Lipooligosaccharides (LOS) isolated from Bordetella pertussis strains 186 and 606 were analyzed by
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and high-resolution magic angle spinning nuclear magnetic resonance (NMR). These analyses distinguished between the LOS of strains 186 and 606, suggesting that the structure of LOS in B.
pertussis is heterogeneous. The pentasaccharide was selectively cleaved from LOS of B.
pertussis strain 186, purified, and covalently linked to a monomer fraction of
tetanus toxoid. Injection of rabbits with the neoglycoconjugate emulsified in complete
Freund's adjuvant yielded
immunoglobulin G antibodies that were reactive with the LOS. These
antibodies reacted strongly with B.
pertussis LOS possessing the complete dodecasaccharide, as determined by an
enzyme-linked
immunosorbent assay, immunoblotting, and flow cytometry with intact, live bacterial cells. The binding
epitope within the pentasaccharide was investigated by saturation transfer difference (STD) NMR spectroscopy.
Protons H-1 and H-4 of the terminal alpha-D-GlcpNAc and
proton H-6 and
protons of an N-methyl group at H-4 of 3-substituted beta-L-FucpNAc4NMe exhibited the largest saturation transfers. STD NMR experiments confirmed that the
immunodominant epitope recognized by the antineoglycoconjugate
antibodies is located predominantly in the distal
trisaccharide of B.
pertussis 186 LOS. The antipentasaccharide
antibodies induced by the conjugate inhibited the secretion of
tumor necrosis factor alpha,
interleukin-6, and NO by LOS-stimulated J774A.1 cells.