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
Authors: Duan Qiangde , Huang Jiachen , Xiao Nan , Seo Hyesuk , Zhang Weiping 
Published: October 2017

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 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
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
-mnLT
R192G/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 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
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.

IMPORTANCE
Enterotoxigenic
Escherichia coli
(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
in vitro
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.