Design of an Ara h 2 hypoallergen from conformational epitopes.

INTRODUCTION: Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). METHODS: X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. RESULTS: A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. CONCLUSION: These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Recombinant T-cell epitope conjugation: A new approach for Dermatophagoides hypoallergen design.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only clinical approach that can potentially cure some allergic diseases by inducing immunological tolerance. Dermatophagoides pteronyssinus is considered as the most important source of mite allergens worldwide, with high sensitization rates for the major allergens Der p 1, Der p 2 and Der p 23. The aim of this work is to generate a hypoallergenic hybrid molecule containing T-cell epitopes from these three major allergens. METHODS: The hybrid protein termed Der p 2231 containing T-cell epitopes was purified by affinity chromatography. The human IgE reactivity was verified by comparing those with the parental allergens. The hybrid was also characterized immunologically through an in vivo mice model. RESULTS: The hybrid rDer p 2231 stimulated in peripheral blood mononuclear cells (PBMCs) isolated from allergic patients with higher levels of IL- 2, IL-10, IL-15 and IFN-γ, as well as lower levels of IL-4, IL-5, IL-13, TNF-α and GM-CSF. The use of hybrid molecules as a therapeutic model in D. pteronyssinus allergic mice led to the reduction of IgE production and lower eosinophilic peroxidase activity in the airways. We found increased levels of IgG antibodies that blocked the IgE binding to the parental allergens in the serum of allergic patients. Furthermore, the stimulation of splenocytes from mice treated with rDer p 2231 induced higher levels of IL-10 and IFN-γ and decreased the secretion of IL-4 and IL-5, when compared with parental allergens and D. pteronyssinus extract. CONCLUSIONS: rDer p 2231 has the potential to be used in AIT in patients co-sensitized with D. pteronyssinus major allergens, once it was able to reduce IgE production, inducing allergen-specific blocking antibodies, restoring and balancing Th1/Th2 immune responses, and inducing regulatory T-cells.

Development of a novel Ara h 2 hypoallergen with no IgE binding or anaphylactogenic activity.

BACKGROUND: To date, no safe allergen-specific immunotherapy for patients with peanut allergy is available. Previous trials were associated with severe side effects. OBJECTIVE: We sought to determine the relative importance of conformational and linear IgE-binding epitopes of the major peanut allergen Ara h 2 and to produce a hypoallergenic variant with abolished anaphylactogenic activity. METHODS: Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopes were produced in insect cells. Conformational IgE epitopes were removed by unfolding these proteins through reduction and alkylation. IgE binding was tested by means of ELISA with sera from 48 Ara h 2-sensitized patients with peanut allergy. Basophil activation and T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by using intraperitoneal challenge of mice sensitized intragastrically to peanut extract. RESULTS: Patients' IgE recognized conformational and linear epitopes in a patient-specific manner. The unfolded mutant lacking both types of epitopes displayed significantly lower IgE binding (median ELISA OD, 0.03; interquartile range, 0.01-0.06) than natural Ara h 2 (median ELISA OD, 0.99; interquartile range, 0.90-1.03; P < .01). Basophil activation by unfolded mutant Ara h 2 was low (median area under the curve, 72 vs 138 for native wild-type Ara h 2; P < .05), but its ability to induce T-cell proliferation was retained. Unfolded mutants without conformational epitopes did not induce anaphylaxis in peanut-sensitized mice. CONCLUSIONS: By removing conformational and linear IgE epitopes, a hypoallergenic Ara h 2 mutant with abolished IgE binding and anaphylactogenic potency but retained T-cell activation was generated.

A hybrid of two major Blomia tropicalis allergens as an allergy vaccine candidate.

INTRODUCTION: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens. AIM: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies caused by B tropicalis. METHODS: Using in silico design of B tropicalis hybrid proteins, we chose two hybrid proteins for heterologous expression. Wild-type Blo t 5/Blo t 21 hybrid molecule and a hypoallergenic version, termed BTH1 and BTH2, respectively, were purified by ion exchange and size exclusion chromatography and characterized by physicochemical, as well as in vitro and in vivo immunological, experiments. RESULTS: BTH1, BTH2 and the parental allergens were purified to homogeneity and characterized in detail. BTH2 displayed the lowest IgE reactivity that induced basophil degranulation using sera from allergic rhinitis and asthmatic patients. BTH2 essentially presented the same endolysosomal degradation pattern as the shortened rBlo t 5 and showed a higher resistance towards degradation than the full-length Blo t 5. In vivo immunization of mice with BTH2 led to the production of IgG antibodies that competed with human IgE for allergen binding. Stimulation of splenocytes from BTH2-immunized mice produced higher levels of IL-10 and decreased secretion of IL-4 and IL-5. In addition, BTH2 stimulated T-cell proliferation in PBMCs isolated from allergic patients, with secretion of higher levels of IL-10 and lower levels of IL-5 and IL-13, when compared to parental allergens. CONCLUSIONS AND CLINICAL RELEVANCE: BTH2 is a promising hybrid vaccine candidate for immunotherapy of Blomia allergy. However, further pre-clinical studies addressing its efficacy and safety are needed.

Modulating Shrimp Tropomyosin-Mediated Allergy: Hypoallergen DNA Vaccines Induce Regulatory T Cells to Reduce Hypersensitivity in Mouse Model.

Shellfish allergy is one of the most common food allergies, with tropomyosin as the major cross-reactive allergen. However, no allergen-specific immunotherapy is clinically available. Recently, we designed two shrimp hypoallergens MEM49 and MED171. This study aimed to examine and compare the efficacy of the MEM49- and MED171-based DNA vaccines (pMEM49 and pMED171) in modulating shrimp allergy in a murine model of shrimp tropomyosin sensitivity. Intradermal immunization of BALB/c mice with pMEM49 or pMED171 effectively down-modulated allergic symptoms, tropomyosin-specific IgE levels, intestinal Th2 cytokines expression, and inflammatory cell infiltration. Both pMEM49 and pMED171 increased the frequency of regulatory T cells, but to a greater extent by pMED171 with upregulation of gut-homing molecules integrin-α4ß7. The functionality of the pMED171-induced Treg cells was further illustrated by anti-CD25-mediated depletion of Treg cells and the adoptive transfer of CD4+CD25+Foxp3+Treg cells. Collectively, the data demonstrate that intradermal administration of pMED171 leads to the priming, activation, and migration of dermal dendritic cells which subsequently induce Treg cells, both locally and systemically, to downregulate the allergic responses to tropomyosin. This study is the first to demonstrate the potency of hypoallergen-encoding DNA vaccines as a therapeutic strategy for human shellfish allergy via the vigorous induction of functional Treg cells.

Development of recombinant stable house dust mite allergen Der p 3 molecules for component-resolved diagnosis and specific immunotherapy.

BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy. OBJECTIVE: The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy. METHODS: Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analysed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization. RESULTS: Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50%) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, rat basophil leukaemia degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH 1-biased immune response that prevented allergic response in mice but retained Der p 3-specific T-cell reactivity. CONCLUSION: rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3.

Epicutaneous immunotherapy with a hypoallergenic Bet v 1 suppresses allergic asthma in a murine model.

BACKGROUND: Due to reduced allergic potency, hypoallergenic variants have been suggested as safer and potentially more efficacious alternative to the corresponding wild-type allergens in allergen-specific immunotherapy. Here, we aimed at investigating the efficacy of recombinant Bet v 1B2, a hypoallergenic folding variant of Bet v 1, in epicutaneous immunotherapy to suppress asthmatic features using a murine model of birch pollen allergy. METHODS AND RESULTS: Before, or after sensitization with rBet v 1 plus ALUMW and intranasal challenges with birch pollen extract, BALB/c mice received epicutaneous immunization (EPI) with rBet v 1, or rBet v 1B2 on their depilated back. Prophylactic EPI with rBet v 1B2, but not with rBet v 1, suppressed serum levels of Bet v 1-specific IgE antibodies and reduced the number of eosinophils and the concentrations of Th2 cytokines in bronchoalveolar lavage. In an established allergic condition, serum levels of Bet v 1-specific IgE antibodies were similar between PBS-treated control mice and EPI-treated mice. However, therapeutic EPI with rBet v 1B2, but not with rBet v 1, significantly suppressed the development of airway inflammation and lung function impairment. CONCLUSION: This study is the first to show the effect of therapeutic EPI with a recombinant form of a hypoallergenic folding variant on the suppression of asthmatic features. Our results suggest that rBet v 1B2 along with its reduced IgE-binding capacity could be a preferred therapeutic allergen than wild-type rBet v 1 in epicutaneous immunotherapy of birch pollen-induced allergic asthma, in particular due to a lower risk of allergic side effect.

Allergens of weed pollen: an overview on recombinant and natural molecules.

Weeds represent a botanically unrelated group of plants that usually lack commercial or aesthetical value. Pollen of allergenic weeds are able to trigger type I reactions in allergic patients and can be found in the plant families of Asteraceae, Amaranthaceae, Plantaginaceae, Urticaceae, and Euphorbiaceae. To date, 34 weed pollen allergens are listed in the IUIS allergen nomenclature database, which were physicochemically and immunologically characterized to varying degrees. Relevant allergens of weeds belong to the pectate lyase family, defensin-like family, Ole e 1-like family, non-specific lipid transfer protein 1 family and the pan-allergens profilin and polcalcins. This review provides an overview on weed pollen allergens primarily focusing on the molecular level. In particular, the characteristics and properties of purified recombinant allergens and hypoallergenic derivatives are described and their potential use in diagnosis and therapy of weed pollen allergy is discussed.

Isolation, expression and characterization of a minor allergen from Penicillium crustosum.

A ribosomal P1 protein, Pen b 26 from Penicillium brevicompactum, was previously identified as a major allergen. A homolog protein was isolated and characterized from Penicillium crustosum which is not known to be allergenic mold. A cDNA library of P. crustosum was constructed and screened using a probe based on the DNA sequence of Pen b 26. A positive clone was isolated, expressed in Escherichia coli, purified and characterized by comparing its immunological and physical properties to Pen b 26. It was designated as Pen cr 26 and had a 321 nt ORF corresponding to 107 amino acids with a MW of 11 kDa. Pen cr 26 had strong sequence homology to Pen b 26 (92% for nucleotides and 86% for amino acids) and its physical and predicted structural properties were similar to the latter. The level of expression of Pen cr 26 was much lower than that of Pen b 26 in the same expression vector. Both proteins were recognized equally well by the IgG class specific antibodies, but Pen cr 26 was poorly recognized by Penicillium-sensitive atopic sera (IgE), suggesting striking antigenic difference in IgE epitopes, i.e., 87% were positive for Pen b 26 while only 23% were positive for Pen cr 26. The allergenicity of Pen cr 26 seems to be minor in nature and it could be a hypoallergenic variant of Pen b 26.

Transgenic rice seeds accumulating recombinant hypoallergenic birch pollen allergen Bet v 1 generate giant protein bodies.

A versatile hypoallergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen-specific immunotherapy. Such a tolerogen should exhibit high efficacy, without side effects, when administered at high doses and should be applicable to several allergens. Tree pollen chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen against birch pollen allergy, was previously selected by DNA shuffling of 14 types of Fagales tree pollen allergens. In this study, transgenic rice seed accumulating TPC7 was generated as an oral vaccine against birch pollen allergy by expressing this protein as a secretory protein using the N-terminal signal peptide and the C-terminal KDEL tag under the control of an endosperm-specific glutelin promoter. The highest level of TPC7 accumulation was approximately 207 µg grain(-1). Recombinant TPC7 is a glycoprotein with high mannose-type N-glycan, but without ß1,2-xylose or α1,3-fucose, suggesting that TPC7 is retained in the endoplasmic reticulum (ER). TPC7 is deposited as a novel, giant spherical ER-derived protein body, >20 µm in diameter, which is referred to as the TPC7 body. Removal of the KDEL retention signal or mutation of a cysteine residue resulted in an alteration of TPC7 body morphology, and deletion of the signal peptide prevented the accumulation of TPC7 in rice seeds. Therefore, the novel TPC7 bodies may have formed aggregates within the ER lumen, primarily due to the intrinsic physicochemical properties of the protein.

Rationally designed hypoallergenic mutant variants of the house dust mite allergen Der p 21.

BACKGROUND: Allergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant. METHODS: Herein, we used a structural bioinformatics and immunoinformatics approach to design hypoallergenic mutant variants of the Der p 21 allergen of Dermatophagoides pteronyssinus, which were then recombinantly expressed in bacteria and tested for their IgE-reactivities. For this, we scanned the wild-type Der p 21 protein for all possible single amino acid substitutions in key IgE-binding regions that could render destabilization of the major epitope regions. RESULTS: Four main substitutions (D82P, K110G, E77G, and E87S) were selected to build mutant variants of the Der p 21 allergen, which were produced in their recombinant forms; two of these variants showed reduced reactivity with IgE. Molecular dynamic simulations and immune simulations demonstrated the overall effects of these mutations on the structural stability of the Der p 21 allergen and on the profile of immune response induced through immunotherapy. CONCLUSIONS: When produced in their recombinant forms, two of the Der p 21 mutant variants, namely proteins K110G and E87S, showed significantly reduced IgE reactivities against sera from HDM-allergic individuals (n = 20; p < 0.001). GENERAL SIGNIFICANCE: This study successfully translated a rational in silico mutagenesis design into low IgE-binding mutant variants of the allergen rDer p 21. These novel hypoallergens are promising to compose next-generation allergen-immunotherapy formulations in near future.

Reducing the Allergenicity of Shrimp Tropomyosin and Allergy Desensitization Based on Glycation Modification.

Shrimp is a major allergic food that could trigger severe food allergy, with the most significant and potent allergen of shrimp referred to as tropomyosin (TM). Glycation modification (Maillard reaction) could reportedly weaken the allergenicity of TM and generate hypoallergenic TM, while up to now, there is still a lack of investigations on the hypoallergenic glycated tropomyosin (GTM) as a candidate immunotherapy for desensitizing the shrimp TM-induced allergy. This study analyzed the effects of glycation modification on decreasing the allergenicity of TM and generated hypoallergenic GTM and how GTM absorbed to the Al(OH)3 function as a candidate immunotherapy for desensitizing allergy. As the results, in comparison to TM, the saccharides of smaller molecular sizes could lead to more advanced glycation end products in GTMs than saccharides of greater molecular sizes, and TM glycated by saccharides of different molecular sizes (glucose, maltose, maltotriose, maltopentaose, and maltoheptaose) exhibited lower allergenicity as a hypoallergen upon activating the allergic reactions of the mast cell and mouse model, while TM glycated by maltose had insignificant allergenicity changes upon activating the allergic reactions of the mast cell and mouse model. In addition, the hypoallergenic GTM + Al(OH)3 was efficient as a candidate immunotherapy; this work intended to offer preclinical data to promote GTM + Al(OH)3 as a candidate allergen-specific immunotherapy for desensitizing the allergy reactions for patients allergic to shrimp food.

Reduced anaphylactic potential of denatured pullulan-conjugated Cry j 1.

Japanese Cedar (JC) pollinosis is the most common seasonal allergic rhinitis in Japan. Throughout the JC pollen season, patients suffer from the allergic symptoms, resulting in a reduction of quality of life. Allergy immunotherapy (AIT) is an established treatment option for a wide range of allergens that unlike symptomatic treatments (e.g. antihistamines) may provide sustained immune tolerance. However, AIT, especially subcutaneous immunotherapy (SCIT) has a fatal anaphylaxis risk due to the use of crude allergen extracts. Consequently, development of allergen derivatives with substantially reduced anaphylactic potential is desirable. An allergen derivative that showed reduced IgE-binding and anaphylactic potential was developed through conjugation of native Cry j 1 (n Cry j 1), a major JC allergen, to the polysaccharide pullulan followed by chemical but non-covalent denaturation. The resulting Cry j 1 allergen derivative, Dn p-Cry j 1, showed reduced IgE-binding and IgE-mediated effector cell activation in vitro using an ELISA competition assay and a mast cell activation model (EXiLE). Reduced anaphylactic potential of Dn p-Cry j 1 in vivo was demonstrated using the rat passive cutaneous anaphylaxis (PCA) assay. The difference in anaphylactic potential of Dn p-Cry j 1 compared to n Cry j 1 in wild-type rats was of the same magnitude as the difference seen in the anaphylaxis reactions obtained with n Cry j 1 in wild-type rats and mast-cell deficient rats, indicating a dramatic reduction in anaphylactic potential of Dn p-Cry j 1. These results indicate that Dn p-Cry j 1 is a promising candidate for next-generation JC AIT.

In vivo Induction of Functional Inhibitory IgG Antibodies by a Hypoallergenic Bet v 1 Variant.

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (104 to 106) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT.

Hypoallergenic mutants of the major oyster allergen Cra g 1 alleviate oyster tropomyosin allergenic potency.

Design of hypoallergen with low IgE reactivity is desirable for allergen-specific immunotherapy. Despite oyster tropomyosin (Cra g 1) is considered as the major allergen, no immunotherapy is available now. In the current research, we generated hypoallergens of Cra g 1 and evaluated their allergenicity. Four hypoallergenic derivatives were constructed by epitope deletion or site-directed mutagenesis on grounds of the identified epitopes. They showed obvious reduction in reactivity towards IgE from oyster-allergic patients and Cra g 1-sensitized BN rats, as well as significant decrease in degranulation and secretion of allergic mediators including histamine, IL-4, IL-6 and TNF-α. In addition, to further investigate the molecular mechanism, we examined the effects of these variants on FcεRI-dependent signalling pathway in IgE-challenged RBL-2H3 cells. We found that the hypoallergenic mutants were able to attenuate FcεRI-mediated signaling cascades in tested cells. These results indicate that the hypoallergenic molecules have ideal characteristics and offer a promising new strategy in clinical immunotherapy for shellfish-allergic subjects.

Perspectives in Allergen-Specific Immunotherapy: Molecular Evolution of Peptide- and Protein-Based Strategies.

Allergen-specific Immunotherapy (AIT), through repetitive subcutaneous or sublingual administrations of allergen extracts, represents up to now the unique treatment against allergic sensitizations. However, the clinical efficacy of AIT can be largely dependent on the quality of natural allergen extracts. Moreover, the long duration and adverse side effects associated with AIT negatively impact patient adherence. Tremendous progress in the field of molecular allergology has made possible the design of safer, shorter and more effective new immunotherapeutic approaches based on purified and characterized natural or recombinant allergen derivatives and peptides. This review will summarize the characteristics of these different innovative vaccines including their effects in preclinical studies and clinical trials.

Immunotherapy of Food Allergy: a Comprehensive Review.

Food allergy imposes a severe global health burden, and thus, there is a dire need for safe and effective treatments. Allergen-specific immunotherapy (AIT) is currently the only approach to restore immune tolerance through administrating increasing doses of allergen extracts. Unfortunately, the development of AIT for food allergies has been impeded by the frequent anaphylactic side effects during the course of treatment. The emergence of component-resolved diagnosis has greatly improved our ability to identify causative allergens and revolutionized the design of AIT. Molecular features such as IgE-binding epitopes and T cell epitopes have been elucidated in most major food allergens, inspiring the use of multiple strategies to manipulate the allergens and design safer alternatives to AIT. Although these allergen-modifying approaches are currently restricted to preclinical characterization and animal studies, the employment of these strategies has certainly paved the way for improving the safety of existing AIT. A safe and effective AIT for food allergy is not far beyond reach.

Synergistic effects of dendritic cell targeting and laser-microporation on enhancing epicutaneous skin vaccination efficacy.

Due to its unique immunological properties, the skin is an attractive target tissue for allergen-specific immunotherapy. In our current work, we combined a dendritic cell targeting approach with epicutaneous immunization using an ablative fractional laser to generate defined micropores in the upper layers of the skin. By coupling the major birch pollen allergen Bet v 1 to mannan from S. cerevisiae via mild periodate oxidation we generated hypoallergenic Bet-mannan neoglycoconjugates, which efficiently targeted CD14+ dendritic cells and Langerhans cells in human skin explants. Mannan conjugation resulted in sustained release from the skin and retention in secondary lymphoid organs, whereas unconjugated antigen showed fast renal clearance. In a mouse model, Bet-mannan neoglycoconjugates applied via laser-microporated skin synergistically elicited potent humoral and cellular immune responses, superior to intradermal injection. The induced antibody responses displayed IgE-blocking capacity, highlighting the therapeutic potential of the approach. Moreover, application via micropores, but not by intradermal injection, resulted in a mixed TH1/TH17-biased immune response. Our data clearly show that applying mannan-neoglycoconjugates to an organ rich in dendritic cells using laser-microporation is superior to intradermal injection. Due to their low IgE binding capacity and biodegradability, mannan neoglycoconjugates therefore represent an attractive formulation for allergen-specific epicutaneous immunotherapy.

Hypoallergenic molecules for subcutaneous immunotherapy.

Although a large part of the population suffers from allergies, a cure is not yet available. Allergen-specific immunotherapy (AIT) offers promise for these patients. AIT has proven successful in insect and venom allergies; however, for food allergy this is still unclear. In this editorial we focus on the recent advances in a proof of concept study in food allergy, FAST (Food allergy specific immunotherapy), which may increase interest within the biomolecular and pharmaceutical industry to embark on similar projects of immunology driven precision medicine within the allergy field.