Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in Chinchillas

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.     

Nontypeable Haemophilus influenzae initiates formation of neutrophil extracellular traps

Nontypeable Haemophilus influenzae (NTHI) is a leading cause of otitis media infections, which are often chronic and/or recurrent in nature. NTHI and other bacterial species persist in vivo within biofilms during otitis media and other persistent infections. These biofilms have a significant host component that includes neutrophil extracellular traps (NETs). These NETs do not mediate clearance of NTHI, which survives within NET structures by means of specific subpopulations of lipooligosaccharides on the bacterial surface that are determinants of biofilm formation in vitro. In this study, the ability of NTHI and NTHI components to initiate NET formation was examined using an in vitro model system. Both viable and nonviable NTHI strains were shown to promote NET formation, as did preparations of bacterial DNA, outer membrane proteins, and lipooligosaccharide (endotoxin). However, only endotoxin from a parental strain of NTHI exhibited equivalent potency in NET formation to that of NTHI. Additional studies showed that NTHI entrapped within NET structures is resistant to both extracellular killing within NETs and phagocytic killing by incoming neutrophils, due to oligosaccharide moieties within the lipooligosaccharides. Thus, we concluded that NTHI elicits NET formation by means of multiple pathogen-associated molecular patterns (most notably endotoxin) and is highly resistant to killing within NET structures. These data support the conclusion that, for NTHI, formation of NET structures may be a persistence determinant by providing a niche within the middle-ear chamber.     

Opacity-Associated Protein A Contributes to the Binding of Haemophilus influenzae to Chang Epithelial Cells

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of Haemophilus influenzae, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable H. influenzae strains with or without OapA. We also showed that OapA was conserved among H. influenzae strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of H. influenzae to Chang epithelial cells, suggesting that the interaction of H. influenzae is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of H. influenzae. Expression of oapA gene in a nonadherent Escherichia coli strain significantly increased the binding to Chang epithelial cells, and disruption of the oapA gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in H. influenzae binding to human conjunctival epithelial cells.     

Nontypeable Haemophilus influenzae as a cause of spontaneous bacterial peritonitis

Haemophilus influenzae rarely causes spontaneous bacterial peritonitis. We describe a typical case of spontaneous bacterial peritonitis in which the causative organism was identified as nontypeable H. influenzae, biotype III. Infection progressed despite the presence of adequate serum bactericidal antibody, probably due to the absence of complement in ascites fluid.     

Role of Pht Proteins in Attachment of Streptococcus pneumoniae to Respiratory Epithelial Cells

Pneumococcal adherence to mucosal surfaces is a critical step in nasopharyngeal colonization, but so far few pneumococcal adhesins involved in the interaction with host cells have been identified. PhtA, PhtB, PhtD, and PhtE are conserved pneumococcal surface proteins that have proven promising as vaccine candidates. One suggested virulence function of Pht proteins is to mediate adherence at the respiratory mucosa. In this study, we assessed the role of Pht proteins in pneumococcal binding to respiratory epithelial cells. Pneumococci were incubated with human nasopharyngeal epithelial cells (Detroit-562) and lung epithelial cells (A549 and NCI-H292), and the proportion of bound bacteria was measured by plating viable counts. Strains R36A (unencapsulated), D39 (serotype 2), 43 (serotype 3), 4-CDC (serotype 4), and 2737 (serotype 19F) with one or more of the four homologous Pht proteins deleted were compared with their wild-type counterparts. Also, the effect of anti-PhtD antibodies on the adherence of strain 2737 to the respiratory epithelial cells was studied. Our results suggest that Pht proteins play a role in pneumococcal adhesion to the respiratory epithelium. We also found that antibody to PhtD is able to inhibit bacterial attachment to the cells, suggesting that antibodies against PhtD present at mucosal surfaces might protect from pneumococcal attachment and subsequent colonization. However, the relative significance of Pht proteins to the ability of pneumococci to bind in vitro to epithelial cells depends on the genetic background and the capsular serotype of the strain.     

Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis

Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.     

Prevalence of the sodC Gene in Nontypeable Haemophilus influenzae and Haemophilus haemolyticus by Microarray-Based Hybridization

The sodC gene has been reported to be a useful marker for differentiating nontypeable (NT) Haemophilus influenzae from Haemophilus haemolyticus in respiratory-tract samples, but discrepancies exist as to the prevalence of sodC in NT H. influenzae. Therefore, we used a microarray-based, “library-on-a-slide” method to differentiate the species and found that 21 of 169 (12.4%) NT H. influenzae strains and all 110 (100%) H. haemolyticus strains possessed the sodC gene. Multilocus sequence analysis confirmed that the 21 NT H. influenzae strains were H. influenzae and not H. haemolyticus. An inactive sodC gene has been reported in encapsulated H. influenzae strains belonging to phylogenetic division II. Capsule-specific Southern hybridization and PCR and a lack of copper/zinc-cofactored superoxide dismutase (CuZnSOD) expression indicated that 6 of the 21 sodC-containing NT H. influenzae strains in our study were likely capsule-deficient mutants belonging to phylogenetic division II. DNA sequence comparisons of the 21 H. influenzae sodC genes with sodC from H. haemolyticus or encapsulated H. influenzae demonstrated that the sodC genes of the six H. influenzae capsule-deficient mutants were, on average, 99% identical to sodC from encapsulated H. influenzae but only 85% identical to sodC from H. haemolyticus. The sodC genes from 2/15 NT H. influenzae strains were similarly more closely related to sodC from encapsulated strains, while sodC genes from 13 NT H. influenzae strains were almost 95% identical to sodC genes from H. haemolyticus, suggesting the possibility of interspecies recombination in these strains. In summary, this study demonstrates that sodC is not completely absent (9.2%) in true NT H. influenzae strains.Haemophilus influenzae asymptomatically colonizes the pharyngeal cavity of humans but may cause either systemic or respiratory-tract infections. Those strains that possess a polysaccharide capsule, serotypes a through f, cause most H. influenzae invasive infections (11), whereas nonencapsulated or nonserotypeable (NT) H. influenzae strains are associated with localized respiratory-tract diseases, such as otitis media and exacerbation of chronic obstructive pulmonary disease (COPD) (19). In addition, a cryptic genospecies of NT H. influenzae is also associated with neonatal invasive infections and adult urogenital infections (36). Phylogenetically, however, the cryptic genospecies is more closely related to Haemophilus haemolyticus than to H. influenzae of the respiratory tract (26, 27).H. haemolyticus is a commensal of the pharyngeal cavity, and H. haemolyticus and NT H. influenzae isolated from the respiratory tract overlap taxonomically and phylogenetically, likely through the exchange of DNA by natural transformation (1, 20, 21, 32). The species have been traditionally differentiated by the ability of H. haemolyticus to hemolyze horse blood (11, 25), but recent studies have identified significant numbers of nonhemolytic H. haemolyticus in NT H. influenzae collections isolated from throat or sputum samples (21, 37). Since sputum samples are used to monitor COPD exacerbation, accurate differentiation of NT H. influenzae from H. haemolyticus is clinically important (5, 21).Fung et al. (9) recently suggested that the presence of the sodC gene or activity of its cognate protein, copper/zinc-cofactored superoxide dismutase (CuZnSOD), can differentiate H. haemolyticus from NT H. influenzae, as sodC was present in 20 H. haemolyticus isolates and absent in 20 NT H. influenzae isolates. In addition, a previous study failed to find the sodC gene in 45 NT H. influenzae disease isolates (18). Prior to these studies, however, 12 of 26 NT H. influenzae strains were found to hybridize weakly with a sodC gene probe, and these strains also displayed CuZnSOD activity (13). The discrepancy between these studies has never been explained. Aside from the questionable absence of sodC in NT H. influenzae of the respiratory tract, the gene and its associated CuZnSOD activity are present in the H. influenzae cryptic genospecies associated with neonatal invasive infections and adult urogenital infections (18). In addition, the gene is present among serotype e encapsulated strains and in strains that belong to the capsular phylogenetic division II, a multilocus enzyme electrophoresis (MLEE)-based division that contains some serotype a and b (Hib) strains and all serotype f (Hif) strains (13, 18, 22). When present, the sodC gene in encapsulated strains is adjacent to the bexA to -D genes of the capsule locus. The sodC gene of encapsulated sodC-containing strains shares significant sequence homology with the gene from Haemophilus parainfluenzae, but unlike H. parainfluenzae, encapsulated H. influenzae lacks CuZnSOD activity, presumably due to a substitution mutation that replaces an active-site histidine with tyrosine (13).In the present study, we further evaluated the potential of the sodC gene to differentiate a large collection of known NT H. influenzae and H. haemolyticus respiratory tract isolates using a whole-genome microarray hybridization technology called library on a slide (39).     

Nontypeable Haemophilus influenzae Clearance by Alveolar Macrophages Is Impaired by Exposure to Cigarette Smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-α) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-α secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-α secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.The human respiratory tract is one of the largest body surfaces in contact with the environment and, therefore, is a main entry portal for microorganisms. In healthy humans, the lungs are sterile due to the combined actions of a repertoire of defense mechanisms. The components of lung innate immunity include mechanical barriers such as the mucociliary barrier, humoral elements present in the fluid in contact with the lung epithelium such as surfactants, complement, antimicrobial peptides, lysozyme, and lactoferrin, and resident innate immunity cells such as alveolar macrophages and dendritic cells (32, 37). Alveolar macrophages are professional phagocytes and antigen-presenting cells which patrol the lungs as sentinels and are endowed with, among other things, a collection of pattern recognition receptors used to recognize microorganisms containing pathogen-associated molecular patterns. As professional phagocytes, alveolar macrophages recognize, ingest, and process foreign material using a phagolysosomal pathway and thus play an essential role in the clearance of infections (18).Cigarette smoke is the main risk factor for the development of lung cancer, chronic obstructive pulmonary disease (COPD), and respiratory infections (26). In this context, the so-called “British hypothesis” states that recurrent bronchial infections were the reason, at least partially, that some smokers developed progressive airway obstruction and others did not (12, 13). Exposure to cigarette smoke markedly alters lung immunity by disruption of the mucociliary function, mucus hypersecretion, and disturbance of the mucosal integrity (31). Cigarette smoke also causes oxidative stress which triggers local lung inflammation by activation of epithelial cells, alveolar macrophages, neutrophils, and T lymphocytes (2). These cells secrete inflammatory cytokines, proteases, and reactive oxygen species, causing necrosis, tissue damage, and further amplification of the inflammatory response with enhanced recruitment of neutrophils into the lung. Tissue damage promotes the release of inflammatory mediators and inhibits lung tissue repair functions, further increasing the tissue damage in the lungs of smokers (35, 38, 39). It is generally accepted, although it has not been formally proven, that these alterations could allow access of microorganisms to the otherwise sterile lungs, thereby leading to microbial colonization (28-30). Supporting this hypothesis, mice exposed to cigarette smoke were impaired in the ability to clear a Pseudomonas aeruginosa infection (10). However, there is currently limited information concerning the effect of cigarette smoke at the molecular and cellular levels on the interaction between pathogens and alveolar macrophages.Glucocorticoids are drugs that are widely used to control many inflammatory and immune diseases, including respiratory diseases. Moreover, adjunctive glucocorticoid therapy is currently being used against a variety of bacterial infections, including otitis media, and COPD (7, 21). However, despite their importance in suppressing inflammatory responses, little is known about the effects of glucocorticoids in host defense against pathogens.Nontypeable Haemophilus influenzae (NTHI) is a frequent gram-negative asymptomatic colonizer of the upper respiratory tract in healthy humans, but it is also an opportunistic bacterial pathogen. NTHI causes invasive diseases such as meningitis and acute respiratory infections such as otitis media with effusion, sinusitis, pneumonia, and bronchitis (24). Moreover, NTHI is the pathogen isolated most frequently from lower respiratory tract secretions from patients suffering from chronic respiratory diseases such as COPD and chronic bronchitis (30). Lipooligosaccharide (LOS) is the main glycolipid on the NTHI cell surface and comprises a membrane-anchoring lipid A molecule linked to oligosaccharide chains that extend from the bacterial cell surface (27). Phosphocholine (PCho) is a substituent frequently present in NTHI LOS chain extensions (36). This modification has been shown to be a virulence factor that is involved in NTHI adhesion and invasion of the respiratory epithelium and hence promotes pathogen persistence on the mucosal surface of the respiratory tract (33, 34).The importance of NTHI as a respiratory pathogen has been extensively demonstrated, and alveolar macrophages play an essential role in the clearance of bacterial infections. However, little is known about the interaction between NTHI and alveolar macrophages and about the influence of PCho on this interaction. It is tempting to speculate that NTHI might be able to escape alveolar macrophage-mediated killing and that PCho could play an important role in this process. In addition, given the association between cigarette smoke and respiratory infections caused by NTHI, we hypothesized that cigarette smoke could modify the characteristics of the interaction between NTHI and alveolar macrophages. In the present study, we investigated the features of the interaction between NTHI and alveolar macrophages. Furthermore, we analyzed the impact of cigarette smoke on the ability of alveolar macrophages to engulf and process this respiratory pathogen and whether glucocorticoids affect this interaction.     

Diversity of Nontypeable Haemophilus influenzae Strains Colonizing Australian Aboriginal and Non-Aboriginal Children

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n = 81) and non-Aboriginal (n = 76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P < 0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P = 0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.     

Comparative Profile of Heme Acquisition Genes in Disease-Causing and Colonizing Nontypeable Haemophilus influenzae and Haemophilus haemolyticus

Nontypeable Haemophilus influenzae (NTHI) are Gram-negative bacteria that colonize the human pharynx and can cause respiratory tract infections, such as acute otitis media (AOM). Since NTHI require iron from their hosts for aerobic growth, the heme acquisition genes may play a significant role in avoiding host nutritional immunity and determining virulence. Therefore, we employed a hybridization-based technique to compare the prevalence of five heme acquisition genes (hxuA, hxuB, hxuC, hemR, and hup) between 514 middle ear strains from children with AOM and 235 throat strains from healthy children. We also investigated their prevalences in 148 Haemophilus haemolyticus strains, a closely related species that colonizes the human pharynx and is considered to be nonpathogenic. Four out of five genes (hxuA, hxuB, hxuC, and hemR) were significantly more prevalent in the middle ear strains (96%, 100%, 100%, and 97%, respectively) than in throat strains (80%, 92%, 93%, and 85%, respectively) of NTHI, suggesting that strains possessing these genes have a virulence advantage over those lacking them. All five genes were dramatically more prevalent in NTHI strains than in H. haemolyticus, with 91% versus 9% hxuA, 98% versus 11% hxuB, 98% versus 11% hxuC, 93% versus 20% hemR, and 97% versus 34% hup, supporting their potential role in virulence and highlighting their possibility to serve as biomarkers to distinguish H. influenzae from H. haemolyticus. In summary, this study demonstrates that heme acquisition genes are more prevalent in disease-causing NTHI strains isolated from the middle ear than in colonizing NTHI strains and H. haemolyticus isolated from the pharynx.     

Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms

Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.     

Nontypeable Haemophilus influenzae and chronic obstructive pulmonary disease: a review for clinicians

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of morbidity and mortality worldwide. In the lower airways of COPD patients, bacterial infection is a common phenomenon and Haemophilus influenzae is the most commonly identified bacteria. Haemophilus influenzae is divided into typeable and nontypeable (NTHi) strains based on the presence or absence of a polysaccharide capsule. While NTHi is a common commensal in the human nasopharynx, it is associated with considerable inflammation when it is present in the lower airways of COPD patients, resulting in morbidity due to worsening symptoms and increased frequency of COPD exacerbations. Treatment of lower airway NTHi infection with antibiotics, though successful in the short term, does not offer long-term protection against reinfection, nor does it change the course of the disease. Hence, there has been much interest in the development of an effective NTHi vaccine. This review will summarize the current literature concerning the role of NTHi infections in COPD patients and the consequences of using prophylactic antibiotics in patients with COPD. There is particular focus on the rationale, findings of clinical studies and possible future directions of NTHi vaccines in patients with COPD.     

Antibodies Specific for the Hia Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity

The Hia autotransporter proteins are highly immunogenic surface adhesins expressed by nontypeable Haemophilus influenzae (NTHI). The objective of our study was to assess the opsonophagocytic activity of anti-Hia antibodies against homologous and heterologous NTHI. A segment of the hia gene that encodes a surface-exposed portion of the H. influenzae strain 11 Hia protein was cloned into a pGEMEX-2 expression vector. Escherichia coli JM101 was transformed with the resulting pGEMEX-Hia BstEII del recombinant plasmid, and recombinant fusion protein was recovered. An immune serum against recombinant GEMEX-Hia (rGEMEX-Hia)-mediated killing of the homologous NTHI strain 11 at a 1:160 titer and five heterologous Hia-expressing strains at titers of ≥1:40. Immune serum did not mediate killing of two Hia-knockout strains whose hia genes were inactivated but did mediate killing of one knockout strain at a high titer after the strain was transformed with a plasmid containing the hia gene. Immune serum did not mediate killing of HMW1/HMW2-expressing NTHI strains, which do not express the Hia adhesin. However, when two representative HMW1/HMW2-expressing strains were transformed with the plasmid containing the hia gene, they expressed abundant Hia and were susceptible to killing by the immune serum. Immune serum did not mediate killing of HMW1/HMW2-expressing strains transformed with the plasmid without the hia gene. Our results demonstrate that the Hia proteins of NTHI are targets of opsonophagocytic antibodies and that shared epitopes recognized by such antibodies are present on the Hia proteins of unrelated NTHI strains. These data argue for the continued investigation of the Hia proteins as vaccine candidates for the prevention of NTHI disease.Otitis media remains a significant health problem for children in this country and elsewhere in the world (10, 11). Most children in the United States have had at least one episode of otitis by their third birthdays, and one-third have had three or more episodes (34). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (33, 41). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for the prevention of otitis media in young children (20). Although the total prevention of disease will be a difficult goal to achieve, the prevention of even a portion of cases would be beneficial, given the magnitude and costs of the problem.Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of the cases of acute otitis media (16, 35). Streptococcus pneumoniae is the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (16, 35). Nontypeable Haemophilus influenzae (NTHI) is the second-most-common bacterium recovered and accounts for 20% to 30% of the cases of acute otitis media and a larger percentage of the cases of chronic and recurrent disease (26). Interestingly, since the introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccine schedule, NTHI has become an even more common cause of acute and recurrent middle ear disease, often surpassing S. pneumoniae in the frequency of recovery from middle ear specimens (12, 26). Many different antigens have been suggested as possible NTHI vaccine candidates (1, 3, 18, 29, 30, 42). Outer membrane proteins appear to be the principal targets of bactericidal and protective antibodies (22), and as a group, they have been the major focus of vaccine development efforts. Table ​Table11 summarizes the relevant characteristics of some of the leading vaccine candidates currently under active investigation.

TABLE 1.

Potential vaccine antigens of NTHI

Infection of Primary Human Bronchial Epithelial Cells by Haemophilus influenzae: Macropinocytosis as a Mechanism of Airway Epithelial Cell Entry

Nontypeable Haemophilus influenzae is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeable H. influenzae strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that H. influenzae cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable H. influenzae can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.     

Absence of an Important Vaccine and Diagnostic Target in Carriage- and Disease-Related Nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi)-associated disease is a major health problem globally. Whole-genome sequence analysis identified the absence of hpd genes encoding Haemophilus protein D in 3 of 16 phylogenetically distinct NTHi isolates. This novel finding is of potential clinical significance, as protein D and hpd represent important NTHi vaccine antigen and diagnostic targets, respectively.