William Melaugh

S15.27 Structural determination of the lipooligosaccharides ofHoemophilus ducreyi and possible therapeutic targets

Haemophilus influenzae type b (Hib) is a major cause of bacterial meningitis in children in the United States and many other countries. To prevent Hib disease, Hib polysaccharide (PS)-protein conjugate vaccines have been developed by a few manufacturers. Measurement of PS content in the conjugate vaccines is required to assure their quality and effectiveness. The PS content in the vaccines is currently measured by ribose, a component of the PS, using the colorimetric orcinol assay. This assay is not useful for a vaccine containing lactose, a sugar stabilizer, due to its interference. We have developed a simple HPLC method to quantitate the PS in the vaccines regardless of the presence of the sugar stabilizer. The PS in the vaccines was depolymerized in sodium hydroxide. The major depolymerized product (82%) was isolated and characterized by composition analysis and 3~p NMR to be a single repeating unit of the PS, ribitol-ribose-phosphate. The PS repeating unit in the alkali-treated vaccines was separated from the large amount of sugar stabilizer by high-performance anionexchange chromatography using CarboPak PA-1 column (Dionex Corp.) and quantitated by pulsed amperometric detection. The procedure is simple and reproducible. This HPLC method can quantitate PS levels as low as 0.1/ag or at a PS concentration of one ~g/ml, and the sensitivity is at least 30-fold higher than that of the orcinol assay. This HPLC method of quantitation may be applied to other bacterial PS or PS-conjugate vaccines after hydrolysis of PS to monosaccharides or oligosaccharides.