Epicutaneous allergen application preferentially boosts specific T cell responses in sensitized patients.
ABSTRACT: The effects of epicutaneous allergen administration on systemic immune responses in allergic and non-allergic individuals has not been investigated with defined allergen molecules. We studied the effects of epicutaneous administration of rBet v 1 and rBet v 1 fragments on systemic immune responses in allergic and non-allergic subjects. We conducted a clinical trial in which rBet v 1 and two hypoallergenic rBet v 1 fragments were applied epicutaneously by atopy patch testing (APT) to 15 birch pollen (bp) allergic patients suffering from atopic dermatitis, 5 bp-allergic patients suffering from rhinoconjunctivitis only, 5 patients with respiratory allergy without bp allergy and 5 non-allergic individuals. Epicutaneous administration of rBet v 1 and rBet v 1 fragments led to strong and significant increases of allergen-specific T cell proliferation (CLA+ and CCR4+T cell responses) only in bp-allergic patients with a positive APT reaction. There were no relevant changes of Bet v 1-specific IgE and IgG responses. No changes were noted in allergic subjects without bp allergy and in non-allergic subjects. Epicutaneous allergen application boosts specific T cell but not antibody responses mainly in allergic, APT-positive patients suggesting IgE-facilitated allergen presentation as mechanism for its effects on systemic allergen-specific immune responses.
Project description:Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity.We sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope-containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD).A clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)(+) and CCR4(+) T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively.rBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1-specific proliferation of CLA(+) and CCR4(+) T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1-specific CLA(+) and CCR4(+) proliferation and cytokine secretion in patients with and without APT reactions.Hypoallergenic rBet v 1 fragments induce T cell-dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.
Project description:Epicutaneous allergen-specific immunotherapy (EPIT) is proposed as an alternative route for allergen-specific immunotherapy (AIT). The induction of allergen-specific blocking IgG antibodies represents an important mechanism underlying AIT, but has not been investigated for EPIT. Here, we compared the induction of allergen-specific blocking IgG in outbred guinea pigs which had been immunized with recombinant birch pollen allergen Bet v 1 using patch delivery system (PDS) with or without heat-labile toxin (LT) from Escherichia coli or subcutaneously with aluminum hydroxide (Alum)-adsorbed rBet v 1. Only subcutaneous immunization with Alum-adsorbed rBet v 1 and epicutaneous administration of rBet v 1 with PDS in combination with LT from E. coli induced allergen-specific IgG antibodies blocking allergic patients' IgE, but not immunization with rBet v 1 via PDS alone. Our results suggest that patch vaccination with rBet v 1 in combination with LT may be a promising strategy for allergen-specific immunotherapy against birch pollen allergy.
Project description:Recombinant Bet v 1a (rBet v 1a) has been used in allergy research for more than three decades, including clinical application of so-called hypoallergens. Quantitative IgE binding to rBet v 1a depends on its native protein conformation, which might be compromised upon heterologous expression, purification, or mutational engineering of rBet v 1a.To correlate experimental/theoretical comparisons of IgE binding of defined molar ratios of folded/misfolded recombinant Bet v 1a variants and to determine accuracy and precision of immuno- and physicochemical assays routinely used to assess the quality of recombinant allergen preparations.rBet v 1a and its misfolded variant rBet v 1aS112P/R145P were heterologously expressed and purified from Escherichia coli. Structural integrities and oligomerisation of the recombinant allergens were evaluated by 1H-nuclear magnetic resonance (1H-NMR), circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). IgE binding of defined combinations of rBet v 1a and rBet v 1aS112P/R145P was assessed using immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA) and mediator release (MR) of humanized rat basophilic leukemia cells sensitized with serum IgE of subjects allergic to birch pollen. Experimental and theoretically expected results of the analyses were compared.1H-NMR spectra of rBet v 1a and rBet v 1aS112P/R145P demonstrate a native and highly disordered protein conformations, respectively. The CD spectra suggested typical alpha-helical and beta-sheet secondary structure content of rBet v 1a and random coil for rBet v 1aS112P/R145P. The hydrodynamic radii (RH) of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) showed monomeric dispersion of both allergens in solution. Serum IgE of birch pollen allergic subjects bound to 0.1% rBet v 1a in the presence of 99.9% of non-IgE binding rBet v 1aS112P/R145P. Immunoblot analysis overestimated, whereas ELISA and mediator release assay underestimated the actual quantity of IgE-reactive rBet v 1a in mixtures of rBet v 1a/rBet v 1aS112P/R145P with a molar ratio of rBet v 1a ? 10%.Valid conclusions on quantitative IgE binding of recombinant Bet v 1a preparations depend on the accuracy and precision of physico- and immunochemical assays with which natively folded allergen is detected.
Project description:The adaptive immunity underlying allergy comprises two components, the allergen-specific antibody (i.e. IgE, IgG) and the T-cell response. These two components are responsible for different disease manifestations and can be targeted by different therapeutic approaches. Here, we investigated the association of allergen-specific antibody and T- as well as B-cell responses in pollen-allergic patients using recombinant (r) major birch pollen allergen rBet v 1 and major timothy grass pollen allergen rPhl p 5 as defined antigens.Allergen-specific IgE and IgG antibody responses were determined by ELISA, and allergen-specific T- and B-cell responses were measured in peripheral blood mononuclear cells using a carboxyfluorescein-diacetate-succinimidylester (CFSE) dilution assay.CFSE staining in combination with T-cell- and B-cell-specific gating allowed discriminating between allergen-specific T-cell and B-cell responses. Interestingly, we identified patients where mainly T cells and others where mainly B cells proliferated in response to allergen stimulation. No association between the level of allergen-specific Ig responses and B- or T-cell proliferation was observed.Purified recombinant allergens in conjunction with CFSE staining allow the dissection of allergen-specific B- and T-cell responses. The dissociation of allergen-specific antibody, and B- and T-cell responses may explain the occurrence of selective IgE- and T-cell-mediated manifestations of allergic inflammation and may be important for the development of diagnostic and therapeutic strategies selectively targeting B cells and T cells.
Project description:BACKGROUND:Subcutaneous allergen-specific immunotherapy is a standard route for the immunotherapy of allergic diseases. It modulates the course of allergy and can generate long-term remission. However, subcutaneous allergen-specific immunotherapy can also induce anaphylaxis in some patients, and therefore additional routes of administration should be investigated to improve the safety and tolerability of immunotherapy. OBJECTIVE:We sought to determine whether epicutaneous treatment with antigen in the presence of a Toll-like receptor 9 agonist can suppress TH2-mediated responses in an antigen-specific manner. METHODS:Epicutaneous immunization was performed by applying a skin patch soaked with ovalbumin (OVA) plus CpG, and its suppressor activity was determined by using the mouse model of atopic dermatitis. Finally, adoptive cell transfers were implemented to characterize the regulatory cells that are induced by epicutaneous immunization. RESULTS:Epicutaneous immunization with OVA and CpG reduces the production of OVA-specific IgE and increases the synthesis of OVA-specific IgG2a antibodies in an antigen-specific manner. Moreover, eosinophil peroxidase activity in the skin and production of IL-4, IL-5, IL-10, and IL-13 are suppressed. The observed reduction of IgE synthesis is transferable with T-cell receptor (TCR) ??(+)CD4(+)CD25(-) cells, whereas IgG2a production is dependent on both TCR??(+) and TCR??(+) T cells. Further experiments show that the described phenomenon is myeloid differentiation primary response 88, IFN-?, and IL-17A dependent. Finally, the results suggest that epicutaneous immunization with OVA and CpG decreases the synthesis of OVA-specific IgE and skin eosinophil peroxidase activity in mice with ongoing skin allergy. CONCLUSION:Epicutaneous application of protein antigen in the presence of adjuvant could be an attractive needle-free and self-administered immunotherapy for allergic diseases.
Project description:Food allergy is a life-threatening allergic disease that is increasing in prevalence with no approved curative therapy. Standard treatment of food allergy is limited to avoidance of the allergen and supportive management of allergic symptoms and anaphylaxis. Current research, however, has been focused on developing therapy that can modify the allergic immune response in both allergen-specific and non-specific methods. This review will provide an overview of these methods including oral immunotherapy, sublingual immunotherapy, epicutaneous immunotherapy, modified food protein vaccines, anti-IgE monoclonal antibody adjuvant therapy, Chinese herbs, and helminth therapy.
Project description:Lactobacilli are able to induce upregulation of co-stimulatory molecules in DCs with Th1 cytokines production and increase in Treg activity. This could explain the observed effectiveness of the prolonged administration of lactobacilli in the prevention of allergic disorders in infants and envisage the possible use of bacteria expressing the allergen for the specific immunotherapy of allergic diseases. Hence, we evaluated Streptococcus thermophilus (ST) expressing rBet v 1 as allergen delivery tool and adjuvant factor for immunotherapy in Betv1-sensitized mice. rBet v 1 gene was introduced and expressed in ST (ST[rBet v 1]). BALB/c mice were sensitized with rBet v 1 and then treated with either ST alone, ST[rBet v 1], or the combination of ST and rBet v 1, for 20 days. After 2 aerosol challenges, Treg frequency, in vitro allergen-induced cytokines, rBet v 1-specific IgE and IgG2a, and bronchial histology were made in harvested spleen, sera, and lung. Results were compared with those obtained from not-treated/sensitized mice. ST[rBet v 1] induced immunological and histological changes typical of successful SIT: increased frequency of Tregs and expression of Foxp3; decreased allergen-specific IgE/IgG2a ratio; decrease of in vitro rBet v 1-induced IL-4 from spleen cells; increased allergen-induced IL-10 and IFN-?; drop of bronchial eosinophilia. ST and ST+rBet v 1 combination, even though induced a slight increase in the frequency of Tregs and moderate allergen-induced IL-10, were ineffective in reducing bronchial eosinophilia, allergen induced IL-4 and rBet v 1-specific IgE/IgG2a ratio. ST[rBet v 1] has tolerogenic and Th-1 skewing properties and efficiently delivers the allergen to the gut immune-system restraining and readdressing the established specific Th2 response toward the allergen in mice.
Project description:Molecular allergology uses pure, mainly recombinant and structurally defined allergen molecules and allergen-derived epitopes to study mechanisms of IgE-associated allergy, to diagnose, and even predict the development of allergic manifestations and to treat and prevent IgE-associated allergies. Atopic dermatitis, a chronic inflammatory skin disease is almost always associated with IgE sensitization to allergens. However, also non-IgE-mediated pathomechanisms seem to be operative in atopic dermatitis and it is often difficult to identify the disease-causing allergens. Here we review recent work showing the usefulness of molecular allergology to study mechanisms of atopic dermatitis, for diagnosis and eventually for treatment and prevention of atopic dermatitis.IgE sensitization to airborne, food-derived, microbial allergens, and autoallergens has been found to be associated with atopic dermatitis. Using defined allergen molecules and non-IgE-reactive allergen derivatives, evidence could be provided for the existence of IgE- and non-IgE-mediated mechanisms of inflammation in atopic dermatitis. Furthermore, effects of epicutaneous allergen administration on systemic allergen-specific immune responses have been studied. Multi-allergen tests containing micro-arrayed allergen molecules have been shown to be useful for the identification of culprit allergens in atopic dermatitis and may improve the management of atopic dermatitis by allergen-specific immunotherapy, allergen avoidance, and IgE-targeting therapies in a personalized medicine approach.Molecular allergology allows for dissection of the pathomechanisms of atopic dermatitis, provides new forms of allergy diagnosis for identification of disease-causing allergens, and opens the door to new forms of management by allergen-specific and T cells-targeting or IgE-targeting interventions in a personalized medicine approach.
Project description:IgE-mediated allergies today affect up to 30 % of the population in industrialized countries. Allergen immunotherapy is the only disease-modifying treatment option with a long-term effect. However, very few patients (<5 %) choose immunotherapy, due to the long treatment duration (between 3-5 years) and possible local and systemic allergic side effects of the allergen administrations. The latter occur when an allergen accidentally reaches the blood circulation. Therefore, the ideal application route for allergen immunotherapy should be characterized by two hallmarks: firstly, by a high number of potent antigen-presenting cells, which enhance efficacy and thus shorten treatment duration. Secondly, the allergen administration site is ideally non-vascularized, so that inadvertent systemic distribution of the allergen and consequent systemic allergic side effects are minimized. The epidermis contains high numbers of potent antigen-presenting Langerhans cells and, as an epithelium, is non-vascularized. Therefore, the epidermis represents an interesting administration route. Historical evidence for the clinical efficacy of epicutaneous allergy immunotherapy (EPIT) has now been strengthened by a number of recent double-blinded placebo-controlled clinical trials performed by independent groups. We review the immunological rationale, history and clinical experience with epicutaneous allergy immunotherapy.
Project description:BackgroundSensitization to Betula verrucosa pollen is common in North Central Europe. In Spain, it is also common in patients from Galicia, a region located in the Northwest of Spain. Birch trees are abundant in this region and several birch species have been described. The main objectives were to determine the differences in the IgE reactivity to B. verrucosa and rBet v 1 between patients from Sweden and Spain, as well as the contribution of the major allergen Bet v 1 to the overall reactivity of B. verrucosa. Allergenicity was also compared using a serum pool derived from sera of North American (USA) patients.MethodsIgE reactivity to B. verrucosa and rBet v 1 was measured in sera from 44 Spanish and 21 Swedish patients. rBet v 1 was produced as N-terminal His-tagged fusion protein and purified as originally described. All of them were sensitized to the pollen of this tree species and suffered from allergic rhinitis during the pollination period. rBet v 1 contribution was determined by inhibition ELISA using 3 pools of sera: from Spanish, North American and Swedish patients. IgE binding pattern was evaluated by Western blots using the same pools of sera. Specific IgE binding was expressed in arbitrary units.ResultsSpecific IgE to B. verrucosa was detected in 50 from the 65 sera analyzed (mean = 3.77 ± 3.71 Units). Specific IgE levels to rBet v 1 were 3.13 ± 3.32 Units. Immunoblot assays confirmed specific IgE binding to rBet v 1, and also to other allergens present in the extract. The Spanish pool presented reactivity to more allergens than the Swedish pool. ELISA Inhibition assays, performed with a native extract and rBet v 1, revealed a significant contribution (>80% inhibition) of rBet v 1 to the allergenicity of the extract, with no differences according to the origin of the sera.ConclusionsBet v 1 has a great importance in birch allergy in Galicia and Sweden. Nevertheless, there are differences in the IgE recognition pattern according to the rest of birch allergens. Mayor allergen from birch, Bet v 1, significantly contributes to the allergenicity of B. verrucosa in Galicia.