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

Document Type: Research article

Authors

1 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

2 Biotechnology Research Center, Department of Biotechnolog, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.

3 Antimicrobial Resistance Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

4 Department of Biochemistry, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.

Abstract

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.

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