An unnatural amino acid dependent, conditional Pseudomonas vaccine prevents bacterial infection
Michael Pigula, Yen‐Chung Lai, Minseob Koh, Christian S. Diercks, Thomas F. Rogers, David A. Dik, Peter G. Schultz
Abstract
Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-l-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10−11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens. In this work, authors develop a live vaccine candidate against Pseudomonas aeruginosa using an unnatural amino acid-based auxotrophic strategy. They provide insight on the vaccine’s in vivo safety profile and protection of mice against lethal P. aeruginosa challenge.