Intestinal immune responses in patients infected with enterotoxigenic Escherichia coli and in vaccinees.
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Immune responses against enterotoxigenic Escherichia coli (ETEC) were examined in Bangladeshi adults with naturally acquired disease and compared to responses in age-matched Bangladeshi volunteers who had been orally immunized with a vaccine consisting of inactivated ETEC bacteria expressing different colonization factor antigens (CFs) and the B subunit of cholera toxin. B-cell responses in duodenal biopsy samples, feces, intestinal washings, and blood were determined. Because most of the patients included in the study were infected with ETEC expressing CS5, immune responses to this CF were studied most extensively. Vaccinees and patients had comparable B-cell responses against this antigen in the duodenum: the median numbers of antibody-secreting cells (ASC) were 3,300 immunoglobulin A (IgA) ASC/10(7) mononuclear cells (MNC) in the patient group (n = 8) and 1,200 IgA ASC/10(7) MNC in the vaccinees (n = 13) (not a significant difference). Similarly, no statistically significant differences were seen in the levels of duodenal B cells directed against enterotoxin among vaccinees and patients. A comparison of the capacities of the various methods used to assess mucosal immune responses revealed a correlation between numbers of circulating B cells and antibody levels in saponin extracts of duodenal biopsy samples (r = 0.58; n = 13; P = 0.04) after vaccination. However, no correlation was seen between blood IgA ASC and duodenal IgA ASC after two doses of vaccine. Still, a correlation between numbers of CF-specific B cells in blood sampled from patients early during infection and numbers of duodenal B cells collected 1 week later was apparent (r = 0.70; n = 10; P = 0.03). (+info)
Safety and immunogenicity of two different lots of the oral, killed enterotoxigenic escherichia coli-cholera toxin B subunit vaccine in Israeli young adults.
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Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea among Israeli soldiers serving in field units. Two double-blind placebo-controlled, randomized trials were performed among 155 healthy volunteers to evaluate the safety and immunogenicity of different lots of the oral, killed ETEC vaccine consisting of two doses of whole cells plus recombinantly produced cholera toxin B subunit (rCTB). The two doses of vaccine lot E005 and the first dose of vaccine lot E003 were well tolerated by the volunteers. However, 5 (17%) vaccinees reported an episode of vomiting a few hours after the second dose of lot E003; none of the placebo recipients reported similar symptoms. Both lots of vaccine stimulated a rate of significant antibody-secreting cell (ASC) response to CTB and to colonization factor antigen I (CFA/I) after one or two doses, ranging from 85 to 100% and from 81 to 100%, respectively. The rate of ASC response to CS2, CS4, and CS5 was slightly lower than the rate of ASC response induced to CTB, CFA/I, and CS1. The second vaccine dose enhanced the response to CTB but did not increase the frequencies or magnitude of ASC responses to the other antigens. The two lots of the ETEC vaccine induced similar rates of serum antibody responses to CTB and CFA/I which were less frequent than the ASC responses to the same antigens. Based on these safety and immunogenicity data, an efficacy study of the ETEC vaccine is under way in the Israel Defense Force. (+info)
Molecular variation among type IV pilin (bfpA) genes from diverse enteropathogenic Escherichia coli strains.
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Typical enteropathogenic Escherichia coli (EPEC) strains produce bundle-forming pili (BFP), type IVB fimbriae that have been implicated in EPEC virulence, antigenicity, autoaggregation, and localized adherence to epithelial cells (LA). BFP are polymers of bundlin, a pilin protein that is encoded by the bfpA gene found on a large EPEC plasmid. Striking sequence variation has previously been observed among type IV pilin genes of other gram-negative bacterial pathogens (e.g., Pseudomonas and Neisseria spp.). In contrast, the established sequences of bfpA genes from two distantly related prototype EPEC strains vary by only a single base pair. To determine whether bundlin sequences vary more extensively, we used PCR to amplify the bfpA genes from 19 EPEC strains chosen for their various serotypes and sites and years of isolation. Eight different bfpA alleles were identified by sequencing of the PCR products. These alleles can be classified into two major groups. The alpha group contains three alleles derived from strains carrying O55, O86, O111, O119, O127, or O128 somatic antigens. The beta group contains five alleles derived from strains carrying O55, O110, O128ab, O142, or nontypeable antigens. Sequence comparisons show that bundlin has highly conserved and variable regions, with most of the variation occurring in the C-terminal two-thirds of the protein. The results of multilocus enzyme electrophoresis support the hypothesis that bfpA sequences have spread horizontally across distantly related clonal lineages. Strains with divergent bundlin sequences express bundlin protein, produce BFP, and carry out autoaggregation and LA. However, four strains lack most or all of these phenotypes despite having an intact bfpA gene. These results have important implications for our understanding of bundlin structure, transmission of the bfp gene cluster among EPEC strains, and the role of bundlin variation in the evasion of host immune system responses. (+info)
Immunoprophylactic potential of cloned Shiga toxin 2 B subunit.
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The Shiga toxins Stx1 and Stx2 contribute to the development of enterohemorrhagic O157:H7 Escherichia coli-mediated colitis and hemolytic-uremic syndrome in humans. The Stx2 B subunit, which binds to globotriaosylceramide (GB3) receptors on target cells, was cloned. This involved replacing the Stx2 B subunit leader peptide nucleotide sequences with those from the Stx1 B subunit. The construct was expressed in the TOPP3 E. coli strain. The Stx2 B subunits from this strain assembled into a pentamer and bound to a GB3 receptor analogue. The cloned Stx2 B subunit was not cytotoxic to Vero cells or apoptogenic in Burkitt's lymphoma cells. Although their immune response to the Stx2 B subunit was variable, rabbits that developed Stx2 B subunit-specific antibodies, as determined by immunoblot and in vitro cytotoxicity neutralization assays, survived a challenge with Stx2 holotoxin. This is thought to be the first demonstration of the immunoprophylactic potential of the Stx2 B subunit. (+info)
Oral administration of formaldehyde-killed recombinant bacteria expressing a mimic of the Shiga toxin receptor protects mice from fatal challenge with Shiga-toxigenic Escherichia coli.
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Gastrointestinal disease caused by Shiga toxin-producing Escherichia coli (STEC) is frequently complicated by life-threatening toxin-induced systemic sequelae, including the hemolytic uremic syndrome. We previously constructed a recombinant bacterium displaying a Shiga toxin receptor mimic on its surface which neutralized Shiga toxins with very high efficiency. Moreover, oral administration of the live bacterium completely protected mice from challenge with virulent STEC. In this study, we investigated the protective capacity of formaldehyde-killed receptor mimic bacteria, as these are likely to be safer for administration to humans. The killed bacteria completely protected STEC-challenged mice when administered three times daily; incomplete protection was achieved using two doses per day. Commencement of therapy could be delayed for up to 48 h after challenge without diminishing protection, depending on the virulence of the challenge strain. Thus, administration of this agent early in the course of human STEC disease may prevent progression to life-threatening complications. (+info)
Active immunization with a detoxified Escherichia coli J5 lipopolysaccharide group B meningococcal outer membrane protein complex vaccine protects animals from experimental sepsis.
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The passive infusion of antibodies elicited in rabbits with a detoxified J5 lipopolysaccharide (LPS)/group B meningococcal outer membrane protein complex vaccine protected neutropenic rats from heterologous lethal gram-negative bacterial infection. In this study, active immunization was studied in neutropenic rats infected with Pseudomonas aeruginosa, in the presence or absence of ceftazidime therapy, and with Klebsiella pneumoniae. This vaccine elicited a > 200-fold increase in anti-J5 LPS antibody, which remained elevated throughout the duration of cyclophosphamide-induced neutropenia and for < or = 3 months. There was improved survival among immunized versus control animals: 48% (13/28) versus 7% (2/29) in Pseudomonas-challenged rats; 61% (11/18) versus 0% (0/10) in Pseudomonas- and ceftazidime-treated rats; and 64% (9/14) versus 13% (2/15) in Klebsiella-challenged rats (P < 0.01 for each comparison). Immunized animals had lower levels of bacteria in organs and lower levels of circulating endotoxin at the onset of fever. In conclusion, active immunization with an anti-endotoxin vaccine improved survival after infection with > or = 2 heterologous, clinically relevant bacterial species in immunocompromised animals. Active immunization with this vaccine merits further investigation. (+info)
Dose-dependent circulating immunoglobulin A antibody-secreting cell and serum antibody responses in Swedish volunteers to an oral inactivated enterotoxigenic Escherichia coli vaccine.
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The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine. (+info)
Construction and characterization of genetically defined aro omp mutants of enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans.
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Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers to countries where the disease is endemic and causes a major disease burden in the indigenous population, particularly children. We describe here the generation and preclinical characterization of candidate strains of ETEC which are intended to provide the basis of a live attenuated oral vaccine to prevent this disease. It has been shown previously that a spontaneously arising toxin-negative variant ETEC strain, E1392/75-2A, could confer 75% protection against challenge when administered to volunteers. Unfortunately this strain induced mild diarrhea in 15% of recipients. To eliminate the unacceptable reactogenicity of strain E1392/75-2A, it was further attenuated by introducing three different combinations of defined deletion mutations into the chromosome. A mouse intranasal model of immunization was developed and used to show that all of the strains were immunogenic. Immune responses against colonization factor antigens (CFAs) were particularly strong when the bacterial inocula were grown on "CFA agar," which induces strong expression of these antigens. Two of the strains were selected for a phase I dose escalation safety study with healthy adult volunteers. Freshly grown organisms were harvested from CFA agar plates and administered to volunteers as a suspension containing from 5 x 10(7) to 5 x 10(9) CFU. The vaccine was well tolerated at all doses and induced significant immune responses in all recipients at the highest dose of either strain. The results provide the basis for further clinical evaluation of these vaccine candidates. (+info)