Immunological evaluation of recombinant chimeric construct from Enterotoxigenic E. coli expressed in hairy roots.

Enterotoxigenic Escherichia coli is the most important bacterial agent causing traveller's diarrhea in developing countries. Enterotoxins (LT & ST) and colonization factors (CFs) are two important factors in the ETEC pathogenesis. In the present study, a recombinant four-part fusion protein containing CFAB*ST, CFAE, and LTB (CCL) was expressed in hairy roots of Nicotiana tabacum. The synthetic gene sequence and gene order were designed based on bioinformatics analysis that predicted the best arrangement for antigenicity and stimulation of the immune response. Codon usage was optimized for expression in tobacco plant, under the control of promoter CaMV35S in plasmid pBI121. CCL was efficiently expressed in tobacco hairy roots to yield 1.11% soluble protein as determined by quantitative ELISA and Western blot. In this study, mice were immunized with purified CCL via the oral and subcutaneous route. Humoral immunity especially mucosal immunity with antigen specific IgG and IgA detected in serum and feces. The ability of CCL to elicit neutralizing antibodies was evaluated in the rabbit ileal loop model, using anti-CCL antibodies derived from immunized mice, and co-incubated with ETEC strains. A decrease in fluid accumulation in the intestinal lumen of rabbit ileal loops challenged with ETEC LT and ST positive strains, correlated with the presence of anti-CCL antibodies capable of toxin neutralization. The ability of these antibodies to neutralize toxin confirmed the recognition of epitopes, either linear or conformational, displayed by the recombinant chimeric protein expressed in transgenic tobacco hairy roots. Transgenic plants containing multivalent immunogenic vaccine candidates have the potential to be used for immunization with protection against gastrointestinal pathogens like ETEC.

In Silico designing and immunogenic production of the multimeric CfaB*ST, CfaE, LTB antigen as a peptide vaccine against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most frequent bacterial cause of diarrhea particularly reported in children of developing countries and also travelers. Enterotoxins and colonization factor antigens (CFAs) are two major virulence factors in ETEC pathogenesis. Colonization factor antigen I (CFA/I) includes major pilin subunit CfaB, and a minor adhesive subunit (CfaE), and enterotoxins consisting of heat-labile toxin subunit B (LTB) and heat-stable toxin (ST). Chimeric proteins (CCL) carrying epitopes and adjuvant sequences increase the possibility of eliciting a broad cellular or effective immune response. In the present study, a chimeric candidate vaccine containing CfaB*ST, CfaE, and LTB (CCL) was designed via in silico techniques. This chimeric gene was synthesized by using codon usage of E. coli for increasing the expression of the recombinant protein. After designing the chimeric construct, it showed a high antigenicity index estimated by the vaxiJen server. Linear and conformational B-cell epitopes were identified and indicated suitable immunogenicity of this multimeric recombinant protein. Thermodynamic analyses for mRNA structures revealed the appropriate folding of the RNA representative good stability of this molecule. In silico scanning was done to predict the 3D structure of the protein, and modeling was validated using the Ramachandran plot analysis. The chimeric protein (rCCL) was expressed in a prokaryotic expression system (E. coli), purified, and analyzed for their immunogenic properties. It was revealed that the production of a high titer of antibody produced in immunized mice could neutralize the ETEC using the rabbit ileal loop tests. The results indicated that the protein inferred from the recombinant protein (rCCL) construct could act as a proper vaccine candidate against three critical causative agents of diarrheal bacteria at the same time.