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.

HIV-1 envelope diversity and sensitivity to broadly neutralizing antibodies across stages of acute HIV-1 infection.

We studied the relationship between viral diversity and susceptibility to broadly neutralizing antibodies (bNAbs) in longitudinal plasma and peripheral blood mononuclear cells from 89 people with HIV who initiated antiretroviral therapy (ART) during acute and early HIV-1 infection (AEHI). HIV-1 diversity and predicted bNAb susceptibility were comparable across AEHI. Diversity evolution was not observed during ART, suggesting (pro)viruses at initiation or during treatment may identify individuals with susceptible virus for bNAb interventional trials.

Evaluation of HIV-1 reservoir size and broadly neutralizing antibody susceptibility in acute antiretroviral therapy-treated individuals.

OBJECTIVE: Persistence of the viral reservoir is the main barrier to curing HIV. Initiation of ART during acute HIV infection can limit the size and diversity of the reservoir. In depth characterization of the reservoir in individuals who initiate ART during acute infection will be critical for clinical trial design and cure strategies. METHODS: Four cohorts with participants who initiated ART during acute infection or during chronic infection were enrolled in a cross-sectional, noninterventional study. Viral reservoir was evaluated by the Intact Proviral DNA Assay (IPDA), the Total HIV DNA Assay (THDA) and the Quantitative Viral Outgrowth Assay (QVOA). Viral diversity and susceptibility to V3-glycan bNAbs were determined by genotyping of the viral envelope gene. RESULTS: Participants who initiated ART during the acute Fiebig I-IV stages had lower level of total HIV DNA than participants who initiated ART during chronic infection whereas no difference was observed in intact HIV DNA or outgrowth virus. Participants who initiated ART during Fiebig I-IV also had lower viral diversity and appeared to have higher susceptibility to bNAbs than participants initiating ART during chronic infection. CONCLUSION: Individuals initiating ART during Fiebig I-IV had small viral reservoirs, low viral diversity, and high susceptibility to bNAbs, and would be an optimal target population for proof-of-concept HIV cure trials.

Evaluation of Broadly Neutralizing Antibody Sensitivity by Genotyping and Phenotyping for Qualifying Participants to HIV Clinical Trials.

BACKGROUND: HIV envelope (env) diversity represents a significant challenge for the use of broadly neutralizing antibodies (bNAbs) in HIV treatment and cure studies. Screening for viral sensitivity to bNAbs to select eligible trial participants will be important to improve clinical efficacy; however, no universal approach has been established. METHODS: Pre-antiretroviral therapy plasma virus from participants in the Zurich Primary HIV Infection (ZPHI) study was genotyped and phenotyped for sensitivity to the bNAbs elipovimab (EVM, formerly GS-9722) and 3BNC117. The genotyping and phenotyping assessments were performed following the Clinical Laboratory Improvement Amendments of 1988 guidelines as required for entry into clinical trials. The genotypic-based prediction of bNAb sensitivity was based on HIV env amino acid signatures identified from a genotypic-phenotypic correlation algorithm using a subtype B database. RESULTS: Genotyping the plasma virus and applying env sensitivity signatures, ZPHI study participants with viral sensitivity to EVM and 3BNC117 were identified. ZPHI study participants with virus sensitive to EVM and 3BNC117 were also identified by phenotyping the plasma virus. Comparison of the genotypic and phenotypic sensitivity assessments showed strong agreement between the 2 methodologies. CONCLUSIONS: The genotypic assessment was found to be as predictive as the direct measurement of bNAb sensitivity by phenotyping and may, therefore, be preferred because of more rapid turnaround time and assay simplicity. A significant number of the participants were predicted to have virus sensitive to EVM and 3BNC117 and could, thus, be potential participants for clinical trials involving these bNAbs.

Genetic conservation of SARS-CoV-2 RNA replication complex in globally circulating isolates and recently emerged variants from humans and minks suggests minimal pre-existing resistance to remdesivir.

Remdesivir (RDV) exhibits potent antiviral activity against SARS-CoV-2 and is currently the only drug approved for the treatment of COVID-19. However, little is currently known about the potential for pre-existing resistance to RDV and the possibility of SARS-CoV-2 genetic diversification that might impact RDV efficacy as the virus continue to spread globally. In this study, >90,000 SARS-CoV-2 sequences from globally circulating clinical isolates, including sequences from recently emerged United Kingdom and South Africa variants, and >300 from mink isolates were analyzed for genetic diversity in the RNA replication complex (nsp7, nsp8, nsp10, nsp12, nsp13, and nsp14) with a focus on the RNA-dependent RNA polymerase (nsp12), the molecular target of RDV. Overall, low genetic variation was observed with only 12 amino acid substitutions present in the entire RNA replication complex in ≥0.5% of analyzed sequences with the highest overall frequency (82.2%) observed for nsp12 P323L that consistently increased over time. Low sequence variation in the RNA replication complex was also observed among the mink isolates. Importantly, the coronavirus Nsp12 mutations previously selected in vitro in the presence of RDV were identified in only 2 isolates (0.002%) within all the analyzed sequences. In addition, among the sequence variants observed in ≥0.5% clinical isolates, including P323L, none were located near the established polymerase active site or sites critical for the RDV mechanism of inhibition. In summary, the low diversity and high genetic stability of the RNA replication complex observed over time and in the recently emerged SARS-CoV-2 variants suggests a minimal global risk of pre-existing SARS-CoV-2 resistance to RDV.

Switching to Bictegravir/Emtricitabine/Tenofovir Alafenamide (B/F/TAF) From Dolutegravir (DTG)+F/TAF or DTG+F/Tenofovir Disoproxil Fumarate (TDF) in the Presence of Pre-existing NRTI Resistance.

BACKGROUND: Study 4030 was a phase 3, randomized, double-blinded study of 565 HIV-1 RNA-suppressed participants switching to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) or dolutegravir (DTG)+F/TAF. Nucleoside reverse transcriptase inhibitor (NRTI), non-NRTI, and protease inhibitor resistance (-R) was allowed, but integrase strand transfer inhibitor-R was excluded. Here, we describe the detailed resistance analysis. METHODS: Historical plasma HIV-1 RNA genotypes and baseline proviral DNA genotypes were analyzed. Documented or investigator-suspected NRTI-R was grouped for stratification into 3 categories of level of resistance. Viral blips were assessed through week 48. Virologic failures had genotypic and phenotypic resistance analyses at week 48, confirmed failure, or last visit, if HIV-1 RNA did not resuppress to <50 copies/mL while on study drug. RESULTS: In total, 83% (470/565) of participants had baseline genotypic data available with NRTI-R detected in 24% (138/565), including 5% (30/565) with K65R/E/N or ≥3 thymidine analog mutations and 19% (108/565) with other NRTI-R mutations. M184V/I was present in 14% (81/565). Pre-existing integrase strand transfer inhibitor-R mutations were found in 4% (20/565) of participants. Primary non-NRTI-R and protease inhibitor-R mutations were present in 21% (118/565) and 7% (38/565) of participants. High rates of viral suppression were maintained in all groups through week 48; blips were observed in only 15 participants (2.7%). Three participants met criteria for resistance analysis (all in DTG+F/TAF arm); none developed treatment-emergent resistance to study drugs. CONCLUSIONS: Participants with baseline NRTI resistance, much of which was previously undocumented, maintained suppression 48 weeks after switching to B/F/TAF or DTG+F/TAF triple therapy. Blips and virologic failure were uncommon using either regimen, with no treatment-emergent resistance.

Sofosbuvir susceptibility of genotype 1 to 6 HCV from DAA-naïve subjects.

High sequence diversity of HCV may lead to variation in susceptibility to antiviral agents amongst different genotypes and subtypes of the virus. We assessed the susceptibility to sofosbuvir of chimeric replicons carrying the full length NS5B coding region from 479 HCV infected, treatment-naïve patients, including 15 subtypes in 6 genotypes. NS5B replicon vectors with subtype 1b, subtype 4a and subtype 6a backbone were modified to support testing of patient samples. We also evaluated sofosbuvir susceptibility in a panel of 331 replicons containing engineered NS5B inhibitor resistance-associated substitutions. The mean 50% effective sofosbuvir concentration (EC50) amongst different genotypes ranged from 32 (subtype 2a) to 130 nM (genotype 4); while some variation in susceptibility amongst patient isolates was observed, the 95th percentile for any genotype did not exceed 189 nM. Levels of resistance to sofosbuvir in replicons containing S282T were between 2.4 and 18 fold-change in EC50; no other single NS5B resistance-associated substitution demonstrated reduced sofosbuvir susceptibility. These data suggest that S282T is the only known substitution that confers detectable resistance to sofosbuvir in vitro. Sofosbuvir displayed potent antiviral activity across a diverse range of NS5B mutants and HCV clinical isolates in multiple subtypes of genotypes 1 to 6.

A web server for comparative analysis of single-cell RNA-seq data.

Single cell RNA-Seq (scRNA-seq) studies profile thousands of cells in heterogeneous environments. Current methods for characterizing cells perform unsupervised analysis followed by assignment using a small set of known marker genes. Such approaches are limited to a few, well characterized cell types. We developed an automated pipeline to download, process, and annotate publicly available scRNA-seq datasets to enable large scale supervised characterization. We extend supervised neural networks to obtain efficient and accurate representations for scRNA-seq data. We apply our pipeline to analyze data from over 500 different studies with over 300 unique cell types and show that supervised methods outperform unsupervised methods for cell type identification. A case study highlights the usefulness of these methods for comparing cell type distributions in healthy and diseased mice. Finally, we present scQuery, a web server which uses our neural networks and fast matching methods to determine cell types, key genes, and more.