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

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 beta-configuration, while in all other RUs the GlcNAc was present in the alpha-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.

Figure 1

BioGel P-10 gel filtration profiles of 2% acetic acid hydrolyzed Shigellae LPSs. F1 – O-SP; more then 10 RU; F2, F3 – core + avr. 2–5 O-SP RU; F4 - core + avr. 1 O-SP RU.

Figure 2

Structures of Shigellae O-SPC fragments of core plus one O-SP repeat unit (RU).

Figure 3

Fragment of COSY (green-cyan) and TOCSY (red) spectra of the core-RU from the LPSs of S. flexneri O2a (top) and S. flexneri O6 (bottom), illustrating O-acetylation of the terminal rhamnose residues (T). Proton signals at the acetylation site are shifted to low field.

Figure 4

Integration of the proton signals of S.flexneri 2a 1 O-SPC fractions for the determination of the average number of O-SP repeat units (RU). Rha methyl signals in ¹H-NMR spectra (1.16–1.31 ppm) in the O-SP relative to the anomeric signals of the core α-Gal K (5.90 ppm) and α-Glc L (5.52 ppm) were used. F1-O-SP; F2-core-3RU; F3-core-2RU; F4-core-1RU; F5-core.

Figure 5

Integration of the anomeric signals of S. dysenteriae 1 O-SPC fractions for the determination of the average number of O-SP repeat units (RU). The anomeric signal of the terminal Rha T (5.08 ppm) relative to that of the substituted Rha T (5.11 ppm) was used. F1-O-SP; F2-core-3RU; F3-core-2RU; F4-core-1RU.