Molnupiravir Exhibits a High Barrier to the Development of SARS-CoV-2 Resistance in vitro

Background. Molnupiravir is an orally available prodrug of the antiviral nucleoside analog N-Hydroxycytidine (NHC). In preclinical studies NHC has shown broad-spectrum antiviral activity against multiple RNA viruses including SARS-CoV-2. Incorporation of NHC by viral polymerases impairs replication by introducing errors into the viral genome. NHC has been shown to have a high barrier to the development of resistance in vitro with RSV, Influenza and Venezualen Equine Encephalitis viruses. In these studies, we have explored the potential for SARS-CoV-2 to develop resistance to NHC in cell culture. Methods. Vero E6 cells were infected with SARS-CoV-2 (WA-1) in triplicate in the presence of NHC or a C3L-protease inhibitor (MRK-A). Culture supernatants from wells with the highest drug concentration exhibiting a cytopathic effect (CPE) score of>=2+ were repassaged and at each passage, IC50 values were estimated based on CPE scoring. At each passage, full genome next generation sequencing (NGS) was performed on the viral RNA Results. No change in susceptibility to NHC (EC50 fold change <= 1.1) was noted in 2 of 3 cultures and a 2-fold change was observed in one culture after 30 passages. In contrast, a 3- to 4-fold decreases in susceptibility to the 3CL protease inhibitor were seen by passage by 12, with increasing resistance of 4.6- to 15.7-fold observed by passage 30. NHC passaged viruses exhibited 53 to 99 amino acid changes, including substitutions and deletions (both in-frame and frameshift), across 25 different viral proteins as compared with 10 to 13 changes in 13 proteins in the MRK-A cultures. With NHC, 3 to 4 changes were observed in the viral polymerase;however, these were randomly distributed, and none were observed more than once. In contrast, the 3CL protease passaged virus had a nsp5 T21I substitution detected in all 3 cultures. Conclusion. No evidence of SARS-CoV-2 phenotypic or genotypic resistance was observed following 30 passages with NHC. A random pattern of amino acid changes were observed across multiple proteins consistent with the mechanism of action of NHC. In the same study, resistance was readily selected to a control 3CL protease inhibitor. Together these data support previous reports demonstrating the high barrier to resistance of NHC.

MOLNUPIRAVIR INCREASES RANDOM SARS-CoV-2 RNA ERRORS WITHOUT SELECTION for RESISTANCE

Background: Molnupiravir (MOV), the orally administered prodrug of the antiviral ribonucleoside analogue, N-hydroxycytidine (NHC) has received emergency use authorization for treatment of COVID-19. NHC inhibits viral replication by introduction of random transition errors across the viral genome, resulting in non-infectious virus. In the Phase II/III (MOVe-OUT) study, non-hospitalized participants received MOV or placebo (PBO) for 5 days and followed to Day 29. Viral RNA was sequenced to determine the rate, distribution and type of viral errors observed. Methods: SARS-CoV-2 RNA isolated from nasopharyngeal swabs was quantified by RT-PCR followed by complete genome NGS using the Ion AmpliSeq SARS-CoV-2 Research panel and Ion Torrent sequencing. To distinguish between nucleotide errors resulting from the mechanism of action of MOV and those potentially associated with reduced susceptibility to NHC, two different analyses were used. To measure impact of MOV on accumulation of low-frequency errors in the viral quasispecies, nucleotide variants were identified using VarScan 2.4 mutation caller with 0.4% minimum variant allele frequency cut-off. Resistance-associated changes were identified as amino acid substitutions occurring in D3 or D5 samples from ≥2 participants with a frequency of ≥5% of NGS reads. Phenotypic analysis of selected amino acid substitutions was performed using a replicon model. Results: NGS results showed a relationship between the number of random errors across the viral genome with increasing MOV dose. By Day 5 the mean number of viral genome errors were 21, 83, 129 and 223 in the PBO, 200, 400 and 800 mg groups, respectively. Among the sequence changes observed, the majority were transitions errors, consistent with MOV's mechanism of action. After MOV treatment, few treatment-emergent amino acid substitutions were identified in the viral replicase genes. These included nsp12 (T731I) and nsp14 (A220S/T/V, V466I, S503L/P);none associated with loss of susceptibility to MOV. Changes in spike protein in both PBO and MOV groups were at sites previously described in circulating variants. Conclusion: Consistent with the mechanism of action, MOV treatment resulted in a dose-dependent increase in transition errors across the SARS-CoV-2 genome. No resistance-associated mutations were identified in the viral replicase and no evidence that MOV treatment selected for unique mutations in spike protein not previously observed in circulating variants.

Treatment with Molnupiravir in the MOVe-In and MOVe-Out Clinical Trials Results in an Increase in Transition Mutations Across the SARS-CoV-2 Genome

Background. Molnupiravir (MOV), (MK-4482, EIDD-2801) is being clinically developed for the treatment of COVID-19 disease caused by SARS-CoV-2. MOV is the orally administered 5-isobutyrate prodrug of the active, antiviral ribonucleoside analogue, N-hydroxycytidine (NHC, EIDD-1931) which inhibits viral replication by induction of mutations in the viral genome, leading to viral error catastrophe. In 2 clinical studies, hospitalized (MOVe-In) and non-hospitalized (MOVe-Out) participants were treated for 5 days with MOV and followed up to Day 29. Viral RNA isolated from nasal swab samples were sequenced to determine the rate, distribution and type of viral mutations observed after MOV treatment. Methods. RNA isolated from nasopharangeal swab samples collected during study conduct was quantified by RT-PCR. Samples containing >22,000 copies/mL of RNA underwent complete genome NGS using the Ion AmpliSeq SARS-CoV-2 research panel and Ion Torrent sequencing. Mutation rates were calculated by determining the number of nucleotide changes observed across the entire genome at Day 3 and/ or Day 5 compared to baseline. Results. Combined data from both studies showed an increase of ~2-4 fold in the viral mutation rate post-baseline in MOV treated compared with placebo. Mutations were distributed across the entire genome with only a minority being observed in more than one sample. The most frequent mutations were transitions of C to U observed in the highest MOV dose group (800 mg/BID). Conclusion. Consistent with the proposed mechanism of action of MOV, an increase in the rate of transition mutations in the virus was observed in post-baseline nasal swab samples from participants treated with MOV compared with placebo.

Molnupiravir Maintains Antiviral Activity Against SARS-CoV-2 Variants in Vitro and in Early Clinical Studies

Background. Molnupiravir (MOV, MK-4482, EIDD-2801) is an orally administered prodrug of N-hydroxycytidine (NHC, EIDD-1931), a nucleoside with broad antiviral activity against a range of RNA viruses. MOV acts by driving viral error catastrophe following its incorporation by the viral RdRp into the viral genome. Given its mechanism of action, MOV activity should not be affected by substitutions in the spike protein present in SARS-CoV-2 variants of concern which impact efficacy of therapeutic neutralizing antibodies and vaccine induced immunity. We characterized MOV activity against variants by assessing antiviral activity in vitro and virologic response from the Phase 2/3 clinical trials (MOVe-In, MOVe-Out) for treatment of COVID-19. Methods. MOV activity against several SARS-CoV-2 variants, was evaluated in an in vitro infection assay. Antiviral potency of NHC (IC50) was determined in Vero E6 cells infected with virus at MOI ~0.1 by monitoring CPE. Longitudinal SARSCoV-2 RNA viral load measures in participants enrolled in MOVe-In and MOVe-Out were analyzed based on SARS-CoV-2 genotype. Sequences of SARS-CoV-2 from study participants were amplified from nasal swabs by PCR and NGS was performed on samples with viral genome RNA of >22,000 copies/ml amplified by primers covering full length genome with Ion Torrent sequencing to identify clades represented in trial participants. SARS-CoV-2 clades were assigned using clade.nextstrain.org. Results. In vitro, NHC was equally effective against SARS-CoV-2 variants B.1.1.7 (20I), B.1351 (20H), and P1 (20J), compared with the original WA1 (19B) isolate. In clinical trials, no discernable difference was observed in magnitude of viral response measured by change from baseline in RNA titer over time across all clades represented including 20A through 20E and 20G to 20I. No participants at the time of the study presented with 20F, 20J, or 21A. Conclusion. Distribution of clades in participants in MOVe-In and MOVe-Out was representative of those circulating globally at the time of collection (Oct 2020 -Jan 2021). Both in vitro and clinical data suggest that spike protein substitutions do not impact antiviral activity of MOV and suggest its potential use for the treatment of SARS-CoV-2 variants.