Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates |
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Authors: | Joanna Kubler-Kielb Evgeny Vinogradov Vince Pozsgay John B. Robbins |
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Affiliation: | a The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA b Institute for Biological Sciences, National Research Council, 100 Sussex Dr., Ottawa ON, Canada K1A 0R6 |
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Abstract: | There is no licensed vaccine for the prevention of shigellosis. Our approach to the development of a Shigella vaccines is based on inducing serum IgG antibodies to the O-specific polysaccharide (O-SP) domain of their lipopolysaccharides (LPS). We have shown that low molecular mass O-SP-core (O-SPC) fragments isolated from Shigella sonnei LPS conjugated to proteins induced significantly higher antibody levels in mice than the full length O-SP conjugates. This finding is now extended to the O-SPC of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1. The structures of O-SPC, containing core plus 1-4 O-SP repeat units (RUs), were analyzed by NMR and mass spectroscopy. The first RUs attached to the cores of S. flexneri 2a and 6 LPS were different from the following RUs in their O-acetylation and/or glucosylation. Conjugates of core plus more than 1 RU were necessary to induce LPS antibodies in mice. The resulting antibody levels were comparable to those induced by the full length O-SP conjugates. In S. dysenteriae type 1, the first RU was identical to the following RUs, with the exception that the GlcNAc was bound to the core in the β-configuration, while in all other RUs the GlcNAc was present in the α-configuration. In spite of this difference, conjugates of S. dysenteriae type 1 core with 1, 2, or 3 RUs induced LPS antibodies in mice with levels statistically higher than those of the full size O-SP conjugates. O-SPC conjugates are easy to prepare, characterize, and standardize, and their clinical evaluation is planned. |
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Keywords: | COSY, correlation spectroscopy TOCSY, total correlation spectroscopy NOESY, nuclear Overhauser enhancement spectroscopy HSQC, heteronuclear single quantum coherence HMBC, heteronuclear multiple bond connectivity LPS, lipopolysaccharide O-SP, O-specific polysaccharide O-SPC, low molecular mass O-SP-core fragments GlcN, glucosamine GlcNAc, N-acetylglucosamine Hep, smallcaps" >l-glycero- smallcaps" >d-manno-heptose Kdo, 3-deoxy- smallcaps" >d-manno-octulosonic acid RU, repeat unit of O-SP ES-MS, electrospray mass spectrometry GLC-MS, gas liquid chromatography mass spectrometry GM, geometric mean SBAP, N-succinimidoyl 3-(bromoacetamido) propionate TFA, trifluoroacetic acid PBS, phosphate buffer saline, pH 7.4 sc, subcutaneously EU, Elisa units GM, geometric mean |
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