SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization

SciScore for 10.1101/2021.02.12.430998: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	Consent: Written consent was obtained from all individuals and the study was approved by the local ethics committee (14/8/20). IRB: Collection of plasma samples from COVID-19 patients treated at the intensive care unit was approved by the Ethic committee of the University Medicine Göttingen (SeptImmun Study 25/4/19 Ü)
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	Contamination: Further, cell lines were routinely tested for contamination by mycoplasma.

Table 2: Resources

Antibodies
Sentences	Resources
Production of recombinant human monoclonal antibodies against SARS-CoV-2 …

SciScore for 10.1101/2021.02.12.430998: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	Consent: Written consent was obtained from all individuals and the study was approved by the local ethics committee (14/8/20). IRB: Collection of plasma samples from COVID-19 patients treated at the intensive care unit was approved by the Ethic committee of the University Medicine Göttingen (SeptImmun Study 25/4/19 Ü)
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	Contamination: Further, cell lines were routinely tested for contamination by mycoplasma.

Table 2: Resources

Antibodies
Sentences	Resources
Production of recombinant human monoclonal antibodies against SARS-CoV-2 spike: VH and VL sequences of Regeneron antibodies Casirivimab/REGN10933, Imdevimab/REGN10987 and REGN10989 (Hansen et al., 2020) were cloned in pCMC3-untagged-NCV (SINO Biologics, Cat: CV011) and produced in 293T cells by SINO Biological (Beijing, China).	REGN10989 suggested: None
The human IgG1 isotype control antibodies IgG1/κ and IgG1/λ were produced by transfecting FreeStyle 293-F or 293T cells (Fisher Scientific, Schwerte, Germany, Cat. no. R790-07) with the respective plasmids using the protocol provided with the FreeStyle 293 Expression System (Thermo Fisher Scientific, Cat. no. K9000-01).	human IgG1 suggested: None
Briefly, 293T cells were stained with the recombinant human IgG1 antibodies in FACS buffer (PBS with 0.5% bovine serum albumin and 1 nmol sodium azide) for 20 minutes in ice, washed, incubated with an Alexa Fluor 647-labeled mouse monoclonal antibody against the human IgG1-Fc (Biolegend, San Diego, USA, cat #409320) and analyzed in a Gallios flow cytometer (Beckman Coulter, Brea, California, USA respectively)	human IgG1-Fc suggested: (Sino Biological Cat# 10702-MM01T-H, RRID:AB_2860221)
Finally, culture medium was added that was supplemented with anti-VSV-G antibody (culture supernatant from I1-hybridoma cells; ATCC no. CRL-2700; not added to cells expressing VSV-G).	anti-VSV-G suggested: None
The upper portion of the membrane was probed with anti-HA tag antibody (mouse, Sigma-Aldrich, H3663) diluted 1:1,000 in 5% skim milk solution, while the lower portion of the membrane was probed with anti-VSV matrix protein antibody (Kerafast, EB0011; loading control) diluted 1:2,500 in 5% skim milk solution.	anti-HA suggested: (Sigma-Aldrich Cat# H3663, RRID:AB_262051) anti-VSV matrix protein suggested: None
Following incubation over night at 4 °C, membranes were washed three times with PBS-T, before being probed with peroxidase-conjugated anti-mouse antibody (Dianova, 115-035-003, 1:5,000) for 1 h at room temperature.	anti-mouse suggested: (Jackson ImmunoResearch Labs Cat# 115-035-003, RRID:AB_10015289)
Experimental Models: Cell Lines
Sentences	Resources
Cell culture: All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 293T (human, kidney; ACC-635, DSMZ), Huh-7 (human, liver; JCRB0403, JCRB; kindly provided by Thomas Pietschmann, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany) and Vero76 cells (African green monkey, kidney; CRL-1586, ATCC; kindly provided by Andrea Maisner, Institute of Virology, Philipps University Marburg, Marburg, Germany) were cultivated in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS, Biochrom), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech).	Huh-7 suggested: None Vero76 suggested: None
Caco-2 (human, intestine; HTB-37, ATCC) and Calu-3 cells (human, lung; HTB-55, ATCC; kindly provided by Stephan Ludwig, Institute of Virology, University of Münster, Germany) were cultivated in minimum essential medium supplemented with 10% FCS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA) and 1 mM sodium pyruvate (Thermo Fisher	Caco-2 suggested: None HTB-37 suggested: None
A549 cells (human, lung; CRM-CCL-185, ATCC) were cultivated in DMEM/F-12 medium with Nutrient Mix (Thermo Fisher Scientific) supplemented with 10% FCS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech).	A549 suggested: None
The following experimental set-ups were used: (i) In case of experiments comparing the efficiency cell entry by WT and mutant SARS-2-S, target cells were inoculated with 100 μl/well of the respective pseudotype particles; (ii) For investigation of inhibition of SARS-2-S-driven cell entry by the serine protease inhibitor Camostat mesylate, Calu-3 cells were preincubated for 1 h with medium (50 μl/well) containing either increasing concentrations of Camostat (0.5, 5 or 50 μM; Tocris) or dimethyl sulfoxide (solvent control) before the respective pseudotype particles were added on top; in order to assess the ability of sol-hACE2-Fc, patient sera and monoclonal antibodies to block SARS-2-S-driven cell entry, pseudotype particles were preincubated for 30 min with medium containing different dilutions of either sol-hACE2-Fc (1:20, 1:200, 1:2,000) or patient serum/plasma (serum: 1:50, 1:100, 1:200, 1:400, 1:800; plasma: 1:25, 1:100, 1:400, 1:1600, 1:6400), or with different concentrations of monoclonal antibody (5, 0.5, 0.05, 0.005, 0.0005 μg/ml), before being inoculated onto Vero76 cells.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
Production of sol-hACE2-Fc: 293T cells were grown in a T-75 flask and transfected with 20 μg of sol-hACE2-Fc expression plasmid.	293T suggested: None
Data normalization was done as follows: (i) In order to assess enhancement of S protein-driven pseudotype entry in BHK-21 cells following directed overexpression of hACE2, transduction was normalized against the assay background (which was determined by using rhabdoviral pseudotypes bearing no viral glycoprotein, set as 1); (ii) To compare efficiency of cell entry driven by the different S protein variants under study, transduction was normalized against SARS-2-S WT (set as 100%); (iii) For experiments investigating inhibitory effects exerted by sol-hACE2-Fc or Camostat Mesylate, patient serum/plasma samples or monoclonal antibodies, transduction was normalized against a reference sample (control-treated cells or pseudotypes, set as 100%).	BHK-21 suggested: None
Software and Algorithms
Sentences	Resources
Sequence alignments were performed using the Clustal Omega online tool (	Clustal Omega suggested: (Clustal Omega, RRID:SCR_001591)
Protein models were designed using the YASARA (http://www.yasara.org/index.html) and UCSF Chimera (version 1.14, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco) software packages, and are either based on PDB: 6XDG (Hansen et al., 2020) or on a template generated by modelling the SARS-2-S sequence on a published crystal structure (PDB: 6XR8, (Cai et al., 2020)) with the help of the SWISS-MODEL online tool (https://swissmodel.expasy.org/).	YASARA suggested: (YASARA, RRID:SCR_017591)
Data normalization and statistical analysis: Data analysis was performed using Microsoft Excel as part of the Microsoft Office software package (version 2019, Microsoft Corporation) and GraphPad Prism 8 version 8.4.3 (GraphPad Software).	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137) GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

The following limitations of our study need to be considered. We employed pseudotyped particles instead of authentic SARS-CoV-2 and we did not determine whether Y453F affects viral inhibition by T cell responses raised against SARS-CoV-2. Further, we did not investigate whether presence of Y453F in the SARS-CoV-2 S protein increases binding to mink ACE2. Nevertheless, our results suggest that the introduction of SARS-CoV-2 into mink allows the virus to acquire mutations that compromise viral control by the humoral immune response in humans. As a consequence, infection of mink and other animal species should be prevented and it should be continuously monitored whether SARS-CoV-2 amplification in other wild or domestic animals occurs and changes critical biological properties of the virus.

Results from TrialIdentifier: No clinical trial numbers were referenced.

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).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from scite Reference Check: We found no unreliable references.

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SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization

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