Reactions And Immunochemistry Of Carbohydrates

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

We continue with our endeavor aimed at developing a conjugate vaccine for cholera from synthetic fragments of the O-PS of Vibrio cholerae O:1. A potent vaccine for the disease is not available. This work is important from the point of view of public health and, because of the involvement of our military in protecting US interests in developing third world countries, the availability of a vaccine for cholera has become of national interest. Cholera is a serious enteric disease caused mainly by two strains of Vibrio cholerae O:1, Ogawa and Inaba. The work towards a potent conjugate vaccine for cholera involves synthesis of oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugation of these antigens to suitable carriers, and serologic evaluation of the immunogenicity of the resulting neoglycoconjugates. Our original protocol for the synthesis of the requisite oligosaccharides involved assembly of the required oligosaccharides from intermediates lacking both the 3-deoxy-L-glycero-tetronic acid side chain and the spacer molecule. This synthetic strategy required many chemical manipulations with the assembled oligosaccharide, in order to arrive at a molecule ready for conjugation. We have previously incorporated a number of improvements in the original protocol, to minimize the number of chemical modifications of the assembled oligosaccharide but realize that further adjustments are necessary. Currently, aiming at further improvements of the synthesis, we are making carbohydrate antigens that mimic the O-PS of Vibrio cholerae O:1 in a fundamentally different way. The new strategy is based on the use of glycosyl donors and glycosyl acceptors having the N-acyl side chain already in place. Also, the glycosyl acceptor is the N-3-deoxy-L-glycero-tetronylated perosamine glycoside of the spacer molecule (methyl 6-hydroxyhexanoate). Thus, when the requisite oligosaccharide is assembled the only chemical manipulation that needs to be carried out to obtain material suitable for conjugation is removal of protecting groups. Within our intention to investigate the role of details in the architecture of neoglycoconjugates upon immunogenicity, we have synthesized conjugates that mimic the O-PS of Vibrio cholerae O:1 involving different length linkers which separate the antigen from the carrier protein. The serological evaluation of sera resulting from immunizing small laboratory animals with our experimental immunogens is in progress. Similar evaluation of neoglycoconjugates prepared from the Inaba oligosaccharides and bovine serum albumin (BSA) showed that these constructs were highly immunogenic but, unlike the hexasaccharide conjugate from the Ogawa hexasaxxharide, the antibodies produced did not show protective capacity. In collaboration with The Department of Bacteriology, Dartmouth Medical School, we are continuing in our efforts to find the most suitable configuration of the neoglycoconjugate that would serve as a potent immunogen for anti Vibrio cholerae O:1 antibodies. Concurrently, we work on other challenges. A new target of our synthetic endeavors is the O-PS, as well as fragments, of a new, recently emerged bacterial strain which causes a serious health problem in developing countries, Vibrio cholerae O:139. The structure of the O-PS is even more difficult to synthesize than that of Vibrio cholerae O:1, as it is a hexasaccharide which consists of five different sugars. One of the constituents sugars is the rare substance collitose (3,6-dideoxy-D-galactose). A prerequisite for making the requisite hexasaccharide antigen is availability of colitose by chemical synthesis. We have improved the existing synthesis of colitose, and use it currently to synthesize fragments that mimic the O-PS of Vibrio cholerae O:139. These substances will be converted to neoglycoconjugates and their immunogenicity will be evaluated. Another project under investigation in our laboratory is aimed at a vaccine for anthrax. Recently, a tetrasaccharide was found to form side chain of the major glycoprotein of Bacillus anthracis exosporium. Its end group is formed by a newly discovered sugar anthrose [methyl 4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-D-glucopyranose]. Our laboratory is the first to ever synthesize this rare sugar. This opens for us ways to proceed with synthesis of immunogens for anti Bacillus anthracis antibodies.