Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy

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1. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.05.02.073411: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethics statement: All procedures involving cells and animals were conducted in Biosafety Level 3 laboratory (BSL-3) and approved by the Animal Experiment Committee of Laboratory Animal Center, Beijing Institute of Microbiology and Epidemiology (approval number: IACUC-DWZX-2020-002).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	A dose of 7.2×105 plaque forming unit (PFU) of SARS-CoV-2 (BetaCoV/Wuhan/AMMS01/2020) was administered intranasally (i.n.) to three anesthetized, 9-month-old female BALB/c mice in a total volume of 40 μL.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	R…

   SciScore for 10.1101/2020.05.02.073411: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethics statement: All procedures involving cells and animals were conducted in Biosafety Level 3 laboratory (BSL-3) and approved by the Animal Experiment Committee of Laboratory Animal Center, Beijing Institute of Microbiology and Epidemiology (approval number: IACUC-DWZX-2020-002).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	A dose of 7.2×105 plaque forming unit (PFU) of SARS-CoV-2 (BetaCoV/Wuhan/AMMS01/2020) was administered intranasally (i.n.) to three anesthetized, 9-month-old female BALB/c mice in a total volume of 40 μL.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Multiplex antibody panels applied in this study are: ACE2 (Abcam, ab108252, 1:200), SARS-CoV Spike (Sinobiological, 40150-T52, 1:2000); β-IV-tubulin IV (Abcam, ab179504, 1:1000), CC10 (Millipore, 07-623, 1:500)	ACE2 suggested: (Abcam Cat# ab108252, RRID:AB_10864415) β-IV-tubulin IV ( Abcam , ab179504 suggested: None CC10 suggested: None
   After staining with FITC-conjugated goat anti-human IgG antibody (1:500; Thermo Fisher Scientific), the cells were analyzed by flow cytometer (BD LSRFortessa™) and Flowjo software.	anti-human IgG suggested: None
   ELISA: ELISA was performed to detect SARS-CoV-2 RBD-specific IgG antibodies in the immunized mouse sera.	RBD-specific IgG suggested: None
   After four washes, the bound antibodies were detected by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (1:5,000, Thermo Fisher Scientific) for 1 h at 37°C.	anti-mouse IgG suggested: None
   Neutralizing antibody titers were determined as the highest dilution of sera that completely inhibited virus-induced CPE in at least 90% of the wells (NT90).	NT90 suggested: (Hytest Cat# 4CNT5-CaNT90, RRID:AB_2858081)
   Experimental Models: Cell Lines
   Sentences	Resources
   Virus and cell: SARS-CoV-2 strain BetaCoV/Wuhan/AMMS01/2020 was originally isolated with Vero cells from a patient returning from Wuhan.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Briefly, the recombinant RBD007 plasmid was transfected using FuGENE 6 transfection reagents (Roche Applied Science, Indianapolis, IN) into CHO-K1 cells precultured in F-12K medium (American Type Culture Collection, Manassas, VA), according to the manufacturer’s instructions.	CHO-K1 suggested: None
   Flow cytometry analysis: Flow cytometry analysis was carried out to detect the binding of SARS-CoV-2 RBD-Fc to hACE2 receptor in hACE2-expressing 293T (hACE2/293T) cells.	293T suggested: None
   Briefly, hACE2/293T cells were incubated with SARS-CoV-2 RBD-Fc protein or Fc control (10 μg/ml) for 20 min at 37°C.	hACE2/293T suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Serial passage of SARS-CoV-2 in aged BALB/c mice: Adaptation of SARS-CoV-2 was achieved by serial passage through lungs of BALB/c mice.	BALB/c suggested: RRID:IMSR_ORNL:BALB/cRl)
   Software and Algorithms
   Sentences	Resources
   After staining with FITC-conjugated goat anti-human IgG antibody (1:500; Thermo Fisher Scientific), the cells were analyzed by flow cytometer (BD LSRFortessa™) and Flowjo software.	Flowjo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analysis: Statistical analyses were carried out using Prism software (GraphPad).	Prism suggested: (PRISM, RRID:SCR_005375) 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 did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 19 and 23. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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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2. 

   ScreenIT

   Jun 6, 2020

   SciScore for 10.1101/2020.05.02.073411: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   All procedures involving cells and animals were conducted in Biosafety Level 3 laboratory ( BSL-3 ) and approved by the Animal Experiment Committee of Laboratory Animal Center , Beijing Institute of Microbiology and Epidemiology ( approval number: IACUC-DWZX2020-002) .

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   A dose of 7.2×105 plaque forming unit ( PFU ) of SARS-CoV-2 ( BetaCoV/Wuhan/AMMS01/2020 ) was administered intranasally ( i.n . ) to three anesthetized , 9-month-old female BALB/c mice in a total volume of 40

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	R…

   SciScore for 10.1101/2020.05.02.073411: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   All procedures involving cells and animals were conducted in Biosafety Level 3 laboratory ( BSL-3 ) and approved by the Animal Experiment Committee of Laboratory Animal Center , Beijing Institute of Microbiology and Epidemiology ( approval number: IACUC-DWZX2020-002) .

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   A dose of 7.2×105 plaque forming unit ( PFU ) of SARS-CoV-2 ( BetaCoV/Wuhan/AMMS01/2020 ) was administered intranasally ( i.n . ) to three anesthetized , 9-month-old female BALB/c mice in a total volume of 40

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Using this novel animal model, we further evaluated the in vivo protective efficacy of an RBD-based SARS-CoV-2 subunit vaccine, which elicited highly potent neutralizing antibodies and conferred full protection against SARS-CoV-2 MACSp6 challenge.	SARS-CoV-2 subunit vaccine, suggested: None
   Notably , the short supply and high cost of hACE2-transgenic mice , as well as the costs involved in purchasing and handling other animals permissive to SARS-CoV-2 , including NHPs , make it very difficult for researchers to evaluate the in vivo protective efficacy of anti-SARS-vaccines , antibodies and therapeutics .	anti-SARS-vaccines , suggested: None
   Overall , our established mouse-adapted SARS-CoV-2 infection mouse model can be conveniently , economically , and effectively used for evaluation of the in vivo protective efficacy of SARS-CoV-2 vaccines as well as anti-SARS-CoV-2 antibodies and therapeutics .	anti-SARS-CoV-2 suggested: None
   RBD-specific IgG antibody detected at the indicated time points ( n=10) .	RBD-specific IgG suggested: None
   Multiplex antibody panels applied in this study are: ACE2 ( Abcam , ab108252 , 1:200) , SARS-CoV Spike ( Sinobiological , 40150-T52 , 1:2000); β-IV-tubulin IV ( Abcam , ab179504 , 1:1000) , CC10 ( Millipore , 07-623 , 1:500)	ACE2 suggested: (LifeSpan Cat# LS-C347-1000, AB_1271963) <div style="margin-bottom:8px"> <div><b>β-IV-tubulin IV ( Abcam , ab179504</b></div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div><b>CC10</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After staining with FITC-conjugated goat anti-human IgG antibody ( 1:500; Thermo Fisher Scientific) , the cells were analyzed by flow cytometer ( BD LSRFortessa™ ) and Flowjo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>anti-human IgG</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After four washes , the bound antibodies were detected by incubation with horseradish peroxidase ( HRP)-conjugated anti-mouse IgG antibody ( 1:5,000 , Thermo Fisher Scientific ) for 1 h at 37℃ .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>anti-mouse IgG</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralizing antibody titers were determined as the highest dilution of sera that completely inhibited virus-induced CPE in at least 90 % of the wells ( NT90) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>NT90</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD-Fc was highly expressed in an established CHO stable cell line and purified with high purity ( fig .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>CHO</b></div> <div>suggested: CLS Cat# 603479/p746_CHO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0213">CVCL_0213</a></div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus was amplified and titrated by standard plaque forming assay on Vero cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Vero</b></div> <div>suggested: CLS Cat# 605372/p622_VERO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0059">CVCL_0059</a></div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly , the recombinant RBD007 plasmid was transfected using FuGENE 6 transfection reagents ( Roche Applied Science , Indianapolis , IN ) into CHO-K1 cells precultured in F-12K medium ( American Type Culture Collection , Manassas , VA) , according to the manufacturer’s instructions .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>CHO-K1</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry analysis was carried out to detect the binding of SARSCoV-2 RBD-Fc to hACE2 receptor in hACE2-expressing 293T ( hACE2/293T ) cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>293T</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly , hACE2/293T cells were incubated with SARS-CoV-2 RBD-Fc protein or Fc control ( 10 μg/ml ) for 20 min at 37℃ .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>hACE2/293T</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">bioRxiv , 2020.2003.2013.990036 ( 2020)10.1101/2020.03.13.990036) . 25</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>bioRxiv</b></div> <div>suggested: (bioRxiv, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003933">SCR_003933</a>)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After staining with FITC-conjugated goat anti-human IgG antibody ( 1:500; Thermo Fisher Scientific) , the cells were analyzed by flow cytometer ( BD LSRFortessa™ ) and Flowjo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Flowjo</b></div> <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses were carried out using Prism software ( GraphPad) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Prism</b></div> <div>suggested: (PRISM, <a href="https://scicrunch.org/resources/Any/search?q=SCR_005375">SCR_005375</a>)</div> </div> <div style="margin-bottom:8px"> <div><b>GraphPad</b></div> <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div> </div> </td></tr></table> Results from Barzooka: We also 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 OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog). About SciScore SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

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3. 

   Version published to 10.1101/2020.05.02.073411v1 on bioRxiv

   May 2, 2020