Allergy models and related assays to test the allergic qualities of Escherichia coli heat labile toxin subunit B
Transgenic maize containing Escherichia coli heat labile toxin subunit B (LT-B) has proven to be a strong mucosal immunogen in animal models and stimulates an IgG1 and IgA antibody response when administered orally. There is concern the T-helper cell 2 response stimulated by LT-B could sensitize the immune system to produce an allergic response. This concern is further compounded by the approximate 80% homology which exists between E. coli heat labile toxin and cholera toxin (CT), a known allergy inducer. Thus, the research was based on ascertaining what possible allergic stimulation, if any, LT-B could have on animal models.
The first experiment was to reproduce an established allergy model as a positive control, using peanuts as the allergen and CT as the adjuvant. The animals were administered 10 ug CT and 5 mg of peanut extract via intragastric gavage per dose and received 4 sensitizing doses. A number of assays were also established to measure various aspects of an allergic response. Mice treated with CT and peanut extract showed higher concentrations of total and peanut specific IgE, peanut and CT specific IgG1, and higher visual scores of allergy symptoms. A second experiment was designed in which animals were fed pellets containing LT-B (100 ug or 20 ug) with peanuts. One control group was fed CT and peanuts. Neither LT-B fed animal group produced an allergic response to peanut; however, the animals fed CT and peanuts also did not produce an allergic response. A third experiment was designed to optimize the parameters of the CT and peanut extract positive control, with CT and peanuts both being gavaged or fed at various concentrations (CT = 20, 10, or 0 ug; peanut = 20, 10, 5, or 0 mg). The route of delivery was the most important factor. Mice treated with CT and peanut both by intragastric gavage showed significant increases of total and peanut specific IgE, and peanut specific and CT IgG1 from the nayve mice. Mice fed CT and peanuts did not show a significant difference from the nayve mice in any measured antibody concentrations.
These experiments demonstrated LT-B does not induce an allergic response to co-fed novel proteins. Cholera toxin needs to be administered via intragastric gavage to induce allergies. There is no difference between 10 ug and 20 ug concentration doses of intragastric gavage CT. When administered via fed pellet, CT does not elicit an immune response. As CT is not excreted in the feces, the toxin is probably broken down before it reaches the intestines.
Peanut is only capable of eliciting an allergic response when both it and the adjuvant CT is administered via intragastric gavage, and the peanut can be gavaged after CT and still elicit an allergic response. There is no difference between 10 mg and 5 mg concentrations of intragastric gavaged peanut. If peanut is gavaged without CT, it will elicit an IgG1 immune response, which is not an indicator of an allergic reaction.