Immunoregulation by Toxoplasma gondii infection prevents allergic immune responses in mice.

Toxoplasma gondii is a ubiquitous intracellular parasite affecting most mammals including humans. In epidemiological studies, infection with T. gondii and allergy development have been postulated to be inversely related. Using a mouse model of birch pollen allergy we investigated whether infection with T. gondii influences allergic immune responses to birch pollen. BALB/c mice were infected with T. gondii oocysts either before or at the end of sensitisation with the major birch pollen allergen Bet v 1 and thereafter aerosol challenged with birch pollen extract. During the acute phase of infection, clinical signs correlated with increased levels of serum TNF-alpha, IL-6, IFN-gamma and anti-Toxoplasma-IgM. In the chronic phase, Toxoplasma-specific serum IgG, brain tissue cysts and high IFN-gamma production in spleen cell cultures were detected. Mice infected prior to allergic sensitisation produced significantly less allergen-specific IgE and IgG1, while IgG2a levels were markedly increased. IL-5 levels in spleen cell cultures and bronchoalveolar lavage fluid were significantly reduced, and airway inflammation was prevented in these mice. Notably, in mice infected at the end of the allergic sensitisation process, systemic and local immune responses to the allergen were markedly reduced. T.gondii infection was associated with up-regulation of Toll-like receptor 2 (TLR2), 4, 9 and 11, as well as T-bet (a differentiation factor for Th1 cells) mRNA expression in splenocytes; moreover, enhanced TGF-beta, IL-10 and Foxp3 mRNA expression in these cells suggested that regulatory mechanisms were involved in suppression of the allergic immune response. Kinetic studies confirmed the induction of Foxp3(+)CD4(+)CD25(+) regulatory T cells preferentially during the chronic phase of T. gondii infection. Our data demonstrate that T. gondii exhibits strong immunomodulating properties which lead to prevention of allergic immune responses and thereby support the hygiene hypothesis.

Naturally occurring hypoallergenic Bet v 1 isoforms fail to induce IgE responses in individuals with birch pollen allergy.

BACKGROUND: Engineered hypoallergens are currently being investigated for specific immunotherapy of allergic diseases in preclinical and clinical studies. Naturally occurring hypoallergens have by and large not been considered as a source of vaccine candidates. OBJECTIVE: Evaluation of the antibody response in atopic individuals induced by birch pollen containing isoforms of the major birch pollen allergen Bet v 1. METHODS: Isoform-specific antibody isotype responses for Bet v 1.0101, Bet v 1.0401, and Bet v 1.1001 were determined for 35 sera of individuals with birch pollen allergy. Isoform structures were compared and related to IgE-binding inhibitory capacities and induction of mediator release in human Fcvarepsilon receptor transformed rat basophilic leukemia cells. RESULTS: Bet v 1.0101 induced a predominant IgE response, whereas the significant highest levels of IgG(4) antibodies were directed against Bet v 1.0401. Bet v 1.1001 induced only a minimal antibody response. Structural comparisons revealed that most of the amino acid differences between the isoforms were located on the protein surfaces. IgE induced by Bet v 1.0101 only partly cross-reacted with the 2 other isoforms and bound to them with notably lower affinity. Bet v 1.0401 and Bet v 1.1001 also were poor inducers of mediator release. CONCLUSION: Bet v 1 isoforms possess highly variant immunogenic and allergenic properties. Bet v 1.0101 acts as the sensitizing agent, whereas Bet v 1.0401 and Bet v 1.1001 can induce only a minimal IgE response.

Targeting antigens to murine and human M-cells with Aleuria aurantia lectin-functionalized microparticles.

Neuraminidases act as a virulence factors for several pathogens that invade the human body through Peyer's patch M-cells. Because of the structural similarity of Aleuria aurantia lectin (AAL) to neuraminidases, we hypothesized that AAL might also target human M-cells. In an in vitro human M-cell co-culture model significantly more particles were transported across the epithelium when microparticles were functionalized with AAL versus those that were not. Moreover, high concentrations of AAL induced no detectable cytotoxic effects on the related intestinal epithelial cell cultures, epithelial Caco2- and HT29-MTX-E12-cells. Upon incubation with AAL, PBMCs of allergic volunteers proliferated in response to AAL and secreted the cytokines, IL-2, IFN-gamma, IL-10 and IL-5 in a concentration-dependent manner, indicating immune-stimulatory properties of the lectin. We conclude that AAL-coated microparticles may have the potential to target entrapped antigens to human M-cells for oral vaccination.

Mucosal targeting of allergen-loaded microspheres by Aleuria aurantia lectin.

Murine intestinal M-cells express alpha-L-fucose residues. We constructed alpha-L-fucose-targeting particles for oral immunotherapy of IgE-mediated allergy. Poly(D,L-lactic-co-glycolic acid)-microspheres were loaded with birch pollen allergens, and functionalised with the alpha-L-fucose specific Aleuria aurantia lectin (AAL). The AAL-microspheres had a size of 1-3 microm, protected the entrapped allergens from gastric degradation and released 46.6+/-1.3% allergen over 21 days in vitro. Oral gavages of AAL-particles to naive BALB/c mice induced birch pollen-specific IgG2a, but not IgG1 antibodies. We conclude that targeting allergens to alpha-L-fucose-receptor bearing cells using AAL-microspheres induces specific Th1-antibody responses possibly counteracting Th2-dominated allergy, and therefore provides a potentially useful formulation for oral immunotherapy.

Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal immunogen and adjuvant.

We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.

Cross-reactive N-glycans of Api g 5, a high molecular weight glycoprotein allergen from celery, are required for immunoglobulin E binding and activation of effector cells from allergic patients.

Allergy diagnosis relying on the determination of specific IgE is frequently complicated by the presence of cross-reacting IgE of unclear clinical relevance. Particularly, the anaphylactogenic activity of IgE directed to cross-reactive carbohydrate moieties of glycoproteins from plants and invertebrates has been a matter of debate. In this study, we present the biochemical and immunological characterization of Api g 5, a glycoprotein allergen from celery with homology to FAD containing oxidases. Carbohydrate analysis of the allergen revealed the presence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. Chemical deglycosylation of the protein completely abolished binding of serum IgE from all 14 patients tested. Likewise, basophils from a patient allergic to mugwort pollen and celery were stimulated only by native Api g 5, whereas the deglycosylated allergen did not trigger release of histamine. IgE inhibition immunoblots showed that native Api g 5 other than the deglycosylated protein completely inhibited IgE binding to high molecular weight allergens in protein extracts from birch pollen, mugwort pollen, and celery. A similar inhibition was accomplished using the IgE binding oligosaccharide, MUXF, coupled to bovine serum albumin. All these observations taken together confer convincing evidence that IgE directed to cross-reactive carbohydrates is capable of eliciting allergic reactions in vivo.

Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier.

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9A using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development.