Immunization with recombinant Streptococcus pneumoniae neuraminidase NanA protects chinchillas against nasopharyngeal colonization.

Immunization with recombinant S. pneumoniae neuraminidase NanA (rNanA) resulted in a significant reduction in pneumococcal colonization in the chinchilla model. The bacteria were eliminated from the nasopharynx 1 week earlier than that from the control cohort. Our data suggest that rNanA affords protection against pneumococcal nasopharyngeal colonization.

Immunization with native or recombinant Streptococcus pneumoniae neuraminidase affords protection in the chinchilla otitis media model.

Streptococcus pneumoniae neuraminidase has been implicated as a virulence factor in the pathogenesis of pneumococcal otitis media. In this study, native neuraminidase was partially purified from cultures of S. pneumoniae by serial chromatography with DEAE-Sepharose and Sephacryl S-200. Recombinant neuraminidase, a 3,038-bp fragment of the neuraminidase A (nanA) gene, was cloned into the pET-28b vector and then expressed at high levels in Escherichia coli. Chinchillas were immunized subcutaneously with either the gel-purified native or recombinant neuraminidase, and all responded with elevated titers of antineuraminidase antibody in serum. Immunization with neuraminidase resulted in a significant reduction in nasopharyngeal colonization as well as in the incidence of otitis media with effusion. These data demonstrate for the first time that neuraminidase affords protection against S. pneumoniae nasopharyngeal colonization and experimental otitis media.

Differential expression of cytokine genes and inducible nitric oxide synthase induced by opacity phenotype variants of Streptococcus pneumoniae during acute otitis media in the rat.

Phase variation in the colonial opacity phenotype of Streptococcus pneumoniae has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal otitis media (OM). The purpose of this study was to determine whether S. pneumoniae opacity variants influence the induction of gene expression for proinflammatory mediators in vivo using the rat model of OM. Both the opaque and transparent phenotype variants induced a significant up-regulation in gene expression for interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-10, tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) compared to saline sham-inoculated controls at both 4 and 24 h postinoculation (P < 0.05 in all cases). Furthermore, whereas a significant difference in gene expression was evident for only IL-6 (greater following challenge with the opaque variant) and IL-1beta (greater following challenge with the transparent variant) at 4 h, by 24 h the opaque variant cohort demonstrated a significant increase in gene expression for IL-1alpha, IL-1beta, IL-6, IL-10, and iNOS relative to animals inoculated with the transparent phenotype variant (P < 0.05 in all cases). Enzyme-linked immunosorbent assay results confirmed the gene expression data as determined by real-time PCR. Moreover, the concentrations of the opaque variant in the middle ear lavage fluid were a full log higher than those of the transparent variant. The aforementioned results indicate that the opaque phenotype variant is more efficient at survival and multiplication within the middle ear space, resulting in the accumulation of more inflammatory cells and the enhanced expression and production of inflammatory mediators. However, when the data were normalized to account for differences in middle ear bacterial titers, it became apparent that the transparent variant of S. pneumoniae is a more potent inducer of inflammation, triggering the accumulation of more inflammatory cells and substantially greater fold increases in the expression and production of inflammatory mediators. Data from this study indicate that S. pneumoniae opacity variants influence the temporal mRNA expression of inflammatory mediators within the middle ear.

Expression of cytokine and chemokine genes by human middle ear epithelial cells induced by influenza A virus and Streptococcus pneumoniae opacity variants.

Real-time PCR and enzyme-linked immunosorbent assay were used to evaluate the ability of influenza A virus and Streptococcus pneumoniae opacity variants, either alone or in combination, to induce cytokine and chemokine genes in primary cultures of human middle ear epithelial (HMEE) cells. Following treatment with influenza A virus, the induction of gene expression, which occurred in a dose- and time-dependent manner, was strong for macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta; moderate for tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8; and weak for IL-1 beta and monocyte chemotactic peptide 1 (MCP-1). Except for TNF-alpha, all the gene products were detected in the cell culture supernatants. In contrast, infection of HMEE cells with S. pneumoniae alone induced low levels of mRNA expression of MIP-1 alpha and MIP-1 beta and did not significantly induce the transcription of the other cytokines and chemokines examined. However, both S. pneumoniae opacity variants increased mRNA expression of MIP-1 alpha, MIP-1 beta, IL-6, and MCP-1 in HMEE cells activated by a prior influenza A virus infection compared to levels in cells treated with either agent alone. Up-regulation of IL-6, IL-8, and MCP-1 mRNA expression and production by the virus in combination with opaque S. pneumoniae was two- to threefold higher than that induced by the virus combined with the transparent S. pneumoniae variant. These data indicate that the activation of HMEE cells by influenza A virus enhances the induction of cytokine and chemokine gene transcripts by S. pneumoniae and that this effect appears to be most pronounced when S. pneumoniae is in the opaque phase.

Comparison of alteration of cell surface carbohydrates of the chinchilla tubotympanum and colonial opacity phenotype of Streptococcus pneumoniae during experimental pneumococcal otitis media with or without an antecedent influenza A virus infection.

Experimental and clinical studies suggest that influenza A virus promotes Streptococcus pneumoniae-induced otitis media; however, the mechanism underlying this synergistic interaction has not been completely defined. In this study, glycoconjugate expression patterns were evaluated on the cell surface in the chinchilla eustachian tube (ET) lumen of a cohort challenged intranasally (i.n.) with S. pneumoniae type 6A, which is predominantly transparent and a cohort with an antecedent influenza A virus infection, followed by i.n. inoculation with S. pneumoniae. The labeling patterns obtained with six lectin probes revealed that the binding of Bandeiraea simplicifolia lectin II, succinylated wheat germ agglutinin, and peanut agglutinin were significantly increased in the lumenal surface of the ET in the cohort infected with both pathogens compared to the cohort inoculated with only S. pneumoniae, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. A significant decreased labeling with Sambucus nigra agglutinin in the combined influenza A virus and pneumococcus infection cohort suggested that there were few sialic acid residues remaining in the ET epithelium. In addition, the colonial opacity of S. pneumoniae during the disease course was examined. The opaque phenotype was predominant among the pneumococcus isolates from the middle-ear fluid in the cohort infected with the both pathogens. Together, these data suggest that the synergic effect of influenza A virus and S. pneumoniae on the changes of the carbohydrate moieties in the ET epithelium and that the selection of the opaque variant may facilitate the pneumococcal invasion of the middle ear.

Comparison of structural changes of cell surface carbohydrates in the eustachian tube epithelium of chinchillas infected with a Streptococcus pneumoniae neuraminidase-deficient mutant or its isogenic parent strain.

Six different lectin probes were used to examine alterations of the cell surface carbohydrates in the chinchilla eustachian tube (ET) lumen subsequent to the intranasal (i.n.) challenge with the Streptococcus pneumoniae parent strain, D39, or its isogenic derivative, DeltaNA1, which is deficient in neuraminidase NanA. The labelling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated wheat germ agglutinin (SWGA) were increased in the lumenal surface of the ET in the D39 inoculated cohort compared to the uninfected control, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. Concurrently, decreased labelling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the ET epithelium subsequent to i.n. inoculation with D39. The DeltaNA1 neuraminidase deficient mutant, however, did not induce any significant changes in the lectin labelling patterns, and was comparable to that of the control cohort. We propose that products of the nanA gene have a significant impact on the changes of the carbohydrate moieties in the ET epithelium and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.

Expression of cytokine and chemokine genes by human middle ear epithelial cells induced by formalin-killed Haemophilus influenzae or its lipooligosaccharide htrB and rfaD mutants.

To define the role of nontypeable Haemophilus influenzae (NTHI) lipooligosaccharide (LOS) in the induction of proinflammatory cytokine gene expression during otitis media, we compared the abilities of formalin-killed NTHI strain 2019 and its LOS htrB and rfaD mutants to stimulate human middle ear epithelial (HMEE) cell cytokine and chemokine gene expression and production in vitro. Strain DK-1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. HMEE cells were incubated with formalin-killed NTHI 2019, B29, or DK-1. The supernatants and the cells were collected at 2, 4, 8, and 24 h after stimulation. Expression of genes for the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin lbeta (IL-1beta), and IL-6 and for the chemokines macrophage inflammatory protein 1beta (MIP-1beta), monocyte chemotactic peptide 1 (MCP-1), and IL-8 was quantitated by real-time PCR. NTHI B29 did not significantly stimulate any cytokine or chemokine mRNA expression in HMEE cells. In striking contrast, NTHI 2019 induced up to 105-, 139-, and 187-fold increases in HMEE cell expression of IL-1beta, TNF-alpha, and MIP-1beta, respectively (P < 0.01 [2019 versus B29]). NTHI 2019 also induced upregulation of IL-8, IL-6, and MCP-1 mRNA expression (by 26-, 44-, and 14-fold, respectively [P < 0.05 (2019 versus B29)]). The significant induction of cytokine genes was confirmed by quantitating the secretion of cytokines in culture supernatants with an enzyme-linked immunosorbent assay. There were no significant differences in mRNA expression of IL-8, IL-6, and MCP-1 between the 2019- and DK-1-treated groups. The low levels of gene transcripts observed after incubation of HMEE cells with B29 indicate that products of the disrupted NTHI htrB LOS gene may play a major role in induction of these particular inflammatory mediators.

Detection of mucin gene expression in normal rat middle ear mucosa by reverse transcriptase-polymerase chain reaction.

Hypersecretion of mucin is a common feature of chronic and mucoid otitis media which may play an important role in hearing loss. The mechanisms controlling mucin secretion in the middle ear are not completely understood. Our reverse transcriptase-polymerase chain reaction results demonstrate that mRNAs of MUC1, MUC2, MUC3, MUC4 and MUC5AC are expressed in normal rat middle ear mucosa. Moreover, the expression of mRNA of the secretory mucins MUC2, MUC3 and MUC5AC was threefold lower in normal middle ear mucosa than that in the intestine or trachea. In contrast, expression of the membrane-bound mucins MUC1 and MUC4 was approximately the same in both middle ear mucosa and the intestine or trachea. MUC5AC proteins were also identified immunohistochemically in normal rat middle car epithelium. The methodology used in this study provides useful baseline information for investigation of the mechanisms of regulation of mucin gene expression during otitis media.

Effect of influenza A virus infection on nasopharyngeal colonization and otitis media induced by transparent or opaque phenotype variants of Streptococcus pneumoniae in the chinchilla model.

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal disease. Additionally, the synergistic effects of influenza A virus and S. pneumoniae in the development of otitis media (OM) have been reported. This study examined the ability of opaque or transparent S. pneumoniae from the same strain in combination with an antecedent influenza A virus infection to colonize the nasopharynx and invade the middle ear in the chinchilla model. Our data indicated that there was no significant difference in the level of nasopharyngeal colonization and induction of OM between the opaque and transparent variants unless there was a prior challenge with influenza A virus. Subsequent to influenza A virus infection, there was a significant difference between the variants in the ability to colonize and persist in the nasopharynx and middle ear. The concentrations of the opaque variant in nasopharyngeal-lavage samples and middle-ear fluid remained consistently higher than those of the transparent variant for 10 days postinoculation. Data from this study indicate that the effects of influenza A virus on the pathogenesis of experimental S. pneumoniae-induced OM differ depending on the opacity phenotype involved.

Effect of adenovirus type 1 and influenza A virus on Streptococcus pneumoniae nasopharyngeal colonization and otitis media in the chinchilla.

Considerable evidence has implicated respiratory tract virus potentiation of bacterial adherence, colonization, and superinfection as a significant factor contributing to the pathogenesis of otitis media (OM). Influenza A and B viruses, adenovirus, and respiratory syncytial virus are the primary respiratory tract viruses associated with this disease. Investigations have established a dramatic increase in the development of experimental OM in chinchillas co-inoculated with influenza A virus and Streptococcus pneumoniae (Spn). The mechanism underlying this phenomenon was suggested to involve, in part, viral compromise of eustachian tube mucosal integrity and function. This study was designed to assess and compare the effect of adenovirus and influenza A virus infection on adherence, the kinetics of colonization, and invasion of the middle ear by Spn in the chinchilla model of OM. Cohorts were inoculated intranasally with adenovirus type 1 or influenza A virus, and then inoculated intranasally 7 days later with Spn 6A. All cohorts were observed over a 14-day period after challenge with Spn, and the incidence and severity of OM were assessed by several methods, including culture of the nasopharynx and middle ear effusions. The data indicated that influenza A virus promotes a significant increase in nasopharyngeal colonization by Spn, an increased incidence and severity of OM, and a sustained presence of Spn in the effusions. Adenovirus infection, however, did not enhance colonization by Spn or result in an increased incidence or severity of OM.

Evaluation of phase variation of nontypeable Haemophilus influenzae lipooligosaccharide during nasopharyngeal colonization and development of otitis media in the chinchilla model.

Nontypeable Haemophilus influenzae (NTHI) has four loci, lic-1 to lic-3 and lgtC, that generate phase-variable lipooligosaccharide (LOS) structures. lic-1, which is required for the expression of phosphorylcholine (ChoP), is the best characterized and is associated with an enhanced ability of H. influenzae to persist within the nasopharynges of infant rats. Recent data indicate that LOS impacts various aspects of NTHI virulence in the chinchilla model of nasopharyngeal colonization and otitis media (OM). In this study the effects of ChoP expression and the sequences of lic-1 to lic-3 and lgtC of NTHI strain 2019 were evaluated in the chinchilla OM model. Nasopharyngeal colonization data showed that a switch from the ChoP(-) to the ChoP(+) phenotype was observed as early as day 3 after intranasal inoculation. Chinchillas colonized by strains with the ChoP(+) phenotype demonstrated a significantly higher level of NTHI 2019 per milliliter of nasal lavage fluid than chinchillas colonized with predominantly the ChoP(-) variant (P < 0.05). The concentration of cells with the ChoP(+) phenotype in the middle ear was 3 log units higher than that of cells with the ChoP(-) variant (P < 0.01). There was a statistically significant association between ChoP(+) expression in the nasal lavage and the development of OM with culture-positive middle ear fluids in this model. These data suggest that expression of the ChoP(+) phenotype promotes enhanced nasopharyngeal colonization and development of OM.

Evaluation of the virulence of a Streptococcus pneumoniae neuraminidase-deficient mutant in nasopharyngeal colonization and development of otitis media in the chinchilla model.

Considerable evidence has implicated Streptococcus pneumoniae neuraminidase in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, an S. pneumoniae neuraminidase-deficient mutant, DeltaNA1, has been constructed by insertion-duplication mutagenesis of the nanA gene of S. pneumoniae strain D39. The relative ability of DeltaNA1 and the D39 parent strain to colonize the nasopharynx and to induce OM subsequent to intranasal inoculation and to survive in the middle ear cleft after direct challenge of the middle ear were evaluated in the chinchilla model. Nasopharyngeal colonization data indicate a significant difference in the ability of the DeltaNA1 mutant to colonize as well as to persist in the nasopharynx. The neuraminidase-deficient mutant was eliminated from the nasopharynx 2 weeks earlier than the D39 parent strain. Both the parent and the mutant exhibited similar virulence levels and kinetics during the first week after direct inoculation of the middle ear. The DeltaNA1 neuraminidase-deficient mutant, however, was then completely eliminated from the middle ear by day 10 postchallenge, 11 days before the D39 parent strain. Data from this study indicate that products of the nanA gene have an impact on the ability of S. pneumoniae to colonize and persist in the nasopharynx as well as the middle ear.

Localization of nontypeable Haemophilus influenzae endotoxin in the middle and inner ear during experimental otitis media.

Nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide, the major component of H. influenzae endotoxin, was localized in the middle and inner ear subsequent to the resolution of experimental otitis media induced by this pathogen. A monoclonal antibody specific for the lipooligosaccharide of this strain was used to probe sections of middle and inner ear tissue and visualized by means of the avidin-biotin peroxidase complex technique. Sixteen to seventeen days post inoculation with either viable or formalin-inactivated NTHi, endotoxin could be localized in both the middle and inner ear at a time when the middle ear was culture negative. Our data demonstrate that endotoxin shed by NTHi during otitis media penetrates the inner ear and binds to both tissue components and inflammatory cells in both the middle and inner ear.

Effect of tumor necrosis factor alpha and interleukin 1-alpha on the adherence of Streptococcus pneumoniae to chinchilla tracheal epithelium.

The trachea whole organ perfusion technique was used to study the effect of tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) on the adherence of otitis media pathogen Streptococcus pneumoniae (Spn) type 6A. Tracheas were removed from chinchillas and divided equally. One-half trachea was activated by incubation with 1-10 ng/ml of either TNF alpha or IL-1 alpha prior to the addition of Spn 6A to the organ culture perfusion chamber. Colony forming units (cfu) of Spn/millimeter trachea were determined for activated tracheas and controls. Dose response and kinetics data were generated for each cytokine. The specificity of each reaction was determined by neutralization studies with specific anti-cytokine antibodies. The data indicate that both TNF alpha and IL-1 alpha increase the adherence of Spn to the respiratory epithelium of this tubal organ and suggest a mechanism which may facilitate enhanced adherence in vivo and thereby contribute to the pathogenesis of otitis media and other upper respiratory tract diseases.

Effect of lacto-N-neotetraose, asialoganglioside-GM1 and neuraminidase on adherence of otitis media-associated serotypes of Streptococcus pneumoniae to chinchilla tracheal epithelium.

The adherence of Streptococcus pneumoniae (Spn) otitis media-associated serotypes 3, 6A and 14 to ciliated chinchilla respiratory epithelium was investigated using a whole organ perfusion technique. We demonstrated that Spn adhere to chinchilla tracheal epithelium within 30 min and exhibit saturation kinetics indicating that the effect being observed is receptor mediated. Inhibition of adherence was achieved by prior incubation of Spn with lacto-N-neotetraose (LNnT) or asialoIganglioside GM1 (aGM1), recognized by glycoconjugate analogs of known Spn receptors. NeurIaminidase treatment of the tracheae increased Spn adherence in vitro and reversed the inhibition effect of LNnT suggesting that neuraminidase treatment resulted in an increase in the number of available receptors for Spn. The chinchilla trachea organ perfusion culture system used in this study imitates eustachian tube conditions more closely than isolated cell culture systems and is a useful model for investigating the role of Spn adherence in vitro in the pathogenesis of OM.

Relationship of endotoxin to tumor necrosis factor-alpha and interleukin-1 beta in children with otitis media with effusion.

Sixty-five middle ear effusions and paired sera from 41 children with chronic otitis media with effusion were assayed for endotoxin and for tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in order to establish whether a correlation exists between the concentrations of endotoxin and of these cytokines. Endotoxin concentration was determined by means of a chromogenic limulus amebocyte lysate assay, and the cytokine concentration by means of a quantitative enzyme-linked immunosorbent assay. Forty percent of the effusions had detectable levels of endotoxin, with a mean concentration of 2.9 +/- 7.8 endotoxin units per milligram of total protein. The mean concentration of TNF-alpha was 1.24 +/- 3.1 pg/mg total protein, and that of IL-1 beta was 18.79 pg/mg total protein. A strong, statistically significant correlation exists between the concentrations of endotoxin and TNF-alpha (r = .89) and IL-1 beta (r = .72). The data indicate that endotoxin may contribute to the pathogenesis of chronic otitis media with effusion by stimulating the sustained production of TNF-alpha and IL-1 beta in the middle ear.

Evaluation of the virulence of nontypeable Haemophilus influenzae lipooligosaccharide htrB and rfaD mutants in the chinchilla model of otitis media.

Considerable evidence has implicated nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, two NTHi LOS-deficient mutants have been created and described. Strain 2019-DK1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain 2019-B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. Each strain's ability to colonize the nasopharynx and to induce OM subsequent to transbullar inoculation was evaluated in the chinchilla model. Nasopharyngeal colonization data indicate that the parent strain and both mutants are able to colonize the nasopharynx and exhibit comparable clearance kinetics. Compared with the parent and each other, however, the mutants demonstrated marked differences in virulence regarding their relative abilities to induce OM and persist in the middle ear post-transbullar inoculation. Strain B29 required a 3-log-greater dose to induce OM than the parent strain and did not exhibit evidence of sustained multiplication but persisted for the same duration as the parent. Conversely, strain-DK1, even when inoculated at a dose 4 logs greater than the parent dose, was eliminated from the middle ear 72 h after challenge. A comparison of the relative pathogenicities of these isolates provides the opportunity to address fundamental questions regarding the contribution of LOS to pathogenesis issues at the molecular level. Specifically, the impact of these LOS gene disruptions on OM pathogenesis can be defined and may thus provide potential new targets for future protection and intervention strategies.

Tumor necrosis factor during experimental lipopolysaccharide-induced otitis media.

Studies in our laboratory and others have indicated that endotoxin is present in a high percentage of human middle ear effusions, including those that are culture negative. Endotoxin has been identified as one of the most potent inducers of tumor necrosis factor (TNF), but this relationship has not been investigated in regard to the pathogenesis of otitis media. The purpose of this study was to determine whether endotoxin induces the production of TNF in the middle ear. Otitis media was induced in chinchillas by the injection of isolated lipopolysaccharide and middle ear fluids (MEFs) and serum harvested and assayed for TNF content by means of a cytolytic assay using L-929 cells and the Cell Titer 96 assay (Promega, Madison, WI). The MEF pools' TNF concentration ranged from 200 to 1,100 pg/mL MEF. Serum pools did not contain any detectable TNF. The results of the present study suggest that endotoxin induces TNF production locally in the middle ear cleft, which may contribute to the pathogenesis of otitis media via any number of established inflammatory pathways.

The effect of antibiotic treatment on the release of endotoxin during nontypable Haemophilus influenzae-induced otitis media in the chinchilla.

The gram negative bacteria, nontypable Haemophilus influenzae (NTHi) was used to induce otitis media in a total of 18 chinchillas. Three days post-inoculation, three cohorts of 6 chinchillas each were treated daily for four days with either ceftriaxone, chloramphenicol, or diluent without antibiotics. Middle ear fluid (MEF) was obtained daily, assayed for endotoxin content by means of the chromogenic limulus amebocyte lysate assay, and concentration of the NTHi/mL MEF determined by standard plate count. The endotoxin concentration per mL MEF from both the antibiotic treated cohorts decreased during the observation period, but increased in the MEF of the untreated control group. The data indicate that, unlike the dramatic increase in endotoxin concentration, after antibiotic treatment in the cerebrospinal fluid (CSF) during experimental Haemophilus influenzae-induced meningitis, there is no demonstrable sustained release of endotoxin in the middle ear subsequent to antibiotic treatment during experimental otitis media.

Immunization with outer membrane protein P6 from nontypeable Haemophilus influenzae induces bactericidal antibody and affords protection in the chinchilla model of otitis media.

The role of nontypeable Haemophilus influenzae (NTHi) outer membrane protein (OMP) P6 in the pathogenesis of otitis media (OM) has not been defined. OMPs, fimbriae, pili, and lipooligosaccharide are several types of surface antigens of NTHi that are currently being evaluated as potential vaccine candidates. P6 is antigenically conserved among both nontypeable and type b H. influenzae strains and elicits bactericidal as well as protective antibodies; however, initial evaluation of a vaccine mixture of P6 combined with other NTHi OMPs failed to induce bactericidal antibody or protection in the chinchilla model of OM. We undertook an assessment of the ability of immunization with isolated P6 lipoprotein alone to confer protection. Chinchillas were immunized with P6 and challenged 10 days after the final immunization with either 3 x 10(3) CFU of NTHi delivered directly into the middle ear to induce OM or 5 x 10(8) CFU of NTHi delivered intranasally to establish nasopharyngeal colonization. All immunized animals responded with elevated serum titers of anti-P6 antibody, which also demonstrated bactericidal activity against homologous as well as a heterologous NTHi isolate. By 14 days post-transbullar challenge, the number of chinchillas with middle ear fluid and the incidence of NTHi culture-positive middle ear fluids were reduced 48 and 51%, respectively, in the P6-immunized chinchillas relative to the sham-immunized cohort. Nasopharyngeal colonization levels were comparable in the two cohorts. These data demonstrate that active immunization with P6 results in the production of NTHi-specific bactericidal antibody in the chinchilla and also affords a reduction in the incidence of NTHi-induced OM; however, parenteral immunization does not appear to affect the extent or duration of nasopharyngeal colonization by NTHi.