Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli

Abstract

g Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity. D iarrhea caused by enterotoxigenic Escherichia coli (ETEC)contributes to the almost 600,000 annual child deaths due to diarrheal disease in low-and middle-income countries (1). In addition, the 280 to 400 million annual episodes of ETEC diarrhea in children Ͻ5 years old (2, 3) contribute to malnutrition and a failure to thrive (4). Thus, the World Health Organization strongly encourages the development of an ETEC vaccine (2). ETEC is also the most common cause of traveler's diarrhea (5).ETEC causes diarrhea by colonizing the small intestine with subsequent expression of heat-labile (LT) and/or heat-stable toxins (ST; variants STh and STp) that elicit a net efflux of salt and water into the intestinal lumen (6). Both LT and ST are potential vaccine targets, in addition to the many surface-exposed antigens, including the colonization factors. ETEC vaccine development has primarily targeted colonization factors and LT (7,8), and the most promising vaccine candidate to date is a killed whole-cell vaccine comprising five different ETEC strains that express the most prevalent colonization factors, coadministered with the cholera toxin B subunit, which is a homologue of the LT B subunit (9). This vaccine was found to be efficacious against serious diarrhea among American travelers to Guatemala (10) but not protective when evaluated in Egyptian children (11).A recently conducted large global multicenter study designed to analyze the etiology of enteric pathogens in children ranked ...

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout

“…22 estP and estH produce two different variants of heat-stable enterotoxins found in enterotoxigenic E. coli, and is a major cause of diarrhea in travelers and infants. [20][21][22] Additionally, we designed another primer for aggR to observe using malachite green aptamer, a red aptamer (λ ex =630 nm, λ em =650 nm). 20,23,24 Reactions were successful using Corn to detect estP and malachite green aptamer to detect aggR (Figure 3A and Supplementary Figure S15).…”

“…Reactions contained 40 mM Tris-acetate, 24 mM Mg-glutamate, 100 mM K-glutamate, 2 mM spermidine, 1 mM DTT, 4 mM ATP, 4 mM GTP, 4 mM UTP, 4 mM CTP, 1 µM overexpressed T7 RNA polymerase (conversely New England Biolabs T7 RNA Polymerase M0251L can be used, but it does not give as great a yield), 1X Pyrophosphatase as used in Heili et al and double stranded DNA templates were used according to Supplementary Table S1. 21 Optimal template concentration was determined empirically and was typically ca. 100 nM for the amplicons used here.…”

Design and characterization of a chimeric multiepitope construct containing CfaB, heat‐stable toxoid, CssA, CssB, and heat‐labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach

“…This chimeric protein consists of 133 residues from a carboxy terminal of CfaB that is linked to the complete sequences of CssA, CssB, and LTB sequence. Furthermore, 14 residues [6][7][8][9][10][11][12][13][14][15][16][17][18][19] from the HT were selected as a toxoid fragment and incorporated into the allowed region of a CfaB protein. According to structural studies of CfaB, 36 residues of each subunit in the assembled form (CFA/I) buried in the stalk of fimbriae and did not expose.…”

“…1). Because of the small size of ST, the 14 residues encoding the toxic domain of detoxified ST (amino acids [6][7][8][9][10][11][12][13][14][15][16][17][18][19] were inserted into a CfaB molecule as an epitope. The pfam database was used to find out the best position for the insertion of a ST epitope.…”

Molecular determinants of enterotoxigenicEscherichia coliheat-stable toxin secretion and delivery

“…Bacteria producing any of the toxins LT, STh, or STp have been linked to diarrheal illness in humans( 20–23 ), and recent studies suggest that ST-producing ETEC are commonly represented among the pathogens that cause severe diarrheal illness among young children in low income countries leading to substantial interest in the development of a vaccine that incorporates ST-toxoids( 24 ).…”