Heat-Labile- and Heat-Stable-Toxoid Fusions (LT R192G -STa P13F ) of Human Enterotoxigenic Escherichia coli Elicit Neutralizing Antitoxin Antibodies

Abstract

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT R192G (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT R192G ) and a full-length STa toxoid (STa P13F ) and genetically fused them to produce LT 192 -STa 13 toxoid fusions. Mice immunized with LT 192 -STa 13 fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa 13 toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT 192 to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT 192 -STa 13 fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea.