COVID-19 Models

Animal models have assisted researchers’ understanding of COVID-19 pathogenesis and host immune response to SARS-CoV-2 infection, and are of great significance in helping the rapid development of disease preventatives, vaccines, and treatments. Although the physiological characteristics and immune regulation of non-human primates are most highly conserved with humans, they are often prohibitively expensive or inaccessible for a majority of SARS-CoV-2 infection research.

Angiotensin-converting enzyme-2 (ACE2) is the most important cell surface receptor for the SARS-CoV-2 virus to invade the human body, but due to differences between species, the receptor-binding domain (RBD) of the SARS-CoV-2 Spike (S) protein cannot bind to the ACE2 receptor of wild-type rodents under natural conditions, but by replacing mouse Ace2 with human ACE2 through gene editing technology, humanized ACE2 mice (hACE2) stably expressing human ACE2 receptors can be obtained. Related studies have proved that hACE2 mice are susceptible to SARS-CoV-2 infection and play a role in different research directions.

Humanized ACE2 mice (hACE2) models, genetically modified to stably express human ACE2 receptors, have quickly become essential animal research models. Numerous studies have demonstrated the susceptibility of hACE2 mice to the SARS-CoV-2 virus, playing a role in various research directions.

COVID-19 Preclinical Research Models

Cyagen has developed ACE2 mice with multiple gene-targeting strategies across three different genetic backgrounds, designed to meet the needs of customers in basic research and new drug development animal models.

▶ hACE2-All CDS-B6J mice

This mouse model uses the human ACE2 CDS to replace the mouse endogenous Ace2 sequence (retaining the mouse Ace2 signal peptide sequence) so that the expression of hACE2 is directed by the mouse endogenous Ace2 regulatory elements. The gene is located on the X chromosome, and homozygous females and hemizygous males are viable and fertile.

▶ K18-hACE2-2A-CreERT2 mice

This mouse model expresses human ACE2 in epithelial cells, including initially infected airway epithelial cells, driven by the human keratin K18 promoter. The mouse model contains the CreERT2 element, which can be time-specifically regulated and expressed using Tamoxifen-inducible Cre mice.

K18-hACE2 mice can be infected with the SARS-CoV-2 virus, causing a severe disease characterized by weight loss, rapid breathing, hunched posture, and inactivity. These mice responded to virus challenge in a dose-dependent manner, allowing the study of severe acute respiratory disease in mice challenged with high viral titers as well as the long-term effects of mild infection at low doses.

COVID-19 Mouse Models: Research Case Studies

1. Study of SARS-CoV-2 Infection Mechanism

“SARS-CoV-2 spike engagement of ACE2 primes S2’site cleavage and fusion initiation.” PNAS (2021) doi.org/10.1073/PNAS.2111199119

Current research has demonstrated that the binding site between the SARS-CoV-2 virus and the ACE2 receptor is the Spike (S) protein of the virus. However, the molecular and cellular mechanisms after their binding remain unclear. More importantly, the currently circulating variants globally have multiple mutations in the S protein, which poses a significant challenge to vaccine development. Therefore, research on the binding of the S protein to the ACE2 receptor and the subsequent molecular and cellular mechanisms will be of utmost importance.

The Spike (S) protein monomer of SARS-CoV-2 consists of two subunits: the amino-terminal S1 subunit, which recognizes and interacts with the host ACE2 receptor, and the carboxyl-terminal S2 subunit, which facilitates the fusion of the virus with the host cell membrane. The S2 subunit drives the release of viral RNA into the cell, where replication occurs in the infected cells. Further investigation is needed into processes such as receptor recognition and membrane fusion. On December 21, 2021, researchers from the Shanghai Pasteur Institute of the Chinese Academy of Sciences revealed that during SARS-CoV-2 infection, the spike protein is cleaved to generate the S2' fragment, which plays a role in the recognition between the virus and the receptor, as well as in membrane fusion. In this study, the researchers used K18-hACE2-2A-CreERT2 mice with an epithelial cell-specific promoter K18 to isolate lung epithelial cells and simulate Spike-driven syncytium formation in primary cells. They demonstrated that the formation of the functional S2' fragment requires the specific recognition of the functional ACE2 receptor.

Figure 1. In vitro cell-to-cell fusion experiments demonstrated that the human ACE2 protein can interact with the viral Spike protein, while the mouse Ace2 protein cannot. In the hACE2 syncytium, researchers not only detected the S2' band but also observed green fluorescence signals indicating syncytium formation. The hACE2 mice carry the "K18 promoter-Kozak-human ACE2 CDS-P2A-CreERT2-rBGpA" cassette, which can be used to study the role of specific host genes (floxed) in the response to SARS-CoV-2 infection.

Subsequent experiments demonstrated that the hydrolysis resulting in the formation of the S2' protein during the involvement of the Spike protein in viral infection is an indispensable molecular event in the SARS-CoV-2 infection process. Specifically, the arginine residue at position 815 is essential for the hydrolytic cleavage, which provides new insights for the development of vaccines and antibody drugs.

2. COVID-19 Vaccine Development

“Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins.” Frontiers in cellular and infection microbiology (2022) doi.org/10.3389/FCIMB.2022.967493

Vaccination is the preferred method for controlling and preventing the COVID-19 pandemic. However, due to factors such as the continuous mutation of the SARS-CoV-2 virus during the pandemic, the short duration of protection provided by existing vaccines, and differences among human populations, the development of long-lasting anti-infection SARS-CoV-2 vaccines remains an urgent task.

VLP (virus-like particle) is a virus capsid that does not contain the viral genome and can be used as a multifunctional, high-safety, and high-immunogenicity vaccine. Compared to subunit vaccines, the repetitive antigen patterns on the surface of VLPs are more easily recognized by antigen-presenting cells, thereby inducing stronger and broader humoral and cellular immune responses. On July 18, 2022, researchers from the Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, and Jilin University developed and validated several SARS-CoV-2 vaccines based on different virus-like particles (VLPs). In this study, researchers used hACE2-All CDS-BALBC to validate the newly developed vaccines and found that these vaccines could induce higher expression of SARS-CoV-2-specific antibodies in mice (Figure 2), demonstrating that vaccines based on mSM virus-like particles and Al/CpG adjuvants are promising candidates for preventing the COVID-19 pandemic.

Figure 2. Immunogenicity evaluation of different VLPs

3. COVID-19 Preventive Drug Screening

“Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models.” bioRxiv (2022) doi.org/10.1101/2022.07.14.499651

ACE2 is the primary receptor for SARS-CoV-2 entry into cells. Although progress has been made in targeting ACE2 to inhibit SARS-CoV-2 binding, effective drugs and methods for preventing SARS-CoV-2 infection are still lacking. Studies have shown that reducing ACE2 protein levels by half can significantly decrease SARS-CoV-2 infection, suggesting that partially lowering ACE2 protein levels may be a potential new strategy for preventing SARS-CoV-2 infection.

On July 15, 2022, researchers from Jiangsu University and the Jiangsu Provincial Key Laboratory of Infection and Immunity identified the natural compound Vitamin C (VitC) through drug screening, finding that it can lower ACE2 protein levels to prevent SARS-CoV-2 infection. They further investigated its mechanism of action. In the study, the authors first demonstrated through in vitro cell experiments that VitC can prevent SARS-CoV-2 infection. They then identified USP50 as a key regulator of ACE2 protein levels. VitC promotes ACE2 degradation by blocking the interaction between USP50 and ACE2, rather than disrupting ACE2 transcriptional expression.

To further study the in vivo anti-infection effects of VitC, the authors used hACE2-All CDS-c.A) mice, and found that VitC treatment strongly blocked the interaction between USP50 and ACE2 in lung tissues of hACE2 mice (Figure 3.B), upregulated ACE2 ubiquitination levels (Figure 3.C), and promoted ACE2 protein degradation (Figure 3.D-E). After infection with SARS-CoV-2-S pseudovirus, they observed that VitC treatment significantly reduced the expression of SARS-CoV-2 S protein (Figure 3.F) and SARS-CoV-2-S RNA (Figure 3.G) in various tissues of hACE2 mice.

Through these experiments, the researchers demonstrated that USP50 and VitC jointly regulate ACE2 protein levels in vivo. VitC treatment effectively reduces ACE2 protein levels and lowers the risk of SARS-CoV-2 infection, revealing the critical role and application of VitC in daily protection against SARS-CoV-2 infection.

Figure 3. VitC treatment reduces ACE2 protein levels in vivo and limits SARS-CoV-2 infection.

4. Research on SARS-CoV-2 Postviral Complications

 "SARS-CoV-2 spike spurs intestinal inflammation via VEGF production in enterocytes." EMBO molecular medicine (2022) doi.org/10.15252/emmm.202114844

SARS-CoV-2 mainly causes clinical symptoms such as lung inflammation and fever through respiratory tract infection but also has various complications such as anosmia, catatonic schizophrenia, digestive diseases, thrombocytopenia, and testicular damage. Learning about the pathogenesis of different complications is also very important for the treatment of the patient's disease. Using hACE2-All CDS-B6J mice (product number: C001191) as a validation model, the researchers determined the molecular mechanism of VEGF overproduction in the gut following SARS-CoV-2 S protein stimulation, proving that ERK/VEGF is a key axis of COVID-19 potential biomarkers and therapeutic targets for intestinal inflammation and disease progression.

5. Antibody Drug Development And Verification

"SARS-CoV-2 Delta and Omicron variants evade population antibody response by mutations in a single spike epitope." Nature Microbiology (2022) doi.org/10.1038/s41564-022-01235-4

The  SARS-CoV-2 virus has constantly mutated throughout the epidemic, and the emergence of multiple pathogenic or transmissible mutant strains such as Delta and Omicron has brought considerable challenges to the prevention and treatment of the disease. Given this, more effective prevention and treatment strategies are urgently needed, and broad-spectrum neutralizing antibodies are one of the strategies to deal with it. Researchers used hACE2-All CDS-B6J mice (product number: C001191) to evaluate the in vivo protective effect of the R1-32 antibody and confirmed that R1-32 has protective activity against SARS-CoV-2 infection.