In vitro and in vivo analyses of constitutive and in vivo-induced promoters in attenuated vaccine and vector strains of Vibrio cholerae

The optimal promoter for in vivo expression of heterologous antigens by liv e, attenuated vaccine vector strains of Vibrio cholerae is unclear; in vitr o analyses of promoter activity may not accurately predict expression of an tigens in vivo. We therefore introduced plasmids expressing the B subunit o f cholera toxin (CtxB) under the control of a number of promoters into V. c holerae vaccine strain Peru2, We evaluated the tac promoter, which is const itutively expressed in V. cholerae, as well as the in vivo-induced V. chole rae heat shock htpG promoter and the in vivo-induced V. cholerae iron-regul ated irgA promoter. The functionality of all promoters was confirmed in vit ro. In vitro antigenic expression was highest in vaccine strains expressing CtxB under the control of the tac promoter (2 to 5 mu g/ml/unit of optical density at 600 nm [OD600]) and, under low-iron conditions, in strains cont aining the irgA promoter (5 mu g/ml/OD600). We orally inoculated mice with the various vaccine strains and used anti-CtxB immune responses as a marker for in vivo expression of CtxB. The vaccine strain expressing CtxB under t he control of the tac promoter elicited the most prominent specific anti-Ct xB responses in vivo (serum immunoglobulin G [IgG], P less than or equal to 0.05; serum IgA, P less than or equal to 0.05; stool IgA, P less than or e qual to 0.05; bile IgA, P less than or equal to 0.05), despite the finding that the tac and irgA promoters expressed equivalent amounts of CtxB in vit ro. Vibriocidal antibody titers were equivalent in all groups of animals, O ur results indicate that in vitro assessment of antigen expression by vacci ne and vector strains of V. cholerae may correlate poorly with immune respo nses in vivo and that of the promoters examined, the tac promoter may be be st suited for expression from plasmids of at least certain heterologous ant igens in such strains.