Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2

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.

Principles of SARS-CoV-2 glycosylation

“…Furthermore, the high degree of N-linked glycosylation of the SARS-CoV-2 S protein suggests a selective pressure on the virus that has driven the density of the glycan shield. Among the many different key functions driving the evolution of the shield, glycans can be aiding S folding and structural integrity [ 33 , 41 ], facilitate the interaction with cellular factors [ 42 , 43 ], or be shielding underlying epitopes [ 33 , 44 ]. Because of this key functional role, the glycan shield evolved significantly along the phylogeny, and it is continually evolving [ 45 ], with the very recent loss of N370 glycosylation due to T372A mutation in SARS-CoV-2.…”

Distinct Core Glycan and O-Glycoform Utilization of SARS-CoV-2 Omicron Variant Spike Protein RBD Revealed by Top-Down Mass Spectrometry

“…Furthermore, S protein glycosylation is critically involved in human receptor ACE2 binding, 6 cell infectivity, 7 and shields epitopes from antibody neutralization. 8 Although S protein N-glycosylation has been characterized in detail 9 with ongoing efforts to understand the impact of N-glycosylation for vaccine development, 10 characterization of O-glycosylation is challenging 11 due to the large microheterogeneity and structural diversity of O-glycans leading to multiple O-glycoforms. 12 To address these challenges we have recently developed a hybrid top-down mass spectrometry (MS) approach 13 that is capable of simultaneous characterization of the molecular structures, the site specificity, and the relative abundance of various glycoforms along with the overall post-translational modification (PTM) compositions of different coappearing ‘proteoforms’ 14 to enable proteoform-resolved analysis 15 of complex glycoproteins.…”

“…Given that the S-RBD is the principal target for neutralizing antibodies and other therapeutics, 4 and that glycosylation plays critical roles in host receptor ACE2 binding and function, [5][6][7][8] it is crucial to decipher the glycoform changes of the Omicron S-RBD as compared to WT and Delta. Although S protein N-glycosylation has been characterized in detail 9,10 with ongoing efforts to understand the impact of N-glycosylation for vaccine development, 11 characterization of O-glycosylation is challenging 12,13 due to the large microheterogeneity and structural diversity of O-glycans leading to multiple O-glycoforms. 14,15 To address these challenges, we have recently developed a hybrid top-down mass spectrometry (MS) approach 16 for comprehensive characterization of O-glycoforms, along with other post-translational modifications (PTMs), to enable proteoform analysis 17,18 of complex glycoproteins.…”