S. flexneri, a type of gram-negative bacteria, produces two different polysaccharides on its outer membrane: lipopolysaccharide (LPS) and enterobacterial common antigen (ECA). These polysaccharides possess a tripartite composition, encompassing lipid A as the proximal membrane anchor, inner and outer core sugars, and a distal chain of O antigen polysaccharides. The diverse functions of LPS in gram-negative pathogens, such as S. flexneri, are well recognized. It helps in maintaining the pathogen's viability, contributing to pathogenesis, and evading immune responses. Strikingly, Shigella bacilli lack a capsule, relying solely on the O antigen to define their serospecificity. The O antigen also plays a critical role in shaping virulence. Notably, the immune response triggered by the O antigen holds tremendous potential for delivering protection, making it a promising candidate in the formulation of shigellosis vaccines, including conjugate vaccines.
Fig.1 Polysaccharide biosynthesis in S. flexneri. (Maczuga, et al., 2022)
To purify PS produced by S. flexneri cultures, CD BioGlyco utilizes a range of traditional methods, including molecular weight cut-off studies, phenol-chloroform extraction, acid precipitation, and detergent approaches. To enhance yield and achieve more efficient purification, innovative techniques such as chromatographic purification and novel silicate methods are subsequently introduced at CD BioGlyco. We utilize high-performance liquid chromatography (HPLC) to scrutinize the average molecular weight distribution of the purified S. flexneri polysaccharides. Moreover, we evaluate distinct sugar compositions within the total polysaccharides of S. flexneri by using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
This polysaccharide we generate at a reduced time is utilized to conjugate with Cross-reacting Material 197 (CRM197) to generate a glycoconjugate. The conjugation of the O antigen polysaccharides and CRM197 is achieved by using the adipic acid dihydrazide (ADH) as the linker, with EDAC/NHS activation facilitating the process. Subsequently, we utilize some analytical techniques such as size exclusion chromatography (HPLC-SEC) and HPAEC-PAD to identify the efficiency and quantity of the resulting glycoconjugate. Finally, we develop a stable S. flexneri polysaccharide-CRM197 conjugate with high molecular weight for the shigellosis vaccine research.
Fig.2 Steps of the conjugation of polysaccharide and CRM197. (CD BioGlyco)
CD BioGlyco possesses the sophisticated Glyco™ Vaccine Development Platform that makes us skillfully produce polysaccharide antigens. To explore the area of Carbohydrate-based Vaccine Development, we develop various approaches to produce polysaccharide-carrier conjugates. If you are interested in what we can provide, please feel free to contact us for more detailed information.
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