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publication, the variables and questions associated with the development of in vivo models are examined and several unpublished results are presented.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.

Risks of allergic reactions to biotech proteins in foods: perception and reality.

In recent years, significant attention has been paid to the use of biotechnology to improve the quality and quantity of the food supply due in part to the projected growth in the world population, plus limited options available for increasing the amount of land under cultivation. Alterations in the food supply induced by classical breeding and selection methods typically involve the movement of large portions of genomic DNA between different plant varieties to obtain the desired trait. This is in contrast to techniques of genetic engineering which allows the selection and transfers specific genes from one species to another. The primary allergy risk to consumers from genetically modified crops may be placed into one of three categories. The first represents the highest risk to the allergic consumer is the transfer of known allergen or cross-reacting allergen into a food crop. The second category, representing an intermediate risk to the consumer, is the potential for replacing the endogenous allergenicity of a genetically-modified crop. The last category involves expression of novel proteins that may become allergens in man and generally represents a relatively low risk to the consumer, although this possibility has received attention of late. In order to mitigate the three categories of potential allergy risk associated with biotech crops, all genes introduced into food crops undergo a series of tests designed to determine if the biotech protein exhibits properties of known food allergens. The result of this risk assessment process to date is that no biotech proteins in foods have been documented to cause allergic reactions. These results indicate that the current assessment process is robust, although as science of allergy and allergens evolves, new information and new technology should help further the assessment process for potential allergenicity.

A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Serological characteristics of peanut allergy in children.

BACKGROUND: Food challenge is considered an excellent clinical tool for the diagnosis of specific food allergy. However in the case of peanut allergy it may be difficult to perform because of the severity of the reactions. The quantitation of a specific immunoglobulin E (IgE) response to different peanut allergens could also contribute to the improvement of the diagnosis. We characterized the IgE response to a whole peanut protein extract and to Ara h 1 and Ara h 2 in different groups of patients classified according to the severity of their allergic reactions. METHODS: Specific serum IgE were analyzed in 96 children by enzyme-linked immunosorbent assay tests using a whole protein extract or purified peanut proteins and anti-human IgE monoclonal antibodies labeled with acetylcholinesterase. RESULTS: A parallel was observed between levels of peanut-specific IgE and the classification in five groups and subgroups of patients upon increasing severity of symptoms, especially within the group of highest severity. Moreover, the highest frequency of positive response and the highest levels of specific IgE were observed with whole peanut protein extract. CONCLUSION: In a retrospective evaluation of peanut allergy in children, we have shown that quantitation of peanut-specific IgE could be used to avoid a food challenge particularly in the case of severe reactions. When compared to Ara h 1 and Ara h 2, whole peanut protein extract appeared to be the most appropriate allergen to perform the test.

Oral administration of IL-12 suppresses anaphylactic reactions in a murine model of peanut hypersensitivity.

There is no satisfactory therapeutic intervention for peanut allergy, which accounts for most life-threatening food allergic reactions. Since IL-12 has been found to inhibit allergic airway responses in a mouse model of asthma and to cure Th2 cytokine-mediated murine schistosomiasis, we hypothesized that IL-12 treatment might also inhibit peanut allergic reactions. Consequently, we investigated the effects of oral IL-12 treatment in a murine model of peanut allergy and found that oral administration of liposome encapsulated rIL-12 could both prevent and reverse peanut hypersensitivity and could reduce histamine release, peanut-specific serum IgE and IgG1, and fecal IgA levels. Oral IL-12 treatment also increased IFN-gamma but did not decrease IL-4 or IL-5 levels. We conclude that oral rIL-12 treatment has therapeutic as well as preventive effects on peanut allergy, which are associated with increased IFN-gamma production.

Effects of cooking methods on peanut allergenicity.

BACKGROUND: Allergy to peanut is a significant health problem. Interestingly, the prevalence of peanut allergy in China is much lower than that in the United States, despite a high rate of peanut consumption in China. In China, peanuts are commonly fried or boiled, whereas in the United States peanuts are typically dry roasted. OBJECTIVE: The aim of this study was to examine whether the method of preparing peanuts could be a factor in the disparity of allergy prevalence between the 2 countries. METHODS: Two varieties of peanuts grown in the United States were roasted, boiled, or fried. Proteins were analyzed by using SDS-PAGE and immunoblotting. Allergenicity was compared by using immunolabeling with sera from 8 patients with peanut allergy. RESULTS: The protein fractions of both varieties of peanuts were altered to a similar degree by frying or boiling. Compared with roasted peanuts, the relative amount of Ara h 1 was reduced in the fried and boiled preparations, resulting in a significant reduction of IgE-binding intensity. In addition, there was significantly less IgE binding to Ara h 2 and Ara h 3 in fried and boiled peanuts compared with that in roasted peanuts, even though the protein amounts were similar in all 3 preparations. CONCLUSION: The methods of frying or boiling peanuts, as practiced in China, appear to reduce the allergenicity of peanuts compared with the method of dry roasting practiced widely in the United States. Roasting uses higher temperatures that apparently increase the allergenic property of peanut proteins and may help explain the difference in prevalence of peanut allergy observed in the 2 countries.

Engineering, characterization and in vitro efficacy of the major peanut allergens for use in immunotherapy.

BACKGROUND: Numerous strategies have been proposed for the treatment of peanut allergies, but despite the steady advancement in our understanding of atopic immune responses and the increasing number of deaths each year from peanut anaphylaxis, there is still no safe, effective, specific therapy for the peanut-sensitive individual. Immunotherapy would be safer and more effective if the allergens could be altered to reduce their ability to initiate an allergic reaction without altering their ability to desensitize the allergic patient. METHODS: The cDNA clones for three major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, have been cloned and characterized. The IgE-binding epitopes of each of these allergens have been determined and amino acids critical to each epitope identified. Site-directed mutagenesis of the allergen cDNA clones, followed by recombinant production of the modified allergen, provided the reagents necessary to test our hypothesis that hypoallergenic proteins are effective immunotherapeutic reagents for treating peanut-sensitive patients. Modified peanut allergens were subjected to immunoblot analysis using peanut-positive patient sera IgE, T cell proliferation assays, and tested in a murine model of peanut anaphylaxis. RESULTS: In general, the modified allergens were poor competitors for binding of peanut-specific IgE when compared to their wild-type counterpart. The modified allergens demonstrated a greatly reduced IgE-binding capacity when individual patient serum IgE was compared to the binding capacity of the wild-type allergens. In addition, while there was considerable variability between patients, the modified allergens retained the ability to stimulate T cell proliferation. CONCLUSIONS: These modified allergen genes and proteins should provide a safe immunotherapeutic agent for the treatment of peanut allergy.

Food allergens.

A number of advances in the scientific knowledge concerning adverse food reactions have been made in the past few years. Understanding about the nature of the food allergen itself, the molecular characterization of the epitopes on these allergens, the pathophysiology of the clinical reaction, and the diagnostic methods have all been significantly enhanced.

A soybean G2 glycinin allergen. 2. Epitope mapping and three-dimensional modeling.

BACKGROUND: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, has been shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 22 kD. These findings suggested that this unique protein fraction from soybean might be responsible, in part, for soybean allergic reactivity. The objective of the present study was to characterize specific B cell epitopes, to determine if any amino acid was critical to IgE binding and to model the 22-kD G2 soybean allergen to the three-dimensional (3-D) phaseolin molecule. METHODS: B cell epitopes were identified using SPOTs peptide analysis. Structural orientation of the IgE-binding regions was mapped to the 3-D phaseolin molecule using molecular modeling of the protein tertiary structure. RESULTS: Eleven linear epitopes, representing 15 amino acid peptide sequences, bound to IgE in the glycinin molecule. These epitopes were predicted to be distributed asymmetrically on the surface of G2 trimers. CONCLUSIONS: Only 1 epitope could be rendered non-IgE binding by alanine substitutions in the peptide. The nonrandom distribution of the IgE binding sites provides new insight into their organization in trimers in 11S complexes of the G2 glycinin allergen.

A soybean G2 glycinin allergen. 1. Identification and characterization.

BACKGROUND: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, was shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 21 kD. These findings suggested that unique proteins in soybeans might be responsible for soybean allergic reactivity. The objective of the present study was to identify unique proteins in soybean extracts that bind to specific IgE from soybean-sensitive individuals, and to characterize the allergen using physicochemical methods and IgE binding. METHODS: Two-dimensional and preparative SDS-PAGE/IgE immunoblot analysis was used to identify a 22-kD soybean-specific allergen from crude soybean extracts. N-terminal sequence analysis was used to determine the identification of the protein binding IgE from soybean-sensitive individuals. RESULTS: IgE immunoblot and amino acid sequence analysis identified the 22-kD protein as a member of the G2 glycinin soybean protein family. Further investigation revealed that the IgEs reacted with basic chains from each member of the glycinin family of soybean storage proteins. CONCLUSIONS: Each of the subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens.

A murine model of peanut anaphylaxis: T- and B-cell responses to a major peanut allergen mimic human responses.

BACKGROUND: Peanut allergy affects 0.6% of the US population. At the present time, allergen avoidance is the only therapeutic option. Animal models of food-induced anaphylaxis would facilitate attempts to design novel immunotherapeutic strategies for the treatment of peanut allergy. OBJECTIVE: The purpose of this study was to develop a murine model of IgE-mediated peanut hypersensitivity that closely mimics human peanut allergy. METHODS: C3H/HeJ mice sensitized orally with freshly ground whole peanut and cholera toxin as adjuvant were challenged orally 3 and 5 weeks later with crude peanut extract. Anaphylactic reactions were determined. T- and B-cell responses to Ara h 1 and Ara h 2, the major peanut allergens, were characterized by evaluating splenocyte proliferative responses and IgE antibody concentrations. Furthermore, IgE antibodies in the sera of patients with peanut allergy and mice were compared for antibody binding to Ara h 2 isoforms and allergenic epitopes. RESULTS: Peanut-specific IgE was induced by oral peanut sensitization, and hypersensitivity reactions were provoked by feeding peanut to sensitized mice. The symptoms were similar to those seen in human subjects. Ara h 1- and Ara h 2-specific antibodies were present in the sera of mice with peanut allergy. Furthermore, these Ara h 2-specific IgE antibodies bound the same Ara h 2 isoforms and major allergenic epitopes as antibodies in the sera of human subjects with peanut allergy. Splenocytes from mice with peanut allergy exhibited proliferative responses to Ara h 1 and Ara h 2. CONCLUSION: This murine model of peanut allergy mimics the clinical and immunologic characteristics of peanut allergy in human subjects and should be a useful tool for developing immunotherapeutic approaches for the treatment of peanut allergy.

Structure of the major peanut allergen Ara h 1 may protect IgE-binding epitopes from degradation.

In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.

Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K.

BACKGROUND: Peanuts and soybeans are 2 foods that have been shown to be responsible for many atopic disorders. Because of their nutritional benefit, soybean proteins are now being used increasingly in a number of food products. Previous studies have documented multiple allergens in soybean extracts, including glycinin, beta-conglycinin, and the P34/Gly m Bd 30K protein. OBJECTIVE: Our overall goal was to identify soybean-specific allergens to begin to understand molecular and immunochemical characteristics of legume proteins. The specific aim of the current investigation was to identify the essential amino acid residues necessary for IgE binding in the 5 distinct immunodominant epitopes of P34/Gly m Bd 30K. METHODS: Serum IgE from 6 clinically sensitive soybean-allergic individuals was used to identify P34/Gly m Bd 30K in the native and single amino acid substituted peptides with use of the SPOTS peptide synthesis technique to determine critical amino acids required for IgE binding. RESULTS: The intensity of IgE binding and epitope recognition by serum IgE from the individuals varied substantially. With use of serum from 6 clinically soybean-sensitive individuals, 2 of the 5 immunodominant epitopes could be mutagenized to non-IgE binding peptides. CONCLUSIONS: Single-site amino acid substitution of the 5 immunodominant epitopes of Gly m Bd 30K with alanine revealed that IgE binding could be reduced or eliminated in epitopes 6 and 16 in the serum obtained from 6 soybean-sensitive patients.

Peanut-induced anaphylactic reactions.

Food allergies, particularly to peanuts, are a common cause of anaphylaxis. Approximately 125 people die each year in the USA secondary to food-induced anaphylaxis. Clinical anaphylaxis is a syndrome of diverse etiology and dramatic presentation of symptoms associated with the classic features of type I, IgE-mediated hypersensitivity [1]. Typically the term anaphylaxis connotes an immunologically-mediated event that occurs after exposure to certain foreign substances. This reaction results from the generation and release of a variety of potent biologically active mediators and their concerted effects on various target organs. Anaphylaxis is recognized by cutaneous, respiratory, cardiovascular, and gastrointestinal signs and symptoms occurring singly or in combination. This article focuses on allergic reactions to peanuts that manifest as signs and symptoms involving multiple target organs or the cardiovascular system alone.

Strain-dependent induction of allergic sensitization caused by peanut allergen DNA immunization in mice.

To investigate the potential application of allergen gene immunization in the modulation of food allergy, C3H/HeSn (C3H) mice received i.m. injections of pAra h2 plasmid DNA encoding one of the major peanut allergens, Ara h2. Three weeks following pDNA immunization, serum Ara h2-specific IgG2a, IgG1, but not IgE, were increased significantly in a dose-dependent manner. IgG1 was 30-fold higher in multiply compared with singly immunized mice. Ara h2 or peanut protein injection of immunized mice induced anaphylactic reactions, which were more severe in multiply immunized mice. Heat-inactivated immune serum induced passive cutaneous anaphylaxis, suggesting that anaphylaxis in C3H mice was mediated by IgG1. IgG1 responses were also induced by intradermal injection of pAra h2, and by i.m. injection of pOMC, the plasmid DNA encoding the major egg allergen protein, ovomucoid. To elucidate whether the pDNA immunization-induced anaphylaxis was a strain-dependent phenomenon, AKR/J and BALB/c mice also received multiple i.m. pAra h2 immunizations. Injection of peanut protein into these strains at weeks 3 or 5 following immunization did not induce reactions. Although IgG2a was increased significantly from week 2 in AKR/J mice and from week 4 in BALB/c mice and remained elevated for at least 6 wk, no IgG1 or IgE was detected. These results indicate that the type of immune responses to pDNA immunization in mice is strain dependent. Consequently, models for studying human allergen gene immunization require careful selection of suitable strains. In addition, this suggests that similar interindividual variation is likely in humans.

Molecular cloning and epitope analysis of the peanut allergen Ara h 3.

Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from approximately 45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions.