Resistance characterization of hepatitis C virus genotype 2 from Japanese patients treated with ombitasvir and paritaprevir/ritonavir

Treatment of HCV genotype (GT) 2‐infected Japanese patients with paritaprevir (NS3/4A inhibitor boosted with ritonavir) and ombitasvir (NS5A inhibitor) without ribavirin for 12 weeks in the phase 2 study M12‐536, and with ribavirin for 16 weeks in phase 3 study GIFT II resulted in SVR rates of 72.2% to 91.5%. Overall, 11 out of 125 patients with GT2a and 37 out of 79 patients with GT2b infection experienced virologic failure. The prevalence of baseline polymorphisms in NS3 and NS5A and their the impact on treatment outcome, as well as the development of viral resistance in GT2‐infected patients experiencing virologic failure were evaluated by HCV NS3 and NS5A population and clonal sequence analyses. Baseline polymorphisms in NS3 that confer resistance to paritaprevir were rare in both GT2a‐ and GT2b‐infected patients, while baseline polymorphisms in NS5A that confer resistance to ombitasvir were detected in 11.2% and 14.1% of the GT2a‐ and GT2b‐infected patients, respectively. There was no significant impact of baseline polymorphisms on treatment outcome in Japanese patients. The most common treatment‐emergent substitutions at the time of virologic failure occurred at amino acid positions 168 in NS3 and 28 in NS5A in both GT2a‐ and GT2b‐infected patients. Although there was a higher rate of virologic failure in patients with GT2b infection, the resistance analyses presented in this report support the conclusion that testing for baseline resistance‐associated polymorphisms is not warranted for HCV GT2‐infected patients treated with a regimen of ombitasvir/paritaprevir/ritonavir + ribavirin for 16 weeks.

patients, of which two-thirds are infected with subtype 2a and onethird are infected with subtype 2b. 4 The majority of the remaining 70% of HCV infections in Japan are caused by GT1b. 4 The genotype distribution of HCV infections in Japan has changed over time, and recent studies have reported a decrease in GT1 prevalence and an increase in GT2 prevalence in the HCV-infected patient population which was correlated with differences in transmission routes, especially for individuals born after 1970 where <50% of infections were reported as GT1b. 4,5 Given that approximately 70% of HCC cases are caused by HCV in Japan, and that HCC prevalence has increased over the past 50 years, 4,6 direct-acting antiviral (DAA) therapies are needed to treat both HCV GT1 and GT2 infections in Japan.
Several DAA-based therapies with high sustained virologic response (SVR) rates at post-treatment week 12 (SVR 12 ) are approved for the treatment of HCV GT1 infection in Japan. [7][8][9][10] For HCV GT2 infection, the standard of care was previously a combination of pegylated interferon alpha (pegIFN) plus ribavirin (RBV) for 24 weeks, which demonstrated SVR rates of 71-80% in large clinical trials worldwide. [11][12][13] The first IFN-free DAA regimen for HCV GT2 infection with high SVR 12 rates of 93-98% was the combination of the NS5B nucleotide polymerase inhibitor sofosbuvir (SOF) plus RBV for 12 weeks, 14,15 approved for the treatment of GT2-infected patients in 2015. 16 A treatment regimen of ombitasvir/paritaprevir/ritonavir (OBV/PTV/r) plus RBV for 16 weeks was recently approved for the treatment of GT2-infected patients in Japan.
Ombitasvir is an HCV NS5A inhibitor 17 and paritaprevir is an HCV NS3/4A inhibitor (identified by AbbVie and Enanta) that is coadministered with the pharmacokinetic enhancer ritonavir. 18 Ombitasvir and paritaprevir have demonstrated in vitro antiviral activity against multiple HCV genotypes, with 50% effective concentration (EC 50 ) values of 12 and 4.3 pM for ombitasvir against GT2a and GT2b, 19 respectively, and EC 50 values of 9.8 and 107 nM for paritaprevir against GT2a and GT2b, respectively. A dose-ranging Japanese phase 2 study (M12-536) reported an SVR 24 rate of 72.2% in treatment-experienced HCV GT2-infected Japanese patients without cirrhosis who received OBV/PTV/r dosed at 25/150/100 mg for 12 weeks. 7 Due to the lower overall SVR 24 rate for the treatment of HCV GT2b infection in study M12-536, extended treatment duration of 16 weeks and the addition of RBV to the treatment regimen were evaluated in phase 3 study GIFT-II.
The Japanese GIFT-II study reported SVR 12 rates of 91.5% and 75.0%, respectively, in treatment-naïve and treatment-experienced HCV GT2-infected Japanese patients without cirrhosis who received co-formulated OBV/PTV/r plus RBV for 16 weeks. 20 The current report was designed as a comprehensive clinical

| Ethics statement
The M12-536 and GIFT-II studies were conducted in accordance with   guidelines of the International Conference of Harmonization, applicable regulations and guidelines governing clinical study conduct, and ethical principles expressed in the Declaration of Helsinki. The study protocols were approved by the relevant institutional review boards and regulatory agencies, and all patients provided written informed consent.

| Study design and patient population
The M12-536 study (ClinicalTrials.gov identifier: NCT01672983) was a randomized, open-label, dose-and duration-ranging, phase 2 study that evaluated the safety and efficacy of ombitasvir and paritaprevir/r without RBV in patients with HCV GT 1b or GT2 infection. The study design, safety, and efficacy results through SVR 24 from patients with HCV GT1b or GT2 infection were previously reported. 7 The GT2infected patient population included non-cirrhotic, pegIFN/RBV treatment-experienced patients (null responders, partial responders, and relapsers) in Japan. Patients with GT2 infection were randomized in a 1:1 ratio to receive once-daily ombitasvir (25 mg) plus either paritaprevir/ritonavir 100/100 mg or 150/100 mg for 12 weeks.

| HCV genotype and subtype determination by phylogenetic analysis
The Versant HCV Genotype Inno-LiPA Assay v2.0 (LiPA 2.0) was used to determine HCV genotype for enrollment of patients with chronic HCV GT2 infection for studies M12-536 and GIFT-II, but was unable to identify the viral subtype for the majority of GT2-infected patients.
The preliminary viral subtype was therefore determined by phylogenetic analysis of a 329 nucleotide (nt) region of the NS5B gene that was PCR amplified from baseline samples of HCV GT2-infected patients. 21,22 Results from this analysis determined the subtype and gene-specific reverse transcriptase (RT)-PCR and nested PCR primer sets for amplification of NS3/4A and NS5A genes from baseline samples. Phylogenetic analyses were subsequently conducted using HCV NS3 (1-543 nt) and NS5A (1-645 nt) nucleotide sequences from baseline samples. Nucleotide sequences for NS3, NS5A, and NS5B were aligned using the MAFFT sequence alignment method. 23 Phylogenetic trees were constructed using the neighbor-joining tree-building method 24,25 with the HKY85 nucleotide substitution model 26 and 1000 bootstrapping replicates. The final HCV GT2 subtype assignment was determined by consensus between NS3, NS5A, and NS5B results. 27

| Patient sample processing and sequence analysis
Viral RNA isolation, RT-PCR, and nested PCR were conducted for the target genes NS3/4A and NS5A on samples with HCV RNA ≥1000 IU/mL, as previously described. 19,28,29 For patients who achieved SVR, population nucleotide sequencing was conducted on the regions encoding HCV NS3 amino acids 1-181 and NS5A amino    3.2 | Lack of impact of baseline polymorphisms in NS3 and NS5A on SVR NS3 D168E in GT2a, which confers 5.3-fold resistance to paritaprevir, was detected in 1 patient; resistance-conferring baseline polymorphisms were not detected in GT2b (Table 2).
The impact of baseline polymorphisms in NS3 and NS5A on treatment outcome was evaluated for studies M12-536 (Supplementary   Table S2) and GIFT-II (Table 3) by comparing SVR rates in patients with and without baseline polymorphisms. In study M12-536, SVR 24 rates were similar in patients with or without an NS3 or NS5A polymorphism at baseline with GT2a or GT2b infection (Supplementary Table S2). In GIFT-II, SVR 12 rates were compared separately by GT2 subtype for treatment-naïve and IFN treatment-experienced patients without cirrhosis, and for patients with cirrhosis, who received 12 or 16 weeks of treatment. For GT2a infection, the SVR 12 rates were similar in noncirrhotic treatment-naïve and treatment-experienced patients with or without an NS3 or NS5A polymorphism at baseline ( Table 3)  baseline. There was no significant difference in SVR 12 rates for GT2ainfected patients with M or L amino acids at position 31 in NS5A.
Although there was an increased rate of virologic failure in patients with HCV GT2b infection in study GIFT-II, SVR 12 rates were similar in GT2b-infected patients with or without an NS3 or NS5A polymorphism at baseline (Table 3). Overall SVR 12 rates were higher for treatment-naïve compared to pegIFN/RBV treatment-experienced GT2b-infected patients without cirrhosis (  (Table 4).
Nine GT2a-infected patients in study GIFT-II experienced VF (Tables 1 and 4

| Persistence of treatment-emergent substitutions through post-treatment week 48
Baseline polymorphisms and treatment-emergent substitutions in NS3 and NS5A were monitored for persistence through post-treatment week 48 by population and clonal sequencing analyses (Figure 1).    GT, genotype; NA, not applicable as there were no GT2a VFs in the study arm; TN, treatment-naïve; TE, treatment-experienced to an IFN-containing regimen (IFN alpha, beta, or pegIFN) with or without RBV. a n = number of patients with the treatment-emergent substitution, N = total number of patients who experienced VF in the designated study Arm. Patients without cirrhosis or with compensated cirrhosis who experienced VF are included in the analysis. b Patients in study M12-536 received OBV/PTV/r for 12 weeks, and all patients were treatment-experienced to pegIFN/RBV.  warranted as a pre-screen for GT2-infected patients seeking treatment with this regimen. The treatment regimen of OBV/PTV/ r + RBV for 16 weeks is approved in Japan for non-cirrhotic patients with HCV GT2 infection.

ACKNOWLEDGMENTS
The authors acknowledge the clinical providers and patients for their study participation, and the study coordinators for assistance provided in the preparation and operation of the study. The design, study conduct, and financial support for these studies were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.  FIGURE 1 Persistence of resistance-associated baseline polymorphisms and treatment-emergent substitutions in GT 2a or 2b-infected patients who experienced virologic failure. The percentage of VF patients with the designated baseline or treatment-emergent substitution is shown for the baseline, time of VF, post-treatment week 24, and post-treatment week 48 time points. Columns denoted as "Any" include patients with any baseline polymorphism or treatment-emergent substitution for NS3 or NS5A at signature resistance-associated amino acid positions. Baseline polymorphisms L/M31 in NS5A were not included in the total "Any" count. Specific amino acid substitutions designated as "(any)" include any amino acid change from wild-type in the total count SCHNELL ET AL.