Lipidated Protein D vaccination elicits humoral and cellular responses and protects mice against challenge with non-typeable <i>Haemophilus influenzae</i>

Nontypeable <i>Haemophilus influenzae</i> (<i>NTHi</i>) is a primary cause of acute otitis media (AOM) in children and lower respiratory tract infections in adults. Vaccines that can reduce nasopharyngeal carriage and prevent disease are needed. We used an adult murine model of nasopharyngeal carriage and AOM infection to evaluate immunogenicity and protection provided by subcutaneous and intramuscular vaccination with lipidated Protein D (L-PD) compared with non-lipidated PD (NL-PD) formulated with and without alum adjuvant. ELISA was used for antibody measurements and flow cytometry to characterize T-cell immunity. L-PD + Alum subcutaneous vaccination induced low levels of PD-specific IgG antibodies and significantly elevated Th17 and T effector memory cell activation in cervical lymph nodes and splenocytes compared with NL-PD + Alum. The addition of alum increased bactericidal antibody activity. Intramuscular vaccination with L-PD with and without alum induced 3- to 4-fold log₁₀ higher PD-specific IgG antibodies than subcutaneous vaccination but did not elicit Th17 or T effector memory cell activation. Subcutaneous vaccination with L-PD + Alum protected mice within both the nasopharynx and middle ear. Middle ear bacterial density reduction correlated with IgG antibody levels after intramuscular vaccination with L-PD + Alum but not L-PD without alum, yet no protection from nasopharyngeal carriage occurred. PD + Alum vaccination increased humoral and cellular immunity and protected mice from <i>NTHi</i> carriage and AOM, whereas NL-PD did not. Activation of Th17 immunity by lipidation of PD and subcutaneous vaccination correlated with protection from nasopharyngeal carriage of <i>NTHi</i>.