Induction of Specific Immunoglobulin A and Th2 Immune Responses to P6 Outer Membrane Protein of NontypeableHaemophilus influenzaein Middle Ear Mucosa by Intranasal Immunization

Abstract

Nontypeable Haemophilus influenzae (NTHI) is a major pathogen of otitis media. One of the outer membrane proteins of NTHI, P6, is an antigen common to all strains and is considered as a candidate for mucosal vaccine. To elucidate the possibility of developing a nasal vaccine against nontypeable Haemophilus influenzae (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. Mice were given with P6 and cholera toxin intranasally as an adjuvant on days 0, 7, and 14 and were killed on day 21. The P6-specific immunoglobulin A (IgA) antibody titer 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 an enzyme-linked immunospot assay. In addition, an increase in memory T cells in middle ear mucosa was detected with flow cytometric analysis after intranasal immunization. Moreover, in vitro stimulation with P6 resulted in proliferation of purified CD4 ؉ T cells from immunized mice, and these T cells expressed Th2 cytokine mRNA. These results indicate that P6-specific IgA-B-cell immune responses and selected Th2 cytokine expressing Th cells were induced in middle ear mucosa by intranasal immunization. These findings suggest that a nasal vaccine is useful for preventing otitis media with effusion.Nontypeable Haemophilus influenzae (NTHI) is a major pathogen of otitis media with effusion (OME) and other upper respiratory tract diseases (10, 30). In patients with OME, this bacterium is frequently isolated from the nasopharynx, as well as from middle ear effusions, and the inhibition of NTHI colonization in the upper respiratory tract is considered effective in preventing OME. Due to the increase of antibiotic-resistant strains of NTHI in recent years, the development of a vaccine against this bacterium is considered an important goal for public health. Since NTHI lacks capsular antigens, the chief antigenicity is present in the outer membrane proteins (OMPs). One of the OMPs of NTHI, P6, is a common antigen to all strains and is considered as a candidate for mucosal vaccine (7,9,10,11,30,31).In the mucosal surface, secretory immunoglobulin A (IgA) plays a major role in protective immunity. We previously demonstrated that intranasal immunization was an effective regimen for inducing mucosal IgA immune responses in the upper respiratory tract (26) and that the nasal mucosal IgA immune responses induced by intranasal immunization were effective for the clearance of bacteria in the nasopharynx.The mucosal immune system is considered as a separate functional entity quite independent of the systemic immune system because the mucosal immune system possesses unique anatomic features and is composed of specialized subsets of lymphoid cells (21,27,34). Despite the recent emphasis on a better understanding of molecular and cellular aspects of the mucosal immune system,...

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