Structural basis for potent antibody neutralization of SARS-CoV-2 variants including B.1.1.529

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1. Version published to 10.1126/science.abn8897

   Apr 22, 2022
2. 

   ScreenIT

   Jan 3, 2022

   SciScore for 10.1101/2021.12.27.474307: (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	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For antibodies where vectors were unavailable (e.g., S309, CB6, REGN10933, REGN10987, COV2-2196, COV2-2130, CT-P59, C144, C135, S2E12) (12, 13, 21, 22, 25, 27–31), published amino acids sequences were used for synthesis and cloning into corresponding pVRC8400 vectors (Genscript) (46, 47).	C144 suggested: (Leinco Technologies Cat# C144, RRID:AB_2828501)
   TMPRSS2 and ACE2 expression profiles in 293 Flpin-TMPSS2-ACE2 were characterized by flow cytometry using a mouse monoclonal antibody …

   SciScore for 10.1101/2021.12.27.474307: (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	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For antibodies where vectors were unavailable (e.g., S309, CB6, REGN10933, REGN10987, COV2-2196, COV2-2130, CT-P59, C144, C135, S2E12) (12, 13, 21, 22, 25, 27–31), published amino acids sequences were used for synthesis and cloning into corresponding pVRC8400 vectors (Genscript) (46, 47).	C144 suggested: (Leinco Technologies Cat# C144, RRID:AB_2828501)
   TMPRSS2 and ACE2 expression profiles in 293 Flpin-TMPSS2-ACE2 were characterized by flow cytometry using a mouse monoclonal antibody against TMPRSS2 (MillliporeSigma) followed by an anti-mouse IgG1 APC conjugate (Jackson laboratories) and a molecular probe containing the SARS-CoV-2 receptor binding domain tagged with biotin (Sino biological) followed by staining with a BV421 conjugated streptavidin probe (BD Biosciences).	TMPRSS2 suggested: None anti-mouse IgG1 suggested: None
   After incubation with the antibodies or ACE2, the cells were washed and incubated with an allophycocyanin conjugated anti-human IgG (709-136-149, Jackson Immunoresearch Laboratories) or BV421 conjugated streptavidin conjugate for another 30 minutes.	ACE2 suggested: None anti-human IgG suggested: (Jackson ImmunoResearch Labs Cat# 709-136-149, RRID:AB_2340526)
   Experimental Models: Cell Lines
   Sentences	Resources
   Proteins were expressed in Expi293 cells by transfection with expression vectors encoding corresponding genes.	Expi293 suggested: RRID:CVCL_D615)
   Generation of 293 Flpin-TMPRSS2-ACE2 cell line: 293 Flpin-TMPRSS2-ACE2 isogenic cell line was prepared by co-transfecting pCDNA5/FRT plasmid encoding TMPRSS2-T2A-ACE2 and pOG44 plasmid encoding Flp recombinase in 293 Flpin parental cell line (Thermo Fisher, Cat R75007)	Flpin-TMPRSS2-ACE2 suggested: None
   transducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and S plasmids from SARS CoV-2 variants into 293T cells using Lipofectamine 3000 transfection reagent (L3000-001, ThermoFisher Scientific, Asheville, NC) (48, 49).	293T suggested: None
   293T-ACE2 cells (provided by Dr. Michael Farzan) or 293 flpin-TMPRSS2-ACE2 cells were plated into 96-well white/black Isoplates (PerkinElmer, Waltham, MA) at 75,00 cells per well the day before infection of SARS CoV-2 pseudovirus.	293T-ACE2 suggested: None
   Cell surface binding: HEK293T cells were transiently transfected with plasmids encoding full length SARS CoV-2 spike variants using lipofectamine 3000 (L3000-001, ThermoFisher) following manufacturer’s protocol.	HEK293T suggested: None
   Recombinant DNA
   Sentences	Resources
   Variable heavy chain sequences were human codon optimized, synthesized and cloned into a VRC8400 (CMV/R expression vector)-based IgG1 vector containing an HRV3C protease site (44) as previously described (45).	IgG1 suggested: None
   Generation of 293 Flpin-TMPRSS2-ACE2 cell line: 293 Flpin-TMPRSS2-ACE2 isogenic cell line was prepared by co-transfecting pCDNA5/FRT plasmid encoding TMPRSS2-T2A-ACE2 and pOG44 plasmid encoding Flp recombinase in 293 Flpin parental cell line (Thermo Fisher, Cat R75007)	pCDNA5/FRT suggested: RRID:Addgene_31984) pOG44 suggested: None
   S-containing lentiviral pseudovirions were produced by co-transfection of packaging plasmid pCMVdR8.2,	pCMVdR8.2 suggested: RRID:Addgene_8455)
   transducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and S plasmids from SARS CoV-2 variants into 293T cells using Lipofectamine 3000 transfection reagent (L3000-001, ThermoFisher Scientific, Asheville, NC) (48, 49).	pHR’ suggested: None TMPRSS2 suggested: RRID:Addgene_53887)
   Software and Algorithms
   Sentences	Resources
   The samples were then acquired in a BD LSRFortessa X-50 flow cytometer (BD biosciences) and analyzed using Flowjo (BD biosciences).	Flowjo suggested: (FlowJo, RRID:SCR_008520)
   Iterative manual model building and real-space refinement were carried out in Coot (48) and in Phenix (51), respectively.	Coot suggested: (Coot, RRID:SCR_014222)
   Data process workflow, including motion correction, CTF estimation, particle picking and extraction, 2D classification, ab initio reconstruction, homogeneous refinement, heterogeneous refinement, non-uniform refinement, local refinement and local resolution estimation, were carried out with C1 symmetry in cryoSPARC 3.3 (50).	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Iterative manual model building and real-space refinement were carried out in Coot (48) and in Phenix 1.19.2 (51), respectively.	Phenix suggested: (Phenix, RRID:SCR_014224)
   Molprobity (52) was used to validate geometry and check structure quality at each iteration step.	Molprobity suggested: (MolProbity, RRID:SCR_014226)
   The graphs were generated in GraphPad Prism.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 49, 53, 54 and 55. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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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3. 

   Version published to 10.1101/2021.12.27.474307v1 on bioRxiv

   Dec 28, 2021