Project Summary Many diseases could be prevented or treated by enlisting the immune system to recognize a specific antigen. Bacterial diseases warrant heightened attention as many bacterial pathogens lack efficacious vaccines and exhibit rising rates of antibiotic resistance. These pathogens often find many ways of evading immune system detection, including varying their most immunogenic antigens. While many virulence-related proteins can be strongly conserved across pathogen serotypes, they often exhibit weak immunogenicity that is insufficient to draw the response of the immune system. In this project, we ask: Are there strategies to shine a light on live bacterial antigens for increased recognition by immune cells? Furthermore, can we couple these strategies to shelf-stable delivery vectors that are simple to administer to patients across the world? We propose a transformational approach to expand the list of candidate antigens for use in live bacterial vaccine vectors by teaching Bacillus subtilis to produce and harness an immunogenic amino acid. This amino acid has been demonstrated to terminate immune self-tolerance when substituted on the surface of autologous proteins in mice. Site-specific introduction of nitrated residues within proteins has resulted in presentation of a neoepitope that is recognized by helper T cells for subsequent activation of B cells that produce polyclonal antibodies. It stands to reason that the immunogenicity of many weakly immunogenic foreign antigens could be increased using this strategy, though this has not yet been tested. One challenge is that prior studies also established a critical but poorly understood role of the MHC Class II locus in enabling immune response to nitrated antigens. Our project will investigate the potential of spore-displayed nitrated antigens as a transformational vaccination platform for bacterial disease, with the Shigella invasion protein antigens as a model system. We will first perform animal studies with Shigella antigens that are weakly immunogenic but strongly conserved across pathogen serotypes to determine if nitration can increase their immunogenicity. To better understand where nitrated residues should be placed for optimal recognition by immune cell machinery, we will develop a high-throughput microbial display platform to screen MHC-II preference towards unnatural peptide ligands. In parallel, we will develop tools to enable site-specific incorporation of the immunogenic amino acid within proteins fused to the spore coat of B. subtilis. Recombinant spores of this non-pathogenic organism can be orally administered and maintain immunization efficacy after exposure to harsh conditions. The spore-based platform has promise to overcome limitations in the manufacture, transport, and administration of vaccines; however, the platform has low immunogenicity. Our strategy to form nitrated residues using this platform could overcome that limitation. From this project, we will gain i...