Genetically modified α-amylase inhibitor peas are not specifically allergenic in mice

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 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI 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 αAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking αAI led to an anti-αAI response due to a cross-reactive response to pea lectin. Our data demonstrate that αAI 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.

Figure 1. Experimental protocols.

A. Intraperitoneal immunization with purified proteins to assess protein immunogenicity. On days 0 and 21 mice were immunized with 10 µg of purified αAIs from the transgenic peas, Tendergreen bean, Pinto bean, or pea lectin purified from non-transgenic peas. On day 28, sera were harvested and evaluated for IgG1, IgE and IgGa2 antibodies to αAI. B. Intranasal immunization with purified proteins to assess differences in protein allergenicity. On the indicated days, mice were instilled with 50 µg of purified αAIs from transgenic peas, Tendergreen bean, Pinto bean and tested for antibody titres and allergic lung responses on day 21. C. Seed meal feeding for the evaluation of immune responses to αAI upon ingestion. Mice were gavaged with 25 mg seed meals from αAI -pea, -cowpea, -chickpea, non-transgenic peas, Pinto bean and Tendergreen bean 8 times on the indicated days. On day 29, mice received an intranasal instillation of 50 µg of αAI purified from αAI pea or Tendergreen bean, and were evaluated on day 32 for antibody titres and allergic lung responses. D. Adjuvant effect of peas and beans on the initiation of OVA-induced allergic lung disease. Mice were gavaged with 25 mg raw or cooked seed meals from αAI -pea, -cowpea, -chickpea, non-transgenic peas, Pinto bean and Tendergreen bean 8 times on the indicated days. Mice were immunized to induce allergic disease with 10 µg of OVA on days 7 and 28. After one week, the mice were nebulized with OVA on days 35 and 36. On day 39, antibody titres and allergic lung responses were measured. E. Adjuvant effect of peas and beans on the exacerbation of OVA-induced allergic lung disease. Mice were induced with allergic disease on days 0 and 21 and aerosolized on days 28 and 29 and then allowed to recuperate. On the indicated days mice were gavaged 8 times with 25 mg raw or cooked seed meals. One day later, mice were nebulized with OVA on 2 consecutive days to induce a disease exacerbation. On day 90, they were evaluated for antibody titres and allergic lung responses.

Figure 2. Immune responses to αAIs upon intranasal and intraperitoneal immunization.

Serum antibody titres for Anti-αAI IgG1, IgE and IgG2a from A. i.p. αAI immunized mice and B. from i.n. αAI immunized mice. The treatment groups for A and B include PBS only ×, purified αAI proteins from Tendergreen bean ▪, Pinto bean □, pea ▴, chickpea ⧫, and cowpea •. Data are expressed as mean OD_{450 nm} ± SEM; n = 8, duplicate samples. For IgE, dilutions are expressed ×10³. C. Eosinophil counts in BAL fluid from mice immunized with i.n. αAIs. D. Eosinophil counts in Luna-stained lung sections from mice immunized with i.n. αAIs. E. Inflammation scores of lung sections from mice immunized with i.n. αAIs. F. Mucus scores in PAS-stained lung sections from mice immunized with i.n. αAIs. Data are expressed as means ± SEM; n = 8. For eosinophil counts in BAL and lungs, data were compared using the Kruskal-Wallis test followed by Dunn's multiple comparison test. For histological scoring, data were compared with the Mann Whitney test. *p<0.05 for all groups above the PBS controls. These are representative data from 2 experiments.

Figure 3. Immune responses following consumption of raw or cooked seed meal from αAI pea and bean.

A. Serum antibody titres for anti-αAI IgG1, IgE and IgG2a from mice gavaged PBS or seed meals. B. Serum antibody titres for anti-pea lectin IgG1 from mice gavaged PBS or seed meals. C. Serum antibody titres for anti-pea lectin IgG1 from mice immunized i.p. with either PBS or pea lectin +. D. Serum IgG1 antibody titres of mice immunized with i.p. with pea lectin against αAI proteins purified from pea, cowpea, chickpea, Pinto bean and Tendergreen bean. Groups include PBS alone ×, Tendergreen bean ▪, Pinto bean □, αAI pea ▴, nGM pea ▵, αAI chickpea ⧫, nGM chickpea ◊, αAI cowpea • and nGM cowpea ○. IgE dilutions are expressed ×10³. Data are expressed as mean OD_{450 nm} ± SEM; n = 8, duplicate samples. Allergic lung inflammation evaluated by E. Eosinophil counts in BAL fluid, F. Eosinophil counts in Luna-stained lung sections, and G. Inflammation scores of lung sections. H. Allergen-induced mucus production is graded using mucus scores in PAS-stained lung sections. Raw (filled bars), cooked (open bars). Data are expressed as means ± SEM; n = 8. For eosinophil counts in BAL and lungs, data were compared using the Kruskal-Wallis test followed by Dunn's multiple comparison test. For histological scoring, data were compared with the Mann Whitney test. *p<0.05 for all groups above the PBS controls. These are representative data from 2 experiments.

Figure 4. Adjuvant effect of αAI pea and bean consumption.

Naïve BALB/c mice were compared with OVA-immunized and challenged mice gavaged with either PBS, or Tendergreen bean, αAI peas, non-transgenic pea seed meal. A. Eosinophil counts in BAL fluid from mice at disease initiation and e. exacerbation. B. Eosinophil counts in Luna-stained lung sections from mice at disease initiation and F. exacerbation. C. Inflammation scores of lung sections from mice at disease initiation and G. exacerbation. D. Mucus scores in PAS-stained lung sections from mice at disease initiation and H. exacerbation. Serum anti-OVA IgG1 and IgE antibody titres for mice at .I. disease initiation or J. disease exacerbation. Groups include naïve mice , PBS alone ×, Tendergreen bean ▪, αAI pea ▴ and nGM pea ▵ gavaged mice. Data are expressed as mean OD_{450 nm} ± SEM; n = 8, duplicate samples. For IgE, dilutions are expressed ×10³. Data are expressed as means ± SEM; n = 8. For eosinophil counts in BAL and lungs, data were compared using the Kruskal-Wallis test followed by Dunn's multiple comparison test. For histological scoring, data were compared with the Mann Whitney test. *p<0.05 for all groups above the PBS controls. These are representative data from 2 experiments.