Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

  1. Samuel A. Theuerkauf
  2. Alexander Michels
  3. Vanessa Riechert
  4. Thorsten J. Maier
  5. Egbert Flory
  6. Klaus Cichutek
  7. Christian J. Buchholz

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Abstract

Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split β-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.15.340604: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Sera & S-Neutralizing Antibodies: Sera donation with informed consent was approved by an ethics vote from the local committee at Frankfurt University Hospital.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    A commercially available neutralizing antibody against SARS-CoV-2 (Sino Biological, 40592-R001) and the corresponding normal control (Sino Biological, CR1) served as a positive control.
    CR1
    suggested: None
    Subsequently, the membranes were incubated with the secondary antibody rabbit anti-mouse conjugated to horseradish peroxidase (Dako, 1:2000) for 90 min at RT.
    anti-mouse
    suggested: None
    S protein was specifically stained with the mouse IgG1 anti-SARS-CoV-2 Spike (Clone: 1A9, GeneTex, 1 μL/105 cells in 100 μL) antibody for 45 min at 4°C followed by the incubation with the secondary antibody anti-IgG1-PE (REA1017, Miltenyi Biotec, 1 μL/105 cells in 100 μL) for 30 min at 4°C.
    mouse IgG1
    suggested: None
    anti-IgG1-PE (REA1017, Miltenyi Biotec, 1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Calu-3 cells (AddexBio, C0016001, lot 0179286) were cultured in Minimal Essential Medium Eagle (Sigma, M2414) supplemented with 10% FBS, 1x L-glutamine, 1x non-essential amino acids (Gibco, 11140-035) and 1x sodium pyruvate (Gibco, 11360-070).
    Calu-3
    suggested: None
    MRC-5 and Calu-3 were subcultured with trypsin in EDTA-PBS every two weeks at ratios between 1:2 and 1:3, medium was exchanged twice weekly.
    MRC-5
    suggested: ICLC Cat# HL95001, RRID:CVCL_0440)
    Fusion assays and neutralization: HEK-293T cells were transfected as described above, in the T75 format.
    HEK-293T
    suggested: None
    Immunofluorescence staining and laser scanning microscopy: Vero E6 cells constitutively expressing GFP were generated by LV mediated transduction and subsequent puromycin selection.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Statistical Analysis: All statistical analyses were carried out in GraphPad Prism version 8.4.2.
    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: 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.
    • 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.

  2. Version published to 10.1101/2020.10.15.340604v1 on bioRxiv