A large part of our research program involves preparation of neoglycoconjugates from synthetic fragments of complex polysaccharides that are part of Gram negative bacteria, e.g. O-specific polysaccharides (O-PS), and evaluation of their immunogenicity. In order to rationally select structures to become antigenic components of these immunogens we have to identify immunologically dominant epitopes of the O-PS. To do so, we chemically synthesize oligosaccharide sequences that mimic the structure of the O-PS, and measure their binding with homologous antibodies. To effect conjugation of antigenic oligosaccharides to suitable protein carriers, we prepare them in the form of glycosides whose aglycons make them suitable for conjugation. Using diferent linkers, different conjugation chemistry and by varying other factors, the conjugates we obtain differ in architectonic details. We then evaluate the immunogenicity of these neoglycoconjugates using small animals as hosts. In continuation of our work on mapping the combining sites of some monoclonal antibodies for Vibrio cholerae O:1, we have synthesized the last of the four theoretically possible specifically monofluorinated analogs of the terminal monosaccharide determinant of the O-PS of Vibrio chorerae O1, serotype Ogawa. We have also prepared a large number of other analogs of the immunologically dominant epitope of Vibrio cholerae O:1 and studied binding of these substances with monoclonal antibodies specific for the Ogawa LPS. The results obtained provided additional information, at the molecular level, on the antigen-antibody interaction in this system. Efficient use in the conjugation process of the labor-intensive, spacer-equipped haptens required method by way of which, a. the conjugation process could be monitored so that the reaction could be terminated when the desired hapten?protein ratio had been reached and, b. a method allowing recovery of the hapten used in excess. We have been able to show that surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) in combination with the ProteinChip System, a technique developed for a different application, allows routine and rapid analysis of the conjugation mixture, and provides real-time information about the increasing molecular weight of the conjugate. We have also developed a protocol allowing recovery of the ligand used in excess during conjgution. The ability to recover haptens from conjugation mixtures improves considerably the overall economy of the conjugation process. Testing of the neoglycoconjugates obtained for their immunogenicity in neonatal mice is in progress.
Showing the most recent 10 out of 28 publications
