Binding specificity for four monoclonal antibodies recognizing terminal Galα1 4Gal residues in Haemophilus influenzae lipopolysaccharide

Abstract

Four murine monoclonal antibodies (MAbs) reactive with the outer-core region of the lipopolysaccharide (LPS) from Haemophilus influenzaewere generated after immunization with azide-killed H. influenzaeRM.7004 AH1-2 and their epitope specificities studied. The monoclonal antibodies: MAHI 6 (IgM), MAHI 5 (IgG2a), MAHI 8 (IgG3), and MAHI 11 (IgG2b) bound to synthetic glycoconjugates or glycolipids with terminal galabiosyl (Galα1 4Galβ1-) or globotriaosyl (Galα1 4Galβ1 1 4GLc) residues as evaluated in enzyme immunoassays (EIA). Glycoconjugates or glycolipids with internally placed galabiose elements were not active, indicating selectively of the MAbs for recognition of the epitope. Nine LPSs from H. influenzaeinhibited the binding of the four MAbs. The presence of the galabiosyl disaccharide element in these nine LPSs was evidenced by the binding of 125I-labeled Shiga toxin isolated from the bacterium Shigella dysenteriaetype 1, reported to have as receptor the Galα1 4Galβ disaccharide (Lindberg et al., J Biol Chem, 1987, 262: 1779–85). Structural studies of these H. influenzaeLPSs were also in accord with the presence of the galabiosyl disaccharide, in addition 1H-NMR spectroscopy showed the presence of O-acetyl groups in the RM.7004 AH1-2 LPS. However, differential binding specificities of the MAbs to modified RM.7004 AH1-2 LPSs were observed. MAHI 6 and MAHI 11 bound equally well to LPS, polysaccharides obtained after mild acidic treatment, and dephosphorylated LPS samples as shown in inhibition EIA. In contrast, both dephosphorylated LPS samples and polysaccharides were poorer inhibitors of the binding of MAHI 5 fu1 and MAHI 8 to native RM.7004 AH1-2 LPS. Neither the de-O-acylated nor the de-O,N-acylated LPSs were effective inhibitors of any of the four MAbs. These results suggest that the MAbs recognition involves Galα1 4Gal and O-acetyl and other saccharide residue(s) from the oligosaccharide moiety of the LPS. The epitopes are also expressed and accessible to recognition in clinical isolates coming from different sources of Neisseriaspp., Haemophilusspp., and Moraxella catarrhalis, but not in Bordetellaspp., Aeromonasspp. or Enterobacteriaceaeas evaluated by whole-bacteria EIA and colony-dot-immunoblotting.