Analysis of SARS-CoV-2 spike glycosylation reveals shedding of a vaccine candidate

Abstract

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses 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 neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, which may have implications for the resulting immune response and future vaccine design.

Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

“…Potential candidates for O-glycosylated residues include Thr 323, Ser 325, Ser 659, Ser 673, Thr 676, Thr 678 and Ser 680 in S1, and Thr 696, Thr 1160 and Ser 1170 in S2. O-linked glycosylation at Thr 323/Ser 325 was reported for soluble S glycoproteins and virion S glycoprotein trimers, but with low occupancy; in some cases, less than 1% of the residues were modified ( 33 , 35 , 36 , 53 ). In this study, the occupancy rate for these two sites is also around 1% ( Table 1 ).…”

“…In Figure 8 , we compare our results with available glycosylation analyses of soluble or solubilized SARS-CoV-2 S glycoproteins. These include wild-type S glycoproteins purified from SARS-CoV-2 virions, as well as soluble and full-length S glycoprotein trimers modified to inhibit furin cleavage and to stabilize a prefusion conformation ( 30 , 33 , 34 , 36 , 51 – 53 ). The glycans on individually expressed SARS-CoV-2 S1 and S2 glycoproteins have also been analyzed ( 35 ).…”

“…In this study, the occupancy rate for these two sites is also around 1% ( Table 1 ). Thr 678 has also been reported to be O-glycosylated in soluble and virion S glycoproteins, with higher occupancy than that for Thr 323/Ser 325 ( 36 , 53 ). In our study, the peptide containing Ser 659, Ser 673, Thr 676, Thr 678 and Ser 680 was found to be occupied by at least one O-linked glycan at a level of about 5%.…”

“…The glycosylation of the SARS-CoV-2 spike has been studied using soluble or detergent-solubilized versions of the uncleaved S glycoprotein trimer, modified to retain a pretriggered conformation ( 30 , 33 – 36 , 51 ). Fewer studies of the glycosylation of S glycoproteins on SARS-CoV-2 virion preparations have been conducted ( 52 , 53 ). Experience with human immunodeficiency virus (HIV-1) indicates that native, membrane-anchored viral envelope glycoproteins can exhibit glycosylation profiles that differ from those of soluble glycoprotein trimers ( 54 – 57 ).…”

Recent Advances in the Chemical Biology of N-Glycans

“…For the development of vaccines against COVID-19, the spike protein is a promising antigen candidate. This protein is heavily glycosylated [ 156 ], but N -glycan modifications of spike proteins have been reported to reduce their antigenicity [ 157 ]. However, N -glycan-modified antigens may induce antibodies against endogenous N -glycans, which should be carefully examined.…”