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.

Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC)

“…Recombinant CfaB-epitope (CfaB-FedF-ep) fusion proteins expressed by E. coli BL21 (DE3) were extracted with bacterial protein extraction reagent (B-PER; Thermo Fisher Scientific, Rochester, NY) and refolded as we previously described (Huang et al, 2018;Nandre et al, 2016;Rausch et al, 2017). Refolded fusion proteins were examined in SDS-PAGE with Coomassie blue staining, and were characterized in Western blot or direct ELISAs with anti-F18 mouse antiserum.…”

“…Blocking attachment of all ETEC fimbriae and neutralizing against enterotoxicity of LT and STs have proven very challenging. However, a recent breakthrough in antigen preparation by using neutralizing epitopes and multiepitope-fusion-antigen (MEFA) technology makes completion of such a task feasible Nandre et al, 2016Ruan et al, 2014a. Additionally, molecular epidemiological studies showed that the vast majority of ETEC strains causing PWD express K88 or F18 fimbriae in conjunction with 2-3 toxins (Frydendahl, 2002;Zhang et al, 2007).…”

Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTa N12S -mnLT G192G/L211A -Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea

“…Plasmids (pET28␣) carrying the chimeric adhesin-toxoid MEFA gene were introduced into E. coli BL21 bacteria to express tagless adhesin-toxoid MEFA protein. Protein expression, extraction, and refolding followed protocols described previously (37,38). Briefly, a single colony of the recombinant strain was cultured in 5 ml lysogeny broth (LB) supplemented with kanamycin (30 g/ml) at 37°C on a shaker (150 rpm).…”

Development of an enterotoxigenic Escherichia coli vaccine based on the heat-stable toxin

“…55 Accordingly, in silico predicted B-cell epitopes from CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21, and EtpA were inserted into the backbone of the colonization factor antigen I (CFA/I) tip subunit CfaE where they replaced surface exposed, but less antigenic peptides. 56 Notably, mice immunized with the chimeric antigen elicited antibodies against all the nine epitopes, and the sera prevented the attachment of ETEC strains to Caco-2 cells in vitro. 56 MEFA CFs were further genetically fused to an ST toxoid and LT antigen to afford even broader protection.…”

“…56 Notably, mice immunized with the chimeric antigen elicited antibodies against all the nine epitopes, and the sera prevented the attachment of ETEC strains to Caco-2 cells in vitro. 56 MEFA CFs were further genetically fused to an ST toxoid and LT antigen to afford even broader protection. 57,58 Immunized mice that were challenged with lethal doses of ETEC (CFA/I + , LT + , ST + ) were protected.…”