VACCINES BASED ON SYNTHETIC GLYCOCONJUGATES? ? ? Our studies are aimed at designing and developing bacterial vaccines. Human bacterial pathogens may have surface exposedsaccharides serving as virulence factors and as protective antigens. such saccharides include capsular polysaccharides (CPS), lipopolysaccharides (LPS) and cell wall polysaccharides and may vary in size and complexity. We are studying synthetic chemical approaches to bacterial surface-exposed saccharide structures that can be used to elicit serum IgG antibodies against the organisms. ? ? ? Pozsgay, Robbins, Ekborg, Kubler-Kielb, Nelson, Schneerson? ? SYNTHETIC VACCINE AGAINST SHIGELLA DYSENTERIAE TYPE 1? Shigella dysenteriae type 1, a Gram-negative human pathogen,causes dysentery and diarrhea in endemic and epidemic forms in developing countries. The lack of a licensed vaccine against this organism and the emergence of strains that are increasingly resistant to available antibiotics call for the development of a vaccine against S. dysenteriae type 1. Our approach is based on immunogens that induce serum IgG antibodies to the O-specific polysaccharide (O-SP) of the LPS. Such an immunogen was shown to be protective against S. sonnei dysentery in adults. The O-SP of S.dysenteriae type 1 cosists of approximately one hundred monosaccharide residues. The repeating unit of the O-SP is a tetrasaccharide that contains two L-rhamnose, one N-acetyl-D-glucosamine, and one D-galactose residue. We have demonstrated that an oligosaccharide as small as an octasaccharide made up of two contiguous Rha-Gal-GlcNAc-Rha sequences can elicit O-SP-specific IgG antibodies in mice when covalently linked to an immunogenic protein. The highest immune responses were obtained with conjugates containing an average of 10-12 copies of dodeca- and hexadeca-saccharides having the above given sequence, per human serum albumin used as a carrier protein. We are also studying the importance of the non-reducing terminal monosaccharide residue in eliciting an O-SP immune response in mice. To address this question we have synthesized spacer-linked hexa- to pentadecasaccharides, corresponding to the sequence given above, exposing each of the component monosaccharides at the non-reducing end of the saccharide constructs. All the synthetic oligosaccharides inhibited 20-25% binding of the LPS to polyclonal IgG mouse anti O-Sp, i.e. neither the saccharide chain length nor the identity of the terminal monosaccharide residue had a significant role in inhibition. However as immunogens, conjugates of the oligosaccharides with bovine serum albumin elicited O-SP serum antibodies in mice in a structure-dependent fashion: the constructs having either N-acetyl-D-glucosamine or D-galactose at their non-reducing end elicited approximately ten times higher IgG levels than those terminating in either of the two rhamnose residues. To further explore the effect of the terminal monosaccharide on O-SP immunogenicity, oligosaccharies of equal length but different terminal sugarswere synthetized and conjugated to the recombinant exotoxin A of Pseudomonas aeruginosa. ? ? SYNTHETIC VACCINE AGAINST BORRELIA BURGDORFEI, THE ETIOLOGICAL AGENT OF LYME DISEASE? B. burgdorferi expresses a unique glycolipid on its surface composed of palmitoyl-galactosyl-cholesterol. Because of its surface location on the bacterium, this unusual glycolipid may be a candidate for vaccine development. Its chemical synthesis has been reported. Now we have studied chemical synthesis of this glycolipid in a form that allows its covalent attachment to proteins. We utilized the previously reported galactosyl cholesterol that was chemically modified allowing the attachment of palmitic acid moiety at position O-6 of the galactose unit. This moiety also featured a latent aldehydo group at its end. After removal of all the protecting groups, the aldehydo-modified glycolipid was coovalently attached to aminooxypropylated BSA, using the reverse micelle technique, to yield a water-soluble four-domain glycolipoprotein incorporating an average of 10 glycolipid chains per BSA molecule. Immunogenicity of this conjugate is being evaluated in mice. ? ? SYNTHETIC VACCINE BASED ON OLIGOMERS OF RIBITOL-PHOSPHATE? Polymers of ribitol phosphate have been found in several human pathogens as part of their capsular and cell wall structures. Vaccines based on the CPS of Haemophilus influenzae type b composed of ribose-ribitol-phosphate have been highly successful. We have synthesized octa- and dodecamers of ribitol phosphate and have covalently attached them to BSA at various saccharide-protein ratios. Immunogenicity and cross-reactivity of these conjugates are being evaluated.
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