Identification of conserved SARS-CoV-2 spike epitopes that expand public cTfh clonotypes in mild COVID-19 patients

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1. Version published to 10.1084/jem.20211327

   Oct 14, 2021
2. 

   ScreenIT

   Mar 25, 2021

   SciScore for 10.1101/2021.03.23.436573: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Human subjects: The Institutional Review Boards of Osaka University (approval number 898-4) and National Institute of Infectious Diseases Consent: Written informed consent was obtained from all participants or designated healthcare surrogates if participants were unable to provide informed consent.
   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
   5 × 105 PBMCs were stimulated in the same medium with inactivated SARS-CoV-2, 1 or 10 μg/ml of recombinant S protein, 1 μg/ml of S peptide pool or 1 μg/ml of M + N (MN) …

   SciScore for 10.1101/2021.03.23.436573: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Human subjects: The Institutional Review Boards of Osaka University (approval number 898-4) and National Institute of Infectious Diseases Consent: Written informed consent was obtained from all participants or designated healthcare surrogates if participants were unable to provide informed consent.
   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
   5 × 105 PBMCs were stimulated in the same medium with inactivated SARS-CoV-2, 1 or 10 μg/ml of recombinant S protein, 1 μg/ml of S peptide pool or 1 μg/ml of M + N (MN) peptide pool for 20 hours, followed by staining with anti-human CD3, CD69, CD137, CD154 and TotalSeq-C Hashtags antibodies.	anti-human CD3 suggested: None CD69 suggested: None CD137 suggested: None
   Cells were then stained with biotin-conjugated anti-PE antibody together with antibodies for surface markers for flow cytometric analysis using AttuneNxT (Thermo Fisher Scientific) and FlowJo 10.5.2 (BD Biosciences).	anti-PE suggested: None
   ), anti-mouse CD3 (17A2), anti-mouse CD69 (H1.2F3), anti-rat CD2 (OX-34) and biotin anti-phycoerythrin (PE)(PE001) antibodies were purchased from BioLegend.	anti-mouse CD3 suggested: None anti-mouse CD69 suggested: None anti-rat CD2 suggested: None anti-phycoerythrin (PE) suggested: None PE001 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Plasma diluted at 1:5 followed by two-fold serial dilutions was incubated with equal volume of solution containing 100 TCID50 of SARS-CoV-2 for 1 hour at 37°C and added to VeroE6/TMPRSS2 cells.	VeroE6/TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   For the expression of S proteins of SARS-CoV-2 and HCoV-OC43, pME18S expression vectors for each protein were transfected into HEK293T cells.	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Software and Algorithms
   Sentences	Resources
   2.0 (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.).	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   Hashtag oligo demultiplexing was performed on CLR-normalized hashtag UMI counts, and clonotypes were matched to the gene expression data through their droplet barcodes, using Python scripts.	Python suggested: (IPython, RRID:SCR_001658)
   Two vectors containing paired TCRα and β chains were transfected together into Phoenix packaging cells using PEI MAX (Polysciences)	Phoenix suggested: (Phoenix, RRID:SCR_003163)
   Cells were then stained with biotin-conjugated anti-PE antibody together with antibodies for surface markers for flow cytometric analysis using AttuneNxT (Thermo Fisher Scientific) and FlowJo 10.5.2 (BD Biosciences).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   After initial phase determination, the model buildings were manually performed using program COOT (Emsley et al., 2010).	COOT suggested: (Coot, RRID:SCR_014222)
   The stereochemical quality of the final model was assessed by program MolProbity (Williams et al., 2018).	MolProbity suggested: (MolProbity, RRID:SCR_014226)
   Statistical analysis: Statistical analysis was performed with t-test using SciPy and p-values were indicated in Figure 3A and 4D.	SciPy suggested: (SciPy, RRID:SCR_008058)

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   Results from OddPub: Thank you for sharing your data.

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

   The current study still has several limitations. 1) We analyzed only PBMCs. Although circulating Tfh cells in the periphery can reflect Tfh cells in germinal center (Crotty, 2019; Heit et al., 2017; Hill et al., 2019; Locci et al., 2013; Schmitt et al., 2014), the analysis of tissue resident Tfh cells of biopsy samples is ideal to fully characterize Tfh clonotype. 2) Although the present study suggests that these Tfh clonotypes contribute to amelioration of COVID-19 in patients recovered from mild symptoms, additional studies on patients with severe symptoms are helpful for further aspects. 3) The restricting HLA of the public clonotypes expanded in the patients were DRA-DRB1*15:01 (8.8%), DRA-DRB1*15:02 (2.5%), DRA-DRB1*15:03 (2.0%), DRA-DRB1*15:04 (0.02%), DRA-DRB4*01:03 (33.9% in local)(González-Quezada et al., 2019), DPA1*01:03 (60.7%)-DPB1*04:02 (16.5%), and DPA1*02:02 (24.7%)-DPB1*05:01 (14.8%), which cover a large part of, but not all, population worldwide (Gonzalez-Galarza et al., 2020). Notwithstanding, the current study proposes that combination of single cell paired TCR sequencing and public database is a potent tool to identify public clonotypes which actually responded to SARS-CoV-2 in patients. The rapid reconstitution and determination platform enables us to identify epitopes and responsible HLA simultaneously. As the identified T cell epitopes are conserved among emerging SARS-CoV-2 mutants (Elbe and Buckland-Merrett, 2017; Singer et al., 2020) (Figure 3C), th...

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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 59 and 55. 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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3. 

   Version published to 10.1101/2021.03.23.436573 on bioRxiv

   Mar 23, 2021