Analysis of the susceptibility of refractory hepatitis C virus resistant to nonstructural 5A inhibitors

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.


Abbreviations
Hepatitis C virus (HCV) is a ribonucleic acid (RNA) virus belonging to the family Flaviviridae that was identified in 1989 by Choo et al. 1 .Approximately 70% of patients infected with HCV develop chronic hepatitis.Persistent liver inflammation leads to liver fibrosis, which progresses to cirrhosis and hepatocellular carcinoma (HCC) 2 .
In 2014, the first interferon-free direct-acting antivirals (DAAs), daclatasvir (a nonstructural (NS) 5A inhibitor) and asunaprevir were authorized for the treatment of chronic hepatitis or compensated cirrhosis in patients with HCV genotype 1 3 .Subsequently, various regimens have been approved [4][5][6][7][8][9] .Currently, DAA therapy is available for chronic hepatitis C for as little as 8 weeks, and regimens for re-treatment and non-compensated cirrhosis

Results
Frequency of NS5A RASs before and after DAA treatment in glecaprevir/pibrentasvir failure We analyzed the amino acid substitutions in the NS5A regions of two patients with glecaprevir/pibrentasvir failure before and after treatment using direct sequencing and ultra-deep sequencing.Table 1 shows the results of ultra-deep sequencing, highlighting the evolution of NS5A RASs over time.In Case 1, most amino acid substitutions were Q24K/L28M/R30Q/A92T or A before daclatasvir and asunaprevir therapy.After the failure of daclatasvir and asunaprevir treatment (prior to glecaprevir/pibrentasvir treatment), R30E or A92K were detected, which were not present before treatment and were caused by single nucleotide substitutions.After glecaprevir/ pibrentasvir failure, most amino acid substitutions changed to Q24K/L28M/R30E/A92K.

Effect of pibrentasvir and sofosbuvir on Q24K/L28M/R30E/A92K
The Q24K/L28M/R30E/A92K was more resistant to all NS5A inhibitors (specifically to pibrentasvir) than the WT and Q24K/L28M/R30Q/A92K.However, among the NS5A inhibitors examined in this study, pibrentasvir showed the weakest resistance to the Q24K/L28M/R30E/A92K RASs.Compared with WT, Q24K/L28M/R30E/ A92K was not resistant to the NS5B inhibitor sofosbuvir.Therefore, we believe that the combination of pibrentasvir and sofosbuvir is most effective against Q24K/L28M/R30E/A92K, and we investigated the effect of this combination on the Q24K/L28M/R30E/A92K RASs (Fig. 3).Ten-centimeter dishes were prepared in which the Q24K/L28M/R30E/A92K RASs grew continuously.Each dish was supplemented with either pibrentasvir alone (Fig. 3a), sofosbuvir alone (Fig. 3b), or a combination of pibrentasvir and sofosbuvir (Fig. 3c).Pibrentasvir and sofosbuvir were added at concentrations of 80 nM and 1 µM, respectively, which were 10 times the EC50 against the Q24K/L28M/R30E/A92K RASs.The agents were added each time the cells were passaged.G418 was not added for the first 2 weeks; subsequently, G418 was added at each passage to select the cells.After three passages, Coomassie Brilliant Blue (CBB) staining showed that the cells almost died in the dish containing both pibrentasvir and sofosbuvir, whereas cell proliferation continued in the dishes containing only pibrentasvir or sofosbuvir (Fig. 3).While single administration of pibrentasvir or www.nature.com/scientificreports/sofosbuvir failed to suppress the proliferation of the Q24K/L28M/R30E/A92K RASs, the combination was able to suppress it.Therefore, it was considered that HCV with multiple mutations of NS5A Q24K/L28M/R30E/A92K could be treated effectively by the combined administration of pibrentasvir and sofosbuvir.

Discussion
In the present report, we revealed the susceptibility of the coexistence of NS5A Q24K/L28M/R30E/A92K RASs in the NS5A region to various agents using subgenomic replicon cells.These RASs were highly resistant to all NS5A inhibitors in vitro.There have been no reports on the susceptibility of coexistence of these RASs to NS5A inhibitors in vitro.In addition, combination treatment with pibrentasvir and sofosbuvir against the coexistence of these RASs was effective in the replicon cells.Additionally, our study was performed using only HCV cells generated from genotype 1b, which may be clinically relevant.
In a nationwide multicenter study in Japan, a total of 1,193 patients with HCV for whom DAA treatment had failed were enrolled.NS5A L28M, R30Q, and A92K RASs were found in 17%, 17%, and 2.4% of patients, respectively 14 .Although this report did not mention the prevalence of the coexistence of Q24K/L28M/R30Q/ A92K or Q24K/L28M/R30E/A92K RASs, it was speculated that cases with such mutations existed.Among the patients enrolled in the domestic phase III study of glecaprevir/pibrentasvir, there were some patients with RASs in NS5A Q24/L28/R30/A92 among those already treated with DAA.All of these patients achieved SVR with glecaprevir/pibrentasvir, except for those with the P32del mutation [15][16][17] .No patient had the coexistence of NS5A Q24K/L28M/R30E/A92K RASs.In actual clinical practice, our group and Sezaki et al. reported that the coexistence of NS5A Q24K/L28M/R30E/A92K RASs was observed in patients for whom glecaprevir/pibrentasvir treatment failed, and R30Q was changed to R30E after virologic failure 12,18 .However, the coexistence of Q24K/ L28M/R30Q/A92K or Q24K/L28M/R30E/A92K RASs has appeared in re-treatment cases and has been reported in small numbers.We believe that to eradicate HCV globally and to prevent HCV-infected individuals from progressing to liver cirrhosis or developing cancer, establishing a treatment for highly resistant HCV is important.
According to the glecaprevir/pibretasvir interview form 17 , in an experiment using the Con-1 HCV strain, which is a replicon cell of genotype 1B, a single mutation of L28M or R30Q RASs was 1.0-fold or 0.5-fold resistant to pibrentasvir, respectively, compared with the WT.No resistance was observed with single mutations.Resistance to single mutations of Q24K, R30E, and A92K and resistance to multiple mutations of Q24K, L28M, R30Q, R30E, and A92K were not examined.Nitta et al. examined the NS5A region of the genotype 2a JFH-1 strain with the genotype 1b Con-1 HCV strain 19 .They generated strains with a single A92K mutation in the NS5A region and multiple RASs of NS5A Q24K, L28M, and R30Q, as reported in cases previously treated with daclatasvir and asunaprevir 16,20 .They examined these strains in cells transduced with Huh7.5.1 and showed that compared with the WT, the A92K RAS was 17,000,000-fold resistant to ledipasvir, 13.7-fold resistant to velpatasvir, and 8600-fold resistant to elbasvir.The R30Q/A92K RAS was 20,000,000-fold resistant to ledipasvir, 620-fold resistant to velpatasvir, and 31,000-fold resistant to elbasvir than the A92K single mutation, and the Q24K/L28M/R30Q/ A92K RAS was more resistant than the R30Q/A92K RASs.However, susceptibility to the coexistence of NS5A Q24K/L28M/R30E/A92K RASs was not examined.
The mechanism by which HCV RASs occur remains unclear.Kai et al. revealed two mechanisms in posttreatment RASs: the selection of pre-existing substitutions among quasispecies and generation of novel mutations during therapy using deep sequencing analysis 21 .Yamauchi et al. suggested that the combination of various mutations, other than the known signature RASs, influences the kinetics of individual HCV quasispecies during DAA treatment 22 .They analyzed single-molecule long-read sequencing using rolling circle amplification to correct sequencing errors in the Oxford Nanopore sequencer.In the present study, we also performed ultra-deep www.nature.com/scientificreports/sequencing analysis using samples taken before and after glecaprevir/pibrentasvir treatment.Case 1 had Q24K/ L28M/R30Q/A92T RASs before daclatasvir and asunaprevir treatment, and novel mutations such as R30E and A92K RASs appeared after daclatasvir and asunaprevir treatment.The pre-existing R30E and A92K RASs before glecaprevir/pibrentasvir treatment induced glecaprevir/pibrentasvir failure.Case 2 also had R30E and A92K RASs before glecaprevir/pibrentasvir treatment.Most amino acid substitutions were Q24K/L28M/R30E/A92K RASs after glecaprevir/pibrentasvir treatment (Table 1).We speculate that the R30Q to R30E mutation we identified also occurred via the above two mechanisms.Several studies have reported the effects of anti-HCV treatment on the subsequent course of patients.Laursen et al. showed that successful DAA therapy was beneficial in advanced liver disease, with an initial rapid resolution of liver inflammation and a subsequent gradual but steady improvement in liver fibrosis, metabolic liver function and reaction time 23 .Furthermore, Seko et al. reported that among patients with HCC who were treated with molecularly targeted agents, the overall survival of patients who achieved SVR was significantly longer than that of those who did not achieve SVR (18.1 months vs. 11.3 months) 24 .Ochi et al. reported that in Child-Pugh grade A patients with a history of hepatectomy or radiofrequency ablation for HCC, the DAA-treated group had a significantly higher survival rate at 48 months and a significantly lower HCC recurrence rate than the untreated group 25 .The SVR rate is generally > 95% owing to the progression of DAA.
Ikeda et al. reported mizoribine, fluvastatin, and teprenone as candidate anti-HCV agents [26][27][28] .Furthermore, they reported that oncostatin M, a member of the interleukin-6 family, had marked anti-HCV activity in an HCV RNA-replicating cell culture system and showed synergistic inhibitory activity against interferon-alpha even at low concentrations 29 .We wondered whether agents other than DAAs with anti-HCV activity could be added to DAAs to obtain an effect against the Q24K/L28M/R30E/A92K and investigated interferon-alpha, fluvastatin, and oncostatin M. The Q24K/L28M/R30Q/A92K and Q24K/L28M/R30E/A92K RASs were mildly resistant to these agents compared with the WT, and no additional treatment was considered (Table 3).The combination of pibrentasvir and sofosbuvir successfully eliminated HCV in the Q24K/L28M/R30E/A92K RASs (Fig. 3).
One possible limitation of this study is that replicon cells may have acquired new HCV mutations other than those at the insertion site during cell passaging, which may have affected the results.We sequenced 3 types of replicon cells after passing them 13 times to confirm that the cells had not acquired any new mutations.Furthermore, Kato et al. analyzed spontaneous genetic mutations in HCV replicon cells, 50-1 cells derived from HuH-7 cells, and sO cells after 9 years of passaging culture.The results showed that genetic mutations in both replicon cells accumulated at 2.3 × 10 3 to 3.1 × 10 3 base substitutions/site/year 30 .The cells used in this study were of the OR6c/1B-4 strain, which differs from those used in previous studies.However, only cells that had been passaged fewer than 20 times were used in the experiment, and the sustained passage period was as short as 2-3 months, suggesting that the acquisition of new mutations during the passage was quite low.

Table 2 .
Susceptibility of NS5A RAS to NS5A inhibitors.The fold change in resistance was compared with that of the WT.EC50 effective concentration required to inhibit 50%, WT wild-type, NS nonstructural, RAS resistance-associated substitution.