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Background The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children. Objective We aimed to evaluate the capacity of EPIT to provide protection against cashew‐induced anaphylaxis in a relevant mouse model. Methods The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew‐sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast‐cell proteases (mMCP)‐1/7, were quantified. Results Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast‐cell reactivity as attested by the reduction of mMCP‐1/7 in plasma, suggesting that EPIT specifically decrease IgE‐mediated anaphylaxis. Conclusion We demonstrate that EPIT markedly reduced IgE‐mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy., This study demonstrates a newly developed robust mouse model of IgE‐mediated cashew anaphylaxis. Cashew‐sensitized mice are treated with epicutaneous patches containing cashew protein extract (cashew patches) or excipient (placebo patches) for 16 weeks. Mice treated with cashew patches present a decrease in cashew‐specific Th2 response and a decrease in mast‐cell reactivity and anaphylaxis symptoms following oral challenge. Abbreviations: EPIT, epicutaneous immunotherapy.

The skin is an immune organ comprised of a large network of antigen-presenting cells such as dendritic cells, making it an attractive target for the development of new vaccines and immunotherapies. Recently, we developed a new innovative and non-invasive vaccination method without adjuvant based on epicutaneous vaccine patches on which antigen forms a dry deposit. Here we describe in mice a method for potentiating the efficacy of our epicutaneous vaccination approach using a minimally invasive and epidermis-limited skin preparation based on laser-induced micro-perforation. Our results showed that epidermal micro-perforation increased trans-epidermal water loss, resulting in an enhancement of antigen solubilization from the surface of the patch, and increased the quantity of antigen delivered to the epidermis. Importantly, this was not associated with an increase in systemic passage of the antigen. Skin micro-perforation slightly activated keratinocytes without inducing an excessive level of local inflammation. Moreover, epidermal micro-perforation improved antigen capture by epidermal dendritic cells and specifically increased the level of Langerhans cells activation. Finally, we observed that epidermal micro-perforation significantly increased the level of the specific antibody response induced by our epicutaneous Pertussis vaccine candidate containing non-adsorbed recombinant Pertussis Toxin and reduced the amount of antigen dose required. Overall, these data confirm the benefit of a minimal and controlled epidermal preparation for improving the effectiveness of an epicutaneous patch-based vaccine, without adversely affecting the safety of the method.

Due to its richness in antigen presenting cells, e.g., dendritic cells (DC), the skin has been identified as a promising route for immunotherapy and vaccination. Several years ago, a skin delivery system was developed based on epicutaneous patches allowing the administration of antigen through intact skin. Using mouse models, we have shown that epicutaneous allergen application leads to a rapid uptake and transport of allergen-positive cells to skin-draining lymph nodes (LN). This occurred primarily in animals previously sensitized to the same allergen. In that context, we sought to better understand the role of the specific preexisting immunity in allergen capture by skin DC and their subsequent migration to LN. Specifically, we investigated the role of humoral immunity induced by sensitization and the involvement of IgG Fc receptors (FcγR). Epicutaneous patches containing fluorescently-labeled ovalbumin (OVA) were applied to naïve mice that had previously received either sera or purified IgG isolated from OVA-sensitized mice. To investigate the involvement of FcγR, animals received 2.4G2 (anti-FcγRII/RIII) blocking antibody, 24 hours before patch application. Mice that received sera or purified IgG originating from OVA-sensitized mice showed an increase in the quantity of OVA-positive DC in skin and LN. Moreover, the blockade of FcγR reduced the number of OVA-positive DC in LN to a level similar to that observed in naïve animals. Overall, these results demonstrate that preexisting specific-IgG antibodies are involved in allergen capture by skin DC following EPIT through the involvement of antigen-specific IgG-FcγR.

Background: The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children. Objective: We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model. Methods: A mouse model of IgE-mediated cashew anaphylaxis was first developed. Based upon this model, the efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. Cashew-specific antibody titers were measured throughout treatment. Treated mice were challenged orally to cashew and anaphylactic symptoms were monitored. Additionally, plasma levels of mast-cell proteases (mMCP)-1/7 were quantified from blood samples collected after challenge to evaluate IgE-induced mast-cell activation. Results: EPIT significantly decreased anaphylactic symptoms following challenge and increased cashew-specific IgG2a (equivalent of human IgG1). Interestingly, this protection was associated with a sharp decrease in mast-cell reactivity. Conclusion: We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

Background: Epicutaneous immunotherapy (EPIT®), currently investigated in the treatment of food allergy, needs the integrity of the skin to warrant safety and efficacy. Mutations in the gene encoding the key epidermal protein filaggrin (FLG) are risk factors for peanut allergy and disrupt the skin intergrity. We investigated the association between FLG deficiency and peanut EPIT® efficacy in a murine model. Methods: FLG mutant mice deficient in filaggrin (FLG-/-) or wild-type (WT) mice were sensitized with peanut protein extract (peanut protein) and cholera toxin. Sensitized mice received a patch per week during 8 weeks for EPIT®, using Viaskin®, and were then submitted to sustained peanut oral exposure. We assessed blood humoral and cellular responses and evaluated eosinophil infiltration in the gut mucosa. The different steps of allergen capture and transportation following deposition on the skin was also analyzed in sensitized mice. Results: Sensitization of mice was confirmed by a significant increase of specific Th2 biaised immunological responses. In sensitized mice, EPIT® significantly reduced IgE levels, splenocytes secretion of Th2 cytokines and recruitment of eosinophils in esophagus, compared to sensitized mice without epicutaneous immunotherapy. The allergen applied onto the skin of FLG-/- mice did not undergo passive skin passage or systemic delivery. Instead, the allergen was captured by skin CD205highDCs, which migrated to afferent lymph nodes, as already described in WT mice. Conclusions: EPIT® was efficient and safe in FLG-/- mice, suggesting that in Humans EPIT® keeps efficacy and safety in the presence of loss of function of FLG.

Allergen-specific immunotherapy has been proposed as an attractive strategy to actively treat food allergy using the following three different immunotherapy routes: oral (OIT), sublingual (SLIT) and epicutaneous (EPIT) immunotherapy. Regulatory T cells (Tregs) have been shown to have a pivotal role in the mechanisms of immunotherapy. The aim of this study was to compare the phenotype and function of Tregs induced in peanut-sensitized BALB/c mice using these three routes of treatment. We show that although EPIT, OIT and SLIT were all able to effectively desensitize peanut-sensitized mice, they induced different subsets of Tregs. Foxp3+ Tregs were induced by the three treatment routes but with greater numbers induced by EPIT. EPIT and OIT also increased the level of LAP+ Tregs, whereas SLIT induced IL-10+ cells. The suppressive activity of EPIT-induced Tregs did not depend on IL-10 but required CTLA-4, whereas OIT acted through both mechanisms and SLIT was strictly dependent on IL-10. Moreover, the three routes influenced the homing properties of induced Tregs differently, with a larger repertoire of chemokine receptors expressed by EPIT-induced Tregs compared with OIT- and SLIT- induced cells, resulting in different protective consequences against allergen exposure. Furthermore, whereas OIT- or SLIT-induced Tregs lost their suppressive activities after treatment was discontinued, the suppressive activities of EPIT-induced Tregs were still effective 8 weeks after the end of treatment, suggesting the induction of a more long-lasting tolerance. In summary, EPIT, OIT and SLIT mediated desensitization through the induction of different subsets of Tregs, leading to important differences in the subsequent protection against allergen exposure and the possible induction of tolerance.

The skin is a major immunologic organ that may induce protection, sensitization or tolerance. Epicutaneous immunotherapy (EPIT) has been proposed as an attractive strategy to actively treat food allergy and has been shown to induce tolerance in sensitized mice through the induction of Foxp3+ regulatory T cells (Tregs), especially CD62L+ Tregs. Among immune cells in the skin, dendritic cells are key players in antigen-specific immune activation or regulation. The role of different populations of skin DCs in tolerance induction remains to be elucidated. Using OVA-sensitized BALB/c mice, we demonstrated that the application of a patch containing OVA-A647 to the skin resulted in allergen uptake by Langerhans cells (LCs) and CD11b+ dermal cDC2 and subsequent migration into skin draining lymph nodes. These 2 populations induced Foxp3 expression in CD4+ cells in vitro. Only LCs induced LAP+ cells and CD62L+ Tregs. Using Langerin-eGFP-DTR mice, we analyzed the role of LCs in the mechanisms of tolerance induction by EPIT in vivo. Following complete depletion of LCs, a dramatic decrease in the number of OVA+ DCs and OVA+ CD11b+ dermal cDC2 was observed in skin draining lymph nodes 48 h after epicutaneous application. Likewise, 2 weeks of EPIT in non-depleted mice induced Foxp3+ Tregs, especially CD62L+, and LAP+ Tregs in skin draining lymph nodes and spleen, whereas no induction of Tregs was observed in LC-depleted mice. Following 8 weeks of treatment, EPIT-treated mice showed significant protection against anaphylaxis accompanied by a significant increase of Foxp3+ Tregs, especially CD62L+ Tregs, which was not seen in the absence of LCs. In summary, although both LCs and CD11b+ dermal cDC2s could induce regulatory T cells, the absence of LCs during EPIT impaired treatment efficacy, indicating their crucial role in skin-induced tolerance.

Background Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT). Methods BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE‐patch or by PPE‐OIT. Another set of peanut‐sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analyzed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. Results Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4th week and a significant hypomethylation of the Foxp3 promoter in CD62L+ Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. Conclusions Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T‐cell compartments with downregulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect.

Introduction L’immunotherapie epicutanee (EPIT) constitue une voie therapeutique nouvelle pour l’allergie alimentaire. La peau contient une grande quantite de cellules presentatrice d’antigenes dont les cellules de Langerhans dans l’epiderme et les cellules dendritiques du derme. Le but de cette etude etait de caracteriser les cellules impliquees dans la prise en charge de l’allergene et la dynamique de leur migration vers les ganglions drainant la peau lors de l’application epicutanee chez des souris sensibilisees a l’ovalbumine. Methodes Des souris sensibilisees a l’ovalbumine (OVA) par voie sous-cutanee ont recu un patch (Viaskin®) contenant de l’OVA couple a un fluorochrome (OVA-A488) sur peau intacte ou abrasee, et les cellules ayant capte l’allergene ont ete analysees par cytometrie en flux apres 2, 6, 24, 48 et 72 h dans la peau et les ganglions drainants et comparee a des souris naives (controle). Resultats Apres deux heures d’application, un passage passif important est detecte dans les ganglions drainants apres application sur peau abrasee contrairement a la peau intacte. Apres application sur peau intacte, l’OVA est prise en charge par les cellules de Langerhans dans l’epiderme plus rapidement chez les souris sensibilisees que chez les souris naives. Chez les souris sensibilisees, le nombre de cellules OVA+ dans l’epiderme diminue significativement apres 24 h et augmente transitoirement dans le derme entre 24 et 48 h, suggerant une migration des cellules de Langerhans vers les ganglions drainants. Apres 48 h, une augmentation significative des cellules dendritiques OVA+ est observee dans les ganglions drainants. Les cellules de Langerhans ayant migre dans les ganglions presentent une forte expression de CMH-II et une augmentation de CD86, PD-L1 et de PD-L2. L’abrasion de la peau induit une expression plus importante de CD86 et PD-L1 mais plus faible de PD-L2. Discussion Ces resultats montrent que l’application epicutanee d’allergene sur peau intacte chez des souris sensibilisees permet sa prise en charge par les cellules de Langerhans, leur maturation et leur migration rapide vers les ganglions drainants avec un profil d’activation tolerogene par rapport a l’application sur peau abrasee.

Introduction L’immunotherapie epicutanee sur peau intacte (EPIT) est une voie therapeutique des allergies alimentaires en cours de developpement. Dans les modeles animaux, la desensibilisation obtenue par l’EPIT est maintenue apres arret du traitement (tolerance) et previent de sensibilisations a d’autres allergenes. Le but de ce travail etait d’evaluer, dans un modele de souris sensibilisees, la cinetique des modifications epigenetiques etayant l’effet therapeutique de l’EPIT et l’induction de tolerance. Methodes Des souris BALB/c femelles sensibilisees a l’arachide ont ete traitees par EPIT ou immunotherapie orale (OIT) ou non traitees pendant 8 semaines. Les prelevements de rate et sang ont ete realises aux semaines 1, 2, 4 et 8 du traitement et 8 semaines apres arret du traitement. Un autre panel de groupes de souris a ete analyse apres une phase de sensibilisation a un autre allergene, l’ovalbumine. La methylation de l’ADN a ete analysee par pyrosequencage dans les cellules CD4+ purifiees de la rate et du sang. Resultats Dans les cellules CD4+ (rate et sang), une hypermethylation des ilots CpG associes au promoteur de Gata3 apparait des la 4e semaine d’EPIT, persiste jusqu’a la fin du traitement (p Conclusion L’EPIT comparee a l’OIT conduit a une signature epigenetique unique et stable permettant une regulation negative de l’expression des genes de la voie du Th2 et une activation des cellules T regulatrices. Ces mecanismes seraient impliques dans l’induction de tolerance par EPIT.

Introduction L’allergie a l’œuf est l’une des trois plus frequentes allergies chez l’enfant (Rance F., Clin exp all, 2005). Bien que 40 % des patients allergiques acquierent une tolerance naturelle a l’âge de 5 ans, l’âge de resolution se developpe de plus en plus tardivement et predispose a d’autres maladies atopiques. L’objectif de cette etude est d’evaluer l’efficacite de l’immunotherapie par voie cutanee (EPIT) chez des souris prealablement sensibilisees a l’œuf. Methodes Des souris BALB/c (n = 50) ont ete sensibilisees aux proteines d’œuf par deux injections intra-peritoneales de 10 μg de proteines d’œuf adsorbees avec 2 mg d’Alum a une semaine d’intervalle. Une partie des souris est ensuite traitee par EPIT a differentes doses : 10, 50, 100 ou 250 μg (n = 10/groupe). Des souris n’ont pas ete traitees (n = 10, Sham) et des souris controles n’ont pas ete sensibilisees ni traitees (n = 10, naives). La reponse humorale a ete analysee apres la sensibilisation et a la fin du traitement (anticorps specifiques IgE, IgG1 et IgG2a). A la fin de l’immunotherapie, les souris ont recu 6 gavages de 20 mg de proteines d’œuf puis un test de provocation par voie orale (TPO) a ete realise (gavage de 80 mg de proteines d’œuf). La temperature rectale a ete mesuree 45 minutes apres le challenge. La reponse cellulaire a ete analysee apres stimulation in vitro des splenocytes. Resultats L’immunotherapie par voie epicutanee induit une baisse significative des IgE specifiques a l’œuf ainsi qu’une production significative d’IgG2a pour tous les groupes traites par rapport aux souris non traitees (Sham). Le TPO entraine la survenue de diarrhees ainsi qu’une baisse de temperature chez les souris non traitees ainsi que chez les souris traitees par EPIT aux faibles doses (10 μg et 50 μg). En revanche, les souris traitees aux doses de 100 μg et de 250 μg ne presentent pas ou moins de signes cliniques lors du TPO. Les cytokines de type Th2 ont egalement ete reduites chez les souris traitees par EPIT aux fortes doses. Conclusion Cette etude demontre pour la premiere fois l’efficacite de l’immunotherapie par voie epicutanee contre l’anaphylaxie induite par l’œuf dans un modele de souris allergique.

Background Allergy to cow's milk increases the risk of sensitization to other foods in young children. Objectives We sought to evaluate the effect of early epicutaneous immunotherapy (EPIT) on further sensitization to peanut or house dust mite (HDM) in a murine model of sensitization to cow's milk. Methods BALB/c mice orally sensitized to milk were epicutaneously treated with a Viaskin patch (DBV Technologies) loaded with milk proteins for 8 weeks. Mice were then sensitized to peanut or HDM. After sensitization to peanut, mice were exposed to a peanut regimen known to induce eosinophilic esophageal inflammation. After sensitization to HDM, mice were challenged with aerosols to HDM, and airway hyperresponsiveness was evaluated by using plethysmography. Humoral response was also analyzed. The role of regulatory T (Treg) cells was evaluated by adoptively transferring Treg cells from milk EPIT–treated mice to naive mice before sensitization to peanut. Protection against anaphylaxis was also investigated. Methylation of the promoter region of transcription factors was analyzed by using PCR assays. Results In milk-sensitized mice specific EPIT prevented further sensitization to peanut or HDM. EPIT significantly modified the humoral response, reduced T H 2 cytokine levels, decreased eosinophilic esophageal infiltration, and suppressed airway hyperresponsiveness. The protective effect was sustained over 2 months. Moreover, the adoptive transfer of milk EPIT Treg cells completely prevented sensitization to peanut and peanut-induced anaphylaxis. Milk EPIT enhanced methylation of the GATA-3 promoter region. Conclusions Our results showed that EPIT influences the natural history of allergy and reduces the risk of further sensitization through a Treg cell–dependent mechanism.

Introduction Les cellules T regulatrices (Tregs) jouent un role primordial dans les mecanismes d’action de l’immunotherapie specifique. La capacite des Tregs a migrer vers les organes cibles est liee a l’expression de recepteurs specifiques. Le but de cette etude est la caracterisation de l’expression des recepteurs de migration vers la peau (CLA) et le tube digestif (CCR9) par les Tregs induit par l’immunotherapie epicutanee (EPIT) qui constitue une voie therapeutique nouvelle pour le traitement des allergies alimentaires. Methodes Des souris BALB/c femelles sensibilisees a l’arachide par voie orale ont ete traitees par EPIT ou non traitees (NT). Apres 8 semaines de traitement, les souris ont ete sacrifiees afin de prelever la rate, les ganglions mesenteriques (mLNs) et les ganglions inguinaux (iLNs) drainant la peau. Apres extractions des cellules, la proportion de Tregs et l’expression des recepteurs de migration specifique CLA + CCR9– (peau), CLA-CCR9+ (tube digestif) et CLA + CCR9+ (double tropisme) ont ete analyses par cytometrie en flux. Resultats Apres 8 semaines de traitement, la proportion de cellules Tregs CD4 + CD25 + Foxp3+ dans la rate et les iLNs est augmentee par EPIT par rapport au NT ( p p p p Discussion Les Tregs Foxp3+ jouent un role essentiel au cours du traitement de l’allergie alimentaire. Le tropisme a la fois digestif et cutane des Tregs induits par l’EPIT aussi bien dans les ganglions drainant la peau que le tube digestif, fait de cette methode un traitement de choix de l’allergie alimentaire.

Results: In spleen, expression of CCR4 increased on Tregs induced by all 3 therapies whereas the expression of CXCR3, CCR6 and CCR8 increased on EPIT-induced Tregs only. Skin homing receptor (CLA) increased on EPITbut not SLITor OIT-induced Tregs. Gut homing receptor CCR9 increased on EPITand OITbut not SLIT-induced Tregs. Interestingly, 81% of the CCR9+ Tregs induced by EPIT expressed CLA whereas only 43% did after OIT. In iLN, Tregs level increased only after EPIT with induction of CLA+CCR9-(22%) and CLA+CCR9+ (12%) Tregs. In mLN, EPIT and OIT significantly induced CCR9+ Tregs, 25% and 18% of them expressing also CLA in EPIT and OIT respectively.

Results: In splenocytes, a significant hypermethylation in GATA-3 CpG islands was induced by EPIT versus Sham, starting from the 4 of treatment (p

Methods: After sensitization to milk, mice were treated by EPIT or Sham, and 2 weeks later, submitted to a peanut-sensitization procedure. Mice were then intravenously challenged with peanut. In a second experiment, CD4+CD25+ T cells were isolated from spleen of milk-sensitized mice after EPIT or Sham, and transferred into naive mice. Recipient mice were submitted to peanut sensitization and intravenously challenged with peanut. In a third experiment, Tregs were obtained from Foxp3+-gfp mice sensitized to peanut and EPIT-treated, then adoptively transferred. Recipient mice were submitted or not to peanut sensitization before transfer, then all sensitized and IV challenged to ovalbumin. Outcome tools were rectal temperature, hypersensitivity reactions and blood mouse mast cell protease-1 (mMCP1).