Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

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1. Version published to 10.1016/j.isci.2021.102479

   May 1, 2021
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   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2021.02.02.428995: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Work was undertaken in accordance with UK Research Ethics Committee (REC) approval for laboratory research projects investigating the immune response to COVID-19 infection with academic collaborators (REC reference 20/SC/0310). IACUC: Animal experiments: Treatment of animals was in accordance with regulations outlined in the U.S. Department of Agriculture (USDA) Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institute of Health, 2011).
   Randomization	Single colonies were randomly picked from the third cycle output and screen of specific binders was performed, using phage ELISA against NTD Vs RBD.
   Blin…

   SciScore for 10.1101/2021.02.02.428995: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Work was undertaken in accordance with UK Research Ethics Committee (REC) approval for laboratory research projects investigating the immune response to COVID-19 infection with academic collaborators (REC reference 20/SC/0310). IACUC: Animal experiments: Treatment of animals was in accordance with regulations outlined in the U.S. Department of Agriculture (USDA) Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institute of Health, 2011).
   Randomization	Single colonies were randomly picked from the third cycle output and screen of specific binders was performed, using phage ELISA against NTD Vs RBD.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Female K18-hACE2 transgenic (B6.Cg-Tg (K18-hACE2)2Prlmn/J HEMI; Jackson Laboratories, USA) were maintained at 20−22 °C and a relative humidity of 50 ± 10% on a 12 hours light/dark cycle, fed with commercial rodent chow (Koffolk Inc.) and provided with tap water ad libitum.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Production of Antibodies: Phagemid DNA of the desired clones were isolated using QIAprep spin Miniprep kit (Qiagen, GmbH, Hilden, Germany), and the entire scFv was cloned into a pcDNA3.1+ based expression vector that was modified, providing the scFv with the human (IgG1) CH2-CH3 Fc fragments, resulting in scFv-Fc antibody format.	IgG1 suggested: None
   For phage ELISA, HRP-conjugated anti-M13 antibody (Sino Biological, USA, Cat# 11973-MM05T-H lot HO13AU601; used at 1:8000 working dilution) was used following detection with TMB substrate (Millipore, USA).	anti-M13 suggested: (Sino Biological Cat# 11973-MM05T-H, RRID:AB_2857928)
   ELISA of both sera and recombinant human antibodies was applied with AP-conjugated Donkey anti-human IgG (Jackson ImmunoResearch, USA, Cat# 709-055-149 lot 130049; used at 1:2000 working dilution) following detection using p-nitrophenyl phosphate (pNPP) substrate (Sigma, Israel).	anti-human IgG suggested: (Jackson ImmunoResearch Labs Cat# 709-055-149, RRID:AB_2340501)
   For L-SIGN binding inhibition assay, NTD-His (10 µg/ml) was immobilized on Ni-NTA sensors, incubated with anti-NTD antibodies washed and incubated with L-SIGN (20 µg/ml).	anti-NTD suggested: None
   Plated peptides were incubated with the individual monoclonal antibodies (5 µg/ml diluted in blocking buffer) and further incubated with donkey anti-human alkaline phosphatase-conjugated secondary antibody (Jackson ImmunoResearch, USA)	anti-human alkaline phosphatase-conjugated secondary suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The original viral isolate was amplified by 5 passages and quantified by plaque titration assay in Vero E6 cells, and stored at −80°C until use.	Vero E6 suggested: RRID:CVCL_XD71)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   For ELISA inhibition assay, primary antibodies (1.5 µg/ml in blocking solution) were pre-incubated for two hours on ice in the presence or absence of inhibitors [blocking buffer for no-inhibition control, 8 mM 2-O-methyl-α-Neu5Ac (Ac2Me), 8 mM D-Glucuronic acid (GlcA; Sigma-Aldrich), or with 0.06 mM sialoglycopeptides (GP) produced from Neu5Gc-deficient Cmah−/− mice sera33 containing 3.4 mM Neu5Ac-GP (validated by DMB-HPLC)].	Cmah−/− suggested: None
   Glycan binding analyses: Cells and virus strains: Vero E6 (ATCC® CRL-1586™) were obtained from the American Type Culture Collection.	Vero E6 suggested: None
   Female K18-hACE2 transgenic (B6.Cg-Tg (K18-hACE2)2Prlmn/J HEMI; Jackson Laboratories, USA) were maintained at 20−22 °C and a relative humidity of 50 ± 10% on a 12 hours light/dark cycle, fed with commercial rodent chow (Koffolk Inc.) and provided with tap water ad libitum.	K18-hACE2 suggested: RRID:IMSR_GPT:T037657) B6.Cg-Tg suggested: None
   Software and Algorithms
   Sentences	Resources
   For the modeling of mAbs recognition sites on SARS-CoV-2 S protein, spike structure with PDB ID 7C2L24 was used and analyzed by The PyMOL Molecular Graphics System (Version 1.7 Schrödinger, LLC).	PyMOL suggested: (PyMOL, RRID:SCR_000305)

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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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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3. 

   Version published to 10.1101/2021.02.02.428995 on bioRxiv

   Feb 2, 2021