Mucosal immune cell priming by intranasally delivered Haemophilus haemolyticus is associated with heterologous protection against influenza and nontypeable Haemophilus influenzae

Introduction Intranasal vaccines offer a needle-free strategy to enhance immunity to respiratory infections. We investigated the mechanism of action of a novel intranasal vaccine using the human respiratory commensal Haemophilus haemolyticus (Hh), previously shown to protect against nontypeable Haemophilus influenzae (NTHi) otitis media and accelerate clearance of influenza A virus (IAV). Methods Mucosal and systemic cellular immune responses were assessed 2–144 hours after intranasal Hh treatment in mice, compared with placebo or the Toll-Like Receptor (TLR)2–6 agonist Pam2CSK4 using spectral flow cytometry. The impact of treatment on subsequent IAV and NTHi challenge was also evaluated. Results Hh induced a distinct, tissue-specific immune signature with rapid recruitment of neutrophils and inflammatory monocytes to the lungs, peaking at 6 hours, earlier than Pam2CSK4. Hh also generated higher proportions of nasal CD103 + CD4 + T cells within 48 hours, which further expanded following sequential IAV and NTHi infection. Discussion These findings demonstrate that Hh primes mucosal immune responses to promote heterologous protection.