first step of HCV-host cell interactions and involves the HCV envelope glycoproteins E1 and E2 as well as several host factors. It is believed that cell-free HCV entry is a highly coordinated multistep process (Fig. 1). Highly sulfated heparan sulfate proteoglycans [
      • Barth H.
      • Schäfer C.
      • Adah M.I.
      • Zhang F.
      • Linhardt R.J.
      • Toyoda H.
      • et al.
      Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.
      ] represent first attachment sites, allowing viral concentration on the basolateral hepatocyte membrane. The virus then interacts with several entry factors including scavenger receptor BI (SR-BI) [
      • Scarselli E.
      • Ansuini H.
      • Cerino R.
      • Roccasecca R.M.
      • Acali S.
      • Filocamo G.
      • et al.
      The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
      ], CD81 [
      • Pileri P.
      • Uematsu Y.
      • Campagnoli S.
      • Galli G.
      • Falugi F.
      • Petracca R.
      • et al.
      Binding of hepatitis C virus to CD81.
      ], claudin-1 (CLDN1) [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ] and occludin (OCLN) [
      • Ploss A.
      • Evans M.J.
      • Gaysinskaya V.A.
      • Panis M.
      • You H.
      • de Jong Y.P.
      • et al.
      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ]. The formation of CD81-CLDN1 complexes is essential for HCV infection [
      • Harris H.J.
      • Farquhar M.J.
      • Mee C.J.
      • Davis C.
      • Reynolds G.M.
      • Jennings A.
      • et al.
      CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry.
      ,
      • Harris H.J.
      • Davis C.
      • Mullins J.G.
      • Hu K.
      • Goodall M.
      • Farquhar M.J.
      • et al.
      Claudin association with CD81 defines hepatitis C virus entry.
      ]. In addition, host cell kinases play an important role in regulating the HCV entry process [
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Trotard M.
      • Lepere-Douard C.
      • Regeard M.
      • Piquet-Pellorce C.
      • Lavillette D.
      • Cosset F.L.
      • et al.
      Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening.
      ,
      • Farquhar M.J.
      • Harris H.J.
      • Diskar M.
      • Jones S.
      • Mee C.J.
      • Nielsen S.U.
      • et al.
      Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity.
      ]. Among them, two cell surface receptor tyrosine kinases (RTKs) have been identified as HCV entry factors: epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2). EGFR and EphA2 promote CD81-CLDN1 co-receptor interaction that is required for HCV entry [
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ]. The Niemann-Pick C1-Like1 (NPC1L1) cholesterol absorption receptor has recently been proposed as another host entry co-factor [
      • Sainz Jr., B.
      • Barretto N.
      • Martin D.N.
      • Hiraga N.
      • Imamura M.
      • Hussain S.
      • et al.
      Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor.
      ]. Given its physiological role, NPC1L1 may promote HCV entry either directly by interacting with the HCV lipoviral particle cholesterol or act as indirect entry factor by modulating cholesterol homeostasis and membrane composition required for HCV entry. HCV is internalized via clathrin- and dynamin-dependent endocytosis and is subsequently delivered to the early endosome [
      • Blanchard E.
      • Belouzard S.
      • Goueslain L.
      • Wakita T.
      • Dubuisson J.
      • Wychowski C.
      • et al.
      Hepatitis C virus entry depends on clathrin-mediated endocytosis.
      ,
      • Codran A.
      • Royer C.
      • Jaeck D.
      • Bastien-Valle M.
      • Baumert T.F.
      • Kieny M.P.
      • et al.
      Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis.
      ,
      • Meertens L.
      • Bertaux C.
      • Dragic T.
      Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles.
      ,
      • Farquhar M.J.
      • Hu K.
      • Harris H.J.
      • Davis C.
      • Brimacombe C.L.
      • Fletcher S.J.
      • et al.
      Hepatitis C virus induces CD81 and claudin-1 endocytosis.
      ]. CD81 and CLDN1 associate during internalization [
      • Farquhar M.J.
      • Hu K.
      • Harris H.J.
      • Davis C.
      • Brimacombe C.L.
      • Fletcher S.J.
      • et al.
      Hepatitis C virus induces CD81 and claudin-1 endocytosis.
      ,
      • Coller K.E.
      • Berger K.L.
      • Heaton N.S.
      • Cooper J.D.
      • Yoon R.
      • Randall G.
      RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
      ], but it remains unclear whether other HCV host factors internalize together with HCV. Although required for CD81-CLDN1 interaction, EGFR does not seem to be essential for CD81 internalization [
      • Farquhar M.J.
      • Hu K.
      • Harris H.J.
      • Davis C.
      • Brimacombe C.L.
      • Fletcher S.J.
      • et al.
      Hepatitis C virus induces CD81 and claudin-1 endocytosis.
      ]. The fusion of the viral and the endosomal membrane is pH-dependent and involves both viral and host proteins [
      • Blanchard E.
      • Belouzard S.
      • Goueslain L.
      • Wakita T.
      • Dubuisson J.
      • Wychowski C.
      • et al.
      Hepatitis C virus entry depends on clathrin-mediated endocytosis.
      ,
      • Tscherne D.M.
      • Jones C.T.
      • Evans M.J.
      • Lindenbach B.D.
      • McKeating J.A.
      • Rice C.M.
      Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.
      ,
      • Lavillette D.
      • Bartosch B.
      • Nourrisson D.
      • Verney G.
      • Cosset F.L.
      • Penin F.
      • et al.
      Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes.
      ,
      • Lavillette D.
      • Pecheur E.I.
      • Donot P.
      • Fresquet J.
      • Molle J.
      • Corbau R.
      • et al.
      Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus.
      ]. Among host entry factors, CD81 and CLDN1 play a role in the HCV envelope glycoprotein-dependent cell–cell fusion process [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Kobayashi M.
      • Bennett M.C.
      • Bercot T.
      • Singh I.R.
      Functional analysis of hepatitis C virus envelope proteins, using a cell–cell fusion assay.
      ], which is regulated by RTK function [
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ].
      An alternative route of viral entry is direct cell–cell transmission, which also requires numerous host factors including CD81, SR-BI, CLDN1, OCLN, EGFR, EphA2 and potentially NPC1L1 [
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Sainz Jr., B.
      • Barretto N.
      • Martin D.N.
      • Hiraga N.
      • Imamura M.
      • Hussain S.
      • et al.
      Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor.
      ,
      • Timpe J.M.
      • Stamataki Z.
      • Jennings A.
      • Hu K.
      • Farquhar M.J.
      • Harris H.J.
      • et al.
      Hepatitis C virus cell–cell transmission in hepatoma cells in the presence of neutralizing antibodies.
      ,
      • Brimacombe C.L.
      • Grove J.
      • Meredith L.W.
      • Hu K.
      • Syder A.J.
      • Flores M.V.
      • et al.
      Neutralizing antibody-resistant hepatitis C virus cell-to-cell transmission.
      ]. As this entry route is resistant to the majority of neutralizing antibodies described so far, direct cell–cell transmission probably represents the main process of viral spread [
      • Timpe J.M.
      • Stamataki Z.
      • Jennings A.
      • Hu K.
      • Farquhar M.J.
      • Harris H.J.
      • et al.
      Hepatitis C virus cell–cell transmission in hepatoma cells in the presence of neutralizing antibodies.
      ,
      • Brimacombe C.L.
      • Grove J.
      • Meredith L.W.
      • Hu K.
      • Syder A.J.
      • Flores M.V.
      • et al.
      Neutralizing antibody-resistant hepatitis C virus cell-to-cell transmission.
      ]. It is worth noting that there is an overlap of host factors required for cell-free and cell–cell transmission, as most of the host factors involved in cell-free entry have also been described to play a role in cell–cell transmission.
      Targeting HCV entry factors may thus allow preventing initiation of HCV infection, such as after LT, and also reduce viral spread and thus maintenance of infection. However, while cell-free HCV entry is strictly dependent on CD81, CD81-independent routes of cell–cell transmission have been described [
      • Witteveldt J.
      • Evans M.J.
      • Bitzegeio J.
      • Koutsoudakis G.
      • Owsianka A.M.
      • Angus A.G.
      • et al.
      CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells.
      ,
      • Jones C.T.
      • Catanese M.T.
      • Law L.M.
      • Khetani S.R.
      • Syder A.J.
      • Ploss A.
      • et al.
      Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system.
      ]. This has to be taken into account for the development of HTA directed against HCV entry factors.
      Viral entry has been shown to play an important role in the pathogenesis of HCV infection, especially during HCV reinfection of the graft after LT [
      • Fafi-Kremer S.
      • Fofana I.
      • Soulier E.
      • Carolla P.
      • Meuleman P.
      • Leroux-Roels G.
      • et al.
      Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation.
      ,
      • Fofana I.
      • Fafi-Kremer S.
      • Carolla P.
      • Fauvelle C.
      • Zahid M.N.
      • Turek M.
      • et al.
      Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.
      ]. Viral entry is thus a very promising target for prevention of HCV infection of the liver graft (Fig. 2). Numerous HTAs directed against host entry factors demonstrated a potent antiviral activity in vitro (reviewed in [
      • Zeisel M.B.
      • Fofana I.
      • Fafi-Kremer S.
      • Baumert T.F.
      Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies.
      ]). Proof-of-concept studies of HTAs targeting HCV entry have been conducted in vivo using the chimeric uPA-SCID mouse model. Antibodies directed against CD81 and SR-BI have both been investigated in prophylactic and post-exposure treatment studies. Administration of 400 μg of either anti-CD81 or anti-SRBI monoclonal antibodies (mAbs) completely protected mice from challenge with HCV [
      • Meuleman P.
      • Hesselgesser J.
      • Paulson M.
      • Vanwolleghem T.
      • Desombere I.
      • Reiser H.
      • et al.
      Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo.
      ,
      • Meuleman P.
      • Catanese M.T.
      • Verhoye L.
      • Desombere I.
      • Farhoudi A.
      • Jones C.T.
      • et al.
      A human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo.
      ,
      • Lacek K.
      • Vercauteren K.
      • Grzyb K.
      • Naddeo M.
      • Verhoye L.
      • Slowikowski M.P.
      • et al.
      Novel human SR-BI antibodies prevent infection and dissemination of HCV in vitro and in humanized mice.
      ]. Noteworthy, only the administration of anti-SRBI mAb was able to reduce viral dissemination [
      • Meuleman P.
      • Catanese M.T.
      • Verhoye L.
      • Desombere I.
      • Farhoudi A.
      • Jones C.T.
      • et al.
      A human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo.
      ,
      • Lacek K.
      • Vercauteren K.
      • Grzyb K.
      • Naddeo M.
      • Verhoye L.
      • Slowikowski M.P.
      • et al.
      Novel human SR-BI antibodies prevent infection and dissemination of HCV in vitro and in humanized mice.
      ]. The clinically approved EGFR inhibitor erlotinib, preventing the formation of CLDN1-CD81 complexes, and NPC1L1 inhibitor ezetimibe, that decreases systemic cholesterol in patients, markedly impaired the establishment of HCV infection in the uPA-SCID mouse model [
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Sainz Jr., B.
      • Barretto N.
      • Martin D.N.
      • Hiraga N.
      • Imamura M.
      • Hussain S.
      • et al.
      Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor.
      ]. Indeed, administration of erlotinib (50 mg/kg/day for 10 days) or ezetimibe (10 mg/kg/day for 2 weeks) prior to viral inoculation significantly delayed the kinetics of HCV infection [
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Sainz Jr., B.
      • Barretto N.
      • Martin D.N.
      • Hiraga N.
      • Imamura M.
      • Hussain S.
      • et al.
      Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor.
      ]. The clinical potential of kinase inhibitors has been emphasized in a recent case report describing rapid virologic response (RVR) after erlotinib monotherapy (150 mg/day for 12 months) in a HCV-positive HCC patient after LT and viral recurrence due to a discontinued SOC treatment [
      • Bardou-Jacquet E.
      • Lorho R.
      • Guyader D.
      Kinase inhibitors in the treatment of chronic hepatitis C virus.
      ]. A clinical trial investigating safety and toxicity of erlotinib in chronically HCV infected patients will soon be conducted to further assess the potential of kinase inhibitors as anti-HCV drugs in combination with DAAs. A phase 1b study assessing the safety of ITX 5061 [
      • Syder A.J.
      • Lee H.
      • Zeisel M.B.
      • Grove J.
      • Soulier E.
      • Macdonald J.
      • et al.
      Small molecule scavenger receptor BI antagonists are potent HCV entry inhibitors.
      ], a small molecule inhibitor targeting the HCV entry factor SR-BI, in HCV-treatment naive patients, is ongoing and an open-label, proof-of-concept phase 1b study assessing the safety and tolerability of ITX 5061 in LT patients has been initiated (Table 1).
      Figure thumbnail fx2

      HCV replication inhibitors

      Following HCV entry, the HCV RNA genome is released into the cytosol. Initiation of HCV translation occurs through binding of the 40S ribosomal subunit to the HCV IRES and this association can be enhanced by miR-122, a liver-specific microRNA (miRNA) [
      • Henke J.I.
      • Goergen D.
      • Zheng J.
      • Song Y.
      • Schuttler C.G.
      • Fehr C.
      • et al.
      MicroRNA-122 stimulates translation of hepatitis C virus RNA.
      ,
      • Jangra R.K.
      • Yi M.
      • Lemon S.M.
      Regulation of hepatitis C virus translation and infectious virus production by the microRNA miR-122.
      ]. miR-122 is also an important host factor for HCV replication [
      • Jopling C.L.
      • Yi M.
      • Lancaster A.M.
      • Lemon S.M.
      • Sarnow P.
      Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA.
      ] and miR-122 sequestration using 122–2′OMe oligomers or miR-122 antisense locked nucleic acid SPC3649 reduces HCV replication in a genotype-independent manner in vitro [
      • Jopling C.L.
      • Yi M.
      • Lancaster A.M.
      • Lemon S.M.
      • Sarnow P.
      Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA.
      ,
      • Li Y.P.
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Bukh J.
      MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.
      ]. Interestingly, weekly intravenous administration of miR-122 antisense locked nucleic acid miravirsen/SPC3649 (5 mg/kg) for 12 weeks to chronically genotype 1 infected chimpanzees led to sustained suppression of HCV viremia, with no evidence of viral resistance [
      • Lanford R.E.
      • Hildebrandt-Eriksen E.S.
      • Petri A.
      • Persson R.
      • Lindow M.
      • Munk M.E.
      • et al.
      Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.
      ]. Given the physiological role of miR-122 in cholesterol metabolism, miravirsen/SPC3649 led to markedly lowered serum cholesterol in animals but no important adverse effects were observed [
      • Lanford R.E.
      • Hildebrandt-Eriksen E.S.
      • Petri A.
      • Persson R.
      • Lindow M.
      • Munk M.E.
      • et al.
      Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.
      ,
      • Elmen J.
      • Lindow M.
      • Schutz S.
      • Lawrence M.
      • Petri A.
      • Obad S.
      • et al.
      LNA-mediated microRNA silencing in non-human primates.
      ,
      • Hildebrandt-Eriksen E.S.
      • Aarup V.
      • Persson R.
      • Hansen H.F.
      • Munk M.E.
      • Orum H.
      A locked nucleic acid oligonucleotide targeting microRNA 122 is well-tolerated in Cynomolgus monkeys.
      ]. Recently, the safety, tolerability and efficacy of miravirsen/SPC3649 have been assessed in a phase 2a study (Table 1). Miravirsen/SPC3649 given as a four-week monotherapy (3, 5 and 7 mg/kg) to treatment-naïve genotype 1 patients was well tolerated and provided robust, dose-dependent antiviral activity that was maintained for more than four weeks after the end of therapy [
      • Janssen H.L.
      • Reesink H.W.
      • Zeuzem S.
      • Lawitz E.
      • Rodriguez-Torres M.
      • Chen A.
      • et al.
      A randomized, double-blind, placebo (plb) controlled safety and anti-viral proof-of-concept study of miravirsen (mir), an oligonucleotide targeting miR122, in treatment naive patients with genotype 1 (gt1) chronic HCV infection.
      ]. Four out of nine patients treated at the highest dose with miravirsen/SPC3649 (7 mg/kg) became HCV RNA undetectable during the study. Although markedly decreased pretreatment miR-122 levels had been reported in livers of chronic HCV infected patients who did not achieve virological response during IFN therapy [
      • Sarasin-Filipowicz M.
      • Krol J.
      • Markiewicz I.
      • Heim M.H.
      • Filipowicz W.
      Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy.
      ], data from this first clinical trial indicate that targeting miR-122 in vivo offers a high barrier to viral resistance and the potential for combination in a future IFN-free regimen [
      • Janssen H.L.
      • Reesink H.W.
      • Zeuzem S.
      • Lawitz E.
      • Rodriguez-Torres M.
      • Chen A.
      • et al.
      A randomized, double-blind, placebo (plb) controlled safety and anti-viral proof-of-concept study of miravirsen (mir), an oligonucleotide targeting miR122, in treatment naive patients with genotype 1 (gt1) chronic HCV infection.
      ]. Most recently, an allosteric self-cleavable ribozyme capable of releasing antisense sequence to miR-122 only in the presence of HCV NS5B was developed in order to minimize potential side effects related to targeting physiological miR-122 functions [
      • Lee C.H.
      • Kim J.H.
      • Kim H.W.
      • Myung H.
      • Lee S.W.
      Hepatitis C virus replication-specific inhibition of microRNA activity with self-cleavable allosteric ribozyme.
      ]. The safety and efficacy of this strategy will next have to be assessed in vivo.
      HCV RNA replication depends on viral protein association with altered intracellular membranes, probably derived from the endoplasmic reticulum (ER), in a so called membranous web (reviewed in [
      • Moradpour D.
      • Penin F.
      • Rice C.M.
      Replication of hepatitis C virus.
      ]). The HCV replication complex, i.e., viral RNA and viral proteins associated to altered host cell membranes, is dependent on the host cell lipid metabolism. Indeed, this complex requires elements of cholesterol and fatty acid synthesis and geranylgeranylation of host proteins, as in vitro HCV replication can be disrupted by treatment with inhibitors of 3-hydroxy-3-methyglutaryl CoA (HMGCoA) reductase – such as the statin lovastatin, L-659,699 or ZA – or with an inhibitor of protein geranylgeranyl transferase I [
      • Ye J.
      • Wang C.
      • Sumpter Jr., R.
      • Brown M.S.
      • Goldstein J.L.
      • Gale Jr., M.
      Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation.
      ,
      • Kapadia S.B.
      • Chisari F.V.
      Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids.
      ]. This is in line with data indicating that HCV replication during acute infection of chimpanzees is associated with the modulation of several genes involved in lipid metabolism [
      • Su A.I.
      • Pezacki J.P.
      • Wodicka L.
      • Brideau A.D.
      • Supekova L.
      • Thimme R.
      • et al.
      Genomic analysis of the host response to hepatitis C virus infection.
      ]. Noteworthy, not all HMGCoA reductase inhibitors also inhibit HCV replication as the statin pravastatin exhibits no anti-HCV activity while fluvastatin has the strongest antiviral effect [
      • Ikeda M.
      • Abe K.
      • Yamada M.
      • Dansako H.
      • Naka K.
      • Kato N.
      Different anti-HCV profiles of statins and their potential for combination therapy with interferon.
      ]. While initial clinical studies indicated that statin monotherapy did either not significantly modulate HCV RNA levels or only modestly reduced HCV RNA in chronic HCV patients [
      • O’Leary J.G.
      • Chan J.L.
      • McMahon C.M.
      • Chung R.T.
      Atorvastatin does not exhibit antiviral activity against HCV at conventional doses: a pilot clinical trial.
      ,
      • Bader T.
      • Fazili J.
      • Madhoun M.
      • Aston C.
      • Hughes D.
      • Rizvi S.
      • et al.
      Fluvastatin inhibits hepatitis C replication in humans.
      ,
      • Patel K.
      • Jhaveri R.
      • George J.
      • Qiang G.
      • Kenedi C.
      • Brown K.
      • et al.
      Open-label, ascending dose, prospective cohort study evaluating the antiviral efficacy of Rosuvastatin therapy in serum and lipid fractions in patients with chronic hepatitis C.
      ], statins may represent interesting adjuvants to SOC. Indeed, fluvastatin (20 mg/day) increased the response to PegIFN-α/RBV, especially in aged women who respond poorly to SOC [
      • Sezaki H.
      • Suzuki F.
      • Akuta N.
      • Yatsuji H.
      • Hosaka T.
      • Kobayashi M.
      • et al.
      An open pilot study exploring the efficacy of fluvastatin, pegylated interferon and ribavirin in patients with hepatitis C virus genotype 1b in high viral loads.
      ]. Moreover, in two recent large retrospective analyses, statin use was associated with an improved sustained virological response (SVR) in patients receiving combination antiviral therapy [
      • Harrison S.A.
      • Rossaro L.
      • Hu K.Q.
      • Patel K.
      • Tillmann H.
      • Dhaliwal S.
      • et al.
      Serum cholesterol and statin use predict virological response to peginterferon and ribavirin therapy.
      ,
      • Rao G.A.
      • Pandya P.K.
      Statin therapy improves sustained virologic response among diabetic patients with chronic hepatitis C.
      ]. However, the addition of fluvastatin (80 mg/day) to PegIFN-α/RBV did not significantly increase SVR rates in HIV/HCV genotype 1 co-infected patients (also receiving highly active antiretroviral (HAART) therapy with a complete suppression of HIV replication) although it did significantly improve the RVR [
      • Milazzo L.
      • Caramma I.
      • Mazzali C.
      • Cesari M.
      • Olivetti M.
      • Galli M.
      • et al.
      Fluvastatin as an adjuvant to pegylated interferon and ribavirin in HIV/hepatitis C virus genotype 1 co-infected patients: an open-label randomized controlled study.
      ]. Taken together, these clinical trials indicate that, with the exception of HIV/HCV co-infected patients, statins may increase the efficacy of SOC in chronic HCV infected patients. Interestingly, most recently small molecule inhibitors of SKI-1/S1P, a lipogenic pathway regulator upstream of HMGCoA reductase, have been described [
      • Blanchet M.
      • Seidah N.G.
      • Labonte P.
      SKI-1/S1P inhibition: a promising surrogate to statins to block Hepatitis C virus replication.
      ]. The most potent inhibitor, PF-429242, inhibited HCVcc replication more efficiently than statins and, in contrast to statins, also reduced infectious particle production [
      • Blanchet M.
      • Seidah N.G.
      • Labonte P.
      SKI-1/S1P inhibition: a promising surrogate to statins to block Hepatitis C virus replication.
      ]. SKI-1/S1P inhibitors may thus also be considered for development of novel antivirals.
      Cyclophilins are also important host factors for HCV replication and CypA has been demonstrated to interact with HCV NS5A [
      • Hanoulle X.
      • Badillo A.
      • Wieruszeski J.M.
      • Verdegem D.
      • Landrieu I.
      • Bartenschlager R.
      • et al.
      Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.
      ,
      • Kaul A.
      • Stauffer S.
      • Berger C.
      • Pertel T.
      • Schmitt J.
      • Kallis S.
      • et al.
      Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.
      ]. Cyclophilins had been identified as host targets for antiviral therapy more than 20 years ago as cyclosporine, a widely used immunosuppressive drug, was demonstrated to inhibit non-A non-B hepatitis virus [
      • Teraoka S.
      • Mishiro S.
      • Ebihara K.
      • Sanaka T.
      • Yamaguchi Y.
      • Nakajima I.
      • et al.
      Effect of cyclosporine on proliferation of non-A, non-B hepatitis virus.
      ]. More recently, cyclosporine analogs lacking immunosuppressive activity and displaying higher in vitro antiviral activity, e.g., alisporivir/Debio 025, NIM811 and SCY-635, have been developed [
      • Paeshuyse J.
      • Kaul A.
      • De Clercq E.
      • Rosenwirth B.
      • Dumont J.M.
      • Scalfaro P.
      • et al.
      The non-immunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replication in vitro.
      ,
      • Chatterji U.
      • Bobardt M.
      • Selvarajah S.
      • Yang F.
      • Tang H.
      • Sakamoto N.
      • et al.
      The isomerase active site of cyclophilin A is critical for hepatitis C virus replication.
      ,
      • Hopkins S.
      • Scorneaux B.
      • Huang Z.
      • Murray M.G.
      • Wring S.
      • Smitley C.
      • et al.
      SCY-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis C virus RNA replication in vitro.
      ]. These compounds disrupted CypA-NS5A interaction [
      • Coelmont L.
      • Hanoulle X.
      • Chatterji U.
      • Berger C.
      • Snoeck J.
      • Bobardt M.
      • et al.
      DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A.
      ,
      • Hopkins S.
      • Bobardt M.
      • Chatterji U.
      • Garcia-Rivera J.A.
      • Lim P.
      • Gallay P.A.
      The cyclophilin inhibitor SCY-635 disrupts HCV NS5A-cyclophilin A complexes.
      ]. Moreover, SCY-635, currently in phase 1 clinical study, enhanced secretion of type I and type III IFNs in replicon cells and increased the expression of IFN response genes [
      • Hopkins S.
      • Dimassimo B.
      • Rusnak P.
      • Heuman D.
      • Lalezari J.
      • Sluder A.
      • et al.
      The cyclophilin inhibitor SCY-635 suppresses viral replication and induces endogenous interferons in patients with chronic HCV genotype 1 infection.
      ]. These data suggest that in addition to inhibiting viral replication, CypA inhibitors may restore the host innate immune responses to HCV inhibitors and thereby enhance their antiviral activity [
      • Hopkins S.
      • Dimassimo B.
      • Rusnak P.
      • Heuman D.
      • Lalezari J.
      • Sluder A.
      • et al.
      The cyclophilin inhibitor SCY-635 suppresses viral replication and induces endogenous interferons in patients with chronic HCV genotype 1 infection.
      ]. Interestingly, alisporivir/Debio 025 has also proven anti-HIV activity in vitro as this molecule inhibits CypA-HIV capsid protein binding [
      • Ptak R.G.
      • Gallay P.A.
      • Jochmans D.
      • Halestrap A.P.
      • Ruegg U.T.
      • Pallansch L.A.
      • et al.
      Inhibition of human immunodeficiency virus type 1 replication in human cells by Debio-025, a novel cyclophilin binding agent.
      ,
      • Daelemans D.
      • Dumont J.M.
      • Rosenwirth B.
      • De Clercq E.
      • Pannecouque C.
      Debio-025 inhibits HIV-1 by interfering with an early event in the replication cycle.
      ]. CypA inhibitors may thus have an additional benefit in HIV/HCV co-infected patients. In a phase 1 study, 14-day oral alipsorivir/Debio 025 (1200 mg twice daily) treatment significantly reduced HCV RNA serum levels in HIV/HCV co-infected patients independently of the HCV genotype (1, 3 and 4) [
      • Flisiak R.
      • Horban A.
      • Gallay P.
      • Bobardt M.
      • Selvarajah S.
      • Wiercinska-Drapalo A.
      • et al.
      The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus.
      ]. However, a potent synergy between alisporivir/Debio 025 (200, 600 and 1200 mg twice a day for one week and then once daily) and PegIFN-α was also observed in a subsequent phase 2 study demonstrating that addition of alisporivir/Debio 025 increased RVR [
      • Flisiak R.
      • Feinman S.V.
      • Jablkowski M.
      • Horban A.
      • Kryczka W.
      • Pawlowska M.
      • et al.
      The cyclophilin inhibitor Debio 025 combined with PEG IFNalpha2a significantly reduces viral load in treatment-naive hepatitis C patients.
      ]. Further phase 2 trials also demonstrated improved efficacy and good tolerance adding alisporivir/Debio 025 to PegIFN-α/RBV without selection of resistant variants (reviewed in [
      • Flisiak R.
      • Jaroszewicz J.
      • Flisiak I.
      • Lapinski T.
      Update on alisporivir in treatment of viral hepatitis C.
      ]). This CypA inhibitor is thus characterized by a high barrier to resistance and is the first HTA that reached phase 3 studies (Table 1). Given three cases of acute pancreatitis, the FDA recently put a clinical hold on this trial before proceeding to the next steps. The fact that the combination of alisporivir/Debio 025 with DAAs resulted in additive antiviral activity in short-term in vitro antiviral assays [
      • Coelmont L.
      • Kaptein S.
      • Paeshuyse J.
      • Vliegen I.
      • Dumont J.M.
      • Vuagniaux G.
      • et al.
      Debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis C virus (HCV) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for HCV (STAT-C) inhibitors.
      ] holds promise for HTAs as part of future IFN-sparing regimen(s) for the treatment of HCV infection.

      HCV assembly/release inhibitors

      Following HCV replication, new infectious virions are assembled in the vicinity of lipid droplets and ER [
      • Miyanari Y.
      • Atsuzawa K.
      • Usuda N.
      • Watashi K.
      • Hishiki T.
      • Zayas M.
      • et al.
      The lipid droplet is an important organelle for hepatitis C virus production.
      ,
      • Boulant S.
      • Targett-Adams P.
      • McLauchlan J.
      Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus.
      ,
      • Roingeard P.
      • Hourioux C.
      • Blanchard E.
      • Prensier G.
      Hepatitis C virus budding at lipid droplet-associated ER membrane visualized by 3D electron microscopy.
      ,
      • Bartenschlager R.
      • Penin F.
      • Lohmann V.
      • Andre P.
      Assembly of infectious hepatitis C virus particles.
      ]. The HCV particle is composed of an encapsidated RNA genome that is surrounded by an envelope composed of the envelope glycoproteins E1 and E2 [
      • Lavie M.
      • Goffard A.
      • Dubuisson J.
      Assembly of a functional HCV glycoprotein heterodimer.
      ,
      • Tews B.A.
      • Popescu C.I.
      • Dubuisson J.
      Last stop before exit – hepatitis C assembly and release as antiviral drug targets.
      ]. E1 and E2 associate as a non-covalent heterodimer and are essential for viral infectivity as they mediate interactions with different host cell factors during viral binding and entry. E1 and E2 are heavily N-glycosylated, contain ER retention signals and are processed within the ER by glucosidases I and II to ensure proper folding and assembly [
      • Lavie M.
      • Goffard A.
      • Dubuisson J.
      Assembly of a functional HCV glycoprotein heterodimer.
      ]. HCV assembly has been suggested to parallel VLDL assembly [
      • Huang H.
      • Sun F.
      • Owen D.M.
      • Li W.
      • Chen Y.
      • Gale Jr, M.
      • et al.
      Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins.
      ,
      • Gastaminza P.
      • Cheng G.
      • Wieland S.
      • Zhong J.
      • Liao W.
      • Chisari F.V.
      Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion.
      ,
      • Jiang J.
      • Luo G.
      Apolipoprotein E but not B is required for the formation of infectious hepatitis C virus particles.
      ]. Microsomal triglyceride transfer protein (MTP), the rate limiting enzyme of VLDL assembly [
      • Jamil H.
      • Chu C.H.
      • Dickson Jr., J.K.
      • Chen Y.
      • Yan M.
      • Biller S.A.
      • et al.
      Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.
      ], probably also contributes to HCV particle assembly [
      • Gastaminza P.
      • Cheng G.
      • Wieland S.
      • Zhong J.
      • Liao W.
      • Chisari F.V.
      Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion.
      ].
      Targeting host glucosidases thus represents a promising strategy to interfere with viral infectivity (Table 1). MX-3253/celgosivir (reviewed in [
      • Durantel D.
      Celgosivir, an alpha-glucosidase I inhibitor for the potential treatment of HCV infection.
      ]), an alpha-glucosidase I inhibitor, induces misfolding of HCV envelope glycoproteins and leads to reduced viral infectivity in vitro [
      • Chapel C.
      • Garcia C.
      • Roingeard P.
      • Zitzmann N.
      • Dubuisson J.
      • Dwek R.A.
      • et al.
      Antiviral effect of alpha-glucosidase inhibitors on viral morphogenesis and binding properties of hepatitis C virus-like particles.
      ,
      • Chapel C.
      • Garcia C.
      • Bartosch B.
      • Roingeard P.
      • Zitzmann N.
      • Cosset F.L.
      • et al.
      Reduction of the infectivity of hepatitis C virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors.
      ]. MX-3253/celgosivir demonstrated modest antiviral efficacy in a phase 2a monotherapy study (200 and 400 mg/day for 12 weeks) in treatment-naive and IFN-intolerant genotype 1 HCV patients [
      • Yoshida E.
      • Kunimoto D.
      • Lee S.E.
      • Sherman M.
      • Heathcote J.E.
      • Enns R.
      Results of a phase II dose ranging study of orally administered celgosivir as monotherapy in chronic hepatitis C genotype-1 patients.
      ]. While MX-3253/celgosivir (400 mg/day for 12 weeks) demonstrated clinical benefit in combination with PegIFN-α/RBV in chronic HCV genotype 1 infected patients [
      • Kaita K.
      • Yoshida E.
      • Kunimoto D.
      • Anderson F.
      • Morris S.
      • Marotta P.
      • et al.
      Phase II Proof of Concept Study of Celgosivir in combination with peginterferon alfa-2b and ribavirin in chronic hepatitis C genotype-1 non-responder patients.
      ], the further development of MX-3253/celgosivir for HCV infection has subsequently been halted.
      Compounds inhibiting VLDL assembly, such as MTP inhibitors, also reduce HCV release from infected cells [
      • Huang H.
      • Sun F.
      • Owen D.M.
      • Li W.
      • Chen Y.
      • Gale Jr, M.
      • et al.
      Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins.
      ,
      • Gastaminza P.
      • Cheng G.
      • Wieland S.
      • Zhong J.
      • Liao W.
      • Chisari F.V.
      Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion.
      ,
      • Jiang J.
      • Luo G.
      Apolipoprotein E but not B is required for the formation of infectious hepatitis C virus particles.
      ]. MTP inhibitors have been developed for treatment of dyslipidemia and currently several MTP inhibitors are in clinical trials for the treatment of hypercholesterolemia or hyperlipidemia (reviewed in [
      • Raval S.K.
      • Raval P.S.
      • Jain M.R.
      Emerging therapies for dyslipidemia: known knowns and known unknowns of MTP inhibitors.
      ]). However, whether MTP inhibitors display an antiviral effect against HCV infection in vivo remains to be determined. Moreover, recent screens revealed that several approved drugs display antiviral activity against HCV by targeting HCV assembly and/or release: these studies identified two anti-cancer drugs, pterostilbene (a methylated form of resveratrol) and torimefene (a derivative of tamoxifene) [
      • Gastaminza P.
      • Whitten-Bauer C.
      • Chisari F.V.
      Unbiased probing of the entire hepatitis C virus life cycle identifies clinical compounds that target multiple aspects of the infection.
      ] as well as quinidine, a class I antiarrhythmic agent [
      • Chockalingam K.
      • Simeon R.L.
      • Rice C.M.
      • Chen Z.
      A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle.
      ] as potential antivirals against HCV. Taken together, these data indicate the further potential of clinical development of HCV assembly inhibitors for the treatment of chronic hepatitis C.

      Clinical perspectives of HTAs interfering with the HCV life cycle

      To date, the main issue of anti-HCV SOC is to avoid viral resistance and severe side effects. Generally speaking, the use of DAAs against different potential highly variable viruses, such as HCV, HIV or influenza virus, is associated with the development of resistance, while HTAs, acting on cellular targets that are less prone to mutations, may impose a higher genetic barrier for resistance (Fig. 3) [
      • Delang L.
      • Vliegen I.
      • Froeyen M.
      • Neyts J.
      Comparative study of the genetic barriers and pathways towards resistance of selective inhibitors of hepatitis C virus replication.
      ,
      • Konig R.
      • Stertz S.
      • Zhou Y.
      • Inoue A.
      • Hoffmann H.H.
      • Bhattacharyya S.
      • et al.
      Human host factors required for influenza virus replication.
      ]. On the other hand, the principle theoretical drawback of using HTAs is their potential greater cellular toxicity. Nevertheless, it has to be pointed out that the development of several DAAs targeting HCV, such as BILN 2061, had to be stopped due to severe side effects [
      • Vanwolleghem T.
      • Meuleman P.
      • Libbrecht L.
      • Roskams T.
      • De Vos R.
      • Leroux-Roels G.
      Ultra-rapid cardiotoxicity of the hepatitis C virus protease inhibitor BILN 2061 in the urokinase-type plasminogen activator mouse.
      ]. Moreover, the majority of current drugs widely used for cardiovascular, neurological or endocrine diseases as well as cancer, target host proteins [
      • Imming P.
      • Sinning C.
      • Meyer A.
      Drugs, their targets and the nature and number of drug targets.
      ,
      • Overington J.P.
      • Al-Lazikani B.
      • Hopkins A.L.
      How many drug targets are there?.
      ,
      • Landry Y.
      • Gies J.P.
      Drugs and their molecular targets: an updated overview.
      ]. Thus, side effects have to be carefully evaluated for novel antiviral strategies against hepatitis C irrespective of the drug target.
      While DAAs allow increasing the virological response of HCV genotype 1 infected patients, a large fraction of chronic HCV patients, especially HIV/HCV co-infected patients and patients undergoing LT, will not be eligible for DAAs given the important drug–drug interactions with anti-retroviral therapy and immunosuppressive agents. Noteworthy, synergy between IFN-α, DAAs and HTAs allowing to decrease the concentrations of the individual compounds [
      • Fofana I.
      • Xiao F.
      • Thumann C.
      • Lupberger J.
      • Leyssen P.
      • Neyts J.H.
      • et al.
      Synergy of entry inhibitors and direct acting antivirals or interferon-alfa identifies novel antiviral combinations for hepatitis C virus infection.
      ,
      • Zhu H.
      • Wong-Staal F.
      • Lee H.
      • Syder A.
      • McKelvy J.
      • Schooley R.T.
      • et al.
      Evaluation of ITX 5061, a scavenger receptor B1 antagonist: resistance selection and activity in combination with other hepatitis C virus antivirals.
      ] holds promise for a variety of possibilities of future combination therapy treatments of hepatitis C infection that may be adapted to the individual patient. Furthermore, given (i) the importance of host entry factors for HCV reinfection of the graft during LT [
      • Fofana I.
      • Fafi-Kremer S.
      • Carolla P.
      • Fauvelle C.
      • Zahid M.N.
      • Turek M.
      • et al.
      Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.
      ], (ii) the broad antiviral activity of entry inhibitors against viral escape variants selected during LT [
      • Fafi-Kremer S.
      • Fofana I.
      • Soulier E.
      • Carolla P.
      • Meuleman P.
      • Leroux-Roels G.
      • et al.
      Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation.
      ,
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ,
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ], and (iii) the synergy between entry inhibitors and neutralizing anti-HCV envelope antibodies [
      • Zahid M.N.
      • Turek M.
      • Xiao F.
      • Dao Thi V.L.
      • Guérin M.
      • Fofana I.
      • et al.
      The post-binding activity of scavenger receptor BI mediates initiation of hepatitis C virus infection and viral dissemination.
      ], entry inhibitors also represent a promising strategy to prevent viral reinfection of the liver graft (Fig. 2).

      Conclusions and perspectives

      The goal of current anti-HCV SOC is sustained viral eradication. However, due to the high variability of HCV, viral resistance and subsequent treatment failure remain major challenges. Moreover, therapeutic strategies for a large fraction of patients, especially HIV/HCV co-infected patients, patients with immunosuppression and co-morbidity and patients undergoing LT remain limited [
      • Feinstone S.M.
      • Hu D.J.
      • Major M.E.
      Prospects for prophylactic and therapeutic vaccines against hepatitis C virus.
      ,
      • Stoll-Keller F.
      • Barth H.
      • Fafi-Kremer S.
      • Zeisel M.B.
      • Baumert T.F.
      Development of hepatitis C virus vaccines: challenges and progress.
      ]. Although early clinical trials have demonstrated impressive outcomes for combinations of DAAs in IFN-free regimens for treatment naïve patients [
      • Sarrazin C.
      • Hezode C.
      • Zeuzem S.
      • Pawlotsky J.M.
      Antiviral strategies in hepatitis C virus infection.
      ] there will be a need for antivirals addressing resistance, treatment of patients with co-morbidity, co-medication or immunosuppression and patients undergoing LT [
      • Pawlotsky J.M.
      Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus.
      ].
      Figure thumbnail fx3
      The recent preclinical and clinical development of HTAs for HCV as well as novel HTA-based strategies for other pathogens including other viruses and bacteria [
      • Nathan C.
      Fresh approaches to anti-infective therapies.
      ] highlights the promise of this approach to address unmet medical needs in the prevention and treatment of virus-induced liver disease.

      Financial support

      The authors acknowledge financial support of their work by the European Union ( ERC-2008-AdG-233130-HEPCENT and INTERREG-IV-2009-FEDER-Hepato-Regio-Net ), Laboratoire d’Excellence HEPSYS (Investissement d’Avenir; ANR-10-LAB-28 ), ANRS ( 2008/354 , 2009/183 , 2011/132 , 2012/239 ), Inserm, the Direction Générale de l’Offre de Soins (A12027MS), University of Strasbourg and the Strasbourg University Hospitals, France.

      Conflict of interest

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

      Acknowledgements

      We would like to thank Prof. H. Wedemeyer (Medizinische Hochschule Hannover, Germany) and Prof. M. Levrero (University of Rome, Italy) for critical reading of the manuscript. We apologize to all authors whose work could not be cited due to space restrictions.

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