An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity

  1. Nathalie M Schmidt
  2. Peter AC Wing
  3. Rory Peters
  4. Rachel Brown
  5. Hao Wang
  6. Leo Swadling
  7. COVIDsortium Investigators
  8. Joseph Newman
  9. Nazia Thakur
  10. Kaho Shionoya
  11. Sophie B Morgan
  12. Timothy SC Hinks
  13. Koichi Watashi
  14. Dalan Bailey
  15. Scott B Hansen
  16. Mala K Maini
  17. Jane A McKeating

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Abstract

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.

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  1. ScreenIT

    SciScore for 10.1101/2022.04.12.487988: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics: The COVIDsortium cohort was approved by the ethical committee of UK National Research Ethics Service (20/SC/0149) and registered at https://ClinicalTrials.gov (NCT04318314).
    Consent: All study participants gave written informed consent prior to inclusion in the study and all storage of samples obtained complied with the Human Tissue Act 2004.
    Sex as a biological variableThe Acute Cohort was recruited from hospitalized patients at the Royal Free Hospital, London, and SARS-CoV-2 infection was confirmed by PCR (n=22; median age 82 years; 45% female, 55% male; 73% white, 4% black, 14% Asian, 9% other).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    On d7, PBMC were restimulated with 1μg/mL peptide + anti-CD28 in the presence of 1μg/ml Brefeldin A (Sigma-Aldrich) for 16h at 37°C, followed by antibody staining and flow cytometric analysis.
    anti-CD28
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 pseudoparticle genesis and infection: Lentiviral pseudoparticles were generated by transfecting 293T cells with p8.91 (Gag-pol), pCSFW
    293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    Viral titres were determined by infecting Calu-3 cells with a serial dilution of virus and 48h later measuring cellular luciferase.
    Calu-3
    suggested: None
    Briefly, VeroE6 cells were seeded into 96-well flat culture plates with transparent-bottom to reach confluency (~ 5 × 104 per well).
    VeroE6
    suggested: None
    Recombinant DNA
    SentencesResources
    SARS-CoV-2 pseudoparticle genesis and infection: Lentiviral pseudoparticles were generated by transfecting 293T cells with p8.91 (Gag-pol), pCSFW
    p8.91
    suggested: None
    (luciferase reporter) and a codon optimised expression construct pcDNA3.1-SARS-CoV-2-Spike, as previously described (Thompson et al., 2020).
    pcDNA3.1-SARS-CoV-2-Spike
    suggested: None
    Software and Algorithms
    SentencesResources
    The full peptide sequences can be found in (Reynolds et al., 2020)) in cRPMI (RPMI 1640 (Thermo Fisher Scientific)+2
    Thermo Fisher Scientific)+2
    suggested: None
    recombinant human IL-2 (PeproTech)+ 5μg/ml anti-CD28 (Invitrogen).
    PeproTech)+
    suggested: None
    All samples were acquired on a BD Biosciences Fortessa-X20 or Fortessa and analysed using FlowJo v.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The Zen software also has many rendering options including removing localization errors and outliers based on brightness and size of fluorescent signals.
    Zen
    suggested: None
    Statistical analysis: Statistical analyses were performed with Prism 7.0 (GraphPad) as indicated in figure legends (Wilcoxon matched-pairs signed-rank test, Mann–Whitney test, Spearman correlation, Kruskall Wallis, unpaired t test) with significant differences marked on all figures.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04318314RecruitingCOVID-19: Healthcare Worker Bioresource: Immune Protection a…

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2022.04.12.487988v1 on bioRxiv