An Integrated Concept for the Assessment of Unintended Immune

Chapter 21

An Integrated Concept for the Assessment of Unintended Immune Responses in Modern Food Biotechnology 1,2

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Downloaded by PENNSYLVANIA STATE UNIV on July 2, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch021

A. G. Haslberger , H. Karlic , M . Handschur , and S. Irez 1

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Department of Nutritional Sciences, Center for Ecology, University of Vienna, Vienna, Austria Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch Hospital, Vienna, Austria

CODEX guidelines have been established for the risk assessment of G M food. The evaluation of allergenicity includes aspects of the source of the transferred gene, unintended effects of the transformation event, stability testing, bioinformatic tools for analysis of sequence homologies with identified allergens, and the use of sera from allergic patients. Discussions of the present concept address its reliability, especially the methods for a comparison with allergen sequences, and the potential need of additional tests. A WHO report on modern food biotechnology additionally asks for a holistic, integrative concept which includes the analysis of potential indirect consequences from the environment. New developments of functional foods, such as probiotics, combating infections, cell mediated or even autoimmune responses, will soon require the establishment of internationally harmonized risk assessment concepts in these areas.

© 2008 American Chemical Society

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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358 Besides foodborne infections, unintended immune responses represent a major hazard in food production. The understanding of the interaction between foods, nutrition, the development of the immune system, and immune responses to foods are an important element in the assessment of adverse reactions to foods. The main strategy for avoiding adverse allergic reactions to foods among individuals sensitized to those foods has been the avoidance of the incriminated food. However, epidemiological studies fail to show simple associations, e.g. between maternal consumption of allergen during pregnancy and lactation and the development of food allergies (/). A low reported prevalence of peanut allergy has been observed in African and Asian countries despite high levels of environmental exposure to peanut. One possible explanation is that oral tolerance induction through early infant feeding protects these populations against allergic sensitization; there is growing understanding that the development and maintenance of immunological tolerance are lifelong processes which start already prenatally through changes of specific T cell responses (/). With the development of modern food biotechnology and the use of genes and constituents from sources with no history of safe food use, the assessment of unintended immune responses to foods became even more important. Global trading and upcoming international regulations for risk assessment, risk management, and approval of foods produced with modern methods of biotechnology have resulted in the need of internationally harmonized concepts for the evaluation of immune responses. Whereas potential adverse immune responses induced by foods containing genetically modified organisms (GMO) have been the subject of intensive research and assessment by national authorities, many foods produced by more traditional methods have entered the markets without such a specified risk assessment according to international concepts.

International Regulations for Foods Derived from Modern Biotechnology In response to the increased delivery of genetically modified (GM) foods to international markets, the Ad Hoc Intergovernmental Task Force on Food Derived from Biotechnology of the Codex Alimentarius Commission (Rome) agreed on principles for the human health risk analysis of G M foods. These principles dictate a case-by-case premarket assessment that includes an evaluation of both direct and unintended effects. They state that safety assessment of G M foods needs to investigate: •

direct health effects (toxicity),


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• • • • •

tendency to provoke allergic reactions (allergenicity), specific components thought to have nutritional or toxic properties, the stability of the inserted gene, nutritional effects associated with genetic modification and any unintended effects that could resultfromthe gene insertion.

Of particular note, the task force broadened risk assessment to encompass not only health-related effects of the food itself, but also indirect effects of food on human health, e.g., potential health risks derived from outcrossing (2). CODEX principles do not have a binding effect on national legislation, but are referred to specifically in the Agreement on the Application of Sanitary and Phytosanitary Measures of the World Trade Organization (SPS Agreement, see WTO 1995), and are often used as a reference in the case of trade disputes. In addition, the Cartagena Protocol of Biosafety (CPB) is a legally binding international instrument that regulates the transboundary movement of living modified organisms (LMOs) resulting from modern biotechnology with the objective of protecting the environment. The backbone of the protocol is the advance informed agreement requiring consent prior to the shipment and introduction of an LMO into the environment of an importing country. The CODEX guidelines include a concept for an assessment of potential allergenicity of G M foods, including a decision tree elaborated in consultations of FAO/WHO. Risk-assessment protocols for food allergy examine four elements: 1. Allergenicity assessment (Is the food or elements in the food a potential allergen?) 2. Dose response assessment (Is there a safe concentration of the allergen?) 3. Exposure assessment (How likely is it that people will encounter the allergen) 4. Susceptible subpopulations (How do individuals prone to allergy react to this new food?) Elements of an allergenicity assessment include a comparison of the sequence of the transferred gene (including the flanking regions at the site of insertion) with sequence motifs of allergenic proteins from databanks, an evaluation of the stability of the newly expressed proteins against digestion, and animal and immune tests, as appropriate (5). Also the guidance document of the scientific panel on genetically modified organisms of the European Food Safety Agency (EFSA) for the risk assessment of genetically modified plants and derived food and feed (4) only considers allergenicity in the context of unintended immune responses and widely follows CODEX principles. This document additionally explains that allergenicity is not an intrinsic and fully predictable property of a given protein but is a biological


360 activity requiring an interaction with individuals with a pre-disposed genetic background. Allergenicity therefore depends upon the genetic diversity and variability in atopic humans. CODEX and EFSA guidance documents mainly focus on the assessment of allergic immune responses and only briefly address other types of immune responses.


The Assessment of Indirect Effects from the Environment Evidence for substantial environmental influences on health and food safety comes from work with environmental health indicators which show that agroenvironmental practices have direct and indirect effects on human health, concluding that "the quality of the environment influences the quality and safety of foods" (5). Recently, a WHO report on modern food biotechnology pointed out the need for a holistic, integrated assessment of G M foods including environmental and socio-economic aspects (6). Therefore, evaluating the link between the environment, food production, and immune responses to foods was suggested to be an important aspect in the assessment of new foods as experiences from conventional methods of food production suggest that changes of immune responses within certain groups of consumers could be mediated by changes in the food production methods (7). Pollen-allergic individuals frequently present allergic symptoms after ingestion of several kinds of plantderived food components presumably by cross-reactive structures. Unexpected immune responses to a naturally non-toxic protein transferred from beans to peas were blamed to be responsible for allergic lung damage observed in mice (5), possibly because of subtle structural changes when the protein was expressed in peas. Allergenic structures that sensitize pollen-allergic patients are also present in grass and weed pollen (9). Allergic potential of grasses and weeds, including sensitization and possibly also induction of tolerance, are well known. For example rice plants contribute a huge pollen load in agricultural fields during flowering which results in a seasonal trigger of hay fever and respiratory allergy in field workers and people living in the vicinity (70, 77). On the other hand, farmers who have grown up on farms present a lower prevalence of atopy (72). Recently detailed analysis of allergen-dependent switching patterns in vivo confirm that the farming environment protects against IgE responses of human B-cells from allergen-dependent, T 2 cell mediated isotype switching. The protective effects of farm exposure were confined to T 2-dependent IgGl, IgG4, and IgE expression and were allergen and switch stage specific. This suggests that distinct mechanisms regulate individual steps within allergen-induced class switching in vivo (13). These results go in line with the hygiene hypothesis which suggests that limited exposure to bacterial and viral pathogens during early childhood results H

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361 in an insufficient stimulation of T 1 cells, which in turn cannot counterbalance the expansion of T 2 cells, resulting in a predisposition to allergy. Exposure to some pathogens may reduce the risk of atopy by more than 60% (14). Whereas the role of T 2 cells producing interleukin IL4, IL-5, and IL-13 in allergic reactions is well-defined. New evidence describing a distinct proinflammatory T cell lineage called T 17 cells, producing IL-17A, a cytokine that induces IL-8 and recruits neutrophils, may bring new aspects to the understanding of certain immune reactions to foods (15,16). H

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Unintended Immune Responses to Microorganisms in Foods In the area of microorganisms and modern food biotechnology, the CODEX guidelines give advice for the conduction of food safety assessment of G M foods (17). In addition to the general CODEX assessment scheme for allergenicity, this document describes aspects of genetically modified microorganisms and mentions that a variety of microorganisms used in food production have a long history of safe use that predates scientific assessment. Few microorganisms have been characterized scientifically regarding the potential risks associated with the food they are used to produce, including, in some instances, the consumption of viable microorganisms. Furthermore, the CODEX principles of risk analysis are primarily intended to apply to discrete chemical entities such as food additives and pesticide residues, or specific chemical or microbial contaminants that have identifiable hazards and risks. Recombinant-DNA microorganisms that remain viable in foods may interact with the immune system in the gastrointestinal tract. Closer examination of these interactions will depend on the types of differences between the recombinant-DNA microorganism and its conventional counterpart; there is a need an assessment of viability and residence of microorganisms in the human gastrointestinal tract. Also the guidance document of the scientific panel of the European Food Safety Authority (EFSA) on genetically modified organisms for the risk assessment of genetically modified microorganisms and their derived products intended for food and feed use (18) only remarks that particular attention should be paid to potential interactions with the gut microbiota and the evaluation of any effect on the digestive physiology and immune response of the host. Internationally harmonized concepts for the safety assessment of complex immune responses induced by novel foods containing microorganisms are urgently desired as functional or dietetic food concepts are developing rapidly. There is growing appreciation for the role of the enteric flora in health and disease. Evidence for the role of commensal gut bacteria in the inflammatory bowel diseases, Crohn's disease, and ulcerative colitis have accumulated (19). Probiotics are established in the maintenance therapy for all these diseases. Most



362 approaches for these diseases currently aim at modulating the immune response. A different concept is to consider probiotic therapy in terms of specific molecules modulating defined targets in the gut mucosa. Western infants have delayed acquisition of several gut microbes and a reduced turnover of strains in the colon, indicating a low exposure to a small variety of environmental bacteria. An immunoflora study has examined how early intestinal colonization affects allergy in Swedish infants. In a meta-analysis of the effect of oral administration of probiotic bacteria on acute infectious diarrhea in children, the use of probiotics was associated with significantly reduced risks of diarrhoea. In addition, antibiotic treatment of respiratory infections could be reduced (79). Data also suggest that probiotics are more effective in preventing relapse of inflammatory process than in suppressing active disease (79). Following these concepts we soon may also see improved bacteria as functional foods or therapies delivering anti-inflammatory cytokines or other biologically active molecules to the gut.

Individual Aspects of Unintended Immune Responses In addition to environmental factors, the genetic disposition of individuals has often been mentioned as a major determinant of unintended immune reactions to foods. Many polymorphisms in molecules involved in the generation of immune responses have been described previously. Nutritional genomic studies have effectively demonstrated the consequences of genetic disposition in the different individual reactions to food ingredients and many single nucleotide polymorphism (SNP) studies have been reported in association with atopy, or allergic diseases. The impact of SNPs in cytokine-related genes on the severity of food allergy and atopic eczema in children has been described (20). Therefore, individual characteristics may need to be an additional aspect in the risk assessment of unintended immune reactions to foods in the future.

Criticisms and Suggestions for Improvements Despite or in response to internationally elaborated concepts, there are continous discussions about these concepts (27, 22) and the possibility that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism, or to different



363 means of exposure. Based on the experience with isoforms of allergens with only slight modification of sequences but significant changes in allergenicity (23) the decision tree endorsed by CODEX may not exclude the allergenicity of a given GMO with certainty. An alternative improvement of the current evaluation was proposed; an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. Furthermore, the suggested assessment procedures should also be applied to natural cultivars in order to establish effective measures for allergy prevention (24). Also a quantitative risk assessment model for allergens based on probabilistic techniques instead of conventional deterministic approaches was suggested, in order to to obtain a more exhaustive risk assessment and more detailed information (25). For bioinformatics comparison of proteins, three comparative approaches have traditionally been used or considered for safety evaluations: 1. 2.

3.

Identifying any short segment (6-8 amino acids) of the protein that exactly matches a known allergen sequence. An overall primary sequence comparison using Basic Local Alignment Search Tool (BLAST) or FASTA to find matches of greater than 35% identity over 80 amino acids. Programs which identify 3-D similarities that might predict potential crossreactivity.

The utility of each of these approaches was recently debated in a bioinformatics workshop (26). The consensus agreement from the expert participants in the workshop was that the short-segment match (e. g., 6-8 amino acids) provides an unacceptably high rate of false positive matches and an uncertain rate of true positive matches, and was not particularly useful for an allergenicity evaluation performed in the context of a comprehensive safety evaluation. There was no consensus regarding the most appropriate bioinformatics method, an acceptable scoring criterion for triggering closer examination subsequent to a positive match, or an acceptable scoring mechanism for ranking the utility of the various 3-D approaches. However, the general consensus was that the most practical approach at this time to evaluate primary sequence identities to known allergens is using either FASTA or BLAST. While there was good agreement that identities of greater than 35% over 80 or more amino acids (recommended by CODEX in 2003) is quite conservative, the conclusion was that additional data or studies would be needed to justify changing this criterion as there is evidence that some individuals sensitized to proteins in evolutionary conserved protein families may experience cross-reactions to proteins sharing approximately 40% identity (26).


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An Integrated Concept for the Assessment of Unintended Immune

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