Highly pathogenic avian influenza (HPAI) H5N1 virus is a major threat to public health as well as to the global poultry industry. Most fatal human infections are caused by contact with infected poultry. Therefore, preventing the virus from entering the poultry population is a priority. This is, however, problematic in emergency situations, e.g. during outbreaks in poultry, as there are currently no mass application methods to effectively vaccinate large numbers of birds within a short period of time. To evaluate the suitability of needle-free pulmonary immunization for mass vaccination of poultry against HPAI H5N1, we performed a proof-of-concept study in which we investigated whether non-adjuvanted spray-freeze-dried (SFD) whole inactivated virus (WIV) can be used as a dry powder aerosol vaccine to immunize chickens. Our results show that chickens that received SFD-WIV vaccine as aerosolized powder directly at the syrinx (the site of the tracheal bifurcation), mounted a protective antibody response after two vaccinations and survived a lethal challenge with HPAI H5N1. Furthermore, both the number of animals that shed challenge virus, as well as the level of virus shedding, were significantly reduced. Based on antibody levels and reduction of virus shedding, pulmonary vaccination with non-adjuvanted vaccine was at least as efficient as intratracheal vaccination using live virus. Animals that received aerosolized SFD-WIV vaccine by temporary passive inhalation showed partial protection (22% survival) and a delay in time-to-death, thereby demonstrating the feasibility of the method, but indicating that the efficiency of vaccination by passive inhalation needs further improvement. Altogether our results provide a proof-of-concept that pulmonary vaccination using an SFD-WIV powder vaccine is able to protect chickens from lethal HPAI challenge. If the efficacy of pulmonary vaccination by passive inhalation can be improved, this method might be suitable for mass application
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