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Comparative Study
. 2020 Dec;9(1):2433-2445.
doi: 10.1080/22221751.2020.1838955.

Comparison of transgenic and adenovirus hACE2 mouse models for SARS-CoV-2 infection

Raveen Rathnasinghe  1   2   3 , Shirin Strohmeier  1   3 , Fatima Amanat  1   3 , Virginia L Gillespie  4 , Florian Krammer  1 , Adolfo García-Sastre  1   2   5   6 , Lynda Coughlan  1   7 , Michael Schotsaert  1   2 , Melissa B Uccellini  1   2
Affiliations
Comparative Study

Comparison of transgenic and adenovirus hACE2 mouse models for SARS-CoV-2 infection

Raveen Rathnasinghe et al. Emerg Microbes Infect. 2020 Dec.

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 the nasal turbinates, lung and brain, with high lethality, and cytokine/chemokine production. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the nasal turbinates and lung, and no clinical signs of infection. The K18-hACE2 model provides a stringent model for testing vaccines and antivirals, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.

Keywords: ACE2; COVID-19; SARS-CoV-2; adenovirus; mouse models.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Validation of Ad-hACE2. (A) A549 cells were transduced with Ad-Empty or Ad-hACE2 at an MOI of 100 for 3 h at 37°C. 24 h post-transduction, surface expression of hACE2 was detected by flow cytometry. RFI = relative fluorescence intensity generated by multiplying % hACE2+ cells by the geometric mean fluorescence. (B) A549 cells were transduced with Ad vectors as described in (A) followed by infection 24 h later with SARS-CoV-2 USA-WA1/2020 at an MOI of 0.1. Virus titers were determined by plaque assay on VeroE6 cells. BALB/c mice were administered intranasally (i.n) with the indicated dose of Ad-Empty, Ad-hACE2, or PBS. Lungs were harvested on day 5 post-transduction, paraffin embedded and 5μm sections stained for H&E (C), or for IHC-P using an isotype control (D) or α-hACE2 monoclonal antibody (E). Regions of the lung anatomy are indicated on isotype control sections; AS = alveolar septa, BE = bronchiolar epithelium, E = endothelium. Scale bar is 100 nm, IHC-P staining is indicated by brown staining for DAB substrate. (F) Separate groups of BALB/c mice administered i.n. with PBS, Ad-Empty or Ad-hACE2 (2.5×108, 1×108, or 7.5×107 PFU) were infected five days later (D5) with 1×104 pfu of SARS-CoV-2 and lung viral titers were determined by plaque assay on D2 and D5 post-SARS-CoV-2 challenge. Note; data points for viral lung titers for BALB/c mice treated with Ad-hACE2 at a dose of 2.5×108 PFU shown in Figure 1(F), are the same group of mice as shown in Figure 2(D) and are shown for visualization purposes to allow a comparison between doses, although all groups were part of the same larger experiment.
Figure 2.
Figure 2.
B6 and BALB/c Ad-hACE2 SARS-CoV-2 infection (A-B-E) B6 mice and (C-D) Balb/c mice were transduced with 2.5×108 PFU of Ad-empty, Ad-hACE2, or PBS. On D5 post-Ad administration mice were infected with 1×104 pfu of SARS-CoV-2 and monitored for weight loss (A and C) and viral titers (B,D, and E) according to the indicated timeline. n = animal number at day 0, as animals were harvested for titers n was reduced according to the diagram. Note; data points for viral lung titers for Balb/c mice treated with Ad-hACE2 at a dose of 2.5×108 PFU shown in Figure 2(D), are the same group of mice as shown in Figure 1(F).
Figure 3.
Figure 3.
B6-K18-hACE2 SARS-CoV-2 infection. B6 K1 8-hACE2 mice were infected with 1×104 PFU of SARS-CoV-2 and monitored for weight loss (A) and viral titers (B and C) according to the indicated timeline. n = animal number at day 0, as animals were harvested for titers n was reduced according to the diagram.
Figure 4.
Figure 4.
Comparison of histology and immunohistochemistry on lung and brain tissue from B6 AdhACE2 or K18-hACE2 animal models following SARS-CoV-2 challenge. (A) Paraffin lung sections (5μm) from B6 mice administered intranasally (i.n) with 2.5×108 PFU Ad-hACE2 and subsequently challenged with 1×104 PFU of SARS-CoV-2 were stained by H&E, or for SARS-CoV-2 N protein (n=3 per group, per timepoint). Lungs were harvested on D0, D2, D5 and D6 post-challenge. Regions of the lung anatomy are indicated on images for negative control polyclonal IgG (pIgG) antibody sections: AS = alveolar septa or BE = bronchiolar epithelium. Black arrow indicates regions of specific dark brown/red anti-SARS-CoV-2 N staining for less obvious regions of positive labelling. (B) Paraffin brain sections (5μm) from B6 Ad-hACE2, as above. Regions of brain anatomy are indicated: OB = olfactory bulb, CC = cerebral cortex. (C) H&E sections and IHC-P for SARS-CoV-2 N was also performed on lung and (D) brain tissue sections from K18-hACE2 transgenic mice at the same time-points post-challenge with SARS-CoV-2. Scale bar on all images is 200 nm, IHC-P staining is indicated by red staining for Nova Red substrate.
Figure 5.
Figure 5.
B6-K18-hACE2, BALB/c Ad-ACE2, and B6 Ad-ACE2 cytokine/chemokine induction. Cytokine/chemokine production in the lung was measured by qPCR at day 2 or 5 post-infection for samples in Figures 2 and 3.
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