Immunoprotective oral whole cell vaccine for enterotoxigenic Escherichia coli diarrhea prepared by in situ destruction of chromosomal and plasmid DNA with colicin E2

Abstract Vaccine regimens which mimic actual infection with bacterial enteropathogens should offer the best opportunity for successful long-term immunoprotection against diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC) or Vibrio cholerae . Based on this principle, we designed and tested an oral whole cell anti-ETEC vaccine consisting of intact cells of ETEC strain H-10407 (ST⁺LT⁺; O78: H11: CFA/I) which were rendered incapable of replication by treatment with a potent DNA endonuclease, colicin E2. Young healthy volunteers were administered two oral doses of either placebo or approx. 3 × 10¹⁰ vaccine cells. In a double-blind study, 9 of 10 vaccinees responded with an increase in CFA/I-specific intestinal IgA antibody, determined as percent of total IgA. Challenge with virulent strain H-10407 (5 × 10⁹ living cells) produced diarrhea in 8 of 9 (89%) of the placebo-treated volunteers and in 2 of 10 (20%) of the vaccinees. Thus, the colicin E2-killed whole cell vaccine afforded both a significant intestinal immune response and significant protection against challenge with the virulent organism. The data presented here suggest that for this vaccine preparation an intestinal anti-CFA/I IgA response is a good indicator of a protective immune response, which most likely involves antibody responses to a number of antigens in addition to CFA/I. We conclude that the colicin E2 method for preparing an oral anti-ETEC vaccine merits further study and that this method may also be applicable to other enteropathogens.     

Non-replicating oral whole cell vaccine protective against enterotoxigenic Escherichia coli (ETEC) diarrhea: stimulation of anti-CFA (CFA/I) and anti-enterotoxin (anti-LT) intestinal IgA and protection against challenge with ETEC belonging to heterologous serotypes

An oral killed (non-replicating) whole-cell anti-ETEC vaccine was prepared by treating enterotoxigenic Escherichia coli strain H-10407 (ST + LT +; 078: H11: CFA/I) with a 100%-lethal amount of colicin E2. Colicin E2 is a potent DNA endonuclease which enters the target bacterial cells without disrupting cellular integrity. Thus the vaccine consists of intact cells lacking chromosomal and plasmid DNA but possessing a normal complement of antigens, including CFA/I and enterotoxin(s), unaltered by chemical- or heat-treatment. Young healthy volunteers were administered two oral doses, one month apart, of approximately 3 x 10(10) vaccine cells. Of 22 vaccinees, 17 (77.3%) showed an intestinal anti-CFA/I IgA response and 19 (86.4%) showed an increase in intestinal anti-LT IgA. Twenty of 22 (90.9%) vaccinees had antibody responses to either CFA/I, LT, or both antigens, demonstrating that colicin E2-treated CFA-positive E. coli cells are an efficient vehicle in terms of delivery of antigens to the gut immune system. We previously demonstrated protection of vaccinees against challenge with the living homologous ETEC (strain H-10407). In this study, two groups of 8 vaccinees were challenged with a diarrheagenic dose of virulent ST + LT + ETEC of heterologous serotype; one group was challenged with a CFA/I-positive 063: H- strain and the other group was challenged with a CFA/II-positive 06: H16 strain. Approximately 75% efficacy was achieved in both challenge groups. None of the 16 vaccinees who had responded to both CFA/I and LT became ill upon challenge while both of the vaccinees who had not responded to either antigen did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine

In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.     

CfaE tip mutations in enterotoxigenic Escherichia coli CFA/I fimbriae define critical human intestinal binding sites

Enterotoxigenic Escherichia coli (ETEC) use colonization factors to attach to the human intestinal mucosa, followed by enterotoxin expression that induces net secretion and diarrhoeal illness. ETEC strain H10407 expresses CFA/I fimbriae, which are composed of multiple CfaB structural subunits and a CfaE tip subunit. Currently, the contribution of these individual fimbrial subunits in intestinal binding remains incompletely defined. To identify the role of CfaE in attachment in the native ETEC background, an R181A single-amino-acid substitution was introduced by recombination into the H10407 genome. The substitution of R181A eliminated haemagglutination and binding of intestinal mucosa biopsies in in vitro organ culture assays, without loss of CFA/I fimbriae expression. Wild-type in trans plasmid-expressed cfaE restored the binding phenotype . In contrast, in trans expression of cfaE containing amino acid 181 substitutions with similar amino acids, lysine, methionine and glutamine did not restore the binding phenotype, indicating that the loss of the binding phenotype was due to localized areas of epitope disruption. R181 appears to have an irreplaceable role in the formation of a receptor-binding feature on CFA/I fimbriae. The results specifically indicate that the CfaE tip protein is a required binding factor in CFA/I-mediated ETEC colonization, making it a potentially important vaccine antigen.     

Detection of attachment of enterotoxigenic Escherichia coli (ETEC) to human small intestinal cells by enzyme immunoassay

Abstract Simple immunoassays were developed to study the binding between enterocytes of the small intestine and other cell types, and enterotoxigenic Escherichia coli (ETEC). CFA/I or CFA/II pilus protein or CFA-positive E. coli bacteria were wells of microtitre plates and incubated with vesicles or crude mucus prepared from human brush border enterocytes. Binding of the cell preparations was detected by adding specific rabbit anti-brush border IgG followed by urease-labelled goat anti-rabbit IgG and urea substrate. The binding of purified CFA/I to human or rabbit small intestine, human oral epithelial cells or Caco-2 cells was detected with specific anti-CFA/I IgG. Both human brush border and mucus-derived preparations were able to attach to ETEC. The binding was CFA-specific and strong enough to withstand several washings. In contrast, CFA/I did not bind to small intestinal cells of non-human small intestinal origin, indicating that there may be important differences in affinity between receptors present on human small intestinal cells and cells of non-human small intestinal origin. Antibodies directed against human small intestinal and non-small intestinal cells did not cross-react with either preparation, indicating that receptors between these different cell sources are different. The EIA proved useful during the identification of a newly-recognised 15 kDa bacterial surface component of ETEC strain H10407P, which may function as a putative attachment factor. The EIAs developed in this study were easy to perform and multiple tests could be performed on small samples, including biopsy samples obtained during endoscopy.     

Immune response to Escherichia coli antigens entrapped in liposomes. I. Immunopathological studies

In this study, we have searched for an effective mucosal delivery system for a purified E. coli antigen which elicits anticolonization and anti-toxic immunity. E. coli colonization factor antigen (CFA/I) and heat-labile enterotoxin (LT) were encapsulated in liposomes. To determine the efficacies of soluble and liposome-encapsulated E. coli antigens young rabbits were mucosally treated with three oral doses of E. coli antigens given 7 days apart. Ten days after the last booster, rabbits were orally challenged with 5 x 10(9) bacterial cells (O78:H11 serotype). The experimental results allow of making some remarks which can be correlated with the protection obtained in vaccinated animals: (a) immunization with E. coli antigens entrapped in liposomes ensured protection against ETEC strains; (b) lower protection against homologous and heterologous CFA/I +(LT- ST+) strains were noticed; (c) adhesion of labelled -3H-leucine-bacteria to the intestinal mucosa revealed a maximum distribution in duodenum-jejunum and minimum in the colonic mucosa; (d) it contributed to the release of inoculated virulent bacteria from intestinal tract; (e) humoral, cellular and histopathological findings confirm the afore mentioned observation. Summing up, these results suggest that liposomes are very good carriers for E. coli antigens and these findings highlight the potential use of LT and CFA/I antigens entrapped in liposomes as mucosal and humoral induction of immune response and make them a candidate for future use in prophylaxis of diarrhoea in man.     

Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166

Entertoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I. Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae. Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae. CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe. Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes. The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production. Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen-Specific Memory B-cell Responses to Enterotoxigenic Escherichia coli Infection in Bangladeshi Adults

Background

Multiple infections with diverse enterotoxigenic E. coli (ETEC) strains lead to broad spectrum protection against ETEC diarrhea. However, the precise mechanism of protection against ETEC infection is still unknown. Therefore, memory B cell responses and affinity maturation of antibodies to the specific ETEC antigens might be important to understand the mechanism of protection.

Methodology

In this study, we investigated the heat labile toxin B subunit (LTB) and colonization factor antigens (CFA/I and CS6) specific IgA and IgG memory B cell responses in Bangladeshi adults (n = 52) who were infected with ETEC. We also investigated the avidity of IgA and IgG antibodies that developed after infection to these antigens.

Principal Findings

Patients infected with ETEC expressing LT or LT+heat stable toxin (ST) and CFA/I group or CS6 colonization factors developed LTB, CFA/I or CS6 specific memory B cell responses at day 30 after infection. Similarly, these patients developed high avidity IgA and IgG antibodies to LTB, CFA/I or CS6 at day 7 that remained significantly elevated at day 30 when compared to the avidity of these specific antibodies at the acute stage of infection (day 2). The memory B cell responses, antibody avidity and other immune responses to CFA/I not only developed in patients infected with ETEC expressing CFA/I but also in those infected with ETEC expressing CFA/I cross-reacting epitopes. We also detected a significant positive correlation of LTB, CFA/I and CS6 specific memory B cell responses with the corresponding increase in antibody avidity.

Conclusion

This study demonstrates that natural infection with ETEC induces memory B cells and high avidity antibodies to LTB and colonization factor CFA/I and CS6 antigens that could mediate anamnestic responses on re-exposure to ETEC and may help in understanding the requirements to design an effective vaccination strategies.     

Characterization of colonization factor antigen CFA/I from an enterotoxigenic Escherichia coli strain (0128:H12)

CFA/I antigen was isolated and purified from E. coli, mutant 279 B-1-14, serotype 0128:H12, and had the following biochemical and biological features: a) amino-acid content was similar to that of purified antigen prepared from strain H10407; b) latex particles sensitization with purified CFA/I antigen produced bovine and human erythrocytes group A/II hemagglutination in carbohydrates presence; c) purified anti-CFA/I specific antibodies agglutinated CFA/I-positive enterotoxigenic E. coli strains; d) 3H-leucine-labelled CFA/I antigen adhered to rabbits intestinal mucosa at significant values; e) intestinal mucosa pretreating with purified CFA/I antigen, followed by 3H-leucine labelled enterotoxigenic bacteria infection, had a least 3 local effects: 1) intestinal mucosa protection against parental enterotoxigenic bacteria; 2) inhibition of CFA/I-positive bacteria adherence to intestinal mucosa; 3) release of approximately 96% intraluminally inoculated bacteria.     

The role of CFA/I in adherence and toxin delivery by ETEC expressing multiple colonization factors in the human enteroid model

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low-to-middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC that causes moderate-to-severe diarrhea and plays an important role in pathogenesis. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and strain-dependent differences in CFA/I and CS21 expression. The critical role of CFA/I in adherence by ETEC strains expressing CFA/I and CS21 was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting a limitation of this static aerobic infection model. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the lack of decrease in transepithelial electrical resistance and the lack of IL-8 cytokines produced during infection. Taken together, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the human enteroid model has significant utility for the study of ETEC pathogenesis and evaluation of vaccine-induced functional antibody responses.     

Directed evaluation of enterotoxigenic Escherichia coli autotransporter proteins as putative vaccine candidates

Background

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies.

Methodology

Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice.

Principal Findings

Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC.

Conclusions

Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.     

Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTa_A14Q-dmLT or 3xSTa_N12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STa_A14Q-dmLT and CFA/I/II/IV-STa_N12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa_-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTa_N12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa_-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTa_N12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.     

Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal–mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.     

Catabolite repression of the colonization factor antigen I (CFA/I) operon ofEscherichia coli

Experiments were performed to study whether the synthesis of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenicEscherichia coli is affected by glucose. The CFA/I-producing strain H-10407 (O78:H11:CFA/I) was grown in CFA medium containing various concentrations of glucose. Addition of 1% glucose into the medium resulted in a pronounced decrease in CFA/I production by H-10407 as assessed by ELISA, hemagglutination, and electron microscopy. The repressive effect of glucose was reversed by the addition of 10 mM cAMP to the medium. Examination of the promoter sequence of thecfaA gene of the CFA/I operon revealed a consensus binding site for the catabolite activator protein-cAMP complex. With a reporter plasmid containing a fusion of thecfaA promoter, a portion of thecfaA gene, and thelacZ gene, it was shown that the activity of this promoter was influenced by glucose. In a wild-typeE. coli strain, addition of 0.5% glucose to the growth medium diminished the promoter activity more than 70%. ThecfaA promoter also exhibited a lower level of activity incya (adenyl cyclase) andcrp (cAMP receptor protein) mutants than in the wild-type strain. The addition of 10 mM cAMP resulted in a marked increase in the expression from thecfaA promoter in thecya but not in thecrp mutant. These results suggest that the suppressive effect of glucose in the CFA/I system is mediated via the mechanism of catabolite repression through thecfaA promoter of the CFA/I operon.     

A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic Escherichia coli Strain H10407

In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.Current dogma suggests the Gram-negative motile bacterium Escherichia coli colonizes the infant gut within hours of birth and establishes itself as the predominant facultative anaerobe of the colon for the remainder of life (3, 59). While the majority of E. coli strains maintain this harmless existence, some strains have adopted a pathogenic lifestyle. Contemporary tenets suggest that pathogenic strains of E. coli have acquired genetic elements that encode virulence factors and enable the organism to cause disease (12). The large repertoire of virulence factors enables E. coli to cause a variety of clinical manifestations, including intestinal infections mediating diarrhea and extraintestinal infections, such as urinary tract infections, septicemia, and meningitis. Based on clinical manifestation of disease, the repertoire of virulence factors, epidemiology, and phylogenetic profiles, the strains causing intestinal infections can be divided into six separate pathotypes, viz., enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), diffuse adhering E. coli (DAEC), and enterotoxigenic E. coli (ETEC) (33, 35, 39).ETEC is responsible for the majority of E. coli-mediated cases of human diarrhea worldwide. It is particularly prevalent among children in developing countries, where sanitation and clean supplies of drinking water are inadequate, and in travelers to such regions. It is estimated that there are 200 million incidences of ETEC infection annually, resulting in hundreds of thousands of deaths in children under the age of 5 (55, 64). The essential determinants of ETEC virulence are traditionally considered to be colonization of the host small-intestinal epithelium via plasmid-encoded colonization factors (CFs) and subsequent release of plasmid-encoded heat-stable (ST) and/or heat-labile (LT) enterotoxins that induce a net secretory state leading to profuse watery diarrhea (20, 62). More recently, additional plasmid-encoded factors have been implicated in the pathogenesis of ETEC, namely, the EatA serine protease autotransporter (SPATE) and the EtpA protein, which acts as an intermediate in the adhesion between bacterial flagella and host cells (23, 32, 42, 46). Furthermore, a number of chromosomal factors are thought to be involved in virulence, e.g., the invasin Tia; the TibA adhesin/invasin; and LeoA, a GTPase with unknown function (14, 21, 22). E. coli {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 is considered a prototypical ETEC strain; it expresses colonization factor antigen 1 (CFA/I) and the heat-stable and heat labile toxins. Loss of a 94.8-kb plasmid encoding CFA/I and a gene for ST enterotoxin from E. coli strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 leads to reduced ability to cause diarrhea (17).Here, we report the complete genome sequence and virulence factor repertoire of the prototypical ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and the nucleotide sequence and gene repertoire of the plasmids from ETEC strain E1392/75, and we describe a novel conserved secretion system associated with the sequenced ETEC strains.     

Linear epitopes of colonization factor antigen I and peptide vaccine approach to enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) cause diarrhea in infants and in travelers to developing countries. The bacteria utilize colonization factors (CF) for adherence to intestinal epithelia, then release toxins causing diarrhea. CF are strong immunogens as well as protective antigens. While 20 ETEC CF have been described in the literature, 11 CF are prominent enough to be considered for vaccine targeting. Of this group, six of the members fall into the CFA/I family of CF. Geysen pin (peptide) linear epitope analysis demonstrated that three regions containing linear epitopes exist in CFA/I, and that both B- and T-cell linear epitopes of CFA/I were concentrated at the N-terminus of the protein. We have determined N-terminal sequence of the CFA/I family members not previously sequenced. Comparison of the protein sequence of the six members of the family showed a strong homology up to residue 36. A peptide of 36 amino acids representing a consensus of the six sequences was synthesized and used to immunize animals. The antibody induced to the peptide was reactive to the peptide as well as cross-reactive to each member of the CFA/I family in Western blots. In addition, this antibody agglutinated three of the six members of the CFA/I family when added to whole cells expressing the native CF. We are currently evaluating different carriers and conjugation methods to maximize production of high titer, agglutinating antibody. It is hoped that this and related research will result in an effective and inexpensive cross-reactive and cross-protective ETEC vaccine. Received 04 October 1996/ Accepted in revised form 14 April 1997     

A live diarrheal vaccine imprints a Th2 cell bias and acts as an anti-inflammatory vaccine

An experimental vaccine for enterotoxigenic Escherichia coli (ETEC) composed of a live, attenuated Salmonella vector-expressing enterotoxigenic E. coli fimbriae, colonization factor Ag I (CFA/I), stimulated a biphasic Th cell response when given orally and suppressed the normally produced proinflammatory response. Such suppression was also evident upon the Salmonella-CFA/I infection of macrophages resulting in diminished TNF-alpha, IL-1, and IL-6 production and suggesting that the CFA/I fimbrial expression by Salmonella may protect against a proinflammatory disease. To test this hypothesis, SJL/J mice were vaccinated with Salmonella-CFA/I construct 1 or 4 wk before induction of experimental autoimmune encephalomyelitis using an encephalitogenic proteolipid protein peptide, PLP(139-151). Mice receiving Salmonella-CFA/I vaccine recovered completely from mild acute clinical disease and showed only mild inflammatory infiltrates in the spinal cord white and gray matter. This protective effect was accompanied by a loss of encephalitogenic IFN-gamma-secreting Th cells and was replaced with an increase in IL-4, IL-10, and IL-13 secretion. Collectively, these data suggested that Salmonella-CFA/I is an anti-inflammatory vaccine that down-regulates proinflammatory cells and confers protection against a proinflammatory disease, experimental autoimmune encephalomyelitis, via immune deviation.     

Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.     

双歧杆菌表达ETEC CFA／I的免疫电镜定位分析

目的研究双歧杆菌表达的CFA／I在细胞中的分布。方法将培养8～10h的重组双歧杆菌固定后制作电镜切片，镜检合格后用镍网捞取3-5个切片与金标抗体作用，镜检CFA／I在双歧杆菌中的表达分布。结果自制的胶体金颗粒均匀，与抗体结合后能够示踪CFA／I在双歧杆菌中的表达分布。结论双歧杆菌表达的CFA／I主要集中在靠近细胞膜的周质腔一侧，利于向外分泌和释放。