Discovery of a SARS-CoV-2 Broadly-Acting Neutralizing Antibody with Activity against Omicron and Omicron + R346K Variants

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   ScreenIT

   Jan 24, 2022

   SciScore for 10.1101/2022.01.19.476998: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Following sera binding and neutralization analysis, two mice were selected for hybridoma fusion and received two final boosts consisting of 50-100 μg of the RBD protein at - 5 and -2 days before being euthanized by IACUC approved methods with spleens harvested and a final bleeding collected for sera analysis (“fusion sera”).
   Sex as a biological variable	Biodistribution Study: Female CD-1-IGS (strain code #022) were obtained from Charles River at 6-8 weeks of age.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Binding was detected using an anti-rat IgG-APC detection antibody and …

   SciScore for 10.1101/2022.01.19.476998: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Following sera binding and neutralization analysis, two mice were selected for hybridoma fusion and received two final boosts consisting of 50-100 μg of the RBD protein at - 5 and -2 days before being euthanized by IACUC approved methods with spleens harvested and a final bleeding collected for sera analysis (“fusion sera”).
   Sex as a biological variable	Biodistribution Study: Female CD-1-IGS (strain code #022) were obtained from Charles River at 6-8 weeks of age.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Binding was detected using an anti-rat IgG-APC detection antibody and samples were run on a high-throughput flow cytometer (Intellicyte High Throughput Flow Cytometer [Intellicyte Corp., Albuquerque, NM].	anti-rat IgG-APC suggested: (SouthernBiotech Cat# 0108-11, RRID:AB_2793937)
   For antibody binding to the cells expressing the Spike proteins, the cells were dispensed into wells of a 96-well V bottom plate (40 µL per well), and an equal volume of 2x final concentration of serially-diluted anti-S1 antibody solution was added.	anti-S1 suggested: None
   Detection of bound antibody was carried out by staining the cells with 50 µL of 1:500 diluted APC AffiniPure F(ab’)₂ Fragment (Goat Anti-Human IgG (H+L).	Anti-Human IgG suggested: None
   The next day, pseudotyped VSV was incubated with anti-spike (concentration as indicated) and anti-VSV-G (1 µg/mL) antibodies for 30 minutes at room temperature and added to the HEK-Blue 293 hACE2-TMPRSS2 cells in triplicate.	anti-spike suggested: None anti-VSV-G (1 µg/mL) suggested: (Thermo Fisher Scientific Cat# PA1-30138, RRID:AB_1961360)
   Anti-human fragment crystallizable region (Fc region) antibody was immobilized on a CM5 sensor chip to approximately 8,000 resonance units (RU) using standard N-hydroxysuccinimide/N-Ethyl-N′-(3- dimethylaminopropyl) carbodiimide hydrochloride (NHS/EDC) coupling methodology.	Anti-human fragment crystallizable region (Fc region) suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   For neutralization, VsV-SARS-spike GFP-expressing reporter virus was pre-incubated with mouse sera (1:100-1,200), hybridoma supernatants (1:10-1:10,000), or purified human monoclonal antibodies (0.1 ng/ml-1 μg/ml) and incubated at 4 °C for 1 hr before the inoculum was added to HEK-293 cells expressing human ACE2 and Transmembrane Serine Protease-2 overnight at 37 °C, 5% CO2 27.	HEK-293 suggested: None
   HEK293 cells were transfected using FuGeneHD transfection reagent according to manufacturer’s protocol (Promega, Cat # E2311).	HEK293 suggested: None
   BHK21/WI-2 cells (Kerafast #EH1011) were maintained in DMEM (Thermo Fisher #11965092) supplemented with 5% fetal bovine serum.	BHK21/WI-2 suggested: RRID:CVCL_HB78)
   293-ACE2 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 200 µg/mL G418 (Invivogen #ant-gn-2).	293-ACE2 suggested: RRID:CVCL_DR94)
   Lambda: G75V, T76I, R246N, Δ247-253, L452Q, F490S, D614G, and T859N. B.1.1.318: T95I, ΔY144, E484K, D614G, P681H, and D796H. Mu: T95I, Y144T, Y145S, ins146N, R346K, E484K, N501Y, D614G, P681H, and D950N. Omicron: A67V, del69-70, T95I, G142D, del143-145, N211D, del212, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T457K, D614G, H665Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F VSV-Spike pseudotype generation: To generate each Spike pseudotyped VSV, 1.2E6 BHK21 cells were nucleofected with 2 µg of Spike plasmid using an Amaxa Nucleofector II with cell line kit L (Lonza #VCA-1005) and program A-031.	BHK21 suggested: None
   medium was removed, and serial dilution of homogenized lungs were added to Vero cells and subsequently incubated for 1 h at 37 °C.	Vero suggested: None
   Cell culture media was removed from cells and sera/virus premix was added to VeroE6 cells at 100 μL/well and incubated for 1 h at 37 °C.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Immunizations were done on eight-to twelve-week-old H2L2 mice interperitoneally with 50-100 μg of a recombinant SARS-CoV2 Spike RBD319-591-Fc fusion protein generated from sequence from the original Wuhan seafood market pneumonia virus isolate (GenBank Accession# MN908947) and cloned in-frame into pcDNA vectors containing human IgG1 and mouse IgG2a Fc tags (GenScript USA Inc., Piscataway, NJ).	H2L2 suggested: None
   Pharmacokinetic Study: Female CD-1-IGS (strain code #022) were obtained from Charles River Laboratories at 6-8 weeks of age.	CD-1-IGS suggested: None
   khACE2 mouse model of COVID-19 infection: K18-hACE2 transgenic mice were purchased from Jackson laboratory and maintained in pathogen-free conditions and handling conforms to the requirements of the National Institutes of Health and the Scripps Research Institute Animal Research Committee.	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   Recombinant DNA
   Sentences	Resources
   Immunizations were done on eight-to twelve-week-old H2L2 mice interperitoneally with 50-100 μg of a recombinant SARS-CoV2 Spike RBD319-591-Fc fusion protein generated from sequence from the original Wuhan seafood market pneumonia virus isolate (GenBank Accession# MN908947) and cloned in-frame into pcDNA vectors containing human IgG1 and mouse IgG2a Fc tags (GenScript USA Inc., Piscataway, NJ).	pcDNA suggested: RRID:Addgene_66792)
   Unique clones were chosen from each clonal family, and DNA was synthesized and cloned in-framed into pcDNA-based vectors containing a human IgG1 constant region and a human kappa light chain constant region (GenScript USA Inc., Piscataway, NJ).	pcDNA-based suggested: None
   Plasmids: All SARS-CoV-2 Spike constructs for pseudotype generation were expressed from plasmid pCDNA3.1 (ThermoFisher #V79020).	pCDNA3.1 suggested: RRID:Addgene_79663)
   Software and Algorithms
   Sentences	Resources
   Cells with a high mean fluorescence intensity (MFI) were identified using FlowJo software (Tree Star, Inc.) and graphed using GraphPad Prism to create a heat map based on mean fluorescence intensity.	FlowJo suggested: (FlowJo, RRID:SCR_008520) GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Purified PCR product was then submitted for Sanger sequencing using 3’ constant gene primers (GeneWiz, South Plainfield, NJ).	GeneWiz suggested: (GENEWIZ, RRID:SCR_003177)
   Pharmacokinetic analysis of the collected ELISA data was performed with the Phoenix WiNnonlin suite of software (version 6.4, Certara) using a non-compartmental approach consistent with an IN-bolus route of administration.	Phoenix suggested: (Phoenix, RRID:SCR_003163)

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

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   Version published to 10.1101/2022.01.19.476998v1 on bioRxiv

   Jan 20, 2022