* International Life Sciences Institute Health and Environmental Sciences Institute, One Thomas Circle NW, Ninth Floor, Washington, District of Columbia 20005; Monsanto Co., 800 North Lindbergh Boulevard, St. Louis, Missouri 63167;
Food Allergy Research and Resource Program, University of Nebraska, 255 Food Industry Building, Lincoln, Nebraska 68583;
Bayer CropScience, Regulatory Toxicology, 355 rue Doistoïevski, Sophia Antipolis, France; ¶ E.I. Dupont De Nemours and Company, Haskell Laboratory for Toxicology and Industrial Medicine, P.O. Box 50 1090 Elkton Road, Newark, Delaware 19714; || Bayer CropScience, P.O. Box 12014, 2 T.W. Alexander Drive, Research Triangle Park, North Carolina 27709; and ||| BASF Plant Science, 26 Davis Drive, Research Triangle Park, North Carolina 27709
1 To whom correspondence should be addressed at One Thomas Circle NW, Ninth Floor, Washington, DC 20005. Fax: (202) 659-3859. E-mail: kthomas{at}ilsi.org.
Received June 14, 2005; accepted August 1, 2005
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ABSTRACT |
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Key Words: allergy; antibody; antigen; IgE; food allergy; immunoglobulin.
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INTRODUCTION |
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Novel Protein Evaluation
Novel proteins introduced into genetically engineered plants are assessed for their potential to induce human allergic responses. A comparison of the amino acid sequence of the novel (introduced) protein to the amino acid sequences of known allergens is one of several criteria used to evaluate product safety (Astwood et al., 2003; Codex Alimentarius, 2003
; Metcalfe et al., 1996
). The purpose of this screen is to determine if the introduced protein shares any sequence similarities with known allergens or gliadins (i.e., proteins derived from wheat, rye, barley, and perhaps oats that are involved in the etiology of celiac disease) that might indicate that the protein could elicit a clinical reaction in a sensitized individual. It is important to note, however, that bioinformatics alone cannot be used to predict whether a protein will be allergenic. If a novel protein demonstrates some similarity to a known allergen, further testing such as IgE-binding studies using sera from appropriately allergic subjects should be considered to confirm the match. The criteria for identifying a protein as a potential allergen have changed as more information about allergens has accumulated and search algorithms have become more sophisticated.
An important consideration for homology comparisons is the availability of representative repositories of protein data that can be used as the basis for the bioinformatics evaluation. Large-scale DNA sequencing efforts have resulted in the characterization and sequencing of a large number of protein allergens that have subsequently been included in a number of allergen databases (a list of current databases is in Table 1). For proteins to be included in allergen databases, participants agreed that the protein must have clear evidence of IgE binding and minimal criteria should be established for inclusion of a protein in a particular database. The workshop group also agreed that standards for database maintenance, management, archival, retrieval, and updating procedures should be designed prior to the development of any database. Additionally, it was agreed that a diversity of database approaches (such as exists now) is desirable and that allergen databases should be publicly accessible.
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A sliding window search was first recommended by Metcalfe et al. (1996) to determine if small regions of proteins that represented IgE-binding epitopes or T-cell epitopes shared identities with known allergens that would be missed in the global sequence alignment. The recommendation of a sliding window search size of eight amino acids was considered a conservative approach that took into account the known sizes of these types of epitopes and the fact that very limited experimental data existed regarding the size and structure of epitopes on food allergens. In 2001, an FAO/WHO scientific advisory panel recommended that the size of the sliding window be decreased due to experimental evidence that IgE-binding epitopes could be as small as six amino acids. However, several publications have demonstrated that matches of six amino acids with any allergen occurs frequently by chance, which limits the utility of this criteria for predicting allergenicity (Hileman et al., 2002
; Stadler and Stadler, 2003
). Information presented at the meeting indicated that more than 150 linear IgE-binding epitopes have now been determined and have been published on a variety of allergens. Sizes of these epitopes ranged from 4 amino acids in length to as much as 23 amino acids long, with the vast majority of epitopes consisting of at least 8 amino acids. Even with this large increase in experimentally determined IgE-binding epitopes, workshop participants were reluctant to recommend that novel proteins be searched using an IgE epitope library, either in conjunction with or as an alternative to the sliding search window approach. Participants cited the variable quality of the IgE epitope data, the variability in the methods used to identify the epitopes, the fact that all epitopes have not been identified, and the recognition that there is very little information available regarding conformational epitopes as reasons for their reluctance to utilize an IgE epitope library. Additionally, data were presented that confirm using a sliding window search of six amino acids resulted in an unacceptable number of false-positive matches. The workshop participants agreed that the 6-mer sliding amino acid window search does not provide any value for the bioinformatics evaluation of novel proteins in a safety assessment. This was in general agreement with the Codex Ad Hoc Inter-governmental Task Force on Foods Derived from Biotechnology Report (2003) that stated any sliding window search should be performed with a scientifically justified search window size. Most participants agreed that further research was required to define a useful window size to use in this type of bioinformatics search.
IgE Testing
IgE-binding studies and methods for assessing functional IgE binding for diagnostic purposes and their utility and weaknesses for use in the safety assessment were also discussed. The general consensus of the workshop participants was that sera screening done for the purpose of public-health decision-making should be performed using individual sera instead of pooled sera. Participants recognized, however, that there might be availability constraints from the standpoint of individual patient sera to conduct such studies. There was also general agreement that in most cases IgE binding to carbohydrate domains is irrelevant to clinical allergy, unless there are multiple carbohydrates on the potentially allergenic protein. The need for careful clinical characterization of serum donors was also noted in order to reduce the potential for false negative or false positive testing. In particular, participants felt it important to establish a link between the sera sample and a double-blind, placebo-controlled food challenge (DBPCFC) study. The need to demonstrate that IgE binding is functional either by in vivo methods (e.g., skin prick testing) or by basophil histamine release assays in vitro was discussed. It was noted that all of these assays can be technically challenging and require method validation in laboratories that would use them to further evaluate whether a protein that has a meaningful bioinformatics match with a known allergen is likely to cause clinical cross-reactivity. The workshop group also discussed the value of targeted sera screening as recommended by FAO/WHO (2001). The consensus of the participants was that the utility of targeted sera screening for predicting protein allergenicity has not been demonstrated, nor have specific methods for this screen been validated.
Conclusion
Overall, there was general consensus from workshop participants that bioinformatics plays a vital role in the allergy assessment of biotech proteins. Together with IgE-binding studies that utilize well-characterized patient sera, bioinformatics plays a critical role in identifying proteins with sequence or structural similarities to allergens, thus identifying proteins with the potential to cross-react with IgE and cause clinical symptoms of allergy. Workshop participants recognized that bioinformatics methods associated with allergy assessment were evolving, and that new methods, once they are tested and validated, may lead to improved methods for identifying potentially allergenic characteristics of novel proteins.
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