Shigella sonnei oligosaccharide-protein conjugates

AbstractSynthetic oligosaccharides composed of several repeats of Shigella dysenteriae type 1 O-specific polysaccharide (O-SP), bound by their reducing ends to a carrier protein (“sun” type configuration), induced in mice significantly higher antibody levels than conjugates of the native O-SP (“lattice” type configuration). Here we present isolation and characterization of low molecular mass oligosaccharides of Shigella sonnei lipopolysaccharides and their conjugation to several carrier proteins. Conjugates were formed by oxime linkages between the terminal Kdo residues of the reducing ends of the saccharides and aminooxy linkers bound to the protein. IgG antibody levels induced in young outbread mice by these conjugates were significantly higher than those prepared with the full length native O-SP. We propose clinical evaluation of the new S. sonnei conjugates

Chemical Structure, Conjugation, and Cross-Reactivity of Bacillus pumilus Sh18 Cell Wall Polysaccharide

Bacillus pumilus strain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy-α-galactopyranose (13%) and 2-acetamido-2-deoxy-α-glucopyranose (6%) on position O-2, were found. A minor component was established to be a polymer of →3-O-(2-acetamido-2-deoxy-β-glucopyranosyl)-1→4-ribitol-1-OPO(3)→. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide (ADH)-derivatized protein, and (ii) binding of the carbodiimide-activated terminal phosphate group of the CWP to ADH-derivatized protein. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with the homologous polysaccharide and with a number of other bacterial polysaccharides containing ribitol and glycerol phosphates, including H. influenzae types a and b and strains of Staphylococcus aureus and Staphylococcus epidermidis

The structure of the Escherichia coli O148 lipopolysaccharide core region and its linkage to the O-specific polysaccahride.

Recently it was demonstrated that Shigella dysenteriae type 1, a cause of severe dysentery epidemics, gained its O-specific polysaccharide (O-SP) from Escherichia coli O148. The O-SPs of these bacteria differ only by a galactose residue in the repeat unit of S. dysenteriae type 1 in place of a glucose residue in E. coli O148. Herein, we analyzed the core structure and its linkage to the O-SP in E. coli O148 LPS. Both were found to be identical to those of S. dysenteriae type 1 structures, further supporting the relatedness of these two bacteria. The following structure of the core with one repeat unit of the O-SP has been assigned (all have D-configuration except L-Rha):Peer reviewed: YesNRC publication: Ye

Additional Conjugation Methods and Immunogenicity of Bacillus anthracis Poly-γ-d-Glutamic Acid-Protein Conjugates

The capsule of Bacillus anthracis, composed of poly-γ-d-glutamic acid (γDPGA), is an essential virulence factor of B. anthracis. The capsule inhibits innate host defense through its antiphagocytic action. γDPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic γDPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between γDPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal γDPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol γDPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 μg of γDPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-γDPGA levels