Mice with Induced Pulmonary Comorbidities Display Severe Lung Inflammation and Mortality following Exposure to SARS-CoV-2

  1. Reut Falach
  2. Liat Bar-On
  3. Shlomi Lazar
  4. Tamar Kadar
  5. Ohad Mazor
  6. Moshe Aftalion
  7. David Gur
  8. Ohad Shifman
  9. Ofir Israeli
  10. Inbar Cohen-Gihon
  11. Galia Zaide
  12. Hila Gutman
  13. Yentl Evgy
  14. Yaron Vagima
  15. Efi Makdasi
  16. Dana Stein
  17. Ronit Rosenfeld
  18. Ron Alcalay
  19. Eran Zahavy
  20. Haim Levy
  21. Itai Glinert
  22. Amir Ben-Shmuel
  23. Tomer Israely
  24. Sharon Melamed
  25. Boaz Politi
  26. Hagit Achdout
  27. Shmuel Yitzhaki
  28. Chanoch Kronman
  29. Tamar Sabo

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Abstract

Severe manifestations of COVID-19 are mostly restricted to people with comorbidities. Here we report that induced mild pulmonary morbidities render SARS-CoV-2-refractive CD-1 mice to be susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low-doses of the acute-lung-injury stimulants bleomycin or ricin caused a severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates of >50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart and serum of low-dose-ricin pretreated, as compared to non-pretreated mice. Notably, the deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against SARS-CoV-2 RBD. Thus, viral cell entry in the sensitized mice seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel mice-based animal model for the study of comorbidity-dependent severe COVID-19.

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  1. Version published to 10.1101/2020.10.28.358614v3 on bioRxiv
  2. Version published to 10.1101/2020.10.28.358614v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.10.28.358614: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Treatment of animals was in accordance with regulations outlined in the USDA Animal Welfare Act and the conditions specified in the National Institute of Health Guide for Care and Use of Laboratory Animals.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAnimal experiments: Animals in this study were female CD-1 mice (Charles River Laboratories Ltd., Margate, UK) weighing 27-32 grams.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Where indicated, mice were administered (intraperitoneal injection, 1 ml) one of the following antibodies: equine-derived polyclonal anti-ricin F(ab)2 fragment (1730 IsNU) (14), monoclonal anti-SARS-CoV-2 RBD (MD65) antibody (1 mg) (18), polyclonal anti-SARS-CoV-2-RBD antibody (SARS-CoV-2 NT50= 1:20,000) or polyclonal anti-SARS-CoV-2 (SARS-CoV-2 NT50= 1:10,000
    anti-ricin F(ab)2 fragment ( 1730 IsNU ) ( 14)
    suggested: None
    anti-SARS-CoV-2 RBD ( MD65
    suggested: None
    anti-SARS-CoV-2-RBD
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    Cells were stained using the following antibodies (eBioscience): CD45-FITC (clone 30-F11), CD31-PE (clone 390), CD326-PerCP (clone G8.8).
    CD45-FITC
    suggested: None
    CD31-PE
    suggested: (SouthernBiotech Cat# 1625-09L, RRID:AB_2795047)
    CD326-PerCP
    suggested: None
    For ACE2 receptor staining, cells were stained using goat anti-mACE2 antibody (R&D, AF3437) followed by donkey anti-goat IgG coupled to AF647.
    anti-mACE2
    suggested: None
    anti-goat IgG
    suggested: (SouthernBiotech Cat# 6158-31, RRID:AB_2796225)
    For TMPRSS2 staining, cells were stained using anti-TMPRSS2 polyclonal antibody coupled to AF647 (Santa Cruz Biotechnology sc-515727).
    anti-TMPRSS2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and virus: African green monkey kidney clone E6 cells (Vero E6, ATCC® CRL-1586™) were grown in Dulbecco’s Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS)
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Animal experiments: Animals in this study were female CD-1 mice (Charles River Laboratories Ltd., Margate, UK) weighing 27-32 grams.
    CD-1
    suggested: None
    Polyclonal anti-SARS-CoV-2-RBD antibody was generated by immunizing a rabbit (female New-Zealand White) with RBD-huFc (150 μg, Complete Freund’s adjuvant) followed by boosting with RBD-huFc (150 μg, Incomplete Freund’s adjuvant) at day 21 and with monomeric RBD (150 μg, Incomplete Freund’s adjuvant) at day 42.
    New-Zealand White
    suggested: None
    Software and Algorithms
    SentencesResources
    RNA-seq quality control was performed using fastQC v0.11.5, checking for per base sequence quality, per sequence quality scores and adapter content.
    fastQC
    suggested: (FastQC, RRID:SCR_014583)
    Analysis of differentially expressed genes under various conditions was performed using the R package DESeq2 v1/18.1, with default parameters.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Fluorescence intensity was quantified using Zen software (version 2.1 Zeiss, Jena, Germany, 2008).
    Zen
    suggested: None
    Cells were analyzed on FACSCalibur (BD Bioscience, San Jose, CA, USA).
    FACSCalibur
    suggested: None
    Statistical analysis was calculated using Prism software (version 5.01,
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    , 2007; GraphPad Software, La Jolla, CA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    One may therefore be tempted to suggest that increased expression of ACE2 can compensate for the relatively poor binding of SARS-CoV-2 spike RBD to this ACE2 species and that overexpression of this receptor in mouse lung cells can overcome this limitation and bring upon a successful viral infection. However, comparative flow cytometry analysis of lung cells derived from sham-treated and LDR mice failed to detect an increase in ACE2 expressing cells following application of low dose ricin. On the other hand, histochemical analysis of lung sections prepared from LDR mice did reveal a significant increase in the binding of viral RBD. The combination of these two findings may imply that in LDR mice (and possibly in bleomycin mice), there is a gain of receptors other than ACE2 that can serve as an effective binding site for SARS-CoV-2, perhaps with the aid of the high level of lung cell surface TMPRSS2 induced by low dose ricin application. Various viruses use junction proteins as receptors and access epithelium cells through apical junction complexes which contain tight junctions (TJs) and adherens junctions (AJs) even if most of these proteins are not readily available, possibly by taking advantage of compromised epithelium (24). Indeed, studies carried out in our laboratory demonstrated that intranasal application of a lethal dose of ricin (7μg/kg) to mice leads to rapid diminution of both adherens and tight junctions in the lungs, thereby precipitating the disruption of the al...

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  4. Version published to 10.1101/2020.10.28.358614v1 on bioRxiv