Vaccines against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children less than 3 years of age in developing countries and in travelers to these areas. The key pathogenic mechanisms that contribute to the pathogenesis of ETEC are the production of colonization factors (CFs) and a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin. To provide broad-spectrum protection, an ETEC vaccine should, most likely, contain the most prevalent fimbrial antigens, that is, CF antigen I and CS1-CS6, and/or a LT toxoid. Different strategies have been taken to deliver ETEC fimbriae and toxin antigens to the human immune system to elicit strong mucosal, in particular, intestinal immune responses that are considered to be of prime importance for protection against ETEC disease. There has been some promise when testing different ETEC candidate vaccines for protection against diarrhea in adult travelers. However, no ETEC candidate vaccine has been shown to be effective in the most important target group, which is infants and young children in endemic areas. Against this background, intense efforts are in progress to try to improve the immunogenicity of different available candidate vaccines, as well as to develop new types of ETEC vaccines.     

Progress and hurdles in the development of vaccines against enterotoxigenic Escherichia coli in humans

Diarrhea is the second leading cause of death in children younger than 5 years. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacterial cause of diarrhea in young children living in endemic countries and children and adults traveling to these areas. Pathogenesis of ETEC diarrhea has been well studied, and the key virulence factors are bacterial colonization factor antigens and enterotoxins produced by ETEC strains. Colonization factor antigens mediate bacteria attachment to host small intestinal epithelial cells and subsequent colonization, whereas enterotoxins including heat-labile and heat-stable toxins disrupt fluid homeostasis in host epithelial cells, which leads to fluid and electrolyte hypersecretion and diarrhea. Vaccines stimulating host anti-adhesin immunity to block ETEC attachment and colonization and also antitoxin immunity to neutralize enterotoxicity are considered optimal for prevention of ETEC diarrhea. Vaccines under development have been designed to stimulate local intestinal immunity and are either oral vaccines or transcutaneous vaccines. A cholera vaccine (Dukoral?) does stimulate anti-heat-labile toxin immunity and is licensed for short-term protection of ETEC diarrhea in travelers in some countries. Newer experimental ETEC vaccine candidates are being developed with hope to provide long-lasting and more broad-based protection against ETEC. Some have shown promising results in safety and immunogenicity studies and are approaching field trials for efficacy. A key problem is the development of a vaccine that is both practical and inexpensive so that it can be affordable for use in poor countries where it is needed.     

Current Progress in Developing Subunit Vaccines against Enterotoxigenic Escherichia coli-Associated Diarrhea

Diarrhea continues to be a leading cause of death in children <5 years of age, and enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of children''s diarrhea. Currently, there are no available vaccines against ETEC-associated diarrhea. Whole-cell vaccine candidates have been under development but require further improvements because they provide inadequate protection and produce unwanted adverse effects. Meanwhile, a newer approach using polypeptide or subunit vaccine candidates focusing on ETEC colonization factor antigens (CFAs) and enterotoxins, the major virulence determinants of ETEC diarrhea, shows substantial promise. A conservative CFA/I adhesin tip antigen and a CFA MEFA (multiepitope fusion antigen) were shown to induce cross-reactive antiadhesin antibodies that protected against adherence by multiple important CFAs. Genetic fusion of toxoids derived from ETEC heat-labile toxin (LT) and heat-stable toxin (STa) induced antibodies neutralizing both enterotoxins. Moreover, CFA-toxoid MEFA polypeptides, generated by fusing CFA MEFA to an STa-LT toxoid fusion, induced antiadhesin antibodies that broadly inhibited adherence of the seven most important ETEC CFAs associated with about 80% of the diarrhea cases caused by ETEC strains with known CFAs. This same antigen preparation also induced antitoxin antibodies that neutralized both toxins that are associated with all cases of ETEC diarrhea. Results from these studies suggest that polypeptide or subunit vaccines have the potential to effectively protect against ETEC diarrhea. In addition, novel adhesins and mucin proteases have been investigated as potential alternatives or, more likely, additional antigens for ETEC subunit vaccine development.     

Outer membrane vesicles induce immune responses to virulence proteins and protect against colonization by enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains are a heterogeneous group of pathogens that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Collectively, these pathogens are responsible for hundreds of thousands of deaths annually in developing countries, particularly in children under the age of 5 years. The heterogeneity of previously investigated molecular targets and the lack of complete sustained protection afforded by antitoxin immunity have impeded progress to date toward a broadly protective vaccine. Many pathogens, including ETEC, have the capacity to form outer membrane vesicles (OMV), which often contain one or more virulence proteins. Prompted by recent studies that identified several immunogenic virulence proteins in outer membrane vesicles of ETEC, we sought to examine the immunogenicity and protective efficacy of these structures in a murine model of infection. Here we demonstrate that immunization with OMV impairs ETEC colonization of the small intestine and stimulates antibodies that recognize the heat-labile toxin and two additional putative virulence proteins, the EtpA adhesin and CexE. Similar to earlier studies with EtpA, vaccination with LT alone also inhibited intestinal colonization. Together, these findings suggest that OMV could be exploited to deliver protective antigens relevant to development of ETEC vaccines.     

Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains

Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease and infant mortality in developing countries. Studies of ETEC pathogenesis relevant to vaccine development have been greatly hampered by the lack of a suitable small-animal model of infection with human ETEC strains. Here, we demonstrate that adult immunocompetent outbred mice can be effectively colonized with the prototypical human ETEC H10407 strain (colonization factor antigen I; heat-labile and heat-stable enterotoxin positive) and that production of heat-labile holotoxin provides a significant advantage in colonization of the small intestine in this model.     

Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli.

Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC.     

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

Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of diarrheal illness worldwide. These pathogens disproportionately afflict children in developing countries, where they cause substantial morbidity and are responsible for hundreds of thousands of deaths each year. Although these organisms are important targets for enteric vaccines, most development efforts to date have centered on a subset of plasmid-encoded fimbrial adhesins known as colonization factors and heat-labile toxin (LT). Emerging data suggest that ETEC undergoes considerable changes in its surface architecture, sequentially deploying a number of putative adhesins during its interactions with the host. We demonstrate here that one putative highly conserved, chromosomally encoded adhesin, EaeH, engages the surfaces of intestinal epithelial cells and contributes to bacterial adhesion, LT delivery, and colonization of the small intestine.     

Enterotoxigenic Escherichia coli virulence factors and vaccine approaches

Enterotoxigenic Escherichia coli (ETEC) is recognized as one of the major causes of infectious diarrhea in developing countries. Worldwide, the incidence of ETEC infections is estimated to result in 650 million cases of diarrhea and 380,000 deaths in children under 5 years of age. ETEC is also an important cause of travelers' diarrhea in people traveling to endemic regions of the world. Although ETEC is an uncommon cause of infections in the USA, there have been 14 reported outbreaks of ETEC in the USA and seven on cruise ships over the 20-year period between 1975 and 1995. ETEC strains are comprised of a large number of serotypes that produce a variety of colonization factors and enterotoxins. On infection, ETEC first establishes itself by adhering to the epithelium of the small intestine via one or more colonization factor antigens or coli surface proteins. Once established, ETEC expresses one or more enterotoxin(s), which results in the production of secretory diarrhea. While the need for an efficacious, easily administered vaccine is great, there are currently no licensed ETEC vaccines available for use in endemic countries or for US travelers.     

Transcutaneous immunization using colonization factor and heat-labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) diarrheal disease is a worldwide problem that may be addressed by transcutaneous delivery of a vaccine. In several human settings, protective immunity has been associated with immune responses to E. coli colonization factors and to the heat-labile toxin that induces the diarrhea. In this set of animal studies, transcutaneous immunization (TCI) using recombinant colonization factor CS6 and cholera toxin (CT) or heat-labile enterotoxin (LT) as the adjuvant induced immunoglobulin G (IgG) and IgA anti-CS6 responses in sera and stools and antibody responses that recognized CS6 antigen in its native configuration. The antitoxin immunity induced by TCI was also shown to protect against enteric toxin challenge. Although immunization with LT via the skin induced mucosal secretory IgA responses to LT, protection could also be achieved by intravenous injection of the immune sera. Finally, a malaria vaccine antigen, merzoite surface protein 1(42) administered with CT as the adjuvant, induced both merzoite surface protein antibodies and T-cell responses while conferring protective antitoxin immunity, suggesting that both antiparasitic activity and antidiarrheal activity can be obtained with a single vaccine formulation. Overall, our results demonstrate that relevant colonization factor and antitoxin immunity can be induced by TCI and suggest that an ETEC traveler's diarrhea vaccine could be delivered by using a patch.     

A combination vaccine consisting of three live attenuated enterotoxigenic Escherichia coli strains expressing a range of colonization factors and heat-labile toxin subunit B is well tolerated and immunogenic in a placebo-controlled double-blind phase I trial in healthy adults

Immune responses against colonization factors (CFs) and the nontoxic B component of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LTB) are considered to be important for immunity against diarrhea caused by ETEC. Individual live attenuated ETEC derivatives that have had their toxin genes removed and whose aroC, ompC, and ompF genes are deleted have shown promise as vaccines against ETEC. The development of such strains has culminated in the testing of a three-strain-combination live attenuated vaccine known as ACE527, comprised of strains ACAM2025 expressing colonization factor antigen I (CFA/I) and LTB; ACAM2022, expressing CS5, CS6, and LTB; and ACAM2027, expressing CS1, CS2, CS3, and LTB. The recombinant CF and LTB genes expressed in the three strains were inserted into the bacterial chromosome to ensure their stable inheritance and expression without the requirement for any selection. ACE527 has been tested in a randomized placebo-controlled, double-blind, phase I safety and immunogenicity study in healthy adult volunteers and proved to be well tolerated and immunogenic at dose levels of 10(10) and 10(11) total CFU. There was no indication of strain interference on the basis of fecal shedding patterns, with all three being detected in the feces of 50% and 83% of low- and high-dose vaccine recipients, respectively. Similarly, strong immune responses to LTB and to CFs expressed on all three constituent strains were induced, with at least 50% of subjects in the high-dose group responding to LTB, CFA/I, CS3, and CS6.     

Vaccination with Dukoral against travelers' diarrhea (ETEC) and cholera

There is currently only one vaccine available that provides protection against diarrhea caused by Vibrio cholerae and, to a lesser degree, enterotoxigenic Escherichia coli (ETEC). Adverse events of this oral whole-cell/recombinant B-subunit vaccine have been negligible. Protective efficacy against cholera is 85%, while protection against the heat-labile toxin of ETEC reaches 67%. There is still a need for data on protection of Western travelers against travelers' diarrhea in general by Dukoral vaccination. However, current studies show a protective effect of up to 43%. Although the vaccine is only licensed for vaccination against cholera in most Western countries, there is mounting evidence that the oral cholera vaccine is a valuable option to those traveling to high-risk endemic areas. Vaccination against cholera and ETEC should be recommended for at-risk travelers, in particular those with high exposure at their travel destination or high personal risks through fluid loss.     

Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously

Transcutaneous immunization (TCI) is a new method for vaccine delivery that has been shown to induce immunity relevant to enteric disease vaccines. We evaluated the clinical safety and immunogenicity of a recombinant subunit vaccine against enterotoxigenic Escherichia coli (ETEC) delivered by TCI. Adult volunteers received patches containing the recombinant ETEC colonization factor CS6, either with heat-labile enterotoxin (LT) or patches containing CS6 alone. The vaccine was administered at 0, 1, and 3 months, and serum antibodies and antibody-secreting cells (ASCs) were assessed. Among the 26 volunteers that completed the trial, there were no responses to CS6 in the absence of LT. In the groups receiving both CS6 and LT, 68 and 53% were found to have serum anti-CS6 immunoglobulin G (IgG) and IgA, respectively; 37 and 42% had IgG and IgA anti-CS6 ASCs. All of the volunteers receiving LT had anti-LT IgG, and 90% had serum anti-LT IgA; 79 and 37% had anti-LT IgG and IgA ASCs. Delayed-type hypersensitivity (DTH), suggesting T-cell responses, was seen in 14 of 19 volunteers receiving LT and CS6; no DTH was seen in subjects receiving CS6 alone. This study demonstrated that protein antigens delivered by a simple patch could induce significant systemic immune responses but only in the presence of an adjuvant such as LT. The data suggest that an ETEC vaccine for travelers delivered by a patch may be a viable approach worthy of further evaluation.     

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children from developing countries and in adult travelers from industrialized countries to the developing world (16, 21). According to the World Health Organization (WHO), ETEC is the second most common cause of diarrhea after rotavirus in children less than 5 years of age and is therefore an important target for vaccine development (11). Diarrhea due to ETEC develops between 8 and 72 h after initial infection, usually due to the ingestion of contaminated food and water (21). The disease varies from a mild illness to one of great severity, usually without leukocytes or fecal blood but often with vomiting and, potentially, dehydration (10).The ability of ETEC to adhere to and colonize the human intestinal mucosa has been correlated with the presence of specific antigenic fimbriae called colonization factors (CFs), which have been designated colonization factor antigens (CFAs), coli surface antigens (CSs), or putative colonization factors (PCFs), followed by a numeric designation. The CFs are mainly fimbrial or fibrillar proteins, although some are not fimbrial in structure (21). To date, over 25 human ETEC CFs have been described. In turn, these CFs have been divided into different families: (i) a CFA/I-like group including CFA/I, CS1, CS2, CS4, CS14, and CS17; (ii) a CS5-like group including CS5, CS7, CS18, and CS20; and (iii) a unique group including CS3, CS6, and CS10 to CS12 (8, 21, 33).Following CF-mediated mucosal adhesion, ETEC elaborates one or both of two enterotoxins: heat-labile toxin (LT), a protein multimer which shares many features with cholera toxin and which binds to intracellular adenylylcyclase, leading to increased cyclic AMP levels, and/or heat-stable toxin (ST), a small-peptide molecule that similarly activates guanylylcyclase and which produces increased intracellular cyclic GMP. For both toxins, the increased chloride secretion resulting from these toxins produces a watery diarrhea (10, 16). Both of these virulence factors are plasmid encoded. ST is encoded by two different genes: estA and st1, which produce STh (originally isolated from ETEC in humans) and STp (originally from a pig isolate), respectively. LT toxin is encoded by the eltA and eltB genes (12). The diagnosis of ETEC infection relies upon the detection of either the genes themselves or their gene products in clinical specimens.Currently, derivatives of LT and the CFs are targets for the development of vaccines against ETEC. However, the great variability of ETEC CFs requires determination of the CF types prevalent in different geographic locations (21, 33). The aims of this study were (i) to determine the clinical and epidemiological characteristics of ETEC diarrhea in Peruvian children, (ii) to determine the presence of ST and LT, (iii) to determine the presence and distribution of colonization factors in these strains, and (iv) to determine the antibiotic susceptibilities of these strains.     

Mutations in the periplasmic chaperone leading to loss of surface expression of the colonization factor CS6 in enterotoxigenic Escherichia coli (ETEC) clinical isolates

Enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by adhesion to human enterocytes by one or more colonization factors (CFs) and secretion of heat-labile (LT) and/or heat-stable (ST) enterotoxins. Expression of coli surface antigen 6 (CS6) on the bacterial surface, usually associated with ETEC strains that produce ST alone or in combination with LT, is rarely found in strains expressing only LT. However, a number of LT-only strains which are genotypically positive but phenotypically negative for CS6 have been identified. In this study, eight such strains from India and Guinea-Bissau belonging to different clones were analysed. The CS6 operon cssABCD was transcribed but protein analyses suggested that the structural subunits CssA and CssB of CS6 were absent in the periplasm. Most strains contained truncating mutations within the periplasmic chaperone-encoding gene cssC and protein modelling indicated that this severely affected the substrate-binding capacity of the chaperone. A single-nucleotide polymorphism (SNP) (A-->T) in the 5'-untranslated region of cssC distinguished the eight strains from ETEC strains that do express CS6 on the surface and may be a potential marker for ETEC strains containing phenotypically silent cssABCD. The study emphasizes the importance of using both genotypic and phenotypic methods in epidemiological studies of ETEC, e.g. for vaccine development.     

Generation and characterization of a live attenuated enterotoxigenic Escherichia coli combination vaccine expressing six colonization factors and heat-labile toxin subunit B

Live attenuated oral enterotoxigenic Escherichia coli (ETEC) vaccines have been demonstrated to be safe and immunogenic in human volunteers and to provide a viable approach to provide protection against this important pathogen. This report describes the construction of new ETEC vaccine candidate strains from recent clinical isolates and their characterization. All known genes for ETEC toxins were removed, and attenuating deletion mutations were made in the aroC, ompC, and ompF chromosomal genes. An isolate expressing coli surface antigen 2 (CS2), CS3, heat-labile toxin (LT), heat-stable toxin (ST), and enteroaggregative Escherichia coli heat-stable toxin 1 (EAST1) was attenuated to generate ACAM2007. The subsequent insertion of the operon encoding CS1 created ACAM2017, and this was further modified by the addition of an expression cassette containing the eltB gene, encoding a pentamer of B subunits of LT (LTB), to generate ACAM2027. Another isolate expressing CS5, CS6, LT, ST, and EAST1 was attenuated to generate ACAM2006, from which a lysogenic prophage was deleted to create ACAM2012 and an LTB gene was introduced to form ACAM2022. Finally, a previously described vaccine strain, ACAM2010, had the eltB gene incorporated to generate ACAM2025. All recombinant genes were incorporated into the chromosomal sites of the attenuating mutations to ensure maximal genetic stability. The expression of the recombinant antigens and the changes in plasmids accompanying the deletion of toxin genes are described. Strains ACAM2025, ACAM2022, and ACAM2027 have been combined to create the ETEC vaccine formulation ACE527, which has recently successfully completed a randomized, double-blind, placebo-controlled phase I trial and is currently undergoing a randomized, double-blind placebo-controlled phase II challenge trial, both in healthy adult volunteers.     

Characterization of enterotoxigenic Escherichia coli isolated from U.S. troops deployed to the Middle East.

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.     

Enterotoxigenic Escherichia coli Secretes a Highly Conserved Mucin-Degrading Metalloprotease To Effectively Engage Intestinal Epithelial Cells

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of death due to diarrheal illness among young children in developing countries, and there is currently no effective vaccine. Many elements of ETEC pathogenesis are still poorly defined. Here we demonstrate that YghJ, a secreted ETEC antigen identified in immunoproteomic studies using convalescent patient sera, is required for efficient access to small intestinal enterocytes and for the optimal delivery of heat-labile toxin (LT). Furthermore, YghJ is a highly conserved metalloprotease that influences intestinal colonization of ETEC by degrading the major mucins in the small intestine, MUC2 and MUC3. Genes encoding YghJ and its cognate type II secretion system (T2SS), which also secretes LT, are highly conserved in ETEC and exist in other enteric pathogens, including other diarrheagenic E. coli and Vibrio cholerae bacteria, suggesting that this mucin-degrading enzyme may represent a shared virulence feature of these important pathogens.     

The EtpA exoprotein of enterotoxigenic Escherichia coli promotes intestinal colonization and is a protective antigen in an experimental model of murine infection

The enterotoxigenic Escherichia coli (ETEC) strains are major causes of morbidity and mortality due to diarrheal illness in developing countries. At present, there is no broadly protective vaccine for this diverse group of pathogens. The EtpA protein, identified in ETEC H10407 in a recent search for candidate immunogens, is a large glycosylated exoprotein secreted via two-partner secretion (TPS). Similar to structurally related molecules, EtpA functions in vitro as an adhesin. The studies reported here use a recently developed murine model of ETEC intestinal colonization to examine the immunogenicity and protective efficacy of EtpA. We report that mice repeatedly exposed to ETEC are protected from subsequent colonization and that they mount immune responses to both EtpA and its presumed two-partner secretion transporter (EtpB) during the course of experimental infection. Furthermore, isogenic etpA deletion mutants were impaired in the colonization of mice, and intranasal immunization of mice with recombinant EtpA conferred protection against ETEC H10407 in this model. Together, these data suggest that EtpA is required for optimal colonization of the intestine, findings paralleling those of previous in vitro studies demonstrating its role in adherence. EtpA and other TPS proteins may be viable targets for ETEC vaccine development.     

Survey in Vanuatu on enterotoxigenic Escherichia coli in children and infants with and without acute diarrhea.

We have studied the incidence of enterotoxigenic Escherichia coli (ETEC) strains isolated from infants with and without diarrheal diseases in Vanuatu, South Pacific. Over a period of 5 months we have isolated enterotoxigenic E. coli strains from 29 (26.6%) of 109 children with acute diarrhea and from 13 (21.6%) of 60 children of the control group. In the group with diarrhea, 7 (6.4%) strains released heat-labile toxin, 7 (6.4%) released heat-stable toxin, and 15 (13.7%) produced both heat-labile and heat-stable toxin. In the control group, only one strain (1.6%) produced heat-stable toxin, 12 (20%) produced heat-labile toxin, and none produced both. Association of strains releasing heat-stable toxin or both heat-labile and heat-stable toxin with diarrhea was highly significant as shown by statistical analysis. The O serogroups and colonization factors CFA/I and CFA/II are presented.