INTRODUCTION

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It is estimated that in 1995, 16.4 million people in the United States suffered from chronic obstructive pulmonary disease (COPD) (226). COPD is also the fourth most common cause of death in the United States (14). Both the prevalence of and mortality from this disease have been increasing worldwide (118, 226, 330). COPD is defined physiologically by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and/or emphysema (14). Chronic bronchitis is defined clinically by the presence of cough with sputum production for most days of at least 3 months a year for 2 consecutive years (200). Other causes of chronic cough need to be excluded. Emphysema is defined pathologically as permanent dilation of the airspaces distal to the terminal bronchioles, accompanied by destruction of the alveolar septa in the absence of fibrosis (292). More than 80% of COPD cases encountered in the Western world are related to tobacco smoke exposure. Occupational exposures and alpha-1 antitrypsin deficiency are uncommon precedents for the development of COPD (177, 234).

Several potential contributions of bacterial infection to the etiology, pathogenesis, and clinical course of COPD can be identified (219). However, the precise role of bacterial infection in COPD has been a source of controversy for several decades (175, 296, 307). Opinion regarding the contribution of bacteria to the pathogenesis of COPD has ranged from the idea that it has a preeminent role (along with mucus hypersecretion) as embodied in the British hypothesis in the 1950s and 1960s, to the idea that it is a mere epiphenomenon in the 1970s and 1980s (200, 296, 307). In the last decade, new research techniques have become available, and traditionally noninfectious diseases such as peptic ulcer have been shown to be of infectious origin (240). This has renewed interest in the area of bacteria and COPD, and these new research methodologies should lead to a precise delineation of the contribution of bacterial infection to this disease.

Five potential pathways by which bacteria could contribute to the course and pathogenesis of COPD can be identified. (i) Childhood lower respiratory tract infection impairs lung growth, reflected in smaller lung volumes in adulthood. (ii) Bacteria cause a substantial proportion of acute exacerbations of chronic bronchitis which cause considerable morbidity and mortality. (iii) Chronic colonization of the lower respiratory tract by bacterial pathogens amplifies the chronic inflammatory response present in COPD and leads to progressive airway obstruction (vicious circle hypothesis). (iv) Bacterial pathogens invade and persist in respiratory tissues, alter the host response to cigarette smoke, or induce a chronic inflammatory response and thus contribute to the pathogenesis of COPD. (v) Bacterial antigens in the lower airway induce hypersensitivity that enhances airway hyperreactivity and induces eosinophilic inflammation. Evidence supporting these roles will be discussed in this review, with an emphasis on information gained from newer research techniques in the last decade. The second part of this review will discuss each of the major pathogens, with emphasis on recent developments related specifically to infections in COPD.

POTENTIAL ROLES OF BACTERIAL INFECTION IN COPD

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Childhood Lower Respiratory Tract Infection and Adult Lung Function

Four recent studies have reported lung function (measured by spirometry) in cohorts of adult patients for whom reliable information was available regarding the incidence of lower respiratory tract infection (bronchitis, pneumonia, or whooping cough) in childhood (<14 years of age) (Table 1) (26, 152, 279, 280). These studies have consistently shown a lower forced expiratory volume in 1 s (FEV₁) and often a lower forced vital capacity among adults who experienced childhood lower respiratory tract infection compared to others in the cohort who did not experience such infection (26, 152, 279, 280). FEV₁ and forced vital capacity are widely used tests of pulmonary function. This association is seen after controlling for confounding factors such as tobacco exposure. The magnitude of this defect in FEV₁ has varied among the studies but tends to be greater in older cohorts. The extent of decrease in FEV₁ is unlikely to cause symptomatic pulmonary disease per se but could make the individual susceptible to the effects of additional injurious agents, such as tobacco smoke, and environmental or occupational exposure to air-borne pollutants. The defect in lung function is not airway obstruction, as the FEV₁/FVC ratio is preserved. Instead, it is consistent with "smaller lungs", suggesting impaired lung growth.

Although the association between childhood lower respiratory tract infection and impaired lung function in adulthood is now well established, there is ongoing debate whether this association reflects a cause-effect relationship. Such a relationship could be explained by damage caused to a vulnerable lung undergoing rapid postnatal growth and maturation by the infectious process. If this were the case, then the effect of the infection on lung function should be seen only in the first 2 years of life, the major period of postnatal lung growth, but not in later childhood (3 to 14 years). However, this has not been a consistent observation in the studies to date (26, 152, 279, 280). An alternative explanation for the observed association between childhood lower respiratory tract infection and impaired lung function in adulthood is that an undetermined genetic factor predisposes these individuals to lower respiratory tract infections in childhood as well as a lower FEV₁ in adulthood. This explanation implies that impaired lung growth antedates the respiratory tract infection, with the infectious episode a result of the vulnerability of smaller lungs to infection in childhood.

The etiology of childhood lower respiratory tract infection was not established in these studies, and therefore whether the impact of viral infection differs from that of bacterial infection is not known. Though it is likely that a substantial proportion of these childhood infections were viral, bacterial infection, especially with Streptococcus pneumoniae and Haemophilus influenzae, is a common cause of severe pneumonia in children (333). The impact of childhood bacterial lower respiratory tract infection on the prevalence of COPD is likely to be greater in developing countries, where these infections are common and are often inadequately treated.

Bacteria are isolated from sputum in 40 to 60% of acute exacerbations of COPD (274). Table 2 shows the sputum bacteriology obtained in 14 clinical trials of antibiotics in acute exacerbation published in the last 4 years (12, 19, 53-55, 71, 74, 127, 170-172, 251, 278, 348). Variation in the relative incidence of specific pathogens is seen and may relate to patient inclusion criteria and sputum culture techniques. The three predominant bacterial species isolated are nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. Other infrequently isolated potential pathogens are Haemophilus parainfluenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and members of the family Enterobacteriaceae.

Whether isolation from sputum of a potential pathogen represents infection of the lower airway causing the exacerbation episode has been a controversial issue for several decades. In the 1950s and 1960s, the British hypothesis of the pathogenesis of COPD included as major contributors recurrent bacterial infection and mucus hypersecretion (200). With the realization of tobacco smoke exposure as the primary pathogenic mechanism of COPD and the emergence of evidence that mucus hypersecretion and worsening airway obstruction were independent, the British hypothesis fell into disfavor (28, 102, 145). Several longitudinal cohort studies in the 1960s and 1970s demonstrated that the incidence of bacterial isolation from sputum during exacerbations of COPD was not different from the incidence during stable COPD (123, 196). These studies also failed to demonstrate a higher bacterial titer in sputum during acute exacerbation than during stable COPD (123). Serological studies conducted in the same time period that compared serum antibody titers to airway bacterial pathogens such as nontypeable H. influenzae in COPD patients with those in controls arrived at confusing and contradictory conclusions (reviewed in reference 219). Results of placebo-controlled antibiotic trials in acute exacerbations have also been inconsistent, demonstrating either small or no benefit with antibiotic therapy (17, 269).

A common interpretation of these observations has been that isolation of bacteria from sputum during exacerbations represents chronic colonization, an innocent bystander role for the bacteria (95, 228, 296, 307). Alternative explanations for the contradictory observations from serological studies and antibiotic therapy should be considered (148, 219). Serological studies of antibodies to bacterial pathogens in exacerbations yielded confusing and contradictory results for the following reasons. (i) Laboratory strains were often used as the antigen instead of homologous patient strains. Thus, the strain variation in the surface antigens of the bacterial pathogens that has only recently been understood was not taken into account in these studies. (ii) The immunological methods employed in these studies often were not specific for antibodies to surface-exposed epitopes on the bacteria. A bacterial pathogen presents several hundred antigenic epitopes to a host, many of which are non-surface exposed and therefore potentially irrelevant to host defense. Furthermore, several of these epitopes are cross-reactive among bacterial species. A protective immune response that develops after an infection may be limited to a few epitopes on the bacterial surface. Detecting this response among the multitude of irrelevant, non-surface-exposed antigen-antibody interactions requires immunological assays that are specific for antibodies that bind to surface-exposed epitopes on the bacteria. Such assays include radioimmunoprecipitation assays, flow cytometry assays with whole bacterial cells as an antigen, and assays that measure functional (bactericidal or opsonophagocytic) antibodies. Immunoblots and whole bacterial immunodots that were used in previous serological studies do not have such specificity (115). (iii) Not all studies used true preinfection sera for comparison with the convalescent-phase serum. Instead, acute-phase serum taken at the time that the patient presents with symptoms was used. The symptoms of an acute exacerbation have often been present for several days, and therefore a serological response to the strain may be missed if an acute-phase serum is substituted for a preinfection serum.

Trials showing no benefit with antibiotics in acute exacerbations also have several potential explanations. (i) An exacerbation is a mucosal infection, and the use of antibiotics in other mucosal infections such as otitis media and sinusitis is also not associated with dramatic efficacy over placebo (345). This does not imply that mucosal infections are nonbacterial. (ii) The expected benefits from antibiotics in a mucosal infection are primarily a more rapid resolution of symptoms and prevention of complications. Unfortunately, most studies of antibiotics in acute exacerbations have not measured the speed of resolution of symptoms. Instead, the endpoint has been whether the treatment was successful at 3 weeks after the onset of the exacerbation. The systemic immune-inflammatory response would be expected to resolve a large proportion of bacterial exacerbations in this time period, disguising any potential effect of antibiotics (17). (iii) Many studies include patients with mild impairment of lung function who are likely to have a low rate of complications, making a difference from the placebo group prone to type 2 error. In other words, the study populations could have contained too few individuals with potential for benefit for a benefit to be observed. (iv) Exacerbations are nonbacterial in 50% of patients, with no expected benefit from antibiotics, again predisposing studies to a type 2 error. (v) Antibiotic resistance in some of these pathogens that may be compounded by lack of penetration into the bronchial tissues and fluids of some of the antibiotics is likely to diminish the effect of antibiotics in exacerbations.

In the last decade, several investigators have reexamined the issue of whether bacteria cause acute exacerbations of COPD using either new diagnostic modalities or new research techniques, including bronchoscopic sampling of the lower respiratory tract (96, 208, 239, 294), immune response to bacterial pathogens in exacerbations (49, 222, 355), molecular epidemiology of bacterial pathogens (116, 220, 270, 271, 288, 289, 290), and airway inflammation measurement and correlation with bacteriology (161, 293). These methods provide new data which contribute to a more rigorous evaluation of the etiology of exacerbations.

Bronchoscopic sampling of lower respiratory tract in exacerbations of COPD. An attractive approach to understanding the role of bacterial infection in exacerbated COPD is sampling of distal airway secretions for quantitative culture by protected specimen brush or by bronchoalveolar lavage (BAL) to determine bacterial concentrations in the distal airways. Such an approach has contributed tremendously to our understanding of nosocomial pneumonia (50). Samples obtained from the distal airways with these techniques have low levels of contamination by upper respiratory tract secretions. Bacterial concentrations above certain thresholds on quantitative culture have been found to correlate with tissue infection in patients with pneumonia (50). Four studies that have used this method in acute exacerbations have been published, and all have consistently shown significant bacterial infection of the distal airways in approximately 50% of patients experiencing an exacerbation (Table 3) (96, 208, 239, 294). The bacterial species isolated in these studies represent the same spectrum of pathogens commonly isolated from sputum cultures of patients with acute exacerbation.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Culture results of bronchoscopic samples obtained from patients with stable COPD and those with acute exacerbation. The number of positive samples at both pathogen concentrations was significantly greater (P < 0.05) in the exacerbation group. Data taken from the work of Monso et al. (208). PSB, protected specimen brush; CFU/ml, CFU of pathogenic bacteria per milliliter of epithelial lining fluid.

Immune responses to bacterial pathogens. Older studies of immune response to nontypeable H. influenzae in COPD had several limitations, as discussed above (219). Recently, we and other investigators have explored the immune response to bacterial pathogens in acute exacerbations of COPD with methods that avoid the pitfalls of earlier studies. These studies are discussed in detail later. These studies have demonstrated the development of specific immune response to infecting strains of nontypeable H. influenzae and M. catarrhalis and support the role of bacterial infection in acute exacerbations of COPD. Similar evidence with other bacterial species (see Table 2) would help us better define their role in acute exacerbations.

Molecular epidemiology of bacterial pathogens. Strains of a bacterial species can differ considerably in their surface antigenic structure. The "bacterial load" model of bacterial infection in COPD assumes that an increase in the titer of a bacterial species in the airway is responsible for the transition from stable COPD to exacerbation of COPD (346). Studies of bacterial titers in the sputum of patients with COPD have not supported this model (123). This model does not take into account the genetic diversity within the bacterial species, including alterations in surface antigenic structure. An alternative model is that infection with a bacterial strain with an antigenic structure new to the host leads to an immune and inflammatory response that presents clinically as an acute exacerbation. Longitudinal studies of patients with COPD in combination with molecular typing of the strains will allow investigators to test this model.

Tobacco smoking cannot be the sole factor responsible for the pathogenesis of COPD, as only a small proportion (15%) of smokers develop chronic bronchitis and an even smaller proportion go on to develop obstructive airway disease (COPD). In the absence of underlying lung disease, the tracheobronchial tree is sterile. In patients with COPD, the tracheobronchial tree is chronically colonized with potential respiratory pathogens, predominantly nontypeable H. influenzae, S. pneumoniae, and M. catarrhalis (124, 173, 208, 358). Several years ago, we proposed a vicious circle hypothesis to explain how chronic bacterial colonization of the lower airways in patients with COPD can perpetuate inflammation and contribute to progression of the disease (Fig. 2) (63, 219). A similar mechanism is believed to contribute to the pathogenesis of lung disease in individuals with cystic fibrosis. Substantial supporting evidence for this hypothesis in COPD, both in vitro and in vivo, has now accumulated and is discussed below.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Diagrammatic representation of the vicious circle hypothesis.

Central to the vicious circle hypothesis is the notion that once bacterial pathogens have gained a foothold in the lower respiratory tract from impaired mucociliary clearance due to tobacco smoking, the bacteria persist by further impairing mucociliary clearance (Fig. 2). This impairment of mucociliary clearance can be due to enhanced mucus secretion, disruption of normal ciliary activity, and airway epithelial injury. Experimental evidence demonstrates that respiratory tract pathogens and their products can cause all of these effects in vitro.

Bacterial infection and chronic mucus hypersecretion. Adler et al. examined the effect of cell-free filtrates of broth cultures of nontypeable H. influenzae, S. pneumoniae, and P. aeruginosa on the secretion of mucous glycoproteins by explanted guinea pig airway tissue (1). Seven of 28 (25%) strains of nontypeable H. influenzae, 10 to 26 (34%) strains of S. pneumoniae, and 12 of 18 (66%) strains of P. aeruginosa stimulated mucin secretion. This stimulation was a true secretory effect and not passive release of preformed intracellular macromolecules due to cellular damage, as ultrastructural assessment (by light, transmission, and scanning electron microscopy) demonstrated an absence of cytotoxicity. The Pseudomonas stimulatory products were proteases of 60 to 100 kDa. The Haemophilus and pneumococcal stimulatory exoproducts were 50 to 300 kDa in size and did not possess proteolytic activity.

Bacterial infection and mucociliary clearance. The tracheobronchial ciliary escalator is of paramount importance in maintaining sterility of the lower respiratory tract by transporting bacteria trapped in mucus towards the pharynx (347). Disruption of this ciliary activity is therefore likely to be important in the establishment of chronic colonization in the tracheobronchial tree. Wilson et al. measured by photometry the effect of cell-free supernatants of nontypeable H. influenzae, P. aeruginosa, and S. aureus on ciliary beat frequency of strips of human nasal ciliary epithelium (349). Rapid inhibition of ciliary beat frequency was seen with nontypeable H. influenzae and P. aeruginosa but not with S. aureus. On direct examination, ciliary dyskinesia and ciliostasis were seen. Human neutrophil elastase inhibits ciliary activity and damages respiratory epithelium (15). Bacterial products in the airways may be a potent stimulus for neutrophil migration into the airways, and elastase released from these neutrophils can act synergistically with bacterial products and cause further inhibition of tracheobronchial ciliary function.

Bacterial infection and airway epithelial injury. An important component of the vicious circle hypothesis is the potentially damaging effects of bacteria and bacterial products on airway epithelial lining cells. Such epithelial injury in the large airways would contribute to bacterial persistence and in the small airways could contribute to the respiratory bronchiolitis that causes progressive airway obstruction (67, 107). In an in vitro tissue culture model of nasal turbinate epithelium, Read et al. have demonstrated that nontypeable H. influenzae causes airway epithelial injury (252). They studied these epithelia after 30 min, 14 h, and 24 h of incubation with a nontypeable H. influenzae strain. At 30 min, the airway epithelium and cilia were intact and the bacteria were associated with the overlying mucus layer. At 14 h, patchy injury developed to the airway epithelium, with bacterial cells now associating with these damaged epithelial cells but not with intact epithelium. At 24 h, detached epithelial cells with adherent bacteria were seen.

Bacterial infection and airway inflammation. The presence of bacteria in the lower airways in patients with stable COPD has been labeled colonization. However, this bacterial presence is definitely abnormal and is not confined to the large airways. Bacteria have been shown to extend to the peripheral airways by cultures of bronchoscopic protected specimen brushings and BAL (208, 358). Even during colonization, bacteria in these airways are in a constant state of turnover, releasing extracellular products, undergoing lysis with release of a variety of proteins, lipooligosaccharide (LOS), and peptidoglycan (121). LOS is a potent inflammatory stimulus; in fact, repeated instillation of LOS can lead to the development of emphysema in hamsters (306). It is therefore quite likely that this colonization is actually a low-grade smoldering infection that induces chronic airway inflammation. In the large airways such inflammation would contribute to mucus production, and in the small airways it could contribute to respiratory bronchiolitis and progressive airway obstruction (79, 310). Recent data that support this hypothesis include in vitro experiments with LOS of nontypeable H. influenzae and a bronchoscopic study that demonstrates that bacterial colonization may be an independent stimulus to airway inflammation in patients with stable COPD, as described below. Furthermore, Hill et al. recently demonstrated an association between bacterial numbers and markers of airway inflammation in stable chronic bronchitis (141a).

Chronic Chlamydia pneumoniae infection in COPD. C. pneumoniae is an obligate intracellular atypical bacterial pathogen. Acute C. pneumoniae infection can cause bronchitis, pneumonia, and acute exacerbations of COPD (see below). Chronic infection with C. pneumoniae is being actively investigated as a cause of several systemic diseases, especially coronary artery disease (178). Von Hertzen et al. studied whether the incidence of chronic C. pneumoniae infection is increased in COPD (332). The presence of chronic C. pneumoniae infection was determined by three different methods: serum antibodies to C. pneumoniae (immunoglobulin G [IgG] and IgA and circulating immune complexes), sputum IgA antibodies to C. pneumoniae, and PCR of sputum for C. pneumoniae DNA. Two of the three methods had to yield positive results for the same patient to demonstrate a chronic C. pneumoniae infection. The incidence of chronic C. pneumoniae infection (as defined above) was 71% in patients with severe COPD, 46% in mild to moderate COPD, and 0% in the control group. Whether this chronic infection contributes to the pathogenesis of COPD as discussed above or is a reflection of compromised local immunity warrants further investigation.

Allergic bronchopulmonary aspergillosis is an infectious disease with predominantly allergic manifestations mediated by a Th2-type immune response and characterized by IgE and eosinophil predominance (158). Inefficient removal of bacteria from the lower respiratory tract is characteristic of chronic bronchitis, resulting in prolonged contact between the airway lymphoid tissue and bacterial antigens. This could lead to the emergence of IgE antibodies to bacterial antigens, which could induce eosinophil infiltration and mast cell degranulation on repeated exposures to the bacterial antigens. An increased number of eosinophils is characteristic of airway inflammation in most patients with COPD, and tissue and airway lumen eosinophilia becomes more prominent during exacerbations (268). Furthermore, a small subgroup of patients with COPD have an eosinophilic bronchitis that is responsive to steroids (132).

The ability of bacterial pathogens to induce histamine release, hypersensitivity, and IgE-mediated inflammation has been investigated sporadically. Mast cells release histamine by non-IgE-mediated and IgE-mediated mechanisms. Clementsen et al. exposed mast cells obtained by BAL from the airways of patients with chronic bronchitis and normal individuals by BAL to Formalin-killed suspensions of nontypeable H. influenzae, S. pneumoniae, M. catarrhalis, and S. aureus. Nontypeable H. influenzae and S. aureus induced non-IgE-mediated and enhanced IgE-mediated histamine release (61). The enhancement of IgE-mediated histamine release appears to be mediated by the endotoxin of nontypeable H. influenzae (62). Histamine increases bronchial epithelium permeability, stimulates mucus secretion, and induces bronchoconstriction.

Patients with acute exacerbations of chronic bronchitis have had basophil-bound IgE and serum IgE to homologous strains of nontypeable H. influenzae and S. pneumoniae isolated from sputum with the acute exacerbation (162). In another study in asthmatics, 29% of patients had serum IgE antibodies to nontypeable H. influenzae and/or S. pneumoniae (238). This sensitization to bacterial antigens may contribute to the bronchoconstriction and airway inflammation seen with acute exacerbations of COPD.

These observations regarding histamine release and IgE to bacterial antigens suggest that bacterial pathogens, either directly or indirectly via a Th2-type immune response, could contribute to the eosinophilia, airway hyperreactivity, and bronchoconstriction seen in patients with COPD. Further investigation in this area is warranted, especially in the group of COPD patients with eosinophilic bronchitis (132).

BACTERIAL PATHOGENS

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Nontypeable Haemophilus influenzae

Dynamics of colonization and molecular epidemiology. Nontypeable H. influenzae strains are common inhabitants of the human upper respiratory tract, being present in up to three-fourths of healthy adults. When serial cultures are performed, the organism can be recovered from the sputum of virtually all patients with chronic bronchitis. Adults with chronic bronchitis are colonized in the lower airways with nontypeable H. influenzae and other bacteria (45, 173). Colonization with nontypeable H. influenzae is a dynamic process, with new strains being acquired and replacing old strains periodically (271). Multiple strains frequently colonize the respiratory tract simultaneously in the setting of chronic bronchitis (116, 220).

(ii) Adherence to respiratory mucosa. Research in the past decade has witnessed the identification and characterization of multiple adhesins expressed by nontypeable H. influenzae (300, 301) (Table 5). Teleologically, the expression of multiple adhesin molecules and the ability to modulate expression of these adhesins support the notion that adherence to the respiratory tract is critical for survival of the bacterium.

(i) Antigenic heterogeneity. Nontypeable H. influenzae expresses six to eight major proteins in its outer membrane. Studies in the 1980s demonstrated that a high degree of variability in the molecular weights of these outer membrane proteins existed among strains of nontypeable H. influenzae (23, 216). OMP P2, which coustitutes approximately half of the protein content of the outer membrane, shows a particularly high degree of size variability among strains (216). P2 is the major porin protein of H. influenzae, allowing small hydrophilic molecules to pass through the outer membrane (318). Analysis of the sequence of the gene which encodes P2 revealed that portions of the protein which are buried within the outer membrane are relatively conserved among strains but that several of the eight loops which are exposed on the bacterial surface show a high degree of sequence variability among strains (31, 84, 283). Since antibodies to P2 elicit strain-specific protection (117, 157, 312), these observations suggested that antigenic heterogeneity of the major surface protein plays a role in the ability of nontypeable H. influenzae to cause recurrent respiratory tract infections in humans.

(ii) Point mutations under immune selective pressure. Analysis of OMP patterns from strains of nontypeable H. influenzae recovered prospectively from patients with chronic bronchitis reveals a high degree of turnover of strains, with frequent infection by new strains in some patients and persistent infection by the same strain in other patients (116). Among the strains which show persistence in the respiratory tract, variants with changes in the molecular weight of P2 but identical DNA fingerprints have been observed (116, 117). To determine the mechanism of this antigenic drift, Duim et al. (85) studied the sequences of the genes encoding P2 in these variants. The antigenic drift resulted from single-base changes in the P2 gene, all generating amino acid changes in surface-exposed loops of the P2 protein (85). Similar single-base changes were observed in the P2 gene from variants selected in subcutaneous cages implanted in rabbits and from a variant which survived antibody-mediated killing in vitro (85, 86, 329). All of the point mutations in the P2 gene were nonsynonymous, since they resulted in amino acid changes. Since all of the substitutions resulted in amino acid changes, these mutations produced a selective advantage for the bacterium. These observations strongly suggested that the accumulation of point mutations under immune selective pressure resulted in antigenic drift of surface-exposed regions of a major OMP. This mechanism of evading an immune response by the host could allow persistent H. influenzae infection in COPD.

(iii) Horizontal transfer of genes. Recent studies in an Aboriginal community in the Northern Territory of Australia reveal another mechanism by which nontypeable H. influenzae alters its P2 molecule to evade host defenses. Rural Aboriginal children are heavily colonized by nontypeable H. influenzae in the nasopharynx at an early age (174). Ribotyping of prospectively recovered isolates has revealed that the children are colonized by multiple strains of H. influenzae simultaneously and that strains are acquired and cleared frequently, resulting in a high rate of turnover (288). By determining the sequences of P2 genes from selected strains, Smith-Vaughn et al. (291) demonstrated the presence of identical P2 genes in strains with different genetic backgrounds. In view of the wide diversity of P2 gene sequences, the authors concluded that horizontal transfer of the P2 gene occurred among strains. The presence in the human respiratory tract of simultaneous, multiple strains of a bacterium which is competent for DNA uptake provides a powerful mechanism for the bacterium to alter expression of surface molecules. This phenomenon is likely to occur in other settings in which multiple strains of nontypeable H. influenzae colonize the respiratory tract, such as cystic fibrosis (206) and chronic bronchitis (220).

(ii) Phase variation. Another mechanism by which H. influenzae alters its LOS is phase variation, which is the ability to regulate the expression of molecules by turning on and off the expression of selected genes. The LOS of H. influenzae demonstrates phase variation, which occurs through a mechanism known as slipped-strand mispairing (141). The lic locus, which is responsible for synthesis of oligosaccharide structures, contains open reading frames which are preceded by multiple tandem repeats of the tetramer 5'-CAAT-3'. Alterations in the number of repeats through the nonrecombinational mechanism of slipped-strand mispairing shift upstream initiation codons into or out of frame, creating a translational switch and resulting in phase variation (141). Multiple oligosaccharide structures undergo phase variation in a complex pattern. Some genes vary independently, and some vary in a coordinate fashion with other genes. As a result, the bacterium has the ability to display a varied array of LOS structures on its surface. This ability enables H. influenzae to adapt to its environment in the various stages of colonization and infection.

(iii) Molecular mimicry of host tissue. The LOS of many strains of H. influenzae contain a terminal digalactoside, Gal--(1-4)--Gal, which is also present in human glycosphingolipids in the urinary tract, intestinal epithelium, and erythrocytes (187). The mimicry of host tissue may be an adaptive mechanism which promotes bacterial survival in the respiratory tract of the host.

(ii) Strain-specific immune responses. OMP P2 is strongly immunogenic in experimental animals and humans (117, 213, 298, 354). Analysis of monoclonal antibodies to P2 which were generated by immunizing mice with whole bacterial cells revealed that most antibodies were directed toward a single surface-exposed loop on the P2 protein (125, 126). All of these antibodies were highly specific for the immunizing strain. This observation suggested that nontypeable H. influenzae expresses an immunodominant, strain-specific epitope on the bacterial surface.

View larger version (76K): [in this window] [in a new window]   FIG. 3.   Immunoblot assay (left panel) and whole-cell radioimmunoprecipitation (RIP) assay (right panel) with preexacerbation serum (lanes A) and postexacerbation serum (lanes B) from an adult with COPD who experienced an exacerbation due to nontypeable H. influenzae. Assays were performed with the homologous infecting strain. The positions of molecular mass standards are noted (in kilodaltons) to the left of each panel. Note that immunoblot assays detect antibodies to many bands with minimal difference between pre- and postexacerbation sera. By contrast, whole-cell radioimmunoprecipitation assays with the same sera show the development of new antibodies to P2 (arrow) and higher-molecular-mass proteins following the exacerbation.

(iii) Antibodies to surface-exposed epitopes. Figure 3 shows that serum from a patient who experienced an exacerbation due to nontypeable H. influenzae had antibodies to many antigens before the exacerbation and in spite of developing new bactericidal antibodies, had no new detectable antibodies by immunoblot assay. This observation is consistent with that of Groeneveld et al. (115), who showed that patients with abundant antibodies to H. influenzae in serum and sputum still experienced infection. Immunoblot assays detect antibodies to many epitopes on OMPs, including those which are buried within the outer membrane and not available for binding on the intact bacterium. Antibodies which bind to epitopes which are not on the bacterial surface are not likely to be protective. The OMPs of H. influenzae share cross-reactive epitopes with the OMPs of many gram-negative bacteria. Most of the cross-reactive epitopes are buried in the membrane and not on the bacterial surface. In order to detect the meaningful and potentially protective immune response, immunoassays which specifically detect antibodies to epitopes on the bacterial surface should be used. Such assays include whole-cell radioimmunoprecipitation assays, flow cytometry, and functional assays such bactericidal and opsonophagocytosis assays. Subjecting the serum in Fig. 3 to whole-cell radioimmunoprecipitation revealed that the patient developed new antibodies to P2 and to higher-molecular-mass proteins, an observation which was missed by immunoblot assay. Adsorption studies further established that new bactericidal antibodies were directed at strain-specific epitopes on the P2 protein (355).

(ii) Vaccine strategies. Nontypeable H. influenzae causes mucosal infections. Therefore, a mucosal immune response may be the most effective in preventing infections (106). One avenue of investigation is the development of technologies for the mucosal delivery of vaccine antigens to generate a mucosal immune response (167, 169, 335, 336). This exciting and potentially fruitful approach is being pursued by several groups of investigators.