These full virology data from pre-specified, exploratory analyses of the MOVe-OUT trial confirm the antiviral efficacy of molnupiravir against SARS-CoV-2 in patients with COVID-19 and are congruent with the previously reported partial virology results from this trial [27], as well as with corresponding observations from other clinical trials [35,36,37]. Molnupiravir consistently led to rapid reduction of infectious SARS-CoV-2. In participants with infectious SARS-CoV-2 isolated at baseline, none receiving molnupiravir subsequently had detectable infectious virus by day 3, whereas infectious SARS-CoV-2 was recovered from some placebo-arm participants up to the end-of-treatment visit. Molnupiravir also showed greater viral RNA reductions than placebo during the early viral replication period, up to study day 10. This finding is important, because SARS-CoV-2 loads at day 5 and day 10 post treatment initiation were previously identified as strong predictors of clinical outcomes in this high-risk patient population [38]. High SARS-CoV-2 RNA titers persisting through days 5 to 10 despite antiviral treatment are associated with an increased risk of hospitalization/death, as well as an increased probability of requiring mechanical ventilation or supplemental oxygen [38]. At later timepoints, on the other hand, viral RNA titers are of limited clinical relevance and generally due to prolonged shedding of RNA fragments unlikely to be associated with infectious virus [32, 33, 39,40,41]. Even in patients with persistently high viral load, infectious virus is generally not detected after day 10 of COVID-19 symptom onset [32, 40, 42, 43], and there is no known threshold SARS-CoV-2 RNA titer at later timepoints that suggests the presence of infectious virus. In our trial, SARS-CoV-2 RNA titers at day 15 and day 29 were low in both study arms (since viral loads naturally decline over time in immunocompetent patients) and were more comparable between arms at those timepoints. Of note, viral infectivity was not evaluated in samples with low RNA titers of < 105 copies/mL; this threshold was predefined on the basis of published data indicating that even with higher titers, infectious SARS-CoV-2 is infrequently isolated from samples with < 106 copies/mL [30, 33], something that we also observed in our own data. Overall, the virologic response to molnupiravir was consistent, irrespective of baseline viral load, presence of baseline SARS-CoV-2 antibodies baseline (indicating a humoral immune response against the virus), or SARS-CoV-2 clade.

In this updated, full virology dataset from MOVe-OUT, about two-thirds of participants had Delta variant sublineages confirmed by NGS, consistent with the increasing global prevalence of Delta during later phases of participant enrollment into MOVe-OUT (i.e., July–October 2021). Given the timing of the trial, the Omicron variant was not detected in any participant. However, data from in vitro studies confirmed that molnupiravir remains active against all Omicron sublineages evaluated to date [18, 44]. When the MOVe-OUT primary endpoint of all-cause hospitalization or death through day 29 was compared by baseline viral clade, molnupiravir generally performed better than placebo, especially among participants infected with Gamma and Mu. Only for the Delta variant was the treatment effect comparable between arms, driven by the markedly lower rate of hospitalization or death observed in the placebo arm among participants with clade 21J (Delta) compared with other common clades, i.e., 8% versus 16–20%, respectively. Of note, molnupiravir clearly maintained antiviral activity in this subgroup of participants with 21J (Delta); when COVID-19 caused by this Delta clade was treated, molnupiravir was associated with a greater reduction in mean change from baseline in SARS-CoV-2 RNA than placebo and also yielded rapid reduction in the number of participants with infectious virus at all post-baseline visits.

Importantly, it does not appear that molnupiravir had reduced clinical efficacy against the no longer circulating Delta variant. Rather, additional analyses of MOVe-OUT trial data suggest that the lower observed effect size with molnupiravir in the final versus the interim analysis coincided with the increasing predominance of Delta but was likely caused by the cumulative effect of minor differences in baseline characteristics known to be prognostic for progression to severe disease. These cumulative differences among the study population increasingly biased outcomes in favor of participants in the placebo arm, especially during the latter parts of the trial, as shown using multivariable logistic regression models [45]. Important examples of such shifts in prognostic baseline factors were greater proportions of participants in the molnupiravir arm ≥ 75 years old and/or with multiple risk factors (both characteristics increase the COVID-19 progression risk) after the interim analysis compared with the interim analysis population; a lower proportion of participants in the placebo arm with moderate COVID-19 (which also increases the risk of progression to severe COVID-19) after the interim analysis; and imbalances favoring placebo in the post-interim analysis cohort that were not present at the interim analysis (i.e., higher proportions of participants with low/undetectable SARS-CoV-2 RNA and/or with anti-SARS-CoV-2 antibodies at baseline, both known to protect from severe disease) [45].

MOVe-OUT is now the second randomized, controlled clinical trial showing rapid decrease of infectious SARS-CoV-2 with molnupiravir as assessed by plaque assay [37], a finding that was also demonstrated in multiple animal models [12,13,14]. Further evidence is needed to assess whether molnupiravir treatment has a clinically relevant impact on SARS-CoV-2 transmission. In MOVe-OUT, only one molnupiravir-treated participant had infectious SARS-CoV-2 isolated after end of treatment, but this participant did not have confirmed SARS-CoV-2 at baseline through day 5. Instead, the participant probably presented with symptoms of a metapneumovirus infection (which was PCR-confirmed at baseline) and a false-positive SARS-CoV-2 PCR result at screening (conducted locally pre-baseline), given that their centrally assessed nasopharyngeal samples did not test positive for SARS-CoV-2 on both qualitative and quantitative assays from baseline until day 10 (and then with a high viral load). The virologic findings observed in MOVe-OUT are also in line with another randomized, placebo-controlled trial, i.e., the AGILE CST-2 phase 2a study (n = 180 participants), which similarly reported greater decreases in viral load at the end of therapy with molnupiravir than placebo [35]. Molnupiravir also exhibited a consistent antiviral effect in the phase 2 component of MOVe-OUT [29].

Molnupiravir does not directly inhibit viral RNA replication by interfering with the activity of the viral polymerase. Instead, its metabolite NHC exerts its antiviral activity via viral error induction, which leads to the production of defective and/or non-infectious virus [12,13,14]. Since non-infectious viral RNA fragments not yet fully cleared from the nasal cavity can result in positive PCR tests, it is not surprising that molnupiravir- and placebo-treated participants had comparable rates of time to PCR negativity when samples were assessed through a sensitive qualitative SARS-CoV-2 PCR assay. The mechanism of viral error induction underlies NHC’s broad activity across SARS-CoV-2 variants (and other RNA viruses) and its demonstrated high barrier to the development of resistance [11,12,13,14,15,16,17,18,19,20,21,22,23]. Sequencing data confirmed that viral error rates across the SARS-CoV-2 genome were higher with molnupiravir than placebo: these nucleotide errors were primarily C-to-U and G-to-A transitions and randomly distributed throughout the viral genome (including across genes encoding for structural and non-structural proteins), as predicted by molnupiravir’s mechanism of action [24, 25]. The same observations were also reported from the AGILE CST-2 trial, in which molnupiravir treatment similarly resulted in a randomly distributed statistically significant increase in the transition: transversion error ratio and was not associated with selection or accumulation of nucleotide errors at specific gene locations, including no apparent induction of potential resistance mutations [36]. In the MOVe-OUT trial, only a small number of molnupiravir-treated participants had treatment-emergent amino acid changes in the viral replicase complex proteins; none of these observed changes (which were likely induced by molnupiravir’s mechanism of action) are currently known to be associated with molnupiravir resistance. Since molnupiravir treatment leads to a rapid reduction in infectious virus, the transmission of virus with treatment-emergent amino acid changes is unlikely.

The data presented here are exclusive to outpatients with mild-to-moderate COVID-19 at antiviral treatment initiation and should not be applied to patients already hospitalized with severe COVID-19, in whom disease progression is largely driven by an overactive host immune response [46]. Patients who progress to severe disease have generally been infected with SARS-CoV-2 for longer than the time window within which initiation of antiviral therapy still confers clinical benefit, and molnupiravir is not indicated in that population. The analyses described in this report also have certain limitations that need to be considered when interpreting the results. First, trial participants had to be unvaccinated, so the virologic impact of molnupiravir in vaccinated patients with breakthrough infections could not be evaluated. On a related note, only a minority of trial participants had serologic evidence of previous COVID-19 infection (which also confers some degree of protective immunity), while pre-existing immunity from vaccination and/or prior infection is now more common worldwide. Second, the occurrence of symptomatic viral rebound was not assessed in the MOVe-OUT trial and is therefore out of scope for this report. Third, the Delta variant, which no longer appears to be circulating, was very prevalent in our trial population. Finally, the first-generation plaque assay (using a single cell type and manual readout) we employed to evaluate SARS-CoV-2 infectivity may have less sensitivity to detect low levels of infectious virus in nasopharyngeal samples than newer infectivity assays based on PCR or immunofluorescence methods. Regardless of this potential limitation (which would have affected both study arms equally), the magnitude of the observed reduction in infectious virus in favor of molnupiravir over placebo was notable, particularly during the first few days after initiation of study intervention. All samples above the prespecified threshold of 105 RNA copies/mL were evaluated for infectivity beyond the 5-day treatment period (i.e., up to study day 29) However, given the potential limitations of the assay, it is possible that we did not detect some cases of low-level infectious SARS-CoV-2 occurring after end of treatment although this is unlikely, especially when considering that about half of the participants in our trial already had 9 or 10 days of COVID-19 symptoms at the end-of-treatment visit and that infectious virus is rarely isolated from nasal swabs more than 10 days after symptom onset [32, 40, 42, 43]. Our results were consistent with those prior reports, with no sample in the molnupiravir arm and only a small number in the placebo arm having infectious virus recovered beyond 10 days post symptom onset. Of note, there is currently no standard approach to measure SARS-CoV-2 infectivity. While there are no direct comparisons of our assay’s sensitivity with that of other methodologies, a phase 2 trial using a PCR-based culture assay (which theoretically may be more sensitive than our method) similarly observed rapid decreases in infectious virus with molnupiravir through the end of 5-day treatment [37], thus lending further support to our results.

Determining virologic outcomes with molnupiravir in the current phase of the pandemic, which is dominated by Omicron sublineages, requires further evaluation, for example through real-world studies. The results from the open-label, randomized, controlled PANORAMIC trial [9], which enrolled over 26,000 participants (about 95% of whom had received ≥ 3 doses of a COVID-19 vaccine) during a period when Omicron had emerged as the predominant variant have recently been reported. PANORAMIC included a virology substudy in which the primary outcome was SARS-CoV-2 viral load on day 7. In the subset of participants from the intensively sampled virology cohort, SARS-CoV-2 RNA levels on day 7 were undetectable in 7/34 participants (21%) in the molnupiravir plus usual care arm compared with 1/39 (3%) in the usual care-only arm. Furthermore, the mean viral RNA load on day 7 was more than tenfold lower with molnupiravir (mean viral RNA load 3.82 log10) than in the control arm (mean viral RNA load 4.93 log10). Other real-world studies conducted in high-risk patients with COVID-19 (including substantial proportions with COVID-19 vaccination and/or prior SARS-CoV-2 infection) during the Omicron era demonstrated that molnupiravir treatment significantly reduced the risk of hospitalization/death [47,48,49] and of post-acute sequelae of SARS-CoV-2 (i.e., “long COVID”) [50] compared to no treatment and that molnupiravir decreased SARS-CoV-2 viral load [51]. These real-world data provide further evidence of molnupiravir’s antiviral activity against SARS-CoV-2 variants (including Omicron) and also illustrate molnupiravir’s clinical benefits for treating breakthrough COVID-19 in vaccinated patients and/or symptomatic reinfection in patients with prior natural immunity to SARS-CoV-2.