A potent neutralizing nanobody against SARS‐CoV‐2 with inhaled delivery potential

  1. Junwei Gai
  2. Linlin Ma
  3. Guanghui Li
  4. Min Zhu
  5. Peng Qiao
  6. Xiaofei Li
  7. Haiwei Zhang
  8. Yanmin Zhang
  9. Yadong Chen
  10. Weiwei Ji
  11. Hao Zhang
  12. Huanhuan Cao
  13. Xionghui Li
  14. Rui Gong
  15. Yakun Wan

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Abstract

The coronavirus disease 2019 (COVID‐19) pandemic has become a serious burden on global public health. Although therapeutic drugs against COVID‐19 have been used in many countries, their efficacy is still limited. We here reported nanobody (Nb) phage display libraries derived from four camels immunized with the SARS‐CoV‐2 spike receptor‐binding domain (RBD), from which 381 Nbs were identified to recognize SARS‐CoV‐2‐RBD. Furthermore, seven Nbs were shown to block interaction of human angiotensin‐converting enzyme 2 (ACE2) with SARS‐CoV‐2‐RBD variants and two Nbs blocked the interaction of human ACE2 with bat‐SL‐CoV‐WIV1‐RBD and SARS‐CoV‐1‐RBD. Among these candidates, Nb11‐59 exhibited the highest activity against authentic SARS‐CoV‐2 with 50% neutralizing dose (ND 50 ) of 0.55 μg/ml. Nb11‐59 can be produced on large scale in Pichia pastoris , with 20 g/L titer and 99.36% purity. It also showed good stability profile, and nebulization did not impact its stability. Overall, Nb11‐59 might be a promising prophylactic and therapeutic molecule against COVID‐19, especially through inhalation delivery.

Article activity feed

  1. Version published to 10.1002/mco2.60
  2. ScreenIT

    SciScore for 10.1101/2020.08.09.242867: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationPeriplasmic extract ELISA (PE-ELISA): In order to identify positive clones, 400 clones from each of the 4 libraries were randomly picked and expressed in microplate for PE-ELISA verification.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The mouse anti-HA antibody (Covance, Princeton, NJ, USA) and goat anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich) were added to the wells for incubation successively.
    anti-HA
    suggested: (Covance Cat# MMS-101P, RRID:AB_2314672)
    Next, the plates were incubated with the mouse anti-HA antibody followed by goat anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich).
    anti-mouse IgG-alkaline
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The HEK 293 were grown in DMEM supplemented with 1% Penicillin-Streptomycin and 10% FBS for Nbs functional analysis, and in CD05 (OPEM, SH, CN) for proteins production.
    HEK 293
    suggested: None
    Activity assay of Nbs blocking SARS-CoV-2-RBD/ACE2: To determine the activity of anti-SARS-CoV-2-RBD Nbs blocking SARS-CoV-2-RBD/ACE2 interaction, ACE2/HEK 293 stable cell line was constructed using a lentiviral packaging system. 3×105 ACE2/HEK 293 cells were incubated with the 2.5 μg/mL purified SARS-CoV-2-RBD labeled with biotin, and a gradient concentration of anti-SARS-CoV-2-RBD Nbs, followed by staining with streptavidin-PE (eBioscience, San Diego, CA, USA).
    293
    suggested: None
    ACE2/HEK 293
    suggested: None
    200 μL of the Nbs-virus mixture was added into a 24-well culture plate containing Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Generation of SARS-CoV-2 spike RBD wildtype and mutant proteins: The coding sequence of SARS-CoV-2 spike RBD was achieved from the UniProt website (https://www.uniprot.org/).
    UniProt
    suggested: (UniProtKB, RRID:SCR_004426)
    https://www.uniprot.org/
    suggested: (Universal Protein Resource, RRID:SCR_002380)
    Phylogenetic tree: The multiple alignment and the construction of the tree was made by Clustal Omega, and pair-wise distance was calculated by the strategy of Neighbor-joining47.
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    The binding interaction model of Nb with SARS-CoV-2 Spike protein were generated by Pymol (https://pymol.org/2/).
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Statistical analysis: Statistical analysis was performed using GraphPad Prism 6 software.
    GraphPad Prism
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04452318RecruitingCOVID-19 Study Assessing the Efficacy and Safety of Anti-Spi…

    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.

  3. Version published to 10.1101/2020.08.09.242867v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.08.09.242867: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.RandomizationPeriplasmic extract ELISA (PE-ELISA) In order to identify positive clones, 400 clones from each of the 4 libraries were randomly picked and expressed in microplate for PE-ELISA verification.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    6.7512 SOURCE IDENTIFIER Wuhan Institute of Virology, ChineseN/A Academy of Science This paper N/A This paper N/A Escherichia coli strain WK6 Pichia pastoris Antibodies The mouse anti-HA antibody Biolegend goat anti-mouse IgG-alkaline phosphatase Sigma-Aldrich streptavidin-PE eBioscience Chemicals, Peptides, and Recombinant Proteins DMEM Gibco Penicillin-Streptomycin Gibco FBS Gibco Terrific Broth medium ThermoFisher IPTG Sigma-Aldrich 10×Sypro Orange protein Gel stain Invitrogen carboxymethyl cellulose Merck crystal violet Merck Experimental Models: Cell Lines HEK 293 ATCC Vero E6 CCTCC Software and Algorithms SWISS-MODEL SWISS-MODEL Z-DOCK
    anti-HA
    suggested: None
    The mouse anti-HA antibody (Covance, Princeton, NJ, USA) and goat anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich) were added to the wells for incubation successively.
    anti-mouse IgG-alkaline
    suggested: None
    Next, the plates were incubated with the mouse antiHA antibody followed by goat anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich).
    antiHA
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    (C) Plaques formed in Vero E6 cells inoculated with 100 PFU SARS-CoV-2 plus 3-fold diluted Nb16-68 or Nb11-59 mixture.
    Vero E6
    suggested: None
    6.7512 SOURCE IDENTIFIER Wuhan Institute of Virology, ChineseN/A Academy of Science This paper N/A This paper N/A Escherichia coli strain WK6 Pichia pastoris Antibodies The mouse anti-HA antibody Biolegend goat anti-mouse IgG-alkaline phosphatase Sigma-Aldrich streptavidin-PE eBioscience Chemicals, Peptides, and Recombinant Proteins DMEM Gibco Penicillin-Streptomycin Gibco FBS Gibco Terrific Broth medium ThermoFisher IPTG Sigma-Aldrich 10×Sypro Orange protein Gel stain Invitrogen carboxymethyl cellulose Merck crystal violet Merck Experimental Models: Cell Lines HEK 293 ATCC Vero E6 CCTCC Software and Algorithms SWISS-MODEL SWISS-MODEL Z-DOCK
    HEK
    suggested: None
    EXPERIMENTAL MODELS AND SUBJECT DETAILS Cells and Viruses The HEK 293 and Vero E6 cells were obtained from the American Type Culture Collection (ATCC) and China Center for Type Culture Collection (CCTCC), respectively.
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Activity assay of Nbs blocking SARS-CoV-2-RBD/ACE2 To determine the activity of anti-SARS-CoV-2-RBD Nbs blocking SARS-CoV-2-RBD/ACE2 interaction, ACE2/HEK 293 stable cell line was constructed using a lentiviral packaging system. 3×105 ACE2/HEK 293 cells were incubated with the 2.5 μg/mL purified SARS-CoV-2-RBD labeled with biotin, and a gradient concentration of anti-SARS-CoV-2-RBD Nbs, followed by staining with streptavidin-PE (eBioscience, San Diego, CA, USA).
    293
    suggested: None
    ACE2/HEK 293
    suggested: None
    Software and Algorithms
    SentencesResources
    ( STAR METHODS KEY RESOURCES TABLE REAGENT or RESOURCE Bacterial and Virus Strains SARS-CoV-2
    STAR
    suggested: (STAR, RRID:SCR_015899)
    6.7512 SOURCE IDENTIFIER Wuhan Institute of Virology, ChineseN/A Academy of Science This paper N/A This paper N/A Escherichia coli strain WK6 Pichia pastoris Antibodies The mouse anti-HA antibody Biolegend goat anti-mouse IgG-alkaline phosphatase Sigma-Aldrich streptavidin-PE eBioscience Chemicals, Peptides, and Recombinant Proteins DMEM Gibco Penicillin-Streptomycin Gibco FBS Gibco Terrific Broth medium ThermoFisher IPTG Sigma-Aldrich 10×Sypro Orange protein Gel stain Invitrogen carboxymethyl cellulose Merck crystal violet Merck Experimental Models: Cell Lines HEK 293 ATCC Vero E6 CCTCC Software and Algorithms SWISS-MODEL SWISS-MODEL Z-DOCK
    ThermoFisher IPTG Sigma-Aldrich
    suggested: None
    Pymol Pymol GraphPad Prism 6.0 Graphpad Cat#901515 Cat#A3562 Cat#12-4317-87 Cat#11965092 Cat#15140122 Cat#10099-141C Cat#A1374301 Cat#367-93-1 Cat#S6651 Cat#C5013 Cat#C0775 Cat#CRL-1573 Cat#GDC146 https://swissmodel.expasy.org/ http://zdock.umassmed.edu https://pymol.org/2/
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    https://www.graphpad.com/
    https://www.graphpad.com/
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Phylogenetic tree The multiple alignment and the construction of the tree was made by Clustal Omega, and pair-wise distance was calculated by the strategy of Neighbor-joining(Madeira et al., 2019)
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)

    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 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.

    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.

  5. Version published to 10.1101/2020.08.09.242867v1 on bioRxiv