A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19

  1. Teodor-D. Brumeanu
  2. Pooja Vir
  3. Ahmad Faisal Karim
  4. Swagata Kar
  5. Dalia Benetiene
  6. Megan Lok
  7. Jack Greenhouse
  8. Tammy Putmon-Taylor
  9. Christopher Kitajewski
  10. Kevin K. Chung
  11. Kathleen P. Pratt
  12. Sofia A. Casares

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Abstract

We report the first Human Immune System (HIS)-humanized mouse model (“DRAGA”: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19 research. This mouse is reconstituted with human cord blood-derived, HLA-matched hematopoietic stem cells. It engrafts human epi/endothelial cells expressing the human ACE2 receptor for SARS-CoV-2 and TMPRSS2 serine protease co-localized on lung epithelia. HIS-DRAGA mice sustained SARS-CoV-2 infection, showing deteriorated clinical condition, replicating virus in the lungs, and human-like lung immunopathology including T-cell infiltrates, microthrombi and pulmonary sequelae. Among T-cell infiltrates, lung-resident (CD103 + ) CD8 + T cells were sequestered in epithelial (CD326 + ) lung niches and secreted granzyme B and perforin, indicating cytotoxic potential. Infected mice also developed antibodies against the SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathology and for testing the safety and efficacy of candidate vaccines and therapeutics.

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    SciScore for 10.1101/2020.08.19.251249: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Infection of HIS-DRAGA mice and harvesting of serum and organs were conducted in a BSL-3 laboratory at Bioqual, Inc. (Rockville, MD, USA) in compliance with local, state, and federal regulations under IACUC protocol #20-019P.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Mice were irradiated (350 rads) and injected intravenously with CD3+ T-cell-depleted cord blood cells (EasySep Human CD3 Positive Selection Kit, Stem Cell Technologies, Vancouver, BC, Canada) containing approximately 105 human CD34+ hematopoietic stem cells (HSC) determined by FACS using a mouse anti-human CD34 antibody (BD Biosciences, San Jose, CA, USA) as described49,52,54.
    anti-human CD34
    suggested: None
    Bound human IgM and IgG antibodies against the His-S trimer protein were then revealed by adding anti-human IgM or IgG antibody-HRP conjugates (Bethyl Laboratories) to the His-S-trimer-coated plates.
    anti-human IgM
    suggested: None
    IgG antibody-HRP conjugates ( Bethyl Laboratories ) to the His-S-trimer-coated plates
    suggested: None
    Due to limited serum volumes, total IgG + IgM antibodies against the His-N protein were revealed by adding anti-human kappa plus lambda antibody-HRP conjugates (Bethyl Laboratories) to the His-N protein coated plates.
    total IgG + IgM antibodies
    suggested: None
    total IgG + IgM
    suggested: None
    anti-human kappa
    suggested: None
    antibody-HRP
    suggested: None
    The positive control (anti-S1(RBD)) antibody from the kit was used as a positive control for both S1(RBD) and S1-trimer binding ELISA assays.
    anti-S1 ( RBD) )
    suggested: None
    Other antibodies to detect antigens of interest were: anti-human CD3-FITC, anti-human CD4-PE, anti-human CD8-PE
    anti-human CD3-FITC
    suggested: (Millipore Cat# 4700-0350, RRID:AB_1977080)
    anti-human CD4-PE , anti-human CD8-PE
    suggested: None
    anti-human
    suggested: (SouthernBiotech Cat# 9535-09, RRID:AB_2796875)
    USA), anti-mouse CD61-PE (Invitrogen), and anti-hACE2 antibody (clone# MM0073—11A3,
    anti-mouse CD61-PE
    suggested: None
    anti-hACE2
    suggested: None
    , anti-human TMPRSS2 monoclonal IgG1 mouse antibody (#clone P5H9-A3, Sigma Aldrich), mouse IgG1/κ-Alexa Fluor 594 anti-human CD31 Antibody (clone WM59, Biolegend), goat F(ab’)2 anti-mouse IgG1-PE conjugate (Southern Biotech), and goat F(ab’)2 IgG anti-mouse IgG2a (Southern Biotech).
    anti-human TMPRSS2
    suggested: None
    IgG1
    suggested: None
    anti-human CD31
    suggested: None
    F(ab’)2 anti-mouse IgG1-PE
    suggested: (SouthernBiotech Cat# 0115-09, RRID:AB_2793975)
    anti-mouse
    suggested: (Santa Cruz Biotechnology Cat# sc-3764, RRID:AB_649117)
    anti-mouse IgG2a
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The SARS-COV-2 stock was expanded in Vero E6 cells, and the challenging virus was collected at day 5 of culture when the infection reached 90% cytopathic effect.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Extraction and quantification of hACE2 protein in HIS-DRAGA and non-HIS-reconstituted DRAGA mouse lungs and in a human lung control: Lungs from 10 non-infected HIS-DRAGA and 10 non-infected, non-HIS reconstituted DRAGA mice were homogenized in the presence of MPER mammalian protein extraction reagent (Fisher Scientific, Waltham, MA, USA) containing complete protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany) using tubes loaded with ceramic beads (MP Biologicals, Irvine, CA, USA) in a Fast-prep homogenizer (MP Biologicals).
    MP Biologicals
    suggested: None
    Standard deviations (+/−SD) for each serum sample in duplicate wells were determined at 99% interval of confidence by SigmaPlot v.
    SigmaPlot
    suggested: (SigmaPlot, RRID:SCR_003210)

    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 32, 33, 36, 37, 38, 39, 40 and 28. 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.

  2. Version published to 10.1101/2020.08.19.251249 on bioRxiv