Summary
Clinical phase II/III studies of the nucleotide analogue HCV NS5B inhibitor sofosbuvir (SOF) have demonstrated high efficacy in HCV‐infected patients in combination therapy. To date, ...resistance to SOF (S282T in NS5B) has rarely been detected in patients. In this study, we investigated the evolution of S282T viral variants detected in one HCV genotype 2b‐infected patient who relapsed following 12 weeks of SOF monotherapy. Deep sequencing of the NS5B gene was performed on longitudinal plasma samples at baseline, days 2 and 3 on SOF, and longitudinal samples post‐SOF treatment through week 48. Intrapatient HCV evolution was analysed by maximum‐likelihood phylogenetic analysis. Deep sequencing analysis revealed a low level pre‐existence of S282T at 0.05% of viral sequences (4/7755 reads) at baseline and 0.03% (6/23 415 reads) at day 2 on SOF. Viral relapse was detected at week 4 post‐treatment where 99.8% of the viral population harboured S282T. Follow‐up analysis determined that S282T levels diminished post‐treatment reaching undetectable levels 24–48 weeks post‐SOF. Phylogenetic analysis together with the persistence of unique post‐treatment mutations in all post‐SOF samples suggested that growth of wild type resulted from reversion of the S282T mutant to a wild type and not outgrowth of the baseline wild‐type population. Our data suggest that a very low level of pre‐existing S282T at baseline in this patient was enriched and transiently detected following SOF monotherapy. Despite relapse with drug resistance to SOF, this patient was successfully retreated with SOF plus ribavirin for 12 weeks and is now cured from HCV infection.
Summary
The study aimed to evaluate the effects of baseline hepatitis C virus (HCV) nonstructural protein 5A (NS5A) resistance‐associated substitutions (RASs) on sustained virologic response to ...ledipasvir (LDV)‐containing regimens in the absence of sofosbuvir (SOF) in patients with HCV genotype (GT) 1 infection across 6 phase 2 clinical studies. We analysed data from 1103 patients who received either LDV + vedroprevir (NS3 protease inhibitor) + tegobuvir (NS5B inhibitor) ± ribavirin or LDV + ribavirin + pegylated interferon. Population sequencing of HCV NS5A was performed at baseline and at virologic failure from patient plasma samples. Of 1045 patients with available baseline sequences, 747 (67.7%) had GT1a, and 298 (26.9%) had GT1b infection. The overall prevalence of NS5A RASs at baseline was 9.4%; 7.6% (57/747) and 13.8% (41/298) of patients with GT1a and GT1b infection, respectively. The majority of GT1a‐infected patients with NS5A RASs at baseline had a single NS5A RAS (78.9%) at NS5A positions K24R, M28T, Q30H/L, L31M and Y93H/N/C/S. The spectrum of NS5A RASs detected in GT1b patients was much less diverse compared to GT1a patients, with all patients harbouring a single NS5A RAS either L31M or Y93H/C. For patients treated with LDV‐containing regimens in the absence of SOF, the presence of baseline NS5A RASs was associated with low SVR rates. In patients with virologic failure, nearly all had either pre‐existing and/or emergent NS5A RASs: 287/287 (100%) and 40/42 (95.2%) patients with GT1a and GT1b infection, respectively. Three novel NS5A substitutions were identified as emergent NS5A RASs: K26E and S38F in GT1a; and L31I in GT1b. In conclusion, the presence of NS5A RASs at baseline reduced the SVR rate in patients treated with LDV in combination vedroprevir + tegobuvir ± ribavirin or ribavirin + pegylated interferon. Virologic failure was associated with the detection of NS5A RASs in nearly all patients. These results suggest that the resistance barrier may differ depending on HCV drug combination and may be more important than that of the individual DAAs.
Summary
GS‐9190 is a NS5B non‐nucleoside analogue with demonstrated effectiveness in a Phase 1 monotherapy study and in combination with other DAAs for treatment of chronic HCV infection. Here, the ...resistance profile of GS‐9190 monotherapy in a Phase 1b study was investigated. Resistance analysis was performed by population sequencing and allele‐specific PCR (AS‐PCR) for Y448H with an assay cut‐off of 0.5%. Phenotypic susceptibility analyses were performed on patient isolates as well as site‐directed mutagenesis of mutations selected during monotherapy. No resistance‐associated variants were observed in patients before or after receiving single doses of GS‐9190 by population sequencing. In contrast, in patients who received GS‐9190 for 8 days, mutations Y448H and Y452H in NS5B were observed by population sequencing in 21/36 (58%) and 2/36 (5.6%) patients, respectively, at Day 8 or Day 14. Among the remaining 15 patients who had no detectable Y448H at Day 8 or Day 14 by population sequencing, low frequencies of Y448H ranging from 1.3 to 9.7% were detected in 14 of 15 patients by AS‐PCR. By AS‐PCR, Y448H remained detectable at reduced frequency in the majority of patients analysed through 4–6 months of follow‐up. Chimeric HCV replicons constructed with the NS5B sequence from patients with Y448H and Y448H + Y452H/Y demonstrated 27‐fold and 78.5‐fold reduced susceptibility to GS‐9190. In conclusion, Y448H was rapidly selected in the majority of patients receiving multiple doses of GS‐9190 as monotherapy, despite undetectable levels in pretreatment samples. Y448H confers reduced susceptibility to GS‐9190 and other NNIs and persisted in most patients for months post‐treatment.
Ultra-deep pyrosequencing (UDPS) is used to identify rare sequence variants. The sequence depth is influenced by several factors including the error frequency of PCR and UDPS. This study investigated ...the characteristics and source of errors in raw and cleaned UDPS data.
UDPS of a 167-nucleotide fragment of the HIV-1 SG3Δenv plasmid was performed on the Roche/454 platform. The plasmid was diluted to one copy, PCR amplified and subjected to bidirectional UDPS on three occasions. The dataset consisted of 47,693 UDPS reads. Raw UDPS data had an average error frequency of 0.30% per nucleotide site. Most errors were insertions and deletions in homopolymeric regions. We used a cleaning strategy that removed almost all indel errors, but had little effect on substitution errors, which reduced the error frequency to 0.056% per nucleotide. In cleaned data the error frequency was similar in homopolymeric and non-homopolymeric regions, but varied considerably across sites. These site-specific error frequencies were moderately, but still significantly, correlated between runs (r=0.15-0.65) and between forward and reverse sequencing directions within runs (r=0.33-0.65). Furthermore, transition errors were 48-times more common than transversion errors (0.052% vs. 0.001%; p<0.0001). Collectively the results indicate that a considerable proportion of the sequencing errors that remained after data cleaning were generated during the PCR that preceded UDPS.
A majority of the sequencing errors that remained after data cleaning were introduced by PCR prior to sequencing, which means that they will be independent of platform used for next-generation sequencing. The transition vs. transversion error bias in cleaned UDPS data will influence the detection limits of rare mutations and sequence variants.
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