Biosynthesis of Conjugate Vaccines Using an O-Linked Glycosylation System.

unlabelledConjugate 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. IMPORTANCE: Recently, the rapid development of synthetic biology has allowed bioconjugate vaccines with N-linked protein glycosylation to become a reality. However, the difficulty of reestablishing the exogenous polysaccharide synthetic pathway in Escherichia coli hinders their application. Here, 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. To facilitate the further design of next-generation bioconjugate vaccines, we optimized a novel short motif consisting of 8 amino acids that is sufficient for glycosylation. Our results expand the application potential of O-linked protein glycosylation and demonstrate a simpler and more robust strategy for producing bioconjugate vaccines against different pathogens. In the future, bacterial antigenic polysaccharides could be attached to major histocompatibility complex binding peptides to improve immunological memory or attached to protein subunit vaccine candidates to provide double immune stimulation.