Escherichia coliK88ac Fimbriae Expressing Heat-Labile and Heat-Stable (STa) Toxin Epitopes Elicit Antibodies That Neutralize Cholera Toxin and STa Toxin and Inhibit Adherence of K88ac FimbrialE. coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, 8 LCSEYRNTQIYTIN 21 ) and an STa toxoid epitope ( 5 CCELCCNPQCAGCY 18 ) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC.Enterotoxigenic Escherichia coli (ETEC) strains that colonize the host small intestine and produce heat-labile (LT) and/or heat-stable (ST) enterotoxins are a major cause of diarrheal disease in humans and farm animals. The virulence determinants of ETEC in diarrhea are bacterial adhesins and enterotoxins (1,7,24,25,37,41,43). Adhesins mediate the attachment of ETEC bacteria to host epithelium cells in the small intestine and facilitate subsequent bacterial colonization. Enterotoxins, including ST and LT toxins (17,18,33), disrupt host fluid homeostasis and cause fluid and electrolyte hypersecretion through activation of adenyl cyclase (by LT) or guanylate cyclase (by STa) in small intestinal epithelial cells (19,23). Recent experimental studies using a porcine model demonstrated that ETEC strains expressing LT or STa as the only toxin are sufficiently virulent to cause diarrhea (7,43,46).No vaccines are currently available to effectively protect humans and animals against ETEC infections. Experimental oral vaccines carrying adhesin antigens alone showed protection against colonization by ETEC strains expressing the same or homologous adhesins (40). Similarly, experimental antitoxin vaccines using toxin antigens, mainly LT toxoids or LT B subunits, could not provide effective protection either. Evidence indicated that LT antigen-based experimental vaccines provided protection against only LT-producing ETEC strains but not against ETEC strains that produce STa toxin (13, 14).As more than two-thirds of human ETEC diarrheal cases and more than one-quarter of porcine ETEC diarrhea cases are caused by STa-producing ETEC strains (15,1...

A Tripartite Fusion, FaeG-FedF-LT192A2:B, of Enterotoxigenic Escherichia coli (ETEC) Elicits Antibodies That Neutralize Cholera Toxin, Inhibit Adherence of K88 (F4) and F18 Fimbriae, and Protect Pigs against K88ac/Heat-Labile Toxin Infection

“…Anti-LT antibodies were titrated in a GM1 ELISA using GM1 and LT B (not CT, which is typically used in GM1 ELISA) as antigens, as described previously (39,40,41). For anti-K88 and anti-F18 antibody titration, each well was coated with 50 ng of heat-extracted K88 fimbriae and F18 fimbriae in a Maxisorb plate (Nunc, Roskilde, Denmark) for ELISA as described previously (39). Mouse serum, fecal suspension, and intestine wash samples (in 1:100 dilution) were used in a binary dilution.…”

“…Horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (1:3,300; Sigma) was used as the secondary antibody. The cutoff OD values in the ELISAs were defined as the A 405 background plus 0.4 (15,39,40,41). The dilution that gave OD values above the cutoff was calculated for antibody titers, and titers were expressed in a log 10 scale.…”

“…The collected porcine serum, fecal suspensions, and intestine wash samples were examined for antibody neutralization against commercial available CT toxin (Sigma) and adherence inhibition against K88 and F18 fimbriae. Neutralization against CT toxin was examined using T-84 cells and a cyclic AMP (cAMP) enzyme immunoassay kit (Assay Design, Ann Arbor, MI) as described previously (39,41). We used CT instead of LT in neutralization assays in this study.…”

A Multiepitope Fusion Antigen Elicits Neutralizing Antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea VirusIn Vitro

“…T-84 cells (ATCC, CCL-248) and an intracellular cyclic GMP (cGMP) enzyme immunoassay (EIA) kit (Assay Design, Ann Arbor, MI) were used to examine antibody neutralization activity against STa toxin (a gift from D. Robertson at Kansas State University) as described previously (22)(23)(24) …”

“…In order to be broadly effective, an ETEC vaccine needs to induce anti-adhesin immunity to block bacterial adherence but also antitoxin immunity to neutralize enterotoxicity (20,21). Recently, studies demonstrated that an STa toxoid, when genetically fused to a strongly immunogenic carrier protein, such as a heat-labile toxoid or E. coli fimbria, elicited protective anti-STa antibodies (22)(23)(24). That indicates that STa antigen can be included as an ETEC vaccine component to induce protective antitoxin immunity.…”