Abstract

Outer membrane protein P4, together with P6, is highly conserved among all typeable and nontypeable strains of Haemophilus influenzae (H. influenzae). Thus, the protein is an attractive antigen for the inclusion in a vaccine against nontypeable H. influenzae (NTHi). However, the ability of P4 to induce antibodies protective against NTHi infections is still controversial. In this study, we investigated the specific mucosal immune responses against NTHi induced by intranasal immunization with the lipidated form of recombinant P4 protein (rP4) and non-fatty acylated recombinant P6 protein (rP6) with or without cholera toxin (CT) in BALB/c mice model. Intranasal immunization with either rP4+CT, a mixture of rP4 and rP6+CT, or rP4 and rP6 without CT elicited anti-rP4 specific IgG antibody in serum of mice. Intranasal immunization with either rP4+CT or a mixture of rP4, rP6+CT elicited anti-rP4 specific IgA antibody in nasopharyngeal washing (NPW), while intranasal immunization with rP4 and rP6 without CT did not induced anti-rP4 specific IgA antibody responses in NPWs. Sera from mice intranasally immunized with rP4+CT and a mixture of rP4, rP6+CT also showed bactericidal activity. Significant clearance of NTHi in nasopharynx was seen 3 days after the inoculation of live NTHi in mice intranasally immunized with rP4+CT. The current findings suggested that P4 would be a useful antigen as the component of the vaccine to induce protective immune responses against NTHi. The use of an intranasal vaccine composed of the different surface protein antigens is an attractive strategy for the development of a vaccine against NTHi.