A Potent Bispecific Nanobody Protects hACE2 Mice Against SARS-CoV-2 Infection via Intranasal Administration

The dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing nanobodies (Nbs) are a potential therapeutic strategy for treating COVID-19. Here, we characterized several RBD-specific Nbs isolated from an Nb library derived from an alpaca immunized with SARS-CoV-2 spike glycoprotein (S); among them, three Nbs exhibited picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and 15 circulating SARS-CoV-2 variants. To improve the efficacy, various configurations of Nbs were engineered. Nb15-NbH-Nb15, a novel trimer constituted of three Nbs, was constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibited sub-ng/ml neutralization potency against the wild-type and currently circulating variants of SARS-CoV-2 with a long half-life in vivo. In addition, we showed that intranasal administration of Nb15H-Nb15 provided effective protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15m-NbH-Nb15 is a potential candidate for both prevention and treatment of SARS-CoV-2 through respiratory administration.Funding: This work was supported by National Science Foundation of China (NSFC) (No. 81803414, 31970149), the Major Research and Development Project (2018ZX10301406), Nanjing University-Ningxia University Collaborative Project (Grant# 2017BN04), Jiangsu Province Natural Science Foundation for Young Scholar (Grant# BK20170653), Key Natural Science Foundation of Jiangsu Province (Grant# ZDA2020014), Jiangsu province “Innovative and Entrepreneurial talent” and Six Talent Peaks Project of Jiangsu Province, the Emergency Prevention and Control Capacity Program for New Severe Infectious diseases of National Institute for Viral Disease Control and Prevention, and the 135 Strategic Program of Chinese Academy of Sciences, the Science and Technology Innovation Committee of Shenzhen Municipality (JCYJ20180228162229889).Declaration of Interest: The authors have declared no conflict of interest. A patent application on the neutralizing Nbs was submitted by XW and ZW as co-inventors.Ethical Approval: The study and the protocol for this research were approved by the Center for Public Health Research, Medical School, Nanjing University. All animal experimental procedures without infection were approved by the Committee on the Use of Live Animals by the Ethics Committee of Nanjing University.