Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

  1. Hua-Long Xiong
  2. Yang-Tao Wu
  3. Jia-Li Cao
  4. Ren Yang
  5. Jian Ma
  6. Xiao-Yang Qiao
  7. Xiang-Yang Yao
  8. Bao-Hui Zhang
  9. Ya-Li Zhang
  10. Wang-Heng Hou
  11. Yang-Shi
  12. Jing-Jing Xu
  13. Liang-Zhang
  14. Shao-Juan Wang
  15. Bao-Rong Fu
  16. Ting Yang
  17. Sheng-Xiang Ge
  18. Jun Zhang
  19. Quan Yuan
  20. Bao-Ying Huang
  21. Zhi-Yong Li
  22. Tian-Ying Zhang
  23. Ning-Shao Xia

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Abstract

The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.08.026948: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written informed consents were obtained from all the volunteers.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cells and Samples: Vero-E6 (American Type Culture Collection [ATCC], CRL-1586), Vero (ATCC, CCL-81), BHK21 (ATCC, CCL-10) and 293T (kindly gifted by Dr. Jiahuai Han) cells were maintained in high glucose DMEM (SIGMA-ALDRICH) supplemented with 10% FBS (GIBCO), penicillin (100 IU/mL), streptomycin (100 μg/mL) in a 5% CO2 environment at 37°C and passaged every 2 days.
    BHK21
    suggested: None
    293T
    suggested: None
    BHK21-hACE2 cell was developed by stably transfection of hACE2-expressing plasmid following puromycin resistance selection.
    BHK21-hACE2
    suggested: None
    Live SARS-CoV-2-based neutralization assay: Seeded Vero cells in 96-well plates.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    The IC50 (the half maximal inhibitory concentration) or ID 50 (plasma/serum dilutions causing a 50% reduction of GFP-positive cell number or RNA level) values were calculated with non-linear regression, i.e. log (inhibitor) vs. normalized response – Variable slope, using GraphPad Prism 7.00 (GraphPad Software, Inc., 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

    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 10.1101/2020.04.08.026948v2 on bioRxiv
  3. Version published to 10.1101/2020.04.08.026948v1 on bioRxiv