Monoclonal antibodies (MAbs) were elicited to the nontypeable Haemophilus influenzae variants dl to d4, which differ in the outer membrane protein P2 to analyze the immunological properties of the variable parts of this protein. Five MAbs reacted in a whole-cell enzymne-linked immunosorbent assay (ELISA) only with the homologous strain and in some cases with its variants, but not with 69 unrelated nonencapsulated H. influenzae isolates; nine MAbs also reacted with some other H. influenzae isolates, and four MAbs showed broad crossreactivity. All of the MAbs reacted with purified protein P2 in ELISAs and immunoblotting. The five MAbs which reacted with the homologous strain d3 and not with the variants dl, d2, and d4 promoted complementdependent bactericidal activity against strain d3. These and four other MAbs reacted with the intact bacteria of strain d3 in immunogold electron microscopy, indicating that they were directed against surface-exposed epitopes of outer membrane protein P2. A mutant of strain d3 was isolated as a survivor from bacterial killing by complement and MAb 30DA5. This mutant had an altered P2 protein on sodium dodecyl sulfatepolyacrylamide gels and had lost its reactivity with all of the five H. influenzae d3-specific MAbs but not with the other MAbs. From these results, we conclude that the variable parts of outer membrane protein P2 of nonencapsulated H. influenzae from the sputum of patients with chronic obstructive pulmonary disease are immunogenic and mostly surface exposed. Only strain-specific MAbs promoted complement-dependent killing

The role of fimbriae in the adherence of Haemophilus influenzae to oropharyngeal epithelial cells and the hemagglutination (HA) of human Anton-positive erythrocytes was examined. HA of bacteria was lost after shearing. Fimbriae purified from the extracellular fluid caused HA and bound to oropharyngeal epithelial cells, as analyzed with immunoperoxidase staining, in a way which was similar to the adherence of bacteria to these cells: binding was over the entire surface of the cells and showed cell-to-cell variation. The specific role of fimbriae in HA .nd adherence was further examined by inhibition experiments with inonoclonal antibodies elicited against the isolated fimbriae. These monoclonal antibodies bound along the entire length of the fimbriae, as seen by immunogold electron microscopy. The monoclonal antibodies and their Fab fragments inhibited HA (reduction in titer from 1:512 to 1:128 and 1:64, respectively) and inhibited the adherence of the homologous H. influenzae strain and of three of eight heterologous H. influenzae strains to oropharyngeal epithelial cells. These results indicate that fimbriae are involved in adherence and HA and that the binding site for the monoclonal antibodies on the fimbriae is not common on all strains.

The structure of the receptor for the fimbriae of Haemophilus influenzae on human oropharyngeal epithelial cells and erythrocytes was determined in inhibition experiments with various sugars, glycolipids, and glycoproteins. Of 30 monosaccharides and disaccharides at a concentration of 0.1 M and of 3 polysaccharides at a concentration of 1 mg/ml, none inhibited fimbria-specific adherence and hemagglutination. Inhibition was obtained with gangliosides GM1, GM2, GM3, and GDla in nanomolar concentrations, whereas the asialo derivative of GM1, sialyl-lactose, and sialoglycoproteins were poor inhibitors. These findings indicate that sialyl-lactosylceramide (GM3) is the minimal structure for the fimbria-dependent binding of H. influenzae to its receptor on oropharyngeal epithelial cells and erythrocytes. As is the case with GM2, substitution of GM3 with N-acetylgalactosamine makes the molecule a 10-fold-better receptor analog.

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Summary. The hypothesis was investigated that tissue tropism of Haemophilus inzuenzae during colonisation and infection is associated with the ability of fimbriate bacteria to bind to the organs and cell types involved. H . inzuenzae type b with fimbriae (strain 770235f') bound to several cell types, including ciliated columnar epithelial cells, pneumocytes, ependymal cells, glial cells, connective tissue fibroblasts, synovial cells, antigen-presenting cells, lymphocytes, erythrocytes and endothelial cells. Binding of H . inzuenzae to kidney, liver and conjunctiva cells was poor. Fimbriae-specific monoclonal antibody (MAb 6HE8) inhibited this binding. Some binding to endothelial cells and macrophages was also observed with non-fimbriate strains. This binding was not inhibited by MAb 6HE8. We conclude that in-vitro binding of fimbriate H . inzuenzae is mainly to those tissues and cells where H . inzuenzae is found during colonisation and infection. The data suggest that a shift to the nonfimbriate form is required for bacteria in the bloodstream to escape clearance mechanisms mediated by blood cells.