key: cord-0794453-ghrlj6b2 authors: Pruijssers, Andrea J.; George, Amelia S.; Schäfer, Alexandra; Leist, Sarah R.; Gralinksi, Lisa E.; Dinnon, Kenneth H.; Yount, Boyd L.; Agostini, Maria L.; Stevens, Laura J.; Chappell, James D.; Lu, Xiaotao; Hughes, Tia M.; Gully, Kendra; Martinez, David R.; Brown, Ariane J.; Graham, Rachel L.; Perry, Jason K.; Du Pont, Venice; Pitts, Jared; Ma, Bin; Babusis, Darius; Murakami, Eisuke; Feng, Joy Y.; Bilello, John P.; Porter, Danielle P.; Cihlar, Tomas; Baric, Ralph S.; Denison, Mark R.; Sheahan, Timothy P. title: Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice date: 2020-04-27 journal: bioRxiv DOI: 10.1101/2020.04.27.064279 sha: c558266cf583cc2c375fff8f4870a2e9f7f0ac21 doc_id: 794453 cord_uid: ghrlj6b2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 μM). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 μM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19. While four endemic human CoV (HCoV-OC43, -229E, -NL63, and -HKU1) typically cause mild 23 respiratory diseases with common cold-like symptoms, SARS-CoV-1, MERS-CoV, and SARS-CoV-2 24 cause severe respiratory disease with respective mortality rates of 11% (Chan-Yeung and Xu, 2003) , 35% 25 (Arabi et al., 2017) , and an estimated 3% (Chen, 2020) . The development of effective broad-spectrum 26 antivirals has been hampered by viral diversity, the capacity of CoVs to adaptively overcome negative 27 selective pressures, and the ability to actively counteract drugs through the action of a proofreading 28 exoribonuclease. We previously reported that remdesivir (RDV), a monophosphoramidate prodrug of the 29 C-adenosine analog GS-441524, potently inhibits replication of a broad spectrum of pre-pandemic bat 30 CoVs and human epidemic CoVs in primary human lung cell cultures (Agostini et Calu3 human lung cells with sub-micromolar EC50 and in primary human airway epithelial cultures 41 (HAEs) with nanomolar EC50. Notably, we have detected comparably lower potency of RDV in 42 established human and monkey cell lines due to their lower metabolic capacity to activate the compound. Mice infected with chimeric SARS-CoV-1 encoding the SARS-CoV-2 RdRp and treated therapeutically 44 with RDV show decreased viral loads in the lungs and increased pulmonary function. These data 45 emphasize the potential of RDV as a promising countermeasure against the ongoing COVID-19 46 pandemic. 47 replication for each cell type, and infectious viral titer and viral genome copy number in the supernatant 109 were quantified by plaque assay and RT-qPCR, respectively. RDV and GS-441524 potently inhibited 110 SARS-CoV-2 replication in a dose-dependent manner in both cell types ( Fig. 2; Table 1 ). In Calu3 cells, 111 both compounds displayed dose-dependent inhibition of viral replication as determined by plaque assay 112 ( Fig. 2B) and RT-qPCR (Fig. 2C) . RDV inhibited SARS-CoV-2 with an EC50 = 0.28 μM and EC90 = 2.48 113 μM. The parent compound GS-441524 was less potent: EC50 = 0.62 μM, EC90 = 1.34 μM ( Fig. 2D ; Table 114 1). EC50 values determined by quantification of viral genome copies were roughly two-fold higher than 115 those obtained by quantification of infectious virus ( Fig. 2E; Table 1 ). Both compounds also displayed 116 dose-dependent inhibition of viral replication in Vero E6 cells as determined by infectious viral titer and 117 genome copy number (Fig 2F) . RDV inhibited SARS-CoV-2 with EC50 = 1.65 μM and EC90 = 2.40 μM, 118 while GS-441524 was more potent (EC50 = 0.47 μM, EC90 = 0.71 μM) ( Fig. 2G and physiology of the human conducting airway (Sims et al., 2005) . Therefore, we evaluated antiviral 125 activity of RDV in this biologically relevant model. In RDV treated HAE, we observed a dose-dependent 126 reduction in infectious virus production, with >100-fold inhibition at the highest tested concentration 127 (Fig. 3A) . Importantly, RDV demonstrate potent antiviral activity with EC50 values of 0.0010 and 0.009 128 µM in two independent experiments (Fig. 3B) . We previously reported that RDV is not cytotoxic at doses 129 at or below 10 µM in this culture system, supporting the conclusion that the observed antiviral effect was 130 virus-specific (Sheahan et al., 2017) . Together, these data demonstrate that RDV is potently antiviral 131 against SARS-CoV-2 in primary human lung cultures with a selectivity index of >1000. 132 (Fig. S4 ) and the efficiency of each step might 138 differ between cell types. Therefore, to reconcile the differences in antiviral activity of RDV and GS-139 441524 observed in our and other studies, we compared intracellular RDV-TP concentrations in Vero E6, 140 Calu3 2B4, and HAEs following incubation with the two compounds. RDV-TP levels per million cells 6 produced after 8-to 48-hour treatment with RDV were substantially higher in primary HAE cultures than 142 either Calu3 2B4 or Vero E6. (Fig 4; Table 1; Tables S1, S2). Given the primary nature of HAE cultures, 143 we used cells from two independent donors with similar demographic profiles. RDV-TP was efficiently 144 formed in both donor cultures following incubation with RDV with a difference of < 3-fold from each 145 other. The lowest levels of RDV-TP were observed following RDV treatment of Vero E6 cells and were 146 approximately 4-and 20-fold lower than those observed in Calu3 2B4 and HAE cultures, respectively. 147 The levels of GS-441524 as well as the intermediate mono-and di-phosphorylated metabolites (RDV-MP 148 and RDV-DP) were readily detected in Calu3 2B4 cultures following treatment with RDV, but were 149 below the limit of quantification in Vero E6 cells at all time points tested (Table S1 ). In addition, 150 incubation of Vero E6 cells with GS-441524 yielded 4-fold higher RDV-TP levels compared to 151 incubation with RDV corresponding to higher antiviral potency of GS-441524 relative to RDV, which is 152 not observed with either Calu3 or HAE cultures. (Table S1, S2). In conclusion, the RDV-TP levels in the 153 different cell types directly correlated with the antiviral potencies of RDV and GS-441524 against SARS-154 CoV-2 with the HAE cultures producing substantially higher levels of RDV-TP that translated into 155 markedly more potent antiviral activity of RDV (Table 1) . Importantly, the metabolism of RDV in Vero 156 E6 cells appeared altered and was less efficient particularly in comparison with the HAE cultures, 157 indicating that Vero E6 cells might not be an adequate cell type to characterize the antiviral activity of 158 RDV and potentially also other nucleotide prodrug-based antivirals. 159 To determine whether RDV exerts antiviral 160 effect on SARS-CoV-2 in vivo, we constructed a chimeric mouse-adapted SARS-CoV-1 variant encoding 161 the target of RDV antiviral activity, the RdRp, of SARS-CoV-2 (SARS1/SARS2-RdRp) (Fig. 5A) . 162 Although other chimeric replicase ORF recombinant CoVs have shown to be viable (Stobart et al., 2013) , 163 this is the first demonstration that the RdRp from a related but different CoV can support efficient 164 replication of another. After recovery and sequence-confirmation ( Fig. 5B ) of recombinant chimeric 165 viruses with and without nanoluciferase reporter, we compared SARS-CoV-1 and SARS1/SARS2-RdRp 166 replication and sensitivity to RDV in Huh7 cells. Replication of both viruses was inhibited similarly in a 167 dose-dependent manner by RDV (SARS-CoV-1 mean EC50 = 0.007 µM; SARS1/SARS2-RdRp mean 168 EC50 = 0.003 µM) ( Fig. 5C and D) . We then sought to determine the therapeutic efficacy of RDV against 169 the SARS1/SARS2-RdRp in mouse models employed for previous studies of RDV (Sheahan et al., 2017) . 170 Mice produce a serum esterase absent in humans, carboxyl esterase 1c (Ces1c), that dramatically reduces 171 half-life of RDV. Thus, to mirror pharmacokinetics observed in humans, mouse studies with RDV must 172 be performed in transgenic C57Bl/6 Ces1c -/mice (Sheahan et al., 2017) . We infected female C57Bl/6 173 Ces1c -/mice with 10 3 PFU SARS1/SARS2-RdRp and initiated subcutaneous treatment with 25 mg/kg 174 RDV BID at one day post-infection (dpi). This regimen was continued until study termination. While 175 weight loss did not differ between vehicle-and RDV-treated animals (Fig. 5E) , lung hemorrhage at five 176 dpi was significantly reduced with RDV treatment (Fig. 5F ). To gain insight into physiologic metrics of 177 disease severity, we measured pulmonary function daily by whole body plethysmography (WBP). The 178 WBP metric, PenH, is a surrogate marker of pulmonary obstruction (Menachery et al., 2015a) . 179 Therapeutic RDV significantly ameliorated loss of pulmonary function observed in the vehicle-treated 180 group (Fig. 5G) . Importantly, RDV treatment dramatically reduced lung viral load (Fig. 5H) . Taken 181 together, these data demonstrate that therapeutically administered RDV can reduce virus replication and versus SARS-CoV-2 isolates used in the previously mentioned studies assessing RDV potency did not 212 reveal consensus changes in nsp12 sequence, suggesting that any isolate-specific variation in RDV 213 sensitivity is not likely due to differences in the RDV-TP interaction with the RdRp. Therefore, the 214 differences in EC50 may be partially explained by intrinsic differences of SARS-CoV-2 virus isolates, 215 quantification methods, and assay conditions such as incubation period and virus input. We thank Dr. Natalie Thornburg at the Centers for Disease Control and Prevention in Atlanta, USA for 293 providing the stock of SARS-CoV-2 used in this study. Finally, we thank VUMC and UNC 294 Environmental Health and Safety personnel for ensuring that our work is performed safely and securely. 295 We also thank Facilities Management personnel for their tireless commitment to excellent facility 296 performance and our grant management teams for their administrative support of our research operations. 297 The authors affiliated with Gilead Sciences, Inc. are employees of the company and own company stock. 300 The other authors have no conflict of interest to report. 301 Transwell-COL (12mm diameter) supports (Corning). Human airway epithelium cultures (HAE) were 404 generated by provision of an air-liquid interface for 6 to 8 weeks to form well-differentiated, polarized 405 cultures that resembled in vivo pseudostratified mucociliary epithelium (Fulcher et al., 2005) . 406 Clinical specimens of SARS-CoV-2 from a case-patient who acquired COVID-19 during travel to China 407 and diagnosed in Washington State, USA upon return were collected as described (Holshue et al., 2020) . 408 Virus isolation from this patient's specimens was performed as described in (Harcourt et al.) . The 409 sequence is available through GenBank (accession number MN985325). A passage 3 stock of the SARS-410 CoV-2 Seattle isolate was obtained from the CDC and passed twice in Vero E6 cells to generate high-titer 411 passage 5 stock for experiments described in this manuscript. CoV-2 N gene positive control plasmid (IDT, cat# 10006625) served as template to PCR-amplify a 1280 502 bp product using forward (5'-TAATACGACTCACTATAGGGATGTCTGATAATGGACCCCA) and 503 reverse (5'-TTAGGCCTGAGTTGAGTCAG) primers that appended a T7 RNA polymerase promoter to 504 the 5' end of the complete N ORF. PCR product was column purified (Promega) for subsequent in vitro 505 transcription of N RNA using mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) according to 506 manufacturer's protocol. N RNA was purified using RNeasy mini kit (Qiagen) according to 507 manufacturer's protocol, and copy number was calculated using SciencePrimer.com cop number 508 calculator. 509 In vitro metabolism of RDV and GS-441524. Calu3 2B4 or Vero E6 cells were seeded in a 6-well plate 510 at 8.0 x 10 5 or 3.5 X 10 5 cells/well, respectively. Twenty-four hours later, cell culture media was replaced 511 with media containing 1 μM RDV (GS-5734) or GS-441524 and incubated at 37˚C. Differentiated HAE 512 cultures from two healthy donors (MatTek Corporation; Ashland, MA) were maintained with media 513 replacement every other day for 1 week. The HAE donors were 56-and 62-year-old females of the same 514 race. At the time of treatment, media was replaced on the basal side of the transwell HAE culture, while the apical surface media was replaced with 200 µL media containing 1 μM RDV. At 8, 24 and 48h post 516 drug addition to all cultures, cells were washed 3 times with ice-cold tris-buffered saline, scraped into 0.5 517 mL ice-cold 70% methanol and stored at -80°C. Extracts were centrifuged at 15,000 x g for 15 minutes 518 and supernatants were transferred to clean tubes for evaporation in a MiVac Duo concentrator (Genevac). 519 Dried samples were reconstituted in mobile phase A containing 3 mM ammonium formate (pH 5) with 10 520 mM dimethylhexylamine (DMH) in water for analysis by LC-MS/MS, using a multi-stage linear gradient 521 from 10% to 50% acetonitrile in mobile phase A at a flow rate of 300 μL/min. Analytes were separated 522 using a 50 x 2 mm, 2.5 μm Luna C18(2) HST column (Phenomenex) connected to an LC-20ADXR 523 (Shimadzu) ternary pump system and HTS PAL autosampler (LEAP Technologies). Detection was 524 performed on a Qtrap 6500+ (AB Sciex) mass spectrometer operating in positive ion and multiple 525 reaction monitoring modes. Analytes were quantified using a 7-point standard curve ranging in 526 concentration from 0.156 to 40 pmol prepared in extracts from untreated cells. For normalization by cell 527 number, multiple untreated Calu3 or Vero E6 culture wells were counted at each timepoint. HAE cells 528 were counted at the 24-h timepoint and the counts for other timepoints were determined by normalized to 529 endogenous ATP levels for accuracy. 530 Formulations for in vivo studies. RDV was solubilized at 2.5 mg/mL in vehicle containing 12% 531 sulfobutylether-β-cyclodextrin sodium salt in water (with HCl/NaOH) at pH 5.0. 532 In vivo efficacy studies. All animal experiments were performed in accordance with the University of 533 North Carolina at Chapel Hill Institutional Animal Care and Use Committee policies and guidelines. To 534 achieve a pharmacokinetic profile similar to that observed in humans, we performed therapeutic efficacy 535 studies in Ces1c -/mice (stock 014096, The Jackson Laboratory), which lack a serum esterase not present 536 in humans that dramatically reduces RDV half-life (Sheahan et al., 2017) . 17 week-old female Ces1c -/-537 mice were anaesthetized with a mixture of ketamine/xylazine and intranasally infected with 10 3 PFU 538 SARS1/SARS2-RdRp in 50 µL. One dpi, vehicle (n = 7) and RDV (n = 7) dosing was initiated (25 mg/kg 539 subcutaneously) and continued every 12 h until the end of the study at five dpi. To monitor morbidity, 540 mice were weighed daily. Pulmonary function testing was performed daily by whole body 541 plethysmography (WBP) (Data Sciences International) (Sheahan et al., 2017) . At five dpi, animals were 542 sacrificed by isoflurane overdose, lungs were scored for lung hemorrhage, and the inferior right lobe was 543 frozen at −80°C for viral titration via plaque assay on Vero E6 cells. Lung hemorrhage is a gross 544 pathological phenotype readily observed by the naked eye and driven by the degree of virus replication, 545 where lung coloration changes from pink to dark red (Sheahan et al., 2017 (Sheahan et al., , 2020a . For the plaque assay, 546 5 x 10 5 Vero E6 cells/well were seeded in 6-well plates. The following day, medium was removed, and 547 monolayers were adsorbed at 37˚C for one h with serial dilutions of sample ranging from 10 -1 to 10 -6 . Cells were overlayed with 1X DMEM, 5% Fetal Clone 2 serum, 1× antibiotic-antimycotic, 0.8% agarose. 549 Viral plaques were enumerated three days later. 550 Mathematical and statistical analyses. 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