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.

Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases. Although nanotechnology has been greatly applied in the field of vaccines, it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers. In this work, an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system. When SpyTag/SpyCatcher system is combined with protein glycosylation technology, bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles (VLPs) in a biocompatible and controlled manner. After confirming the excellent lymph node targeting and humoral immune activation abilities, these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model. These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines. To the best of the researchers' knowledge, this is the first time VLP-based nanoconjugate vaccines are produced efficiently, and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.

Klebsiella pneumoniae has emerged as a severe opportunistic pathogen with multiple drug resistances. Finding effective vaccines against this pathogen is urgent. Although O-polysaccharides (OPS) of K. pneumoniae are suitable antigens for the preparation of vaccines given their low levels of diversity, the low immunogenicity (especially serotype O2) limit their application. In this study, a general Escherichia coli host system is developed to produce a nanoscale conjugate vaccine against K. pneumoniae using the Nano-B5 self-assembly platform. The experimental data illustrate that this nanoconjugate vaccine can induce an efficient humoral immune response in draining lymph nodes (dLNs) and elicit high titers of the IgG antibody against bacterial lipopolysaccharide (LPS). The ideal prophylactic effects of these nanoconjugate vaccines are further demonstrated in mouse models of both systemic and pulmonary infection. These results demonstrate that OPS with low immunogenicity can be changed into an effective antigen, indicating that other haptens may be applicable to this strategy in the future. To the knowledge, this is the first study to produce biosynthetic nanoconjugate vaccines against K. pneumoniae in E. coli, and this strategy can be applied to the development of other vaccines against pathogenic bacteria.