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

Abstract

Enterotoxigenic Escherichia coli (ETEC)-induced diarrhoea is the second most commoncause of death in children in the developing countries. Heat labile toxin (LT) is responsible forETEC-induced diarrhoea. In the present study, a novel live ETEC vaccine based on subunitB of LT (LTB) expression in attenuated PhoPc Salmonella strain was developed. Herein, weaimed to compare the in-vitro activity of promoters including constitutive tac, IPTG inducibletrc, and in-vivo-inducible (nirB and nirB78-23) in PhoPc. Additionally, the ability of theserecombinant PhoPc/pLTBs to induce LTB-specific antibody responses in BALB/c mice afternasal immunization was evaluated. In-vitro studies demonstrated that PhoPc has the abilityto produce rLTB. Furthermore, nirB promoter directed significantly more LTB expression inPhoPc/pnirBLTB under anaerobic condition without induction compared to the amount of rLTBsecreted by PhoPc/ptrcLTB in bacterial soup under uninduced condition (6.06 ± 0.05 vs. 1.4 ±0.46 μg/109 cfu, p < 0.01). In addition, the constitutive rLTB expression from tac promoter wasmore than its expression from uninduced trc promoter in bacterial soup (4.2 ± 0.92 vs. 1.4 ±0.46 (μg/109 cfu)) and pellet (27.4 ± 0.89 vs. 13.4 ± 1.42 (μg/109 cfu), p < 0.0001). However,the mice immunized with PhoPc/ptrcLTB elicited the superior anti-LTB responses among thePhoPc containing the examined prompters, which were significantly higher than those inducedby PhoPc/pnirB78-23LTB and PhoPc/pnirB, 6 weeks after the first immunization. Totally, itcould be concluded that in-vitro analysis of promoters for LTB expression in PhoPc may notnecessarily predict the recombinant PhoPc immunogenicity.