Outer Membrane Protein P2 Research Articles

Serum concentrations of antibodies against outer membrane protein P6 and its T-and B-combined antigenic epitopes of nontypeable Haemophilus influenzae in children and adults of different ages

Objective To measure the concentrations of antibodies against outer membrane protein P6 and its T-and B-combined antigenic epitopes of nontypeable Haemophilus influenzae （ NTHi） in children and adults of different ages and to evaluate the differences among different subjects for further investigation on NTHi multiple antigenic peptide vaccine .Methods A prokaryotic expression system was established to ex-press the recombinant outer membrane protein P 6 of NTHi.The expressed protein was purified by using Ni-NTA affinity chromatography .T-and B-cell epitopes in protein P6 were predicted with Epitope prediction software 1.0 and ANTIGENIC program and were used to synthesize T-and B-combined antigenic epitopes .A total of 605 subjects aged from 1 day to 103 years old were recruited from October 2013 to March 2014 .Ser-um concentrations of antibodies against protein P 6 and its T-and B-combined antigenic epitopes were meas-ured by using ELISA .Mann-Whitney U test was used to analyze the differences between groups .Pearson product-moment correlation coefficient was used for correlation analysis .Results Four T-and B-combined antigenic epitopes including P 6-2, P6-61, P6-95 and P6-122 were predicted and synthesized .The levels of antibodies against NTHi P6 and P6-2, P6-61, P6-95 and P6-122 were significant lower in the <1 months group than those in the 1-6 months group （all P〈0.001） and 7 months-3 years group （all P〈0.001）.Three groups including 7 months-3 years group , 4-6 years group and 7-14 years group showed significant differ-ences regarding to the antibodies levels , among the 7 months-3 years group showed the highest levels , fol-lowed by the 4-6 years group and the 7-14 years group.However, no significant difference was found be-tween other adjacent groups .Concentrations of antibodies against P 6-2, P6-61, P6-95 and P6-122 were pos-itively correlated with the level of antibody against P 6 （P〈0.0001）.Conclusion The distribution of anti-bodies against T-and B-combined antigenic epitopes in P6 was highly in accord with those against P6, which indicated good immunogenicity of those epitopes .The highest antibodies levels were found in subjects aged 7 months to 3 years old , which might correlate with the high risk of NTHi infection at that stage . Key words: Nontypeable Haemophilus influenza; Epitope; Antibody

Binding of human factor H to outer membrane protein P5 of non-typeable Haemophilus influenzae contributes to complement resistance.

Non-typeable Haemophilus influenzae is an opportunistic pathogen of the human upper respiratory tract and is often found to cause inflammatory diseases that include sinusitis, otitis media and exacerbations of chronic obstructive pulmonary disease. To persist in the inflammatory milieu during infection, non-typeable H. influenzae must resist the antimicrobial activity of the human complement system. Here, we used Tn-seq to identify genes important for resistance to complement-mediated killing. This screen identified outer membrane protein P5 in evasion of the alternative pathway of complement activation. Outer membrane protein P5 was shown to bind human complement regulatory protein factor H directly, thereby, preventing complement factor C3 deposition on the surface of the bacterium. Furthermore, we show that amino acid variation within surface-exposed regions within outer membrane P5 affected the level of factor H binding between individual strains.

The outer membrane protein P2 (OmpP2) of Haemophilus parasuis induces proinflammatory cytokine mRNA expression in porcine alveolar macrophages

Porins, expressed by Gram-negative bacteria, have several biological effects on host tissues or cells. The outer membrane protein P2 (OmpP2), a member of the porin family, has been identified as a multifunctional protein involved in the pathogenicity of Haemophilus parasuis. In the present study, it was shown that OmpP2 (0.5–10μg/mL) from H. parasuis Nagasaki strain up-regulated mRNA expression of interleukin (IL)-1α, IL-1β, IL-6 and IL-8 in porcine alveolar macrophages (PAM, 3D4/31) in vitro, in a dose-dependent manner. Moreover, OmpP2 porin induced a more prolonged cytokine response in PAM than that of the lipooligosaccharide (LOS) from this microorganism. The data demonstrate that H. parasuis OmpP2 can stimulate proinflammatory cytokine mRNA expression, suggesting that this particular porin might play an important role in the pathogenesis of disease caused by H. parasuis.

Outer Membrane Protein P5 Is Required for Resistance of Nontypeable Haemophilus influenzae to Both the Classical and Alternative Complement Pathways

The complement system is an important first line of defense against the human pathogen Haemophilus influenzae. To survive and propagate in vivo, H. influenzae has evolved mechanisms for subverting this host defense, most of which have been shown to involve outer surface structures, including lipooligosaccharide glycans and outer surface proteins. Bacterial defense against complement acts at multiple steps in the pathway by mechanisms that are not fully understood. Here we identify outer membrane protein P5 as an essential factor in serum resistance of both H. influenzae strain Rd and nontypeable H. influenzae (NTHi) clinical isolate NT127. P5 was essential for resistance of Rd and NT127 to complement in pooled human serum. Further investigation determined that P5 expression decreased cell surface binding of IgM, a potent activator of the classical pathway of complement, to both Rd and NT127. Additionally, P5 expression was required for NT127 to bind factor H (fH), an important inhibitor of alternative pathway (AP) activation. Collectively, the results obtained in this work highlight the ability of H. influenzae to utilize a single protein to perform multiple protective functions for evading host immunity.

Improving patient care via development of a protein-based diagnostic test for microbe-specific detection of chronic rhinosinusitis.

The hypothesis is that signature bacterial proteins can be identified in sinus secretions via high-throughput, proteomic based techniques. Nontypeable Haemophilus influenzae (NTHI) is the most common bacterial pathogen associated with sinusitis and serves as proof of principle pathogen for identifying biomarkers. In vitro and in vivo studies using proteomic-based analysis of cultures of NTHI and a novel, experimental chinchilla polymicrobial sinusitis model. Nano-liquid chromatography /tandem mass spectrometry (nano-LC-MS/MS) was performed to annotate the secretome from an NTHI biofilm. A model of NTHI-induced sinusitis was developed in a chinchilla, and NTHI proteins were detected in chinchilla secretions. A reference standard RT-PCR-based assay was adapted to allow for sensitivity and specificity testing of the identified signature biomarkers in human patients. Outer membrane proteins P2 (OMP-P2) and P5 (OMP-P5) were identified as promising candidates for the detection of NTHI biofilms and positively detected in nasopharyngeal secretions of chinchillas experimentally infected with NTHI. An RT-PCR based test for the presence of NTHI biofilms demonstrated 100% sensitivity and 100% specificity when tested against eight unique strains commonly found in human bacterial rhinosinusitis. Proteomic analysis was successful in identifying signature proteins for possible use as a biomarker for chronic rhinosinusitis (CRS). OMP-P2 and OMP-P5 were validated as promising candidates and were positively detected from nasopharyngeal secretions from chinchillas experimentally infected with NTHI. Collectively, these data support the use of OMP-P2 and OMP-P5 as biomarkers for a human clinical trial to develop a point-of-care medical diagnostic test to assist in the diagnosis and treatment of CRS.

Absence of high molecular weight proteins 1 and/or 2 is associated with decreased adherence among non-typeable Haemophilus influenzae clinical isolates

High molecular weight (Hmw) proteins 1 and 2, type IV pilin protein (PilA), outer-membrane protein P5 (OmpP5), Haemophilus protein D (Hpd) and Haemophilus adhesive protein (Hap) are surface proteins involved in the adherence of non-typeable Haemophilus influenzae. One hundred clinical isolates were evaluated for the presence of the genes encoding these proteins by PCR and for their adherence capacity (AC) to Detroit 562 nasopharyngeal cells (D562). The majority of isolates were from blood (77/100); other sites were also represented. Confluent D562 monolayers (1.2×10(5) cells per well) were inoculated with standardized minimal infective doses (m.o.i.) of 10(2), 10(3) or 10(4) c.f.u. per well. The AC was categorized as low (<10 %) or high (≥10 %) depending on the percentage of c.f.u. adhering per well. All the isolates evaluated showed adherence: 69/100 (69 %) demonstrated high adherence, while 31/100 (31 %) showed low adherence. Of all the genes evaluated, hmw1A and/or hmw2A were detected in 69/100 (69 %) of isolates. The presence of hmw1A and/or hmw2A was associated with increased adherence to D562 cells (P≤0.001). Dot immunoblots were performed to detect protein expression using mAbs 3D6, AD6 and 10C5. Among the high-adherence isolates (n = 69), 72 % reacted with 3D6 and 21 % with 10C5. Our data indicate that the absence of Hmw1 and/or Hmw2 was associated with decreased adherence to D562 cells.

Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica.

Describing the dual orientation of vaccine candidate P6

Nontypable Haemophilus influenzae (NTHi) are pathogenic bacteria that cause several illnesses, including otitis media (ear infections). The outer membrane protein P6 is currently a top vaccine candidate for NTHi. Past studies have demonstrated that P6 can interact with molecules both inside AND outside of the cell, but its sequence and structure suggests that it does not cross the outer cellular membrane. P6 is a peptidoglycan associated lipoprotein and therefore known to interact with peptidoglycan inside the cell. To demonstrate surface exposure of P6, we utilized flow cytometry and confocal microscopy. In both experiments, whole NTHi cells were incubated with P6 monoclonal antibody and a secondary antibody conjugated to a fluorophore. Fluorescently labeled cells were detected using flow cytometry and visualized via confocal microscopy. Results from our flow cytometry experiments demonstrate that ~17% of NTHi cells express surface exposed P6. Confocal microscopy corroborates that P6 is indeed on the cell surface of intact cells. We conclude that P6 is expressed in two orientations in NTHi: one in which it is surface exposed and able to interact with extracellular antibodies, and one in which it is internally localized and able to interact with peptidoglycan. This study was funded by the Rochester Institute of Technology, NIH NIDCD RO1 08671 (to MEP), and an ASBMB UAN Undergraduate Research Award to Joy Snyder.

MALDI-TOF MS Distinctly Differentiates Nontypable Haemophilus influenzae from Haemophilus haemolyticus

Nontypable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus exhibit different pathogenicities, but to date, there remains no definitive and reliable strategy for differentiating these strains. In this study, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a potential method for differentiating NTHi and H. haemolyticus. The phylogenetic analysis of concatenated 16S rRNA and recombinase A (recA) gene sequences, outer membrane protein P6 gene sequencing and single-gene PCR were used as reference methods. The original reference database (ORD, provided with the Biotyper software) and new reference database (NRD, extended with Chinese strains) were compared for the evaluation of MALDI-TOF MS. Through a search of the ORD, 76.9% of the NTHi (40/52) and none of the H. haemolyticus (0/20) strains were identified at the species level. However, all NTHi and H. haemolyticus strains used for identification were accurately recognized at the species level when searching the NRD. From the dendrogram clustering of the main spectra projections, the Chinese and foreign H. influenzae reference strains were categorized into two distinct groups, and H. influenzae and H. haemolyticus were also separated into two categories. Compared to the existing methods, MALDI-TOF MS has the advantage of integrating high throughput, accuracy and speed. In conclusion, MALDI-TOF MS is an excellent method for differentiating NTHi and H. haemolyticus. This method can be recommended for use in appropriately equipped laboratories.

Pseudomonas syringaepv.actinidiaedetection in kiwifruit plant tissue and bleeding sap

AbstractThe rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method forPseudomonas syringaepv.actinidiae(Psa) detection, to find possible inoculum sources and limit the pathogen spreading. APCRmethod, using new primers designed on the gene encoding a putative outer membrane proteinP1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCRwas also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both forPCRassays and for pathogen isolation and identification. ThePCRand nestedPCRmethods were able to detect Psa presence at very low concentration from plant and pollen extracts;RFLPanalyses withBclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion withBfmI andAluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.

Natural IgG antibodies in normal rabbit serum are involved in killing of the ompP2 mutant of Haemophilus parasuis SC096 strain via the classical complement pathway

Serum resistance in Haemophilus parasuis strain SC096 has been shown to be dependent on expression of the outer membrane protein P2 (OmpP2) and loss of the ompP2 gene results in significantly greater sensitivity to both porcine and rabbit sera. However, the mechanism of complement activation by the serum sensitive ΔompP2 strain is unknown. In this study, the classical complement pathway is demonstrated to be the main pathway for killing the H. parasuis ΔompP2 strain, and not the mannan-binding lectin (MBL) or alternative pathway. In addition, absorption of antibodies against ΔompP2 strain or depletion of IgGs from serum inhibited serum killing activity, which could be restored by addition of heat-inactivated serum or purified IgGs. Western blot analysis indicated that the OmpP2 mutant could bind significantly more IgGs than the wild type strain SC096 when incubated with serum. Finally, IgGs in normal rabbit serum targeted to the OMPs, but not lipooligosaccharide (LOS) in the OmpP2 mutant strain were found to be involved in bacterial killing indicating that the bactericidal epitope(s) is in the outer membrane proteins.

Kinetic analysis and evaluation of the mechanisms involved in the resolution of experimental nontypeable Haemophilus influenzae-induced otitis media after transcutaneous immunization

Transcutaneous immunization (TCI) is a simple and needle-free method with which to induce protective immune responses. Using a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media (OM), we examined the efficacy afforded by TCI with a novel chimeric immunogen called ‘chimV4’ which targets two critical adhesins expressed by NTHI, outer membrane protein P5 and the majority subunit of NTHI Type IV pilus, PilA. Experimental OM was first established in cohorts of animals, and then TCI performed via a therapeutic immunization regime by rubbing vaccine formulations on hydrated pinnae. The kinetics of resolution of established experimental disease was evaluated by clinically-relevant assessments of OM, bacterial culture of planktonic and adherent NTHI within the middle ear and gross examination of the relative amount of NTHI mucosal biofilms within the middle ear space. Within seven days after primary TCI, a significant reduction in the signs of OM, significantly fewer NTHI adherent to the middle ear mucosa and significant resolution of mucosal biofilms was detected in animals that received chimV4+ the adjuvant LT(R192G-L211A), compared to animals administered LT(R192G-L211A) alone or saline by TCI (p<0.05) with eradication of NTHI within an additional seven days. The mechanism for rapid disease resolution involved efflux of activated dermal dendritic cells from the pinnae after TCI, secretion of factors chemotactic for CD4+ T-cells, induction of polyfunctional IFNγ- and IL-17-producing CD4+ T-cells and secretion of host defense peptide within the middle ear. These data support TCI as a therapeutic intervention against experimental NTHI-induced OM and begin to elucidate the host response to immunization by this noninvasive regimen.

Enhanced adherence to and invasion of PUVEC and PK-15 cells due to the overexpression of RfaD, ThyA and Mip in the ΔompP2 mutant of Haemophilus parasuis SC096 strain

In Gram-negative bacteria, porins not only contribute to bacterial homeostasis, but also are involved in adherence to and invasion of host cells. Haemophilus parasuis outer membrane protein P2 (OmpP2), a member of the porin family, is an important surface protein involved in serum resistance. To further determine the features of OmpP2, the ability of ompP2 deficient mutant (ΔompP2) of a H. parasuis SC096 strain to interact with porcine umbilicus veins endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15) was evaluated in this study. The ΔompP2 mutant exhibited dramatically increased ability to adhere to and invade PUVEC and PK-15 cells. Conversely, pretreatment of cell lines with purified native OmpP2 porins significantly inhibited adhesion and invasion of the SC096 strain to the both host cells. To explain the unexpected phenomenon, a 2-dimensional gel electrophoresis-based proteomics comparison was performed between the wild-type SC096 and ΔompP2 mutant strains. There were 55 differentially expressed proteins identified from mutant. Among them, three overexpressed proteins of the ADP-l-glycero-d-mannoheptose-6-epimerase RfaD, thymidylate synthase ThyA and putative macrophage infectivity potentiator-related protein Mip were confirmed as molecular targets that modulated adherence and invasion capacities of the ΔompP2 mutant. Collectively, the OmpP2 of the H. parasuis SC096 strain mediated the adherence to and invasion of cells and loss of OmpP2 expression resulted in promoted cell-adherence and invasion properties which were due to the overexpression of RfaD, ThyA and Mip.

Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

Protease digestion of P6: Demonstrating a novel dual orientation of P6 in Nontypable Haemophilus influenzae

Nontypable Haemophilus influenzae (NTHi) causes otitis media (ear infections) and other respiratory illnesses. Currently, NTHi's outer membrane protein P6 is a leading vaccine candidate for the pathogenic bacteria. We have recently demonstrated that P6 is surface exposed, but not a transmembrane protein. In this study, we performed a protease digestion experiment to show that P6 is also present on the inside of NTHi cells. We exposed whole and lysed NTHi cells to a serine protease called Proteinase K and collected time resolved samples to test for the digestion of P6. P6 present on the inside of whole cells were protected from Proteinase K digestion, while P6 in lysed cells were readily digested by Proteinase K. This experiment, in light of other studies from our lab, has demonstrated that P6 exists in two orientations in the outer membrane of NTHi. Additionally, we have made progress toward quantifying the amount of P6 present on the inside versus the outside of the cell. This study was funded by the Rochester Institute of Technology and by NIH NIDCD RO1 08671 (to MEP).

Comparison of ompP5 sequence-based typing and pulsed-filed gel electrophoresis for genotyping of Haemophilus parasuis

In this study, comparison of the outer membrane protein P5 gene (ompP5) sequence-based typing with pulsed-field gel electrophoresis (PFGE) for the genotyping of Haemophilus parasuis, the 15 serovar reference strains and 43 isolates were investigated. When comparing the two methods, 31 ompP5 sequence types (STs) and 43 PFGE types were identified withompP5 STs A16 and A28 being the dominant ompP5 ST types, while no predominant PFGE type was found. The discriminatory indices were 0.95 for ompP5 sequence-based method and 0.98 for PFGE, and the two techniques were proved to be 70.7% congruent. Therefore,ompP5 sequence-based typing was a simple and inexpensive method, enabling it as a preliminary technique to research the molecular differentiation of H. parasuis. Key words: Haemophilus parasuis, ompP5-based typing method, PFGE, molecular typing.

The omp2 Gene of HPS-type Bacteria Cloning and Sequence Analysis Isolates from Sichuan Province

In order to compare the homology and antigen of Haemophilus parasuis (HPS)4 outer membrane protein P2(omp2), we design a test with specific primers, using PCR amplification of isolates of Haemophilus parasuis from Sichuan Province(HP Sch2010), ompP2 gene will be cloned into the pGM-T vector, and transformed into E. coli DH5α. Identified by PCR and sequencing and analysis, the sequencing results showed that the published 4 HPS SW124 strains omp2 gene (1077bp), compared with the amplified 1086bp purpose fragment(containing omp2 genes), is relatively stable, with the nucleotide homology level 97% and amino acid homology level of 92.5%. The variable regions are mainly concentrated in the three base sequences: 40-65,110-156,180-202.

Serum resistance in Haemophilus parasuisSC096 strain requires outer membrane protein P2 expression

Haemophilus parasuis outer membrane protein P2 (OmpP2), the most abundant protein in the outer membrane, has been identified as an antigenic protein and a potential virulence factor. To study the precise function of OmpP2, an ompP2-deficient mutant (ΔompP2) of a H.parasuis serovar 4 clinical strain SC096 was constructed by a modified natural transformation system. Compared with the wild-type SC096 strain, the ΔompP2 mutant showed a pronounced growth defect and exhibited significantly greater sensitivity to the bactericidal action of porcine and rabbit sera, whereas the complemented strain could restore the growth and serum resistance phenotypes. The results indicated that H.parasuis OmpP2 from SC096 strain is an important surface protein involved in serum resistance.

Molecular cloning, sequencing, and expression of the outer membrane protein P2 gene of Haemophilus parasuis

Haemophilus parasuis is the etiological agent of Glässer’s disease characterized by fibrinous polyserositis, polyarthritis, and meningitis in young pigs. But it is difficult to develop universal serological diagnostic tools and effective vaccines against this disease because of the serovar diversity of the isolates. In this study, enterobacterial repetitive intergenic consensus-polymerase chain reaction, were performed to investigate the gene profile of 111 isolates of H. parasuis from China. And a specific common gene of H. parasuis was cloned and identified as the outer-membrane protein (OMP) P2 gene. Sequencing results of OMP P2 genes of 22 isolates showed that they had high homology and could be divided into 2 genetic types. Moreover, the OMPP2 protein was expressed in Escherichia coli expressing system. And the purified recombinant protein provided partial protection against H. parasuis infection in mice. It suggested the OMP P2 was an immunogenic protein and had great potential to serve as a vaccine and diagnostic antigen.

Th17 cells contribute to nontypeable Haemophilus influenzae-specific protective immunity induced by nasal vaccination with P6 outer membrane protein and α-galactosylceramide

Nasal vaccination is an effective therapeutic means of preventing upper respiratory infection. Recently, nasal vaccination with P6 outer membrane protein of nontypeable Haemophilus influenzae (NTHi) and alpha-galactosylceramide (α-GalCer) was reported to induce NTHi-specific protective immunity. The present study investigated the role of the Th17 cells induced by nasal vaccination. Mice were immunized with P6 and α-GalCer, and their P6-specific immune responses were examined. Cytokine-producing cells were analyzed by flow cytometry, and expression of cytokines in P6-specific CD4+ T cells was determined by reverse transcription-polymerase chain reaction. Bacterial challenges were performed with live NTHi. To examine the role of Th17 cells, bacterial clearance was also evaluated after interleukin (IL)-17 neutralization. P6-specific nasal wash immunoglobulin (Ig) A and serum IgG were increased after immunization with P6 and α-GalCer. Specific IgA-producing cells increased markedly in the nasal passages (NPs) of the immunized mice. In addition to P6-specific Th1 and Th2 cells, IL-17-producing Th17 cells were induced in the NPs and spleen. Bacterial clearance was enhanced by nasal vaccination. Interestingly, impaired NTHi clearance was shown after IL-17 neutralization. These findings suggest that nasal vaccination with P6 and α-GalCer is an effective regimen for the induction of NTHi-specific protective immunity in the upper respiratory tract. In addition to antigen-specific secretory-IgA, specific Th17 cells induced by nasal vaccination contribute to protection against NTHi.