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Genetic variation of SARS-CoV-2 has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern or interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV, VEKLURY(R)) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein ELISA and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with EC50 values 0.31 to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.15 to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.
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The nucleoside analog remdesivir (RDV) is an FDA-approved antiviral for the treatment of SARS- CoV-2 infections, and as such it is critical to understand potential genetic determinants and barriers to RDV resistance. In this study, SARS-CoV-2 was subjected to 13 passages in cell culture with increasing concentrations of GS-441524, the parent nucleoside of RDV. At passage 13 the RDV resistance of the lineages ranged from 2.7-to 10.4-fold increase in EC50. Sequence analysis of the three lineage populations identified non-synonymous mutations in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I and C799F/R. Two of the three lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first then combined with S759A. Introduction of the S759A and V792I substitutions at homologous nsp12 positions in viable isogenic clones of the betacoronavirus murine hepatitis virus (MHV) demonstrated their transferability across CoVs, up to 38-fold RDV resistance in combination, and a significant replication defect associated with their introduction. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a [~]10- fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, while nsp12-V792I diminished the UTP concentration needed to overcome the template-dependent inhibition associated with RDV. The in vitro selected substitutions here identified were rare or not detected in the >6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in various clinical settings. One Sentence SummarySARS-CoV-2 develops in vitro resistance to remdesivir by distinct and complementary mutations and mechanisms in the viral polymerase
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The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.
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There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35{degrees}C than 37{degrees}C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN ({beta}1 and {lambda}1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.
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Improving the standard of clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAb) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of COVID-19. However, it is not known if combination RDV/mAb will improve outcomes over single agent therapies or whether antibody therapies will remain efficacious against variants. In kinetic studies in a mouse-adapted model of ancestral SARS-CoV-2 pathogenesis, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 hours after infection. The same antibody combination was also effective in prevention and therapy against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared to single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and support the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.
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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.
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A high-throughput platform would greatly facilitate COVID-19 serological testing and antiviral screening. Here we report a nanoluciferase SARS-CoV-2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. We demonstrate that the optimized reporter virus assay in Vero E6 cells can be used to measure neutralizing antibody activity in patient sera and produces results in concordance with a plaque reduction neutralization test (PRNT). Compared with the low-throughput PRNT (3 days), the SARS-CoV-2-Nluc assay has substantially shorter turnaround time (5 hours) with a high-throughput testing capacity. Thus, the assay can be readily deployed for large-scale vaccine evaluation and neutralizing antibody testing in humans. Additionally, we developed a high-throughput antiviral assay using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2). When tested against this reporter virus, remdesivir exhibited substantially more potent activity in A549-hACE2 cells compared to Vero E6 cells (EC50 0.115 vs 1.28 M), while this difference was not observed for chloroquine (EC50 1.32 vs 3.52 M), underscoring the importance of selecting appropriate cells for antiviral testing. Using the optimized SARS-CoV-2-Nluc assay, we evaluated a collection of approved and investigational antivirals and other anti-infective drugs. Nelfinavir, rupintrivir, and cobicistat were identified as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 M). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 M. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 M). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 M) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.
