Our strategy was to add lipids to recombinant proteins. 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 CDH5 from mice vaccinated with lipidated protein constructs experienced higher levels of interleukin-17 (IL-17), IL-22, and CD4+T-cell memory. Passive transfer of sera from L-OMP26NL-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 contamination by NTHi in mice. KEYWORDS:Haemophilus influenzae, P6, OMP26, lipidated recombinant proteins, antibodies, nasopharyngeal colonization, acute otitis media, Th17 immunity, TLR2 == INTRODUCTION == Nontypeable (noncapsulated)Haemophilus influenzae(NTHi) causes respiratory infections in children and adults that lead to high morbidity and mortality worldwide. NTHi has become the most common cause of acute otitis media (AOM) in children, following the introduction of pneumococcal conjugate vaccines (1). It is the predominant pathogen of prolonged AOM, recurrent AOM, and AOM treatment failures (24) and is a common cause of acute sinusitis and conjunctivitis in children and adults, and acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults (58). Invasive diseases are also caused by NTHi (9,10), and antibiotic resistance to NTHi is usually increasing (11,12). Protein D from NTHi is included in Phenylephrine HCl a pneumococcal conjugate vaccine as a carrier protein (PhiD-CV [not approved by the U.S. Food and Drug Administration]). PhiD-CV produced a 15% reduction in AOM caused by NTHi in children (13) but did not reduce nasopharyngeal (NP) colonization (14,15), a key goal to achieve herd immunity from vaccination. Early-phase clinical studies in adults have evaluated NTHi proteins D, E, and PilA for security and immunogenicity (16,17), and an efficacy trial in adults to prevent acute exacerbations of COPD is usually nearing completion (18). Our group has been studying protein D, P6, and OMP26, with a focus on Phenylephrine HCl natural immunity induced by colonization and AOM contamination in young children, including those who are otitis prone to NTHi (19),Streptococcus pneumoniae(20), andMoraxella catarrhalis(21) and have deficiencies in immunity response (22). Several groups have evaluated potential NTHi proteins, including proteins P4, P6, D, E, F, OMP26, PilA, Hap, HMW, and ZnuA, as vaccine candidates in animals (2326). It is apparent from our prior studies in children that natural exposure to NTHi and the immune response that follows are not sufficiently protective in prevention of subsequent NP colonization or AOM contamination in either otitis prone or nonprone children during early life (25,2730). Also, recent clinical trials of pneumococcal recombinant proteins PhtD, PhtE, and PlyD1 (NCT01446926) and PhtD and Ply (NCT01262872) in human subjects failed to demonstrate protection from NP colonization (31) or AOM (32,33), a desired end result from vaccines targeting respiratory bacteria in order to provide herd immunity protection. Therefore, we sought a novel method to enhance immunogenicity of NTHi protein antigens as vaccines by making lipidated proteins (acylation at the N terminus). A lipid moiety stimulates the immune response via TLR2 receptor with TLR1 or TLR6 depending upon the triacyl or diacyl lipid nature of the ligands, respectively, and then downstream signaling via NFK (34,35). Lipidated protein antigens fromStreptococcus pneumoniaehave been shown to stimulate Th17 cell responses (36). We selected two highly conserved outer membrane and surface-exposed NTHi proteins (P6 and OMP26) as vaccine antigens that play different functions in pathogenesis. Of various vaccine candidates, we have evaluated (P6, PD, OMP26, PF, and Eftu), and we selected P6 Phenylephrine HCl since it is the most immunogenic in young children following NP colonization and AOM infections (19,27,37) and elicits bactericidal antibody in otitis-prone children despite their generally poor immune responsivity (27). P6 is usually naturally lipidated and has been shown to be protective in a rat lung challenge model (38,39). OMP26 is not a naturally lipidated protein. In chinchillas immunized.