Vaccines Based on Synthetic Glycoconjugates:? Our studies are aimed at developing bacterial vaccines. Human bacterial pathogens may have surface exposed saccharides serving as virulence factors and as protective antigens. Such saccharides include capsular polysaccharides, lipopolysaccharides (LPS) and cell wall polysaccharides and may vary in size and complexity. We are studying synthetic chemical approaches to bacterial surface-exposed saccharides that can be used to elicit serum IgG antibodies against these organisms.? ? SYNTHETIC GLYCOCONJUGATES AS EXPERIMENTAL VACCINES? Shigella dysenteriae type 1 is the causative organism of endemic and epidemic dysentery in many parts of the world. Despite its discovery more than a hundred years ago, there is no licensed vaccine against this organism. S. dysenteriae type 1 acquired resistance to most available antibiotics. Evidence indicates that antibodies to the O-Specific polysaccharide (O-SP) of the LPS of this bacterium may be protective. The O-SP consists of a tetrasaccharide repeating unit containing L-rhamnose, D-galactose, and N-acetyl-D-glucosamine units having the →3)-α-L-Rhap-(1→2)-α-D-Galp-(1→3)-α-D-GlNAcp-(1→3)-α-L-Rhap-(1→ linear tetrasaccharide structure. We have shown that covalent conjugates of bovine serum albumin to oligosaccharides corresponding to the O-SP in the 8-16-mer range elicit O-SP-specific antibodies in mice. The highest levels were achieved when the non-reducing terminus was occupied either by a D-galactose or by an N-acetyl-D-glucosamine residue whereas those oligosaccharides that contained an L-rhamnose residue at that terminus produced low levels of anti-saccharide antibodies. With this finding, we have redesigned our previous synthetic sequence and prepared a decamer and an undecamer containing the GlcNAc-Rha-Rha-Gal-GlcNAc-Rha-Rha-Gal-GlcNAc-Rha and the Gal-GlcNAc-Rha-Rha-Gal-GlcNAc-Rha-Rha-Gal-GlcNAc-Rha structures, in amounts exceeding 300 mg. These oligosaccharides contain a linker at their reducing end for covalent attachment to a medically acceptable protein using our oxime-based conjugation method. Evaluation of the conjugate-induced anti-O-SP antibody levels in volunteers is planned. ? It has been proposed that Shigellae are descendants of E. coli and that E. coli O-148 is the precursor to S. dysenteriae type 1. Similarly to S. dysenteriae type 1, E. coli O-148 is a cause of diarrhea in children, amongst soldiers and in travelers. The repeating unit of the O-SP in E. coli O-148 is almost identical to that of S. dysenteriae type 1, the only difference being that the galactose residue in the latter is replaced by D-glucose. To establish cross-reactivity between these two bacteria, we have synthesized chemically oligosaccharide fragments of the O-SP of E. coli 148. These probes are composed of one, two, and three consecutive repeating units containing 4, 8, and 12 monosaccharide residues. The saccharide constructs also contain a spacer equipped with a keto moiety. Using our oxime-based conjugation technique, we have prepared human serum albumin conjugates containing up to an average of 28 oligosaccharide chains per protein molecule. We will investigate the immunogenicity of these conjugates to study cross-reactivity with the S. dysenteriae type 1 O-SP.? ? SYNTHETIC VACCINE AGAINST BORRELIA BURGDORFERI, THE ETIOLOGIAL AGENT OF LYME DISEASE? BORRELIA BURGDORFERI expresses two major glycolipids on its surface termed BBGL-1 and 2. Both constructs contain a galactose residue, in addition to palmitoyl and oleoyl moieties. BBGL-1 also has a cholesterol unit whereas BBGL-2 has a glycerol residue acylated by the palmitic and oleoic acids. The location of these glycolipids on the bacterial surface makes them a target for vaccine development. We have synthezised chemically BBGL-1 both in its native and in bioconjugatable forms. The native BBGL-1 or its protein conjugate elicited low amounts of anti-BBGL-1 antibodies in mice. As a continuation of this project, we are examining the immunogenicity of BBGL-2. First, we embarked on its chemical synthesis. Starting from a commercially available optically active glycerol derivative, we attached a fully protected galactose residue stereospecifically to the free hydroxyl of the glycerol moiety. Next, the original protecting groups on the glycerol were removed. The diol so obtained was regioselectively acylated with palmitic acid followed by the placement of the oleoyl moiety at the secondary hydroxyl group of the glycerol residue. Chemoselective removal of the protecting groups on the galactose moiety yielded BBGL-2. We will adapt this methodology for the synthesis of a conjugatable derivative of BBGL-2 which will be linked to a protein to evaluate the feasibility to enhance immunogenicity by conjugation to an immunogenic protein.
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