Synthetic Vaccine Against Human Pathogenic Bacteria

Child Health And Human Development

Investigators

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Abstract

Shigella dysenteriae type 1 and Shigella sonnei Shigella dysenteriae type 1 and Shigella sonnei are Gram-negative human pathogens that cause endemic and epidemic dysentery, an acute inflammation of the lower intestines, worldwide. In spite of their discoveries many years ago, there are still no licensed vaccine against these bacteria that have developed resistance to most available antibiotics. Our approach to vaccine development against these bacteria is based on the demonstration that serum antibodies the O-specific polysaccharides (OSPs) of Gram-negative bacteria are important for host protection. While OSPs are non-immunogenic presumably because of their low molecular weight, covalent conjugates of an immunogenic protein and the O-specific polysaccharide of S. dysenteriae type 1 and Shigella sonnei have been shown to elicit significant anti-OSP antibody levels of IgG isotype that may be boosted by repeated injections. We surmised that an improved vaccine might be constructed from chemically defined oligosaccharide fragments of the OSPs that are devoid of biological contamination, have a uniform molecular weight and can be characterized by physico-chemical methods. We have chemically synthesized oligosaccharide fragments of the native OSP of Shigella dysenteriae type 1 up to a tetracosasaccharide consisting of 24 monosaccharide residues. These constructs are attached to a spacer that makes bioconjugation possible through the termini of the spacer moieties (i. e. at a single site). We have been developing a new technique for chemical synthesis of extended oligosaccharides that circumvents the difficulties associated with the conventional, liquid-phase coupling of oligosaccharide building blocks and also avoids the problems inherent in the solid-phase approaches. Briefly, our method employs lipophilic protecting groups in one of the blocks while uses conventional protecting groups in the others. The presence of the lipophilic groups in the targeted intermediate but not in the side-products allows the isolation of the products by the solid-phase extraction technique that uses recyclable chromatographic materials and environmentally friendly, alcoholic solvents. We have designed and synthesized several new protecting groups including 4-dodecyloxybenzyl, 4-dodecylbenzyl, and 4-octadecylbenzyl moieties and have demonstrated their utility in oligosaccharide synthesis. We are currently using our new technology for the synthesis of the oligosaccharides related to the OSP of Shigella dysenteriae type 1 under clean laboratory conditions for Phase I clinical trials. So far we have demonstrated that the immunogenicity of the synthetic saccharide-human serum albumin conjugates depends on the saccharide chain length and the saccharide density on the protein. Current work is directed to determine the role, if any, of the monosaccharide terminating the oligosaccharide chain. To this end we have synthesized oligosaccharide fragments of the O-SP that differ in the terminal unit, i. e. in the repeating unit frame. We designed experiments towards a polysaccharide mimic that might be more immunogenic when conjugated to proteins than the saccharides synthesized so far. Briefly, we designed linearly arranged clusters of the synthetic oligosaccharides that are interconnected through pre-defined sites by a spacer. We demonstrated the feasibility of this approach by interconnecting six monosaccharide units. Next, we extended this approach to a synthetic octasaccharide. So far we succeded to interconnect three such units leading to a polymer containing 24 monosaccharide residues. Current experiments are directed at synthesizing even longer constructs as their protein conjugates for immunological experiments. A new technique was developed for reagentless attachment of oligosaccharides to proteins. In short, the technique is based on the Diels-Alder cycloaddition between an activated double bond and a conjugated diene. The activated double bond component was introduced in the protein using the commercial reagent 3-sulfosuccinimidyl 4-maleimidobutyrate. The carbohydrate sector was equipped with various linkers containing a 1,3 conjugated diene system. When solutions of the functionalized components were combined, conjugation took place at room temperature. pH has a marked effect on the efficiency of the coupling: lower pH (e. g. pH 5.7) favors conjugation relative to higher pH (e. g. 9.5). The conjugation was as effective in water as in pH 5.7 buffer. On the other hand, the structure of the diene system has little effect on the conjugation reaction. Unreacted saccharide can be recovered with its linker suitable for reuse in a subsequent conjugation experiment. Group A Neisseria meningitidis Group A Neisseria meningitidis causes endemic and epidemic meningococcal meningitis worldwide. Although a vaccine containing the purified capsular polysaccharide (CPS) of this bacterium has been available for years, it is not sufficiently immunogenic in infants that are at the highest risk. Our program to develop a more efficient vaccine is based on the assumption that protein conjugates of relatively short fragments of the CPS of N. meningitidis will be more immunogenic in infants than the currently available vaccine. The CPS of Group A N. meningitidis consists of alpha-linked N-acetyl-mannosaminyl residues that are interconnected by an anomerically located phosphodiester linkage. The native polysaccharide is non-stoichiometrically O-acetylated at the O-3 position. In our first approach to study the requirements of synthetic oligosaccharides related to this polymer, we targeted the synthesis of non-O-acetylated congeners. We have designed a route to the mannosaminyl H-phosphonate unit. Iterative condensation of this unit allowed the synthesis of a dimer and trimer of the polysaccharide's repeating unit in bioconjugatable form. Also synthesized were the mannosaminyl spacer and its phosphate ester. Covalent attachment of the synthetic subunits to human serum albumin using our conjugation technology afforded glycoconjugates containing up to 30 saccharide units per molecule of HSA. The antigenicity of these conjugates were demonstrated by the precipitin formation with the serum of a horse that was immunized with formalin-killed Group A Neisseria meningitidis. These experiments showed that the serum recognizes as small a component of the polysaccharide as a monosaccharide. Second, the formation of precipitation also indicates that the presence of O-acetyl groups in the polymer is not essential for antigenicity. Current work is directed towards the synthesis of higher oligomers with and without O-acetyl groups.