Therapeutically administered ribonucleoside analogue MK-4482/EIDD-2801 blocks SARS-CoV-2 transmission in ferrets

Abstract

The coronavirus disease 2019 (COVID-19) pandemic is having a catastrophic impact on human health¹. Widespread community transmission has triggered stringent distancing measures with severe socio-economic consequences. Gaining control of the pandemic will depend on the interruption of transmission chains until vaccine-induced or naturally acquired protective herd immunity arises. However, approved antiviral treatments such as remdesivir and reconvalescent serum cannot be delivered orally^2,3, making them poorly suitable for transmission control. We previously reported the development of an orally efficacious ribonucleoside analogue inhibitor of influenza viruses, MK-4482/EIDD-2801 (refs. ^4,5), that was repurposed for use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently in phase II/III clinical trials (NCT04405570 and NCT04405739). Here, we explored the efficacy of therapeutically administered MK-4482/EIDD-2801 to mitigate SARS-CoV-2 infection and block transmission in the ferret model, given that ferrets and related members of the weasel genus transmit the virus efficiently with minimal clinical signs^6-9, which resembles the spread in the human young-adult population. We demonstrate high SARS-CoV-2 burden in nasal tissues and secretions, which coincided with efficient transmission through direct contact. Therapeutic treatment of infected animals with MK-4482/EIDD-2801 twice a day significantly reduced the SARS-CoV-2 load in the upper respiratory tract and completely suppressed spread to untreated contact animals. This study identified 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.

Ferrets were inoculated intranasally with 1×10⁴ (blue) or 1×10⁵ pfu (black) of 2019-nCoV/USA-WA1/2020. 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 biologically independent animals) and 10 (n=2 biologically independent animals) days after infection. The number of independent biological repeats is shown. Symbols represent independent biological repeats (individual animals).

Extended Data Fig. 2. Experimental means and intergroup comparison of in vivo efficacy study results of MK-4482/EIDD-2801 in SARS-CoV-2-infected ferrets.

a, Comparison of SARS-CoV-2 titers in nasal lavages of SARS-CoV-2 infected ferrets treated with different doses and dosing regimens. Mean virus titers ± SD are shown for different study days (days pI). The limit of detection (LoD) was 10 pfu (a-b). Samples below LoD were plotted as 9 pfu. b, SARS-CoV-2 titers in nasal turbinates of ferrets from (a). Mean virus titers ± SD are shown. LoD was 10 pfu (a-b). c, Viral RNA detected in nasal turbinates of ferrets from (a). Mean RNA copy numbers ± SD are shown. Statistical significance was determined between different treatment groups. LoD was 1 RNA copy, samples below LoD were plotted as 1 RNA copy. Statistical analysis was performed by two-way (a) or one-way (b-c) ANOVA with Tukey’s multiple comparison post-hoc test. P values are shown. (*) denotes samples where one or two repeats were below LoD. (**) denotes all three repeats for at least one dosing group were below LoD.

Extended Data Fig. 3. Interferon induction and cytokine profiling of SARS-CoV-2 ferrets treated with MK-4482/EIDD-2801.

Ferrets (n=3 biologically independent animals) were infected intranasally with 1×10⁵ pfu 2019-nCoV/USA-WA1/2020 and either gavaged with vehicle (black (a-f)) or treated b.i.d. with MK-4482/EIDD-2801 commencing 12 (5 mg/kg (blue (a-f)) and 15 mg/kg (orange (a-f)) or 36-hours (15 mg/kg; red (a-f)) after infection. 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. The number of independent biological repeats (n; individual animals) is shown. In all panels, symbols represent independent biological repeats (individual animals), lines connect group means ± SD.

Extended Data Fig. 4. Complete blood count of SARS-CoV-2 ferrets treated with MK-4482/EIDD-2801.

Ferrets (n=3 biologically independent animals) were infected intranasally with 1×10⁵ pfu 2019-nCoV/USA-WA1/2020 and either gavaged with vehicle (black (a-f)) or treated b.i.d. with MK-4482/EIDD-2801 commencing 12 (5 mg/kg (blue (a-f)) and 15 mg/kg (orange (a-f)) or 36-hours (15 mg/kg; red (a-f)) after infection. 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. The number of independent biological repeats (n; individual animals) is shown for each subpanel. Symbols represent independent biological repeats (individual animals), lines connect group means ± SD.

Fig. 1.

SARS-CoV-2 infects the upper respiratory tract of ferrets. Ferrets (n=4 biologically independent animals) were inoculated intranasally with 1×10⁴ (blue throughout) or 1×10⁵ pfu (black throughout) 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/USA-WA1/2020 RNA in rectal swabs of ferrets inoculated with 1×10⁵ pfu. RNA was extracted from rectal swabs and absolute copy numbers were determined by RT-qPCR. 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-l, 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 ifn-β (h) and ifn-γ (i). il-6 was moderately elevated only in some animals (j). Interferon stimulated genes (mx1 (k; p=0.0192 on day 4) and isg15 (l; p=0.009 and p<0.0001 on days 2 and 4, respectively)) showed a sharp peak at day 4 after infection. The number of independent biological repeats (n; individual animals) is shown for each subpanel. 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 means ± SD (a,e-l), and bar graphs show means only (b-d).

Fig. 2.

Therapeutic MK-4482/EIDD-2801 is orally efficacious against SARS-CoV-2 in ferrets. a, Dose-response inhibition test of NHC against SARS-CoV-2 in Vero E6 cells (MOI 0.1 pfu/cell; n=3 biologically independent experiments). Effective concentrations (EC₅₀ and EC₉₀, shown with upper 95% confidence interval limit in parenthesis) are derived from four-parameter variable slope regression modeling. b, Therapeutic efficacy study schematic. Ferrets (n=3 biologically independent animals) were infected intranasally with 1×10⁵ pfu 2019-nCoV/USA-WA1/2020 and either gavaged with vehicle (black (c-e)) or treated b.i.d. with MK-4482/EIDD-2801 commencing 12 (5 mg/kg (blue (c-e)) and 15 mg/kg (orange (c-e)) or 36-hours (15 mg/kg; red (c-e)) after infection. Nasal lavages were collected twice daily. Blood was collected every other day. c, 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. d-e, Quantitation of infectious particles (d) and virus RNA copy numbers (e) 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. The number of independent biological repeats is shown for each subpanel. P values are shown. In all panels, symbols represent independent biological repeats (individual animals), lines connect group means ± SD (a,c) and bar graphs show means ± SD (d-e).

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 biologically independent animals 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.; blue (b-f)) or vehicle (black (b-f)) starting 12 hours after infection. At 30 hours after infection, each source ferret was co-housed with two uninfected, untreated contact ferrets (light blue and red for MK-4482 treated or vehicle treated source ferrets, respectively (b-f)). 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 (p<0.0001 for intergroup comparison of contact animals (c); p=0.0002 and p<0.0001 for intergroup comparison of source and contract animals, respectively (d)). 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. Statistical analysis by one-way (e) or two-way (f) ANOVA with Sidak’s multiple comparison post-hoc test. Samples being compared in post-hoc tests (c-ef) are color coded black or red for vehicle treated source and contact ferrets, respectively). The number of independent biological repeats is shown for each subpanel. P values are shown. In all panels, symbols represent independent biological repeats (individual animals), lines connect group means ± SD (b,f), and bar graphs show means ± SD (c-e).