Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike

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   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2021.02.03.429355: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Both approvals were granted under the terms of the Infectious Disease Biobank’s ethics permission (reference 19/SC/0232) granted by the South Central Hampshire B Research Ethics Committee in 2019.
   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
   Fab/Fc ELISA: 96-well plates (Corning, 3690) were coated with goat anti-human Fc IgG antibody at 3 μg/mL overnight at 4°C.	anti-human Fc IgG suggested: None
   The plates were washed twice in FACS buffer and stained with 50 μl/well of 1:200 dilution of PE-conjugated mouse …

   SciScore for 10.1101/2021.02.03.429355: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Both approvals were granted under the terms of the Infectious Disease Biobank’s ethics permission (reference 19/SC/0232) granted by the South Central Hampshire B Research Ethics Committee in 2019.
   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
   Fab/Fc ELISA: 96-well plates (Corning, 3690) were coated with goat anti-human Fc IgG antibody at 3 μg/mL overnight at 4°C.	anti-human Fc IgG suggested: None
   The plates were washed twice in FACS buffer and stained with 50 μl/well of 1:200 dilution of PE-conjugated mouse anti-human IgG Fc antibody (BioLegend) on ice in dark for 1h.	anti-human IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Biotinylated Spike was expressed in 1L of HEK293F cells (Invitrogen) at a density of 1.5 × 106 cells ml-1.	HEK293F suggested: None
   SARS-CoV-2 (wild-type and mutants) and SARS-CoV pseudotyped virus preparation: Pseudotyped HIV virus incorporating the SARS-Cov-2 wild-type or mutants (D614G, N501Y, D614G+Del69/70 and B.1.1.7) or SARS-CoV spike protein was produced in a 10 cm dish seeded the day prior with 5×106 HEK293T/17 cells in 10 ml of complete Dulbecco’s Modified Eagle’s Medium (DMEM-C, 10% foetal bovine serum (FBS) and 1% Pen/Strep (100 IU/ml penicillin and 100 mg/ml streptomycin)).	HEK293T/17 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
   Infectious virus strain and propagation: Vero-E6 cells (Cercopithecus aethiops derived epithelial kidney cells, provided by Prof Wendy Barclay, Imperial College London) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with GlutaMAX, 10% fetal bovine serum (FBS), 20 μg/mL gentamicin, and incubated at 37°C with 5% CO2.	Vero-E6 suggested: None
   The media was removed from the pre-plated Vero-E6 cells and the serum-virus mixtures were added to the Vero E6 cells and incubated at 37°C for 24 h.	Vero E6 suggested: None
   Gibson assembly products were directly transfected into HEK-293T cells and transformed under ampicillin selection.	HEK-293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   IgG expression and purification: Ab heavy and light plasmids were co-transfected at a 1:1 ratio into HEK-293F cells (Thermofisher) using PEI Max 40K (1mg/mL, linear polyethylenimine hydrochloride, Polysciences, Inc.) at a 3:1 ratio (PEI max:DNA).	HEK-293F suggested: RRID:CVCL_6642)
   Monoclonal antibody binding to Spike using flow cytometry: HEK293T cells were plated in a 6-well plate (2×106 cells/well).	HEK293T suggested: None
   HeLa and HeLa-ACE2 cells alone and with SARS-CoV-2 Spike only were used as background and positive controls, respectively.	HeLa suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030) HeLa-ACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   PBMCs were stained with live/dead (fixable Aqua Dead, Thermofisher), anti-CD3-APC/Cy7 (Biolegend), anti-CD8-APC-Cy7 (Biolegend), anti-CD14-BV510 (Biolegend), anti-CD19-PerCP-Cy5.5 (Biolegend), anti-IgM-PE (Biolegend), anti-IgD-Pacific Blue (Biolegend) and anti-IgG-PeCy7 (BD) and Spike-Alexa488 (Thermofisher Scientific, S32354) and Spike-APC (Thermofisher Scientific, S32362).	Thermofisher suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
   After another two washes, stained cells were analyzed using flow cytometry, and the binding data were generated by calculating the percent (%) PE-positive cells using FlowJo 10 software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The sequences were aligned with Clustal W and clustered via PhyML to produce maximum likelihood phylogenetic trees which were visualised and annotated using FigTree.	PhyML suggested: (PhyML, RRID:SCR_014629) FigTree suggested: (FigTree, RRID:SCR_008515)

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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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   Version published to 10.1101/2021.02.03.429355 on bioRxiv

   Feb 3, 2021