Labile Toxin Research Articles

Use of a multiplex PCR system for the simultaneous detection of heat labile toxin I and heat stable toxin II Escherichia coli cells in environmental waters near Taichung City

Use of a multiplex PCR system for the simultaneous detection of heat labile toxin I and heat stable toxin II Escherichia coli cells in environmental waters near Taichung City

Vaccine adjuvant choice regulates antigen-specific B cell activation and phenotype

Abstract There has been a global rise of antibiotic resistant bacteria that are causing a significant increase in morbidity and mortality. Vaccines offer one of the best ways to combat and prevent these infections. Antibody responses are often the most effective outcomes of vaccine-induced immunity; however, the role of different vaccine adjuvants has not been fully explored in terms of antigen-specific B cell responses. Using a B cell antigen tetramer, we sought to determine how a novel adjuvant, double mutant heat labile toxin from enterotoxigenic E. Coli (dmLT), compared with those currently used in vaccines. We show that either intradermal or intramuscular immunization of mice with a model vaccine formulated using dmLT induces greater antigen specific B cell numbers in the injection site draining lymph nodes and spleen compared to antigen alone. These B cells were also antibody isotype switched and were phenotypically germinal center B cells. We further demonstrate that vaccine-specific B cells migrate from the injection site draining lymph nodes to more distal lymphoid organs. When we compared dmLT to the most commonly used human vaccine adjuvant, alum, we found dmLT was better at inducing a B cell response. Lastly, we show that dmLT is a more potent B cell adjuvant compared to the TLR4 agonist monophosphoryl lipid A (MPL-A) and that the combination of these two adjuvants is superior to either adjuvant alone. Interestingly, MPL-A induced greater antigen specific IgG compared to dmLT despite dmLT inducing a more robust B cell expansion. These results demonstrate that dmLT is a potent B cell activating adjuvant and that combining dmLT with existing adjuvants has the potential to greatly improve vaccine responses.

Adjuvant combinations activate dendritic cells and contribute to antigen-specific CD4 T cell expansion

Abstract Adjuvants are important mediators of immune responses and enhance the activation of innate immune cells, leading to protective T and B cell responses. Only a few vaccine adjuvants are currently approved for use in the US and, along with the discovery of novel adjuvants, combination adjuvants are likely to have a major impact on future vaccine development. Here, we investigated the ability of both the double mutant Escherichia coli heat labile toxin R192/L211A (dmLT) and the TLR4 agonist monophosphoryl lipid A (MPL-A) to drive innate and adaptative immune responses independently and in combination. We found that the combination adjuvant induces more robust activation of dendritic cells (DCs) than either adjuvant alone, leading to the formation of the NLRP3 inflammasome complex and increased secretion of IL-1β, as well as other hallmark pro-inflammatory cytokines. Furthermore, RNA sequencing and ingenuity pathway analysis support that the combination adjuvant may allow these DCs to polarize a multi-faceted Th1/2/17 CD4 T cell response. To determine how this adjuvant combination might regulate CD4 T cell responses, we intradermally immunized mice with a model T cell antigen in conjunction with dmLT or MPL-A alone or combined and, using MHC class II tetramers, found that vaccination with the combination demonstrated the greatest expansion and activation of endogenous, vaccine-specific CD4+ T cells in all lymphoid tissues assessed which included a Th1/Th17 response. These results reveal how combination adjuvants can be used to not only potentiate better vaccine responses, but to potentially bias the immune response to one that is more capable of combating infection.

Identification and synthesis of an efficient multivalent E. coli heat labile toxin inhibitor __ A dynamic combinatorial chemistry approach

A polymer based dynamic combinatorial library (DCL) was generated through condensation between aldehyde functionalized linear poly(glycidol) (APG) and galactose containing acylhydrazide derivatives. Pentameric E. coli heat labile enterotoxin B subunit (LTB) was subsequently applied to the DCL as external stimulus, resulting in amplification of a specific acylhydrazone side chain that was further used for the synthesis of a multivalent LTB inhibitor. In the in vitro biological evaluation, this inhibitor exhibited strong inhibition properties as well as low cytotoxicity.

Assessment of disease specific immune responses in enteric diseases using dried blood spot (DBS).

BackgroundBlood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits evaluation of immune responses in natural infections and vaccination in field studies. We developed methods to measure antigen specific antibody responses using dried blood spot (DBS) in cholera, ETEC and typhoid fever patients as well as recipients of oral cholera vaccine (OCV).Methodology/Principle findingsWe processed heparinized blood for preparing DBS and plasma specimens from patients with, cholera, ETEC and typhoid as well as OCV recipients. We optimized the conventional vibriocidal method to measure vibriocidal antibody response in DBS eluates. We measured responses in DBS samples and plasma (range of titer of 5 to 10240). Vibriocidal titer showed strong agreement between DBS eluates and plasma in cholera patients (ICC = 0.9) and in OCV recipients (ICC = 0.8) using the Bland-Altman analysis and a positive correlation was seen (r = 0.7, p = 0.02 and r = 0.6, p = 0.006, respectively). We observed a strong agreement of lipopolysaccharide (LPS) and cholera toxin B (CTB)-specific antibody responses between DBS eluates and plasma in cholera patients and OCV recipients. We also found agreement of heat labile toxin B (LTB) and membrane protein (MP)-specific antibody responses in DBS eluates and plasma specimen of ETEC and typhoid patients respectively.ConclusionOur results demonstrate that dried blood specimens can be used as an alternate method for preservation of samples to measure antibody responses in enteric diseases and vaccine trials and can be applied to assessment of responses in humanitarian crisis and other adverse field settings.

Development of a novel multiplex electrochemiluminescent-based immunoassay to aid enterotoxigenic Escherichia coli vaccine development and evaluations

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea both among children in low and middle income countries and in travelers to these regions. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. The most advanced ETEC vaccine candidates include multiple colonization factors along with the heat labile toxin B subunit. In the absence of known correlates of protection, and to understand the mechanism of protection, monitoring immune responses to a majority of the vaccine associated antigens using various types of samples is needed. Unfortunately, a traditional ELISA is time consuming, labor intensive and requires substantial amounts of antigens and sample volumes. To address these constraints, we developed and validated a novel high throughput electrochemiluminescent (ECL) - based multiplex immunoassay using Meso Scale Discovery (MSD) platform for analyzing immune responses to ETEC antigens. The ETEC multiplex ECL assay is an 8-plex assay which includes the ETEC colonization factor antigens (CFA/I, CS1, CS2, CS3, CS5 and CS6) along with the two subunits of heat labile toxin (LTA and LTB). Our data suggested that a single dilution of sample provides a quantifiable result for a wide range of sample titers. To compare ETEC multiplex ECL with ELISA, we carried out assays using the same antigens with the two immunoassay platforms using a common sample set of serum and ALS (antibodies in lymphocyte supernatant) specimens. The MSD platform achieved excellent correlations with ELISA for the antigens tested, consistently detecting comparable antibody levels in the samples. The ETEC multiplex ECL can serve as a fundamental platform in evaluating performances of candidate ETEC vaccines in future field trials.

Molecular Characterization of Diarrhoegenic Escherichia coli Isolated from Neonatal Goat-Kids

Faecal samples (n=300) from diarrhoeic neonatal goat-kids of different livestock sheds of ICAR-CIRG, Makhdoom, and field goat-kids of Mathura, UP were aseptically collected, and used for E. coli isolation. On the basis of cultural, morphological, biochemical and molecular characteristics, a total of 193 E. coli isolates were identified from 300 fecal samples. Out of 140 E. coli isolates, only 90 isolates could be serotyped at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, and the most common serogroups responsible for neonatal diarrhoea were found as O88 (n=11), O22 (n=10), O11 (n=8) and O83 (n=7). Congo red dye agar test was done to determine invasiveness of the isolates, and 77.20% (149/193)E. coli isolates showed Congo red binding activity. Identification of shiga toxin producing E. coli (STEC) was done by PCR amplification of its stx-1 gene, and 5.69% (11/193) isolates were identified as STEC. Pathotype specific primers were used to amplify bundle forming pilus (bfpA) gene of enteropathogenic E. coli (EPEC), and 35.23% (68/193) isolates were identified as EPEC. A multiplex PCR was performed to detect labile toxin producing enterotoxigenic E. coli (ETEC-lt), stable toxin producing enterotoxigenic E. coli (ETEC-st) and enteroinvasive E. coli (EIEC), and 24.35% (47/193), 2.59% (5/193) and 2.07% (4/193) isolates were determined as ETEC-st, ETEC-lt and EIEC, respectively. EPEC and ETEC-st were found as the most prevalent pathotypes associated with neonatal diarrhoea in goat-kids whereas; O88 and O22 were observed as the most common serogroups in causing diarrhoea in the neonatal goat-kids.

Diarrheal bacterial pathogens and multi-resistant enterobacteria in the Choqueyapu River in La Paz, Bolivia.

Water borne diarrheal pathogens might accumulate in river water and cause contamination of drinking and irrigation water. The La Paz River basin, including the Choqueyapu River, flows through La Paz city in Bolivia where it is receiving sewage, and residues from inhabitants, hospitals, and industry. Using quantitative real-time PCR (qPCR), we determined the quantity and occurrence of diarrheagenic Escherichia coli (DEC), Salmonella enterica, Klebsiella pneumoniae, Shigella spp. and total enterobacteria in river water, downstream agricultural soil, and irrigated crops, during one year of sampling. The most abundant and frequently detected genes were gapA and eltB, indicating presence of enterobacteria and enterotoxigenic E. coli (ETEC) carrying the heat labile toxin, respectively. Pathogen levels in the samples were significantly positively associated with high water conductivity and low water temperature. In addition, a set of bacterial isolates from water, soil and crops were analyzed by PCR for presence of the genes blaCTX-M, blaKPC, blaNDM, blaVIM and blaOXA-48. Four isolates were found to be positive for blaCTX-M genes and whole genome sequencing identified them as E. coli and one Enterobacter cloacae. The E. coli isolates belonged to the emerging, globally disseminated, multi-resistant E. coli lineages ST648, ST410 and ST162. The results indicate not only a high potential risk of transmission of diarrheal diseases by the consumption of contaminated water and vegetables but also the possibility of antibiotic resistance transfer from the environment to the community.

Evaluation of the Effect of Promoter Type on the Immunogenicity of the Live Recombinant Salmonella Vaccines Expressing Escherichia Coli Heat-labile Enterotoxins (LTB).

Enterotoxigenic Escherichia coli (ETEC)-induced diarrhoea is the second most common cause of death in children in the developing countries. Heat labile toxin (LT) is responsible for ETEC-induced diarrhoea. In the present study, a novel live ETEC vaccine based on subunit B of LT (LTB) expression in attenuated PhoPc Salmonella strain was developed. Herein, we aimed to compare the in-vitro activity of promoters including constitutive tac, IPTG inducible trc, and in-vivo-inducible (nirB and nirB78-23) in PhoPc. Additionally, the ability of these recombinant PhoPc/pLTBs to induce LTB-specific antibody responses in BALB/c mice after nasal immunization was evaluated. In-vitro studies demonstrated that PhoPc has the ability to produce rLTB. Furthermore, nirB promoter directed significantly more LTB expression in PhoPc/pnirBLTB under anaerobic condition without induction compared to the amount of rLTB secreted by PhoPc/ptrcLTB in bacterial soup under uninduced condition (6.06 ± 0.05 vs. 1.4 ± 0.46 μg/109 cfu, p < 0.01). In addition, the constitutive rLTB expression from tac promoter was more than its expression from uninduced trc promoter in bacterial soup (4.2 ± 0.92 vs. 1.4 ± 0.46 (μg/109 cfu)) and pellet (27.4 ± 0.89 vs. 13.4 ± 1.42 (μg/109 cfu), p < 0.0001). However, the mice immunized with PhoPc/ptrcLTB elicited the superior anti-LTB responses among the PhoPc containing the examined prompters, which were significantly higher than those induced by PhoPc/pnirB78-23LTB and PhoPc/pnirB, 6 weeks after the first immunization. Totally, it could be concluded that in-vitro analysis of promoters for LTB expression in PhoPc may not necessarily predict the recombinant PhoPc immunogenicity.

Modelling paralytic shellfish toxins (PST) accumulation in Crassostrea gigas by using Dynamic Energy Budgets (DEB)

As other filter-feeders, Crassostrea gigas can concentrate paralytic shellfish toxins (PST) by consuming dinoflagellate phytoplankton species like Alexandrium minutum. Intake of PST in oyster tissues mainly results from feeding processes, i.e. clearance rate, pre-ingestive sorting and ingestion that are directly influenced by environmental conditions (trophic sources, temperature). This study aimed to develop a mechanistic model coupling the kinetics of PST accumulation and bioenergetics in C. gigas based on Dynamic Energy Budget (DEB) theory. For the first time, the Synthesizing Units (SU) concept was applied to formalize the feeding preference of oysters between non-toxic and toxic microalgae. Toxin intake and accumulation were both dependent on the physiological status of oysters. The accumulation was modelled through the dynamics of two toxin compartments: (1) a compartment of ingested but non-assimilated toxins, with labile toxins within the digestive gland eliminated via faeces production; (2) a compartment of assimilated toxins with a rapid detoxification rate (within a few days). Firstly, the DEB-PST model was calibrated using data from two laboratory experiments where oysters have been exposed to A. minutum. Secondly, it was validated using data from another laboratory experiment and from three field surveys carried out in the Bay of Brest (France) from 2012 to 2014. To account for the variability in PST content of A. minutum cells, the saxitoxin (STX) amount per energy units in a toxic algae (ρPST) was adjusted for each dataset. Additionally, the effects of PST on the oyster bioenergetics were calibrated during the first laboratory experiment. However, these effects were shown to depend on the strain of A. minutum. Results of this study could be of great importance for monitoring agencies and decision makers to identify risky conditions (e.g. production areas, seawater temperature), to properly assess detoxification step (e.g. duration, modalities) before any commercialization or to improve predictions regarding closing of shellfish areas.

Rotavirus VP6 protein mucosally delivered by cell wall-derived particles from Lactococcus lactis induces protection against infection in a murine model.

Rotaviruses are the primary cause of acute gastroenteritis in children worldwide. Although the implementation of live attenuated vaccines has reduced the number of rotavirus-associated deaths, variance in their effectiveness has been reported in different countries. This fact, among other concerns, leads to continuous efforts for the development of new generation of vaccines against rotavirus.In this work, we describe the obtention of cell wall-derived particles from a recombinant Lactococcus lactis expressing a cell wall-anchored version of the rotavirus VP6 protein. After confirming by SDS-PAGE, Western blot, flow cytometry and electronic immunomicroscopy that these particles were carrying the VP6 protein, their immunogenic potential was evaluated in adult BALB/c mice. For that, mucosal immunizations (oral or intranasal), with or without the dmLT [(double mutant Escherichia coli heat labile toxin LT(R192G/L211A)] adjuvant were performed. The results showed that these cell wall-derived particles were able to generate anti-rotavirus IgG and IgA antibodies only when administered intranasally, whether the adjuvant was present or not. However, the presence of dmLT was necessary to confer protection against rotavirus infection, which was evidenced by a 79.5 percent viral shedding reduction.In summary, this work describes the production of cell wall-derived particles which were able to induce a protective immune response after intranasal immunization. Further studies are needed to characterize the immune response elicited by these particles as well as to determine their potential as an alternative to the use of live L. lactis for mucosal antigen delivery.

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1-independent manner.

The primary virulence factor of Vibrio cholerae, cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhoea characteristic of cholera. Secreted CT binds to GM1 on the surface of host cells to facilitate internalisation. Many bacterial toxins, including CT, have been shown to be additionally delivered via outer membrane vesicles (OMVs). A fraction of the closely related heat labile toxin produced by enterotoxigenic Escherichia coli has been demonstrated to reside on the surface of OMVs, where it binds GM1 to facilitate OMV internalisation by host cells. In this work, we investigated whether OMV-associated CT is likewise trafficked to host cells in a GM1-dependent mechanism. We demonstrated that a majority of CT is secreted in its OMV-associated form and is located exclusively inside the vesicle. Therefore, the toxin is unable to bind GM1 on the host cell surface, and the OMVs are trafficked to the host cells in a GM1-independent mechanism. These findings point to a secondary, noncompeting mechanism for secretion and delivery of CT, beyond its well-studied secretion via a Type II secretion system and underscore the importance of focusing future studies on understanding this GM1-independent delivery mechanism to fully understand Vibrio cholerae pathogenesis.

Liquid chromatography-ultraviolet detection and quantification of heat-labile toxin produced by enterotoxigenic E. coli cultured under different conditions

Enterotoxigenic Escherichia coli (ETEC) is the main bacterial cause of dehydrating infant diarrhoea in less-developed countries. Labile toxin (LT) is the major virulent factor of ETEC. Easy diagnostic tests are necessary to reduce the number of cases. Immunological methods have some drawbacks and also have important limitations. For that reason, a Liquid Chromatography coupled to UV detector technique (LC-UV) has been optimize to a rapid identification and quantification of LT from bacteria cultures. It is also important to know optimal conditions for LT and with this purpose several enterotoxigenic E. coli strains have been studied to determine the influence of glucose concentration and different culture media on LT production.LC-UV technique demonstrated to be a good method for LT quantification showing values of 15 ng/mL and 45 ng/mL for limits of detection and quantification respectively. LT quantification revealed that toxin production is directly related to the concentration of glucose added in the broth medium. Tryptic Soy Broth is the most efficient culture medium for E. coli growth and enterotoxin production.

Structural Characterization and Physicochemical Stability Profile of a Double Mutant Heat Labile Toxin Protein Based Adjuvant

A novel protein adjuvant double-mutant Escherichia coli heat-labile toxin, LT (R192G/L211A) or dmLT, is in preclinical and early clinical development with various vaccine candidates. Structural characterization and formulation development of dmLT will play a key role in its successful process development, scale-up/transfer, and commercial manufacturing. This work describes extensive analytical characterization of structural integrity and physicochemical stability profile of dmLT from a lyophilized clinical formulation. Reconstituted dmLT contained a heterogeneous mixture of intact holotoxin (AB5, ∼75%) and free B5 subunit (∼25%) as assessed by analytical ultracentrifugation and hydrophobic interaction chromatography. Intact mass spectrometry (MS) analysis revealed presence of Lys84 glycation near the native sugar-binding site in dmLT, and forced degradation studies using liquid chromatography-MS peptide mapping demonstrated specific Asn deamidation and Met oxidation sites. Using multiple biophysical measurements, dmLT was found most stable between pH 6.5 and 7.5 and at temperatures ≤50°C. In addition, soluble aggregates and particle formation were observed upon shaking stress. By identifying the physicochemical degradation pathways of dmLT using newly developed stability-indicating analytical methods from this study, we aim at developing more stable candidate formulations of dmLT that will minimize the formation of degradants and improve storage stability, as both a frozen bulk substance and eventually as a liquid final dosage form.

Global burden of diarrheal diseases among children in developing countries: Incidence, etiology, and insights from new molecular diagnostic techniques.

The Global Enteric Multicenter Study (GEMS) demonstrated that Shigella and enterotoxigenic Escherichia coli (ETEC) producing heat stable toxin (ST) (either alone or in combination with heat labile toxin) are among the most important pathogens associated with moderate-to-severe diarrhea (MSD) in children younger than 5years of age living in developing countries. To inform the design of vaccines and other interventions, we reviewed published data and new results from GEMS characterizing the burden of Shigella and ST-ETEC infections. Clinical parameters were assessed to examine the value of various case definitions as indicators of MSD associated with Shigella and ST-ETEC for use in clinical trials. We discussed advantages and disadvantages of culture-based and culture-independent molecular diagnostics for detecting clinically and epidemiologically relevant disease. Shigella serotyping data from GEMS were examined to identify desirable components of Shigella and ETEC vaccines likely to confer broad protection. These findings can inform the development and implementation of vaccines to prevent these important infections among infants and children in developing countries.

Development of a candidate stabilizing formulation for bulk storage of a double mutant heat labile toxin (dmLT) protein based adjuvant.

This work describes the formulation design and development of a novel protein based adjuvant, a double mutant of heat labile toxin (dmLT), based on knowledge of the protein’s structural integrity and physicochemical degradation pathways. Various classes of pharmaceutical excipients were screened for their stabilizing effect on dmLT during exposure to thermal and agitation stresses as monitored by high throughput analytical assays for dmLT degradation. Sucrose, phosphate, sodium chloride, methionine and polysorbate-80 were identified as potential stabilizers that protected dmLT against either conformational destabilization, aggregation/particle formation or chemical degradation (e.g., Met oxidation and Lys glycation). Different combinations and concentrations of the selected stabilizers were then evaluated to further optimize dmLT stability while maintaining pharmaceutically acceptable ranges of solution pH and osmolality. The effect of multiple freeze-thaw (FT) cycles on the physical stability of candidate bulk formulations was also examined. Increasing the polysorbate-80 concentration to 0.1% in the lead candidate bulk formulation mitigated the loss of protein mass during FT. This formulation development study enabled the design of a new bulk formulation of the dmLT adjuvant and provides flexibility for future use in combination with a variety of different vaccine dosage forms with different antigens.

Development of live attenuated bacterial vaccines targeting Escherichia coli heat-labile and heat-stable enterotoxins

Enterotoxigenic Escherichia coli (ETEC), defined by the production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of diarrhea in animals, children in developing countries and to travelers. No broadly protective ETEC vaccine is available, largely because of the difficulty in inducing immunity to the small ST molecule. To take advantage of the demonstration (Liu et al., 2011; Zhang et al., 2013, 2010) that genetically produced fusions of mutant ST with LT subunits can induce effective immunity against both toxins, we engineered a live attenuated vaccine vector strain of E. coli (ZCR533), expressing the immunogenic LT-ST fusions. To present the LT-ST fusions to the mucosal immune system, we used restriction-free cloning to incorporate them into the passenger domain of the autotransporter protein (EspP) expressed on a medium copy number plasmid. This versatile system permits expression of incorporated antigens in either surface-bound or secreted forms by the ZCR533 vector, for delivery to the mucosal inductive sites. Incorporation of the fusions into EspP plasmids was confirmed by PCR and DNA sequencing. Protein expression was confirmed by Western blot of whole cell lysates and culture supernatents using polyclonal antisera to LT. Expression of the surface-targeted fusion on the surface of ZCR533 was confirmed by immuno-fluorescent staining. These studies show that antigenic LT-ST fusions can be surface-expressed or secreted, by our attenuated E. coli ZCR533 vaccine vector via the EspP autotransporter. These constructs could serve as broadly protective vaccine candidates to protect against both LT- and ST-producing ETEC.

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions

BackgroundEnterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach.ResultsStrong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated.ConclusionsETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

Immunization with Salmonella Enteritidis secreting mucosal adjuvant labile toxin confers protection against wild type challenge via augmentation of CD3+CD4+ T-cell proliferation and enhancement of IFN-γ, IL-6 and IL-10 expressions in chicken

The protective efficacy and immunological profiles of chickens immunized with an attenuated Salmonella Enteritidis (SE) constitutively secreting double mutant heat labile enterotoxin (dmLT) were investigated. The dmLT is a detoxified variant of Escherichia coli heat labile toxin and is a potent mucosal adjuvant capable of inducing both humoral and cell-mediated immunity. In this study, four-week-old chickens were inoculated with SE-dmLT strain JOL1641, parental SE strain JOL1087 or phosphate buffered saline control. Peripheral blood mononuclear cells of SE-dmLT inoculated birds showed significant proliferation upon stimulation with SE antigens as compared to the control and JOL1087 groups (P⩽0.05). One week post-challenge, the ratio of CD3+CD4+ to CD3+CD8+ T-cells showed a significant increase in the immunized groups. Significant increases in IFN-γ levels were observed in JOL1641 birds immunized via oral and intramuscular routes. While immunizations with the JOL1087 strain via the intramuscular route also induced significant increases in IFN-γ, immunization via the oral route did not trigger significant changes. Pro-inflammatory cytokine IL-6 was also elevated significantly in immunized birds; a significant elevation of IL-10 was observed only in oral immunization with JOL1641 (P⩽0.05). JOL1641 immunized birds showed significant reduction of challenge bacterial-organ recovery as compared to JOL1087 and non-immunized birds. Collectively, our results revealed that immunization with the adjuvant-secreting S. Enteritidis confers protection against wild type SE challenge via induction of strong cell proliferative response, augmentation of CD3+CD4+: CD3+CD8+ T-cells ratio and enhancement of IFN-γ, IL-6 and IL-10 cytokine secretion.

The Mucosal Adjuvant Activities of ADP-Ribosylating Bacterial Enterotoxins.

The bacterial enterotoxins, cholera toxin and the heat labile toxin of E. coli, are well known adjuvants for mucosal immune response. Their common A chain mediates the toxigenic mechanism by causing ADP ribosylation of G proteins and subsequent elevation of cAMP in target cells. A large IgA and IgG antibody response to admixed protein antigen (Ag) is the hallmark of these adjuvants and is clearly associated with the A chain activity. Expansion of Ag-specific B and T cells, alteration of T cell cytokine production, and changes in regulatory T cells have been reported as adjuvant mechanisms. The B chain derivatives of these toxins can also weakly enhance immune response, especially if covalently associated with Ag and used for nasophyrangeal immunization. Importantly, these toxins or their B chain derivatives can alter the normal immune regulation that produces oral tolerance. This indicates that they modulate mechanisms operative between the mucosal and systemic immune systems. There are some discrepancies between in vitro models of CT or LT activity and in vivo manifestations of their adjuvant activities. Interpretation of current data regarding in vivo mechanism is hampered by an incomplete understanding of how mucosal B and T cells can interact with systemic lymphoid tissue and vice versa. More important, there is no clear understanding of the early effects of the toxins on the local (and draining) mucosal lymphoid tissues. This is especially true in the critical areas of antigen presentation, T and B cell activation, and cytokine production.