Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants

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1. Version published to 10.1128/jvi.00685-21

   Sep 9, 2021
2. 

   ScreenIT

   Mar 24, 2021

   SciScore for 10.1101/2021.03.17.435802: (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	Animal caretakers and pathology personnel were blinded for the treatment groups.
   Power Analysis	not detected.
   Sex as a biological variable	In vivo hamster model: Syrian hamsters (Mesocricetus auratus) RjHan:AURA strain, male and females 4-10 weeks old, were individually caged in a human biosafety level 3 laboratory.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Monocyte isolation was determined with the following flow cytometry antibody panel after 10 min incubation with anti-human CD32 (StemCell – 18520): APC anti-human CD14 (Biolegend – 301808), PE-Cy7 anti-human CD3 (Biolegend – …

   SciScore for 10.1101/2021.03.17.435802: (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	Animal caretakers and pathology personnel were blinded for the treatment groups.
   Power Analysis	not detected.
   Sex as a biological variable	In vivo hamster model: Syrian hamsters (Mesocricetus auratus) RjHan:AURA strain, male and females 4-10 weeks old, were individually caged in a human biosafety level 3 laboratory.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Monocyte isolation was determined with the following flow cytometry antibody panel after 10 min incubation with anti-human CD32 (StemCell – 18520): APC anti-human CD14 (Biolegend – 301808), PE-Cy7 anti-human CD3 (Biolegend – 344186), AF488 anti-human CD20 (Biolegend – 302316) and live/dead Sytox Blue stain (Invitrogen – S34857).	anti-human CD32 suggested: None anti-human CD14 suggested: (BioLegend Cat# 348805, RRID:AB_2889063) anti-human CD3 suggested: (BioLegend Cat# 348805, RRID:AB_2889063) anti-human CD20 suggested: (BioLegend Cat# 302316, RRID:AB_493227)
   The following flow cytometry antibody panel was used to determine monocyte differentiation and M1 polarization after 10 min incubation with anti-human CD32: APC anti-human CD14 (Biolegend – 344186) BV421 anti-human CD80 (Biolegend – 305222), PE anti-human CCR7 (Biolegend 353204), APC/Fire750 anti-human CD209 (330116) and 7-AAD viability staining solution at 5 μl/1×106 cells.	anti-human CD32: APC anti-human CD14 suggested: None anti-human suggested: (BioLegend Cat# 305222, RRID:AB_2564407) anti-human CD80 suggested: (BioLegend Cat# 305222, RRID:AB_2564407) anti-human CCR7 suggested: (BioLegend Cat# 353204, RRID:AB_10913813) APC/Fire750 anti-human CD209 suggested: None
   REGN-COV2 antibody cocktail was generated as a 1:1 mix of REGN10933 and REGN10987.	REGN10987 suggested: None
   Bound Fc-tagged proteins were detected with anti-human HRP-conjugated secondary antibodies (Jackson ImmunoResearch – 109-035-088) at 1:3000 dilution in PBS 0.5% BSA.	anti-human HRP-conjugated secondary suggested: None
   Briefly, after incubation with kit’s ViraBind™ reagents and virus inactivation, samples were incubated in microwell plates precoated with anti-p24 antibodies followed by a subsequent incubation with secondary FITC-conjugated anti-HIV p24 monoclonal antibody (1:1000).	anti-p24 suggested: None anti-HIV suggested: None
   Subsequently, well were exposed to HRP-conjugated anti- FITC monoclonal antibody (1:1000).	anti- FITC suggested: None
   Antibody-virus mixtures were then cultured with 3 x 104 HEK-293T cells previously genetically engineered to express human ACE2 and TMPRSS2, in the presence of 8 μg/mL of polybrene, in 48-well plates with a final volume of 0.5 mL per well.	TMPRSS2 suggested: None
   Plates were washed with 0.05% v/v PBS-Tween and sequentially incubated with mouse anti-SARS-CoV-2 N protein antibody (The Native Antigen Company – MAB12183-100) at 1:500 dilution and HRP-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch – 115-035-146) at 1:5000 dilution in 3% w/v milk in 0.05%PBS-Tween.	anti-SARS-CoV-2 N protein suggested: None anti-mouse IgG suggested: (DSHB Cat# LEP100 IgG, RRID:AB_528124)
   Experimental Models: Cell Lines
   Sentences	Resources
   Sup-T1 (ATCC – CRL-1942), U937 (ATCC – CRL-1593.2) and K562 (ATCC – CCL-243) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco – 21875034) supplemented with 10% Foetal Calf Serum (FCS, Biosera – FB 1001/500) and 2 mM GlutaMAX™ (Gibco – 35050061) at 37°C with 5% CO2.	U937 suggested: ATCC Cat# CRL-1593, RRID:CVCL_0007) K562 suggested: ATCC Cat# CCL-243, RRID:CVCL_0004)
   Sup-T1 cells were γ-retrovirally transduced to express the S glycoprotein of SARS-CoV-2 Wuhan Hu-1 strain co-expressed with eBFP as a marker gene.	Sup-T1 suggested: BCRC Cat# 60191, RRID:CVCL_1714)
   Supernatant from transfected CHO cells was purified using 1 ml HiTrap MabSelect PrismA (GE Healthcare – 17549851) affinity chromatography with in-line dialysis in PBS via HiTrap 5 ml desalting columns (GE Healthcare – 29048684) using an Akta™ Pure system (GE Healthcare), following manufacturer’s recommendations.	CHO suggested: None
   Binding capacity of ACE2(HH:NN) Fc, LALA Fc and LALA-PG Fc to SupT1 expressing wild-type SARS-CoV-2 full length spike was assessed via incubation of test protein at 45.6 nM with 2-fold serial dilutions for 30 mins at RT, followed by secondary incubation with anti-Human IgG (H+L) AF647 (Invitrogen – A21445) for 20 mins at RT in the dark.	SupT1 suggested: None
   Antibody-virus mixtures were then cultured with 3 x 104 HEK-293T cells previously genetically engineered to express human ACE2 and TMPRSS2, in the presence of 8 μg/mL of polybrene, in 48-well plates with a final volume of 0.5 mL per well.	HEK-293T suggested: None
   SARS-CoV-2 virus neutralisation assay: Vero cells (ATC-CCL81) cultured in Dulbecco’s MEM (Sigma, Cat. No. D6546) with 10% FCS and 2 mM L-Glutamine (Sigma, Cat No. G7513) and 1% penicillin/streptomycin (Invitrogen cat no. 15140148) were seeded the day prior to infection at 2 x104 cells per well in 96-well flat bottom plate.	Vero suggested: ATCC Cat# CCL-81, RRID:CVCL_0059)
   HEK293 cells were used for reverse transfection and expression.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   Software and Algorithms
   Sentences	Resources
   Data analysed with GraphPad Prism 8 (GraphPad software).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Stained samples were acquired using a MacsQuant10 instrument (Miltenyi Biotec) and analyzed on FlowJo software (BD).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Data analysed with Graph Prism 8 (GraphPad software).	Graph Prism suggested: None
   Hit detected by fluorescent secondary antibody using ImageQuant software (GE Healthcare).	ImageQuant suggested: (ImageQuant, RRID:SCR_014246)

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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: 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 10.1101/2021.03.17.435802v1 on bioRxiv

   Mar 17, 2021