Our long-term goal is to understand the pathogenesis of bacterial otitis media and to develop a multivalent vaccine that reduces the incidence of otitis media caused by all major bacterial pathogens in children. ? ? Lipooligosaccharide (LOS), a major bacterial component of the outer membrane of Moraxella catarrhalis, is a possible virulence factor in the pathogenesis of otitis meda caused by the organism. To gain an insight into the biological activities of the LOS molecule of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35Elgt3 LOS mutant. MALDI-TOF MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo2 (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, while the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity. ? ? In addition, we have used the bacterial surface antigen, LOS, as a component to develop new conjugate vaccines against serotype C M. catarrhalis. Previous studies suggest that only three major serotypes (A, B, and C) have been identified among clinical isolates. Our former laboratory data revealed that serotype A and B LOS-based conjugate vaccines were safe and immunogenic in animal models. Our current testing in vitro as well as in animal experiments with the newly developed serotype C LOS based conjugate vaccines for M. catarrhalis demonstrated that the vaccines were safe and effective to elicit an immune response that inhibited bacterial growth. All conjugates were highly immunogenic in both mouse and rabbit models. Three subcutaneous injections with each conjugate elicited >700-fold rise of serum anti-LOS IgG in mice, and >2,000-fold rise in rabbits. The resulting mouse and rabbit anti-serum showed complement-mediated bactericidal activity against the homologous strain. In addition, a representative rabbit anti-serum showed bactericidal activity against 14 of 18 testable strains, and such bactericidal activity could be 100% inhibited by the serotype C or A LOS but only 30% inhibited by the serotype B LOS. These studies are significant advances towards future clinical trials to test these candidate vaccines for safety and efficacy in humans. ? ? In an effort to identify the epitope/epitopes on the LOS molecule recognized by a rabbit bactericidal anti-LOS antiserum elicited by the above serotype C conjugate vaccine, a mutant that showed no binding activity to the functional rabbit serum, was created from M. catarrhalis strain 26404 by random transposon mutagenesis. The mutant was identified as an lgt2 gene inactivated strain by sequence analysis, which encodes β(1-4) galactosyltransferase. An isogenic mutant 26404lgt2 was then constructed. A mass spectrometric analysis indicated that the mutant produced a truncated LOS structure lacking terminal galactose residues from both 4-link and 6-link oligosaccharide chains of the wild type strain. Both parental and mutant strains or their purified LOSs were tested with the recognition by the anti-LOS rabbit serum in varies assays. The data showed that the mutant or its LOS lost or significantly reduced the binding activity in Western blotting，ELISA, or flow cytometry assay, compared with the parental strain or its LOS. In the bactericidal assay, the anti-LOS rabbit serum lost the activity to the mutant, whereas it showed a titer of 1:80 to the wild type strain. These studies suggest that the terminal galactose residues on the LOS of M. catarrhalis strain 26404 form the functional epitope recognized by the bactericidal rabbit anti-LOS serum elicited by the conjugate vaccine.
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