Allergenic Potential Of Novel Proteins Research Articles

Novel foods: allergenicity assessment of insect proteins.

Insects represent a promising source of proteins and have been reported as a great potential for being used as novel food and feed proteins. This makes them a valuable source of nutrients to face the increasing demand of food necessitated by the growing global population. The current European food legislation on novel food (EU Reg. 2015/2283), which entered into force in 2018, provides the provisions that should be considered in the applications for the authorisation of novel foods in the European market. Insects, intended as an alternative source of food proteins for human consumption, are considered novel foods. Since food allergens are mostly proteins, the analysis and identification of the potential allergenicity of novel proteins should be a fundamental activity that enables the applicants to fulfil the requirements for the application and authorisation to bring a novel food into the European market and ensures a high level of food safety for the European consumers. The main aims of the work of the EU-FORA fellow were to: (i) Review, assess and identify gaps in the current strategies for predicting allergenicity of novel foods and new alternative protein sources; and (ii) Familiarise, understand and perform an allergenicity assessment of a novel food protein source by: (a) Working on an allergenicity assessment case study of insect proteins from black soldier fly larva (Hermetia Illucens); and (b) Taking into consideration other risk assessment aspects of insects as novel food, including toxicological, nutritional and microbial risks. The project contributed to the continuous learning of the fellow on practical assays and methodologies for the in silico, invitro and invivo analysis principles and complemented personal skills related to the food risk assessment requirement for the preparation and submission of an application for authorisation of a novel food.

Still SDAPing Along: 20 Years of the Structural Database of Allergenic Proteins.

The introduction of plant extracts to mitigate the symptoms of “hay fever”, about a century ago, led to discoveries beginning sixty years ago on determining the sequences and eventually structures of allergenic proteins. As more proteins were cloned, there was a need to rapidly identify and categorize those with significant similarity to known allergens. The Structural Database of Allergenic Proteins (SDAP) was created at the beginning of the 21st century as the first cross-referenced website to allow rapid overview of the structures and sequences of allergenic proteins. SDAP provides a way to identify sequence and functional similarities between these proteins, despite the complex nomenclature system based on the Latin names of their different sources. A rapid FASTA search simplifies grouping allergens from the same structural or functional family. SDAP also provides an overview of the rapidly expanding literature on the sequence, structure and epitopes of allergenic proteins and a way to estimate the potential allergenicity of novel proteins based on rules provided by the IUIS. Twenty years and a pandemic later, the list of allergenic proteins and their attributes continues to grow. SDAP is expanding and improving to allow rapid access to all this information.

Allergenicity prediction of novel and modified proteins: Not a mission impossible! Development of a Random Forest allergenicity prediction model

Alternative and sustainable protein sources (e.g., algae, duckweed, insects) are required to produce (future) foods. However, introduction of new food sources to the market requires a thorough risk assessment of nutritional, microbial and toxicological risks and potential allergic responses. Yet, the risk assessment of allergenic potential of novel proteins is challenging. Currently, guidance for genetically modified proteins relies on a weight-of-evidence approach. Current Codex (2009) and EFSA (2010; 2017) guidance indicates that sequence identity to known allergens is acceptable for predicting the cross-reactive potential of novel proteins and resistance to pepsin digestion and glycosylation status is used for evaluating de novo allergenicity potential. Other physicochemical and biochemical protein properties, however, are not used in the current weight-of-evidence approach. In this study, we have used the Random Forest algorithm for developing an in silico model that yields a prediction of the allergenic potential of a protein based on its physicochemical and biochemical properties. The final model contains twenty-nine variables, which were all calculated using the protein sequence by means of the ProtParam software and the PSIPred Protein Sequence Analysis program. Proteins were assigned as allergenic when present in the COMPARE database.Results show a robust model performance with a sensitivity, specificity and accuracy each greater than ≥85%. As the model only requires the protein sequence for calculations, it can be easily incorporated into the existing risk assessment approach. In conclusion, the model developed in this study improves the predictability of the allergenicity of new or modified food proteins, as demonstrated for insect proteins.

Allergenic potential of novel proteins – What can we learn from animal production?

Currently, risk assessment of the allergenic potential of novel proteins relies heavily on evaluating protein digestibility under normal conditions based on the theory that allergens are more resistant to gastrointestinal digestion than non-allergens. There is also proposed guidance for expanded in vitro digestibility assay conditions to include vulnerable sub-populations. One of the underlying rationales for the expanded guidance is that current in vitro assays do not accurately replicate the range of physiological conditions. Animal scientists have long sought to predict protein and amino acid digestibility for precision nutrition. Monogastric production animals, especially swine, have gastrointestinal systems similar to humans, and evaluating potential allergen digestibility in this context may be beneficial. Currently, there is no compelling evidence that the mechanisms sometimes postulated to be associated with allergenic sensitization, e.g. antacid modification of stomach pH, are valid among production animals. Furthermore, examples are provided where non-biologically representative assays are better at predicting protein and amino acid digestibility compared with those designed to mimic in vivo conditions. Greater emphasis should be made to align in vitro assessments with in vivo data.

Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

Food allergy is a major health problem in the Western countries, affecting 3–8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.

Study on animal models for food allergy

Animal models play an important role in study on food allergy.This paper reviews the potential benefits and pitfalls of different animal models,including animal species and strains,test proteins,routes of exposure,the incorpration of adjuvants and evaluting indictor of allergic models,which can provide theoretical basis for evaluting the potential allergenicity of novel proteins and preventing food allergic diseases. Key words: Food allergy; Animal models; Allergen; Adjuvants

Heat stability, its measurement, and its lack of utility in the assessment of the potential allergenicity of novel proteins

Thermal stability has been reported as a shared characteristic among some of the major food allergens and appears to have originated from the observation that some cooked foods retain their ability to cause allergic reactions by Immunoglobulin E (IgE) binding and the subsequent cascade of events that mediate allergic reactions. Based on this observation, the thermal stability of novel food proteins, like those in transgenic crops, is considered correlative with allergenic risk and has prompted requests from some regulatory agencies for additional testing to address safety concerns. Because human testing and serum IgE screening are not feasible nor are they necessarily useful for evaluating the thermal stability of a novel food protein, a protein function assay is often used to assess the thermal stability in the context of an allergenicity risk assessment. Some regulatory authorities also require immunodetection using polyclonal IgG antibodies and gel based methods. Here we review why heat stability as measured by these functional and immunodetection assays does not correlate with allergenicity and provides no useful safety information in assessing the allergenic potential of novel food proteins.

In Vivo Digestion of a Thaumatin-Like Kiwifruit Protein in Rats

Food allergens must exhibit sufficient gastrointestinal stability to reach the intestinal mucosa where absorption and sensitization can occur. Therefore, investigation of protein stability within the gastrointestinal tract may provide a prospective test for the allergenic potential of novel proteins. The aim of this work was to examine the effect of the fruit matrix and purified pectin on the digestion in vivo of kiwifruit allergens in the rat gastrointestinal system. The major kiwi allergen, Act d 2, was quantified in several compartments of the gastrointestinal tract by a monoclonal antibody-based ELISA. Protein intactness was demonstrated by immunoblot. Under conditions of complex food digestion in vivo, a pepsin-labile protein survived passage from the stomach to the caecum during a 3-h period. Decay of Act d 2 in the rat gut exhibited an exponential pattern. Ingestion of kiwifruit was followed by a decrease in both total and specific pepsin activity. When purified, Act d 2 allergen was consumed together with pure apple pectin; both gastric acidity as well as specific and total pepsin activity declined and thus protected 23% of the ingested allergen from digestion for 90 min. In conclusion, ingestion of pectin-rich fruits and particularly pectin supplements may have a protective action on pepsin-labile allergens and prolong their survival in both gastric and duodenal juices, enabling efficient uptake and presentation to the immune system.

Inbred Chinese Wuzhishan (WZS) Minipig Model for Soybean Glycinin and β-Conglycinin Allergy

Validated murine models have been built to assess the potential allergenicity of novel proteins. Large animals, such as pigs, share more similarities to humans in physiology and immunology than murine. Among Chinese minipigs, Wuzhishan (WZS) minipigs have the highest inbreeding coefficient, more stable heredity, and less variability, which were gastrically sensitized and excitated with diets containing 4% glycinin or 4% beta-conglycinin or neither to induce anaphylactic reactions in the present study. In glycinin- and beta-conglycinin-sensitized animals, diarrhea symptoms and skin wheal and flare responses were observed. In comparison to the control, after oral excitation with glycinin or beta-conglycinin, the serum IgE was increased by 34.4 or 38.4% and the serum histamine was increased by 42.1 or 46.9%, respectively. In addition, the serum IFN-gamma were reduced by 12.4 or 30.0%, respectively. The jejunum histamine level of beta-conglycinin-sensitized animals was increased by 196.6%, while the number of mast cells in the submucosa of jejunum and ileum of the glycinin-sensitized animals declined by 48.1 and 45.0%. In conclusion, the WZS minipig allergy models induced by soybean glycinin and beta-conglycinin represent type-I hypersensitivity reactions mediated by IgE, which could potentially be useful in determining the potential allergenicity of novel proteins.

Value of eight-amino-acid matches in predicting the allergenicity status of proteins: an empirical bioinformatic investigation

The use of biotechnological techniques to introduce novel proteins into food crops (transgenic or GM crops) has motivated investigation into the properties of proteins that favor their potential to elicit allergic reactions. As part of the allergenicity assessment, bioinformatic approaches are used to compare the amino-acid sequence of candidate proteins with sequences in a database of known allergens to predict potential cross reactivity between novel food proteins and proteins to which people have become sensitized. Two criteria commonly used for these queries are searches over 80-amino-acid stretches for >35% identity, and searches for 8-amino-acid contiguous matches. We investigated the added value provided by the 8-amino-acid criterion over that provided by the >35%-identity-over-80-amino-acid criterion, by identifying allergens pairs that only met the former criterion, but not the latter criterion. We found that the allergen-sequence pairs only sharing 8-amino-acid identity, but not >35% identity over 80 amino acids, were unlikely to be cross reactive allergens. Thus, the common search for 8-amino-acid identity between novel proteins and known allergens appears to be of little additional value in assessing the potential allergenicity of novel proteins.

Review of animal models designed to predict the potential allergenicity of novel proteins in genetically modified crops

The safety assessment of genetically modified crops involves the evaluation of the potential allergenicity of novel proteins by using several in silico and in vitro endpoints. In this publication, the variables and questions associated with the development of in vivo models are examined and several unpublished results are presented. Both rodent and non-rodent (dog and pig) models have been investigated using various routes of administration with purified proteins or food extracts, with or without the use of an adjuvant. The ideal model should be simple, reproducible across laboratories over time, specific and sensitive enough for distinguishing a threshold beyond which relevant allergenicity would be predicted and, for ranking proteins correlated with the allergic responses in humans, and acceptable under animal care. Preliminary data suggest that a few appear promising; however, further evaluation of these models is required. In particular, more extensive validation testing with additional allergenic and non-allergenic material should be performed before using them in the safety assessment of genetically modified crops.

Approaches to Assess the Allergenic Potential of Novel Proteins: Are We on the Right Track?

Approaches to Assess the Allergenic Potential of Novel Proteins: Are We on the Right Track?

Animal models of protein allergenicity: potential benefits, pitfalls and challenges

Food allergy is an important health issue. With an increasing interest in novel foods derived from transgenic crop plants, there is a growing need for the development of approaches suitable for the characterization of the allergenic potential of proteins. There are methods available currently (such as homology searches and serological testing) that are very effective at identifying proteins that are likely to cross-react with known allergens. However, animal models may play a role in the identification of truly novel proteins, such as bacterial or fungal proteins, that have not been experienced previously in the diet. We consider here the potential benefits, pitfalls and challenges of the selection of various animal models, including the mouse, the rat, the dog and the neonatal swine. The advantages and disadvantages of various experimental end-points are discussed, including the measurement of specific IgE by ELISA, Western blotting or functional tests such as the passive cutaneous anaphylaxis assay, and the assessment of challenge-induced clinical symptoms in previously sensitized animals. The experimental variables of route of exposure to test proteins and the incorporation of adjuvant to increase the sensitivity of the responses are considered also. It is important to emphasize that currently none of these approaches has been validated for the purposes of hazard identification in the context of a safety assessment. However, the available evidence suggests that the judicious use of an accurate and robust animal model could provide important additional data that would contribute significantly to the assessment of the potential allergenicity of novel proteins.

Characteristic motifs for families of allergenic proteins

The identification of potential allergenic proteins is usually done by scanning a database of allergenic proteins and locating known allergens with a high sequence similarity. However, there is no universally accepted cut-off value for sequence similarity to indicate potential IgE cross-reactivity. Further, overall sequence similarity may be less important than discrete areas of similarity in proteins with homologous structure. To identify such areas, we first classified all allergens and their subdomains in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to their closest protein families as defined in Pfam, and identified conserved physicochemical property motifs characteristic of each group of sequences. Allergens populate only a small subset of all known Pfam families, as all allergenic proteins in SDAP could be grouped to only 130 (of 9318 total) Pfams, and 31 families contain more than four allergens. Conserved physicochemical property motifs for the aligned sequences of the most populated Pfam families were identified with the PCPMer program suite and catalogued in the webserver MotifMate (http://born.utmb.edu/motifmate/summary.php). We also determined specific motifs for allergenic members of a family that could distinguish them from non-allergenic ones. These allergen specific motifs should be most useful in database searches for potential allergens. We found that sequence motifs unique to the allergens in three families (seed storage proteins, Bet v 1, and tropomyosin) overlap with known IgE epitopes, thus providing evidence that our motif based approach can be used to assess the potential allergenicity of novel proteins.

Inter‐laboratory comparisons of assessment of the allergenic potential of proteins in mice

Assessment of the potential allergenicity of novel proteins, including those expressed in genetically modified plants, is an important issue. In previous studies, we have shown that the IgE measurement induced by systemic exposure of BALB/c mice to a range of proteins correlates broadly with what is known of their allergenic potential in humans. The approach used a homologous passive cutaneous anaphylaxis (PCA) assay that reflects IgE-dependent biological activity and is of sufficient sensitivity to detect IgE production in the absence of adjuvant. In previous studies, the immunization phase was conducted independently in two separate facilities, and the subsequent analytical work (PCA) conducted in a single facility. The purpose here was to further evaluate the transferability of this approach. To this end, BALB/c mice were exposed to a range of doses of peanut agglutinin or ovalbumin, allergenic proteins of peanut and hen's egg, respectively, in two independent laboratories. Serial doubling dilutions of serum pooled for each treatment group were analyzed for specific IgE. At higher doses of allergen very similar, or identical, IgE titers were achieved in both laboratories, although at lower doses, responses were somewhat more variable. These data demonstrate that, although technically demanding, the measurement of protein allergen-induced IgE antibody production in mice using PCA is relatively robust and is transferable and reproducible between laboratories. This approach may provide a useful tool for the safety assessment of novel proteins and suggests that continued evaluation of the approach with a wider range of protein allergens and non-sensitising proteins is justified.

Current and future methods for evaluating the allergenic potential of proteins: International workshop report 23–25 October 2007

The International Life Science Institute’s Health and Environmental Sciences Institute’s Protein Allergenicity Technical Committee hosted an international workshop October 23–25, 2007, in Nice, France, to review and discuss existing and emerging methods and techniques for improving the current weight-of-evidence approach for evaluating the potential allergenicity of novel proteins. The workshop included over 40 international experts from government, industry, and academia. Their expertise represented a range of disciplines including immunology, chemistry, molecular biology, bioinformatics, and toxicology. Among participants, there was consensus that (1) current bioinformatic approaches are highly conservative; (2) advances in bioinformatics using structural comparisons of proteins may be helpful as the availability of structural data increases; (3) proteomics may prove useful for monitoring the natural variability in a plant’s proteome and assessing the impact of biotechnology transformations on endogenous levels of allergens, but only when analytical techniques have been standardized and additional data are available on the natural variation of protein expression in non-transgenic bred plants; (4) basophil response assays are promising techniques, but need additional evaluation around specificity, sensitivity, and reproducibility; (5) additional research is required to develop and validate an animal model for the purpose of predicting protein allergenicity.

Review of the development of methodology for evaluating the human allergenic potential of novel proteins

Safety assessment of novel proteins in genetic-engineered foods is a key component of the overall safety evaluation for these products. Since allergens are typically proteins, assessment of the potential allergenicity of the novel proteins in genetically engineered foods is critical. This article reviews methods available to assess the potential allergenicity of novel proteins, as well as problems and deficiencies in the existing methods. The role of bioinformatics and knowledge of allergenic epitopes in developing new approaches to this problem is discussed.

Pollen allergens are restricted to few protein families and show distinct patterns of species distribution

Inhalative allergies are elicited predominantly by pollen of various plant species. However, a classification of the large number of identified pollen allergens is still missing. To analyze pollen allergen sequences with respect to protein family membership, taxonomic distribution of protein families, and interspecies variability. Protein family memberships of all plant allergen sequences from the Allergome database were determined by using the Protein Families Database of Alignments and Hidden Markov Models. The taxonomic distribution of pollen allergens was established from the Integrated Taxonomic Information System. Members of abundant pollen allergen families were compared with allergenic and nonallergenic homologues by database similarity searches and multiple sequence alignments. Pollen allergens were classified into 29 of 7868 protein families. Expansins, profilins, and calcium-binding proteins constitute the major pollen allergen families, whereas most plant food allergens belong to the prolamin, cupin, or profilin families. Pollen allergens were revealed to be ubiquitous (eg, profilins), present in certain plant families (eg, pectate lyases), or limited to a single taxon (eg, thaumatin-like proteins). Allergenic plant profilins constitute a highly conserved family with sequence identities of 70% to 85% among each other but low identities of 30% to 40% with nonallergenic profilins from other eukaryotes, including human beings. Similarly, allergenic polcalcins possess sequence identities of 64% to 92% but show low identities of 39% to 42% to related nonallergenic calmodulins and calmodulin-like proteins from vegetative plant tissues and man. This classification of pollen allergens into protein families will aid in predicting cross-reactivity, designing comprehensive diagnostic devices, and assessing the allergenic potential of novel proteins.

In Silico Methods for Evaluating Human Allergenicity to Novel Proteins: International Bioinformatics Workshop Meeting Report, 23–24 February 2005

The ILSI Health and Environmental Sciences Institute (HESI) hosted an expert workshop 22-24 February 2005 in Mallorca, Spain, to review the state-of-the-science for conducting a sequence homology/bioinformatics evaluation in the context of a comprehensive allergenicity assessment for novel proteins, to obtain consensus on the value and role of bioinformatics in evaluating novel proteins, and to discuss the utility and methods of allergen-specific IgE testing in the diagnosis of food allergy. The workshop participants included over forty international experts from academia, industry, and government. The workshop was hosted by the HESI Protein Allergenicity Technical committee, which has established a long-term program whose mission is to advance the scientific understanding of the relevant parameters for characterizing the allergenic potential of novel proteins.

Studies on BN rats model to determine the potential allergenicity of proteins from genetically modified foods

To develop a Brown Norway (BN) rat model to determine the potential allergenicity of novel proteins in genetically modified food. The allergenicity of different proteins were compared, including ovalbumin (OVA), a potent respiratory and food allergen, bovine serum albumin (BSA), a protein that is considered to have a lesser allergenic potential, and potato acid phosphatase (PAP), a non-allergenic protein when administered to BN rats via different routes of exposure (intraperitoneally or by gavage). IgG and IgE antibody responses were determined by ELISA and PCA, respectively. An immunoassay kit was used to determine the plasma histamine level. In addition, possible systemic effect of allergens was investigated by monitoring blood pressure. OVA provoked very vigorous protein-specific IgG and IgE responses, low grade protein-specific IgG and IgE responses were elicited by BSA, while by neither route did PAP elicit anything. In either routes of exposure, plasma histamine level in BN rats sensitized with OVA was higher than that of BSA or PAP. In addition, an oral challenge with BSA and PAP did not induce any effect on blood pressure, while a temporary drop in systolic blood pressure in few animals of each routes of exposure was found by an oral challenge with OVA. BN rat model might be a useful and predictive animal model to study the potential allergenicity of novel food proteins.