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Infection and Immunity, November 2005, p. 7381-7389, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7381-7389.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Epitope of the Vaccine-Type Bordetella pertussis Strain 186 Lipooligosaccharide and Antiendotoxin Activity of Antibodies Directed against the Terminal Pentasaccharide-Tetanus Toxoid Conjugate

Tomasz Niedziela,1,3* Iwona Letowska,2 Jolanta Lukasiewicz,1 Marta Kaszowska,1 Anna Czarnecka,1 Lennart Kenne,3 and Czeslaw Lugowski1

L. Hirszfeld Institute of Immunology and Experimental Therapy, R. Weigla 12, PL-53-114 Wroclaw, Poland,1 National Institute of Public Health, Chelmska 30/34, PL-00-725 Warsaw, Poland,2 Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden3

Received 25 May 2005/ Returned for modification 28 June 2005/ Accepted 22 July 2005

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 resonsnace (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 ß-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.

* Corresponding author. Mailing address: Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, PL-53-114 Wroclaw, Poland. Phone: 48 71 337 11 72. Fax: 48 71 337 13 82. E-mail: tomasz.niedziela{at}iitd.pan.wroc.pl.

{dagger} Supplemental material for this article may be found at http://iai.asm.org.

Editor: J. D. Clements

Infection and Immunity, November 2005, p. 7381-7389, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7381-7389.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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