Coli Heat-labile Enterotoxin Research Articles

Potent protective cellular immune responses generated by a DNA vaccine encoding HSV-2 ICP27 and the E. coli heat labile enterotoxin

A mouse model was employed to evaluate protective cellular immune responses induced by an immediate early antigen of HSV-2. Particle-mediated DNA vaccination of mice with a DNA plasmid-encoding ICP27 resulted in the induction of ICP27-specific IFN-γ and TNF-α production in Balb/c mice, but little protection to intranasal challenge with wild type HSV-2. However, when the DNA vaccine was supplemented with as little as 50ng of a vector encoding the A and B subunits of the Escherichia coli heat labile enterotoxin (LT), animals were profoundly protected from morbidity and mortality. The ICP27+LT-mediated protection was correlated with a large increase in ICP27-specific IFN-γ and TNF-α production but cytokine-specific monoclonal antibody treatment at the time of challenge showed that protection was mediated predominantly by IFN-γ. Furthermore, depletion of T cell subsets prior to infectious challenge demonstrated that removal of either CD8+ or CD4+ T cells impaired protection with CD8+ T cells appearing to play a direct effector role. These data demonstrate that augmented cellular immune responses resulting from LT vector plus antigen vector administration to the skin are biologically significant, leading to enhanced protection against mucosal pathogenic challenge.

The B subunit of Escherichia coli heat-labile enterotoxin induces both caspase-dependent and -independent cell death pathways in CD8+ T cells.

The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule that acts both as an adjuvant and to stimulate immune deviation processes, resulting in the suppression of Th1-associated inflammatory responses. The ability of EtxB to alter immune reactivity is dependent on its ability to modulate immune cell function through binding to cell surface molecules, the principal receptor of which is the ubiquitous GM1-ganglioside. EtxB activates B cells and antigen-presenting cells and induces the selective apoptosis of murine CD8+ T cells. We postulated that these effects are mediated by the induction of intracellular signaling pathways following EtxB-receptor interaction. We have previously shown that CD8+ T-cell apoptosis induced by EtxB results from the activation of the transcription factor NF-kappaB and caspases. Here we report that while caspase activity is required for apoptosis, additional features of cell death are caspase independent. EtxB induces a rapid loss of mitochondrial membrane potential and cell viability that are unaffected by caspase inhibitors. In addition, our data suggest that these processes are independent of the activity of Bax and Bcl-2 but are mediated by nitric oxide synthase.

Nasal delivery of antigen with the B subunit of Escherichia coli heat-labile enterotoxin augments antigen-specific T-cell clonal expansion and differentiation.

Escherichia coli heat-labile enterotoxin has unique immunogenic and adjuvant properties when administered mucosally to mice. These properties have revealed the potential for its use in the development of mucosal vaccines, an area of increasing interest. However, the inherent toxicity mediated by the A subunit precludes its widespread use. This problem has led to attempts to dissociate toxicity from adjuvant function by use of the B subunit. The ability of the B subunit of E. coli heat-labile enterotoxin (EtxB) to enhance responses against antigens coadministered intranasally is demonstrated here with the use of the DO11.10 adoptive-transfer model, in which ovalbumin (OVA)-specific adoptively transferred T cells can be monitored directly by flow cytometry. Intranasal delivery of OVA with EtxB resulted in increased T-cell proliferative and systemic antibody responses against antigens. The increased Th2 cytokine production detected following in vitro restimulation of splenocyte and cervical lymph node (CLN) cells from the immunized mice correlated with increased OVA-specific immunoglobulin G1 antibody production. Flow cytometric analysis of T cells from mice early after immunization directly revealed the ability of EtxB to support antigen-specific clonal expansion and differentiation. Furthermore, while responses were first detected in the CLNs, they rapidly progressed to the spleen, where they were further sustained. Examination of CD69 expression on dividing cells supported the notion that activation induced by the presence of antigens is not sufficient to drive T-cell differentiation. Furthermore, a lack of CD25 expression on dividing cells suggested that EtxB-mediated T-cell clonal expansion may occur without a sustained requirement for interleukin 2.

Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horses.

Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (alpha2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.

Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant.

The receptor binding domains of the most potent mucosal adjuvants, bacterial toxins and plant lectins, are organized in repeat units to recognize specific sugar residues. The lectin-like structure of the C-terminal region of Clostridium difficile toxin A prompted us to investigate the mucosal adjuvant properties of a nontoxigenic peptide corresponding to amino acids 2394 to 2706 (TxA(C314)). We compared TxA(C314) adjuvant activity to those of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin subunit B (EtxB) coadministered orally or nasotracheally with poor peptide antigens (keyhole limpet hemocyanin [KLH] and hen egg lysozyme [HEL]). Levels of anti-KLH-specific serum immunoglobulin G (IgG) and IgA as well as that of mucosal IgA were significantly higher in animals immunized orally with TxA(C314) plus KLH than with KLH alone, CT plus KLH, or EtxB plus KLH. Following intranasal immunization with TxA(C314) plus HEL, levels of serum- and mucosa-specific antibodies were comparable to those induced by coadministering HEL with CT or EtxB. The TxA(C314) adjuvant effect following oral, but not intranasal, immunization was dose dependent. The analysis of the subclasses of anti-KLH-specific IgG isotypes and the cytokines released from splenocytes of immunized mice challenged in vitro with KLH indicates the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch. We conclude that TxA(C314) enhances immune responses against mucosa-coadministered foreign antigens and represents a promising mucosal adjuvant, especially because its ability to stimulate mixed Th1/Th2 responses with a strong a Th1 component is extremely worthwhile against intracellular pathogens.

The B subunit of Escherichia coli heat labile enterotoxin abrogates oral tolerance, promoting predominantly Th2-type immune responses.

Mucosal antigen encounter usually results in a state of systemic non-responsiveness (tolerance). This failure to mount a protective response is a major hurdle to mucosal vaccine development. Hence, the identification of safe and effective mucosal adjuvants promoting protective immunity is of critical importance. The non-toxic B subunit of Escherichia coli heat labile enterotoxin(EtxB) is a potent nasal adjuvant; however, its usefulness following oral delivery is unconfirmed. We used DO11.10 chimeric mice to assess whether EtxB could abrogate tolerance to oral OVA. We show that admixing EtxB with OVA for oral immunization abrogates oral tolerance and results in a weak anti-OVA immune response. Importantly, EtxB profoundly modulated the nature of the response to subsequent parenteral challenge, promoting IgG1 in favor of IgG2a antibodies and depressing IFN-gamma production while elevating TGF-beta secretion. The addition of EtxB promoted T cell division, as assessed by loss of staining with carboxyfluorescein diacetate succinimidyl ester. Enhanced cell division promoted by EtxB was associated with T cell differentiation (increased numbers of CD45RBlow cells) in vivo, although dividing OVA-specific T cells were CD25-. These data suggest that although EtxB is a weak oral adjuvant, it can profoundly modulate the nature of the immune response to admixed antigen.

Zonula occludens toxin acts as an adjuvant through different mucosal routes and induces protective immune responses.

Zonula occludens toxin (Zot) is produced by Vibrio cholerae and has the ability to increase mucosal permeability by reversibly affecting the structure of tight junctions. Because of this property, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. Here we show that Zot acts as a mucosal adjuvant to induce long-lasting and protective immune responses upon mucosal immunization of mice. Indeed, the intranasal delivery of ovalbumin with two different recombinant forms of Zot in BALB/c mice resulted in high Ag-specific serum immunoglobulin G titers that were maintained over the course of a year. Moreover, His-Zot induced humoral and cell-mediated responses to tetanus toxoid in C57BL/6 mice and protected the mice against a systemic challenge with tetanus toxin. In addition, we found that Zot also acts as an adjuvant through the intrarectal route and that it has very low immunogenicity compared to the adjuvant Escherichia coli heat-labile enterotoxin. Finally, by using an octapeptide representing the putative binding site of Zot and of its endogenous analogue zonulin, we provide evidence that Zot may bind a mucosal receptor on nasal mucosa and may mimic an endogenous regulator of tight junctions to deliver Ags in the submucosa. In conclusion, Zot is a novel and effective mucosal adjuvant that may be useful for the development of mucosal vaccines.

Mutant Escherichia coli heat-labile toxin B subunit that separates toxoid-mediated signaling and immunomodulatory action from trafficking and delivery functions.

The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function. Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane. Here we describe the properties of EtxB(H57S), a mutant B subunit with a His-->Ser substitution at position 57. The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8(+)-T-cell apoptosis, (ii) activate nuclear translocation of NF-kappaB in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant. However, its GM1 binding, cellular uptake, and delivery functions remained intact. This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line. These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB. Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking. Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits.

Expression of the Plasmodium falciparum immunodominant epitope (NANP)(4) on the surface of Salmonella enterica using the autotransporter MisL.

Gram-negative bacterial proteins which are exported from the cytosol to the external environment by the type V secretion system are also known as autotransporters. Once translocated to the periplasmic compartment by the sec-dependent general secretory pathway, their C-terminal domain forms a pore through which the N-terminal domain travels to the outer membrane without the need of other accessory proteins. MisL (protein of membrane insertion and secretion) is a protein of unknown function located in the pathogenicity island SPI-3 of Salmonella enterica and classified as an autotransporter due to its high homology to Escherichia coli AIDA-I. In the present work, the MisL C-terminal translocator domain was used to display the immunodominant B-cell epitope of the circumsporozoite protein (CSP) from Plasmodium falciparum on the surface of Salmonella enterica serovar Typhimurium (serovar Typhimurium SL3261) and serovar Typhi (serovar Typhi CVD 908). The MisL beta domain was predicted by alignment with AIDA-I, amplified from serovar Typhimurium SL3261, cloned in a plasmid fused to four repeats of the tetrapeptide NANP behind the Escherichia coli heat-labile enterotoxin B subunit signal peptide to ensure periplasmic traffic, and expressed under the control of the anaerobically inducible nirB promoter. The fusion protein was translocated to the outer membrane of both bacterial strains, although the foreign epitope was displayed more efficiently in serovar Typhimurium SL3261, which elicited a better specific antibody response in BALB/c mice. More importantly, antibodies were able to recognize the native CSP in P. falciparum sporozoites. These results confirm that MisL is indeed an autotransporter and that it can be used to express foreign immunogenic epitopes on the surface of gram-negative bacteria.

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines.

Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.

Anchor-Based Design of Improved Cholera Toxin and E. coli Heat-Labile Enterotoxin Receptor Binding Antagonists that Display Multiple Binding Modes

The action of cholera toxin and E. coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1. We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure. In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG. Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC). Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives. The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity.

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Mucosal Delivery of Inactivated Influenza Vaccine Induces B-Cell-Dependent Heterosubtypic Cross-Protection against Lethal Influenza A H5N1 Virus Infection

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.

Human papillomavirus virus-like particles are efficient oral immunogens when coadministered with Escherichia coli heat-labile enterotoxin mutant R192G or CpG DNA.

Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129-2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant.

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

Inhibition of T-cell response by Escherichia coli heat-labile enterotoxin-treated epithelial cells.

Escherichia coli heat-labile enterotoxin (LT) is an extensively studied adjuvant of mucosal responses. Nevertheless, its mode of action as an adjuvant remains incompletely understood. In this study, we describe a simplified in vitro model with which to look at some aspects of immunoregulation by LT. The interaction of LT with the apical surface of a monolayer of CaCo-2 epithelial cells induces the release of a soluble factor which inhibits the antigen-induced release of interleukin-2 by T cells cultured at the basolateral side of the cells. The release of this factor requires the ADP-ribosylating activity of LT since the isolated B subunit, as well as an enzymatically silent LT mutant, loses biological activity in this model. The inhibitory activity is likely to be due to prostaglandin release, since it is blocked by indomethacin. The contribution of LT-induced prostaglandin release to the complex immunoregulatory activity of LT is discussed.

Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni.

A recombinant protein comprising the maltose-binding protein (MBP) of Escherichia coli fused to amino acids 5 to 337 of the FlaA flagellin of Campylobacter coli VC167 was evaluated for immunogenicity and protective efficacy against challenge by a heterologous strain of campylobacter, Campylobacter jejuni 81-176, in two murine models. The sequence of the flaA gene of strain 81-176 revealed a predicted protein which was 98.1% similar to that of VC167 FlaA over the region expressed in the fusion protein. Mice were immunized intranasally with two doses of 3 to 50 microgram of MBP-FlaA, given 8 days apart, with or without 5 microgram of the mutant E. coli heat-labile enterotoxin (LT(R192G)) as a mucosal adjuvant. The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum immunoglobulin G (IgG) responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA (>/=25 microgram) or coadministration of lower doses with the adjuvant. When vaccinated mice were challenged intranasally 26 days after immunization, the best protection was seen in animals given 50 microgram of MBP-FlaA plus LT(R192G). The protective efficacies of this dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 microgram of MBP-FlaA plus LT(R192G) intranasally were challenged orally with 8 x 10(10), 8 x 10(9), or 8 x 10(8) cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively.

The sigma ligand, igmesine, inhibits cholera toxin andEscherichia coli enterotoxin induced jejunal secretion in the rat

BACKGROUNDCholera toxin, andEscherichia coli heat labile (LT) and heat stable (STa) enterotoxins induce small intestinal secretion in part by activating enteric nerves. Igmesine is a novel sigma receptor ligand that...

Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E. coli heat-labile enterotoxin B subunit

Peptides KDYGASTGSSL (Epi1), SLLRRRRNGEDVSV (Epi3) and DDEIYGNDGHP (Epi6), predicted to constitute immunogenic epitopes of the hemolysin-cytotoxin ApxIA of Actinobacillus pleuropneumoniae were inserted into a surface-exposed loop of the B subunit of the E. coli heat-labile enterotoxin (EtxB). The resulting chimeric proteins were recognized by monospecific antibodies against purified native ApxI and by convalescent sera of pigs that were positive for A. pleuropneumoniae serotype 1. Mice anti-sera against chimeric proteins EtxB::ApxIAEpi3 and EtxB::ApxIAEpi6 reacted with purified ApxI. These results indicate that Epi3 and Epi6 regions constitute linear epitopes of the structural ApxIA protein toxin. Epitope Epi6 which is located in the structure of the glycine rich repeats in ApxI elicits the formation of hemolysin neutralizing antibodies when introduced into mice in the form of a chimeric EtxB fusion protein. We suggest that fusion of peptide sequences to EtxB is a useful tool for the analysis of epitopes of complex proteins such as RTX toxins.

Display of an inhibin epitope in a surface-exposed loop of the E. coli heat-labile enterotoxin B subunit

In vitro gene manipulation was used to develop a novel chimeric antigen consisting of the non-toxic B subunit (EtxB) of an E. coli enterotoxin and the first 14 N-terminal amino acid residues of the carboxy-terminal portion of the α subunit of bovine inhibin (bINH 1–14). Rabbits immunized subcutaneously (s.c.) or intravenously (i.v.) with EtxB::bINH 1–14, with or without Freund's adjuvant, developed significant titres of antibodies that recognized an inhibin peptide fragment containing bINH 1–14, native inhibins, and EtxB during separate enzyme-linked immunosorbent assay (ELISA). Passive immunization of mice with the rabbit anti-EtxB::bINH 1–14 serum increased concentrations of follicle-stimulating hormone (FSH) in serum twofold compared with controls, whereas serum concentrations of luteinizing hormone (LH) were unaltered. Since FSH is the primary hormone from the pituitary gland that stimulates ovarian follicle growth and spermatogenesis, the results of this study demonstrate that EtxB::bINH 1–14 has potential as antigen for development of inhibin-based fertility vaccines.