Glycan Masking of Epitopes in the NTD and RBD of the Spike Protein Elicits Broadly Neutralizing Antibodies Against SARS-CoV-2 Variants

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1. Version published to 10.3389/fimmu.2021.795741

   Dec 2, 2021
2. 

   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.11.01.466834: (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	Mouse immunization: Groups of female BALB/c mice (6 to 8 weeks old) (n=5 per group) were obtained from the National Laboratory Animal Center, Taipei, Taiwan.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   SARS-CoV-2 S proteins were probed with anti-SARS-CoV-2 primary antibodies (GTX135356, GeneTex) overnight at 4°C, and detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (KPL) for 1 h at room temperature.	anti-SARS-CoV-2 suggested: None GTX135356 suggested: (GeneTex Cat# GTX135356, RRID:AB_2887482) anti-rabbit IgG suggested: None

   SciScore for 10.1101/2021.11.01.466834: (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	Mouse immunization: Groups of female BALB/c mice (6 to 8 weeks old) (n=5 per group) were obtained from the National Laboratory Animal Center, Taipei, Taiwan.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   SARS-CoV-2 S proteins were probed with anti-SARS-CoV-2 primary antibodies (GTX135356, GeneTex) overnight at 4°C, and detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (KPL) for 1 h at room temperature.	anti-SARS-CoV-2 suggested: None GTX135356 suggested: (GeneTex Cat# GTX135356, RRID:AB_2887482) anti-rabbit IgG suggested: None
   Following this, the plates were incubated with 100 μl of HRP) conjugated anti-mouse IgG antibody (1:30000 in dilution buffer) for 1 h at room temperature.	anti-mouse IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   To determine the Ad titers, HEK293A cells were seeded into 6-well plates at a density of 106 cells/well and incubated at 37°C overnight.	HEK293A suggested: None
   SARS-CoV-2 pseudotyped lentivirus neutralization assay: To produce SARS-CoV-2 pseudoviruses, a plasmid expressing the full-length S protein (Wuhan-Hu-1, B.1.1.7, or B.1.351) of SARS-CoV-2 was co-transfected into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   The mixture was then inoculated with an equal volume of 10,000 HEK-293T cells stably expressing the ACE2 gene in 96-well plates, which were seeded one day before infection.	HEK-293T suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse immunization: Groups of female BALB/c mice (6 to 8 weeks old) (n=5 per group) were obtained from the National Laboratory Animal Center, Taipei, Taiwan.	BALB/c suggested: RRID:IMSR_ORNL:BALB/cRl)
   Recombinant DNA
   Sentences	Resources
   Wild-type S and glycan-masking S genes were first cloned into the pENTR1A vector (Invitrogen), and then cloned into the adenoviral plasmid pAd/CMV/V5-DEST (Invitrogen) using LR ClonaseTM II Enzyme Mix (Invitrogen) to produce the Ad plasmid expressing SARS-CoV-2 S gene.	pENTR1A suggested: RRID:Addgene_37431) pAd/CMV/V5-DEST suggested: RRID:Addgene_66112)
   SARS-CoV-2 pseudotyped lentivirus neutralization assay: To produce SARS-CoV-2 pseudoviruses, a plasmid expressing the full-length S protein (Wuhan-Hu-1, B.1.1.7, or B.1.351) of SARS-CoV-2 was co-transfected into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc.	pCMVΔ8.91 suggested: None pLAS2w.FLuc suggested: None
   Software and Algorithms
   Sentences	Resources
   These cells were grown in Dulbecco’s modified Eagle medium (DMEM) (Thermo Scienific) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 units/ml penicillin/streptomycin (P/S), and maintained in an incubator at 37°C with 5% CO2.	Thermo Scienific suggested: None
   The exposed loops or the protruding sites of the exposed loops on the NTD and RBD of the S protein were examined using PyMol (The PyMol Molecular Graphics System, version 4.0; Schrödinger, LLC).	PyMol suggested: (PyMOL, RRID:SCR_000305)
   The IC50 values of the neutralization were obtained from the fitting curves using GraphPad Prism v6.01	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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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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3. 

   Version published to 10.1101/2021.11.01.466834v1 on bioRxiv

   Nov 2, 2021