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Second generation direct-acting antivirals – Do we expect major improvements?

  • Jordan J. Feld
    Correspondence
    Corresponding authors. Addresses: Toronto Centre for Liver Disease, Sandra Rotman Centre for Global Health, University Health Network, University of Toronto, 200 Elizabeth Street, 9EN, M5G 2C4, Canada. Tel.: +1 4163404584; fax: +1 4166035472 (J. Feld), or Queen Mary University of London, The Blizard Institute, 4 Newark Street, London E1 4AT, UK. Tel.: +44 207 882 7242; fax: +44 207 882 2191 (G. Foster).
    Affiliations
    Toronto Centre for Liver Disease, Sandra Rotman Centre for Global Health, University of Toronto, Toronto, Canada
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  • Graham R. Foster
    Correspondence
    Corresponding authors. Addresses: Toronto Centre for Liver Disease, Sandra Rotman Centre for Global Health, University Health Network, University of Toronto, 200 Elizabeth Street, 9EN, M5G 2C4, Canada. Tel.: +1 4163404584; fax: +1 4166035472 (J. Feld), or Queen Mary University of London, The Blizard Institute, 4 Newark Street, London E1 4AT, UK. Tel.: +44 207 882 7242; fax: +44 207 882 2191 (G. Foster).
    Affiliations
    Queen Mary University London, London, UK
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      Summary

      The rapid progress in the development of direct-acting antiviral agents for hepatitis C has allowed the vast majority of patients to receive all oral therapy that will eliminate their virus. The success of the new regimens has led many to question the need for further developments in this field. Major improvements in drugs for hepatitis C are unlikely but we predict incremental improvements in the next few years. We hope that the next generation of drugs will address the unresolved issues for patients with genotype 3 infection where current treatments are still not entirely satisfactory and we anticipate improvements in the management of patients with renal failure. Shorter duration treatments, perhaps with novel modes of action, may allow simplified ‘one-dose’ treatments that will greatly expand our ability to treat patients who have difficulty accessing current services and we anticipate that the clinical community will better define the patients with advanced disease who will benefit from therapy prior to liver transplantation.

      Abbreviations:

      HCV (hepatitis C virus), G3 (genotype 3), SVR (sustained virological response), NS5A (non-structural 5A), PI (protease inhibitor)

      Keywords

      Linked Article

      Introduction

      The development of direct-acting antivirals (DAAs) has revolutionized the treatment of hepatitis C virus (HCV) infection. These small molecule inhibitors of different viral proteins have dramatically improved both response rates and the tolerability of treatment. The evolution of DAA therapy has moved at a remarkable pace from initial combinations with interferon (IFN) and ribavirin being replaced by extremely well tolerated single or multi-tablet regimens combining DAAs of different classes within just a few years. Current regimens deliver rates of sustained virological response (SVR) above 90% in most patient populations, raising the prospect of widespread treatment leading to local elimination or, possibly, even global eradication of HCV as a public health problem.
      Despite the incredible success of the first oral DAA regimens, development of new DAAs and new DAA combinations continues at a rapid pace. Future agents hold promise to address the remaining therapeutic holes and perhaps more importantly, to further simplify therapy, a key factor if treatment is going to move out of specialty clinics. While high SVR rates are critical to the success of HCV therapy for the individual patient, major improvements in all aspects of the cascade of care right from diagnosis through engagement in treatment must improve to address HCV at the population level. Simplified, less expensive therapies hold the promise of treatment being delivered in primary care settings using novel models of care, which has the potential to lead to the increases in treatment capacity which will be required to deliver the promise of HCV elimination [
      • Dore G.J.
      • Feld J.J.
      Hepatitis C virus therapeutic development: in pursuit of “perfectovir”.
      ].
      Here we will briefly review current drugs and discuss the remaining therapeutic challenges before examining DAAs in development, with a focus on how these regimens will address existing therapeutic and logistical gaps. Novel approaches to treatment and the future of HCV therapy will also be explored.

      First generation DAAs

      The first oral agents developed for HCV targeted the HCV non-structural 3 (NS3) serine protease [
      • Lamarre D.
      • Anderson P.C.
      • Bailey M.
      • Beaulieu P.
      • Bolger G.
      • Bonneau P.
      • et al.
      An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus.
      ]. While potent inhibitors of HCV replication, the first generation protease inhibitors (PIs), telaprevir and boceprevir, had important deficiencies including a low barrier to resistance, multiple drug-drug interactions and numerous side effects, which were particularly problematic when combined with pegylated interferon (PegIFN) and ribavirin [
      • Pawlotsky J.M.
      • Feld J.J.
      • Zeuzem S.
      • Hoofnagle J.H.
      From non-A, non-B hepatitis to hepatitis C virus cure.
      ]. These agents were quickly replaced by a second wave of first generation PIs including simeprevir, asunaprevir and paritaprevir, which primarily improved the side effect profile with only modest changes in the other attributes of this class of agents.
      In addition to the NS3 protease, the NS5B RNA-dependent RNA polymerase has been targeted and two approaches have been used. Classical nucleotide analogues that are incorporated into replicating HCV RNA and lead to chain termination of nascent viral genomes have been developed and differ substantially from so-called non-nucleotide polymerase inhibitors that target regions of the enzyme outside of the active site, inhibiting its function through steric hindrance [
      • Kumar S.
      • Jacobson I.M.
      Antiviral therapy with nucleotide polymerase inhibitors for chronic hepatitis C.
      ,
      • Feld J.J.
      Interferon-free strategies with a nucleoside/nucleotide analogue.
      ]. Despite the evaluation of numerous nucleotide inhibitors (NIs) in clinical trials, only sofosbuvir has been approved to date, with other agents in the class failing for a combination of lack of efficacy (e.g. mericitabine) and/or toxicity (PSI-938) [
      • Kumar S.
      • Jacobson I.M.
      Antiviral therapy with nucleotide polymerase inhibitors for chronic hepatitis C.
      ,
      • Feld J.J.
      Interferon-free strategies with a nucleoside/nucleotide analogue.
      ]. Sofosbuvir has proven highly effective with an excellent safety profile, pan-genotypic activity and most importantly, a very high barrier to resistance [
      • Feld J.J.
      Interferon-free strategies with a nucleoside/nucleotide analogue.
      ]. The only significant limitation to sofosbuvir is the fact that its major metabolite is renally cleared, preventing its use in patients with significant renal impairment [
      • Kirby B.J.
      • Symonds W.T.
      • Kearney B.P.
      • Mathias A.A.
      Pharmacokinetic, pharmacodynamic, and drug-interaction profile of the hepatitis C virus NS5B polymerase inhibitor sofosbuvir.
      ]. Unlike NIs, non-nucleotide polymerase inhibitors (NNIs) have a very low barrier to resistance and a very restricted genotype specificity [
      • 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.
      ]. To date, only dasabuvir, a ‘Thumb’ I inhibitor, which is only active against genotype 1 HCV, has been approved for clinical use, in combination with paritaprevir (PI) and ombitasvir (NS5A inhibitor) [
      • Kati W.
      • Koev G.
      • Irvin M.
      • Beyer J.
      • Liu Y.
      • Krishnan P.
      • et al.
      In vitro activity and resistance profile of dasabuvir, a nonnucleoside hepatitis C virus polymerase inhibitor.
      ].
      Despite an incomplete understanding of its function, the NS5A protein has proven to be an excellent therapeutic target. Agents discovered through compound screening with potent activity against HCV replicons selected for variants with nucleotide changes in the NS5A sequence, suggesting that these novel agents were targeting this protein which is involved in viral assembly and, probably, other aspects of the replication cycle [
      • 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.
      ,
      • Guedj J.
      • Dahari H.
      • Rong L.
      • Sansone N.D.
      • Nettles R.E.
      • Cotler S.J.
      • et al.
      Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life.
      ]. With extremely potent antiviral activity, wide genotypic coverage and little potential for drug-drug interactions, NS5A inhibitors quickly became an important component of most combination DAA regimens. The major limitation of the first generation agents in this class, including daclatasvir, ombitasvir and ledipasvir, is their very low barrier to antiviral resistance [
      • Nguyen L.T.
      • Hall N.
      • Sheerin D.
      • Carr M.
      • De Gascun C.F.
      Naturally occurring HCV NS5A/B inhibitor resistance-associated mutations to direct-acting antivirals.
      ]. Variants with resistance to NS5A inhibitors are generally very fit, allowing them to emerge even in untreated patients and to persist long-term in those who have failed an NS5A-inibitor-containing regimen [
      • Krishnan P.
      • Tripathi R.
      • Schnell G.
      • Reisch T.
      • Beyer J.
      • Dekhtyar T.
      • et al.
      Long-term follow-up of treatment-emergent resistance-associated vairantes in NS3, NS5A and NS5B with paritaprevir, ombitasvir and dasabuvir-based regimens.
      ]. Some troublesome resistant variants, such as the Y93H polymorphism persist in untreated populations, albeit at low frequency, compromising the efficacy of some regimens, particularly those of short duration [
      • Sulkowski M.
      • Hezode C.
      • Gerstoft J.
      • Vierling J.M.
      • Mallolas J.
      • Pol S.
      • et al.
      Efficacy and safety of 8 weeks versus 12 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin in patients with hepatitis C virus genotype 1 mono-infection and HIV/hepatitis C virus co-infection (C-WORTHY): a randomised, open-label phase 2 trial.
      ].
      Combinations of the four DAA classes have been evaluated in clinical trials and proven highly effective across a wide array of patient populations, including groups that were very difficult to cure with IFN-based therapy. Combinations approved to date include: PI + NI (simeprevir + sofosbuvir), PI + NS5A + NNI (paritaprevir/r, ombitasvir and dasabuvir), PI + NS5A (grazoprevir and elbasvir) and NS5A + NI (ledipasvir/sofosbuvir and daclatasvir + sofosbuvir).
      First generation DAA regimens are highly successful. However, there are specific patient populations for whom these therapies are suboptimal.

      Therapeutic challenges with existing regimens

      Clinical trials have reported SVR rates above 95% in most populations using the licensed combinations of first generation DAAs and remarkably, early data from real-world registries suggest that SVR rates in clinical practice are only marginally inferior [
      • Sulkowski M.S.
      • Vargas H.E.
      • Di Bisceglie A.M.
      • Kuo A.
      • Reddy K.R.
      • Lim J.K.
      • et al.
      Effectiveness of simeprevir plus sofosbuvir, with or without ribavirin, in real-world patients with HCV genotype 1 infection.
      ,
      • Terrault N.A.
      • Zeuzem S.
      • Di Bisceglie A.M.
      • Lim J.K.
      • Pockros P.
      • Frazier L.M.
      • et al.
      Treatment outcomes with 8, 12 and 24 week regimens of ledipasvir/sofosbuvir for the treatment of hepatitis C infection: analysis of a multicenter prospective, observational study.
      ]. However, despite their remarkable success, there are specific populations for whom currently approved regimens remain suboptimal.

      Genotype 3

      Of approved DAAs, only sofosbuvir and daclatasvir have significant activity against genotype 3 HCV, although other protease and NS5A inhibitors, such as the combination of grazoprevir and elbasvir may have clinical value, particularly in combination with a nucleotide. The combination of sofosbuvir and ribavirin for 24 weeks is highly effective for patients who do not have cirrhosis, but SVR rates drop significantly in those with cirrhosis [
      • Zeuzem S.
      • Dusheiko G.M.
      • Salupere R.
      • Mangia A.
      • Flisiak R.
      • Hyland R.H.
      • et al.
      Sofosbuvir and ribavirin in HCV genotypes 2 and 3.
      ]. The effect is most pronounced in patients with cirrhosis who have failed prior treatment with PegIFN and ribavirin in whom clinical trials report SVR rates of just 60% and real-world studies document SVR rates below 50% [
      • Zeuzem S.
      • Dusheiko G.M.
      • Salupere R.
      • Mangia A.
      • Flisiak R.
      • Hyland R.H.
      • et al.
      Sofosbuvir and ribavirin in HCV genotypes 2 and 3.
      ,
      • Alqahtani S.
      • Zeuzem S.
      Safety and effectiveness of sofosbuvir-based regimens for the treatment of hepatitis C genotype 3 and 4 infections: internal analysis of a prospective observational study.
      ]. Even for treatment-naïve patients with cirrhosis, SVR rates from real-world studies are as low as 58%, in contrast to trial data showing SVR rates of 79% [
      • Alqahtani S.
      • Zeuzem S.
      Safety and effectiveness of sofosbuvir-based regimens for the treatment of hepatitis C genotype 3 and 4 infections: internal analysis of a prospective observational study.
      ]. Sofosbuvir has also been evaluated in combination with daclatasvir in patients with genotype 3. When given for 12 weeks, this combination is highly effective in patients without cirrhosis, yielding SVR rates of 97% and 98% in treatment-naïve and treatment-experienced populations, respectively. However, in patients with cirrhosis, SVR rates are much less encouraging, falling to 57% and 63% in treatment-naïve and treatment-experienced cohorts [
      • Nelson D.R.
      • Cooper J.N.
      • Lalezari J.P.
      • Lawitz E.
      • Pockros P.J.
      • Gitlin N.
      • et al.
      All-oral 12-week treatment with daclatasvir plus sofosbuvir in patients with hepatitis C virus genotype 3 infection: ALLY-3 phase III study.
      ]. A small trial extending therapy to 16 weeks and adding ribavirin resulted in SVR rates of 86% in patients with cirrhosis who had previously failed IFN-based therapies, however the small number of patients and the lack of difference compared to patients treated for 12 weeks make this trial difficult to interpret [
      • Leroy V.
      • Angus P.
      • Bronowicki J.P.
      • Dore G.J.
      • Hezode C.
      • Pianko S.
      • et al.
      Daclatasvir, sofosbuvir, and ribavirin for hepatitis C virus genotype 3 and advanced liver disease: a randomized phase III study (ALLY-3+).
      ]. Although real-world studies extending daclatasvir and sofosbuvir with or without ribavirin have reported SVR rates of above 80% in patients with genotype 3 and cirrhosis, including those with decompensated cirrhosis, there are no clinical trial data evaluating this approach [
      • Welzel T.M.
      • Peterson J.
      • Ferenci P.
      • Gschwantler M.
      • Herzer K.
      • Cornberg M.
      • et al.
      Safety and efficacy of daclatasvir plus sofosbuvir with or without ribavirin for the treatment of chronic HCV genotype 3 infection: interim results of a multicenter european compassionate use program.
      ].
      Given the disappointing results with all oral therapies for patients with genotype 3 HCV and cirrhosis, studies were carried out evaluating the addition of PegIFN to sofosbuvir and ribavirin. In the BOSON trial, twelve weeks of sofosbuvir/ribavirin with PegIFN proved more effective than sofosbuvir/ribavirin alone for 16 or 24 weeks in treatment-naïve and treatment-experienced patients with and without cirrhosis [
      • Foster G.R.
      • Pianko S.
      • Brown A.
      • Forton D.
      • Nahass R.G.
      • George J.
      • et al.
      Efficacy of sofosbuvir plus ribavirin with or without peginterferon-alfa in patients with hepatitis C virus genotype 3 infection and treatment-experienced patients with cirrhosis and hepatitis C virus genotype 2 infection.
      ]. The most marked effect was seen in patients with cirrhosis in whom sofosbuvir/ribavirin with PegIFN led to an SVR rate of 91% in treatment-naïve and 86% in treatment-experienced patients, compared to 82% and 77% with 24 weeks of sofosbuvir/ribavirin respectively. However, despite the fact that PegIFN was well tolerated in this trial with only one patient discontinuing due to adverse effects, the uptake of this approach has been very limited in clinical practice with less than 10% of those treated in the large HCV-TARGET registry opting for this regimen despite the evidence of its superior efficacy [
      • Alqahtani S.
      • Zeuzem S.
      Safety and effectiveness of sofosbuvir-based regimens for the treatment of hepatitis C genotype 3 and 4 infections: internal analysis of a prospective observational study.
      ]. Clearly the need to revert to IFN-based therapy is poorly tolerated by both patients and clinicians, highlighting the need for improved therapeutic options for patients with genotype 3 infection.
      It is unclear why genotype 3 has proven so difficult to cure with DAAs. Genotype 3 HCV has always behaved rather differently when compared to other genotypes with multiple studies now confirming its association with more rapid progression of fibrosis and earlier development of liver malignancy. Although genotype 3 has long been associated with marked intrahepatic steatosis perhaps surprisingly it appears to have a reduced impact on insulin resistance when compared to other genotypes [
      • Pianko S.
      • Zeuzem S.
      • Chuang W.L.
      • Foster G.R.
      • Sarin S.K.
      • Flisiak R.
      • et al.
      Randomized trial of albinterferon alfa-2b every 4 weeks for chronic hepatitis C virus genotype 2/3.
      ,
      • Thompson A.J.
      • Patel K.
      • Chuang W.L.
      • Lawitz E.J.
      • Rodriguez-Torres M.
      • Rustgi V.K.
      • et al.
      Viral clearance is associated with improved insulin resistance in genotype 1 chronic hepatitis C but not genotype 2/3.
      ]. In addition this genotype has different impacts upon host cholesterol metabolism when compared to other genotypes [
      • Clark P.J.
      • Thompson A.J.
      • Vock D.M.
      • Kratz L.E.
      • Tolun A.A.
      • Muir A.J.
      • et al.
      Hepatitis C virus selectively perturbs the distal cholesterol synthesis pathway in a genotype-specific manner.
      ] and recent data from Younossi and colleagues have shown that patients with genotype 3 HCV infection have lower levels of metabolites from late in the cholesterol biosynthesis pathway than patients infected with other HCV genotypes [
      • Younossi Z.M.
      • Stepanova M.
      • Estep M.
      • Negro F.
      • Clark P.J.
      • Hunt S.
      • et al.
      Dysregulation of distal cholesterol biosynthesis in association with relapse and advanced disease in CHC genotype 2 and 3 treated with sofosbuvir and ribavirin.
      ]. Specifically, they found that lower baseline levels of the metabolite lathosterol and the change in levels during therapy were associated with relapse after sofosbuvir/ribavirin therapy [
      • Younossi Z.M.
      • Stepanova M.
      • Estep M.
      • Negro F.
      • Clark P.J.
      • Hunt S.
      • et al.
      Dysregulation of distal cholesterol biosynthesis in association with relapse and advanced disease in CHC genotype 2 and 3 treated with sofosbuvir and ribavirin.
      ]. These findings were more pronounced in patients with cirrhosis, however the underlying mechanisms explaining how these findings affect response to antiviral therapy remain unclear. Multiple hypotheses for the reduced responses seen in patients with genotype 3 HCV and cirrhosis have been proposed ranging from alterations in drug delivery and impaired drug metabolism to reduced innate and adaptive immune function [
      • Al Marzooqi S.H.
      • Feld J.J.
      Sorting out cirrhosis: mechanisms of non-response to hepatitis C therapy.
      ]. It is important to note that all of the current DAAs were developed by screening for activity in genotype 1 replicons and only very limited studies have been performed in laboratory strains of genotype 3 HCV [
      • Hussain S.
      • Barretto N.
      • Uprichard S.L.
      New hepatitis C virus drug discovery strategies and model systems.
      ]. Given the divergence of genotype 3 and genotype 1 HCV and the wide geographical distribution of genotype 3 with its large range of different quasispecies, it is perhaps not surprising that drugs developed with high antiviral potency against genotype 1 HCV have reduced activity against some of the naturally occurring genotype 3 strains. It remains unclear why these drugs with slightly reduced activity against genotype 3 are highly potent in the absence of cirrhosis but surprisingly ineffective in its presence. Further work is clearly required to address this issue but it is probable that an unholy trinity of reduced activity, reduced drug delivery to the cirrhotic liver and impaired host immunity combine to impair responses. Whatever the mechanism of the poor efficacy of genotype 3 drugs in cirrhosis it is clear that future studies should focus on these patients, and physicians and pharmaceutical companies should not be lulled into a false sense of security based upon high response rates in patients with genotype 3 infection and mild disease – the true test of any new regimen is treatment-experienced patients with cirrhosis as this represents the major unmet clinical need. Fig. 1 highlights the efficacy of current antiviral regimens in patients with G3 HCV and cirrhosis who have previously failed to respond to PegIFN and ribavirin.
      Figure thumbnail gr1
      Fig. 1The challenge of G3 cirrhosis. SVR rates are shown with currently and soon-to-be approved regimens for patients with genotype 3 and cirrhosis. Although encouraging progress has been made, results for this sub-group of patients remain suboptimal compared to other populations.

      Genotype 2, 4, 5 and 6 with cirrhosis

      Differences in geographical distribution of HCV sub-types require taylor made treatment strategies to be developed for individual countries.
      Genotypes 2, 4 5 and 6 have been studied much less extensively than genotypes 1 and 3, largely due to their relatively low prevalence in Western countries [
      • Gower E.
      • Estes C.
      • Blach S.
      • Razavi-Shearer K.
      • Razavi H.
      Global epidemiology and genotype distribution of the hepatitis C virus infection.
      ]. Although currently approved DAA regimens have activity against all of these genotypes, clinical trial data, particularly in patients with cirrhosis are limited. For patients with genotype 2, sofosbuvir and ribavirin is the only approved regimen, however there are very limited data for patients with cirrhosis and the optimal duration of therapy remains unclear with SVR rates ranging from 60 to 88% with 12 weeks, 78 to 87% with 16 weeks and 100% with 24 weeks of therapy [
      • Zeuzem S.
      • Dusheiko G.M.
      • Salupere R.
      • Mangia A.
      • Flisiak R.
      • Hyland R.H.
      • et al.
      Sofosbuvir and ribavirin in HCV genotypes 2 and 3.
      ,
      • Foster G.R.
      • Pianko S.
      • Brown A.
      • Forton D.
      • Nahass R.G.
      • George J.
      • et al.
      Efficacy of sofosbuvir plus ribavirin with or without peginterferon-alfa in patients with hepatitis C virus genotype 3 infection and treatment-experienced patients with cirrhosis and hepatitis C virus genotype 2 infection.
      ]. Guidelines also recommend sofosbuvir and daclatasvir however the limited clinical trial data available do not include patients with cirrhosis. Similarly, for genotype 4, although approved regimens (sofosbuvir/ledipasvir and ombitasvir/paritaprevir/r with ribavirin for 12 weeks) are effective, there are very limited data in patients with cirrhosis and the highly divergent genotype 4 strains that predominate in central Africa remain understudied with some trials suggesting that efficacy may be reduced [
      • Schnell G.
      • Tripathi R.
      • Beyer J.
      • Reisch T.
      • Krishnan P.
      • Lu L.
      • et al.
      Hepatitis C virus genotype 4 resistance and subtype demographic characterization of patients treated with ombitasvir plus paritaprevir/ritonavir.
      ,
      • Asselah T.
      • Bourliere M.
      Hepatitis C virus: current and evolving treatments for genotype 4.
      ]. Very few patients with genotypes 5 and 6, infection, particularly with cirrhosis, have been included in registration trials, making it very difficult to know how to manage these patients in clinical practice [
      • Abergel A.
      • Asselah T.
      • Metivier S.
      • Kersey K.
      • Jiang D.
      • Mo H.
      • et al.
      Ledipasvir-sofosbuvir in patients with hepatitis C virus genotype 5 infection: an open-label, multicentre, single-arm, phase 2 study.
      ]. Once again the relatively limited subset of viral strains studied to date makes it difficult to generalise and predict response rates in more diverse, real-world settings.

      Decompensated cirrhosis

      The approved regimens for genotype 1 have been studied extensively in patients with compensated cirrhosis. Although slight alterations in the duration of therapy and/or the addition of ribavirin are required for specific populations with cirrhosis, the efficacy and tolerability of both sofosbuvir/ledipasvir and paritaprevir/r/ombitasvir with dasabuvir are excellent in patients with compensated cirrhosis and comparable to those with earlier stage disease [
      • Poordad F.
      • Hezode C.
      • Trinh R.
      • Kowdley K.V.
      • Zeuzem S.
      • Agarwal K.
      • et al.
      ABT-450/r-ombitasvir and dasabuvir with ribavirin for hepatitis C with cirrhosis.
      ,
      • Reddy K.R.
      • Bourliere M.
      • Sulkowski M.
      • Omata M.
      • Zeuzem S.
      • Feld J.J.
      • et al.
      Ledipasvir and sofosbuvir in patients with genotype 1 hepatitis C virus infection and compensated cirrhosis: An integrated safety and efficacy analysis.
      ]. Patients with more advanced cirrhosis were rarely treated with PegIFN-based therapy because of the risk of further decompensation [
      • Everson G.T.
      • Trotter J.
      • Forman L.
      • Kugelmas M.
      • Halprin A.
      • Fey B.
      • et al.
      Treatment of advanced hepatitis C with a low accelerating dosage regimen of antiviral therapy.
      ]. Initial studies with sofosbuvir/ribavirin and sofosbuvir/ledipasvir with ribavirin for 12 or 24 weeks in patients with Child-Pugh B and C cirrhosis have yielded surprisingly good results with SVR rates reported to be as high as 88% [
      • 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.
      ]. While certainly a major advance for these very sick patients, current therapies leave room for improvement. In addition to the fact that SVR rates are generally below 90%, some safety concerns have been raised with a small number of reports of patients decompensating further on sofosbuvir-based therapy, some of which have led to liver transplantation and/or death [
      • Welker M.W.
      • Luhne S.
      • Lange C.M.
      • Vermehren J.
      • Farnik H.
      • Herrmann E.
      • et al.
      Lactic acidosis in patients with hepatitis C virus cirrhosis and combined ribavirin/sofosbuvir treatment.
      ,
      • Dyson J.K.
      • Hutchinson J.
      • Harrison L.
      • Rotimi O.
      • Tiniakos D.
      • Foster G.R.
      • et al.
      Liver toxicity associated with sofosbuvir, an NS5A inhibitor and ribavirin use.
      ]. Reports of liver failure and death in patients treated with paritaprevir/r/ombitasvir with dasabuvir with or without ribavirin led the US Food and Drug Administration (FDA) to issue a warning stating that this regimen is contraindicated in patients with Child-Pugh B or C cirrhosis [

      (FDA) FDA. FDA Drug Safety Communication: FDA warns of serious liver injury risk with hepatitis C treatments Viekira Pak and Technivie. In; 2015.

      ]. Even for patients who do safely complete therapy and achieve SVR, it remains unclear which patients will improve and which will remain in a decompensated state [
      • 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.
      ]. If patients are ultimately going to require liver transplantation despite SVR, it may be easier to treat them in the post-transplant setting when therapy is highly effective and cirrhosis is no longer present. For patients with decompensated cirrhosis due to non-genotype 1 infection, treatment options are extremely limited with almost no trial data available. Clearly patients with decompensated cirrhosis remain a significant unmet medical need with current therapies, and data identifying patient groups who are likely to benefit from viral elimination and regain liver function are urgently required. The risk of eliminating the virus and leaving patients ineligible for transplantation but with a reduced quality of life (‘MELD purgatory’) is very real and large scale analyses to address this critical issue must be prioritised.

      Chronic kidney disease

      DAA therapies have eliminated many of the previous ‘difficult to cure’ populations. Patients with CKD, however remain a challenge with currently approved therapies. The major metabolite of sofosbuvir, GS-331007, is entirely renally cleared and only partially removed with dialysis. In patients with estimated glomerular filtration rates (eGFR) below 30 cc/minute, levels of GS-331007 accumulate to extremely high levels [
      • Kirby B.J.
      • Symonds W.T.
      • Kearney B.P.
      • Mathias A.A.
      Pharmacokinetic, pharmacodynamic, and drug-interaction profile of the hepatitis C virus NS5B polymerase inhibitor sofosbuvir.
      ]. The clinical significance of this accumulation remains unknown but has led to sofosbuvir being contraindicated in patients with eGFR below this level. Reports from real-world studies have suggested that use of sofosbuvir in patients with eGFR <30 cc/minute may further worsen renal function and cause other adverse events [
      • Terrault N.A.
      • Zeuzem S.
      • Di Bisceglie A.M.
      • Lim J.K.
      • Pockros P.
      • Frazier L.M.
      • et al.
      Treatment outcomes with 8, 12 and 24 week regimens of ledipasvir/sofosbuvir for the treatment of hepatitis C infection: analysis of a multicenter prospective, observational study.
      ]. Reducing the dose of sofosbuvir may be safe, however the antiviral efficacy is likely to be impaired based on initial dose-finding studies and as such, this approach is not recommended.
      Paritaprevir/r/ombitasvir and dasabuvir are all completely metabolized by the liver and therefore are expected to be safe in renal failure. A small trial of 20 patients showed that this regimen was effective (SVR 18 out of 20) and safe in patients with advanced renal disease, including patients on dialysis [
      • Pockros P.
      • Reddy K.R.
      • Mantry P.S.
      • Cohen E.
      • Bennett M.
      • Sulkowski M.
      • et al.
      RUBY-I: Ombitasvir/paritaprevir/ritonavir + dasabuvir ± RIBAVIRIN in non-cirrhotic HCV genotype 1-infected patients with severe renal impairment or end-stage renal disease.
      ]. However, the need to include ribavirin for patients with genotype 1a infection proved problematic, with the majority of patients needing to stop ribavirin despite the starting dose of just 200 mg per day [
      • Pockros P.
      • Reddy K.R.
      • Mantry P.S.
      • Cohen E.
      • Bennett M.
      • Sulkowski M.
      • et al.
      RUBY-I: Ombitasvir/paritaprevir/ritonavir + dasabuvir ± RIBAVIRIN in non-cirrhotic HCV genotype 1-infected patients with severe renal impairment or end-stage renal disease.
      ]. Although these data are promising, the very small size of the study and the issues with ribavirin limit its application.

      DAA treatment failures

      The introduction of DAAs into clinical practice has led to enormous treatment uptake and early real-world data suggest that SVR rates have approached those reported in clinical trials. However, even with SVR rates above 90%, some patients do not respond to current treatment. For these unfortunate individuals, current options are extremely limited. Although small clinical trials have reported on various retreatment strategies, there are no currently approved therapies for patients who have failed an all oral DAA regimen. Understanding the optimal approach to managing these patients is clearly a high priority for regimens in development.

      Complicated therapy

      The initial introduction of DAAs into antiviral therapy significantly complicated treatment paradigms. The need for different durations for different populations, variable stopping rules and response-guided therapy made therapy challenging [
      • Jacobson I.M.
      • Pawlotsky J.M.
      • Afdhal N.H.
      • Dusheiko G.M.
      • Forns X.
      • Jensen D.M.
      • et al.
      A practical guide for the use of boceprevir and telaprevir for the treatment of hepatitis C.
      ]. All oral therapies have greatly simplified treatment, however there are still a number of subtleties to treatment regimens that make it difficult for clinicians without experience to enter the field. Currently regimens vary by genotype, subtype, treatment history and the presence or absence of cirrhosis [
      • Poordad F.
      • Sievert W.
      • Mollison L.
      • Bennett M.
      • Tse E.
      • Brau N.
      • et al.
      Fixed-dose combination therapy with daclatasvir, asunaprevir, and beclabuvir for noncirrhotic patients with HCV genotype 1 infection.
      ,
      EASL recommendations on treatment of hepatitis C 2015.
      ]. In addition, there are still a number of drug-drug interactions that need to be considered in all patients [
      EASL recommendations on treatment of hepatitis C 2015.
      ]. Although on-treatment monitoring is now quite straightforward, there is no consensus on the frequency or the nature of testing that should be done. Streamlining therapy to a ‘one-size-fits-all’ or at least a ‘one-size-fits-most’ approach would make it much easier to introduce treatment in primary care settings or even extend to pharmacies and other ‘user friendly’ treatment delivery options. Simplified regimens would not only improve overall treatment capacity but also enables treatment of patients who may not access specialist care or who would prefer to be treated by providers with specific expertise tailored to their other comorbidities, such as ongoing injection drug use [
      • Dore G.J.
      • Feld J.J.
      Hepatitis C virus therapeutic development: in pursuit of “perfectovir”.
      ]. Patients with advanced liver disease should probably still be managed in specialized centres, but non-invasive assessment of fibrosis needs to be simplified and made widely available so that clinicians can easily identify patients with cirrhosis who require more intensive monitoring and subsequent long-term follow-up in specialist services. It is easy to envisage a simple treatment algorithm where patients with high platelet counts or normal APRI scores receive a standardised regimen in the community and those with low platelet counts or high APRI scores are referred to specialist centres. Such an approach will require genuine ‘pan-genotypic’ regimens with minimal side effects and few drug-drug interactions alongside affordable pricing approaches [
      • Dore G.J.
      • Feld J.J.
      Hepatitis C virus therapeutic development: in pursuit of “perfectovir”.
      ]. Even with very simple regimens, adherence will still be a critical issue, which may be a significant challenge in certain populations such as some groups of people who are actively using drugs and have unstable social situations. For such patients depot preparations may be preferable.
      Another area for potential improvement is the duration of therapy. Although uniform durations for all patients would be helpful, there is great interest in identifying populations for whom therapy can be shortened. While patients and providers would prefer shorter therapy, duration cannot come at the expense of optimal SVR rates, particularly given the incredibly good tolerability of current regimens. Many argue that shortening therapy is a potential way to save significant amounts of money [
      • O’Brien T.R.
      • Feld J.J.
      • Kottilil S.
      • Pfeiffer R.M.
      No scientific basis to restrict 8 weeks of treatment with ledipasvir/sofosbuvir to patients with HCV RNA <6,000,000 IU/ml.
      ], however this is an artificial construct of current drug pricing. The price of therapy has little to do with the cost of production of the medications [
      • van de Ven N.
      • Fortunak J.
      • Simmons B.
      • Ford N.
      • Cooke G.S.
      • Khoo S.
      • et al.
      Minimum target prices for production of direct-acting antivirals and associated diagnostics to combat hepatitis C virus.
      ]. It would be preferable to simply lower the per pill price of the medications and treat long enough to maximize SVR rates than to shorten therapy for the purpose of saving money. However, such an approach will require significant drug pricing reform in the US and many other jurisdictions.

      Treatment access

      Limited access to DAA drugs and low diagnosis rates need to be addressed in order to apply an ‘Access for All‘ philosophy.
      Perhaps the greatest failing of first generation DAAs is the limitation on access to these potentially life-saving medications. In most regions of the world, treatment access is highly restricted. Many low and middle-income countries have no access to DAAs at all and in most middle and high-income countries access is limited to those with advanced liver disease. Other restrictions, including those based on alcohol and drug use, have no evidentiary basis and should be abandoned [
      • Barua S.
      • Greenwald R.
      • Grebely J.
      • Dore G.J.
      • Swan T.
      • Taylor L.E.
      Restrictions for medicaid reimbursement of sofosbuvir for the treatment of hepatitis C virus infection in the United States.
      ,
      • Grebely J.
      • Dore G.J.
      • Greenwald R.
      • Swan T.
      • Barua S.
      • Taylor L.E.
      Hepatitis C virus treatment and persons who inject drugs.
      ]. Efforts by international health bodies, advocacy groups, as well as competition among manufacturers will hopefully bring prices down and improve access to treatment. However improved access is not only a price issue. In most high-income countries, diagnosis rates remain below 50% and in low and middle-income countries a tiny percentage of those infected have been diagnosed [
      • Wedemeyer H.
      • Dore G.J.
      • Ward J.W.
      Estimates on HCV disease burden worldwide - filling the gaps.
      ]. Greater awareness and higher diagnosis rates need to be coupled with increased training of healthcare providers to deliver HCV treatment alongside political will to effect change [
      • Dore G.J.
      • Feld J.J.
      Hepatitis C virus therapeutic development: in pursuit of “perfectovir”.
      ]. Concerted efforts to reach marginalized populations with high HCV prevalence, particularly people who inject drugs, will need to be a priority to curb the spread of disease. While issues of access apply equally to all ‘generations’ of DAA therapy, this will hopefully be a greater priority as new agents are developed and approved. Current international guidelines already state that all infected individuals should be considered candidates for HCV therapy [
      EASL clinical practice guidelines: management of hepatitis C virus infection.
      ,
      Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus.
      ] and as treatments continue to improve, this guiding philosophy of ‘Access for All’ will become easier to implement.

      The goal of next generation DAAs

      With the remarkable success of current therapy for most patient populations, the bar is set extremely high for the development and approval of new DAAs. Regimens with extremely high cure rates, excellent tolerability, low pill burden and few drug-drug interactions are already available. To improve upon current options, new treatment approaches need to address the existing therapeutic and logistical challenges. Ideally regimens will overcome all of the remaining issues, but it is more likely that advances will be incremental, targeting specific, rather than all, challenges. The first goal will be to address the few true therapeutic gaps such as CKD and genotype 3 infection with cirrhosis (Fig. 2). This will be closely followed by attempts to develop truly pan-genotypic regimens that are equally effective in all groups, eliminating the need for genotyping and most on-treatment monitoring. While it would be ideal if a uniform duration were effective for all groups, at least initially, clinicians are comfortable tailoring therapy, particularly for patients with more advanced liver disease. Shortening therapy will continue to be a goal, however the merits of this goal should be carefully considered because with the outstanding tolerability of all regimens, the only downside to slight over-treatment is cost, which, as discussed previously, could be addressed through drug pricing reform. Finally, salvage regimens for those who fail first and even second generation DAA combinations are clearly needed. While these regimens will only serve a very small minority of the HCV-infected population, for those individuals and potentially for true elimination/eradication efforts, they will be critical. It is probable that ‘salvage regimens’ will differ substantially from ‘primary regimens’ in terms of treatment duration and cost and it may be appropriate to encourage this and reduce potential overuse of rescue treatments to avoid the development of multi-drug resistant viral strains.
      Figure thumbnail gr2
      Fig. 2Schematic representation of DAA development in order to reach global HCV elimination. The vast majority of patients who receive all oral DAA therapy will achieve sustained virological response (green). However, a second generation of drugs will address the unresolved issues for patients where current treatments are still not satisfactory (red; blue bubbles). Refinement of drug delivery approaches will be necessary to achieve ‘cure-all’ treatments.

      Next generation DAAs in development for specific populations

      Chronic kidney disease

      The combination of grazoprevir, a second generation PI, with elbasvir, a second generation NS5A inhibitor, has recently been approved in North America. Both agents are completely hepatically metabolized and pharmacokinetic data showed them to be safe in patients with advanced renal disease including those on hemodialysis [
      • 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.
      ]. The C-SURFER study evaluated a combination of these 2 agents in patients with advanced CKD, with genotype 1a or 1b HCV infection. In total, 224 patients were recruited, 76% of whom were on hemodialysis [
      • 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.
      ]. Patients were randomized to immediate treatment for 12 weeks or placebo for 12 weeks followed by delayed treatment. The SVR rate was 94% with only 1 virological failure in the entire study. Perhaps even more impressive than the SVR rate was the tolerability of treatment. Adverse events were almost identical in patients on active treatment as on placebo, with no significant safety signals identified, despite the difficult study population. Grazoprevir and elbasvir are also active against genotype 4 and to a lesser extent genotype 6 [
      • Zeuzem S.
      • Ghalib R.
      • Reddy K.R.
      • Pockros P.J.
      • Ben Ari Z.
      • Zhao Y.
      • et al.
      Grazoprevir-elbasvir combination therapy for treatment-naive cirrhotic and noncirrhotic patients with chronic hepatitis C virus genotype 1, 4, or 6 infection: a randomized trial.
      ], however they are not effective for genotypes 2 or 3, leaving patients with these genotypes and renal disease no immediate option for treatment. The second generation protease inhibitor ABT-493 and NS5A inhibitor ABT-530 are also renally cleared and are active against all genotypes, including genotype 3 [
      • Muir A.J.
      • Strasser S.
      • Wang S.
      • et al.
      High SVR rate with ABT-493+ABT530 for 8 weeks in non-cirrhotic patients with HCV genotype 3 infection.
      ], holding promise that non-sofosbuvir-based regimens safe in patients with CKD will be available in the relatively near future. In the interim, for patients who require sofosbuvir-based therapy, small studies and case series have documented high efficacy and good safety in patients with advanced CKD treated with sofosbuvir-containing regimens, including those on dialysis [
      • Desnoyer A.
      • Pospai D.
      • Le M.P.
      • Gervais A.
      • Heurgue-Berlot A.
      • Laradi A.
      • et al.
      Pharmacokinetics, safety and efficacy of a full dose sofosbuvir-based regimen given daily in hemodialysis patients with chronic hepatitis C.
      ,
      • Nazario H.E.
      • Ndungu M.
      • Modi A.A.
      Sofosbuvir and simeprevir in hepatitis C genotype 1-patients with end-stage renal disease on haemodialysis or GFR <30 ml/min.
      ]. Although worsening renal function during treatment has been reported in some series [
      • Saxena V.
      • Koraishy F.M.
      • Sise M.E.
      • Lim J.K.
      • Schmidt M.
      • Chung R.T.
      • et al.
      Safety and efficacy of sofosbuvir-containing regimens in hepatitis C-infected patients with impaired renal function.
      ], this has not been found in all studies and a recent detailed pharmacokinetic study of patients on hemodialysis receiving daily full dose sofosbuvir showed limited accumulation of GS-331007 with 53% removal with dialysis [
      • Desnoyer A.
      • Pospai D.
      • Le M.P.
      • Gervais A.
      • Heurgue-Berlot A.
      • Laradi A.
      • et al.
      Pharmacokinetics, safety and efficacy of a full dose sofosbuvir-based regimen given daily in hemodialysis patients with chronic hepatitis C.
      ]. These data suggest that patients with advanced CKD who require urgent therapy with sofosbuvir can be treated but close monitoring is required and safety may be less of concern in those already on dialysis in whom renal deterioration is less of an issue (Table 1).
      Table 1Remaining issues and possible solutions in patients with chronic HCV infection.

      Genotype 3

      Velpatasvir, formerly known as GS-5816, is a pan-genotypic NS5A inhibitor with picomolar activity against genotype 3 HCV. Velpatasvir was combined with sofosbuvir in a fixed-dose single-tablet regimen given for 12 weeks and compared to sofosbuvir plus ribavirin for 24 weeks in patients with genotype 3 infection in the ASTRAL 3 study [
      • 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.
      ]. In patients without cirrhosis, SVR rates with sofosbuvir/velpatasvir were 98% in treatment-naïve patients and 91% in those who had failed previous treatment with PegIFN and ribavirin. More importantly, in patients with cirrhosis, 40 of 43 (93%) treatment-naïve patients and 34 of 37 (89%) treatment-experienced patients achieved SVR [
      • 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.
      ]. Results were superior to those seen with sofosbuvir/ribavirin for 24 weeks in all populations. The anticipated approval of sofosbuvir/velpatasvir holds promise for an IFN and ribavirin-free 12-week regimen with very good but still not optimal efficacy in patients with genotype 3 cirrhosis. To get SVR rates up to the 95% seen in most other populations for patients with genotype 3 infection and cirrhosis, a third agent or possibly ribavirin may be required. Small studies have evaluated adding the pan-genotypic protease inhibitor GS-9857 to sofosbuvir/velpatasvir. In non-cirrhotic patients, this triple combination given for only 6 weeks led to SVR in all 21 genotype 3 treatment-naïve patients treated, and was also highly effective across all genotypes when given for 8 weeks [
      • Gane E.
      • Nguyen M.
      • Kwo P.
      • Kowdley K.V.
      • Reau N.
      • Jacobson I.
      • et al.
      Short-duration treatment with sofosbuvir/velpatasvir plus GS-9857 in treatment-naïve genotype 1–6 HCV-infected patients with or without cirrhosis.
      ]. Extending the duration to 12 weeks in treatment-experienced patients with or without cirrhosis led to SVR in 34 of 35 (97%) patients [
      • Gane E.
      • Poordad F.
      • Wells J.T.
      • Hyland R.
      • Yang Y.
      • Dvory-Sobol H.
      • 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.
      ]. Phase 3 trials of this regimen are ongoing.
      ABT-493, a second generation pan-genotypic PI, has been combined with ABT-530, a second generation pan-genotypic NS5A inhibitor. Both agents are highly active against genotype 3 replicons and they appear safe, well tolerated and highly effective in small clinical trials [
      • Ng T.
      • Krishnan P.
      ABT-530, an HCV NS5A inhibitor with potent pangenotypic activity and high genetic barrier to resistance.
      ,
      • Poordad F.
      • Felizarta F.
      • Asatryan A.
      • Hassanein T.
      • Aguilar H.
      • Lalezari J.
      • et al.
      100% SVR4 in HCV genotype 1 non-cirrhotic treatment-naïve or -experienced patients with the combination of ABT-493 and ABT-530 for 8 weeks (SURVEYOR-I) [8 weeks].
      ]. In a dose-ranging study of patients with genotype 3 infection, there was a single patient with virological failure at the highest dose of both agents leading to an overall SVR rate of 93% (28 of 30) [
      • Kwo P.
      • Bennett M.
      • Wang S.
      • Vargas H.E.
      • Wyles D.
      • Overcash J.S.
      • et al.
      SURVEYOR-II: High SVR4 rates achieved with the next generation NS3/4A protease inhibitor ABT-493 and NS5A inhibitor ABT-530 in non-cirrhotic treatment-naïve and treatment-experienced patients with HCV genotype 3 infection.
      ]. Perhaps more importantly, when this combination was given to patients with genotype 3 infection and compensated cirrhosis for 12 weeks, SVR rates were 100%, with (24 of 24) or without ribavirin (24 or 24) [
      • Kwo P.
      • Wyles D.
      • Wang S.
      • Poordad F.
      • Gane E.
      • Maliakkal B.
      • et al.
      100% SVR with ABT-493 and ABT-530 with or without ribavirin in treatment-naive HCV genotype 3-infected patients with cirrhosis.
      ].
      Other regimens with activity against genotype 3 are also in development. Grazoprevir is active against genotype 3 but doses of 200 mg or higher are required for optimal efficacy and these higher doses have been associated with hepatotoxicity [
      • Caro L.
      • Du L.
      • Huang S.P.
      • Wenning L.
      • Su J.
      • Hwang P.
      • et al.
      Relationship Between Transaminase Levels and Plasma Pharmacokinetics Following Administration of MK-5172 With Pegylated Interferon Alfa-2b and Ribavirin to HCV Genotype 1 Treatment-Naïve Patients.
      ]. At the approved 100 mg dose, genotype 3 is not well covered. To broaden activity, grazoprevir has been studied with elbasvir or alternatively with MK-8408, another second generation pan-genotypic NS5A inhibitor, in combination with sofosbuvir. With this triple drug regimen given for 12 weeks, small studies have confirmed efficacy against genotype 3 with 14 of 14 non-cirrhotic patients and 10 of 11 patients with compensated cirrhosis achieving SVR [
      • Poordad F.
      • Lawitz E.
      • Gutierrez J.A.
      • Evans B.
      • Howe A.
      • Hwa-Ping F.
      • et al.
      C-SWIFT: grazoprevir/elbasvir + sofosbuvir in cirrhotic and noncirrhotic, treatment-naive patients with hepatitis C virus genotype 1 infection, for durations of 4, 6 or 8 weeks and genotype 3 infection for durations of 8 or 12 weeks.
      ]. These data provide proof of concept that grazoprevir combined with an NS5A inhibitor and a nucleotide polymerase inhibitor will be broadly active, including genotype 3, and set the stage for phase 3 studies of grazoprevir and MK-8408 combined with MK-3682, a novel nucleotide analogue, which are currently underway.

      Genotype 2, 4, 5 and 6 with cirrhosis

      The combination of sofosbuvir/velpatasvir has been evaluated in genotypes 1 through 6. In the ASTRAL 2 study, 266 patients with genotype 2 infection were randomized to sofosbuvir/velpatasvir or sofosbuvir plus ribavirin for 12 weeks [
      • 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.
      ]. Only one of the 134 patients in the sofosbuvir/velpatasvir arm did not achieve SVR and this patient discontinued treatment after one dose of therapy due to adverse effects. The 99% SVR rate compared favourably to that seen in the sofosbuvir plus ribavirin group in which 94% achieved SVR while 6 patients relapsed and 2 were lost to follow-up. Only 19 patients in each arm had compensated cirrhosis, but notably all 19 achieved SVR with sofosbuvir/velpatasvir therapy [
      • 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.
      ]. Sofosbuvir/velpatasvir was also evaluated in patients with genotypes 1, 4, 5 and 6 in the ASTRAL 1 study [
      • 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.
      ]. Of the 624 patients in the trial, only 2 relapsed, with SVR rates of 97 to 100% across the genotypes evaluated. A total of 20% of included patients had compensated cirrhosis, including 10 with genotype 2, 27 with genotype 4 but only 5 with genotype 5 and 6 with genotype 6. Notably, all of these patients achieved SVR, as did 72 of the 73 patients with genotype 1 and 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.
      ]. Importantly in the ASTRAL 1 study, 20% of patients were randomized to initial treatment with a placebo to evaluate the safety of the regimen. The therapy was extremely well tolerated with a side effect profile almost indistinguishable from placebo, including in patients with cirrhosis. Collectively, the combination of sofosbuvir/velpatasvir holds promise of a highly effective pan-genotypic regimen that can be used with a ‘one-size-fits-all’ 12-week approach, including patients with compensated cirrhosis, theoretically obviating the need for pre-treatment genotyping. The other pan-genotypic regimens in development, including ABT-493/ABT-530 and grazoprevir/MK-8048/MK-3682, have also shown promise in small studies including patients with compensated cirrhosis.

      Decompensated cirrhosis

      For patients with decompensated cirrhosis, real-world studies with sofosbuvir and current NS5A inhibitors have confirmed the overall safety of these regimens and shown that in some patients there is an improvement in MELD scores. In the English early access program, of 409 treated patients there was a median reduction in MELD score of 0.85 with 18% of patients developing a new decompensating event over a period of 24 weeks [
      • Foster G.R.
      • Irving W.L.
      • Cheung M.C.
      • Walker A.J.
      • Hudson B.E.
      • Verma S.
      • et al.
      Cohort study of the impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis.
      ]. In an untreated control cohort of 261 patients the median MELD score increased by 0.75 and 28% developed a decompensating event over a 24-week period. The overall SVR rate differed by genotype but over 80% of patients eliminated virus. A medium term follow-up study showed that the early benefits were sustained for at least one year with a reduction in the incidence of liver cancer and other adverse events [
      • Cheung M.
      • Foster G.R.
      • Irving W.
      • Walker A.J.
      • Hudson B.
      • Verma S.
      • et al.
      Antiviral treatment in patients with advanced HCV cirrhosis using sofosbuvir and ledipasvir/daclatasvir with or without ribavirin - 6 and 12 month outcomes compared to untreated patients.
      ]. However, patients with Childs-Pugh class C cirrhosis had relatively poor outcomes with only 20% of patients remaining well and free from complications 12 months after completion of therapy. Similar virological response rates have been reported by other groups and indicate that more effective regimens and better patient selection will be required for patients with genotype 3 infection and decompensated cirrhosis [

      Welzel TM, Herzer K, Ferenci P, Petersen J, Gschwantler M, Cornberg M, Berg T, et al. Daclatasvir plus sofosbuvir with or without ribavirin for the treatment of HCV in patients with severe liver disease: interim results of a multicenter compassionate use program international liver congress, European Association for the Study of the Liver 2015, Vienna, Austria, 2015.

      ,
      • Pol S.
      • Bourliere M.
      • Lucier S.
      • de Ledinghen V.
      • Zoulim F.
      • Dorival C.
      • et al.
      Safety and efficacy of the combination daclatasvir-sofosbuvir in HCV genotype 1-mono-infected patients from the French observational cohort ANRS CO22 HEPATHER.
      ]. In the ASTRAL 4 study patients with decompensated cirrhosis were treated with sofosbuvir/velpatasvir with and without ribavirin for 12 or 24 weeks [
      • 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 overall virological response was optimal (94%) in patients receiving 12 weeks sofosbuvir/velpatasvir and ribavirin, with lower response rates in the groups without ribavirin for 12 (83%) or 24 weeks (86%). Although therapy was generally well tolerated in these sick patients, serious adverse events occurred in close to 20% of patients [
      • 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.
      ]. Toxicity of treatment is hard to tease apart because of the high risk of adverse events due to the natural history of advanced cirrhosis, but reports suggest that at least some patients appear to do worse on-treatment with even the relatively safe sofosbuvir/NS5A regimens [
      • Welker M.W.
      • Luhne S.
      • Lange C.M.
      • Vermehren J.
      • Farnik H.
      • Herrmann E.
      • et al.
      Lactic acidosis in patients with hepatitis C virus cirrhosis and combined ribavirin/sofosbuvir treatment.
      ,
      • Dyson J.K.
      • Hutchinson J.
      • Harrison L.
      • Rotimi O.
      • Tiniakos D.
      • Foster G.R.
      • et al.
      Liver toxicity associated with sofosbuvir, an NS5A inhibitor and ribavirin use.
      ]. Due to their hepatic metabolism, PI are not recommended in these patients, which may pose a problem for retreatment strategies in patients who fail an initial regimen [

      (FDA) FDA. FDA Drug Safety Communication: FDA warns of serious liver injury risk with hepatitis C treatments Viekira Pak and Technivie. In; 2015.

      ]. Overall, the results in patients with decompensated cirrhosis are very encouraging, suggesting that viral elimination is possible in most patients, however patient selection remains a significant challenge. Understanding which patients will benefit from viral clearance at this advanced stage of disease is a major research priority, as some individuals would be served better by delaying treatment until after liver transplantation. It will be particularly important to assess the risk of hepatocellular carcinoma (HCC) in patients with cirrhosis who achieve SVR. Given the higher risk of HCC in patients with more advanced cirrhosis, some of the reduction in HCC seen with HCV clearance using IFN-based therapy may not be seen when patients with CP-B and C are cured. The recent report of frequent and early recurrence of HCC after SVR in patients treated with IFN-free therapy has even raised concerns that viral clearance may even promote HCC development [
      • Reig M.
      • Marino Z.
      • Perello C.
      • Inarrairaegui M.
      • Ribeiro A.
      • Lens S.
      • et al.
      Unexpected early tumor recurrence in patients with hepatitis C virus -related hepatocellular carcinoma undergoing interferon-free therapy: a note of caution.
      ]. Notably however, an increase in the incidence of liver cancer was not seen in the English EAP study where patients with decompensated cirrhosis who did not have pre-existing liver cancer were successfully treated with all oral regimens with a reduction in the incidence of liver cancer [
      • Cheung M.
      • Foster G.R.
      • Irving W.
      • Walker A.J.
      • Hudson B.
      • Verma S.
      • et al.
      Antiviral treatment in patients with advanced HCV cirrhosis using sofosbuvir and ledipasvir/daclatasvir with or without ribavirin - 6 and 12 month outcomes compared to untreated patients.
      ]. To date there are no data to support an increased rate of de novo HCC after SVR in patients with cirrhosis, however long-term follow-up studies will clearly be required to ensure that this does not occur.

      Simplified shorter regimens

      Refining regimens and shortening treatment length will improve the effectiveness of both existing and future HCV treatments.
      The key to treatment simplification will be introduction of truly pan-genotypic regimens that ideally utilize the same duration for all populations. As noted, sofosbuvir/velpatasvir is an effective pan-genotypic regimen but requires 12 weeks of therapy with shorter durations showing reduced efficacy [
      • Tran T.T.
      • Morgan T.R.
      • Thuluvath P.J.
      • Etzkorn K.
      • Hinestrosa F.
      • Tong M.
      • et al.
      Safety and efficacy of treatment with sofosbuvir + GS-5816 ± ribavirin for 8 or 12 weeks in treatment-naïve patients with genotype 1–6 HCV infection.
      ]. The addition of a third DAA (a novel protease inhibitor – GS-9857) to this regimen is being studied and may, allow further reductions in the duration of therapy. Early studies involving this triple therapy for 6 weeks showed that 14 of 15 non-cirrhotic treatment-naïve patients achieved SVR, but this dropped to 20 of 30 among treatment-experienced patients with cirrhosis [
      • Gane E.J.
      • Hyland R.
      • Ying Y.
      • Svarovskaia E.
      • Stamm L.M.
      • Brainard D.
      • et al.
      Safety and efficacy of short-duration treatment with GS-9857 combined with sofosbuvir/GS-5816 in treatment-naïve and DAA-experienced genotype 1 patients with and without cirrhosis.
      ]. Shortening further to 4 weeks proved entirely unsuccessful with only 4 of 15 (27%) treatment-naïve non-cirrhotic patients achieving SVR. Similarly, attempts to shorten therapy with the potent combination of grazoprevir/elbasvir and sofosbuvir were not very successful. When given for 8 weeks, 17 of 18 (94%) of patients with cirrhosis achieved SVR but shortening to 6 weeks reduced SVR rates to 80% (16 of 20) in patients with cirrhosis and 87% (26 of 30) even in those without cirrhosis. Pushing further to 4 weeks resulted in an SVR rate of only 33% (10 of 30) in a treatment-naïve non-cirrhotic genotype 1 population [
      • Poordad F.
      • Lawitz E.
      • Gutierrez J.A.
      • Evans B.
      • Howe A.
      • Hwa-Ping F.
      • et al.
      C-SWIFT: grazoprevir/elbasvir + sofosbuvir in cirrhotic and noncirrhotic, treatment-naive patients with hepatitis C virus genotype 1 infection, for durations of 4, 6 or 8 weeks and genotype 3 infection for durations of 8 or 12 weeks.
      ].
      Clearly pan-genotypic regimens will soon be available. Early attempts at shortening therapy suggest that 8 weeks may be the shortest possible duration that would be adequate for most, or ideally, all patients, even with very potent multi-DAA regimens. Specific populations may be able to be cured with shorter treatments. One ‘proof of concept study’ reported 100% efficacy with just 3 weeks of therapy in treatment-naïve non-cirrhotic patients with genotype 1b infection who had HCV RNA suppressed below 50 IU/ml by day 2 of a PI, NS5A and nucleotide analogue [
      • Lau G.K.
      • Benhamou Y.
      • Chen C.
      • Schinazi R.F.
      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).
      ]. The question will then be whether to tailor therapy, allowing for shorter regimens for specific populations, or to simplify therapy using a ‘one-size-fits-all’ approach that may be highly effective and easy to implement but will lead to over-treatment of many individual patients. Clearly understanding how these different approaches will affect the overall cost of therapy and treatment uptake at the population level will need to be carefully evaluated.

      Alternative approaches to shorten therapy

      Shortening therapy to 4 weeks does not seem likely to be effective in the majority of patients, even combining the most potent DAA combinations. Reaching 4 weeks or shorter may require solutions beyond more potent DAAs. Host-targeting agents may be an alternative or complementary approach to achieve this goal. To date, the most effective host-targeting agents have included micro (mi)RNA (miR122) and cyclophilin inhibitors.
      miR122 is a non-coding small RNA that binds directly to the HCV genome and is required for its replication cycle. Sequestration of miR122 with complementary oligonucleotide technology has shown to be effective at suppressing HCV replication. A Phase II study of miravirsen, a locked-nucleic acid that sequesters miR122, showed potent HCV RNA suppression with weekly subcutaneous dosing [
      • 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.
      ]. More recently, an improved miR122 inhibitor, RG-101, has been developed that combines the complementary oligo to a GalNac linker leading to increased hepatocyte uptake and more potent miR122 sequestration. Single dose studies of RG-101 (4 mg/kg IV) showed potent suppression, with 9 of 12 patients achieving undetectable HCV RNA that lasted out to 20 weeks in 4 individuals [
      • Van Der Ree M.
      • de Vree M.L.
      • Stelma F.
      • Willemse S.
      • ven der Valk M.
      • Rietdijk S.
      • et al.
      A single dose of RG-101, a GalNAc-conjugated oligonucleotide targeting mir-122, results in undetectable HCV RNA levels in chronic hepatitis C patients at week 28 of follow-up.
      ]. While this agent is likely inadequate on its own, its very high barrier to resistance and long-lasting activity raise the prospect of using this agent in combination with DAAs. Ongoing studies are evaluating an approach of giving an initial injection of RG-101 followed by 4 weeks of potent combination DAA therapy and completed with a final dose of RG-101 that will hopefully mop up any residual virus, allowing for a 4-week regimen with high efficacy. Preliminary studies with this approach showed high efficacy even with a single DAA given for 4 weeks between doses of RG-101, however complete SVR12 data are not yet available [
      • Horvath G.
      • Papatheodoridis M.
      • Fabri M.
      • Makara M.
      • Grint P.
      • Huang M.
      • et al.
      RG-101 in combination with 4 weeks of oral diract acting antiviral therapy achieves high virologic response rates in treatment-naive gneotype 1 and 4 chronic hepatitis C patients: interim results from a randomised, multicenter, phase 2 study.
      ]. In addition to shortening therapy, this type of approach may be very useful in populations in which adherence with DAAs is a concern, particularly those with active substance use and mental health issues. Similar strategies combining host-targeting agents, particularly those with long-lasting activity, with DAAs, may prove effective, however with all host-targeting therapies, safety issues will have to be carefully evaluated.

      Salvage regimens for those who fail DAA therapy

      Fortunately, the vast majority of patients respond to currently available regimens. However, some individuals do not respond, and numbers may increase as treatment expands to more marginalized and harder-to-reach populations where treatment adherence may be more of an issue. To date retreatment studies are limited, however small studies have been informative and suggest that the agents in development will be adequate to cure most individuals who fail initial DAA regimens.
      Emergence of treatment resistant strains of HCV may develop in patients who require retreatment after DAA failure.
      The major challenge with retreatment is the emergence of resistance-associated variants (RAVs) that may complicate future treatment attempts. The lack of inter-class cross-resistance means that ideally retreatment should aim to include DAAs of a different class or classes than the first course of therapy. Changing DAA class has proven very effective with those who failed first generation PI-based therapy, with these patients responding similarly to sofosbuvir/NS5A inhibitor combinations as patients who were DAA-naïve [
      • Afdhal N.
      • Reddy K.R.
      • Nelson D.R.
      • Lawitz E.
      • Gordon S.C.
      • Schiff E.
      • et al.
      Ledipasvir and sofosbuvir for previously treated HCV genotype 1 infection.
      ,
      • Sulkowski M.S.
      • Gardiner D.F.
      • Rodriguez-Torres M.
      • Reddy K.R.
      • Hassanein T.
      • Jacobson I.
      • et al.
      Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection.
      ].
      As first-line regimens combine multiple classes of DAAs, completely avoiding retreatment with the same DAA class may not be possible. Some second generation DAA regimens include agents with activity against RAVs selected by first generation agents. For example, grazoprevir, a second generation PI potently inhibits the R155K mutant that is highly resistant to first generation agents in this class [
      • Xue W.
      • Ban Y.
      • Liu H.
      • Yao X.
      Computational study on the drug resistance mechanism against HCV NS3/4A protease inhibitors vaniprevir and MK-5172 by the combination use of molecular dynamics simulation, residue interaction network, and substrate envelope analysis.
      ]. However, this issue is more challenging with NS5A inhibitors, which are included in most approved regimens and in all regimens in late-stage development. Even highly resistant NS5A variants are very fit and persist long-term after a course of unsuccessful therapy [
      • Krishnan P.
      • Tripathi R.
      • Schnell G.
      • Reisch T.
      • Beyer J.
      • Dekhtyar T.
      • et al.
      Long-term follow-up of treatment-emergent resistance-associated vairantes in NS3, NS5A and NS5B with paritaprevir, ombitasvir and dasabuvir-based regimens.
      ]. While second wave NS5A inhibitors generally have a higher barrier to resistance than the first agents in this class, they do not cover the highly resistant variants like those with substitutions at the Y93 position. Fortunately, small studies suggest that even if second-line regimens also include an NS5A inhibitor, SVR is possible, particularly with extended treatment durations that often include ribavirin. Interestingly, a recent mechanistic study suggests that surprisingly, combining NS5A inhibitors may resensitize even highly resistant virus to this class of agents, possibly by interactions between adjacent NS5A molecules and the inhibitors [
      • 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.
      ]. Whether this will be successful in patients is currently unknown but if effective, it could be an important salvage approach.
      To overcome resistance, adding another DAA class (or even another agent of the same class [
      • 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.
      ]), extending duration of therapy and adding ribavirin all appear to be useful approaches and from the available data to date, it would seem that at least two of these strategies are required to maximize SVR. Patients who had failed a course of ledipasvir-based therapy were retreated with sofosbuvir/ledipasvir for longer but without a new class of agent or ribavirin and the SVR rates were disappointing at 70% [
      • Lawitz E.
      • Flamm S.L.
      • 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.
      ]. However, patients who relapsed after a short course of sofosbuvir (NI) with grazoprevir (PI) and elbasvir (NS5A) were retreated with the same regimen for longer with the addition of ribavirin and all 23 patients achieved SVR [
      • Lawitz E.
      • Poordad F.
      • Gutierrez J.A.
      • Wells J.T.
      • Landaverde C.E.
      • Reiling J.R.
      • et al.
      C-SWIFT retreatment (Part B): 12 weeks of elbasvir/grazoprevir with sofosbuvir and ribavirin successfully treated G1-infected subjects who failed short-duration all-oral therapy.
      ]. Small studies of patients who failed DAA regimens using ABT-493/ABT-530 or sofosbuvir/velpatasvir/GS-9857 have shown near 100% SVR rates without the need for ribavirin [
      • Gane E.
      • Poordad F.
      • Wells J.T.
      • Hyland R.
      • Yang Y.
      • Dvory-Sobol H.
      • 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.
      ,
      • Poordad F.
      • Gordon S.
      • Asatryan A.
      • Felizarta F.
      • Reindollar R.
      • Landis C.
      • et al.
      High efficacy of ABT-493 and ABT-530 in HCV genotype 1 infected patients who have failed direct-acting antviral-containing regimens: The Magellan-I Study.
      ] suggesting that potent combinations of second generation agents will likely work as salvage regimens for most patients.
      Resistance testing is being evaluated as an approach to help tailor second-line regimens. Retreatment studies have shown that the presence of RAVs significantly affects the outcome of the second course of therapy [
      • Lawitz E.
      • Flamm S.L.
      • 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.
      ]. If simpler regimens are effective for patients who fail therapy without emergence of highly resistant variants, resistance testing may be particularly useful. Alternatively, If the potent regimens in development prove to be highly effective in all patients, resistance testing may not be necessary except for exceptional circumstances, which will certainly simplify therapy for most treating clinicians. Certainly until the optimal regimens are identified, RAV testing should be considered to help guide retreatment strategies [
      Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus.
      ].

      Conclusions

      The last few years have seen extraordinary advances in the management of patients with chronic HCV infection. However, hepatitis C is a worthy adversary with a long history of rapid evolution and adaptation to a wide variety of innate defense mechanisms. The virus has a tendency to infect relatively inaccessible populations making its elimination problematic. The immediate challenges in eliminating chronic HCV remain the need for true pan-genotypic regimens that are effective in both advanced renal and liver disease. Incremental advances are on the near horizon with grazoprevir/elbasvir offering a highly effective and safe regimen for patients with genotype 1 or 4 and advanced renal disease and sofosbuvir/velpatasvir providing the first truly pan-genotypic regimen with high efficacy and safety even in patients with genotype 3 and advanced cirrhosis. Future regimens will also be pan-genotypic and will allow for shortening of therapy for most patients. Once these challenges have been solved we will need management guidelines to optimise the timing of therapy and carefully evaluated salvage regimens that avoid the nightmare scenario of multi-drug resistant virus. These technological challenges are the subject of intensive ongoing research and it is probable that they will be resolved in the near future. The development of highly effective ‘cure-all’ treatments will allow the academic community to focus on the final challenge of finding and treating all those who suffer from this infection. This will require a combination of innovative drug delivery approaches along with improved pathways to care that will allow all citizens of our planet to take advantage of these wonderful new drugs. It is probable that these approaches will need to be tailored to individual countries’ requirements and technological advances in both diagnostic and drug delivery systems may be needed before we can finally declare hepatitis C a dead virus.

      Conflict of interest

      JJF reports receiving honoraria and/or research funds from Abbvie, Bristol-Myers Squibb, Gilead, Janssen, Merck, Theravance. GRF reports receiving honoraria and/or research funds from Abbvie, Alnylam, Bristol-Myers Squibb, Roche, GSK, Gilead, Janssen, Merck, Springbank.

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