scholarly journals 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

2015 ◽  
Vol 23 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Taufiqur Rahman Bhuiyan ◽  
Mohammad Rubel Hoq ◽  
Naoshin Sharmin Nishat ◽  
Deena Al Mahbuba ◽  
Rasheduzzaman Rashu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Vibrio Cholerae ◽  
Elispot Assay ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Content Type ◽  
A Cell ◽  
Mucosal Immune Responses ◽  
Antibody Secreting Cells

ABSTRACTVibrio choleraeand enterotoxigenicEscherichia 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.

scholarly journals Characterization of Mucosal Immune Responses to Enterotoxigenic Escherichia coli Vaccine Antigens in a Human Challenge Model: Response Profiles after Primary Infection and Homologous Rechallenge with Strain H10407

2015 ◽  
Vol 23 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Subhra Chakraborty ◽  
Clayton Harro ◽  
Barbara DeNearing ◽  
Malathi Ram ◽  
Andrea Feller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Response ◽  
Immune Responses ◽  
Vaccine Development ◽  
Lavage Fluid ◽  
Antibody Responses ◽  
Enterotoxigenic Escherichia Coli ◽  
Effective Vaccine ◽  
Content Type ◽  
Mucosal Immune Responses

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) bacteria are the most common bacterial cause of diarrhea in children in resource-poor settings as well as in travelers. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. A significant challenge to successful vaccine development is our poor understanding of the immune responses that correlate best with protection against ETEC illness. In this study, ETEC-specific mucosal immune responses were characterized and compared in subjects challenged with ETEC strain H10407 and in subjects rechallenged with the homologous organism. IgA responses to lipopolysaccharide (LPS), heat-labile toxin B subunit (LTB), and colonization factor antigen I (CFA/I) in antibody in lymphocyte supernatant (ALS), feces, lavage fluid, and saliva samples were evaluated. In all assay comparisons, ALS was the most sensitive indicator of a local immune response, but serum IgA was also a useful indirect marker of immune response to oral antigens. Volunteers challenged and then rechallenged with strain H10407 were protected from illness following rechallenge. Comparing mucosal antibody responses after primary and homologous rechallenge, protection against disease was reflected in reduced antibody responses to key ETEC antigens and in reduced fecal shedding of the H10407 challenge strain. Subjects challenged with strain H10407 mounted stronger antibody responses to LPS and LTB than subjects in the rechallenge group, while responses to CFA/I in the rechallenge group were higher than in the challenge group. We anticipate that this study will help provide an immunological benchmark for the evaluation of ETEC vaccines and immunization regimens in the future.

scholarly journals Enterotoxigenic Escherichia coli Prevents Host NF-κB Activation by Targeting IκBα Polyubiquitination

2012 ◽  
Vol 80 (12) ◽  
pp. 4417-4425 ◽  
Author(s):  
Xiaogang Wang ◽  
Philip R. Hardwidge
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Diarrheal Disease ◽  
Innate Immune ◽  
Host Cells ◽  
Enterotoxigenic Escherichia Coli ◽  
Host Responses ◽  
Innate Immune Responses ◽  
Content Type ◽  
Heat Stable

ABSTRACTThe NF-κB pathway regulates innate immune responses to infection. NF-κB is activated after pathogen-associated molecular patterns are detected, leading to the induction of proinflammatory host responses. As a countermeasure, bacterial pathogens have evolved mechanisms to subvert NF-κB signaling. EnterotoxigenicEscherichia coli(ETEC) causes diarrheal disease and significant morbidity and mortality for humans in developing nations. The extent to which this important pathogen subverts innate immune responses by directly targeting the NF-κB pathway is an understudied topic. Here we report that ETEC secretes a heat-stable, proteinaceous factor that blocks NF-κB signaling normally induced by tumor necrosis factor (TNF), interleukin-1β, and flagellin. Pretreating intestinal epithelial cells with ETEC supernatant significantly blocked the degradation of the NF-κB inhibitor IκBα without affecting IκBα phosphorylation. Data from immunoprecipitation experiments suggest that the ETEC factor functions by preventing IκBα polyubiquitination. Inhibiting clathrin function blocked the activity of the secreted ETEC factor, suggesting that this yet-uncharacterized activity may utilize clathrin-dependent endocytosis to enter host cells. These data suggest that ETEC evades the host innate immune response by directly modulating NF-κB signaling.

scholarly journals Mucosal and Systemic Immune Responses in Patients with Diarrhea Due to CS6-Expressing Enterotoxigenic Escherichia coli

2007 ◽  
Vol 75 (5) ◽  
pp. 2269-2274 ◽  
Author(s):  
Firdausi Qadri ◽  
Tanvir Ahmed ◽  
Firoz Ahmed ◽  
M. Saruar Bhuiyan ◽  
Mohammad Golam Mostofa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Protein A ◽  
Enterotoxigenic Escherichia Coli ◽  
Severe Dehydration ◽  
Colonization Factor ◽  
Disease Spectrum ◽  
Iga Antibodies ◽  
Iga Antibody ◽  
Antibody Secreting Cells

ABSTRACTColonization factor CS6 expressed by enterotoxigenicEscherichia coli(ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n= 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.

scholarly journals TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

2015 ◽  
Vol 83 (5) ◽  
pp. 1893-1903 ◽  
Author(s):  
Daniela Gutiérrez ◽  
Mirka Pardo ◽  
David Montero ◽  
Angel Oñate ◽  
Mauricio J. Farfán ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Coli Strain ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Temperature Sensitive ◽  
Content Type ◽  
E Coli ◽  
Colonization Factor ◽  
Adhesion Level

EnterotoxigenicEscherichia coli(ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avianE. colistrains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with atleAmutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression oftleAconferred the capacity for adherence to nonadherentE. coliHB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.

scholarly journals Genetically modified enterotoxigenic Escherichia coli vaccines induce mucosal immune responses without inflammation

Gut ◽  
2007 ◽  
Vol 56 (11) ◽  
pp. 1550-1556 ◽  
Author(s):  
A. Daley ◽  
R. Randall ◽  
M. Darsley ◽  
N. Choudhry ◽  
N. Thomas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Genetically Modified ◽  
Enterotoxigenic Escherichia Coli ◽  
Mucosal Immune Responses

scholarly journals A Combination Vaccine Consisting of Three Live Attenuated Enterotoxigenic Escherichia coli Strains Expressing a Range of Colonization Factors and Heat-Labile Toxin Subunit B Is Well Tolerated and Immunogenic in a Placebo-Controlled Double-Blind Phase I Trial in Healthy Adults

2011 ◽  
Vol 18 (12) ◽  
pp. 2118-2127 ◽  
Author(s):  
Clayton Harro ◽  
David Sack ◽  
A. Louis Bourgeois ◽  
R. Walker ◽  
Barbara DeNearing ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Phase I ◽  
Enterotoxigenic Escherichia Coli ◽  
High Dose ◽  
Double Blind ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Heat Labile ◽  
Colonization Factors

ABSTRACTImmune responses against colonization factors (CFs) and the nontoxic B component of the enterotoxigenicEscherichia coli(ETEC) heat-labile toxin (LTB) are considered to be important for immunity against diarrhea caused by ETEC. Individual live attenuated ETEC derivatives that have had their toxin genes removed and whosearoC,ompC, andompFgenes are deleted have shown promise as vaccines against ETEC. The development of such strains has culminated in the testing of a three-strain-combination live attenuated vaccine known as ACE527, comprised of strains ACAM2025 expressing colonization factor antigen I (CFA/I) and LTB; ACAM2022, expressing CS5, CS6, and LTB; and ACAM2027, expressing CS1, CS2, CS3, and LTB. The recombinant CF and LTB genes expressed in the three strains were inserted into the bacterial chromosome to ensure their stable inheritance and expression without the requirement for any selection. ACE527 has been tested in a randomized placebo-controlled, double-blind, phase I safety and immunogenicity study in healthy adult volunteers and proved to be well tolerated and immunogenic at dose levels of 1010and 1011total CFU. There was no indication of strain interference on the basis of fecal shedding patterns, with all three being detected in the feces of 50% and 83% of low- and high-dose vaccine recipients, respectively. Similarly, strong immune responses to LTB and to CFs expressed on all three constituent strains were induced, with at least 50% of subjects in the high-dose group responding to LTB, CFA/I, CS3, and CS6.

scholarly journals Concomitant Enterotoxigenic Escherichia coli Infection Induces Increased Immune Responses to Vibrio cholerae O1 Antigens in Patients with Cholera in Bangladesh

2010 ◽  
Vol 78 (5) ◽  
pp. 2117-2124 ◽  
Author(s):  
Fahima Chowdhury ◽  
Yasmin A. Begum ◽  
Mohammad Murshid Alam ◽  
Ashraful I. Khan ◽  
Tanvir Ahmed ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Enterotoxigenic Escherichia Coli ◽  
Secretory Diarrhea ◽  
Immunological Responses ◽  
Heat Stable ◽  
Escherichia Coli Infection ◽  
Antibody Titers

ABSTRACT Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) are major bacterial pathogens that cause dehydrating disease requiring hospitalization of children and adults. The cholera toxin (CT) produced by V. cholerae O1 and the heat-labile toxin (LT) and/or heat-stable toxin (ST) of ETEC are responsible for secretory diarrhea. We have observed that about 13% of hospitalized diarrheal patients are concomitantly infected with V. cholerae O1 and ETEC. In order to understand the outcome of such dual infections on the clinical and immunological responses in cholera patients, we studied patients infected with V. cholerae O1 (group VC; n = 25), those infected with both V. cholerae O1 and ETEC (group VCET; n = 25), and those infected with ETEC only (group ET; n = 25). The VCET group showed more severe dehydration and had a higher intake of intravenous fluid and more vomiting than the ETEC group (P = 0.01 to 0.003). The VCET patients showed higher vibriocidal responses and increased antibody titers to cholera toxin and lipopolysaccharide (LPS) in plasma than did the V. cholerae O1 patients (P = 0.02 to <0.001). All responses in the V. cholerae O1 and in the VCET groups were more robust than those seen in the group infected with ETEC only (P = 0.01 to <0.001). We thus show that concomitant colonization with ETEC induces immune responses to V. cholerae antigens that are more robust than those seen with V. cholerae O1 infection alone. It is possible that LT or other factors expressed by ETEC may play a role as a mucosal adjuvant in enhancing the immune responses to V. cholerae O1.

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