Allergenicity assessment of new or modified protein-containing food sources and ingredients.

The growing world population, changing dietary habits, and increasing pressure on agricultural resources are drivers for the development of novel foods (including new protein sources as well as existing protein sources that are produced or used in an alternative way or in a different concentration). These changes, coupled with consumer inclination to adopt new dietary trends, may heighten the intake of unfamiliar proteins, or escalate consumption of specific ones, potentially amplifying the prevalence of known and undiscovered food allergies. Assessing the allergenicity of novel or modified protein-based foods encounters several challenges, including uncertainty surrounding acceptable risks and assessment criteria for determining safety. Moreover, the available methodological tools for gathering supportive data exhibit significant gaps. This paper synthesises these challenges, addressing the varied interpretations of "safe" across jurisdictions and societal attitudes towards allergenic risk. It proposes a comprehensive two-part framework for allergenicity assessment: the first part emphasises systematic consideration of knowledge and data requirements, while the second part proposes the application of a generic assessment approach, integrating a Threshold of Allergological Concern. This combined framework highlights areas that require attention to bridge knowledge and data gaps, and it delineates research priorities for its development and implementation.

Parvalbumin Gene: A Valuable Marker for Pike Authentication and Allergen Risk Assessment.

Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.

Identifying Region-Specific Allergy Sensitization Clusters to Optimize Diagnosis and Reduce Costs.

OBJECTIVE: To delineate quantitatively the allergen sensitization patterns in a large pediatric cohort and inform the selection of a region-specific panel of allergen tests for timely and cost-effective in vitro atopy screening. STUDY DESIGN: IgE levels for specific allergens from patients in the Texas Children's Health System were analyzed retrospectively. Statistical and network analyses were conducted to reveal sensitization patterns. RESULTS: Network analysis of 114 distinct allergens among 12 065 patients identified 2 main groups of allergens: environmental and food. Approximately 67.5% of patients were sensitized to environmental allergens, 47.2% to food allergens, and 7.3% to at least 1 allergen from both groups. We identified a novel panel of 13 allergens that could detect sensitization in 95% of patients, whereas panels of 7 allergens within each category effectively identified sensitization in 95% of patients with specific sensitivities. This data-driven approach is estimated to reduce overall testing costs by 52%. In agreement with literature, we observed correlations among allergens within specific categories, such as pollen, shellfish, nuts, and dairy allergens. CONCLUSIONS: This study provides insights into allergen sensitization patterns informing an algorithmic testing approach tailored for primary care settings. The use of a region and population-specific test panel can efficiently identify atopy, leading to more targeted testing. This strategy has the potential to refine laboratory testing, reduce costs, and improve the appropriateness of referrals to allergy specialists, ultimately enhancing diagnostic accuracy and resource allocation.

Regulatory Requirements for the Quality of Allergen Products for Allergen Immunotherapy of Food Allergy.

PURPOSE OF REVIEW: Medicinal products for allergen immunotherapy (AIT) of food allergies have gained enormous momentum in recent years. With this new class of products entering marketing authorization procedures, compliance to regulatory requirements becomes a critical element. Here, an overview is provided on specific requirements and aspects concerning the quality control and manufacturing of these products. RECENT FINDINGS: Recent developments in the field of AIT for food allergies are divers, including products for oral, epicutaneous, and subcutaneous application, most notably targeting egg, milk, and peanut allergy. As the source materials for food AIT product are typically produced for food consumption and not for medicinal purposes, unique challenges arise in the manufacturing processes and controls of these medicinal products. Individual approaches are needed to assure acceptable quality, including control of relevant quantitative and qualitative characteristics. Major characteristics for quality verification include determination of protein content, total allergenic activity, and major allergen content. The applied manufacturing processes need to be established such that relevant process parameters are kept within justified limits and consistency of produced batches is assured. Allergen products for food AIT present specific challenges with respect to quality aspects that differentiate them from other commonly available AIT products. While established regulation is available and provides clear guidance for most aspects, other issues require consideration of new and individual settings relevant here. Consequently, as experience grows, respective amendments to currently available guidance may be needed.

History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk.

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

Biologics for the Treatment of Food Allergies.

Food allergy is increasingly prevalent and poses a life-threatening risk to those afflicted. The health care costs associated with food allergies are also increasing. Current and emerging treatments for food allergies aim at protecting against reactions caused by accidental ingestion and increasing the food allergen reaction threshold, although this protection is often temporary. In the future, ideal biologic therapies would target key mediators of the type II immune pathway, essential in development of the atopic march to prevent development of food allergies. Biologics offering long-term protection against allergic reactions to food are needed, and several agents are already in development.

Milk allergy most burdensome in multi-food allergic children.

BACKGROUND: Food allergy is a substantial health burden, which disproportionately affects children. Among children with food allergy, as many as 70% have multiple food allergies. Whereas the overall burden of food allergy on quality of life has been described, little is known about the burden of individual allergens. We aimed to examine the perception of burden among families with multiple food-allergic children. METHODS: Parents of children with 1 + children with multiple food allergies including milk responded to online questions, including both open-ended and closed-ended questions on food allergy-related burdens of time, financial costs, social restrictions, and emotional demands. RESULTS: Overall, 64 children (69.8% boys) of whom (73.0%) most were aged 10 and younger were included. Most had been diagnosed with food allergy in infancy and by a (pediatric) allergist. Other common allergies included peanut (65.6%), tree nuts (57.8%), egg (76.6%), and sesame (31.3%). Quantitatively, milk allergy was reported as carrying the most burden, including most socially limiting (81.5%), requiring the most planning (75.9%), causing the most anxiety (68.5%), most challenging to find "safe" or allergy-friendly foods (72.2%), and costly (81.5%). Qualitatively, we identified five themes that captured burdens associated with costs, marketing of milk products to children, risk of cross-contamination, ubiquity of milk/dairy and public confusion with lactose intolerance, and an unwillingness of others to accommodate the allergy. CONCLUSION: Parents whose children have multiple food allergies, including milk, report milk as the allergy associated with the greatest time, financial, social, and emotional burdens.

A multi-center assessment to compare residual allergenicity of partial hydrolyzed whey proteins in a murine model for cow's milk allergy - Comparison to the single parameter guinea pig model.

INTRODUCTION: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas. AIM: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers. METHODS: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study. RESULTS: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers. CONCLUSIONS: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account.

Updated population minimal eliciting dose distributions for use in risk assessment of 14 priority food allergens.

Food allergy and allergen management are important global public health issues. In 2011, the first iteration of our allergen threshold database (ATDB) was established based on individual NOAELs and LOAELs from oral food challenge in roughly 1750 allergic individuals. Population minimal eliciting dose (EDp) distributions based on this dataset were published for 11 allergenic foods in 2014. Systematic data collection has continued (2011-2018) and the dataset now contains over 3400 data points. The current study provides new and updated EDp values for 14 allergenic foods and incorporates a newly developed Stacked Model Averaging statistical method for interval-censored data. ED01 and ED05 values, the doses at which 1%, and respectively 5%, of the respective allergic population would be predicted to experience any objective allergic reaction were determined. The 14 allergenic foods were cashew, celery, egg, fish, hazelnut, lupine, milk, mustard, peanut, sesame, shrimp (for crustacean shellfish), soy, walnut, and wheat. Updated ED01 estimates ranged between 0.03 mg for walnut protein and 26.2 mg for shrimp protein. ED05 estimates ranged between 0.4 mg for mustard protein and 280 mg for shrimp protein. The ED01 and ED05 values presented here are valuable in the risk assessment and subsequent risk management of allergenic foods.

Shotgun proteomics, in-silico evaluation and immunoblotting assays for allergenicity assessment of lesser mealworm, black soldier fly and their protein hydrolysates.

Since 2018, insects have belonged the category of Novel Foods and the presence of allergens represents one of the main hazards connected to their consumption, also due to the potential cross-reactivity with Arthropoda pan-allergens. In the present work, the allergenicity assessment of black soldier fly and lesser mealworm was performed with a shotgun bottom-up proteomic approach combined with in-silico assessment, followed by IgG- and IgE-immunoblotting experiments. The peptides identified, filtered for their abundance and robustness, belonged mainly to muscle proteins, which represented the most abundant protein group. The relevant potential allergens were in-silico identified by sequence similarity to known allergens, and among them tropomyosin resulted the most abundant insect allergen. IgG-immunoblotting analysis with anti-Tropomyosin I antibodies and IgE-immunoblotting assay with serum from patient allergic to crustacean tropomyosin were performed in order to assess the immunoreactivity in both insects. The immunoassays were carried out also on protein hydrolysates extracted by treating insects with Protease from Bacillus licheniformis (1%, 60 °C, pH 7.5). While IgG-immunoblotting demonstrated the loss of immunoreactivity for both hydrolysates, IgE-immunoblotting showed a partial immunoreactivity preservation, also after hydrolysis, in the case of black soldier fly hydrolysate, and a total loss of immunoreactivity for lesser mealworm hydrolysate.

Food allergy: how expensive are they?

PURPOSE OF REVIEW: Food allergy remains a disease of global public health significance well known to impact social, emotional, and financial well being. This review aims to summarize the existing literature focused on the direct, indirect, and intangible costs of food allergy at the household and healthcare system levels, and begin to discuss how emerging treatment and prevention strategies may be leveraged to comprehensively care for the food allergic population with the efficient use of health resources. RECENT FINDINGS: Food allergy imposes significant costs to multiple stakeholders and largely impact families at the household level. Recent studies elucidate the need to balance the household management of food allergy with the efficient use of health resources. Overall, it remains critical that safe foods and medications remain affordable while further exploring the cost-effectiveness of early introduction, emerging food allergy therapies, and the wider use of stock epinephrine to adequately meet the public health needs of the food-allergic community. SUMMARY: Better understanding how the cost of food allergy impacts populations will help to inform more progressive policies aimed at lessening socioeconomic disparities and ultimately improve quality of life for children and adults with food allergies.

Food allergy in adults in Europe: what can we learn from geographical differences?

PURPOSE OF REVIEW: The aim of this article is to characterize the present state-of-the-art on the topic of food allergies across Europe. RECENT FINDINGS: A systematic review and metaanalysis on the epidemiology of food allergy in Europe have been performed by the Food Allergy and Anaphylaxis Guidelines Group of the European Academy of Allergology and Clinical Immunology. The authors had made an extensive search of four different electronic databases which retrieved thousands of hits. A critical appraisal of the documents reduced their number to just over 100 articles covering the period 2000-2012, revealing striking methodological inhomogeneity and blank areas on the map of the continent, particularly for the adult population. A major new development intending to fill in the gaps in the field of food allergy is the launch and implementation of the European Union-funded project 'Prevalence, Cost and Basis of Food Allergy Across Europe,' acronym 'EuroPrevall.' Among the deliverable of the project are several seminal articles on food allergy in adults which are presented in this review. SUMMARY: The EuroPrevall project confirmed much more reliably and in more detail the existing inhomogeneity in the prevalence of food allergy, which reflects environmental and climate differences between the separate countries, but possibly also the level of public awareness.

Vitamin D: can the sun stop the atopic epidemic?

PURPOSE OF REVIEW: To review recent evidence on the capacity of vitamin D to prevent atopic disease, focussing on food allergy and asthma, and potential underlying mechanisms. RECENT FINDINGS: The incidence of allergic disease continues to increase worldwide. Vitamin D status is influenced by sun exposure and dietary intake. Vitamin D deficiency is linked to an increased incidence of allergic disease and asthma. These associations are generally strongest in early life. The capacity of vitamin D to enhance antimicrobial pathways, promote peripheral immunological tolerance and maintain mucosal barrier integrity may underlie these associations. Interventional studies have addressed the capacity of vitamin D supplementation in utero and early life to reduce the incidence of disease. Ancillary studies have provided insights into potential biological mechanisms linked to these effects. SUMMARY: Observational studies show an inverse association between vitamin D levels and development of food allergy and asthma. Secondary analyses of two recent interventional studies suggest that achieving vitamin D sufficiency throughout pregnancy reduces the incidence of asthma/recurrent wheeze at 3 years. Longitudinal studies of vitamin D requirements in utero and postnatally, better understanding of factors that influence bioavailability of vitamin D and mechanistic insights into vitamin D effects on neonatal-specific immune pathways are awaited.

Frequentist and Bayesian approaches for food allergen risk assessment: risk outcome and uncertainty comparisons.

Peer-reviewed probabilistic methods already predict the probability of an allergic reaction resulting from an accidental exposure to food allergens, however, the methods calculate it in different ways. The available methods utilize the same three major input parameters in the risk model: the risk is estimated from the amount of food consumed, the concentration of allergen in the contaminated product and the distribution of thresholds among allergic persons. However, consensus is lacking about the optimal method to estimate the risk of allergic reaction and the associated uncertainty. This study aims to compare estimation of the risk of allergic reaction and associated uncertainty using different methods and suggest improvements. Four cases were developed based on the previous publications and the risk estimations were compared. The risk estimation was found to agree within 0.5% with the different simulation cases. Finally, an uncertainty analysis method is also presented in order to evaluate the uncertainty propagation from the input parameters to the risk.

Can dietary strategies in early life prevent childhood food allergy? A report from two iFAAM workshops.

Food allergy affects a small but significant number of children and adults. Food allergy is responsible for considerable morbidity and is the commonest cause of anaphylaxis in children. One of the aims of the European Union-funded "Integrated Approaches to Food Allergen and Allergy Risk Management" (iFAAM) project was to improve our understanding of the best way to prevent the development of food allergy. Groups within the project worked on integrating the current prevention evidence base as well as generating new data to move our understanding forward. This paper from the iFAAM project is a unique addition to the literature on this topic as it not only outlines the recently published randomized controlled trials (as have previous reviews) but also summarizes two iFAAM-associated project workshops. These workshops focused on how we may be able to use dietary strategies in early life to prevent the development of food allergy and summarized the range of opinions amongst experts in this controversial area.

Identifying and managing patients at risk of severe allergic reactions to food: Report from two iFAAM workshops.

Food allergy affects a small but important number of children and adults. Much of the morbidity associated with food allergy is driven by the fear of a severe reaction and fatalities continue to occur. Foods are the commonest cause of anaphylaxis. One of the aims of the European Union-funded Integrated Approaches to Food Allergen and Allergy Risk Management (iFAAM) project was to improve the identification and management of children and adults at risk of experiencing a severe reaction. A number of interconnected studies within the project have focused on quantifying the severity of allergic reactions; the impact of food matrix, immunological factors on severity of reactions; the impact of co-factors such as medications on the severity of reactions; utilizing single-dose challenges to understand threshold and severity of reactions; and community studies to understand the experience of patients suffering real-life allergic reactions to food. Associated studies have examined population thresholds and co-factors such as exercise and stress. This paper summarizes two workshops focused on the severity of allergic reactions to food. It outlines the related studies being undertaken in the project indicating how they are likely to impact on our ability to identify individuals at risk of severe reactions and improve their management.

The relevance of a digestibility evaluation in the allergenicity risk assessment of novel proteins. Opinion of a joint initiative of COST action ImpARAS and COST action INFOGEST.

The current allergenicity assessment of novel proteins is based on the EFSA GMO guidance. Recently, EFSA launched a new guidance document on allergenicity assessment of GM plants (2017). This document describes, amongst other topics, the new scientific and regulatory developments on in vitro protein digestibility tests. The EFSA GMO Panel stated that for in vitro protein digestibility tests, additional investigations are needed before any additional recommendation in the form of guidance can be provided. To this end, an interim phase is considered necessary to evaluate the revisions to the in vitro gastrointestinal digestion test, proposed by EFSA. This prompted the establishment of a joint workshop through two COST Action networks: COST Action ImpARAS and COST Acton INFOGEST. In 2017, a workshop was organised to discuss the relevance of digestion in allergenicity risk assessment and how to potentially improve the current methods and readouts. The outcome of the workshop is that there is no rationale for a clear readout that is predictive for allergenicity and we suggest to omit the digestion test from the allergenicity assessment strategy for now, and put an effort into filling the knowledge gaps as summarized in this paper first.

Proteomic and Bioinformatic Analyses for the Identification of Proteins With Low Allergenic Potential for Hazard Assessment.

Use of botanicals and natural substances in consumer products has increased in recent years. Such extracts can contain protein that may theoretically represent a potential risk of IgE-mediated allergy. No method has yet been generally accepted or validated for assessment of the allergenic potential of proteins. For development of suitable methods datasets of allergenic and nonallergenic (or low allergenic) proteins are required that can serve, respectively, as positive and negative controls. However, data are unavailable on proteins that lack or have low allergenic potential. Here, low allergenic potential proteins are identified based on the assumption that proteins with established human exposure, but with a lack of an association with allergy, possess low allergenic potential. Proteins were extracted from sources considered to have less allergenic potential (corn, potato, spinach, rice, and tomato) as well as higher allergenic potential (wheat) regarding common allergenic foods. Proteins were identified and semi-quantified by label-free proteomic analysis conducted using mass spectrometry. Predicted allergenicity was determined using AllerCatPro (https://allercatpro.bii.a-star.edu.sg/). In summary, 9077 proteins were identified and semi-quantified from 6 protein sources. Within the top 10% of the most abundant proteins identified, 178 characterized proteins were found to have no evidence for allergenicity predicted by AllerCatPro and were considered to have low allergenic potential. This panel of low allergenic potential proteins provides a pragmatic approach to aid the development of alternative methods for robust testing strategies to distinguish between proteins of high and low allergenic potential to assess the risk of proteins from natural or botanical sources.