Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

Tyrosine-directed conjugation of large glycans to proteins via copper-free click chemistry.

We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide-alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called reverse vaccinology. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens.

Simultaneous quantification of hyoscyamine and scopolamine using HPLC-DAD in four Solanaceae: Hyoscyamus niger, Datura stramonium, Atropa belladonna and Mandragora officinarum.

Alkaloids are a group of secondary metabolites that generate great interest since ancient times. Numerous Solanaceae plants are rich sources of tropane alkaloids as hyoscyamine and scopolamine which are obtained mainly from Hyoscyamus niger, Datura stramonium, Atropa belladonna, Mandragora officinarum. In the present study it was developed an HPLC-DAD using an XBridge Phenyl column for the quantification of scopolamine and hyoscyamine, molecules used in pharmaceutical industry to treat stomach or intestinal disorders. A. belladonna presented hyoscyamine and scopolamine, the first one ranged from 1466 to 5117 mg/Kg DW while the second one ranged from 140 to 1743 mg/Kg DW. In D. stramonium, hyoscyamine was not found while scopolamine ranged from 430 to 8980 mg/Kg DW. On the contrary H. niger and M. officinarum did not contain any trace of these alkaloids. This is the first work in which different parts of four Solanaceae were analysed for their hyoscyamine and scopolamine content.