Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

Abstract

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

Keywords: COVID-19; SARS-CoV-2; mouse model; pathogenesis; therapeutics; vaccine.

Graphical abstract

Figure 1

Development of Mice Sensitized to SARS-CoV-2 Infection (A and B) To assess hACE2 expression and surface localization, 17CL-1 cells were transduced with Ad5-hACE2 or Ad5-Empty at MOI of 100 at 37°C for 4 h. hACE2 expression was monitored by western blot assay (A) or flow cytometry (B). (C) Ad5-hACE2 transduced 17CL-1 cells were infected with SARS-CoV-2 at MOI of 0.5 at 48 h post transduction, and virus titers were determined by foci forming assay (FFA) at 24, 48, and 72 hours post infection (h.p.i.). (D) Five days after transduction with 2.5 × 10⁸ FFU of Ad5-hACE2 or Ad5-Empty in 75 μL of DMEM intranasally, lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with an anti-hACE2 antibody (brown color). hACE2 protein (brown color) was detected only in Ad-hACE2-treated mice and was predominantly localized to alveolar epithelial cells. Scale bars, 467 and 94 μm, top and bottom panels, respectively. (E and F) Ad5-hACE2- or Ad5-Empty-transduced BALB/c or C57BL/6 mice were intranasally infected with 1 × 10⁵ PFU of SARS-CoV-2 in 50 μL of DMEM. Weight changes in 6-to-8-week old BALB/c (E) and C57BL/6 (F) mice were monitored daily (n = 5 mice per group). To obtain virus kinetics in BALB/c (E) and C57BL/6 (F) mice, lungs were harvested and homogenized at the indicated time points, and virus was titered by plaque assay. Titers are expressed as PFU/g lung tissue (n = 3 mice per group per time point). Data are representative of two independent experiments. (G) 2 d.p.i., lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with anti-SARS-CoV-2 N protein. Scale bar, 476 μm. (H) Representative Hematoxylin-eosin (HE) staining of lungs from BALB/c and C57BL/6 mice harvested at the indicated time points p.i. Scale bars, 443 and 88 μm, top and bottom panels, respectively. Asterisk, edema. (I) Summary histology scores determined at the indicated time points (n = 4 to 5 mice per group). PMN, neutrophils. (J) Photographs of lung specimens isolated from infected mice at indicated time points are shown. Arrowheads indicate regions with vascular congestion and hemorrhage.

Figure 2

The Role for IFN and STAT1 Signaling in SARS-CoV-2 Infection (A) 5 days after transduction with 2.5 × 10⁸ FFU of Ad5-hACE2, C57BL/6 mice were intranasally infected with 1 × 10⁵ PFU of SARS-CoV-2. Weight changes were monitored daily (n = 5 mice per group), and virus titers in the lungs were measured at the indicated time points using FFA (n = 3–4 mice per group per time point). Titers are expressed as FFU/g tissue. (B) Sections of paraffin embedded lungs from SARS-CoV-2-infected Ad5-hACE2-transduced, wild-type and genetically modified C57BL/6 mice at 4 d.p.i. were stained with hematoxylin/eosin. Scale bar, 100 μm. (C) Photographs of gross pathological lung specimens isolated from infected C57BL/6 mice at 4 d.p.i. Arrowheads indicate regions with vascular congestion and hemorrhage. (D) Ad5-hACE2-transduced C57BL/6 mice were treated with 80 μg of poly I:C in 50 μL of PBS 6 h before intranasal infection with SARS-CoV-2. Weight changes were monitored daily, and viral titers in lungs were measured at the indicated time points. ^∗p values ≤ 0.05; ^∗∗p values ≤ 0.005; ^∗∗∗p values ≤ 0.0005; ^∗∗∗∗p values ≤ 0.0001.

Figure 3

Differentially Expressed Genes in the Lungs of SARS-CoV-2-Infected Mice (A) SARS-CoV-2 viral RNA detected by RNA-seq in Ad5-Empty- and Ad5-ACE2-transduced mouse lungs. Data are expressed as normalized read counts. (B) Volcano plot showing differentially expressed genes in the lungs of Ad5-ACE2-transduced mice compared with Ad5-Empty-transduced mice. A total of 3,056 transcripts were differentially regulated. (C) Gene ontology (GO) analysis showing the differentially expressed genes from (B). (D) CD4⁺ (Cd4), CD8⁺ (Cd8a) T cell and B cell (Cd79b), macrophage (Cd68), and monocyte (Cd14) lineage marker expression. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test. (E) Selected cytokines and chemokines differentially regulated in the lungs of Ad5-Empty- and Ad5-ACE2-transduced BALB/c mice at 2 d.p.i., obtained from the RNA-seq data. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test.

Figure 4

Requirements for T Cells and Antibodies for SARS-CoV-2 Clearance and Protection from Subsequent Challenge Ad5-hACE2-transduced mice were infected with 1 × 10⁵ PFU of SARS-CoV-2. (A) For systemic depletion of CD4⁺ or CD8⁺ T cells, mice were injected intraperitoneally (i.p.) with 0.5 mg anti-CD4 antibody (clone GK1.5) and/or 0.5 mg anti-CD8 antibody (clone 2.43) or 0.5 mg rat IgG at days −2 and 0 p.i. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as FFU/g tissue (n = 4 mice per group per time point) (B–D) To identify SARS-CoV-2 T cell responses, single-cell suspensions were prepared from the BALF of transduced/infected BALB/c mice and stimulated with 2 μM structural protein peptide pools for 5–6 h in the presence of brefeldin A. Flow plots (B, 7 d.p.i), and summary of frequencies and cell numbers of SARS-CoV-2-N pool specific CD4⁺ T cells (C) and S1 pool specific CD8⁺ T cells (D) (determined by IFN-γ intracellular staining) are shown (n = 3 to 4 mice per time point). (E) PRNT₅₀ titers in the sera of transduced/infected C57BL/6 mice at indicated time points p.i. are shown. (F) BALB/c and C57BL/6 mice were immunized with 1 × 10⁵ infectious units (IU) of VRP-S intranasally in 50 μL of PBS. Mice were transduced and infected with 1 × 10⁵ PFU of SARS-CoV-2 3 weeks after vaccination. Virus titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point). (G) For adoptive transfer of serum, BALB/c mice were immunized with 1 × 10⁵ IU of VRP in the footpad in 50 μL of PBS and boosted with the same dose 3 weeks later. Sera were obtained 1–2 weeks after VRP booster. Then, 150 μL of serum was transferred into transduced mice intravenously (i.v.) 1 day before SARS-CoV-2 infection (n = 3 mice per group per time point). ^∗p values ≤ 0.05; ^∗∗p values ≤ 0.005; ^∗∗∗p values ≤ 0.0005; ^∗∗∗∗p values ≤ 0.0001.

Figure 5

Convalescent Plasma from COVID-19 Patients and Remdesivir Protect Mice from SARS-CoV-2 Infection (A and B) For plasma adoptive transfer, Ad5-hACE2-transduced mice were injected with 150 μL of plasma i.v. from a healthy donor or COVID-19, MERS, or SARS convalescent patients, at −1 d.p.i. Weight and virus titers in lung tissues were monitored (A) and expressed as FFU/g tissue (n = 4 mice per group per time point). Sections of paraffin embedded lungs from plasma adoptive transferred and infected mice were stained with HE at day 4 p.i. (B). Scale bar, 100 μm. (C and D) For remdesivir treatment, Ad5-hACE2-transduced mice were treated with remdesivir (25 mg/kg, bid s.c.) or vehicle at −1 d.p.i. Weight loss of infected mice and virus titers in the lungs were monitored (C), and hematoxylin/eosin staining of sections of paraffin-embedded lungs is shown at 4 d.p.i. (D) (n = 4 mice per group per time point). Data are representative of two independent experiments. Scale bar, 100 μm. ^∗p values ≤ 0.05; ^∗∗p values ≤ 0.005; ^∗∗∗p values ≤ 0.0005; ^∗∗∗∗p values of ≤ 0.0001.