IgE binding epitope mapping with TL1A tagged peptides.

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis.

Expression, purification, characterization, and patient IgE reactivity of new macadamia nut iso-allergen.

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2.

STAT5B restrains human B-cell differentiation to maintain humoral immune homeostasis.

BACKGROUND: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. OBJECTIVES: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. METHODS: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4+ T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. RESULTS: IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21loTbet+ B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. CONCLUSIONS: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

A positive feedback loop reinforces the allergic immune response in human peanut allergy.

Food allergies are a leading cause of anaphylaxis, and cellular mechanisms involving antigen presentation likely play key roles in their pathogenesis. However, little is known about the response of specific antigen-presenting cell (APC) subsets to food allergens in the setting of food allergies. Here, we show that in peanut-allergic humans, peanut allergen drives the differentiation of CD209+ monocyte-derived dendritic cells (DCs) and CD23+ (FcєRII) myeloid dendritic cells through the action of allergen-specific CD4+ T cells. CD209+ DCs act reciprocally on the same peanut-specific CD4+ T cell population to reinforce Th2 cytokine expression in a positive feedback loop, which may explain the persistence of established food allergy. In support of this novel model, we show clinically that the initiation of oral immunotherapy (OIT) in peanut-allergic patients is associated with a decrease in CD209+ DCs, suggesting that breaking the cycle of positive feedback is associated with therapeutic effect.

Can the biomolecular corona induce an allergic reaction?-A proof-of-concept study.

Ferumoxytol nanoparticles are being used clinically for the treatment of anemia and molecular imaging in patients. It is well documented that while most patients tolerate ferumoxytol well, a small percentage of patients (i.e., 0.01%) develop severe allergic reactions. The purpose of our proof-of-concept study was to determine whether patients with or without hypersensitivity reactions have specific protein corona profiles around ferumoxytol nanoparticles. In a retrospective, institutional review board approved pilot study, we enrolled 13 pediatric patients (5 girls, 8 boys, mean age 16.9 ± 8.2 years) who received a ferumoxytol-enhanced magnetic resonance imaging and who did (group 1, n = 5) or did not (group 2, n = 8) develop an allergic reaction. Blood samples of these patients were incubated with ferumoxytol, and the formation of a hard protein corona around ferumoxytol nanoparticles was measured by dynamic light scattering, zeta potential, and liquid chromatography-mass spectrometry. We also performed in vitro immune response analyses to randomly selected coronas from each group. Our results provide preliminary evidence that ex vivo analysis of the biomolecular corona may provide useful and predictive information on the possibility of severe allergic reactions to ferumoxytol nanoparticles. In the future, patients with predisposition of an allergic reaction to ferumoxytol may be diagnosed based on the proteomic patterns of the corona around ferumoxytol in their blood sample.

Targeted DNA methylation profiling reveals epigenetic signatures in peanut allergy.

DNA methylation (DNAm) has been shown to play a role in mediating food allergy; however, the mechanism by which it does so is poorly understood. In this study, we used targeted next-generation bisulfite sequencing to evaluate DNAm levels in 125 targeted highly informative genomic regions containing 602 CpG sites on 70 immune-related genes to understand whether DNAm can differentiate peanut allergy (PA) versus nonallergy (NA). We found PA-associated DNAm signatures associated with 12 genes (7 potentially novel to food allergy, 3 associated with Th1/Th2, and 2 associated with innate immunity), as well as DNAm signature combinations with superior diagnostic potential compared with serum peanut-specific IgE for PA versus NA. Furthermore, we found that, following peanut protein stimulation, peripheral blood mononuclear cell (PBMCs) from PA participants showed increased production of cognate cytokines compared with NA participants. The varying responses between PA and NA participants may be associated with the interaction between the modification of DNAm and the interference of environment. Using Euclidean distance analysis, we found that the distances of methylation profile comprising 12 DNAm signatures between PA and NA pairs in monozygotic (MZ) twins were smaller than those in randomly paired genetically unrelated individuals, suggesting that PA-related DNAm signatures may be associated with genetic factors.

Aging and CMV discordance are associated with increased immune diversity between monozygotic twins.

BACKGROUND: Broadly, much of variance in immune system phenotype has been linked to the influence of non-heritable factors rather than genetics. In particular, two non-heritable factors: aging and human cytolomegavirus (CMV) infection, have been known to account for significant inter-individual immune variance. However, many specific relationships between them and immune composition remain unclear, especially between individuals over narrower age ranges. Further exploration of these relationships may be useful for informing personalized intervention development. RESULTS: To address this need, we evaluated 41 different cell type frequencies by mass cytometry and identified their relationships with aging and CMV seropositivity. Analyses were done using 60 healthy individuals, including 23 monozygotic twin pairs, categorized into young (12-31 years) and middle-aged (42-59 years). Aging and CMV discordance were associated with increased immune diversity between monozygotic twins overall, and particularly strongly in various T cell populations. Notably, we identified 17 and 11 cell subset frequencies as relatively influenced and uninfluenced by non-heritable factors, respectively, with results that largely matched those from studies on older-aged cohorts. Next, CD4+ T cell frequency was shown to diverge with age in twins, but with lower slope than in demographically similar non-twins, suggesting that much inter-individual variance in this cell type can be attributed to interactions between genetic and environmental factors. Several cell frequencies previously associated with memory inflation, such as CD27- CD8+ T cells and CD161+ CD4+ T cells, were positively correlated with CMV seropositivity, supporting findings that CMV infection may incur rapid aging of the immune system. CONCLUSIONS: Our study confirms previous findings that aging, even within a relatively small age range and by mid-adulthood, and CMV seropositivity, both contribute significantly to inter-individual immune diversity. Notably, we identify several key immune cell subsets that vary considerably with aging, as well as others associated with memory inflation which correlate with CMV seropositivity.

Transcriptional changes in peanut-specific CD4+ T cells over the course of oral immunotherapy.

Oral immunotherapy (OIT) can successfully desensitize allergic individuals to offending foods such as peanut. Our recent clinical trial (NCT02103270) of peanut OIT allowed us to monitor peanut-specific CD4+ T cells, using MHC-peptide Dextramers, over the course of OIT. We used a single-cell targeted RNAseq assay to analyze these cells at 0, 12, 24, 52, and 104 weeks of OIT. We found a transient increase in TGFß-producing cells at 52 weeks in those with successful desensitization, which lasted until 117 weeks. We also performed clustering and identified 5 major clusters of Dextramer+ cells, which we tracked over time. One of these clusters appeared to be anergic, while another was consistent with recently described TFH13 cells. The other 3 clusters appeared to be Th2 cells by their coordinated production of IL-4 and IL-13, but they varied in their expression of STAT signaling proteins and other markers. A cluster with high expression of STAT family members also showed a possible transient increase at week 24 in those with successful desensitization. Single cell TCRαß repertoire sequences were too diverse to track clones over time. Together with increased TGFß production, these changes may be mechanistic predictors of successful OIT that should be further investigated.

Mass cytometry reveals cellular fingerprint associated with IgE+ peanut tolerance and allergy in early life.

IgE-mediated peanut allergic is common, often serious, and usually lifelong. Not all individuals who produce peanut-specific IgE will react upon consumption of peanut and can eat the food without adverse reactions, known as sensitized tolerance. Here, we employ high-dimensional mass cytometry to define the circulating immune cell signatures associated with sensitized tolerance and clinical allergy to peanut in the first year of life. Key features of clinical peanut allergic are increased frequency of activated B cells (CD19hiHLADRhi), overproduction of TNFα and increased frequency of peanut-specific memory CD4 T cells. Infants with sensitized tolerance display reduced frequency but hyper-responsive naive CD4 T cells and an increased frequency of plasmacytoid dendritic cells. This work demonstrates the utility and power of high-dimensional mass cytometry analysis to interrogate the cellular interactions that are associated with allergic sensitization and clinical food allergy in the first year of life.

Phase 2a randomized, placebo-controlled study of anti-IL-33 in peanut allergy.

BACKGROUNDIL-33, found in high levels in participants with allergic disorders, is thought to mediate allergic reactions. Etokimab, an anti-IL-33 biologic, has previously demonstrated a good safety profile and favorable pharmacodynamic properties in many clinical studies.METHODSIn this 6-week placebo-controlled phase 2a study, we evaluated the safety and the ability of a single dose of etokimab to desensitize peanut-allergic adults. Participants received either etokimab (n = 15) or blinded placebo (n = 5). Clinical tests included oral food challenges and skin prick tests at days 15 and 45. Blood samples were collected for IgE levels and measurement of ex vivo peanut-stimulated T cell cytokine production.RESULTSEfficacy measurements for active vs. placebo participants at the day 15 and 45 food challenge (tolerating a cumulative 275 mg of peanut protein, which was the food challenge outcome defined in this paper) demonstrated, respectively, 73% vs. 0% (P = 0.008) to 57% vs. 0% (ns). The etokimab group had fewer adverse events compared with placebo. IL-4, IL-5, IL-9, IL-13, and ST2 levels in CD4+ T cells were reduced in the active vs. placebo arm upon peanut-induced T cell activation (P = 0.036 for IL-13 and IL-9 at day 15), and peanut-specific IgE was reduced in active vs. placebo (P = 0.014 at day 15).CONCLUSIONThe phase 2a results suggest etokimab is safe and well tolerated and that a single dose of etokimab could have the potential to desensitize peanut-allergic participants and possibly reduce atopy-related adverse events.TRIAL REGISTRATIONClinicalTrials.gov NCT02920021.FUNDINGThis work was supported by NIH grant R01AI140134, AnaptysBio, the Hartman Vaccine Fund, and the Sean N. Parker Center for Allergy and Asthma Research at Stanford University.

Sustained outcomes in oral immunotherapy for peanut allergy (POISED study): a large, randomised, double-blind, placebo-controlled, phase 2 study.

BACKGROUND: Dietary avoidance is recommended for peanut allergies. We evaluated the sustained effects of peanut allergy oral immunotherapy (OIT) in a randomised long-term study in adults and children. METHODS: In this randomised, double-blind, placebo-controlled, phase 2 study, we enrolled participants at the Sean N Parker Center for Allergy and Asthma Research at Stanford University (Stanford, CA, USA) with peanut allergy aged 7-55 years with a positive result from a double-blind, placebo-controlled, food challenge (DBPCFC; ≤500 mg of peanut protein), a positive skin-prick test (SPT) result (≥5 mm wheal diameter above the negative control), and peanut-specific immunoglobulin (Ig)E concentration of more than 4 kU/L. Participants were randomly assigned (2·4:1·4:1) in a two-by-two block design via a computerised system to be built up and maintained on 4000 mg peanut protein through to week 104 then discontinued on peanut (peanut-0 group), to be built up and maintained on 4000 mg peanut protein through to week 104 then to ingest 300 mg peanut protein daily (peanut-300 group) for 52 weeks, or to receive oat flour (placebo group). DBPCFCs to 4000 mg peanut protein were done at baseline and weeks 104, 117, 130, 143, and 156. The pharmacist assigned treatment on the basis of a randomised computer list. Peanut or placebo (oat) flour was administered orally and participants and the study team were masked throughout by use of oat flour that was similar in look and feel to the peanut flour and nose clips, as tolerated, to mask taste. The statistician was also masked. The primary endpoint was the proportion of participants who passed DBPCFCs to a cumulative dose of 4000 mg at both 104 and 117 weeks. The primary efficacy analysis was done in the intention-to-treat population. Safety was assessed in the intention-to-treat population. This trial is registered at ClinicalTrials.gov, NCT02103270. FINDINGS: Between April 15, 2014, and March 2, 2016, of 152 individuals assessed, we enrolled 120 participants, who were randomly assigned to the peanut-0 (n=60), peanut-300 (n=35), and placebo groups (n=25). 21 (35%) of peanut-0 group participants and one (4%) placebo group participant passed the 4000 mg challenge at both 104 and 117 weeks (odds ratio [OR] 12·7, 95% CI 1·8-554·8; p=0·0024). Over the entire study, the most common adverse events were mild gastrointestinal symptoms, which were seen in 90 of 120 patients (50/60 in the peanut-0 group, 29/35 in the peanut-300 group, and 11/25 in the placebo group) and skin disorders, which were seen in 50/120 patients (26/60 in the peanut-0 group, 15/35 in the peanut-300 group, and 9/25 in the placebo group). Adverse events decreased over time in all groups. Two participants in the peanut groups had serious adverse events during the 3-year study. In the peanut-0 group, in which eight (13%) of 60 participants passed DBPCFCs at week 156, higher baseline peanut-specific IgG4 to IgE ratio and lower Ara h 2 IgE and basophil activation responses were associated with sustained unresponsiveness. No treatment-related deaths occurred. INTERPRETATION: Our study suggests that peanut OIT could desensitise individuals with peanut allergy to 4000 mg peanut protein but discontinuation, or even reduction to 300 mg daily, could increase the likelihood of regaining clinical reactivity to peanut. Since baseline blood tests correlated with week 117 treatment outcomes, this study might aid in optimal patient selection for this therapy. FUNDING: National Institute of Allergy and Infectious Diseases.

Almond (Prunus dulcis) Allergen Pru du 8, the First Member of a New Family of Food Allergens.

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.

Identification of Almond ( Prunus dulcis) Vicilin As a Food Allergen.

Almond is one of the tree nuts listed by U.S. FDA as a food allergen source. A food allergen identified with patient sera has been debated to be the 2S albumin or the 7S vicilin. However, neither of these proteins has been defined as a food allergen. The purpose of this study was to clone, express, and purify almond vicilin and test whether it is a food allergen. Western blot experiment was performed with 18 individual sera from patients with double-blind, placebo-controlled clinical almond allergy. The results showed that 44% of the sera contained IgE antibodies that recognized the recombinant almond vicilin, indicating that it is an almond allergen. Identifying this and additional almond allergens will facilitate the understanding of the allergenicity of seed proteins in tree nuts and their cross-reactivity.

Purification and Characterization of a Black Walnut (Juglans nigra) Allergen, Jug n 4.

Tree nuts as a group cause a significant number of fatal anaphylactic reactions to foods. Walnuts (Juglans spp.) are one of the leading causes of allergic reactions to tree nuts in the U.S. and Japan. The purpose of this study was to purify and characterize potential food allergens from black walnut. Here, we report the isolation of the black walnuts allergen Jug n 4 (an 11S globulin) by ammonium sulfate precipitation, hydrophobic interaction, and size exclusion chromatography. Reducing SDS-PAGE analysis indicated that purified Jug n 4 consists of three major bands. N-Terminal sequencing data of these bands indicated that they were the results of a post-transcriptional protease cleavage of the mature protein at a site that consists of a known conserved protease recognition motif, NGXEET. Western blot experiments revealed that 32% of the sera from 25 patients with double-blind, placebo-controlled clinical walnut allergy contained IgE antibodies that recognized Jug n 4, indicating that it is a walnut allergen. Identifying this and additional allergens may facilitate the understanding of the allergenicity of seed storage proteins in tree nuts and their cross-reactivity.