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Future landscape of hepatitis C research – Basic, translational and clinical perspectives

  • Darius Moradpour
    Correspondence
    Corresponding authors. Addresses: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Rue du Bugnon 44, Switzerland. Tel.: +41 21 314 47 14; fax: +41 21 314 83 60 (D. Moradpour), or Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine and Yerkes National Primate Research Center, Emory Vaccine Center, Atlanta, GA, USA. Tel.: +1 404 727 5850; fax: +1 404 727 7768 (A. Grakoui), or Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany. Tel.: +49 0511 532 3305; fax: +49 0511 532 4896 (M.P. Manns).
    Affiliations
    Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland
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  • Arash Grakoui
    Correspondence
    Corresponding authors. Addresses: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Rue du Bugnon 44, Switzerland. Tel.: +41 21 314 47 14; fax: +41 21 314 83 60 (D. Moradpour), or Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine and Yerkes National Primate Research Center, Emory Vaccine Center, Atlanta, GA, USA. Tel.: +1 404 727 5850; fax: +1 404 727 7768 (A. Grakoui), or Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany. Tel.: +49 0511 532 3305; fax: +49 0511 532 4896 (M.P. Manns).
    Affiliations
    Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine and Yerkes National Primate Research Center, Emory Vaccine Center, Atlanta, GA, USA
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  • Michael P. Manns
    Correspondence
    Corresponding authors. Addresses: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Rue du Bugnon 44, Switzerland. Tel.: +41 21 314 47 14; fax: +41 21 314 83 60 (D. Moradpour), or Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine and Yerkes National Primate Research Center, Emory Vaccine Center, Atlanta, GA, USA. Tel.: +1 404 727 5850; fax: +1 404 727 7768 (A. Grakoui), or Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany. Tel.: +49 0511 532 3305; fax: +49 0511 532 4896 (M.P. Manns).
    Affiliations
    Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany

    German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
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      Summary

      With the latest all-oral interferon- and ribavirin-free regimens based on direct acting antivirals against the hepatitis C virus (HCV), sustained virological response rates of >90% are achieved, which is equivalent to cure. This has become possible for all genotypes and all subgroups of patients, including many of the most difficult-to-treat populations so far. Since a prophylactic HCV vaccine is not yet available, control of HCV infection will for the time being have to rely on the use of effective and safe antiviral treatments as well as their accessibility and affordability. Different approaches may apply to different parts of the world, eradication of HCV representing a major long-term goal. Whether hepatitis C becomes the first chronic viral infection to be eradicated without a prophylactic vaccine remains to be shown. Here, we briefly summarize advances in the molecular virology of hepatitis C, highlight lessons of biological relevance that were learned through the study of HCV, and its translational and clinical implications. We have also listed selected unsolved challenges, emphasizing that HCV is a unique model and that advances in this direction may yield knowledge of broad biological significance, novel technologies and insights into related important human pathogens.

      Keywords

      Introduction

      Hepatitis C virus (HCV) research represents a success of modern medicine: from the discovery of an uncultured infectious agent that chronically infects an estimated 80 to 180 million persons worldwide, to the development of oral combination therapies, which allow to cure the majority, in 25 years (reviewed in [
      • Bartenschlager R.
      • Lohmann V.
      • Penin F.
      The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection.
      ,
      • Manns M.P.
      • von Hahn T.
      Novel therapies for hepatitis C – one pill fits all?.
      ,
      • Pawlotsky J.M.
      • Feld J.J.
      • Zeuzem S.
      • Hoofnagle J.H.
      From non-A, non-B hepatitis to hepatitis C virus cure.
      ,
      • Scheel T.K.
      • Rice C.M.
      Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.
      ]). This progress has been made possible through intense research efforts in academia as well as the pharmaceutical industry. Selected milestones on this extraordinary journey are illustrated in Fig. 1.
      Figure thumbnail gr1
      Fig. 1Milestones in HCV research (selection). DAA, direct acting antiviral; IFN, interferon; PI, protease inhibitor; RBV, ribavirin.
      Research on HCV has yielded a detailed view of the viral life cycle (reviewed in [

      Poulot A and Penin F. The HCV life cycle. Available from: http://hcvlifecycle.univ-lyon1.fr.

      ,
      • Moradpour D.
      • Penin F.
      • Rice C.M.
      Replication of hepatitis C virus.
      ,

      Bartenschlager R, editor. Hepatitis C virus: from molecular virology to antiviral therapy. Curr Top Microbiol Immunol. Springer; 2013.

      ]) and allowed to define targets for antiviral intervention. In addition, it has also provided lessons of broad significance to the fields of virology as well as human biology and immunology. A selection of such general lessons is listed in Table 1.
      Table 1HCV as a teacher of cell biology and immunology. General lessons learned from studying HCV (selection).
      CRE, cis-acting replication element; IFN-λ3/-λ4, interferon- λ3/- λ4; IRES, internal ribosome entry site; miR122, microRNA-122; PI4KIIIα, phosphatidylinositol 4-kinase IIIα; RIG-I, retinoic acid inducible gene I.
      As we enter the post-drug discovery era, doctoral students, postdoctoral fellows, research group leaders and funding agencies may ask whether it is still worthwhile to engage in basic research on HCV. The answer, in our view, is a clear yes for as long as questions of general biological relevance are asked. Indeed, HCV is one of the best-characterized positive-strand RNA viruses and a unique model of a persistent infection in an immunocompetent host that can be eliminated by effective antiviral therapy. Powerful experimental model systems have been developed to study HCV, detailed structure-function analyses of the viral proteins have been performed, and there is a broad array of tools to probe their functions as well as interactions with host factors. Viruses in general and HCV in particular are excellent teachers of cell biology and immunology as well as drivers of technological innovation. Hence, understanding fundamental mechanisms of HCV replication should yield new insight into our own functioning and provide new opportunities to successfully target other viruses. Among these, the related Flaviviridae family members Dengue, West Nile and Zika virus are prominent examples of threats to human health [
      • Haug C.J.
      • Kieny M.P.
      • Murgue B.
      The Zika Challenge.
      ].

      Challenges in basic research on HCV

      An in-depth discussion of open questions in basic research on HCV would exceed the space allowed for this review. Some aspects were elegantly discussed recently [
      • Lindenbach B.D.
      What’s next for hepatitis C virus research?.
      ]. In addition, Table 2 lists some of the current challenges in the field. We will briefly summarize below selected virological aspects and challenges to overcome in the near future.
      Progress in the understanding of the molecular virology of hepatitis C has translated into highly effective antiviral therapies. However, important challenges remain and new ones emerge.
      Table 2Challenges in basic research on HCV (selection).
      HCC, hepatocellular carcinoma.

      Taxonomy

      HCV is a member of the hepacivirus genus within the Flaviviridae family which also comprises the flavi-, pesti- and pegiviruses. The hepaci- and pegivirus genera have expanded recently, with the identification of closely related viruses in horses, certain primates, bats, cattle and rodents [
      • Firth C.
      • Bhat M.
      • Firth M.A.
      • Williams S.H.
      • Frye M.J.
      • Simmonds P.
      • et al.
      Detection of zoonotic pathogens and characterization of novel viruses carried by commensal Rattus norvegicus in New York City.
      ,
      • Scheel T.K.
      • Simmonds P.
      • Kapoor A.
      Surveying the global virome: identification and characterization of HCV-related animal hepaciviruses.
      ,
      • Corman V.M.
      • Grundhoff A.
      • Baechlein C.
      • Fischer N.
      • Gmyl A.
      • Wollny R.
      • et al.
      Highly divergent hepaciviruses from African cattle.
      ]. These discoveries yield interesting questions on the origin of HCV in humans as well as opportunities for the development of novel model systems for HCV and related viruses.

      Model systems

      In vitro and in vivo model systems, most notably the replicon and cell culture-derived HCV systems [
      • Lohmann V.
      • Körner F.
      • Koch J.-O.
      • Herian U.
      • Theilmann L.
      • Bartenschlager R.
      Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.
      ,
      • Wakita T.
      • Pietschmann T.
      • Kato T.
      • Date T.
      • Miyamoto M.
      • Zhao Z.
      • et al.
      Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.
      ], have been key to the study of HCV (reviewed in [
      • Billerbeck E.
      • de Jong Y.
      • Dorner M.
      • de la Fuente C.
      • Ploss A.
      Animal models for hepatitis C.
      ,
      • Steinmann E.
      • Pietschmann T.
      Cell culture systems for hepatitis C virus.
      ]). In addition, insight has been gained into viral and host determinants for efficient HCV replication in cell culture ([
      • Yamane D.
      • McGivern D.R.
      • Wauthier E.
      • Yi M.
      • Madden V.J.
      • Welsch C.
      • et al.
      Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.
      ,
      • Saeed M.
      • Andreo U.
      • Chung H.Y.
      • Espiritu C.
      • Branch A.D.
      • Silva J.M.
      • et al.
      SEC14L2 enables pan-genotype HCV replication in cell culture.
      ] and references therein). However, an immunocompetent small animal model for HCV still represents a challenge to overcome.

      HCV particle

      It has been difficult to directly visualize HCV particles in a consistent manner. HCV is believed to circulate in association with lipoproteins, either as hybrid “lipo-viro-particles” or tethered to lipids via apolipoproteins (reviewed in [
      • Bartenschlager R.
      • Penin F.
      • Lohmann V.
      • Andre P.
      Assembly of infectious hepatitis C virus particles.
      ,
      • Lindenbach B.D.
      • Rice C.M.
      The ins and outs of hepatitis C virus entry and assembly.
      ]) Molecular tagging and the use of affinity grids have allowed visualization of HCV by electron microscopy [
      • Catanese M.T.
      • Uryu K.
      • Kopp M.
      • Edwards T.J.
      • Andrus L.
      • Rice W.J.
      • et al.
      Ultrastructural analysis of hepatitis C virus particles.
      ]. In addition, lipidomic and proteomic studies have allowed analyzing the viral particle [
      • Merz A.
      • Long G.
      • Hiet M.S.
      • Brugger B.
      • Chlanda P.
      • Andre P.
      • et al.
      Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome.
      ,
      • Lussignol M.
      • Kopp M.
      • Molloy K.
      • Vizcay-Barrena G.
      • Fleck R.A.
      • Dorner M.
      • et al.
      Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis.
      ]. However, a detailed biophysical characterization and a high-resolution structure of the virion remain elusive.

      HCV entry

      Key entry factors for HCV have been identified, allowing to develop genetically humanized mice as a small animal model for HCV infection [
      • Dorner M.
      • Horwitz J.A.
      • Donovan B.M.
      • Labitt R.N.
      • Budell W.C.
      • Friling T.
      • et al.
      Completion of the entire hepatitis C virus life cycle in genetically humanized mice.
      ] and entry inhibitors as a novel class of antivirals ([
      • de Jong Y.P.
      • Dorner M.
      • Mommersteeg M.C.
      • Xiao J.W.
      • Balazs A.B.
      • Robbins J.B.
      • et al.
      Broadly neutralizing antibodies abrogate established hepatitis C virus infection.
      ,
      • Mailly L.
      • Xiao F.
      • Lupberger J.
      • Wilson G.K.
      • Aubert P.
      • Duong F.H.
      • et al.
      Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.
      ] and references therein). However, additional and unexpected players in viral entry continue to emerge ([
      • Zona L.
      • Lupberger J.
      • Sidahmed-Adrar N.
      • Thumann C.
      • Harris H.J.
      • Barnes A.
      • et al.
      HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex.
      ,
      • Gerold G.
      • Meissner F.
      • Bruening J.
      • Welsch K.
      • Perin P.M.
      • Baumert T.F.
      • et al.
      Quantitative proteomics identifies serum response factor binding protein 1 as a host factor for hepatitis C virus entry.
      ,
      • Ujino S.
      • Nishitsuji H.
      • Hishiki T.
      • Sugiyama K.
      • Takaku H.
      • Shimotohno K.
      Hepatitis C virus utilizes VLDLR as a novel entry pathway.
      ] and references therein) and a full picture of the entry and egress pathways in cells that reflect polarized human hepatocytes within the liver is still under intense investigation.

      HCV RNA

      The organization of the viral RNA genome has been delineated shortly after the discovery of HCV but it took several years until the correct 3′ end was identified and a functional molecular clone could be constructed [
      • Kolykhalov A.A.
      • Agapov E.V.
      • Blight K.J.
      • Mihalik K.
      • Feinstone S.M.
      • Rice C.M.
      Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.
      ]. Elegant work has resulted in structural and functional insights into the viral internal ribosome entry side (reviewed in [
      • Fraser C.S.
      • Doudna J.A.
      Structural and mechanistic insights into hepatitis C viral translation initiation.
      ,
      • Niepmann M.
      Hepatitis C virus RNA translation.
      ]). The discoveries of cis-acting replication elements within the protein-coding region and of the crucial interaction of HCV RNA with microRNA-122 (miR122) were important breakthroughs [
      • You S.
      • Stump D.D.
      • Branch A.D.
      • Rice C.M.
      A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA 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.
      ]. The latter observation has led to clinical proof-of-concept studies of an antagomir as an antiviral agent against hepatitis C [
      • 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.
      ,
      • Janssen H.L.
      • Reesink H.W.
      • Lawitz E.J.
      • Zeuzem S.
      • Rodriguez-Torres M.
      • Patel K.
      • et al.
      Treatment of HCV infection by targeting microRNA.
      ]. Exciting discoveries continue to be generated, including the identification of additional layers of information within the genomic RNA (“codes within the code”) [
      • Pirakitikulr N.
      • Kohlway A.
      • Lindenbach B.D.
      • Pyle A.M.
      The coding region of the HCV genome contains a network of regulatory RNA structures.
      ], mechanisms regulating the turnover of viral RNA and its interactions with host proteins and microRNAs [
      • Li Y.
      • Yamane D.
      • Masaki T.
      • Lemon S.M.
      The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA.
      ], and the discovery of HCV RNA as a ’microRNA sponge’, with potential implications for the pathogenesis of hepatitis C and of HCV-related hepatocellular carcinoma (HCC) [
      • Luna J.M.
      • Scheel T.K.
      • Danino T.
      • Shaw K.S.
      • Mele A.
      • Fak J.J.
      • et al.
      Hepatitis C virus RNA functionally sequesters miR-122.
      ].

      Structural biology

      Great advances have been made in the structural biology of hepatitis C, with atomic resolution structural information available for the targets of current direct acting antivirals (DAAs), i.e., the NS3-4A protease, NS5A and the NS5B RNA-dependent RNA polymerase, as well as the p7 viroporin, the NS2 protease and the NS3 helicase (reviewed in [
      • Moradpour D.
      • Penin F.
      Hepatitis C virus proteins: from structure to function.
      ]). However, only partial structures are available for the core protein, the envelope glycoproteins E1 and E2 as well as NS4B. The partial structures of the envelope glycoproteins E1 and E2 solved recently [
      • Kong L.
      • Giang E.
      • Nieusma T.
      • Kadam R.U.
      • Cogburn K.E.
      • Hua Y.
      • et al.
      Hepatitis C virus E2 envelope glycoprotein core structure.
      ,
      • El Omari K.
      • Iourin O.
      • Kadlec J.
      • Sutton G.
      • Harlos K.
      • Grimes J.M.
      • et al.
      Unexpected structure for the N-terminal domain of hepatitis C virus envelope glycoprotein E1.
      ,
      • Khan A.G.
      • Whidby J.
      • Miller M.T.
      • Scarborough H.
      • Zatorski A.V.
      • Cygan A.
      • et al.
      Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2.
      ] identified novel and unexpected folds. Hence, completing the structure of the E1-E2 heterodimer may yield a basis for rational vaccine design and may at the same time reveal a novel mechanism of membrane fusion (reviewed in [
      • Khan A.G.
      • Miller M.T.
      • Marcotrigiano J.
      HCV glycoprotein structures: what to expect from the unexpected.
      ]). In addition, a complete structure of the integral membrane protein NS4B, a major challenge for the field, may yield important insights into this protein’s function in the establishment of the viral replication complex (reviewed in [
      • Gouttenoire J.
      • Penin F.
      • Moradpour D.
      Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory.
      ]). Obtaining a higher-order structure of the viral replicase (i.e., of NS3-4A, NS4B, NS5A and NS5B in concert) represents a major aim that would provide unprecedented insights into the functional architecture of a positive-strand RNA virus replication complex.

      HCV RNA replication

      Much has been learned about the mechanism of HCV RNA replication as well as the host factors and cellular structures involved in this process ([
      • Yamane D.
      • McGivern D.R.
      • Wauthier E.
      • Yi M.
      • Madden V.J.
      • Welsch C.
      • et al.
      Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.
      ,
      • Saeed M.
      • Andreo U.
      • Chung H.Y.
      • Espiritu C.
      • Branch A.D.
      • Silva J.M.
      • et al.
      SEC14L2 enables pan-genotype HCV replication in cell culture.
      ,
      • Reiss S.
      • Harak C.
      • Romero-Brey I.
      • Radujkovic D.
      • Klein R.
      • Ruggieri A.
      • et al.
      The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A.
      ,
      • Kazakov T.
      • Yang F.
      • Ramanathan H.N.
      • Kohlway A.
      • Diamond M.S.
      • Lindenbach B.D.
      Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of viral nonstructural proteins.
      ,
      • Romero-Brey I.
      • Berger C.
      • Kallis S.
      • Kolovou A.
      • Paul D.
      • Lohmann V.
      • et al.
      NS5A domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis C virus replication.
      ,
      • Shulla A.
      • Randall G.
      Spatiotemporal analysis of hepatitis C virus infection.
      ] and references therein) (reviewed in [
      • Lohmann V.
      Hepatitis C virus RNA replication.
      ,
      • Paul D.
      • Madan V.
      • Bartenschlager R.
      Hepatitis C virus RNA replication and assembly: living on the fat of the land.
      ]). However, there is room to learn about the mechanisms regulating HCV RNA translation vs. replication vs. virion packaging as well as the biogenesis of the viral replication complex.

      Viral assembly and release

      The late steps of the viral life cycle involve the coordinated interplay between viral structural and nonstructural proteins as well as a number of host factors [
      • Lindenbach B.D.
      • Rice C.M.
      The ins and outs of hepatitis C virus entry and assembly.
      ]. Lipid droplets and an egress pathway related to lipoprotein secretion have been found to play central roles. However, a more detailed view is still required concerning packaging of the viral genome as well as assembly and release of viral particles, especially the functional and spatial relation between replication and assembly sites as well as the exact roles of the p7 viroporin, the NS2 protease, and the other nonstructural proteins which are all involved also in the late steps of the viral life cycle [
      • Lindenbach B.D.
      • Rice C.M.
      The ins and outs of hepatitis C virus entry and assembly.
      ].

      Drug discovery

      The NS3-4A protease and the NS5B RNA-dependent RNA polymerase were pursued as classical antiviral targets since their identification in the early 1990s, resulting in the development of protease and polymerase inhibitors as key DAAs [
      • Bartenschlager R.
      • Lohmann V.
      • Penin F.
      The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection.
      ,
      • Scheel T.K.
      • Rice C.M.
      Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.
      ]. In addition, the serendipitous discovery of NS5A inhibitors as mainstay of all currently approved antiviral combination therapies, represents a beautiful example of investigation in the replicon system that translated to successful clinical use [
      • Gao M.
      • Nettles R.E.
      • Belema M.
      • Snyder L.B.
      • Nguyen V.N.
      • Fridell R.A.
      • et al.
      Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.
      ]. However, the exact mode of action of NS5A inhibitors as well as of synergistic compounds able to overcome resistance is still unknown [
      • Ascher D.B.
      • Wielens J.
      • Nero T.L.
      • Doughty L.
      • Morton C.J.
      • Parker M.W.
      Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA.
      ,
      • Berger C.
      • Romero-Brey I.
      • Radujkovic D.
      • Terreux R.
      • Zayas M.
      • Paul D.
      • et al.
      Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication.
      ,
      • McGivern D.R.
      • Masaki T.
      • Williford S.
      • Ingravallo P.
      • Feng Z.
      • Lahser F.
      • et al.
      Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors.
      ,
      • Nettles J.H.
      • Stanton R.A.
      • Broyde J.
      • Amblard F.
      • Zhang H.
      • Zhou L.
      • et al.
      Asymmetric binding to NS5A by daclatasvir (BMS-790052) and analogs suggests two novel modes of HCV inhibition.
      ,
      • Barakat K.H.
      • Anwar-Mohamed A.
      • Tuszynski J.A.
      • Robins M.J.
      • Tyrrell D.L.
      • Houghton M.
      A Refined Model of the HCV NS5A protein bound to daclatasvir explains drug-resistant mutations and activity against divergent genotypes.
      ,
      • Sun J.H.
      • O’Boyle 2nd, D.R.
      • Fridell R.A.
      • Langley D.R.
      • Wang C.
      • Roberts S.B.
      • et al.
      Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A.
      ]. Advances in these directions may yield new insight into basic principles of RNA and membrane (protein) biology on the one hand and may allow to develop potent antivirals against other positive-strand RNA viruses on the other.

      Lessons from hepatitis A, B and E viruses

      Hepatitis virus research has taught us repeatedly that it is worthwhile to pursue open questions in a given field. Who would have thought a few years ago that we would learn important and unexpected new lessons on how the hepatitis A virus (HAV) circulates in the blood? In fact, we know today that HAV (and the same applies to the hepatitis E virus [HEV]) does not circulate as a naked particle but wrapped (’quasi-enveloped’) in cellular membranes; a finding with implications for the transmission, pathogenesis and vaccine protection from hepatitis A and E [
      • Feng Z.
      • Hensley L.
      • McKnight K.L.
      • Hu F.
      • Madden V.
      • Ping L.
      • et al.
      A pathogenic picornavirus acquires an envelope by hijacking cellular membranes.
      ] (reviewed in [
      • Feng Z.
      • Hirai-Yuki A.
      • McKnight K.L.
      • Lemon S.M.
      Naked viruses that aren’t always naked: quasi-enveloped agents of acute hepatitis.
      ,
      • Walker C.M.
      • Feng Z.
      • Lemon S.M.
      Reassessing immune control of hepatitis A virus.
      ]). Additionally, textbook chapters dealing with HEV had to be constantly updated over the last years, and it is likely that unexpected discoveries will continue to be made in this exciting area (reviewed in [
      • Dalton H.R.
      • Webb G.W.
      • Norton B.C.
      • Woolson K.L.
      Hepatitis E virus: time to change the textbooks.
      ,
      • Debing Y.
      • Moradpour D.
      • Neyts J.
      • Gouttenoire J.
      Update on hepatitis E virology: implications for clinical practice.
      ]). Finally, highly protective and well-tolerated vaccines against the hepatitis B virus (HBV) were approved in the 1980s and effective antivirals were successively introduced as of the 1990s, reducing interest in HBV research. However, an estimated 250 million people are still infected today, and it is only in the last few years that HBV research has gained new momentum, with key discoveries that may facilitate efforts towards (functional) cure of hepatitis B (reviewed in [
      • Zeisel M.B.
      • Lucifora J.
      • Mason W.S.
      • Sureau C.
      • Beck J.
      • Levrero M.
      • et al.
      Towards an HBV cure: state-of-the-art and unresolved questions–report of the ANRS workshop on HBV cure.
      ,
      • Liang T.J.
      • Block T.M.
      • McMahon B.J.
      • Ghany M.G.
      • Urban S.
      • Guo J.T.
      • et al.
      Present and future therapies of hepatitis B: From discovery to cure.
      ,
      • Revill P.
      • Testoni B.
      • Locarnini S.
      • Zoulim F.
      Global strategies are required to cure and eliminate HBV infection.
      ]). Hence, addressing the many challenges in the molecular virology of hepatitis C will in all likelihood yield additional fundamental lessons about human biology and will at the same time allow to be prepared for unexpected developments.

      Immunology of viral hepatitis C

      It is still unclear what specific characteristics during the acute phase of infection can predict infection outcome. We do know that the memory T cell response that is generated following the spontaneous resolution of the primary response, greatly reduces the chance of persistence upon re-exposure to homologous and/or heterologous infections [
      • Dahari H.
      • Feinstone S.M.
      • Major M.E.
      Meta-analysis of hepatitis C virus vaccine efficacy in chimpanzees indicates an importance for structural proteins.
      ,
      • Sacks-Davis R.
      • Grebely J.
      • Dore G.J.
      • Osburn W.
      • Cox A.L.
      • Rice T.M.
      • et al.
      Hepatitis C virus reinfection and spontaneous clearance of reinfection–the InC3 study.
      ]. Previous studies have highlighted the importance of the T cell response for viral clearance and attributed persistent infection to an insufficient and/or early loss of T cell responsiveness, specifically in the cluster of differentiation (CD)4+ T helper subset [
      • Gerlach J.T.
      • Diepolder H.M.
      • Jung M.C.
      • Gruener N.H.
      • Schraut W.W.
      • Zachoval R.
      • et al.
      Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C.
      ]. These antigen-specific CD4+ T cells are lost abruptly from circulation and signal the early demise of immune control of the infection [
      • Gerlach J.T.
      • Diepolder H.M.
      • Jung M.C.
      • Gruener N.H.
      • Schraut W.W.
      • Zachoval R.
      • et al.
      Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C.
      ,
      • Ulsenheimer A.
      • Lucas M.
      • Seth N.P.
      • Tilman Gerlach J.
      • Gruener N.H.
      • Loughry A.
      • et al.
      Transient immunological control during acute hepatitis C virus infection: ex vivo analysis of helper T-cell responses.
      ,
      • Neumann-Haefelin C.
      • Thimme R.
      Adaptive immune responses in hepatitis C virus infection.
      ]. While still technically challenging, a number of studies have nicely shown a reduction in the frequency of antigen-specific CD4+ T cells in circulation as acute infection progresses to the chronic phase [
      • Schulze Zur Wiesch J.
      • Ciuffreda D.
      • Lewis-Ximenez L.
      • Kasprowicz V.
      • Nolan B.E.
      • Streeck H.
      • et al.
      Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence.
      ,
      • Day C.L.
      • Seth N.P.
      • Lucas M.
      • Appel H.
      • Gauthier L.
      • Lauer G.M.
      • et al.
      Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers.
      ,
      • zur Wiesch J. Schulze
      • Lauer G.M.
      • Day C.L.
      • Kim A.Y.
      • Ouchi K.
      • Duncan J.E.
      • et al.
      Broad repertoire of the CD4+ Th cell response in spontaneously controlled hepatitis C virus infection includes dominant and highly promiscuous epitopes.
      ]. Interestingly, initially during the acute infection the number of epitopes targeted by these CD4+ T cells seem to be equivalent but their responses diverge significantly when the infection becomes chronic [
      • Schulze Zur Wiesch J.
      • Ciuffreda D.
      • Lewis-Ximenez L.
      • Kasprowicz V.
      • Nolan B.E.
      • Streeck H.
      • et al.
      Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence.
      ]. It has been suggested that HCV infection may induce CD4+ T cells to differentiate into other regulatory (Treg) and inflammatory (Th17) cells [
      • Cabrera R.
      • Tu Z.
      • Xu Y.
      • Firpi R.J.
      • Rosen H.R.
      • Liu C.
      • et al.
      An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection.
      ,
      • Sugimoto K.
      • Ikeda F.
      • Stadanlick J.
      • Nunes F.A.
      • Alter H.J.
      • Chang K.M.
      Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection.
      ,
      • Kared H.
      • Fabre T.
      • Bedard N.
      • Bruneau J.
      • Shoukry N.H.
      Galectin-9 and IL-21 mediate cross-regulation between Th17 and Treg cells during acute hepatitis C.
      ] that then influence infection outcome. However, their functional responsiveness directly ex vivo from the site of active viral replication, the liver, is not known.
      Although 90% of patients may now be cured by direct-acting antiviral agents, those few patients that fail to achieve sustained virological response are a next treatment challenge.
      While antigen-specific CD8+ T cells can readily be observed even during chronic infection, where these cells exhibit an exhausted phenotype and likely target an escape epitope, antigen-specific CD4+ T cells are much more difficult to identify. Additionally, epitope escape from a CD4+ T cell is not observed [
      • Fuller M.J.
      • Shoukry N.H.
      • Gushima T.
      • Bowen D.G.
      • Callendret B.
      • Campbell K.J.
      • et al.
      Selection-driven immune escape is not a significant factor in the failure of CD4 T cell responses in persistent hepatitis C virus infection.
      ] and it is much more difficult to expand these cells from the liver by in vitro stimulation. Whether antigen-specific CD4+ T cells are deleted from the repertoire or are rendered functionally unresponsive to any stimuli is difficult to ascertain at this time. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection of mice suggest that the transcriptional signature profile of exhausted CD4+ T cells, that includes such genes as Helios, type I IFN signaling, and various sets of coinhibitory and costimulatory molecules, is distinct when compared to that of exhausted CD8+ T cells [
      • Crawford A.
      • Angelosanto J.M.
      • Kao C.
      • Doering T.A.
      • Odorizzi P.M.
      • Barnett B.E.
      • et al.
      Molecular and transcriptional basis of CD4(+) T cell dysfunction during chronic infection.
      ]. This suggests that mechanisms of T cell failure during the chronic infection may be different between the two cell subsets. While direct examination between antigen-specific CD4+ and CD8+ T cells would allow for the establishment of differences in their respective phenotype and function, limited access to the human liver during the acute phase of infection slows progress on these analyses.
      As to why persistence ensues in the majority of the cases, failure to sustain a T cell response (particularly CD4+ T helper activity) during acute hepatitis C is a reliable predictor of a chronic outcome [
      • Ulsenheimer A.
      • Lucas M.
      • Seth N.P.
      • Tilman Gerlach J.
      • Gruener N.H.
      • Loughry A.
      • et al.
      Transient immunological control during acute hepatitis C virus infection: ex vivo analysis of helper T-cell responses.
      ,
      • Schulze Zur Wiesch J.
      • Ciuffreda D.
      • Lewis-Ximenez L.
      • Kasprowicz V.
      • Nolan B.E.
      • Streeck H.
      • et al.
      Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence.
      ,
      • Diepolder H.M.
      • Zachoval R.
      • Hoffmann R.M.
      • Jung M.C.
      • Gerlach T.
      • Pape G.R.
      The role of hepatitis C virus specific CD4+ T lymphocytes in acute and chronic hepatitis C.
      ,
      • Lucas M.
      • Ulsenheimer A.
      • Pfafferot K.
      • Heeg M.H.
      • Gaudieri S.
      • Gruner N.
      • et al.
      Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection.
      ]. In addition, several studies suggest that the intrahepatic HCV-specific T cell response in chronically infected patients does not mirror the response in the peripheral blood and that the liver-infiltrating T cells are phenotypically exhausted and dysfunctional [
      • Radziewicz H.
      • Ibegbu C.C.
      • Fernandez M.L.
      • Workowski K.A.
      • Obideen K.
      • Wehbi M.
      • et al.
      Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.
      ,
      • Kroy D.C.
      • Ciuffreda D.
      • Cooperrider J.H.
      • Tomlinson M.
      • Hauck G.D.
      • Aneja J.
      • et al.
      Liver environment and HCV replication affect human T-cell phenotype and expression of inhibitory receptors.
      ]. The quality of the T cell response can also be impaired at all stages, including priming, expansion, differentiation, homing, cytotoxic T lymphocyte escape and maintenance [
      • Park S.H.
      • Rehermann B.
      Immune responses to HCV and other hepatitis viruses.
      ]. In addition, antibody-mediated depletion of CD4+ T cells just before HCV re-challenge of immune chimpanzees provided direct evidence that loss of helper cell activity results in CD8+ T cell exhaustion, emergence of escape variants, and virus persistence [
      • Grakoui A.
      • Shoukry N.H.
      • Woollard D.J.
      • Han J.H.
      • Hanson H.L.
      • Ghrayeb J.
      • et al.
      HCV persistence and immune evasion in the absence of memory T cell help.
      ]. HCV-specific CD8+ T cells in the peripheral blood of patients have impaired proliferative capacity that is associated with, and reversed by antibody blockade of the inhibitory molecule programmed cell death protein 1 (PD-1) both in vitro [
      • Radziewicz H.
      • Ibegbu C.C.
      • Fernandez M.L.
      • Workowski K.A.
      • Obideen K.
      • Wehbi M.
      • et al.
      Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.
      ,
      • Golden-Mason L.
      • Palmer B.
      • Klarquist J.
      • Mengshol J.A.
      • Castelblanco N.
      • Rosen H.R.
      Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction.
      ,
      • Urbani S.
      • Amadei B.
      • Tola D.
      • Massari M.
      • Schivazappa S.
      • Missale G.
      • et al.
      PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion.
      ] and in vivo [
      • Fuller M.J.
      • Callendret B.
      • Zhu B.
      • Freeman G.J.
      • Hasselschwert D.L.
      • Satterfield W.
      • et al.
      Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1).
      ]. Importantly, PD-1 and other markers of functional exhaustion (such as Tim3, 2B4 and lag3) and apoptosis are further upregulated on intrahepatic T cells [
      • Radziewicz H.
      • Ibegbu C.C.
      • Fernandez M.L.
      • Workowski K.A.
      • Obideen K.
      • Wehbi M.
      • et al.
      Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.
      ,
      • McMahan R.H.
      • Golden-Mason L.
      • Nishimura M.I.
      • McMahon B.J.
      • Kemper M.
      • Allen T.M.
      • 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.
      ]. A recent publication indicates that epigenetic modification of the PD-1 gene (PDCD1) regulatory element is critical to CD8+ T cell exhaustion and memory development [
      • Youngblood B.
      • Oestreich K.J.
      • Ha S.J.
      • Duraiswamy J.
      • Akondy R.S.
      • West E.E.
      • et al.
      Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8(+) T cells.
      ]. Specifically, transient demethylation of CpG dinucleotides immediately upstream of the PDCD1 gene resulted in PD-1 expression on effector cells during acute LCMV infection [
      • Youngblood B.
      • Oestreich K.J.
      • Ha S.J.
      • Duraiswamy J.
      • Akondy R.S.
      • West E.E.
      • et al.
      Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8(+) T cells.
      ]. In contrast to fully differentiated memory T cells, remethylation of PDCD1 did not occur in CD8+ T cells progressing towards exhaustion. Despite innumerable studies contributing to great progress in our understanding of HCV infection, features of intrahepatic T cell immunity that determine resolution vs. persistence of the virus remain incompletely understood.

      Challenges for translational and clinical hepatitis C research

      More than 90% of patients with chronic hepatitis C can now be cured with interferon (IFN)-free all-oral regimens, the so-called DAA regimens. Real-life data from around the world confirm the results obtained in phase 3 trials. Among the remaining challenges in HCV treatment is the management of those who failed previous all-oral regimens, the so-called DAA failures (Table 3). Failure to respond to previous pegylated IFNα (PegIFNα) plus ribavirin (RBV) treatment, no longer represent a challenge.
      Table 3Clinical unmet needs in hepatitis C.
      DAA, direct acting antiviral.

      Resistance-associated variants

      Preexisting NS5A resistance-associated variants (RAVs) which were recently also termed resistance associated substitutions (RAS) turned out to be of particular relevance and remain for the moment a major obstacle to overcome. Prevalence of pretreatment NS5A RAVs varies around the world, overall around 15%, and depends on the HCV genotype or subtype [
      • Manns M.
      • Pol S.
      • Jacobson I.M.
      • Marcellin P.
      • Gordon S.C.
      • Peng C.Y.
      • et al.
      All-oral daclatasvir plus asunaprevir for hepatitis C virus genotype 1b: a multinational, phase 3, multicohort study.
      ,

      Chu C, et al. Poster P006 (M13–767). APASL STC 2016; 2016; Taiwan.

      ,

      Chu C, et al. Poster P007 (M14–491). APASL STC 2016; 2016; Taiwan.

      ]. These patients are at risk of treatment failure. In addition, NS5A RAVs exhibit long-term persistence [
      • Wyles D.L.
      • Ruane P.
      • Sulkowski M.S.
      • Dieterich D.
      • Luetkemeyer A.F.
      • Morgan T.R.
      • et al.
      Daclatasvir plus sofosbuvir for treatment of HCV genotypes 1–4 in Hiv-Hcv coinfection: the ally-2 study.
      ,
      • Pawlotsky J.M.
      • Hepatitis C.
      Virus resistance to direct-acting antiviral drugs in interferon-free regimens.
      ]. Pretreatment NS5A RAV testing is recommended in the US for the new dual DAA regimen comprising grazoprevir plus elbasvir according to FDA labelling. In Japan and Korea pretreatment NS5A RAV testing has become an everyday practice since the 24-week regimen comprising asunaprevir plus daclatasvir became widely used. This regimen is highly efficacious and cost effective for genotype 1b patients without preexisting NS5A RAVs. Population sequencing with a sensitivity of ∼15% is preferred over deep sequencing that has a sensitivity of ∼1%. Retreatment with the same regimen for longer time and/or addition of RBV is successful in a proportion of patients with DAA failures [
      • Lawitz E.
      • Flamm S.
      • Yang J.C.
      • Pang P.S.
      • Zhu Y.
      • Svarovskaia E.
      • et al.
      Retreatment of patients who failed 8 or 12 weeks of ledipasvir/sofosbuvir- based regimens with ledipasvir/sofosbuvir for 24 weeks.
      ]. The fixed dose combination of velpatasvir plus sofobuvir (VEL/SOF) is the latest approved by FDA for the US and EMA for EU for genotypes 1-6. High SVR12 rates were reported [
      • Curry M.P.
      • O’Leary J.G.
      • Bzowej N.
      • Muir A.J.
      • Korenblat K.M.
      • Fenkel J.M.
      • et al.
      Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis.
      ,
      • Feld J.J.
      • Jacobson I.M.
      • Hezode C.
      • Asselah T.
      • Ruane P.J.
      • Gruener N.
      • et al.
      Sofosbuvir and velpatasvir for HCV genotype 1, 2, 4, 5, and 6 infection.
      ,
      • Foster G.R.
      • Afdhal N.
      • Roberts S.K.
      • Brau N.
      • Gane E.J.
      • Pianko S.
      • et al.
      Sofosbuvir and velpatasvir for HCV genotype 2 and 3 infection.
      ] and analysis of treatment failures for the Astral 1-4 phase III studies were reported as well [
      • Hezode C.R.N.
      • Svarovskaia E.S.
      • Doehle B.
      • Chodavarapu K.
      • Dvory-Sobol H.
      • McNally J.
      • et al.
      Resistance analysis in 1284 patients with genotype 1–6 HCV infection treated with sofosbuvir/velpatasvir in the phase 3 ASTRAL-1, ASTRAL-2, ASTRAL-3, and ASTRAL-4 studies.
      ]. NS 5A RAVs at position Y93 seem to matter in particular for genotype 3 patients and decompensated liver disease, however, the relevance of RAVs for this VEL/SOF combination needs to be confirmed including real world experiences.
      At the moment several triple drug regimens are in phase 2 and 3 development for patients who failed to previous DAA-based regimens. These triple regimens include drugs against the main antiviral targets within the HCV replicase, i.e., a protease inhibitor, an NS5A inhibitor and a nucleos(t)idic HCV polymerase inhibitor. Encouraging preliminary results have already been reported [
      • Poordad F.
      • Bennett M.
      • Sepe T.E.
      • Cohen E.
      • Reindollar R.W.
      • Everson G.
      • et al.
      Ombitasvir/paritaprevir/R, dasabuvir and sofosbuvir treatment of patients with HCV genotype 1 infection who failed a prior course of DAA therapy: the QUARTZ-1 Study.
      ,
      • Poordad F.
      • Felizarta F.
      • Wang S.
      • Asatryan A.
      • Hassanein T.
      • Aguilar H.
      • et al.
      High SVR rates with the combination of ABT-493 + ABT-530 for 8 weeks in non-cirrhotic patients with HCV genotype 1 or 2 infection.
      ,
      • Lawitz E.
      • Kowdley K.
      • Curry M.
      • Reau N.
      • Nguyen M.
      • Kwo P.
      • et al.
      High efficacy of sofosbuvir/velpatasvir plus GS-9857 for 12 weeks in treatment-experienced genotype 1-6 HCV-infected patients, including those previously treated with direct-acting antivirals.
      ].

      Chimeric viruses

      Recently, surprisingly low sustained virological response (SVR) rates were observed in some cohorts of patients treated with sofosbuvir plus RBV for HCV genotype 2 infection, in particular in cohorts including significant numbers of Russian patients [
      • Tacke F.
      • Günther R.
      • Buggisch P.
      • Klinker H.
      • Schober A.
      • John C.
      • et al.
      Treatment of HCV genotype 2 with sofosbuvir and ribavirin results in low SVR rates in a real world cohort (German Hepatitis C Registry, DHC-R).
      ]. Molecular analyses resulted in the identification of recombinant HCV viruses comprising structural and nonstructural regions from genotypes 2k and 1b, respectively [
      • Susser S.
      • Dietz J.
      • Barak M.
      • Schlevogt B.
      • Daniel R.
      • Piazzolla V.
      • et al.
      Prevalence and clinical importance of hepatitis C virus genotype 2k/1b chimeras.
      ]. While the 2k segment encodes for the 5’ noncoding and core regions responsible for the genotyping result, the remaining segment encodes the nonstructural proteins that confer treatment response based on the genotype 1 sequences. Therefore, patients carrying this chimeric virus are wrongly typed as genotype 2 while their treatment response mirrors that of HCV genotype 1. This recombinant HCV was originally termed the St. Petersburg variant; it is nowadays not only observed in Russia but also in other parts of the world with a strong representation of migrants, including Israel and Germany.

      Renal insufficiency

      A further challenge is the management of patients with renal insufficiency, in particular since sofosbuvir-based regimens are limited to patients with a glomerular filtration rate (GFR) >30 ml/min. The so-called 3D regimen (ritonavir-boosted paritaprevir, ombitasvir and dasabuvir) ± RBV can be used in patients with a GFR <30 [
      • Pockros P.J.
      • Reddy K.R.
      • Mantry P.S.
      • Cohen E.
      • Bennett M.
      • Sulkowski M.S.
      • et al.
      Efficacy of direct-acting antiviral combination for patients with hepatitis C virus genotype 1 infection and severe renal impairment or end-stage renal disease.
      ]. However, RBV has to be used with this regimen in patients with HCV genotype 1a. The grazoprevir plus elbasvir combination, recently approved and available in the US and Switzerland, is also approved for patients with renal insufficiency, including patients with end-stage renal disease and on dialysis [
      • Roth D.
      • Nelson D.R.
      • Bruchfeld A.
      • Liapakis A.
      • Silva M.
      • Monsour Jr, H.
      • et al.
      Grazoprevir plus elbasvir in treatment-naive and treatment-experienced patients with hepatitis C virus genotype 1 infection and stage 4–5 chronic kidney disease (the C-SURFER study): a combination phase 3 study.
      ].

      HCV genotype 3

      Another difficult-to-treat population are the patients with HCV genotype 3 infection. However, it seems that the next generation of DAAs may overcome this problem. One such combination, comprising the new pangenotypic NS5A inhibitor velpatasvir and sofosbuvir, has completed the phase 3 program [
      • Curry M.P.
      • O’Leary J.G.
      • Bzowej N.
      • Muir A.J.
      • Korenblat K.M.
      • Fenkel J.M.
      • et al.
      Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis.
      ,
      • Feld J.J.
      • Jacobson I.M.
      • Hezode C.
      • Asselah T.
      • Ruane P.J.
      • Gruener N.
      • et al.
      Sofosbuvir and velpatasvir for HCV genotype 1, 2, 4, 5, and 6 infection.
      ,
      • Foster G.R.
      • Afdhal N.
      • Roberts S.K.
      • Brau N.
      • Gane E.J.
      • Pianko S.
      • et al.
      Sofosbuvir and velpatasvir for HCV genotype 2 and 3 infection.
      ] and has been approved in the US and EU in mid 2016.

      Decompensated liver disease

      Maybe the biggest remaining challenge is the treatment of patients with decompensated cirrhosis. Two regimens, ledipasvir plus sofosbuvir and daclatasvir plus sofosbuvir, are approved for decompensated liver disease; each with RBV for 12 weeks and without RBV for 24 weeks, both for genotype 1 and 4, daclatasvir plus sofosbuvir also for genotype 3 [
      EASL recommendations on treatment of hepatitis C 2015.
      ,
      • Chung R.T.
      • Davis G.L.
      • Jensen D.M.
      • Masur H.
      • Saag M.S.
      • Thomas D.L.
      • et al.
      Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus.
      ]. These regimens are efficacious and safe in this population of patients but can only be given if GFR is >30 ml/min, a limitation related to sofosbuvir. The SOLAR-1 and -2 studies, forming the basis for drug approval for ledipasvir plus sofosbuvir, compared 12 to 24 weeks and used RBV in all arms [
      • Charlton M.
      • Everson G.T.
      • Flamm S.L.
      • Kumar P.
      • Landis C.
      • Brown Jr, R.S.
      • et al.
      Ledipasvir and sofosbuvir plus ribavirin for treatment of HCV infection in patients with advanced liver disease.
      ,
      • Manns M.
      • Samuel D.
      • Gane E.J.
      • Mutimer D.
      • McCaughan G.
      • Buti M.
      • et al.
      Ledipasvir and sofosbuvir plus ribavirin in patients with genotype 1 or 4 hepatitis C virus infection and advanced liver disease: a multicentre, open-label, randomised, phase 2 trial.
      ]. Twelve-week results were comparable to 24 weeks; however, the role of RBV remains unclear in decompensated liver disease. The majority of patients treated with decompensated cirrhosis Child-Pugh B or C, before or after liver transplantation, improved both Child-Pugh as well as model for end-stage liver disease (MELD) scores. Similar results were seen with the recently approved combination of velpatasvir and sofosbuvir in a phase 3 study including patients with decompensated cirrhosis Child-Pugh B [
      • Curry M.P.
      • O’Leary J.G.
      • Bzowej N.
      • Muir A.J.
      • Korenblat K.M.
      • Fenkel J.M.
      • et al.
      Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis.
      ]. The ASTRAL-4 study of this program contained also RBV-free arms. Interestingly, 12 weeks of velpatasvir and sofosbuvir plus RBV were numerically superior to 12 weeks without RBV but also to velpatasvir and sofosbuvir without RBV for 24 weeks. This observation needs to be confirmed by additional prospective and adequately powered studies. However, a minority of patients with decompensated liver disease deteriorated their liver function despite achievement of SVR. Therefore, the point of no return until when the treatment of decompensated cirrhosis is beneficial needs to be defined. We urgently need longer follow up data also to identify risk factors for deterioration of liver disease following clearance of the virus; SVR 12 certainly is not enough. Finally, regimen for this patient population are needed that can be used in patients with renal insufficiency, e.g., GFR <30 ml/min).

      Hepatocellular carcinoma

      The role of HCV therapy in the management of HCC is a topic of increasing importance. HCV-related cirrhosis is associated with a high risk of HCC development, similar to HBV and alcohol. Control of chronic hepatitis B under HBV polymerase inhibitor treatment (e.g., lamivudine, entecavir, tenofovir) reduces but does not eliminate the risk of HCC development in patients with cirrhosis [
      • Liaw Y.F.
      • Sung J.J.
      • Chow W.C.
      • Farrell G.
      • Lee C.Z.
      • Yuen H.
      • et al.
      Lamivudine for patients with chronic hepatitis B and advanced liver disease.
      ,
      • Papatheodoridis G.V.
      • Lampertico P.
      • Manolakopoulos S.
      • Lok A.
      Incidence of hepatocellular carcinoma in chronic hepatitis B patients receiving nucleos(t)ide therapy: a systematic review.
      ]. Patients with HCV-associated cirrhosis who achieve an SVR following IFN-based therapies also show a reduced risk of HCC development [
      • van der Meer A.J.
      • Veldt B.J.
      • Feld J.J.
      • Wedemeyer H.
      • Dufour J.F.
      • Lammert F.
      • et al.
      Association between sustained virological response and all-cause mortality among patients with chronic hepatitis C and advanced hepatic fibrosis.
      ,
      • Morgan R.L.
      • Baack B.
      • Smith B.D.
      • Yartel A.
      • Pitasi M.
      • Falck-Ytter Y.
      Eradication of hepatitis C virus infection and the development of hepatocellular carcinoma: a meta-analysis of observational studies.
      ]. Recent data indicate that this may be different or at least controversial for patients with cirrhosis eliminating HCV after DAA-based therapies. The risk for recurrent HCC after curative or non-curative HCC therapies was increased in patients achieving SVR following DAA therapy [
      • Reig M.
      • Marino Z.
      • Perello C.
      • Inarrairaegui M.
      • Ribeiro A.
      • Lens S.
      • et al.
      Unexpected early tumor recurrence in patients with HCV-related HCC undergoing interferon-free therapy: a note of caution.
      ,
      • Kozbial K.
      • Stern R.
      • Freissmuth C.
      • Beinhardt S.
      • Stättermayer A.F.
      • Munda P.
      • et al.
      High risk for hepatocellular carcinoma in cirrhotic patients with SVR following IFN-free DAA treatment within 1 year follow-up.
      ]. In contrast, recurrent HCC was not increased in patients after curative HCC therapies in another study [
      • Pol S.
      Lack of evidence of an effect of direct-acting antivirals on the recurrence of hepatocellular carcinoma: The ANRS collaborative study group on hepatocellular carcinoma (ANRS CO22 HEPATHER, CO12 CIRVIR and CO23 CUPILT cohorts).
      ]. De novo HCC development after successful DAA therapy was decreased in a large early access program of patients with decompensated liver disease [
      • Foster G.R.
      • Irving W.L.
      • Cheung M.C.
      • Walker A.J.
      • Hudson B.E.
      • Verma S.
      • et al.
      Impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis.
      ]. Patients with HCV-related cirrhosis need to be followed long-term and the risk in these patients needs to be defined prospectively. Beneficial tumor suppressive effects of IFNα have been reported in the past. Low-dose PegIFNα treatment in patients with HCV cirrhosis has been explored prospectively some years ago, e.g., in the HALT-C trial in the US [
      • Lok A.S.
      • Everhart J.E.
      • Wright E.C.
      • Di Bisceglie A.M.
      • Kim H.Y.
      • Sterling R.K.
      • et al.
      Maintenance peginterferon therapy and other factors associated with hepatocellular carcinoma in patients with advanced hepatitis C.
      ] and the global European prospective investigation into cancer and nutrition (EPIC) studies [
      • Bruix J.
      • Poynard T.
      • Colombo M.
      • Schiff E.
      • Burak K.
      • Heathcote E.J.
      • et al.
      Maintenance therapy with peginterferon alfa-2b does not prevent hepatocellular carcinoma in cirrhotic patients with chronic hepatitis C.
      ]. Perhaps ongoing HCV infection induces IFN-inducible genes that are beneficial in suppressing HCC development. However, further studies are required to assess the risk of de novo and recurrent HCC development in HCV-related cirrhosis after successful DAA-based therapies and potential mechanisms need to be explored in future prospective studies. In this context DAA treatment provides a unique opportunity to study innate and adaptive immune events after antigen removal in a chronic human viral infection [
      • Hengst J.
      • Strunz B.
      • Deterding K.
      • Ljunggren H.G.
      • Leeansyah E.
      • Manns M.P.
      • et al.
      Nonreversible MAIT cell-dysfunction in chronic hepatitis C virus infection despite successful interferon-free therapy.
      ,
      • Meissner E.G.
      • Kohli A.
      • Virtaneva K.
      • Sturdevant D.
      • Martens C.
      • Porcella S.F.
      • et al.
      Achieving sustained virologic response after interferon-free hepatitis C virus treatment correlates with hepatic interferon gene expression changes independent of cirrhosis.
      ].

      Shortening treatment duration

      Another debate is how far we will be able to shorten treatment duration for all or subpopulations of patients with chronic hepatitis C. Eight weeks already seems possible for most non-cirrhotic patients, six weeks seems realistic for the future while four weeks at the moment seems to be too short; a challenge even for the next generation of antivirals [
      • Gane E.J.
      • Hyland R.H.
      • Ying Y.
      • Svarovskaia E.
      • Stamm L.M.
      • Brainard D.M.
      • et al.
      Safety and efficacy of short-duration treatment with Gs-9857 combined with sofosbuvir/Gs-5816 in treatment-naive and Daa-experienced genotype 1 patients with and without cirrhosis.
      ,
      • Lawitz E.
      • Poordad F.
      • Gutierrez J.A.
      • Evans B.
      • Hwang P.
      • Robertson M.
      • et al.
      C-SWIFT: MK-5172+MK-8742+sofosbuvir in treatment-naive patients with hepatitis C virus genotype 1 infection, with and without cirrhosis, for durations of 4, 6, or 8 weeks.
      ]. Six weeks has already become the reality for monoinfected patients with acute hepatitis C [
      • Deterding K.
      • Spinner C.
      • Schott E.
      • Welzel T.
      • Gerken G.
      • Klinker H.
      • et al.
      Six weeks of sofosbuvir/ledipasvir are sufficient to treat acute hepatitis C virus genotype 1 monoinfection: the HEPNET acute HCV IV study.
      ]. Studies are ongoing to evaluate the shortest possible duration of treatment. Recent data have shown that response-guided therapy may experience a revival at least for some subpopulations of patients with chronic hepatitis C in resource-limited areas. In a small series of Chinese treatment-naïve non-cirrhotic genotype 1b patients all achieved SVR12 after 3 weeks of triple therapy with different combinations of approved DAAs once they had achieved an ultrarapid viral response, i.e., <500 IU/ml HCV RNA in serum after 48 h [
      • Lau G.K.
      • Benhamou Y.
      • Chen G.F.
      • Li J.
      • Shao Q.
      • Ji D.
      • et al.
      Complete cure after three weeks of all-oral triple-direct acting antiviral (DAA) regimens in non-cirrhotic chronic hepatitis C genotype 1b Chinese subjects (SODAPI STUDY).
      ]. Therefore, the debate on future HCV therapies will go on: “one pill fits all” vs. individualized therapies based on (very) early viral kinetics [
      • Cornberg M.
      • Manns M.
      Hepatitis C: individualised medicine versus one pill fits all.
      ].

      Screening for HCV

      There will be no control of hepatitis C without national screening programs. In most countries so far, there are no screening programs in place apart from screening blood donations for anti-HCV and HCV RNA which represents one of the greatest successes in preventive medicine thus far.
      One strategy is screening of particular risk groups such as those who have received blood or blood products before 1990 as well as individuals with a history of intravenous (i.v.) drug use. Screening of certain birth cohorts in addition to risk-based screening is recommended in the US. In fact, those born between 1945 and 1965, the so-called baby boomer generation, have been identified as carrying the highest burden of HCV infections in the US. German guidelines recommend screening in all patients with elevated alanine aminotransferase (ALT) levels and in those with the risk factors above. The German Liver Foundation together with other stakeholders tries to convince the government to introduce ALT screening as part of the “Check-up 35” program covered by health insurance. For specific patient populations, like patients who inject drugs (PWIDs), and resource limited settings like World Health Organisation (WHO) low income countries, rapid point-of-care diagnostics and dry blood spot technology may be applied to assess the disease burden and improve treatment uptake [
      • Razavi H.
      • Waked I.
      • Sarrazin C.
      • Myers R.P.
      • Idilman R.
      • Calinas F.
      • et al.
      The present and future disease burden of hepatitis C virus (HCV) infection with today’s treatment paradigm.
      ].

      Treatment as prevention: Elimination of HCV in special patient populations

      A major source of further spread of HCV are certain risk groups with a high prevalence of infection. Therefore, one short-term goal is to eliminate HCV infection from specific patient populations. One such risk group – maybe the most important one – is patients who inject drugs (PWIDs). The population of i.v. drug users or PWIDs is an enormous challenge due to the risk of reinfection even after successful antiviral treatment. Another risk group are prison inmates. These two risk groups certainly overlap. It is well known that in some countries like Australia the major source for de novo HCV infections are prison inmates who continue to inject drugs. Reinfection after achieving SVR while continuing or reinitiating i.v. drug abuse is a major source of new HCV infections [
      • Midgard H.
      • Bjoro B.
      • Maeland A.
      • Konopski Z.
      • Kileng H.
      • Damas J.K.
      • et al.
      Hepatitis C reinfection after sustained virological response.
      ]. A prophylactic vaccine inducing protective immunity against HCV would be of particular relevance for this patient population. Maybe as long as a prophylactic vaccine is not available novel therapies like depot administrations of existing DAAs or long-acting miR122 antagomirs may be a solution for these specific hard to treat populations like PWIDs. Another approach to prevent transmission of the virus consists in identifying and treating acute hepatitis C. Recently, short-term DAA treatment for 6 weeks was shown to cure all monoinfected patients [
      • Deterding K.
      • Spinner C.
      • Schott E.
      • Welzel T.
      • Gerken G.
      • Klinker H.
      • et al.
      Six weeks of sofosbuvir/ledipasvir are sufficient to treat acute hepatitis C virus genotype 1 monoinfection: the HEPNET acute HCV IV study.
      ]. However, lower response rates were reported in human immunodeficiency virus (HIV)-coinfected patients with acute hepatitis C [

      Rockstroh J, Bhagani S, Hyland RH, Yun C, Zhang W, Brainard DM, et al. Ledipasvir/sofosbuvir for 6 weeks in HIV-infected patients with acute HCV infection. CROI 2016; Boston.

      ]. Other patient populations that soon should become HCV-free are those with end-stage renal disease or on dialysis as well as those with hematological disorders such as thalassemia and hemophilia.
      Effective and far-reaching screening programs as well as linkage to care will be essential.

      Patients on liver transplantation waiting list

      Treating patients who are on the liver transplant waiting list and those with recurrent HCV infection after transplantation is essential and also a realistic short-term goal. In the years to come all transplant patients, liver and non-liver, should be free of hepatitis C. This implies treating all HCV-positive transplant candidates while they are on the waiting list for as long as their liver disease is not too advanced [
      • Manns M.
      • Samuel D.
      • Gane E.J.
      • Mutimer D.
      • McCaughan G.
      • Buti M.
      • et al.
      Ledipasvir and sofosbuvir plus ribavirin in patients with genotype 1 or 4 hepatitis C virus infection and advanced liver disease: a multicentre, open-label, randomised, phase 2 trial.
      ,
      • Charlton M.
      • Gane E.
      • Manns M.P.
      • Brown R.S.
      • Curry M.P.
      • Kwo P.Y.
      • et al.
      Sofosbuvir and ribavirin for treatment of compensated recurrent hepatitis C virus infection after liver transplantation.
      ]. This could also mean using organs from HCV-positive donors. Live (e.g., kidney) donors should be treated and cured before transplantation. For cadaveric donors this would mean using HCV-positive organs and curing HCV after transplantation. Excellent results were reported for various regimens used before or after liver transplantation. Recently, a large series of post-renal transplant patients were shown to be 100% cured by ledipasvir plus sofosbuvir [
      • Colombo M.A.A.
      • Liu H.
      • Dvory-Sobol H.
      • Hyland R.
      • Yun C.
      • Brainard D.M.
      • et al.
      Ledipasvir/sofosbuvir (LDV/SOF) for 12 or 24 weeks is safe and effective in kidney transplant recipients with chronic genotype 1 or 4 HCV infection.
      ]. Twelve weeks were as good as 24 weeks, each without RBV. The limitation of this regimen is that only patients with a GFR >30 ml/min could be enrolled.
      Work is now underway to ensure that all HCV populations can be successfully treated with the new treatment regimens.

      Extrahepatic manifestations

      Expanding treatment to patients with extrahepatic manifestations in the absence of liver disease is another future challenge [
      • Negro F.
      • Forton D.
      • Craxi A.
      • Sulkowski M.S.
      • Feld J.J.
      • Manns M.P.
      Extrahepatic morbidity and mortality of chronic hepatitis C.
      ]. It is well documented that successful treatment of hepatitis C reduces not only liver related but also overall mortality [
      • van der Meer A.J.
      • Wedemeyer H.
      • Feld J.J.
      • Dufour J.F.
      • Zeuzem S.
      • Hansen B.E.
      • et al.
      Life expectancy in patients with chronic HCV infection and cirrhosis compared with a general population.
      ]. This includes mortality due to cardiovascular causes and cancer. Cure of hepatitis C also means reduction of a general inflammatory response that may increase the risk for cardiovascular complications such as myocardial infarction and cerebral insults as well as cancer. There is also an association between diabetes mellitus and hepatitis C. In HCV patients the presence of type 2 diabetes mellitus increases the incidence of HCC while controlling glycemia reduces the HCC risk [
      • Tacke F.
      • Günther R.
      • Buggisch P.
      • Klinker H.
      • Schober A.
      • John C.
      • et al.
      Treatment of HCV genotype 2 with sofosbuvir and ribavirin results in low SVR rates in a real world cohort (German Hepatitis C Registry, DHC-R).
      ]. Overall, HCV-associated insulin resistence is correlated with poor outcomes including hepatic fibrosis progression, development of HCC, type 2 diabetes mellitus and cardiovascular sequelae. Eliminating HCV improves insulin resistance [
      • Kawaguchi T.
      • Ide T.
      • Taniguchi E.
      • Hirano E.
      • Itou M.
      • Sumie S.
      • et al.
      Clearance of HCV improves insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate 1 and 2.
      ]. The association of hepatitis C and neurological manifestations is controversial. Fatigue is prevalent in patients with chronic hepatitis C and is regarded as an extrahepatic symptom or syndrome as is cognitive impairment. Theories have attributed this phenomenon to intracerebral replication of HCV. Future studies will have to show whether fatigue represents an indication for treatment of chronic hepatitis C independent from the stage of liver fibrosis. These approaches are encouraged by data showing that while under treatment with IFN- and RBV-free regimens patient-related outcomes and quality of life measures improve [
      • Younossi Z.M.
      • Stepanova M.
      • Feld J.
      • Zeuzem S.
      • Jacobson I.
      • Agarwal K.
      • et al.
      Sofosbuvir/velpatasvir improves patient-reported outcomes in HCV patients: Results from ASTRAL-1 placebo-controlled trial.
      ]. The relationship of hepatitis C with mixed cryoglobulinemia is well established, mechanisms are well understood and benefits for patients achieving SVR following treatment with (Peg)IFNα and RBV were demonstrated many years ago. Results for treating patients with HCV-associated mixed cryoglobulinemia with an all-oral DAA therapy are emerging [
      • Saadoun D.
      • Thibault V.
      • Si Ahmed S.N.
      • Alric L.
      • Mallet M.
      • Guillaud C.
      • et al.
      Sofosbuvir plus ribavirin for hepatitis C virus-associated cryoglobulinaemia vasculitis: VASCUVALDIC study.
      ,
      • Sise M.E.
      • Bloom A.K.
      • Wisocky J.
      • Lin M.V.
      • Gustafson J.L.
      • Lundquist A.L.
      • et al.
      Treatment of hepatitis C virus-associated mixed cryoglobulinemia with direct-acting antiviral agents.
      ].

      HCV and lymphoproliferative disorders

      There are data on the relationship between HCV infection and lymphoproliferative disorders, namely non-Hodgkin lymphoma (NHL) [
      • Tacke F.
      • Günther R.
      • Buggisch P.
      • Klinker H.
      • Schober A.
      • John C.
      • et al.
      Treatment of HCV genotype 2 with sofosbuvir and ribavirin results in low SVR rates in a real world cohort (German Hepatitis C Registry, DHC-R).
      ]. Cure of HCV following interferon based therapies may lead to regression of low grade HCV-related NHL and may prevent relapse of B cell lymphomas [
      • Hermine O.
      • Lefrere F.
      • Bronowicki J.P.
      • Mariette X.
      • Jondeau K.
      • Eclache-Saudreau V.
      • et al.
      Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection.
      ,
      • La Mura V.
      • De Renzo A.
      • Perna F.
      • D’Agostino D.
      • Masarone M.
      • Romano M.
      • et al.
      Antiviral therapy after complete response to chemotherapy could be efficacious in HCV-positive non-Hodgkin’s lymphoma.
      ]. Benefits of DAA therapies on the natural course of HCV-associated NHL are not well studied yet. However, lymphomas should be screened for HCV and treated if positive to aid in therapy and prevent relapse. There might be at least a subpopulation of NHL that regresses once HCV infection is cured, as can be seen in Helicobacter pylori-associated mucosa-associated lymphoid tissue (MALT) lymphoma.
      This list of extrahepatic manifestations associated with hepatitis C is long and may further expand [
      • Negro F.
      • Forton D.
      • Craxi A.
      • Sulkowski M.S.
      • Feld J.J.
      • Manns M.P.
      Extrahepatic morbidity and mortality of chronic hepatitis C.
      ]. However, the level of scientific evidence for such a relationship varies for the individual extrahepatic manifestation. Perhaps a response to DAA therapies will prove or rebut certain proposed causal relationships.
      Access to treatments is key to the elimination of HCV globally.

      Access to HCV therapy

      Most important is to improve access to treatment with the effective and safe DAA regimens that are now available. On February 17, 2016, the Hepatitis B and C Public Policy Association (HEPBCPPA) has presented to the public and published the manifesto “Hepatitis C Elimination in Europe” that was signed in Brussels by the EU Commissioner for Health and Food Safety, Dr. Vytenis Andriukaitis (http://www.hcvbrusselssummit.eu).
      Drugs are currently approved in the US by the FDA and in the EU by the EMA as well as several other national regulatory authorities. For many countries, however, approval does not necessarily mean that these therapies are immediately reimbursed. In some countries reimbursement is limited to patients with a certain fibrosis stage. However, this limitation was recently abandoned in Germany and France.
      While in the past the UK has usually been very restrictive towards the use of IFN-based therapies for HCV, the benefits of the newly approved oral HCV drugs were acknowledged by the National Institute for Health and Care Excellence (NICE) and the new therapies are recommended and reimbursed by the National Health Service.
      The pharmaceutical industry is also part of models for access to treatment. Since the high costs of the new DAAs limit accessibility, novel approaches to paying for these drugs are inevitable. Some examples already exist. For example, 600,000 therapies are provided to those with an Egyptian passport and residency in Egypt at <1% of the price in Western countries such as the US and Germany. In the meantime, generic as well as the original products are available in Egypt. In Portugal, after pressure by patient associations, the government negotiated a price with the industry for one regimen that is continuously reduced with the increase in numbers of patients being treated. So the overall effort is to cure hepatitis C in all patients with chronic hepatitis C in Portugal. Additional access programs are in place, in particular in low income countries with a high HCV prevalence such as Georgia, Mongolia and others aiming at HCV elimination. In India, more than seven pharmaceutical companies provide generic sofosbuvir and other DAAs for much less than 10% of the cost in Western countries. Other low income countries have followed, and the number of countries continues to expand, including Pakistan, Indonesia and others. There are also examples in some Western countries like Australia where prices drop or are even waved once a certain number of treatments is purchased at the regular price which also means a significant reduction in the overall cost burden. Health economic studies need to further prove cost effectiveness for HCV therapies in particular in preventing end-stage liver diseases like decompensated cirrhosis, liver cancer and liver transplantations but also non-liver related mortality. Such data would be very helpful to encourage governments to promote access to HCV therapies and treatment uptake. Treatment uptake has a much greater impact on the disease burden than increased efficacy of HCV drugs.

      Challenges in vaccine development for HCV

      Theoretically, the effectiveness of DAAs should substantially decrease the global burden of HCV infection and potentially eliminate viral transmission [
      • Hagan L.M.
      • Wolpe P.R.
      • Schinazi R.F.
      Treatment as prevention and cure towards global eradication of hepatitis C virus.
      ]. However, DAA regimens alone are likely not enough to facilitate eradication of this virus. HCV infection is asymptomatic and the number of individuals that are persistently infected may be underestimated. Many of those infected are unaware of their infection status. Furthermore, infection rates are increasing in adolescents and young adults who inject drugs, as illustrated by a recent study of the problem in the US [
      • Kim A.Y.
      • Onofrey S.
      • Church D.R.
      An epidemiologic update on hepatitis C infection in persons living with or at risk of HIV infection.
      ]. In 2002, the age distribution of newly reported cases of HCV infection in Massachusetts showed a single peak at ∼45 years of age. Data from 2011 showed a biphasic pattern with an additional second peak of new infection incidence at 24 years of age [
      • Kim A.Y.
      • Onofrey S.
      • Church D.R.
      An epidemiologic update on hepatitis C infection in persons living with or at risk of HIV infection.
      ]. While DAAs are highly effective for known HCV infection, the arduous task remains that one must be able to first identify those with new infections. This poses quite a challenge to keep up with the rate that undiagnosed HCV infection occurs.
      A preventive vaccine is needed to stop HCV transmission to uninfected individuals, and to those who are cured with DAA but remain at risk for re-exposure to the virus. However, vaccine development is complicated by our poor understanding of the adaptive immune responses to the virus [
      • Rehermann B.
      • Nascimbeni M.
      Immunology of hepatitis B virus and hepatitis C virus infection.
      ]. Studies in both humans and chimpanzees have shown that resolution of the primary infection can significantly reduce the chance of developing persistent infection upon subsequent re-exposures [
      • Walker C.M.
      • Grakoui A.
      Hepatitis C virus: why do we need a vaccine to prevent a curable persistent infection?.
      ]. T cell immunity has emerged as an important correlate of protective immunity in both species [
      • Grakoui A.
      • Shoukry N.H.
      • Woollard D.J.
      • Han J.H.
      • Hanson H.L.
      • Ghrayeb J.
      • et al.
      HCV persistence and immune evasion in the absence of memory T cell help.
      ,
      • Shoukry N.H.
      • Grakoui A.
      • Houghton M.
      • Chien D.Y.
      • Ghrayeb J.
      • Reimann K.A.
      • et al.
      Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection.
      ,
      • Klenerman P.
      • Thimme R.
      T cell responses in hepatitis C: the good, the bad and the unconventional.
      ,
      • Osburn W.O.
      • Fisher B.E.
      • Dowd K.A.
      • Urban G.
      • Liu L.
      • Ray S.C.
      • et al.
      Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.
      ]. Based on these premises, recent work has shown that vaccination of animals with a recombinant adenovirus that expresses the nonstructural proteins of HCV leads to both lower amplitude and duration of viremia in challenged animals [
      • Folgori A.
      • Capone S.
      • Ruggeri L.
      • Meola A.
      • Sporeno E.
      • Ercole B.B.
      • et al.
      A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.
      ]. As a result of this important observation, a similar vaccine is now in a phase II human clinical trial (Clinical Trials.gov NCT01296451). The results in naïve volunteers have already shown that the vaccine elicits a strong T cell immunity [
      • Swadling L.
      • Capone S.
      • Antrobus R.D.
      • Brown A.
      • Richardson R.
      • Newell E.W.
      • et al.
      A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory.
      ] and therefore holds great promise in providing protection from developing HCV persistence.
      Studies that define antiviral T cell immunity during acute HCV infection will be important for efficacious vaccine development. However, important obstacles remain; will governments introduce universal vaccination programs for HCV and pay for it once they would be available? Recent modeling suggests that in patient populations with a HCV prevalence <25% eradication can be achieved by medical therapy alone. However, if the prevalence in a particular risk group increases to 50%, such as in active i.v. drug users, only a vaccine will eliminate the virus from this population cohort [
      • Scott N.
      • McBryde E.
      • Vickerman P.
      • Martin N.K.
      • Stone J.
      • Drummer H.
      • et al.
      The role of a hepatitis C virus vaccine: modelling the benefits alongside direct-acting antiviral treatments.
      ]. Unfortunately, at the present moment, there is a paucity of larger vaccine industries that are investing in HCV vaccine development programs. However, a vaccine is still necessary in order to successfully eliminate this virus globally.

      Conclusion

      We have come a long way from non A, non B hepatitis to the identification of the HCV and, finally, all-oral combination therapies leading to cure in 95 to 100%. The story of HCV infection with a hand in hand development of diagnostic tests as well as highly effective and safe therapies is a masterpiece in translational research. This success story of modern medicine is a wonderful example of how the interplay of basic and translational science can lead to the control of a major global disease burden (Fig. 1). There is still a long way to go until this global health burden will be controlled and eradication of HCV will be achieved. For the time being, this final goal has to be approached without a prophylactic vaccine. Particular challenges to be addressed are elimination of HCV from particular risk groups like PWIDs, transplant populations, decompensated liver disease and HIV/HCV coinfections by medical therapies only. Once drug costs are reduced and access to care is improved, treatment will be extended to all patients with chronic HCV infection independent of liver fibrosis stage which on the long run will not only reduce liver related but also overall mortality. This also includes prevention of reinfection in risk groups like PWIDs which is crucial to prevent further spread of the virus. There is still a debate whether we need new drugs, whether in the future we will still have individualized treatment or whether “one pill fits all”. Until a prophylactic vaccine becomes available, reducing drug costs, improving access to treatment and, in particular, treatment uptake is crucial to combat this global health threat. By no means should further investment into hepatitis C research be abandoned. It is necessary to control this infection first by antiviral treatment and finally a prophylactic vaccine in order to prevent end-stage liver disease, liver cancer and the need of liver transplantation (Fig. 2). HCV is a unique model that yields knowledge of broad significance beyond hepatitis C.
      Figure thumbnail gr2
      Fig. 2Future landscape of hepatitis C research.

      Conflict of interest

      M.M., received grants from Roche, Bristol Myers Squibb, Gilead, Boehringer Ingelheim, Novartis, Merck (MSD), Janssen, GlaxoSmithKline, Biotest, AbbVie; and personal fees from Roche, Bristol Myers Squibb, Gilead, Boehringer Ingelheim, Novartis, Merck (MSD), Janssen, GlaxoSmithKline, Biotest, AbbVie, Achillion.
      D.M. participated in advisory boards organized by AbbVie, BMS, Gilead, Janssen, MSD and Roche, and received research support from BMS, Gilead and Roche.
      A.G., has nothing to declare.

      Acknowledgments

      We would like to acknowledge invaluable discussions with Drs. Markus Cornberg, Christopher M. Walker as well as Charles M. Rice and the support by Swiss National Science Foundation grant 31003A-156030 to D.M., NIH grants R01AI070101 , R01AI124680 , R01AI126890 and 1R21AI118337 to A.G., ORIP/OD P51OD011132 (formerly NCRR P51RR000165 ) to the Yerkes National Primate Research Center and the German Center for Infection Research (DZIF) funded by the German Ministry for Education and Research ( BMBF ) to MM. The editorial support by Drs. Svenja Hardtke and Philipp Solbach is highly appreciated.

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