A bivalent protein targeting glycans and HR1 domain in spike protein potently inhibited infection of SARS-CoV-2 and other human coronaviruses

Our previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection. In this study, we aimed to design and construct a bivalent protein consisting of GRFT and EK1 components and evaluate its inhibitory activity and mechanism of action against infection by SARS-CoV-2 and its mutants, as well as other human coronaviruses (HCoVs). The bivalent proteins were expressed in E. coli and purified with Ni-NTA column. HIV backbone-based pseudovirus (PsV) infection and HCoV S-mediated cell–cell fusion assays were performed to test their inhibitory activity. ELISA and Native-PAGE were conducted to illustrate the mechanism of action of these bivalent proteins. Five-day-old newborn mice were intranasally administrated with a selected bivalent protein before or after HCoV-OC43 challenge, and its protective effect was monitored for 14 days. Among the three bivalent proteins purified, GL25E exhibited the most potent inhibitory activity against infection of SARS-CoV-2 PsVs expressing wild-type and mutated S protein. GL25E was significantly more effective than GRFT and EK1 alone in inhibiting HCoV S-mediated cell–cell fusion, as well as infection by SARS-CoV-2 and other HCoVs, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. GL25E could inhibit authentic SASR-CoV-2, HCoV-OC43 and HCoV-229E infection in vitro and prevent newborn mice from authentic HCoV-OC43 infection in vivo. GL25E could bind to glycans in the S1 subunit and HR1 in the S2 subunit in S protein, showing a mechanism of action similar to that of GRFT and EK1 alone. Since GL25E showed highly potent and broad-spectrum inhibitory activity against infection of SARS-CoV-2 and its mutants, as well as other HCoVs, it is a promising candidate for further development as a broad-spectrum anti-HCoV therapeutic and prophylactic to treat and prevent COVID-19 and other emerging HCoV diseases.