Modeling SARS-CoV-2 Infection in Mice Using Lentiviral hACE2 Vectors Infers Two Modes of Immune Responses to SARS-CoV-2 Infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1^-/-) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19.

Keywords: COVID-19; SARS-CoV-2; hACE2; immune response; lentivirus; mouse model.

Figure 1

Expression of hACE2 in lentiviral-transduced murine cells allows for productive SARS-CoV-2 infection. (A) Western blotting analysis of lentiviral-transduced LET1 cells shows the expression of hACE2 in LET1-hACE2-transduced cells. (B) Immunofluorescence staining analysis of hACE2 of Lent-hACE2 and Lenti-control-transduced LET1 cells. Scale bar—20 µm. (C) Deconvolution microscopy of Lenti-hACE2 and Lenti-control-transduced LET1 cells infected with SARS-CoV-2. Top and lower rows depict a maximum-intensity projection of three-dimensional (3D) stacks, following deconvolution. Top row, cells were transduced with hACE-2 and infected with SARS-CoV-2; lower row, cells were transduced with control lentivirus and infected with SARS-CoV-2. Bottom micrograph is a 3D rendition of the inset appearing in the top row. 10 µm bar in upper rows, 10 µm grid in 3D rendition.

Figure 2

SARS-CoV-2 infection in Lenti-hACE2-transduced IFNAR^−/− mice. (A) 12–14 weeks old IFNAR^−/− C57BL/6 mice were intranasally transduced with hACE2 or control lentiviral vectors (Lenti-hACE2 and Lenti-control, respectively) and 3 days later infected intranasally with 2 × 10⁵ pfu SARS-CoV-2. Mice body weight was monitored daily for 5 days post SARS-CoV-2 infection (dpi). Mice were sacrificed at 5 dpi for plaque assays, and at 2 and 4 dpi for RNA-seq analysis. (B) Weight of Lenti-hACE2 (n = 5) and Lenti-control (n = 5) mice infected with SARS-CoV-2 was recorded for 5 days. Values are presented as the mean ± SEM p < 0.01 according to the area under the curve (AUC) analysis. (C) Virus titers in the lung were determined on Vero E6 cells. Values are presented as the mean ± SEM, n = 5, * p < 0.05 according to one-way ANOVA corrected for multiple comparisons. Dashed line represents the limit of detection. (D) hACE2 and SARS-CoV-2 viral genes mean expression levels ± SEM calculated from RNA-seq data of lung homogenates collected from Lenti-control (n = 2), Lenti-hACE2 (n = 1), Lenti-control infected with SARS-CoV-2 (n = 2; 1 at 2 dpi and 1 at 4 dpi) and Lenti-hACE2 infected with SARS-CoV-2 (n = 2; 1 at 2 dpi and 1 at 4 dpi) mice.

Figure 3

The exposure model: SARS-CoV-2 infection elicits an immune response in IFNAR^−/− mice. RNA-seq data of lung homogenates collected from Lenti-control (n = 2), Lenti-hACE2 (n = 1), Lenti-control infected with SARS-CoV-2 (n = 2; 1 at 2 dpi and 1 at 4 dpi) and Lenti-hACE2 infected with SARS-CoV-2 (n = 2; 1 at 2 dpi and 1 at 4 dpi) mice. (A) Principal component analysis (PCA) of RNA-seq data. (B) Heat map of significantly up- and down-regulated genes in Lenti-control and Lenti-hACE2-transduced SARS-CoV-2-infected mice versus not infected controls. (C) Up-regulated pathways in SARS-CoV-2-infected mice vs. not infected controls according to Reactome and Hallmark collections, FDR < 0.001. (D) Immune cell subtypes enriched in lungs of SARS-CoV-2-infected mice according to ImmGen classification, FDR < 0.001.

Figure 4

The infection model: hACE2 lentiviral transduction effect on SARS-CoV-2 infection in IFNAR^−/− mice. RNA-seq data of lung homogenates collected from Lenti-control (n = 2), Lenti-hACE2 (n = 1), Lenti-control infected with SARS-CoV-2 (n = 2; 1 at 2 dpi and 1 at 4 dpi) and Lenti-hACE2 infected with SARS-CoV-2 (n = 2; 1 at 2 dpi and 1 at 4 dpi) mice. Heat map of significantly up- and down-regulated genes in Lenti-hACE2-transduced SARS-CoV-2-infected mice versus control mice that were not exposed to SARS-CoV-2 (see Materials and Methods for inclusion criteria).