Evaluation of the Effect of Promoter Type on the Immunogenicity of the Live Recombinant Salmonella Vaccines Expressing Escherichia Coli Heat-labile Enterotoxins (LTB)

Enterotoxigenic Escherichia coli (ETEC)-induced diarrhoea is the second most common cause of death in children in the developing countries. Heat labile toxin (LT) is responsible for ETEC-induced diarrhoea. In the present study, a novel live ETEC vaccine based on subunit B of LT (LTB) expression in attenuated PhoP(c) Salmonella strain was developed. Herein, we aimed to compare the in-vitro activity of promoters including constitutive tac, IPTG inducible trc, and in-vivo-inducible (nirB and nirB78-23) in PhoP(c). Additionally, the ability of these recombinant PhoP(c)/pLTBs to induce LTB-specific antibody responses in BALB/c mice after nasal immunization was evaluated. In-vitro studies demonstrated that PhoP(c) has the ability to produce rLTB. Furthermore, nirB promoter directed significantly more LTB expression in PhoP(c)/pnirBLTB under anaerobic condition without induction compared to the amount of rLTB secreted by PhoP(c)/ptrcLTB in bacterial soup under uninduced condition (6.06 ± 0.05 vs. 1.4 ± 0.46 μg/10(9) cfu, p < 0.01). In addition, the constitutive rLTB expression from tac promoter was more than its expression from uninduced trc promoter in bacterial soup (4.2 ± 0.92 vs. 1.4 ± 0.46 (μg/10(9) cfu)) and pellet (27.4 ± 0.89 vs. 13.4 ± 1.42 (μg/10(9) cfu), p < 0.0001). However, the mice immunized with PhoP(c)/ptrcLTB elicited the superior anti-LTB responses among the PhoP(c) containing the examined prompters, which were significantly higher than those induced by PhoP(c)/pnirB78-23LTB and PhoP(c)/pnirB, 6 weeks after the first immunization. Totally, it could be concluded that in-vitro analysis of promoters for LTB expression in PhoP(c )may not necessarily predict the recombinant PhoP(c )immunogenicity.