All targeting vectors were constructed using gene synthesis and standard molecular cloning approaches (Zeng et al., 2016). The hACE2 coding sequence was used as the template to amplify hACE2 using the following primers: hACE2-Clon-F: 5′-GAATTCGCCACCATGTCAAGCTCTTC-3′; hACE2-Clon-R: 5′-GCGGCCGCCTAAAAGGAGGTCT-3′. The pCAG-EGFP-N1 plasmid and the amplified hACE2 product were digested with EcoRI (R3101S, NEB) and NotI (R3189S, NEB) restriction enzymes. The target DNA fragments were subject to gel electrophoresis and recovered by subsequent gel extraction (DP214, Tiangen). The DNA fragments of pCAG-EGFP-N1 and hACE2 were enzymatically ligated to obtain the pCAG-hACE2-N1 plasmid vector. The pCAG-hACE2-N1 plasmid vector was digested with SpeI (R3133S, NEB) and AflII (R0520S, NEB) restriction enzymes and the linearized DNA fragments were obtained. C57BL/6 mice were purchased from Institute for Laboratory Animal N/A Resources, NIFDC, China. The wide type C57BL/6 mice were used to generated hACE2-Tg mice. The specific targeting sgRNA and Cas9 mRNA were microinjected into wide-type C57BL/6 zygotes, and then these zygotes were subsequently implanted into pseudopregnant mice obtain the founder mice. hACE2-positive founder mice were generated and backcrossed with the wide type C57BL/6 mice to produce generation F1. Starting from F1, hACE2 positive mice in each lineage were mated, and mixing across lineages was not allowed.

Genomic DNA was extracted from each mouse tail using a tissue DNA extraction kit (DP304, Tiangen) (Zeng et al., 2016). Genotyping was carried out using the following primers: hACE2-F: 5′-TGCTGGTTATTGTGCTGTCTCATCA-3′, and hACE2-R: 5′-CAGGTCTTCGGCTTCGTGGTTAA-3′. Polymerase chain reactions (PCR) were performed using Green Taq Mix (P131, Vazyme). PCR cycling conditions consisted of an initial activation step at 95°C for 15 min, followed by 30 cycles of denaturation at 95°C for 30 s, primer annealing at 58°C for 30 s, and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min. PCR products were visualized on a 1% (w/v) agarose gel and the genotype of each animal was determined.

All mice involved live SARS-CoV-2 were housed in an approved animal biosafety level-3 facility and given access to standard pellet feed and water ad libitum. Mice were anaesthetized intraperitoneally with sodium pentobarbital (50 mg/kg) and infected intranasally with 3 × 10⁴ TCID₅₀ (for naive infection) of SARS-CoV-2 (Wuhan strain) in 50 μL of Dulbecco’s Modified Eagle Medium (DMEM) per mouse. Twenty-four 6–8 week-old male hACE2-Tg mice were used in the infection experiment. Mice are randomly assigned to be sacrificed on days 1, 3, and 5 post-infection and for daily monitoring lasting 7–8 days, based on animal age and body weight. Sample sizes were determined on the basis of previous studies and experimental experience. When mice are on the verge of death or unable to move, or do not respond to gentle stimulation, or lose 20% of their pre-experimental body weight, they are considered to have reached the humane endpoint and euthanasia is performed immediately.

Mouse samples for virus titer detection were obtained by the following methods: Mice were dissected at 1, 3, and 5 dpi to collect lung, turbinalia, trachea, liver, brain, spleen, small intestine, kidney, and heart tissues to screen virus titer. Tissues homogenates (1 g/mL) were prepared by homogenizing tissues using an electric homogenizer for 2 min in 2% FBS-DMEM. The homogenates were centrifuged at 3,000 rpm for 10 min at 4°C. The supernatant was collected and stored at −80°C for viral titer detection. Samples were tested for live virus by TCID₅₀ assay in a 96-well plate format. Serial 10-fold dilutions of homogenate supernatants were added to 96-well plates of Vero E6 cells. Plates were incubated at 37°C and scored for cytopathic effect at 72 h post-infection. TCID₅₀ was calculated using the method of Reed and Muench (Ramakrishnan, 2016).

Serum samples for cytokine expression detection were acquired by the following methods: Whole blood was collected by orbital venous puncture and allowed to clot for 30 min at 25°C. Then, samples were centrifuged for 15 min at a force of 2,000 g, and the supernatant was transferred to a new tube. High-throughput liquid-phase protein chip technology was used to detect the cytokine expression in the sera of mice in the uninfected and SARS-CoV-2 infected groups. The experiment was divided into four groups. The first group consisted of sera from three uninfected hACE2-Tg mice, the second group consisted of the sera from 3 to 5 hACE2-Tg mice at 1 day post-infection, the third group consisted of sera from three hACE2-Tg mice at 3 days post-infection, and the fourth group consisted of sera from three or four hACE2-Tg mice at 5 days post-infection. Cytokine detection was performed using an EPX360-26092-901 kit and Luminex 200, according to the manufacturer’s instructions.

All tissues were immersed in 4% paraformaldehyde for 4–6 h and transferred to 70% ethanol. Individual lobes of tissues were placed in processing cassettes, dehydrated in a serially diluted alcohol gradient, and embedded in paraffin wax blocks (Gu et al., 2020). Before immunostaining, 5-μm-thick tissue sections were dewaxed in xylene, rehydrated in decreasing concentrations of ethanol, and washed in PBS. Then, the tissue sections were stained with H&E. Sections were dehydrated in increasing concentrations of ethanol and xylene.

The 5-μm-thick tissue sections were subject to immunohistochemical analysis by incubating with recombinant anti-ACE2 antibody (ab108209, Abcam) at 4°C overnight. The sections were washed, incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (ZB-2301, ZSBIO, China) for 20 min at room temperature, and then treated with DAB reagent (ZLI-9018, ZSBIO, China). After staining, sections were dehydrated in increasing concentrations of ethanol and xylene (Niu et al., 2018).

Anti-RBD antibody MW06 was prepared just as before (Jiang et al., 2021). mCD24-anti-RBD antibody, mCD24-MW06, was constructed as follows: the encoding genes of mouse CD24 (1-56aa) were cloned before the N terminal of MW06 light chain genes into the expression vector. The vector of MW06-H and mCD24-MW06-L were transferred into HEK293 cells by using 293fectin (cat: 12347019, Life Technologies) for expression of recombinant mCD24-MW06. Purified MAb proteins were obtained by protein A purification.

Affinity measurement was performed on Octet Red 384 system (Pall ForteBio, United States) using BLI strategy. Antibody (4 μg/mL) was loaded onto the AHC biosensors (Cat. 18–5060, FORTEBIO). Following a short baseline in kinetics buffer, the loaded biosensors were exposed to recombinant S1 of SARS-CoV-2 at concentration of 60 nM and background subtraction was used to correct for sensor drifting. ForteBio’s data analysis software was used to fit the data to a 1:1 binding model to extract the association and dissociation rates. The K_d was calculated using the ratio K_off/K_on.

The pseudovirus neutralization assays were conducted using Huh-7 cell lines, which were cultured in DMEM with high glucose in a 5% CO₂ environment at 37°C. The antibodies were diluted in complete DMEM culture media in a 96-well plate, with a total of nine gradients. Subsequently, the virus solution with 1.5 × 10⁴ TCID₅₀/mL was added. After 1-h incubation at 37°C and 5% CO₂, the 96-well plates were seeded with 100 μL of Huh-7 cell (2 × 10⁴ cells/mL). After 48-h incubation, each well was supplemented with 100 μL of Bright-LiteTM luciferase assay system (Vazyme, DD1204-02), and the luminescence was measured using a microplate luminometer (PerkinElmer, Ensight). The IC₅₀ were determined by a four-parameter non-linear regression using GraphPad Prism 8.5.

Mice were intraperitoneally injected with either anti-RBD (MW06) or mCD24-anti-RBD (mCD24-MW06) at a dose of 20 mg/kg body weight (Chi et al., 2022). For prophylactic and therapeutic effect assessment after low-dose viral infections, one day after/before the two antibodies were injected, all mice were intranasally exposed to 2 × 10³ TCID₅₀ SARS-CoV-2 in 30 μL DMEM per mouse, and the survival and body weight changes in mice were detected. For prophylactic effect evaluation after high-dose viral infections, one day after the two antibodies were injected, all mice were intranasally exposed to 3 × 10⁴ TCID₅₀ SARS-CoV-2 in 30 μL DMEM, and the survival and body weight changes were identified. At 5 days after viral exposure, half of the mice were euthanized and equivalent portions of tissues, including lung, trachea, brain, small intestine, liver and heart, were collected for viral titers analysis, and the rest mice were remained for monitoring survival and body weight. Viral exposure was conducted at the ABSL-3 facility.

Three independent biological replicates were used for all experiments unless indicated otherwise. Statistical analysis was performed using GraphPad Prism 8.5. Viral titers were compared among anti-RBD, mCD24-anti-RBD and hACE2-Tg groups of mice and tested for significance of differences by unpaired two-tailed Student’s t-tests. One-way ANOVA was performed to compare other groups. Statistical significance was set to p < 0.05. p*, p < 0.05; p**, p < 0.01; p***, p < 0.001; p****, p < 0.0001.

Previous studies have shown that transgenic mice expressing hACE2 are highly susceptible to SARS-CoV-2 infection (Bao et al., 2020; Golden et al., 2020; Jiang et al., 2020). In this study, a COVID-19 hACE2-Tg mouse model overexpressing hACE2 was successfully generated utilizing random integration technology. Genotyping results showed that hACE2 was successfully inserted in two lines of hACE2-Tg mice (Figure 1B). Both two lines were used for next reproduction, and after several generations, both two showed similar phenotypes with the wide type mouse. In addition, line 1 demonstrated a higher clarity of the target band compared to line 3. This observation suggested a greater abundance of target DNA fragments in line 1, thereby line 1 was chosen to reproduce and establish the hACE2 overexpression lineages. Also, there were no obvious abnormalities in the weight growth of hACE2-Tg mice compared with that of C57BL/6 mice, suggesting transgene has no effect on the growth and development of mice (Figure 1C). Immunohistochemical results showed that the hACE2 protein was highly expressed in some tissues, such as the lungs, heart, and kidneys, while other tissues express lower hACE2 protein, including spleen, brain and liver (Figure 1D). All the above results showed successful introduction of hACE2 into C57BL/6 mice, which were consistent with other mouse models previously reported (Niu et al., 2018; Gu et al., 2020).

Then, hACE2-Tg mice were infected with SARS-CoV-2 to study the pathogenicity of SARS-CoV-2. First, hACE2-Tg mice were challenged with SARS-CoV-2 at doses of 3 × 10⁴ TCID₅₀ virus via intranasal inoculation. Survival and body weight change were recorded and mice were euthanized at various days post-infection (dpi) to measure infectious viral titers as well as to determine pathology change. Compared with C57BL/6 mice, hACE2-Tg mice began to die at 4 dpi and all mice died at 5 dpi, suggesting that hACE2-Tg mice were highly susceptible to SARS-CoV-2 (Figure 2A). We found that hACE2-Tg mice infected with SARS-CoV-2 showed dramatic loss of body weight starting from 4 dpi. About 10–15% body weight was lost for most hACE2-Tg mice (Figure 2B).

To explore viral replication in the hACE2-Tg mice, 9 kinds of tissue samples, including lung, turbinalia, trachea, live, brain, spleen, small intestine, kidney, and heart, were collected from hACE2-Tg mice challenged with SARS-CoV-2 at 1, 3, and 5 dpi. Viral titers were determined by TCID₅₀ assay using Vero E6 cells for different tissues from SARS-CoV-2 infected mice. As shown in Figure 2C, viral titers were high in lung, turbinalia, and trachea at just 1 dpi, but not in brain, liver, and small intestine, showing that the respiratory system was the main viral replication site at earlier stages of infection. From 3 dpi, brain and small intestine began to have high viral titers, consistent with respiratory organs including lung, turbinalia, and trachea, indicating that virus gradually transmitted to the nervous system and the digestive system from the respiratory system. It was worth noting that viral titers in spleen were very low, particularly for 3 and 5 dpi, which might result from that large amounts of immune cells in spleen degrade live viral particles.

HE-staining was analyzed from mice infected with SARS-CoV-2 to assess the disease severity (Figure 3). The lungs were the most severely damaged tissue, and its manifestation included diffuse alveolar damage with pneumocyte injury, alveolar hemorrhage, alveolar septal thickening, peripheral parenchymal collapse, and hyaline membrane formation. Extrapulmonary manifestations, namely mild congestion mainly occurred in brain tissue, with other tissues, such as spleen, liver, heart, and kidney tissues, appearing less significant or normal histopathological damage. These findings provided evidence of widespread viral pneumonia and serious acute lung injury in the CAG-hACE2 transgenic mice when infected with SARS-CoV-2 (Winkler et al., 2020; Yinda et al., 2021; Dong et al., 2022; Gawish et al., 2022; Yu et al., 2022).

Systematic “cytokine storm” was one of the characteristic pathological features for severely ill patients suffered from COVID-19 (Bishop et al., 2022). To investigate the host immune response of this lethal hACE2-Tg mouse model infected with SARS-CoV-2 infection, we examined expressions of 32 cytokines in the blood samples (Figure 4). At 1 dpi, SARS-CoV-2 infection significantly induced the upregulation of 26 cytokines expression, including tumor necrosis factor-alpha (TNF-α), interferon- alpha (IFN-α), interferon-gamma (IFN-γ), interleukin 6 (IL-6), IL-22, C–X–C motif chemokine ligand 10 (CXCL10), chemokine ligand 2 (CCL2), CCL7, CCL3, CCL4, CXCL2, CCL5, interleukin 1-beta (IL-1β), chemoattractant granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), IL-5, leukemia inhibitory factor (LIF), IL-28, IL-31, IL-9, IL-13, IL-4, IL-2, IL-3, IL-10, and IL-15. Five cytokines, including IL-23, IL-12p70, IL-1α, CSF-3, cytolytic T lymphocyte-associated antigen 8 (CTLA-8) did not show increasing and one cytokine, CCL11, was downregulated up to zero. These showed that SARS-CoV-2 infection could induce systematic inflammatory responses in hACE2-Tg mouse within a short period, which was consisted with rapid lethal pathogenicity of SARS-CoV-2 in this model. Surprisingly, at 3 dpi and 5 dpi, some key pro-inflammatory cytokines and chemokines, including TNF-α, IFN-γ, IL -22, CXCL10, CCL2, CCL7, CCL3, CXCL2, CCL5, IL-1β, GM-CSF, M-CSF, and IL-5, did not show significant upregulation compared with the uninfected group. These results were consistent with some previous reports (Winkler et al., 2020; Yu et al., 2022), which might indicate the lethality of extensive inflammatory responses immediately after infection.

One of the important applications of SARS-CoV-2 infection mouse model was to assess the efficacy of prophylactic and therapeutic interventions, such as chemical drugs and neutralizing antibody treatments (Asaka et al., 2021; Renn et al., 2021). Research findings have demonstrated that CD24 effectively mitigated systemic inflammation and restored immune homeostasis in individuals infected with SARS-CoV-2 (Song et al., 2022). Therefore, we used hACE2-Tg mice to evaluate the protective and therapeutic effects of two antibodies, MW06 and mCD24-MW06. mCD24-MW06 was an antibody constructed on the basis of MW06, and the molecular structure of mCD24-MW06 MAb was schematically shown in Figure 5A. The mCD24 expression gene was located at the N-terminal end of the light chain variable region of MW06. Protein electrophoresis results indicated an increase in the molecular weight of the light chain in mCD24-MW06 compared to MW06 (Figure 5B), with the size aligning closely with the anticipated theoretical molecular weight.

To better characterize antibodies, we have performed experiments to assess their antigen-binding affinities and pseudoviral neutralizing activities. Affinity measurement between antibodies and the recombinant S1 of SARS-CoV-2 was conducted using BLI strategy. As shown in Figure 5C and Supplementary Figure S1, the dissociation constant, Kd, was determined to be 7.50 × 10⁻¹⁰ M for MW06 and 1.01 × 10⁻⁹ M for mCD24-MW06, suggesting that the affinity of both antibodies toward the S1 protein was practically identical. Figure 5D demonstrated the pseudoviral neutralization results of two antibodies. Both antibodies achieved 100% neutralization and neutralized the virus in a dose-dependent manner. The IC₅₀ values of MW06 and mCD24-MW06 were determined to be 184.8 ng/mL and 290.1 ng/mL, respectively, indicating that there was minimal disparity in their capacity to neutralize pseudoviruses. The above results indicated that the affinity and neutralizing activity of our developed antibody, mCD24-MW06, were comparable to those of MW06 toward the antigen.

After antibody characterization, the prophylactic and therapeutic effects of the antibodies after low-dose and high-dose viral infections was evaluated. Prophylactic effects of the antibodies after low-dose viral infections (2 × 10³ TCID₅₀) were first analyzed. As shown in Supplementary Figure S2A, mice that were pre-injected with either MW06 or mCD24-MW06 antibody exhibited survival even at 14 days post-infection. Meanwhile, mice administered with antibodies prior to viral challenge did not demonstrate any significant reduction in body weight (Supplementary Figure S2B). This observation implied that the early administration of antibodies demonstrated effective prophylactic effects after low-dose viral infections. Subsequently, therapeutic effects of the antibodies were assessed and the results were shown in Supplementary Figures S2C,D. Following viral infection, all mice that received the antibody treatment exhibited 100% survival rate after a 14-day period. Moreover, the administration of the antibody effectively ameliorated the weight loss of the mice. The above results suggested that our mCD24-MW06 antibody demonstrated proper prophylactic and therapeutic effects following low-dose viral infections.

Next, an investigation into the prophylactic effects of antibodies after high-dose viral infections (3 × 10⁴ TCID₅₀) was conducted. As shown in Figures 6A,B, when mice were exposed to a high dose of virus (3 × 10⁴ TCID₅₀), all mice in mock died on day 5 post-infection, whereas those mice treated with MW06 or mCD24-MW06 injections both died on day 8 post-infection. These findings indicated that antibody injection effectively extended the survival time in the face of a high viral load, yet failed to confer protection against mortality. Although the lethality was not significantly improved for mCD24-MW06 treatment, viral titers were significantly reduced in tissues including lung, trachea, brain, small intestine, and liver, while those in heart were similar between MW06 and mCD24-MW06 treatment (Figure 6C). These findings revealed that hACE2-Tg mice could be used as a tool to assess therapeutic potentials of treatments with neutralizing antibodies or plasma from convalescent patients after infection.