Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Abstract

dEnterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonization in vivo and toxin delivery to epithelial cells in vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development. Enterotoxigenic Escherichia coli (ETEC) strains comprise a genetically and phenotypically diverse group of organisms that share the ability to produce and effectively deliver heat-labile (LT) and/or heat-stable (ST) toxins to effector sites in the small intestine (14). Effective delivery of these toxins results in increases of intracellular concentrations of cyclic nucleotides, cyclic AMP (cAMP) and cGMP, respectively. Both toxins stimulate cellular kinases responsible for the phosphorylation of the cystic fibrosis transmembrane regulatory channel (CFTR) in the cell membrane (7,17). Activation of the CFTR in turn leads to efflux of chloride into intestinal lumen with commensurate salt and water losses responsible for the watery diarrhea associated with these pathogens.ETEC strains are a leading cause of diarrheal illness in developing countries, where they are responsible for hundreds of thousands of deaths, largely among young children. Although ETEC strains were discovered more than 40 years ago, the development of a broadly protective vaccine has been hampered by a number of factors (5, 35), including (i) the lack of complete sustained protection afforded by anti-LT immunity, (ii) the poor inherent immunogenicity of ST molecules, typically short peptides, and (iii) the antigenic heterogeneity of plasmid-encoded fimbrial colonization factors (20), one of the principle targets of ETEC vaccines to date.Despite these challenges, the development of immunity following either a naturally occurring (4, 34) or an experimental (22) infection with ETEC has suggested that the development of an ETEC vaccine is technically feasible. In addition, recent molecular (30, 31) and immunoproteomic (26) studies have demonstrated that the pathogenesis of these organisms is considerably more complex than previously appreciated, and conseque...

Contribution of the Highly Conserved EaeH Surface Protein to Enterotoxigenic Escherichia coli Pathogenesis

“…Theoretically, genes that promote interaction with the intestinal epithelium might also impact the delivery of LT. In accord with other recently described virulence factors that affect epithelial cell adhesion (18,22), eaeH mutants were impaired in delivery of heat-labile toxin, and antibodies against this surface protein significantly inhibited bacterial activation of cAMP in target epithelial cells (Fig. 4c) in vitro.…”

“…To examine the role of eaeH in delivery of heat-labile toxin, ETEC H10407 or mutants were used to infect Caco-2 cells grown to semiconfluence in 96-well plates. After 3 h, the plates were washed with prewarmed tissue culture media, incubated for an additional 2.5 h, and then lysed and processed for cAMP determination by using an enzyme-linked immunosorbent assay (Arbor Assays) as previously described (18,22).…”

“…More recent investigations have highlighted a number of novel putative virulence factors (16, 17), unique interactions of ETEC with the epithelium (18)(19)(20), and an intricate orchestration of multiple pathogen-host events (21) that culminate in successful toxin delivery to epithelial cell targets (22). Collectively, the emerging data suggest that these sophisticated interactions of ETEC strains with their host might be exploited in outlining novel strategies for vaccine development (23).…”

Immunogenicity and Protective Efficacy against Enterotoxigenic Escherichia coli Colonization following Intradermal, Sublingual, or Oral Vaccination with EtpA Adhesin

“…These features and the molecular and functional similarity of EtpA to filamentous hemagglutinin (36), a component of acellular pertussis vaccines (37), have prompted preclinical investigation of the utility of EtpA as a protective antigen. Multiple studies with mice have now demonstrated that EtpA vaccination protects against colonization of the small intestine (14,19,20), thought to be a critical determinant of ETEC diarrheal illness. Another consideration driving the present studies is that ETEC and Shigella are among the most common bacterial pathogens causing serious diarrheal illness among young children in developing countries; therefore, we questioned whether subunit approaches presently being developed for Shigella effectors (38, 39) could be also applied to novel secreted ETEC antigens, including EtpA.…”

“…Early proof-of-principal studies with EtpA were conducted using an intranasal route of vaccination with Protollin (14,(17)(18)(19) or heat-labile toxin (LT) (20). However, because LT is not safe for intranasal vaccination in humans (21,22), and because an LT toxoid will likely be a key component of next-generation vaccines, here we investigated the immunogenicity and protective efficacy of EtpA when delivered by other routes using a double mutant (R192G L211A) heat-labile toxin (dmLT) as the adjuvant.…”