Neutralizing Anti-Heat-Stable Toxin (STa) Antibodies Derived from Enterotoxigenic Escherichia coli Toxoid Fusions with STa Proteins Containing N12S, L9A/N12S, or N12S/A14T Mutations Show Little Cross-Reactivity with Guanylin or Uroguanylin

Abstract

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 STa_L9A/N12S, STa_L9A/A14H, STa_N12S/A14T and STa_{L9A/N12S/A14H}. We then fused each STa mutant (three copies) to a monomeric LT mutant (mnLT_R192G/L211A) for toxoid fusions 3xSTa_L9A/N12S-mnLT_R192G/L211A, 3xSTa_L9A/A14H-mnLT_R192G/L211A, 3xSTa_N12S/A14T-mnLT_R192G/L211A and 3xSTa_{L9A/N12S/A14H}-mnLT_R192G/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 3xSTa_N12S-mnLT_R192G/L211A, 3xSTa_L9A/N12S-mnLT_R192G/L211A or 3xSTa_N12S/A14T-mnLT_R192G/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 3xSTa_N12S-mnLT_R192G/L211A, 3xSTa_L9A/N12S-mnLT_R192G/L211A or 3xSTa_N12S/A14T-mnLT_R192G/L211A neutralized STa toxin and had little cross-reactivity with guanylin and uroguanylin, suggesting these toxoid fusions are suitable antigens for ETEC vaccines.