Development of Rice-Seed-Based Oral Allergy Vaccines Containing Hypoallergenic Japanese Cedar Pollen Allergen Derivatives for Immunotherapy.

Allergen-specific immunotherapy is the only available curative treatment for IgE-mediated allergen diseases. A safe hypoallergenic allergen derivative with high efficiency is required as a tolerogen to induce immune tolerance to the causitive allergens. In this study, to generate a rice-based oral allergy vaccine for Japanese cedar (JC) pollinosis, the tertiary structures of major JC pollen allergens, Cry j 1 and Cry j 2, were more completely destructed by shuffling than the previous ones without losing immunogenicity and then were specifically expressed in the endosperm of transgenic rice seed. They accumulated at high levels and were deposited in endoplasmic reticulum (ER) and ER-derived protein bodies. The low allergenicity of these deconstructed Cry j 1 and Cry j 2 allergens was evaluated by examining their binding activities to the specific IgE antibody and by the basophil degranulation test.

Identification of biomarker sets for predicting the efficacy of sublingual immunotherapy against pollen-induced allergic rhinitis.

Sublingual immunotherapy (SLIT) is effective against allergic rhinitis, although a substantial proportion of individuals is refractory. Herein, we describe a predictive modality to reliably identify SLIT non-responders (NRs). We conducted a 2-year clinical study in 193 adult patients with Japanese cedar pollinosis, with biweekly administration of 2000 Japanese allergy units of cedar pollen extract as the maintenance dose. After identifying high-responder (HR) patients with improved severity scores and NR patients with unchanged or exacerbated symptoms, differences in 33 HR and 34 NR patients were evaluated in terms of peripheral blood cellular profiles by flow cytometry and serum factors by ELISA and cytokine bead array, both pre- and post-SLIT. Improved clinical responses were seen in 72% of the treated patients. Pre-therapy IL-12p70 and post-therapy IgG1 serum levels were significantly different between HR and NR patients, although these parameters alone failed to distinguish NR from HR patients. However, the analysis of serum parameters in the pre-therapy samples with the Adaptive Boosting (AdaBoost) algorithm distinguished NR patients with high probability within the training data set. Cluster analysis revealed a positive correlation between serum Th1/Th2 cytokines and other cytokines/chemokines in HR patients after SLIT. Thus, processing of pre-therapy serum parameters with AdaBoost and cluster analysis can be reliably used to develop a prediction method for HR/NR patients.

Induced Treg Cells Augment the Th17-Mediated Intestinal Inflammatory Response in a CTLA4-Dependent Manner.

Th17 cells and Foxp3+ regulatory T cells (Tregs) are thought to promote and suppress inflammatory responses, respectively. However, whether they counteract each other or synergize in regulating immune reactions remains controversial. To determine their interactions, we describe the results of experiments employing mouse models of intestinal inflammation by transferring antigen-specific Th cells (Th1, Th2, and Th17) differentiated in vitro followed by the administration of the cognate antigen via enema. We show that cotransfer of induced Tregs (iTregs) suppressed Th1- and Th2-mediated colon inflammation. In contrast, colon inflammation induced by transfer of Th17 cells, was augmented by the cotransfer of iTregs. Furthermore, oral delivery of antigen potentiated Th17-mediated colon inflammation. Administration of a blocking antibody against cytotoxic T lymphocyte-associated antigen 4 (CTLA4) abrogated the effects of cotransfer of iTregs, while the injection of a soluble recombinant immunoglobulin (Ig) fusion protein, CTLA4-Ig substituted for the cotransfer of iTregs. These results suggest that antigen-specific activation of iTregs in a local environment stimulates the Th17-mediated inflammatory response in a CTLA4-dependent manner.

Concentrated protein body product derived from rice endosperm as an oral tolerogen for allergen-specific immunotherapy--a new mucosal vaccine formulation against Japanese cedar pollen allergy.

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.

Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis.

Transgenic rice accumulating the modified major Japanese cedar pollen allergens, Cryptomeria japonica 1 (Cry j 1) and Cryptomeria japonica 2 (Cry j 2), which were deconstructed by fragmentation and shuffling, respectively, in the edible part of the seed was generated by transformation of a good-tasting rice variety, 'Koshihikari'. These modified cedar pollen antigens were deposited in ER-derived protein bodies (PB-I), which are suitable for delivery to the mucosal immune system in gut-associated lymphoid tissue when orally administered because antigens bioencapsulated in PB-I are resistant against hydrolysis by intestinal enzymes and harsh environments. Mice fed transgenic seeds daily for three weeks and then challenged with crude cedar pollen allergen showed marked suppression of allergen-specific CD4(+) T-cell proliferation, IgE and IgG levels compared with mice fed nontransgenic rice seeds. As clinical symptoms of pollinosis, sneezing frequency and infiltration of inflammatory cells such as eosinophils and neutrophils were also significantly reduced in the nasal tissue. These results imply that oral administration of transgenic rice seeds containing the structurally disrupted Cry j 1 and Cry j 2 antigens, serving as universal antigens, is a promising approach for specific immunoprophylaxis against Japanese cedar pollinosis.

Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides.

Mucosal delivery of peptide/protein therapeutics via the oral route is a desirable strategy in human immunotherapy. A key step for enhancing the bioavailability of orally administered therapeutics is to protect them from enzymatic digestion in the gastrointestinal tract. Here, we generated transgenic rice seeds accumulating allergen-derived T cell epitopes, a model tolerogen for the control of pollen allergy, in either ER-derived protein body-I (PB-I) or protein storage vacuole protein body-II (PB-II). Compared with PB-II-localized or chemically synthesized forms, PB-I-localized T cell epitopes showed higher resistance to enzymatic digestion in simulated gastric fluid. Moreover, the dose of T cell epitope required for suppression of allergen-specific IgE in mice was about 20-fold lower when fed in PB-I localized form than when unprotected. These findings demonstrate the potential of bioencapsulation in PB-I for broad applications as a viable strategy to achieve efficient mucosal delivery of oral peptide/protein therapeutics.

Efficient induction of oral tolerance by fusing cholera toxin B subunit with allergen-specific T-cell epitopes accumulated in rice seed.

Cholera toxin B (CTB) subunit is an efficient mucosal carrier molecule for induction of oral tolerance to antigens and allergens. Here, T-cell epitopes of Cry j 1 and Cry j 2, major allergens in Japanese cedar pollen, were expressed in rice seed as a fusion protein with either CTB or rice glutelin as a control. Feeding mice with rice seed containing CTB-fused T-cell epitopes suppressed allergen-specific IgE responses and pollen-induced clinical symptoms at 50-fold lower doses of T-cell epitopes than required when using control seed. Our findings present a novel potential strategy for immunotherapy of type-I allergy.

Peptide immunotherapy for allergic diseases using a rice-based edible vaccine.

PURPOSE OF REVIEW: Plant pollens are the most common cause of seasonal allergic disease. The number of patients undergoing treatment for allergies to the pollen of Japanese cedar (major antigens: Cry j 1 and Cry j 2) has increased steadily each year. Integration of an effective, safe and inexpensive clinical program would be greatly improved by addressing deficiencies in systemically delivered immunotherapy. RECENT FINDINGS: We have demonstrated that feeding mice transgenic rice seeds accumulating the T-cell epitope peptides of Cry j 1 and Cry j 2 before systemic challenge with total protein of cedar pollen inhibits the development of allergen-specific IgE, IgG and CD4 T-cell proliferative responses. The levels of allergen-specific CD4 T-cell-derived allergy-associated T-helper 2 cytokine of IL-4, IL-5, and IL-13 and histamine release in serum were also significantly decreased. Moreover, clinical symptoms were inhibited in an experimental sneezing-mouse model. SUMMARY: Plant-based edible vaccine has been shown to be effective for treatment of Japanese cedar pollinosis. When rice seeds containing T-cell epitopes derived from cedar pollen allergens were orally administered to mice, immune tolerance leading to reduction of allergen-specific IgE, T-cell proliferative reaction and histamine could be induced, resulting in suppression of allergic-specific symptoms such as sneezing.

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

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.