Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

Ting Gao; Mingdong Hu; Xiaopeng Zhang; Hongzhen Li; Lin Zhu; Hainan Liu; Qincai Dong; Zhang Zhang; Zhongyi Wang; Yong Hu; Yangbo Fu; Yanwen Jin; Kaitong Li; Songtao Zhao; Yongjiu Xiao; Shuping Luo; Lufeng Li; Lingfang Zhao; Junli Liu; Huailong Zhao; Yue Liu; Weihong Yang; Jing Peng; Xiaoyu Chen; Ping Li; Yaoning Liu; Yonghong Xie; Jibo Song; Lu Zhang; Qingjun Ma; Xiuwu Bian; Wei Chen; Xuan Liu; Qing Mao; Cheng Cao

The pathogenicity of N protein was further investigated using Masp2 knockout (KO) mice (KO region was indicated by Fig. S3B ) with MASP-2 protease activity deficiency by the 10 same way. Mice were pre-infected with Ad-SARS N or Ad-MERS N (1×10 9 PFU) for 5 days, and then challenged with LPS. Compared with wild-type mice, Masp2 KO mice survived longer and had a higher survival rate (Fig. 3F ), which may be attributed to a compromised complement activation because of MASP-2 deficiency. 15 Because the SARS-CoV and SARS-CoV-2 induce only mild lung damage in mice, and investigation with live SARS-CoV, including mouse adapted SARS-CoV, is not allowed by related regulations in China, we try to get the clinical evidence that the over-activation of complement LP pathway occurred in COVID-19 patients. The paraformaldehyde-fixed lung tissue of patients who died of COVID-19 were collected and subjected to IHC staining with 20 MBL, MASP-2, C4alpha, C3 or C5b-9 antibodies. All these complement cascade components demonstrated strong IHC staining signals in the patient lung tissue ( Fig. 4A-4E ), which suggested that the complement components were deposited in type I and type II alveolar epithelia cells, as well as inflammatory cells, some hyperplastic pneumocytes, and exudates in alveolar spaces with necrotic cell debris. Notably, SARS-CoV-2 N protein was also observed in 25 the lung tissue of died patients by autopsy (31) . Further, significantly increased serum C5a level was also observed in COVID-19 patient, particularly in severe cases (Fig. 4F) . These results collectively indicated that complement pathways were aggressively activated in the lungs of COVID-19 patients, which may be attributed to SARS-CoV-2 N protein. 30 It has been widely accepted that the excessive inflammatory and cytokine storm greatly contribute to the severity and lethality of COVID-19, which may be attributed to the unrestrained activation of complement pathway. Therefore, downregulation of MASP-2 as well as its downstream signal molecules, such as the potent anaphylatoxin C5a, may provide a new approach to control the pneumonia induced by the SARS-CoV-2. 35 Based on our finding and its potential and tempting application in COVID-19 therapy, a recombinant C5a antibody (BDB-001 injection by Staidson (Beijing) Biopharmaceuticals Co., Ltd), which was in phase I clinical trial for hidradenitis suppurativa, was rapidly approved by National Medical Product Administration (NMPA) for clinical trials for the treatment of COVID-19 (2020L00003). "A multicenter, randomized double blind placebo-controlled trial in 40 mild COVID-19 patients" and an open label "two-cohort clinical trial in patients with severe and critical COVID-19" were carried out simultaneously under the approval from ethic committee of Huoshensan Hospital with the informed consent of the patients. While the larger dataset from All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.