In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation

Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering. In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production. The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.