Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

  1. Lamiaa El-Shennawy
  2. Andrew D. Hoffmann
  3. Nurmaa Khund Dashzeveg
  4. Kathleen M. McAndrews
  5. Paul J. Mehl
  6. Daphne Cornish
  7. Zihao Yu
  8. Valerie L. Tokars
  9. Vlad Nicolaescu
  10. Anastasia Tomatsidou
  11. Chengsheng Mao
  12. Christopher J. Felicelli
  13. Chia-Feng Tsai
  14. Carolina Ostiguin
  15. Yuzhi Jia
  16. Lin Li
  17. Kevin Furlong
  18. Jan Wysocki
  19. Xin Luo
  20. Carolina F. Ruivo
  21. Daniel Batlle
  22. Thomas J. Hope
  23. Yang Shen
  24. Young Kwang Chae
  25. Hui Zhang
  26. Valerie S. LeBleu
  27. Tujin Shi
  28. Suchitra Swaminathan
  29. Yuan Luo
  30. Dominique Missiakas
  31. Glenn C. Randall
  32. Alexis R. Demonbreun
  33. Michael G. Ison
  34. Raghu Kalluri
  35. Deyu Fang
  36. Huiping Liu

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, β, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.

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  1. Version published to 10.1038/s41467-021-27893-2
  2. Version published to 10.21203/rs.3.rs-141729/v1 on Research Square
  3. ScreenIT

    SciScore for 10.1101/2020.12.03.407031: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Human subject study and biosafety approvals: All research activities with human blood specimens of pre-COVID-19 and COVID-19 convalescent patients were implemented under NIH guidelines for human subject studies and the protocols approved by the Northwestern University Institutional Review Board (STU00205299 and #STU00212371) as well as the Institutional Biosafety Committee.
    IACUC: Human subject study and biosafety approvals: All research activities with human blood specimens of pre-COVID-19 and COVID-19 convalescent patients were implemented under NIH guidelines for human subject studies and the protocols approved by the Northwestern University Institutional Review Board (STU00205299 and #STU00212371) as well as the Institutional Biosafety Committee.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: Cells were tested for mycoplasma contamination before culturing.

    Table 2: Resources

    Antibodies
    SentencesResources
    Flow cytometry: Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (R&D systems, FAB9332R),
    anti-human ACE2
    suggested: None
    The antibodies, ACE2 (R&D systems, AF933), CD81 (GeneTex, CTX101766), CD63 (Proteintech, 25682-1-AP), GRP94 (Proteintech, 1H10B7), TSG101 (Proteintech, 14497-1-AP) and β-actin (Sigma, A5441) were used as primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Promega (Rabbit W401B and Mouse W402B), and the substrate ECL was detected by Pierce ECL2 solution (Thermo Fisher Scientific, 1896433A)
    ACE2
    suggested: (R and D Systems Cat# AF933, RRID:AB_355722)
    CD81
    suggested: None
    CTX101766
    suggested: None
    CD63
    suggested: (Proteintech Cat# 25682-1-AP, RRID:AB_2783831)
    GRP94
    suggested: (Proteintech Cat# 60012-1-Ig, RRID:AB_2119056)
    TSG101
    suggested: (Proteintech Cat# 14497-1-AP, RRID:AB_2208090)
    14497-1-AP
    suggested: (Proteintech Cat# 14497-1-AP, RRID:AB_2208090)
    β-actin
    suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
    Results were normalized to the CR3022 antibody with known affinity to RBD of SARS-CoV-2 42.
    CR3022
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: The parent ACE2− human embryonic kidney HEK-293 cells (HEK) or human cervical cancer HeLa cells (HeLa) are transduced with lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2+ exosomes. Dr. Thomas Gallagher of Stritch Medical School, Loyola University kindly provided HeLa and HeLa-ACE2 cells via the Hope group.
    HEK-293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    HeLa-ACE2
    suggested: None
    RBD bait that was incubated with PBS, or with ACE2− exosomes, non-fluorescent RBD bait (mock control) and ACE2− cells were used as controls.
    ACE2−
    suggested: None
    Software and Algorithms
    SentencesResources
    After motion correction of the movies 38, exosomes were identified manually using ImageJ 39.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Plasma was run in quadruplicate and reported as the average.
    Plasma
    suggested: None
    Biotin conjugated RBD protein (ACROBiosystems, SPD-C82E9) were coupled with magnetic beads (CELLection Biotin Binder Kit, Thermo Fisher Sceintific, 11533D), and 5 μg of anti-ACE2 antibody (R&D systems, AF933) or isotope control IgG (R&D systems) were conjugated to dynabeads (Dynabeads Co-Immunoprecipitation Kit, Thermo Fisher Scientific, 14321D)
    Thermo Fisher Sceintific
    suggested: None
    Statistical Analysis: GraphPad Prism 6.0 Software was used to perform statistical analyses and calculate the IC50.
    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: 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 16 and 21. 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.
    • 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.

  4. Version published to 10.1101/2020.12.03.407031v1 on bioRxiv