Using innovative glycochemical technologies, we design and synthesize oligosaccharide- and glycolipid-based conjugate vaccines for the prevention of human diseases. We develop high-yielding, mild, and experimentally simple methods for covalent conjugation of glycans to proteins. Human bacterial pathogens may have surface-exposed saccharides that serve as both virulence factors and protective antigens. Such saccharides include CPs, LPSs, and cell-wall polysaccharides varying 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 such pathogens. Synthetic glycoconjugates as experimental vaccines. Shigella dysenteriae as acquired resistance to most available antibiotics. Evidence suggests that antibodies to the 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 with the 3)-alpha-L-RhaP-(1-2)-alpha-D-GalP-(1-3)-alpha-D-GlNAcP-(1-3)-alpha-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- to 16-mer range elicit O-SP specific antibodies in mice. The highest antibody levels were achieved when the non-reducing terminus was occupied by either a D-galactose or an N-acetyl-D-glucosamine residue, whereas 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 for clinical use. For use with our oxime-based conjugation method, these oligosaccharides contain a linker at their reducing end. It has been proposed that Shigellae are descendants of E. coli and that E. coli O-148 is the precursor of S. dysenteriae type 1. Similar to S. dysenteriae type 1, E. coli O-148 causes diarrhea in children, soldiers, and 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 is that D-glucose replaces the galactose residue in the latter. To establish cross-reactivity between the two bacteria, we synthesized chemically oligosaccharide fragments of the O-SP of E. coli 148. These probes are composed of 1, 2, and 3 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. The immunogenicity of these conjugates will be studied. Synthetic vaccine against Borrelia burgdorferi. Borrelia burgdorferi, the etiological agent of Lyme disease, expresses two major glycolipids on its surface, termed BBGL-1 and BBGL-2. Both 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 the two glycolipids on the bacterial surface makes them a suitable target for vaccine development. We have synthesized chemically BBGL-1 both in its native and conjugatable forms. Native BBGL-1 or its protein conjugate elicited low levels of antiCBBGL-1 antibodies in mice. We are now examining the immunogenicity of BBGL-2. First, we undertook 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, we removed the original protecting groups on the glycerol. We acylated the resulting diol regioselectively with palmitic acid followed by 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 we will link to a protein to evaluate its immunogenicity.

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Pozsgay, Vince; Kubler-Kielb, Joanna; Coxon, Bruce et al. (2012) Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens. J Org Chem 77:5922-41
Pozsgay, Vince; Kubler-Kielb, Joanna; Coxon, Bruce et al. (2011) Synthesis and antigenicity of BBGL-2 glycolipids of Borrelia burgdorferi, the causative agent of Lyme disease. Carbohydr Res 346:1551-63
Kubler-Kielb, Joanna; Vinogradov, Evgeny; Mocca, Christopher et al. (2010) Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates. Carbohydr Res 345:1600-8
Robbins, John B; Kubler-Kielb, Joanna; Vinogradov, Evguenii et al. (2009) Synthesis, characterization, and immunogenicity in mice of Shigella sonnei O-specific oligosaccharide-core-protein conjugates. Proc Natl Acad Sci U S A 106:7974-8