Intranasal Immunization Enhances Clearance of NontypeableHaemophilus influenzaeand Reduces Stimulation of Tumor Necrosis Factor Alpha Production in the Murine Model of Otitis Media

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media (OM). One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is considered a candidate for mucosal vaccine. We have previously reported that intranasal immunization with P6 and cholera toxin (CT) could induce P6-specific immunoglobulin A (IgA) antibodies in the middle ear. In the present study, we assessed the effect of intranasal immunization for the protection against NTHi-induced OM. Mice were immunized intranasally with P6 and CT as an adjuvant on days 0, 7, and 14. Control mice were given phosphate-buffered saline (PBS) without antigen. One week after the final immunization, a suspension of live NTHi (10 7 CFU) was injected into the tympanic cavity to induce experimental OM. On days 3 and 7 after bacterial challenge, mice were killed and middle ear effusions (MEEs) were collected. All immunized mice showed elevated titers of P6-specific antibodies in MEEs. The rank order of specific antibody included, from highest to lowest levels, IgG, IgA, and IgM. In addition, immunized mice showed enhanced clearance of NTHi from the middle ear and the number of NTHi in MEEs of immunized mice was reduced by 97% on day 3 and by 92% on day 7 after bacterial challenge relative the number in the MEEs of control mice. The protective effect of intranasal immunization on the incidence of NTHi-induced experimental OM was evident on day 7 after challenge. By day 7, the number of MEEs in immunized mice was 64% less than that in control mice and the incidence of NTHi culture-positive MEEs in immunized mice was 56% less than that in control mice. Less stimulation of tumor necrosis factor alpha (TNF-␣) production in the middle ear was evident on day 3 after challenge. Immunized mice showed lower concentrations of TNF-␣ in MEEs. These results indicate that intranasal immunization affords protection against experimental OM as evidenced by enhanced clearance of NTHi and less stimulation of TNF-␣ production in the middle ear. These findings suggest that a nasal vaccine might be useful for preventing OM.Otitis media (OM) is one of the most common infectious diseases in children, and the peak incidence of this disease occurs in early childhood. Nontypeable Haemophilus influenzae (NTHi) is a major causative pathogen of OM and is often isolated from middle ear effusions (MEEs) and the nasopharynx (12). Because of the increased incidence of antibioticresistant strains of NTHi in recent years, the development of a vaccine against this bacterium is considered an important goal for public health (14, 15). Recent efforts to develop an effective vaccine candidate against NTHi have focused on P6, an outer membrane protein of NTHi and a common antigen to all strains (32, 33).The middle ear mucosa is capable of producing local and systemic responses after an appropriate antigenic stimulus (30). Local immunity in the middle ear, in conjunction with systemic immunity, plays an important role in OM. Antigenspecific antibodies appear in MEE ...

Characterization of Salivary Immunoglobulin A Responses in Children Heavily Exposed to the Oral Bacterium Streptococcus mutans : Influence of Specific Antigen Recognition in Infection

“…For example, a peak of immune response to a potential vaccine target of H. influenzae, the outer membrane protein P6 (29), was detected early in life (1 to 24 months) among children with short persistence of colonization, while low response to P6 occurred in children colonized for several distinct strains during extended periods (12). Persistent and recurrent colonization by nontypeable H. influenzae strains in otitis-prone children was also associated to a poor local immune response to P6 (41).…”

Construction of a Mutant and Characterization of the Role of the Vaccine Antigen P6 in Outer Membrane Integrity of NontypeableHaemophilus influenzae

“…P6 induces protective immune responses in a variety of animal model systems, including the infant rat model of invasive infection (17,33,53), a rat pulmonary clearance model (27), otitis media models in the chinchilla and mouse (12,18,48), and nasopharyngeal colonization models (6,21,23,32). P6 is the target of bactericidal antibodies from rats, chinchillas, rabbits, and humans (12,17,27,38).…”

Respiratory Bacterial Vaccines

“…Moreover, mucosal (i.n.) immunization of mice with recombinant P6 combined with cholera toxin stimulated P6-specific mucosal IgA and systemic IgG responses, and enhanced clearance of NTHi from the nasopharynx and middle ear (Hotomi et al, 2002;Sabirov et al, 2001). Thus, these data suggest that the design of future NTHi vaccines should be directed toward mucosal immunization with H. influenzae surface lipoproteins and possibly other NTHi antigens, such as lipooligosaccharide, fimbriae, and outer membrane proteins, to limit colonization and disease.…”