Quantification of Allergic Crustacean Tropomyosin Using Shared Signature Peptides in Processed Foods with a Mass Spectrometry-Based Proteomic Strategy.

Crustacean shellfish are major allergens in East Asia. In the present study, a major allergic protein in crustaceans, tropomyosin, was detected accurately using multiple reaction monitoring mode-based mass spectrometry, with shared signature peptides identified through proteomic analysis. The peptides were deliberately screened through thermal stability and enzymatic digestion efficiency to improve the suitability and accuracy of the developed method. Finally, the proposed method demonstrated a linear range of 0.15 to 30 mgTM/kgfood (R2 > 0.99), with a limit of detection of 0.15 mgTM/kg food and a limit of quantification of 0.5mgTM/kgfood and successfully applied to commercially processed foods, such as potato chips, biscuits, surimi, and hot pot seasonings, which evidenced the applicability of proteomics-based methodology for food allergen analysis.

T-Cell Epitope Immunotherapy in Mouse Models of Food Allergy.

Food allergy has been rising in prevalence over the last two decades, affecting more than 10% of the world population. Current management of IgE-mediated food allergy relies on avoidance and rescue medications; research into treatments that are safer and providing guaranteed and durable curative effects is, therefore, essential. T-cell epitope-based immunotherapy holds the potential for modulating food allergic responses without IgE cross-linking. In this chapter, we describe the methods in evaluating the therapeutic capacities of immunodominant T-cell epitopes in animal models of food allergy. Moreover, we explain in detail the methods to measure the allergen-specific antibody levels, prepare single-cell suspension from spleen, and prepare small intestine for immunohistochemical analysis of eosinophils and Foxp3+ cells.

Shrimp and cockroach co-sensitization in Southern China: Association with moth sensitization.

Background: Moth is a common allergen in southern China. Shrimp sensitization might be related to the moth allergen. Objective: This study investigated sensitization to moth allergen in patients in southern China sensitized to shrimp and explored the effect of moth sensitization on different allergic diseases. Methods: Serum samples from 212 patients sensitized to shrimp were tested for specific immunoglobulin E (sIgE) to Dermatophagoides pteronyssinus, crab, cockroach, and moth. Results: The patients sensitized to shrimp were co-sensitized to D. pteronyssinus (88.7%), crab (85.4%), cockroach (89.2%), and moth (92.0%). Overall, 75% of the patients sensitized to shrimp tested positive to the above allergens; only four patients were sensitized to shrimp alone. The median (interquartile range [IQR]) concentrations of sIgE to shrimp (2.66 kU/L [1.02-6.11 kU/L] versus 1.61 kU/L [0.70-3.67 kU/L]), crab (2.35 kU/L [0.83-4.18 kU/L] versus 1.30 kU/L [0.59-3.14 kU/L]), cockroach (3.78 kU/L [0.98-6.91 kU/L] versus 1.56 kU/L [0.85-3.17 kU/L]), and moth (4.70 kU/L [2.98-9.62 kU/L] versus 2.85 kU/L [1.16-7.01 kU/L]) in patients with skin allergic diseases was significantly higher than in patients with respiratory allergic diseases (all p < 0.05). The median (IQR) concentration of sIgE to cockroach in the young adults (2.33 kU/L [0.86-5.56 kU/L]) was the highest among all age groups as well as to moth (young adults: 4.14 kU/L [1.93-8.24 kU/L]). With the increasing positive class of shrimp allergen, the sIgE concentration of moth, cockroach, and crab also increased, and the optimal scaling analysis showed that the sIgE of crab, cockroach, and moth had a strong correlation with sIgE to shrimp (Cronbach α = 93.8%). Conclusion: This study found a high rate of co-sensitization between moth, D. pteronyssinus, cockroach, and crab among patients sensitized to shrimp and a strong correlation between shrimp, moth, and cockroach. Shrimp and cockroach co-sensitization might be related to moth allergens.

IgE-binding epitope mapping of tropomyosin allergen (Exo m 1) from Exopalaemon modestus, the freshwater Siberian prawn.

Exopalaemon modestus (EM) is a shrimp delicacy that could cause food allergy, the major allergen of EM is Exo m 1. The amino acid (AA) sequence, IgE-binding epitopes and allergenic peptides in gastrointestinal (GI) digests of Exo m 1, and their effects on basophil function were investigated. Exo m 1 has an AA-sequence of high similarity with other shrimp tropomyosins, while not 100% matching. The IgE-binding epitopes of Exo m 1 are epitope 1 (43-59, VHNLQKRMQQLENDLDS), epitope 2 (85-105, VAALNRRIQLLEEDLERSEER), epitope 3 (131-164, ENRSLSDEERMDALENQLKEARFLAEEADRKYDE), epitope 4 (187-201, ESKIVELEEELRVVG) and epitope 5 (243-280, ERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSE). Among the thirty-three peptides of Exo m 1 identified in GI digests, two were highly recognized by IgE, twenty-four moderately or weakly bound IgE, and seven had no IgE-reactivities. These IgE-binding epitopes and GI digestion induced-allergenic peptides could activate basophil degranulation, and CD63 and CD203c expression, they could be potential peptide-based immunotherapy for shrimp allergic individuals.

Homologous tropomyosins from vertebrate and invertebrate: Recombinant calibrator proteins in functional biological assays for tropomyosin allergenicity assessment of novel animal foods.

BACKGROUND: Novel foods may provide new protein sources for a growing world population but entail risks of unexpected food-allergic reactions. No guidance on allergenicity assessment of novel foods exists, while for genetically modified (GM) crops it includes comparison of sequence identity with known allergens, digestibility tests and IgE serum screening. OBJECTIVE: As a proof of concept, to evaluate non-/allergenic tropomyosins (TMs) regarding their potential as new calibrator proteins in functional biological in vitro assays for the semi-quantitative allergy risk assessment of novel TM-containing animal foods with mealworm TM as an example. METHODS: Purified TMs (shrimp, Penaeus monodon; chicken Gallus gallus; E coli overexpression) were compared by protein sequencing, circular dichroism analysis and in vitro digestion. IgE binding was quantified using shrimp-allergic patients' sera (ELISA). Biological activities were investigated (skin testing; titrated basophil activation tests, BAT), compared to titrated biological mediator release using humanized rat basophil leukaemia (RBL) cells. RESULTS: Shrimp and chicken TMs showed high sequence homology, both alpha-helical structures and thermal stability. Shrimp TM was stable during in vitro gastric digestion, chicken TM degraded quickly. Both TMs bound specific IgE from shrimp-allergic patients (significantly higher for shrimp TM), whereas skin reactivity was mostly positive with only shrimp TM. BAT and RBL cell assays were positive with shrimp and chicken TM, although at up to 100- to 1000-times lower allergen concentrations for shrimp than chicken TM. In RBL cell assays using both TM as calibrators, an activation of effector cells by mealworm TM similar to that by shrimp TM confirmed the already reported high allergenic potency of mealworm TM as a novel protein source. CONCLUSIONS & CLINICAL RELEVANCE: According to current GM crops' allergenicity assessment, non-allergenic chicken TM could falsely be considered an allergen on a weight-of-evidence approach. However, calibrating allergenic potency in functional BAT and RBL cell assays with clinically validated TMs allowed for semi-quantitative discrimination of novel food protein's allergenicity. With TM calibration as a proof of concept, similar systems of homologous protein might be developed to scale on an axis of allergenicity.

Modulating Shrimp Tropomyosin-Mediated Allergy: Hypoallergen DNA Vaccines Induce Regulatory T Cells to Reduce Hypersensitivity in Mouse Model.

Shellfish allergy is one of the most common food allergies, with tropomyosin as the major cross-reactive allergen. However, no allergen-specific immunotherapy is clinically available. Recently, we designed two shrimp hypoallergens MEM49 and MED171. This study aimed to examine and compare the efficacy of the MEM49- and MED171-based DNA vaccines (pMEM49 and pMED171) in modulating shrimp allergy in a murine model of shrimp tropomyosin sensitivity. Intradermal immunization of BALB/c mice with pMEM49 or pMED171 effectively down-modulated allergic symptoms, tropomyosin-specific IgE levels, intestinal Th2 cytokines expression, and inflammatory cell infiltration. Both pMEM49 and pMED171 increased the frequency of regulatory T cells, but to a greater extent by pMED171 with upregulation of gut-homing molecules integrin-α4ß7. The functionality of the pMED171-induced Treg cells was further illustrated by anti-CD25-mediated depletion of Treg cells and the adoptive transfer of CD4+CD25+Foxp3+Treg cells. Collectively, the data demonstrate that intradermal administration of pMED171 leads to the priming, activation, and migration of dermal dendritic cells which subsequently induce Treg cells, both locally and systemically, to downregulate the allergic responses to tropomyosin. This study is the first to demonstrate the potency of hypoallergen-encoding DNA vaccines as a therapeutic strategy for human shellfish allergy via the vigorous induction of functional Treg cells.

Allergenicity of acrolein-treated shrimp tropomyosin evaluated using RBL-2H3 cell and mouse model.

BACKGROUND: Food processing effects can modify protein functional properties. However, protein was oxidized inevitably by lipid peroxidation during food processing. Acrolein, a primary by-product of lipid peroxidation, can modify the structural and functional properties of protein. The aim of the research was to analyze the effect of acrolein on allergenicity of TM, a major allergen in shrimp. RESULTS: The overall allergenic effects of acrolein-treated TM were evaluated using female BALB/c mice and a mediator-releasing RBL-2H3 cell line. Acrolein-treated TM significantly decreased TM-specific immunoglobulin E/G1 levels, and histamine and mMCP-1 release in mouse serum. Release of inflammatory mediators such as ß-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 was clearly suppressed after acrolein treatment. CONCLUSION: These results indicate that acrolein-induced tropomyosin modification can decrease the allergenicity of TM. This reduction contributes to allergenic potential changes in shrimp during processing and preservation. © 2018 Society of Chemical Industry.

Assessment of the sensitizing capacity and allergenicity of enzymatic cross-linked arginine kinase, the crab allergen.

SCOPE: The enzymatic cross-linking of an allergen by food processing may alter its sensitization potential. In this study, the IgE-binding activity and allergenicity of cross-linked thermal polymerized arginine kinase (CL-pAK) were investigated. METHODS AND RESULTS: The IgE-binding activity and stability of CL-pAK were analyzed by immunological and proteomics methods. The sensitization and potency to induce oral tolerance of CL-pAK were tested using in vivo assays and a cell model. According to the results of inhibition of ELISA, the half inhibitory concentration of AK after cross-linking changed from 1.13 to 228.36 µg/mL. The results of in vitro digestion demonstrated that CL-pAK showed more resistance to gastrointestinal digestion than native AK. Low allergenicity and capacity to induce oral tolerance in mice were shown by the sera levels of AK-specific antibodies and T-cell cytokine production. Exposure of RBL-2H3 cells to CL-pAK compared with AK, resulted in lower levels of mast degranulation and histamine. CONCLUSION: Enzymatic cross-linking with thermal polymerization of AK by tyrosinase and caffeic acid had high potential in mitigating IgE-binding activity and allergenicity, which were influenced by altering the molecular and immunological features of the shellfish protein.