Lipidation of Haemophilus influenzae Antigens P6 and OMP26 Improves Immunogenicity and Protection against Nasopharyngeal Colonization and Ear Infection.

Nontypeable Haemophilus influenzae (NTHi) causes respiratory infections that lead to high morbidity and mortality worldwide, encouraging development of effective vaccines. To achieve a protective impact on nasopharyngeal (NP) colonization by NTHi, enhanced immunogenicity beyond that achievable with recombinant-protein antigens is likely to be necessary. Adding a lipid moiety to a recombinant protein would enhance immunogenicity through Toll-like receptor 2 signaling of antigen-presenting cells and Th17 cell response in the nasal-associated lymphoid tissue (NALT). We investigated effects of lipidation (L) of recombinant proteins P6 and OMP26 compared to nonlipidated (NL) P6 and OMP26 and as fusion constructs (L-OMP26ϕNL-P6 and L-P6ϕNL-OMP26) in a mouse model. After intraperitoneal or intranasal vaccination, antibody responses were compared and protection from NP colonization and middle ear infection were assessed. L-P6 and L-OMP26 induced approximately 10- to 100-fold-higher IgG antibody levels than NL-P6 and NL-OMP26. Fusion constructs significantly increased IgG antibody to both target proteins, even though only one of the proteins was lipidated. NP colonization and middle ear bullae NTHi density was 1 to 4 logs lower following vaccination with L-P6 and L-OMP26 than with NL-P6 and NL-OMP26. Fusion constructs also resulted in a 1- to 3-log-lower NTHi density following vaccination. NALT cells from mice vaccinated with lipidated protein constructs had higher levels of interleukin-17 (IL-17), IL-22, and CD4+ T-cell memory. Passive transfer of sera from L-OMP26ϕNL-P6-vaccinated mice to recipient infant mice reduced NP colonization and ear bulla NTHi density. We conclude that L-P6, L-OMP26, and fusion constructs generate enhanced antibody responses and protection from NP colonization and middle ear infection by NTHi in mice.