A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

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1. Version published to 10.1038/s41467-020-19055-7

   Oct 15, 2020
2. 

   ScreenIT

   Jun 24, 2020

   SciScore for 10.1101/2020.06.22.165712: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   On the next day, cells were wash three times with PBS to remove any residual FBS and followed by 1-hour treatment with goat anti-human ACE2 antibody (R&D Systems) or anti-hDDP4 antibody (R&D Systems) (both antibodies were prepared in OptiMEM medium to the given concentrations).	anti-human ACE2 suggested: None anti-hDDP4 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: African …

   SciScore for 10.1101/2020.06.22.165712: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: All cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   On the next day, cells were wash three times with PBS to remove any residual FBS and followed by 1-hour treatment with goat anti-human ACE2 antibody (R&D Systems) or anti-hDDP4 antibody (R&D Systems) (both antibodies were prepared in OptiMEM medium to the given concentrations).	anti-human ACE2 suggested: None anti-hDDP4 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: African green monkey kidney epithelial cells Vero E6 (ATCC®CRL-1586) were purchased from the American Type Culture Collection (ATCC, Bethesda, MD) and maintained in a high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, South Logan, UT) and 1% penicillin/streptomycin (P/S).	Vero E6 suggested: None
   Human alveolar epithelial cell line (A549) and human embryonic kidney cells (HEK293) were maintained in a high-glucose DMEM supplemented with 10% fetal bovine serum, 1% P/S and 1% HEPES (ThermoFisher Scientific).	A549 suggested: None
   The A549-hACE2 and HEK293-hACE2 cells that stably express human angiotensin-converting enzyme 2 (hACE2)79 were grown in the culture medium supplemented with 10 μg/mL Blasticidin S.	HEK293-hACE2 suggested: None
   SARS-CoV-2-Nluc antiviral assay: Vero or A549-hACE2 cells (12,000 cells per well in phenol-red free medium containing 2% FBS) were plated into a white opaque 96-well plate (Corning).	A549-hACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   The concentration that reduces the 50% luciferase signal (NT50) were estimated by using a four-parameter logistic regression model from the Prism 8 software (GraphPad Software Inc.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Despite the strengths of high throughput and speed, the current rapid neutralization assay must be performed in a biosafety level 3 (BSL-3) facility, representing a major limitation. Experiments are ongoing to attenuate SARS-CoV-2-Nluc so that the assay could be performed in a BSL-2 laboratory. Aligned with the same premise, BSL-2 lab compatible neutralization assays have been reported using VSV pseudotyped with SARS-CoV-2 spike protein21,21. We additionally optimized and validated the recombinant SARS-CoV-2-Nluc for high-throughput antiviral screening. Our results demonstrate that cell type could significantly affect a compound’s EC50 value, underscoring the importance of using biologically relevant cells for drug discovery. The extent of EC50 discrepancy from different cells was dependent on the compound’s mode of action. Remdesivir EC50 values differed by >10-fold when the assay used Vero E6 and A549-hACE2 cells. In another study, remdesivir was shown to be even more potent (EC50 0.01 μM) when tested on primary human airway epithelial (HAE) cells13. The potency differences seen between cell types are due to the differential metabolism of remdesivir in various cells. Host metabolic enzymes are required to convert the remdesivir prodrug to a monophosphate substrate, which is further metabolized by host kinases to its active triphosphate form that incorporates into viral RNA for chain termination. Vero E6 cells are less efficient in forming the active triphosphate than A549-h...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We 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).

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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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3. 

   Version published to 10.1101/2020.06.22.165712v1 on bioRxiv

   Jun 23, 2020