Anaphylaxis is an acute, potentially life-threatening reaction, often triggered by foods and largely mediated by immunoglobulin (Ig)E. The human microbiota is known to influence oral tolerance, but the microbial mechanisms directly involved in IgE-mediated anaphylaxis remain unknown. Here, we demonstrate that human saliva and jejunum harbor peanut (PN)-degrading bacteria that metabolize immunodominant allergens (Ara h 1 and 2), reducing IgE binding and anaphylaxis. Isolated Rothia and Staphylococcus species degraded PN allergens in vitro, generating proteins with reduced IgE binding and limited mast cell activation. Mice colonized with Rothia showed reduced local and systemic Ara h 1 and 2 levels and dampened anaphylaxis upon PN challenge. In clinical studies, common PN-degrading bacteria, including Rothia, are more abundant in PN-allergic patients who exhibit better tolerance to allergen exposure. Altogether, these results demonstrate that human microbiota modulates IgE-mediated reactions to foods through allergen metabolism, highlighting potential avenues to prevent or reduce the severity of IgE-mediated anaphylaxis.

The double-blind placebo-controlled oral food challenge (DBPCFC) has been the "gold standard" for diagnosing IgE-mediated food allergy for the past 50 years. Despite tremendous strides in our understanding of basic mechanisms and clinical features of food allergy, we remain dependent on refinements of old technologies, i.e., skin tests and serum IgE measurements, to complement clinical history in making an accurate diagnosis of food allergies. However, recent technical advances in two effector cell assays, i.e., the basophil and mast cell activation tests, may soon bring these diagnostic biomarkers to the clinic. In addition, new advances in antibody assays, T-cell assays, transcriptomics, and metabolomics in conjunction with machine learning and artificial intelligence may soon enable us to diagnose food allergy and accurately monitor immunotherapeutic outcomes without the need for an oral food challenge.

Durable tolerance, or the ability to safely tolerate allergen ingestion through preventative intervention or modulation of an existing immunoglobulin E (IgE)-mediated food allergy, is vital to the treatment of food allergy. Recent insights through clinical trials and fundamental research have discovered that interventions at younger ages underlie higher frequency and durability of tolerance after treatment. Here, we review highly successful preventive and clinical trials of oral, sublingual, and epicutaneous immunotherapy for food allergy and the evidence that intervention at a younger age is highly successful at the induction of durable tolerance. In parallel, our ability to interrogate the mechanisms of IgE-mediated food allergy has become increasingly sophisticated. Work on IgE diversity has repeatedly established that greater IgE diversity and immune progression with time are associated with poorer clinical outcomes. Durable tolerance, on the other hand, is associated with neutralizing antibodies that block multiple IgE clones, thereby providing protection from a diversified IgE repertoire. Taken together, these advances not only pave an evidence-based pathway for the treatment of IgE-mediated food allergies but also help us understand the mechanism of these interventions, allowing us for the first time to begin to address the wide range of heterogeneity underlying IgE-mediated food allergy to advance therapeutic implementation.

As an ex-vivo test of allergic effector cell activation, basophil activation testing (BAT) to allergen enables quantification of the in-vivo IgE-mediated allergic response. BAT thus holds promise in the diagnosis and monitoring of peanut and tree nut allergies. Recent systematic analyses and expert recommendations support a role for BAT in the diagnosis of peanut and tree nut allergy. Diagnostic cut-offs for BAT in peanut and tree nut allergy have been identified. Consistently, BAT can discriminate with high sensitivity and specificity between allergy and tolerance when measured against oral food challenges. Furthermore, the utilization of BAT has can increase the sensitivity and specificity of peanut allergy and tree nut allergy diagnosis, both alone and in conjunction with specific IgE testing and skin prick testing. BAT is a promising tool in the diagnosis of peanut and tree nut allergy.

BACKGROUND:Human milk provides an infant one of the first routes of exposure to dietary antigens. A better understanding of the levels and functional capacity of maternal dietary proteins in human milk (HM) and their impact on infant food allergy (FA) is needed. OBJECTIVE:To measure the quantity and biologic activity of maternal dietary proteins in HM and determine factors associated with their presence. METHODS:HM samples from mothers of infants with or without FA were collected before and serially after consumption of a meal with prespecified amounts of peanut, egg, and cow's milk protein. Ara h 2 from peanut, egg ovalbumin, and bovine β-lactoglobulin in HM was detected using ELISA. IgE-crosslinking capacity of Ara h 2 was assessed by the basophil activation test (BAT). Maternal intestinal permeability was measured with the lactulose/mannitol test. RESULTS:Thirty-nine mothers were included and 69% had measurable Ara h 2 in HM. Secretion of food allergens was variable between different mothers and across allergens, but did not correlate with intestinal permeability. Atopic mothers' milk had a higher peak Ara h 2 concentration (median 246 pg/mL, range 2.0-1634) than nonatopic mothers (median 0 pg/mL, range 0-135, p = 0.017). In samples with the most BAT reactivity, there was a significant correlation between BAT reactivity and Ara h 2 level (R = 0.72, p = 2 × 10-8). Multiple monoclonal antibodies directed at different epitopes detected Ara h 2 at similar levels. CONCLUSION:Ara h 2 is secreted in HM as intact protein or significant parts of it that are capable of IgE-crosslinking, found in higher levels in atopic mothers. HM levels of peanut lack correlation with other dietary proteins, which suggests antigen specificity of the secretion of dietary protein into HM.

B-cell epitope specificity on food allergens is highly relevant to understanding both the pathogenesis of IgE and IgG-mediated clinical tolerance after immunotherapy. The molecular binding interactions between the paratopes of antibodies and the epitopes of food allergens drive IgE-crosslinking, the subsequent activation of allergen effector cells, and the symptoms of systemic and life-threatening anaphylaxis. Various characteristics of allergen-specific IgE, such as high affinity and clonal diversity, can contribute to the persistence of food allergy and the severity of anaphylaxis upon allergen exposure. On the other hand, the induction of epitope-specific IgG antibodies in allergic individuals can inhibit IgE binding to food allergens. A subset of these can be neutralizing antibodies that effectively disrupt multiple allergen-IgE interactions to suppress effector responses. This review details the crucial role of epitope specificity and antibody clonality in both the pathogenesis of food allergy and in the development of clinical tolerance.

Humans develop immunoglobulin G (IgG) antibodies to the foods they consume. In the context of food allergy, allergen-specific IgG antibodies can sequentially class-switch to pathogenic IgE. However, the mechanism underlying the antigenicity of food proteins remains uncharacterized. Here, we identified convergent antibodies arising from different antibody gene rearrangements that bind to the immunodominant peanut allergen Ara h 2 and characterized allelic and junctional constraints on germline antibody specificity. Structurally, we found similar epitope-paratope interactions across multiple gene rearrangements. We demonstrate that these germline-encoded epitope-specific convergent antibodies to peanut occur commonly in the population because of the worldwide prevalence of the relevant gene rearrangements, allelic independence, and junctional malleability. As a result, serum IgG to this public epitope is prevalent among diverse cohorts of nonallergic peanut-consuming infants and peanut-allergic children and adults. This work demonstrates that IgG recognition of dietary antigens can be intrinsically programmed by the germline antibody repertoire.

Eosinophilic esophagitis (EoE) is a chronic allergic disease driven by exposure to culprit antigens. Due to the local nature of the inflammation, diagnosis and assessment are limited to invasive procedures. Based on prior single-cell RNA sequencing (scRNA-seq) data linking peripheral GPR15+ pathogenic effector Th2 (peTh2) cells to esophageal tissue peTh2s, we hypothesized the direct involvement of GPR15+ peTh2 cells in EoE pathogenesis and aimed to further evaluate their association with EoE disease status. We subjected samples from subjects with or without EoE to flow cytometry (n = 74 peripheral blood, 17 biopsy) and scRNA-seq (n = 27 peripheral blood, 10 biopsy). Expression of GPR15 by peripheral peTh2 cells was increased in EoE, and these cells expressed increased CD38 in active EoE--findings recapitulated in esophageal biopsies. We also identified a peTh2-associated, CD38-containing gene expression program that peripheral GPR15+ peTh2 cells upregulated in active EoE. The level of upregulation was distinct from other circulating peTh2 cells and was more similar to that seen in esophageal peTh2 cells. An association between expression of GPR15 by peripheral peTh2 cells, the aryl hydrocarbon receptor was strongest in subjects with EoE, suggesting an environmental exposure or susceptibility. The magnitude of GPR15 expression by peripheral peTh2 cells could effectively in discriminate active EoE from no EoE in our study population (AUC 0.93). Our data suggest that EoE-related peTh2 cells are identifiable and accessible in the peripheral blood, and could be exploited in both clinical practice as a non-invasive biomarker and continued investigation into mechanisms driving EoE. One sentence summary:GPR15 marks a subset of peripheral blood pathogenic effector Th2 cells associated with eosinophilic esophagitis (EoE) that upregulate CD38 during active disease - an observation that has potential to be used for non-invasive diagnosis and monitoring of EoE and that has suggests new mechanisms driving this increasingly prevalent allergic disease.

Anaphylaxis is an acute, potentially life-threatening reaction, often triggered by foods and largely mediated by IgE. A critically important aspect of anaphylaxis pertains to the factors that modulate its severity. The human microbiota is known to influence oral tolerance, but the microbial mechanisms directly involved in IgE-mediated anaphylaxis remain unknown. Here, we demonstrate that human saliva harbors peanut-degrading bacteria that metabolize immunodominant allergens (Ara h 1 and Ara h 2) and alter IgE binding. Additionally, we provide in vivo evidence showing that oral bacteria metabolize peanut allergens, influencing systemic allergen exposure and the severity of anaphylaxis. Finally, in a clinical study, we observe that common peanut-degrading bacteria, such as Rothia, from the oral cavity, are more abundant in peanut-allergic patients who exhibit better tolerance to allergen exposure. Altogether, these results demonstrate the role of the human microbiota in modulating IgE-mediated reactions through allergen metabolism. These findings reveal a novel microbial mechanism with potential to prevent, or reduce, the severity of IgE-mediated anaphylaxis.

Aberrant and pathological IgE responses to food allergens, whether persisting for a year or for a lifetime, are rooted in long-lived immunological memory. Unconventionally, IgE humoral memory is short-lived relative to protective immunity against pathogens, placing emphasis on cellular memory in the maintenance of allergic reactivity. Studies in mice and humans have uncovered a central interplay between IgG+ memory B cells and type 2 polarized CD4+ T cells that replenishes the transient IgE pool. These strides in our understanding of cellular phenotype, localization, and molecular crosstalk are critical for transforming therapeutic strategies to target allergen-specific memory.

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.

Bone marrow plasma cells (BMPCs) produce durable, protective IgM, IgG, and IgA antibodies, and in some cases, pro-allergic IgE antibodies, but their properties and sources are unclear. We charted single BMPC transcriptional and clonal heterogeneity in food-allergic and non-allergic individuals across CD19 protein expression given its inverse correlation to BMPC longevity. Transcriptional and clonal diversity revealed distinct functional profiles. Additionally, distribution of somatic hypermutation and intraclonal antibody sequence variance suggest that CD19low and CD19high BMPCs arise from recalled memory and germinal center B cells, respectively. Most IgE BMPCs were from peanut-allergic individuals; two out of 32 from independent donors bound peanut antigens in vitro and in vivo. These findings shed light on BMPC origins and highlight the bone marrow as a source of pathogenic IgE in peanut allergy.

Background: Clinical efficacy of oral immunotherapy (OIT) has been associated with the induction of blocking antibodies, particularly those capable of disrupting IgE-allergen interactions. Previously, we identified mAbs to Ara h 2 and structurally characterized their epitopes. Objective: We investigated longitudinal changes during OIT in antibody binding to conformational epitopes and correlated the results with isotype and clinical efficacy. Methods: We developed an indirect inhibitory ELISA using mAbs to block conformational epitopes on immobilized Ara h 2 from binding to serum immunoglobulins from peanut -allergic patients undergoing OIT. We tested the functional blocking ability of mAbs using passive cutaneous anaphylaxis in mice with humanized Fc epsilon RI receptors. Results: Diverse serum IgE recognition of Ara h 2 conformational epitopes are similar before and after OIT. Optimal inhibition of serum IgE occurs with the combination of 2 neutralizing mAbs (nAbs) recognizing epitopes 1.2 and 3, compared to 2 nonneutralizing mAbs (non-nAbs). After OIT, IgG 4 nAbs, but not IgG 1 or IgG 2 nAbs, increased in sustained compared to transient outcomes. Induction of IgG 4 nAbs occurs after OIT only in those with sustained efficacy. Murine passive cutaneous anaphylaxis after sensitization with pooled human sera is significantly inhibited by nAbs compared to non-nAbs. Conclusions: Serum IgE conformational epitope diversity remains unchanged during OIT. However, IgG 4 nAbs capable of uniquely disrupting IgE-allergen interactions to prevent effector cell activation are selectively induced in OIT-treated individuals with sustained clinical efficacy. Therefore, the induction of neutralizing IgG 4 antibodies to Ara h 2 are clinically relevant biomarkers of durable efficacy in OIT. (J Allergy Clin Immunol 2024;153:1611-20.)

In peanut allergy, Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea 6 (Ara h 6) are two clinically relevant peanut allergens with known structural and sequence homology and demonstrated cross-reactivity. We have previously utilized X-ray crystallography and epitope binning to define the epitopes on Ara h 2. We aimed to quantitatively characterize the cross-reactivity between Ara h 2 and Ara h 6 on a molecular level using human monoclonal antibodies (mAbs) and structural characterization of allergenic epitopes. We utilized mAbs cloned from Ara h 2 positive single B cells isolated from peanut-allergic, oral immunotherapy-treated patients to quantitatively analyze cross-reactivity between recombinant Ara h 2 (rAra h 2) and Ara h 6 (rAra h 6) proteins using biolayer interferometry and indirect inhibitory ELISA. Molecular dynamics simulations assessed time-dependent motions and interactions in the antibody-antigen complexes. Three epitopes-conformational epitopes 1.1 and 3, and the sequential epitope KRELRNL/KRELMNL-are conserved between Ara h 2 and Ara h 6, while two more conformational and three sequential epitopes are not. Overall, mAb affinity was significantly lower to rAra h 6 than it was to rAra h 2. This difference in affinity was primarily due to increased dissociation of the antibodies from rAra h 6, a phenomenon explained by the higher conformational flexibility of the Ara h 6-antibody complexes in comparison to Ara h 2-antibody complexes. Our results further elucidate the cross-reactivity of peanut 2S albumins on a molecular level and support the clinical immunodominance of Ara h 2.

BackgroundThe immunometabolic mechanisms underlying variable responses to oral immunotherapy (OIT) in patients with IgE-mediated food allergy are unknown.ObjectiveTo identify novel pathways associated with tolerance in food allergy, we used metabolomic profiling to find pathways important for food allergy in multiethnic cohorts and responses to OIT.MethodsUntargeted plasma metabolomics data were generated from the VDAART healthy infant cohort (N = 384), a Costa Rican cohort of children with asthma (N = 1040), and a peanut OIT trial (N = 20) evaluating sustained unresponsiveness (SU, protection that lasts after therapy) versus transient desensitization (TD, protection that ends immediately afterward). Generalized linear regression modeling and pathway enrichment analysis identified metabolites associated with food allergy and OIT outcomes.ResultsCompared with unaffected children, those with food allergy were more likely to have metabolomic profiles with altered histidines and increased bile acids. Eicosanoids (e.g., arachidonic acid derivatives) (q = 2.4 x 10-20) and linoleic acid derivatives (q = 3.8 x 10-5) pathways decreased over time on OIT. Comparing SU versus TD revealed differing concentrations of bile acids (q = 4.1 x 10-8), eicosanoids (q = 7.9 x 10-7), and histidine pathways (q = .015). In particular, the bile acid lithocholate (4.97 [1.93, 16.14], p = .0027), the eicosanoid leukotriene B4 (3.21 [1.38, 8.38], p = .01), and the histidine metabolite urocanic acid (22.13 [3.98, 194.67], p = .0015) were higher in SU.ConclusionsWe observed distinct profiles of bile acids, histidines, and eicosanoids that vary among patients with food allergy, over time on OIT and between SU and TD. Participants with SU had higher levels of metabolites such as lithocholate and urocanic acid, which have immunomodulatory roles in key T-cell subsets, suggesting potential mechanisms of tolerance in immunotherapy.image

Prior studies of food allergy have identified metabolomic pathways involved in immune function and allergy that are associated with having food allergies, but there is limited data around oral immunotherapy (OIT), desensitization, and immune tolerance. We aim to characterize metabolomic changes during desensitization through OIT.

Abstract Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells, but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited, especially from widely-used 3′-barcoded libraries. Here, we report a method that can recover paired, full-length variable region sequences of BCRs from 3′-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate, full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys, indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination.