Search

The proportion of patients with type I allergy in the world population has been increasing and with it the number of people suffering from allergic symptoms. Recently we showed that prophylactic cell therapy employing allergen-expressing bone marrow (BM) cells or splenic B cells induced allergen-specific tolerance in naïve mice. Here we investigated if cell therapy can modulate an established secondary allergen-specific immune response in pre-immunized mice. We sensitized mice against the grass pollen allergen Phl p 5 and an unrelated control allergen, Bet v 1, from birch pollen before the transfer of Phl p 5-expressing BM cells. Mice were conditioned with several combinations of low-dose irradiation, costimulation blockade, rapamycin and T cell-depleting anti-thymocyte globulin (ATG). Levels of allergen-specific IgE and IgG1 in serum after cell transfer were measured via ELISA and alterations in cellular responses were measured via an in vitro proliferation assay and transplantation of Phl p 5+ skin grafts. None of the tested treatment protocols impacted Phl p 5-specific antibody levels. Transient low-level chimerism of Phl p 5+ leukocytes as well as a markedly prolonged skin graft survival were observed in mice conditioned with high numbers of Phl p 5+ BMC or no sensitization events between the day of cell therapy and skin grafting. The data presented herein demonstrate that a pre-existing secondary allergen-specific immune response poses a substantial hurdle opposing tolerization through cell therapy and underscore the importance of prophylactic approaches for the prevention of IgE-mediated allergy. [ABSTRACT FROM AUTHOR]

Introduction: Prophylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells (HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4+ T cells, CD8+ T cells, and CD19+ B cells) to induce allergen-specific tolerance in naive mice and identified CD19+ B cells as promising candidates for allergen-specific cell therapy. Methods: For this purpose, CD19+ B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG1 levels by RBL assay and ELISA, respectively. In vivo allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing. Results: The transfer of purified Phl p 5-expressing CD19+ B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG1 antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19+ B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice. Discussion: Thus, we demonstrated that the transfer of Phl p 5-expressing CD19+ B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans. [ABSTRACT FROM AUTHOR]

Background: Immunoglobulin E (IgE)-mediated cow's milk allergy (CMA) can be life-threatening and affects up to 3% of children. Hypoallergenic infant formulas based on hydrolyzed cow's milk protein are increasingly considered for therapy and prevention of cow's milk allergy. The aim of this study was to investigate the allergenic activity and ability to induce T cell and cytokine responses of an infant formula based on extensively hydrolyzed cow's milk protein (whey) (eHF, extensively hydrolyzed formula) supplemented with Galactooligosaccharides (GOS) and Limosilactobacillus fermentum CECT5716 (LF) to determine its suitability for treatment and prevention of CMA. Methods: eHF and standard protein formula based on intact cow's milk proteins (iPF) with or without Galactooligosaccharide (GOS) and Limosilactobacillus fermentum CECT5716 (LF) were investigated with allergen-specific antibodies and tested for IgE reactivity and allergenic activity in basophil degranulation assays with sera from cow's milk (CM)-allergic infants/children. Their ability to stimulate T cell proliferation and cytokine secretion in cultured peripheral blood mononuclear cells (PBMC) from CM-allergic infants and children was studied with a FACS-based carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay and xMAP Luminex fluorescent bead-based technology, respectively. Results: An eHF supplemented with GOS and LF exhibiting almost no IgE reactivity and allergenic activity was identified. This eHF induced significantly lower inflammatory cytokine secretion as compared to an intact protein-based infant formula but retained T cell reactivity. Conclusions: Due to strongly reduced allergenic activity and induction of inflammatory cytokine secretion but retained T cell reactivity, the identified eHF may be used for treatment and prevention of CMA by induction of specific T cell tolerance. [ABSTRACT FROM AUTHOR]

Abstract The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Background: Early introduction of food allergens into children's diet is considered as a strategy for the prevention of food allergy. The major fish allergen parvalbumin exhibits high stability against gastrointestinal digestion. We investigated whether resistance of carp parvalbumin to digestion affects oral tolerance induction. Methods: Natural Cyp c 1, nCyp c 1, and a gastrointestinal digestion‐sensitive recombinant Cyp c 1 mutant, mCyp c 1, were analyzed for their ability to induce oral tolerance in a murine model. Both antigens were compared by gel filtration, circular dichroism measurement, in vitro digestion, and splenocyte proliferation assays using synthetic Cyp c 1‐derived peptides. BALB/c mice were fed once with high doses of nCyp c 1 or mCyp c 1, before sensitization to nCyp c 1. Immunological tolerance was studied by measuring Cyp c 1‐specific antibodies and cellular responses by ELISA, basophil activation, splenocyte proliferations, and intragastric allergen challenge. Results: Wild‐type and mCyp c 1 showed the same physicochemical properties and shared the same major T‐cell epitope. However, mCyp c 1 was more sensitive to enzymatic digestion in vitro than nCyp c 1. A single high‐dose oral administration of nCyp c 1 but not of mCyp c 1 induced long‐term oral tolerance, characterized by lack of parvalbumin‐specific antibody and cellular responses. Moreover, mCyp c 1‐fed mice, but not nCyp c 1‐fed mice developed allergic symptoms upon challenge with nCyp c 1. Conclusion: Sensitivity to digestion in the gastrointestinal tract influences the capacity of an allergen to induce prophylactic oral tolerance. [ABSTRACT FROM AUTHOR]

Background: Skin testing represents a commonly used first diagnostic method in clinical practice, but allergen extracts may vary in composition and often contain cross‐reactive allergens and therefore do not always allow the precise identification of the sensitizing allergen source. Our aim was to investigate the suitability of a single recombinant hybrid molecule, consisting of the four major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Phl p 6) for in vivo diagnosis of genuine grass pollen allergy in children suffering from pollinosis. Methods: Sixty‐four children aged from 6 to 17 years with a positive skin reaction and/or specific IgE to grass pollen extract and respiratory symptoms of pollinosis as well as 9 control children with allergy to other allergen sources were studied. SPT was performed with the recombinant hybrid, the four recombinant timothy grass pollen allergens, and grass pollen extract. Specific IgE reactivity to 176 micro‐arrayed allergen molecules was determined using ImmunoCAP ISAC technology. IgE reactivity to the hybrid was detected by non‐denaturing RAST‐based dot blot assay. Results: Genuine grass pollen sensitization was confirmed in 94% of the children with positive SPT to grass pollen extract by SPT and IgE reactivity to the hybrid. The four hybrid‐negative children showed IgE reactivity to cross‐reactive allergens such as Phl p 4, Phl p 11, and Phl p 12 and had also sensitizations to pollen allergens from unrelated plants. Conclusions: The recombinant hybrid molecule represents a useful tool for in vivo diagnosis of genuine grass pollen sensitization. [ABSTRACT FROM AUTHOR]

Background: In high‐risk populations, allergen‐specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus‐like nanoparticles (VNP) expressing surface‐exposed or shielded allergens. Methods: Full‐length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results: Virus‐like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface‐exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1‐specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen‐specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface‐exposed but not shielded allergen induced allergen‐specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen‐protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg‐dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion: Virus‐like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization. Allergen‐expressing VNP, which are produced in HEK 293T cells with the help of MoMLV structural proteins, are unable to stimulate allergen‐specific B cells or sensitized effector cells (basophils, mast cells). Instead, upon i.n. application, they are being taken up by lung‐resident CD103+ DC and alveolar macrophages, which unpack their (allergic) cargo, expand Foxp3+ Treg cells and prevent from sensitization. [ABSTRACT FROM AUTHOR]