Broad neutralizing nanobody against SARS-CoV-2 engineered from pre-designed synthetic library

  1. Qianyun Liu
  2. Chenguang Cai
  3. Yanyan Huang
  4. Li Zhou
  5. Yanbin Guan
  6. Shiying Fu
  7. Youyou Lin
  8. Ting Yang
  9. Nanyan Wan
  10. Fengzhi Zhang
  11. Qi Sun
  12. Ying Bai
  13. Yu Chen
  14. Xiaohua Liang
  15. Huan Yan
  16. Zhen Zhang
  17. Ke Lan
  18. Yu Chen
  19. Xiang Li
  20. Shin-Chen Hou
  21. Yi Xiong

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Abstract

SARS-CoV-2 infection is initiated with Spike glycoprotein binding to the receptor of human angiotensin converting enzyme 2 via its receptor binding domain. Blocking this interaction is considered as an effective approach to inhibit virus infection. Here we report the discovery of a neutralizing nanobody, VHH60, directly produced from a humanized synthetic nanobody library. VHH60 competes with human ACE2 to bind the receptor binding domain of the Spike protein with a K D of 2.56 nM, inhibits infections of both live SARS-CoV-2 and pseudotyped viruses harboring wildtype, escape mutations and prevailing variants at nanomolar level. VHH60 also suppresses SARS-CoV-2 infection and propagation 50-fold better and protects mice from death two times longer than that of control group after live virus inoculation on mice. VHH60 therefore is a powerful synthetic nanobody with a promising profile for disease control against COVID19.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.07.455523: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All animal experiments were approved by the Animal Care and Use Committee of Wuhan University.
    Sex as a biological variableAge-matched (9-10-week-old) female mice were grouped for infection of nanobodies (0.5mg/kg).
    RandomizationAfter 2–3 rounds of selection-amplification cycle, single colonies were randomly selected into deep 96-well culture plate containing 850 μL/well of 2YT (100 μg/mL ampicillin) and shook at 37°C for 3h.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    RBD-His binding to the plate was detected with anti-His tag mouse monoclonal antibody (1:3000 dilution, SinoBiological, 105327-MM02T) and followed by an HRP conjugated anti-mouse IgG (H+L) Goat antibody (Beyotime, A0216).
    anti-His tag
    suggested: (Sino Biological Cat# 105327-MM02T, RRID:AB_2857924)
    anti-mouse IgG
    suggested: (Beyotime Cat# A0216, RRID:AB_2860575)
    For IFA, Anti-hACE2 antibody and anti-SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (Cat: 10108-RP01 and 40143-MM05, SinoBiological) were added as primary antibodies.
    Anti-hACE2
    suggested: None
    anti-SARS-CoV/SARS-CoV-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and viruses: Vero-E6 (ATCC® CRL-1586), CaCO2(ATCC® HTB-37) and 293T (ATCC® CRL-3216) cell are cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermal Fisher, # 12430112), supplied with 10% fetal bovine serum (Thermal Fisher, # 26140079),1%
    Vero-E6
    suggested: None
    293T
    suggested: None
    Vero E6 (ATCC number: CRL-1586) cells were cultured to determinate viral titer.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Protection of K18-hACE2 transgenic mice against SARS-CoV-2: K18-hACE2 transgenic mice expressing human ACE2 driven by the human epithelial cell cytokeratin-18 (K18) promoter, were purchased from Gempharmatech and housed in ABSL-3 pathogen-free facilities under 12-h light-dark cycles with ad libitum access to food and water.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Recombinant DNA
    SentencesResources
    The whole coding cassette was ligated into a pCMV3 expression vector with a signal peptide of MEFGLSWVFLVALFRGVQC at the N-terminal, and either a 6-his tag (for RBD-his) or a human IgG1 Fc fragment with (GSSSS)3 linker at C-terminal.
    pCMV3
    suggested: RRID:Addgene_161029)
    Briefly, pseudovirus bearing wildtype SARS-CoV-2 S protein or mutants were produced by co-transfection with plasmids expressing corresponding protein and backbone plasmid pNL-4-3-Luc.-
    pNL-4-3-Luc
    suggested: None
    To accurately quantify the absolute number of SARS-CoV-2 genomes, a standard curve was prepared by measuring the SARS-CoV-2 N gene constructed in the pCMV-N plasmid.
    pCMV-N
    suggested: None
    Software and Algorithms
    SentencesResources
    ExpiCHO Expression System was purchased from Thermal Fisher (#A29133).
    Thermal Fisher
    suggested: None
    Protein expression and purification: The constructs of VHHs were selected from phage display, RBD fragment (aa319-541) of SARS-CoV-2 S protein (GenBank: MN908947.3) was synthesized by Genewiz Inc (GENEWIZ, Suzhou, China), the extracellular domain of human ACE2 (1-740 aa) (GenBank: NM_021804.1) was amplified from a plasmid (HG10108-ACG, Sinobiologic, Beijing, China).
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    The acid eluted fraction was neutralized with 1M Tris-HCl, pH9.0 and was concentrated and desalted into PBS with Amicon® Ultra-15, PLTK Ultracel-PL membrane (MilliporeSigma Life Science Center, Burlington, Massachusetts, USA) with appropriate MWCO.
    Amicon®
    suggested: None
    The IC50 values were calculated with non-linear regression using GraphPad Prism 8 (GraphPad Software, Inc.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Protection of K18-hACE2 transgenic mice against SARS-CoV-2: K18-hACE2 transgenic mice expressing human ACE2 driven by the human epithelial cell cytokeratin-18 (K18) promoter, were purchased from Gempharmatech and housed in ABSL-3 pathogen-free facilities under 12-h light-dark cycles with ad libitum access to food and water.
    Gempharmatech
    suggested: (GemPharmatech, RRID:SCR_017239)
    All data was analyzed by XLfit (IDBS, Boston, MA 02210) or Prism 5(GraphPad Software, San Diego, CA 92108).
    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.08.07.455523v1 on bioRxiv