Novel Salmonella vectors with in vivo amplifiable plasmids for vaccination and tumor therapy

Live attenuated Salmonella are highly potential vectors for the delivery of proteins and DNA with applications in vaccination and tumor therapy. Conventional expression systems are based on maintenance of the plasmid at multi-copy numbers representing a high metabolic burden for the bacteria that results in a decreased performance of the vector. In this work, a system for in vivo inducible amplification of expression plasmids was established in Salmonella enterica serovar Typhimurium strain SL7207. Amplifiable plasmids contain two replication origins. The first replicon ensures the minimal-copy number state under non-inducing conditions. A second medium-copy replicon was placed under control of Salmonella-specific in vivo inducible promoters PsifA, Pstm1630 and PphoN or the PBAD promoter. PsifA exhibited strong amplification of the plasmid copy number from 1-5 to 50-100 copies and enhanced expression of plasmid-encoded antigen in induction medium, upon infection of macrophages and mice. However, in vivo imaging demonstrated that PsifA is activated already early in the mouse intestine resulting in impaired invasion efficiency of orally administered plasmid amplifying strains. Nevertheless, after intravenous immunization, plasmid amplification resulted in substantial ovalbumin-specific CD4+ T cell responses. In the second part of this work, plasmid amplification could be activated by the L-arabinose inducible PBAD promoter. This remote control was possible in culture, upon infection of macrophages and in tumor, spleen and liver of tumor-bearing mice. Administration of the sugar resulted in high reporter activity especially when plasmid amplification and reporter expression were simultaneously driven by PBAD. In addition, repeated induction of reporter expression could be demonstrated in vivo. In summary, inducible plasmid amplification should contribute to the optimization of Salmonella-mediated delivery of prophylactic and therapeutic molecules.