Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPS) or the O-specific polysaccharide (O-SP) of lipopolysaccharides (LPS), serve both as essential virulence factors and as protective antigens. Covalent binding of these saccharides to medically-useful proteins to form conjugates both increases their immunogenicity and confers to them T-cell dependence making them suitable vaccines for infants and children. The O-SP of Shigella sonnei bound to recombinant non toxic P. aeruginosa exoprotein A (rEPA) had an efficacy of over 70% in young adults exposed to 6-14% attack rates. This conjugate and of S. flexneri 2a bound to the succinylated exoprotein A (rEPA-succ) were safe and induced IgG antibodies to the homologous LPS in 1-4 year-olds. A randomized, blinded, phase 3 study of these conjugates in 1-4 year-olds with each conjugate serving as a control for the other showed these vaccines to be safe. Immunogenicity and efficacy were age related with little efficacy in 1-2 years old, about 70% in 3-4 years old. Fold increases in antibody levels were, as before, similar to those of adults but the actual levels achieved were lower.? ? The effect of these children's immune sera and IgG isolated from them upon Shigella invasion into epithelial intestinal cells was studied in vitro using Caco-2 and HeLa cells. The sera inhibited Shigella invasion in a type specific manner. Pretreatment of the sera or of Caco-2 cells with O-SP abrogated these effects in a type specific and dose dependent manner.? ? To enhance the immunogenicity of these conjugates S. sonnei and S. flexneri 2a O-SPs were bound to additional carrier proteins: tetanus toxoid (TT) and recombinant protective antigen (rPA). Mice were injected twice, with the same or a different carrier the second time. For S. flexneri 2a the TT conjugate injected twice induced the highest (>4-fold) antibody levels. For S. sonnei, the TT conjugate followed by the rPA conjugate induced significantly higher levels than the other combinations. ? ? We have shown that synthetic oligosaccharides of S. dysenteriae type 1 O-SP bound by their reducing end to a carrier protein (sun configuration) induced significantly higher antibody levels than conjugates of the native O-SP bound to protein by multiple point attachments (lattice configuration). Synthesis of S. sonnei oligosaccharides was not successful. Therefore, the low molecular mass O-SP-core (O-SPC) fragments of the native O-SP were isolated and used to bind to proteins by oxime linkages between the terminal Kdo residues of the reducing end core and aminoxy linkers bound to BSA or to a non toxic diphtheria toxin mutant. The coupling reaction was done at a neutral pH, room temperature and in a short time. IgG anti S. sonnei LPS levels induced by these conjugates in young outbred mice were significantly higher than those induced by the full length O-SP conjugates.? ? The O-SP of Plesiomonas shigelloides O17 is structurally identical to that of S. sonnei but the core structure of S. sonnei only is published. We investigated the core structure of P. shigelloides O17, including its linkage to the O-SP by NMR and mass spectroscopy. The structure was assigned.
| 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 |
| Kubler-Kielb, Joanna; Vinogradov, Evgeny; Chu, Chiayung et al. (2007) O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a. Carbohydr Res 342:643-7 |
| Pozsgay, Vince; Kubler-Kielb, Joanna; Schneerson, Rachel et al. (2007) Effect of the nonreducing end of Shigella dysenteriae type 1 O-specific oligosaccharides on their immunogenicity as conjugates in mice. Proc Natl Acad Sci U S A 104:14478-82 |
