key: cord-0836957-dgg7hbos authors: Rosenke, Kyle; Okumura, Atsushi; Lewis, Matthew C.; Feldmann, Friederike; Meade-White, Kimberly; Bohler, W. Forrest; Griffin, Amanda; Rosenke, Rebecca; Shaia, Carl; Jarvis, Michael A.; Feldmann, Heinz title: Molnupiravir (MK-4482) is efficacious against Omicron and other SARS-CoV-2 variants in the Syrian hamster COVID-19 model date: 2022-02-23 journal: bioRxiv DOI: 10.1101/2022.02.22.481491 sha: cbc4899bacde644cca7912376a726a4e0438a4ed doc_id: 836957 cord_uid: dgg7hbos 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. One Sentence Summary MK-4482 inhibits replication of multiple SARS-CoV-2 variants of concern, including Omicron, in the Syrian hamster COVID-19 model 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. 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. Syrian hamsters were randomly divided into vehicle-or MK-4482-treatment groups, and infected 65 intranasally with 10 3 TCID50 as previously established (11) 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 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) . (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 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) Omicron SARS-CoV-2 variant: a new chapter in the 274 COVID-19 pandemic Preliminary data indicate COVID-19 vaccines remain 276 effective against severe disease and hospitalisation caused by the Omicron variant Omicron escapes the majority of existing SARS-CoV-2 neutralizing 279 antibodies SARS-CoV-2 Omicron VOC Transmission in Danish Households Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis Molnupiravir for Oral Treatment of Covid-19 in Nonhospitalized 285 Patients Emergency Use Authorization 108: Letter in response to Merck request that the 287 FDA issue an EUA for the emergency use of molnupiravir for the treatment of mild-to-288 moderate COVID-19 in certain adults who are at high-risk for progression to severe 289 COVID-19 Efficacy of Antibodies and Antiviral Drugs against Covid-19 Online ahead of print Hydroxychloroquine prophylaxis and treatment is ineffective in 294 macaque and hamster SARS-CoV-2 disease models Targeting Ebola virus replication through 296 pharmaceutical intervention Defining the Syrian hamster as a highly susceptible preclinical model 298 for SARS-CoV-2 infection Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the 300 Syrian hamster model Online ahead of print. 303 14. World Health Organization. Tracking SARS-CoV-2 variants Mutational cascade of SARS-CoV-2 leading to evolution and 307 emergence of omicron variant. bioRxiv Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome Characterization of orally efficacious influenza drug with high resistance 311 barrier in ferrets and human airway epithelia Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance Evaluation of drugs for potential repurposing against COVID-19 316 using a tier-based scoring system Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. 318 Syrian hamsters as a small animal model for SARS-CoV-2 infection and 320 countermeasure development Animal models for COVID-19 The Roborovski Dwarf Hamster Is A Highly Susceptible Model for a 323 Rapid and Fatal Course of SARS-CoV-2 Infection SARS-CoV-2 variant of concern type and biological sex affect 328 efficacy of molnupiravir in dwarf hamster model of severe COVID-19. bioRxiv SARS-CoV-2 disease severity and transmission efficiency is increased 330 for airborne compared to fomite exposure in Syrian hamsters Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-333 PCR 336 The authors thank staff members of the Laboratory of Virology and the Genomics Unit of the 337 Research Technology Branch (both National Institutes of Allergy and Infectious Diseases 338 (NIAID) and National Institutes of Health (NIH) for their efforts in providing workable CoV-2 stocks. The authors also wish to thank the Rocky Mountain Veterinary Branch (NIAID, 340 NIH) for animal care and husbandry. The authors are thankful to Anita Mora, Visual and 341 Medical Arts Unit, NIAID, NIH) for help with graphical design. Isolate SARS-CoV-342 2_2hCOV_19_England_204820464_2020 isolate (B.