Molecular analysis of interactions between IgE antibody and allergen allows the structural basis of IgE recognition to be defined. Human IgE (hIgE) epitopes of respiratory lipocalin allergens, including Can f 1, remain elusive due to a lack of IgE-allergen complexes. This study aims to map the structure of allergenic epitopes on Can f 1. The fragment antigen-binding (Fab) regions of Can f 1 specific human IgE monoclonal antibodies (hIgE mAb) were used to determine the structures of IgE epitopes. Epitope mutants were designed to target Can f 1 epitopes. Immunoassays and a human FcεRIα transgenic mouse model of passive anaphylaxis in vivo were used to assess the functional activity of epitope mutants. Crystal structures of natural or recombinant Can f 1 complexed with two hIgE mAb 1J11 and 12F3 Fabs, respectively, were determined. The hIgE mAb bound to two partially overlapping epitopes and recognized two different Can f 1 conformations. The hIgE mAb 12F3 showed an unusual mode of binding by protruding its heavy chain CDR3 inside the Can f 1 calyx. Epitope mutants generated based on the structural analyses displayed a 64%-89% reduction in IgE antibody binding and failed to induce passive anaphylaxis in a human FcεRIα transgenic mouse model. In summary, the structures of Can f 1-hIgE Fab complexes revealed two unique and partially overlapping epitopes on Can f 1. The modification of the identified IgE epitopes provides a pathway for the design of hypoallergens to treat dog allergies.

Identification of IgE epitopes on major dog allergen Can f 1 provides a rationale for the epitope-based design of hypoallergens that may be used for immunotherapy. This process is facilitated by using human IgE monoclonal antibodies (hIgE mAb) from dog-allergic individuals.

Allergen measurements are widely used for validation of molecular allergy diagnostics and allergy therapeutics. However, few standardized food allergen reference materials have been developed. While NIST and MoniQA food standards are characterized extensively for biochemical and nutritional composition, data on allergen content are lacking. The aim was to produce standardized food-flour-proteins with defined allergen content that could serve as reference materials for allergy diagnostics or therapeutics.

Background: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire. Objective: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2. Methods: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (Fc epsilon RI alpha)-transgenic mouse model of passive systemic anaphylaxis. Results: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 angstrom. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human Fc epsilon RI alpha-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link Fc epsilon RI epsilon RI to induce anaphylaxis when challenged with the epitope mutants. C onclusions: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics.

Successful allergen-specific immunotherapy (AIT) induces T-cell responses that suppress Th2 inflammation. However, quantifying T-cell modulatory effects of candidate AIT molecules is challenging using standard in vitro assays, owing to the rarity of allergen-specific T-cells. We describe a high-dimensional single-cell method for quantifying T-cell skewing induced by a variant of Der p 2 (2F10) containing 3 amino acid mutations within a major IgE epitope. PBMCs from allergic and non-allergic subjects were cultured for 7 days with 2F10 (<0.01 EU/μg) or wild type (WT) Der p 2. Molecular signatures of dust-mite reactive (CellTrace™ Violet-low) T-cells were determined using a 20-marker spectral flow cytometry panel (Cytek® Northern Lights™) and marker enrichment modeling. Differences in numbers of rare T-cell subsets induced by 2F10 and WT Der p 2 were analyzed using the algorithm Tracking Responders Expanding (T-REX). The Der p 2 variant 2F10 induced enhanced proliferation as compared with WT, confirming that CD4+ T-cell epitopes were intact (frequency of memory CD4+ T-cells: 4%±3 versus 1%±0.8, n=7). T-REX identified 2 "Th1-like" subsets that increased in frequency by ≥85% in response to 2F10 versus WT allergen, regardless of allergic status (frequency of memory CD4+ T-cells: 1%±0.80 versus 0.06%±0.05, n=7). These included multi-cytokine producing cells (IFN-γ+6TNF-α+6CD25+2CTV+1 and IL-17A+7.5IFN-γ+7TNF-α+6CD25+2CTV+1). Notably absent were Th2 signatures, which were nonetheless induced by house dust mite extract in cultures from allergic subjects only (IL-4+5IL-5+5CD25+3CTV+1). Our single-cell method highlights rare, atypical, and complex T-cell types induced by Der p 2 and the potential for a novel allergen variant to boost their numbers.

Abstract Immunoglobulin E (IgE) antibody is a critical effector molecule for adaptive allergen-induced immune responses, which affect up to 40% of the population worldwide. Allergens are usually innocuous molecules but induce IgE antibody production in allergic subjects. Allergen cross-linking of IgE bound to its high affinity receptor (FcεRI) on mast cells and basophils triggers release of histamine and other mediators that cause allergic symptoms. Little is known about the direct allergen–IgE antibody interaction due to the polyclonal nature of serum IgE and the low frequency of IgE-producing B cells in blood. Here, we report the X-ray crystal structure of a house dust mite allergen, Der p 2, in complex with Fab of a human IgE monoclonal antibody (mAb) isolated by hybridoma technology using human B cells from an allergic subject. This IgE mAb, 2F10, has the correct pairing of heavy and light chains as it occurs in vivo. Key amino acids forming the IgE epitope on Der p 2 were identified. Mutation of these residues ablated their functional ability to cross-link IgE in a mouse model of passive systemic anaphylaxis. These analyses revealed an important conformational epitope associated with the IgE antibody repertoire to a major mite allergen.

Our goal was to analyze the structure of a human IgE mAb recognizing the mite allergen Der-p-2, given the current lack of allergen-IgE antibody PDB-structures.

Recent statistical evidence suggests that allergens are more stable and more highly expressed than other proteins from an allergen source. These factors may influence human exposure and, on a smaller scale, stability may affect processing by sentinel dendritic cells skewing the immune response toward allergy. Interestingly, for cockroach allergens purified from frass, the allergen stabilities were not statistically greater than their non-allergen counterparts, likely due to the high mean stability of the latter. To investigate if this was a statistical sampling anomaly, the stability of additional recombinant cockroach allergens was studied. The stability of 6 recombinant cockroach allergens was measured as a function of guanidinium chloride concentration using HNSB and SPROX labeling. Additionally, distant relatives of Bla g 1 (MA proteins) were compared for their thermal stability using temperature dependent circular dichroism as a function of lipid ligand concentration. Bla g 1, 4, 6, and 9 showed elevated stabilities compared to the mean of non-allergen cockroach proteins. However, the differences between allergens and non-allergens was not statistically significant. Additional MA homologues of Bla g 1 demonstrated similar melting temperatures to the latter with no ligand present. However, the stability enhancement due to lipid loading was not present or not as great for other MA proteins compared to Bla g 1 loaded with lipids. Cockroach allergens are highly stable proteins compared to proteins from other allergen sources. However, compared to non-allergens from cockroaches there is not a significant difference, even with data from additional recombinant allergens.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

This study addresses the significant variability in allergen content among different sources of cockroach extracts, which poses a challenge in terms of extract standardization for clinical use, and compares their potency for IgE reactivity in individual patients. Twelve German cockroach extracts were compared for content and potency of IgE reactivity. Levels of Bla g 1, Bla g 2, Bla g 5 and flagellin were measured by immunoassays. Endotoxin was measured by Limulus-Amebocyte-Lysate-test. IgE antibody levels to 7 purified recombinant allergens from groups 1, 2, 4, 5, 7, 9 and 11 were measured by ImmunoCAP. IgE antibody binding inhibition assays were performed to assess extract potency (IC30) in five cockroach allergic patients. Endotoxin and allergen levels were highly variable (endotoxin: 45,118±98,472 EU/ml, Bla g 1: 39±47 μg/ml, Bla g 2: 17±20 μg/ml, and Bla g 5: 75±93 ng/ml). In addition to Bla g 2 and Bla g 5, two new major allergens (groups 9 and 11), were identified, with 50-63% IgE prevalences (n=16). Commercial extracts (5/9) showed the best potencies (IC30: 0.4-2.9 μg/ml). Within two groups of patients identified, good correlations of the IC30s between patient-pairs were observed (r >0.9), and the allergen-specific IgE levels correlated better than between two patients from different groups. The potency of German cockroach extracts for IgE reactivity depends on allergen content and allergen-specific IgE titers of the cockroach allergic patient. These factors are relevant for the selection of potent extracts to be used for immunotherapy and the design and interpretation of data from immunotherapy trials.

Background: Cockroach allergens are an important cause of IgE-mediated sensitization in inner-city asthmatic patients. However, cockroach extracts used for diagnosis and immunotherapy are not standardized. Objective: We sought to determine the allergen content of nonstandardized German cockroach extracts and the levels of sensitization to an expanded set of cockroach allergens as determinants of in vitro extract potency for IgE reactivity. Methods: Twelve German cockroach extracts were compared for allergen content and potency of IgE reactivity. Bla g 1, Bla g 2, and Bla g 5 were measured by using immunoassays. IgE antibody levels to 8 purified recombinant allergens from groups 1, 2, 4, 5, 6, 7, 9, and 11 were measured by using ImmunoCAP. IgE antibody binding inhibition assays were performed to assess extract in vitro potencies (concentration inhibiting 30% of the total IgE antibody-binding inhibition) relative to an arbitrarily selected reference extract in 5 patients with cockroach allergy. Results: Allergen levels were highly variable. Three new major allergens (groups 6, 9, and 11), were identified among highly cockroach-sensitized subjects (CAP class > 3). Sensitization profiles were unique per subject without immunodominant allergens. The sum of IgE to 8 allergen components showed a good correlation with cockroach-specific IgE levels (r = 0.88, P<.001). In vitro potencies varied among different extracts per subject and among subjects for each extract. Conclusions: The in vitro potency of German cockroach extracts for IgE reactivity depends on allergen content and allergen-specific IgE titers of patients with cockroach allergy. These factors are relevant for selection of potent extracts to be used for immunotherapy and for the design and interpretation of data from immunotherapy trials.

Der p 1 and Der f 1 are major allergens from Dermatophagoides pteronyssinus and D. farinae, respectively. An analysis of antigenic determinants on both allergens was performed by site-directed mutagenesis. The analysis was based on the x-ray crystal structures of the allergens in complex with Fab fragments of three murine mAbs that interfere with IgE Ab binding: the two Der p 1–specific mAbs 5H8 and 10B9, and the cross-reactive mAb 4C1. On one hand, selected residues in the epitopes for mAb 5H8 and mAb 4C1 were substituted with amino acids that resulted in impaired Ab binding to Der p 1. On the other hand, an epitope for the Der p 1–specific mAb 10B9, which partially overlaps with mAb 4C1, was created in Der f 1. The mutation of 1–3 aa residues in Der f 1 was sufficient to bind mAb 10B9. These residues form hydrogen bonds with CDRs of the Ab other than H CDR3. This observation unveils an exception to the dominant role of H CDR3 commonly observed in Ag recognition. Overall, this study resulted in the identification of important residues for mAb and IgE Ab recognition in group 1 mite allergens. This information can be used to engineer allergen mutants with reduced IgE Ab binding for immunotherapy.

Highly purified, well-characterized allergens can be used for molecular allergy diagnostics and as reference materials. Reference materials are important for standardization of allergen extracts, determination of potency of allergy vaccines and validation of molecular diagnostics. The objective of this study was to analyze purity and isoallergen distribution in affinity-purified dust mite allergen Der p 1. Isoform distribution of Der p 1 in mite culture, during bioprocessing and in preparations of purified Der p 1 was compared by LC-MS/MS. Data from digests were analyzed against individual Der p 1 isoform sequences. Der p 1 was lyophilized using different buffer conditions. Real time stability data were collected from frozen liquid allergens and lyophilized allergens. Patterns of Der p 1 isoforms were identical in mite culture and purified allergens. Based on diagnostic peptides, five isoforms of Der p 1 (0101, 0102, 0106, 0108, and 0124) were identified and 17 Der p 1 isoforms could be excluded. Purified Der p 1 was free of contaminants. Real time stability tests of frozen liquid allergens and of frozen lyophilized allergens showed comparable potency in allergen-specific ELISA and no signs of degradation on SDS-PAGE. Mass Spectrometry is a valuable tool to assess purity and isoform composition of purified allergens. Affinity-purification of natural Der p 1 does not affect the original isoform distribution found in mite culture. Bioprocessing pathways have been established to yield high purity mite allergens with homogenous isoform profiles. Purified natural Der p 1 can be used as molecular reference material for allergen standardization.