Outer Membrane Protein P2 Research Articles

Pore Characteristics of Nontypeable Haemophilus influenzae Outer Membrane Protein P5 in Planar Lipid Bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22°C, LDS-P5 formed narrow pores (58 ± 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 ± 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures ≥40°C; the process took 4–6 h at 40°C or 35–45 min at 42°C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.

Construction of a mutant and characterization of the role of the vaccine antigen P6 in outer membrane integrity of nontypeable Haemophilus influenzae.

Outer membrane protein P6 is the subject of investigation as a vaccine antigen to prevent infections caused by nontypeable Haemophilus influenzae, which causes otitis media in children and respiratory tract infections in adults with chronic lung disease. P6 induces protective immune responses in animal models and is the target of potentially protective immune responses in humans. P6 is a 16-kDa lipoprotein that shares homology with the peptidoglycan-associated lipoproteins of gram-negative bacteria and is highly conserved among strains of H. influenzae. To characterize the function of P6, an isogenic mutant was constructed by replacing the P6 gene with a chloramphenicol resistance cassette. The P6 mutant showed altered colony morphology and slower growth in vitro than that of the parent strain. By electron microscopy, the P6 mutant cells demonstrated increased size, variability in size, vesicle formation, and fragility compared to the parent cells. The P6 mutant showed hypersensitivity to selected antibiotics with different mechanisms of action, indicating increased accessibility of the agents to their targets. The P6 mutant was more sensitive to complement-mediated killing by normal human serum. Complementation of the mutation in trans completely or partially restored the phenotypes. We concluded that P6 plays a structural role in maintaining the integrity of the outer membrane by anchoring the outer membrane to the cell wall. The observation that the absence of expression of P6 is detrimental to the cell is a highly desirable feature for a vaccine antigen, supporting further investigation of P6 as a vaccine candidate for H. influenzae.

Identification of human T-cell epitopes and highly immunogenic analog peptides on the non-typeable Haemophilus influenzae P6 outer membrane protein

P6 outer membrane protein is one of the candidates for a vaccine formulation against non-typeable Haemophilus influenzae (NTHi) infection. However, otitis-prone children who have recurrent episodes of acute otitis media due to NTHi fail to respond adequately to P6. An innovative approach to vaccination is therefore required to augment such children's immune response. To develop an effective peptide vaccine, we established P6-specific CD4 + T-cell lines (TCLs) restricted by the human histocompatibility leukocyte antigen (HLA)-DR9 molecule, and revealed a human T-cell epitope on P6 and its core peptide sequence (p77–85; EYNIALGQR). Furthermore, we found that 3 analog peptides, E77D (the substitution of E at position 77 with D), N79G, and R85K, induced high proliferative responses as well as marked cytokine production when compared to the T-cell epitope peptide. These peptides may be candidates for a peptide vaccine formulation effective against NTHi infections, even in otitis-prone children.

Maternal intranasal immunization with outer membrane protein P6 maintains specific antibody level of derived offspring

Acute otitis media is one of the most common infectious diseases in children younger than 2 years of age. Immunological studies in young children have revealed that immature antibody-responses to major pathogens, such as S. pneumoniae, H. influenzae, would cause the vulnerability to upper respiratory tract infections. Thus, it is very important to induce effective protective immunity among children younger than 2 years of age. In this study, we evaluated the capacity of maternal immunization with P6 of H. influenzae to evoke specific antibody to P6 and to transfer it to offspring. We intranasally immunized mother mice with P6 and investigated the induction of specific antibody in sera and breast milk. The specific antibody among offspring delivered by immunized mother was also investigated according to the nursing status to evaluate the importance of breast feedings by immunized mothers. Anti-P6 specific IgG in sera were high at delivery and maintained during nursing periods among P6-immunized mother mice. Anti-P6 specific IgG were predominantly induced in breast milk. IgG subclass induced in sera and breast milk from P6-immunized mother mice were IgG2b, followed by IgG1 and IgG2a subclass. Offspring delivered by P6-immunized mothers had anti-P6 specific IgG in sera at the birth. The levels of anti-P6 specific IgG in sera from offspring breast-fed by P6-immunized mothers were then increased until day 14 and then decreased on day 21. The anti-P6 specific IgG in sera from offspring breast-fed by sham-immunized mothers were rapidly decreased after birth. The current findings strongly suggest that maternal intranasal immunization with P6 would be an attractive strategy against NTHi infections during early childhood. It can supply protective antibodies via transplacental transfer during pregnancy and via breast milk after birth.

Impaired Alveolar Macrophage Response to Haemophilus Antigens in Chronic Obstructive Lung Disease

Interactions of nontypeable Haemophilus influenzae (NTHI) with macrophages are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immunologic mechanisms that mediate NTHI-macrophage inflammation are poorly understood. Outer membrane protein (OMP) P6 and lipooligosaccharide (LOS) of NTHI are potent immunomodulators. We theorized that alveolar macrophages in COPD possess fundamental immune defects that permit NTHI to evade host responses. To test this hypothesis, we obtained human alveolar and blood macrophages from exsmokers with COPD, exsmokers without COPD, and nonsmokers. Alveolar and blood macrophages from each donor were incubated with purified LOS and OMP P6 and with OMP P2 and the total outer membrane preparation (0.1-1 microg/ml). Supernatants (24 h) were assayed for IL-1beta, TNF-alpha, IL-10, IL-12, and IL-8 by multianalyte multiplexed flow cytometry. Comparative induction of COPD and non-COPD alveolar macrophages by LOS and OMP P6 revealed diminished IL-8, TNF-alpha, and IL-1beta responses of COPD alveolar macrophages (p < or = 0.03 for each). COPD alveolar macrophages also had diminished responses to total outer membrane (p < or = 0.03 for each). In contrast, COPD blood macrophages had no significant differences among donor groups in IL-8, TNF-alpha, or IL-1beta responsiveness to NTHI antigens. Diminished IL-12 responses of COPD blood macrophages to NTHI antigens, compared with nonsmokers, could not be independently dissociated from group differences in age and pack-years. These findings support a paradigm of defective immune responsiveness of alveolar macrophages, but not blood macrophages, in COPD.

Distribution of Bacterial Proteins in Biofilms Formed by Non-typeable Haemophilus influenzae

The ability to preserve the fragile ultrastructural organization of bacterial biofilms using cryo-preparation methods for electron microscopy has enabled us to probe sections through non-typeable Haemophilus influenzae (NTHi) biofilms and determine the localization of NTHi-specific lipooligosaccharide (LOS) and proteins within these structures. Some of the proteins we examined are currently being considered as candidates for vaccine development, so it is important that their distribution and accessibility within the biofilms formed by NTHi be determined. We have localized LOS to the extracellular matrix (ECM) of the biofilm and the P6 outer membrane protein to the membrane of what appear to be viable bacteria within the biofilm. The Hap and HWM1/HMW2 adhesive proteins were associated with bacteria within the biofilm and were present in the biofilm ECM. The IgA1 protease is a secreted protein that was also associated with NTHi in the biofilm and was in the ECM, but was more concentrated in the top region of the biofilm, suggesting a role in protecting biofilm bacteria from antibody attack.

Safety and Efficacy of Nasal Application of CpG Oligodeoxynucleotide as a Mucosal Adjuvant

Nasal vaccination is an effective regimen to prevent upper respiratory infections. An appropriate adjuvant is required for the development of a nasal vaccine. The safety and efficacy of CpG oligodeoxynucleotide (ODN) as a mucosal adjuvant was examined. Mice were nasally administered various doses of CpG ODN weekly, a total of three times. Histologic changes in the spleen and the nasal mucosa were examined, and the alterations in cell subpopulations were analyzed by flow cytometry. In addition, the mice were nasally immunized with P6 outer membrane protein of nontypeable Haemophilus influenzae (NTHi) and CpG ODN, and P6-specific immune responses were examined. No inflammation or tissue damage was observed locally or systemically after nasal administration, even with a high dose of CpG ODN. A high dose of CpG ODN induced an increase in CD8+ T cells in the nasal mucosa and B cells in the spleen. When CpG ODN was coadministered with P6, P6-specific mucosal and systemic immune responses were effectively induced, since high levels of the specific IgA and IgG were detected in the nasal wash and serum, respectively. These findings suggest that CpG ODN is a safe and effective mucosal adjuvant. Further, nasal vaccination with P6 and CpG ODN might be an effective regimen to prevent upper respiratory infections.

Antibodies directed at a conserved motif in loop 6 of outer membrane protein P2 of nontypeableHaemophilus influenzaerecognize multiple strains in immunoassays

The P2 porin is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae. Analysis of P2 sequences from a limited number of strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. We have previously shown that antibodies raised against the loop 6 sequence of P2 from strain 5657 are bactericidal against multiple isolates. In this study, we determined the nucleotide sequence of the loop 6 region of the P2 molecule from 108 strains of nontypeable H. influenzae in order to assess more rigorously the degree of conservation of loop 6. Based on this analysis, we identified a conserved sequence, different from that of strain 5657, that occurs in approximately one-third of the strains sequenced. To assess the potential of this peptide as a vaccine antigen, antibodies raised to a multiple antigenic peptide corresponding to this sequence were characterized with respect to specificity for the P2 molecule and reactivity with heterologous strains in immunoblot assay, flow cytometry and bactericidal assays. Antibodies were reactive to the P2 molecule of 16 of 20 strains tested by immunoblot assay. Antibodies recognized nine of the 20 strains in a flow cytometry assay, and 13 of 20 demonstrated complement-mediated killing in bactericidal assays. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.

Eustachian tube possesses immunological characteristics as a mucosal effector site and responds to P6 outer membrane protein of nontypeable Haemophilus influenzae

The eustachian tube (ET) plays an important role in the pathogenesis of otitis media (OM). To better understand its biology and to develop a nasal vaccine for preventing OM, mucosal lymphocytes in the ET were analyzed, and the ET's immunological function was investigated. Mononuclear cells were isolated from murine ET, and lymphocyte subsets were analyzed by flow cytometry. Antibody-producing cells were determined by enzyme-linked immunospot assay. The expression of cytokine mRNA in ET CD4 + T cells was determined by RT-PCR. Results in naive mice showed that the ET contained many immunocompetent cells, including a relative large number of IgA-producing cells and Th2 cytokine-expressing T cells. Next, we investigated antigen-specific immune responses in the ET. Mice were immunized intranasally with the P6 outer membrane of nontypeable Haemophilus influenzae (NTHi) and cholera toxin (CT), and P6-specific immune responses in the ET were examined. P6-specific IgA producing cells markedly increased in the ET. Moreover, in vitro stimulation with P6 of purified CD4 + T cells from immunized mice resulted in the proliferation of CD4 + T cells that expressed Th2 cytokine mRNA. These results indicate that the ET might be characterized as a mucosal effector site and that antigen-specific IgA and Th2 immune responses could be induced in the ET by intranasal immunization. These findings suggest that the ET might be a key immunological organ in the pathogenesis of OM, and in the development of a nasal vaccine.

Investigation of nontypeable Haemophilus influenzae outer membrane protein P6 as a new carrier for lipooligosaccharide conjugate vaccines

Nontypeable Haemophilus influenzae (NTHi) outer membrane protein P6 was used as a new protein carrier for NTHi detoxified lipooligosaccharide (dLOS) conjugates due to its conservation and potential to elicit bactericidal antibodies. P6 was covalently conjugated to dLOS of strain 9274 through adipic acid dihydrazide with different ratios of dLOS to P6, which resulted in two conjugate formulations with weight ratios of dLOS to P6 of 3.7 for dLOS–P6 (I) and 1.6 for dLOS–P6 (II). Binding activity of the conjugates was examined by an enzyme-linked immunosorbent assay with mouse monoclonal antibodies specific to LOS and P6 and a rabbit anti-P6 serum. The results showed that the conjugates bound not only to the LOS antibody but also to both P6 antibodies, suggesting that the conjugates retained epitopes of both LOS and P6 antigens. Animal studies revealed that dLOS–P6 (II) induced high levels of anti-LOS and anti-P6 IgGs in mice and rabbits. However, dLOS–P6 (I) induced lower levels of anti-LOS IgGs in mice and rabbits and anti-P6 IgGs in rabbits with no anti-P6 IgGs in mice. In addition, all rabbit, but not mouse, antisera elicited by the conjugates showed bactericidal activity against the homologous strain, and two of them elicited by each conjugate plus Ribi adjuvant showed cross-bactericidal activity against three of five major serotype stains. These data indicate that P6 could serve as an effective carrier for dLOS or other carbohydrate conjugates and that the ratio of carbohydrate to P6 might contribute to immune responses in vivo.

Outer Membrane Protein P6 of NontypeableHaemophilus influenzaeIs a Potent and Selective Inducer of Human Macrophage Proinflammatory Cytokines

Interactions of nontypeable Haemophilus influenzae (NTHI) with human macrophages contribute to the pathogenesis of NTHI-induced infection in humans. However, the immunologic mechanisms that initiate and perpetuate NTHI-mediated macrophage responses have not been well explored. Outer membrane protein (OMP) P6 is a conserved lipoprotein expressed by NTHI in vivo that possesses a Pam(3)Cys terminal motif, characteristic of immunoactive bacterial lipoproteins associated with Toll-like receptor signaling. We theorized that OMP P6 is a potent immunomodulator of human macrophages. To test this hypothesis, we purified OMP P6 as well as OMP P2, the predominant NTHI outer membrane protein, and lipooligosaccharide (LOS), the specific endotoxin of NTHI, from NTHI strain 1479. Human blood monocyte-derived macrophages, purified from healthy donors, were incubated with each outer membrane constituent, and cytokine production of macrophage supernatants interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), IL-10, IL-12, and IL-8 was measured. OMP P6 selectively upregulated IL-10, TNF-alpha, and IL-8. While OMP P6 (0.1 mug/ml for 8 h) elicited slightly greater concentrations of IL-10, it resulted in over ninefold greater concentrations of TNF-alpha and over fourfold greater concentrations of IL-8 than did OMP P2. OMP P6 at doses as low as 10 pg/ml was still effective at induction of macrophage IL-8, while OMP P2 and LOS were not. OMP P6 of NTHI is a specific trigger of bacteria-induced human macrophage inflammatory events, with IL-8 and TNF-alpha as key effectors of P6-induced macrophage responses.

Differential Expression of Porins OmpP2A and OmpP2B of Haemophilus ducreyi

Haemophilus ducreyi is a strict human pathogen and the causative agent of the sexually transmitted disease chancroid. The genome of the human-passaged strain of H. ducreyi (35000HP) contains two homologous genes whose protein products have estimated molecular masses of 46 and 43 kDa. A comparative analysis of the deduced amino acid sequences revealed that these proteins share 27 to 33% identity to the outer membrane protein P2 (OmpP2), a major porin of Haemophilus influenzae. Therefore, these proteins have been designated OmpP2A and OmpP2B, respectively. The detection of ompP2A and ompP2B transcripts by reverse transcriptase PCR indicated that these genes were independently transcribed in H. ducreyi 35000HP. Western blot analysis of outer membrane proteins isolated from a geographically diverse collection of H. ducreyi clinical isolates revealed that OmpP2A and OmpP2B were differentially expressed among these strains. Although PCR analysis suggested that ompP2A and ompP2B were conserved among the strains tested, the differential expression observed was due to nucleotide additions and partial gene deletions. Purified OmpP2A and OmpP2B were isolated under nondenaturing conditions, and subsequent analysis demonstrated that these two proteins exhibited porin activity. OmpP2A and OmpP2B are the first porins described for H. ducreyi.

Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2.

The expression of inducible antimicrobial peptides, such as human beta-defensin-2 (HBD-2) by epithelia, comprises a component of innate pulmonary defenses. We hypothesized that HBD-2 induction in airway epithelia is linked to pattern recognition receptors such as the Toll-like receptors (TLRs). We found that primary cultures of well-differentiated human airway epithelia express the mRNA for TLR-4, but little or no MD-2 mRNA, and display little HBD-2 expression in response to treatment with purified endotoxin +/- LPS binding protein (LBP) and soluble CD14. Expression of endogenous MD-2 by transduction of airway epithelial cells with an adenoviral vector encoding MD-2 or extracellular addition of recombinant MD-2 both increased the responses of airway epithelia to endotoxin + LBP and sCD14 by >100-fold, as measured by NF-kappaB-luciferase activity and HBD-2 mRNA expression. MD-2 mRNA could be induced in airway epithelia by exposure of these cells to specific bacterial or host products (e.g., killed Haemophilus influenzae, the P6 outer membrane protein from H. influenzae, or TNF-alpha + IFN-gamma). These findings suggest that MD-2, either coexpressed with TLR-4 or secreted when produced in excess of TLR-4 from neighboring cells, is required for airway epithelia to respond sensitively to endotoxin. The regulation of MD-2 expression in airway epithelia and pulmonary macrophages may serve as a means to modify endotoxin responsiveness in the airway.

Intranasal immunization with outer membrane protein P6 and cholera toxin induces specific sinus mucosal immunity and enhances sinus clearance of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is one of the leading pathogens in sinusitis. One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is an attractive candidate for a subunit bacterial vaccine. In this study, we characterized normal sinus mucosa (SM) and investigated the potential of intranasal immunization with P6 and cholera toxin (CT) for induction of mucosal protective immunity against NTHi in the maxillary sinuses of rats. Intranasal immunization induced P6-specific sinus mucosal and systemic immunological responses, mainly of the IgA and IgG isotype. The protective effect of intranasal immunization was demonstrated by enhancement of sinus clearance of NTHi. The present study showed that unilateral intranasal immunization has a capacity to induce protective immunity against NTHi in the bilateral maxillary sinuses. Systemic administration of the vaccine did not affect sinus clearance of NTHi. These findings suggest that a nasal vaccine might be useful for preventing sinusitis.

Natural materno-fetal transfer of antibodies to PspA and to PsaA.

PspA and PsaA are Streptococcus pneumoniae surface proteins and potential pneumococcal vaccine antigens. The aim of this study was to characterize the transplacental transfer of antibodies to PspA and to PsaA. Paired mother and cord blood sera were obtained at delivery from 28 women. Concentrations of antibodies against PspA, PsaA, tetanus toxoid (vaccine-induced antibodies) and P6-outer membrane protein (OMP) of nontypeable Haemophilus influenzae were determined by ELISA. Antibodies to PspA of the IgG, IgG1 and IgG2 antibodies were also determined. The geometric mean percentage (GM%) of the paired infant:mother antibody were calculated. The GM% of the infant:mother antibody concentrations against PspA, PsaA and P6-OMP antibodies were 64.7% (3.3 micro g/ml in infants vs. 5.1 micro g/ml in mothers), 50.4% (6.8 micro g/ml vs. 13.5 micro g/ml) and 66.7% (5.6 micro g/ml vs. 8.4 micro g/ml), respectively; the GM% of antibodies against tetanus toxoid was 104.5% (4.6 micro g/ml vs. 4.4 micro g/ml). Transplacental transfer of IgG1 was more efficient than that of IgG2 (approximately 120%vs. 65%). A transplacental transfer of antibodies to PspA and to PsaA exist. Moreover, these data suggest an active placental transfer of IgG1 antibodies to PspA since the concentration of these antibodies were consistently higher in cord sera than in the mother's sera.

Epitope analysis of the P6 outer membrane protein of non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHI) is a common pathogen of upper airway infection, such as tonsilitis, pharyngitis, sinusitis and acute otitis media. P6 is an outer membrane protein that is highly conserved among strains of NTHI and is the target antigen of protective antibody. P6 is referred to as one of the leading vaccine antigens to NTHI infection. In recent years, attention has been paid to the vaccine treatment using peptides containing T cell and B cell epitopes. It is thought that peptide vaccine can guide a more effective immune response and has fewer side effects. The aim of our study was to investigate the epitope of P6. We established CD4+ T cell line specific to P6 using the dendritic cells (DC) as antigen presenting cells. The lymphocyte proliferative response to overlapping peptide of P6 was analyzed. The T cell line specific to P6 responded to the eighth overlapping peptide. The proliferative response of this T cell line was restricted by HLA-DR9. In our study, one of the T cell epitopes existed in the eighth overlapping peptide of P6. To DR9 holder, the eightth overlapping peptide of P6 was considered to be one of the candidates for peptide vaccine to NTHI infection.

Circulating IgA, IgG, and IgM class antibody against Haemophilus parainfluenzae antigens in patients with IgA nephropathy.

We previously demonstrated a close relationship between the outer membranes of Haemophilus parainfluenzae (HP) antigens (OMHP) and IgA nephropathy (IgAN). Our objective was to clarify the relationship among IgA, IgG, and IgM class antibody against OMHP in the sera of 44 patients with IgAN and 62 patients with other glomerular diseases (OGD) by ELISA. Patients with IgAN showed a significantly higher level of IgA antibodies (P less than 0.0005) and IgG antibodies (P less than 0.001) against OHMP, than did patients with OGD. Positive correlations were observed between IgA and IgG antibodies, between IgA and IgM antibodies, and between IgG and IgM antibodies against OMHP in the sera of patients with IgAN. Immunoblotting showed that IgA, IgG, or IgM antibodies against OMHP in the sera of all patients with IgAN bound to components of OMHP. Amino acid sequences of three components of OMHP recognized by the sera from patients with IgAN revealed homology with those reported for outer membrane protein (OMP) P6 precursor, OMP P5, and P2 porin protein of H. influenzae. Results suggest that patients with IgAN have glomerular deposits of OMP P6 precursor, OMP P5, or P2 porin protein of HP, and a specific increase in the production of IgA antibodies against OMHP via polyclonal activation against these, with switching of production from one isotype to another, e.g. from IgM to IgA.

Prevention of otitis media by nasal vaccine—an animal study

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen of otitis media. P6 outer membrane protein is a common antigen to all strains and is considered as a candidate for mucosal vaccine. To elucidate the possibility of developing a nasal vaccine against NTHi and to investigate mucosal immune responses in the middle ear, mice were immunized intranasally with the P6 outer membrane protein of NTHi, and P6-specific immune responses in the middle ear mucosa were examined. P6-specific IgA antibody titre in ear wash was significantly elevated. Mononuclear cells were isolated from middle ear mucosa, and an increase in P6-specific IgA-producing cells was shown with ELISPOT assay. In addition, in vitro stimulation with P6 resulted in proliferation of purified CD4+ T cells from immunized mice, and these T cells expressed Th2 cytokine mRNA. Moreover, bacterial challenge with live NTHi into the middle ear was performed after immunization, and the clearance of NTHi from the middle ear was enhanced by nasal immunization. These results indicate that P6-specific protective immunity was induced in middle ear mucosa by nasal immunization. These findings suggest that nasal vaccine is useful for preventing otitis media.

Intranasal immunization with recombinant outer membrane protein P6 induces specific immune responses against nontypeable Haemophilus influenzae

Objective: Nontypeable Haemophilus influenzae (NTHi) is one of the leading causative pathogens for otitis media. The outer membrane protein P6 of NTHi is highly conserved among the strains and is an attractive candidate for a preventive vaccine. However, for the production of a relatively small amount P6 containing lipopolysaccharides, the development of a recombinant version of this protein is required. This study was designed to investigate the specific mucosal immunity induced by intranasal immunization of recombinant P6 (rP6) with cholera toxin (CT). Methods: BALB/c mice were immunized with of rP6 (30 μg) and CT (2 μg) intranasally every 2 days for 2 weeks. Anti-rP6 specific IgG, IgA and IgM antibodies and the subclass of anti-rP6 specific IgG antibody were determined by enzyme linked immunosorbent assay (ELISA). Anti-rP6 specific IgA in nasopharyngeal washings were also determined by ELISA. Nasopharyngeal clearance of inoculated NTHi after the intranasal immunization were assessed. All statistical differences between the two groups were assessed by ANOVA parametric test. Results: Intranasal immunization with rP6 and CT evoked rP6-specific mucosal IgA immune response as well as the systemic IgG immune response against rP6 and enhanced nasopharyngeal clearance of inoculated live NTHi. Conclusion: These results indicate the good immunogenicities of rP6 to induce specific immune responses against NTHi. Intranasal immunization with rP6 will be an effective approach to protect infections of NTHi.

Sequence stability of the gene encoding outer membrane protein P2 of nontypeable Haemophilus influenzae in the human respiratory tract.

Nontypeable Haemophilus influenzae (NTHI) is an important cause of lower respiratory tract infections in patients with chronic obstructive pulmonary disease. Recent findings suggest that the major outer membrane protein P2 should be reconsidered as a vaccine candidate for NTHI. A P2-based vaccine would require a relative degree of sequence stability of the gene encoding P2 (ompP2) during colonization. To characterize the sequence stability of ompP2 during colonization of the human respiratory tract, ompP2 genes from 13 sets of isolates that persisted in patients with chronic obstructive pulmonary disease (mean colonization, 7 months) were sequenced. In 9 sets of isolates, ompP2 did not change. Sequence changes were noted in 4 sets of isolates. Most of these changes occurred within areas of repetitive DNA, suggesting that this type of DNA has a role in antigenic variation of P2. The sequence of ompP2 is relatively stable during persistence of NTHI in the human host.