Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV

Abstract

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

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

“…To construct adhesin tip MEFA gene, we first used web-based B-cell epitope prediction software to in silico identify B-cell epitopes from the adhesin tips of six adhesins (CFA/I, CS1-CS5) and adhesive subunits of three adhesins (CS6, CS21, EtpA), as previously described [41]. We then selected CFA/I tip CfaE gene ( cfa E) the backbone, truncated eight nucleotide fragments coding surface-exposed but less antigenic peptides from the cfaE gene, and replaced them with nucleotide fragments coding the most antigenic epitope of the other eight adhesin tips or adhesive subunits.…”

“…For serum of the control mice, dilutions of 1:200 were also included in ELISAs. Each adhesin tip or subunit recombinant protein (400 ng per well), in 100 μl coating buffer [41], was added to wells of Immulon 2HB 96-well plates (Thermo Fisher Scientific, Rochester, NY). Plates incubated at 37°C for 1 h and then 4°C overnight were washed three times with PBS-0.05% Tween 20 (PBST).…”

“…To develop a vaccine preventing attachment and colonization from CFA/I, CS1-CS6, CS21 and EtpA ETEC adhesins, in this study we in silico identified antigenic B-cell epitopes from adhesin tips or adhesive subunits of these nine adhesins, and applied MEFA (multiepitope fusion antigen) approach [41] to integrate the most antigenic epitope predicted from each of these nine adhesin tips or adhesive subunits into a single MEFA protein. This adhesin tip MEFA was then used to immunize mice and examined for anti-adhesin antigenicity, and was assessed for potential application in ETEC vaccine development.…”

Current Progress in Developing Subunit Vaccines against Enterotoxigenic Escherichia coli-Associated Diarrhea

“…This CFA MEFA was strongly immunogenic, as it induced high titers of antibodies specific to all seven of these CFAs and probably to other CFAs homologous to CFA/I. More importantly, the antibodies induced significantly blocked the in vitro adherence of E. coli strains expressing these seven CFAs (80). Thus, this CFA MEFA represents a strong candidate for antiadhesin subunit vaccine development.…”

“…Then we replaced surfaceexposed, but less immunogenic, epitopes of CfaB with the most immunogenic B-cell epitopes from other clinically important ETEC CFAs, including CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6). The end product was a CFA MEFA that induced broadly protective anti-CFA antibodies (80). Since ETEC strains expressing these seven CFAs are responsible for approximately 80% of the diarrhea cases caused by ETEC strains with known CFAs and are generally responsible for the moderate-to-severe cases (40), a vaccine protecting against these seven CFAs should be broadly effective against ETEC diarrhea.…”