We are pleased to introduce the collection Frontiers in Allergy – Highlights in Allergens and Allergen Immunotherapy. This collection will welcome and showcase a selection of high impact articles authored by leaders in the field. The work presented here highlights the broad diversity of research performed across the Allergens and Allergen Immunotherapy section, and aims to put a spotlight on the main areas of interest. This collection aims to further support Frontiers’ strong community by shining a spotlight on our authors' highly impactful research.

Medical University of Vienna

Michigan Medicine, University of Michigan

Reims University

University of Salzburg

School of Medicine, Ajou University

Broad range of topics shedding light on influencial studies in allergens and allergen immunotherapy

. The research topic of allergy and immunotherapy has been explored in various studies, with the following highlights: <ul> <li>Human IgE monoclonal antibodies have potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics.</li> <li>Pet lipocalins have a central hydrophobic ligand-binding cavity called a calyx, and research has provided insights into the determinants of selectivity and candidate ligands.</li> <li>Type 1 regulatory T (Tr1) cells have been identified as a mechanism of allergen-specific immunotherapy (AIT), which can suppress allergic symptoms.</li> <li>A database has been established to provide evidence-based causative peptides and proteins for Celiac disease risk assessment.</li> <li>A murine model of asthma has been established to study adjuvant effects of immune modulatory substances on immunotherapy.</li> <li>Liposomes can be used as an adjuvant to increase antigen uptake after intradermal injection.</li> </ul>

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Frontiers in Allergy

Food Allergy

Allergens

Allergen Immunotherapy

Highlights in Allergens and Allergen Immunotherapy

IntroductionAllergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity.</sec>Materials and methodsThe IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3–18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing β-hexosaminidase release from a humanized rat basophilic cell line.</sec>ResultsHuman IgE monoclonal antibodies (n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450–1,702,500 kU/L) with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of β-hexosaminidase (35%–80%) from a humanized rat basophilic cell line.</sec>ConclusionsHuman IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes.</sec>

Introduction: Allergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity.Materials and methods: The IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3–18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing β-hexosaminidase release from a humanized rat basophilic cell line.Results: Human IgE monoclonal antibodies (n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450–1,702,500 kU/L) with ratios of specific IgE to total IgE ranging from &lt;0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (&lt;1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of β-hexosaminidase (35%–80%) from a humanized rat basophilic cell line.Conclusions: Human IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes.

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease

IntroductionPet lipocalins are respiratory allergens with a central hydrophobic ligand-binding cavity called a calyx. Molecules carried in the calyx by allergens are suggested to influence allergenicity, but little is known about the native ligands.</sec>MethodsTo provide more information on prospective ligands, we report crystal structures, NMR, molecular dynamics, and florescence studies of a dog lipocalin allergen Can f 1 and its closely related (and cross-reactive) cat allergen Fel d 7.</sec>ResultsStructural comparisons with reported lipocalins revealed that Can f 1 and Fel d 7 calyxes are open and positively charged while other dog lipocalin allergens are closed and negatively charged. We screened fatty acids as surrogate ligands, and found that Can f 1 and Fel d 7 bind multiple ligands with preferences for palmitic acid (16:0) among saturated fatty acids and oleic acid (18:1 cis-9) among unsaturated ones. NMR analysis of methyl probes reveals that conformational changes occur upon binding of pinolenic acid inside the calyx. Molecular dynamics simulation shows that the carboxylic group of fatty acids shuttles between two positively charged amino acids inside the Can f 1 and Fel d 7 calyx. Consistent with simulations, the stoichiometry of oleic acid-binding is 2:1 (fatty acid: protein) for Can f 1 and Fel d 7.</sec>DiscussionThe results provide valuable insights into the determinants of selectivity and candidate ligands for pet lipocalin allergens Can f 1 and Fel d 7.</sec>

Introduction: Pet lipocalins are respiratory allergens with a central hydrophobic ligand-binding cavity called a calyx. Molecules carried in the calyx by allergens are suggested to influence allergenicity, but little is known about the native ligands.Methods: To provide more information on prospective ligands, we report crystal structures, NMR, molecular dynamics, and florescence studies of a dog lipocalin allergen Can f 1 and its closely related (and cross-reactive) cat allergen Fel d 7.Results: Structural comparisons with reported lipocalins revealed that Can f 1 and Fel d 7 calyxes are open and positively charged while other dog lipocalin allergens are closed and negatively charged. We screened fatty acids as surrogate ligands, and found that Can f 1 and Fel d 7 bind multiple ligands with preferences for palmitic acid (16:0) among saturated fatty acids and oleic acid (18:1 cis-9) among unsaturated ones. NMR analysis of methyl probes reveals that conformational changes occur upon binding of pinolenic acid inside the calyx. Molecular dynamics simulation shows that the carboxylic group of fatty acids shuttles between two positively charged amino acids inside the Can f 1 and Fel d 7 calyx. Consistent with simulations, the stoichiometry of oleic acid-binding is 2:1 (fatty acid: protein) for Can f 1 and Fel d 7.Discussion: The results provide valuable insights into the determinants of selectivity and candidate ligands for pet lipocalin allergens Can f 1 and Fel d 7.

Structural and ligand binding analysis of the pet allergens Can f 1 and Fel d 7

Allergen-specific immunotherapy (AIT) is the only causative treatment for allergic diseases by modification of the immune response to allergens. A key feature of AIT is to induce immunotolerance to allergens by generating antigen-specific regulatory T (Treg) cells in allergic patients. Type 1 regulatory T (Tr1) cells and forkhead box protein 3 (Foxp3)-expressing Treg cells are well known among Treg cell subsets. Foxp3 was identified as a master transcription factor of Treg cells, and its expression is necessary for their suppressive activity. In contrast to Foxp3+ Treg cells, the master transcription factor of Tr1 cells has not been elucidated. Nevertheless, Tr1 cells are generally considered as a distinct subset of Treg cells induced in the periphery during antigen exposure in tolerogenic conditions and can produce large amounts of anti-inflammatory cytokines such as interleukin-10 and transforming growth factor-β, followed by down-regulation of the function of effector immune cells independently of Foxp3 expression. Since the discovery of Tr1 cells more than 20 years ago, research on Tr1 cells has expanded our understanding of the mechanism of AIT. Although the direct precursors and true identity of these cells continues to be disputed, we and others have demonstrated that Tr1 cells are induced in the periphery by AIT, and the induced cells are re-activated by antigens, followed by suppression of allergic symptoms. In this review, we discuss the immune mechanisms for the induction of Tr1 cells by AIT and the immune-suppressive roles of Tr1 cells in AIT.

Allergen-specific immunotherapy (AIT) is the only causative treatment for allergic diseases by modification of the immune response to allergens. A key feature of AIT is to induce immunotolerance to allergens by generating antigen-specific regulatory T (Treg) cells in allergic patients. Type 1 regulatory T (Tr1) cells and forkhead box protein 3 (Foxp3)-expressing Treg cells are well known among Treg cell subsets. Foxp3 was identified as a master transcription factor of Treg cells, and its expression is necessary for their suppressive activity. In contrast to Foxp3+ Treg cells, the master transcription factor of Tr1 cells has not been elucidated. Nevertheless, Tr1 cells are generally considered as a distinct subset of Treg cells induced in the periphery during antigen exposure in tolerogenic conditions and can produce large amounts of anti-inflammatory cytokines such as interleukin-10 and transforming growth factor-β, followed by down-regulation of the function of effector immune cells independently of Foxp3 expression. Since the discovery of Tr1 cells more than 20 years ago, research on Tr1 cells has expanded our understanding of the mechanism of AIT. Although the direct precursors and true identity of these cells continues to be disputed, we and others have demonstrated that Tr1 cells are induced in the periphery by AIT, and the induced cells are re-activated by antigens, followed by suppression of allergic symptoms. In this review, we discuss the immune mechanisms for the induction of Tr1 cells by AIT and the immune-suppressive roles of Tr1 cells in AIT.

Roles of type 1 regulatory T (Tr1) cells in allergen-specific immunotherapy

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E-scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods.

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E-scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods.

Development of a Sequence Searchable Database of Celiac Disease-Associated Peptides and Proteins for Risk Assessment of Novel Food Proteins

Background: The use of ovalbumin as a model allergen in murine models of allergic asthma is controversially discussed since it is not an aeroallergen and sensitization can only be achieved by using strong Th2-inducing adjuvants. Therefore, in this study, a murine model of asthma has been established in which sensitization against the major grass pollen allergen Phl p5b was performed without using aluminum hydroxide (alum). We used this model for specific immunotherapy.Methods: Female, 5–6-week-old mice were sensitized by six subcutaneous (s.c.) injections of 20 μg Phl p5b followed by four provocations to induce allergic airway inflammation. For desensitization, 1 mg of Phl p5b was injected subcutaneously during allergen challenge for one to a maximum of four times. Three days after the last challenge, the allergic immune response was analyzed.Results: Sensitized and challenged animals showed a significant infiltration of eosinophils into the airways, and the production of interleukin-5 (IL-5) by in vitro re-stimulated splenocytes could be detected. Furthermore, hyper-responsiveness of the airways was verified by invasive measurement of airway resistance in methacholine-challenged animals. Desensitized animals showed a significant reduction of all parameters.Conclusion: In this study, a murine model of asthma has successfully been established by sensitization against the clinically relevant allergen Phl p5b without using alum. S.c. injection of allergen dose dependently led to desensitization of sensitized mice. We suggest that this model is useful to study adjuvant effects of immune modulatory substances on immunotherapy without the interference of alum.

Background: The use of ovalbumin as a model allergen in murine models of allergic asthma is controversially discussed since it is not an aeroallergen and sensitization can only be achieved by using strong Th2-inducing adjuvants. Therefore, in this study, a murine model of asthma has been established in which sensitization against the major grass pollen allergen Phl p5b was performed without using aluminum hydroxide (alum). We used this model for specific immunotherapy.Methods: Female, 5–6-week-old mice were sensitized by six subcutaneous (s.c.) injections of 20 μg Phl p5b followed by four provocations to induce allergic airway inflammation. For desensitization, 1 mg of Phl p5b was injected subcutaneously during allergen challenge for one to a maximum of four times. Three days after the last challenge, the allergic immune response was analyzed.Results: Sensitized and challenged animals showed a significant infiltration of eosinophils into the airways, and the production of interleukin-5 (IL-5) by in vitro re-stimulated splenocytes could be detected. Furthermore, hyper-responsiveness of the airways was verified by invasive measurement of airway resistance in methacholine-challenged animals. Desensitized animals showed a significant reduction of all parameters.Conclusion: In this study, a murine model of asthma has successfully been established by sensitization against the clinically relevant allergen Phl p5b without using alum. S.c. injection of allergen dose dependently led to desensitization of sensitized mice. We suggest that this model is useful to study adjuvant effects of immune modulatory substances on immunotherapy without the interference of alum.

Specific Immunotherapy in a Murine Model of Grass Pollen (Phl p5b)-Induced Airway Inflammation

The skin is an attractive alternative administration route for allergy vaccination, as the skin is rich in dendritic cells (DCs) and is easily accessible. In the skin multiple subsets of DCs with distinct roles reside at different depths. In this study antigen (=allergen for allergy) formulations were injected in ex vivo human skin in a depth-controlled manner by using a hollow microneedle injection system. Biopsies were harvested at the injection site, which were then cultured for 72 h. Subsequently, the crawled-out cells were collected from the medium and analyzed with flow cytometry. Intradermal administration of ovalbumin (OVA, model antigen) solution at various depths in the skin did not affect the migration and maturation of DCs. OVA was taken up efficiently by the DCs, and this was not affected by the injection depth. In contrast, Bet v 1, the major allergen in birch pollen allergy, was barely taken up by dermal DCs (dDCs). Antigens were more efficiently taken up by CD14+ dDCs than CD1a+ dDCs, which in turn were more efficient at taken up antigen than Langerhans cells. Subsequently, both OVA and Bet v 1 were formulated in cationic and anionic liposomes, which altered antigen uptake drastically following intradermal microinjection. While OVA uptake was reduced by formulation in liposomes, Bet v 1 uptake in dDCs was increased by encapsulation in both cationic and anionic liposomes. This highlights the potential use of liposomes as adjuvant in intradermal allergy vaccine delivery. In conclusion, we observed that antigen uptake after intradermal injection was not affected by injection depth, but varied between different antigens and formulation.

The skin is an attractive alternative administration route for allergy vaccination, as the skin is rich in dendritic cells (DCs) and is easily accessible. In the skin multiple subsets of DCs with distinct roles reside at different depths. In this study antigen (=allergen for allergy) formulations were injected in ex vivo human skin in a depth-controlled manner by using a hollow microneedle injection system. Biopsies were harvested at the injection site, which were then cultured for 72 h. Subsequently, the crawled-out cells were collected from the medium and analyzed with flow cytometry. Intradermal administration of ovalbumin (OVA, model antigen) solution at various depths in the skin did not affect the migration and maturation of DCs. OVA was taken up efficiently by the DCs, and this was not affected by the injection depth. In contrast, Bet v 1, the major allergen in birch pollen allergy, was barely taken up by dermal DCs (dDCs). Antigens were more efficiently taken up by CD14+ dDCs than CD1a+ dDCs, which in turn were more efficient at taken up antigen than Langerhans cells. Subsequently, both OVA and Bet v 1 were formulated in cationic and anionic liposomes, which altered antigen uptake drastically following intradermal microinjection. While OVA uptake was reduced by formulation in liposomes, Bet v 1 uptake in dDCs was increased by encapsulation in both cationic and anionic liposomes. This highlights the potential use of liposomes as adjuvant in intradermal allergy vaccine delivery. In conclusion, we observed that antigen uptake after intradermal injection was not affected by injection depth, but varied between different antigens and formulation.