Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants

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1. Version published to 10.1038/s41467-021-26539-7

   Nov 2, 2021
2. 

   ScreenIT

   Apr 24, 2021

   SciScore for 10.1101/2021.04.19.440481: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: The original studies to obtain blood samples after written informed consent were previously described and had been approved by the Ethics Board of ChongQing Medical University24. IRB: The original studies to obtain blood samples after written informed consent were previously described and had been approved by the Ethics Board of ChongQing Medical University24.
   Sex as a biological variable	Six-to eight-week-old female hACE2 mice were treated with 58G6 or 510A5 monoclonal antibody at a concentration of 10 mg/kg by intraperitoneal route, respectively.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Se…

   SciScore for 10.1101/2021.04.19.440481: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: The original studies to obtain blood samples after written informed consent were previously described and had been approved by the Ethics Board of ChongQing Medical University24. IRB: The original studies to obtain blood samples after written informed consent were previously described and had been approved by the Ethics Board of ChongQing Medical University24.
   Sex as a biological variable	Six-to eight-week-old female hACE2 mice were treated with 58G6 or 510A5 monoclonal antibody at a concentration of 10 mg/kg by intraperitoneal route, respectively.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Sequence analysis of antigen-specific mAbs: IMGT/V-QUEST (http://www.imgt.org/IMGT_vquest/vquest) and IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/), MIXCR (https://mixcr.readthedocs.io/en/master/) and VDJtools (https://vdjtools-doc.readthedocs.io/en/master/overlap.html) tools were used to do the variable region analysis and annotation for each antibody clone.	antigen-specific mAbs: IMGT/V-QUEST ( http://www.imgt.org/IMGT_vquest/vquest ) suggested: None
   A CM5 chip (GE Healthcare) was linked with anti-human IgG-Fc antibody to capture about 9000 response units of the neutralizing Abs.	anti-human IgG-Fc suggested: None
   The next days, the membranes were washed with TBST and incubated with HRP-conjugated Goat-anti-human Fc antibody (Abcam, ab99759, 1:10000) for 1 h at RT.	ab99759 suggested: (Abcam Cat# ab99759, RRID:AB_10673762)
   The ELISA plates were washed 4 times by blocking buffer and 50 μL Goat F(ab’)2 Anti-Human IgG (Fab’)2 secondary antibody conjugated with ALP (Abcam, ab98532, 1:2000) was incubated with the plate at RT for 30 mins.	Anti-Human IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   After the incubation, the mixtures were then transferred into 96-well plates, which were seeded with Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   HEK293T cells were grown to 80% confluency before transfection with VSV-G pseudotyped ΔG-luciferase, pWPXL and pSPAX2.	HEK293T suggested: None
   After 72 hrs, the luciferase activities of infected HEK293T/ACE2 cells were detected by the Bright-Luciferase Reporter Assay System (Promega, E2650)	HEK293T/ACE2 suggested: None
   Expi293 cells (Thermo Fisher Scientific, USA) cultured in Freestyle 293 Expression Medium (Thermo Fisher Scientific, USA) were maintained at 37 °C.	Expi293 suggested: RRID:CVCL_D615)
   Authentic SARS-CoV-2 and B.1.351 viruses and animal study: Authentic SARS-CoV-2 (WIV04) and B.1.351 (NPRC 2.062100001) viruses were propagated on the Vero-E6 cells and titrated by single layer plaque assay with standard procedure.	Vero-E6 suggested: None
   Recombinant DNA
   Sentences	Resources
   Production of pseudovirus bearing S protein: pVSVG expressing SARS-CoV-2 S protein was constructed as previously described29.	pVSVG suggested: RRID:Addgene_85140)
   HEK293T cells were grown to 80% confluency before transfection with VSV-G pseudotyped ΔG-luciferase, pWPXL and pSPAX2.	pSPAX2 suggested: RRID:Addgene_12260)
   Protein expression and purification: To express the prefusion S ectodomain, the gene encoding residues 1-1208 of SARS-CoV-2 S (GenBank: MN908947.3) with a C-terminal T4 fibritin trimerization motif, an HRV-3C protease cleavage site, a Twin-Strep-tag and an 8 × His-tag was synthesized, and cloned into the mammalian expression vector pcDNA3.1, which was a kind gift from L.	pcDNA3.1 suggested: RRID:Addgene_79663)
   Software and Algorithms
   Sentences	Resources
   The IC50 and IC80 of the evaluated mAbs was and calculated by a four-parameter logistic regression using GraphPad Prism 8.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Sequence analysis of antigen-specific mAbs: IMGT/V-QUEST (http://www.imgt.org/IMGT_vquest/vquest) and IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/), MIXCR (https://mixcr.readthedocs.io/en/master/) and VDJtools (https://vdjtools-doc.readthedocs.io/en/master/overlap.html) tools were used to do the variable region analysis and annotation for each antibody clone.	IgBLAST suggested: (IgBLAST, RRID:SCR_002873)
   2605 micrographs were collected in a single session with a defocus range comprised between 1.0 and 2.8 μm using SerialEM.	SerialEM suggested: (SerialEM, RRID:SCR_017293)
   Cryo-EM data processing: All dose-fractioned images were motion-corrected and dose-weighted by MotionCorr2 software32 and their contrast transfer functions were estimated by cryoSPARC patch CTF estimation33.	MotionCorr2 suggested: None
   The following 2D, 3D classifications, and refinements were all performed in cryoSPARC.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Both of the 58G6 and 13G9 Fab models were first predicted using Phyre226 and then manually built in Coot 0.936 with the guidance of the cryo-EM electron density maps, and overall real-space refinements were performed using Phenix 1.1837.	Coot suggested: (Coot, RRID:SCR_014222) Phenix suggested: (Phenix, RRID:SCR_014224)

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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 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: 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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   Read the original source
3. 

   Version published to 10.1101/2021.04.19.440481v1 on bioRxiv

   Apr 20, 2021
4. 

   Version published to 10.21203/rs.3.rs-215131/v1 on Research Square

   Feb 12, 2021