Characterization of immunological cross-reactivity between enterotoxigenic Escherichia coli heat-stable toxin and human guanylin and uroguanylin

Abstract

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.

FIG 1

Structure and sequence comparisons of STp, STh, uroguanylin, and guanylin. (A) Surface representation of structural models of STp and STh and experimental structures of uroguanylin (PDB accession no. 1UYA) and guanylin (PDB accession no. 1GNA). The shared cysteines are shown in light blue, other residues shared by at least three of the peptides are in dark blue, and the ST-specific disulfide bridge is in dark gray. Each structure is shown from two opposite sides. (B) Sequence alignment of STp, STh, uroguanylin, and guanylin, colored as described above for the structures. The ST disulfide bonding pattern is shown above the alignment, and the (uro)guanylin pattern is shown below.

FIG 2

Anti-ST antibodies neutralize ST-induced intracellular cGMP production in the T-84 cell assay. (A) Ten nanograms of STh was preincubated with different dilutions of polyclonal anti-STh and anti-STp antibodies (horizontal axis) prior to the T-84 cell assay. Normalized cGMP levels are given as percentages of the positive control containing only STh peptide (vertical axis). Two unrelated polyclonal antibodies were included as negative controls. (B) Ten nanograms of STp was preincubated with different molar ratios of three anti-STp monoclonal antibodies, clone 29, clone 30, and clone 31 (horizontal axis), prior to the T-84 cell assay. Normalized cGMP levels are given as percentages of the positive control containing only STp peptide (vertical axis). The results from three independent experiments are shown, with the means shown as horizontal lines.

FIG 3

Anti-STp monoclonal antibody clone 31 specifically neutralizes induction of intracellular cGMP production mediated by STp but not by STh in the T-84 cell assay. Ten nanograms of STp and 10 ng of STh were preincubated with different molar ratios of clone 31 (horizontal axis) prior to the T-84 cell assay. Normalized cGMP levels are given as percentages of the positive control containing only STp peptide (vertical axis). The results from three independent experiments are shown, with the means shown as horizontal lines.

FIG 4

Anti-STh (A), anti-STp (B), antiuroguanylin (C), and antiguanylin (D) polyclonal antibodies were tested for their abilities to bind to the STh, STp, uroguanylin (UGN), and guanylin (GN) peptides in competitive ELISAs. Each panel to the left displays data from three independent experiments where serial dilutions of the four peptides (horizontal axis) were tested in triplicate. Points represent the means of all three experiments, and the vertical lines outline the maximum and minimum values. The vertical axis represents the ability of the peptides to inhibit the binding of antibody to the ELISA coating, given as a percentage of maximum binding (activity) measured in the absence of a competing peptide. The middle panels repeat the mean values of the highest peptide doses, along with P values for comparisons of the test peptides and the negative control (nc). Sequence alignments are shown to the right of the panels, where the GC-C ligand domain residues are shaded to indicate identity to those of the cognate peptide for each antibody. The sequence identities are given, and the dendrograms reflect similarities.

FIG 5

Competitive ELISA to estimate the abilities of STh, STp, uroguanylin, and guanylin peptides to bind to the anti-STh ST:G8 monoclonal antibody. Three independent experiments were performed with serial dilutions of all peptides in triplicate (horizontal axis; logarithmic scale). Points represent the means of all three experiments, and the vertical lines outline the maximum and minimum values. The vertical axis represents the ability of the peptides to inhibit the binding of antibody to the ELISA coating, given as a percentage of maximum binding (activity) measured in the absence of a competing peptide. The maximum inhibition values calculated by regression analysis from these data are 97% ± 1% for STh, 95% ± 1% for STp, and 94% ± 2% for uroguanylin. Calculated half-maximal inhibitory concentrations (IC₅₀s) are 0.0027 ± 0.0001 μM for STh, 0.0120 ± 0.0006 μM for STp, and 0.21 ± 0.01 μM for uroguanylin.