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

Abstract

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.

Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis To Confer Protection against Mycobacterium tuberculosis

“…Similar to the pattern of humoral responses to Ag85A, the antibody response against ESAT-6 was inversely related to the copy number of the plasmids, suggesting that a metabolic burden was imposed on the RASV by the synthesis of the protective antigens. A similar observation has been described previously, where the immune response to antigens delivered by Salmonella Typhi vaccines was improved when the ClyA-PA83 antigen was expressed from low-copy-number plasmids (pSC101 ori), which induced the highest antibody responses (32). In this study, the highest anti-ESAT-6 IgG titers were observed in the mice vaccinated with 11021(pYA4894 [p15A ori]) alone or in mice vaccinated simultaneously with both 11021(pYA4894) and 11021(pYA4892 [pSC101 ori]).…”

“…Stability of the recombinant plasmids was determined for approximately 50 generations of bacterial growth in LB medium under selective and nonselective (presence of DAP) conditions as described previously (22,32,57,67).…”

Characterization of Intracellular Growth Regulator icgR by Utilizing Transcriptomics To Identify Mediators of Pathogenesis in Shigella flexneri

“…This was digested with EcoRI and ligated into pSEC10. This plasmid carries the hok-sok genes for stable maintenance in the absence of antibiotics and is related to pSEC10S-83 (32).…”

Low-pH Rescue of Acid-Sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-Regulated Arginine Decarboxylase System

“…An additional advantage of the P rhaBAD promoter was its decreased level of AdiA protein synthesis. Protein overproduction has been shown to impair the efficacy of Salmonella vaccines (48,49). For these reasons, we selected the ⌬P adiA276 ::TT rhaSR P rhaBAD adiA mutation for use in further studies.…”