Grantee Research Project Results

2013 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, 2012 through September 14,2013
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 beta-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, beta-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:

1. Protein Purification:

Method development for purification of the peanut allergen, Ara h 2, from roasted peanut flour was completed during Year 3 of the grant. Approximately 2 g of Ara h 2 was obtained through a series of large scale purifications. Each pool was homogenously mixed and lyophilized to ensure stability during storage. The purified Ara h 2 was found to be 90-95% pure based on 1D-SDS PAGE and densitometry analysis. This material will be used for sensitization of conventional and germ free C3H/HeN mice in early 2014.

Purified Sigma BLG was used along with whey protein for sensitization of conventional and germ free mice as described later in this report. The lipopolysaccharide (LPS) content of the Sigma BLG was measured using the Limulus Amebocyte Lysate (LAL) assay. High levels of LPS have been reported to skew the mouse immune response to a Th1, non-allergic response. A level 2561 EU of LPS per mg of Sigma BLG was detected. Due to this high level of LPS, a second source of BLG from Arla Foods was sourced. The Arla Foods BLG had low levels of LPS (<0.005 EU/mg protein) and was used for subsequent comparison of sensitization of germ-free and conventional C3H/HeN.

Purified soybean lypoxygenase was acquired with a purity of >90% as assessed by SDS-PAGE and densitometry. The LPS content of this protein was measured using the LAL assay and was found to be 70 EU/mg protein. The LPS content of the purified Ara h 2 is currently being assessed.

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

Both germ-free and conventional mice that were challenged with BLG or whey protein intragastrically (IG) showed more variation in clinical scores, biomarker levels and temperature upon challenge than compared to the mice challenged by an intraperitoneal (IP) route. Among the IG challenged mice, IP sensitization provided slightly more consistency in clinical scores, although a significant difference was not observed. Germ-free mice that were IG challenged also showed less variability. Mice that were IP sensitized and IP challenged showed markedly greater clinical scores and temperature, with less variability than the IG challenged mice. Overall, germ-free mice that were IP sensitized and IP challenged had best allergic responses upon challenge with less variability among mice. Due to these factors, the primary focus on evaluation of a mouse model for prediction of novel protein sensitization will focus on IP sensitization and IP challenge.

The level of LPS (bacterial endotoxin) in the purified protein sources was assessed as high levels could result in skewing the immune response to a Th1 response (non-allergenic immune response) and perhaps contribute to the variability observed in both the germ free and conventional mice. Overall, germ-free mice that were sensitized and challenged with the low LPS BLG exhibited a more pronounced decrease in body temperature upon challenge (decrease of 7.5°C ) compared to germ-free mice that were sensitized and challenged with the Sigma BLQ (high LPS; decrease in body temperature of 5.9°C). Clinical scores of germ-free mice sensitized and challenged with the low LPS BLG also had consistently higher clinical scores with less variability compared to the mice sensitized with high LPS BLG. The LPS content of BLG did not have a significant impact in temperature drop and clinical score for conventional C3H/HeN mice. The LPS content of the protein extract used for sensitization analysis appears to be an important factor in germ-free mice. If germ-free mice are found to provide the best predictive model for prediction of allergic sensitization of novel protein sources, the content of LPS in the novel protein extract will need to be taken into consideration.

Preliminary experiments were conducted to compare the allergic sensitization and elicitation potential of soy lipoxygenase (LOX) in germ-free and conventional C3H/HeN mice. LOX is a protein that has been shown to have a very low level of allergenicity and would therefore be predicted to have low allergic response in our animal model as compared to BLG. When comparing the temperature drop and clinical scores of mice sensitized with low LPS BLG compared to LOX, a more pronounced allergic response was observed with BLG as compared to LOX as we had hypothesized. The average temperature drop observed in BLG sensitized mice was 8°C compared to 2°C in mice sensitized with LOX. The clinical scores were more pronounced with BLG as well with an average score of 4 compared to a score of 1 with LOX. These preliminary results suggest that our germ-free C3H/HeN mouse model may provide a viable tool for differentiate between allergenic protein for prediction of sensitization potential.

Future Activities:

Evaluation of the sensitization and elicitation potential of purified peanut Ara h 2 will be conducted in Year 4 of the grant. Serological work will continue to evaluate levels of specific IgE and mast cell protease from mice challenged with BLG, LOX and Ara h 2. Evaluation of the food matrix (peanut and soy) on the sensitization potential to Ara h 2 and LOX will also be evaluated in 2014.

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

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.