Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

  1. Katharine H.D. Crawford
  2. Rachel Eguia
  3. Adam S. Dingens
  4. Andrea N. Loes
  5. Keara D. Malone
  6. Caitlin R. Wolf
  7. Helen Y. Chu
  8. M. Alejandra Tortorici
  9. David Veesler
  10. Michael Murphy
  11. Deleah Pettie
  12. Neil P. King
  13. Alejandro B. Balazs
  14. Jesse D. Bloom

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Abstract

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.20.051219: (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
    The transduced cells were stained with anti-human ACE-2 polyclonal goat IgG (AF933, R&D Systems) primary antibody at 1 ug/mL and donkey anti-goat IgG conjugated to Alexa Fluor 488 (ab150129, Abcam) secondary antibody at a 1:2500 dilution and sorted based on antibody staining.
    anti-human ACE-2
    suggested: None
    anti-goat IgG
    suggested: (Abcam Cat# ab150129, RRID:AB_2687506)
    For verifying expression via flow cytometry, cells were harvested with enzyme-free dissociation buffer (ThermoFisher, 13151014) and stained with anti-human ACE-2 polyclonal goat IgG primary antibody at 2 ug/mL and donkey anti-goat IgG (Alexa Fluor 488) secondary antibody at a 1:1000 dilution.
    IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    4.2 Creation of 293T ACE2 cells: VSV G-pseudotyped lentivirus packaging the human ACE2 was generated via co-transfecting 293T cells (ATCC, CRL-3216) with the pHAGE2-EF1aInt-ACE2-WT plasmid (File S1) and lentiviral helper plasmids (HDM-VSVG, HDM-Hgpm2, HDM-tat1b, and pRC-CMV-Rev1b).
    293T
    suggested: None
    The 293T-ACE2 cells are available from BEI Resources as catalog number NR-52511.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Software and Algorithms
    SentencesResources
    The plasmids themselves are available in BEI Resources (https://www.beiresources.org/) with the following catalog numbers:
    https://www.beiresources.org/
    suggested: (BEI Resource Repository, RRID:SCR_013698)

    Results from OddPub: Thank you for sharing your code and data.

    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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  2. Version published to 10.1101/2020.04.20.051219v1 on bioRxiv