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.

Figure 1

Purification and glycan analysis of the SARS-CoV-2 spike glycoprotein. (A) Schematic representation of spike purification from SARS-CoV-2 infected Calu-3 cells by immunoaffinity purification using the S1 targeting CR3022 antibody. Spike S1 and S2 subunits are colored dark and light blue, respectively, with receptor binding domain (RBD), N-terminal domain (NTD), furin cleavage site (FCS), connecting domain (CD), heptad repeat 2 (HR2), transmembrane domain (TM), and cytoplasmic tail (CT) labeled. (B) SDS-PAGE showing the presence of S1 and S2 subunits of virus-derived spike. Quantitative UHPLC N-glycan analysis showing the distribution of oligomannose and complex-type glycans on S1_virus (C) and S_{recombinant-trimer} (D). (E) N-glycan maturation showing color coding for degree of glycan processing from oligomannose (green) to hybrid (yellow) to complex (purple). (F) Quantitative site-specific N- and O-glycosylation by bottom-up glycoproteomics of S1_virus. Pie charts depict the degree of N-glycan processing depicted in part E.

Figure 2

Glycosylation and assembly of a non-stabilized spike with a tPA leader sequence.. (A) SDS-PAGE of CR3022 purified S1_{vaccine-antigen}. (B) Mass photometry of monomeric S1_{vaccine-antigen} (∼120 kDa) and S_{recombinant-trimer} (∼550 kDa). (C) Quantitative UHPLC N-glycan analysis of S1_{vaccine-antigen} showing the degree of glycan processing. (D) Site-specific N- and O-glycosylation of S1_{vaccine-antigen} (see Figure 1E for the pie chart legend).

Figure 3

Correlation of spike cellular location and macromolecular assembly with N234 and T678 glycan processing. (A) Structural position and orientation of the S1 N-glycan N234 (shown as Man₅GlcNAc₂) in a pocket formed by the RBD (top-right corner) and NTD of the same protomer, and the neighboring RBD (top-left corner). The GLYCAM web server (http://glycam.org) was used to model the glycan onto the PDB 6VXX and rendered using PyMOL. (B) Percentage change in oligomannose content of the N234 N-glycan of S_{recombinant-trimer}, S1_virus, S1_{vaccine-antigen}, and S1_recombinant. (C) Location of the S1 O-glycan T678 (shown as disialylated core-1 structure) located in the subdomain (SD) near the furin cleavage site between S1 and S2 (modeled on PDB 6VXX using the GLYCAM web server) and rendered using PyMOL. (D) Changes in T678 O-glycan occupancy across samples tested. (E) Flow cytometry analysis of nontransfected and S_{recombinant-trimer} and S_{vaccine-antigen} transfected HEK293F cells stained positive for S1 or (F) S2 solely on the cell surface or in permeabilized cells. Data are shown as mean ± SEM (n = 2).

Figure 4

Differential expression and glycan processing of virions and non-stablized spike glycoproteins. SARS-CoV-2 binds to its receptor ACE-2 and infects cells, leading to the release of the viral genome and translation of viral proteins. Spike protein is cotranslationally N-glycosylated and forms trimers in the ER that traffic to the ERGIC where they are incorporated into budding virions. Individual virions continue through the secretory pathway to the trans-Golgi prior to following a lysosomal egress route. For any vaccine delivering DNA/RNA that results in a non-stabilized trimer, the spike is synthesized in the ER, where it is N-glycosylated and trimerizes as before, but as it is not incorporated into a budding virion in the ERGIC, it continues through the secretory pathway and, via lysosomes, to the plasma membrane. In both cases, the spike glycoproteins have access to both the N- and O-linked host glycosylation machinery. Upon furin cleavage in the trans-Golgi, S1 and S2 of the virus stay noncovalently associated, whereas furin cleavage of the non-stabilized spike results in shedding of monomeric S1_{vaccine-antigen}. Glycomic signature analysis of these two proteins shows that the N-linked glycosylation occupancy levels, which are determined in the ER, are comparable for S1_virus and S1_{vaccine-antigen} whereas the attached glycoforms vary reflecting their different accessibility to glycan processing enzymes. S1_{vaccine-antigen} not only carries higher levels of complex N-glycans but is also extensively O-glycosylated after furin cleavage in the trans-Golgi, when most S1_{vaccine-antigen} is shed and secreted in a soluble monomeric form. Some S1 and S2_{vaccine-antigen} are displayed on the cell surface, presumably as trimers.