A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses

Abstract

Peptide immunotherapy using multiple predominant allergen-specific T cell epitopes is a safe and promising strategy for the control of type I allergy. In this study, we developed transgenic rice plants expressing mouse dominant T cell epitope peptides of Cry j I and Cry j II allergens of Japanese cedar pollen as a fusion protein with the soybean seed storage protein glycinin. Under the control of the rice seed storage protein glutelin GluB-1 promoter, the fusion protein was specifically expressed and accumulated in seeds at a level of 0.5% of the total seed protein. Oral feeding to mice of transgenic rice seeds expressing the T cell epitope peptides of Cry j I and Cry j II before systemic challenge with total protein of cedar pollen inhibited the development of allergen-specific serum IgE and IgG antibody and CD4(+) T cell proliferative responses. The levels of allergen-specific CD4(+) T cell-derived allergy-associated T helper 2 cytokine production of IL-4, IL-5, and IL-13 and histamine release in serum were significantly decreased. Moreover, the development of pollen-induced clinical symptoms was inhibited in our experimental sneezing mouse model. These results indicate the potential of transgenic rice seeds in production and mucosal delivery of allergen-specific T cell epitope peptides for the induction of oral tolerance to pollen allergens.

Fig. 1.

Expression of A1aB1b-Crp-1 and -2 in transgenic rice. (A) Schematic representation of the transformation plasmid. The DNA fragment coding for the A1aB1b-Crp-1 and -2 protein was placed under the control of rice seed major storage protein glutelin 2.3-kb GluB-1 promoter. The hpt gene was used for the selection of transgenic rice plants. GluB-1, rice glutelin GluB-1; 35S, cauliflower mosaic virus 35S promoter; hpt, hygromycin phosphotransferase gene; pAg7, agropine synthase polyadenylation signal sequence; RB, right border; LB, left border. (B) Northern blot analysis. Total RNA was isolated from leaves (L), roots (R), or developing seeds (S) of nontransgenic and A1aB1b-Crp-1 and -2 transgenic lines 9 and 12. (C) Western blot analysis of total protein extracted from seeds. Lane 1, nontransgenic rice; lane 2, A1aB1b-Crp-1 and -2 transgenic line 9; lane 3, A1aB1b-Crp-1 and -2 transgenic line 12. Anti-glycinin antibody, anti-Crp-1 antibody, or anti-Crp-2 antibody was used for the detection of A1aB1b-Crp-1 and -2 protein. (D) Southern blot analysis. Genomic DNA isolated from young leaves of rice plants was digested with SacI and fractionated by electrophoresis on 0.8% agarose gel. Lane 1, nontransgenic rice; lane 2, A1aB1b-Crp-1 and -2 transgenic line #9; lane 3, A1aB1b-Crp-1 and -2 transgenic line 12.

Fig. 2.

Inhibition of allergen-specific serum IgE, IgG, and CD4⁺ T cell responses by oral administration of A1aB1b-Crp-1 and -2 rice seeds. Levels of allergen-specific IgE (A), total IgE (B), and allergen-specific IgG (C) were examined in serum of mice fed with PBS, nontransgenic rice seeds, or A1aB1b-Crp-1 and -2 rice seeds before systemic challenge with total protein extracts of pollen. Allergen-specific splenic CD4⁺ T responses (D) were expressed as stimulation index calculated as the ratio of [cpm of cells cultured in the presence of allergen]/[cpm of cells cultured in the absence of allergen]. Data are expressed as mean ± SD. ^*, P < 0.01 for the group of mice fed with A1aB1b-Crp-1 and -2 rice seeds in comparison with the group of mice fed with PBS or nontransgenic rice seeds.

Fig. 3.

Inhibition of allergen-induced Th2 cytokine production by splenic CD4⁺ T cells isolated from mice fed with A1aB1b-Crp-1 and -2 rice seeds. Splenic CD4⁺ T cells were cultured with or without total protein extracts of pollen as described earlier. Levels of Th1 and Th2 cytokines in cell-free culture supernatants of CD4⁺ T cells were assayed by ELISA. Data are presented as mean ± SD. ^*, P < 0.01 for the group of mice fed with transgenic rice seeds in comparison with the group of mice fed with PBS or nontransgenic rice seeds.

Fig. 4.

Serum histamine levels (A) and the number of sneezes (B) were inhibited in the group of mice fed with A1aB1b-Crp-1 and -2 rice seeds. The number of sneezes was counted in the 5 min after the last nasal challenge at week 8 (white bars). Sham-challenged mice were nasally administered with 20 μl of PBS in the same manner (black bars). Data are expressed as mean ± SD. ^*, P < 0.01 for the group of mice fed with A1aB1b-Crp-1 and -2 seeds in comparison with the group of mice fed with nontransgenic rice seeds.