Comparison of Gnotobiotic and Conventional Mice for Predicting the Allergenic Potential of Proteins Introduced into Genetically Engineered PlantsEPA Grant Number: RD834824
Title: Comparison of Gnotobiotic and Conventional Mice for Predicting the Allergenic Potential of Proteins Introduced into Genetically Engineered Plants
Investigators: Baumert, Joseph L , Goodman, Richard E. , Peterson, Daniel H
Institution: University of Nebraska at Lincoln
EPA Project Officer: McOliver, Cynthia
Project Period: September 15, 2010 through September 14, 2013 (Extended to September 14, 2014)
Project Amount: $423,546
RFA: Approaches to Assessing Potential Food Allergy from Genetically Engineered Plants (2009) RFA Text | Recipients Lists
Research Category: Food Allergy , Health
The proposed research focuses on the development of a more reliable, practical and predictive animal model that can be used to evaluate the allergenic potential of proteins introduced into genetically engineered plants. The objectives of this research project are 1) to evaluate sensitization responses in germ-free mice and mice having different defined intestinal microflora, to orally presented purified proteins (potent allergen, peanut Ara h 2; moderate allergen, egg-white lysozyme and non-allergenic soybean lipoxygenase); 2) to evaluate the importance of the food matrix (peanut, egg-white and soybean) on sensitizing potential of pure proteins; and 3) to test for differences in absorption (serum concentrations) of sensitizing proteins (Ara h 2, lysozyme and soybean lipoxygenase) in 1) and 2), as a potential source of differences in allergic sensitization.
C57BL/6 mice of the same age and genetic lineage that are germ-free or inoculated with defined proallergenic or conventional microflora will be orally sensitization with pure proteins (Ara h 2, lysozyme and lipoxygenase), natural matrices (peanut, egg white or soybean) and artificially mixed matrices (Ara h 2 and lysozyme in soybean). Doses of antigen will be optimized and given once per week, 3 consecutive weeks. Sensitization will be measured by clinical responses to injected allergen as well as by antigen specific testing for IgE (and other immunoglobulin isotypes) responses in B cells and cytokine responses to lamina propria T cells using ELISPOT assays. Serum from mice of each treatment group will be tested for the amount of allergen (Ara h 2, lysozyme and lipoxygenase) absorbed at 2, 4 and 24 hours after the final sensitizing dose using immunoassay (Ara h 2) and peptide analysis (all three proteins) with triple quadrapole (QQQ)-HPLC-MS.
The current allergenicity assessment of genetically modified crops (Codex, 2003) could be improved to evaluate certain product types (highly abundant proteins, pepsin-stable proteins, minor allergens and novel proteins) if a predictive animal model of allergenicity was available. Based on current knowledge of susceptible populations for food allergy (highest incidence in infants and young children), mechanisms of oral tolerance and sensitization, the germ-free mice and mice inoculated with a specific proallergenic bacterial strain are expected to be more readily sensitized and more predictive of allergenic potential than those with conventional microflora. Knowledge of absorption rates of these proteins in the controlled microfloral environments should help in understanding the predictive value of stability of proteins to pepsin and should provide a tool for evaluating the safety of proteins that are only partially digested by pepsin.