Use of a Liver-Targeting Immune-Tolerogenic mRNA Lipid Nanoparticle Platform to Treat Peanut-Induced Anaphylaxis by Single- and Multiple-Epitope Nucleotide Sequence Delivery
While oral desensitization is capable of alleviating peanut allergen anaphylaxis, long-term immune tolerance is the sought-after goal. We developed a liver-targeting lipid nanoparticle (LNP) platform to deliver mRNA-encoded peanut allergen epitopes to liver sinusoidal endothelial cells (LSECs), which function as robust tolerogenic antigen-presenting cells that induce FoxP3+ regulatory T-cells (Tregs). The mRNA strand was constructed by including nucleotide sequences encoding for nonallergenic MHC-II binding T-cell epitopes, identified in the dominant peanut allergen, Ara h2. These epitopes were inserted in the mRNA strand downstream of an MHC-II targeting sequence, further endowed in vitro with 5′ and 3′ capping sequences, a PolyA tail, and uridine substitution. Codon-optimized mRNA was used for microfluidics synthesis of LNPs with an ionizable cationic lipid, also decorated with a lipid-anchored mannose ligand for LSEC targeting. Biodistribution to the liver was confirmed by in vivo imaging, while ELISpot assays demonstrated an increase in IL-10-producing Tregs in the spleen. Prophylactic administration of tandem-repeat or a combination of encapsulated Ara h2 epitopes induced robust tolerogenic effects in C3H/HeJ mice, sensitized to and subsequently challenged with crude peanut allergen extract. In addition to alleviating physical manifestations of anaphylaxis, there was suppression of Th2-mediated cytokine production, IgE synthesis, and mast cell release, accompanied by increased IL-10 and TGF-β production in the peritoneum. Similar efficacy was demonstrated during LNP administration postsensitization. While nondecorated particles had lesser but significant effects, PolyA/LNP-Man lacked protective effects. These results demonstrate an exciting application of mRNA/LNP for treatment of food allergen anaphylaxis, with the promise to be widely applicable to the allergy field. https://doi.org/10.1021/acsnano.2c12420
The protective effect of moderate maternal peanut consumption on peanut sensitization and allergy
Background: The Learning Early About Peanut Allergy or LEAP trial found that the early introduction of peanuts in the diet of infants at risk for peanut allergies prevents peanut allergy. The effect of maternal consumption of peanuts on subsequent peanut sensitization or peanut allergy in the LEAP trial has not been studied to date. Objective: To determine whether maternal consumption of peanut protein while breastfeeding protects against peanut-allergic outcomes in the absence of peanut consumption in infants. Methods: We performed an analysis of the data from the peanut avoidance arm of the LEAP study to discern the effects of maternal consumption of peanuts while pregnant and breastfeeding on an infant's peanut-allergic outcomes. Results: Of the 303 infants in the avoidance group, 31 mothers consumed more than 5 g of peanut per week, 69 consumed less than 5 g of peanut per week and 181 did not consume peanut while breastfeeding. Peanut sensitization (P = .03) and peanut allergy (P = .07) occurred less frequently in infants whose mothers consumed a moderate amount of peanuts while breastfeeding when compared with those who either did not consume peanuts while breastfeeding or those who consumed a large amount of peanuts when breastfeeding. Ethnicity (odds ratio [OR], 0.47; P = .046, 95% confidence interval [CI], 0.22-0.99), baseline peanut skin prick test stratum (OR, 4.87; P < .001, 95% CI, 2.13-11.12), no maternal peanut consumption while breastfeeding (OR, 3.25; P = .008, 95% CI, 1.36-7.77), and baseline SCORing Atopic Dermatitis greater than 40 (OR, 2.78; P = .007, 95% CI, 1.32-5.85) were all significant contributors to peanut sensitization or allergy at 60 months of age. Conclusion: Moderate consumption (<5 grams per week) of peanuts while breastfeeding provides a significant protective effect against peanut sensitization and a noticeable but not statistically significant protective effect against peanut allergy later on in life in high-risk infants in the context of delayed peanut introduction. https://doi.org/10.1016/j.anai.2023.04.012
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. https://doi.org/10.1016/j.pep.2022.106211
Cross-Serological Reaction of Glandless Cottonseed Proteins to Peanut and Tree Nut Allergic IgE.
Food allergy is a potentially life-threatening health concern caused by immunoglobulin E (IgE) antibodies that mistakenly recognize normally harmless food proteins as threats. Peanuts and tree nuts contain several seed storage proteins that commonly act as allergens. Glandless cottonseed, lacking the toxic compound gossypol, is a new food source. However, the seed storage proteins in cottonseed may act as allergens. To assess this risk, glandless cottonseed protein extracts were evaluated for IgE binding by peanut and tree nut allergic volunteers. ELISA demonstrated that 25% of 32 samples had significant binding to cottonseed extracts. Immunoblot analysis with pooled sera indicated that IgE recognized a pair of bands migrating at approximately 50 kDa. Excision of these bands and subsequent mass-spectrometric analysis demonstrated peptide matches to cotton C72 and GC72 vicilin and legumin A and B proteins. Further, in silico analysis indicated similarity of the cotton vicilin and legumin proteins to peanut vicilin (Ara h 1) and cashew nut legumin (Ana o 2) IgE-binding epitopes among others. The observations suggest both the cotton vicilin and legumin proteins were recognized by the nut allergic IgE, and they should be considered for future allergen risk assessments evaluating glandless cottonseed protein products. https://doi.org/10.3390/molecules28041587
Identification and Structure of Epitopes on Cashew Allergens Ana o 2 and Ana o 3 Using Phage Display.
Background: Cashew (Anacardium occidentale L.) is a commercially important plant. Cashew nuts are a popular food source that belong to the tree nut family. Tree nuts are one of the eight major food allergens identified by the Food and Drug Administration in the USA. Allergies to cashew nuts cause severe and systemic immune reactions. Tree nut allergies are frequently fatal and are becoming more common. Aim: We aimed to identify the key allergenic epitopes of cashew nut proteins by correlating the phage display epitope prediction results with bioinformatics analysis. Design: We predicted and experimentally confirmed cashew nut allergen antigenic peptides, which we named Ana o 2 (cupin superfamily) and Ana o 3 (prolamin superfamily). The Ana o 2 and Ana o 3 epitopes were predicted using DNAstar and PyMoL (incorporated in the Swiss-model package). The predicted weak and strong epitopes were synthesized as peptides. The related phage library was built. The peptides were also tested using phage display technology. The expressed antigens were tested and confirmed using microtiter plates coated with pooled human sera from patients with cashew nut allergies or healthy controls. Results: The Ana o 2 epitopes were represented by four linear peptides, with the epitopes corresponding to amino acids 108-111, 113-119, 181-186, and 218-224. Furthermore, the identified Ana o 3 epitopes corresponding to amino acids 10-24, 13-27, 39-49, 66-70, 101-106, 107-114, and 115-122 were also screened out and chosen as the key allergenic epitopes. Discussion: The Ana o 3 epitopes accounted for more than 40% of the total amino acid sequence of the protein; thus, Ana o 3 is potentially more allergenic than Ana o 2. Conclusions: The bioinformatic epitope prediction produced subpar results in this study. Furthermore, the phage display method was extremely effective in identifying the allergenic epitopes of cashew nut proteins. The key allergenic epitopes were chosen, providing important information for the study of cashew nut allergens. https://doi.org/10.3390/molecules28041880
TreEAT trial: Protocol for a randomized controlled trial investigating the efficacy and safety of early introduction of tree nuts for the prevention of tree nut allergy in infants with peanut allergy.
Introduction: Children with peanut allergy are at increased risk of developing tree nut allergies, which can be severe and for most lifelong. Introduction of peanut in the first year of life can reduce the risk of peanut allergy; however, prevention strategies for tree nut allergies have not been established. We aimed to test the efficacy and safety of a novel strategy, a supervised multi-nut oral food challenge (OFC) compared with standard care for tree nut allergy prevention in infants at high risk of developing tree nut allergy, TreEAT. Methods and analysis: TreEAT is a 2-armed, open-label, randomized, controlled trial (RCT). Infants (n = 212) aged 4-11 months with peanut allergy will be randomized 1:1 at peanut allergy diagnosis to either a hospital-based multi-tree nut (almond, cashew, hazelnut, and walnut) OFC using multi-nut butter or standard care (home introduction of individual tree nuts). All infants will be assessed at age 18 months, with questionnaires and SPT to peanut and tree nuts. Peanut and tree nut OFCs will be performed as required to determine the allergy status for each nut. The primary outcome is tree nut allergy at age 18 months. Secondary outcomes include peanut allergy resolution, proportion, and severity of adverse events related to tree nut ingestion, number and frequency of tree nuts ingested, quality of life and parental anxiety, and allergy-related healthcare visits from randomization to 18 months of age. Analyses will be performed on an intention-to-treat basis. Ethics and dissemination: TreEAT was approved by the Royal Children's Hospital Human Research Ethics Committee (#70489). Outcomes will be presented at scientific conferences and disseminated through publication. https://doi.org/10.1111/pai.13930
Natural History of Hazelnut Allergy and Current Approach to Its Diagnosis and Treatment.
Hazelnut allergy is the most prevalent type of nut allergy in Europe, with symptoms that can range from mild, such as hives and itching, to severe, such as anaphylaxis, particularly in patients who are sensitized to highly stable allergens, such as storage proteins. Compared to other types of food allergies, allergies to tree nuts, including hazelnuts, tend to persist throughout life. Although symptoms can appear in early childhood, they often continue into adulthood, with a minority of cases improving during adolescence. Currently, there is no curative treatment available for hazelnut allergy, and patients must adhere to a restrictive diet and carry autoinjective epinephrine. However, oral allergen immunotherapy (AIT) is a promising treatment option. Patients can be categorized based on their risk for severe reactions using various clinical, in vivo, and in vitro tests, including component-resolved diagnosis and oral food challenge. This review aims to provide an overview of the current knowledge of the natural history of hazelnut allergy and new approaches for its diagnosis and management. https://doi.org/10.3390/children10030585
Peanut Allergenicity: An Insight into Its Mitigation Using Thermomechanical Processing.
Peanuts are the seeds of a legume crop grown for nuts and oil production. Peanut allergy has gained significant attention as a public health issue due to its increasing prevalence, high rate of sensitization, severity of the corresponding allergic symptoms, cross-reactivity with other food allergens, and lifelong persistence. Given the importance of peanuts in several sectors, and taking into consideration the criticality of their high allergic potential, strategies aiming at mitigating their allergenicity are urgently needed. In this regard, most of the processing methods used to treat peanuts are categorized as either thermal or thermomechanical techniques. The purpose of this review is to provide the reader with an updated outlook of the peanut’s allergens, their mechanisms of action, the processing methods as applied to whole peanuts, as well as a critical insight on their impact on the allergenicity. The methods discussed include boiling, roasting/baking, microwaving, ultrasonication, frying, and high-pressure steaming/autoclaving. Their effectiveness in alleviating the allergenicity, and their capacity in preserving the structural integrity of the treated peanuts, were thoroughly explored. Research data on this matter may open further perspectives for future relevant investigation ultimately aiming at producing hypoallergenic peanuts. https://doi.org/10.3390/foods12061253
Open-label study of the efficacy, safety and durability of peanut sublingual immunotherapy in peanut-allergic children.
Background: Studies are limited on the efficacy of peanut sublingual immunotherapy (SLIT). The durability of desensitization after SLIT has not been well described. Objective: To evaluate the efficacy and safety of 4 mg peanut SLIT and persistence of desensitization after SLIT discontinuation. Methods: Challenge proven peanut-allergic 1-11 year old children were treated with open-label 4 mg peanut SLIT for 48 months. Desensitization after peanut SLIT was assessed by 5000 mg double-blind, placebo-controlled food challenge (DBPCFC). A novel randomly assigned avoidance period between 1-17 weeks was followed by a DBPCFC. Skin prick testing (SPT), immunoglobulins, basophil activation testing (BAT), TH1, TH2, and IL-10 cytokines were measured longitudinally. Safety was assessed through patient-reported home diaries. Results: Fifty-four participants were enrolled and 47 (87%) completed peanut SLIT and the 48-month DBPCFC per protocol. Mean successfully consumed dose (SCD) during DBPCFC increased from 48 mg to 2723 mg peanut protein after SLIT (p<0.0001), with 70% achieving clinically significant desensitization (SCD >800 mg) and 36% full desensitization (SCD = 5000 mg). Modeled median time to loss of clinically significant desensitization was 22 weeks. Peanut SPT; peanut-specific IgE, IgG4, IgG4/IgE ratio; and peanut-stimulated BAT, IL-4, IL-5, IL-13, IFN-gamma, and IL-10 changed significantly compared to baseline with changes seen as early as 6 months. Median rate of reaction per dose was 0.5% with transient oropharyngeal itching most common and no dosing symptoms requiring epinephrine. Conclusion: In this open-label, prospective study, peanut SLIT was safe and induced clinically significant desensitization in the majority of children lasting more than 17 weeks after discontinuation of therapy. https://doi.org/10.1016/j.jaci.2023.01.036
Mitigating the allergenicity of peanut allergen Ara h 1 by cold atmospheric pressure argon plasma jet.
Background: Peanut allergy is recognized as a major food allergy that triggers severe and even fatal symptoms. Avoidance of peanuts in the diet is the main option for current safety management. Processing techniques reducing peanut allergenicity are required to develop other options. Cold plasma is currently considered as a novel non-thermal approach to alter protein structure and has the potential to alleviate immunoreactivity of protein allergen. Results: The application of a cold argon plasma jet to peanut protein extract could reduce the amount of a 64 kDa protein band corresponding to a major peanut allergen Ara h 1 using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but the overall protein size distribution did not change significantly. A decrease in peanut protein solubility was a possible cause that led to the loss of protein content in the soluble fraction. Immunoblotting and enzyme-linked immunosorbent assay elucidated that the immunoreactivity of Ara h 1 was significantly decreased with the time treated with plasma. Ara h 1 antigenicity reduced by 38% after five scans (approximately 3 min) of cold argon plasma jet treatment, and the reduction was up to 66% after approximately 15 min of treatment. Conclusion: The results indicate that cold argon plasma jet treatment could be a suitable platform for alleviating the immunoreactivity of peanut protein. This work demonstrates an efficient, compact, and rapid platform for mitigating the allergenicity of peanuts, and shows great potential for the plasma platform as a non-thermal technique in the food industry. © 2023 Society of Chemical Industry. https://doi.org/10.1002/jsfa.12454