Isolation of cross-reactive monoclonal antibodies against divergent human coronaviruses that delineate a conserved and vulnerable site on the spike protein

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   ScreenIT

   Oct 22, 2020

   SciScore for 10.1101/2020.10.20.346916: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: The use of human materials was approved by the local medical ethical committee (MEC approval: 2014-414).
   Randomization	Order of these peptides was randomized, when synthesized on mini-cards.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (goat anti-human HRP conjugate, Southern Biotech, cat. no.: 2010-05 or goat anti-lama HRP conjugate, Abcore, cat. no. AC15-0354) for 1 h at 25°C.	anti-human HRP suggested: …

   SciScore for 10.1101/2020.10.20.346916: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: The use of human materials was approved by the local medical ethical committee (MEC approval: 2014-414).
   Randomization	Order of these peptides was randomized, when synthesized on mini-cards.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (goat anti-human HRP conjugate, Southern Biotech, cat. no.: 2010-05 or goat anti-lama HRP conjugate, Abcore, cat. no. AC15-0354) for 1 h at 25°C.	anti-human HRP suggested: (SouthernBiotech Cat# 2010-05, RRID:AB_2795564) anti-lama suggested: (Acris Antibodies GmbH Cat# AM39052SU-N, RRID:AB_11216596)
   Antibody binding to the peptides was determined using a goat anti-human IgG HRP conjugate (ITK Southern Biotech) for 1 h at RT and tetramethylbenzidine substrate (BioFX).	anti-human IgG suggested: None
   The potent neutralizing anti-MERS-S1 control antibody 7.7G6 43 and an isotype matched negative control mAb were taken along as controls.	anti-MERS-S1 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Spike ectodomains were stably produced in Drosophila S2 cell line, as previously described 28.	S2 suggested: None
   Recombinant antibodies were purified using Protein A sepharose (IBA) according to the manufacturer’s instruction. mAb 1.6C7 and 7.7G6 used in the animal experiments were produced in Chinese hamster ovary (CHO) cells as previously described 61.	Chinese hamster ovary ( CHO ) suggested: None
   In brief, HEK-293T cells at 70~80% confluency were transfected with the pCAGGS expression vectors encoding full-length MERS-S, SARS-S, SARS2-S or OC43-S with a C-terminal cytoplasmic tail truncation to increase cell surface expression levels.	HEK-293T suggested: None
   Pseudotyped VSV virus were titrated on monolayer African green monkey kidney VeroCCL81 cells (MERS-S pseudotyped VSV)	VeroCCL81 suggested: None
   In the virus neutralization assay, serially diluted mAbs were pre-incubated with an equal volume of virus at RT for 1 h, and then inoculated on Vero/HRT-18 cells, and further incubated at 37℃.	Vero/HRT-18 suggested: None
   The mixture was then added to Huh-7 cells (MERS-CoV) or VeroE6 cells (SARS-CoV and SARS-CoV-2) and incubated for 1 hr, after which the cells were washed and further incubated in medium for 8 h.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Huh-7 cells were seeded one day before reaching a confluency of 70-80%.	Huh-7 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   In a second experiment, the prophylactic efficacy of mAb 28D9 was tested against MERS-CoV and SARS-CoV infection in the K18 TghDpp4 mouse model.	TghDpp4 suggested: None
   Software and Algorithms
   Sentences	Resources
   proteins: Coronavirus spike ectodomains of MERS-CoV (residues 19–1262; strain EMC; GenBank accession number (GB): YP_009047204.1), SARS-CoV (residues 15–1182; strain Urbani; GB: AY278741.1), MHV (residues 15–1231; strain A59; UniProt accession number: P11224) and the S2 ectodomain of MHV (residues 718–1252; strain A59; UniProt accession number: P11224) fused with a C-terminal T4/GCN4 trimerization motif, a thrombin cleavage site and a Strep-tag purification tag were cloned in-frame into pMT\Bip\V5\His expression vector.	UniProt suggested: (UniProtKB, RRID:SCR_004426)
   Half-maximum effective concentration (EC50) binding values were calculated by 4-parameter logistic regression on the binding curves using GraphPad Prism version 7.04.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The results were analysed by FlowJo (version 10) and percentage of GFP+Alexa Fluor 594+ cells over GFP+ cells were calculated.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The half maximal inhibitory concentrations (IC50) were determined using 4-parameter logistic regression (GraphPad Prism v7.0).	GraphPad 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 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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   Version published to 10.1101/2020.10.20.346916v1 on bioRxiv

   Oct 20, 2020