Conjugate vaccines of polysaccharides (PS; including lipopolysaccharides or LPS) utilize a carrier protein as a vehicle to convert the T- independent immunogen PS to a T-dependent protein-PS conjugate, and thus afford the protection to an otherwise PS-non-responding population such as infants and the elderly. In this research, we investigate the issues related to the chemistries of conjugate vaccine synthesis and their impacts to the vaccine immunogenicity and effectiveness. This includes the studies of: 1) the structures of PS and LPS, carrier proteins and peptides, spacer molecules and the conjugates, 2) conditions of various chemical reactions for conjugate formation, and 3) characterization of the conjugates in the aspects of physical and chemical properties and immunology. We have synthesized PS-tetanus toxoid (TT) conjugates for pneumococcal types 4, 6B, 9V, 14, 18C, 19F and 23F. The immunogenicity of these singular and combined 7-valent conjugates were investigated in mice in parallel to the corresponding PS-CRM197 conjugates of the 7- valent pneumococcal conjugate vaccine Prevnar of Wyeth-Lederle Vaccines and Pediatrics. It was found that the PS-CRM197 conjugates were more immunogenic than the PS-TT conjugates. The conjugates in singular form were more effective as an immunogen than in the combined 7-valent form except for T4 PS-CRM197 and T23F PS-TT. Presence of free unconjugated carried protein reduced the immunogenicity of the conjugates. Pre- immunization of the mice with the carrier protein might increase or decrease the immunogenicity of the conjugate depending on the carrier proteins. The elicited antisera exhibited opsonophagocytosis activity to the homologous pneumococci. This activity, an in vitro test of the biological function for antibody, correlated with the antibody content. In the second line of research, we have conjugated LPS from mutants of group B menigococci and B. pertussis to TT. These conjugates exhibit T- dependent immunogenicity against homologous LPS, and have a remarkable reduction of LAL activity. The bactericidal activities of the antibodies induced by these conjugates are currently being measured in vitro and in vivo. In the third line of research, we have adapted and developed purpald assay methods for quantification of LPS and PS. These methods provide: 1) a conenient means (96-well tissue culture plates/room temperature vs. temperature higher than 90 degree/test tubes) for measurement of LPS and PS, 2) a measurement for LPS from Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae, 3) a determination of epitope preservation in conjugate vaccines.
