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.

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In its own right, vaccinology has been undergoing a revolution, and there are now a large number of innovative projects seeking to develop both prophylactic and therapeutic vaccines against diseases such as Hepatitis B, influenza, HIV, and cancers. [4-6] Generally speaking, the major advantages conferred by nanovaccines include improving stability by protecting antigens from premature degradation, providing good adjuvant properties, and assisting in the targeted delivery of an antigen to antigen-presenting cells (APCs). [7] A large variety of nanoscale materials have been deployed in nanovaccine designs. Seminal work with inorganic nanoparticles (NPs, e.g., gold, carbon, and silica) established the capacity of nanovaccine-bound antigens to elicit desired immune responses. Subsequent technologies have elaborated beyond inorganic NPs, for example, use of inorganic/ organic hybrid NPs (e.g., PEI adopted silica NPs and biomimetic magnetosomes) to enhance antigen immunogenicity. [8,9] Recently, new types of organic NPs (e.g., lipoprotein-mimicking nanodisks, pickering emulsions, and nanogels) have also received great attention for their applications in vaccines. [10-16] Recent years have seen enormous advances in nanovaccines for both prophylactic and therapeutic applications, but most of these technologies employ chemical or hybrid semi-biosynthetic production methods. Thus, production of nanovaccines has to date failed to exploit biology-only processes like complex sequential post-translational biochemical modifications and scalability, limiting the realization of the initial promise for offering major performance advantages and improved therapeutic outcomes over conventional vaccines. A Nano-B5 platform for in vivo production of fully protein-based, self-assembling, stable nanovaccines bearing diverse antigens including peptides and polysaccharides is presented here. Combined with the self-assembly capacities of pentamer domains from the bacterial AB 5 toxin and unnatural trimer peptides, diverse nanovaccine structures can be produced in common Escherichia coli strains and in attenuated pathogenic strains. Notably, the chassis of these nanovaccines functions as an immunostimulant. After showing excellent lymph node targeting and immunoresponse elicitation and safety performance in both mouse and monkey models, the strong prophylactic effects of these nanovaccines against infection, as well as their efficient therapeutic effects against tumors are further demonstrated. Thus, the Nano-B5 platform can efficiently combine diverse modular components and antigen cargos to efficiently generate a potentially very large diversity of nanovaccine structures using many bacterial species.

Shigella flexneri 2a is an important pathogen causing bacillary dysentery in humans. In order to investigate any potential vaccine candidate proteins present in outer membrane proteins (OMPs) and extracellular proteins of S. flexneri 2a 2457T, we use the proteome mapping and database analyzing techniques. A subproteome map and database of OMPs were established first. One hundred and nine of the total 126 marked spots were cut out and processed to MALDI-TOF-MS and PMF. Eighty-seven spots were identified and they represented 55 OMP entries. Furthermore, immunoproteomics analysis of OMPs and extracellular proteins were performed. Total of 34 immunoreactive spots were identified, in which 22 and 12 were from OMPs and extracellular proteins, respectively. Eight novel antigens were found and some of these antigens may be potential vaccine candidate proteins. These results are useful for future studying of pathogenicity, vaccine, and novel antibacterial drugs. Maps and tables of all identified proteins are available on the Internet at www.proteomics.com.cn.

Enteric fever, mainly caused by Salmonella enterica serovar Paratyphi A, remains a common and serious infectious disease worldwide. As yet, there are no licensed vaccines against S. Paratyphi A. Biosynthesis of conjugate vaccines has become a promising approach against bacterial infection. However, the popular biosynthetic strategy using N-linked glycosylation systems does not recognize the specialized O-polysaccharide structure of S. Paratyphi A. Here, we describe an O-linked glycosylation approach, the only currently available glycosylation system suitable for an S. Paratyphi A conjugate vaccine. We successfully generated a recombinant S. Paratyphi A strain with a longer O-polysaccharide chain and transformed the O-linked glycosylation system into the strain. Thus, we avoided the need for construction of an O-polysaccharide expression vector. In vivo assays indicated that this conjugate vaccine could evoke IgG1 antibody to O-antigen of S. Paratyphi A strain CMCC 50973 and elicit bactericidal activity against S. Paratyphi A strain CMCC 50973 and five other epidemic strains. Furthermore, we replaced the peptides after the glycosylation site (Ser) with an antigenic peptide (P2). The results showed that the anti-lipopolysaccharide antibody titer, bactericidal activity of serum, and protective effect during animal challenge could be improved, indicating a potential strategy for further vaccine design. Our system provides an easier and more economical method for the production of S. Paratyphi A conjugate vaccines. Modification of the glycosylation site sequon provides a potential approach for the development of next-generation “precise conjugate vaccines.”