Genetically Modified a-Amylase Inhibitor Peas Are Not Specifically Allergenic in Mice Rui-Yun Lee 1. , Daniela Reiner 1. , Gerhard Dekan 2 , Andrew E. Moore 3 , T. J. V. Higgins 3 , Michelle M. Epstein 1 * 1 Division of Immunology, Allergy and Infectious Diseases, Experimental Allergy, Department of Dermatology, Medical University of Vienna, Vienna, Austria, 2 Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria, 3 CSIRO Plant Industry, Canberra, ACT, Australia Abstract Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (aAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. aAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity in mice of the transgenic pea but not the bean. Here, we observed that transgenic aAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking aAI led to an anti-aAI response due to a cross-reactive response to pea lectin. Our data demonstrate that aAI transgenic peas are not more allergenic than beans or non-transgenic peas in mice. This study illustrates the importance of repeat experiments in independent laboratories and the potential for unexpected cross-reactive allergic responses upon consumption of plant products in mice. Citation: Lee R-Y, Reiner D, Dekan G, Moore AE, Higgins TJV, et al. (2013) Genetically Modified a-Amylase Inhibitor Peas Are Not Specifically Allergenic in Mice. PLoS ONE 8(1): e52972. doi:10.1371/journal.pone.0052972 Editor: Muriel Moser, Universite ´ Libre de Bruxelles, Belgium Received September 14, 2012; Accepted November 23, 2012; Published January 9, 2013 Copyright: ß 2013 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was provided the GMSAFOOD project (grant nu,ber 211820), through the 7th Framework grant, administered by the European Commission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michelle.epstein@meduniwien.ac.at . These authors contributed equally to this work. Introduction Genetically modified (GM) crop areas have increased rapidly since their introduction in 1996 [1]. New approaches to generate plants that are resistant to insect infestation are being actively sought, especially to reduce reliance on chemical insecticides. For example, genetically modified peas (Pisum sativum), chickpeas (Cicer arietinum) and cowpeas (Vigna unguiculata) expressing the gene for alpha-amylase inhibitor-1 (aAI) from the common bean (Phaseolus vulgaris) cultivar Tendergreen are completely protected from weevil destruction [2,3,4]. aAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system [5]. The excellent insecticidal effect of aAI [6] and the long-term safe consumption of beans containing aAI [7] make it a promising gene to insert into insect-susceptible legumes. However, one study suggested that aAI peas expressed a variant protein resulting in allergic responses in mice to the peas but not the beans [8]. They found that mice consuming aAI peas developed elevated levels of aAI-specific IgG1 but not IgE antibodies, had enhanced delayed-type hypersensitivity responses and increased reactivity to other allergens (adjuvant effect) whereas mice fed non-transgenic peas and Pinto beans had no aAI reaction. Mass spectrometry results revealed differences in post- translational modifications, which the authors suggested led to the reported allergenicity. These results were received with some skepticism including an editorial in Nature Biotechnology [9]. More recently, a comparison using high-resolution mass spectrometry of aAI from bean and transgenic legume sources revealed heterogeneous structural variations in peas and beans due to differences in glycan and carboxypeptidase processing, but the transgenic versions were within the range of those observed from several bean varieties [5]. Moreover, when purified aAIs from beans and transgenic peas were used to immunize mice, all elicited Th1 and Th2- type aAI-specific antibodies [5]. This questions the reported enhanced aAI transgenic pea-specific immunogenicity and allergenicity compared with the naturally occurring protein in beans. The objective of this study was to evaluate allergenicity of aAI peas, cowpeas and chickpeas and compare them to non-transgenic controls, Pinto and Tendergreen beans (the latter was the source of aAI gene) in mice. To achieve this aim, we evaluated the immunogenicity and allergenicity of aAIs from these transgenic legumes to determine whether the transgenic aAIs were more allergenic than the aAIs from Pinto and Tendergreen beans. The evaluation included a comparison of antibody titres to aAIs from each source. Additionally, we tested the antibody response to twice weekly consumption of the pea, cowpea, chickpea and bean meals for 4 weeks. After the feeding period, we challenged the respiratory tract with aAI to evaluate in vivo T lymphocyte responses. Lastly, we assessed the adjuvant effect of aAI pea consumption on the initiation and exacerbation of non-cross-reactive ovalbumin (OVA)-induced allergic lung disease. PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e52972