Efficacy and safety of oral immunotherapy in children aged 1-3 years with peanut allergy (the Immune Tolerance Network IMPACT trial): a randomised placebo-controlled study.

BACKGROUND: For young children with peanut allergy, dietary avoidance is the current standard of care. We aimed to assess whether peanut oral immunotherapy can induce desensitisation (an increased allergic reaction threshold while on therapy) or remission (a state of non-responsiveness after discontinuation of immunotherapy) in this population. METHODS: We did a randomised, double-blind, placebo-controlled study in five US academic medical centres. Eligible participants were children aged 12 to younger than 48 months who were reactive to 500 mg or less of peanut protein during a double-blind, placebo-controlled food challenge (DBPCFC). Participants were randomly assigned by use of a computer, in a 2:1 allocation ratio, to receive peanut oral immunotherapy or placebo for 134 weeks (2000 mg peanut protein per day) followed by 26 weeks of avoidance, with participants and study staff and investigators masked to group treatment assignment. The primary outcome was desensitisation at the end of treatment (week 134), and remission after avoidance (week 160), as the key secondary outcome, were assessed by DBPCFC to 5000 mg in the intention-to-treat population. Safety and immunological parameters were assessed in the same population. This trial is registered on ClinicalTrials.gov, NCT03345160. FINDINGS: Between Aug 13, 2013, and Oct 1, 2015, 146 children, with a median age of 39·3 months (IQR 30·8-44·7), were randomly assigned to receive peanut oral immunotherapy (96 participants) or placebo (50 participants). At week 134, 68 (71%, 95% CI 61-80) of 96 participants who received peanut oral immunotherapy compared with one (2%, 0·05-11) of 50 who received placebo met the primary outcome of desensitisation (risk difference [RD] 69%, 95% CI 59-79; p<0·0001). The median cumulative tolerated dose during the week 134 DBPCFC was 5005 mg (IQR 3755-5005) for peanut oral immunotherapy versus 5 mg (0-105) for placebo (p<0·0001). After avoidance, 20 (21%, 95% CI 13-30) of 96 participants receiving peanut oral immunotherapy compared with one (2%, 0·05-11) of 50 receiving placebo met remission criteria (RD 19%, 95% CI 10-28; p=0·0021). The median cumulative tolerated dose during the week 160 DBPCFC was 755 mg (IQR 0-2755) for peanut oral immunotherapy and 0 mg (0-55) for placebo (p<0·0001). A significant proportion of participants receiving peanut oral immunotherapy who passed the 5000 mg DBPCFC at week 134 could no longer tolerate 5000 mg at week 160 (p<0·001). The participant receiving placebo who was desensitised at week 134 also achieved remission at week 160. Compared with placebo, peanut oral immunotherapy decreased peanut-specific and Ara h2-specific IgE, skin prick test, and basophil activation, and increased peanut-specific and Ara h2-specific IgG4 at weeks 134 and 160. By use of multivariable regression analysis of participants receiving peanut oral immunotherapy, younger age and lower baseline peanut-specific IgE was predictive of remission. Most participants (98% with peanut oral immunotherapy vs 80% with placebo) had at least one oral immunotherapy dosing reaction, predominantly mild to moderate and occurring more frequently in participants receiving peanut oral immunotherapy. 35 oral immunotherapy dosing events with moderate symptoms were treated with epinephrine in 21 participants receiving peanut oral immunotherapy. INTERPRETATION: In children with a peanut allergy, initiation of peanut oral immunotherapy before age 4 years was associated with an increase in both desensitisation and remission. Development of remission correlated with immunological biomarkers. The outcomes suggest a window of opportunity at a young age for intervention to induce remission of peanut allergy. FUNDING: National Institute of Allergy and Infectious Disease, Immune Tolerance Network.

Tick salivary gland extract induces alpha-gal syndrome in alpha-gal deficient mice.

INTRODUCTION: Alpha-gal syndrome (AGS) is characterized by delayed hypersensitivity to non-primate mammalian meat in people having specific immunoglobulin E (sIgE) to the oligosaccharide galactose-alpha-1,3-galactose. AGS has been linked to tick bites from Amblyomma americanum (Aa) in the U.S. A small animal model of meat allergy is needed to study the mechanism of alpha-gal sensitization, the effector phase leading to delayed allergic responses and potential therapeutics to treat AGS. METHODS: Eight- to ten-weeks old mice with a targeted inactivation of alpha-1,3-galactosyltransferase (AGKO) were injected intradermally with 50 µg of Aa tick salivary gland extract (TSGE) on days 0, 7, 21, 28, 42, and 49. Total IgE and alpha-gal sIgE were quantitated on Day 56 by enzyme-linked immunosorbent assay. Mice were challenged orally with 400 mg of cooked pork kidney homogenate or pork fat. Reaction severity was assessed by measuring a drop in core body temperature and scoring allergic signs. RESULTS: Compared to control animals, mice treated with TSGE had 190-fold higher total IgE on Day 56 (0.60 ± 0.12 ng/ml vs. 113.2 ± 24.77 ng/ml; p < 0.001). Alpha-gal sIgE was also produced in AGKO mice following TSGE sensitization (undetected vs. 158.4 ± 72.43 pg/ml). Further, sensitized mice displayed moderate clinical allergic signs along with a drop in core body temperature of ≥2°C as an objective measure of a systemic allergic reaction. Interestingly, female mice had higher total IgE responses to TSGE treatment but male mice had larger declines in mean body temperature. CONCLUSION: TSGE-sensitized AGKO mice generate sIgE to alpha-gal and demonstrate characteristic allergic responses to pork fat and pork kidney. In keeping with the AGS responses documented in humans, mice reacted more rapidly to organ meat than to high fat pork challenge. This mouse model establishes the central role of tick bites in the development of AGS and provides a small animal model to mechanistically study mammalian meat allergy.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

Hypoallergenic Proteins for the Treatment of Food Allergy.

PURPOSE OF REVIEW: Food allergy is a growing health problem worldwide that impacts millions of individuals. Current treatment options are limited and strict dietary avoidance remains the standard of care. Immunotherapy using whole, native allergens is under active clinical investigation but harbors the risk of severe side effects including anaphylaxis. Newer food-specific therapies with hypoallergenic proteins may potentially offer safer treatment alternatives, and this review seeks to investigate the evidence supporting the use of these modalities. RECENT FINDINGS: The utilization of different methods to alter allergen structure and IgE binding leads to reduced allergenicity and decreases the risk for systemic reactions, making the use of potential therapies including extensively heated egg/milk, peptide immunotherapy, recombinant allergen immunotherapy, and DNA vaccines safe and possibly efficacious forms of treatment in food allergy. However, for the majority of these treatment modalities, limited data currently exists looking at the safety and efficacy in human subjects with food allergy. This review provides a comprehensive overview of the current evidence examining the safety and efficacy of hypoallergenic proteins in the treatment of food allergies.

Antigenic Liposomes for Generation of Disease-specific Antibodies.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a naïve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Characterization of the B-cell receptor repertoires in peanut allergic subjects undergoing oral immunotherapy.

B-cell receptors (BCRs) play a critical role in adaptive immunity as they generate highly diverse immunoglobulin repertoires to recognize a wide variety of antigens. To better understand immune responses, it is critically important to establish a quantitative and rapid method to analyze BCR repertoire comprehensively. Here, we developed "Bcrip", a novel approach to characterize BCR repertoire by sequencing millions of BCR cDNA using next-generation sequencer. Using this method and quantitative real-time PCR, we analyzed expression levels and repertoires of BCRs in a total of 17 peanut allergic subjects' peripheral blood samples before and after receiving oral immunotherapy (OIT) or placebo. By our methods, we successfully identified all of variable (V), joining (J), and constant (C) regions, in an average of 79.1% of total reads and 99.6% of these VJC-mapped reads contained the C region corresponding to the isotypes that we aimed to analyze. In the 17 peanut allergic subjects' peripheral blood samples, we observed an oligoclonal enrichment of certain immunoglobulin heavy chain alpha (IGHA) and IGH gamma (IGHG) clones (P = 0.034 and P = 0.027, respectively) in peanut allergic subjects after OIT. This newly developed BCR sequencing and analysis method can be applied to investigate B-cell repertoires in various research areas, including food allergies as well as autoimmune and infectious diseases.

Mast cell desensitization inhibits calcium flux and aberrantly remodels actin.

Rush desensitization (DS) is a widely used and effective clinical strategy for the rapid inhibition of IgE-mediated anaphylactic responses. However, the cellular targets and underlying mechanisms behind this process remain unclear. Recent studies have implicated mast cells (MCs) as the primary target cells for DS. Here, we developed a murine model of passive anaphylaxis with demonstrated MC involvement and an in vitro assay to evaluate the effect of DS on MCs. In contrast with previous reports, we determined that functional IgE remains on the cell surface of desensitized MCs following DS. Despite notable reductions in MC degranulation following DS, the high-affinity IgE receptor FcεRI was still capable of transducing signals in desensitized MCs. Additionally, we found that displacement of the actin cytoskeleton and its continued association with FcεRI impede the capacity of desensitized MCs to evoke the calcium response that is essential for MC degranulation. Together, these findings suggest that reduced degranulation responses in desensitized MCs arise from aberrant actin remodeling, providing insights that may lead to improvement of DS treatments for anaphylactic responses.

Peanut Allergy: New Developments and Clinical Implications.

Food allergies have increased in prevalence over the past 20 years, now becoming an important public health concern. Although there are no therapies currently available for routine clinical care, recent reports have indicated that immunotherapies targeting the mucosal immune system may be effective. Oral immunotherapy is conducted by administering small, increasing amounts of food allergen; it has shown promise for desensitizing individuals with peanut, egg, or milk allergies. Sublingual immunotherapy also desensitizes allergic patients to foods-two major studies have examined the effects of sublingual immunotherapy in subjects with peanut allergies. We review the complex nature of IgE-mediated food allergies and the therapies being evaluated in clinical trials. We focus on the diagnosis and management of food allergies and investigational therapies.

Diagnosis, management, and investigational therapies for food allergies.

Food allergies have increased in prevalence over the past 20 years, now becoming an important public health concern. Although there are no therapies currently available for routine clinical care, recent reports have indicated that immunotherapies targeting the mucosal immune system may be effective. Oral immunotherapy is conducted by administering small, increasing amounts of food allergen; it has shown promise for desensitizing individuals with peanut, egg, or milk allergies. Sublingual immunotherapy also desensitizes allergic patients to foods-2 major studies have examined the effects of sublingual immunotherapy in subjects with peanut allergies. We review the complex nature of IgE-mediated food allergies and the therapies being evaluated in clinical trials. We focus on the diagnosis and management of food allergies and investigational therapies.

Novel strategy to create hypoallergenic peanut protein-polyphenol edible matrices for oral immunotherapy.

Peanut allergy is an IgE-mediated hypersensitivity. Upon peanut consumption by an allergic individual, epitopes on peanut proteins bind and cross-link peanut-specific IgE on mast cell and basophil surfaces triggering the cells to release inflammatory mediators responsible for allergic reactions. Polyphenolic phytochemicals have high affinity to bind proteins and form soluble and insoluble complexes with unique functionality. This study investigated the allergenicity of polyphenol-fortified peanut matrices prepared by complexing various polyphenol-rich plant juices and extracts with peanut flour. Polyphenol-fortified peanut matrices reduced IgE binding to one or more peanut allergens (Ara h 1, Ara h 2, Ara h 3, and Ara h 6). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) suggested changes in secondary protein structure. Peanut protein-cranberry polyphenol fortified matrices triggered significantly less basophil degranulation than unmodified flour in an ex vivo assay using human blood and less mast cell degranulation when used to orally challenge peanut-allergic mice. Polyphenol fortification of peanut flour resulted in a hypoallergenic matrix with reduced IgE binding and degranulation capacity, likely due to changes in protein secondary structure or masking of epitopes, suggesting potential applications for oral immunotherapy.

Type B CpG oligodeoxynucleotides induce Th1 responses to peanut antigens: modulation of sensitization and utility in a truncated immunotherapy regimen in mice.

SCOPE: Peanut allergy stems from a Th2-biased immune response to peanut allergens leading to IgE production and allergic reactions upon ingestion. METHODS AND RESULTS: A model of peanut allergy in C3H/HeJ mice was used to assess whether type A, B, or C CpG oligodeoxynucleotide (ODN) molecules would be effective in: (i) a prophylactic approach to prevent peanut allergy when administered simultaneously with a Th2-skewing adjuvant, and (ii) a therapeutic model to allow for shortened immunotherapy. Type B ODNs were extremely effective in inhibiting anaphylaxis in the sensitization protocol as evidenced by differences in symptom scores, body temperature, and mouse mast cell protease 1 release compared to sham treatment. In the therapeutic model, co-administration of type B ODN plus peanut proteins was highly effective in reducing anaphylactic reactions in mice with established peanut allergy. The therapeutic effect was accompanied by an increase in IFN-γ and peanut-IgG2a, without a significant decrease in peanut IgE or IL-4 responses. CONCLUSION: CpG ODNs, especially type B, were highly effective in inducing Th1 responses in mice undergoing induction of peanut allergy, as well as in mice undergoing therapy for established peanut allergy. Interestingly, the IgE response was not significantly altered, suggesting that IgG antibodies may be enough to prevent peanut-induced anaphylaxis.

Oral immunotherapy for food allergy: clinical and preclinical studies.

Food allergies affect approximately 5% of the U.S. population and have increased in the last decade. In recent years, oral immunotherapy (OIT) has been tested in clinical trials for peanut, milk, and egg allergies in young children. OIT appears to be fairly well tolerated by most subjects and leads to desensitization with a greatly increased threshold of allergen required to induce reactions. Further approaches being investigated in preclinical studies in mouse models indicate the potential for using adjuvants, such as TLR9 agonists in combination with OIT; peptide OIT; and non-allergen specific applications such as herbal formulations. Further questions about OIT remain, including the optimal dosing and length of treatment; whether tolerance can be developed; and the exact cellular mechanisms resulting in protection following OIT. With many clinical trials underway across the United States and other countries, and a growing pipeline of preclinical research with translational potential, there is great hope for a widely applicable food allergy treatment.

Pepsinized cashew proteins are hypoallergenic and immunogenic and provide effective immunotherapy in mice with cashew allergy.

BACKGROUND: IgE-mediated allergic reactions to cashews and other nuts can trigger life-threatening anaphylaxis. Proactive therapies to decrease reaction severity do not exist. OBJECTIVES: We aimed to determine the efficacy of pepsin-digested cashew proteins used as immunotherapy in a murine model of cashew allergy. METHODS: Mice were sensitized to cashew and then underwent challenges with digested or native cashew allergens to assess the allergenicity of the protein preparations. Using native or pepsinized cashew proteins, mice underwent oral or intraperitoneal sensitization protocols to determine the immunogenic properties of the protein preparations. Finally, cashew-sensitized mice underwent an immunotherapy protocol with native or pepsinized cashew proteins and subsequent provocation challenges. RESULTS: Pepsinized cashew proteins elicited weaker allergic reactions than native cashew proteins but importantly retained the ability to stimulate cellular proliferation and cytokine production. Mice sensitized with pepsinized proteins reacted on challenge with native allergens, demonstrating that pepsinized allergens retain immunogenicity in vivo. Immunotherapy with pepsinized cashew allergens significantly decreased allergic symptoms and body temperature decrease relative to placebo after challenge with native and pepsinized proteins. Immunologic changes were comparable after immunotherapy with native or pepsinized allergens: T(H)2-type cytokine secretion from splenocytes was decreased, whereas specific IgG(1) and IgG(2a) levels were increased. CONCLUSIONS: Pepsinized cashew proteins are effective in treating cashew allergy in mice and appear to work through the same mechanisms as native protein immunotherapy.

A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response.

BACKGROUND: Open-label oral immunotherapy (OIT) protocols have been used to treat small numbers of patients with peanut allergy. Peanut OIT has not been evaluated in double-blind, placebo-controlled trials. OBJECTIVE: To investigate the safety and effectiveness of OIT for peanut allergy in a double-blind, placebo-controlled study. METHODS: In this multicenter study, children ages 1 to 16 years with peanut allergy received OIT with peanut flour or placebo. Initial escalation, build-up, and maintenance phases were followed by an oral food challenge (OFC) at approximately 1 year. Titrated skin prick tests (SPTs) and laboratory studies were performed at regular intervals. RESULTS: Twenty-eight subjects were enrolled in the study. Three peanut OIT subjects withdrew early in the study because of allergic side effects. During the double-blind, placebo-controlled food challenge, all remaining peanut OIT subjects (n = 16) ingested the maximum cumulative dose of 5000 mg (approximately 20 peanuts), whereas placebo subjects (n = 9) ingested a median cumulative dose of 280 mg (range, 0-1900 mg; P < .001). In contrast with the placebo group, the peanut OIT group showed reductions in SPT size (P < .001), IL-5 (P = .01), and IL-13 (P = .02) and increases in peanut-specific IgG(4) (P < .001). Peanut OIT subjects had initial increases in peanut-specific IgE (P < .01) but did not show significant change from baseline by the time of OFC. The ratio of forkhead box protein 3 (FoxP3)(hi): FoxP3(intermediate) CD4+ CD25+ T cells increased at the time of OFC (P = .04) in peanut OIT subjects. CONCLUSION: These results conclusively demonstrate that peanut OIT induces desensitization and concurrent immune modulation. The current study continues and is evaluating the hypothesis that peanut OIT causes long-term immune tolerance.

Single-tree nut immunotherapy attenuates allergic reactions in mice with hypersensitivity to multiple tree nuts.

BACKGROUND: Allergic reactions to tree nuts are often severe and are outgrown in less than 10% of diagnosed patients. OBJECTIVES: To determine whether treatment of underlying tree nut sensitization will prevent allergic reactions to cross-reacting tree nuts and to determine the effects of single-tree nut immunotherapy on true multi-tree nut sensitization. METHODS: Cross-reactivity model: Cashew-sensitized mice underwent immunotherapy with cashew and were subsequently challenged with cashew and pistachio. Multisensitization model: Cashew plus walnut-sensitized mice were treated with cashew alone, walnut alone, or both cashew and walnut and then underwent challenges to cashew and walnut. Challenges were assessed on the basis of symptoms, changes in body temperature, and mouse mast cell protease-1 release. RESULTS: In the cross-reactivity model, cashew immunotherapy completely prevented allergic reactions on challenges with cashew or the cross-reactive pistachio. In the multisensitization model, mice with cashew plus walnut allergy were significantly protected from anaphylactic reactions on cashew challenge in both the cashew-alone and walnut-alone immunotherapy groups. Results from the walnut challenge demonstrated significantly decreased allergic responses in the walnut immunotherapy group, whereas mice in the cashew immunotherapy group experienced significantly lower symptoms. In the cross-reactivity model, immunotherapy effectively decreased IL-4 and IL-5 production and increased IL-12 relative to placebo while also inducing a 5-fold increase in specific IgG(1). CONCLUSION: Single-tree nut immunotherapy can effectively decrease allergic responses in both the cross-reactivity and multisensitization mouse models. Further studies are needed to determine which single-tree nut immunotherapies will be most effective for specific multi-tree nut allergy profiles.