Effective Aerosol Inoculation of Dose-Escalated Seasonal Influenza H3N2 Virus in Controlled Human Infection Model

Human challenge models (CHIMs) are instrumental in advancing influenza research but have traditionally relied on intranasal inoculation, which does not mimic the natural aerosol transmission of the virus. We conducted a dose-escalation influenza CHIM study to evaluate the safety and feasibility of two modern aerosol delivery systems: a flow-focusing monodisperse aerosol generator (FMAG) and a medical nebulizer. Fourteen healthy adults aged 18-49 years were exposed to influenza A/Perth/16/2009 (H3N2) in a controlled inpatient setting. Infection rates were 75% (3/4) with FMAG and 50% (2/4) with the nebulizer at the higher dose. Infections were self-limited, with sinus congestion, rhinorrhea, and cough being the most common symptoms. No serious adverse events occurred. Viral shedding was reproducible across respiratory sites, and seroconversion occurred in 33% of infected participants. Symptom timing and viral kinetics were comparable to those observed in prior intranasal CHIMs. Participants receiving nebulizer-delivered virus showed earlier viral detection in the oral cavity, suggesting broader airway deposition. These findings demonstrate that aerosol influenza challenge is both safe and effective and can simulate natural infection more accurately than intranasal delivery. This reintroduction of aerosolized influenza challenge provides a robust platform for studying transmission dynamics, tissue-specific immune responses, and for evaluating next-generation vaccines and therapeutics under conditions that better approximate real-world exposure. One Sentence SummaryWe demonstrated controlled human infection with dose-escalated H3N2 influenza virus via two safe and effective aerosolization routes that provide more accurate simulation of real-world exposure than intranasal delivery for studying influenza transmission, tissue-specific immune responses, and vaccines/therapeutics.