Nontypeable Haemophilus influenzae (NTHi) strains form a major group of pathogenic bacteria that colonizes the nasopharynx and causes otitis media in young children. At present, there is no licensed vaccine for NTHi. Because NTHi colonizes the upper respiratory tract and forms biofilms that cause subsequent infectious events, a nasal vaccine that induces NTHi-specific secretory IgA capable of preventing biofilm formation in the respiratory tract is desirable. Here, we developed a cationic cholesteryl pullulan-based (cCHP nanogel) nasal vaccine containing the NTHi surface antigen P6 (cCHP-P6) as a universal vaccine antigen, because P6 expression is conserved among 90% of NTHi strains. Nasal immunization of mice with cCHP-P6 effectively induced P6-specific IgA in mucosal fluids, including nasal and middle ear washes. The vaccine-induced P6-specific IgA showed direct binding to the NTHi via the surface P6 proteins, resulting in the inhibition of NTHi biofilm formation. cCHP-P6 nasal vaccine thus protected mice from intranasal NTHi challenge by reducing NTHi colonization of nasal tissues and eventually eliminated the bacteria. In addition, the vaccine-induced IgA bound to different NTHi clinical isolates from patients with otitis media and inhibited NTHi attachment in a three-dimensional in vitro model of the human nasal epithelial surface. Therefore, the cCHP-P6 nanogel nasal vaccine induced effective protection in the airway mucosa, making it a strong vaccine candidate for preventing NTHi-induced infectious diseases, such as otitis media, sinusitis, and pneumonia.

Respiratory syncytial virus (RSV) is a leading cause of upper and lower respiratory tract infection, especially in children and the elderly. Various vaccines containing the major transmembrane surface proteins of RSV (proteins F and G) have been tested; however, they have either afforded inadequate protection or are associated with the risk of vaccine-enhanced disease (VED). Recently, F protein-based maternal immunization and vaccines for elderly patients have shown promising results in phase III clinical trials, however, these vaccines have been administered by injection. Here, we examined the potential of using the ectodomain of small hydrophobic protein (SHe), also an RSV transmembrane surface protein, as a nasal vaccine antigen. A vaccine was formulated using our previously developed cationic cholesteryl-group-bearing pullulan nanogel as the delivery system, and SHe was linked in triplicate to pneumococcal surface protein A as a carrier protein. Nasal immunization of mice and cotton rats induced both SHe-specific serum IgG and mucosal IgA antibodies, preventing viral invasion in both the upper and lower respiratory tracts without inducing VED. Moreover, nasal immunization induced greater protective immunity against RSV in the upper respiratory tract than did systemic immunization, suggesting a critical role for mucosal RSV-specific IgA responses in viral elimination at the airway epithelium. Thus, our nasal vaccine induced effective protection against RSV infection in the airway mucosa and is therefore a promising vaccine candidate for further development.