Synthetic Vaccine Against Human Pathogenic Bacteria

Child Health And Human Development

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Abstract

SYNTHETIC APPROACH TO DEVELOP IMPROVED CONJUGATE VACCINES Synthetic vaccines against Shigella dysenteriae Type 1 Shigella dysenteriae type 1 is a Gram-negative human pathogen that causes endemic and epidemic dysentery throughout the world. Despite its discovery many years ago, there are still no licensed vaccines against this pathogen, which has developed resistance to most available antibiotics. Our approach to vaccine development against this bacterium is based on the demonstration that serum antibodies against the O-specific polysaccharides (O-SPs) of Gram-negative bacteria are important for host protection. While O-SPs are nonimmunogenic, presumably because of their low molecular weight, covalent conjugates of an immunogenic protein and the O-SP of S. dysenteriae type 1 has been shown to elicit significant anti?O-SP antibody levels of the IgG isotype, which may be boosted by repeated injections. We surmised that an improved vaccine might be constructed from chemically defined oligosaccharide fragments of the O-SP, which are devoid of biological contamination, carry a uniform molecular weight, and can be characterized by physicochemical methods. In earlier studies we have found that oligosaccharides as small as an octasaccharide can generate substantial amounts of O-SP specific antibodies in mice against S. dysenteriae type 1 when coupled to a protein, even if administered without an adjuvant. Based on this finding, most of our efforts in the past year have been directed towards the chemical synthesis of octa-, dodeca-, and hexadecasaccharides under clean-laboratory conditions in sufficient quantities. Our earlier synthetic experience served as the basis of this work. However, numerous chemical problems were encountered during the scale-up that necessitated further synthetic explorations. A particular difficulty was caused by the need for stereoselective construction of the alpha-glucosamidine linkage. Earlier, in small-scale syntheses, we have circumvented this problem by repeated chromatographic purifications of the product mixture. On the large scale, this would be laborious and prohibitively expensive. Based on the assumption that a rigid glucosamine donor, as opposed to a flexible one, might lead to higher proportions of the alpha interglycosidic linkage, we have prepared numerous 4,6-O-acetal-protected 2-azido-glucose derivatives and tested their reactions with a rhamnose acceptor. We found that the benzophenone acetal protecting group in a azido-glucose donor moiety provided high stereoselectivity, presumably by cooperative factors including rigidity of the hexopyranose ring and steric crowding on the beta-side. Using this approach, we were able to achieve more than 95 % stereoselectivity in the construction of alpha-glucosamidine interglycosidic linkages. The synthetic improvements allow us to scale up the synthetic operations. As a result, we now have the targeted spacer-equipped synthetic polysaccharides in quantities of 300 to 400 mg. These preparations will be conjugated to a medically acceptable protein for evaluating their immunogenicities in humans. Synthetic vaccine against the etiological agent of Lyme disease Work in this laboratory by Ben-Menachem and co-workers established the structures of major glycolipids in Borrelia burgdorferi as cholesteryl 6-O-palmitoyl/oleoyl-b-D-galactopyranosides. Based on the assumption that antibodies to these glycolipids, assumed to be functional equivalents of a lipopolysaccharide, might offer protection against this bacterium, a synthetic project was initiated to produce the glycolipids and their protein conjugates in quantities and purity suitable for immunization experiments. We have so far synthesized the native glycolipids and their structural congeners including the 2-O and 4-O-palmitoyl derivatives as well as the parent compound cholesteryl b-D-galactopyranoside. All the glycolipids reacted with antisera raised against the native glycolipids, the weakest reaction occurred with the unnatural isomers. We assume that conjugation to a protein would be most suitable through the methyl terminus of the palmitoyl moiety. In order to demonstrate that chemical functionalization at this site will not abolish antigenicity, we synthesized the 16-hydroxy-palmitoyl derivative. The reaction of this material with the antiserum demonstrated that chemical changes at the terminal position does not abolish antigenicity. Based on this finding we are currently synthesizing a spacer-equipped derivative of the native material for chemical attachment to protein that will be used in immunization experiments.