During persistence of nonencapsulated Haemophilus influenzae in the respiratory tracts of patients with chronic bronchitis, the major outer membrane proteins (MOMPs) P2 and P5 show antigenic drift. The hypothesis that appearance of antigenic variants is the consequence of antibody-dependent selection was tested in a rabbit model. Persistence of H. influenzae d1 was achieved in subcutaneous tissue cages for up to 948 days. During persistence in the rabbits, similar changes in MOMP P2 of H. influenzae occurred, as observed in isolates from chronic bronchitis patients. In rabbits vaccinated with strain d3 and in nonvaccinated rabbits, antigenic drift occurred later than in rabbits vaccinated with strain d1. High titers of antibodies against H. influenzae were measured in tissue cage fluid and serum. Vaccination of the rabbits with H. influenzae d1 or d3, an antigenic variant of strain d1, resulted neither in eradication of H. influenzae d1 nor in increased antibody titers in serum and tissue cage fluid. The sera of nonvaccinated rabbits during persistence had no strain d1-specific bactericidal activity in the presence of complement. Vaccination with H. influenzae d1 induced serum bactericidal activity against strain d1 in the presence of complement. However, a variant of strain d1 appearing in the tissue cages was not killed by this serum bactericidal activity. We conclude that immunological pressure leads to the selection of MOMP variants of H. influenzae and that these variants escape the antibody-mediated strain-specific bactericidal activity against H. influenzae.

The occurrence of fimbria gene clusters in nonencapsulatedHaemophilus influenzae strains from chronic bronchitis patients (n = 58), patients with acute otitis media (n = 13), and healthy carriers (n = 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate H. influenzae type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting ofhifA to hifE inserted in the chromosome betweenpurE and pepN, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (n = 2), leading to optimal positioning between the −10 and −35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity.hifA appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between hifA andpurE has not been found in H. influenzae type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genespurE and pepN suggest that the fimbria gene cluster was originally contained on a mobile element.

Since nonencapsulated Haemophilus influenzae persists in the lower respiratory tracts of patients with chronic bronchitis despite the presence of specific antibodies, complement, and polymorphonuclear leukocytes (PMNs), opsonophagocytosis of H. influenzae was analyzed. Nonencapsulated H. influenzae isolated from the sputa of chronic bronchitis patients was labeled with fluorescein isothiocyanate and incubated with human PMNs in the presence of complement and antibodies for 30 min at 37 degrees C. Candida albicans was added to each sample as an internal standard, and the reduction of the number of bacteria was determined by flow cytometry. Fluorescence quenching with ethidium bromide was used to discriminate between intracellular and extracellular bacteria. Opsonophagocytosis of viable H. influenzae d1 was 17% +/- 29% in the presence of complement and human pooled sera containing high titers of strain-specific antibodies. Opsonophagocytosis of six other H. influenzae strains was also poor. Under the same conditions, opsonophagocytosis of Staphylococcus aureus was 90% +/- 5%, and opsonophagocytosis of C. albicans was 55% +/- 23%. About half of the number of H. influenzae bacteria associated with PMNs was internalized. Opsonophagocytosis of heat-killed H. influenzae d1 (41% +/- 20%) was higher than that of viable bacteria of the same strain (P < 0.05). This result suggests that the accessibility of epitopes on H. influenzae for opsonizing antibodies is better on killed than on viable bacteria. We conclude that viable nonencapsulated H. influenzae is poorly opsonophagocytized in the presence of strain-specific antibodies and complement.

Since the ability of bacteria to compete with lactoferrin for iron contributes to the pathogenesis of mucosal infections, the presence of lactoferrin receptor activity in non-encapsulated Haemophilus influenzae was investigated. The growth of 18 H. influenzae isolates from the sputum samples of chronic bronchitis patients and of six of seven H. influenzae throat isolates from healthy adults was stimulated by iron saturated human lactoferrin. Apo-lactoferrin did not stimulate the growth of H. influenzae. Human lactoferrin binding to iron limited bacteria was detected for 16 H. influenzae strains from chronic bronchitis patients and for five of seven isolates from healthy adults. We conclude that the majority of H. influenzae isolates tested bind human lactoferrin and that the iron from lactoferrin is used for growth.

Airway inflammation during infection is associated with increased transudation of serum proteins and increased production of protein by the airway epithelium. We therefore, assessed whether Haemophilus influenzae infections in patients with chronic bronchitis are associated with increased levels of transferrin and lactoferrin in the sputum compared to uninfected patients.Sputum sol phase and serum samples from 14 infected and 13 uninfected patients with chronic bronchitis and from 12 bronchial asthma patients were included in the study.The median Q-values (the concentration in sputum sol phase/the concentration in serum) × 10 3 of transferrin appeared increased in chronic bronchitis patients with an H. influenzae infection (26.0, n=13) compared to uninfected controls (9.5, n=11) and bronchial asthma patients (4.5, n=6). The ratio of the Q(transferrin)/Q(albumin) was >1 in infected chronic bronchitis patients, indicating local production of transferrin. Growth of H. influenzae was stimulated more in sputum from infected and uninfected patients with chronic bronchitis than in sputum from patients with bronchial asthma. The concentrations of lactoferrin were not significantly different in infected (n=14) and uninfected (n=13) chronic bronchitis patients and bronchial asthma patients (n=12) (median 137.4, 84.6, 87.1 mg·L -1 , respectively).We conclude that in patients with chronic bronchitis with Haemophilus influenzae infections, the levels of transferrin are increased and the levels of lactoferrin are not associated with infections.