Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice.

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.

Expression of chemokine receptors in the feline reproductive tract and large intestine.

Feline immunodeficiency virus (FIV) is a lentivirus that causes feline acquired immunodeficiency syndrome. Infection can be transmitted experimentally via the vagina and rectum, making the cat a useful model for human immunodeficiency virus (HIV) infection. Some strains of FIV use the CXCR4 chemokine receptor in vitro to gain entry to feline cell lines, thymocytes and peripheral blood leucocytes (PBLs). In this study, the tissue expression of messenger ribonucleic acid (mRNA) encoding the CCR3, CXCR4 and CCR5 receptors was examined by reverse transcriptase polymerase chain reaction (RT-PCR). mRNA encoding each receptor was expressed by two feline T-cell lines (Mya-1 and FeTJ), a feline kidney fibroblast cell line (FKCU) and PBLs. Mesenteric lymph node, colon, rectum, uterus, cervix and vagina all expressed mRNA for CXCR4 and CCR5 whilst only lymph node expressed CCR3 mRNA. In order to locate this receptor mRNA expression, in-situ hybridization studies were performed with DNA probes specific for the chemokine receptor mRNAs. CCR5 and CXCR4 receptor mRNA was expressed by epithelial cells and some lamina propria cells of the colon and rectum. Epithelial cell expression of chemokine receptor mRNA was reduced in intensity towards the base of the crypts. Expression of CXCR4 receptor was also demonstrated immunohistochemically on some lamina propria and intraepithelial cells. The expression of these receptor molecules may be important in mucosal infection with FIV.

A cholera toxin B-subunit variant that binds ganglioside G(M1) but fails to induce toxicity.

Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit. The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1). Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity. CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT. The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts). Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro. Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization. These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.

Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc.

Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB. When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

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.

Is immune cell activation the missing link in the pathogenesis of post-diarrhoeal HUS?

Haemolytic uraemic syndrome (HUS), which is caused by Shiga toxin (Stx)-producing Escherichia coli, is the commonest cause of acute renal failure in childhood. It is widely believed that HUS develops following the release of Stx, an AB5 toxin that inhibits protein synthesis and has a direct toxic effect on the kidney endothelium. There remains, however, a mismatch between the current understanding of the pathogenesis of HUS and the evolution of the clinical signs of the disease. Our hypothesis is that Stx-mediated immune cell activation in the gut is the missing link in the pathogenesis of this condition, initiating the characteristic renal pathology of HUS either alone or in synergy with Stx. Validation of this hypothesis could lead to a targeted anti-inflammatory approach aimed at modulating immune cell function in HUS.

Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its vlosely related homologue, the B subunit of cholera toxin.

Although cholera toxin (Ctx) and Escherichia coli heat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.

Cholera toxin and Escherichia coli enterotoxin B-subunits inhibit macrophage-mediated antigen processing and presentation: evidence for antigen persistence in non-acidic recycling endosomal compartments.

Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.

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.

Floating cholera toxin into epithelial cells: functional association with caveolae-like detergent-insoluble membrane microdomains.

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and likely endoplasmic reticulum (ER) (Lencer et al., J. Cell Biol. 131, 951-962 (1995)). We have recently found that the toxin's apical membrane receptor ganglioside GM1 acts specifically in this signal transduction pathway, likely by coupling CT with caveolae or caveolae-related membrane domains (lipid rafts) (Wolf et al., J. Cell Biol. 141, 917-927 (1998)). Work in progress shows that 1) cholesterol depletion uncouples the CT-GM1 receptor complex from signal transduction, a characteristic of lipid rafts; 2) the GM1 acyl chains rather than the carbohydrate head groups appear to account for the structural basis of ganglioside specificity in toxin trafficking; and 3) intestinal epithelial cells obtained from normal adult humans exhibit lipid rafts which differentiate between CT-GM1 and LTIIb-GD1a complexes and which contain caveolin 1.

Production and secretion of collagen-binding proteins from Aeromonas veronii.

Collagen-binding protein (CNBP) synthesized by Aeromonas veronii is located conserved within the subcellular fraction. The results of this study show that 98% of the total CNBP produced by Aer. veronii is present in the extracellular medium, and that the remaining CNBP is distributed either on the cell surface, within the periplasm or anchored on the outer membrane. CNBP is specifically secreted from Aer. veronii into the culture medium, because all the beta-lactamase activity was located in the cells and could be released by polymixin B extraction of periplasmic proteins. CNBP was produced at growth temperatures from 12 degrees C to 42 degrees C, but not at 4 degrees C. The findings indicate that the level of CNBP in the medium increases during the exponential growth phase and reaches a maximum during the early stationary phase. There was less CNBP production in poor nutrient MMB medium than in the rich LB nutrient medium. CNBP secretion, in contrast to aerolysin secretion, was unaffected by the exeA mutation of Aer. hydrophila. It is concluded that CNBP secretion from Aer. veronii must be achieved by a mechanism different from that reported for aerolysin secretion.

Cholera toxin and related enterotoxins: a cell biological and immunological perspective.

Cholera toxin (Ctx) from Vibrio cholerae and the closely related Escherichia coli heat-labile enterotoxin (Etx) are the primary virulence factors responsible for causing cholera and traveller's diarrhea, respectively. Studies on the mode of action of these toxins on gut epithelial cells have revealed important insights into the mechanisms of toxin uptake and trafficking in eukaryotic cells. However, of perhaps even greater fascination have been the discoveries that Ctx and Etx exhibit remarkable immunological properties. When either of these toxins is administered via mucosal routes, it triggers a potent mucosal and systemic anti-toxin immune response. By contrast, local or systemic immunization with other soluble protein antigens usually stimulates only a meagre immune response, or results in a state of immunological tolerance. Even more striking are the findings that when Ctx or Etx are mixed with heterologous antigens, they function as adjuvants, leading to stimulation of mucosal responses to the admixed antigen, and the abrogation of oral tolerance. In addition, recent observations have shown that the receptor-binding component of these toxins can down-regulate inflammatory diseases associated with the induction of autoimmune disorders such as rheumatoid arthritis, diabetes, and multiple sclerosis. While the underlying mechanisms responsible for these remarkable properties have yet to be resolved, it is clear that the toxins' ability to bind to cell surface receptors plays an important role in their potent immunogenicity, adjuvanticity, and immunotherapeutic properties. This review provides an overview of the latest developments within the Ctx/Etx field, with a special emphasis on the cell entry mechanisms and immunomodulatory action of Ctx/Etx and their component subunits.

Membrane traffic and the cellular uptake of cholera toxin.

In nature, cholera toxin (CT) and the structurally related E. coli heat labile toxin type I (LTI) must breech the epithelial barrier of the intestine to cause the massive diarrhea seen in cholera. This requires endocytosis of toxin-receptor complexes into the apical endosome, retrograde transport into Golgi cisternae or endoplasmic reticulum (ER), and finally transport of toxin across the cell to its site of action on the basolateral membrane. Targeting into this pathway depends on toxin binding ganglioside GM1 and association with caveolae-like membrane domains. Thus to cause disease, both CT and LTI co-opt the molecular machinery used by the host cell to sort, move, and organize their cellular membranes and substituent components.

Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin.

We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein-protein interactions and may be developed to target specific cellular compartments.

Construction and murine immunogenicity of recombinant Bacille Calmette Guérin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin.

Three recombinant strains of Mycobacterium bovis Bacille Calmette Guerin (rBCG) were prepared in which the immunogenic B subunit of human Escherichia coli heat labile enterotoxin (LT-Bh) was expressed either as a cytoplasm protein, a cell wall associated lipoprotein or a secreted protein. Intraperitoneal immunisation of mice with these rBCG induced IgG and IgA antibodies to LT-Bh and shifted the serum IgG subclass response to subsequent challenge with purified LT-Bh from IgG1 to an IgG2a. Oral administration of recombinant BCG induced mucosal and serum IgA antibodies to LT-Bh which peaked four months after immunisation. Antibody responses were greater when LT-Bh was expressed as a secreted protein or lipoprotein rather than in the cytoplasm. Oral vaccination with recombinant BCG may be an effective approach, particularly to induce mucosal IgA and prime for a serum TH1 recall response.

Biofilm formation by Helicobacter pylori.

Helicobacter pylori NCTC 11637 produces a water-insoluble biofilm when grown under defined conditions with a high carbon:nitrogen ratio in continuous culture and in 10% strength Brucella broth supplemented with 3 g l-1 glucose. Biofilm accumulated at the air/liquid interface of the culture. Light microscopy of frozen sections of the biofilm material showed few bacterial cells in the mass of the biofilm. The material stained with periodic acid Schiff's reagent. Fucose, glucose, galactose, and glycero-manno-heptose, N-acetylglucosamine and N-acetylmuramic acid were identified in partially purified and in crude material, using gas chromatography and mass spectrometry. The sugar composition strongly indicates the presence of a polysaccharide as a component of the biofilm material. Antibodies (IgG) to partially purified material were found in both sero-positive and sero-negative individuals. Treatment of the biofilm material with periodic acid reduced or abolished immunoreactivity. Treatment with 5 mol l-1 urea at 100 degrees C and with phenol did not remove antigenic recognition by patient sera. The production of a water-insoluble biofilm by H. pylori may be important in enhancing resistance to host defence factors and antibiotics, and in microenvironmental pH homeostasis facilitating the growth and survival of H. pylori in vivo.

Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic.

Cholera toxin and its close relative, Escherichia coli heat-labile enterotoxin, are potent immunogens and mucosal adjuvants. The recent findings that their B subunits can promote tolerance highlights the complexity of their interactions with the immune system. Here, Neil Williams and colleagues review the mechanisms by which these molecules modulate leukocyte populations and seek to explain the paradox.

Structural basis for the differential toxicity of cholera toxin and Escherichia coli heat-labile enterotoxin. Construction of hybrid toxins identifies the A2-domain as the determinant of differential toxicity.

Cholera toxin (Ctx) and E. coli heat-labile enterotoxin (Etx) are structurally and functionally similar AB5 toxins with over 80% sequence identity. When their action in polarized human epithelial (T84) cells was monitored by measuring toxin-induced Cl- ion secretion, Ctx was found to be the more potent of the two toxins. Here, we examine the structural basis for this difference in toxicity by engineering a set of mutant and hybrid toxins and testing their activity in T84 cells. This revealed that the differential toxicity of Ctx and Etx was (i) not due to differences in the A-subunit's C-terminal KDEL targeting motif (which is RDEL in Etx), as a KDEL to RDEL substitution had no effect on cholera toxin activity; (ii) not attributable to the enzymatically active A1-fragment, as hybrid toxins in which the A1-fragment in Ctx was substituted for that of Etx (and vice versa) did not alter relative toxicity; and (iii) not due to the B-subunit, as the replacement of the B-subunit in Ctx for that of Etx caused no alteration in toxicity, thus excluding the possibility that the broader receptor specificity of EtxB is responsible for reduced activity. Remarkably, the difference in toxicity could be mapped to a 10-amino acid segment of the A2-fragment that penetrates the central pore of the B-subunit pentamer. A comparison of the in vitro stability of two hybrid toxins, differing only in this 10-amino acid segment, revealed that the Ctx A2-segment conferred a greater stability to the interaction between the A- and B-subunits than the corresponding segment from Etx A2. This suggests that the reason for the relative potency of Ctx compared with Etx stems from the increased ability of the A2-fragment of Ctx to maintain holotoxin stability during uptake and transport into intestinal epithelia.