ABSTRACT
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 167 million confirmed cases and over 3 million deaths so far. This global pandemic has led to great efforts directed toward the study of this virus and its infection mechanism as well as development of effective means to control this devastating infectious disease. Like many other viral surface proteins, the trimeric SARS-CoV-2 spike (S) protein is heavily glycosylated with 22 N- and 2 O-glycosites per monomer which are likely to influence S protein folding and evade host immune response. More than one million S protein sequences with over 1,000 sites of mutation in its 1,273 amino acids have been reported to the GISAID database, including the highly transmissible variant strains found in the UK and South Africa. This high frequency of transmission and mutation is a major challenge in the development of broadly protective vaccines to control the pandemic. We have studied the impact of glycosylation on receptor-ligand interaction through evaluation of ACE2 and S protein expressed in different cell lines. Of different S protein glycoforms, the one expressed from lung epithelial cells, the primary cells for infection, has more complex-type glycans and higher binding avidity to the receptor as compared with the S protein from HEK293T cells which have more high-mannose or hybrid-type glycoforms. We also found that most of the S protein glycosites are highly conserved and the glycosites at positions 801 and 1194 are essential for viral entry. In addition, the RBD of S1 and the HR regions of S2 contain most of highly conserved sequences, and removal of each glycosite on pseudotyped SARS-CoV-2 virus for evaluation of the impact on structure and function provides insights into the design of broadly protective vaccines. In an effort to develop such universal vaccines, we found that mice immunized with monoglycosylated S protein (Smg) elicited better antibody responses capable of neutralizing not only the wild type but also the variants from the UK and South Africa than those with the fully-glycosylated S protein (Sfg), and strikingly, Smg vaccination provides better survival for hACE2 transgenic mice when challenged with lethal dose of SARS-CoV-2. Moreover, using single B cell technology, we isolated a monoclonal antibody from Smg immunized mice which was also able to neutralize the wild type and variants, suggesting that removal of unnecessary glycans from S protein to better expose the highly conserved sequences is an effective approach to developing broadly protective vaccines against SARS-CoV-2 and variants.