A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike

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1. Version published to 10.1038/s41467-022-32232-0

   Aug 4, 2022
2. 

   ScreenIT

   Dec 7, 2021

   SciScore for 10.1101/2021.12.01.470767: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Horseradish peroxidase (HRP)-labelled secondary antibody (goat anti-human IgG 1:3000) in casein was then incubated for 1 h at RT.	anti-human IgG suggested: None
   Barcoded antibody mix, anti-CD19-AF700, Live/DEAD dye, and labelled SARS-CoV-2 S (AF647 and BV421) were added to the cells and stained for 30min at 4 °C.	anti-CD19-AF700 suggested: None BV421 suggested: None
   Computational analyses for single cell sequencing data: Demultiplexing of raw base call (BCL) files, alignment, read filtering, …

   SciScore for 10.1101/2021.12.01.470767: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Horseradish peroxidase (HRP)-labelled secondary antibody (goat anti-human IgG 1:3000) in casein was then incubated for 1 h at RT.	anti-human IgG suggested: None
   Barcoded antibody mix, anti-CD19-AF700, Live/DEAD dye, and labelled SARS-CoV-2 S (AF647 and BV421) were added to the cells and stained for 30min at 4 °C.	anti-CD19-AF700 suggested: None BV421 suggested: None
   Computational analyses for single cell sequencing data: Demultiplexing of raw base call (BCL) files, alignment, read filtering, barcode and UMI counting of the gene expression and feature barcode (10 hashtag oligos (HTOs) to discriminate individual donors and two antibody-derived tags (ADTs) for IgD/CD27 phenotyping) libraries was performed with the 10X Genomics Cell Ranger analysis pipeline using ‘cellranger count’	antibody-derived tags suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   In short, lentiviruses were produced by co-transfecting the expression plasmid with pMDL, pVSV-g and pRSV-Rev into HEK293T cells using lipofectamine 2000 (Invitrogen).	HEK293T suggested: None
   Two days post transfection, IgM-negative Ramos B cells cultured in RPMI10 were transduced with filtered (0.45 μm) and concentrated (100 kDa molecular weight cutoff, GE Healthcare) HEK293T supernatant.	Ramos B suggested: RRID:CVCL_JL65)
   Ramos cells were first gated based on the morphology (FSC-A/SSC-A) and doublets were removed.	Ramos suggested: CLS Cat# 302007/p32087_Ramos, RRID:CVCL_0597)
   Antibody-dependent cellular trogocytosis: HEK293F cells (Invitrogen) at a density of 1×106cells/mL were transfected using SARS-CoV-2 S plasmid and PEImax (1 µg/µl) in a 3:1 ratio in OptiMEM.	HEK293F suggested: None
   Flow cytometry was used to measure the double positive, PKH26+CFSE+THP-1 cells.	PKH26+CFSE+THP-1 suggested: None
   ADCT was calculated by the fraction of THP-1 cells that received membrane fragments from the HEK293F cells.	THP-1 suggested: None
   Recombinant DNA
   Sentences	Resources
   In short, lentiviruses were produced by co-transfecting the expression plasmid with pMDL, pVSV-g and pRSV-Rev into HEK293T cells using lipofectamine 2000 (Invitrogen).	pMDL suggested: None pVSV-g suggested: RRID:Addgene_138479) pRSV-Rev suggested: RRID:Addgene_12253)
   Software and Algorithms
   Sentences	Resources
   Probe preparation and staining: Biotinylated protein antigens were individually multimerized with fluorescently labeled streptavidin (BB515, BD Biosciences; BUV615, BD Biosciences; AF647, Biolegend; BV421, Biolegend) as described previously (Brouwer et al. 2020).	BD Biosciences suggested: (BD Biosciences, RRID:SCR_013311)
   Analysis was performed using DIVA and Flowjo 10 software (BD Biosciences)	Flowjo suggested: (FlowJo, RRID:SCR_008520)
   10X Genomics library construction: Single-cell embedding in gel beads-in-emulsion (GEMs) and generation of barcoded cDNAwas performed on a 10X Genomics Chromium Controller, following the 10X Genomics v1.1 single-cell V(D)J Next GEM with Feature Barcoding technology for cell surface protein chemistry.	Genomics suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)
   Resulting filtered feature-barcode matrices were imported in R (version 4.0.3) using the Seurat package (version 4.0.2)	Seurat suggested: (SEURAT, RRID:SCR_007322)
   Sequences assembled by CellRanger were aligned to the IMGT gene database (Giudicelli, Chaume, and Lefranc 2005) with BWA (default settings) (Li and Durbin 2010).	BWA suggested: (BWA, RRID:SCR_010910)
   Variants were called with samtools mpileup (Li et al. 2009) and VarScan (Koboldt et al. 2009) with minimum coverage set to 1 read to avoid missing mutations from antibody parts covered by a single sequence read.	samtools suggested: (SAMTOOLS, RRID:SCR_002105) VarScan suggested: (VARSCAN, RRID:SCR_006849)
   Figures were made in UCSF Chimera and Adobe Photoshop (Pettersen et al. 2004).	Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)
   Data analysis and visualization: Data visualization and statistical analyses were performed in GraphPad Prism software (version 8.3), while sequence handling, analysis and visualization were performed in RStudio (version 1.3.1093, R 4.0.3).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   A Mann-Whitney U test to compare CDRH3 lengths was performed in RStudio 1.3.1093 (R 4.0.3).	RStudio suggested: (RStudio, RRID:SCR_000432)

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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:

   Indeed, recent studies highlighted the contribution of Fc effector function in the protection, clearance or limitation of symptoms in COVID-19, in which one of the studied Abs was a non-neutralizing targeting S2 (Shiakolas et al. 2021; Beaudoin-Bussières et al. 2021). Moreover, it has been shown that Fc effector functions are a key determinant of protection after vaccination in a macaque model (Gorman et al. 2021). We report that the S2-targeting IGHV1-69/IGKV3-11 MAbs elicit effector functions and that they cross-react with multiple distinct S glycoproteins, including variants of concern and SARS-CoV-like viruses isolated from bats, but not endemic HCoVs. Overall, such antibody responses could help to mount a quicker adaptive immune response against new variants and potential de novo SARS-CoV-like outbreaks. In contrast, exposure of immunodominant non-NAb epitopes might disfavor the induction of NAbs, which currently represent the most convincing correlate of protection for SARS-CoV-2 vaccines (Corbett et al. 2021; Earle et al. 2021; Feng et al. 2021). Thus, an argument can be made to engineer away non-NAb epitopes such as the one described here, in order to favor the induction of NAbs. Indeed, stabilization of viral glycoproteins in the native prefusion conformation has brought substantial improvements in the induction of NAb responses against the respective viruses, in particular RSV and HIV-1 (de Taeye et al. 2015; McLellan et al. 2013). Similarly, several studies have sh...

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

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3. 

   Version published to 10.1101/2021.12.01.470767 on bioRxiv

   Dec 3, 2021