Characterization of Heat-Stable (STa) Toxoids of Enterotoxigenic Escherichia coli Fused to Double Mutant Heat-Labile Toxin Peptide in Inducing Neutralizing Anti-STa Antibodies

Abstract

A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteria causing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin type Ib (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vaccine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safe for vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studies demonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies and suggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, we selected 14 STa toxoids from a mini-STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies, and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions. These toxoid fusions were used to immunize mice and were characterized for induction of anti-STa antibody response. The results showed that different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T, and STaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated that antibodies induced by the 3×STaN12S-dmLT fusion antigen exhibited the greatest neutralizing activity against STa toxin. These results suggested 3×STaN12S-dmLT is a preferred fusion antigen to induce an anti-STa antibody response and provided long-awaited information for effective ETEC vaccine development.