ACTIVATION OF SOLUBLE POLYSACCHARIDES WITH 1-CYANO-4-DIMETHYLAMINOPYRIDINIUM TETRAFLUOROBORATE FOR USE IN PROTEIN-POLYSACCHARIDE CONJUGATE VACCINES AND IMMUNOLOGICAL REAGENTS

Neonates have poor immune responses to type 2 T-cell independent antig ens (TI-2), such as polysaccharides and immunization of human infants with these antigens does not induce protective levels of serum antibod ies. Conjugating proteins to TI-2 antigens converts the immune respons e to one which is T-cell dependent. We used an organic cyanylating rea gent, 1-cyano-4-dimethylaminopyridinium tetrafluroborate (CDAP), to ac tivate polysaccharides, in water, and subsequently react them with hex anediamine, in preparation of coupling proteins to the polysaccharide. CDAP activation of polysaccharide is rapid (<2 min) and efficient. CD AP can be used to activate polysaccharides of diverse chemical natures , including dextrans and pneumococcal types 6, 14, 19 and 23. The crit ical parameters in CDAP activation of polysaccharides were the reagent concentrations and the pH. Activation can be performed over a broad a lkaline pH range, with an optimum of pH 9-10. Furthermore, proteins ca n be coupled to CDAP-activated polysaccharides without the use of a sp acer. Direct conjugation of protein to CDAP-activated polysaccharides can be performed under mildly alkaline conditions (pH 7-9). These cond itions allow CDAP to be used with alkaline-sensitive polysaccharides a nd proteins. Mice immunized with BSA-pneumococcal type 14 polysacchari des (Pn14) conjugates, prepared either by direct conjugation or via a spacer, and had anti-Pn14 and anti-BSA serum antibody IgG1 titers, whe reas no IgG1 antibody was induced to the unconjugated components. The case of sue and mild activating conditions should prove of value in us ing CDAP to prepare conjugate vaccines, as well as other immunological ly useful reagents.