Mouse intestinal innate immune responses altered by enterotoxigenic Escherichia coli (ETEC) infection

Enterotoxigenic Escherichia coli (ETEC) is an important cause of human and porcine morbidity and mortality. The current study was conducted to identify intestinal immunity that is altered in a mouse model of ETEC infection. Innate immune responses and inflammation were analyzed. The activation of signal transduction pathways, including toll like receptor 4 (TLR-4)-nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK), was analyzed using immunoblotting and PCR array analyses. We found that ETEC infection promoted the expression of pro-inflammatory cytokines through the activation of the NF-κB and MAPK pathways. Meanwhile, ETEC infection affected sIgA transportation and Paneth cell function. These data improve our understanding of how ETEC causes disease in animals.     

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.     

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.     

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

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 X 10(10) 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 X 10(9) 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.     

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.     

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.     

An in silico chimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant

Enteric infections resulting in diarrheal diseases remain as major global health problems. Among bacteria, enterotoxigenic Escherichia coli (ETEC) causes the largest number of diarrheal cases. There is a great interest in developing an effective ETEC vaccine. An ETEC vaccine could focus on virulence factors present in ETEC pathogens and nontoxic Heat-labile B subunit (LTB). Chimeric proteins carrying epitopes, or adjuvant sequences increase the possibility of eliciting a broad cellular or humoral immune response. In-silico tools are highly suited to study, design and evaluate vaccine strategies. Colonization factors are among the virulence factor studied in the present work employing bioinformatic tools. A synthetic chimeric gene, encoding CfaB, CstH, CotA, and LTB was designed. Modeling was done to predict the 3D structure of protein. This model was validated using Ramachandran plot statistics. The predicted B-cell epitopes were mapped on the surface of the model. Validation result showed that 97.2% residues lie in favored or additional allowed region of Ramachandran plot. VaxiJen analysis of the protein showed high antigenicity. Linear and conformational B-cell epitopes were identified. The identified T-cell epitopes are apt to bind MHC molecules. The epitopes in the chimeric protein are likely to induce both the B-cell and T-cell mediated immune responses.     

CFA/I-ST plasmids: comparison of enterotoxigenic Escherichia coli (ETEC) of serogroups O25, O63, O78, and O128 and mobilization from an R factor-containing epidemic ETEC isolate

Colonization factor antigen I (CFA/I) plays an important role in the pathogenesis of diarrhea due to enterotoxigenic Escherichia coli. In this study, we examined 11 CFA/I+ enterotoxigenic E. coli from serogroups O25, O63, O78, and O128 and found that with all strains, spontaneous loss of CFA/I was associated with the loss of heat-stable toxin (ST) and with the loss of a single plasmid ranging in size from 54 to 60 megadaltons; when heat-labile toxin was lost, this was associated with the loss of another plasmid. The R factor of one strain, TX432 (O78:H12:CFA/I+; ST+), was found to mobilize the CFA/I-ST plasmid into E. coli K-12 at a frequency of 20%. These studies provide further evidence that CFA/I production is plasmid mediated in enterotoxigenic E. coli belonging to serogroups O25, O63, O78, and O128.     

肠毒素大肠杆菌定居因子CFA/Ⅰ和CS6在减毒福氏志贺氏菌中的共表达

定居因子CFA/I和CS6是肠毒素大肠杆菌 (ETEC)中重要的两种优势抗原 ,是ETEC疫苗研制的首选组分。采用基因重组技术将二者构建在以asd基因为选择标记的重组质粒上 ,与asd基因缺失突变型减毒福氏志贺氏菌FWL0 1构成宿主 载体平衡致死系统。实验结果表明 ,重组疫苗候选株能够稳定表达CFA/I和CS6抗原 ,并可在菌体表面形成相应菌毛。重组菌口服免疫BALB/c小鼠后 ,可诱生相应的抗CFA/I和CS6的特异性血清抗体IgG和分泌型抗体sIgA ,说明以志贺氏菌为载体 ,可以构建同时表达多个定居因子抗原的ETEC多价菌苗     

Strategies to overexpress enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveler's diarrhea (TD). Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. The most extensively studied ETEC candidate vaccine is the rCTB-CF ETEC vaccine, containing recombinantly produced cholera B subunit and the most commonly encountered ETEC CFs on the surface of whole inactivated bacteria. Initial clinical trials with this vaccine showed significant immune responses against the key antigens in different age groups in Bangladesh and Egypt and protection against more severe TD in Western travelers. However, when tested in a phase-III trial in Egyptian infants, the protective efficacy of the vaccine was found to be low, indicating the need to improve the immunogenicity of the vaccine, e.g., by increasing the levels of the protective antigens. This review describes different strategies for the construction of recombinant nontoxigenic E. coli and Vibrio cholerae candidate vaccine strains over-expressing higher amounts of ETEC CFs than clinical ETEC isolates selected to produce high levels of the respective CF, e.g., those ETEC strains which have been used in the rCTB-CF ETEC vaccine. Several different expression vectors containing the genes responsible for the expression and assembly of the examined CFs, all downstream of the powerful tac promoter, which could be maintained either with or without antibiotic selection, were constructed. Expression from the tac promoter was under the control of the lacI ^q repressor present on the plasmids. Following induction with isopropyl-β-d-thiogalactopyranoside, candidate vaccine strains over-expressing single CFs, unnatural combinations of two CFs, and also hybrid forms of ETEC CFs were produced. Specific monoclonal antibodies against the major subunits of the examined CF were used to quantify the amount of the surface-expressed CF by a dot-blot assay and inhibition ELISA. Oral immunization with formalin- or phenol-inactivated recombinant bacteria over-expressing the CFs was found to induce significantly higher antibody responses compared to immunization with the previously used vaccine strains. We therefore conclude that our constructs may be useful as candidate strains in an oral whole-cell inactivated CF ETEC vaccine.     

Antibody response against plasmid-encoded toxin (Pet) and the protein involved in intestinal colonization (Pic) in children with diarrhea produced by enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of pediatric and adult travellers diarrhea. The mechanism by which EAEC induce diarrhea is not completely known. Two serine protease autotransporter proteins, named Pet and Pic have been identified in EAEC strains. Pet has enterotoxic and cytotoxic activities, while the role of Pic in pathogenesis may lie on its mucinolytic activity. Little is known about Pet and Pic biological activities in vivo. In this study the antibody responses against these autotransporter proteins in convalescent children is investigated. Fifteen (83%) children showed specific antibodies against Pet or Pic in their sera. IgG and IgM antibodies were the main isotype found. Specific antibodies against Pic, but not against Pet, were detected in sera from age-matched control group. These data show that specific anti-Pet and anti-Pic antibodies are produced during the course of a natural EAEC infection in children.