Accepted Manuscript

Review

The importance of resistance to direct antiviral drugs in HCV in clinical practice

Christoph Sarrazin

PII: S0168-8278(15)00629-7 DOI: http://dx.doi.org/10.1016/j.jhep.2015.09.011 Reference: JHEPAT 5836

To appear in: Journal of Hepatology

Received Date: 22 April 2015 Revised Date: 15 September 2015 Accepted Date: 15 September 2015

Please cite this article as: Sarrazin, C., The importance of resistance to direct antiviral drugs in HCV infection in clinical practice, Journal of Hepatology (2015), doi: http://dx.doi.org/10.1016/j.jhep.2015.09.011

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The importance of resistance to direct antiviral drugs in HCV infection in clinical practice

Christoph Sarrazin

J. W. Goethe-University Hospital, Medizinische Klinik 1, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany

Phone: +49-69-6301-5122 Fax: +49-69-6301-83112 E-Mail: [email protected]

Electronic word count: 9067

Number of tables and figures: 11

Keywords: C virus, chronic , direct acting antiviral agents, resistance- associated variants, antiviral therapy

Abbreviations: HCV, ; DAA, direct acting antiviral agent; NS3, non- structural protein 3; NS5A, non-structural protein 5A; NS5B, non-structural protein 5B; RAVs, resistance-associated-variants; IP-10, -gamma-inducible protein 10; IFNL4, interferon lambda 4; IL28B, interleukin 28B; HIV, human immunodefiency virus; HBV, hepatitis B virus; PEG/R, pegylated-interferon plus ; PI, protease inhibitor; SVR, sustained virological response; EC50, half maximal effective concentration; die, Latin: dies, day; IU, international unit; GT, genotype; TID, Latin: ter in die, three times daily ; QD; Latin: quaque die, once daily; BID, Latin: bis in die; twice daily; IC50, half maximal inhibitory concentration; PEG-IFN, pegylated-interferon; BOC, ; TVR, ; SMV, , DCV, ; ASV, ; SOF, ; PTV/r, -boosted ; OMV, ; DSV, ; ISG, interferon-stimulated genes; TN, treatment naive; TE, treatment experienced; R, ribavirin; bl, baseline; pts, patients

Conflict of interest: Advisory Committees or Review Panels: Abbvie, Abbott, Achillion, Boehringer Ingelheim, BMS, Janssen, Merck/MSD, Gilead, Roche. Grant/Research Support: Abbott, Roche, Merck/MSD, Gilead, Janssen, Siemens, Qiagen. Speaking and Teaching: Bristol-Myers Squibb, Gilead, Abbott, Abbvie, Roche, Merck/MSD, Janssen, Siemens, Boehringer Ingelheim.

Financial support: CS was supported by a research grant “Deutsches Zentrum für Infektionsforschung (DZIF), TTU Hepatitis”.

Abstract Treatment of chronic hepatitis C virus (HCV) infection with direct acting antiviral agents (DAA) is associated with high rates of sustained virologic response. Remaining factors associated with treatment failure include advanced stages of liver fibrosis, response to previous antiviral therapy and viral factors such as baseline viral load and suboptimal interaction of the DAA with the target based on viral variants. Heterogeneity within NS3, NS5A and NS5B areas interacting with DAAs exist between HCV geno- and subtypes as well as HCV isolates of the same geno- and subtype and amino acid polymorphisms associated with suboptimal efficacy of DAAs are termed resistance associated variants (RAVs). RAVs may be associated with virologic treatment failure. However, virologic treatment failure typically occurs only if other negative predictive host or viral factors are present at the same time, susceptibility to additional antiviral agents is reduced or duration of treatment is suboptimal. In this review geno- and phenotypic resistance testing as well as clinical data on the importance of RAVs for conventional triple therapies with sofosbuvir, simeprevir and daclatasvir and available interferon-free DAA combinations are discussed.

Introduction Before discovery in 1989 transmission of hepatitis C virus (HCV) mainly through medical procedures (transfusion of blood products, vaccination etc.) but also by other means like drug abuse, tattooing or sexual intercourse has led to a significant prevalence of chronic infection between 0.4-3% in the majority of countries and to a huge epidemic with a prevalence between 6-10% in some countries like Pakistan, Egypt and Mongolia [1, 2]. Based on a large data base the global prevalence of viraemic HCV infection recently was re-estimated between 64 and 103 million patients [2]. In the absence of cofactors like heavy alcohol consumption or other liver diseases chronic hepatitis C is characterized by slow progression leading to liver cirrhosis and its sequelae typically after 20-30 years only [3]. As the majority of took place before the discovery of the virus now in many countries an increasing number of patients who present with late stage disease is recognized [4]. Thus, highly effective antiviral therapy is urgently required for many patients. Treatment with (pegylated) interferon alfa and ribavirin is relatively ineffective with a viral eradication rate of approx. 50% only. Furthermore, interferon alfa-based antiviral therapy is associated with a number of severe adverse events, which excluded up to 50% of patients upfront from antiviral therapy [5]. Increasing knowledge on HCV molecular biology and establishment of robust HCV replication and cell culture systems since 1999 facilitated the development of direct-acting antiviral agents (DAAs) for treatment of HCV infection. Currently, 4 classes of DAAs 2 targeting three HCV proteins, which are discussed in detail for their structural and functional properties in a recent review, are approved for treatment in many countries [6]. Different to other chronic viral infections like HBV and HIV the aim of therapy in HCV infection is viral eradication which is observed already spontaneously in a significant proportion of patients (20-40%) [7]. Patients with chronic HCV infection are characterized by different levels of immunological failure to spontaneously eradicate the virus. The underlying mechanisms are not completely understood but several surrogate host parameters like IFNL4 genotype (formerly known as IL28B), IP-10 levels and others are associated with spontaneous and treatment induced viral eradication [8, 9]. The individual variability of efficacy to control HCV infection also explains the huge variation of efforts required to eradicate the virus in single patients. While for some patients weak antivirals and short term treatments are sufficient others require combination therapies with several highly active antivirals for longer durations. In addition the HCV genotype and stage of liver fibrosis have been established as independent parameters associated with virologic treatment response (Figure 1). Based on the principal possibility and the individual differences of susceptibility to viral eradication in HCV infection described above it is clear that also resistance associated variants (RAVs) to DAAs will play a different role in clinical practice as in HIV and HBV infection. On the one hand in HCV infected patients with pre-existence and selection of RAVs during treatment viral eradication has been reported for example with the NS3 protease inhibitor telaprevir in combination with PEG/R and short term treatment or even as monotherapy [10, 11]. On the other hand viral breakthrough or relapse was observed in patients without detectable pre- existence of RAVs and treatment with highly active DAA combination therapies [12]. In this review the importance of RAVs will be discussed in the context of influencing parameters like prevalence of RAVs, their frequencies within the HCV quasispecies, antiviral activities and barrier to resistance of DAAs, duration of antiviral therapy and host parameters like IFNL4 genotype, stage of liver fibrosis and gender.

Genotypic resistance analysis Genotypic resistance analysis is based on DNA sequencing technologies. Below the detection limits of available assays it is unknown whether certain variants exist and persist although due to the error prone replication of HCV it is likely that all possible single and double variants are continuously generated [13]. In the following the term pre-existence of certain variants always is related to detectability by a certain sequencing method. In the majority of cases population sequencing of the HCV genome with a sensitivity to detect viral variants at an approx. 20% frequency within the HCV quasispecies is performed to 3 assess the presence or absence of RAVs to a certain DAA. Clonal and deep sequencing technologies allow reliable detection of viral variants with a frequency down to 0.5-1% [14]. However, currently it is unclear which frequency cut-off is clinically relevant for prediction of virologic treatment failure. Generally, due to the high heterogeneity of HCV isolates and methodological restrictions all sequencing technologies may miss detection of RAVs due to non-amplification based on HCV RNA secondary structures, primer selection and low frequencies within HCV quasispecies.

NS3 protease Mainly dependent on the replicative fitness associated with the presence of RAVs the likeliness of pre-existence of HCV isolates which confer resistance to certain DAAs is highly variable. Many RAVs to NS3 protease inhibitors are associated with a replicative impairment which explains a relatively low likeliness of detectable pre-existence as well as a relatively rapid replacement by wild-type virus after stopping NS3 PI-containing antiviral therapy. For the majority of NS3 protease inhibitors the frequency of natural occurrence of single RAVs in HCV genotype 1 infected patients is between 0.1% and 3.1% (Table 1). An exception is the Q80K variant with no loss of replicative fitness in many patients and a relative high probability of pre-existence. The Q80K variant is associated with resistance to some approved NS3 protease inhibitors only (simeprevir, asunaprevir, paritaprevir). Interestingly, this variant is almost exclusively detected in HCV subtype 1a isolates with important regional differences based on the presence of clade 1 of HCV subtype 1a in the population. While in North America up to 48% of patients with HCV genotype 1a infection harbor the Q80K variant the frequencies in South America and Europe are around 9% and 19%, respectively, with significant variation in the different countries of these continents [15]. For other HCV genotypes only very few data have been published on the natural occurrence of viral variants. However, reduced antiviral activity of NS3 protease inhibitors in different HCV genotypes seems to be in part associated with the natural occurrence of RAVs which present the inherent sequence in these genotypes. For example S122R associated with medium level resistance to simeprevir is present as natural variant in HCV genotype 2 isolates and D168Q which confers high level resistance to simeprevir was observed in all HCV genotype 3 infected patients [16, 17]. Furthermore, Q80K seems to be the natural variant in genotype 5 isolates which may explain again reduced susceptibility to simeprevir. In contrast no variants known to be associated with resistance to NS3 protease inhibitors have been observed in genotype 4 and 6 isolates and here high antiviral activities of simeprevir in clinical studies have been observed [16, 17]. However, the absence of known RAVs for a

4 certain NS3 protease inhibitor does not guarantee a high antiviral activity. For example telaprevir had no efficacy in HCV genotype 3 infected patients in a clinical study although pre-existing variants known to confer resistance to telaprevir have not been observed [18]. Here, obviously so far unknown variants obviously impair proper binding of the inhibitor. Median times to loss of detectability of RAVs by population sequencing have been estimated for different NS3 protease inhibitors. For boceprevir, telaprevir and simeprevir in genotype 1a (1b) infected patients half-life of 14.0 (12.5), 10.6 (0.9) and 8.3 (5.5) months have been reported [19-21]. For paritaprevir analysis is restricted to HCV genotype 1a isolates. Here, NS3 RAVs have been observed in 46% and 9% of patients after 24 and 48 weeks of follow- up, respectively [22]. Few studies investigated long-term follow-up persistence by clonal or deep-sequencing analysis. Here, only in single patients NS3 RAVs have been observed after a follow-up of 4-5 years [23, 24].

NS5A gene Overall, in comparison to the majority of NS3 RAVs, variants conferring resistance to NS5A inhibitors generally are more frequently detected as natural variants in HCV genotype 1 infected patients not exposed to DAA-based antiviral therapies. For single RAVs the rate of natural occurrence was estimated between 0.3% and 3.5% in different studies by population sequencing (Table 1). However, there are two exceptions for HCV genotype 1b isolates. L31M conferring low to medium level resistance to daclatasvir and was observed in 2.1-6.3% of patients and the most frequently detected RAV was Y93H with 3.8-14.1% prevalence in HCV genotype 1b isolates. This variant confers medium to high level resistance to all three approved NS5A inhibitors. If more sensitive methods are applied also the frequency of pre-existent RAVs is increasing. Analysis of more than 2000 patients enrolled in a large phase 2/3 study program by deep sequencing revealed NS5A RAVs to ledipasvir in 15.7% and 16.4% of HCV genotype 1a and 1b infected patients, respectively [25]. Limited data are available on the geographical distribution of naturally occurring RAVs. While no major differences have been observed for the majority of RAVs Y93H seems to be more frequently detected in European HCV genotype 1b infected patients (15.0%) in comparison to the US (9.3%) [25]. For other HCV genotypes only very limited data are available. Generally, in part different variants in comparison to HCV genotype 1 have been attributed for resistance in other HCV genotypes. Moreover, several of these variants were detected at high frequencies in untreated patients (Table 1). Interestingly, in contrast to NS3 protease RAVs, available data suggest that long term persistence of RAVs to NS5A inhibitors is more likely as viral fitness seems not to

5 be impaired. In clinical studies persistence of NS5A RAVs over 1-2 years after treatment failure in over 85% of patients has been reported [22, 26, 27]. The reasons for the unexpected low natural frequency of RAVs despite a lack of impaired viral fitness are unknown but may include subtle differences, initial immunological selection or other so far unknown mechanisms.

NS5B polymerase Currently, only one non-nucleoside inhibitor of the NS5B polymerase binding to the palm I site is approved for treatment of HCV infection. Pre-existence of RAVs to dasabuvir was reported in 0.2%-3.1% of cases with HCV genotype 1 infection (Table 1). An exception is C316N which occurs only in HCV genotype 1b isolates and was observed at a frequency of 10.9 to 35.6% as naturally occurring variant. In addition, also S556G was detected more frequently in genotype 1b in comparison to 1a infected patients (7.0-25% versus 3-6%). Both variants are associated with low level resistance to dasabuvir. Interestingly, HCV genotype 1b isolates harboring C316N (32% versus 5%) and S556G (25% versus 6%) were more frequently observed in Europe in comparison to the United States [28]. Again, data for other genotypes are limited. However, for HCV genotypes 2, 3, 4 and 5 S556G is the natural variant with a prevalence of 97-100%. Moreover, also naturally occurring variants at other positions within the NS5B polymerase (i.e. M289I/L,C316N) were observed and this together with the S556G variant most likely explains the lack of antiviral activity of dasabuvir in non HCV genotype 1 infected patients [29]. Data on viral fitness of variants associated with resistance to dasabuvir and the potential of persistence are sparse. However, preliminary data suggest that at least some RAVs (i.e. M414T, S556G) may tend to persist during long term follow-up for at least one year after treatment failure. Overall, after 24 and 48 weeks in 75% and 57% of patients, respectively, RAVs to dasabuvir were still detectable. Interestingly, the persistence rate of NS5B RAVs which occur together with NS5A RAVs seems to be higher in comparison to isolated NS5B RAVs [22]. Finally, the natural presence of a RAV to the nucleotide analogue sofosbuvir detected by in vitro selection was not reported so far (Table 1). This is explained by a marked impairment of the replicative fitness of HCV isolates containing the S282T variant. The loss of replicative fitness of the S282T variant also explains that so far no viral break-through in patients on treatment with sofosbuvir was observed. In single patients with relapse after the end-of- treatment and detection of the S282T variant reversion to wild-type virus was reported during

6 few weeks of follow-up [30, 31]. This seems to be especially the case in patients with repeated and long exposure to sofosbuvir after failure to a previous sofosbuvir-containing regimen [32]. Whether also other variants with higher replicative fitness and higher natural occurrence (L159F, C316N, and V321A) are associated with resistance to sofosbuvir needs further investigation [33]. In patients with relapse after SOF-based regimens L159F and V321A have been observed at increasing frequencies although in replicon based assays no resistance to sofosbuvir could be observed [34].

Importance of persistence of RAVs Although it is likely that in all patients with virologic treatment failure RAVs have been selected, in a significant rate of patients with treatment failure no RAVs have been detected by sequence analysis. In patients with virologic break-through during treatment almost always RAVs have been observed while in relapse patients the detection rate of RAVs varies between 53% and 91% dependent on the duration of treatment, the DAA class and regimen [20, 21, 25, 35, 36]. Un-detectability of RAVs is explained most likely by the sensitivity of sequencing technology applied, potential rapid reversion to wild-type between end-of-treatment and the day of blood sampling for sequence analysis and a very low frequency of isolates containing RAVs within HCV quasispecies. Moreover, in patients with short duration of DAA-based antiviral therapies wild type virus may not be completely eradicated yet which also explains relapse with a predominantly wild type variant. Long-term persistence of RAVs selected during DAA-based antiviral therapies and subsequent virologic treatment failure is known for HIV and HBV infection which both have the potential of archiving RAVs based on their replication with stable DNA intermediates. As for HCV, some RAVs are associated with high replicative fitness and frequently additional, compensatory mutations have been observed responsible for increased replicative fitness. Due to impaired replicative fitness the frequency of many isolates with RAVs within the quasispecies rapidly decline to very low levels undetectable by population sequencing after stopping DAA treatment. However, despite low frequencies within the HIV quasispecies rapid re-selection of isolates with RAVs upon re-treatment with the same drug or a drug of the same group was observed [37]. Deep sequence analysis together with bioinformatical assessment of nucleotide sequence backbone polymorphisms showed re-selection of persistent isolates which have been archived rather than de novo selection of identical RAVs [37]. For HCV no stable DNA genome forms exist, viral replication showed a rapid turnover (1010 to 1012 virions per day) with a short half-life of virions (2-5 hours) and the error rate during replication with 10-3 to 10-5 mutations per nucleotide per genomic replication was 7 estimated very high in comparison to HIV and HBV [38]. Few studies exist with re-exposure of patients to the same DAA and results are contradictory. While in some studies no evidence for long term persistence and re-selection of isolates with RAVs during re-treatment with the same drug was observed in others indirect evidence pointed to the possibility of persistence and re-selection [39-41]. Given the large number of patients who currently receive all oral DAA therapies and the extension from well-defined HCV geno- and subtypes in clinical studies to a broad heterogeneity of existing HCV subtypes and isolates even with a SVR rate of 90-95% the question on persistence, transmission and reselection of isolates with RAVs will become more important in the near future [42]. Most recently, a study on re-treatment of patients with failure to sofosbuvir plus ledipasvir with and without ribavirin was presented. After 24 weeks of sofosbuvir plus ledipasvir as retreatment the SVR rate was 80% in patients with 8 weeks and only 46% in patients with 12 weeks initial treatment. In patients with detectable NS5A RAVs at baseline the overall SVR rate was 60% which explains the importance of persistent NS5A RAVs for selection of effective re-treatment options [32].

Phenotypic resistance analysis RAVs are typically associated with a change of the shape of the binding or interaction site of DAAs to HCV target proteins. Due to different locations within the sites of interaction and due to different chemical structures of DAAs targeting the same site on the same HCV protein not all RAVs confer the same level of resistance. To assess the level of resistance RAVs typically are introduced as single point mutations into the backbone of the HCV genome within an existing cell-culture/replicon or enzyme based assay. Isolates harboring these RAVs are then challenged by DAAs at increasing concentrations and fold changes based on EC/IC50 and EC/IC90 values are determined for inhibition of replication or enzyme activity in comparison to wild-type virus. Table 2 summarizes EC50/fold change values of different RAVs and DAAs. As for genotypic resistance analysis the majority of values exist for HCV genotype 1 and on genotype 1a and/or genotype 1b backbone isolates only. Only few assays have been established to assess the phenotypic sensitivity to a certain DAA based on the entire HCV quasispecies of a given patient and currently it is unclear whether these assays are required to determine suitable DAAs for patients with resistance to multiple DAAs [43, 44]. For DAA-based antiviral therapy the level of resistance of a certain RAV is not necessarily directly associated with treatment failure. For conventional triple therapy with simeprevir, pegylated interferon alfa and ribavirin for example Q80K as a low level NS3 protease 8 inhibitor RAV significantly influences virologic treatment outcome while for the combination therapy of simeprevir with the nucleotide inhibitor sofosbuvir the importance of the Q80K variant seems to be more limited [45-47]. In fact, for DAA combination therapies not only pre-existing RAVs but also the presence of other predictors of virologic treatment response seems to be of importance. For the combination therapy of sofosbuvir and the NS5A inhibitor ledipasvir for example only high level resistance NS5A variants together with other features seem to have an impact on response to antiviral therapy (see below) [25].

Antiviral activities and barrier to resistance of single DAAs Currently, five NS3 protease inhibitors (boceprevir, telaprevir, simeprevir, asunaprevir, paritaprevir), three NS5A inhibitors (daclatasvir, ledipasvir, ombitasvir), one non-nucleoside (dasabuvir) and one nucleotide NS5B inhibitor (sofosbuvir) are approved globally for treatment of chronic hepatitis C. Additional NS3 protease- (, , , ABT-493), NS5A- (, /GS-5816, /ACH-3102, ABT- 530; MK-8408), non-nucleoside (, GS-9669) and nucleos(t)ide NS5B inhibitors (IDX-21437, ACH-3422) are in phase 2-3 clinical development [48, 49]. Some of them are termed second generation DAAs (grazoprevir, ABT-493, velpatasvir/GS-5816, elbasvir, ABT-530, MK-8408) as they aim to overcome restrictions in terms of resistance profile of the drug class as well concerning the coverage of HCV geno- and subtypes. While for all DAAs short term monotherapy studies have been performed in HCV genotype 1 infected patients for assessment of antiviral activities and barrier to resistance for other HCV genotypes and subtypes antiviral activities are heterogeneous and often no clinical data are available. However, inhibitory concentrations for suppression of replication of many HCV geno- and subtypes have been assessed in vitro and these data may be helpful for selection of active DAAs in patients with failure to multiple DAA-based therapies.

Genetic barrier to resistance It has been recognized already during the clinical development of the first DAAs (boceprevir and telaprevir) that despite an identical amino acid at a certain position within the NS3 protease of HCV subtype 1a and 1b the likeliness to observe a treatment-induced mutation was highly different between these HCV subtypes [50, 51]. This is explained by different nucleotide triplets encoding the same amino acid. While in HCV subtype 1b isolates at codon position 155 for generation of R155K two nucleotide changes are required in HCV subtype 1a isolates one exchange is sufficient. As a consequence distinct resistance patterns are observed

9 in HCV subtype 1a and 1b infected patients after failure to a protease inhibitor based antiviral therapy [52, 53]. Similar differences have been observed also for the generation of other RAVs in other HCV genes [54, 55]. Beside the number of nucleotide changes required for an amino acid exchange as definition of the genetic barrier to resistance also the type of exchange seems to be of importance in HCV infection. The HCV NS5B RNA polymerase was shown to favor the generation of nucleotide transitions in comparison to transversions [56]. This may explain that some RAVs are rarely observed at all or generated only after a longer DAA exposure (for example S282T within NS5B or L31M within NS5A) [32, 54].

NS3 protease inhibitors The antiviral activity of the approved dosing of boceprevir (3x800mg/die) applied as monotherapy was never assessed in a . However, mean maximum decline of HCV RNA concentration in patients infected with HCV genotype 1 was relatively low with half of the approved daily dose (2.1 log IU/ml) [57]. For all other NS3 protease inhibitors high antiviral activities (3.1-4.6 log IU/ml decline) have been described in phase 1 monotherapy trials in HCV genotype 1 infected patients (Table 3). For other HCV genotypes in vitro antiviral activities are heterogeneous and only few clinical studies have been performed (Table 3). All NS3 protease inhibitors bind to the active site of the enzyme, which demonstrate a significant variation between HCV geno- and subtypes explaining heterogeneous affinities of the different PIs and the challenge to design a pan-genotypic NS3 protease inhibitor. While for paritaprevir no clinical monotherapy studies outside of HCV genotype 1 have been performed for simeprevir detailed analysis of antiviral activities and binding profiles to the different HCV geno- and subtypes is available [16, 58]. Here, high antiviral activities in HCV genotype 1, 4 and 6 infected patients could have been demonstrated, while for HCV genotypes 2, 3 and 5 mainly due to the natural occurrence of RAVs low to medium mean maximum viral declines in monotherapy studies were described (Table 3). For grazoprevir the antiviral activity in HCV genotype 3 infected patients was assessed in a 7 days monotherapy study [59]. Here, for the currently used dose of 100mg once daily a moderate antiviral activity was observed (Table 3). Higher doses of grazoprevir were discontinued from further development due elevated liver enzymes in a significant proportion of patients.

10

Indirect evidence for antiviral activities of NS3 protease inhibitors against different HCV genotypes and subtypes can be taken from in vitro studies. Here, data from the developing pharmaceutical companies and independent research groups are available and summarized in Table 4. All currently approved NS3 protease inhibitors were optimized for binding to HCV genotype 1 and all have substantially reduced potency against HCV genotype 3. The three newer NS3 protease inhibitors, simeprevir, paritaprevir, and asunaprevir showed high antiviral activities also against HCV genotype 4a isolates in vitro. For the other HCV geno- and subtypes in vitro activities are heterogeneous (Table 4). The second generation protease inhibitor grazoprevir in principle should be active against all HCV genotypes but data on in vitro studies are only available for genotype 1, 2 and 3 isolates [60]. The barrier to resistance is low for all currently approved NS3 protease inhibitors with broad cross resistance between the different substances. Sequence analysis early during therapy showed rapid selection of isolates harboring RAVs and in the majority of patients after initial viral decline a viral break-through or plateau phase was observed during monotherapy studies for 3-14 days [50, 57, 61-66]. A higher barrier of resistance was observed for grazoprevir due to a higher antiviral activity against typical NS3 RAVs although variants at the same positions as for first generation NS3 protease inhibitors were observed in patients with treatment failure (R155, A156, D168) [67].

NS5A inhibitors NS5A inhibitors typically have broader antiviral activity against different HCV genotypes which is explained by a more conserved interaction site within the NS5A protein [6, 68]. Unfortunately, for all three approved NS5A inhibitors phase 1 monotherapy studies have been performed only in HCV genotype 1 infected patients (Table 3). However, data from in vitro and clinical studies with DAA combination therapies showed antiviral activities also for other HCV genotypes. Moreover, indirect evidence of an antiviral activity of a specific DAA against a certain HCV genotype administered in combination with other drugs results from the detectability of RAVs in patients with virologic treatment failure. The antiviral activity of the first approved NS5A inhibitor daclatasvir is broad with little differences between HCV genotypes in replicon studies [69]. For HCV genotype 3 isolates an approx. 10-fold lower antiviral activity was observed (Table 4). Also for ledipasvir a broad genotypic coverage was observed in vitro [70]. However, for HCV genotype 2 and 3 isolates approx. 1000-fold higher concentrations of ledipasvir were required for inhibition of replication in vitro (EC50), which indicates an insufficient antiviral activity of ledipasvir for these HCV genotypes (Table 4). Ombitasvir is characterized by a coverage of HCV genotypes 1 to 5 with high antiviral

11 activities [71]. However, a 10 to 100-fold lower activity against HCV genotype 6 was observed and ombitasvir is available only in a fixed dosed combination with the protease inhibitor paritaprevir which shows high antiviral activities in HCV genotype 1 and 4 isolates only (Table 4). For HCV genotypes 2 to 6 a large number of HCV subtypes exist in clinical practice with significant amino acid sequence variation and little is known about antiviral activities of NS5A inhibitors against the different HCV subtypes. An example for potentially important variations is ledipasvir. Here, a 1000-fold difference in the antiviral activity was described between HCV genotype 6a and 6e isolates in vitro (Table 4). Also for NS5A inhibitors a low barrier to resistance and a broad cross resistance between the different approved substances with rapid selection of RAVs mainly at positions 28, 30, 31, 58 and 93 has been observed in short term monotherapy studies [71-74]. For the second generation NS5A inhibitor elbasvir high antiviral activities in monotherapy studies in HCV genotype 1 and 3 infected patients have been demonstrated and a higher barrier to resistance in comparison to other NS5A inhibitors was reported from in vitro studies [75, 76].

Non-nucleoside NS5B inhibitors Dasabuvir currently is the only approved non-nucleoside inhibitor of the NS5B polymerase with binding to the palm I site. During a clinical study with monotherapy of different doses of dasabuvir a mean maximum decline of 1.08 HCV RNA log IU/ml was observed indicating a low to medium antiviral activity [77]. In vitro, only data for HCV subtypes 1a and 1b are available (Table 4). However, evidence from biochemical assays indicates that dasabuvir is inactive towards HCV polymerases from HCV genotypes 2, 3 and 4 [78]. The barrier of dasabuvir to resistance is considered to be low although no sequencing studies in patients on monotherapy have been presented. However, a number of RAVs have been selected in HCV replicon studies as well as in patients with failure to combination therapies with other DAAs [36, 77, 78].

Nucleos(t)ide NS5B inhibitors Nucleos(t)ide inhibitors interact with the active site of the HCV NS5B polymerase which is highly conserved between all HCV geno- and subtypes. Therefore, a broad coverage of all HCV isolates is likely for this class of DAAs. For the nucleotide polymerase inhibitor sofosbuvir clinical data are available with monotherapy for 2 to 12 weeks in HCV genotype 1, 2 and 3 infected patients (Table 3) [79, 80]. In addition, in vitro studies showed an almost equal antiviral activity against HCV genotypes 1 to 6 (Table 4) [81]. Moreover, a high barrier to resistance was observed in clinical studies with no viral breakthrough during mono- or

12 combination therapies with other DAAs [79, 80, 82-84]. With the exception of few cases even in patients with virologic treatment failure no major RAVs have been detected [31].

Viral resistance and approved combination therapies

Protease inhibitor plus pegylated interferon and ribavirin For conventional triple therapies with boceprevir or telaprevir in combination with ribavirin the probability of pre-existing RAVs is low and no correlation with treatment response was observed in treatment naïve patients (Figure 2) [35, 85-89]. Hence, no baseline testing was recommended. However, for patients with poor interferon-responsiveness with and without other negative predictors a correlation of pre-existing RAVs with treatment response has been described [88-92]. In contrast, for conventional triple therapy with simeprevir baseline resistance testing is recommended by international guidelines. Here, RAVs at position 43, 122, 155, 168 and the Q80R variant were observed in a low rate of patients only (1.3%) and thus no clear correlation with virologic treatment outcome was possible. However, one additional variant

(Q80K) causing medium level resistance to simeprevir (approx. 8-fold change in EC50) is associated with a much higher natural prevalence [15, 21]. The Q80K variant was almost exclusively detected in HCV subtype 1a isolates with a global prevalence from phase 2/3 studies of approx. 30% (see above) [21]. SVR rates in treatment-naïve HCV genotype 1a infected patients with and without the Q80K variant were 58% versus and 84% (Figure 2). The SVR rate of 58% in patients with Q80K who received simeprevir-based triple therapy was statistically not different from 52% in patients who received pegylated interferon alfa and ribavirin alone [45, 46]. Similar results were obtained in previous null-responders and partial responders and re-treatment with simeprevir-based triple therapy [93]. Therefore, administration of simeprevir-based triple therapy is not recommended in patients with detectable Q80K variant at baseline.

NS5A inhibitor plus pegylated interferon alfa and ribavirin Conventional triple therapy with the NS5A inhibitor daclatasvir, pegylated interferon alfa and ribavirin was investigated in clinical studies in HCV genotype 1-4 infected patients. Overall, higher SVR rates were observed in comparison to dual combination therapy with pegylated interferon alfa, ribavirin and placebo. For genotype 1 infected patients an overall SVR rate of 59-60% was achieved for daclatasvir-based triple therapy with significant differences between HCV subtypes which reflects the overall lower antiviral activity of daclatasvir in HCV

13 subtype 1a versus 1b isolates (55-57% for HCV subtype 1a versus 76-77% in HCV subtype 1b). In 22 of 365 patients (6%) baseline NS5A inhibitor RAVs (L31M/V and/or Y93H/N/S) were observed. SVR rates in patients with pre-existent RAVs were 33% for HCV subtype 1a and 80% for HCV subtype 1b [94]. Given the low frequency and similar SVR rates in patients with and without baseline variants no clear impact of pre-existent RAVs for virologic treatment response could be demonstrated (Figure 2). For HCV genotype 2, 3, and 4 only small cohorts of patients infected with these HCV genotypes were enrolled in clinical studies (n=6-12 per treatment group) not allowing definite conclusions on the importance of baseline RAVs [94, 95]. However, generally similar viral variants associated with resistance were observed in comparison with HCV genotype 1. Furthermore, in HCV genotype 3 infected patients pre-existence of Y93H or A30K may increase the risk of subsequent relapse. Here, virologic treatment failure was observed in 50% (4/8) of patients with Y93H or A30K at baseline in comparison to 16% (8/43) without pre-existence of these variants [95].

Sofosbuvir plus pegylated interferon alfa and ribavirin Sofosbuvir in combination with pegylated interferon alfa and ribavirin led to an SVR rate of 89% in a large phase 3 study on treatment naïve HCV genotype 1 infected patients. Interestingly, a substantial difference was observed between HCV genotype 1 subtypes with an SVR rate of 92% in HCV subtype 1a versus 82% in HCV subtype 1b [96]. This was unexpected as for NS3 protease- and NS5A-inhibitor based conventional triple therapies always lower SVR rates in HCV subtype 1a infected patients were observed which is attributed to the lower antiviral activity of these drug classes in HCV subtype 1a. S282T as a known sofosbuvir resistance mutation from in vitro studies was not observed in any HCV isolate before initiation of antiviral therapy or at treatment failure [96]. This is explained by the low replicative fitness of this variant. Furthermore, no other variants with a clear reduction of susceptibility to sofosbuvir in cell-culture assays are known. A potential explanation for the difference between HCV subtype 1a and 1b comes from a variant at position 316 within the HCV NS5B polymerase which is highly conserved in subtype 1a (C316) but polymorphic in subtype 1b isolates (C316N/H). In a structural assessment it was predicted that C316N/H may alter the ability of sofosbuvir to interact with the active site of the NS5B polymerase [33]. Dependent on geographical differences, between 10% and 30% of HCV genotype 1b isolates harbor C316N as natural variant which may explain the observed differences between subtype 1a and 1b infected patients and treatment with sofosbuvir-based conventional triple therapy [33, 97]. However, the numbers of HCV genotype 1b infected patients in the approval trial was too small to draw definite conclusions and further studies are needed. 14

Sofosbuvir plus ribavirin Due to the limited antiviral efficacy only few HCV genotype 1 infected patients have been enrolled in studies for evaluation of the nucleotide polymerase inhibitor sofosbuvir in combination with ribavirin [79, 98]. In contrast, for HCV genotype 2 infected patients very high SVR rates were achieved with this combination leaving only few patients with virologic failure for resistance analysis [79, 99]. Interestingly, in some of the few patients with HCV genotype 2 infection failure to sofosbuvir plus ribavirin may be explained by the presence of a viral chimera. Here, the structural HCV genes are encoded by HCV genotype 2 and the non- structural genes are coming from an HCV genotype 1 isolate which may explain reduced susceptibility to sofosbuvir plus ribavirin [100]. However, for HCV genotype 3 a relative large number of treatment failure patients could be evaluated for the potential importance of viral resistance. Based on refined selection criteria for variants potentially associated with resistance, L159F, V321A, and S282R have been observed in patients with virologic relapse. Although these variants were not associated with reduced susceptibility against sofosbuvir in cell culture, affection of the interaction of the inhibitor with the NS5B polymerase was predicted by structural bioinformatics [33]. Most likely, in addition to baseline RAVs other factors associated with virologic treatment response like stage of fibrosis, IFNL4 genotype or previous responsiveness to interferon alfa-based antiviral therapies are of importance and large studies in HCV genotype 2 and 3 infected patients for which sofosbuvir plus ribavirin is a standard treatment are required to understand the importance of viral resistance with this regimen.

NS3 protease inhibitor and NS5A inhibitor Asunaprevir plus Daclatasvir The combination of the NS3 protease inhibitor asunaprevir together with the NS5A inhibitor daclatasvir was shown to be highly effective in HCV genotype 1b infected patients while for HCV genotype 1a a high rate of viral break-through was observed in the initial study [101]. Subsequently combination therapies with these two targets were evaluated in larger studies and currently asunaprevir plus daclatasvir is approved as standard treatment in HCV genotype 1b infected patients in Japan. In the pivotal study treatment of HCV genotype 1b infected patients (n=643) with asunaprevir plus daclatasvir for 24 weeks provided SVR rates between 82 and 91% [102]. In 13% of patients pre-existing variants associated with resistance to asunaprevir and or daclatasvir (positions D168 within NS3 and L31, Y93 within NS5A) were

15 observed. The SVR rate of these patients was significantly reduced to 39% while in 92% of patients without baseline RAVs treatment-induced viral eradication was achieved (Figure 3). Simeprevir plus Daclatasvir In addition, clinical study data are available for the combination of the NS3 protease inhibitor simeprevir plus daclatasvir. Here, with treatment of a relative small number of HCV genotype 1b infected patients (n=147) for 12-24 weeks SVR rates between 65 and 95% were obtained [103]. Unfortunately, no data on the importance of baseline RAVs have been presented so far for this study but a significant influence on the rates of viral break-through and relapse is likely. Furthermore, also a small number of HCV genotype 1a infected patients were explored in this study. While 67% of treatment-naïve patients achieved SVR, 7 out of 9 previous null- responders experienced viral break-through. Beside the lower antiviral activity of this regimen in HCV genotype 1a infected patients it is also likely that baseline RAVs are of importance for prediction of virologic treatment response. Grazoprevir plus Elbasvir Recently, phase 3 study results have been reported also for combination of grazoprevir and elbasvir and treatment duration of 12 weeks. RAVs in the NS3 protease had no impact on SVR. Relevant RAVs to the NS5A inhibitor elbasvir were observed in 10% and 13% at baseline in HCV genotype 1a and 1b infected patients. For HCV genotype 1b infected patients the presence of baseline NS5A RAVs had no importance for treatment response. However, in treatment-naïve HCV genotype 1a infected patients SVR rates with and without baseline RAVs were 22% and 98% [104]. Similar results were obtained in treatment- experienced patients. Here, independent of the duration (12 vs 16 weeks) and the addition of ribavirin in HCV genotype 1a infected patients with and without baseline RAVs SVR rates of 52% and 99% were reported [105]. Interestingly, for treatment-naïve and -experienced patients only RAVs with a >5-fold resistance level were of importance (Figure 3) [104, 105]. For patients with HCV genotype 4 and 6 infection number were too small for valid resistance analysis.

Taken together, the barrier to resistance of first wave protease inhibitors like asunaprevir or simeprevir in combination with an NS5A inhibitor is relatively low. Based on a relative high frequency of patients with pre-existing NS3 and / or NS5A RAVs a significant impact on virologic treatment response is obvious. Thus, HCV genotype 1a infected patients should not be treated with this regimen and baseline resistance testing should be mandatory for this DAA combination in genotype 1b infected patients. For treatment with grazoprevir plus elbasvir due to a higher barrier to resistance treatment is highly effective in genotype 1b infected 16 patients without importance of baseline RAVs. However, baseline resistance testing seems to be required in HCV genotype 1a infected patients.

Interestingly, very recent data suggested an association of a major NS5A RAV (Y93H) with beneficial IFNL4 genotypes [106, 107]. If confirmed in additional studies, this would also explain the unexpected inverse correlation of SVR rates with IFNL4-rs12979860 genotype in the dual combination asunaprevir plus daclatasvir combination phase 3 study. Here, SVR rates in patients with the beneficial CC genotype were 76-89% versus 86-96% in those with the TT genotype [102].

NS3 protease inhibitor plus nucleos(t)ide NS5B inhibitor The combination of simeprevir and sofosbuvir with and without the additional application of ribavirin for 12 or 24 weeks was initially investigated in a small prospective randomized phase 2 study [47]. Here, in null-responder patients with early liver fibrosis stages as well as in treatment naïve patients and previous null-responders with advanced fibrosis or liver cirrhosis high SVR rates were reported (92%). Pre-existing RAVs are very rarely observed in HCV genotype 1b infected patients while for HCV genotype 1a the rate of a naturally occurring Q80K variant varies between 10 and 50% with important regional differences (see above) [15, 21]. In line with this, virologic treatment failure was not observed in HCV genotype 1b infected patients while the pre-existence of Q80K in 4 out of 6 HCV genotype 1a infected patients with virologic relapse point to an importance for treatment failure. For subsequent phase 3 studies the combination of simeprevir plus sofosbuvir without ribavirin was explored. Here in HCV genotype 1 infected patients without liver cirrhosis the overall SVR rate was 97% and 83% for 12 and 8 weeks treatment duration. While for a duration of 12 weeks the presence of Q80K at baseline in genotype 1a infected patients had no impact (96% versus 97% SVR) in the group with 8 weeks treatment SVR rates were 73% and 84% with and without Q80K at baseline (Figure 4) [108]. Similar results were obtained in HCV genotype 1a infected patients with cirrhosis and 12 weeks treatment duration (74% versus 92% SVR with and without Q80K) (Figure 4) [109].

NS5A inhibitor plus nucleos(t)ide inhibitor Daclatasvir plus Sofosbuvir In the phase 2 study program only a limited number of HCV genotype 1 infected patients (n=167) was treated with heterogeneous regimens of sofosbuvir and daclatasvir with and without ribavirin for 12 (n=82) or 24 weeks (n=85). Furthermore, patients with liver cirrhosis 17 were excluded and after elimination of non-virologic treatment failure patients the SVR rate was 100% [110]. The subsequent phase 3 studies enrolled non-cirrhotic HCV genotype 1-6 infected patients with HIV coinfection and those with liver cirrhosis or after liver transplantation. Overall high SVR rates in HCV genotype 1 infected patients were obtained (82%-98%). Virologic treatment failure mainly was associated with HCV subtype 1a infection in combination with shortened treatment duration to 8 weeks (and suboptimal dosing of daclatasvir) or the presence of cirrhosis. Unfortunately, full data are not yet available on the precise importance of pre-existent resistance for different types of patients and HCV geno- / subtypes. However, the presence of baseline NS5A RAVs also seems to influence the chance of SVR only if additional negative predictors like liver cirrhosis are present (Figure 5). Ledipasvir plus Sofosbuvir For the combination of sofosbuvir plus ledipasvir with and without the addition of ribavirin more than 2100 patients were enrolled in different phase 2 and 3 studies [82-84, 111]. Sequence analysis of the NS5A gene was successfully performed in more than 99% of baseline samples. In addition and different to the standard analysis in other studies where population sequencing was used, in the vast majority of patients (89%) deep sequence analysis was performed. The rate of pre-existing NS5A RAVs in the entire HCV genotype 1 infected population was 17% with a 1% cut-off and 8% with a 20% cut-off within the HCV quasispecies. The SVR rate in patients with and without baseline NS5A resistance was only slightly reduced but due to the large sample size this result was significant (93% versus 97%, respectively). Similar data were obtained when HCV genotype 1a and 1b patients were analyzed separately [25]. Further analysis was performed based on the level of resistance caused by the different RAVs and treatment duration. RAVs with a less than 100-fold shift of EC50ies had no influence on SVR independent of pre-treatment status and treatment duration (8, 12, 24 weeks) (Table 2). However, in patients with highly resistant RAVs (>100 fold shift EC50) and shorter treatment durations a more significant decline of SVR rates was observed. In treatment naïve patients with 8 weeks the SVR rate was 83% in comparison to 95% in patients with highly resistant RAVs and those without baseline RAVs (Figure 5). In a combined analysis of treatment-naïve and -experienced patients a reduced SVR of 87% with 12 weeks treatment was observed compared to 97% in patients without highly resistant baseline RAVs (Figure 5). SVR rates ranged between 84% and 97% in patients with RAVs at position 24, 28, 30, 31 and 93 of the NS5A protein and were similar for different frequencies of RAVs within the HCV quasispecies. In patients with liver cirrhosis also reduced SVR rates have been reported due to the presence of baseline RAVs (Figure 5) [112].

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Concerning sofosbuvir resistant variants as described also in other studies S282T as major in vitro RAV was not observed at baseline in any patient. Furthermore, also no correlation of other variants within the NS5B polymerase including N142T, L159F, S282G, C316N, and L320F with virologic treatment response was observed. The overall prevalence of these RAVs was 2.5% and the SVR rate 100%. Finally, as to be expected RAVs to NS3 protease inhibitors had no influence on treatment response to sofosbuvir plus ledipasvir [25]. In 76% of patients who failed to achieve SVR NS5A RAVs were observed [25]. In a recent presentation long term persistence of NS5A RAVs has been reported in 86% of patients after failure to different treatment regimens with ledipasvir and 96 weeks of follow-up [27].

Studies in non-genotype 1 patients For treatment of patients with sofosbuvir in combination with an NS5A inhibitor like daclatasvir or ledipasvir infected with other HCV genotypes the experience is very limited. Given the high SVR rates obtained only few patients with treatment failure are available for resistance analysis so far [110, 113]. For sofosbuvir plus daclatasvir relapse was reported in one patient with HCV genotype 3 infection from the phase 2 study. Here, a resistance associated NS5A polymorphism (A30K) was detected at baseline and at the time of relapse [110]. Furthermore, 16 HCV genotype 3 infected patients experienced virologic relapse after 12 weeks sofosbuvir plus daclatasvir in the phase 3 Ally 3 study (n=152) [113]. Here, in 6 patients the resistance variant Y93H was detectable at baseline as well as at treatment failure and in all other patients NS5A RAVs (mainly Y93H and one L31I) emerged at relapse. The overall rate of pre-existing Y93H in the entire cohort of treated patients was 9% (n=13). The SVR rate in patients with Y93H was clearly reduced to 54% in comparison with 89% in the entire cohort. However, for patients with pre-existing Y93H also the additional presence of liver cirrhosis seems to be of importance. For patients without cirrhosis the SVR rate was moderately reduced to 67% (6/9 patients) while patients with cirrhosis and baseline Y93H achieved an SVR in 25% (1/4 patients) only (Figure 6). Variants at positions L31, M28 and A30 very not or rarely observed at baseline and no clear effect on virologic treatment response was visible [113]. Resistance analysis for the combination of sofosbuvir plus ledipasvir has not been published so far.

NS3 protease-, NS5A- and non-nucleoside NS5B inhibitor Paritaprevir/r plus Ombitasvir plus Dasabuvir Triple DAA combination therapy with paritaprevir/r, ombitasvir and dasabuvir with and without ribavirin was approved based on a large clinical study program with more than 2500 19 patients. Baseline resistance analysis based on population sequencing is available from approx. 700 patients only [36]. Here, the prevalence of RAVs within one and two targets was 18% and 0.4% in HCV subtype 1a and 33% and 3% in HCV subtype 1b infected patients, respectively. None of the patients had RAVs within all three targets. As known from other studies within the NS5A protein M28 variants (8% in HCV subtype 1a) and Y93H variants (8% in HCV subtype 1b) were observed most frequently. For RAVs to the non-nucleoside NS5A inhibitor dasabuvir mainly C316N and S556G were observed in genotype 1b (15-17%) and S556G/N/R (3%) in HCV genotype 1a infected patients. For the NS3 protease Q80K is considered a low level resistant variant to paritaprevir and other RAVs were rarely observed at baseline (<1%). Frequencies of SVR in patients with and without baseline RAVs are available from a phase 2 study only (n=391). For the entire group of HCV genotype 1 infected patients after excluding Q80K variants no difference was visible (91 versus 91%). As the vast majority of patients with virologic treatment failure (91%) was infected with HCV subtype 1a further sub-analysis in this subgroup of patients may be of interest. Here, a slightly reduced overall SVR rate in patients with versus those without baseline RAVs in the NS3, NS5A and / or the NS5B genes was reported (87% versus 92%) (Figure 7) [28]. Moreover, in the 8 weeks treatment arm, SVR rates were reduced in patients with baseline Q80K (74% versus 87%), suggesting a relevance of this polymorphism also for this triple DAA regimen (Figure 7). So far no data are available on SVR rates in patients with RAVs to two targets and other treatment predictors like 12 versus 24 weeks of therapy and liver cirrhosis [36]. In 85% of patients with treatment failure RAVs to at least one target of the triple DAA therapy were observed and 58% of virologic failure patients had RAVs against all three targets. Long term follow-up data have been presented recently. While NS3 RAVs were detectable in only 9% of patients by population sequencing after approx. 1 year RAVs within NS5A and NS5B tend to persist with detectability in 96% and 57%, respectively[22]. Asunaprevir plus Daclatasvir plus Beclabuvir Other triple DAA combination therapies without a nucleos(t)ide inhibitor have been investigated in clinical studies. The combination of the protease inhibitor asunaprevir, the NS5A inhibitor daclatasvir and the non-nucleoside thumb 1 inhibitor beclabuvir without the additional application of ribavirin has been administered for 12 weeks in a large phase 3 study in non-cirrhotic patients. Here, SVR rates in HCV genotype 1a infected patients with (n=34) and without (n=195) baseline NS5A RAVs were 74% versus 93% (Figure 7) [114]. Interestingly, in patients with liver cirrhosis and evaluation of the same triple DAA regimen no apparent differences between patients with and without baseline NS5A RAVs were observed which may be explained by the addition of ribavirin in half of the patients (Figure 7) 20

[115]. Thus, a relative low importance and frequency of single RAVs in a regimen targeting 3 HCV proteins for virologic treatment failure may be further reduced by the addition of ribavirin. However, further data especially in difficult-to-treat HCV genotype 1a infected patients are required for understanding of the importance of pre-existing resistance.

Salvage therapy Virologic treatment response of patients who failed to respond to triple therapies with boceprevir or telaprevir has been evaluated in clinical studies with DAA combination therapies without NS3 protease inhibitors. For both regimens, daclatasvir plus sofosbuvir as well as ledipasvir plus sofosbuvir high SVR rates have been reported (94-100%) with no influence of NS3 RAVs on virologic treatment response [83, 110]. Insufficient data are available for determination of the importance of NS3 RAVs for re-treatment with protease inhibitor containing regimens. Data for patients with failure to the different all oral DAA combination therapies are sparse. In one study 14 HCV genotype 1 infected patients with failure to sofosbuvir plus ribavirin for 24 weeks were retreated with sofosbuvir plus ledipasvir for 12 weeks and all patients achieved SVR [116]. This cohort included 7 patients with advanced fibrosis / cirrhosis. In one patient transiently early after termination of the initial therapy with sofosbuvir and ribavirin the S282T variant was detectable while in all other patients wild type NS5B polymerase sequences were observed [116]. In another study, patients who failed 12 to 24 weeks of sofosbuvir plus ribavirin mainly infected with HCV gentoype 3 were enrolled for retreatment with either 12 weeks pegylated interferon, ribavirin and sofosbuvir or 24 weeks sofosbuvir plus ribavirin. An interim analysis of this study showed a high efficacy of conventional triple therapy (SVR12 92%) while another course of sofosbuvir plus ribavirin combination therapy led to an SVR rate of 63% only [117]. A single HCV genotype 1 infected patient who failed to respond to sofosbuvir plus ledipasvir for 8 weeks received successful retreatment with the same regimen in combination with ribavirin for 24 weeks despite the presence of highly resistant NS5A RAVs and the S282T variant which causes major resistance to sofosbuvir [118]. However, in 41 patients with failure to 8-12 weeks of sofosbuvir plus ledipasvir with or without ribavirin salvage treatment with sofosbuvir plus ledipasvir for 24 weeks was initiated. The overall SVR rate was 71%. In the subgroup of patients with failure to 12 weeks previous treatment only 5/11 (45%) achieved SVR and the presence of NS5A resistance was associated with treatment outcome [32]. Further studies investigating salvage therapies for DAA failure patients are ongoing. 21

Currently, it seems that longer re-treatment with the same class of drugs may be effective in some patients although a reduced antiviral efficacy is obvious. Most likely, additional predictors like the stage of liver fibrosis, pre-treatment status, duration of initial therapy and others will be of importance to select optimal salvage therapies. Given the high rates of the presence of multiple RAVs in DAA treatment failures, the known trend to slightly lower SVR rates in patients with pre-existing RAVs, and a high probability of accumulation of negative predictive factors in DAA-failure patients it is likely that resistance analysis will be useful to determine suitable DAAs for re-treatment options. Also due to the high costs of DAAs it would be important to select the most effective re-treatment option. For the time being guidelines recommend to wait for results of clinical studies or in patients with urgent need of re-treatment if possible to switch the used class of DAAs and to take data of resistance analysis into account [119].

Perspective High efficacy of all-oral DAA combination therapies have been obtained in clinical studies with SVR rates above 90% in the majority of patients with chronic hepatitis C. For the combination of a first generation NS3 protease and NS5A inhibitor with a relative low barrier to resistance the presence of baseline resistance has a great impact with bisection of SVR rates. Here, baseline resistance testing is mandatory. However, for regimens with high antiviral activities and high genetic barrier to resistance based on single DAAs or the combination of different drug classes the presence of baseline resistance leads only to a small reduction of SVR rates. Here, additional predictors of response are of importance. While no general recommendation for baseline resistance testing can be given in subgroups of patients with certain HCV geno- or subtypes, in patients with shortened treatment duration or those with liver cirrhosis resistance testing may be used to select optimal DAA regimens. Future studies have to explore whether due the high costs of DAA regimens in regions with economical restriction baseline resistance testing for initial DAA combination regimens with the aim to avoid virologic treatment failure and the need of retreatment may be cost effective. A number of second generation DAAs are in development. Here, unmet medical needs mainly include the development of an effective treatment in genotype 3 infected patients and on the same time to ensure coverage of all different HCV geno- and subtypes. Achievement of the latter aim would facilitate treatment of chronic hepatitis C significantly but due to the large number of HCV subtypes represents a major challenge with the risk of improvement of antiviral activity in one subtype but loosing of efficacy in other HCV subtypes.

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Currently, effective treatment options in patients with failure to all-oral DAA regimens are not defined yet. Despite high SVR rates around 95% given the large number of patients with chronic hepatitis C overall the number of patients with virologic treatment failure will be high. Based on available data more than 80% of these patients will harbor HCV isolates with resistance to one, two or three DAA classes. Also in patients with failure to DAA-based regimens an accumulation of other negative response factors is likely. One approach is re- treatment with the same DAAs for a longer duration. Indeed in patients who received initial therapies for 24 weeks relapse rates have been extremely low. However, preliminary data in small patient cohorts showed reduced efficacies with this approach. Alternatively, a switch of drug classes can be explored. Due to the low likeliness of selection of major RAVs against the nucleotide inhibitors this class of drugs can be re-used. Here, one limitation could be the broad use of NS5A inhibitors in first line DAA regimens together with the high likeliness of selection of NS5A RAVs and persistence of these RAVs. Thus, for patients with virologic treatment failure to all oral DAA-based regimens a switch of DAA classes, longer treatment durations, the addition of ribavirin and finally in patients with multiple resistance also a combination with pegylated interferon alfa may be explored in clinical studies.

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References

[1] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362. [2] Gower E, Estes C, Blach S, Razavi-Shearer K, Razavi H. Global epidemiology and genotype distribution of the hepatitis C virus infection. J Hepatol 2014;61:S45-57. [3] Wiese M, Fischer J, Lobermann M, Gobel U, Grungreiff K, Guthoff W, et al. Evaluation of liver disease progression in the German hepatitis C virus (1b)-contaminated anti-D cohort at 35 years after infection. Hepatology 2014;59:49-57. [4] Razavi H, Waked I, Sarrazin C, Myers RP, Idilman R, Calinas F, et al. The present and future disease burden of hepatitis C virus (HCV) infection with today's treatment paradigm. J Viral Hepat 2014;21 Suppl 1:34-59. [5] Niederau C, Huppe D, Zehnter E, Moller B, Heyne R, Christensen S, et al. Chronic hepatitis C: treat or wait? Medical decision making in clinical practice. World journal of gastroenterology : WJG 2012;18:1339-1347. [6] Bartenschlager R, Lohmann V, Penin F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 2013;11:482- 496. [7] Deterding K, Gruner N, Buggisch P, Wiegand J, Galle PR, Spengler U, et al. Delayed versus immediate treatment for patients with acute hepatitis C: a randomised controlled non- inferiority trial. The Lancet infectious diseases 2013;13:497-506. [8] Park SH, Rehermann B. Immune responses to HCV and other hepatitis viruses. Immunity 2014;40:13-24. [9] Heim MH. Innate immunity and HCV. J Hepatol 2013;58:564-574. [10] Suzuki F, Suzuki Y, Akuta N, Sezaki H, Yatsuji H, Arase Y, et al. Sustained virological response in a patient with chronic hepatitis C treated by monotherapy with the NS3-4A protease inhibitor telaprevir. J Clin Virol 2010;47:76-78. [11] Bronowicki JP, Hezode C, Bengtsson L, Pol S, Bourliere M, Serfaty L, et al. 100% Svr in Il28b Cc Patients Treated with 12 Weeks of Telaprevir, Peginterferon and Ribavirin in the Prove2 Trial. Journal of Hepatology 2012;56:S430-S431. [12] Wyles DL, Rodriguez-Torres M, Lawitz E, Shiffman ML, Pol S, Herring RW, et al. All-Oral Combination of Ledipasvir, Vedroprevir, Tegobuvir, and Ribavirin in Treatment- Naive Patients With Genotype 1 HCV Infection. Hepatology 2014;60:56-64. [13] Rong L, Dahari H, Ribeiro RM, Perelson AS. Rapid emergence of protease inhibitor resistance in hepatitis C virus. SciTranslMed 2010;2:30ra32. [14] Dietz J, Schelhorn SE, Fitting D, Mihm U, Susser S, Welker MW, et al. Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients. J Virol 2013;87:6172-6181. [15] Sarrazin C, Lathouwers E, Peeters M, Daems B, Buelens A, Witek J, et al. Prevalence of the hepatitis C virus NS3 polymorphism Q80K in genotype 1 patients in the European region. Antiviral Res 2015;116:10-16. [16] Lenz O, Vijgen L, Berke JM, Cummings MD, Fevery B, Peeters M, et al. Virologic response and characterisation of HCV genotype 2-6 in patients receiving TMC435 monotherapy (study TMC435-C202). JHepatol 2013;58:445-451. [17] Vallet S, Viron F, Henquell C, Le Guillou-Guillemette H, Lagathu G, Abravanel F, et al. NS3 protease polymorphism and natural resistance to protease inhibitors in French patients infected with HCV genotypes 1-5. Antivir Ther 2011;16:1093-1102. [18] De Meyer S, Ghys A, Foster GR, Beumont M, Van Baelen B, Lin TI, et al. Analysis of genotype 2 and 3 hepatitis C virus variants in patients treated with telaprevir demonstrates a consistent resistance profile across genotypes. J Viral Hepat 2013;20:395-403.

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[19] Howe AY, Long JM, Thompson S, Barnard RJ, Alves K, Howe JA, et al. Analysis of the Durability of Response and Persistance of Resistance Associated Variants during Long Term Follow Up after Boceprevir plus Pegylated Interferon/Ribavirin Therapy-3 Year Analysis. Hepatology 2013;58:1095a-1095a. [20] Sullivan JC, De Meyer S, Bartels DJ, Dierynck I, Zhang EZ, Spanks J, et al. Evolution of treatment-emergent resistant variants in telaprevir phase 3 clinical trials. Clin Infect Dis 2013;57:221-229. [21] Lenz O, Verbinnen T, Fevery B, Tambuyzer L, Vijgen L, Peeters M, et al. Virology analyses of HCV isolates from genotype 1-infected patients treated with simeprevir plus peginterferon/ribavirin in Phase IIb/III studies. J Hepatol 2015;62:1008-1014. [22] Krishnan P, Tripathi R, Schnell G, Reisch T, Beyer J, Dekhtyar T, et al. Long-term follow-up of treatment-emergent resistance-associated variants in NS3, NS5A and NS5B with paritaprevir/r, ombitasvir- and dasabuvir-based regimens. J Hepatol 2015;62:S220-S220. [23] Susser S, Vermehren J, Forestier N, Welker MW, Grigorian N, Fuller C, et al. Analysis of long-term persistence of resistance mutations within the hepatitis C virus NS3 protease after treatment with telaprevir or boceprevir. J ClinVirol 2011. [24] Thomas XV, de Bruijne J, Kieffer TL, Sullivan JC, Rebers SP, deVries M, et al. Long- term follow-up of chronic hepatitis C infected patients treated with telaprevir: evaluation of persistence of resistant variants by ultra-deep sequencing. JHepatol 2011;54(Suppl. 1):S490- S491. [25] Sarrazin C, Dvory-Sobol H, Svarovskaia ES, Doehle B, McCarville JF, Pang PS, et al. Baseline and Post-baseline Resistance Analyses of Phase 2/3 Studies of Ledipasvir/Sofosbuvir +/- RBV. Hepatology 2014;60:1128a-1128a. [26] McPhee F, Hernandez D, Yu F, Ueland J, Monikowski A, Carifa A, et al. Resistance analysis of hepatitis C virus genotype 1 prior treatment null responders receiving daclatasvir and asunaprevir. Hepatology 2013;58:902-911. [27] Dvory-Sobol H, Wyles D, Ouyang W, Chodavarapu K, McNally J, Cheng W, et al. Long-term persistence of HCV NS5A variants after treatment with NS5A inhibitor ledipasvir. Journal of Hepatology 2015;62:S221-S221. [28] Krishnan P, Tripathi R, Schnell G, Reisch T, Beyer J, Irvin M, et al. Resistance Analysis of Baseline and Treatment-Emergent Variants in Hepatitis C Virus Genotype 1 in the AVIATOR Study with Paritaprevir-Ritonavir, Ombitasvir, and Dasabuvir. Antimicrob Agents Chemother 2015;59:5445-5454. [29] Di Maio VC, Cento V, Mirabelli C, Artese A, Costa G, Alcaro S, et al. Hepatitis C virus genetic variability and the presence of NS5B resistance-associated mutations as natural polymorphisms in selected genotypes could affect the response to NS5B inhibitors. Antimicrob Agents Chemother 2014;58:2781-2797. [30] Hedskog C, Dvory-Sobol H, Gontcharova V, Martin R, Ouyang W, Han B, et al. Evolution of the HCV viral population from a patient with S282T detected at relapse after sofosbuvir monotherapy. J Viral Hepat 2015. [31] Svarovskaia ES, Dvory-Sobol H, Parkin N, Hebner C, Gontcharova V, Martin R, et al. Infrequent Development of Resistance in Genotype 1-6 Hepatitis C Virus-Infected Subjects Treated With Sofosbuvir in Phase 2 and 3 Clinical Trials. Clin Infect Dis 2014;59:1666-1674. [32] Lawitz E, Flamm S, Yang J, Pang PS, Zhu Y, Svarosvskaia ES, et al. Retreatment of patients who failed 8 or 12 weeks of ledipasvir/sofosbuvir-based regimens with ledipasvir/sofosbuvir for 24 weeks. Journal of Hepatology 2015;62:S192-S192. [33] Donaldson EF, Harrington PR, O'Rear JJ, Naeger LK. Clinical evidence and bioinformatics characterization of potential hepatitis C virus resistance pathways for sofosbuvir. Hepatology 2015;61:56-65. [34] Svarovskaia ES, Dvory-Sobol H, Doehle B, Gane EJ, Jacobson IM, Nelson DR, et al. L159F and V321A sofosbuvir treatment-emergent HCV NS5B substitutions. Hepatology 2014;60:218a-218a. 25

[35] Barnard RJ, Howe JA, Ogert RA, Zeuzem S, Poordad F, Gordon SC, et al. Analysis of boceprevir resistance associated amino acid variants (RAVs) in two phase 3 boceprevir clinical studies. Virology 2013;444:329-336. [36] Krishnan P, Tripathi R, Schnell G, Reisch T, Beyer J, Irvin M, et al. Pooled analysis of resistance in patients treated with ombitasvir/ABT-450/r and dasabuvir with or without ribavirin in Phase 2 and Phase 3 clinical trials. Hepatology 2014;60:1134a-1135a. [37] Tang MW, Shafer RW. HIV-1 antiretroviral resistance: scientific principles and clinical applications. Drugs 2012;72:e1-25. [38] Herrmann E, Sarrazin C. Hepatitis C-virus --virus kinetics and resistance mechanisms. ZGastroenterol 2004;42:387-396. [39] Vermehren J, Susser S, Lange CM, Forestier N, Karey U, Hughes E, et al. Mutations selected in the hepatitis C virus NS3 protease domain during sequential treatment with boceprevir with and without pegylated interferon alfa-2b. J Viral Hepat 2011:epub. [40] Lenz O, de Bruijne J, Vijgen L, Verbinnen T, Weegink C, Van Marck H, et al. Efficacy of re-treatment with TMC435 as combination therapy in hepatitis C virus-infected patients following TMC435 monotherapy. Gastroenterology 2012;143:1176-1178 e1171- 1176. [41] Susser S, Flinders M, Reesink HW, Zeuzem S, Lawyer G, Ghys A, et al. Evolution of Hepatitis C Virus Quasispecies during Repeated Treatment with the NS3/4A Protease Inhibitor Telaprevir. Antimicrob Agents Chemother 2015;59:2746-2755. [42] Franco S, Tural C, Nevot M, Molto J, Rockstroh JK, Clotet B, et al. Detection of a sexually transmitted hepatitis C virus protease inhibitor-resistance variant in a human immunodeficiency virus-infected homosexual man. Gastroenterology 2014;147:599-601 e591. [43] Qi X, Bae A, Liu S, Yang H, Sun SC, Harris J, et al. Development of a replicon-based phenotypic assay for assessing the drug susceptibilities of HCV NS3 protease genes from clinical isolates. Antiviral Res 2009;81:166-173. [44] Rupp D, Dietz J, Sikorski AM, Sierra S, Brown R, Pietschmann T, et al. A phenotypic NS3-protease inhibitor resistance assay to characterize resistance-associated mutations in patients. J Viral Hepatitis 2015;22:93-93. [45] Jacobson IM, Dore GJ, Foster GR, Fried MW, Radu M, Rafalsky VV, et al. Simeprevir with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients with chronic hepatitis C virus genotype 1 infection (QUEST-1): a phase 3, randomised, double- blind, placebo-controlled trial. Lancet 2014;384:403-413. [46] Manns M, Marcellin P, Poordad F, de Araujo ES, Buti M, Horsmans Y, et al. Simeprevir with pegylated interferon alfa 2a or 2b plus ribavirin in treatment-naive patients with chronic hepatitis C virus genotype 1 infection (QUEST-2): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet 2014;384:414-426. [47] Lawitz E, Sulkowski MS, Ghalib R, Rodriguez-Torres M, Younossi ZM, Corregidor A, et al. Simeprevir plus sofosbuvir, with or without ribavirin, to treat chronic infection with hepatitis C virus genotype 1 in non-responders to pegylated interferon and ribavirin and treatment-naive patients: the COSMOS randomised study. Lancet 2014. [48] Pawlotsky JM. New hepatitis C virus (HCV) drugs and the hope for a cure: concepts in anti-HCV . Semin Liver Dis 2014;34:22-29. [49] Sarrazin C, Hezode C, Zeuzem S, Pawlotsky JM. Antiviral strategies in hepatitis C virus infection. JHepatol 2012;56 Suppl 1:S88-100. [50] Sarrazin C, Kieffer TL, Bartels D, Hanzelka B, Muh U, Welker M, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology 2007;132:1767-1777. [51] Kieffer TL, Sarrazin C, Miller JS, Welker MW, Forestier N, Reesink HW, et al. Telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients. Hepatology 2007;46:631-639. 26

[52] Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. NEnglJ Med 2011;364:1207-1217. [53] Poordad F, McCone J, Jr., Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. NEnglJ Med 2011;364:1195- 1206. [54] Fridell RA, Qiu D, Wang C, Valera L, Gao M. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob Agents Chemother 2010;54:3641-3650. [55] Gotte M. The distinct contributions of fitness and genetic barrier to the development of resistance. Curr Opin Virol 2012;2:644-650. [56] Powdrill MH, Tchesnokov EP, Kozak RA, Russell RS, Martin R, Svarovskaia ES, et al. Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance. Proc Natl Acad Sci U S A 2011;108:20509-20513. [57] Sarrazin C, Rouzier R, Wagner F, Forestier N, Larrey D, Gupta SK, et al. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology 2007;132:1270-1278. [58] Moreno C, Berg T, Tanwandee T, Thongsawat S, Van Vlierberghe H, Zeuzem S, et al. Antiviral activity of TMC435 monotherapy in patients infected with HCV genotypes 2-6: TMC435-C202, a phase IIa, open-label study. J Hepatol 2012;56:1247-1253. [59] Petry AS, Fraser IP, O'Mara E, Van Dyck K, Nachbar RB, De Lepeleire IM, et al. Safety and antiviral activity of MK-5172, a next generation HCV NS3/4A protease inhibitor with a broad HCV genotypic activity spectrum and potent activity against known resistance mutants, in genotype 1 and 3 HCV-infected patients. Hepatology 2011;54:531a-531a. [60] Summa V, Ludmerer SW, McCauley JA, Fandozzi C, Burlein C, Claudio G, et al. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrob Agents Chemother 2012;56:4161-4167. [61] Susser S, Welsch C, Wang Y, Zettler M, Domingues FS, Karey U, et al. Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients. Hepatology 2009;50:1709-1718. [62] Reesink HW, Fanning GC, Farha KA, Weegink C, Van VA, Van 't KG, et al. Rapid HCV-RNA decline with once daily TMC435: a phase I study in healthy volunteers and hepatitis C patients. Gastroenterology 2010;138:913-921. [63] Pilot-Matias T, Tripathi R, Cohen D, Gaultier I, Dekhtyar T, Lu L, et al. In vitro and in vivo antiviral activity and resistance profile of the hepatitis C virus NS3/4A protease inhibitor ABT-450. Antimicrob Agents Chemother 2015;59:988-997. [64] Pasquinelli C, McPhee F, Eley T, Villegas C, Sandy K, Sheridan P, et al. Single- and multiple-ascending-dose studies of the NS3 protease inhibitor asunaprevir in subjects with or without chronic hepatitis C. Antimicrob Agents Chemother 2012;56:1838-1844. [65] McPhee F, Friborg J, Levine S, Chen C, Falk P, Yu F, et al. Resistance analysis of the hepatitis C virus NS3 protease inhibitor asunaprevir. Antimicrob Agents Chemother 2012;56:3670-3681. [66] Reesink HW, Zeuzem S, Weegink CJ, Forestier N, van Vliet A, van de Wetering de R, et al. Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-controlled, randomized study. Gastroenterology 2006;131:997-1002. [67] Howe AY, Black S, Curry S, Ludmerer SW, Liu R, Barnard RJ, et al. Virologic resistance analysis from a phase 2 study of MK-5172 combined with pegylated interferon/ribavirin in treatment-naive patients with hepatitis C virus genotype 1 infection. Clin Infect Dis 2014;59:1657-1665. [68] 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

27 membranous replication factories, independent of RNA replication. Gastroenterology 2014;147:1094-1105 e1025. [69] Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010;465:96-100. [70] Cheng G, Peng B, Corsa A, Yu M, Nash M, Lee YJ, et al. Antiviral Activity and Resistance Profile of the Novel Hcv Ns5a Inhibitor Gs-5885. Journal of Hepatology 2012;56:S464-S464. [71] Krishnan P, Beyer J, Mistry N, Koev G, Reisch T, DeGoey D, et al. In vitro and in vivo antiviral activity and resistance profile of ombitasvir, an inhibitor of hepatitis C virus NS5A. Antimicrob Agents Chemother 2015;59:979-987. [72] Nettles RE, Gao M, Bifano M, Chung E, Persson A, Marbury TC, et al. Multiple ascending dose study of BMS-790052, a nonstructural protein 5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1. Hepatology 2011;54:1956- 1965. [73] Fridell RA, Wang C, Sun JH, O'Boyle DR, 2nd, Nower P, Valera L, et al. Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in humans: in vitro and in vivo correlations. Hepatology 2011;54:1924-1935. [74] Lawitz EJ, Gruener D, Hill JM, Marbury T, Moorehead L, Mathias A, et al. A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C. Journal of Hepatology 2012;57:24-31. [75] Yeh WW, Lipardi C, Jumes P, De Lepeleire IM, Van den Bulk N, Caro L, et al. MK- 8742, a HCV NS5A inhibitor with a broad spectrum of HCV genotypic activity, demonstrates potent antiviral activity in genotype-1 and -3 HCV-infected patients Hepatology 2013;58:438a-439a. [76] Lahser F, Liu R, Bystol K, Xia E, Raubertas R, Asante-Appiah E, et al. A combination containing MK-5172 (HCV NS3 protease inhibitor) and MK-8742 (HCV NS5A inhibitor) demonstrates high barrier to resistance in HCV replicons Hepatology 2012;56:236a-236a. [77] Gaultier I, Cohen DE, Dumas LM, Larsen L, Podsadecki T, Bernstein B. 8-week efficacy and safety of ABT-333 or ABT-972 with standard-of-care, follwing 3-day monotherapy in genotype 1 HCV-infected treatment-naive subjects. Hepatol Int 2011;5. [78] 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. Antimicrob Agents Chemother 2015;59:1505-1511. [79] Gane EJ, Stedman CA, Hyland RH, Ding X, Svarovskaia E, Symonds WT, et al. Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. NEnglJMed 2013;368:34-44. [80] Lawitz EJ, Rodriguez-Torres M, Denning J, Mathias A, Mo H, Gao B, et al. All-oral therapy with nucleotide inhibitors sofosbuvir and GS-0938 for 14 days in treatment-naive genotype 1 hepatitis C (nuclear). JViral Hepat 2013;20:699-707. [81] Lam AM, Espiritu C, Bansal S, Micolochick Steuer HM, Niu C, Zennou V, et al. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother 2012;56:3359-3368. [82] Afdhal N, Zeuzem S, Kwo P, Chojkier M, Gitlin N, Puoti M, et al. Ledipasvir and sofosbuvir for untreated HCV genotype 1 infection. NEnglJMed 2014;370:1889-1898. [83] Afdhal N, Reddy KR, Nelson DR, Lawitz E, Gordon SC, Schiff E, et al. Ledipasvir and sofosbuvir for previously treated HCV genotype 1 infection. The New England journal of medicine 2014;370:1483-1493. [84] Kowdley KV, Gordon SC, Reddy KR, Rossaro L, Bernstein DE, Lawitz E, et al. Ledipasvir and sofosbuvir for 8 or 12 weeks for chronic HCV without cirrhosis. NEnglJMed 2014;370:1879-1888. 28

[85] Kuntzen T, Timm J, Berical A, Lennon N, Berlin AM, Young SK, et al. Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naive patients. Hepatology 2008;48:1769-1778. [86] Gaudieri S, Rauch A, Pfafferott K, Barnes E, Cheng W, McCaughan G, et al. Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy. Hepatology 2009;49:1069-1082. [87] Bartels DJ, Zhou Y, Zhang EZ, Marcial M, Byrn RA, Pfeiffer T, et al. Natural prevalence of hepatitis C virus variants with decreased sensitivity to NS3.4A protease inhibitors in treatment-naive subjects. JInfectDis 2008;198:800-807. [88] Dierynck I, Thys K, Ghys A, Sullivan JC, Kieffer TL, Aerssens J, et al. Deep- sequencing analysis of the gene encoding the hepatitis C virus nonstructural 3-4A protease confirms a low prevalence of telaprevir-resistant variants at baseline and the end of the REALIZE study. The Journal of infectious diseases 2014;210:1871-1880. [89] Howe JA, Long JM, Black S, Chase R, McMonagle P, Curry S, et al. Pooled Clinical Trial Analyses of Detectable Baseline HCV NS3/4A Resistance Associated Variants on the Efficacy of Boceprevir plus Pegylated Interferon/Ribavirin Therapy. Hepatology 2013;58:1097a-1097a. [90] De Meyer S, Dierynck I, Ghys A, Beumont M, Daems B, Van Baelen B, et al. Characterization of telaprevir treatment outcomes and resistance in patients with prior treatment failure: results from the REALIZE trial. Hepatology 2012;56:2106-2115. [91] Trimoulet P, Pinson P, Papuchon J, Foucher J, Vergniol J, Chermak F, et al. Dynamic and rapid changes in viral quasispecies by UDPS in chronic hepatitis C patients receiving telaprevir-based therapy. Antivir Ther 2013;18:723-727. [92] Dietz J, Rupp D, Perner D, Berkowski C, Räder M, Susser S, et al. Analysis of NS3 protease resistance-associated variants and phenotypes for the prediction of treatment response to HCV triple therapy. J Hepatol 2015;62:S643-S643. [93] Reddy KR, Zeuzem S, Zoulim F, Weiland O, Horban A, Stanciu C, et al. Simeprevir versus telaprevir with peginterferon and ribavirin in previous null or partial responders with chronic hepatitis C virus genotype 1 infection (ATTAIN): a randomised, double-blind, non- inferiority phase 3 trial. The Lancet infectious diseases 2015;15:27-35. [94] Hezode C, Hirschfield GM, Ghesquiere W, Sievert W, Rodriguez-Torres M, Shafran SD, et al. Daclatasvir plus peginterferon alfa and ribavirin for treatment-naive chronic hepatitis C genotype 1 or 4 infection: a randomised study. Gut 2014. [95] Dore GJ, Lawitz E, Hezode C, Shafran SD, Ramji A, Tatum HA, et al. Daclatasvir plus peginterferon and ribavirin is noninferior to peginterferon and ribavirin alone, and reduces the duration of treatment for HCV genotype 2 or 3 infection. Gastroenterology 2015;148:355-366 e351. [96] Lawitz E, Mangia A, Wyles D, Rodriguez-Torres M, Hassanein T, Gordon SC, et al. Sofosbuvir for previously untreated chronic hepatitis C infection. NEnglJMed 2013;368:1878-1887. [97] Dietz J, Susser S, Berkowski C, Perner D, Zeuzem S, Sarrazin C. Consideration of viral resistance for optimization of direct antiviral therapy of hepatitis C virus genotype 1- infected patients. PloS one 2015;accepted. [98] Lalezari JP, Nelson DR, Hyland RH, Lin M, Ross SJ, Symonds WT, et al. Once-daily sofosbuvir plus ribavirin given 12 or 24 weeks in treatment-naive patients with HCV infection: the Quantum study. JHepatol 2013;58:845A. [99] Zeuzem S, Dusheiko GM, Salupere R, Mangia A, Flisiak R, Hyland RH, et al. Sofosbuvir and ribavirin in HCV genotypes 2 and 3. NEnglJMed 2014;370:1993-2001. [100] Hedskog C, Doehle B, Chodavarapu K, Gontcharova V, Crespo Garcia J, De Knegt R, et al. Characterization of hepatitis C virus intergenotypic recombinant strains and associated virological response to sofosbuvir/ribavirin. Hepatology 2015;61:471-480.

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[101] Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib R, et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. NEnglJMed 2012;366:216-224. [102] Manns M, Pol S, Jacobson IM, Marcellin P, Gordon SC, Peng CY, et al. All-oral daclatasvir plus asunaprevir for hepatitis C virus genotype 1b: a multinational, phase 3, multicohort study. Lancet 2014. [103] Zeuzem S, Hezode C, Bronowicki JP, Loustad-Ratti V, Gea F, Buti M, et al. Daclatasvir in combination with simeprevir ± ribavirin for hepatitis C virus genotype 1 infection. Top Antivir Med 2014;22:103-103. [104] Zeuzem S, Ghalib R, Reddy KR, Pockros PJ, Ari ZB, 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 Ann Intern Med 2015;163:1-13. [105] Kwo P, E. Gane E, Peng C-Y, Pearlman B, Vireling J, Serfaty L, et al. Efficacy and safety of grazoprevir/elbasvir +/- RBV for 12 weeks in patients with HCV G1 or G4 infection who previously failed peginterferon/RBV: C-EDGE treatment-experienced trial. J Hepatol 2015;62:S674-S674. [106] Miura M, Maekawa S, Sato M, Komatsu N, Tatsumi A, Takano S, et al. Deep sequencing analysis of variants resistant to the non-structural 5A inhibitor daclatasvir in patients with genotype 1b hepatitis C virus infection. Hepatol Res 2014;44:E360-367. [107] Akamatsu S, Hayes CN, Ochi H, Uchida T, Kan H, Murakami E, et al. Association between variants in the interferon lambda 4 locus and substitutions in the hepatitis C virus non-structural protein 5A. J Hepatol 2015. [108] Kwo P, Gitlin N, Nahass R, Bernstein D, Rojter S, Schiff E, et al. A phase 3, randomised, open-label study to evaluate the efficacy and safety of 8 and 12 weeks of simeprevir (SMV) plus sofosbuvir (SOF) in treatment-naive and -experienced patients with chronic HCV genotyope 1 infection without cirrhosis: Optimist-1. J Hepatol 2015;62:S270- S270. [109] Lawitz E, Matusow G, DeJesus E, Yoshida E, Felizarta F, Ghalib R, et al. A phase-3 open-label, single-arm study to evaluate the efficacy and safety of 12 weeks of simeprevir (SMV) plus sofosbuvir (SOF) in treatment-naive or -experienced patients with chronic HCV genotype 1 infection and cirrhosis: Optimist-2. J Hepatol 2015;62:S264-S265. [110] Sulkowski MS, Gardiner DF, Rodriguez-Torres M, Reddy KR, Hassanein T, Jacobson I, et al. Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection. The New England journal of medicine 2014;370:211-221. [111] Lawitz E, Poordad FF, Pang PS, Hyland RH, Ding X, Mo H, et al. Sofosbuvir and ledipasvir fixed-dose combination with and without ribavirin in treatment-naive and previously treated patients with genotype 1 hepatitis C virus infection (LONESTAR): an open-label, randomised, phase 2 trial. Lancet 2014;383:515-523. [112] Sarrazin C, Dvory-Sobol H, Svarovskaia ES, Doehle B, Martin R, Zeuzem S, et al. The prevalence and the effect of HCV NS5A resistance associated variants in subjects with compensated cirrhosis treated with ledipasvir/sofosbuvvir +/− RBV. J Hepatol 2015;62:S620- S620. [113] Nelson DR, Cooper JN, Lalezari JP, Lawitz E, Pockros PJ, 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 3 Study. Hepatology 2015. [114] 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. JAMA 2015;313:1728-1735. [115] Muir AJ, Poordad F, Lalezari J, Everson G, Dore GJ, Herring R, et al. Daclatasvir in combination with asunaprevir and beclabuvir for hepatitis C virus genotype 1 infection with compensated cirrhosis. JAMA 2015;313:1736-1744. 30

[116] Osinusi A, Kohli A, Marti MM, Nelson A, Zhang X, Meissner EG, et al. Re-treatment of chronic hepatitis C virus genotype 1 infection after relapse: an open-label pilot study. Ann Intern Med 2014;161:634-638. [117] Esteban R, Nyberg L, Lalezari J, Ni L, Doehle B, Kanwar B, et al. Successful retreatment with sofosbuvir-containing regimens for HCV genotype 2 or 3 infected patients who failed prior sofosbuvir plus ribavirin therapy. . Journal of Hepatology 2014;60:S4-S5. [118] Lawitz E, Poordad F, Brainard D, Hyland RH, An D, Symonds WT, et al. Sofosbuvir in combination with PEG-IFN and ribavirin for 12 weeks provides high SVR rates in HCV infected genotype 2 or 3 treatment-experienced patients with and without compensated cirrhosis: results from the Lonestar-2 study. Hepatology 2013;58:1380a-1380a. [119] EASL. EASL Clinical Practice Guidelines: management of hepatitis C virus infection. J Hepatol 2015. [120] Bartels DJ, Sullivan JC, Zhang EZ, Tigges AM, Dorrian JL, De Meyer S, et al. Hepatitis C virus variants with decreased sensitivity to direct-acting antivirals (DAAs) were rarely observed in DAA-naive patients prior to treatment. J Virol 2013;87:1544-1553. [121] Howe JA, Long J, Black S, Chase R, McMonagle P, Curry S, et al. Clinical Implications of Detectable Baseline Hepatitis C Virus-Genotype 1 NS3/4A-Protease Variants on the Efficacy of Boceprevir Combined With Peginterferon/Ribavirin. Open Forum Infectious Diseases 2014;1:1-7. [122] Palanisamy N, Danielsson A, Kokkula C, Yin H, Bondeson K, Wesslen L, et al. Implications of baseline polymorphisms for potential resistance to NS3 protease inhibitors in Hepatitis C virus genotypes 1a, 2b and 3a. Antiviral Res 2013;99:12-17. [123] Kitrinos KM, Wyles D, Dvory-Sobol H, Worth A, Han B, Brainard D, et al. Evaluation of the Resistance Profile of Ledipasvir, a Nonstructural Protein 5A Inhibitor, in Genotype 1 Chronically Infected HCV Subjects Treated With Ledipasvir-Containing Regimens Without Sofosbuvir. Hepatology 2014;60:1143a-1144a. [124] Plaza Z, Soriano V, Vispo E, del Mar Gonzalez M, Barreiro P, Seclen E, et al. Prevalence of natural polymorphisms at the HCV NS5A gene associated with resistance to daclatasvir, an NS5A inhibitor. Antivir Ther 2012;17:921-926. [125] Nakamoto S, Kanda T, Wu S, Shirasawa H, Yokosuka O. Hepatitis C virus NS5A inhibitors and drug resistance mutations. World journal of gastroenterology : WJG 2014;20:2902-2912. [126] Wang C, Jia L, Huang H, Qiu D, Valera L, Huang X, et al. In vitro activity of BMS- 790052 on hepatitis C virus genotype 4 NS5A. Antimicrob Agents Chemother 2012;56:1588- 1590. [127] Hernandez D, Zhou N, Ueland J, Monikowski A, McPhee F. Natural prevalence of NS5A polymorphisms in subjects infected with hepatitis C virus genotype 3 and their effects on the antiviral activity of NS5A inhibitors. J Clin Virol 2013;57:13-18. [128] Charlton M, Gane E, Manns MP, Brown RS, Curry MP, Kwo PY, et al. Sofosbuvir and Ribavirin for Treatment of Compensated Recurrent Hepatitis C Virus Infection After Liver Transplantation. Gastroenterology 2015;148:108-117. [129] Tong X, Le Pogam S, Li L, Haines K, Piso K, Baronas V, et al. In vivo emergence of a novel mutant L159F/L320F in the NS5B polymerase confers low-level resistance to the HCV polymerase inhibitors and sofosbuvir. The Journal of infectious diseases 2014;209:668-675. [130] Krishnan P, Tripathi R, Irvin M, Beyer J, Reisch T, Schnell G, et al. Lack of impact of baseline resistance-associated variants (RAVs) on treatment outcome in the aviator study with ABT-450/r, ABT-333 and ABT-267, +/- ribavirin. Journal of Hepatology 2014;60:S498. [131] Jiang M, Mani N, Lin C, Ardzinski A, Nelson M, Reagan D, et al. In vitro phenotypic characterization of hepatitis C virus NS3 protease variants observed in clinical studies of telaprevir. Antimicrob Agents Chemother 2013;57:6236-6245.

31

[132] Lenz O, Verbinnen T, Lin TI, Vijgen L, Cummings MD, Lindberg J, et al. In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435. Antimicrob Agents Chemother 2010;54:1878-1887. [133] Howe JA, Long J, Black S, Chase R, McMonagle P, Curry S, et al. Pooled Clinical Trial Analyses of the Effects of Detectable Baseline HCV NS3/4a Resistance-Associated Variants on the Efficacy of Boceprevir + Pegylated Interferon/Ribavirin Therapy. Hepatology 2013;58:1097a-1097a. [134] Wang C, Jia L, O'Boyle DR, 2nd, Sun JH, Rigat K, Valera L, et al. Comparison of daclatasvir resistance barriers on NS5A from hepatitis C virus genotypes 1 to 6: implications for cross-genotype activity. Antimicrob Agents Chemother 2014;58:5155-5163. [135] Fridell RA, Qiu D, Valera L, Wang C, Rose RE, Gao M. Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS- 790052. J Virol 2011;85:7312-7320. [136] DeGoey DA, Randolph JT, Liu D, Pratt J, Hutchins C, Donner P, et al. Discovery of ABT-267, a pan-genotypic inhibitor of HCV NS5A. Journal of medicinal chemistry 2014;57:2047-2057. [137] Wong KA, Worth A, Martin R, Svarovskaia E, Brainard DM, Lawitz E, et al. Characterization of Hepatitis C virus resistance from a multiple-dose clinical trial of the novel NS5A inhibitor GS-5885. Antimicrob Agents Chemother 2013;57:6333-6340. [138] Pelosi LA, Voss S, Liu M, Gao M, Lemm JA. Effect on hepatitis C virus replication of combinations of direct-acting antivirals, including NS5A inhibitor daclatasvir. Antimicrob Agents Chemother 2012;56:5230-5239. [139] Middleton T, He Y, Beyer J, Menon R, Klein C, Cohen D, et al. Resistance Profile of Abt-333 and Relationship to Viral Load Decrease in Patients Treated in Combination with Peg-Interferon and Ribavirin for 28 Days. Journal of Hepatology 2010;52:S296-S297. [140] Sarrazin C, Zeuzem S. Resistance to Direct Antiviral Agents in Patients With Hepatitis C Virus Infection. Gastroenterology 2010;138:447-462. [141] Silva MO, Treitel M, Graham DJ, Curry S, Frontera MJ, McMonagle P, et al. Antiviral activity of boceprevir monotherapy in treatment-naive subjects with chronic hepatitis C genotype 2/3. J Hepatol 2013;59:31-37. [142] Benhamou Y, Moussalli J, Ratziu V, Lebray P, De Backer K, De Meyer S, et al. Telaprevir activity in treatment-naive patients infected hepatitis C virus genotype 4: a randomized trial. The Journal of infectious diseases 2013;208:1000-1007. [143] Foster GR, Hezode C, Bronowicki JP, Carosi G, Weiland O, Verlinden L, et al. Telaprevir Alone or With Peginterferon and Ribavirin Reduces HCV RNA in Patients With Chronic Genotype 2 But Not Genotype 3 Infections. Gastroenterology 2011. [144] Tong X, Bogen S, Chase R, Girijavallabhan V, Guo Z, Njoroge FG, et al. Characterization of resistance mutations against HCV ketoamide protease inhibitors. Antiviral Res 2008;77:177-185. [145] Victrelis. Prescribing information. MSD. 2011. [146] Li YP, Ramirez S, Humes D, Jensen SB, Gottwein JM, Bukh J. Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors. Gastroenterology 2014;146:812-821 e814. [147] Lin C, Lin K, Luong YP, Rao BG, Wei YY, Brennan DL, et al. In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms. J BiolChem 2004;279:17508-17514. [148] Incivo. Prescribing information. Janssen. 2011. [149] McPhee F, Sheaffer AK, Friborg J, Hernandez D, Falk P, Zhai G, et al. Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032). Antimicrob Agents Chemother 2012;56:5387-5396. [150] Fevery B, Verbinnen T, Peeters M, Zucchetto M, Witek J, Jessner W, et al. Virology analyses of HCV isolates from genotype 4 patients treated with simeprevir in combination 32 with peginterferon/ribavirin in the Phase III RESTORE study. Hepatology 2014;60:1060a- 1060a. [151] Verbinnen T, Fevery B, Vijgen L, Picchio G, De Meyer S, Lenz O. Phenotypic characterization of genotype 1 hepatitis C NS3 protease variants from clinical studies with simeprevir. Antivir Ther 2014;19:A108. [152] Harvoni. Prescribing information. Gilead. 2014. [153] Kieffer TL, De Meyer S, Bartels DJ, Sullivan JC, Zhang EZ, Tigges A, et al. Hepatitis C viral evolution in genotype 1 treatment-naive and treatment-experienced patients receiving telaprevir-based therapy in clinical trials. PLoS One 2012;7:e34372. [154] Zeuzem S, Ghalib R, Reddy KR, Pockros PJ, Ari ZB, Zhao Y, et al. The phase 3 C- EDGE treatment-naive (TN) study of a 12-weeek oral regimen of grazoprevir (GZR, MK- 5172)/elbasvir (EBR, MK-8742) in patients with chronic HCV genotype (GT) 1, 4 or 6 infection. J Hepatol 2015;62:S213-S213. [155] Wyles DL, Ruane PJ, Sulkowski MS, Dieterich D, Luetkemeyer A, Morgan TR, et al. Daclatasvir plus Sofosbuvir for HCV in Patients Coinfected with HIV-1. The New England journal of medicine 2015;Epub ahead of print. [156] Wyles D, Ruane P, Sulkowski M, Dieterich D, Luetkemeyer A, Morgan T, et al. Daclatasvir in Combination With Sofosbuvir for HIV/HCV Coinfection: ALLY-2 Study. Top Antivir Med 2015;23:62-62. [157] Poordad F, Schiff ER, Vierling JM, Landis C, Fontana RJ, Yang R, et al. Daclatasvir, sofosbuvir, and ribavirin combination for HCV patients with advanced cirrhosis or posttransplant recurrence: phase 3 Ally-1 study. J Hepatol 2015;62:S261-S261. [158] European Medicines Agency. Assesment report Viekirax; 2015.

Acknowledgements The author thanks Dr. Julia Dietz for search of literature and collection of data.

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Table 1. Natural prevalence of NS3, NS5A, NS5B nucleotide and non-nucleoside inhibitor RAVs detected by population sequencing (RAVs with a > 2 fold change in comparison to wildtype in a phenotypic assay)**

Variant HCV Resistant to Natural Prevalence in HCV genotype References gene 1a 1b 2 3 4 V36A/C/G NS3 BOC/TVR/PTV n.o. n.o. n.d. n.d. n.d. [85, 120] V36M NS3 BOC/TVR 0.2% - 0.6% 0.1% n.d. n.d. n.d. [85, 120, 121] F43I/L/S/V NS3 SMV/ASV/PTV n.o. n.o. n.d. n.d. n.d. [17, 86, 97, 120] T54A NS3 BOC/TVR 0.1% -1.9% n.o n.d. n.d. n.d. [120-122] T54S NS3 BOC/TVR 0.4% - 3.1% 1.2% - 2.0% n.d. n.d. n.d. [85, 86, 120, 121] V55A NS3 BOC/TVR 2.8% 0.4% n.d. n.d. n.d. [120] Y56H NS3 PTV n.o. n.o. n.d. n.d. n.d. [97, 120] Q80K NS3 SMV/ASV/PTV 4.8% - 75.0% 0.5% - 1.2% n.d. n.d. n.d. [15] Q80R NS3 SMV/ASV 0.8% 0.6% - 0.7% n.d. n.d. n.d. [21, 97, 120] S122R NS3 SMV/ASV n.o. n.o. n.d. n.d. n.d. [97, 122] R155K NS3 BOC/TVR/SMV/ 0.2% - 0.9% n.o. n.d. n.d. n.d. [85, 97, 120, ASV/PTV 121] R155I/G/K/L/M/ NS3 BOC/TVR/SMV/ n.o. n.o. n.d. n.d. n.d. [85, 120] T/Q/S ASV/PTV A156F/N/S/T/V NS3 BOC/TVR/SMV/ n.o n.o. n.d. n.d. n.d. [17, 21, 85, 86, ASV/PTV 97, 120] V158I NS3 BOC n.o 0.1% n.d. n.d. n.d. [120, 121] D168E NS3 SMV/ASV/PTV 0.2% - 0.3% 0.1% - 1.4% n.d. n.d. n.d. [17, 85, 86, 97, 120] D168G/H/V/TY NS3 SMV/ASV/PTV n.o n.o. n.d. n.d. n.d. [17, 97, 120] V170A NS3 BOC/TVR n. a. 0.1% n.d. n.d. n.d. [85, 120] M175L NS3 BOC n. a. 0.8% - 1.1% n.d. n.d. n.d. [120, 121] K24G/N NS5A LDV n.o. n.a. n.d. n.d. n.d. [25] K24R NS5A LDV <1% - 1.5% n.a. n.d. n.d. n.d. [25, 123] M28A NS5A DCV/LDV 0.5% n.a. n.d. n.d. n.d. [25] M28G NS5A LDV n.o n.a. n.d. n.d. n.d. [25, 97] M28T NS5A DCV/LDV/OMV 0.4% - 1.8% n.a. n.d. n.o. 82.0% (M28L) [25, 120, 124] M28V NS5A OMV 3.5% n.a. n.d. n.d. n.d. [97] F28S NS5A DCV n.a. n.a. n.o. n.d. n.d. [125] L28F/T NS5A DCV/OMV n.a. n.o. 8.0% (L28F) n.d. n.d. [97, 120, 125] Q30H/R/E/L/T NS5A DCV/LDV/OMV 0.3% - 1.3% n.a. n.d. 90.4% - 100% 50.0% - 100% [25, 36, 97, (Q30A) (Q30R) 120, 124] R30H NS5A DCV n.a. 0.4% n.d. n.d. n.o. [120, 126] R30S NS5A DCV n.a. n.a. n.d. n.d. 10% [126] R30G/H NS5A DCV n.a. n.a. n.d. n.d. n.o. [126] A30K NS5A DCV n.a. n.a. n.d. 2.3 - 6.3% n.d. [127] L31M NS5A DCV/LDV 0.9% - 1.8% 2.1% - 6.3% 74.0 – 85.0% 1% 92.5% - 100% [97, 120, 124- 127] L31I/F/V NS5A DCV/LDV/OMV n.o. 0.7% - 1% n.d. n.o. n.d. [25, 97, 120, 124, 127] P32L NS5A DCV/LDV n.o <0.5% n.d. n.o. n.o. [25, 97, 120, 124] S38F NS5A LDV n.o. n.o. n.d. n.d. n.d. [25, 97] H58D NS5A DCV/LDV/OMV <1% n.a. n.d. n.d. n.d. [25, 97, 120, 123] P58D NS5A LDV n.a. n.o. n.d. n.d. n.d. [25, 97] A92K NS5A LDV n.o. n.o. n.d. n.d. n.d. [25, 97] A92T NS5A LDV n.o 2.8% n.d. n.d. n.d. [25, 97] C92R NS5A DCV n.a. n.a. n.o. n.d. n.d. [125] Y93C/F/N NS5A DCV/LDV/OMV n.o. - 0.6% n.o. - 0.7% n.d. n.o. n.d. [25, 97, 120, 124] Y93H NS5A DCV/LDV/OMV <1.5% 3.8%-14.1% n.o. 1.3 - 8.3% 5% - 13% [25, 36, 97, 120, 124-127] Y93S NS5A LDV <0.5% <0.5% n.d. n.d. n.d. [25] S282T NS5B SOF n.o. n.o. n.o. n.o. n.d. [31, 85, 99, 34

120, 128, 129] M289I/L NS5B SOF n.o. 1.8% 3.5% n.d. n.d. [31] C316Y NS5B DSV 0.2% - 1.2% n.o. n.d. n.d. n.d. [36, 97, 120] C316N NS5B DSV/SOF n.o. 10.9% - 35.6% n.d. n.d. 7.9% [29, 36, 97, 120] C316H NS5B DSV/SOF n.o. 1.9% - 2.1% n.d. n.d. n.d. [36, 97] L320F NS5B SOF n.o. n.o. n.d. n.d. n.d. [31] S368T NS5B DSV n.o. n.o. n.d. n.d. n.d. [97] N411S NS5B DSV n.o. n.o. n.d. n.d. n.d. [85, 97] M414T NS5B DSV 0.5% 0.4% n.d. n.d. n.d. [120, 130] M414I NS5B DSV n.o n.o n.d. n.d. n.d. [85, 120] E446K/Q NS5B DSV n.o. n.o. n.d. n.d. n.d. [36] Y448C NS5B DSV n.o. n.o. n.d. n.d. n.d. [85, 97, 120] Y448H NS5B DSV 0.2% 1.3% n.d. n.d. n.d. [120] A553I/T/V NS5B DSV 6% n.o. n.d. n.d. n.d. [36] G554S/D* NS5B DSV n.o. n.o. n.d. n.d. n.d. [85, 97, 120] S556G NS5B DSV 0.6% - 3.1% 7.0% - 16% 100% 100% 97% [29, 97, 130]

S556N/R NS5B DSV 0.6% - 1.2% n.o. n.d. n.d. n.d. [97, 120, 130] G558R* NS5B DSV n.o. n.o. n.d. n.d. n.d. [36] D559G NS5B DSV n.o. n.o. n.d. n.d. n.d. [85, 97, 120] Y561H NS5B DSV n.o. n.o. n.d. n.d. n.d. [36]

GT, genotype; ASV, asunaprevir; BOC, boceprevir; PTV, paritaprevir; SMV, simeprevir; TVR, telaprevir; DCV, daclatasvir; LDV, ledipasvir; OMV, ombitasvir; DSV, dasabuvir; SOF, sofosbuvir. n.a.: not applicable because of different natural amino acid sequence in the respective HCV geno-/subtype (NS3: V170 and M175 are the dominant amino acids in GT1b. NS5A: K24, M28, Q30, H58 are the dominant amino acids in GT1a; F28 is the dominant amino acid in subtype 2a and L28 in subtype 2b; A30 is the dominant amino acid in GT3) n.o.: not observed n.d.: no data available *variants reported to be selected in patients with PTV/OMV/DSV treatment failure, EC50 n.d. ** fold-change resistance values provide an indication but depend on the assay and HCV backbone isolate used and are not directly comparable between different studies

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Table 2. Level of resistance of NS3, NS5A and NS5B nucleotide/non-nucleoside inhibitor RAVs (variants with a >2 fold-change resistance to PIs (NS3 protease inhibitors), NS5A- and NS5B inhibitors)***

Variant HCV DAA EC50 [fold-change compared to WT replicon] References gene GT1a GT1b GT2 GT3a GT4a GT5a GT6a V36A/C/G/M NS3 BOC/TVR//PTV 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [63, 121, 131, 132] F43S NS3 ASV/SMV n.d. 2-20 n.d. n.d. n.d. n.d. n.d. [65, 132] F43I/V NS3 SMV n.d. 20-100 n.d. n.d. n.d. n.d. n.d. [132] F43L NS3 PTV 2-20 n.d. n.d. n.d. n.d. n.d. n.d. [63] T54A/S NS3 BOC/TVR 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [121, 131] V55A NS3 BOC/TVR 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [121, 131] Y56H NS3 PTV 2-20 n.d. n.d. n.d. n.d. n.d. n.d. [36] Q80K NS3 ASV/PTV/SMV 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [21, 65, 130] Q80R NS3 ASV/SMV 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [21, 65] S122R NS3 ASV/SMV 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [16, 65] R155K* NS3 TVR/BOC/SMV/ASV/PTV 2-100 2-100 n.d. n.d. n.d. n.d. n.d. [21, 63, 65, 89, 131, 132] R155G/T NS3 BOC/TVR/PTV 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [63, 131] R155I/M/S/W NS3 TVR/PTV 2-20 2-100 n.d. n.d. n.d. n.d. n.d. [63, 131] A156F/G/S NS3 BOC/TVR/ASV n.d. 2-20 n.d. n.d. n.d. n.d. n.d. [65, 131, 132] A156T/V NS3 BOC/TVR/ASV/SMV/GZR 2-20 20- >100** n.d. n.d. n.d. n.d. n.d. [63, 65, 132] V158I NS3 BOC n.r. 2-20 n.d. n.d. n.d. n.d. n.d. [133] D168A NS3 ASV/PTV/SMV/GZR 20-100 20-100 n.d. n.d. n.d. n.d. n.d. [63, 65, 132] D168C/E NS3 ASV/PTV/SMV/GZR 2-100 2-100 n.d. n.d. n.d. n.d. n.d. [63, 65, 132] D168G/N NS3 ASV/PTV/SMV/GZR 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [63, 65, 132] D168H/T/K NS3 ASV/PTV/SMV/GZR 20-100 20- >100 n.d. n.d. n.d. n.d. n.d. [63, 65, 132] D168V/Y NS3 ASVPTV/SMV 20- >100 >100 n.d. n.d. n.d. n.d. n.d. [63, 65, 132] D168Y NS3 GZR n.d. 4 n.d. n.d. n.d. n.d. n.d. [60, 67] V170A NS3 BOC/TVR n.a. 2-20 n.d. n.d. n.d. n.d. n.d. [131, 132] M175L NS3 BOC n.a. 2-20 n.d. n.d. n.d. n.d. n.d. [133] K24G/N/R NS5A LDV 2-100 n.a. n.d. n.d. n.d. n.d. n.d. [25] Q24H NS5A DCV n.a. n.d. n.d. n.d. n.d. n.d. 20-100 [134] T24A NS5A OMV n.a. n.a. 2-100 n.d. n.d. n.d. n.d. [71] F28S# NS5A DCV/OMV n.a. n.a. >100 n.d. n.d. n.d. n.d. [71, 135] L28F# NS5A OMV n.a. n.d. >100 n.d. n.d. n.d. n.d. [71] L28I NS5A OMV n.a. n.d. n.d. n.d. n.d. 20-100 n.d. [71] L28T NS5A DCV/OMV n.a. 20- >100 n.d. n.d. n.d. n.d. n.d. [54, 136] L28V NS5A OMV n.a. n.d. n.d. n.d. 20-100 n.d. n.d. [71] M28A/G NS5A DCV/LDV >100 n.a. n.d. n.d. n.d. n.d. n.d. [25, 73] M28T NS5A DCV/LDV/OMV 20 - >100 n.a. n.d. >100 n.d. n.d. n.d. [25, 54, 71, 136, 137] M28V NS5A OMV 20-100 n.a. n.d. n.d. n.d. n.d. n.d. [136] A30K NS5A DCV n.a. n.a. n.d. 20-100 n.d. n.d. n.d. [127] L30H NS5A DCV n.a. n.a. n.d. n.d. >100 n.d. n.d. [126] R30H NS5A DCV n.a. 2-20 n.d. n.d. n.d. n.d. n.d. [138] Q30E NS5A DCV/LDV/OMV >100 n.a. n.d. n.d. n.d. n.d. n.d. [25, 36, 73] Q30H NS5A DCV/LDV/OMV 2- >100## n.a. n.d. n.d. n.d. n.d. n.d. [71, 73, 74] Q30R NS5A DCV/LDV/OMV >100 n.a. n.d. n.d. n.d. n.d. n.d. [25, 73, 136] Q30G/K NS5A LDV >100 n.a. n.d. n.d. n.d. n.d. n.d. [25] Q30L/T NS5A LDV 2-100 n.a. n.d. n.d. n.d. n.d. n.d. [25] L31I NS5A LDV 2-100 n.d. n.d. n.d. n.d. n.d. n.d. [25] L31F NS5A DCV/OMV n.d. 2-20 n.d. 20-100 n.d. >100 n.d. [54, 71, 134, 136] L31M NS5A DCV/LDV >100 2- >100 >100 >100 n.d. n.d. >100 [25, 73, 74, 127, 134, 135] L31V NS5A DCV/LDV/OMV >100 2-100 >100 >100 n.d. >100 20-100 [25, 71, 73, 36

127, 130, 134, 136] P32L/S NS5A DCV/LDV >100 2-100 n.d. n.d. n.d. n.d. >100 [25, 54, 134] S38F NS5A LDV 2-100 n.d. n.d. n.d. n.d. n.d. n.d. [25] H58D NS5A DCV/LDV/OMV >100 n.a. n.d. n.d. n.d. n.d. n.d. [25, 73, 136] P58D NS5A LDV n.a. >100 n.d. n.d. n.d. n.d. n.d. [25] T58A/N/S NS5A DCV/OMV n.a. n.a. n.d. n.d. n.d. n.d. 20->100 [71, 134] A92K NS5A LDV n.d. >100 n.d. n.d. n.d. n.d. n.d. [25] A92T NS5A LDV 2-100 n.d. n.d. n.d. n.d. n.d. n.d. [25] C92R NS5A DCV n.a. n.a. 20-100 n.d. n.d. n.d. n.d. [135] Y93C NS5A DCV/LDV/OMV >100 n.d. n.d. n.d. n.d. n.d. n.d. [73, 74, 136] Y93F NS5A LDV 2-100 n.d. n.d. n.d. n.d. n.d. n.d. [25] Y93H NS5A DCV/LDV/OMV >100 20->100 >100 >100 >100 n.d. n.d. [71, 73, 74, 126, 127, 135, 136] Y93N NS5A DCV/LDV/OMV >100 n.d. n.d. n.d. n.d. n.d. n.d. [25, 73, 136] Y93R NS5A DCV n.d. n.d. n.d. n.d. >100 n.d. n.d. [126] Y93S NS5A LDV 2-100 n.d. n.d. n.d. n.d. n.d. n.d. [25] S282T NS5B SOF 2-20 2-20 2-20 n.d. n.d. n.d. n.d. [81, 129] M289L NS5B SOF n.a. n.a. 2-20 n.d. n.d. n.d. n.d. [81] C316H/Y+ NS5B DSV >100 >100 n.d. n.d. n.d. n.d. n.d. [78, 130] C316N+ NS5B DSV n.d. 2-20 n.d. n.d. n.d. n.d. n.d. [78] L320F NS5B SOF 2-20 n.r. n.d. n.d. n.d. n.d. n.d. [129] S368T NS5B DSV n.d. >100 n.d. n.d. n.d. n.d. n.d. [78] N411S NS5B DSV n.d. 2-20 n.d. n.d. n.d. n.d. n.d. [78] M414T NS5B DSV 20-100 20-100 n.d. n.d. n.d. n.d. n.d. [78] M414I NS5B DSV n.d. 2-20 n.d. n.d. n.d. n.d. n.d. [36] E446K/Q NS5B DSV 2-100 n.d. n.d. n.d. n.d. n.d. n.d. [36] Y448C NS5B DSV >100 >100 n.d. n.d. n.d. n.d. n.d. [78] Y448H NS5B DSV >100 20-100 n.d. n.d. n.d. n.d. n.d. [78] A553T NS5B DSV >100 n.d. n.d. n.d. n.d. n.d. n.d. [36] A553V NS5B DSV n.d. >100 n.d. n.d. n.d. n.d. n.d. [78] G554S++ NS5B DSV n.d. n.d. n.d. n.d. n.d. n.d. n.d. [36] S556G NS5B DSV 2-20 2-20 n.d. n.d. n.d. n.d. n.d. [78] S556N NS5B DSV 20-100 n.d. n.d. n.d. n.d. n.d. n.d. [78] S556R NS5B DSV >100 n.d. n.d. n.d. n.d. n.d. n.d. [130] G558R++ NS5B DSV n.d. n.d. n.d. n.d. n.d. n.d. n.d. [36] D559G NS5B DSV >100 >100 n.d. n.d. n.d. n.d. n.d. [139] D559 I/N/V++ NS5B DSV n.d. n.d. n.d. n.d. n.d. n.d. n.d. [36] Y561H NS5B DSV 20-100 n.d. n.d. n.d. n.d. n.d. n.d. [36]

GT, genotype; ASV, asunaprevir; BOC, boceprevir; GZR, grazoprevir; PTV, paritaprevir; SMV, simeprevir; TVR, telaprevir; DCV, daclatasvir; LDV, ledipasvir; OMV, ombitasvir; DSV, dasabuvir; SOF, sofosbuvir. n.a.: not applicable because of different natural amino acid sequence in the respective HCV geno-/subtype n.r.: not resistant n.d.: no data available

*R155K is frequently observed in combination with V36 variants leading to improved viral fitness **A156T confers only a 2-20 fold resistance towards asunaprevir in GT1b #F28 is the dominant amino acid in subtype 2a and L28 in subtype 2b ##Q30H conferred a 2-20 fold resistance towards OMV and a >100 fold resistance towards LDV and DCV. +association with failure to SOF treatment unclear ++variants reported to be selected in patients with PTV/OMV/DSV treatment failure, EC50 n.d *** fold-change resistance values provide an indication but depend on the assay and HCV backbone isolate used and are not directly comparable between different studies

37

Table 3. Clinical antiviral activities of approved DAAs (mean or median maximum HCV viral load decline after 3-14 days monotherapy [log10 IU/mL], no head to head comparisons).

Substance Target Dosing GT1 GT2 GT3 GT4 GT5 GT6 References BOC NS3 400 mg TID* 2.1 1.4 1.7 - - - [61, 140, 141] TVR NS3 750 mg TID 4.4 3.7 0.5 0.8 - - [66, 142, 143] SMV NS3 200 mg QD** 3.9 2.7 0.04 3.5 2.2 4.4 [58, 62] PTV/r NS3 100/100 mg QD*** 4.0 - - - - - [63] ASV NS3 100 mg BID 3.1 - - - - - [64] GZR NS3 100mg QD 4.6 - 2.5 - - - [59] DCV NS5A 60 mg QD 3.8 - - - - - [72] LDV NS5A 90 mg QD 3.1 - - - - - [74] OMV NS5A 25 mg QD 3.0 - - - - - [71] EBR NS5A 50mg QD 4.6 - 3.1 - - - [75] DSV NS5B 400 mg BID**** 1.08 - - - - - [77] SOF NS5B 400 mg QD 4.7 4.8# 4.8# - - - [79, 80]

GT, genotype; ASV, asunaprevir; BOC, boceprevir; GZR, grazoprevir; PTV, paritaprevir; SMV, simeprevir; TVR, telaprevir; DCV, daclatasvir; EBR, elbasvir; LDV, ledipasvir; OMV, ombitasvir; DSV, dasabuvir; SOF, sofosbuvir.

* approved standard dose: 800 mg TID ** approved standard dose: 150 mg QD *** approved standard dose: 150mg/100 mg QD **** approved standard dose: 250mg BID # data for GT2 and 3 were presented together

38

Table 4. In vitro antiviral activities of DAAs against wildtype isolates from different HCV genotypes (no head to head comparisons)

EC50 against different genotypes [nM] Substa Gene GT1a GT1b GT2a GT3a GT4a GT5a GT6a References nce BOC NS3 368 356 385 (2a) 803 n.d. n.d. n.d. [141] 750 (2b) BOC NS3 200 [144] BOC NS3 900+ 200 - 600 [145] BOC NS3 65 - 425 n.d. 399 - 503 1215 - 1417 1387 403 - 875 141 - 435 [146] TVR NS3 402 [147] TVR NS3 280 354 [148] TVR NS3 413 653 649 (2a) 3312 (3a) [141] 1119 (2b) TVR NS3 149 - 456 n.d. 493 - 644 2000 - 2745 1949 539 - 695 124 - 412 [146] TVR NS3 995 266 - 427 108 – 229 (2a) 2731 1137 38* 86* [149] 1236 (2b) SMV NS3 2.8 (without 1.8 n.d. (2a) 10250 1.4 (4a) n.d. n.d. [16, 150, 151] Q80K) 187 (2b/i/k) 0.8 (4d) 40 (with Q80K) 2.0 (4, 4c/f/h/k/o/q/r) SMV NS3 10 – 45 n.d. 77 - 91 2366 - 2476 5 109 - 127 56 - 78 [146] PTV/r NS3 1.0 0.2 5.3 19 0.09 n.d. 0.69 [63] ASV NS3 4.0 1.2 -1.3 67 – 230 (2a) 1162 1.8 1.7* 0.9* [149] 480 (2b) ASV NS3 31 - 64 n.d. 67 - 159 2143 - 3712 37 79 - 82 55 - 96 [146] GZR NS3 2 0.5 8 0.9* n.d. n.d. n.d. [60] DCV NS5A 0.05 0.009 0.07 – 0.10 0.146 0.012 0.033 n.d. [69] DCV NS5A 0.03 – 0.06 n.d. 0.09 – 0.1 0.54 – 0.91 0.02 0.03 – 0.04 0.03 – 0.07 [146] LDV NS5A 0.034 0.004 21 – 210 (2a) 35 0.11 (4a) 0.15 0.12 (6a) [70, 152] 16 – 530 (2b) 0.60 (4d) 264 (6e) OMV NS5A 0.014 0.005 0.0008 (2a) 0.019 0.0017 0.0032 0.366 [71] DSV NS5B 7.7 1.8 n.d. n.d. n.d. n.d. n.d. [78] SOF NS5B 44 48 37 – 47 (2a) 16 40 (4a) 15 14 [81, 152] 20 (2b)

GT, genotype; ASV, asunaprevir; BOC, boceprevir; GZR, grazoprevir; PTV, paritaprevir; SMV, simeprevir; TVR, telaprevir; DCV, daclatasvir; LDV, ledipasvir; OMV, ombitasvir; DSV, dasabuvir; SOF, sofosbuvir.

* enzyme IC50

Figure Legends

Figure 1. Susceptibility to antiviral therapy. Suppression of viral load required for HCV eradication according to treatment-associated, viral- and host-related features. According to this hypothesis depending on the used antiviral agents, duration of treatment and a number of viral- and host-related factors different levels of suppression of HCV RNA are required for final eradication of the virus by the immune system.

ISG, interferon-stimulated genes; IP-10, interferon-gamma-inducible protein 10

Figure 2. SVR rates for conventional triple therapy of HCV genotype 1 infected patients according to the presence of baseline RAVs. PEG-IFN (PEG)/ribavirin (R) in combination with different DAAs for 24 to 48 weeks in treatment naive (TN) and/or experienced patients (TE). Boceprevir (BOC) 800 TID, phase 2/3 studies (n=2241). Baseline RAVs at positions 36, 54, 55, 107, 155, 158, 170, 175 of NS3 protease were taken into account [133]. Telaprevir (TVR) 750mg TID, phase 3 studies (n=1414). Baseline RAVs at positions 36, 54, 155, of NS3 protease were taken into account [153]. Simeprevir (SMV) 150mg QD, phase 3 study in treatment naive patients (n=521). Data for RAVs in HCV genotype 1b patients are not available. For HCV genotype 1a analysis was performed according to the NS3 Q80K variant [21]. Daclatasvir (DCV) 20 or 60mg QD, phase 2 study (n=293). Baseline RAVs at positions 31 and 93 of the NS5A protein were taken into account [94]. No head-to-head comparisons. TN, treatment naive; TE, treatment experienced.

Figure 3. SVR rates to NS3 protease inhibitor plus NS5A inhibitor combination regimens in HCV genotype 1 infected patients according to the presence of baseline RAVs. Asunaprevir (ASV) 100mg BID plus Daclatasvir 60mg QD for 24 weeks, phase 3 study (n=643). Baseline RAVs at positions 168 of NS3 protease and 31, 93 of the NS5A protein were taken into account. Due to low barrier to resistance no HCV genotype 1a patients were enrolled [102]. Simeprevir (SMV) 150mg QD plus Daclatasvir (DCV) 30mg QD for 12 to 24 weeks phase 2 study [103]. Grazoprevir (GZR) 100mg QD plus Elbasvir (EBR) 50mg QD for 12 or 16 weeks, phase 3 studies (n=841. The following baseline RAVs for HCV subtype 1a isolates causing >5-fold resistance were taken into account M/L28T/A, Q/R30E/H/RG/K/L/D, L31M/V/F, H58D, Y93C/H. No correlation of baseline RAVs with SVR in HCV subtype 1b infected patients [105, 154]. No head-to-head comparisons. TN, treatment naive; TE, treatment experienced. Figure 4. SVR rates for NS3 protease inhibitor plus nucleos(t)ide NS5B inhibitor combination regimens in HCV genotype 1 infected patients according to the presence of baseline RAVs. Simeprevir (SMV) 150mg QD plus Sofosbuvir 400mg QD +/- ribavirin for 8 or 12 weeks, phase 3 studies (n=413). Analysis according to the absence or presence of Q80K in HCV genotype 1a infected patients [108, 109]. No head-to-head comparisons. TN, treatment naive; TE, treatment experienced.

Figure 5. SVR rates NS5A inhibitor plus nucleos(t)ide NS5B inhibitor DAA combination regimens in HCV genotype 1 infected patients according to the presence of baseline RAVs. Daclatasvir (DCV) 30-90mg QD plus sofosbuvir (SOF) 400mg QD for 8 or 12 weeks in HIV co-infected patients, phase 3 (n=203). This analysis includes small numbers of non-genotype 1 infected patients [155, 156]. Daclatasvir (DCV) 60mg QD plus sofosbuvir (SOF) 400mg QD + ribavirin for 12 weeks in patients with advanced cirrhosis (Child A-C), phase 3 study (n=59). This analysis includes small numbers of non-genotype 1 infected patients. NS5A RAVs at positions 28, 30, 31 and 93 are taken into account [157]. Ledipasvir (LDV) 90mg QD plus sofosbuvir (SOF) 400mg QD for 8 or 12 weeks, phase 2/3 studies (n=2137). Ledipasvir (LDV) 90mg QD plus sofosbuvir (SOF) 400mg QD with and without ribavirin for 12 or 24 weeks, phase 2/3 studies (n=510). NS5A RAVs with >100-fold resistance are taken into account (M28A/G, Q30E/H/G/K/R, L31I/M/V, P32L, H58D, A92K, Y93C/H/N/S) and in the majority of patients deep sequence analysis was performed [25, 112]. No head-to-head comparisons. TN, treatment naive; TE, treatment experienced.

Figure 6. SVR rates according to pre-existent NS5A RAVs in HCV genotype 3 infected patients treated with sofosbuvir plus daclatasvir. Daclatasvir (DCV) 60mg QD plus Sofosbuvir (SOF) 400mg QD for 12 weeks, phase 3 study (n=152) Baseline RAVs at positions M28V, A30 polymorphisms (A30X), and Y93H of the NS5A protein were analyzed [113]. TN, treatment naive; TE, treatment experienced

Figure 7. SVR rates for NS3 protease, NS5A- and NS5B non-nucleoside inhibitor combination regimens in HCV genotype 1 infected patients according to the presence of baseline RAVs. Paritaprevir/r (PTV/r) 150/100mg QD plus Ombitasvir (OMV) 25mg QD plus Dasabuvir (DSV) 250mg BID with and without Ribavirin (R), phase 2 study (n=406). Baseline RAVs at positions 80 and 168 within NS3, 28, 30, 31 and 93 within NS5A and 556 and 316 within NS5B were taken into account [28]. In a subanalysis (ITT) analysis 49 patients who were treated for 8 weeks were investigated and Q80K of the NS3 protein was 41 taken into account [158]. Asunaprevir (ASV) 200mg BID plus Daclatasvir (DCV) 30mg BID plus Beclabuvir (BCV) 75mg BID with or without Ribavirin (R), phase 3 studies (n=415). Baseline RAVs at positions 28, 30, 31 and 93 within NS5A were taken into account. No head- to-head comparisons. TN, treatment naive; TE, treatment experienced.

42

Figure 1

1012 1011 1010 viral decline 109 108 Examples for different intensities 107 of antiviral therapies ~ detection limit of HCV RNA assays Plasma HCV viral load Plasma viral HCV 106 Easy to treat patient (IL28B CC, low ISG, low IP10, low IFN mono 105 fibrosis, low age, female gender, low baseline viral load, HCV genotype 2, NUC mono 4 10 no viral resistance…) NUC + Riba or NS5A + PI

Total body HCV viral load body HCVviral Total 3 10 Difficult to treat patient NUC + NS5A/PI or 3D 102 (IL28B TT, high ISG, high IP10, cirrhosis, high age, male gender, high baseline NUC + NS5A/PI + RBV 101 viral load, HCV genotype 1, baseline viral resistance …) NUC + NS5A + PI +/- longer duration

Antiviral therapy [time] Figure 2

SVR without bl. RAVs SVR with bl. RAVs 100 84 72 70 80 64 65 58 60 60 55 40

SVR (%) SVR n.a. 20 0 BOC + PEG/R (TN+TE, TVR + PEG/R (TN+TE, SMV + PEG/R GT1a SMV + PEG/R GT1b DCV + PEG/R (TN, phase 2/3) phase 3) phase 2)

Frequency of pts. 8% 5% 30% n.a. 8% with baseline RAVs Figure 3

SVR without bl. RAVs SVR with bl. RAVs 98 100 92 99 80 60 52 39 40 No data published yet 22 SVR (%) SVR 20 0 ASV + DCV (GT1b, SMV + DCV (GT1, GZR + EBR (GT1a, TN, GZR + EBR ± R (GT1a, TN+TE, phase 3) phase 2) phase 3) TE, phase 3)

Frequency of pts. 13% 6% 10% with baseline RAVs

Figure 4

SVR without bl. Q80K SVR with bl. Q80K 97 96 92 100 84 80 73 74 60 40 SVR (%) SVR 20 0 SMV + SOF (GT1a, TN+TE, SMV + SOF (GT1a, TN+TE, SMV + SOF (GT1a, TN+TE, phase 3) phase 3) phase 3) 8 weeks 12 weeks 12 weeks no cirrhosis cirrhosis Frequency of pts. 42% 40% 47% with baseline RAVs Figure 5

SVR without bl. RAVs SVR with bl. RAVs

98 98 100 96 95 97 87 87 87 83 78 80 71 67 60

40 SVR (%) SVR

20

0 DCV + SOF (GT1- DCV + SOF (GT1- DCV + SOF + R (GT1- LDV + SOF (GT1, TN, LDV + SOF (GT1, LDV + SOF + R (GT1, 3/HIV, TN, phase 3) 4/HIV, TN+TE, phase 4, Child A-C, phase phase2/3) 8 wks TN+TE, phase2/3) cirrhosis, phase2/3) 8 wks 3) 12 wks 3) 12 wks 12 wks 12-24 wks

Frequency of pts. 18% 16% 24% 12% 12% 12% with baseline RAVs Figure 6

all no cirrhosis cirrhosis 100 100 96 89 80 63 67 68 60 54 40 33

SVR (%) SVR 25 20 0 DCV + SOF (TN+TE, phase 3) DCV + SOF (TN+TE, phase 3) DCV + SOF (TN+TE, phase 3) A30X Y93H

Frequency of pts. 10% 9% with baseline RAVs Figure 7

SVR without bl. RAVs SVR with bl. RAVs 100 93 92 87 92 87 87 80 74 74

60

40 SVR (%) SVR

20

0 PTV/r+OMV+DSV ± R (GT1a, PTV/r + OMV + DSV ± R ASV + DCV + BCV (GT1a, ASV + DCV + BCV ± R (GT1a, TN, phase 2) 8 wks (GT1a, TN+TE, phase 2) 8-24 TN+TE, no cirrhosis, phase 3) TN+TE, cirrhosis, phase 3) 12 wks 12 wks wks Frequency of pts. 39% 52% 11% 10% with baseline RAVs