New Generation of Stable, Nonantibiotic, Low-Copy-Number Plasmids Improves Immune Responses to Foreign Antigens in Salmonella enterica Serovar Typhi Live Vectors

We hypothesized that adequately engineered attenuated Salmonella enterica serovar Typhi strains can serve as multivalent mucosal live vector vaccines to immunize against unrelated human pathogens. Toward this ultimate goal, we have developed a novel genetic stabilization system for antigen-expressing plasmids, engineered to encode the single-stranded binding protein (SSB), an essential protein involved in DNA metabolism which was deleted from the live vector chromosome. We utilized full-length protective antigen (PA83) of anthrax toxin from Bacillus anthracis as a foreign antigen and expressed PA83 as a fusion with the ClyA export protein, which allows export of ClyA-PA83 to the surface of S. Typhi live vectors. A series of SSB-encoding multicopy expression plasmids were introduced into reengineered S. Typhi strains previously tested in clinical trials, i.e., CVD 908-htrA and its less attenuated parent CVD 908. Immunogenicity was examined using a mouse model of intranasal immunization with live vector, followed by parenteral boosting with purified PA83. PA-specific antibody responses markedly improved as the copy number of the SSB-encoding plasmids decreased, and this effect was dramatically enhanced when the foreign antigen was delivered by the less attenuated live vector CVD 908ssb. These results suggest that antibody responses to antigens delivered by S. Typhi live vectors are inversely related to the metabolic burden imposed by expression of the foreign antigen and that these responses can be improved when antigens are expressed from low-copy-number plasmids and exported out of the cytoplasm of less attenuated live vectors.