SUMMARY Burkholderia pseudomallei, the causative agent of melioidosis, is a saprophytic bacterium readily isolated from soil and stagnant water of tropical countries. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders. Infection by these organisms typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Melioidosis and glanders are difficult to diagnose and require prolonged antibiotic therapy that has a low success rate. There is no vaccine to protect against these highly pathogenic bacteria, and there is concern regarding their use as biological warfare agents given that B. mallei has already been utilized in this manner on multiple occasions. For these reasons, the Federal Select Agent Program has classified B. pseudomallei and B. mallei as Tier 1 agents and the development of medical countermeasures is a priority. Parainfluenza virus 5 (PIV5), a paramyxovirus, is thought to be a contributing factor of kennel cough. Several characteristics of the virus make it an excellent vector for developing countermeasures including safety, replication in mammalian cell lines approved for vaccine production, and efficacy (single dose immunization provides protective immunity against multiple infectious agents). Moreover, intranasal immunization of PIV5-based vaccines elicits robust mucosal immune responses, thus providing an ideal platform for vaccinating against respiratory pathogens. In this application, we present preliminary data demonstrating that single dose immunization with PIV5 expressing the Burkholderia autotransporter protein BatA provides excellent protective immunity against lethal respiratory infection with B. pseudomallei and B. mallei. Based on these results, we propose to use PIV5 to develop a vaccine for these Tier 1 select agents focusing on 2 specific aims. In Aim 1, we will examine the mechanisms of protection by PIV5-BatA and improve efficacy. In Aim 2, we will develop a PIV5-based vaccine containing multiple Burkholderia high-value antigens.