The Long View on COVID-19 Theranostics and Oral Antivirals: Living with Endemic Disease and Lessons from Molnupiravir

Abstract Background New treatments are needed to reduce the risk of progression of coronavirus disease 2019 (Covid-19). Molnupiravir is an oral, small-molecule antiviral prodrug that is active against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods We conducted a phase 3, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of treatment with molnupiravir started within 5 days after the onset of signs or symptoms in nonhospitalized, unvaccinated adults with mild-to-moderate, laboratory-confirmed Covid-19 and at least one risk factor for severe Covid-19 illness. Participants in the trial were randomly assigned to receive 800 mg of molnupiravir or placebo twice daily for 5 days. The primary efficacy end point was the incidence hospitalization or death at day 29; the incidence of adverse events was the primary safety end point. A planned interim analysis was performed when 50% of 1550 participants (target enrollment) had been followed through day 29. Results A total of 1433 participants underwent randomization; 716 were assigned to receive molnupiravir and 717 to receive placebo. With the exception of an imbalance in sex, baseline characteristics were similar in the two groups. The superiority of molnupiravir was demonstrated at the interim analysis; the risk of hospitalization for any cause or death through day 29 was lower with molnupiravir (28 of 385 participants [7.3%]) than with placebo (53 of 377 [14.1%]) (difference, −6.8 percentage points; 95% confidence interval, −11.3 to −2.4; P=0.001). In the analysis of all participants who had undergone randomization, the percentage of participants who were hospitalized or died through day 29 was lower in the molnupiravir group than in the placebo group (6.8% [48 of 709] vs. 9.7% [68 of 699]; difference, −3.0 percentage points; 95% confidence interval, −5.9 to −0.1). Results of subgroup analyses were largely consistent with these overall results; in some subgroups, such as patients with evidence of previous SARS-CoV-2 infection, those with low baseline viral load, and those with diabetes, the point estimate for the difference favored placebo. One death was reported in the molnupiravir group and 9 were reported in the placebo group through day 29. Adverse events were reported in 216 of 710 participants (30.4%) in the molnupiravir group and 231 of 701 (33.0%) in the placebo group. Conclusions Early treatment with molnupiravir reduced the risk of hospitalization or death in at-risk, unvaccinated adults with Covid-19. (Funded by Merck Sharp and Dohme; MOVe-OUT ClinicalTrials.gov number, NCT04575597.)

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β- d - N 4 -hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir. Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.

To the Editor: In November 2021, the B.1.1.529 (omicron) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in South Africa. 1 Since then, omicron has rapidly spread around the world. On November 26, 2021, the World Health Organization designated omicron as a variant of concern. The omicron variant was found to have at least 33 mutations (29 amino acid substitutions, 1 insertion of three amino acids, and 3 small deletions) in its spike (S) protein, as compared with early SARS-CoV-2 strains identified in Wuhan, China. 2 Notably, 15 of the 29 substitutions were in the receptor-binding domain of the S protein, which is the primary target for monoclonal antibody–based therapy. This finding suggests that the monoclonal antibodies that have been approved by the Food and Drug Administration (FDA) may be less effective against the omicron variant. Accordingly, we examined the neutralizing ability of FDA-approved and investigational therapeutic monoclonal antibodies (individually and in combination) against omicron and other variants of concern. Using a live-virus focus reduction neutralization assay (FRNT), we assessed the neutralizing activities of monoclonal antibodies against hCoV-19/Japan/NC928-2N/2021 (omicron; NC928), which was isolated from a traveler who arrived in Japan from Namibia; SARS-CoV-2/UT-NC002-1T/Human/2020/Tokyo (NC002), an early SARS-CoV-2 strain from February 2020; SARS-CoV-2/UT-HP127-1Nf/Human/2021/Tokyo (alpha; HP127); hCoV-19/USA/MD-HP01542/2021 (beta; HP01542); hCoV-19/Japan/TY7-503/2021 (gamma; TY7-503); and hCoV-19/USA/WI-UW-5250/2021 (delta; UW5250). Whole-genome sequencing analysis of the NC928 omicron virus stock revealed that the variant had the 15 substitutions that are characteristic of omicron in the receptor-binding domain of the S protein, as compared with the Wuhan/Hu-1/2019 reference strain (Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org). We validated the reactivity of all seven monoclonal antibodies by means of enzyme-linked immunosorbent assay (ELISA) coated with recombinant S protein derived from the early Wuhan reference strain, as well as from representative alpha, beta, gamma, and delta variants. The results were consistent with published data 3 (Table S2). These monoclonal antibodies neutralized the early strain (NC002) and the alpha (HP127) and delta (UW5250) variants with a low FRNT50 value (1.34 to 150.38 ng per milliliter), except for LY-CoV555 (marketed as bamlanivimab), which showed markedly higher FRNT50 values against the delta variant than against the early strain and the alpha variant (Table 1). This result was consistent with a previous study that showed an almost complete loss of activity for bamlanivimab against the delta variant, whereas LY-CoV016 (marketed as etesevimab), REGN10987 (marketed as imdevimab), and REGN10933 (marketed as casirivimab) inhibited this variant. 4 Etesevimab did not neutralize the omicron (NC928), beta (HP01542), or gamma (TY7-503) variants even at the highest FRNT50 value (>50,000 ng per milliliter) that was tested. Bamlanivimab showed reduced neutralizing activity against the beta and gamma variants and did not neutralize omicron. Imdevimab had high neutralizing activity against the beta and gamma variants but lost activity against omicron. Casirivimab neutralized beta, gamma, and omicron with a high FRNT50 value (187.69 to 14,110.70 ng per milliliter); however, the FRNT50 value for omicron was higher by a factor of 18.6 than that for beta and higher by a factor of 75.2 than that for gamma. COV2-2196 (marketed as tixagevimab), COV2-2130 (marketed as cilgavimab), and S309 (precursor of drug marketed as sotrovimab) also retained neutralizing activity against beta, gamma, and omicron; however, the FRNT50 values of these monoclonal antibodies were higher by a factor of 3.7 to 198.2 for omicron than for beta or gamma. All the combinations of monoclonal antibodies that were tested (i.e., etesevimab plus bamlanivimab, imdevimab plus casirivimab, and tixagevimab plus cilgavimab) neutralized the early strain and the alpha and delta variants. The combination of etesevimab plus bamlanivimab showed remarkably reduced neutralizing activity against gamma and lost neutralizing activity against omicron and beta. The imdevimab–casirivimab combination retained activity against beta and gamma but lost inhibitory capability against omicron. The tixagevimab–cilgavimab combination inhibited beta, gamma, and omicron; however, the FRNT50 values of this combination were higher by a factor of 24.8 to 142.9 for omicron than for beta or gamma, respectively. The omicron variant has mutations in both the RNA-dependent RNA polymerase (RdRp) and the main protease of SARS-CoV-2, which are targets for antiviral drugs such as RdRp inhibitors (remdesivir and molnupiravir) and the main protease inhibitor PF-07304814, 5 which arouses concern regarding the decreased effectiveness of these drugs against omicron. Thus, we tested three different antiviral compounds (i.e., remdesivir, molnupiravir, and PF-07304814) for their efficacy against omicron. The in vitro 50% inhibitory concentration (IC50) values of each compound were determined against NC928, NC002, HP127, HP01542, TY7-503, and UW5250. The susceptibilities of omicron to the three compounds were similar to those of the early strain (i.e., IC50 values for remdesivir, molnupiravir, and PF-07304814 that differed by factors of 1.2, 0.8, and 0.7, respectively) (Table 1). These results suggest that all three of these compounds may show efficacy for treating patients infected with the omicron variant. The potential limitations of our study include the lack of clinical data on the efficacy of these monoclonal antibodies and antiviral drugs for the treatment of patients infected with omicron. Additional studies are needed to determine whether these antiviral therapies are indeed effective against infection with the omicron variant. Collectively, our findings show that therapeutic options may be available to combat the omicron variant of SARS-CoV-2; however, some therapeutic monoclonal antibodies may not be effective against this variant.