Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry

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1. Version published to 10.1126/science.abm8143

   Jan 28, 2022
2. 

   ScreenIT

   Oct 22, 2021

   SciScore for 10.1101/2021.10.13.464254: (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	In brief, female Syrian hamsters (Mesocricetus auratus) of 6-8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 μL containing 1×104 TCID50 Beta B.1.351 (derived from hCoV-19/Belgium/rega-1920/2021; EPI_ISL_896474, 2021-01-11).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibody isolation and recombinant production: Antigen specific IgG+ memory B cells were isolated and cloned from PBMC of SARS-CoV-2 convalescent individuals.	Antigen specific IgG+ suggested: None
   goat anti-human IgG secondary …

   SciScore for 10.1101/2021.10.13.464254: (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	In brief, female Syrian hamsters (Mesocricetus auratus) of 6-8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 μL containing 1×104 TCID50 Beta B.1.351 (derived from hCoV-19/Belgium/rega-1920/2021; EPI_ISL_896474, 2021-01-11).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibody isolation and recombinant production: Antigen specific IgG+ memory B cells were isolated and cloned from PBMC of SARS-CoV-2 convalescent individuals.	Antigen specific IgG+ suggested: None
   goat anti-human IgG secondary antibody (Southern Biotech, 2040-04) was added and incubated for 45 min at room temperature.	goat anti-human IgG secondary antibody suggested: None anti-human IgG suggested: (SouthernBiotech Cat# 2040-04, RRID:AB_2795643)
   After 2 h, infected cells were washed an additional five times with DMEM prior to adding media supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) to reduce parental background.	anti-VSV-G suggested: None
   Cells were fixed with 4% PFA (Electron Microscopy Sciences, #15714S), permeabilized with Triton X-100 (SIGMA, #X100-500ML) and stained with an antibody against the viral nucleocapsid protein (Sino Biologicals, #40143-R001) followed by a staining with the nuclear dye Hoechst 33342 (Fisher Scientific, # H1399) and a goat anti-rabbit Alexa Fluor 647 antibody (Invitrogen, #A-21245).	#X100-500ML suggested: None anti-rabbit suggested: (Molecular Probes Cat# A-21245, RRID:AB_141775)
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T-ACE2, Vero-TMPRSS2) (32)	HEK293T-ACE2 suggested: None
   Pseudotyped viruses were prepared using Lenti-X 293 cells seeded in 15-cm dishes.	293 suggested: None
   For neutralization experiments, HEK-293T cells expressing hACE2 (Crawford et al. 2020) in DMEM supplemented with 10% FBS and 1% PenStrep were seeded at 20,000 cells per well into clear bottom, white manually poly-D-lysine coated 96 well plates and incubated at 37°C.	HEK-293T suggested: None
   Neutralization of authentic SARS-CoV-2 viruses: Vero-TMPRSS2 cells were seeded into black-walled, clear-bottom 96-well plates at 2 × 104 cells/well and cultured overnight at 37°C.	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
   Cell-surface mAb-mediated S1 shedding: CHO cells stably expressing the prototypic SARS-CoV-2 Spike protein were harvested, washed in wash buffer (PBS 1% BSA 2 mM EDTA) and resuspended in PBS.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   Viral replication fitness assays: Vero E6 cells (ATCC, CRL-1586) were seeded at 1×106 cells per well in 6-well plates.	Vero E6 suggested: None
   After a 15-minute incubation, Jurkat cells stably expressing FcγRIIIa receptor (V158 variant) or FcγRIIa receptor (H131 variant) and NFAT-driven luciferase gene (effector cells) were added at an effector to target ratio of 6:1 for FcγRIIIa and 5:1 for FcγRIIa.	Jurkat suggested: None
   Recombinant DNA
   Sentences	Resources
   fold-on trimerization motif, and an 8× His tag in the pCMV vector.	pCMV suggested: RRID:Addgene_20783)
   Software and Algorithms
   Sentences	Resources
   Using the Database IMGT (http://www.imgt.org), the VH and VL gene family and the number of somatic mutations were determined by analyzing the homology of the VH and VL sequences to known human V, D and J genes.	http://www.imgt.org suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)
   UCA sequences of heavy and light variable regions were constructed using IMGT/V-QUEST.	IMGT/V-QUEST suggested: (IMGT/V-QUEST, RRID:SCR_010749)
   After 45 min incubation, absorbance at 405 nm was measured by a plate reader (Biotek) and data plotted using Prism GraphPad.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   After a 30 sec stabilization step in KB, biosensors were moved in SARS-CoV or SARS-CoV-2 :2 dilution series (starting concentration: 18.5 nM) for the 600 sec association step, and then moved back in KB to record dissociation signals for 540 sec.	SARS-CoV-2 suggested: (BioLegend Cat# 946101, RRID:AB_2892515)
   The data were baseline subtracted, results fitted using the Pall FortéBio/Sartorius analysis software (version 12.0) and plotted using GraphPad Prism (version 9.1.1	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Data were analyzed and visualized with Prism (Version 9.1.1).	Prism suggested: (PRISM, RRID:SCR_005375)
   Micrographs were recorded using the Leginon software on a 120 kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k	Leginon suggested: (Leginon, RRID:SCR_016731)
   3D refinements were carried out using non-uniform refinement along with per-particle defocus refinement in CryoSPARC (57).	CryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Model building and refinement: UCSF Chimera (61) and Coot (62) were used to fit atomic models into the cryoEM maps.	Coot suggested: (Coot, RRID:SCR_014222)
   This variant was originally isolated in house from nasopharyngeal swabs taken from travelers returning to Belgium (baseline surveillance) and were subjected to sequencing on a MinION platform (Oxford Nanopore) directly from the nasopharyngeal swabs (65).	MinION suggested: (MinION, RRID:SCR_017985)

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   Results from OddPub: Thank you for sharing your code and 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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. 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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   Read the original source
3. 

   Version published to 10.1101/2021.10.13.464254 on bioRxiv

   Oct 14, 2021