Grantee Research Project Results

2012 Progress Report: Comparison of Gnotobiotic and Conventional Mice for Predicting the Allergenic Potential of Proteins Introduced into Genetically Engineered Plants

EPA Grant Number: R834824
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: Aja, Hayley
Project Period: September 15, 2010 through September 14, 2013 (Extended to September 14, 2014)
Project Period Covered by this Report: September 15, 2011 through September 14,2012
Project Amount: $423,546
RFA: Approaches to Assessing Potential Food Allergy from Genetically Engineered Plants (2009) RFA Text |  Recipients Lists
Research Category: Human Health

Objective:

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 a conventional intestinal microflora, to orally presented purified proteins (potent allergen, peanut Ara h 2; moderate allergen, bovine ß-lactoglobulin and non-allergenic soybean lipoxygenase);
2. to evaluate the importance of the food matrix (peanut, whey powder and soybean) on sensitizing potential of pure proteins; and
3. to test for differences in absorption (serum concentrations) of sensitizing proteins (Ara h 2, ß-lactoglobulin and soybean lipoxygenase) in mice used in experiments outlined for objectives 1 and 2 to determine if intestinal microflora and food matrix have potential impacts on  allergic sensitization.

Progress Summary:

This progress report covers research activities completed or in progress during the second year (September 2011 through September 2012) under the EPA STAR Grant # RD834824. Funding was received on October 30, 2010 and research commenced at that time. A summary of the research activities for this period are provided below.

1. Protein Purification:

   Method development for purification of the peanut allergens, Ara h 2 and Ara h 6, from roasted peanut flour commenced in January of 2012 after the final installation of a GE Healthcare AKTA Avant FPLC purification instrument was finalized. The purification protocol includes an initial extraction of soluble peanut proteins from the partially defatted, light roasted peanut flour using 1.0 M NaCl in 0.01 M PBS. Following centrifugation to remove insoluble matter, ammonium sulfate precipitation is used to fractionalize the proteins based upon solubility in this salt buffer. Fractions containing the Ara h 2 and Ara h 6 (as visualized using SDS-PAGE) are collected for subsequent chromatographic purification. Hydrophobic interaction chromatography (HIC) followed by anion exchange chromatography (AE) are used in the purification of Ara h 2 and Ara h 6 as represented Figure 1. Pooled fractions of purified Ara h 2 and Ara h 6 were run on 18% Tris-glycine gels under reducing conditions to evaluate the purity of the respective protein samples based on densitometry analysis (Figure 2). Approximately 90-93% purity of Ara h 2 and 85-90% purity of Ara h 6 was achieved using this purification method. Currently low mg quantities of the purified peanut proteins are available. This method will be applied to larger HIC and AE columns to achieve purification of gram quantities of Ara h 2 and Ara h 6. Purified Ara h 2 and Ara h 6 along with peanut extract will be used for sensitization of conventional and germ free C3H/HeN mice.

2. Development of a Sensitized Mouse Model for Allergy Assessment of Proteins:

   Conventional and germ-free C3H/HeN mice were sensitized with BLG or whey protein either 5x orally or 3x by IP injection on weekly intervals in the presence of cholera toxin (10 ug) or alum adjuvant (1:1 ratio of protein to alum), respectively. One week following the final sensitization an oral challenge of 60 mg BLG was given. One hour post-challenge clinical scores were assigned and body temperatures recorded. Body temperatures dropped an average of 1°C more in germ-free mice compared to conventional mice post-challenge. In addition, germ-free clinical reactions were consistently more pronounced. Conventional mice that were orally sensitized and later orally challenged did not have a significant drop in body temperature and clinical scores were more variable but less severe than observed in IP sensitized conventional and GF mice.

   It appears from this work that removing the microbial signals normally present in the gut resulted in a skewing of the immune response to a more allergenic profile, based on change in body temperature and clinical scores in mice sensitized IP and challenged orally. Serum was collected from each mouse approximately 1 hour after the oral challenge. Serum specific BLG IgE, IgG2a and IgG1 (along with total IgE, IgG2a and IgG1) and mast cell protease levels will be determined using ELISA.

Future Activities:

Protein purification and characterization/identification will continue in Year 3 of the grant to obtain sufficient quantities of peanut Ara h 2 and Ara h 6 (and soybean lipoxygenase if needed).  All purified proteins have been and will continue to be analyzed using SDS-PAGE analysis, immunoblot analysis when antibodies directed against the protein of interest are available, and confirmatory tandem mass spectrometry analysis will be conducted.  The purified Ara h 2, Ara h 6 and soybean lipoxygenase proteins along with peanut extract and soybean extract will be used for oral sensitization of conventional and germ free C3H/H3N mice in 2013.   Serological work will continue to evaluate levels of specific IgE, IgG2a and IgG1 to Ara h 2, Ara h 6 and soybean lipoxygenase.  As the mice from these experiments are euthanized, sera will be collected to determine if digestion resistant peptides of these allergenic proteins can be measured with both ELISA methods and by tandem mass spectrometry as outline in Objective 3 of the proposal. 

Journal Articles:

No journal articles submitted with this report: View all 10 publications for this project

Supplemental Keywords:

Anaphylaxis, antibody, GM, risk assessment, sensitive population, human health risk, food allergenicity, oral allergy syndrome, allergic sensitization

Progress and Final Reports:

Original Abstract

2011 Progress Report

2013 Progress Report

Final Report