Assessing Antigen Structural Integrity through Glycosylation Analysis of the SARS-CoV-2 Viral Spike

Abstract

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of March 29, 2021, has claimed 2 776 175 lives worldwide. Vaccine development efforts focus on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralization, and antigen efficacy is influenced by spike glycoprotein production in vivo. Therefore, immunogen integrity is important for glycoprotein-based vaccine candidates. Here, we show how site-specific glycosylation differs between virus-derived spikes, wild-type, non-stabilized spikes expressed from a plasmid with a CMV promoter and tPA signal sequence, and commonly used recombinant, engineered spike glycoproteins. Furthermore, we show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, including shedding of spike monomeric subunits for the non-stabilized wild-type spike tested, which may have implications for the resulting immune response and vaccine design.

Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation

“…Despite the observed differences, we note that the site-specific glycosylation of the virally derived material outlined here is consistent with previously reported glycan analysis from virally derived S protein produced in Calu-3 lung epithelial cells. 90 …”

Glycosylation of SARS-CoV-2: structural and functional insights

“…While sites N331 and N343 in the RBD region showed predominantly high mannose glycans when S protein is expressed in individual subunits S1 and S2, the same sites showed complex-type glycans (with 98% fucosylation) on trimeric form of S protein expressed on HEK293 cells [ 6 , 10 ]. A recent quantitative N-glycan analysis on S protein subunit S1 isolated from SARS-CoV-2-infected Calu-3 cells via immunoaffinity purification showed high prevalence of complex-type N-glycans (79%) and 21% high mannose and/or hybrid structures [ 16 ]. The same study compared the different glycans on vaccine candidates and recombinant S protein with the wild-type virus S protein with an aim to help the vaccine design strategies and thereby enable high-quality immune response through correct immunogen presentation.…”

“…Recent structural analysis of the SARS-CoV-2 S protein by cryo-EM shows that it is extensively glycosylated, similar to SARS-CoV-1 S protein [ 7 – 9 ]. Moreover, the site-specific glycosylation analysis of S protein by our group and other groups through mass spectrometry revealed both N- and O-glycosylation [ 10 – 16 ]. Based on recent reports, each trimeric spike presents up to 66 N-linked glycosylation sites and several O-linked glycosylation sites [ 12 , 16 ].…”

“…Moreover, the site-specific glycosylation analysis of S protein by our group and other groups through mass spectrometry revealed both N- and O-glycosylation [ 10 – 16 ]. Based on recent reports, each trimeric spike presents up to 66 N-linked glycosylation sites and several O-linked glycosylation sites [ 12 , 16 ]. Site-specific glycosylation on virus-derived, wild-type non-stabilized and recombinant stabilized spike glycoproteins was compared in a latest study [ 16 ].…”

“…Based on recent reports, each trimeric spike presents up to 66 N-linked glycosylation sites and several O-linked glycosylation sites [ 12 , 16 ]. Site-specific glycosylation on virus-derived, wild-type non-stabilized and recombinant stabilized spike glycoproteins was compared in a latest study [ 16 ].…”