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Chemical Structure, Conjugation, and Cross-Reactivity of Bacillus pumilus Sh18 Cell Wall Polysaccharide

Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

Received 5 March 2004/ Accepted 15 July 2004

Bacillus pumilus strain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy--galactopyranose (13%) and 2-acetamido-2-deoxy--glucopyranose (6%) on position O-2, were found. A minor component was established to be a polymer of 3-O-(2-acetamido-2-deoxy-ß-glucopyranosyl)-14-ribitol-1-OPO₃. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide (ADH)-derivatized protein, and (ii) binding of the carbodiimide-activated terminal phosphate group of the CWP to ADH-derivatized protein. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with the homologous polysaccharide and with a number of other bacterial polysaccharides containing ribitol and glycerol phosphates, including H. influenzae types a and b and strains of Staphylococcus aureus and Staphylococcus epidermidis.