(625r) Preparation and Confirmation of Fla-B Conjugated Capsular Polysaccharide of Streptococcus Pneumoniae

Streptococcus pneumoniae causes many types of pneumococcal infection; especially, the incidence of pneumococcal disease which is the highest in infants under 2 years and in people over 60 years of age. Capsular polysaccharides (CPS) are the primary basis for pathogenicity, antigenic and serotypes. The large numbers of serotypes (over 90 characterized serotypes are known until now) have required the development of vaccine products composed of polysaccharides. Two types of vaccines are currently in clinical use: polysaccharide vaccine, and pneumococcal conjugate vaccines. Among the more than 90 different S. pneumoniae serotypes, serotype 19A, 4 and 3 are globally very prevalent. In this study, we investigated a simple process for the production and purification by four simple steps of 19A CPS to make conjugated CPS. A simplified purification procedure including adjustment of cell lysate pH to 4.5, fractionation with 50%–80% ethanol and dialysis obtained capsular polysaccharide (CPS) in a yield of 31.32 ± 3.11 mg from 1 L culture (75% recovery after lyses). The product contained only 69.6 μg protein (99.78% purity) and 0.8 mg (sum of the precipitants from 50~60, 60~70 and 70~80%) nucleic acid (97.45% purity). The size of capsular polysaccharide was reduced by 10 mM acid hydrolysis and separated to different sizes using ethanol fractionation (30~70%). Each product showed elevated purity. Furthermore we conjugated CPS serotypes of 19A, 4, or 3 with Flagellin B (FlaB), respectively, and confirmed conjugation by Western blot analysis using FlaB antibody and CPS antibody.