S282T Substitution Research Articles

Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro

Sofosbuvir, a licensed nucleotide analog targeting hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), has been recently evaluated as a broad anti-Flavivirus lead candidate revealing activity against Zika and Dengue viruses both in vitro and in animal models. In this study, the in vitro antiviral activity of sofosbuvir against West Nile virus (WNV) was determined by plaque assay (PA) and Immunodetection Assay (IA) in human cell lines and by enzymatic RdRp assay. By PA, the sofosbuvir half-maximal inhibitory concentration (IC50) was 1.2 ± 0.3 μM in Huh-7, 5.3 ± 0.9 μM in U87, 7.8 ± 2.5 μM in LN-18 and 63.4 ± 14.1 μM in A549 cells. By IA, anti-WNV activity was confirmed in both hepatic (Huh-7, 1.7 ± 0.5 μM) and neuronal (U87, 7.3 ± 2.0 μM) cell types. Sofosbuvir was confirmed to inhibit the purified WNV RdRp (IC50 11.1 ± 4.6 μM). In vitro resistance selection experiments were performed by propagating WNV in the Huh-7 cell line with two-fold increasing concentrations of sofosbuvir. At 80 μM, a significantly longer time for viral breakthrough was observed compared with lower concentrations (18 vs. 7–9 days post infection; p = 0.029), along with the detection of the S604T mutation, corresponding to the well-known S282T substitution in the motif B of HCV NS5B, which confers resistance to sofosbuvir. Molecular docking experiments confirmed that the S604T mutation within the catalytic site of RdRp affected the binding mode of sofosbuvir. To our knowledge, this is the first report of the antiviral activity of sofosbuvir against WNV as well as of selection of mutants in vitro.

Analysis of Host and Viral-Related Factors Associated to Direct Acting Antiviral Response in Hepatitis C Virus Patients.

Hepatitis C virus (HCV) signifies an important health issue as it is a globally prevalent pathogen and poses a great threat to human health. Direct antiviral therapy became a landmark in treatment against chronic HCV infection as they have proven to increase sustained virological response (SVR) rate, provide shortened and simplified regimens. This study aimed to evaluate efficacy of Sofosbuvir and classify factors of treatment success and their function in therapy continuation decision. We studied host and viral factors in 310 patients who received Sofosbuvir (DAA) at a dose of 400 mg daily along with ribavirin 10 mg/kg body weight for 24 weeks and followed by 12 weeks after completion of treatment. A total of 302 patients (97.42%) showed end-of-treatment response. However, 8 patients (2.58%) were virological nonresponders and 286 patients (94.70%) achieved SVR. Among 310 HCV positive, the percentage of viral genotype 3-infected patients was 89.6%, whereas only 10.32% were affected by HCV genotype 1. Multivariable analysis showed that baseline HCV RNA (≤8 × 106 IU/mL vs. >8 × 106 IU/mL) [95% confidence interval (CI), odds ratio (OR) 3.0, 2.0 to 8.4; p = 0.004], age (<50 vs. ≥50 years) (95% CI, OR 1.9, 3.0 to 10.1; p = 0.03), liver texture (normal liver vs. coarse) are related to virological response. None of the patients included in the study has S282T substitution in NS5b of HCV. No significant difference in response was observed in patients with different genotypes of rs12979860. Liver texture, age, and viral load are predictors of SVR. In this real-life data no substitution of S282T in NS5b was observed related to failure of therapy. Oral Sofosbuvir generally has a high tolerance and can effortlessly beat the safety concerns of past regimens.

THU-386 - Persistence and risk of transmission of the hepatitis C virus NS5B S282T substitution in a HIV-positive man who has sex with men

THU-386 - Persistence and risk of transmission of the hepatitis C virus NS5B S282T substitution in a HIV-positive man who has sex with men

The emergence of NS5B resistance associated substitution S282T after sofosbuvir-based treatment.

S282T in NS5B is the primary amino acid substitution associated with resistance to sofosbuvir (SOF) but has rarely been detected in patients treated with a SOF‐based regimen. Here, the emergence and fitness of the S282T substitution in virologic failure patients administered SOF‐based regimens across the SOF and ledipasvir (LDV)/SOF phase 2 and 3 programs was evaluated. Plasma samples collected at baseline and at virologic failure were amplified and deep sequenced (1% cutoff). To date, over 12,000 patients have been treated in SOF or LDV/SOF phase 2 and 3 studies. Of these, deep sequencing was available at baseline in 8598 patients (62.4% genotype [GT] 1, 10.7% GT2, 20.9% GT3, and 6.0% GT4‐6) and at virologic failure in 901 patients. In the 8598 patients, no S282T substitution was detected at baseline; at virologic failure, 10 of the 901 (1%) patients had S282T detected. The SOF‐based regimen associated with treatment‐emergent S282T was SOF monotherapy in two patients, retreatment with LDV/SOF in prior LDV/SOF failures in three patients, LDV/SOF for 8 weeks in 1 GT1 patient, LDV/SOF for 12 weeks in 1 patient each with GT3, GT4, and GT5, and LDV/SOF + ribavirin for 12 weeks in 1 GT6 patient. Nine of 10 patients with emergent S282T received an SOF‐based retreatment regimen, eight of whom achieved sustained virologic response 12 weeks after treatment and one of whom failed retreatment. Conclusion: The emergence of S282T substitution was rare in patients who fail SOF‐based regimens. Successful retreatment of prior SOF failure patients is possible in the presence of S282T substitution with SOF in combination with various direct‐acting antiviral agents. (Hepatology Communications 2017;1:538–549)

Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors.

The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 μM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 μM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

In vitro selection of resistance to sofosbuvir in HCV replicons of genotype-1 to -6.

Sofosbuvir is a nucleoside analogue inhibitor of the HCV NS5B polymerase approved for treatment of HCV-infected patients in combination with ribavirin or with other antivirals. It has activity against all genotypes of HCV. Resistance to sofosbuvir in genotype-1 and -2 HCV is conferred by the S282T substitution in NS5B. To begin to define the correlates of resistance to sofosbuvir in other genotypes, we performed selection experiments in cell culture using cell lines containing subgenomic replicons derived from genotypes-1b, -2a, -3a and -4a, or chimeric replicons in a genotype-1b background but encoding genotype-2b, -5a and -6a NS5B polymerase. In every case, S282T was selected following passage in the presence of increasing concentrations of sofosbuvir for 10 to 15 weeks. When introduced as a site-directed mutant, S282T conferred reductions in sofosbuvir susceptibility of between 2.4 and 19.4-fold. Other substitutions observed during the selections had relatively less impact on susceptibility, such as N237S in genotype-6a (2.5-fold). Replication capacity was affected by the introduction of S282T in all genotypes to variable extents (3.2% to 22% of wild type). These results confirm that S282T is the primary sofosbuvir resistance-associated substitution and that replication capacity is reduced when it is present in all genotypes of HCV.

Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-D-2'-deoxy-2'-α-fluoro-2'-β-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.

1238 CULTURE LONG-TERM TREATMENT WITH IMINOSUGARS-BASED HCV ASSEMBLY INHIBITORS DOES NOT LEAD TO VIRAL BREAKTHROUGH IN TISSUE CULTURE

Methods: Double-blind studies were conducted in genotype 1 HCVinfected, treatment-naive subjects. In trial 003, subjects received placebo (n =8) or IDX184 (n =33) at doses of 25 to 100mg/day for 3 days. In trial 004, subjects received placebo (n =16) or IDX184 (n =63) at doses of 50 to 200mg/day for 14 days. Samples obtained at baseline, 1 day and 2 weeks after final dosing were analyzed by NS5B population sequencing. Two 003 subjects were further evaluated by clonal sequencing. Results: No S282T mutation was detected by population sequencing in any baseline samples in the two studies. No S282T emerged following either 3 or 14 days of treatment with IDX184 or placebo. Clonal analysis of samples from the 2 003 subjects who had modest rebound (0.5 to 0.7 log10 IU/mL) during IDX184 treatment did not detect the S282T mutation in any of approximately 200 baseline or post-treatment samples. Evaluation of all NS5B codons across all samples from both trials revealed a number of treatment-emergent changes in both placebo and IDX184 subjects. These appeared to be random and generally occurred at sites that were polymorphic or showed mixed residues at baseline. No net change at any residue was attributed to treatment with IDX184. These results accord with preclinical resistance selection studies in cultured cells where the S282T mutation took an average of over 7 weeks to appear and S282T replicon-bearing cells exhibited 5% of wild-type replication capacity. Conclusions: In 3and 14-day clinical trials with IDX184, no pre-existing S282T variant was detected. No S282T substitution or other treatment-emergent variants were detected after IDX184 treatment. These results, along with the results of in vitro preclinical studies, are consistent with the concept of a high genetic barrier to resistance for nucleoside agents such as IDX184.

Selected Replicon Variants with Low-Level In Vitro Resistance to the Hepatitis C Virus NS5B Polymerase Inhibitor PSI-6130 Lack Cross-Resistance with R1479

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.

Replication Fitness and NS5B Drug Sensitivity of Diverse Hepatitis C Virus Isolates Characterized by Using a Transient Replication Assay

The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.