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. 2020 Apr 29;12(541):eabb5883.
doi: 10.1126/scitranslmed.abb5883. Epub 2020 Apr 6.

An Orally Bioavailable Broad-Spectrum Antiviral Inhibits SARS-CoV-2 in Human Airway Epithelial Cell Cultures and Multiple Coronaviruses in Mice

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Free PMC article

An Orally Bioavailable Broad-Spectrum Antiviral Inhibits SARS-CoV-2 in Human Airway Epithelial Cell Cultures and Multiple Coronaviruses in Mice

Timothy P Sheahan et al. Sci Transl Med. .
Free PMC article

Abstract

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

Figures

Fig. 1
Fig. 1
NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication. (A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data are combined from 3 independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in A. Data are combined from 2 independent experiments. (C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with at an MOI of 0.1 for 30 min, washed and exposed to a dose response of NHC in triplicate per condition. 72 hours post infection, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in panel C. Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For A-D, the symbol is at the mean and the error bars represent the standard deviation.
Fig. 2
Fig. 2
NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures. (A) HAE were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate after which virus was removed and cultures were washed in incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to A. qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in C for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. (C) Studies performed as in A but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean and the error bars represent the standard deviation.
Fig. 3
Fig. 3
Remdesivir (RDV) resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. (A) Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV, or MERS-CoV. (B) The variation encompassed in panel A was modeled onto the RdRp structure of the SARS-CoV RdRp. (C) Amino acid sequence of CoV in panel A at known resistance alleles to antiviral drug RDV. (D) Virus titer reduction assay in DBT cells across a range of NHC with recombinant MHV bearing resistance mutations to RDV. Data shown are combined from three independent experiments performed with biological duplicates or triplicates per condition. Asterisks indicate statistically significant differences by Mann-Whitney test as indicated on the graph.
Fig. 4
Fig. 4. NHC is effective against multiple genetically distinct Bat-CoV.
Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Mock, mock-treated. Representative data from two separate experiments with two different cell donors are displayed.
Fig. 5
Fig. 5
NHC antiviral activity is associated with increased viral mutation rates. (A) HAE cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV, or NHC for 48 hours, after which apical washes were collected for virus titration. Data are combined from two independent studies. The boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. (B) Schematic of Primer ID deep sequencing for single RNA genomes of MERS-CoV. (C) The total error rate for MERS-CoV RNA isolated from cultures in panel A as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated group by two-way ANOVA with a Dunnett’s multiple comparison test. (D) Description of potential NHC mutational spectra on both positive and negative sense viral RNA. (E) Nucleotide transitions in cDNA derived from MERS-CoV genomic RNA.
Fig. 6
Fig. 6
Prophylactic and therapeutic EIDD-2801 reduces SARS-CoV replication and pathogenesis. Equivalent numbers of 25-29 week old male and female C57BL/6 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at -2 hours, +12, +24 or +48 hours post infection and every 12 hours thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 1E+04 PFU mouse-adapted SARS-CoV MA15 strain. (A) Percent starting weight. Asterisks indicate differences from vehicle treated by two-way ANOVA with Tukey’s multiple comparison test. (B) Lung hemorrhage in mice from panel A scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. (C) Virus lung titer in mice from panel A as determined by plaque assay. Asterisks in both panel B and C indicate differences from vehicle by one-way ANOVA with a Dunnett’s multiple comparison test. (D) Pulmonary function by whole body plethysmography was performed daily on five animals per group. Asterisks indicate differences from vehicle by two-way ANOVA with a Dunnett’s multiple comparison test. (E) The histological features of acute lung injury (ALI) were blindly scored using the American Thoracic Society Lung Injury Scoring system and a Diffuse Alveolar Damage Scoring System. Three randomly chosen high power (60X) fields of diseased lung were assessed per mouse. The numbers of mice scored per group: Vehicle N = 7, -2 hours N = 9, +12 hours N = 9, +24 hours N = 10, +48 hours N = 9. Asterisks indicate statistical significance compared to vehicle by Kruskal-Wallis with a Dunn’s multiple comparison test. For all panels, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. *, -2 hours and +12 hours compared to vehicle; **, +24 hours compared to vehicle; ***, +48 hours compared to vehicle.
Fig. 7
Fig. 7
Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates. Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at -2 hours, +12, +24 or +48 hours post infection and every 12 hours thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. (A) Percent starting weight. Asterisks indicate differences between -2 hours and +12 hours group from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (B) Lung hemorrhage in mice from panel A scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. (C) Virus lung titer in mice from panel A as determined by plaque assay. Asterisks in both panel B and C indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. (D) MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. (E) Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (F) Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. (G) Number of template consensus sequences (TCS) for MERS-CoV nsp10 and ISG15. (H) Total error rate in MERS-CoV nsp10 and ISG15. (I) The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels G-I, asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (J) Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. For all panels, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range.

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