Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD103-HgR

摘要

Live, attenuated Vibrio cholerae vaccines can induce potent immune responses after only a single oral dose. The strategy of harnessing these strains to present antigens from heterologous pathogens to the mucosal immune system shows great promise. To fully realize this possibility, V. cholerae strains must be created which stably express antigens in vivo in sufficient quantity to generate an immune response. In vivo-induced promoters have been shown to increase stability and immunogenicity of foreign antigens expressed from multicopy plasmids. We report the construction of a series of genetically stabilized plasmids expressing luciferase as a heterologous protein from the following in vivo-induced promoters: V. cholerae PargC, PfhuC, and Pvca1008, and S. Typhi PompC. We demonstrate that several of these expression plasmids meet two critical criteria for V. cholerae live vector vaccine studies. First, the plasmids are highly stable in the V. cholerae vaccine strain CVD103-HgR at low copy number, in the absence of selective pressure. Second, real time bioluminescent imaging (BLI) demonstrates inducible in vivo expression of the promoters in the suckling mouse model of V. cholerae colonization. Moreover, the use of BLI allows for direct quantitative comparison of in vivo expression from four different promoters at various timepoints.