Synthetic Vaccine Against Human Pathogenic Bacteria

Child Health And Human Development

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Abstract

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. There are no licensed vaccines against this pathogen, which has developed resistance to most available antibiotics. While O-specific polysaccharides (O-SPs) are nonimmunogenic, covalent conjugates of an immunogenic protein and the O-SP of S. dysenteriae type 1 have been shown to elicit IgG anti-O-SP, which may be boosted by repeated injections. An improved vaccine might be constructed from chemically defined oligosaccharide fragments of the O-SP, devoid of biological contamination, have a uniform molecular weight, and can be characterized by physicochemical methods. We found that oligosaccharides as small as an octasaccharide can generate O-SP specific antibodies in mice against S. dysenteriae type 1 when coupled to a protein and administered without an adjuvant. Most of our efforts in the past year focused on the chemical synthesis of octa-, dodeca-, and hexadecasaccharides under clean-laboratory conditions in sufficient quantities. In our earlier studies, we synthesized a linear tetrasaccharide corresponding to a repeating unit of the O-SP of the lipopolysaccharide. This unit was oligomerized in a stepwise fashion to make the larger oligosaccharides. To map the importance of the nature of the terminal monosaccharide, we synthesized a series of spacer-linked oligosaccharides ranging from hexa- to trideca-saccharides, and covalently linked them to bovine serum albumin. The repeating unit is made up of four monosaccharides, so four isomeric structures can exist for any given chain length. In order to probe this variable, we synthesized two additional, frame-shifted decasaccharides. All of the synthetic oligosaccharides inhibited binding of anti-S. dysenteriae antibodies with the homologous lipopolysaccharide. The immunogenicity of their protein conjugates will be evaluated. Currently we have approx. 1.3 g of each of the spacer-linked dodeca- and hexadeca-saccharides for clinical evaluation. We have developed a new, efficient, and mild protocol for the coupling of oligosaccharides to proteins. We have introduced aminooxy groups in the protein through thioether linkages using an aminooxy-thiol linker. Condensation of aminooxylated protein and aldehydo/keto-derivatized carbohydrates created covalent saccharide-protein constructs. In this method the uncoupled saccharide could be recovered in its original, reactive form. We have demonstrated the scope and utility of our method by coupling a number of neutral and charged oligosaccharides to proteins. BORRELIA BURGDORFERI: SYNTHETIC VACCINE AGAINST THE ETIOLOGICAL AGENT OF LYME DISEASE; Cholesteryl 6-O-palmitoyl/oleoyl-beta-D-galactopyranosides are major glycolipids in Borrelia burgdorferi. Antibodies to these glycolipids, assumed to be functional equivalents of a lipopolysaccharide, might offer protection against this bacterium, so a project was initiated to synthesize the glycolipids and their protein conjugates in quantities and purity suitable for immunization experiments. We have optimized synthetic routes to 6-O-palmitoyl-galactosyl-cholesterol and the corresponding 6-O-oleoyl derivative, in a reproducible and reliable manner. To enhance the immunogenicity of these glycolipids, their covalent attachment to proteins is planned. We assume that conjugation to a protein would be most suitable by extending the methyl terminus of the palmitoyl moiety. We have prepared 15-azido-palmitic acid, which was transformed to the corresponding amino acid, and will function both as the fatty acid at O-6 of the galactose moiety and a linker to proteins either directly or through a secondary spacer moiety. We are converting this amino acid to a keto derivative that will allow conjugation to a protein using our above-described procedure. SYNTHETIC VACCINE BASED ON OLIGOMERS OF RIBITOL-PHOSPHATE: Polymers of D-ribitol-phosphate are major cell-wall components of a variety of pathogenic bacteria including Staphylococcus aureus. To test the hypothesis that antibodies against this substructure might offer protection against several bacteria, we initiated a project to synthesize a range of ribitol-phosphate oligomers suitable for covalent attachment to proteins. We optimized the synthesis of a building unit suitable for polymerization and, using this unit, have synthesized an octamer and a dodecamer of ribitol phosphate in spacer-linked form, using solution-phase chemistry. We covalently conjugated the oligomers to bovine serum albumin and tetanus toxoid, using our oxime-methodology. The ribitol phosphate-protein conjugates were precipitated by Haemophilus influenzae type b antibodies. Immunogenicity studies of these conjugates in mice are planned to explore cross-reactivity of anti-ribitol-phosphate antibodies.