Cross-reactive serum and memory B-cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

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1. Version published to 10.1038/s41467-021-23074-3

   May 19, 2021
2. 

   ScreenIT

   Sep 28, 2020

   SciScore for 10.1101/2020.09.22.308965: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   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
   CR3022, a SARS-CoV-1 and SARS-CoV-2 spike binding antibody, and dengue antibody, DEN3, were used as positive and negative controls for the assay, respectively.	DEN3 suggested: None
   ELISA binding: 96-well half-area plates (Corning cat. #3690, Thermo Fisher Scientific) were coated overnight at 4°C with 2 ug/ml of mouse anti-His-tag antibody (Invitrogen cat. #MA1-21315-1MG	anti-His-tag suggested: None
   After the washes, a secondary antibody conjugated with alkaline …

   SciScore for 10.1101/2020.09.22.308965: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   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
   CR3022, a SARS-CoV-1 and SARS-CoV-2 spike binding antibody, and dengue antibody, DEN3, were used as positive and negative controls for the assay, respectively.	DEN3 suggested: None
   ELISA binding: 96-well half-area plates (Corning cat. #3690, Thermo Fisher Scientific) were coated overnight at 4°C with 2 ug/ml of mouse anti-His-tag antibody (Invitrogen cat. #MA1-21315-1MG	anti-His-tag suggested: None
   After the washes, a secondary antibody conjugated with alkaline phosphatase (AffiniPure goat anti-human IgG Fc fragment specific, Jackson ImmunoResearch Laboratories cat. #109-055-008) diluted 1:1000 in 1% BSA/PBS-T, was added to each well.	AffiniPure goat anti-human IgG Fc fragment specific suggested: (Jackson ImmunoResearch Labs Cat# 109-005-008, RRID:AB_2337534) anti-human IgG suggested: (Jackson ImmunoResearch Labs Cat# 109-055-008, RRID:AB_2337601)
   A mixture of fluorescently labeled antibodies to cell surface markers was prepared, including antibodies specific for the T cell markers CD3(APC Cy7, BD Pharmingen #557757), CD4(APC-Cy7, Biolegend #317418) and CD8(APC-Cy7, BD Pharmingen	CD3 suggested: (Fitzgerald Industries International Cat# 61R-CD3gHUCY5, RRID:AB_1283253) CD4 suggested: (BioLegend Cat# 317418, RRID:AB_571947) APC-Cy7 suggested: None CD8 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T cells were transfected with plasmids encoding full-length coronavirus spikes including SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63 and HCoV-229E.	HCoV-NL63 suggested: RRID:CVCL_RW88)
   Neutralization assay: Under BSL2/3 conditions, MLV-gag/pol and MLV-CMV plasmids were co-transfected into HEK293T cells along with full-length or variously truncated SARS-CoV1 and SARS-COV2 spike plasmids using Lipofectamine 2000 to produce single-round of infection competent pseudo-viruses.	HEK293T suggested: None
   . 293T cells were plated in advance overnight with DMEM medium +10% FBS + 1% Pen/Strep + 1% L-glutamine.	293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Following the infection, HeLa-hACE2 cells were lysed using 1x luciferase lysis buffer (25mM Gly-Gly pH 7.8, 15mM MgSO4, 4mM EGTA, 1% Triton X-100)	HeLa-hACE2 suggested: None
   20µL of the supernatant was transferred to a 384-well plate seeded with 2E3 HeLa-ACE2 cells and incubated for an additional 24 hours at 34°C.	HeLa-ACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   The extend of biotinylation was evaluated by BioLayer Interferometry binding value using streptavidin biosensors.	BioLayer suggested: (Harvard Medical School Center for Macromolecular Interactions Core Facility, RRID:SCR_018270)
   After another two washes, stained cells were analyzed using flow cytometry (BD Lyrics cytometers), and the binding data were generated by calculating the percent (%) PE-positive cells for antigen binding using FlowJo 10 software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The absorbance was measured after 8, 20, and 30 minutes, and was recorded at an optical density of 405 nm (OD405) using a VersaMax microplate reader (Molecular Devices), where data were collected using SoftMax software version 5.4.	SoftMax suggested: None
   Gibson assembly products were finally transformed into competent E.coli cells and single colonies were picked for sequencing and analysis on IMGT V-Quest online tool (http://www.imgt.org) as well as downstream plasmid production for antibody expression.	http://www.imgt.org suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)
   10µL of human polyclonal sera diluted 1:500 in Perm/Wash Buffer (BD Biosciences)was added to the plate and incubated at RT for 2 hours.	BD Biosciences)was suggested: None
   The PFU/mL of the monocyte plate supernatant was calculated and graphed using Prism 8 software.	Prism suggested: (PRISM, RRID:SCR_005375)
   The Leginon software 37 was used to automate data collection on a FEI Tecnai Spirit (120keV), paired a FEI Eagle 4k x 4k camera.	Leginon suggested: (Leginon, RRID:SCR_016731)
   RELION 3.0 40 was used to generate the 2D class averages.	RELION suggested: (RELION, RRID:SCR_016274)

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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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   Read the original source
3. 

   Version published to 10.1101/2020.09.22.308965v1 on bioRxiv

   Sep 23, 2020