Infrequent development of resistance in genotype 1-6 hepatitis C virus-infected subjects treated with sofosbuvir in phase 2 and 3 clinical trials.
ABSTRACT: BACKGROUND:Sofosbuvir is a chain-terminating nucleotide analogue inhibitor of the hepatitis C virus (HCV) NS5B RNA polymerase that is efficacious in subjects with HCV genotype 1-6 infection. Sofosbuvir resistance is primarily conferred by the S282T substitution in NS5B. METHODS:NS5B sequencing and susceptibility testing of HCV from subjects infected with genotypes 1-6 who participated in phase 2 and 3 sofosbuvir clinical trials was performed. RESULTS:No NS5B variants present at baseline among 1645 sofosbuvir-treated subjects were associated with treatment failure; sofosbuvir susceptibility was within 2-fold of reference. Among 282 subjects who did not achieve sustained virologic response, no novel sofosbuvir resistance-associated variants were identified, and the NS5B changes observed did not confer significant reductions in sofosbuvir susceptibility. In 1 subject with S282T observed at relapse 4 weeks after sofosbuvir monotherapy, the resistant variant (13.5-fold reduced sofosbuvir susceptibility, replication capacity <2% of control) became undetectable by deep sequencing 12 weeks after treatment. L159F and V321A were identified as treatment-emergent variants but did not confer resistance to sofosbuvir in the replicon system. CONCLUSIONS:These data demonstrate a uniform susceptibility of subject-derived HCV to sofosbuvir, and also show that selection of sofosbuvir-resistant HCV is exceedingly rare and is associated with a significant reduction in viral fitness.
Project description:BACKGROUND:Sofosbuvir (SOF) exhibits a high barrier to resistance, with no S282T NS5B substitution or phenotypic resistance detected in phase 3 registration studies. METHODS:Here, emergence of the NS5B variants L159F and V321A and possible association with resistance was evaluated in 8 studies of SOF (NEUTRINO, FISSION, POSITRON, FUSION, VALENCE, PHOTON-1, PHOTON-2, and P7977-2025) and 5 studies of combination ledipasvir (LDV) and SOF (LDV/SOF; LONESTAR, ELECTRON [LDV/SOF arms], ION1, ION2, and ION3), using deep sequencing. RESULTS:Deep sequencing detected L159F in 15% (53 of 353) and V321A in 5% (17 of 353) of patients with virologic failure in the SOF studies. Intensification of SOF treatment with LDV reduced the emergence of L159F or V321A to 2% (1 of 50 each) at virologic failure. L159F and V321A did not influence the outcome of retreatment with SOF, ribavirin, and pegylated interferon. At baseline, L159F was detected only in genotype 1-infected patients (1%) and was only associated with increased virologic failure in patients treated for short durations with SOF and ribavirin. CONCLUSIONS:Deep-sequencing analysis confirmed that NS5B variants L159F and V321A emerged in a subset of patients treated with SOF at virologic failure. These variants had no impact on retreatment outcome with SOF, ribavirin, and pegylated interferon. Baseline L159F in genotype 1 did not affect the treatment outcome with LDV/SOF.
Project description:The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2'-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2'-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively.
Project description:Hepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.A total of 3.5 million high-quality reads of ? 200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.
Project description:BACKGROUND: Direct-acting antiviral (DAA) agents target HCV proteins; some of these have already been approved for the treatment of HCV infection, while others are in development. However, selection of DAA-resistant viral variants may hamper treatment. The aim of this study was to illustrate potential natural DAA-resistance mutations in the HCV NS5A and NS5B regions of HCV genotypes 1a and 1b from DAA-naïve patients. METHODS: Direct sequencing of HCV NS5A and NS5B regions was performed in 32 patients infected with HCV genotype 1a and 30 patients infected with HCV genotype 1b; all subjects were naïve to DAAs. RESULTS: In genotype 1a strains, resistance mutations in NS5A (M28V, L31M and H58P) were observed in 4/32 (12.5%) patients, and resistance mutations in NS5B (V321I, M426L, Y448H, Y452H) were observed in 4/32 (12.5%) patients. In genotype 1b, resistance mutations in NS5A (L28V, L31M, Q54H, Y93H and I280V) were observed in 16/30 (53.3%) patients, while resistance mutations in NS5B (L159F, V321I, C316N, M426L, Y452H, R465G and V499A) were observed in 27/30 (90%) patients. CONCLUSIONS: Mutations conferring DAA resistance were detected in NS5A and NS5B of HCV genotypes 1a and 1b from DAA-naïve patients. Although some mutations confer only a low level of resistance, the presence at baseline of mutated HCV variants should be taken into consideration in the context of DAA therapy.
Project description:The introduction of highly efficient therapies with direct-acting antivirals (DAA) for patients with chronic hepatitis C virus (HCV) infection offers exceptional opportunities to globally control this deadly disease. For achieving this ambitious goal, it is essential to prevent antiviral resistance against the most optimal first-line and retreatment DAA choices. We performed independent comparisons of the efficacy and barrier to resistance of pangenotypic DAA regimens for HCV genotype 2 infections, using previously and newly developed efficient cell culture-adapted strains of subtypes 2a, 2b, and 2c. With the applied experimental cell culture conditions, combination treatment with the sofosbuvir-velpatasvir or glecaprevir-pibrentasvir DAA regimen was efficient in eradicating HCV infections; in contrast, single-drug treatments frequently led to viral escape. Sequence analysis of drug targets from recovered viruses revealed known resistance-associated substitutions (RAS) emerging in the NS3 protease or NS5A after treatment failure. These RAS were genetically stable after viral passage, and viruses with these RAS exhibited significant phenotypic resistance. After sofosbuvir treatment failure, only a genotype 2a virus harbored NS5B RAS S282T and thus had decreased susceptibility to nucleotide analogs (nucs). However, in most cases, viral escape from sofosbuvir led to other NS5B substitutions but drug susceptibility was maintained, and in one case, no changes in NS5B were detected. For a genotype 2b virus, after treatment failure with sofosbuvir-velpatasvir, the efficacy of retreatment with glecaprevir-pibrentasvir was maintained due to the high barrier to resistance and low cross-resistance of pibrentasvir. Our findings suggest the slight superiority of glecaprevir-pibrentasvir against genotype 2b in culture, which could have potential therapeutic interest meriting more definitive investigations in the clinic.
Project description:BACKGROUND:The interferon (IFN)-free regimen of sofosbuvir and ribavirin for 24 weeks was recently approved to treat chronic hepatitis C virus (HCV) genotype 1 (GT-1) infection for patients ineligible for IFN. However, sofosbuvir plus ribavirin therapy is associated with relapse in 15% to 30% of patients with HCV GT-1. Neither the mechanism of relapse nor the optimal re-treatment strategy for these patients is defined. OBJECTIVE:To assess the safety and efficacy of sofosbuvir plus ledipasvir in patients with chronic HCV GT-1 that relapsed after sofosbuvir plus ribavirin therapy. DESIGN:Phase 2a, open-label study. (ClinicalTrials.gov: NCT01805882). SETTING:Single U.S site. PATIENTS:14 patients with HCV GT-1 that relapsed after treatment with sofosbuvir plus ribavirin for 24 weeks were re-treated with sofosbuvir plus ledipasvir for 12 weeks. MEASUREMENTS:HCV RNA concentration and population sequencing to detect NS5B S282T mutations. RESULTS:All 14 patients treated with sofosbuvir plus ledipasvir for 12 weeks achieved a sustained virologic response, including 7 with advanced liver disease (Knodell Histology Activity Index score of 3 or 4) and 1 with a detectable NS5B S282T mutation after sofosbuvir plus ribavirin therapy. Sofosbuvir plus ledipasvir was well-tolerated with few adverse events. Four grade 3 events (elevated serum creatinine in a patient with baseline renal insufficiency, hypercholesterolemia, and hypophosphatemia) occurred. There were no grade 4 events or treatment discontinuations. LIMITATION:Small sample size. CONCLUSION:The fixed-dose combination of sofosbuvir plus ledipasvir was efficacious in a small cohort of patients with HCV GT-1 that relapsed after sofosbuvir plus ribavirin therapy, even in the setting of advanced liver disease. Larger studies are needed to confirm these preliminary efficacy results. PRIMARY FUNDING SOURCE:National Institute of Allergy and Infectious Diseases, National Institutes of Health, National Cancer Institute, and Gilead Sciences.
Project description:PSI-6130 (beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine) is a selective inhibitor of hepatitis C virus (HCV) replication that targets the NS5B polymerase. R7128, the prodrug of PSI-6130, has shown antiviral efficacy in patients chronically infected with HCV genotype 1a (GT-1a) and GT-1b. We observed that the compound exhibited potent in vitro activity against laboratory-optimized HCV replicons as well as against a panel of replicons containing NS5B HCV polymerases derived from GT-1a and GT-1b clinical isolates. We used the HCV replicon cell system to examine the emergence of variants with reduced sensitivity to PSI-6130. Short-term treatment of cells harboring the HCV subgenomic replicon with PSI-6130 cleared the replicon without generating resistant variants. Long-term culture of the cells under the compound selection generated the S282T substitution in a complex pattern with other amino acid substitutions in the NS5B polymerase. The presence of the coselected substitutions did not increase the moderate three- to sixfold loss of sensitivity to PSI-6130 mediated by the S282T substitution; however, their presence enhanced the replication capacity compared to the replication levels seen with the S282T substitution alone. We also observed a lack of cross-resistance between PSI-6130 and R1479 and demonstrated that long-term culture selection with PSI-6130 in replicon cells harboring preexisting mutations resistant to R1479 (S96T/N142T) results in the emergence of the S282T substitution and the reversion of S96T to wild-type serine. In conclusion, PSI-6130 presents a high barrier to resistance selection in vitro, selects for variants exhibiting only low-level resistance, and lacks cross-resistance with R1479, supporting the continued development of the prodrug R7128 as a therapeutic agent for the treatment of HCV infection.
Project description:PSI-353661, a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5'-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O(6)-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration.
Project description:PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of ?-D-2'-deoxy-2'-?-fluoro-2'-?-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.
Project description:Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant nucleoside inhibitors (NIs)/nonnucleoside inhibitors (NNIs). The study included 1,145 NS5B polymerase sequences retrieved from the Los Alamos HCV database and GenBank. The genetic barrier was calculated for drug resistance emergence. Prevalence and genetic barrier were calculated for 1 major NI and 32 NNI resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity toward the diverse HCV genotypes. Overall, NS5B polymerase was moderately conserved among all HCV genotypes, with 313/591 amino acid residues (53.0%) showing ?1% variability and 83/591 residues (14.0%) showing high variability (?25.1%). Nine NNI resistance variants (2 major variants, 414L and 423I; 7 minor variants, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV genotype 4 (HCV-4) (n = 14/38, 36.8%) and in all HCV-5 sequences (n = 17, 100%), respectively. Regardless of HCV genotype, the 282T major NI resistance variant and 10 major NNI resistance variants (316Y, 414L, 423I/T/V, 448H, 486V, 495L, 554D, and 559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier score development among the six HCV genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations. The genetic variability among HCV genotypes, particularly with the presence of polymorphisms at NNI resistance positions, could affect their responsiveness to NS5B inhibitors. A pretherapy HCV NS5B sequencing could help to provide patients with the full efficacy of NNI-containing regimens.