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Food allergies have increased at an alarming rate over the past 2 decades, indicating that environmental factors are driving disease progression. It has been postulated that sensitization to foods, in particular, peanut, occurs through impaired skin. Peanut allergens have been quantified in household dust and may be the culprit source. Indeed, TH2 cell–skewing innate cytokines can be driven by application of food antigens on both intact and impaired skin of mice, resulting in antigen-specific IgE production and anaphylaxis following allergen exposure. However, allergy induction through the skin can be prevented by induction of oral tolerance before skin exposure. These observations led to the dual allergen exposure hypothesis, according to which oral exposure to food antigens leads to tolerance and antigen exposure on impaired skin leads to allergy. Here, we propose the airway as an alternative route of sensitization in the dual allergen exposure hypothesis that leads to food allergy. Specifically, we will provide evidence from mouse models and human cell–based studies that together implicate the airway as a plausible route of sensitization.

Introduction Previous studies in humans support the dual‐allergen exposure hypothesis, and several studies in mouse models have demonstrated that cutaneous exposure to disrupted or intact skin can lead to sensitization to peanut. However, the field lacks definitive evidence that cutaneous exposure leads to peanut allergy in humans or other primates. Methods Peanut extract was applied to the shaved back of the neck of four male and four female African green monkeys three times per week for 4 weeks. An oral food challenge (OFC) was performed the following week by gavage of 200 mg of peanut protein, and vital signs were monitored for 30 minutes post‐OFC. Blood was collected at baseline, day 11, day 32, and 30 minutes post‐OFC. Total IgE, and peanut‐specific immunoglobulin E (IgE) and immunoglobulin G (IgG) were quantified in serum collected throughout the 4 weeks. Histamine was measured in serum collected 30 minutes post‐OFC. Results Peanut‐specific IgE was undetectable at any time points in any of the monkeys, and there was no consistent increase in total IgE. During the oral challenge, none of the monkeys experienced allergic symptoms and histamine levels did not change. However, seven of the eight monkeys produced increasing peanut‐specific IgG by day 32, indicating that repeated skin exposure to peanut is immunogenic. Conclusions Skin exposure to peanut did not lead to sensitization in this study, and monkeys did not experience anaphylaxis upon peanut challenge. However, monkeys produced increased peanut‐specific IgG throughout peanut exposure, indicating that repeated skin exposure to peanut is immunogenic., Peanut proteins were applied to the skin of African green monkeys thrice per week for four weeks. Peanut‐specific IgE was not detected in serum throughout peanut exposure, and monkeys did not experience anaphylaxis upon peanut challenge, indicating that monkeys were not sensitized to peanut. However, peanut‐specific IgG was increased in seven of eight monkeys over the exposure period, indicating that repeated skin exposure to peanut is immunogenic.

BACKGROUND: For patients with peanut allergy, there are currently no methods to predict who will develop sustained unresponsiveness (SU) after oral immunotherapy (OIT). OBJECTIVE: Assess IgE binding to peanut (PN), Ara h 2 and to specific linear epitopes of Ara h 2 as predictors of the important clinical parameters: eliciting dose threshold and attainment of SU following OIT. METHODS: Samples and clinical data were collected from children undergoing OIT. PN- and Ara h 2-sIgE were quantified by ImmunoCap(®). IgE binding to linear peptides of Ara h 2 and Ara h 6 was measured with peptide microarrays. RESULTS: Lower values of PN-sIgE correlated with eliciting dose (P = 0.001) and with a higher likelihood of achieving SU (P < 0.0001) but these relationships were lost at higher values for PN-sIgE (≥ 14 kIU for eliciting dose and ≥ 35 kIU/L for SU). In subjects with PN-sIgE ≥ 14kIU/L, binding of IgE to epitopes 5 & 6 of Ara h 2 was associated with a lower eliciting dose at baseline challenge (P < 0.001; Pc < 0.02). In subjects with PN-sIgE ≥ 35kIU/L, a combined model of IgE binding to epitopes 1, 5 and 6 with PN-sIgE was highly predictive of attainment of SU (AUC of 0.86; P = 0.0067). CONCLUSION: In young patients with peanut allergy, measurement of PN-sIgE and IgE binding to specific linear epitopes of Ara h 2 in baseline samples may allow stratification of patients regarding sensitivity to challenge and outcome of OIT.

Background There is growing evidence that environmental peanut exposure through non-oral routes, including the skin and respiratory tract, can result in peanut sensitization. Environmental adjuvants in indoor dust can promote sensitization to inhaled antigens, but whether they contribute to peanut allergy development is unclear. Objective We investigated whether indoor dust promotes airway sensitization to peanut and peanut allergy development in mice. Methods Female and male C57BL/6J mice were exposed via the airways to peanut, indoor dust extract, or both for 2 weeks. Mice were then challenged with peanut and assessed for anaphylaxis. Peanut-specific immunoglobulins, peanut uptake by lung conventional dendritic cells (cDCs), lung innate cytokines, and T cell differentiation in lung-draining lymph nodes were quantified. Innate cytokine production by primary human bronchial epithelial cells exposed to indoor dust was also determined. Results Inhalational exposure to low levels of peanut in combination with indoor dust, but neither alone, resulted in production of peanut-specific IgE and development of anaphylaxis upon peanut challenge. Indoor dust triggered production of innate cytokines in murine lungs and in primary human bronchial epithelial cells. Additionally, inhaled indoor dust stimulated maturation and migration of peanut-laden lung type 1 cDCs to draining lymph nodes. Inhalational exposure to peanut and indoor dust induced peanut-specific T helper 2 cell differentiation and accumulation of T follicular helper cells in draining lymph nodes, which were associated with increased B cell numbers and peanut-specific immunoglobulin production. Conclusions & clinical relevance Indoor dust promotes airway sensitization to peanut and development of peanut allergy in mice. Our findings suggest that environmental adjuvants in indoor dust may be determinants of peanut allergy development in children.

Background Mechanisms underlying oral immunotherapy (OIT) are unclear and the effects on immune cells at varying maintenance doses are unknown. Objective We aimed to determine the immunologic changes caused by peanut OIT in preschool aged children and determine the effect on these immune responses in groups ingesting low or high-dose peanut OIT (300 mg or 3000 mg, respectively) as maintenance therapy. Methods Blood was drawn at several time-points throughout the OIT protocol and PBMCs isolated and cultured with peanut antigens. Secreted cytokines were quantified via multiplex assay, whereas Treg and peanut-responsive CD4 T cells were studied with flow cytometry. Basophil activation assays were also conducted. Results Th2-, Th1-, Th9- and Tr1-type cytokines decreased over the course of OIT in groups on high- and low-dose OIT. There were no significant differences detected in cytokine changes between the high- and low-dose groups. The initial increase in both the number of peanut-responsive CD4 T cells and the number of Tregs was transient and no significant differences were found between groups. Basophil activation following peanut stimulation was decreased over the course of OIT and associated with increased peanut-IgG4/IgE ratios. No differences were found between high- and low-dose groups in basophil activation at the time of desensitization or sustained unresponsiveness oral food challenges. Conclusions and clinical relevance Peanut OIT leads to decreases in pro-allergic cytokines, including IL-5, IL-13, and IL-9 and decreased basophil activation. No differences in T cell or basophil responses were found between subjects on low or high-dose maintenance OIT, which has implications for clinical dosing strategies.

Background Peanut sublingual immunotherapy (SLIT) for 1 year has been shown to induce modest clinical desensitization in allergic children. Studies of oral immunotherapy, epicutaneous immunotherapy, and SLIT have suggested additional benefit with extended treatment. Objective We sought to investigate the safety, clinical effectiveness, and immunologic changes with long-term SLIT in children with peanut allergy. Methods Children with peanut allergy aged 1 to 11 years underwent extended maintenance SLIT with 2 mg/d peanut protein for up to 5 years. Subjects with peanut skin test wheals of less than 5 mm and peanut-specific IgE levels of less than 15 kU/L were allowed to discontinue therapy early. Desensitization was assessed through a double-blind, placebo-controlled food challenge (DBPCFC) with up to 5000 mg of peanut protein after completion of SLIT dosing. Sustained unresponsiveness was further assessed by using identical DBPCFCs after 2 to 4 weeks without peanut exposure. Results Thirty-seven of 48 subjects completed 3 to 5 years of peanut SLIT, with 67% (32/48) successfully consuming 750 mg or more during DBPCFCs. Furthermore, 25% (12/48) passed the 5000-mg DBPCFC without clinical symptoms, with 10 of these 12 demonstrating sustained unresponsiveness after 2 to 4 weeks. Side effects were reported with 4.8% of doses, with transient oropharyngeal itching reported most commonly. Side effects requiring antihistamine treatment were uncommon (0.21%), and no epinephrine was administered. Peanut skin test wheals, peanut-specific IgE levels, and basophil activation decreased significantly, and peanut-specific IgG4 levels increased significantly after peanut SLIT. Conclusion Extended-therapy peanut SLIT provided clinically meaningful desensitization in the majority of children with peanut allergy that was balanced with ease of administration and a favorable safety profile.

The gut associated lymphoid tissue has effective mechanisms in place to maintain tolerance to food antigens. These can be exploited to induce antigen-specific tolerance for the prevention and treatment of autoimmune diseases and severe allergies and to prevent serious immune responses in protein replacement therapies for genetic diseases. An oral tolerance approach for the prevention of peanut allergy in infants proved highly efficacious and advances in treatment of peanut allergy have brought forth an oral immunotherapy drug that is currently awaiting FDA approval. Several other protein antigens made in plant cells are in clinical development. Plant cell-made proteins are protected in the stomach from acids and enzymes after their oral delivery because of bioencapsulation within plant cell wall, but are released to the immune system upon digestion by gut microbes. Utilization of fusion protein technologies facilitates their delivery to the immune system, oral tolerance induction at low antigen doses, resulting in efficient induction of FoxP3+ and latency-associated peptide (LAP)+ regulatory T cells that express immune suppressive cytokines such as IL-10. LAP and IL-10 expression represent potential biomarkers for plant-based oral tolerance. Efficacy studies in hemophilia dogs support clinical development of oral delivery of bioencapsulated antigens to prevent anti-drug antibody formation. Production of clinical grade materials in cGMP facilities, stability of antigens in lyophilized plant cells for several years when stored at ambient temperature, efficacy of oral delivery of human doses in large animal models and lack of toxicity augur well for clinical advancement of this novel drug delivery concept.

Toll-like receptor ligands in indoor dust act as environmental adjuvants to promote sensitization to inhaled peanut in mice. The timing of exposure to environmental adjuvants relative to peanut is a determinant of peanut allergy development.

Animal models of disease enable the study of the pathology, biomarkers, and treatments for the disease being studied. These models become particularly useful in the study of diseases, such as peanut allergy, that currently have no FDA-approved therapy options. Here, we describe a mouse model of peanut allergy using a peanut extract and cholera toxin that can be applied to both BALB/c and C3H/HeJ mouse strains. Sensitization is induced through the gastrointestinal tract resulting in elevated levels of peanut-specific IgE and anaphylaxis upon challenge with peanut proteins. This model has been used to study the cells and molecules involved in the development of peanut allergy and to evaluate novel immunotherapy approaches and the underlying mechanisms of immunotherapy. Potential utilities of this model are numerous and may include studies on microbial influences on peanut allergy and discovery of biomarkers of anaphylaxis.

Peanut (Arachis hypogaea L.) is an important crop grown worldwide for food and edible oil. The surge of peanut allergy in the past 25 years has profoundly impacted both affected individuals and the peanut and related food industries. In response, several strategies to mitigate peanut allergy have emerged to reduce/eliminate the allergenicity of peanuts or to better treat peanut-allergic individuals. In this review, we give an overview of peanut allergy, with a focus on peanut proteins, including the impact of thermal processing on peanut protein structure and detection in food matrices. We discuss several strategies currently being investigated to mitigate peanut allergy, including genetic engineering, novel processing strategies, and immunotherapy in terms of mechanisms, recent research, and limitations. All strategies are discussed with considerations for both peanut-allergic individuals and the numerous industries/government agencies involved throughout peanut production and utilization.

Background Improved animal models are needed to understand the genetic and environmental factors that contribute to food allergy. Objective We sought to assess food allergy phenotypes in a genetically diverse collection of mice. Methods We selected 16 Collaborative Cross (CC) mouse strains, as well as the classic inbred C57BL/6J, C3H/HeJ, and BALB/cJ strains, for screening. Female mice were sensitized to peanut intragastrically with or without cholera toxin and then challenged with peanut by means of oral gavage or intraperitoneal injection and assessed for anaphylaxis. Peanut-specific immunoglobulins, T-cell cytokines, regulatory T cells, mast cells, and basophils were quantified. Results Eleven of the 16 CC strains had allergic reactions to intraperitoneal peanut challenge, whereas only CC027/GeniUnc mice reproducibly experienced severe symptoms after oral food challenge (OFC). CC027/GeniUnc, C3H/HeJ, and C57BL/6J mice all mounted a TH2 response against peanut, leading to production of IL-4 and IgE, but only the CC027/GeniUnc mice reacted to OFC. Orally induced anaphylaxis in CC027/GeniUnc mice was correlated with serum levels of Ara h 2 in circulation but not with allergen-specific IgE or mucosal mast cell protease 1 levels, indicating systemic allergen absorption is important for anaphylaxis through the gastrointestinal tract. Furthermore, CC027/GeniUnc, but not C3H/HeJ or BALB/cJ, mice can be sensitized in the absence of cholera toxin and react on OFC to peanut. Conclusions We have identified and characterized CC027/GeniUnc mice as a strain that is genetically susceptible to peanut allergy and prone to severe reactions after OFC. More broadly, these findings demonstrate the untapped potential of the CC population in developing novel models for allergy research.

Background: Peanut flour is a high-protein, low-oil, powdered material prepared from roasted peanut seed. In addition to being a well-established food ingredient, peanut flour is also the active ingredient in peanut oral immunotherapy trials. Enzymatic hydrolysis was evaluated as a processing strategy to generate hydrolysates from peanut flour with reduced allergenicity. Methods: Soluble fractions of 10% (w/v) light roasted peanut flour dispersions were hydrolyzed with the following proteases: Alcalase (pH 8.0, 60°C), pepsin (pH 2.0, 37°C) or Flavourzyme (pH 7.0, 50°C) for 60 min. Western blotting, inhibition ELISA and basophil activation tests were used to examine IgE reactivity. Results: Western blotting experiments revealed the hydrolysates retained IgE binding reactivity and these IgE-reactive peptides were primarily Ara h 2 fragments regardless of the protease tested. Inhibition ELISA assays demonstrated that each of the hydrolysates had decreased capacity to bind peanut-specific IgE compared with nonhydrolyzed controls. Basophil activation tests revealed that all hydrolysates were comparable (p > 0.05) to nonhydrolyzed controls in IgE cross-linking capacity. Conclusions: These results indicate that hydrolysis of peanut flour reduced IgE binding capacity; however, IgE cross-linking capacity during hydrolysis was retained, thus suggesting such hydrolysates are not hypoallergenic.

Background Oral immunotherapy (OIT) is an investigational therapeutic approach for the treatment of food allergies. Characterization of the drug product used in oral immunotherapy trials for peanut allergy has not been reported. Objective To quantify relative amounts of the major peanut allergens and microbial load present in peanut flour used in OIT trials and assess whether these parameters change over a 12-month period. We also anticipate that this report will serve as a guide for investigators seeking to conduct OIT trials under Food and Drug Administration–approved Investigational New Drug applications. Methods Densitometric scanning of Ara h 1 and Ara h 2 resolved on SDS-PAGE gels was used to assess allergen content in peanut flour extracts. Microbial testing was conducted on peanut flour under US Pharmacopeia guidelines for the presence of Escherichia coli , salmonella, yeast, mold, and total aerobic bacteria. In addition, aflatoxin was quantified in peanut flour. Reported results were obtained from 4 unique lots of peanut flour. Results Relative amounts of the major peanut allergens were similar between different lots of peanut flour and remained stable over a 12-month period. E coli and salmonella were absent from all lots of flour. Yeast, mold, total aerobic bacteria, and aflatoxin were within established US Pharmacopeia guidelines on all lots tested and remained within the criteria over a 12-month period. Conclusions Peanut flour used as a drug product contains the major peanut allergens and has low levels of potentially harmful microbes. Both these parameters remain stable over a 12-month period.

Capsule Summary Peanut-specific IgA in saliva correlates with DBPCFC outcomes following peanut SLIT, suggesting that peanut-specific salivary IgA may be a potential biomarker for SLIT used to treat peanut allergy.

Oral immunotherapy (OIT) is an effective experimental food allergy treatment that is limited by treatment withdrawal and the frequent reversibility of desensitization if interrupted. Newly diagnosed preschool children may have clinical and immunological characteristics more amenable to treatment.We sought to test the safety, effectiveness, and feasibility of early OIT (E-OIT) in the treatment of peanut allergy.We enrolled 40 children aged 9 to 36 months with suspected or known peanut allergy. Qualifying subjects reacted to peanut during an entry food challenge and were block-randomized 1:1 to receive E-OIT at goal maintenance doses of 300 or 3000 mg/d in a double-blinded fashion. The primary end point, sustained unresponsiveness at 4 weeks after stopping early intervention oral immunotherapy (4-SU), was assessed by double-blinded, placebo-controlled food challenge either upon achieving 4 prespecified criteria, or after 3 maintenance years. Peanut-specific immune responses were serially analyzed. Outcomes were compared with 154 matched standard-care controls.Of 40 consented subjects, 3 (7.5%) did not qualify. Overall, 29 of 37 (78%) in the intent-to-treat analysis achieved 4-SU (300-mg arm, 17 of 20 [85%]; 3000 mg, 12 of 17 [71%], P = .43) over a median of 29 months. Per-protocol, the overall proportion achieving 4-SU was 29 of 32 (91%). Peanut-specific IgE levels significantly declined in E-OIT-treated children, who were 19 times more likely to successfully consume dietary peanut than matched standard-care controls, in whom peanut-specific IgE levels significantly increased (relative risk, 19.42; 95% CI, 8.7-43.7; P .001). Allergic side effects during E-OIT were common but all were mild to moderate.At both doses tested, E-OIT had an acceptable safety profile and was highly successful in rapidly suppressing allergic immune responses and achieving safe dietary reintroduction.

Patients with peanut allergy have highly stable pathologic antibody repertoires to the immunodominant B-cell epitopes of the major peanut allergens Ara h 1 to 3.We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires.Measurements of total peanut-specific IgE (psIgE) and peanut-specific IgG(4) (psIgG(4)) were made with CAP-FEIA. We analyzed sera from 22 patients with OIT and 6 control subjects and measured serum specific IgE and IgG(4) binding to epitopes of Ara h 1 to 3 using a high-throughput peptide microarray technique. Antibody affinity was measured by using a competitive peptide microarray, as previously described.At baseline, psIgE and psIgG(4) diversity was similar between patients and control subjects, and there was broad variation in epitope recognition. After a median of 41 months of OIT, polyclonal psIgG(4) levels increased from a median of 0.3 μg/mL (interquartile range [25% to 75%], 0.1-0.43 μg/mL) at baseline to 10.5 μg/mL (interquartile range [25% to 75%], 3.95-45.48 μg/mL; P.0001) and included de novo specificities. psIgE levels were reduced from a median baseline of 85.45 kU(A)/L (23.05-101.0 kU(A)/L) to 7.75 kU(A)/L (2.58-30.55 kU(A)/L, P.0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated patients, several subjects generated new IgE specificities, even as the total psIgE level decreased. Global epitope-specific shifts from IgE to IgG(4) binding occurred, including at an informative epitope of Ara h 2.OIT differentially alters Ara h 1 to 3 binding patterns. These changes are variable between patients, are not observed in control subjects, and include a progressive polyclonal increase in IgG(4) levels, with concurrent reduction in IgE amount and diversity.

Background Although peanut oral immunotherapy (OIT) has been conclusively shown to cause desensitization, it is currently unknown whether clinical protection persists after stopping therapy. Objective Our primary objective was to determine whether peanut OIT can induce sustained unresponsiveness after withdrawal of OIT. Methods We conducted a pilot clinical trial of peanut OIT at 2 US centers. Subjects age 1 to 16 years were recruited and treated for up to 5 years with peanut OIT. The protocol was modified over time to permit dose increases to a maximum of 4000 mg/d peanut protein. Blood was collected at multiple time points. Clinical end points were measured with 5000-mg double-blinded, placebo-controlled food challenges once specific criteria were met. Results Of the 39 subjects originally enrolled, 24 completed the protocol and had evaluable outcomes. Twelve (50%) of 24 successfully passed a challenge 1 month after stopping OIT and achieved sustained unresponsiveness. Peanut was added to the diet. At baseline and the time of challenge, such subjects had smaller skin test results, as well as lower IgE levels specific for peanut, Ara h 1, and Ara h 2 and lower ratios of peanut-specific IgE/total IgE compared with subjects not passing. There were no differences in peanut IgG 4 levels or functional activity at the end of the study. Conclusions This is the first demonstration of sustained unresponsiveness after peanut OIT, occurring in half of subjects treated for up to 5 years. OIT favorably modified the peanut-specific immune response in all subjects completing the protocol. Smaller skin test results and lower allergen-specific IgE levels were predictive of successful outcome.

To the Editor: There has been considerable recent interest in developing therapies for food allergy, an increasingly common and highly morbid disorder for which strict dietary elimination and ready access to epinephrine remain the standard of care.(1) While both oral immunotherapy (OIT)(2-4) and sublingual immunotherapy (SLIT)(5) have been shown to induce clinical desensitization to foods (reviewed in 6), no head-to-head comparative analysis of the two treatments has been published. We conducted a retrospective study of two previously published protocols for peanut allergy(2,3). This new analysis includes additional subjects, compares the 12-month oral food challenge outcomes, and extends analysis of immunologic parameters out to 24 months. Eligible peanut-allergic subjects were recruited into one of two concurrent clinical trials: OIT (maintenance dose of 4000 mg/day and cumulative double-blind, placebo-controlled challenge (DBPCFC) dose of 5000 mg); or SLIT, (2 mg/day, and 2500 mg, respectively) [all quantities refer to peanut protein]. Although the optimal immunotherapy dose remains unknown, the doses chosen in these trials were based on preliminary data from pilot studies. Of note, unique properties of the oral mucosal immune response are hypothesized to account for SLIT’s efficacy at log-fold lower doses (reviewed in 7). Both trials utilized randomized, double-blind, placebo-controlled designs. Mechanistic studies were performed longitudinally as previously described using blood drawn from subjects within 24 hours of their last immunotherapy dose.(2, 3) At 12 months, subjects underwent DBPCFC to assess clinical desensitization; OIT subjects received a maximal 5000 mg cumulative protein dose, and for safety reasons SLIT challenges were limited to 2500 mg (Online Repository Tables E1/E2). We compared laboratory data between OIT and SLIT at baseline, 12 months, and 24 months, as well as DBPCFC pass/fail outcomes, using the Wilcoxon signed rank test (STATA 12; College Station, TX) and Mann-Whitney U test (GraphPad Prism; La Jolla, CA). Twenty-three subjects on OIT and 27 subjects on SLIT were evaluated after receiving 2 years of treatment (Table 1). We did not undertake a formal comparison of safety parameters between the two studies, and upcoming interval reports of each study will include these data. However, there were no serious adverse events reported in either study. No SLIT and two OIT subjects (one active, one placebo) required four total doses of epinephrine for dose-related reactions. At baseline, the peanut-specific IgE was similar between OIT and SLIT subjects (Fig 1A). Twelve months of treatment led to higher median peanut-specific IgE levels in the OIT group compared to the SLIT group (204.5 kU/L versus 66.7 kU/L, p=0.0382); however, levels were not significantly different between the groups at 24 months (Fig 1A). While peanut-specific IgG4 increased over time in both groups (Fig 1B), the effect was greater with OIT at 12 (20.1 mg/L versus 3.1 mg/L) and 24 months (20.3 mg/L versus 7.9 mg/L, p