Viral Resistance Analyses From the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1).

BACKGROUND: Remdesivir is approved for treatment of coronavirus disease 2019 (COVID-19) in nonhospitalized and hospitalized adult and pediatric patients. Here we present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance analyses from the phase 3 ACTT-1 randomized placebo-controlled trial conducted in adult participants hospitalized with COVID-19. METHODS: Swab samples were collected at baseline and longitudinally through day 29. SARS-CoV-2 genomes were sequenced using next-generation sequencing. Phenotypic analysis was conducted directly on participant virus isolates and/or using SARS-CoV-2 subgenomic replicons expressing mutations identified in the Nsp12 target gene. RESULTS: Among participants with both baseline and postbaseline sequencing data, emergent Nsp12 substitutions were observed in 12 of 31 (38.7%) and 12 of 30 (40.0%) participants in the remdesivir and placebo arms, respectively. No emergent Nsp12 substitutions in the remdesivir arm were observed in more than 1 participant. Phenotyping showed low to no change in susceptibility to remdesivir relative to wild-type Nsp12 reference for the substitutions tested: A16V (0.8-fold change in EC50), P323L + V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N + V792I (3.4-fold). CONCLUSIONS: The similar rate of emerging Nsp12 substitutions in the remdesivir and placebo arms and the minimal change in remdesivir susceptibility among tested substitutions support a high barrier to remdesivir resistance development in COVID-19 patients. Clinical Trials Registration. NCT04280705.

Sequence characterization of extracellular HBV RNA in patient plasma.

Antiviral nucleos(t)ide analogue therapies inhibit HBV replication and suppress the HBV DNA levels in patients with chronic HBV infection. Since HBV RNAs are expressed from cccDNA or HBV integrated sequences, independently of viral genome replication, levels of HBV RNAs in plasma may remain high following treatment with nucleos(t)ide analogue. Thus, HBV RNAs have been proposed to be used as a viral biomarker for treatment outcome and disease progression. Recent investigations of plasma HBV RNAs described the presence of full length as well as subgenomic forms of RNA. To support the usage of plasma HBV RNAs as a viral biomarker, further understanding of HBV RNA composition in clinical samples is needed. Here, sequence of extracellular HBV RNAs was characterized in plasma samples of patients with chronic HBV infection using two independent RNA amplification methods that do not use HBV-specific primers for amplification: total RNA (NuGEN RNAseq) and mRNA (TruSeq RNAseq). Sequencing coverage was obtained across the full length of HBV genome for both methods, confirming the presence of full-length HBV RNA in plasma. The sequence of HBV RNA was nearly identical to plasma HBV DNA sequence in each sample with only 0-14 (median 4) mismatches over 3 kb. Thus, sequence of HBV RNA plasma reflects the intrahepatic viral reservoir and can be used for monitoring of sequence variants such as resistance in clinical trials. Additionally, RNA splice forms, different polyA tails start positions and presence of HBV-human chimeric transcript were identified.

Hepatitis B virus genome diversity in adolescents: Tenofovir disoproxil fumarate treatment effect and HBeAg serocon version.

More systematic analysis of hepatitis B virus (HBV) genome diversity, linked with tenofovir disoproxil fumarate (TDF) treatment and HBeAg seroconversion, are needed. GS-US-174-0115 was a double-blind, placebo-controlled, Phase 3, 192-week clinical trial that evaluated TDF in adolescents with chronic hepatitis B (CHB). HBV full-genome deep sequencing was performed using Illumina MiSeq at baseline (BL; n = 85), Week 8 (W8; n = 80), Week 72 (W72; PBO only, n = 42), and treatment-free follow-up (TDF only, n = 25). The viral diversity was calculated using Shannon entropy and population nucleotide diversity with a 2% variant cutoff. Our data showed (i) a higher viral diversity in the X region at baseline than the core/polymerase/surface regions, (ii) higher core/surface viral diversity at baseline for patients with seroconversion, (iii) an expected reduction in viral diversity after 8 weeks of TDF treatment, and (iv) a drop in viral diversity at W72 for patients receiving placebo with a seroconversion (n = 7). The higher viral diversity in X was associated with higher baseline alanine aminotransferase (ALT) levels (p < .001). Patients with greater reduction of diversity at W8 of TDF treatment had higher baseline ALT levels. For placebo patients who seroconverted, the drop in viral diversity at W72 (p = .04) coincided with reduction of serum HBV DNA (average change from baseline = -4.10 log10 copies/ml) and unique combinations of variants were enriched in a patient's viral population post seroconversion. The basal core promoter (BCP) variants, A1762T and G1764A, and the pC variant, G1896A, were most often enriched at or after seroconversion.

Real-world safety and effectiveness of ledipasvir/sofosbuvir for the treatment of chronic hepatitis C virus genotype 1 in Japan.

As patients with chronic hepatitis C virus (HCV) tend to be older and/or have advanced liver disease in Japan, real-world data are needed to evaluate safe and effective treatment options. The study aim was to assess safety and effectiveness of ledipasvir/sofosbuvir (LDV/SOF) in a real-world cohort of Japanese patients with HCV genotype (GT) 1 infection overall and by patient subgroups: elderly, compensated cirrhotic, advanced fibrotic and those with hepatocellular carcinoma (HCC). A large prospective observational study was conducted, enrolling adult patients treated for HCV GT1 infection with LDV/SOF at clinical sites across Japan. Patients were observed for safety outcomes during and 4 weeks after treatment, and for sustained virologic response at 12-weeks post-treatment (SVR12). Incidence rates (IRs) of adverse drug reactions (ADRs) and serious ADRs (SADRs) and SVR12 rates were assessed overall and by subgroups. ADR and SADR IRs were low (2.26 and 0.17 per 100 person-months, respectively) and did not significantly differ in elderly patients or those with presence of compensated cirrhosis, worsening fibrosis or HCC. SVR12 rates were high overall (98.5%) and across subgroups investigated (≥94%), including patients who were elderly (98.2%), treatment-experienced (97.6%), advanced fibrotic (≥95.8%), had existing NS5A resistance-associated substitutions reported pre-treatment (95.0%), compensated cirrhosis (95.7%), HCC (94.0%) and other chronic liver diseases (96.1%). In this large, real-world observational study of Japanese patients with HCV GT1 infection, LDV/SOF treatment resulted in low incidence of adverse events, with high real-world effectiveness, even among patients with potentially higher risks of adverse safety outcomes and treatment failure.

HIV co-infection is associated with increased transmission risk in patients with chronic hepatitis C virus.

Molecular epidemiological analysis of viral pathogens can identify factors associated with increased transmission risk. We investigated the frequency of genetic clustering in a large data set of NS34A, NS5A, and NS5B viral sequences from patients with chronic hepatitis C virus (HCV). Within a subset of patients with longitudinal samples, Receiver Operator Characteristic (ROC) analysis was applied which identified a threshold of 0.02 substitutions/site as most appropriate for clustering. From the 7457 patients with chronic HCV infection included in this analysis, we inferred 256 clusters comprising 541 patients (7.3%). We found that HCV/HIV co-infection, young age, and high HCV viral load were all associated with increased clustering frequency, an indicator of increased transmission risk. In light of previous work on HCV/HIV co-infection in acute HCV cohorts, our results suggest that patients with HCV/HIV co-infection may disproportionately be the source of new HCV infections and treatment efforts should be geared towards viral elimination in this vulnerable population.

Identification of a Novel Hepatitis C Virus Genotype From Punjab, India: Expanding Classification of Hepatitis C Virus Into 8 Genotypes.

Background: Hepatitis C virus (HCV) exhibits great genetic diversity and is classified into 7 genotypes (GTs), with varied geographic prevalence. Until the recent development of pangenotypic direct-acting antiviral regimens, the determination of HCV GT was necessary to inform optimal treatment. Methods: Plasma samples with unresolved GT using standard commercial genotyping methods were subjected to HCV full-genome sequencing, and phylogenetic analysis was performed to assign GT. Results: Four patients, previously classified as GT5 by LiPA or Abbott RealTime polymerase chain reaction assays, were identified as infected with a novel HCV GT. This novel HCV GT, GT8, is genetically distinct from previously identified HCV GT1-7 with >30% nucleotide sequence divergence to the established HCV subtypes. All 4 patients were originally from Punjab, India, but now reside in Canada and are epidemiologically unlinked. Despite presence of baseline resistance-associated substitutions within the GT8 virus of all 4 patients (NS3: V36L, Q80K/R; NS5A: Q30S, Y93S), all patients achieved a sustained virologic response; 2 treated with sofosbuvir/velpatasvir/voxilaprevir for 8 weeks, 1 with sofosbuvir/ledipasvir plus ribavirin for 24 weeks and 1 with sofosbuvir plus daclatasvir for 12 weeks. Conclusions: The discovery of a novel HCV GT8 confirms the circulation of this newly identified lineage in the human population.

Resistance analysis in patients with genotype 1-6 HCV infection treated with sofosbuvir/velpatasvir in the phase III studies.

BACKGROUND & AIMS: The fixed-dose combination of sofosbuvir/velpatasvir was highly efficacious in patients infected with genotype (GT)1-6 hepatitis C virus (HCV) in the ASTRAL studies. This analysis evaluated the impact of baseline resistance-associated substitutions (RASs) on treatment outcome and emergence of RASs in patients infected with HCV GT1-6 who were treated with sofosbuvir/velpatasvir. METHODS: Non-structural protein 5A and 5B (NS5A and NS5B) deep sequencing was performed at baseline and at the time of relapse for all patients treated with sofosbuvir/velpatasvir for 12 weeks (n = 1,778) in the ASTRAL-1-3, ASTRAL-5 and POLARIS-2-3 studies. RESULTS: Patients with 37 known and 19 novel HCV subtypes were included in these analyses. Overall, 28% (range 9% to 61% depending on genotype) had detectable NS5A class RASs at baseline, using a 15% sequencing assay cut-off. There was no significant effect of baseline NS5A class RASs on sustained virologic response at week 12 (SVR12) with sofosbuvir/velpatasvir; the SVR12 rate in the presence of NS5A class RASs was 100% and 97%, in patients with GT1a and GT1b infection, respectively, and 100% in patients with GT2 and GT4-6 infections. In GT3 infection, the SVR rate was 93% and 98% in patients with and without baseline NS5A class RASs, respectively. The overall virologic failure rate was low (20/1,778 = 1.1%) in patients treated with sofosbuvir/velpatasvir. Single NS5A class resistance was observed at virologic failure in 17 of the 20 patients. CONCLUSIONS: Sofosbuvir/velpatasvir taken for 12 weeks once daily resulted in high SVR rates in patients infected with GT1-6 HCV, irrespective of baseline NS5A RASs. NS5A inhibitor resistance, but not sofosbuvir resistance, was detected in the few patients with virologic failure. These data highlight the high barrier to resistance of this regimen for the treatment of chronic HCV across all genotypes in the vast majority of patients. LAY SUMMARY: Sofosbuvir/velpatasvir taken once daily for 12 weeks resulted in high sustained virologic response rates in patients infected with HCV, irrespective of the presence of NS5A resistance-associated variants prior to treatment. Single class NS5A inhibitor resistance, but not sofosbuvir resistance, was detected in the few patients with virologic failure. These data highlight the high barrier to resistance of this regimen for the treatment of chronic HCV across all genotypes in the vast majority of patients.

Identification of hepatitis C virus 2k/1b intergenotypic recombinants in Georgia.

BACKGROUND AND AIMS: This study aimed to evaluate the prevalence of the hepatitis C virus intergenotype recombinant strain RF1_2k/1b in Georgia, confirm viral recombination by full genome sequencing, and determine a genetic relationship with previously described recombinant hepatitis C viruses. METHODS: We retrospectively analysed data from 1421 Georgian patients with chronic hepatitis C. Genotyping was performed with the INNO-LiPA VERSANT HCV Genotype 2.0 Assay. RESULTS: Virus isolates were assigned to nonspecific hepatitis C genotypes 2a/2c (n = 387) as performed by sequencing of core and NS5B genes. Subsequently, sequencing results classified the core region as genotype 2k and the NS5B region as genotype 1b for 72% (n = 280) of genotype 2 patients, corresponding to 19.7% of hepatitis C patients in Georgia. Eight samples were randomly selected for full genome sequencing which was successful in 7 of 8 samples. Analysis of the generated consensus sequences confirmed that all 7 viruses were 2k/1b recombinants, with the recombination breakpoint located within 73-77 amino acids before the NS2-NS3 junction, similar to the previously described RF1_2k/1b virus. Phylogenetic analysis revealed clustering of the Georgian 2k/1b viruses and RF1_2k/1b, suggesting that they are genetically related. CONCLUSIONS: The 19.7% prevalence of RF1_2k/1b in Georgia patients is far higher than has generally been reported to date worldwide. Identification of recombinants in low income countries with a high prevalence of HCV infection might be reasonable for choosing the most cost-effective treatment regimens.

Late Relapse Versus Hepatitis C Virus Reinfection in Patients With Sustained Virologic Response After Sofosbuvir-Based Therapies.

BACKGROUND: The development of direct-acting antivirals in recent years has dramatically enhanced rates of viral eradication to >90% in patients with chronic hepatitis C virus (HCV) infection. To determine true treatment efficacy and define the most appropriate retreatment, it is important to distinguish virologic relapse from reinfection when patients in whom HCV is eradicated during treatment become infected with a new HCV strain after treatment. METHODS: We investigated the prevalence of late recurrent viremia (patients with sustained virologic response 12 weeks after the end of treatment but detectable HCV RNA at follow-up week 24) and used refined phylogenetic analysis of multiple HCV genes to distinguish virologic relapse from reinfection. RESULTS: Across 11 phase 3 clinical trials of ledipasvir-sofosbuvir (SOF) and SOF, only 12 of 3004 patients had detectable HCV RNA following sustained virologic response 12 weeks after the end of treatment. Of these 12 patients with late recurrent viremia, 11 had the same HCV genotype/subtype at baseline and at recurrence. Phylogenetic analysis demonstrated that 58% (7 of 12) of these patients were successfully treated with the SOF-based regimen, with HCV eradication achieved, but became reinfected with a different HCV strain after treatment. The remaining 5 patients with late recurrent viremia had virologic relapse in which the HCV present at baseline persisted in the liver or another compartment and reemerged in the blood 24 weeks after treatment. CONCLUSIONS: The incidence of late recurrent viremia was low. Distinguishing reinfection from virologic relapse has implications for determining true treatment efficiency and selecting optimal retreatment strategies.

Global epidemiology of HCV subtypes and resistance-associated substitutions evaluated by sequencing-based subtype analyses.

BACKGROUND & AIMS: HCV genotype, subtype, and presence of resistance-associated substitutions (RASs) are key determinants for the selection of direct-acting antiviral (DAA) treatment regimens. However, current HCV genotyping assays have limitations in differentiating between HCV subtypes, and RAS prevalence is largely undefined. The aim of this study was to investigate HCV epidemiology in 12,615 patient samples from 28 different countries across five geographic regions. METHODS: We compared HCV genotype and subtypes using INNO-LiPA 2.0 vs. amplicon sequencing among 8,945 patients from phase II/III clinical trials of DAAs. Global HCV molecular epidemiology in 12,615 patients was investigated. Subtype RAS prevalence was determined by population or deep sequencing, and phylogenetic analyses investigating subtype diversity were performed. RESULTS: Although there was high concordance between INNO-LiPA and sequencing for genotype determination, INNO-LiPA was insufficient for subtype determination for genotype 2, 3, 4, and 6. Sequencing provided subtype refinement for 42%, 10%, 81%, and 78% of genotype 2, 3, 4, and six patients, respectively. Genotype discordance (genotype 2-genotype 1) was observed in 28 of 950 (3%) genotype 2 patients, consistent with inter-genotype recombinants. Sequencing-based analyses demonstrated variations in regional subtype prevalence, notably within genotype 2, 4 and 6. RAS prevalence varied by subtype, with the clinically relevant NS3 RAS Q80K found in genotype 1a, 5a and 6a and the NS5A RAS Y93H in genotype 1b, 3a, 4b, 4r and 7. CONCLUSIONS: Together, these analyses provide an understanding of subtyping accuracy and RAS distribution that are crucial for the implementation of global HCV treatment strategies. LAY SUMMARY: Hepatitis C virus (HCV) is highly variable, with seven genotypes and 67 subtypes characterized to date. The aim of this study was to i) compare two different methods of discriminating between genotypes; ii) investigate the prevalence of HCV subtypes for each genotype around the world; iii) find the prevalence of resistance-associated substitutions (RASs) in different subtypes. We found that both methods showed high concordance in genotype discrimination, but specific subtypes were not always identified accurately. Sequencing-based analyses demonstrated variations in regional subtype prevalence for some genotypes, notably within GT2, 4 and 6. RAS prevalence also varied by subtype. These variations could determine how successful different drugs are for treating HCV.

NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome.

BACKGROUND & AIMS: The efficacy of NS5A inhibitors for the treatment of patients chronically infected with hepatitis C virus (HCV) can be affected by the presence of NS5A resistance-associated substitutions (RASs). We analyzed data from 35 phase I, II, and III studies in 22 countries to determine the pretreatment prevalence of various NS5A RASs, and their effect on outcomes of treatment with ledipasvir-sofosbuvir in patients with genotype 1 HCV. METHODS: NS5A gene deep sequencing analysis was performed on samples from 5397 patients in Gilead clinical trials. The effect of baseline RASs on sustained virologic response (SVR) rates was assessed in the 1765 patients treated with regimens containing ledipasvir-sofosbuvir. RESULTS: Using a 15% cut-off, pretreatment NS5A and ledipasvir-specific RASs were detected in 13% and 8% of genotype 1a patients, respectively, and in 18% and 16% of patients with genotype 1b. Among genotype 1a treatment-naïve patients, SVR rates were 91% (42/46) vs. 99% (539/546) for those with and without ledipasvir-specific RASs, respectively. Among treatment-experienced genotype 1a patients, SVR rates were 76% (22/29) vs. 97% (409/420) for those with and without ledipasvir-specific RASs, respectively. Among treatment-naïve genotype 1b patients, SVR rates were 99% for both those with and without ledipasvir-specific RASs (71/72 vs. 331/334), and among treatment-experienced genotype 1b patients, SVR rates were 89% (41/46) vs. 98% (267/272) for those with and without ledipasvir-specific RASs, respectively. CONCLUSIONS: Pretreatment ledipasvir-specific RASs that were present in 8-16% of patients have an impact on treatment outcome in some patient groups, particularly treatment-experienced patients with genotype 1a HCV. LAY SUMMARY: The efficacy of treatments using NS5A inhibitors for patients with chronic hepatitis C virus (HCV) infection can be affected by the presence of NS5A resistance-associated substitutions (RASs). We reviewed results from 35 clinical trials where patients with genotype 1 HCV infection received treatments that included ledipasvir-sofosbuvir to determine how prevalent NS5A RASs are in patients at baseline, and found that ledipasvir-specific RASs were present in 8-16% of patients prior to treatment and had a negative impact on treatment outcome in subset of patient groups, particularly treatment-experienced patients with genotype 1a HCV.

Hepatitis C Virus RNA Persists in Liver Explants of Most Patients Awaiting Liver Transplantation Treated With an Interferon-Free Regimen.

We assessed the presence of hepatitis C virus (HCV) RNA in liver explants from 39 patients awaiting liver transplantation who were treated with an interferon-free regimen and had undetectable serum HCV RNA at the time of liver transplantation. Interestingly, HCV RNA was detected in most liver explants (67%). Patients with HCV RNA-positive explants had received shorter courses of treatment, and HCV RNA was undetectable in serum for shorter periods before transplantation compared to patients with HCV RNA-negative explants (P = .014 and P = .013, respectively). Levels of HCV RNA in explants were significantly higher in patients with a relapse of HCV infection than patients who responded to treatment (P = .016), but most patients (85%) with residual HCV-RNA in the explant achieved a sustained virologic response after receiving their liver transplant.

L159F and V321A Sofosbuvir-Associated Hepatitis C Virus NS5B Substitutions.

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.

Characterization of hepatitis C virus intergenotypic recombinant strains and associated virological response to sofosbuvir/ribavirin.

UNLABELLED: To date, intergenotypic recombinant hepatitis C viruses (HCVs) and their treatment outcomes have not been well characterized. This study characterized 12 novel HCV recombinant strains and their response to sofosbuvir in combination with ribavirin (SOF/RBV) treatment. Across the phase II/III studies of SOF, HCV samples were genotyped using both the Siemens VERSANT HCV Genotype INNO-LiPA 2.0 Assay (Innogenetics, Ghent, Belgium) and nonstructural (NS)5B sequencing. Among these patient samples, genotype assignment discordance between the two methods was found in 0.5% of all cases (12 of 2,363), of which all were identified as genotype 2 by INNO-LiPA (12 of 487; 2.5%). HCV full-genome sequences were obtained for these 12 samples by a sequence-independent amplification method coupled with next-generation sequencing. HCV full-genome sequencing revealed that these viruses were recombinant HCV strains, with the 5' part corresponding to genotype 2 and the 3' part corresponding to genotype 1. The recombination breakpoint between genotypes 2 and 1 was consistently located within 80 amino acids of the NS2/NS3 junction. Interestingly, one of the recombinant viruses had a 34-amino-acid duplication at the location of the recombination breakpoint. Eleven of these twelve patients were treated with a regimen for genotype 2 HCV infection, but responded as if they had genotype 1 infection; 1 patient had received placebo. CONCLUSION: Twelve new HCV intergenotypic recombinant genotype 2/1 viruses have been characterized. The antiviral response to a 12- to 16-week course of SOF/RBV treatment in these patients was more similar to responses among genotype 1 patients than genotype 2 patients, consistent with their genotype 1 NS5B gene.

Genotype- and Subtype-Independent Full-Genome Sequencing Assay for Hepatitis C Virus.

Hepatitis C virus (HCV) exhibits a high genetic diversity and is classified into 6 genotypes, which are further divided into 66 subtypes. Current sequencing strategies require prior knowledge of the HCV genotype and subtype for efficient amplification, making it difficult to sequence samples with a rare or unknown genotype and/or subtype. Here, we describe a subtype-independent full-genome sequencing assay based on a random amplification strategy coupled with next-generation sequencing. HCV genomes from 17 patient samples with both common subtypes (1a, 1b, 2a, 2b, and 3a) and rare subtypes (2c, 2j, 3i, 4a, 4d, 5a, 6a, 6e, and 6j) were successfully sequenced. On average, 3.7 million reads were generated per sample, with 15% showing HCV specificity. The assembled consensus sequences covered 99.3% to 100% of the HCV coding region, and the average coverage was 6,070 reads/position. The accuracy of the generated consensus sequence was estimated to be >99% based on results from in vitro HCV replicon amplification, with the same extrapolated amount of input RNA molecules as that for the patient samples. Taken together, the HCV genomes from 17 patient samples were successfully sequenced, including samples with subtypes that have limited sequence information. This method has the potential to sequence any HCV patient sample, independent of genotype or subtype. It may be especially useful in confounding cases, like those with rare subtypes, intergenotypic recombination, or multiple genotype infections, and may allow greater insight into HCV evolution, its genetic diversity, and drug resistance development.

Remdesivir for COVID-19 in Hospitalized Children: A Phase 2/3 Study.

OBJECTIVES: Remdesivir decreases the risk of SARS-CoV-2 infection progressing to severe disease in adults. This study evaluated remdesivir safety and pharmacokinetics in infants and children. METHODS: This was a phase 2/3, open-label trial in children aged 28 days to 17 years hospitalized for polymerase chain reaction-confirmed SARS-CoV-2 infection. Participants received for ≤10 days once-daily intravenous remdesivir doses defined using physiologically based pharmacokinetic modeling (for ≥40 kg, 200 mg day 1, then 100 mg/day; for age ≥28 days and ≥3 to <40 kg, 5 mg/kg day 1, then 2.5 mg/kg/day). Sparse pharmacokinetic samples were analyzed using population-pharmacokinetic approaches for remdesivir and metabolites GS-704277 and GS-441524. RESULTS: Among 53 participants, at enrollment the median (Q1, Q3) number of days of COVID-19 symptoms was 5 (3, 7) and hospitalization was 1 (1, 3). Underlying conditions included obesity in 19 (37%), asthma in 11 (21%), and cardiac disorders in 11 (21%). Median duration of remdesivir treatment was 5 days (range, 1-10). Remdesivir treatment had no new apparent safety trends. Two participants discontinued treatment because of adverse events including elevated transaminases; both had elevated transaminases at baseline. Three deaths occurred during treatment (and 1 after). When compared with phase 3 adult data, estimated mean pediatric parameters (area under the concentration-time curve over 1 dosing interval, AUCτ, Cmax, and Cτ) were largely overlapping but modestly increased (remdesivir, 33%-129%; GS-704277, 37%-124%; GS-441524, 0%-60%). Recovery occurred for 62% of participants on day 10 and 83% at last assessment. CONCLUSIONS: In infants and children with COVID-19, the doses of remdesivir evaluated provided drug exposure similar to adult dosing. In this study with a small sample size, no new safety concerns were observed.

No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Consistent Effects of Early Remdesivir on Symptoms and Disease Progression Across At-Risk Outpatient Subgroups: Treatment Effect Heterogeneity in PINETREE Study.

INTRODUCTION: In the PINETREE study, early remdesivir treatment reduced risk of coronavirus disease 2019 (COVID-19)-related hospitalizations or all-cause death versus placebo by 87% by day 28 in high-risk, non-hospitalized patients. Here we report results of assessment of heterogeneity of treatment effect (HTE) of early outpatient remdesivir, focusing on time from symptom onset and number of baseline risk factors (RFs). METHODS: PINETREE was a double-blind, placebo-controlled trial of non-hospitalized patients with COVID-19 who were randomized within 7 days of symptom onset and had ≥ 1 RF for disease progression (age ≥ 60 years, obesity [body mass index ≥ 30], or certain coexisting medical conditions). Patients received remdesivir intravenously (200 mg on day 1 and 100 mg on days 2 and 3) or placebo. RESULTS: In this subgroup analysis, HTE of remdesivir by time from symptom onset at treatment initiation and number of baseline RFs was not detected. Treatment with remdesivir reduced COVID-19-related hospitalizations independent of stratification by time from symptom onset to randomization. Of patients enrolled ≤ 5 days from symptom onset, 1/201 (0.5%) receiving remdesivir and 9/194 (4.6%) receiving placebo were hospitalized (hazard ratio [HR] 0.10; 95% confidence interval [CI] 0.01-0.82). Of those enrolled at > 5 days from symptom onset, 1/78 (1.3%) receiving remdesivir and 6/89 (6.7%) receiving placebo were hospitalized (HR 0.19; 95% CI 0.02-1.61). Remdesivir was also effective in reducing COVID-19-related hospitalizations when stratified by number of baseline RFs for severe disease. Of patients with ≤ 2 RFs, 0/159 (0.0%) receiving remdesivir and 4/164 (2.4%) receiving placebo were hospitalized; of those with ≥ 3 RFs, 2/120 (1.7%) receiving remdesivir and 11/119 (9.2%) receiving placebo were hospitalized (HR 0.16; 95% CI 0.04-0.73). CONCLUSIONS: In the outpatient setting, benefit of remdesivir initiated within 7 days of symptoms appeared to be consistent across patients with RFs. Therefore, it may be reasonable to broadly treat patients with remdesivir regardless of comorbidities. TRIAL REGISTRATION: ClinicalTrials.gov number NCT04501952.

Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 That Exhibits Antiviral Efficacy in SARS-CoV-2-Infected African Green Monkeys.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 (2) into lung cells, thereby forming the bioactive triphosphate 2-NTP. 2-NTP, an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP. Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP, leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2, which supports development of 3 as a promising COVID-19 treatment.

Prevalence of resistance-associated substitutions and phylogenetic analysis of hepatitis C virus infection in Russia.

OBJECTIVES: Due to limited hepatitis C virus (HCV) sequence availability from patients in Russia, the relationship between subtypes and baseline resistance-associated substitutions (RAS) to direct antiretroviral treatment outcome is not fully understood. METHODS: Deep sequencing of HCV NS3, NS5A, and NS5B sequences was performed on plasma HCV samples from 412 direct-acting antiviral (DAA)-naïve patients from Russia. Phylogenetic analysis was performed to investigate sequence similarities between HCV strains from Russia, Asia, Europe, and North America. Pretreatment HCV RAS was assessed with a 15% cutoff. RESULTS: HCV genotype GT1b and GT3a sequences in Russia were related to strains in Europe and Asia. The prevalence of GT1a and GT2a was low in Russia. In GT1b, the prevalence of NS5A Y93H was lower in Russia (6%) compared with Asia (15%). The prevalence of NS5B L159F was similar between Russia and Europe (26-39%). GT3a RAS prevalence was similar between Russia and Asia, Europe, and North America. The 2k/1b recombinant strain in Russia was related to strains from Europe. A higher prevalence of the NS5A RAS L31M (10%) was observed in 2k/1b sequences compared to GT1b (1-6%). CONCLUSIONS: The prevalence of RASs and the phylogenetic analysis showed similarities in HCV strains between Russia, Europe, and North America. This information may be useful for HCV regimens in Russia.