Surface polysaccharides of Gram-negative enteric pathogens in the form of capsule or lipopolysaccharide function as both essential virulence factors and protective antigens. Serum IgG antibodies to these surface polysaccharides confer immunity by killing or inactivating the inoculum of the pathogen. We enhanced the immunogenicity of the polysaccharides by binding them to carrier proteins.
Synthetic approach to developing improved conjugate vaccines
Pozsgay, Robbins, Ekborg, Nelson, Fekete, Kubler-Kielb; in collaboration with Schneerson
SYNTHETIC VACCINES AGAINST SHIGELLA DYSENTERIA 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 against most available antibiotics. While O-specific polysaccharides (O-SPs) are nonimmunogenic, we have shown that covalent conjugates of an immunogenic protein and the O-SP of S. dysenteriae type 1 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, which are 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 conjugated with a protein and administered without an adjuvant. Most of our efforts during the past year focused on the chemical synthesis of octa-, dodeca-, and hexadecasaccharides in sufficient quantities under clean-laboratory conditions. In our earlier studies, we synthesized a linear tetrasaccharide corresponding to a repeating unit of the O-SP of the lipopolysaccharide. We oligomerized the unit 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 tridecasaccharides and covalently linked them to bovine serum albumin. The repeating unit consists of four monosaccharides so that four isomeric structures can exist for any given chain length. To identify the relevant structure, we synthesized two additional frame-shifted decasaccharides. All the synthetic oligosaccharides inhibited binding of anti–S. dysenteriae antibodies with the homologous lipopolysaccharide. We plan to evaluate the immunogenicity of the oligosaccharides’ protein conjugates. Currently, we have approximately 1.3 g of each of the spacer-linked dodeca- and hexadecasaccharides for such an evaluation.
We have developed a new, efficient, and mild protocol for the coupling of oligosaccharides to proteins. We have introduced amino-oxy groups into the protein through thioether linkages by using an amino-oxy-thiol linker. Condensation of amino-oxylated protein and aldehydo/keto-derivatized carbohydrates created covalent saccharide-protein constructs. With this method, we were able to recover the uncoupled saccharide in its original, reactive form. We have demonstrated the scope and utility of our method by coupling several neutral and charged oligosaccharides to proteins.
SYNTHETIC VACCINE AGAINST BORRELIA BURGDORFERI, THE ETIOLOGICAL AGENT OF LYME DISEASE. Cholesteryl 6-O-palmitoyl/oleoyl-beta-D-galactopyranosides are major glycolipids in Borrelia burgdorferi. Antibodies against these glycolipids, assumed to be functional equivalents of a lipopolysaccharide, might offer protection against this bacterium. We therefore initiated a project 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 the glycolipids, we plan to attach them covalently to proteins. 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 by using our procedure as described above.
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 the unit, 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 by using our oxime methodology. We precipitated the ribitol phosphate–protein conjugates by Haemophilus influenzae type b antibodies. To explore cross-reactivity of anti–ribitol-phosphate antibodies, we plan immunogenicity studies of these conjugates in mice.
Kubler-Kielb J, Pozsgay V. A new method for conjugation of carbohydrates to proteins using an aminooxy-thiol heterobifunctional linker. J Org Chem 2005;70:6987-6990.
Pozsgay V, Kubler-Kielb J, Coxon B, Ekborg G. Synthesis of two glycolipid antigens of the causative agent of Lyme disease. Tetrahedron 2005;61:10470-10481.
1Oak Ridge Senior Fellow Program at NIH
Collaborator
Rachel Schneerson, MD, Laboratory of Developmental and Molecular Immunity, NICHD, Bethesda, MD
For further information, contact robbinsj@nichd.nih.gov.