Molnupiravir (MK-4482) is efficacious against Omicron and other SARS-CoV-2 variants in the Syrian hamster COVID-19 model

The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC) containing a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here we assessed the efficacy of MK-4482 against the earlier Alpha, Beta and Delta VOCs and Omicron in the Syrian hamster COVID-19 model. Omicron replication and associated lung disease in vehicle treated hamsters was reduced compared to the earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of Alpha, Beta and Delta VOC infected hamsters. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious virus titers compared to viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.

Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more 32 pronounced inhibitory effect on infectious virus titers compared to viral RNA genome load. 33 Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the 34 level of lung disease and viral antigen load in infected hamsters across all VOCs examined. 35 Together, our data indicate the potential of MK-4482 as an effective antiviral against known 36 SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants. (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 24, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 INTRODUCTION 43 Now in its third year, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 44 coronavirus disease 2019  pandemic has become characterized by the serial 45 emergence of variants of concern (VOCs) that rapidly and globally replace earlier, previously 46 predominant strains. In November 2021, Omicron (B.1.1.529) emerged to rapidly replace Delta 47 (B.1.617.2), the predominant VOC at the time (1). Omicron has shown reduced pathogenicity 48 (2) but enhanced transmissibility primarily associated with an increased ability to evade 49 immunity associated with infection by earlier VOCs or through vaccination (3,4). The 50 emergence of such VOCs with immune evasive ability poses a continuous and considerable 51 threat to global control strategies based on present spike-based vaccines, as well as monoclonal 52 antibody-based therapeutics for treatment of more severe  Molnupiravir (MK-4482) is an orally available antiviral nucleoside analogue that targets SARS-54 CoV-2 polymerase fidelity rather than the spike protein (5). It has been authorized for emergency 55 use against SARS-CoV-2 in high-risk adults (6, 7). Results from recent in vitro studies suggest 56 that molnupiravir retains activity against multiple VOCs including Omicron (8). However, 57 experience with therapeutics targeting SARS-CoV-2 (9), as well as other emerging viruses (10) 58 have shown a frequent disconnect between efficacy observed in vitro compared to therapeutic in 59 vivo effect. In the present study we investigate the ability of MK-4482 to inhibit several SARS-60 CoV-2 VOCs (Alpha, Beta, Delta and Omicron) in the Syrian hamster COVID-19 model.

MK-4482 treatment inhibits replication of multiple VOCs, including Omicron
64 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 24, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 4 Syrian hamsters were randomly divided into vehicle-or MK-4482-treatment groups, and infected 65 intranasally with 10 3 TCID50 as previously established (11) to test in vivo efficacy of MK-4482 66 against the SARS-CoV-2 Alpha, Beta, Delta and Omicron VOCs. Treatment using 250mg/kg 67 MK-4482, administered by oral gavage, was initiated 12h after infection. Treatment was 68 continued every 12h for the established total daily dose of 500mg/kg (12). Hamsters were 69 monitored daily for clinical signs and oral swabs were collected 2 and 4 days post-infection 70 (dpi). At 4 dpi hamsters were euthanized and tissues collected for viral RNA load and infectious 71 titer determination (Fig. 1A). 72 Hamsters in vehicle and MK-4482 treatment groups remained largely asymptomatic with 73 noticeable but not significant weight loss (3-4%) in vehicle treated animals over the 4-day study 74 period ( Fig. 1B). Quantitative RT-PCR (qRT-PCR) targeting subgenomic viral E gene RNA 75 (sgE) in oral swabs and lung tissue was used to quantify replication in the upper and lower 76 respiratory tract, respectively. sgE loads in oral swabs from vehicle treated animals on 2 and 4 77 dpi ranged from 5 to 8 log10 copies/mL confirming infection by all VOCs with the exception of a 78 single Omicron animal (Fig. 1C). MK-4482 treatment resulted in lower replication in oral swabs 79 for all VOCs except Delta, a result that was only statistically significant for Omicron at 2 dpi 80 ( Fig. 1C). sgE loads in lung tissue ranged from 7 to 10 log10 copies/g for Alpha, Beta and Delta 81 in vehicle treated groups, but was notably lower (0-7 log10 copies/g) for Omicron (Fig. 1D). MK-82 4482 treatment resulted in a reduction in sgE lung loads for all VOCs and was below the limit of 83 detection for Omicron, a result that was statistically significant (Fig. 1D). Similarly, infectious 84 virus titers in lung tissue ranged from 6-11 log10 TCID50/g for Alpha, Beta and Delta vehicle 85 treated groups that was significantly lower for Omicron (0 to 6 log10 TCID50/g) (Fig. 1E). 86 Infectious titers were substantially more reduced by MK-4482 than sgE loads. This was observed 87 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 24, 2022.

Inhibitory effect of MK-4482 on Omicron VOC resists higher virus challenge
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(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC We repeated the MK-4482 treatment study with a higher Omicron challenge dose (10 4 TCID50) 110 to account for the decreased replication of the Omicron VOC in lung tissue (13). The study 111 design remained the same as described above (Fig. 1A), but trachea tissue was collected at 4 dpi 112 as an additional target tissue. The higher Omicron challenge dose did not increase clinical 113 disease severity and weight loss remained similar to animals challenged with the lower 10 3 114 TCID50 dose (Figs. 1B,3A). At 2 and 4 dpi, oral swab sgE loads from vehicle treated animals 115 ranged from 5 to 7 log10 copies/mL, which were lower in MK-4482 treated animals (<1 log10 116 copies/mL), a difference that was not statistically significant (  S2). Interestingly, sgE loads were several log10 higher in trachea (9 to 11 log10 127 copies/g) compared to lung tissue of vehicle treated hamsters. sgE loads were less than a log10 128 lower in trachea tissue from MK-4482 treated animals (Fig. 3D). However, infectious virus in 129 trachea tissue dropped precipitously from a median of 5 log10 TCID50/g in vehicle treated 130 animals to below the limit of detection in MK-4482 animals (Fig. 3D,E). 131 132 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 24, 2022.  (19) may also help to reduce the development of such 165 drug resistance. 166 We found a marked discrepancy between viral sgRNA loads and infectious titers for all VOCs. 167 Given the mechanism of action of MK-4482 (5) this may not be surprising as mutated, 168 replication incompetent viral RNA will still be detected by qRT-PCR analysis but not by 169 infectivity assays. This observation emphasizes the need to consider the specific method of 170 analysis being used to assess antiviral activity, as viral sgRNA loads are often used as the single 171 measure for SARS-CoV-2 replication. Our results strongly suggest that infectious titers are 172 critical for a complete investigation and required to determine the efficacy of antiviral drugs and 173 likely also vaccines. Histopathology serves as an important confirmation for virus replication and 174 provides helpful pathologic images. 175 Our study has limitations. The hamster model does not accurately represent COVID-19 disease 176 for all human age-and comorbidity-associated subpopulations as the animals only develop mild-177 to-moderate disease (11,20,21), but this is a drawback of most current animal models (22). The 178 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 24, 2022. ; https://doi.org/10.1101/2022.02.22.481491 doi: bioRxiv preprint 9 more recently identified dwarf hamster model is associated with higher disease severity, but the 179 extremely acute disease progression with animals reaching clinical endpoints within as quickly 180 as 3 dpi (23)  and volume as VOC infection groups. All groups were infected intranasally with 10 3 or 10 4 222 TCID50 (high dose) of SARS-CoV-2 (25 µL/nare). Animal weights were collected once daily, 223 and animals were monitored twice daily for disease signs and progression. All procedures were 224 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 24, 2022.  Statistical analyses. Statistical analysis was performed in Prism 9. The difference in weight, 267 viral load and infectious titers between study arms was assessed by ordinary one-way ANOVA. 268 269 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC