Combined Molnupiravir and Nirmatrelvir Treatment Improves the Inhibitory Effect on SARS-CoV-2 in Rhesus Macaques (preprint)

The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have each recently been approved as monotherapy for use in high risk COVID-19 patients. As preclinical data are only available for rodent and ferret models, we originally assessed the efficacy of MK-4482 and PF-07321332 alone and then in combination Against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs. Combined treatment resulted in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated here in the closest COVID-19 surrogate model.

SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta (preprint)

Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Due to confounding factors in the human population, such as pre-existing immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were upregulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggests that in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.

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

ABSTRACT 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

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine (preprint)

In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Initial studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. We report here the rapid development of a replicating RNA vaccine expressing the B.1.1.529 spike and show that this B.1.1.529-targeted vaccine is immunogenic in mice and hamsters. Interestingly, we found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529- targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine.

Prior SARS-CoV-2 Infection Prevents Acute Disease and Lung Pathology in Reinfected Syrian Hamsters but not Virus Replication in the Upper Respiratory Tract (preprint)

Human cases of SARS-CoV-2 reinfection have been documented throughout the pandemic but are likely underreported. In the current study, we used the Syrian hamster SARS-CoV-2 model to assess reinfection with homologous (Washington WA1) and heterologous (United Kingdom B.1.1.7 (alpha) and South Africa B.1.351 (beta)) SARS-CoV-2 variants over time. Upon primary infection with SARS-CoV-2 WA1, hamsters rapidly developed a strong and long-lasting humoral immune response. Following reinfection with homologous and heterologous SARS-CoV-2 variants, this immune response protected hamsters from clinical disease, virus replication in the lower respiratory tract and acute lung pathology. However, reinfection led to SARS-CoV-2 replication in the upper respiratory tract with the potential for virus shedding. Our findings indicate that reinfection results in restricted SARS-CoV-2 replication despite substantial levels of humoral immunity, denoting the potential for transmission through reinfected asymptomatic individuals.Funding: This study was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH). Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: Work with infectious SARS-CoV-2 was approved by the Institutional Biosafety Committee (IBC) and performed in the high biocontainment facilities at Rocky Mountain Laboratories (RML), NIAID, NIH. Sample removal from high biocontainment followed IBC-approved Standard Operating Protocols (44). Animal work was approved by the Institutional Animal Care and Use Committee and performed by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility. Work followed the institution’s guidelines for animal use, the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

UK B.1.1.7 variant exhibits increased respiratory replication and shedding in nonhuman primates (preprint)

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, African green monkeys were infected intranasally with either a contemporary D614G or the UK B.1.1.7 variant. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tract tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases.

Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the Syrian hamster model (preprint)

The COVID-19 pandemic progresses unabated in many regions of the world. An effective antiviral against SARS-CoV-2 that could be administered orally for use following high-risk exposure would be of substantial benefit in controlling the COVID-19 pandemic. Herein, we show that MK-4482, an orally administered nucleoside analog, inhibits SARS-CoV-2 replication in the Syrian hamster model. The inhibitory effect of MK-4482 on SARS-CoV-2 replication was observed in animals when the drug was administered either beginning 12 hours before or 12 hours following infection in a high-risk exposure model. These data support the potential utility of MK-4482 to control SARS-CoV-2 infection in humans following high-risk exposure as well as for treatment of COVID-19 patients.

Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino-oligomers. (preprint)

Background: SARS-CoV-2 is the causative agent of COVID-19 and a pathogen of immense global public health importance. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense agents composed of a phosphordiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. Objectives and Methods: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method and viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays. Results: PPMO designed to base-pair with sequence in the 5'-terminal region or the leader transcription regulatory sequence-region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titers by up to 4-6 log10 in cell cultures at 48-72 hours post-infection, in a non-toxic and dose-responsive manner. Conclusion: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further pre-clinical development.

Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection (preprint)

Following emergence in late 2019, SARS-CoV-2 rapidly became pandemic and is presently responsible for millions of infections and hundreds of thousands of deaths worldwide. There is currently no approved vaccine to halt the spread of SARS-CoV-2 and only very few treatment options are available to manage COVID-19 patients. For development of preclinical countermeasures, reliable and well-characterized small animal disease models will be of paramount importance. Here we show that intranasal inoculation of SARS-CoV-2 into Syrian hamsters consistently caused moderate broncho-interstitial pneumonia, with high viral lung loads and extensive virus shedding, but animals only displayed transient mild disease. We determined the infectious dose 50 to be only five infectious particles, making the Syrian hamster a highly susceptible model for SARS-CoV-2 infection. Neither hamster age nor sex had any impact on the severity of disease or course of infection. Finally, prolonged viral persistence in interleukin 2 receptor gamma chain knockout hamsters revealed susceptibility of SARS-CoV-2 to adaptive immune control. In conclusion, the Syrian hamster is highly susceptible to SARS-CoV-2 making it a very suitable infection model for COVID-19 countermeasure development. One Sentence SummaryThe Syrian hamster is highly susceptible to SARS-CoV-2 making it an ideal infection model for COVID-19 countermeasure development.

Hydroxychloroquine Proves Ineffective in Hamsters and Macaques Infected with SARS-CoV-2 (preprint)

We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in two animal models. When used for prophylaxis or treatment neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Similarly, HCQ prophylaxis/treatment (6.5 mg/kg) did not significantly benefit clinical outcome nor reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. In conclusion, our preclinical animal studies do not support the use of HCQ in prophylaxis/treatment of COVID-19.One Sentence Summary Hydroxychloroquine prophylaxis/treatment showed no beneficial effect in SARS-CoV-2 hamster and macaque disease models.Competing Interest StatementThe authors have declared no competing interest.View Full Text