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 crossreactivity with guanylin or uroguanylin

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 has accelerated ETEC vaccine development. However, concern remains regarding whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two guanylate cyclase C (GC-C) ligands regulating 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 crossreactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the 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 heat-labile toxin (LT) mutant (mnLT_R192G/L211A) for the toxoid fusions 3×STa_L9A/N12S-mnLT_R192G/L211A, 3×STa_L9A/A14H-mnLT_R192G/L211A, 3×STa_N12S/A14T-mnLT_R192G/L211A, and 3×STa_{L9A/N12S/A14H}-mnLTR192G/L211A; examined each fusion for anti-STa immunogenicity; and assessed the 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 3×STa_N12S-mnLT_R192G/L211A, 3×STa_L9A/N12S-mnLT_R192G/L211A, or 3×STa_N12S/A14T-mnLT_R192G/L211A prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzymelinked immunosorbent assays (ELISAs) showed that guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that antibodies derived from 3×STa_N12S-mnLT_R192G/L211A, 3×STa_L9A/N12S-mnLT_R192G/L211A, or 3×STa_N12S/A14T-mnLT_R192G/L211A neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines.