Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain

  1. Xiaojing Chi
  2. Xiuying Liu
  3. Conghui Wang
  4. Xinhui Zhang
  5. Xiang Li
  6. Jianhua Hou
  7. Lili Ren
  8. Qi Jin
  9. Jianwei Wang
  10. Wei Yang

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads worldwide and leads to an unprecedented medical burden and lives lost. Neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. Here, using SARS-CoV-2 spike receptor-binding domain (RBD) as a bait, we generate a panel of humanized single domain antibodies (sdAbs) from a synthetic library. These sdAbs reveal binding kinetics with the equilibrium dissociation constant ( K D ) of 0.99–35.5 nM. The monomeric sdAbs show half maximal neutralization concentration (EC 50 ) of 0.0009–0.07 µg/mL and 0.13–0.51 µg/mL against SARS-CoV-2 pseudotypes, and authentic SARS-CoV-2, respectively. Competitive ligand-binding experiments suggest that the sdAbs either completely block or significantly inhibit the association between SARS-CoV-2 RBD and viral entry receptor ACE2. Fusion of the human IgG1 Fc to sdAbs improve their neutralization activity by up to ten times. These results support neutralizing sdAbs as a potential alternative for antiviral therapies.

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  1. Version published to 10.1038/s41467-020-18387-8
  2. ScreenIT

    SciScore for 10.1101/2020.04.14.042010: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies were obtained from ThermoFisher for anti-His-HRP, anti-human IgG-HRP, anti-His-488 and anti-CM13.
    anti-His-HRP
    suggested: None
    anti-human IgG-HRP
    suggested: None
    anti-His-488
    suggested: None
    Library design and construction: A synthetic sdAb phage display library was used for the screening of SARS-CoV-2 neutralizing antibodies.
    SARS-CoV-2 neutralizing antibodies .
    suggested: None
    After 4 rounds of panning, phage ELISA identification was performed with 480 individual colonies using Anti-CM13 antibody [B62-FE2] (HRP).
    Anti-CM13
    suggested: None
    Maxipreped plasmids were transiently transfected into 293-F cells (Thermofisher) and the cells were further cultured in suspension for 6 days before harvesting antibody-containing supernatant.
    antibody-containing supernatant .
    suggested: None
    The primary antibody used for Western blot was a horseradish peroxidase conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA)
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and reagents: The Vero (African green monkey kidney), HEK293T (human kidney epithelial), 293F, Calu-3 (human lung adenocarcinoma) cells were obtained from China Infrastructure of Cell Line Resource (Beijing, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, ThermoFisher, Waltham, MA, USA) supplemented with 2-10% fetal bovine serum (FBS,
    Calu-3
    suggested: None
    Production of SARS-CoV-2 spike pseudotyped particle (SARS-CoV-2pp) and virus entry assay: To produce SARS-CoV-2pp, HEK293T cells were seeded 1 day prior to transfection at 2.5×106 cells in a 10-cm plate.
    HEK293T
    suggested: None
    To conduct the virus entry assay, Vero E6 or other cells were seeded in a 96-well plate at 1 day prior to transduction.
    Vero E6
    suggested: None
    For antibody neutralization assay, Vero cells were seeded in 96-well plates at 1 day prior to infection.
    Vero
    suggested: None
    Immunofluorescence microscopy and Western blot: Cultured 293T cells on coverslips were transfected with either SARS-CoV-2 S expression plasmid or empty vector for 24 h and then fixed using 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min.
    293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analysis: Data were analyzed using GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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).

    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: We did not find any issues relating to colormaps.

    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

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  3. Version published to 10.1101/2020.04.14.042010v1 on bioRxiv