Comparison of Transgenic and Adenovirus hACE2 Mouse Models for SARS-CoV-2 Infection

  1. Raveen Rathnasinghe
  2. Shirin Strohmeier
  3. Fatima Amanat
  4. Virginia L. Gillespie
  5. Florian Krammer
  6. Adolfo García-Sastre
  7. Lynda Coughlan
  8. Michael Schotsaert
  9. Melissa Uccellini

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Abstract

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2)(1-3). Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in both the lung and brain leading to lethality. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the lung, and no clinical signs of infection with a challenge dose of 10 4 plaque forming units. The K18-hACE2 model provides a stringent model for testing the ability of vaccines and antivirals to protect against disease, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.06.190066: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Icahn School of Medicine at Mount Sinai (ISMMS).
    Randomizationnot detected.
    BlindingThe pathologist evaluated and photographed both IHC-P for hACE2, and H&E sections, and was blinded to the treatment groups.
    Power Analysisnot detected.
    Sex as a biological variableMice: Hemizygous 6-week old female K18-hACE2 mice on the C57BL/6J background (Jax strain 034860), were compared to age and sex-matched wildtype (WT) C57BL/6J (Jax strain 000664) and WT BALB/cJ (Jax strain 000651) mice.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    A monoclonal rabbit isotype control [clone #SP137; Abcam, Cambridge, MA] or monoclonal rabbit anti-human ACE2 antibody [clone #EPR4436; Abcam Cambridge, MA) diluted in blocking buffer (see above) were added to one section on each slide at a final concentration of 1.33μg/mL for 1h at room temperature.
    anti-human ACE2
    suggested: None
    Biotinylated anti-rabbit secondary antibody was prepared as instructed by guidelines for the VECTASTAIN® Elite ABC-HRP (
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and culture media: T-REx™-293 cells (Life Technologies, Carlsbad, CA) were maintained in high glucose (4500mg/L
    T-REx™-293
    suggested: RRID:CVCL_D585)
    Vero-E6 cells (ATCC® CRL-1586™, clone E6) were grown in DMEM containing 10% FBS, non-essential amino acids, 2-[4-(
    Vero-E6
    suggested: None
    A549 (ATCC® CCL-185™) cells were cultured in Kaighn’s Modification of Ham’s F-12 (F-12K) containing 10% FBS and penicillin-streptomycin, as above.
    A549
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Hemizygous 6-week old female K18-hACE2 mice on the C57BL/6J background (Jax strain 034860), were compared to age and sex-matched wildtype (WT) C57BL/6J (Jax strain 000664) and WT BALB/cJ (Jax strain 000651) mice.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)

    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: We detected the following sentences addressing limitations in the study:
    Other caveats of the Ad-hACE2 animal model include the non-uniform transduction of the lung epithelium (and consequently, non-uniform expression of hACE2 in the mouse lung), the possibility of triggering non-specific inflammatory responses if used at doses higher than 2.5×108 PFU – or with poorly prepared Ad stocks which have large quantities of empty capsids, and the potential lack of suitability for use of the Ad-hACE2 model in immunization studies using non-replicating Ad vaccines for SARS-CoV-2. However, with this in mind, we describe the successful use of a dose of Ad-hACE2 (7.5×107 PFU) which supports equivalent replication of SARS-CoV-2 in the lung to the previously reported higher dose (33, 61). Most importantly, the flexibility of the Ad-hACE2 model allows studies of multiple mouse strains immediately, without time-consuming breeding to a hACE2 transgenic or knock-in background. In conclusion, further refinement and development of these models and additional small animal models will be critical for studying disease pathogenesis, and for evaluating novel therapeutics and vaccines to protect against SARS-CoV-2 infection.

    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 10.1101/2020.07.06.190066v1 on bioRxiv
  3. Version published to 10.1080/22221751.2020.1838955