A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

  1. Xuping Xie
  2. Antonio E. Muruato
  3. Xianwen Zhang
  4. Kumari G. Lokugamage
  5. Camila R. Fontes-Garfias
  6. Jing Zou
  7. Jianying Liu
  8. Ping Ren
  9. Mini Balakrishnan
  10. Tomas Cihlar
  11. Chien-Te K. Tseng
  12. Shinji Makino
  13. Vineet D. Menachery
  14. John P. Bilello
  15. Pei-Yong Shi

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Abstract

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC 50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.

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  1. Version published to 10.1038/s41467-020-19055-7
  2. ScreenIT

    SciScore for 10.1101/2020.06.22.165712: (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 AuthenticationContamination: All cell lines were tested negative for mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    On the next day, cells were wash three times with PBS to remove any residual FBS and followed by 1-hour treatment with goat anti-human ACE2 antibody (R&D Systems) or anti-hDDP4 antibody (R&D Systems) (both antibodies were prepared in OptiMEM medium to the given concentrations).
    anti-human ACE2
    suggested: None
    anti-hDDP4
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: African green monkey kidney epithelial cells Vero E6 (ATCC®CRL-1586) were purchased from the American Type Culture Collection (ATCC, Bethesda, MD) and maintained in a high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, South Logan, UT) and 1% penicillin/streptomycin (P/S).
    Vero E6
    suggested: None
    Human alveolar epithelial cell line (A549) and human embryonic kidney cells (HEK293) were maintained in a high-glucose DMEM supplemented with 10% fetal bovine serum, 1% P/S and 1% HEPES (ThermoFisher Scientific).
    A549
    suggested: None
    The A549-hACE2 and HEK293-hACE2 cells that stably express human angiotensin-converting enzyme 2 (hACE2)79 were grown in the culture medium supplemented with 10 μg/mL Blasticidin S.
    HEK293-hACE2
    suggested: None
    SARS-CoV-2-Nluc antiviral assay: Vero or A549-hACE2 cells (12,000 cells per well in phenol-red free medium containing 2% FBS) were plated into a white opaque 96-well plate (Corning).
    A549-hACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    The concentration that reduces the 50% luciferase signal (NT50) were estimated by using a four-parameter logistic regression model from the Prism 8 software (GraphPad Software Inc.
    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: We detected the following sentences addressing limitations in the study:
    Despite the strengths of high throughput and speed, the current rapid neutralization assay must be performed in a biosafety level 3 (BSL-3) facility, representing a major limitation. Experiments are ongoing to attenuate SARS-CoV-2-Nluc so that the assay could be performed in a BSL-2 laboratory. Aligned with the same premise, BSL-2 lab compatible neutralization assays have been reported using VSV pseudotyped with SARS-CoV-2 spike protein21,21. We additionally optimized and validated the recombinant SARS-CoV-2-Nluc for high-throughput antiviral screening. Our results demonstrate that cell type could significantly affect a compound’s EC50 value, underscoring the importance of using biologically relevant cells for drug discovery. The extent of EC50 discrepancy from different cells was dependent on the compound’s mode of action. Remdesivir EC50 values differed by >10-fold when the assay used Vero E6 and A549-hACE2 cells. In another study, remdesivir was shown to be even more potent (EC50 0.01 μM) when tested on primary human airway epithelial (HAE) cells13. The potency differences seen between cell types are due to the differential metabolism of remdesivir in various cells. Host metabolic enzymes are required to convert the remdesivir prodrug to a monophosphate substrate, which is further metabolized by host kinases to its active triphosphate form that incorporates into viral RNA for chain termination. Vero E6 cells are less efficient in forming the active triphosphate than A549-h...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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 10.1101/2020.06.22.165712v1 on bioRxiv