An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2

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   ScreenIT

   Mar 30, 2022

   SciScore for 10.1101/2022.03.28.486075: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	Animals with sex- and age-matched littermates were randomly included in experiments.
   Blinding	Animal experiments were carried out in a blinded fashion whenever feasible.
   Power Analysis	For the number of animals needed to achieve statistically significant results, we conducted an a priori power analysis.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For assays examining the avid binding of dimeric sACE22-IgG1 and monoclonal antibodies, cells were resuspended in PBS-BSA containing 1:150 polyclonal chicken anti-MYC-FITC (Immunology Consultants Laboratory) and 1:300 anti-human IgG-APC (clone …

   SciScore for 10.1101/2022.03.28.486075: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	Animals with sex- and age-matched littermates were randomly included in experiments.
   Blinding	Animal experiments were carried out in a blinded fashion whenever feasible.
   Power Analysis	For the number of animals needed to achieve statistically significant results, we conducted an a priori power analysis.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For assays examining the avid binding of dimeric sACE22-IgG1 and monoclonal antibodies, cells were resuspended in PBS-BSA containing 1:150 polyclonal chicken anti-MYC-FITC (Immunology Consultants Laboratory) and 1:300 anti-human IgG-APC (clone HP6017, BioLegend).	anti-MYC-FITC (Immunology Consultants Laboratory) suggested: None anti-human IgG-APC suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HeLa-hACE2-11 (a stable human ACE2 HeLa clone) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (4500 mg/l) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C, 5% CO2.	HeLa clone suggested: None
   Calu-3 (ATCC HTB-55) cells were grown in Modified Eagle’s Medium high glucose (4500 mg/l) with 10% FBS, 4 mM L-Glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% CO2.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   Monoclonal antibodies, sACE2-8his proteins, and RBD-8his were expressed in Expi293F cells transfected using Expifectamine (Thermo Fisher) according to the manufacturer’s instructions.	Expi293F suggested: RRID:CVCL_D615)
   Virus titers were quantitated by a plaque forming assay using Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   Pseudovirions were created following a polyethylenimine (PEI)-based transient co-transfection on 293T cells.	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   Pseudovirus Inhibition Assay: HeLa-hACE2-11 cells were seeded (5×103 cells/well) onto white-bottomed 96-well tissue culture plates (100 μL/well) and incubated for 16 h, 37 °C, 5% CO2.	HeLa-hACE2-11 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   HeLa-hACE2-11 (a stable human ACE2 HeLa clone) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (4500 mg/l) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C, 5% CO2.	HeLa-hACE2-11 suggested: None
   Hemizygous K18-hACE2 mice (strain 034860: B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from The Jackson Laboratory.	K18-hACE2 suggested: RRID:IMSR_GPT:T037657) B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860)
   Recombinant DNA
   Sentences	Resources
   Plasmids for sACE22-IgG1 (Addgene #154104), sACE22.v2.4-IgG1 (#154106), sACE2(18-615)-8his (#149268), sACE2(18-615).	sACE22-IgG1 suggested: None
   Mutations for the CDY14 decoy receptor were introduced into the wild type sACE2 plasmids using extension overlap PCR and confirmed by Sanger sequencing.	sACE2 suggested: None
   S Binding Assay: Human codon-optimized genes encoding N-terminal myc-tagged S proteins of BA.1 and BA.2 omicron were synthesized (Integrated DNA Technologies) and cloned into the NheI-XhoI sites of pcDNA3.1(+) (Invitrogen).	pcDNA3.1(+) suggested: RRID:Addgene_129020)
   Pseudovirion Production: Pseudoviruses were created using plasmids for SARS-CoV-2 Omicron B.1.1.529 (BA.1) S and HIV-1 proviral vector pNL4-3.	pNL4-3 suggested: None
   Software and Algorithms
   Sentences	Resources
   Images were taken by Aperio ImageScope 12.4.3 and analyzed by Zen software (Zeiss).	ImageScope suggested: (ImageScope, RRID:SCR_014311) Zen suggested: None
   IC50 values were determined by fitting dose-response curves with four-parameter logistic regression in GraphPad Prism 8 software.	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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04335136	Completed	Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) a…

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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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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2022.03.28.486075 on bioRxiv

   Mar 28, 2022