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A team of scientists from the University of Oxford, UK, has revealed that site-specific glycosylation differs between spike proteins derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and a candidate vaccine based on a viral vector. A significantly high amount of non-physiological glycosylation can affect the ability of a candidate vaccine to induce the desired immune responses. The study is currently available at bioRxiv* prepress server.
SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is a positive sense single-stranded RNA virus with a genome size of approximately 30 kb. The virus spreads rapidly from person to person, mainly through respiratory droplets. In addition to implementing non-pharmacological control measures (use of masks, hand washing / disinfection, and movement restrictions), several attempts have been made to develop effective therapies and vaccines. Although many potential vaccine candidates are currently being developed, it is not yet known how effective these vaccines are in terms of inducing and sustaining SARS-CoV-2 specific immune responses.
Mechanically, the interaction between the viral peak protein and the host’s angiotensin converting enzyme (ACE2) facilitates the entry of SARS-CoV-2 into host cells. The spike protein is a viral surface protein that undergoes N-linked glycosylation (addition of N-glycans to 22 N-glycosylation sites) after its synthesis in the endoplasmic reticulum of the host cell. The addition of O-glycans occurs in the Golgi.
Glycosylation of the spike protein is essential to maintain the stability of the viral protein, influence viral infectivity, and facilitate immune evasion. Within host cells, viral spikes are the molecules most frequently targeted by neutralizing antibodies. Therefore, most candidate vaccines are designed using viral spike protein as the antigen to induce the desired immune responses. However, both N-linked and O-linked glycans on the surface of the spike protect the underlying viral epitopes from attack by host neutralizing antibodies. Sometimes these glycans act as viral epitopes to trigger host cell immune responses. Therefore, adequate knowledge about the peak protein glycosylation process is essential to develop an effective vaccine candidate.
Current study design
The scientists aimed to compare the glycosylation process of the SARS-CoV-2 derived spike protein and a candidate vaccine based on a viral vector. They used lung epithelial cells to grow SARS-CoV-2 and subsequently used an anti-SARS-CoV-2 monoclonal antibody to purify the spike protein.
Differential expression and processing of virion glycans and vaccine-derived peak glycoproteins. SARS-CoV-2 binds to its ACE-2 receptor and infects cells, causing the release of the viral genome and the translation of viral proteins. The spike protein is co-translationally N-glycosylated and forms trimers in the ER that are transferred to the ERGIC, where they are incorporated into budding virions. Individual virions continue through the secretory pathway to the trans-Golgi before following a lysosomal exit route. For the candidate vaccine, the tip DNA is delivered via an adenovirus vector system and the tip protein is synthesized in the ER, where it is N-glycosylated and trimerized as before, but not incorporated into a budding virion in the ERGIC, continues through the secretory pathway and, through the lysosomes, to the plasma membrane. In both cases, the spike glycoproteins have access to the host’s glycosylation machinery linked to both N and O. After furin cleavage in the trans-Golgi, S1 and S2 of the virus remain non-covalently associated, whereas furin cleavage of the vaccine antigen results in the elimination of the monomeric S1 vaccine antigen. Analysis of the glycomic signature of these two proteins shows that the N-linked glycosylation occupancy levels, which are determined in the ER, are comparable for the S1 virus and S1 vaccine antigen, while the attached glycoforms vary reflecting its different accessibility to glycan processing enzymes. Not only does the S1 vaccine antigen carry higher levels of complex N-glycans, but it is also highly O-glycosylated after furin cleavage in the trans-Golgi, when most of the vaccine antigen S1 is cleared and secreted in a soluble monomeric form. Some antigens from the S1 and S2 vaccines show up on the cell surface, presumably as trimers.
Important remarks
Ultra-performance liquid chromatography-based analysis of virus-derived N-glycans showed 79% complex-type N-glycans and 21% oligomannose / hybrid N-glycans. In contrast, analysis of vaccine-derived N-glucans showed 89% complex-type N-glucans and only 11% oligomannose / hybrid N-glucans. These findings indicate that the glycan processing of the SARS-CoV-2 derived spike protein differs significantly from that of the vaccine-derived spike protein.
The scientists conducted mass spectrometry analysis to further evaluate the peak protein glycan processing. They observed that the N-glycan processing of the S1 subunit of the spike protein is comparable between the virus and the vaccine candidate. However, they observed O-linked glycosylation at the T678 site in the virus-derived spike protein, which was absent in the vaccine-derived spike protein. This finding indicates that the viral spike protein maintains a more flexible configuration than the vaccine-derived spike protein.
Furthermore, the scientists expressed spike protein (similar to the vaccine candidate) in mammalian cells and compared its glycosylation process with the spike protein derived from the virus. Interestingly, the S1 subunit of the expressed peak protein showed 96% complex-type N-glycans and only 4% oligomannose-type N-glycans. This indicates that, compared to the S1 subunit of the viral peak protein, the S1 subunit of the expressed peak protein is extensively processed by glycosylation enzymes.
They observed that extensive processing of N-glucans at the N234 site is avoided due to spatial and temporal assembly of the spike protein in the host endoplasmic reticulum and in the Golgi. For the vaccine and virus derived S1 subunits, this site remained completely threading (100% oligomannose); however, for the S1 subunit derived from the expressed protein, some level of glucan processing (oligomannose 75%) was observed at this site. With a more detailed analysis, the scientists observed that the dissociation between the S1 and S2 subunits of the expressed peak protein occurs in the trans-Golgi and not in the cell plasma membrane. By individually expressing the recombinant S1 subunit that cannot trimerize, they observed 100% complex-type N-glycans at the N234 site and 100% O-glycans at the T678 site.
Taken together, the study’s findings indicate that the processing of viral proteins with glucans needs to be critically reviewed before developing SARS-CoV-2 vaccine candidates. A significantly high amount of complex N-glycans can potentially cover viral epitopes, which in turn can inhibit the antibody-epitope interaction and prevent the induction of desired immune responses. According to the scientists, a candidate vaccine containing prefusion stabilized spike protein without a proteolytic cleavage site is optimal for inducing strong and sustained immune responses. Inhibition of S1 subunit clearance by abolishing the proteolytic cleavage site is important for proper immunogen presentation of a vaccine candidate.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.
