Document Type: Research article
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Biotechnology Research Center, Department of Biotechnolog, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Antimicrobial Resistance Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Department of Biochemistry, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
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 PhoPc 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 PhoPc. Additionally, the ability of these
recombinant PhoPc/pLTBs to induce LTB-specific antibody responses in BALB/c mice after
nasal immunization was evaluated. In-vitro studies demonstrated that PhoPc has the ability
to produce rLTB. Furthermore, nirB promoter directed significantly more LTB expression in
PhoPc/pnirBLTB under anaerobic condition without induction compared to the amount of rLTB
secreted 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 was
more 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 the
PhoPc containing the examined prompters, which were significantly higher than those induced
by PhoPc/pnirB78-23LTB and PhoPc/pnirB, 6 weeks after the first immunization. Totally, it
could be concluded that in-vitro analysis of promoters for LTB expression in PhoPc may not
necessarily predict the recombinant PhoPc immunogenicity.