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BJO Online First, published on January 6, 2015 as 10.1136/bjophthalmol-2014-305842 Laboratory science
Prevention of allergic conjunctivitis in mice by a rice-based edible vaccine containing modified Japanese cedar pollen allergens Ken Fukuda,1 Waka Ishida,1 Yosuke Harada,1 Yuhya Wakasa,2 Hidenori Takagi,2 Fumio Takaiwa,2 Atsuki Fukushima1 1
Department of Ophthalmology and Visual Science, Kochi Medical School, Nankoku City, Kochi, Japan 2 Functional Transgenic Crop Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan Correspondence to Ken Fukuda, Department of Ophthalmology and Visual Science, Kochi Medical School, Kohasu, Oko-cho, Nankoku City, Kochi 783-8505, Japan;
[email protected] Received 16 July 2014 Revised 21 November 2014 Accepted 14 December 2014
ABSTRACT Background/aims To determine whether oral immunotherapy with transgenic rice seeds expressing hypoallergenic modified antigens suppresses cedar pollen-induced allergic conjunctivitis by eliciting immune tolerance in mice. Methods BALB/c mice were fed once a day for 20 days with 220 mg of transgenic rice expressing modified Japanese cedar pollen allergens Cry j 1 and Cry j 2 or with non-transgenic rice seeds as a control. They were then sensitised with two intraperitoneal injections of Japanese cedar pollen in alum before challenge twice with pollen in eye drops. Twenty-four hours after the second challenge, the conjunctiva, spleen, and blood were isolated for histological analysis, cytokine production assays, and measurement of serum immunoglobulin E concentrations, respectively. Results The numbers of eosinophils and total inflammatory cells in the conjunctiva were significantly lower in mice fed the transgenic rice than in those fed non-transgenic rice. The clinical score evaluated at 15 min after antigen challenge was also significantly lower in mice fed the transgenic rice than in those fed non-transgenic rice. The serum concentrations of both total and allergen-specific immunoglobulin E were also significantly lower in mice fed the transgenic rice. Oral vaccination with transgenic rice resulted in significant down-regulation of the allergen-induced production of interleukin (IL)-2, IL-4, IL-5, IL-12p70, interferon-γ, and IL-17A by splenocytes. Conclusions Oral immunotherapy with transgenic rice expressing modified Japanese cedar pollen allergens suppressed pollen-induced experimental allergic conjunctivitis in mice by eliciting immune tolerance. This novel prophylactic approach is potentially safe and effective for allergen-specific oral immunotherapy in allergic conjunctivitis.
INTRODUCTION
To cite: Fukuda K, Ishida W, Harada Y, et al. Br J Ophthalmol Published Online First: [ please include Day Month Year] doi:10.1136/bjophthalmol2014-305842
Allergic conjunctivitis induced by pollen is one of the most widespread ocular diseases globally. The pollinosis caused by pollen of Japanese cedar (Cryptomeria japonica) is a predominant allergic disease in Japan, with a prevalence of more than 26.5%.1 The treatment of allergic conjunctivitis includes topical application of eye drops containing a mast cell stabiliser or antihistamine, or both, regardless of the causative antigen. Such treatment relieves symptoms only transiently, however, and is not curative. Allergen-specific immunotherapy by subcutaneous or sublingual administration of antigen is the
only disease-modifying treatment for allergy in which the clinical effects persist over the long term. Such therapy requires several years, however, and can be accompanied by severe adverse reactions such as anaphylactic shock. The development of more effective, safer, and more convenient treatments for allergic diseases is thus warranted. Oral administration of antigen has several advantages including its convenience and lack of pain. We have previously shown that prophylactic and early therapeutic antigen feeding suppresses ovalbumin (OVA)-induced allergic conjunctivitis in a mouse model.2 A reduced allergenicity of antigen is a key factor in improving the efficacy of immunotherapy, given that a hypoallergenic antigen can be administered at a higher dose. We have developed ricebased edible vaccines including antigens of Japanese cedar pollen, birch pollen, or house dust mites.3 The efficacy of a rice-based peptide vaccine for cedar pollen allergy was demonstrated in a mouse model of rhinitis.4 We have also recently established transgenic rice expressing entire molecules of the major cedar pollen allergens Cry j 1 and Cry j 2 after their molecular fragmentation or shuffling in order to treat a broader range of patients with different genetic backgrounds.5 We have now examined whether oral immunotherapy with transgenic rice seeds expressing these hypoallergenic modified antigens suppresses cedar pollen-induced allergic conjunctivitis by eliciting immune tolerance in mice.
MATERIALS AND METHODS Mice Inbred wild-type BALB/c mice were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan) and were maintained under specific pathogen-free conditions at the animal facility of Kochi Medical School. Age- and sex-matched mice were subjected to experiments at 6–12 weeks of age. This study was approved by the Committee for Care and Use of Laboratory Animals at Kochi University ( permit no. F-00063) and was performed in strict accordance with the Statement on the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology.
Transgenic rice Transgenic rice that accumulates in the edible part of the seed-modified forms of the major Japanese cedar pollen allergens Cry j 1 and Cry j 2, which had been deconstructed by fragmentation and molecular shuffling, respectively, was generated by
Fukuda K, et al. Br J Ophthalmol 2015;0:1–5. doi:10.1136/bjophthalmol-2014-305842
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Laboratory science transformation as described previously.5 The modified Cry j 1 and Cry j 2 antigens are deposited in endoplasmic reticulumderived protein bodies and are thereby rendered resistant to hydrolysis by intestinal enzymes and to harsh environments. They are thus suitable for oral delivery to the mucosal immune system in gut-associated lymphoid tissue.6
Feeding and sensitisation of mice Mice were fed orally once a day for 10 or 20 days with 220 mg of finely ground transgenic rice seeds (or non-transgenic rice seeds as a control) suspended in 1.0 mL of phosphate-buffered saline (PBS) with the use of a gastric tube. The mice also had access to normal chow and drinking water ad libitum. Experimental allergic conjunctivitis due to Japanese cedar pollen was induced by a modified version of a previously described protocol.7 In brief, 0.2 mg of Japanese cedar pollen (Hayashibara, Okayama, Japan) was mixed with alum (2.5 mg) and injected intraperitoneally twice with an interval of 7 days between injections. Seven days after the second sensitisation, the eyes of the mice were challenged with Japanese cedar pollen in PBS (1.2 mg per 2 μL per eye). Two days later, the mice were challenged again with cedar pollen in eye drops. At 15 min after the second challenge, clinical symptoms such as lid swelling, tear production or discharge, chemosis, and redness were evaluated in a double-blind manner on the basis of previously described criteria.8 Twenty-four hours after the second challenge, the eyes, blood, and spleen of the mice were isolated for histological analysis, measurement of serum immunoglobulin E (IgE), and cytokine production assays, respectively.
Histological analysis The eye, including the conjunctiva, was fixed in 10% buffered formalin. Vertical sections with a thickness of 2 μm were cut and stained with Giemsa solution. Infiltrating eosinophils and total inflammatory cells in the lamina propria mucosa of the tarsal and bulbar conjunctiva in sections of the central portion of the eye, including the pupil and optic nerve head, were counted by two observers in a blinded manner.9
Measurement of serum IgE Serum isolated from blood samples was assayed for both total and Cry j-specific IgE with the use of ELISAs, essentially as described previously.10 In brief, 96-well EIA plates (Corning-Costar, Corning, New York, USA) were coated overnight at 4°C with either affinity-purified rat antibodies to mouse IgE (2 μg/mL; Southern Biotech, Birmingham, Alabama, USA) or Japanese cedar pollen extract (5 μg/mL; Cosmo Bio, Tokyo, Japan). The plates were then washed, incubated with blocking buffer (1% bovine serum albumin in PBS) for 3 h at room temperature, and washed again before the addition of samples or IgE standard (TNP-KLH-specific IgE; BD Biosciences, San Jose, California, USA). After incubation for 2 h at room temperature, the plates were washed and biotin-conjugated rat antibodies to mouse IgE (BD Biosciences) were added to each well. The plates were incubated for 1 h at room temperature and washed before the addition of avidin-conjugated alkaline phosphatase (Sigma-Aldrich, St Louis, Missouri, USA) and incubation for an additional 1 h. After another wash, the p-nitrophenyl phosphate substrate (Sigma-Aldrich) was added to each well, the plates were incubated for 15 min, and absorbance was measured at 405 nm. The concentration of total IgE was determined by reference to the known concentrations of the IgE standard. For Cry j-specific IgE, the results were expressed as absorbance units because a Cry j-specific IgE standard is not available. 2
Assay of cytokine production by splenocytes Splenocyte preparations depleted of red blood cells were incubated for 48 h at a density of 1×107 cells/mL with Japanese cedar pollen extract (Cosmo Bio) at a concentration of 20 μg/ mL in 96-well flat-bottom plates containing 0.2 mL per well of RPMI 1640 medium supplemented with 20% fetal bovine serum and 0.1 mM 2-mercaptoethanol. The concentrations of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-17A, and interferon-γ (IFN-γ) in the culture supernatants were then measured with the use of a Bioplex system (Bio-Rad, Hercules, California, USA).11
Statistical analysis Quantitative data are presented as means±SEM, unless indicated otherwise, and were compared between groups with the use of the unpaired Student’s t test. A value of p
