LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants

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   ScreenIT

   May 3, 2021

   SciScore for 10.1101/2021.04.30.442182: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Single cells secreting target-specific antibodies were identified and isolated using three assay types (55): a multiplexed bead assay using multiple optically-encoded beads, each conjugated to the soluble pre-fusion stabilized S protein of either SARS-CoV-2 or WIV1 S with T4-foldon domain, 3C protease cleavage site, 6x His-tags, and twin-strep tags (34), the SARS-CoV-2 S1 subunit or negative controls (bovine serum albumin [BSA] His-tag and T4 FoldOn trimerization domain), and a live cell assay using passively dyed suspension-adapted Chinese hamster ovary (CHO) cells transiently transfected to …

   SciScore for 10.1101/2021.04.30.442182: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Single cells secreting target-specific antibodies were identified and isolated using three assay types (55): a multiplexed bead assay using multiple optically-encoded beads, each conjugated to the soluble pre-fusion stabilized S protein of either SARS-CoV-2 or WIV1 S with T4-foldon domain, 3C protease cleavage site, 6x His-tags, and twin-strep tags (34), the SARS-CoV-2 S1 subunit or negative controls (bovine serum albumin [BSA] His-tag and T4 FoldOn trimerization domain), and a live cell assay using passively dyed suspension-adapted Chinese hamster ovary (CHO) cells transiently transfected to surface-express full-length SARS-CoV-2 S protein (GenBank ID MN908947.3) with a green fluorescent protein (GFP) reporter, and non-transfected cells as a negative control.	GFP suggested: None
   Antibodies were recombinantly produced by transient transfection in either human-embryonic kidney (HEK293) or CHO cells as described in Supplemental Methods.	HEK293 suggested: None
   Samples were prepared by mixing each antibody in 10-fold molar excess with antigen (1:1 freshly prepared mix of 400 nM antibody and 40 nM antigen, both diluted in 1X HBSTE + 0.05% BSA running buffer).	antigen suggested: (W. Sieghart, Center for Brain Research, Medical University of Vienna; Vienna; Austria Cat# Beta1 (375-400, RRID:AB_2827800)
   To test the antibodies’ ability to block ACE2, antibodies coupled to the HC-30M chip as described above were exposed to SARS-CoV-2 S protein:ACE2 complex.	ACE2 suggested: None
   Multi-cycle kinetics on Biacore: The capture molecule, an anti-human IgG (Fc) antibody, was immobilized on a Biacore CM5 chip by direct coupling.	anti-human IgG suggested: None
   CHO cells were washed, and binding was detected by using a fluorescently labeled anti-human secondary antibody.	anti-human secondary antibody . suggested: None
   Median fluorescence intensity of each antibody was normalized over the median fluorescence intensity of the human isotype control for respective antigens.	human isotype control for respective antigens . suggested: None
   Infectious titers were determined for a subset of virus preparations by infection of VeroE6 cells (ATCC CRL-1586) with serially diluted virus followed by staining with an anti-luciferase antibody (Novus Cat # NB600-307PEATT594) and analysis by fluorescence-activated cell sorting using a Becton Dickinson LSRFortessaTM X-20	anti-luciferase suggested: None NB600-307PEATT594 suggested: None
   Infected cells were detected using a primary detection antibody recognizing SARS-CoV-2 nucleocapsid protein (Sino Biological) following staining with secondary detection antibody (goat α-rabbit) conjugated to AlexaFluor 488.	SARS-CoV-2 nucleocapsid protein suggested: (Bioss Cat# bsm-41414M, RRID:AB_2848129) α-rabbit suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The extracellular domain (ECD, residues 18 to 618) of ACE2 was expressed in CHO cells as an Fc-fusion protein containing a TEV-protease recognition site.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   Single cells secreting target-specific antibodies were identified and isolated using three assay types (55): a multiplexed bead assay using multiple optically-encoded beads, each conjugated to the soluble pre-fusion stabilized S protein of either SARS-CoV-2 or WIV1 S with T4-foldon domain, 3C protease cleavage site, 6x His-tags, and twin-strep tags (34), the SARS-CoV-2 S1 subunit or negative controls (bovine serum albumin [BSA] His-tag and T4 FoldOn trimerization domain), and a live cell assay using passively dyed suspension-adapted Chinese hamster ovary (CHO) cells transiently transfected to surface-express full-length SARS-CoV-2 S protein (GenBank ID MN908947.3) with a green fluorescent protein (GFP) reporter, and non-transfected cells as a negative control.	Chinese hamster ovary ( CHO ) suggested: None
   Briefly, 293T cells were transfected with individual mutant spike expression plasmids, and 16 to 20 hours later, transfected cells were infected with VSV-G-pseudotyped ΔGluciferase rVSV.	293T suggested: None
   Infectious titers were determined for a subset of virus preparations by infection of VeroE6 cells (ATCC CRL-1586) with serially diluted virus followed by staining with an anti-luciferase antibody (Novus Cat # NB600-307PEATT594) and analysis by fluorescence-activated cell sorting using a Becton Dickinson LSRFortessaTM X-20	VeroE6 suggested: None
   For neutralization assay serial dilutions (2 dilutions at 10 and 1 μg/ml for the initial screen assay or 8 dilutions for the full curve at 10-0.0006 μg/ml) of monoclonal antibodies were mixed with titrated pseudovirus, incubated for 45 minutes at 37 °C and added to pre-seeded 293T-ACE2 cells (provided by Dr. Michael Farzan) in triplicate in 96-well white/black Isoplates (Perkin Elmer).	293T-ACE2 suggested: RRID:CVCL_YZ65)
   Vero-76 cells were inoculated with SARS-CoV-2 (GenBank MT020880.1) at a MOI = 0.01 and incubated at 37°C with 5% CO2 and 80% humidity.	Vero-76 suggested: None
   The antibody-virus mixture was applied to monolayers of Vero-E6 cells in a 96-well plate and incubated for 1 hour at 37°C in a humidified incubator.	Vero-E6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Single-cell sequencing, bioinformatic analysis, and cloning: Single cell polymerase chain reaction (PCR) and custom molecular biology protocols generated NGS sequencing libraries (MiSeq, Illumina) using automated workstations (Bravo, Agilent).	MiSeq suggested: (A5-miseq, RRID:SCR_012148)
   Plasmids were verified by Sanger sequencing to confirm the original sequence previously identified by NGS.	NGS suggested: (PM4NGS, RRID:SCR_019164)
   Neutralization IC50, IC80 and IC90 titers were calculated using GraphPad Prism 8.0.2.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Model building was performed with Coot (CCP4) and final structure validation with MolProbity (Chen et al., 2010) and CCP4 validation tools.	Coot suggested: (Coot, RRID:SCR_014222) MolProbity suggested: (MolProbity, RRID:SCR_014226)
   , 2019.0101; Chemical Computing Group ULC), and detailed contact analysis with CCP4 CONTACT(Winn et al., 2011) and custom shell/Perl scripts.	CCP4 suggested: (CCP4, RRID:SCR_007255)
   We aligned the genomes to the SARS-CoV-2 reference genome (Genbank file MN908947.3) using BWA-MEM (Danecek et al., 2021), and performed variant calling and annotation using SAMTools.	BWA-MEM suggested: (Sniffles, RRID:SCR_017619) SAMTools suggested: (SAMTOOLS, RRID:SCR_002105)
   All further analysis (i.e. slicing by time or geographic region) was performed using custom-written Python scripts.	Python suggested: (IPython, RRID:SCR_001658)

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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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   Version published to 10.1101/2021.04.30.442182 on bioRxiv

   Apr 30, 2021