Flagellin is the main component of flagellar filament that is composed of four structural domains, including D0, D1, D2, and D3. D0/D1 domains primarily constitute the core and consist of the highly conserved N- and C-termini. In contrast, D2/D3 domains make up the outer surface of the filament. Besides, they contain a highly variable central region of flagellin. Both extracellular and intracellular flagellin are recognized by different receptors to conduct immune responses. Extracellular flagellin is identified by TLR5, resulting in the activation of the NFκB pathway. Meanwhile, intracellular flagellin is identified by NAIP5. Notably, flagellin has emerged as a potent immune activator. Moreover, it is considered a protective antigen during microbial infections. The host's innate immune TLR5 recognizes it as a crucial target, which recognizes the flagellar polymer's interior core. Furthermore, flagellin fusion has shown increased immunogenicity in different antigens.
Fig.1 The schematic diagram of flagellin and the cross-sectional and top views of the flagellar filament. (Lu & Swartz, 2016)
At CD BioGlyco, we provide flagellin as a carrier for producing a conjugate compound that has an effect against Salmonella. We combine lipopolysaccharide (LPS)-derived core oligosaccharide (COPS) from Salmonella with flagellin to prepare conjugates.
We utilize two methods to synthesize the conjugates: CDAP chemistry and aminooxy thioether chemistry. Our methods limit coupling to a single point at the terminal 3-deoxy-d-manno-octulosonic acid (KDO) region. The COPS molecules at the saccharide terminus conjugate to some available lysine residues on the protein by this coupling process. That results in the formation of conjugates. In addition, we construct various conjugates to test the impact of different factors affecting immunogenicity. Unconjugated free polysaccharides pose a threat to the efficiency of polysaccharide-protein conjugate vaccines. Since they could weaken anti-polysaccharide immune responses, we remove those free polysaccharides to make the coupling work better.
Fig.2 Flow charts of the preparation of COPS-flagellin conjugates. (CD BioGlyco)
We utilize size exclusion chromatography (SEC) and anion-exchange chromatography (IEX) to purify the conjugate by eliminating unreacted polysaccharides and proteins. Then, we measure the saccharide content in the conjugate constructs by using the resorcinol sulfuric acid assay, which utilizes Salmonella CVD 1941 LPS as standard. Additionally, the protein concentration in conjugates is evaluated by the bicinchoninic acid (BCA) assay kit. The assay kit utilizes unconjugated protein as the reference standard. Our conjugation services offer promising insights for the development of a conjugate vaccine with superior immunological characteristics against Salmonella in mice.
CD BioGlyco is a leading provider of carbohydrate-based vaccine development services utilizing our innovative Glyco™ Vaccine Development Platform. Our team of experts is highly skilled in Carbohydrate-based Vaccine Development by synthesizing and conjugating complex carbohydrates to produce available vaccines against bacteria. With a focus on quality and customization, we are committed to providing comprehensive vaccine development solutions to meet the unique needs of our clients. If our services catch your attention, feel free to contact us for more comprehensive information.
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