Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice
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Abstract
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 (EC 50 = 0.01 μM). Weaker activity was observed in Vero E6 cells (EC 50 = 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.
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SciScore for 10.1101/2020.04.27.064279: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Human tracheobronchial epithelial cells provided by Dr. Scott Randell were obtained from airway specimens resected from patients undergoing surgery under University of North Carolina Institutional Review Board-approved protocols (#03-1396) by the Cystic Fibrosis Center Tissue Culture Core.
IACUC: In vivo efficacy studies: All animal experiments were performed in accordance with the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee policies and guidelines.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable The HAE donors were 56- and 62-year-old females of the same race. Cel… SciScore for 10.1101/2020.04.27.064279: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Human tracheobronchial epithelial cells provided by Dr. Scott Randell were obtained from airway specimens resected from patients undergoing surgery under University of North Carolina Institutional Review Board-approved protocols (#03-1396) by the Cystic Fibrosis Center Tissue Culture Core.
IACUC: In vivo efficacy studies: All animal experiments were performed in accordance with the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee policies and guidelines.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable The HAE donors were 56- and 62-year-old females of the same race. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cells, viruses, and compounds: Vero (ATCC CCL-81) and Vero E6 (ATCC CRL-1586) cells were purchased from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, ThermoFisher Scientific) or 10% FCS fetal clonal serum (FCS)(HyClone, GE Life Sciences), 100 U/ml penicillin and streptomycin (Gibco, ThermoFisher Scientific), and 0.25 μM amphotericin B ( Verosuggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)Antiviral activity assays: Vero E6 cells were seeded at 1 x 105 cells per well, and Calu3 2B4 cells were seeded at 2 x 105 cells per well in 24-well plates (Corning). Calu3 2B4suggested: RRID:CVCL_YZ47)Huh7 cells were plated at a density of 8 x 104 cells per well. Huh7suggested: NoneCytotoxicity Assays: Cells were seeded at a density of 15,000 cells/well (Vero E6) or 30,000 cells/well Vero E6suggested: RRID:CVCL_XD71)Experimental Models: Organisms/Strains Sentences Resources To achieve a pharmacokinetic profile similar to that observed in humans, we performed therapeutic efficacy studies in Ces1-/- mice (stock 014096, The Jackson Laboratory), which lack a serum esterase not present in humans that dramatically reduces RDV half-life (Sheahan et al., 2017). Ces1-/-suggested: NoneSoftware and Algorithms Sentences Resources All statistical tests were executed using GraphPad Prism 8. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.04.27.064279: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Human tracheobronchial epithelial cells provided by Dr. Scott Randell were obtained from airway specimens resected from patients undergoing surgery under University of North Carolina Institutional Review Board-approved protocols ( #03-1396 ) by the Cystic Fibrosis Center Tissue Culture Core. Randomization Prior to the emergence of pandemic SARS-CoV-2 , RDV was evaluated in phase 1 clinical trials as well as phase 2 randomized controlled trial trials to treat acute Ebola virus disease in the Democratic Republic of Congo ( DRC) , and human safety data are available ( Mulangu et al. , 2019) . Blinding not detected. Power Analysis not detected. Sex as a … SciScore for 10.1101/2020.04.27.064279: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Human tracheobronchial epithelial cells provided by Dr. Scott Randell were obtained from airway specimens resected from patients undergoing surgery under University of North Carolina Institutional Review Board-approved protocols ( #03-1396 ) by the Cystic Fibrosis Center Tissue Culture Core. Randomization Prior to the emergence of pandemic SARS-CoV-2 , RDV was evaluated in phase 1 clinical trials as well as phase 2 randomized controlled trial trials to treat acute Ebola virus disease in the Democratic Republic of Congo ( DRC) , and human safety data are available ( Mulangu et al. , 2019) . Blinding not detected. Power Analysis not detected. Sex as a biological variable We infected female C57Bl/6 Ces1c-/- mice with 103 PFU SARS1/SARS2-RdRp and initiated subcutaneous treatment with 25 mg/kg 6 RDV BID at one day post-infection ( dpi) . Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Broad-spectrum antiviral drugs , antibodies , and vaccines are needed to combat the current pandemic and those that will emerge in the future. antiviral drugs ,suggested: NoneTherapies combining direct-acting antivirals ( DAAs ) such as RDV and NHC , along with other DAAs such as antibodies and protease inhibitors that target different stages of the viral replication cycle , could be considered for counteracting resistance if it emerges in patients treated with antiviral monotherapy . antiviral monotherapy .suggested: NoneExperimental Models: Cell Lines Sentences Resources To determine if RDV and GS-441524 inhibit SARS-CoV-2 replication in established cell lines , Calu3 2B4 human lung adenocarcinoma cells and Vero E6 African green monkey kidney cells were infected with the SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 and treated with a range of RDV or GS-441524 concentrations . Vero E6suggested: NoneIn addition , SARS-CoV-1 infects Huh7 cells at low frequency at the MOI used in this study and does not appear to spread throughout the culture over the course of the experiment . Huh7suggested: CLS Cat# 300156/p7178_HuH7, CVCL_0336Calu3 cells were infected with 0.1 PFU/cell SARS-CoV-2 and Vero cells were infected with 0.01 PFU/cell SARS-CoV-2 and treated with RDV , GS-441524 ( ‘524) , or DMSO only ( control ) in cell culture medium . Calu3suggested: BCRJ Cat# 0264, CVCL_0609<div style="margin-bottom:8px"> <div><b>Vero</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 cells were seeded at 1 x 105 cells per well , and Calu3 2B4 cells were seeded at 2 x 105 cells per well in 24-well plates ( Corning) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Calu3 2B4</b></div> <div>suggested: None</div> </div> </td></tr></table>Results from Barzooka: We also found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from OddPub: Thank you for sharing your data.
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