An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

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1. Version published to 10.1016/j.celrep.2020.108528

   Dec 1, 2020
2. 

   ScreenIT

   Sep 21, 2020

   SciScore for 10.1101/2020.09.16.300319: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Human sera: Control and recovered patient sera were collected from patients through the NYU Vaccine Center with written consent under I.R.B. approval (IRB 20-00595 and IRB 18-02037) and were deidentified.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cell-line ACE2 expression levels were quantified by staining with anti-ACE2 antibody (NOVUS) and Alexa-fluor 594-conjugated goat anti-mouse IgG (Biolegend) and pacific blue viability dye.	anti-ACE2 suggested: None
   The proteins were analyzed on an immunoblot …

   SciScore for 10.1101/2020.09.16.300319: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Human sera: Control and recovered patient sera were collected from patients through the NYU Vaccine Center with written consent under I.R.B. approval (IRB 20-00595 and IRB 18-02037) and were deidentified.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cell-line ACE2 expression levels were quantified by staining with anti-ACE2 antibody (NOVUS) and Alexa-fluor 594-conjugated goat anti-mouse IgG (Biolegend) and pacific blue viability dye.	anti-ACE2 suggested: None
   The proteins were analyzed on an immunoblot probed with mouse anti-6XHis antibody (Invitrogen) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich).	anti-6XHis suggested: None anti-mouse IgG suggested: None
   The proteins were transferred to polyvinylidene difluoride membranes and probed with anti-HA mAb (Covance), mouse anti-His mAb (Invitrogen) and anti-GAPDH mAb (Life Technologies) followed by goat anti-mouse HRP-conjugated second antibody (Sigma).	anti-HA suggested: None anti-His suggested: None anti-GAPDH suggested: None anti-mouse suggested: None
   The eluted proteins were separated by SDS-PAGE and analyzed on an immunoblot probed with anti-p24 antibody (AG3.0) followed by goat anti-mouse HRP-conjugated second antibody.	anti-p24 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: Vero E6, CaCO2, A549, ACE2 A549, BHK, Huh7 293T, Vero and CHME3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S) at 37°C in 5% CO2.	A549 suggested: None Huh7 293T suggested: None Vero suggested: None CHME3 suggested: ATCC Cat# CRL-3304, RRID:CVCL_II76)
   U937 cells were cultured in RPMI/10% FBS/ with P/S. ExpiCHO-S (Thermo Fisher Scientific) were cultured in ExpiCHO expression medium at 37 °C in 8% CO2.	U937 suggested: None
   SARS-CoV-2 pseudotype reporter virus assay: SARS-CoV-2 S protein pseudotyped lentiviral stocks were produced by cotransfecting 293T cells (4 × 106) with pMDL, pLenti.GFP-NLuc, S protein expression vector and pRSV.	293T suggested: None
   The mixture was added to ACE2.293T cells in a 96 well tissue culture dish containing 1 × 104 cells/well.	ACE2.293T suggested: None
   Protein purification and molecular mass determination: 293F cells (Thermo Fisher) at a density of 2.5 × 106 cells/ml were transfected with microbody expression vector plasmid DNA using polyethyleneimine (Polysciences, Inc) at a 1:3 plasmid:PEI ratio.	293F suggested: None
   The infectious virus titer was determined by plaque assay on Vero E6 cells after staining with crystal violet.	Vero E6 suggested: RRID:CVCL_XD71)
   Software and Algorithms
   Sentences	Resources
   Data were analyzed by flow cytometry with Flowjo software.	Flowjo suggested: (FlowJo, RRID:SCR_008520)
   The molecular mass of each protein was determined by analysis with ASTRA 6 software.	ASTRA suggested: (ASTRA, RRID:SCR_016255)
   Data analysis and statistics: All experiments were performed in technical duplicates or triplicates and data were analyzed using GraphPad Prism (Version 7 7.0e).	GraphPad Prism 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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56 and 53. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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 10.1101/2020.09.16.300319v1 on bioRxiv

   Sep 17, 2020