Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA

Abstract

Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers' diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea.

Keywords: Adhesin tip; Diarrhea; ETEC (enterotoxigenic Escherichia coli); MEFA (multiepitope fusion antigen); Vaccine.

Figure 1

Schematic illustration of the constructed ETEC adhesin tip MEFA protein. Amino acid sequences of epitopes representing each adhesin tip or adhesive subunit were showed in the top panel. Bottom left panel showed results of Western blot with mouse anti-CFA/I antiserum. 9450 was the adhesin tip MEFA protein (10 and 15 μl), and (−) was the total proteins from BL21 E. coli. Bottom right panel showed Coomassie blue staining of refolded solubilized adhesin tip MEFA proteins (lane 1: protein marker; lane 2: 10 μl tip MEFA protein; lane 3: 15 μl tip MEFA protein).

Figure 2

Mouse serum anti-CfaE (CFA/I), -CooD (CS1), -CotD (CS2), -CstH (CS3), -CsaE (CS4), -CsfD (CS5), -CssB (CS6), anti-LngA (CS21) and anti-EtpA (EtpA) IgG antibody titers (log₁₀). Solid circles (●) were titers of mice intraperitoneally immunized with the adhesin tip MEFA protein; empty circles (○) were of titers of the control mice. Each circle represented the IgG titer from an individual mouse. Bars indicated the mean titers of the group specific to each adhesin.