Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

  1. Sabari Nath Neerukonda
  2. Russell Vassell
  3. Rachel Herrup
  4. Shufeng Liu
  5. Tony Wang
  6. Kazuyo Takeda
  7. Ye Yang
  8. Tsai-Lien Lin
  9. Wei Wang
  10. Carol D. Weiss

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Abstract

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

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  1. Version published to 10.1371/journal.pone.0248348
  2. ScreenIT

    SciScore for 10.1101/2020.12.26.424442: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingA serum and plasma proficiency panel (focused concordance samples) with high, medium, and low neutralizing titers against SARS-COV-2 and a blinded serum and plasma panel developed for the SARS-CoV-2 neutralization assay concordance survey (SNACS) were provided by Dr. David Montefiori (Duke University, Durham, NC).
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and sera: Mouse mAb 10G6H5 against SARS-COV2 S protein was purchased from GenScript (Piscataway, NJ)
    SARS-COV2 S protein
    suggested: (PhosphoSolutions Cat# CoV2-5G8, RRID:AB_2868396)
    The antibody dilution or mAb concentration causing a 50% and 80% reduction of RLU compared to control (ID50 and ID80 or IC50 and IC80, respectively) were reported as the neutralizing antibody titers.
    ID80
    suggested: None
    IC80
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The 293T, Vero, Vero E6, A549, Caco-2, Calu-3 and Huh-7 cells were maintained at 37°C in Dulbecco’s modified eagle medium (DMEM) supplemented with high glucose, L-Glutamine, minimal essential media (MEM) non-essential amino acids, penicillin/streptomycin and 10% fetal bovine serum (FBS)
    Vero E6
    suggested: RRID:CVCL_XD71)
    A549
    suggested: None
    Caco-2
    suggested: None
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    Huh-7
    suggested: None
    Pseudovirus production and neutralization: Pseudoviruses bearing the S glycoprotein and carrying a firefly luciferase (FLuc) reporter gene were produced in 293T cells.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Generation of transient and stable 293T-ACE2.TMPRSS2 cells: The 293T-ACE2.TMPRSS2t cells transiently expressing low, medium, and high levels of TMPRSS2 were generated by co-transfection of ACE2-TM and pCAGGS-TMPRSS2 plasmids in 2μg, 4μg and 8μg, respectively.
    293T-ACE2.TMPRSS2t
    suggested: None
    SARS-CoV-2 mNG infection and confocal microscopy: Vero E6 cells, 293T-hACE2s, and 293T-ACE2.TMPRSS2s cells were seeded on poly-L-lysine-coated coverslips one day prior to infection.
    293T-ACE2.TMPRSS2s
    suggested: None
    Software and Algorithms
    SentencesResources
    , Zanetta E. Morrow, and Bruana Streets (Quest Diagnostics).
    Quest
    suggested: (QUEST, RRID:SCR_005210)
    Titers were calculated using a nonlinear regression curve fit (GraphPad Prism software Inc., La Jolla, CA).
    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: 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 37. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • 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 10.1101/2020.12.26.424442v1 on bioRxiv