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.     

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.     

Enumeration of Gut-Homing β7-Positive,Pathogen-Specific Antibody-Secreting Cells in Whole Blood from Enterotoxigenic Escherichia coli- and Vibrio cholerae-Infected Patients,Determined Using an Enzyme-Linked Immunosorbent Spot Assay Technique

Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates β7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. β7 is a cell surface marker associated with mucosal homing. We isolated β7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a significant increase in toxin-specific gut-homing ASCs at day 7 compared to the levels at days 2 and 30 after onset of illness and to the levels in healthy controls. Similar elevations of responses to the ETEC colonization factors (CFs) CS6 and CFA/I were observed in patients infected with CS6- and CFA/I-positive ETEC strains. Antigen-specific gut-homing ASCs to the B subunit of cholera toxin and cholera-specific lipopolysaccharides (LPS) were also observed on day 7 after the onset of cholera using this approach. This study demonstrates that a simple ELISPOT assay can be used to study the mucosal immunity to specific antigens using a cell-sorting protocol to isolate mucosal homing cells, facilitating measurement of mucosal responses in children following infection or vaccination.     

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.     

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.     

Recent progress toward an enterotoxigenic Escherichia coli vaccine

Enterotoxigenic Escherichia coli(ETEC) is the most common cause of bacterial diarrhea in children in Africa, Asia and Latin America and in travelers to these regions. Despite this, no effective vaccine for ETEC is available. ETEC causes disease by colonizing the small intestine with colonization factors, most of which are fimbriae, and production of heat-labile and/or heat-stable enterotoxins. Antibodies against heat-labile enterotoxin and the colonization factors have been shown to be protective, and local immunity in the gut seems to be of prime importance for protection. Hence, several inactivated and live candidate ETEC vaccines consisting of toxin antigens, alone or together with colonization factors, have been evaluated in clinical trials. In this review, the authors describe ETEC vaccine development in progress and the rationale for constructing different types of vaccines. They also discuss possibilities of enhancing immune responses to candidate ETEC vaccines, particularly in children.     

Enterotoxigenic Escherichia coli with STh and STp genotypes is associated with diarrhea both in children in areas of endemicity and in travelers

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea among children in developing countries and in travelers to areas of ETEC endemicity. ETEC strains isolated from humans may produce a heat-labile enterotoxin (LT) and two types of the heat-stable enterotoxin STa, called STh and STp, encoded by the estA gene. Two commonly used assay methods for the detection of STa, the infant mouse assay or different enzyme-linked immunosorbent assays, are unable to distinguish between the two subtypes of ST. Different genotypic methods, such as DNA probes or PCR assays, may, however, allow such discrimination. Using gene probes, it has recently been reported that ETEC strains producing STp as the only enterotoxin are not associated with diarrhea. In this study, we have used highly specific PCR methods, including newly designed primers for STh together with previously described STp primers, to compare the relative distribution of STh and STp in ETEC isolated from children with diarrhea in three different geographically distinct areas, i.e., Bangladesh, Egypt, and Guatemala, and from travelers to Mexico and Guatemala. It was found that ETEC strains producing STp were as commonly isolated from cases of diarrhea as strains producing STh both in Egypt and Guatemala, whereas STp strains were considerably less common in Bangladesh. No difference was found in the relative distribution of STh and STp in ETEC strains isolated from travelers with diarrhea and from asymptomatic carriers. Irrespective of ST genotype, the disease symptoms were also similar in both children and travelers.     

A tripartite fusion, FaeG-FedF-LT(192)A2:B, of enterotoxigenic Escherichia coli (ETEC) elicits antibodies that neutralize cholera toxin, inhibit adherence of K88 (F4) and F18 fimbriae, and protect pigs against K88ac/heat-labile toxin infection

Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT(192)A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT(192)A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.     

Context-dependent activation kinetics elicited by soluble versus outer membrane vesicle-associated heat-labile enterotoxin

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler's diarrhea and children's diarrhea worldwide. Among its virulence factors, ETEC produces heat-labile enterotoxin (LT). Most secreted LT is associated with outer membrane vesicles that are rich in lipopolysaccharide. The majority of prior studies have focused on soluble LT purified from ETEC periplasm. We investigated the hypothesis that the extracellular vesicle context of toxin presentation might be important in eliciting immune responses. We compared the polarized epithelial cell responses to apically applied soluble LT and LT-containing vesicles (LT(+) vesicles) as well as controls using a catalytically inactive mutant of LT and vesicles lacking LT. Although vesicle treatments with no or catalytically inactive LT induced a modest amount of interleukin-6 (IL-6), samples containing catalytically active LT elicited higher levels. A combination of soluble LT and LT-deficient vesicles induced significantly higher IL-6 levels than either LT or LT(+) vesicles alone. The responses to LT(+) vesicles were found to be independent of the canonical LT pathway, because the inhibition of cyclic AMP response element (CRE)-binding protein (CREB) phosphorylation did not lead to a decrease in cytokine gene expression levels. Furthermore, soluble LT caused earlier phosphorylation of CREB and activation of CRE compared with LT(+) vesicles. Soluble LT also led to the activation of activator protein 1, whereas LT(+) vesicle IL-6 responses appeared to be mediated by NF-κB. In summary, the results demonstrate that soluble LT and vesicle-bound LT elicit ultimately similar cytokine responses through distinct different activation pathways.     

Protection by milk immunoglobulin concentrate against oral challenge with enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli is a common cause of traveler's diarrhea. Prophylaxis against traveler's diarrhea has been associated with side effects from bismuth subsalicylate and the development of resistance to antimicrobial agents. We undertook a double-blind controlled trial in which a bovine milk immunoglobulin concentrate with high titers of antibodies against enterotoxigenic E. coli was used as prophylaxis against E. coli challenge in volunteers. Lyophilized milk immunoglobulins were prepared from the colostrum of cows immunized with several enterotoxigenic E. coli serotypes and fimbria types, E. coli heat-labile enterotoxin, and cholera toxin. As a control, an immunoglobulin concentrate with no anti-E. coli activity was prepared. Ten volunteers received buffered immunoglobulin concentrate against enterotoxigenic E. coli, and 10 received the control immunoglobulin concentrate, dissolved in water, three times a day. No side effects were observed. On the third day of immunoglobulin prophylaxis, the volunteers were given 10(9) colony-forming units of enterotoxigenic E. coli H10407 (O78:H11). This strain produces colonization factor antigen I and heat-labile and heat-stable enterotoxins. None of the 10 volunteers receiving the immunoglobulin concentrate against E. coli had diarrhea, but 9 of the 10 controls did (P less than 0.0001). All volunteers excreted E. coli H10407. We conclude from these preliminary results that milk immunoglobulin concentrate may be an effective prophylaxis against traveler's diarrhea.     

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.     

Detection of diarrheagenic Escherichia coli by multiplex PCR

Background: Diarrheagenic E.coli (DEC) are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus) forEnteropathogenic E.coli (EPEC), hlyA for Enterohemorrhagic E.coli (EHEC), elt and stla for Enterotoxigenic E.coli (ETEC), CVD432 for Enteroaggregative E.coli (EAEC) and ial for Enteroinvasive E.coli (EIEC). Results: In 200 children with diarrhea 52 (26%) DEC infections were found. Among 100 controls 8 (8%) DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%)and controls (5,5%), followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5%) and 3 (1.5%) of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.     

Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of infectious diarrhea worldwide. Four categories of antigens have been commonly studied: O serogroup, H serogroup, colonization factor antigens (CFA), and toxins. A database has been complied from published reports of nearly 1,000 ETEC isolates from 18 locations and analyzed to determine the occurrence, distribution, and associations of O serogroup, H serogroup, CFA, and toxin type. Tables listing the associations of antigens are presented. This analysis documents the widespread nature and variety of ETEC. Even the most common combination of antigens, O6:H16 CFA/II LTST, accounted for only 11% of the ETEC isolates in the database. It was isolated from 12 locations. Many phenotypes occurred only once. CFA detection based on enzyme-linked antibodies with polyclonal sera is suggested as the preferred assay. A combination of CFA and toxin-based antigens is suggested as the most practical vaccine.     

Enterotoxigenic Escherichia coli in central Canada.

During epidemiological studies carried out in urban and rural areas of the midwestern Canadian province of Manitoba, we cultured enterotoxigenic Escherichia coli (ETEC) from 16 (1.7%) of 945 diarrheal stools and 4 (0.3%) of 1,282 normal stools. ETEC was found in not more than 2.3% of diarrheal stools obtained from any population during any season. Diarrhea associated with ETEC persisted for a mean of 9 days. Two children were dehydrated and required intravenous fluid therapy, and one adult suffered a cholera-like syndrome. Half of the children required hospitalization for management of their diarrhea. Two adults and two children who harbored ETEC were completely asymptomatic. The pattern of toxin production correlated with serotype and the serotypes encountered were (with a few exceptions) similar to those found in other areas. We conclude that ETEC is an uncommon cause of diarrhea, both in rural and urban areas of central Canada. However, the possibility that ETEC might cause severe sporadic cases or epidemics of gastroenteritis remains.     

Test-of-cure stool cultures for traveler''s diarrhea.

Whether enteropathogens were eradicated or persisted in test-of-cure stool cultures from 251 patients with traveler's diarrhea, the durations of diarrhea were similar within the antimicrobial agent-treated (32 versus 33 h) and placebo-treated (82 versus 96 h) groups. Routine test-of-cure stool cultures can be useful for evaluating treatment failures and for assessing asymptomatic carriage of enteropathogens after treatment, but they are not mandated in the design of placebo-controlled antimicrobial treatment trials in traveler's diarrhea when the focus of the trial is clinical efficacy.     

Antibiotic resistance pattern of enterotoxigenicEscherichia coli isolated from infants and young adults in Israel

The aim of this study was to describe antibiotic resistance rates of enterotoxigenicEscherichia coli in Israel in order to facilitate the empirical choice of antibiotic treatment or prophylaxis for traveler's diarrhea and infantile diarrhea in our region. A total of 281 enterotoxigenicEscherichia coli isolates were tested: 144 from Bedouin infants and 137 from Israeli soldiers. Antibiotic-resistant isolates were prevalent in both groups, but higher resistance rates were found in the pediatric group. Strains producing heat-labile toxin showed higher resistance rates than strains producing heat-stable toxin. The results obtained in Israel preclude the use of many commonly used antibiotics for the treatment of traveler's diarrhea. Quinolones, however, are still effective.     

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.     

Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II,and CFA/IV

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.     

A comparative genomic analysis of diverse clonal types of enterotoxigenic Escherichia coli reveals pathovar-specific conservation

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in children less than 5 years of age in low- and middle-income nations, whereas it is an emerging enteric pathogen in industrialized nations. Despite being an important cause of diarrhea, little is known about the genomic composition of ETEC. To address this, we sequenced the genomes of five ETEC isolates obtained from children in Guinea-Bissau with diarrhea. These five isolates represent distinct and globally dominant ETEC clonal groups. Comparative genomic analyses utilizing a gene-independent whole-genome alignment method demonstrated that sequenced ETEC strains share approximately 2.7 million bases of genomic sequence. Phylogenetic analysis of this "core genome" confirmed the diverse history of the ETEC pathovar and provides a finer resolution of the E. coli relationships than multilocus sequence typing. No identified genomic regions were conserved exclusively in all ETEC genomes; however, we identified more genomic content conserved among ETEC genomes than among non-ETEC E. coli genomes, suggesting that ETEC isolates share a genomic core. Comparisons of known virulence and of surface-exposed and colonization factor genes across all sequenced ETEC genomes not only identified variability but also indicated that some antigens are restricted to the ETEC pathovar. Overall, the generation of these five genome sequences, in addition to the two previously generated ETEC genomes, highlights the genomic diversity of ETEC. These studies increase our understanding of ETEC evolution, as well as provide insight into virulence factors and conserved proteins, which may be targets for vaccine development.