Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy

  1. Xin Zeng
  2. Lingfang Li
  3. Jing Lin
  4. Xinlei Li
  5. Bin Liu
  6. Yang Kong
  7. Shunze Zeng
  8. Jianhua Du
  9. Huahong Xiao
  10. Tao Zhang
  11. Shelin Zhang
  12. Jianghai Liu

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Abstract

The infection of the novel coronavirus SARS-CoV-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His recombinant protein was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His proteins were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 for recognizing RBD at the same or similar epitopes can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of a synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Nevertheless, top 1 lead from the standard biopanning of Lib AB1, can only bind to RBD in vitro but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2020.03.21.990770: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study approval: This study received approval from the Research Ethics Committee of Shenzhen Third People’s Hospital, China (approval number: 2020-084).
    Consent: The Research Ethics Committee waived the requirement informed consent before the study started because of the urgent need to collect epidemiological and clinical data.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and NP proteins: The recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight.
    SARS-CoV-2 NP protein (Sino Biological, Beijing)
    suggested: None
    The third staining at 4 °C for 30min involved either: Streptavidin-APC (eBioscience) and/or Streptavidin-PE (BD Biosciences) to target the Strep tag of RBD, or anti-his-APC and anti-his-PE antibodies (Abcam) to target the His tag of RBD.
    anti-his-APC
    suggested: (Miltenyi Biotec Cat# 130-101-322, RRID:AB_2800415)
    anti-his-PE
    suggested: (Miltenyi Biotec Cat# 130-092-691, RRID:AB_1103227)
    Single B cell PCR, cloning and expression of mAbs: The IgG heavy and light chain variable genes were amplified by nested PCR and cloned into linear expression cassettes or expression vectors to produce full IgG1 antibodies as previously described 29,41.
    full IgG1
    suggested: None
    The PCR products were purified and cloned into the backbone of antibody expression vectors containing the constant regions of human IgG1.
    human IgG1
    suggested: None
    SARS-CoV antibodies (S230 and m396) previously isolated by others 42 were synthesized and sequences verified before expression in 293T cells and purification by protein A chromatography.
    SARS-CoV
    suggested: None
    HIV-1 antibody VRC01 was a broadly neutralizing antibody directly isolated from a patient targeting the CD4 binding site of envelope glycoprotein 40.
    CD4 binding site of envelope glycoprotein 40
    suggested: None
    The cells were then stained with PE labeled anti-human IgG Fc secondary antibody (Biolegend) at a 1:20 dilution in 50 μl staining buffer at room temperature for 30 minutes.
    anti-human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV antibodies (S230 and m396) previously isolated by others 42 were synthesized and sequences verified before expression in 293T cells and purification by protein A chromatography.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    HEK 293T cells without transfection were also stained as background control.
    HEK 293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Huh7 cells (ATCC) (approximately 1.5 × 104 per well) were added in duplicate to the virus-antibody mixture.
    Huh7
    suggested: None
    The isolate was amplified in Vero cell lines to make working stocks of the virus (1 × 105 PFU/ml).
    Vero
    suggested: None
    To analyze the mAb neutralizing activities, Vero E6 cells were seeded at 104/well in 96-well culture plates and cultured at 37 °C to form a monolayer.
    Vero E6
    suggested: None
    The genes encoding the heavy and light chains of isolated antibodies were separately cloned into expression vectors containing IgG1 constant regions and the vectors were transiently transfected into HEK293T or 293F cells using polyethylenimine (PEI) (Sigma).
    293F
    suggested: RRID:CVCL_D615)
    Software and Algorithms
    SentencesResources
    Finally, the cells were re-suspended and analyzed with FACS Calibur instrument (BD Biosciences, USA) and FlowJo 10 software (FlowJo, USA).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Half-maximal inhibitory concentrations (IC50) of the evaluated mAbs were determined by luciferase activity 48h after exposure to virus-antibody mixture using GraphPad Prism 6 (GraphPad Software Inc.).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The IgG heavy and light chain variable genes were aligned using Clustal W in the BioEdit sequence analysis package (https://bioedit.software.informer.com/7.2/).
    BioEdit
    suggested: (BioEdit, RRID:SCR_007361)
    Phylogenetic analyses were performed by the Maximum Likelihood method using MEGA X (Molecular Evolutionary Genetics Analysis across computing platforms).
    MEGA X
    suggested: None

    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

    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.

  2. Version published to 10.1101/2020.04.19.049643v1 on bioRxiv