Comparative assessment of multiple criteria for the in silico prediction of cross-reactivity of proteins to known allergens.

Genetically modified crops are becoming important components of a sustainable food supply and must be brought to market efficiently while also safeguarding the public from cross-reactivity of novel proteins to known allergens. Bioinformatic assessments can help to identify proteins warranting further experimental checks for cross-reactivity. This study is a large-scale in silico evaluation of assessment criteria, including searches for: alignments between a query and an allergen having ≥ 35% identity over a length ≥ 80; any sequence (of some minimum length) found in both a query and an allergen; any alignment between a query and an allergen with an E-value below some threshold. The criteria and an allergen database (AllergenOnline) are used to assess 27,243 Viridiplantae proteins for potential allergenicity. (A protein is classed as a "real allergen" if it exceeds a test-specific level of identity to an AllergenOnline entry; assessment of real allergens in the query set is against a reduced database from which the identifying allergen has been removed.) Each criterion's ability to minimize false positives without increasing false negative levels of current methods is determined. At best, the data show a reduction in false positives to ∼6% (from ∼10% under current methods) without any increase in false negatives.

Assessment of possible allergenicity of hypothetical ORFs in common food crops using current bioinformatic guidelines and its implications for the safety assessment of GM crops.

Before a genetically modified (GM) crop can be commercialized it must pass through a rigorous regulatory process to verify that it is safe for human and animal consumption, and to the environment. One particular area of focus is the potential introduction of a known or cross-reactive allergen not previously present within the crop. The assessment of possible allergenicity uses the guidelines outlined by the Food and Agriculture Organization (FAO) and World Health Organization's (WHO) Codex Alimentarius Commission (Codex) to evaluate all newly expressed proteins. Some regulatory authorities have broadened the scope of the assessment to include all DNA reading frames between stop codons across the insert and spanning the insert/genomic DNA junctions. To investigate the utility of this bioinformatic assessment, all naturally occurring stop-to-stop frames in the non-transgenic genomes of maize, rice, and soybean, as well as the human genome, were compared against the AllergenOnline (www.allergenonline.org) database using the Codex criteria. We discovered thousands of frames that exceeded the Codex defined threshold for potential cross-reactivity suggesting that evaluating hypothetical ORFs (stop-to-stop frames) has questionable value for making decisions on the safety of GM crops.

Bioinformatics and the allergy assessment of agricultural biotechnology products: industry practices and recommendations.

Bioinformatic tools are being increasingly utilized to evaluate the degree of similarity between a novel protein and known allergens within the context of a larger allergy safety assessment process. Importantly, bioinformatics is not a predictive analysis that can determine if a novel protein will ''become" an allergen, but rather a tool to assess whether the protein is a known allergen or is potentially cross-reactive with an existing allergen. Bioinformatic tools are key components of the 2009 CodexAlimentarius Commission's weight-of-evidence approach, which encompasses a variety of experimental approaches for an overall assessment of the allergenic potential of a novel protein. Bioinformatic search comparisons between novel protein sequences, as well as potential novel fusion sequences derived from the genome and transgene, and known allergens are required by all regulatory agencies that assess the safety of genetically modified (GM) products. The objective of this paper is to identify opportunities for consensus in the methods of applying bioinformatics and to outline differences that impact a consistent and reliable allergy safety assessment. The bioinformatic comparison process has some critical features, which are outlined in this paper. One of them is a curated, publicly available and well-managed database with known allergenic sequences. In this paper, the best practices, scientific value, and food safety implications of bioinformatic analyses, as they are applied to GM food crops are discussed. Recommendations for conducting bioinformatic analysis on novel food proteins for potential cross-reactivity to known allergens are also put forth.

Further evaluation of the utility of "sliding window" FASTA in predicting cross-reactivity with allergenic proteins.

FAO/WHO has recommended that IgE cross-reactivity between a transgenic protein and allergen be considered when there is greater than 35% identity over a sliding "window" of 80 amino acids. In a previous work, we evaluated the false positive and negative rates observed using the FAO/WHO criteria versus conventional, whole protein FASTA analyses [Ladics, G.S., Bannon, G.A., Silvanovich, A., Cressman, R.F., 2007. Comparison of conventional FASTA identity searches with the 80 amino acid sliding window FASTA search for the elucidation of potential identities to known allergens. Mol. Nutr. Food Res. 51 (8), 985-998]. A number of protein sequence datasets were used as queries against the FARRP 7 allergen database. Results indicated that conventional FASTA analysis produced fewer false positives then the "sliding window" search proposed by FAO/WHO. Further, both methods were able to identify the potential for cross-reactivity between the Bet v 1 family of proteins, indicating that the conventional FASTA search possessed sufficient sensitivity. Recently, collections of protein sequences from multiple crop species (corn, soy, barley, lettuce, sugar beets, and spinach) were subjected to the same screen against the FARRP7 allergen dataset. In all cases, the conventional FASTA search returned fewer above threshold matches than the sliding window search. Examination of the matches not recognized by the conventional search revealed two scenarios: (1) "true" false positives consisting of low statistical significance (as measured by E score, i.e., a measure of the potential random occurrence of aligned sequences used to evaluate the significance of an observed alignment) alignments not contained in the conventional FASTA outputs, and (2) above-threshold sliding window alignments that fell below the 35% identity threshold with the conventional FASTA analysis. Although some alignments within this second group were between regions of low sequence complexity, where there was little/no change in E score, the majority of the alignments displayed more significance (lower E scores) under the conventional FASTA algorithm, yet did not meet the threshold defined by FAO/WHO. These data question the utility of the FAO/WHO recommended sliding window FASTA compared to the traditional whole sequence FASTA analysis coupled with appropriate statistical analysis.

A gene-shuffled glyphosate acetyltransferase protein from Bacillus licheniformis (GAT4601) shows no evidence of allergenicity or toxicity.

The glyphosate acetyltransferase (gat) gene from Bacillus licheniformis was subjected to multiple rounds of gene shuffling to optimize kinetics of corresponding GAT proteins to acetylate the herbicide active ingredient glyphosate. Genetically modified soybeans expressing the gat4601 gene (356043 soybeans) are tolerant to the application of glyphosate. The current manuscript reports the outcome of the allergenicity and toxicity assessment for the GAT4601 protein. Bioinformatic comparison of the amino acid sequence of GAT4601 did not identify similarities to known allergenic or toxic proteins. In vitro studies conducted with heterologously produced GAT4601 protein demonstrated that it was rapidly degraded in simulated gastric fluid containing pepsin (< 30 s) and in simulated intestinal fluid containing pancreatin (< 2 min) and completely inactivated at temperatures above 56 degrees C. The GAT4601 protein expressed in planta is not glycosylated and similar protein profiles were observed in flour extracts from 356043 soybeans and nontransgenic near isoline comparator soybeans (Jack) using serum from soy allergic persons. No evidence of adverse effects was observed in mice following acute oral exposure to 2000 mg/kg of GAT4601 protein or in a repeated dose dietary exposure study at doses of 800-1000 mg/kg/day. This comprehensive assessment demonstrates that the GAT4601 protein does not present a risk for adverse effects in humans when used in the context of agricultural biotechnology.

Comparison of conventional FASTA identity searches with the 80 amino acid sliding window FASTA search for the elucidation of potential identities to known allergens.

Food and Agriculture Organization/World Health Organization (FAO/WHO) recommended that IgE cross-reactivity between a transgenic protein and allergen be considered when there is >or= 35% identity over a sliding "window" of 80 amino acids. Our objective was to evaluate the false positive and negative rates observed using the FAO/WHO versus conventional FASTA analyses. Data used as queries against allergen databases and analyzed to assess false positive rates included: 1,102 hypothetical corn ORFs; 907 randomly selected proteins; 89 randomly selected corn proteins; and 97 corn seed proteins. To evaluate false negative rates of both methods: Bet v 1a along with several crossreacting fruit/vegetable allergens and a bean alpha-amylase inhibitor were used as queries. Both methods were also evaluated for their ability to detect a putative nonallergenic test protein containing a sequence derived from Ara h 1. FASTA versions 3.3t0 and 3.4t25 were utilized. Data indicate a conventional FASTA analysis produced fewer false positives and equivalent false negative rates. Conventional FASTA versus sliding window derived E scores were generally more significant. Results suggest a conventional FASTA search provides more relevant identity to the query protein and better reflects the functional similarities between proteins. It is recommended that the conventional FASTA analysis be conducted to compare identities of proteins to allergens.

Lack of cross-reactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust mite Der p7 protein with human sera positive for Der p7-IgE.

Cry1F protein, derived from Bacillus thuringiensis, is effective at controlling lepidopteran pests and a synthetic Cry1F transgene was transferred into maize. For the safety assessment of genetically modified food crops, the allergenic potential of the introduced novel trait(s) is evaluated. Because no single parameter is currently predictive of allergic potential, a 'weight of evidence' approach has been proposed. As part of this assessment, the amino acid (aa) sequence of the Cry1F protein was compared to a database of known allergens using recommended criteria. The Cry1F protein did not show significant similarity or a match of eight contiguous identical aa with any allergen. However, a single six contiguous aa match was identified between Cry1F and the Der p7 protein of the dust mite, Dermatophagoides pteronyssinus. To investigate whether Cry1F was cross-reactive with Der p7, sera from 10 dust mite allergic patients containing Der p 7-specific IgE antibody were used to compare IgE-specific binding. No evidence of cross-reactivity was observed between Cry1F and Der p7. This study provides in vitro IgE sera screening data, that when considered in the context of other bioinformatic data [Hileman R.E., Silvanovich, A., Goodman R.E., Rice E.A., Holleschak G., Astwood J.D., Hefle S.L., 2002. Bioinformatic methods for allergenicity assessment using a comprehensive allergen database. Int. Arch. Allergy Immunol. 128, 280-291; Stadler, M.B., Stadler, B.M., 2003. Allergenicity prediction by protein sequence. FASEB J. 17, 1141-1143.], adds further evidence arguing against the use of a six contiguous identical amino acid search to identify potential cross-reactive allergens. Cry1F is heat labile, rapidly hydrolyzed in an in vitro pepsin resistance assay, not glycosylated and not from an allergenic source. Taken together, these data indicate a lack of allergenic concern for Cry1F.