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J. Biol. Chem., Vol. 278, Issue 28, 25618-25627, July 11, 2003

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Identification of a Cross-reactive Epitope Widely Present in Lipopolysaccharide from Enterobacteria and Recognized by the Cross-protective Monoclonal Antibody WN1 222-5^*

Sven Müller-Loennies   ¶, Lore Brade  , C. Roger MacKenzie ||, Franco E. Di Padova ** and Helmut Brade 

From the Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany, the ^||Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada, and ^**Novartis Pharma AG, CH-4002 Basel, Switzerland

Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R₁--D-Glcp-(13)-[L--D-Hepp-(17)]-L--D-Hepp 4P-(13)-R₂ (where R₁ represents additional sugars of the outer core and R₂ represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (K_D values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (K_D values) determined by SPR were in the range of 3.6 x 10^–5 to 3.2 x 10^–8 M, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest K_D values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5–10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding.

Received for publication, March 21, 2003 , and in revised form, April 23, 2003.

^* This work was supported by Deutsche Forschungsgesellschaft Grant SFB 470, C1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^¶ To whom correspondence should be addressed: Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany. Tel.: 49-4537-188 (ext. 467); Fax: 49-4537-188 (ext. 419); E-mail: sml{at}fz-borstel.de.

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