Grantee Research Project Results
Final Report: Predicting Food Protein Allergenicity Using a Mouse Model
EPA Grant Number: R834822Title: Predicting Food Protein Allergenicity Using a Mouse Model
Investigators: Gangur, Venugopal
Institution: Michigan State University
EPA Project Officer: Aja, Hayley
Project Period: September 15, 2010 through September 14, 2014
Project Amount: $424,919
RFA: Approaches to Assessing Potential Food Allergy from Genetically Engineered Plants (2009) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
There is growing recognition that food allergy is a critical public health problem of international significance that has reached an epidemic proportion (Sicherer and Sampson 2014). Specific reasons for this alarming rise in food allergies are not completely understood at present. Whether genetically engineered (GE) foods contribute to this problem is largely unknown. Assessment of allergenic potential of GE foods is a major challenge facing the international and national regulatory agencies and the agro-biotech industry (Selgrade et al. 2009). Although expert panels suggest the use of animal models as one of the methods for assessment of allergenic potential of GE foods, widely accepted and validated animal models are not available at present (Ladics and Selgrade 2009).
This project was conducted during the period September 15, 2010, to September 14, 2014. Currently, validated animal models to assess the allergenicity of novel dietary proteins, such as those present in GE foods, are not available. We previously published a novel mouse model of near-fatal food allergy that involves transdermal allergen sensitization followed by oral elicitation (TS/OE) of systemic anaphylaxis (Birmingham et al. 2005, 2007; Navuluri et al. 2006; Parvataneni et al. 2009; Gonipeta et al. 2010). The data collected during a previously funded U.S. EPA project (R833133) demonstrated that: (1) the allergenicity readouts in this mouse model are dependent on the number of exposures to the allergenic protein; (2) seven human allergenic proteins (hazelnut, cashew nut, sesame seed, milk, shellfish, egg, fish) produce all three readouts of allergenicity in this mouse model (Birmingham et al. 2005, 2007; Navuluri et al. 2006; Parvataneni et al. 2009; Gonipeta et al. 2010); (3) six human non-allergenic proteins (kidney bean, Pinto bean, blueberry, sorghum, pigeon pea, mung bean) were non-allergenic in this model based on clinical scores and hypothermia readouts; (4) amaranth seed protein, which has no history of human allergenicity and is therefore presumed to be non-allergenic, tested positive for all three readouts of allergenicity (Fig. 1); (5) hazelnut allergy in this mouse model once established is long-lasting similar to human hazelnut allergy (Gonipeta et al. 2010); and (6) food allergy is genetically controlled in this model similar to human food allergy (Parvataneni et al. 2009).
These data suggested that the TS/OE mouse model simulates many aspects of human food allergy and that it has very promising positive and negative predictive value for assessment of food protein allergenicity (Selgrade et al. 2009; Gonipeta et al. 2015). These data also warranted further validation of this mouse model towards the long-term goal of using it for predicting allergenic potential novel food proteins. Consequently, in the current project funded by U.S. EPA, the overall goal of the project was to determine the threshold oral elicitation doses (NOAEL and the LOAEL) of food proteins (whole protein extracts and pure protein) with high versus low/no allergenic potential in this model.
Summary/Accomplishments (Outputs/Outcomes):
First year: Major accomplishments during the first year (2010–11) of the project were as follows: (1) recruitment and training of personnel for the project; (2) establishment of the oral elicitation dose-response curves for hazelnut in TS/OE mouse model using three different sensitization protocols; (3) comparison of the oral elicitation dose-response curves for raw versus boiled hazelnut; (4) optimization and validation of a methodology for measurement of mouse mast cell protease 1 (mMCP-1) as a potential quantifiable biomarker of systemic anaphylaxis in this model; (5) evaluation of an infrared transdermal scanning thermometry as a non-invasive alternative to rectal thermometry in this model; and (6) optimization of a method to prepare pigeon pea protein extract for use in the project.
Second year: Major accomplishments during the second year (2011–12) were: (1) completion of additional experiments on dose-response curves for hazelnut protein with more mice per group and with more doses for hazelnut; (2) establishment of percent responder curves and estimation of NOAEL and LOAEL for hazelnut in this model; (3) determination of human equivalent threshold doses for hazelnut using threshold doses from this mouse model; (4) production of pigeon pea protein extract using optimized in-house methods; (5) evaluating the allergenic potential of in-house produced pigeon pea protein extract in TS/OE model and establishment of dose-response curves and percent responder curves; and (6) optimization of a method for preparing high-quality sesame seed protein extract for use in this project.
Third year: Major accomplishments during the third year (2012–13) were: (1) determination of the allergenicity and oral elicitation threshold doses for hazelnut protein prepared in our laboratory using an in-house method; (2) production of sesame seed (SS) protein extracts using an optimized method in our laboratory and characterization of the quality using SDS PAGE analysis and comparison with the SS protein extracts purchased from Greer Laboratories; (3) evaluation of the allergenicity of our laboratory-prepared SS protein extract and that obtained from the Greer Laboratories in the TS/OE model; (4) determination of oral elicitation dose responses, establishment of percent responder curves and determination of the threshold oral elicitation doses (LOAEL and NOAEL) for these two sources of SS proteins in TS/OE model; and (5) determination of the human equivalent threshold doses for SS proteins from both sources.
Final year of no-cost-extension (NCE) period: Major accomplishments during the NCE period (2013–14) were: (1) evaluation of the allergenicity of extrusion processing-derived hazelnut protein extract in this model; and demonstration that this type of food processing can potentially reduce the oral allergy elicitation potency via enhancing oral threshold dose; (2) identification of IL-6 and CXCL2/MIP-2 as potential novel biomarkers of systemic anaphylaxis upon oral allergen challenge in this model; (3) evaluation of oral allergenicity of pure protein ovalbumin in this model; (4) establishment of oral-dose response curves, percent responder curves and estimation of oral elicitation threshold doses (LOAEL and NOAEL) for ovalbumin in this model; (5) evaluation of allergenicity of ovalbumin via intraperitoneal systemic challenge; (6) evaluation of mMCP-1 as a reliable biomarker of systemic reactions upon allergen challenge; (7) dissemination of the data from the project: (a) presentation of data during the American Association of Immunologists, Annual meeting, May 4, 2014, Pittsburgh, PA; (b) invited seminar presentations at the NIH/National Institutes of Allergy and Infectious Diseases, the Georgia State University (January 21, 2013), U.S. FDA/University of Maryland, Center for Food Safety and Applied Nutrition (CFSAN) (April 7, 2014), and Nestle Purina Pet Care, St. Louis (November 3, 2014); (c) publication of two papers, one Master’s thesis and one abstract, one additional paper accepted for publication (November 14, 2014), two papers and one abstract are in preparation; and (8) preparation of five grant proposals using data from the project as preliminary data (two proposals submitted to NIH; one proposal in preparation; one proposal submitted to industry; one proposal to Ag Bio Research/NIFA/MSU).
During this project with the generous support of the U.S. EPA STAR grant, we evaluated the utility of a novel transdermal sensitization/oral elicitation (TS/OE) mouse model of food allergy for determination of oral elicitation threshold doses for food proteins with high/low allergenic potential.
There are several major findings during this project, including the following:
(1) Establishment of dose-response curves and percent responder curves using hypothermia shock response as a quantifiable marker of oral elicitation of allergic reactions in this mouse model for different types of food proteins with high/low allergenic potential. Determination of oral elicitation threshold doses (NOAEL, LOAEL) for these food proteins in mouse model. The oral elicitation threshold doses determined for various food proteins in this mouse model are summarized in Table 1.
(2) Using the method recommended in the literature (Reagan Shaw, et al. 2008), the mouse threshold doses obtained in this model were translated to the human equivalent threshold doses. These results are summarized in the Table 2.
(3) Degranulation of mucosal mast cells and elevation of circulating levels of murine mast cell protease (mMCP)-1 protein is a useful biomarker of allergic reaction in this model (Gonipeta, et al. 2014)
(4) A commercially obtained pigeon pea protein extract was found to be of inconsistent quality/safety to use in this mouse model; earlier batch material was non-toxic, but later batches obtained were consistently toxic to mice when orally administered; consequently, these data demonstrated that the quality and safety of the food protein to be tested in this model for allergenicity is very critical and must be evaluated and confirmed before using the food extracts in this mouse model for oral threshold dose establishment.
(5) An in-house prepared pigeon pea protein extract that was non-toxic was found be tolerated very well in this mouse model upon oral challenge; with highest dose of 15 mg/mouse elicited very little reactions, suggesting low allergenic potential; these data are similar to the human situation where pigeon pea protein is also considered to be a food protein with low allergenicity potential in humans.
(6) Purified commercial ovalbumin (MP Biomedical) elicits robust systemic IgE antibody responses upon transdermal exposure in this mouse model. However, it did not elicit oral hypothermia shock reactions (Fig. 2A, B). Nevertheless, pure ovalbumin elicited hypothermia shock reaction when administered by intraperitoneal injection (Fig. 2C, D). Whether purified ovalbumin protein that we used in this study is allergenic by oral route in egg-sensitized humans is unknown.
There was a delay in the project during the third year that resulted in requesting a no-cost extension of 1 year for the project. This delay was primarily due to a sudden change in the personnel working on the project due to the research associate moving to another job. However, we quickly identified an excellent graduate student who started working on this project (Ms. Tina Ortiz). We also were able to hire a research assistant to help with the studies (Mr. Michael Reitmeyer). The PI (Dr. Gangur) continued to work on this project during this NCE period (100% efforts during the summer and 40% efforts during the academic year). Thus, with our quick action we were able to complete the project during the NCE period despite the challenge from staff changes.
There were four surprising but significant discoveries during the project:
As compared to raw hazelnut protein extract, extrusion processing-derived hazelnut protein extract exhibited significantly reduced potency in eliciting oral allergic reactions as quantified by hypothermia shock responses as well as elevation of circulating mMCP-1 protein levels (a marker of mucosal mast cell degranulation response) (Ortiz et al. 2014). These data suggest that oral elicitation threshold doses for food allergens will depend on whether raw versus processed food proteins are used in oral challenge testing.
Oral allergen challenge (OC) with hazelnut (HN) in TS/OE mouse model leads to immediate elevation of circulating levels of IL-6 cytokine (Fig. 3A) and CXCL2/MIP-2 chemokine (Fig. 3B). We also found that a hypoallergenic hazelnut protein (HNAD) produced using extrusion processing technology completely lacks the ability to elicit IL-6 and partially loses ability to induce CXCL2/MIP-2 upon oral challenge. These data suggest that these mediators may serve as potential novel biomarkers of oral allergic reactions in this model.
As opposed to raw egg protein extract, oral administration of purified ovalbumin does not elicit hypothermia shock responses in transdermal-sensitized mice (Ortiz et al. in preparation). These data suggest that oral elicitation threshold doses for food allergens will depend on whether whole food protein extract or purified single protein is used in oral challenge testing. They also suggest a potential role of the food matrix in triggering oral allergic reactions in a sensitized host.
Finally, we found cardiac mMCP-4+ mast cell expansion and elevation of IL-6 cytokine and CXCR2, CCR1/CCR3 signaling chemokines in this mouse model (Gonipeta et al. 2015). These data demonstrate that the heart is a direct target of oral allergic reaction to hazelnut (Fig. 4). Consequently, they also suggest that establishing oral elicitation threshold doses for food allergens need to take into account not only hypothermia shock responses but also cardiac tissue response as a critical readout of oral allergenicity of food proteins.
We established the Quality Assurance (QA) and Quality Control Project Plan and QA oversight following the expert guidance and valuable advice provided by the U.S. EPA Project Officer (Dr. Irwin Baumel). Completely randomized study design was used for the experiments. Adult mice were purchased from the Jackson Laboratory. Food proteins were purchased from commercial sources (GREER; Sigma) and used in the project; in addition, in-house food proteins were produced, quality and safety assessed, and then used in the project. All data were entered in Excel and maintained on the server with additional backup. All plasma samples from the project were stored at −70oC in aliquots. Readouts were measured at least twice to ensure reproducibility.
Conclusions:
During this project, we evaluated the utility of a novel adjuvant-free mouse model of food allergy for determination of oral elicitation threshold doses for various food proteins with high/low allergenic potential in humans. The data together suggest that the mouse model can be used to establish oral elicitation threshold doses such as NOAELs and LOAELs for allergenic food protein extracts. This is the first demonstration of the utility of a mouse model of food allergy towards food allergen threshold dose determination.
One critical outcome from this project is the further validation of an adjuvant-free mouse model for potential oral allergenicity hazard identification of novel dietary proteins, including pesticidal proteins used in GE foods. On the long-term, this outcome is expected to enhance our ability to quantitatively assess oral allergenic potency of novel proteins (including those in genetically engineered foods) relative to known allergenic and non-allergenic dietary proteins in a rational, reproducible and cost-effective manner. Furthermore, this research has advanced our basic knowledge on how environmental dietary proteins interact with the immune system and could adversely impact human health.
References:
Birmingham N, Gangur V, Samineni S, Navuluri L, Kelly C. “Hazelnut allergy: evidence that hazelnut can directly elicit specific IgE antibody response via activating type 2 cytokines in mice.” International Archives of Allergy and Immunology 2005;137(4):295-302.
Birmingham NP, Parvataneni S, Hassan HM, Harkema J, Samineni S, Navuluri L, et al. “An adjuvant-free mouse model of tree nut allergy using hazelnut as a model tree nut. International Archives of Allergy and Immunology 2007;144 (3):203-210.
Gonipeta B, Parvataneni S, Paruchuri P, Gangur V. “Long-term characteristics of hazelnut allergy in an adjuvant-free mouse model.” International Archives of Allergy and Immunology 2010;152(3):219-225.
Gonipeta B, Duriancik D, Kim E, Gardner E, Gangur V. “Identification of T- and B-cell subsets that expand in the central and peripheral lymphoid organs during the establishment of nut allergy in an adjuvant-free mouse model.” ISRN Allergy 2013;2013:509427.
Gonipeta B, Kim EJ and Gangur V. “Mouse models of food allergy: how well do they simulate the human disorder?” Critical Reviews in Food Science and Nutrition 2015;55(3):437-452.
Gonipeta B, Para R, He Y, Srkalovic I, Ortiz T, Kim EJ, Parvataneni S, Gangur V. “Cardiac mMCP-4+ mast cell expansion and elevation IL-6, and CCR1/3 and CXCR2 signaling chemokines in an adjuvant-free mouse model of tree nut allergy.” Immunobiology 2015;220(5):663-672.
Ladics GS, Selgrade MK. “Identifying food proteins with allergenic potential: evolution of approaches to safety assessment and research to provide additional tools.” Regulatory Toxicology and Pharmacology 2009;54(3 Suppl):S2-S6.
Navuluri L, Parvataneni S, Hassan H, Birmingham NP, Kelly C, Gangur V. “Allergic and anaphylactic response to sesame seeds in mice: identification of Ses i 3 and basic subunit of 11s globulins as allergens.” International Archives of Allergy and Immunology 2006;140(3):270-276.
Parvataneni S, Birmingham NP, Gonipeta B, Gangur V. “Dominant, non-MHC genetic control of food allergy in an adjuvant-free mouse model.” International Journal of Immunogenetics 2009;36(5):261-267.
Reagan Shaw S, et al. Dose translation from animal to human studies revisited. FASEB Journal 2008;22(3):659-661.
Selgrade MK, Bowman CC, Ladics GS, Privalle L, Laessig SA. “Safety assessment of biotechnology products for potential risk of food allergy: implications of new research.” Toxicological Sciences 2009;110(1):31-39.
Sicherer SH, Sampson HA. “Food allergy: epidemiology, pathogenesis, diagnosis, and treatment.” Journal of Allergy and Clinical Immunology 2014;133(2):291-307.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 19 publications | 5 publications in selected types | All 5 journal articles |
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Gonipeta B, Duriancik D, Kim E, Gardner E, Gangur V. Identification of T-and B-cell subsets that expand in the central and peripheral lymphoid organs during the establishment of nut allergy in an adjuvant-free mouse model. ISRN Allergy 2013;509427. |
R834822 (Final) R833133 (Final) |
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Gonipeta B, Para R, He Y, Srkalovic I, Ortiz T, Kim EJ, Parvataneni S, Gangur V. Cardiac mMCP-4+ mast cell expansion and elevation IL-6, and CCR1/3 and CXCR2 signaling chemokines in an adjuvant-free mouse model of tree nut allergy. Immunobiology 2015;220(5):663–672. |
R834822 (Final) |
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Gonipeta B, Kim E, Gangur V. Mouse models of food allergy: how well do they simulate the human disorder? Critical Reviews in Food Science and Nutrition 2015;55(3):437-452. |
R834822 (Final) R833133 (Final) |
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Ortiz T, Para R, Gonipeta B, Reitmeyer M, He Y, Srkalovic I, Ng PKW, Gangur V. Effect of extrusion processing on immune activation properties of hazelnut protein in a mouse model. International Journal of Food Sciences and Nutrition 2016;67(6):660-669. |
R834822 (Final) |
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Parvataneni S, Gonipeta B, Acharya HG, Gangur V. An Adjuvant-Free Mouse Model of Transdermal Sensitization and Oral Elicitation of Anaphylaxis to Shellfish. International Archives of Allergy and Immunology 2015;168(4):269-276. |
R834822 (Final) R833133 (Final) |
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Supplemental Keywords:
Food allergy, food safety, allergen, systemic anaphylaxis, exposure, hazard assessment, mouse model, potency testing, dose-response, genetic engineering, allergenicityRelevant Websites:
Evaluation of a Mouse Model of Food Allergy for Determination of Oral Elicitation Threshold Doses for Hazelnut (PDF) (21 pp, 146 K, About PDF) ExitProgress and Final Reports:
Original AbstractThe 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.