Reactions And Immunochemistry Of Carbohydrates

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

Our research aimed at developing a conjugate vaccine for cholera from synthetic fragments of the O-PS of Vibrio cholerae O:1 continues. A potent vaccine for the disease is not available. This work is important from the point of view of public health. Also, during the last few years, when our military protects 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, among other things, conjugation to proteins of synthetic oligosaccharides that mimic the structure of O-specific polysaccharides (O-PS) of the relevant Gram-negative bacteria, and evaluation of the immunogenicity of the resulting neoglycoconjugates. Haptens required for conjugation result from multi step, sophisticated chemical syntheses, which is a formidable, very laborious process. For example, the synthesis of the spacer-equipped hexasaccharide fragment representing the terminus of the O-PS of Vibrio cholerae O:1, serotype Ogawa involves over thirty synthetic steps. According to the protocol developed by us, the synthesis starts with the commercially available methyl a-D-mannopyranoside, which is converted to a disaccharide glycosyl acceptor and a disaccharide glycosyl donor. It involves first preparation of 2-(trimethylsilylethyl) (SE) glycoside of the hexasaccharide from building blocks lacking the N-acyl group. When the targeted oligosaccharide is assembled, the six azido groups present in the intermediate are reduced to amino groups and the resulting amines are N-acylated with a suitable derivative of 3-deoxy-L-glycero-tetronic acid. Subsequently, the SE functionality is exchanged for an aglycon (spacer), which makes the hapten amenable for chemical linking to a carrier. Since any chemical transformation with a larger molecule such as a hexasaccharide can be problematic, generally, the drawback of such an approach is that it requires many chemical manipulations to be performed with the assembled oligosaccharide. We have now developed a new approach to the synthesis of tetra- and hexasaccharide fragments of the O-PS of Vibrio cholerae O:1, serotype Inaba, from intermediates having the N-acyl side chain already in place. Also, the glycoside of methyl 6-hydroxyhexanoate was used as the initial glycosyl acceptor. 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. The O-PS of the two strains of Vibrio cholerae O:1, which are the main causes of cholera, have very similar structures. Therefore, the problem we have to deal with when developing a vaccine for the disease is the cross-reactivity of anti Inaba/Ogawa antibodies with the respective lipopolysaccharides present on the surface of the pathogenic bacteria. To obtain antibodies specific for the Inaba O-PS and, at the same time, be able to study the effect of the size of the antigen and the hapten-carrier ratio upon immunogenicity we have prepared neoglycoconjugates from bovine serum albumin (BSA) and the di-, tetra- and the hexasaccharides that mimic the terminal epitopes of the O-PS of Vibrio cholerae O:1. In collaboration with The Department of Bacteriology, Dartmouth Medical School, these materials were used to immunize mice. The serological evaluation of the antibodies raised is currently under investigation.