The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B. 1.1. 529, also named Omicron, is becoming the main circulating strain in many countries worldwide and brings new challenges to preventing COVID-19 [1-5]. The latest data show that the Omicron variant contains more than 50 mutations. Among them, there are 32 mutations in the spike protein [6], which is the key component that determines the infectivity and antigenicity of the virus. Furthermore, 15 mutations are located in the receptor-binding domain (RBD), which is key for viral-cell interactions mediated by angiotensin-converting enzyme 2 (ACE-2)[7, 8]. Due to the large number of mutations, especially in the RBD domain, there is concern that the Omicron variant can escape antibody neutralization induced by the current coronavirus disease 2019 (COVID-19) vaccines or neutralizing antibodies. This concern is supported by the fact that the Omicron variant exhibits significantly more neutralization resistance [2-4, 9]. In particular, RBD mutations are most prevalent. As reported previously, the RBD region of the S1 protein plays a critical role in novel coronavirus-infected cells, directly binding to the host receptor ACE2 [7, 8]. Therefore, almost all neutralizing antibodies have been designed to target the S1 protein [10]. However, in addition to the S1 subunit, the SARS-CoV-2 surface S protein comprises the S2 subunit, which mediates viral cell membrane fusion, though the probability of mutation in the S2 region is relatively low [11]. Based on these findings, we screened and validated S2 protein antibodies against the Omicron variant and explored the possibility of developing an S2 antibody to protect against SARS-CoV-2 variants.

Antibody phage/yeast display libraries are a powerful tool for generating human antibodies against tumors and infectious diseases [12-14]. Previously, we successfully used this technology to develop diagnostic and therapeutic reagents against a series of tumor targets [15, 16] and neutralizing antibodies against the RBD and NTD of SARS-CoV-2 (unpublished data). In this work, we screened a specific scFv against spike protein S2 of SARS-CoV-2. By validating specific binding with S2 and neutralization of Omicron pseudovirus, we identified an S2-specific antibody, HCLC-031, that efficiently neutralizes the Omicron variant in vitro. Our findings indicate that the S2 subunit is a potential target for the development of neutralizing antibodies and vaccines against SARS-CoV-2 Omicron.