Therapeutic MK-4482/EIDD-2801 Blocks SARS-CoV-2 Transmission in Ferrets

Abstract

The COVID-19 pandemic is having a catastrophic impact on human health. Widespread community transmission has triggered stringent distancing measures with severe socioeconomic consequences. Gaining control of the pandemic will depend on interruption of transmission chains until protective herd immunity arises. Ferrets and related members of the weasel genus transmit SARS-CoV-2 efficiently with minimal clinical signs, resembling spread in the young-adult population. We previously reported an orally efficacious nucleoside analog inhibitor of influenza viruses, EIDD-2801 (or MK-4482), that was repurposed against SARS-CoV-2 and is in phase II/III clinical trials. Employing the ferret model, we demonstrate in this study high SARS-CoV-2 burden in nasal tissues and secretions that coincides with efficient direct-contact transmission. Therapeutic treatment of infected animals with twice-daily MK-4482/EIDD-2801 significantly reduced upper respiratory tract SARS-CoV-2 load and completely suppressed spread to untreated contact animals. This study identifies oral MK-4482/EIDD-2801 as a promising antiviral countermeasure to break SARS-CoV-2 community transmission chains.

Extended Data Fig. 1.. SARS-CoV-2 does not progress to the ferret lower respiratory tract.

a, Analysis of bronchioalveolar lavages (BALF) and four lung lobes (right (R.) and left (L.) cranial and caudal) per ferret. BALF and tissues samples were harvested 4 (n=2) and 10 (n=2) days after infection. Symbols represent independent biological repeats (individual animals).

Extended Data Fig. 2.. Interferon induction and cytokine profiling of SARS-CoV-2 ferrets treated with MK-4482/EIDD-2801.

a-f, Selected interferon and cytokine expression levels in PBMCs relative to day 0. Blood samples of animals treated with MK-4482/EIDD-2801 or vehicle as specified were collected every two days after infection and PBMCs analyzed by RT-qPCR. Statistical analysis of changes relative to day 0 by two-way ANOVA with Dunnett’s post-hoc multiple comparison test. In all panels, symbols represent independent biological repeats (individual animals), lines connect group medians ± SD.

Extended Data Fig. 3.. Complete blood count of SARS-CoV-2 ferrets treated with MK-4482/EIDD-2801.

a-d, Blood samples were collected every two days after infection and complete blood counts determined. No abnormal values were observed in all parameters tested, including total WBCs (a), lymphocytes (b), neutrophils (c), and platelets (d). The shaded green areas represent normal Vetscan HM5 lab values. Symbols represent independent biological repeats (individual animals), lines connect group medians ± SD.

Fig. 1.. SARS-CoV-2 infects the upper respiratory tract of ferrets.

Ferrets (n=4) were inoculated intranasally with 1×10⁴ or 1×10⁵ pfu of 2019-nCoV/USA-WA1/2020. a, Virus titer in nasal lavages collected daily. b-f, At 4 and 10 days post infection, 2 ferrets were sacrificed in each group and infection was characterized. b, Infectious virus particles in nasal turbinates. c, Viral RNA was present in the nasal turbinates of all infected ferrets. d, RT-qPCR quantitation of viral RNA copies in selected organs, two lung lobes (right (R.) and left (L.) cranial) per animal, and small (SI) and large (LI) intestine samples extracted from infected ferrets four or 10 days after infection. e, Detection of 2019-nCoV/USAWA1/2020 RNA in rectal swabs of ferrets inoculated with 1×10⁵ pfu. f, Bodyweight of ferrets, measured daily and expressed as % of weight at day 0. g, Complete blood count analysis, performed every second day. No noticeable differences were detected for all parameters tested, including total WBCs, lymphocytes, neutrophils, and platelets. The shaded green areas represent normal Vetscan HM5 lab values. h, Selected interferon and cytokine responses in PBMCs harvested every two days after infection. Analysis by qPCR for animals infected with 1×10⁵ pfu of 2019-nCoV/USA-WA1/2020. Infected ferrets displayed elevated expression of interferon stimulated genes (mx1 and isg15 (h; left)), ifn-β and ifn-γ (h; center), and il-6 (h; right). Statistical analysis by two-way ANOVA with Dunnett’s post-hoc multiple comparison test. In all panels, symbols represent independent biological repeats (individual animals), lines connect group medians ± SEM (a,e) or SD (f-h), and bar graphs (b-d) show means ± range.

Fig. 2.. Therapeutic MK-4482/EIDD-2801 is orally efficacious against SARS-CoV-2 in ferrets.

a, Therapeutic efficacy study schematic. Ferrets (n=3) were infected intranasally with 1×10⁵ pfu 2019-nCoV/USA-WA1/2020 and either gavaged with vehicle or treated b.i.d. with MK-4482/EIDD-2801 commencing 12 (5 mg/kg and 15 mg/kg) or 36-hours (15 mg/kg) after infection. Nasal lavages were collected twice daily. Blood was collected every other day. b, Viral nasal lavage titers in infected ferrets from (a). Treatment with MK-4482/EIDD-2801 significantly reduced virus titers within 12 hours dosing onset in all treatment groups. Statistical analysis by two-way ANOVA with Dunnett’s multiple comparison post-hoc test. P values are shown. c-d, Quantitation of infectious particles (c) and virus RNA copy numbers (d) in nasal turbinates of infected ferrets extracted four days after infection. Statistical analysis by one-way ANOVA with Dunnett’s multiple comparison post-hoc test. P values are shown. In all panels, symbols represent independent biological repeats (individual animals), lines connect group medians ± SEM (b), and bar graphs (c-d) show means ± SD.

Fig. 3.. Therapeutic oral treatment with MK-4482/EIDD-2801 prevents contact transmission.

a, Contact transmission study schematic. Two groups of source ferrets (n=3 each) were infected with 1×10⁵ pfu of 2019-nCoV/USA-WA1/2020 and received MK-4482/EIDD-2801 treatment (5 mg/kg b.i.d.) or vehicle starting 12 hours after infection. At 30 hours after infection, each source ferret was co-housed with two uninfected, untreated contact ferrets. After three days, source animals were euthanized and contact ferrets isolated and monitored for four days. Nasal lavages and rectal swabs were collected once daily and blood sampled at 0, 4, and 8 days post infection. b, Source ferrets treated with MK-4482/EIDD-2801 had significantly lower virus titers 12 hours after treatment onset (p=0.0003) than vehicle animals. Contacts of vehicle-treated sources began to shed 2019-nCoV/USA-WA1/2020 within 20 hours of co-housing. No virus was detectable in untreated contact of MK-4482/EIDD-2801-treated source ferrets. Statistical analysis by two-way ANOVA with Sidak’s multiple comparison post-hoc test. P values are shown. c-d, Quantitation of infectious particles (c) and virus RNA copy numbers (d) in nasal turbinates of source and contact ferrets from (b), extracted four and eight days after study start, respectively. Statistical analysis by one-way ANOVA with Sidak’s multiple comparison post-hoc test. e-f, Quantitation of virus RNA copy numbers in small (SI) and large (LI) intestines (e) and rectal swabs (f). Samples of MK-4482/EIDD-2801-treated source ferrets and their contacts were PCR-negative for viral RNA. In all panels, symbols represent independent biological repeats (individual animals), lines connect group medians ± SEM (b) or SD (f), and bar graphs (c-e) show means ± SD.