Abstract
ABSTRACT Conjugate vaccines are known to be one of the most effective and safest types of vaccines against bacterial pathogens. Previously, vaccine biosynthesis has been performed by using N-linked glycosylation systems. However, the structural specificity of these systems for sugar substrates has hindered their application. Here, we report a novel protein glycosylation system (O-linked glycosylation via Neisseria meningitidis) that can transfer virtually any glycan to produce a conjugate vaccine. We successfully established this system in Shigella spp., avoiding the construction of an expression vector for polysaccharide synthesis. We further found that different protein substrates can be glycosylated using this system and that the O-linked glycosylation system can also effectively function in other Gram-negative bacteria, including some strains whose polysaccharide structure was not suitable for conjugation using the N-linked glycosylation system. The results from a series of animal experiments show that the conjugate vaccine produced by this O-linked glycosylation system offered a potentially protective antibody response. Furthermore, we elucidated and optimized the recognition motif, named MOOR, for the O-glycosyltransferase PglL. Finally, we demonstrated that the fusion of other peptides recognized by major histocompatibility complex class II around MOOR had no adverse effects on substrate glycosylation, suggesting that this optimized system will be useful for future vaccine development. Our results expand the glycoengineering toolbox and provide a simpler and more robust strategy for producing bioconjugate vaccines against a variety of pathogens.
Highlights
Conjugate vaccines are known to be one of the most effective and safest types of vaccines against bacterial pathogens
Establishment of an O-linked glycosylation system in the attenuated Shigella flexneri strain 301DWP. Escherichia coli strains, such as CLM24 [16], are the bacteria that have been most commonly employed as host strains in biomethods to produce conjugate vaccines, and multiple plasmids are required to express carrier proteins, PglB, and the glycan gene cluster together in these strains
Blue native-polyacrylamide gel electrophoresis (BN-PAGE) was performed using a Ready-Gel with a linear 4 to 15% gradient according to the manufacturer’s instructions, and the results show that the molecular mass of the polymer was more than 1,000 kDa, while the molecular mass of the CTB4573C-OPS monomer was only about 40 kDa (Fig. 3F)
Summary
Conjugate vaccines are known to be one of the most effective and safest types of vaccines against bacterial pathogens. We show that an O-linked protein glycosylation system from Neisseria meningitidis, which has a lower structure specificity for sugar substrates, could be engineered directly in attenuated pathogens to produce effective conjugate vaccines. PglB, which is homologous to the Stt component of the oligosaccharyltransferase (OTase) complex in eukaryotic cells [14], was the first to be used to produce conjugate vaccines because its glycosylation sequon was clear, a conserved pentapeptide motif, D/EX1-N-X2-S/T (where X1 and X2 are any residues except proline), unlike the tripeptide motif NXS/T (where X can be any amino acid except proline) that is present in eukaryotic cells [15] This motif can be fused to a carrier protein to generate a glycoprotein [16]. In cases where the recognition sequence for O-link glycosylation is known, the bacterial antigenic polysaccharide can be attached to major histocompatibility complex (MHC) binding peptides to further enhance vaccine efficacy
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