SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

  1. Abigael Chaouat
  2. Hagit Achdout
  3. Inbal Kol
  4. Orit Berhani
  5. Gil Roi
  6. Einat B. Vitner
  7. Sharon Melamed
  8. Boaz Politi
  9. Eran Zahavy
  10. Ilija Brizic
  11. Tihana Lenac Rovis
  12. Or Alfi
  13. Dana Wolf
  14. Stipan Jonjic
  15. Tomer Israely
  16. Ofer Mandelboim

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic, causing health and economic problems. Currently, as dangerous mutations emerge there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus membrane binds to the angiotensin converting enzyme 2 (ACE2) receptor on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in vitro and in vivo SARS-CoV-2 infection, better than ACE2-Ig. Mechanistically we show that anti-spike antibodies generation, ACE2 enzymatic activity and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus concentration infection. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

Author Summary

SARS-CoV-2 infection caused serious socio-economic and health problems around the globe. As dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. SARS-CoV-2 infection starts via binding of SARS-CoV-2 spike protein receptor binding domain (RBD) to its receptor, ACE2, on host cells. To intercept this binding, we generated Ig-fusion proteins. ACE2-Ig was generated to possibly block RBD by binding to it and RBD-Ig to block ACE2. We indeed showed that the fusion proteins bind to their respective target. We found that it is more efficient to inhibit SARS-CoV-2 infection by blocking ACE2 receptor with RBD-Ig. We also showed that RBD-Ig does not interfere with ACE2 activity or surface expression. Importantly, as our treatment does not target the virus directly, it may be efficient against any emerging variant. We propose here that RBD-Ig physically blocks virus infection by binding to ACE2 and thus it may be used for the treatment of SARS-CoV-2-infected patients.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.18.440302: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Study approval: Animal experiments involving SARS-CoV-2 were conducted in a BSL3 facility and treatment of animals was in accordance with regulations outlined in the U.S. Department of Agriculture (USDA) Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institute of Health, 2011).
    Sex as a biological variableMice: Homozygous female outbred K18-hACE2 transgenic mice (2B6.Cg-Tg(K18-ACE2)2Prlmn/J, Stock No: 034860, Jackson laboratory) 6-8 weeks old were maintained at 20-22°C with relative humidity of 50 ± 10% on a 12hrs light/dark cycle.
    RandomizationMice were randomly assigned to experimental groups of 7-8 mice per group.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Purified anti-DYKDDDDK Tag Antibody (Cat#637302, BioLegend)
    anti-DYKDDDDK
    suggested: (BioLegend Cat# 637302, RRID:AB_1134268)
    The following secondary antibodies were used: Alexa Fluor 647-conjugated Goat Anti-Rabbit IgG (Cat#111-606-144, Jackson ImmunoResearch Laboratories)
    Anti-Rabbit IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 111-606-144, RRID:AB_2338083)
    Afterwards, cells were washed in FACS buffer and stained with Alexa Fluor 647-conjugated anti-human IgG secondary antibody.
    anti-human IgG
    suggested: None
    Cells were then washed, and an Alexa Fluor 647 Anti-Mouse IgG secondary antibody was added.
    Anti-Mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Generation of 293T-Spike cells: First, 293T cells were grown overnight in 6-well plates (2.2X105 cells/well).
    293T
    suggested: None
    The 293T-ACE2 cells lysate was prepared and 10 ug of it was incubated with RBD-Ig according to the manufacturer instructions.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Vero E6 cell monolayers were washed once with DMEM and 200µl of each dilution of protein-virus mixture was added in triplicates for 1 hour at 37°C.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Sera were diluted to 1:500, 1:1K, 1:5K, 1:10K per well and added to 50,000 293T-Parental cells or 293T-Spike cells in a 96-U-well plate for 1 hour at 4°C.
    293T-Parental
    suggested: None
    Blocking with mice sera: Sera from the various immunized mice groups was diluted to 1:100 per well and added to 50K 293T-Parental cells or 293T-Spike cells in a 96-U-well plate for 1 hour at 4°C.
    293T-Spike
    suggested: None
    Recombinant DNA
    SentencesResources
    The flag-tagged ACE2 was cloned into the plasmid pHAGE-DsRED(-) GFP(+).
    pHAGE-DsRED(-
    suggested: None
    As a control we performed the same co-transfection but with the VSV-G envelope plasmid.
    VSV-G
    suggested: RRID:Addgene_138479)
    Software and Algorithms
    SentencesResources
    Statistics: Statistical analysis were performed using either Prism 8 (GraphPad) or Excel (Microsoft).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Excel
    suggested: None

    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.
    • 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.

  2. Version published to 10.1101/2021.04.18.440302 on bioRxiv