The lethal K18-hACE2 knock-in mouse model mimicking the severe pneumonia of COVID-19 is practicable for antiviral development

ABSTRACT

Animal models of COVID-19 facilitate the development of vaccines and antivirals against SARS-CoV-2. The efficacy of antivirals or vaccines may differ in different animal models with varied degrees of disease. Here, we introduce a mouse model expressing human angiotensin-converting enzyme 2 (ACE2). In this model, ACE2 with the human cytokeratin 18 promoter was knocked into the Hipp11 locus of C57BL/6J mouse by CRISPR – Cas9 (K18-hACE2 KI). Upon intranasal inoculation with high (3 × 10⁵ PFU) or low (2.5 × 10² PFU) dose of SARS-CoV-2 wildtype (WT), Delta, Omicron BA.1, or Omicron BA.2 variants, all mice showed obvious infection symptoms, including weight loss, high viral loads in the lung, and interstitial pneumonia. 100% lethality was observed in K18-hACE2 KI mice infected by variants with a delay of endpoint for Delta and BA.1, and a significantly attenuated pathogenicity was observed for BA.2. The pneumonia of infected mice was accompanied by the infiltration of neutrophils and pulmonary fibrosis in the lung. Compared with K18-hACE2 Tg mice and HFH4-hACE2 Tg mice, K18-hACE2 KI mice are more susceptible to SARS-CoV-2. In the antivirals test, REGN10933 and Remdesivir had limited antiviral efficacies in K18-hACE2 KI mice upon the challenge of SARS-CoV-2 infections, while Nirmatrelvir, monoclonal antibody 4G4, and mRNA vaccines potently protected the mice from death. Our results suggest that the K18-hACE2 KI mouse model is lethal and stable for SARS-CoV-2 infection, and is practicable and stringent to antiviral development.

KEYWORDS:

• SARS-CoV-2
• mouse model
• K18-hACE2 knock-in mouse
• severe pneumonia
• Omicron
• antivirals

Acknowledgments

We are grateful to Beijing Taikang Yicai Foundation for their great support to this work. We are grateful to Dr. Kun Cai and Yongzhong Jiang, Hubei Provincial Center for Disease Control and Prevention, for providing samples and advice. We are grateful to GemPharmatech Co., Ltd. (Nanjing, China) for their great support to this work.

Disclosure statement

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

Author contributions

Y.C., K.L., K.X., and Z.S. conceived the study and designed the experiments. Z.Z., L.Z., Q.L., Y.Z., X.T., Z.H., M.G., X.W., X.C., and S.L. performed the live SARS-CoV-2 study, animal infection experiments, and sample determinations. W.L., K.S., K.Y., J.L., Q.L., Y.Z., S.Y., Z.C., Q.X., M.D., and Z.S. assisted with the experiments. Z.Z., L.Z., and Q.L. analyzed the figures. Z.Z., L.Z., and Q.L. wrote the initial draft of the manuscript. K.X., K.L., and Y.C. revised the manuscript. All the authors read and commented on the manuscript.

Data availability statement

All data obtained for this study is included in the article.

Additional information

Funding

This work was supported by the National Key R&D Program of China (2021YFF0702000 to Y.C. 2021YFC2300700 to L.Z.), National Natural Science Foundation of China (82172243, 82341061 and 82372223 to Y.C., 82272307 to K.X.), Fundamental Research Funds for the Central Universities (2042023kf1028 and 2042022dx0003 to Y.C.), Special Fund for COVID-19 Research of Wuhan University, Innovation Team Research Program of Hubei Province (2020CFA015 to K.X.), Application & Frontier Research Program of the Wuhan Government (2019020701011463 to K.X.), Advanced Customer Cultivation Project of Wuhan National Biosafety Laboratory (2021ACCP-MS10 to Y.C.), and Natural Science Foundation of Hubei Province (2023AFB201 to Z.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.