Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

  1. Lihong Liu
  2. Pengfei Wang
  3. Manoj S. Nair
  4. Jian Yu
  5. Micah Rapp
  6. Qian Wang
  7. Yang Luo
  8. Jasper F.-W. Chan
  9. Vincent Sahi
  10. Amir Figueroa
  11. Xinzheng V. Guo
  12. Gabriele Cerutti
  13. Jude Bimela
  14. Jason Gorman
  15. Tongqing Zhou
  16. Zhiwei Chen
  17. Kwok-Yung Yuen
  18. Peter D. Kwong
  19. Joseph G. Sodroski
  20. Michael T. Yin
  21. Zizhang Sheng
  22. Yaoxing Huang
  23. Lawrence Shapiro
  24. David D. Ho

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Abstract

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  1. Version published to 10.1038/s41586-020-2571-7
  2. Version published to 10.1101/2020.06.17.153486v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.06.17.153486: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Afterwards, the cells were washed again and incubated with a cocktail of flow cytometry and hashtag antibodies, containing CD3 PE-CF594 (BD Biosciences), CD19 PE-Cy7 (Biolegend), CD20 APC-Cy7 (Biolegend), IgM V450 (BD Biosciences), CD27 PerCP-Cy5.5 (BD Biosciences), anti-His PE (Biolegend), and human Hashtag 3 (Biolegend) at 4°C for 1 hr.
    CD19
    suggested: (BioLegend Cat# 348805, RRID:AB_2889063)
    CD27
    suggested: None
    anti-His PE
    suggested: (BioLegend Cat# 362603, RRID:AB_2563634)
    human Hashtag 3
    suggested: (BioLegend Cat# 394665, RRID:AB_2801033)
    Antibody Transcript Annotation and Selection Criteria: Antigen-specific antibody transcripts were processed using our bioinformatics pipeline SONAR for quality control and annotation26.
    Antigen-specific
    suggested: None
    annotation26
    suggested: None
    Analysis of S Trimer-Specific Antibody Repertoire: Because 88% of the S trimer-specific antibodies were IgG isotype, we therefore compared the repertoire features to IgG repertoires from three healthy donors31 (17,243 H chains, 27,575 kappa L chains, 20,889 lambda L chains).
    S trimer-specific antibodies were IgG
    suggested: None
    Next, 100 μL of 10,000-fold diluted Peroxidase AffiniPure goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) were added into each well and incubated for 1 hr at 37°C.
    anti-human IgG
    suggested: None
    For all the competition ELISA experiments, the relative binding of biotinylated antibodies or ACE2 to the S trimer in the presence of competitors was normalized by comparing to competitor-free controls.
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using FuGENE 6 (Promega).
    HEK293T
    suggested: None
    In brief, Vero E6 cells (ATCC) were seeded in a 96-well plate at a concentration of 2 × 104 cells per well.
    Vero E6
    suggested: None
    Post incubation, the virus-antibody mixture was transferred onto a monolayer of Vero-E6 cells grown overnight.
    Vero-E6
    suggested: None
    Cell-Surface Competition Binding Assay: Expi293 cells were co-transfected with vectors encoding pRRL-cPPT-PGK-GFP (Addgene) and pCMV3-SARS-CoV-2 (2019-nCoV) Spike (Sino Biological) at a ratio of 1:1.
    Expi293
    suggested: RRID:CVCL_D615)
    Software and Algorithms
    SentencesResources
    Afterwards, the cells were washed again and incubated with a cocktail of flow cytometry and hashtag antibodies, containing CD3 PE-CF594 (BD Biosciences), CD19 PE-Cy7 (Biolegend), CD20 APC-Cy7 (Biolegend), IgM V450 (BD Biosciences), CD27 PerCP-Cy5.5 (BD Biosciences), anti-His PE (Biolegend), and human Hashtag 3 (Biolegend) at 4°C for 1 hr.
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    The sorted cells were mixed with mononuclear cells from the same donor, labeled with Hashtag 1, and loaded into the 10X Chromium chip of the 5’ Single Cell Immune Profiling Assay (10X Genomics) at the Columbia University Human Immune Monitoring Core (HIMC; RRID:SCR_016740).
    University Human Immune Monitoring
    detected: Columbia University Human Immune Monitoring Core Facility ( RRID:SCR_016740)
    For H chain transcripts, the constant domain 1 (CH1) sequences were used to assign isotype using BLAST with default parameters against a database of human CH1 genes obtained from IMGT.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    The results were then converted into percentage neutralization at a given mAb concentration, and the averages ± SEM were plotted using a five-parameter dose-response curve in GraphPad Prism 8.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The mean fluorescence intensity of APC in GFP-positive cells was analyzed using FlowJo and the relative binding of 2-43 to S trimer in the presence of competitors was calculated as the percentage of the mean fluorescence intensity compared to that of the competitor-free controls.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The model was then fitted interactively using ISOLDE 1.0b540 and COOT 0.8.9.241, and using real space refinement in Phenix 1.1842.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Code Availability: Next-generation sequencing data of antibody repertoires were processed using Cell ranger v3.1.0, SONAR V1, BLAST v2.2.25, CLUSTALO1.2.3, and USEARCH v9.2.64.
    USEARCH
    suggested: (mubiomics, RRID:SCR_006785)
    Cryo-EM data was collected using Leginon 3.4.beta.
    Leginon
    suggested: (Leginon, RRID:SCR_016731)
    Cryo-EM data was processed using cryoSPARC v2.14.2, MotionCor2, Topaz v0.2.4, 3DFSC v3.0, UCSF Chimera v1.13.1, ChimeraX v0.93, ISOLDE v1.0b5, Phenix v1.18, and COOT v0.8.9.2.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    ChimeraX
    suggested: (UCSF ChimeraX, RRID:SCR_015872)

    Results from OddPub: Thank you for sharing your data.

    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.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 49 and 47. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    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 checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.06.17.153486v1 on bioRxiv