Prophylactic and Therapeutic Effects of Oral Immunotherapy on Birch Pollen-Induced Allergic Conjunctivitis in Mice with a Rice-Based Edible Vaccine Expressing a Hypoallergenic Birch Pollen Allergen

Abstract

We investigated the prophylactic and therapeutic effects of the oral administration of transgenic rice seeds expressing a hypoallergenic Bet v 1 derivative of allergic birch pollen conjunctivitis in mice. Transgenic rice seed depositing a chimeric molecule called TPC7 (tree pollen chimera 7) created by DNA shuffling of Bet v 1 family sequences from birch, alder and hazel in protein bodies of endosperm was generated. BALB/c mice were sensitized to birch pollen in alum and challenged with pollen in eyedrops. They were fed TPC7 transgenic or non-transgenic (control) rice seeds for 14 d before sensitization (prophylactic protocol) or 17 d after sensitization (therapeutic protocol). The clinical score and number of conjunctival eosinophils were significantly lower in TPC7-fed mice than in the control mice based on both the prophylactic and therapeutic protocols. Serum concentration of allergen-specific IgE did not differ between TPC7-fed and control groups in either protocol. Prophylactic administration of TPC7 downregulated the production of IL-4 and IFN-γ, whereas therapeutic administration of TPC7 upregulated the production of IFN-γ by allergen-stimulated splenocytes. Prophylactic or therapeutic oral administration of transgenic rice expressing TPC7 suppressed birch pollen-induced allergic conjunctivitis in mice. Feeding transgenic rice is a potentially effective approach as an allergen-specific immunotherapy for allergic conjunctivitis.

Keywords: allergic conjunctivitis; birch pollen; cytokines; immunotherapy; regulatory T cells; transgenic rice.

Figure 1

Schematic representation for the protocol of oral administration with Tg rice seed and induction of experimental allergic conjunctivitis. Mice were fed Tg or non-Tg control rice seed either orally ad libitum for 14 d before sensitization (prophylactic protocol, (a)) or 17 d after sensitization (therapeutic protocol, (b)). The Tg rice fed group was administered 3 g non-Tg rice seed containing 0.2 g Tg rice seed per day. The control group was fed 3 g non-Tg rice seed per day. To induce EAC, 0.1 mg of birch pollen was mixed with 1.3 mg of alum and intraperitoneally injected twice at an interval of 7 d. Seven days (prophylactic protocol, (a)) or 21 d (therapeutic protocol, (b)) after the second sensitization, birch pollen in PBS (1.2 mg per 4 μL per eye) was administered onto the eyes of the mice for three consecutive days.

Figure 2

Prophylactic administration with Tg rice suppresses the development of allergic conjunctivitis. Mice were fed with Tg rice seed or control (non-Tg) rice seed ad libitum for 14 d before the first sensitization with birch pollen. At 20 min after the last birch pollen challenge, clinical appearance was evaluated. ** p < 0.01, Tg vs. non-Tg rice-fed group according to an unpaired Student’s t test (seven mice per group) (a). At 24 h after the third challenge, the eyes were isolated for histological analysis according to the number of eosinophils in the conjunctiva (b–d). ** p < 0.01, Tg vs. non-Tg rice-fed group according to the Mann–Whitney U test (non-Tg rice group: seven mice per group and Tg rice group: five mice per group). The dots and bars within (a,d) indicate mean values for the two eyes of each mouse and the overall median values, respectively.

Figure 3

Serum concentration of total IgE (A) and Bet v 1-specific IgE (B) in mice fed Tg or non-Tg rice. Data are presented as ng/mL or absorbance units. * p < 0.05, Tg vs. non-Tg rice-fed group according to the unpaired Student’s t test (three mice per group).

Figure 4

Release of IFN-γ (a) and IL-4 (b) by the antigen-stimulated splenocytes from mice fed Tg or non-Tg rice. Data are the means ± SEM. * p < 0.05, Tg vs. non-Tg rice-fed group according to unpaired Student’s t test (three mice per group).

Figure 5

Therapeutic administration with Tg rice suppresses the development of EAC. Mice were fed Tg rice seed or control (non-Tg) rice seed ad libitum for 17 d after the sensitization with birch pollen. At 20 min after the last birch pollen challenge, clinical appearances were evaluated. ** p < 0.01, Tg vs. Non-Tg rice-fed group according to unpaired Student’s t test (seven mice per group) (a). At 24 h after the third challenge, the eyes were isolated for histological analysis of the number of eosinophils in the conjunctiva (b–d). ** p < 0.01, Tg vs. Non-Tg rice-fed group according to the Mann–Whitney U test (seven mice per group). The dots and bars within (a,d) indicate mean values for the two eyes of each mouse and the overall median values, respectively.

Figure 6

Serum concentration of total IgE (a) and Bet v 1-specific IgE (b) in mice fed with Tg or non-Tg rice. Data are presented as ng/mL or absorbance units. * p < 0.05, Tg vs. non-Tg rice-fed group according to unpaired Student’s t test (three mice per group).

Figure 7

The release of IFN-γ (a) and IL-4 (b) by the antigen-stimulated splenocytes from mice fed with Tg or non-Tg rice. Data are means ± SEM. * p < 0.05, Tg vs. non-Tg rice-fed group according to unpaired Student’s t test (three mice per group).

Figure 8

Prophylactic and therapeutic administration with Tg rice expressing Bet v 1 suppresses EAC development. Mice were fed Bet v 1 Tg rice seed or control (non-Tg) rice seed ad libitum for 14 d before the first sensitization ((a,b) prophylactic administration) or for 17 d after the sensitization ((c,d), therapeutic administration) with birch pollen. At 20 min after the last birch pollen challenge, clinical appearances were evaluated. * p < 0.05, ** p < 0.01, Tg vs. non-Tg rice-fed group according to unpaired Student’s t test (seven female mice per group) (a,c). At 24 h after the third challenge, the eyes were isolated for histological analysis of the number of eosinophils in the conjunctiva (b,d). ** p < 0.01, Tg vs. non-Tg rice-fed group according to the Mann–Whitney U test (seven female mice per group). The dots and bars indicate the mean values for the two eyes of each mouse and the overall median values, respectively.

Figure 9

Serum concentration of total IgE (a,c) and Bet v 1-specific IgE (b,d) in mice fed with Bet v 1 Tg or non-Tg rice in prophylactic (a,b) and therapeutic (c,d) protocol. Data are presented as ng/mL or absorbance units. * p < 0.05, ** p < 0.01, Bet v 1 Tg vs. non-Tg rice-fed group according to unpaired Student’s t test (three female mice per group).