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 (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 cross-reactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the double and triple mutants STa, STa, STa, and STa We then fused each STa mutant (three copies) to a monomeric heat-labile toxin (LT) mutant (mnLT) for the toxoid fusions 3×STa-mnLT, 3×STa-mnLT, 3×STa-mnLT, and 3×STa-mnLT; examined each fusion for anti-STa immunogenicity; and assessed the derived antibodies for 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-mnLT, 3×STa-mnLT, or 3×STa-mnLT prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzyme-linked 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-mnLT, 3×STa-mnLT, or 3×STa-mnLT neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines. Enterotoxigenic (ETEC) strains are a leading cause of children's 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 without cross-reacting with guanylin and uroguanylin, two important ligands controlling homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies that neutralize STa enterotoxicity and do not cross-react with guanylin or uroguanylin. These fusions have become the preferred antigens for the development of ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.

Significance of Enterotoxigenic Escherichia coli (ETEC) Heat-Labile Toxin (LT) Enzymatic Subunit Epitopes in LT Enterotoxicity and Immunogenicity

“…Portions (10 g) of each refolded epitope-ovalbumin fusion protein were examined by using sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. Electrophoresed proteins were visualized with Coomassie blue straining and characterized in a Western blot using anti-LT mouse serum or anti-CT rabbit polyclonal antiserum (Sigma) and IRDye-labeled goat anti-mouse or anti-rabbit IgG secondary antibodies (1:10,000 dilution; LI-COR, Lincoln, NE), as described previously (31,47,48).…”

“…Epitope-ovalbumin fusion-induced anti-LT IgG antibody titration. The anti-LT IgG antibody response was examined from each mouse serum sample as described previously (32,47,48). Briefly, LT (a gift from John Clements, Tulane University; 200 ng per well) was coated in wells of Immulon 2HB 96-well plates (Thermo Fisher Scientific).…”

“…Wells were washed with PBST, incubated with HRP-conjugated goat anti-mouse IgG secondary antibodies (1:3,000; Sigma), and then washed with PBST and incubated with TMB Microwell peroxidase substrate (KPL). OD 650 readings were used to calculate anti-LT antibody titers, as previously described (32,33,(47)(48)(49).…”

Development of an enterotoxigenic Escherichia coli vaccine based on the heat-stable toxin

“…88 Genetic fusion approaches to ST-based vaccine development has, therefore, been an important strategy for developing ST toxoid-based ETEC vaccines. 57,81,[89][90][91][92] Three copies of an STh-N12S mutant genetically fused with dmLT, 3xSTh N12S -dmLT, was identified as a promising vaccine candidate that may engender neutralizing antibodies against both ST and LT. 84,85,92 Notably, piglets born from 3xSTh N12S -dmLT immunized pregnant gilts seemed to be passively protected from ETEC diarrhea during challenge. 85 The 3xSTh N12S -dmLT construct was further genetically fused to seven putative immunodominant colonization factor epitopes from prevalent ETEC strains (CFA/I/II/IV).…”

“…68 The single STh mutant, N12S, and two double mutants, L9A/N12S, and N12S/A14T, were recently reported to elicit neutralizing antibodies in mice that showed little immunological cross-reactions to the endogenous peptides, when genetically fused in triplicate to monomeric-LT R192G/L211A. 91 However, when the authors assessed the immunological cross-reactions using a competitive ST ELISA, the ratio between free and bound peptide competing for binding to antibody was kept constant, in contrast to our previously published approach where we varied the ratio between free and bound peptides. 39 In our opinion, a proper dose-response experiment must be conducted in order to convincingly describe possible immunological cross-reactions or the lack thereof.…”