The Effect of Birch Pollen Immunotherapy on Apple and rMal d 1 Challenges in Adults with Apple Allergy.

BACKGROUND: A proportion of patients allergic to birch pollen are also allergic to pit fruit. The objective of this study was to investigate the effect of immunotherapy with birch pollen on birch-pollen-related apple allergy. METHOD: Patients with birch pollen immunotherapy underwent a skin-prick test with birch pollen, apple and rMal d 1, global assessments and nasal challenges with birch pollen, open food challenge with apple and a double-blind, placebo-controlled test with rMal d 1 at the start of and during the immunotherapy. Measurements of specific IgE in response to Bet v 1 and rMal d 1 and IgG4 in response to Bet v 1 and rMal d 1 took place. RESULTS: Six of eight patients demonstrated an improvement of nasal challenge test results and all patients improved on global assessment during the immunotherapy. The median oral dose of apple required to elicit a reaction increased but was not statistically significant. The patients showed a decrease in skin-prick test values in response to birch pollen (1.05 to 0.36), apple (0.78 to 0.25) and rMal d 1 (0.51 to 0.10) with p-values of 0.04, 0.03 and 0.06, respectively and a decrease of specific IgE in response to Bet v 1 (10.66 kU/L to 5.19 kU/L) and rMal d 1 (0.99 to 0.61 kU/L) with p-values of 0.01 and 0.05, respectively. Only the median specific IgG4 value to Bet v 1 increased from 0.05 to 1.85 mg/L (p-value of 0.02) and not to IgG4 rMal d 1 (0.07 to 0.08 kU/L). CONCLUSION: The beneficial effects of immunotherapy for birch pollen were accompanied by a limited effect on apple allergy.

Efficacy and safety of 4 months of sublingual immunotherapy with recombinant Mal d 1 and Bet v 1 in patients with birch pollen-related apple allergy.

BACKGROUND: Birch pollen-related apple allergy is among the most prevalent food allergies in adolescent/adult subjects and mainly results from sensitization to the major birch pollen allergen Bet v 1 and subsequent cross-reaction with the apple protein Mal d 1. However, specific immunotherapy with birch pollen has inconsistent effects on apple allergy. OBJECTIVE: We sought to compare the safety and efficacy of sublingual immunotherapy (SLIT) with 2 formulations containing either rMal d 1 or rBet v 1 on birch pollen-related apple allergy. METHODS: Sixty participants with birch pollen-related apple allergy were randomized to daily sublingual application of placebo (n = 20) or 25 µg of rMal d 1 (n = 20) or rBet v 1 (n = 20) for 16 weeks. Adverse events were regularly recorded. Sublingual challenges with standardized doses of rMal d 1, skin prick tests with recombinant allergens, and measurements of allergen-specific IgE and IgG4 antibodies were performed before and after treatment. RESULTS: Both formulations caused comparable, mainly local adverse events. No systemic reactions occurred. Compared with the placebo and rBet v 1-treated groups, SLIT with rMal d 1 reduced rMal d 1-induced oral symptoms (P = .001 and P = .038) accompanied by longitudinally reduced rMal d 1-specific cutaneous reactions (P = .022) and enhanced IgG4/IgE ratios (P = .012). SLIT with rBet v 1 neither improved the clinical reactivity to rMal d 1 nor enhanced rMal d 1-specific IgG4/IgE ratios. Participants receiving placebo showed no allergen-specific changes. CONCLUSION: Sublingual treatment with a recombinant food allergen was safe and clinically effective, as determined by using standardized challenges. We present a promising approach for the effective treatment of birch pollen-related apple allergy.

Critical role of mammalian target of rapamycin for IL-10 dendritic cell induction by a flagellin A conjugate in preventing allergic sensitization.

BACKGROUND: Fusion proteins incorporating the Toll-like receptor 5 ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. OBJECTIVE: We studied the mechanisms of immune modulation by a flagellin:allergen fusion protein containing the Toll-like receptor 5 ligand flagellin A from Listeria monocytogenes and the birch pollen allergen Bet v 1 (recombinant flagellin A [rFlaA]:Betv1). METHODS: BALB/c mice were vaccinated with rFlaA:Betv1 in an experimental Bet v 1 sensitization model. Myeloid dendritic cells (mDCs) were differentiated from mouse bone marrow, and PBMCs were isolated from subjects with birch pollen allergy. Cells were stimulated with equimolar amounts of rFlaA, rBet v 1, rFlaA plus rBet v 1, or the rFlaA:Betv1 conjugate and analyzed for cell activation, cytokine secretion, and metabolic state. RESULTS: rFlaA:Betv1 displayed strong immune-modulating properties both in vivo and in vitro, as characterized by secretion of both proinflammatory and anti-inflammatory cytokines from murine mDCs and PBMCs from patients with birch allergy. rFlaA:Betv1 suppressed TH2 responses from Bet v 1-specific CD4+ T cells and prevented allergic sensitization in a mouse allergy model. Aggregation of rFlaA:Betv1 resulted in stronger protein uptake accompanied by an increased resistance to microsomal digestion. Remarkably, rFlaA:Betv1 induced activation of mammalian target of rapamycin, which increased the metabolic activity of the stimulated mDCs. rFlaA:Betv1-mediated IL-10 secretion, but not proinflammatory cytokine secretion, was inhibited by rapamycin in mDCs. CONCLUSION: These results provide evidence that mammalian target of rapamycin is a key player involved in prevention of TH2 responses by flagellin A conjugate vaccines.

T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy. METHODS: Domains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT. RESULTS: The IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected. CONCLUSION: Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

Tackling Bet v 1 and associated food allergies with a single hybrid protein.

BACKGROUND: Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1-mediated birch pollen and associated food allergies, a single wild-type allergen does not provide a complete solution. OBJECTIVE: We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4. METHODS: After identification of T cell epitope-containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule. RESULTS: MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients' serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T-cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross-reactive IgG antibodies, which were able to block the binding of human serum IgE. CONCLUSION: Directed epitope rearrangements combined with a knowledge-based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies.

Glutathione-S-transferase: a minor allergen in birch pollen due to limited release from hydrated pollen.

BACKGROUND: Recently, a protein homologous to glutathione-S-transferases (GST) was detected in prominent amounts in birch pollen by proteomic profiling. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST). METHODOLOGY: bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. Sera from 217 birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The mediator-releasing activity of bGST was analysed with IgE-loaded rat basophil leukaemia cells (RBL) expressing human FcεRI. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses to either protein were assessed. IgE-cross-reactivity between bGST with GST from house dust mite, Der p 8, was studied with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen were assessed in water, simulated lung fluid, 0.9% NaCl and PBS. Eluted proteins were quantified by ELISA and analysed by immunoblotting. PRINCIPLE FINDINGS: Only 13% of 217 birch pollen-allergic patients showed IgE-reactivity to bGST. In RBL assays bGST induced mediator release. Immunization of mice with bGST induced specific IgE and a Th2-dominated cellular immune response comparably to immunization with Bet v 1. bGST did not cross-react with Der p 8. In contrast to Bet v 1, only low amounts of bGST were released from pollen grains upon incubation in water and the different physiological solutions. CONCLUSION/SIGNIFICANCE: Although bGST is abundant in birch pollen, immunogenic in mice and able to induce mediator release from effector cells passively loaded with specific IgE, it is a minor allergen for birch pollen-allergic patients. We refer this discrepancy to its limited release from hydrated pollen. Hence, bGST is an example demonstrating that allergenicity depends mainly on rapid elution from airborne particles.

Oral exposure to Mal d 1 affects the immune response in patients with birch pollen allergy.

BACKGROUND: Antibodies and T cells specific for the major birch pollen allergen Bet v 1 cross-react with structurally related food allergens, such as Mal d 1 in apple. OBJECTIVE: We sought to evaluate the effects of oral uptake of Mal d 1 on the allergen-specific immune response in patients with birch pollen allergy. METHODS: Patients received 50 µg of rBet v 1 sublingually on 2 consecutive days outside of the birch pollen season. One year later, equal amounts of rMal d 1 were administered. Blood samples were collected before and after oral exposure, as well as before and after the intermediate birch pollen season. Allergen-specific IgE levels were determined by using ImmunoCAP. Proliferation of allergen-stimulated PBMCs was assessed, as well as the expression of IL-5, IL-13, IL-10, IFN-γ, and forkhead box protein 3 (Foxp3) in isolated T cells (real-time PCR). Allergen-specific T-cell lines were analyzed for epitope recognition. RESULTS: Orally administered Bet v 1 transiently reduced Bet v 1-specific serum IgE levels, as well as Bet v 1- and Mal d 1-induced T-cell proliferation, and enhanced the expression of IL-5, IL-10, and Foxp3. Orally applied Mal d 1 significantly decreased Bet v 1- and Mal d 1-specific IgE levels and induced IL-5 and IL-10 but no Foxp3 expression. In contrast to Bet v 1, Mal d 1 triggered IFN-γ production and T cells with a different epitope repertoire. Inhalation of birch pollen significantly enhanced allergen-specific IgE levels, T-cell proliferation, and IL-5, IL-10, IL-13, and Foxp3 expression. CONCLUSION: Two sublingual administrations of 50 µg of Mal d 1 were well tolerated and induced transient immune responses seen during peripheral tolerance development. Thus recombinant Mal d 1 might be suitable and relevant for sublingual treatment of birch pollen-related apple allergy.