Group 2 innate lymphoid cells are key in lipid transfer protein allergy pathogenesis.

Background: Immunopathology in food allergy is characterized by an uncontrolled type 2 immune response and specific-IgE production. Recent studies have determined that group 2 innate lymphoid cells (ILC2) participate in the food allergy pathogenic mechanism and their severity. Our objective was to investigate the role of ILC2 in peach-allergic patients due to non-specific lipid transfer protein (Pru p 3) sensitization. Methods: The immune response in peripheral blood mononuclear cells was characterized in lipid transfer protein-allergic patients and healthy controls. We have analyzed the Pru p 3 uptake on ILC2, the expression of costimulatory molecules, and their involvement on the T-cell proliferative response and cytokine production under different experimental conditions: cytokines involved in group 2 innate lymphoid cell activation (IL-33 and IL-25), Pru p 3 as main food allergen, and the combination of both components (IL-33/IL-25+Pru p 3) using cell sorting, EliSpot, flow cytometry, and confocal microscopy. Results: Our results show that Pru p 3 allergen is taken up by group 2 innate lymphoid cells, regulating their costimulatory molecule expression (CD83 and HLA-DR) depending on the presence of Pru p 3 and its combination with IL-33/IL-25. The Pru p 3-stimulated ILC2 induced specific GATA3+Th2 proliferation and cytokine (IL-4, IL-5, and IL-13) production in lipid transfer protein-allergic patients in a cell contact-dependent manner with no changes in Tbet+Th1- and FOXP3+Treg cell differentiation. Conclusions: The results indicate that in lipid transfer protein-allergic patients, the responsible allergen, Pru p 3, interacts with group 2 innate lymphoid cells, promoting a Th2 cell response. Our results might be of interest in vivo, as they show a role of group 2 innate lymphoid cells as antigen-presenting cells, contributing to the development of food allergy. Consequently, group 2 innate lymphoid cells may be considered as potential therapeutic targets.

Trimeric Bet v 1-specific nanobodies cause strong suppression of IgE binding.

Background: Around 20% of the population in Northern and Central Europe is affected by birch pollen allergy, with the major birch pollen allergen Bet v 1 as the main elicitor of allergic reactions. Together with its cross-reactive allergens from related trees and foods, Bet v 1 causes an impaired quality of life. Hence, new treatment strategies were elaborated, demonstrating the effectiveness of blocking IgG antibodies on Bet v 1-induced IgE-mediated reactions. A recent study provided evidence for the first time that Bet v 1-specific nanobodies reduce patients´ IgE binding to Bet v 1. In order to increase the potential to outcompete IgE recognition of Bet v 1 and to foster cross-reactivity and cross-protection, we developed Bet v 1-specific nanobody trimers and evaluated their capacity to suppress polyclonal IgE binding to corresponding allergens and allergen-induced basophil degranulation. Methods: Nanobody trimers were engineered by adding isoleucine zippers, thus enabling trimeric formation. Trimers were analyzed for their cross-reactivity, binding kinetics to Bet v 1, and related allergens, and patients' IgE inhibition potential. Finally, their efficacy to prevent basophil degranulation was investigated. Results: Trimers showed enhanced recognition of cross-reactive allergens and increased efficiency to reduce IgE-allergen binding compared to nanobody monomers. Furthermore, trimers displayed slow dissociation rates from allergens and suppressed allergen-induced mediator release. Conclusion: We generated high-affine nanobody trimers that target Bet v 1 and related allergens. Trimers blocked IgE-allergen interaction by competing with IgE for allergen binding. They inhibited IgE-mediated release of biological mediators, demonstrating a promising potential to prevent allergic reactions caused by Bet v 1 and relatives.

Insect Cell-Expressed Major Ragweed Allergen Amb a 1.01 Exhibits Similar Allergenic Properties to Its Natural Counterpart from Common Ragweed Pollen.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Structural and Immunological Features of PR-10 Allergens: Focusing on the Major Alder Pollen Allergen Aln g 1.

Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can significantly increase their allergenicity potential. It has been recently shown that not only the birch pollen allergen Bet v 1 but also the alder pollen allergen Aln g 1, might act as a true sensitizer of the immune system. The current investigation is aimed at the further study of the allergenic and structural features of Aln g 1. By using qPCR, we showed that Aln g 1 was able to upregulate alarmins in epithelial cells, playing an important role in sensitization. With the use of CD-spectroscopy and ELISA assays with the sera of allergic patients, we demonstrated that Aln g 1 did not completely restore its structure after thermal denaturation, which led to a decrease in its IgE-binding capacity. Using site-directed mutagenesis, we revealed that the replacement of two residues (Asp27 and Leu30) in the structure of Aln g 1 led to a decrease in its ability to bind to both IgE from sera of allergic patients and lipid ligands. The obtained data open a prospect for the development of hypoallergenic variants of the major alder allergen Aln g 1 for allergen-specific immunotherapy.

Effects of Chlorogenic Acid on Lowering IgE-Binding Capacity of Soybean 7S: Comparison between Covalent and Noncovalent Interaction.

Allergenicity of soybean 7S protein (7S) troubles many people around the world. However, many processing methods for lowering allergenicity is invalid. Interaction of 7S with phenolic acids, such as chlorogenic acid (CHA), to structurally modify 7S may lower the allergenicity. Hence, the effects of covalent (C-I, periodate oxidation method) and noncovalent interactions (NC-I) of 7S with CHA in different concentrations (0.3, 0.5, and 1.0 mM) on lowering 7S allergenicity were investigated in this study. The results demonstrated that C-I led to higher binding efficiency (C-0.3:28.51 ± 2.13%) than NC-I (N-0.3:22.66 ± 1.75%). The C-I decreased the α-helix content (C-1:21.06%), while the NC-I increased the random coil content (N-1:24.39%). The covalent 7S-CHA complexes of different concentrations had lower IgE binding capacity (C-0.3:37.38 ± 0.61; C-0.5:34.89 ± 0.80; C-1:35.69 ± 0.61%) compared with that of natural 7S (100%), while the noncovalent 7S-CHA complexes showed concentration-dependent inhibition of IgE binding capacity (N-0.3:57.89 ± 1.23; N-0.5:46.91 ± 1.57; N-1:40.79 ± 0.22%). Both interactions produced binding to known linear epitopes. This study provides the theoretical basis for the CHA application in soybean products to lower soybean allergenicity.

Localization of G1A1a Allergenic Domain Destroyed by Thermal Processing.

Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I (151SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG181) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 (157DQMPRRFYLANGNQE170) showed allergenicity, and after replacing it with alanine, it was found that amino acids D157, Q158, M159, and Y164 were the key amino acids that affected its antigenicity, while Q158, M159, R162, and N168 affected allergenicity.

Validation of the NUT CRACKER Diagnostic Algorithm and Prediction for Cashew and Pistachio Co-Allergy.

BACKGROUND: Because of the high cross-sensitization among tree nuts, the NUT CRACKER (Nut Co-reactivity-Acquiring Knowledge for Elimination Recommendations) study proposed a diagnostic algorithm to minimize the number of required oral food challenges (OFCs). OBJECTIVE: To validate the algorithm for cashew and pistachio allergy and determine markers for allergic severity. METHODS: Patients (n = 125) with a median age of 7.8 (interquartile range, 5.9-11.2) years with suspected tree nut allergy were evaluated prospectively with decision tree points on the basis of skin prick test (SPT), basophil activation test (BAT), and knowledge of the coincidence of allergies. Validation of allergic status was determined by OFC. Markers of clinical severity were evaluated using the combined original and prospective cohort (n = 187) in relationship to SPT, BAT, and Ana o 3-sIgE. RESULTS: Reactivity to cashew in SPT, BAT, and Ana o 3-sIgE and the incidence of abdominal pain on challenge were significantly higher in dual-allergic cashew/pistachio patients (n = 82) versus single cashew allergic patients (n = 18) (P = .001). All 3 diagnostic tests showed significant inverse correlation with log10 reaction doses for positive cashew OFC. The algorithm reduced overall the total number of OFCs by 72.0%, with a positive predictive value and negative predictive value of 93.0% and 99.0%, respectively. Cashew false-positives were observed primarily in hazelnut-allergic patients (P = .026). In this population, Ana o 3-specific IgE could diagnose cashew allergy with a sensitivity of more than 90% and a specificity of more than 95%. CONCLUSIONS: The NUT CRACKER diagnostic algorithm was validated and reduced the number of diagnostic OFCs required. Markers for severity phenotypes may guide oral immunotherapy protocols, improving the risk/benefit ratio for patients.

Serologic measurements for peanut allergy: Predicting clinical severity is complex.

Allergist-immunologists use serologic peanut allergy testing to maximize test sensitivity and specificity while minimizing cost and inconvenience. Recent advances toward this goal include a better understanding of specific IgE (sIgE) and component testing, epitope-sIgE assays, and basophil activation testing. Predicting reaction severity with serologic testing is challenged by a range of co-factors that influence reaction severity, such as the amount and form of any allergen consumed and comorbid disease. In 2020, the Allergy Immunology Joint Task Force on Practice Parameters recommended Ara h 2-sIgE as the most cost-effective diagnostic test for peanut allergy because of its superior performance, when compared with skin prick testing and serum IgE. Basophil activation testing, a functional test of allergic response not evaluated in the Joint Task Force on Practice Parameters guideline, is a promising option for both allergy diagnosis and prognosis. Similarly, epitope-sIgE testing may improve prediction of reaction thresholds, but further validation is needed. Despite advances in food allergy testing, many of these tools remain limited by cost, accessibility, and feasibility. In addition, there is a need for further research on how atopic dermatitis may be modifying serologic food allergy severity assessments. Given these limitations, allergy test selection requires a shared decision-making approach so that a patient's values and preferences regarding financial impact, inconvenience, and psychological effects are considered in the context of clinician expertise on the timing and use of optimized testing.

NLRP3 priming due to skin damage precedes LTP allergic sensitization in a mouse model.

Allergic sensitization is initiated by protein and epithelia interaction, although the molecular mechanisms leading this encounter toward an allergic phenotype remain unknown. Here, we apply the two-hit hypothesis of inflammatory diseases to the study of food allergy sensitization. First, we studied the effects of long-term depilation in mice by analyzing samples at different time points. Several weeks of depilation were needed until clear immunological changes were evidenced, starting with upregulation of NLRP3 protein levels, which was followed by overexpression of Il1b and Il18 transcripts. Secondly, we assessed the effects of allergen addition (in this case, Pru p 3 in complex with its natural lipid ligand) over depilated skin. Systemic sensitization was evaluated by intraperitoneal provocation with Pru p 3 and measure of body temperature. Anaphylaxis was achieved, but only in mice sensitized with Prup3_complex and not treated with the NLRP3 inhibitor MCC950, thus demonstrating the importance of both hits (depilation + allergen addition) in the consecution of the allergic phenotype. In addition, allergen encounter (but not depilation) promoted skin remodeling, as well as CD45+ infiltration not only in the sensitized area (the skin), but across several mucosal tissues (skin, lungs, and gut), furtherly validating the systemization of the response. Finally, a low-scale study with human ILC2s is reported, where we demonstrate that Prup3_complex can induce their phenotype switch (↑CD86, ↑S1P1) when cultured in vitro, although more data is needed to understand the implications of these changes in food allergy development.

Structure, Immunogenicity, and IgE Cross-Reactivity among Walnut and Peanut Vicilin-Buried Peptides.

Vicilin-buried peptides (VBPs) from edible plants are derived from the N-terminal leader sequences (LSs) of seed storage proteins. VBPs are defined by a common α-hairpin fold mediated by conserved CxxxCx(10-14)CxxxC motifs. Here, peanut and walnut VBPs were characterized as potential mediators of both peanut/walnut allergenicity and cross-reactivity despite their low (∼17%) sequence identity. The structures of one peanut (AH1.1) and 3 walnut (JR2.1, JR2.2, JR2.3) VBPs were solved using solution NMR, revealing similar α-hairpin structures stabilized by disulfide bonds with high levels of surface similarity. Peptide microarrays identified several peptide sequences primarily on AH1.1 and JR2.1, which were recognized by peanut-, walnut-, and dual-allergic patient IgE, establishing these peanut and walnut VBPs as potential mediators of allergenicity and cross-reactivity. JR2.2 and JR2.3 displayed extreme resilience against endosomal digestion, potentially hindering epitope generation and likely contributing to their reduced allergic potential.

HLA alleles and sustained peanut consumption promote IgG4 responses in subjects protected from peanut allergy.

We investigated the interplay between genetics and oral peanut protein exposure in the determination of the immunological response to peanut using the targeted intervention in the LEAP clinical trial. We identified an association between peanut-specific IgG4 and HLA-DQA1*01:02 that was only observed in the presence of sustained oral peanut protein exposure. The association between IgG4 and HLA-DQA1*01:02 was driven by IgG4 specific for the Ara h 2 component. Once peanut consumption ceased, the association between IgG4-specific Ara h 2 and HLA-DQA1*01:02 was attenuated. The association was validated by observing expanded IgG4-specific epitopes in people who carried HLA-DQA1*01:02. Notably, we confirmed the previously reported associations with HLA-DQA1*01:02 and peanut allergy risk in the absence of oral peanut protein exposure. Interaction between HLA and presence or absence of exposure to peanut in an allergen- and epitope-specific manner implicates a mechanism of antigen recognition that is fundamental to driving immune responses related to allergy risk or protection.

Aptamer based point of care diagnostic for the detection of food allergens.

Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With many food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families.

Pattern of sensitization to Juniperus oxycedrus 4EF-hand polcalcin, Jun o 4, compared with the 2EF-hand grass homolog Phl p 7 in a general Italian population of subjects suffering from pollinosis.

Cupressaceae pollen causes allergic reactions worldwide with long-lasting symptomatic periods. Currently, no cypress polcalcin is available for diagnostic purposes. With the aim to investigate the pattern of sensitization to a cypress polcalcin, a synthetic gene of Jun o 4, the Juniperus oxycedrus 4EF-hand polcalcin, was cloned and expressed in Escherichia coli. Its features were investigated in comparison with the grass 2EF-hand Phl p 7. Rhinitis was the symptom most frequently reported in a cohort of Italian patients sensitized to rJun o 4 and/or rPhl p 7. The detection of many pollen allergic patients sensitized to the cypress polcalcin, but negative to Phl p 7, indicates that Phl p 7 cannot be further considered a marker of sensitization towards all the polcalcins. A 4EF-hand cypress polcalcin claims the inclusion in allergy diagnostic tests. In addition, the sensitivity of polcalcins to gastrointestinal digestion is reported and discussed for the first time.

Detection of Wheat Lipid Transfer Protein (Tri a 14) Sensitization: Comparison between Currently Available Diagnostic Tools.

BACKGROUND: Wheat lipid transfer protein (LTP; Tri a 14) and ω5-gliadin have been described as major allergens in wheat allergy (WA) and relevant in wheat-induced anaphylaxis, frequently associated with cofactors. OBJECTIVE: The objective of this study was to compare tools currently available in routine diagnosis to detect Tri a 14 sensitization, its clinical relevance, and cosensitization to ω5-gliadin and other LTPs. METHODS: One hundred eighteen adults sensitized to rTri a 14 by ImmunoCAP® (cutoff ≥0.1 kUA/L) identified among 210 LTP allergic patients were included. We evaluated (1) wheat skin prick test (SPT), (2) specific IgE (sIgE) to wheat, rTri a 14, rTri a 19, peach, apple, walnut, hazelnut, and peanut LTPs using ImmunoCAP® and microarray ImmunoCAP®ISAC (cutoff ≥0.3I SU), and (3) wheat-related symptoms. RESULTS: Wheat SPT and sIgE were positive in 31% and 85% of subjects, respectively. rTri a 14 by microarray was detected in 25%. Eight percent showed cosensitization to ω5-gliadin. Thirty percent referred symptoms (gastrointestinal [13%], urticaria [11%], and anaphylaxis [8%]). Cofactors (45%) were significantly associated with systemic reactions. CONCLUSION: WA due to Tri a 14 is frequently related with systemic reactions and because are frequently related to cofactors, the culprit may not be suspected. Together with the poor performance to identify Tri a 14 sensitization of the current routine diagnostic tools based on the analysis of whole wheat extract, such as wheat SPT or sIgE, there is a high risk that WA may be overlooked. Thus, when WA is suspected, sIgE Tri a 14 assessment is recommended, together with wheat and ω5-gliadin, preferably in the singleplex format, and carefully evaluated considering ≥0.1 kUA/L as a cutoff.

Targeting immunodominant Bet v 1 epitopes with monoclonal antibodies prevents the birch allergic response.

BACKGROUND: Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response. OBJECTIVES: This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response. METHODS: Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model. RESULTS: REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- and birch pollen extract-induced basophil activation ex vivo and mast cell degranulation in vivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1. CONCLUSIONS: These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.

Novel antibody cocktail targeting Bet v 1 rapidly and sustainably treats birch allergy symptoms in a phase 1 study.

BACKGROUND: The efficacy of an allergen-specific IgG cocktail to treat cat allergy suggests that allergen-specific IgG may be a major protective mechanism elicited by allergen immunotherapy. OBJECTIVES: Extending these findings, we tested a Bet v 1-specific antibody cocktail in birch-allergic subjects. METHODS: This was a phase 1, randomized, double-blind, study with 2 parts. Part A administered ascending doses of the Bet v 1-specific antibody cocktail REGN5713/14/15 (150-900 mg) in 32 healthy adults. Part B administered a single subcutaneous 900-mg dose or placebo in 64 birch-allergic subjects. Total nasal symptom score response to titrated birch extract nasal allergen challenge and skin prick test (SPT) with birch and alder allergen were assessed at screening and days 8, 29, 57, and 113 (SPT only); basophil activation tests (n = 26) were conducted. RESULTS: Single-dose REGN5713/14/15 significantly reduced total nasal symptom score following birch nasal allergen challenge relative to baseline. Differences in total nasal symptom score areas under the curve (0-1 hour) for subjects treated with REGN5713/14/15 versus those given placebo (day 8: -1.17, P = .001; day 29: -1.18, P = .001; day 57: -0.85, P = .024) and titration SPT with birch difference in area under the curve of mean wheal diameters for subjects treated with REGN5713/14/15 versus placebo (all P < .001) were sustained for ≥2 months; similar results were observed with alder SPT. REGN5713/14/15 was well tolerated. Basophil responsiveness to birch-related allergens was significantly decreased in subjects treated with REGN5713/14/15 versus those given placebo on days 8, 57, and 113 (all P < .01). CONCLUSIONS: Single-dose REGN5713/14/15 was well tolerated and provided a rapid (1 week) and durable (2 months) reduction in allergic symptoms after birch allergen nasal allergen challenge, potentially offering a new paradigm for the treatment of birch allergy symptoms.

Preventive Administration of Non-Allergenic Bet v 1 Peptides Reduces Allergic Sensitization to Major Birch Pollen Allergen, Bet v 1.

IgE-mediated allergy to birch pollen affects more than 100 million patients world-wide. Bet v 1, a 17 kDa protein is the major allergen in birch pollen responsible for allergic rhinoconjunctivitis and asthma in birch pollen allergic patients. Allergen-specific immunotherapy (AIT) based on therapeutic administration of Bet v 1-containing vaccines is an effective treatment for birch pollen allergy but no allergen-specific forms of prevention are available. We developed a mouse model for IgE sensitization to Bet v 1 based on subcutaneous injection of aluminum-hydroxide adsorbed recombinant Bet v 1 and performed a detailed characterization of the specificities of the IgE, IgG and CD4+ T cell responses in sensitized mice using seven synthetic peptides of 31-42 amino acids length which comprised the Bet v 1 sequence and the epitopes recognized by human CD4+ T cells. We then demonstrate that preventive systemic administration of a mix of synthetic non-allergenic Bet v 1 peptides to 3-4 week old mice significantly reduced allergic immune responses, including IgE, IgG, IgE-mediated basophil activation, CD4+ T cell and IL-4 responses to the complete Bet v 1 allergen but not to the unrelated major grass pollen allergen Phl p 5, without inducing Bet v 1-specific allergic sensitization or adaptive immunity. Our results thus demonstrate that early preventive administration of non-allergenic synthetic T cell epitope-containing allergen peptides could be a safe strategy for the prevention of allergen-specific IgE sensitization.