Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single supersite

  1. Gabriele Cerutti
  2. Yicheng Guo
  3. Tongqing Zhou
  4. Jason Gorman
  5. Myungjin Lee
  6. Micah Rapp
  7. Eswar R. Reddem
  8. Jian Yu
  9. Fabiana Bahna
  10. Jude Bimela
  11. Yaoxing Huang
  12. Phinikoula S. Katsamba
  13. Lihong Liu
  14. Manoj S. Nair
  15. Reda Rawi
  16. Adam S. Olia
  17. Pengfei Wang
  18. Baoshan Zhang
  19. Gwo-Yu Chuang
  20. David D. Ho
  21. Zizhang Sheng
  22. Peter D. Kwong
  23. Lawrence Shapiro

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

No abstract available

Article activity feed

  1. Version published to 10.1016/j.chom.2021.03.005
  2. ScreenIT

    SciScore for 10.1101/2021.01.10.426120: (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
    These surfaces were generated using the His-capture kit (Cytiva, MA) according to the instructions of the manufacturer, resulting in approximately 10,000 RU of anti-his antibody over each surface.
    anti-his
    suggested: None
    The binding affinities of full IgG antibodies to SARS-CoV-2 spike protein were determined using surface plasmon resonance (SPR) and a BIAcore T200 instrument (GE Healthcare) at 25°C.
    SARS-CoV-2 spike protein
    suggested: None
    DMEM supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added to the infected cells and they were cultured overnight as described above.
    anti-VSV-G
    suggested: None
    I1
    suggested: None
    C α distance calculation: The Cα distances per residue between superimposed ligand-free and antibody-bound NTD were calculated by Python 3.8.
    antibody-bound NTD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    They were expressed in Human Embryonic Kidney (HEK) 293 Freestyle cells (Invitrogen) in suspension culture using serum-free media (Invitrogen) and transfected into HEK293 cells using polyethyleneimine (Polysciences).
    HEK
    suggested: None
    The NTD construct was transiently transfected into HEK293 GnTI-Freestyle cells suspension culture in serum-free media using polyethyleneimine.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (kindly provided by Dr. Peihui Wang, Shandong University, China) using FuGENE 6 (Promega).
    HEK293T
    suggested: None
    In brief, Vero E6 cells were seeded in a 96-well plate at a concentration of 2 × 104 cells per well.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    The data was processed and fit to 1:1 single cycle model using the Scrubber 2.0 (BioLogic Software)
    Scrubber
    suggested: (Scrubber2, RRID:SCR_015745)
    The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software and were plotted using Graphpad.
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    The IC50 values were calculated using non-linear regression in GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    To identify somatic hypermutations, each antibody sequence was aligned to the assigned germline gene using MUSCLE v3.8.31 (Edgar, 2004)
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    Symmetry expansion and focused classification in RELION 3.1 (Scheres, 2012) was used for S2P spike complex with 2-51.
    RELION
    suggested: (RELION, RRID:SCR_016274)
    Geometry validation and structure quality assessment were performed using EMRinger (Barad et al., 2015) and Molprobity (Davis et al., 2004).
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Map-fitting cross correlation (Fit-in-Map tool) and figures preparation were carried out using PyMOL and UCSF Chimera (Pettersen et al., 2004) and Chimera X (Pettersen et al., 2021).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Diffraction data were processed with XDS (Kabsch, 2010) and scaled using AIMLESS (Evans and Murshudov, 2013) from the CCP4 software suite (Winn et al., 2011)
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    Structure refinement was performed with a 3.65 Å high-resolution cutoff using Phenix refine (Adams et al., 2010) and PDB-redo (Joosten et al., 2014) alternated with manual model building using Coot.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    C α distance calculation: The Cα distances per residue between superimposed ligand-free and antibody-bound NTD were calculated by Python 3.8.
    Python
    suggested: (IPython, RRID:SCR_001658)
    Sequence entropy of betacoronavirus spike: Human coronavirus reference amino acid sequences of OC43 (UniProt ID: P36334), HKU1 (UniProt ID: Q5MQD0), SARS (UniProt ID: P59594), and MERS (UniProt ID: W5ZZF5), as well as the initial SARS-CoV-2 (GenBank: QHO60594.1) sequence reported in Washington state were aligned using MAFFT software with default parameters (Katoh et al., 2002).
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Cryo-EM data were processed and analyzed using cryoSPARC and Relion.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Cryo-EM and crystallographic structural statistics were analyzed using Phenix, Molprobity, EMringer and Chimera.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)

    Results from OddPub: Thank you for sharing your code and 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 page 45. 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.

  3. Version published to 10.1101/2021.01.10.426120v1 on bioRxiv