Citation

Heat-stable toxin (STa)-producing enterotoxigenic Escherichia coli (ETEC) strains are a top cause of moderate-to-severe diarrhea in children from developing countries and a common cause of travelers’ diarrhea. Recent progress in using STa toxoids and toxoid fusions to induce neutralizing anti-STa antibodies accelerates ETEC vaccine development. However, concern remains whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two GC-C ligands regulating the fluid and electrolyte transportation in human intestinal and renal epithelial cells. To further divert STa from guanylin and uroguanylin structurally and antigenically and to eliminate anti-STa antibody cross-reactivity with guanylin and uroguanylin, we mutated STa toxin at the 9(th) (leucine), the 12(th) (asparagine) and the 14(th) (alanine) residues for double and triple mutants STaL9A/N12S, STaL9A/A14H, STaN12S/A14T and STaL9A/N12S/A14H We then fused each STa mutant (three copies) to a monomeric LT mutant (mnLTR192G/L211A) for toxoid fusions 3xSTaL9A/N12S-mnLTR192G/L211A, 3xSTaL9A/A14H-mnLTR192G/L211A, 3xSTaN12S/A14T-mnLTR192G/L211A and 3xSTaL9A/N12S/A14H-mnLTR192G/L211A, examined each fusion for anti-STa immunogenicity, and assessed derived antibodies for in vitro neutralization activity against STa toxicity and for cross-reactivity with guanylin and uroguanylin. Mice subcutaneously immunized with each fusion protein developed anti-STa antibodies, and the antibodies derived from 3xSTaN12S-mnLTR192G/L211A, 3xSTaL9A/N12S-mnLTR192G/L211A or 3xSTaN12S/A14T-mnLTR192G/L211A prevented STa from stimulation of intracellular cGMP in T-84 cells. Competitive ELISAs showed guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that the antibodies derived from 3xSTaN12S-mnLTR192G/L211A, 3xSTaL9A/N12S-mnLTR192G/L211A or 3xSTaN12S/A14T-mnLTR192G/L211A neutralized STa toxin and had little cross-reactivity with guanylin and uroguanylin, suggesting these toxoid fusions are suitable antigens for ETEC vaccines.Importance Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children’ diarrhea and travelers’ diarrhea. Currently, there is no licensed vaccine against ETEC diarrhea. One key challenge is to identify safe antigens to induce antibodies neutralizing the key STa toxin without cross-reacting with guanylin and uroguanylin, two important ligands controlling the homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies neutralizing STa enterotoxicity in vitro and not cross-reacting with guanylin or uroguanylin. These fusions become the preferred antigens for developing ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.