Infectious diarrhoea remains an emerging problem in the world health program. Among diarrheagenic agents, Vibrio cholerae and enterotoxigenic and enterohemorrhagic Escherichia coli are critical enteropathogens. AB5 toxin produced by these bacteria, heat-labile enterotoxin (LT), cholera enterotoxin (CT), and shiga-like cytotoxin (STX) can target the immune system and are subunit vaccine candidates. A chemically-synthesized chimeric construct composed of the binding subunits of these toxins (LTB, STXB, and CTXB) was developed based on bioinformatics studies. The whole chimeric protein (rLSC) and each of the segments (rLTB, rSTXB, and rCTXB) were expressed in a prokaryotic expression system (E. coli), purified, and analysed for their immunogenic properties. The results indicate that these recombinant proteins were effectively able to present appropriate epitopes to an animal model of the immune system which could result in and increase IgG in serum and immune responses that protect against the binding activity of these toxins. The immunological assays revealed that the sera of immunized mice prevented toxins from binding to their specific receptors and neutralized their toxic effects. The proposed construct should be considered as a potent immunogen to prevent toxicity and diarrhoea.

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion.

Introduction: The production and purification cost of a recombinant protein in plants is lower than other conventional systems, such as bacteria. Comparing different parts of the plant, the hairy root is known to be a suitable bioreactor to produce recombinant proteins including vaccine candidate immunogens. Among the most important causes of diarrhea, E. coli and Vibrio cholerae cause the disease by producing a toxin. The B subunit of this toxin is an appropriate candidate for vaccine development because of its non-toxicity and exposure to the immune system. Materials and Methods: To investigate the efficiency of transgenic hairy roots in the production of recombinant protein, two methods were considered: direct (induction of hairy root with recombinant Agrobacterium rhizogenes) and indirect (production of transgenic plant with Agrobacterium tumefaciens and then polluting it with non-transgenic A. rhizogenes). To compare the 2 methods, GUS protein expression was used as a reporter and chimeric protein LSC (consisting of ltB-stxB-ctxB) as an antigenic protein. Transformation of tobacco hairy root was confirmed by genomic Polymerase chain reaction (PCR). Gene expression comparison was investigated by semi-quantitative ELISA and enzymatic activity. Results: The results show that the activity of GUS reporter enzyme in the indirect method is seven times more than that of the direct method. Likewise, the expression of LSC recombinant protein in the indirect transformation was 1.5 times more than in direct method. Conclusions: Comparing of these two methods indicated that the hairy roots in the indirect method yield higher protein content than in the direct method.

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