PROJECT SUMMARY A recent study showed that there were ~4.95 million deaths associated with bacterial antimicrobial resistance (AMR) in 2019 which included ~1.27 million deaths attributable to bacterial AMR. Klebsiella pneumoniae, which commonly causes pneumonia and sepsis in hospitalized, immunosuppressed, and chronically ill patients, was responsible for 17.5% of the deaths associated with AMR and 19.9% of the deaths attributable to AMR. Due to the dwindling antibiotic pipeline, other approaches to target these infections are needed. We propose to develop K. pneumoniae vaccines to prevent these infections from occurring, thereby reducing the need for antimicrobial therapy and interrupting pathogen transmission. Here, we describe development of a live attenuated K. pneumoniae vaccine with excellent safety and tolerability while maintaining immunogenicity. The main goal of the current application is to show that we can develop live oral K. pneumoniae vaccines with excellent safety due to reduced potential for transmission, designated here as Live Attenuated Non-Transmissible (LANT) vaccines. To achieve this goal, we will genetically engineer a live attenuated K. pneumoniae candidate vaccine in which arabinose-controlled synthesis of the essential single stranded DNA-binding protein (SSB) strictly limits post-vaccination replication. We will also determine whether we can achieve broad protection mediated by subcapsular antigens. We will complete pre-clinical safety and efficacy studies assessing the in vivo persistence, immunogenicity, and protective efficacy of candidate K. pneumoniae LANT vaccines in mice. At the conclusion of this project, we anticipate demonstrating that an optimized K. pneumoniae LANT vaccine stops replicating in the absence of arabinose in vitro as well as in vivo but is still immunogenic and can protect animals against challenge with wild-type K. pneumoniae. If we are successful, these results will pave the way for development of a broadly protective, safe and immunogenic K. pneumoniae vaccine.