Transgenic Res (2014) 23:573–584 DOI 10.1007/s11248-014-9790-3
ORIGINAL PAPER
Development of a rice-based peptide vaccine for Japanese cedar and cypress pollen allergies Fumio Takaiwa • Lijun Yang
Received: 8 December 2013 / Accepted: 5 March 2014 / Published online: 18 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Peptide immunotherapy using dominant T-cell epitopes is a safe treatment alternative to conventional subcutaneous injection of natural crude allergen extract, which is sometimes accompanied by anaphylactic shock. For Japanese cedar pollinosis (JCP), hybrid peptides composed of six to seven major T-cell epitopes (7Crp peptide) from the causative allergens Cry j 1 and Cry j 2 have been developed on the basis of different human leukemia antigen class II restrictions, because of the diversity of patients’ genetic backgrounds. However, other dominant T-cell epitopes that are produced in some patients are not covered by these peptides. To develop a more universal peptide vaccine for JCP, we generated transgenic rice seeds containing seven new T-cell epitopes (Crp3) in addition to the T-cell epitopes used in the 7Crp peptide. Next, we co-expressed unique T-cell epitopes (6Chao) from the Japanese cypress pollen allergens Cha o 1 and Cha o 2 in transgenic rice seeds, with 7Crp and Crp3. These transgenic rice seeds, containing many highly homologous T-cell epitopes derived from cedar and cypress allergens, are
Electronic supplementary material The online version of this article (doi:10.1007/s11248-014-9790-3) contains supplementary material, which is available to authorized users. F. Takaiwa (&) L. Yang Functional Transgenic Crop Research Unit, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan e-mail: [email protected]
expected to be applicable to a wide range of patients suffering from these pollen allergies. Keywords Allergy vaccine Oral vaccine Peptide immunotherapy Pollinosis Transgenic rice seeds Abbreviations ER Endoplasmic reticulum GALT Gut-associated lymphoid tissue HLA Human leukemia antigen JCP Japanese cedar pollinosis PBMC Peripheral blood mononuclear cells PB Protein body 2-MER 2-Mercaptoethanol
Introduction Japanese cedar pollen allergy is an important public health problem in Japan, with approximately 27 % of Japanese people afflicted by this pollinosis from February to April each year (Baba and Nakae 2008). Allergen-specific immunotherapy, the only curative treatment used to control this prevalent allergic disease (Frew 2010), is achieved by subcutaneous injection of increasing doses of crude allergen extract over a period of three to five years to induce immune tolerance to specific CD4? T-cells against the causative allergens. This curative treatment leads to
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reduction of allergen-specific IgE, an increase in blocking IgG4, down-regulation of allergen-specific T-cell responsiveness, and suppression of effector cells (Larche et al. 2006; Holgate and Polosa 2008). However, conventional immunotherapy is sometimes accompanied by the adverse effect of anaphylactic shock, because the allergen can cross-link with specific IgEs on mast cells and basophils. Therefore, a simpler, safer, more convenient allergen-specific immunotherapy is needed. To improve the efficacy and safety of allergy treatment, the route of administration and immunological properties of tolerogens must be changed. In particular, it is crucial to change from administering native crude extract to administering a hypoallergenic tolerogen to reduce side effects and enhance safety (Valenta et al. 2010). One strategy that addresses this requirement is peptide immunotherapy (PIT) using major T-cell epitopes derived from the causative allergens (Larche 2007). T-cell epitopes efficiently bind to human leukemia antigen II (HLA II) to induce T-cell responses and can be used to generate T-cell tolerance, while their short length and lack of tertiary conformational structures do not facilitate IgE cross-linking. Many T-cell epitopes characteristic of individuals with different HLA II haplotypes should be included when generating a universal T-cell epitope peptide. Another strategy that would improve allergy treatment is to replace subcutaneous injection with oral administration, eliminating patient discomfort and making administration more convenient. A model experiment has been performed in which mouse major T-cell epitopes from the Japanese cedar pollen allergens Cry j 1 and Cry j 2 were expressed as a fusion protein with the soybean seed protein glycinin in transgenic rice seeds (Takagi et al. 2005a). The feasibility of using a rice seed-based peptide vaccine was demonstrated by feeding mice with transgenic rice seeds containing mouse T-cell epitopes from Japanese cedar pollen allergens. Not only did the animals manifest immunological signs, for example reduced levels of allergen-specific IgE, T-cell proliferative responses, and secretion of IL-4, IL-5, and IL13 cytokines by Th2 cells, alleviation of such allergy symptoms as sneezing was also observed (Takagi et al. 2005a). Next, transgenic rice seeds containing a 7Crp hybrid peptide composed of seven major human T-cell epitopes from the Cry j 1 and Cry j 2 sequences were used to control Japanese cedar pollinosis (JCP). The
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7Crp peptide was designed to prevent IgE-mediated adverse effects, because the B-cell epitopes were eliminated from this peptide (Hirahara et al. 2001). Notably, the 7Crp peptide has the same immunogenicity as the inherent Cry j 1 and Cry j 2 allergens; it is, thus, an ideal, safe tolerogen. Peripheral blood mononuclear cells (PBMC) from 44 of 48 JCP patients (92 %) manifested proliferative responses to this hybrid peptide (Hirahara et al. 2001). Furthermore, the 7Crp peptide does not bind to the specific IgE in sera from JCP patients, confirming the safety of this peptide vaccine. When the 7Crp peptide is expressed in transgenic rice seeds under the control of seedspecific promoters, it accumulates predominantly in endoplasmic reticulum (ER)-derived protein bodies (PBs) (Takagi et al. 2005b; Takaiwa et al. 2009). The 7Crp peptide in rice seeds is bioencapsulated within the cell walls and PBs, which protects it from digestion by gastrointestinal enzymes, in contrast with naked peptides (Takagi et al. 2010; Takaiwa 2011). Thus, bioencapsulation of antigens provides an effective delivery system to gut-associated lymphoid tissue (GALT) (Takaiwa 2013). To date, Cry-consensus peptide and 7Crp have been created as universal hybrid T-cell epitope peptides by linking six or seven major T-cell epitopes derived from Japanese cedar pollen allergens (Hirahara et al. 2001; Tsunematsu et al. 2007). However, proliferative responses to these hybrid peptides are not observed for some T-cell lines from JCP patients. To expand the applicability of such treatment to JCP patients, it is important to include as many T-cell epitopes derived from Cry j 1 and Cry j 2 as possible. In this study, we selected the 14 major T-cell epitopes in cedar pollen allergen Cry j 1 and Cry j 2 sequences to produce an improved JCP treatment. Moreover, 70–80 % of JCP patients are also afflicted by cypress pollen allergy (Yasueda et al. 2001). Because the major cypress pollen allergens Cha o 1 and Cha o 2 are highly homologous (75–80 %) with Cry j 1 and Cry j 2, respectively (Suzuki et al. 1996; Mori et al. 1999), we selected six major T-cell epitopes unique to the cypress allergens Cha o 1 and Cha o 2 and expressed these epitopes in transgenic rice seeds, together with those derived from cedar pollen allergens. When T-cell epitope peptides were produced in transgenic rice seeds, they were highly expressed and were deposited into digestive enzyme-resistant PBs via interaction with cysteine-rich prolamins. The
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transgenic rice seeds produced in the study are expected to act as oral tolerogens by induction of immune tolerance against cedar and cypress pollen allergens. Transgenic plants are used as a promising means of production of high-value recombinant proteins. Higher levels of accumulation of recombinant proteins can be achieved by using strong specific promoters, optimizing codon usage, and intracellular targeting into PBs (Qu and Takaiwa 2004; Takaiwa 2007; Wakasa and Takaiwa 2013). We have previously generated many transgenic rice plants that successfully accumulated functional recombinant proteins in seeds by using endosperm-specific promoters of 2.3kb glutelin B1 (GluB-1) and 1.4-kb glutelin B4 (GluB4) and 10-kD prolamin promoters, and by fusing the N-terminal signal peptide of rice seed proteins such as GluB-1 signal peptide and the C terminal KDEL ER retention sequences (Takagi et al. 2005a, b; Takaiwa et al. 2009; Yang et al. 2012). It should be noted that although recombinant proteins are specifically produced and accumulated in PBs, the yields vary substantially, because of the inherent structural and functional properties of the proteins chosen. To obtain a better yield of the proteins it is, therefore, important to test the different promoters and to choose most suitable.
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GluC, or 1.8 kb 10 kD prolamin endosperm-specific promoters containing DNA sequences encoding the corresponding signal peptides (Qu and Takaiwa 2004), and the C-termini KDEL ER retention sequences of the genes were followed by 0.65 kb of the GluB-1 terminator, 0.7 kb of the GluB-4 terminator, 0.64 kb of the GluC terminator, or 0.35 kb of the 10 kD prolamin terminator. The entry clones containing gene cassettes of 12Crp, Crp3, 6Cha o, and 7Crp were obtained (Fig. 1). For construction of expression plasmids for rice genome transformation (pA in Fig. 1), the aforementioned entry clones having gene cassettes of 12Crp, Crp3, 6Chao, and 7Crp were cloned into the destination binary vector p35SHPTAg7-GW by using the LR clonase reactions of the MutiSite Gateway system (Wakasa et al. 2006); the resulting plasmids were designated pA, pB1, pB2, pC, pD, pE, and pF (Fig. 1). The expression plasmids were introduced into the rice genome (Oryza sativa cv. Kita-ake) via Agrobacterium tumefaciens-mediated transformation. Transgenic plants resistant to hygromycin were selected, as described elsewhere (Goto et al. 1999). The transgenic plants were grown in a controlled greenhouse (28 °C, 12 h light/dark cycle) and more than 20 plants with each structure were subjected to transformation analysis. Preparation of antibodies
Materials and methods Plasmid construction and rice transformation In the same way as the 7Crp gene reported by Takagi et al. (2005b), the DNA sequences coding for 12Crp, Crp3, and 6Chao were optimized for codons that are frequently used in rice seed storage protein genes. ATrich sequences, including potential mRNA destabilizing sequences, polyadenylation signals, putative intron splicing sites, and rare codons such as AAA (Lyn), ACG (Thr) TCG (Ser) and CGA (Arg), etc., were avoided among their coding regions (Wakasa and Takaiwa 2013). After further checking of the DNA sequences for these artificial genes by use of Genetyp (Hewlett–Packard), genes having DNA sequences encoding the KDEL ER retention at their C-termini were synthesized by GenScript (NJ, South Plainfield, USA). The genes were then ligated downstream of the 2.3 kb GluB-1, 1.4 kb GluB-4, 1.5 kb
E. coli-expressed purified 12Crp and Crp3 were used to raise rabbit anti-12Crp and anti-Crp3 antibodies (Qiagen, Japan). For detection of 6Chao, a peptide (GPSPKEFESSGKNEG) was synthesized and used to prepare a peptide antibody against 6Chao (Qiagen). Antibodies to 7Crp, glutelin A (GluA), and cysteine rich 13 kD prolamin (RM1) had previously been prepared in our laboratory (Takagi et al. 2006). Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). SDS-PAGE and immunoblot analysis Mature seeds were ground into a fine powder by use of a multibead shocker (Yasui Kikai, Tokyo, Japan). Total protein was extracted from the powder of a single grain by use of 600 ll urea–SDS buffer (50 mM Tris–HCl, pH 6.8, 8 M Urea, 4 % SDS,
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Fig. 1 Diagrammatic representation of the structures used for expression of T-cell epitope peptides in transgenic rice seeds. The genes encoding 7Crp, 12Crp, Crp3, and 6Chao were expressed under the control of the endosperm-specific 2.3 kb GluB-1, 1.4 kb GluB-4, 1.5 kb GluC, or 1.8 kb 10 kD prolamin promoters. Ag7, Agrobacterium gene 7 terminator; hpt,
hygromycin phosphotransferase coding region; 35S P, cauliflower mosaic virus 35S promoter. GluB1 pro SP, GluB4 pro SP, GluC pro SP, and 10 K pro SP represent GluB-1, GluB-4, GluC, and 10 kD prolamin promoters and their signal sequences, whereas GluB1 T, GluB4 T, GluC T, and 10 kD T are terminators of the corresponding genes. KDEL, ER retention signal
5 % 2-mercaptoethanol (2-ME), 20 % glycerol) as described elsewhere (Tada et al. 2003). After separation by 12 % SDS-PAGE, the proteins were visualized by Coomassie brilliant blue (CBB)-R250 staining or transferred to PVDF membranes (Millipore, Billerica, MA, USA) for immunodetection with the corresponding antibodies. Accumulation levels of the peptides were estimated on the basis of the intensity of bands stained with CBB, using Bio-Rad protein Assay Standard II-BSA as calibration control (Bio-Rad Laboratories, Hercules, CA, USA). For proteins with low accumulation levels, immunoblot analysis was performed with the corresponding antibodies using E. coli-expressed, purified recombinant proteins as a controls. Band density was quantified with NIH image J software (National Institutes of Health, Washington, DC, USA). For immunoblot analysis, the membranes were reacted with primary antibodies against 12Crp, Crp3, 6Chao, and 7Crp (all at dilution 1:10,000), then with secondary antibody of horseradish peroxidase-conjugated anti-rabbit IgG (dilution 1:5,000). The bands or dots were visualized with the ECL detection kit (GE healthcare, UK).
In-vitro digestion of transgenic rice seeds by pepsin
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Transgenic rice seeds were subjected to pepsin digestion as described elsewhere (Takagi et al. 2005b). Briefly, 150 ll of reaction buffer containing 0.1 % (w/v) pepsin (Sigma, USA) and 30 mM NaCl (pH 1.2) was added to 5 mg seed powder and incubated at 37 °C. The reaction was terminated by neutralization with NaOH after 0, 2, 5, 15, 30, 60, 120, and 180 min. After addition of 150 ll of urea– SDS buffer, the digested samples were analyzed by 12 % SDS-PAGE then immunoblot analysis. Step-wise extraction of seed proteins Rice seed storage proteins were extracted from pulverized mature seeds by use of the step-wise extraction method described elsewhere (Takaiwa et al. 2009). Extraction buffer containing 0.5 M NaCl and 10 mM Tris–HCl (pH 7.5) was used to extract albumin and globulin. The residues were then treated with 60 % (v/v) n-propanol for extraction of Cys-poor prolamins and further resuspended in 60 % (v/v) n-propanol containing 5 % 2-ME for extraction of
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Cys-rich prolamins. Finally, the residues were treated with 1 % ((v/v) lactic acid for extraction of glutelins and peptide proteins. For all steps, the residues were extracted twice with the same solution.
Results Generation of transgenic rice seeds accumulating T-cell epitope peptides derived from Japanese cedar and cypress pollen allergens The artificial gene encoding the universal T-cell epitope peptide comprising seven major and five additional T-cell epitope peptides, named 12Crp, was first synthesized by using codons frequently used in many rice seed storage protein genes. Additional T-cell epitopes were selected as shown in Supplementary Table 1. The KDEL ER retention signal was attached to the C termini of the 12Crp peptide. The 12Crp peptide is composed of 193 amino acids with a predicted molecular mass of 21,436 Da (Fig. 2a). The codon-optimized synthetic gene encoding 12Crp containing the KDEL signal at the C terminus was ligated to the 2.3 kb GluB-1 or 1.4 kb GluB-4 endospermspecific promoter containing the signal peptide denoted pB1 and pB2, respectively (Fig. 1) and expressed as a secretory protein in transgenic rice seeds after introduction into the rice genome via Agrobacterium-mediated transformation. A total of 20 and 53 independent transgenic rice plants were regenerated for pB1 and pB2 structures, respectively. As shown in Fig. 3b, the 12Crp peptide was detected in mature seeds by immunoblot analysis using a 12Crp-specific antibody. However, the accumulation level (1.6 and 3.6 lg/grain for lines 11 and 16) was much lower than that of 7Crp (23.2 and 29.6 lg/grain for lines 10 and 34), despite that fact that the strong endosperm-specific GluB-1 or GluB-4 promoter was used for expression (Supplementary Table 2). Seven new T-cell epitopes that were not included among the seven T-cell epitopes used in the 7Crp peptide were the selected from the major T-cell epitopes characterized in the Cry j 1 and Cry j 2 molecules (Supplementary Table 1) (Hashiguchi et al. 1996; Sone et al. 1998; Masuyama et al. 2009). For the Cry j 1 allergen, two T-cell epitopes, at positions 80–94 and 108–132, were used in the Cry-consensus peptide, and two other epitopes (at positions 61–79 and 337–350)
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were selected. Furthermore, three other T-cell epitopes, at positions 25–39, 158–175, and 245–264, were selected from the Cry j 2 sequence. These seven T-cell epitopes were linked together, to form a hybrid peptide named Crp3 (Fig. 2b) which is 129 amino acids long with a predicted molecular mass of 14,171 Da. First, we examined whether the Crp3 peptide can strongly accumulate in rice seeds in a similar manner to 7Crp. The gene coding for the Crp3 peptide (including the KDEL ER retention signal at the C terminus) was ligated to the 2.3 kb GluB-1, 1.5 kb GluC, or 1.8 kb 10 kD prolamin endosperm-specific promoter containing the individual signal peptide, and these three expression cassettes were inserted into a binary vector by use of the multisite Gateway system, resulting in pC, which was then introduced into the rice genome. Twenty-four independent transgenic rice plants containing Crp3 were obtained and the Crp3 peptide was strongly accumulated in seeds (80 and 72 lg/grain for lines 1 and 8), and was detected as a visible band on CBB-stained SDS-PAGE gels (Fig. 3c). This result indicates that the Crp3 peptide can be highly produced in rice seeds, as is the 7Crp peptide (Supplementary Table 2). Many JCP patients have specific IgEs to pollen allergens of both Japanese cypress and Japanese cedar. The Cry j 1 and Cry j 2 major pollen allergens from Japanese cedar are approximately 80 and 74 % identical with Cha o 1 and Cha o 2, respectively, from cypress (Suzuki et al. 1996; Mori et al. 1999). Dominant T-cell epitopes have been identified in the Cha o 1 and Cha o 2 sequences (Sone et al. 2005, 2009). Notably, several T-cell epitopes are identical in these cedar and cypress pollen allergens (Cry j 1 vs. Cha o 1) whereas some epitopes are unique to cypress pollen allergens. Therefore, if T-cell epitopes common to the Cha o and Cry j allergens and specific T-cell epitopes unique to the Cha o allergen are selected and expressed together in transgenic rice seeds, these seeds should be highly applicable as edible vaccines for patients suffering from seasonal Japanese cedar and cypress pollinosis. Four T-cell epitopes (at positions 11–30, 211–230, 251–270, and 331–350) in the Cha o1 sequence are common to Cry j 1, and four T-cell epitopes (at positions 61–80, 71–90, 311–330, and 321–340) are unique to Cha o 1. On the other hand, three T-cell epitopes (at positions 41–61, 141–170, and 350–390) are characteristic of Cha o 2 (Sone et al. 2005, 2009) (Supplementary Table 1).
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Fig. 2 The amino acid sequences of 12Crp, Crp3, and 6Chao. The major T-cell epitopes derived from Cry j 1, Cry j 2, Cha o 1, and Cha o 2 are indicated below the corresponding sequences. Amino acid position numbers of individual T-cell epitope in the
mature Cry j 1 and Cry j 2 are in accordance with those determined by Sone et al. (1994) and Namba et al. (1994), and position numbers in parentis are in accordance with the Cry j 2 sequence determined by Komiyama et al. (1994)
We selected six unique T-cell epitopes from the Cha o 1 and Cha o 2 sequences, which were linked together to generate the 6Chao hybrid peptide. The 6Chao peptide is composed of 154 amino acids, and its predicted molecular mass is 17,033 Da (Fig. 2c). When the 6Chao peptide was produced in transgenic
rice seeds under the control of the 2.3 kb GluB-1 promoter containing its signal peptide (plasmid pD), this peptide could not be detected among any of the 23 transgenic rice lines assayed by CBB staining after SDS-PAGE (Fig. 3d). Accumulation levels were 7.2 and 6.6 lg/grain for lines 1 and 8, respectively.
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Fig. 3 Expression of T-cell epitope peptides in transgenic rice seeds. Total proteins were extracted with 600 ll urea–SDS buffer from non-transgenic O. sativa cv. Kita-ake (NT) and transgenic rice seeds containing 7Crp, 12Crp, Crp3, and 6Chao, and 2 ll of each sample was loaded on to 12 % SDS-PAGE, and the gels (left panels) were CBB-stained. The accumulated T-cell epitope peptides (right panels) were immune-detected with the
corresponding antibodies. Molecular markers are shown on the left. Open and solid arrowheads and the solid arrow indicate the 6Chao, 7Crp, and 12Crp bands, respectively, and the asterisk indicates Crp3. Dimers of 7Crp, 12Crp, and Crp3 are indicated by open arrows, and arabic numerals represent transgenic line numbers. For 12Crp, lines 11 and 45 are from pB1 and pB2, respectively
The 6Chao peptide expression cassette, together with the 7Crp expression cassette or the 7Crp and Crp3 expression cassettes, was introduced into a binary vector by use of the Gateway system, resulting plasmid pE and pF, respectively, as shown in Fig. 1. We generated 24 independent transgenic rice plants expressing 6Chao and 7Crp double peptides, and another 24 independent plants expressing 6Chao, 7Crp, and Cryp3 triple peptides (Fig. 3e, f). As shown in Fig. 3f, the 6Chao peptide was detected as a weak band by CBB staining, when expressed together with the 7Crp and Crp3 peptides. The 7Crp, Crp3, and 6Chao hybrid peptides accumulated at relatively high levels (32.4, 55.0, and 12.0 lg/grain for 7Crp, Crp3, and 6Chao, respectively) in transgenic rice seeds containing the triple peptides (Supplementary Table 2). Therefore, many T-cell epitopes accumulated in rice seeds, and are expected to act as tolerogens to induce immune tolerance against the Japanese cedar and cypress pollen allergens when patients are fed these transgenic rice seeds.
Resistance to pepsin digestion in transgenic rice seeds accumulating T-cell epitopes We previously reported that 7Crp peptide predominantly accumulates in ER-derived PBs (PB-Is) via an interaction with Cys-rich prolamins at their disulfide bonds (Takaiwa et al. 2009). For oral-peptide vaccines, resistance to digestion by enzymes in the gastrointestinal tract and to harsh acidic conditions in the stomach is critical for the delivery of these vaccines to GALT to induce immune tolerance (Takaiwa 2011). When transgenic rice seeds accumulating Crp3, 6Chao, and 7Crp/Crp3/6Chao were subjected to in-vitro digestion with pepsin, there was no significant difference in the time required for complete digestion compared with rice seeds containing 7Crp. Notably, as shown in Fig. 4, resistance of the 6Chao and Crp3 peptides in rice seeds to pepsin digestion was higher than that of the endogenous glutelins deposited in storage vacuole-derived PBs (PB-IIs), although the Cys-rich 13 kD prolamin (RM1)
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Fig. 4 In-vitro digestibility of transgenic rice seed powder containing 7Crp (pA) and 6Chao/Crp3/7Crp (pF). Seed powder (5 mg) was added to the reaction mixture containing 0.1 % pepsin and incubated at 37 °C for 0, 2, 5, 15, 30, 60, 120, or 180 min. Total proteins in each reaction mixture were extracted
with urea–SDS buffer and examined by immunoblotting with the corresponding antibodies. RM1 and GluA denote Cys-rich 13 kD prolamin and glutelin A, respectively. Two bands in the GluA panel represent glutelin A precursor and its acidic subunit
deposited in ER-derived PB (PB-I) in the same transgenic rice seeds was much more resistant to pepsin digestion. Specifically, the hybrid peptides were completely digested with pepsin within 30 min, whereas 15 min was required for complete digestion of glutelins. These results suggest that these hybrid peptides (comprising several T-cell epitopes) are deposited into ER-derived PBs,
which is also true for the 7Crp peptide (Takaiwa et al. 2009). The interaction via disulfide bonds is especially critical for the 6Chao peptide, because no 6Chao peptide could be extracted before removal of the Cysrich prolamins. This finding also supports the notion that these peptides, composed of T-cell epitopes from Japanese cedar and cypress pollen allergens, are mainly deposited as aggregates in ER-derived PBs (PB-Is) through interactions with Cys-rich prolamins via disulfide bonds.
Interaction of T-cell epitope peptides with cysteine-rich prolamins The Crp3 and 6Chao peptides contain four and two cystein residues, respectively. We previously showed by immune-electron-microscopy that the 7Crp peptide is predominantly targeted to PB-I (Takaiwa et al. 2009). In this study we found that the 7Crp peptide could be extracted only after removal of Cys-rich prolamins, which suggests there is an interaction between Cys-rich prolamins, for example 10 kD, 16 kD, and Cys-rich 13 kD prolamins, within PB-Is. We then examined whether there was any interaction between the Cys-rich prolamins and the 6Chao or Crp3 peptide. As shown in Fig. 5, when seed proteins were extracted with different extraction buffers in a step-wise manner, these peptides could be extracted only after removal of various endogenous Cys-rich prolamins with 60 % propanol containing 2 % 2-ME,
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Discussion PIT using major T-cell epitopes is a safe and effective treatment strategy, without side effects, because T-cell epitopes are unable to cross-link specific IgE molecule on mast cells and basophils, which leads to anaphylaxis shock, and they cannot activate effecter cells (Larche 2007). Furthermore, high-dose administration of these peptides beginning at the initial stage of treatment shortens the treatment period. However, characterizing T-cell epitopes from the target antigens is a prerequisite for use of PIT. Furthermore, PIT is not applicable to all patients suffering from allergies, because T-cell epitopes differ among humans as a result of genetic diversity. To compensate for this genetic diversity, several major T-cell epitopes
Transgenic Res (2014) 23:573–584 Fig. 5 SDS-PAGE and immune-blot analyses of peptides Crp3, 6Chao, and 7Crp extracted with 1 % lactic acid after preextraction with different solvents. A Crp3 (pC), B 6Cha o (pD), and C Crp3/ 6Cha o/7Crp (pF). T total seed proteins; plus and minus signs indicate with and without pre-extraction, respectively. The upper panel gels are CBB-stained and molecular markers are indicated on the left
derived from the causative antigen sequences can be linked together to yield a hybrid peptide. However, because it is difficult to cover this amount of genetic diversity perfectly, it may be necessary to use many T-cell epitopes to increase coverage for applicable patients. The 7Crp and Cry-consensus human T-cell epitope hybrid peptide derived from cedar pollen allergens Cry j 1 and Cry j 2 manifests proliferative responses to approximately 90 % of PBMCs from JCP patients (Sone et al. 1998; Hirahara et al. 2001). Four T-cell epitopes used in this hybrid epitope peptide are fundamentally identical with each other. To enhance coverage, we included two Cry j 1 T-cell epitopes used in the Cry-consensus peptide and five additional major T-cell epitopes in the new hybrid peptide, Crp3, produced in this study. The sequence common to the 7Crp and Crp3 peptides comprises 235 amino acids, which accounts for approximately 31 % of the total Cry j 1 and Cry j 2 sequences (742 amino acids). On the other hand, 70–80 % of Japanese cedar pollen patients are cross-reactive to Japanese cypress pollen allergens, as diagnosed by the radio allergosorbent test (RAST) or by skin testing (Yasueda et al. 2001). This may be because the major allergens of these pollens are highly homologous (75–80 %) with each other at the amino acid level. Major T-cell
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epitopes in these allergens are well characterized, as shown in Supplementary Table 1. Some major T-cell epitopes in cedar and cypress allergens are identical; unique T-cell epitopes characteristic of the cypress allergens Cha o 1 and Cha o 2 have also been well characterized (Sone et al. 2005, 2009). T-cells from JCP patients recognize Cry j 1 p211–230 and also respond to Cha o 1 p209–228 (Oono et al. 2000), because the minimum peptide sequences (p213–230) of the cross-reacting T-cell epitopes in Cry j 1 and Cha o 1 are identical. There are also three additional common T-cell epitopes (at p11–30, p251–270, and p331–350) between Cha o 1 and Cry j 1 (Sone et al. 2005); thus common antigenicity at the T-cell level between Japanese cedar and cypress pollen allergens may be because of the existence of identical T-cell epitopes, leading to cross-reactivity. Indeed, cedar pollinosis patients continue to have allergic symptoms even after the end of the cedar pollen season, which is followed by the cypress pollen season. Therefore, transgenic rice seeds containing the common and unique T-cell epitopes derived from both the major cedar and cypress pollen allergens at levels of 0.6–2.75 mg/g mature seed for individual hybrid peptides (32.4, 55.0, and 12.0 lg/grain for 7Crp, Crp3 and 6Chao, respectively) are expected to be
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applicable to these patients as oral tolerogens to induce immune tolerance against the causative antigens. It is notable that levels of accumulation of these hybrid peptides were quite different, irrespective of use of the same promoter for their expression. Because lower levels of the 6Chao peptide than of the 7Crp or 3Crp peptides were observed, even when they were introduced into a single same binary vector and used as independent binary vectors, accumulation levels of individual hybrid peptide products may depend on their inherent structural and functional properties. Furthermore, when the 3Crp gene was expressed under the control of three different endosperm-specific promoters, its level of accumulation increased compared with that directed by one endosperm-specific promoter. This finding suggests that expression by different promoters in the same cell may result in greater accumulation. Because the hybrid T-cell epitope peptides generated in this study are specialized for PIT of Japanese cedar and cypress pollinosis among humans it is difficult to evaluate the efficacy of these treatments by use of animal models. Notably, one epitope (Cry j 1 p211–230) in the 7Crp peptide is identical to a major T-cell epitope in the Cry j 1 sequence found in B10.S mice (Yoshitomi et al. 2002). When transgenic rice seeds accumulating 7Crp peptide were fed orally to B10.S mice, allergenspecific T-cell proliferation and IgE accumulation were significantly suppressed compared with those fed nontransgenic rice seeds, indicating that immune tolerance against Cry j 1 can be induced by oral administration of transgenic seeds containing the specific T-cell epitope (Takagi et al. 2005b). Moreover, when the Cryconsensus peptide consisting of six major T-cell epitopes containing Cry j 1 p211–230 was injected subcutaneously into B10.S mice immunized with Cry j 1, reduction of the number of sneezes and of infiltration of eosinophils into nasal tissue, and down-regulation of the specific IgE and Th2-type cytokines IL-4 and IL-5, were observed (Tsunematsu et al. 2007). These results indicate that a major T-cell epitope can function as a safe tolerogen in place of native antigen (Cry j 1), which can cause side effects. Transgenic rice seeds containing T-cell epitopes derived from Japanese cedar and/or cypress pollen allergens have been developed as an oral tolerogen to induce immune tolerance. When T-cell epitope peptides are administered orally, resistance to digestive enzymes in the gastrointestinal tract is highly critical
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for efficacy. In our previous experiments, deposition of the target antigen into ER-derived PBs (PB-I) resulted in a requirement of approximately 33 % less transgenic rice seed for the suppression of allergenspecific IgE compared with antigen deposited into protein-storage vacuoles (PB-IIs), indicating the importance of having a deposition site with greater resistance to digestive enzymes (Takagi et al. 2010). Thus, it is important to examine whether the synthesized hybrid T-cell epitope peptides are bioencapsulated in BP-I or PB-II. The resistance to pepsin digestion of the Crp3 and 6Chao hybrid peptides produced in transgenic rice seeds was similar to that of the 7Crp peptide, as shown in Fig. 4. The greater resistance of the hybrid epitope peptide compared with that of endogenous glutelins may be attributed to their different intracellular location sites, because glutelins are deposited into protein storage vacuoles (PB-IIs). Ligation of the KDEL ER retention signal to these peptides at the C termini may result in preferential sorting into ER-derived PBs (PBIs) when the peptides are produced as secretory proteins in rice seeds. We previously showed by immuneelectron microscopy observation that the 7Crp peptide is predominantly deposited into ER-derived PBs (Takaiwa et al. 2009). This selective trafficking pattern and stable deposition are affected by the interaction of this peptide with cysteine-rich prolamins via disulfide bonds, because most of the 7Crp peptide could be extracted after removal of Cys-rich prolamins. By use of step-wise extraction experiments we demonstrated that the Crp3 and 6Chao peptides interact with Cys-rich prolamins via disulfide bonds (Fig. 5). These findings suggest that these hybrid epitope peptides may accumulate in ER-derived PBs (PB-Is) via formation of aggregates between these peptides and Cys-rich prolamins, although immune-electron microscopy observation is not reported for this study, because of the poor quality of the antibodies. The high resistance of hybrid peptides to pepsin compared with endogenous glutelins also supports the notion that hybrid peptides are deposited into PB-I. These results suggest that, unlike 7Crp, Crp3 and 6Chao bioencapsulated in PBs may be efficiently delivered to GALT without being severely degraded. A recent example of the efficacy of peptide vaccine against pollinosis involves immunotherapy for JCP using sublingual application of 7Crp peptide to JCP allergy patients (Yamanaka et al. 2009). This
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treatment resulted in up-regulation of IL-10 produced by Tr1 cells. The subgroup with increased levels of iTreg cells (IL-10 producing Tr1 cells) had lower clinical symptom scores than the subgroup patients with lower levels of Tr1 cells, who did not receive this PIT. In the current study we developed transgenic rice seeds containing many T-cell epitope peptides derived from Japanese cedar and cypress allergens, which my be suitable for cedar and cypress pollen allergy patients. In the near future it will be necessary to examine (in a clinical study) whether the rice seedbased peptide allergy vaccine produced in this study is effective when administered orally. Conclusion We have generated a rice seed-based peptide vaccine that accumulates most major T-cell epitopes from Japanese cedar and cypress pollen allergens by expressing hybrid peptides composed of T-cell epitopes under the control of endosperm-specific promoters. Large amounts of hybrid peptides accumulated in PBs and were resistant to digestive enzymes. Acknowledgments We thank Ms M. Utsuno, Y. Ikemoto, K. Miyashita, and Y. Yajima for technical assistance. This work was supported by a grant from the Agri-Health Translational Research Project grant from the Ministry of Agriculture Forestry and Fisheries of Japan.
References Baba K, Nakae K (2008) Epidemiology of nasal allergy through Japan. Prog Med 28:2001–2012 (in Japanese) Frew AJ (2010) Allergen immunotherapy. J Allergy Clin Immunol 125(Suppl 2):S306–S313 Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17:282–286 Hashiguchi S, Hino K, Taniguchi Y, Kurimoto M, Fukuda K, Ohyama M, Fujiyoshi T, Sonoda S, Nishimura Y, Yamada G, Sugimura K (1996) Immunodominance of seven regions of a major allergen, Cry j 2, of Japanese cedar pollen for T-cell immunity. Allergy 51:621–632 Hirahara K, Tatsuta T, Takatori T, Otsuka M, Kirinaka H, Kawaguchi J, Serizawa N, Taniguchi Y, Saito S, Sakaguchi M, Inouye S, Shiraishi A (2001) Preclinical evaluation of an immunotherapeutic peptide comprising 7 T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen allergens. J Allergy Clin Immunol 108:94–100 Holgate S, Polosa R (2008) Treatment strategies for allergy and asthma. Nat Rev Immunol 8:218–230
583 Komiyama N, Sone T, Shimizu K, Morikubo K, Kino K (1994) cDNA cloning and expression of Cry j II, the second major allergen of Japanese cedar pollen. Biochem Biophys Res Commun 201:1021–1028 Larche M (2007) Peptide immunotherapy for allergic diseases. Allergy 62:325–331 Larche M, Akdis CA, Valenta R (2006) Immunological mechanisms of allergen-specific immunotherapy. Nat Rev Immunol 6:761–771 Masuyama K, Chikamatsu K, Ikegawa S, Matsuoka T, Takahashi G, Yamamoto T, Endo S (2009) Analysis of helper T cell responses to Cry j 1-derived peptides in patients with nasal allergy: candidate for peptide-based immunotherapy of Japanese cedar pollinosis. Allergol Int 58:63–70 Mori T, Yokoyama M, Komiyama N, Okano M, Kino K (1999) Purification, identification, and cDNA cloning of Cha o 2, the second major allergen of Japanese cypress pollen. Biochem Biophys Res Commun 263:166–171 Namba M, Kurose M, Torigoe K, Hino K, Taniguchi Y, Fukuda S, Usui M, Kurimoto M (1994) Molecular cloning of the second major allergen, Cry j II, from Japanese cedar pollen. FEBS Lett 353:124–128 Oono N, Ide T, Sakaguchi M, Inouye S, Saito S (2000) Common antigenicity between Japanese cedar (Cryptomeria japonica) pollen and Japanese cypress (Chamaecyparis obtuse) pollen, II. Determination of the cross-reacting T-cell epitope of Cry j 1 and Cha o 1 in mice. Immunology 99:630–634 Qu LQ, Takaiwa F (2004) Tissue specific expression and quantitative potential evaluation of seed storage component gene promoters in transgenic rice. Plant Biotech J 2:113–125 Sone T, Komiyama N, Shimizu K, Kusakabe T, Morikubo K, Kino K (1994) Cloning and sequencing of cDNA coding for Cry j I, a major allergen of Japanese cedar pollen. Biochem Biophys Res Commun 199:619–625 Sone T, Morikubo K, Miyahara M, Komiyama N, Shimizu K, Tsunoo H, Kino K (1998) T cell epitopes in Japanese cedar (Cryptomeria japonica) pollen allergens: choice of major T cell epitopes in Cry j 1 and Cry j 2 toward design of the peptide-based immunotherapeutics for the management of Japanese cedar pollinosis. J Immunol 161:448–457 Sone T, Dairiki K, Morikubo K, Shimizu K, Tsunoo H, Mori T, Kino K (2005) Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level. Clin Exp Allergy 35:664–671 Sone T, Dairiki K, Morikubo K, Shimizu K, Tsunoo H, Mori T, Kino K (2009) Recognition of T cell epitopes unique to Cha o 2, the major allergen in Japanese cypress pollen, in allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen. Allergol Int 58:237–245 Suzuki M, Komiyama N, Ithoh M, Itoh H, Sone T, Kino K, Takagi I, Ohta N (1996) Purification, characterization and molecular cloning of Cha o 1, a major allergen of Chamaecyparis obtuse (Japanese cypress) pollen. Mol Immunol 33:451–460 Tada Y, Utsumi S, Takaiwa F (2003) Foreign gene products can be enhanced by introduction into low storage protein mutants. Plant Biotech J 1:411–422
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584 Takagi H, Hiroi T, Yang L, Tada Y, Yuki Y, Takamura K, Ishimitsu R, Kawauchi H, Kiyono H, Takaiwa F (2005a) A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE response. Proc Natl Acad Sci USA 102:17525–17530 Takagi H, Saito S, Yang L, Nagasaka S, Nishizawa N, Takaiwa F (2005b) Oral immunotherapy against a pollen allergy using seed-based peptide vaccine. Plant Biotech J 3:521–533 Takagi H, Hirose S, Yasuda H, Takaiwa F (2006) Biochemical safety evaluation of transgenic rice seeds expressing T cell epitopes of Japanese cedar pollen allergens. J Agric Food Chem 54:9901–9905 Takagi H, Hiroi T, Hirose S, Yang L, Takaiwa F (2010) Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides. Peptides 31:1421–1425 Takaiwa F (2007) Transgenic rice seed as a nutriceutical delivery system. CAB Rev Perspect Agric Vet Sci Nutr Nat Resour 2:1–9 Takaiwa F (2011) Seed-based oral vaccines as allergen-specific immunotherapies. Hum Vaccine 7:357–366 Takaiwa F (2013) Update on the use of transgenic rice seeds in oral immunotherapy. Immunotherapy 5:301–312 Takaiwa F, Hirose S, Takagi H, Yang L, Wakasa Y (2009) Deposition of a recombinant peptide in ER-derived protein bodies by retention with cysteine-rich prolamins in transgenic rice seed. Planta 229:1147–1158 Tsunematsu M, Yamaji T, Kozutsumi D, Murakami R, Kimura S, Kino K (2007) Effect of Cry-consensus peptide, novel recombinant peptide for immunotherapy of Japanese cedar
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ORIGINAL PAPER
Development of a rice-based peptide vaccine for Japanese cedar and cypress pollen allergies Fumio Takaiwa • Lijun Yang
Received: 8 December 2013 / Accepted: 5 March 2014 / Published online: 18 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Peptide immunotherapy using dominant T-cell epitopes is a safe treatment alternative to conventional subcutaneous injection of natural crude allergen extract, which is sometimes accompanied by anaphylactic shock. For Japanese cedar pollinosis (JCP), hybrid peptides composed of six to seven major T-cell epitopes (7Crp peptide) from the causative allergens Cry j 1 and Cry j 2 have been developed on the basis of different human leukemia antigen class II restrictions, because of the diversity of patients’ genetic backgrounds. However, other dominant T-cell epitopes that are produced in some patients are not covered by these peptides. To develop a more universal peptide vaccine for JCP, we generated transgenic rice seeds containing seven new T-cell epitopes (Crp3) in addition to the T-cell epitopes used in the 7Crp peptide. Next, we co-expressed unique T-cell epitopes (6Chao) from the Japanese cypress pollen allergens Cha o 1 and Cha o 2 in transgenic rice seeds, with 7Crp and Crp3. These transgenic rice seeds, containing many highly homologous T-cell epitopes derived from cedar and cypress allergens, are
Electronic supplementary material The online version of this article (doi:10.1007/s11248-014-9790-3) contains supplementary material, which is available to authorized users. F. Takaiwa (&) L. Yang Functional Transgenic Crop Research Unit, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan e-mail: [email protected]
expected to be applicable to a wide range of patients suffering from these pollen allergies. Keywords Allergy vaccine Oral vaccine Peptide immunotherapy Pollinosis Transgenic rice seeds Abbreviations ER Endoplasmic reticulum GALT Gut-associated lymphoid tissue HLA Human leukemia antigen JCP Japanese cedar pollinosis PBMC Peripheral blood mononuclear cells PB Protein body 2-MER 2-Mercaptoethanol
Introduction Japanese cedar pollen allergy is an important public health problem in Japan, with approximately 27 % of Japanese people afflicted by this pollinosis from February to April each year (Baba and Nakae 2008). Allergen-specific immunotherapy, the only curative treatment used to control this prevalent allergic disease (Frew 2010), is achieved by subcutaneous injection of increasing doses of crude allergen extract over a period of three to five years to induce immune tolerance to specific CD4? T-cells against the causative allergens. This curative treatment leads to
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reduction of allergen-specific IgE, an increase in blocking IgG4, down-regulation of allergen-specific T-cell responsiveness, and suppression of effector cells (Larche et al. 2006; Holgate and Polosa 2008). However, conventional immunotherapy is sometimes accompanied by the adverse effect of anaphylactic shock, because the allergen can cross-link with specific IgEs on mast cells and basophils. Therefore, a simpler, safer, more convenient allergen-specific immunotherapy is needed. To improve the efficacy and safety of allergy treatment, the route of administration and immunological properties of tolerogens must be changed. In particular, it is crucial to change from administering native crude extract to administering a hypoallergenic tolerogen to reduce side effects and enhance safety (Valenta et al. 2010). One strategy that addresses this requirement is peptide immunotherapy (PIT) using major T-cell epitopes derived from the causative allergens (Larche 2007). T-cell epitopes efficiently bind to human leukemia antigen II (HLA II) to induce T-cell responses and can be used to generate T-cell tolerance, while their short length and lack of tertiary conformational structures do not facilitate IgE cross-linking. Many T-cell epitopes characteristic of individuals with different HLA II haplotypes should be included when generating a universal T-cell epitope peptide. Another strategy that would improve allergy treatment is to replace subcutaneous injection with oral administration, eliminating patient discomfort and making administration more convenient. A model experiment has been performed in which mouse major T-cell epitopes from the Japanese cedar pollen allergens Cry j 1 and Cry j 2 were expressed as a fusion protein with the soybean seed protein glycinin in transgenic rice seeds (Takagi et al. 2005a). The feasibility of using a rice seed-based peptide vaccine was demonstrated by feeding mice with transgenic rice seeds containing mouse T-cell epitopes from Japanese cedar pollen allergens. Not only did the animals manifest immunological signs, for example reduced levels of allergen-specific IgE, T-cell proliferative responses, and secretion of IL-4, IL-5, and IL13 cytokines by Th2 cells, alleviation of such allergy symptoms as sneezing was also observed (Takagi et al. 2005a). Next, transgenic rice seeds containing a 7Crp hybrid peptide composed of seven major human T-cell epitopes from the Cry j 1 and Cry j 2 sequences were used to control Japanese cedar pollinosis (JCP). The
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7Crp peptide was designed to prevent IgE-mediated adverse effects, because the B-cell epitopes were eliminated from this peptide (Hirahara et al. 2001). Notably, the 7Crp peptide has the same immunogenicity as the inherent Cry j 1 and Cry j 2 allergens; it is, thus, an ideal, safe tolerogen. Peripheral blood mononuclear cells (PBMC) from 44 of 48 JCP patients (92 %) manifested proliferative responses to this hybrid peptide (Hirahara et al. 2001). Furthermore, the 7Crp peptide does not bind to the specific IgE in sera from JCP patients, confirming the safety of this peptide vaccine. When the 7Crp peptide is expressed in transgenic rice seeds under the control of seedspecific promoters, it accumulates predominantly in endoplasmic reticulum (ER)-derived protein bodies (PBs) (Takagi et al. 2005b; Takaiwa et al. 2009). The 7Crp peptide in rice seeds is bioencapsulated within the cell walls and PBs, which protects it from digestion by gastrointestinal enzymes, in contrast with naked peptides (Takagi et al. 2010; Takaiwa 2011). Thus, bioencapsulation of antigens provides an effective delivery system to gut-associated lymphoid tissue (GALT) (Takaiwa 2013). To date, Cry-consensus peptide and 7Crp have been created as universal hybrid T-cell epitope peptides by linking six or seven major T-cell epitopes derived from Japanese cedar pollen allergens (Hirahara et al. 2001; Tsunematsu et al. 2007). However, proliferative responses to these hybrid peptides are not observed for some T-cell lines from JCP patients. To expand the applicability of such treatment to JCP patients, it is important to include as many T-cell epitopes derived from Cry j 1 and Cry j 2 as possible. In this study, we selected the 14 major T-cell epitopes in cedar pollen allergen Cry j 1 and Cry j 2 sequences to produce an improved JCP treatment. Moreover, 70–80 % of JCP patients are also afflicted by cypress pollen allergy (Yasueda et al. 2001). Because the major cypress pollen allergens Cha o 1 and Cha o 2 are highly homologous (75–80 %) with Cry j 1 and Cry j 2, respectively (Suzuki et al. 1996; Mori et al. 1999), we selected six major T-cell epitopes unique to the cypress allergens Cha o 1 and Cha o 2 and expressed these epitopes in transgenic rice seeds, together with those derived from cedar pollen allergens. When T-cell epitope peptides were produced in transgenic rice seeds, they were highly expressed and were deposited into digestive enzyme-resistant PBs via interaction with cysteine-rich prolamins. The
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transgenic rice seeds produced in the study are expected to act as oral tolerogens by induction of immune tolerance against cedar and cypress pollen allergens. Transgenic plants are used as a promising means of production of high-value recombinant proteins. Higher levels of accumulation of recombinant proteins can be achieved by using strong specific promoters, optimizing codon usage, and intracellular targeting into PBs (Qu and Takaiwa 2004; Takaiwa 2007; Wakasa and Takaiwa 2013). We have previously generated many transgenic rice plants that successfully accumulated functional recombinant proteins in seeds by using endosperm-specific promoters of 2.3kb glutelin B1 (GluB-1) and 1.4-kb glutelin B4 (GluB4) and 10-kD prolamin promoters, and by fusing the N-terminal signal peptide of rice seed proteins such as GluB-1 signal peptide and the C terminal KDEL ER retention sequences (Takagi et al. 2005a, b; Takaiwa et al. 2009; Yang et al. 2012). It should be noted that although recombinant proteins are specifically produced and accumulated in PBs, the yields vary substantially, because of the inherent structural and functional properties of the proteins chosen. To obtain a better yield of the proteins it is, therefore, important to test the different promoters and to choose most suitable.
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GluC, or 1.8 kb 10 kD prolamin endosperm-specific promoters containing DNA sequences encoding the corresponding signal peptides (Qu and Takaiwa 2004), and the C-termini KDEL ER retention sequences of the genes were followed by 0.65 kb of the GluB-1 terminator, 0.7 kb of the GluB-4 terminator, 0.64 kb of the GluC terminator, or 0.35 kb of the 10 kD prolamin terminator. The entry clones containing gene cassettes of 12Crp, Crp3, 6Cha o, and 7Crp were obtained (Fig. 1). For construction of expression plasmids for rice genome transformation (pA in Fig. 1), the aforementioned entry clones having gene cassettes of 12Crp, Crp3, 6Chao, and 7Crp were cloned into the destination binary vector p35SHPTAg7-GW by using the LR clonase reactions of the MutiSite Gateway system (Wakasa et al. 2006); the resulting plasmids were designated pA, pB1, pB2, pC, pD, pE, and pF (Fig. 1). The expression plasmids were introduced into the rice genome (Oryza sativa cv. Kita-ake) via Agrobacterium tumefaciens-mediated transformation. Transgenic plants resistant to hygromycin were selected, as described elsewhere (Goto et al. 1999). The transgenic plants were grown in a controlled greenhouse (28 °C, 12 h light/dark cycle) and more than 20 plants with each structure were subjected to transformation analysis. Preparation of antibodies
Materials and methods Plasmid construction and rice transformation In the same way as the 7Crp gene reported by Takagi et al. (2005b), the DNA sequences coding for 12Crp, Crp3, and 6Chao were optimized for codons that are frequently used in rice seed storage protein genes. ATrich sequences, including potential mRNA destabilizing sequences, polyadenylation signals, putative intron splicing sites, and rare codons such as AAA (Lyn), ACG (Thr) TCG (Ser) and CGA (Arg), etc., were avoided among their coding regions (Wakasa and Takaiwa 2013). After further checking of the DNA sequences for these artificial genes by use of Genetyp (Hewlett–Packard), genes having DNA sequences encoding the KDEL ER retention at their C-termini were synthesized by GenScript (NJ, South Plainfield, USA). The genes were then ligated downstream of the 2.3 kb GluB-1, 1.4 kb GluB-4, 1.5 kb
E. coli-expressed purified 12Crp and Crp3 were used to raise rabbit anti-12Crp and anti-Crp3 antibodies (Qiagen, Japan). For detection of 6Chao, a peptide (GPSPKEFESSGKNEG) was synthesized and used to prepare a peptide antibody against 6Chao (Qiagen). Antibodies to 7Crp, glutelin A (GluA), and cysteine rich 13 kD prolamin (RM1) had previously been prepared in our laboratory (Takagi et al. 2006). Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). SDS-PAGE and immunoblot analysis Mature seeds were ground into a fine powder by use of a multibead shocker (Yasui Kikai, Tokyo, Japan). Total protein was extracted from the powder of a single grain by use of 600 ll urea–SDS buffer (50 mM Tris–HCl, pH 6.8, 8 M Urea, 4 % SDS,
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Fig. 1 Diagrammatic representation of the structures used for expression of T-cell epitope peptides in transgenic rice seeds. The genes encoding 7Crp, 12Crp, Crp3, and 6Chao were expressed under the control of the endosperm-specific 2.3 kb GluB-1, 1.4 kb GluB-4, 1.5 kb GluC, or 1.8 kb 10 kD prolamin promoters. Ag7, Agrobacterium gene 7 terminator; hpt,
hygromycin phosphotransferase coding region; 35S P, cauliflower mosaic virus 35S promoter. GluB1 pro SP, GluB4 pro SP, GluC pro SP, and 10 K pro SP represent GluB-1, GluB-4, GluC, and 10 kD prolamin promoters and their signal sequences, whereas GluB1 T, GluB4 T, GluC T, and 10 kD T are terminators of the corresponding genes. KDEL, ER retention signal
5 % 2-mercaptoethanol (2-ME), 20 % glycerol) as described elsewhere (Tada et al. 2003). After separation by 12 % SDS-PAGE, the proteins were visualized by Coomassie brilliant blue (CBB)-R250 staining or transferred to PVDF membranes (Millipore, Billerica, MA, USA) for immunodetection with the corresponding antibodies. Accumulation levels of the peptides were estimated on the basis of the intensity of bands stained with CBB, using Bio-Rad protein Assay Standard II-BSA as calibration control (Bio-Rad Laboratories, Hercules, CA, USA). For proteins with low accumulation levels, immunoblot analysis was performed with the corresponding antibodies using E. coli-expressed, purified recombinant proteins as a controls. Band density was quantified with NIH image J software (National Institutes of Health, Washington, DC, USA). For immunoblot analysis, the membranes were reacted with primary antibodies against 12Crp, Crp3, 6Chao, and 7Crp (all at dilution 1:10,000), then with secondary antibody of horseradish peroxidase-conjugated anti-rabbit IgG (dilution 1:5,000). The bands or dots were visualized with the ECL detection kit (GE healthcare, UK).
In-vitro digestion of transgenic rice seeds by pepsin
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Transgenic rice seeds were subjected to pepsin digestion as described elsewhere (Takagi et al. 2005b). Briefly, 150 ll of reaction buffer containing 0.1 % (w/v) pepsin (Sigma, USA) and 30 mM NaCl (pH 1.2) was added to 5 mg seed powder and incubated at 37 °C. The reaction was terminated by neutralization with NaOH after 0, 2, 5, 15, 30, 60, 120, and 180 min. After addition of 150 ll of urea– SDS buffer, the digested samples were analyzed by 12 % SDS-PAGE then immunoblot analysis. Step-wise extraction of seed proteins Rice seed storage proteins were extracted from pulverized mature seeds by use of the step-wise extraction method described elsewhere (Takaiwa et al. 2009). Extraction buffer containing 0.5 M NaCl and 10 mM Tris–HCl (pH 7.5) was used to extract albumin and globulin. The residues were then treated with 60 % (v/v) n-propanol for extraction of Cys-poor prolamins and further resuspended in 60 % (v/v) n-propanol containing 5 % 2-ME for extraction of
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Cys-rich prolamins. Finally, the residues were treated with 1 % ((v/v) lactic acid for extraction of glutelins and peptide proteins. For all steps, the residues were extracted twice with the same solution.
Results Generation of transgenic rice seeds accumulating T-cell epitope peptides derived from Japanese cedar and cypress pollen allergens The artificial gene encoding the universal T-cell epitope peptide comprising seven major and five additional T-cell epitope peptides, named 12Crp, was first synthesized by using codons frequently used in many rice seed storage protein genes. Additional T-cell epitopes were selected as shown in Supplementary Table 1. The KDEL ER retention signal was attached to the C termini of the 12Crp peptide. The 12Crp peptide is composed of 193 amino acids with a predicted molecular mass of 21,436 Da (Fig. 2a). The codon-optimized synthetic gene encoding 12Crp containing the KDEL signal at the C terminus was ligated to the 2.3 kb GluB-1 or 1.4 kb GluB-4 endospermspecific promoter containing the signal peptide denoted pB1 and pB2, respectively (Fig. 1) and expressed as a secretory protein in transgenic rice seeds after introduction into the rice genome via Agrobacterium-mediated transformation. A total of 20 and 53 independent transgenic rice plants were regenerated for pB1 and pB2 structures, respectively. As shown in Fig. 3b, the 12Crp peptide was detected in mature seeds by immunoblot analysis using a 12Crp-specific antibody. However, the accumulation level (1.6 and 3.6 lg/grain for lines 11 and 16) was much lower than that of 7Crp (23.2 and 29.6 lg/grain for lines 10 and 34), despite that fact that the strong endosperm-specific GluB-1 or GluB-4 promoter was used for expression (Supplementary Table 2). Seven new T-cell epitopes that were not included among the seven T-cell epitopes used in the 7Crp peptide were the selected from the major T-cell epitopes characterized in the Cry j 1 and Cry j 2 molecules (Supplementary Table 1) (Hashiguchi et al. 1996; Sone et al. 1998; Masuyama et al. 2009). For the Cry j 1 allergen, two T-cell epitopes, at positions 80–94 and 108–132, were used in the Cry-consensus peptide, and two other epitopes (at positions 61–79 and 337–350)
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were selected. Furthermore, three other T-cell epitopes, at positions 25–39, 158–175, and 245–264, were selected from the Cry j 2 sequence. These seven T-cell epitopes were linked together, to form a hybrid peptide named Crp3 (Fig. 2b) which is 129 amino acids long with a predicted molecular mass of 14,171 Da. First, we examined whether the Crp3 peptide can strongly accumulate in rice seeds in a similar manner to 7Crp. The gene coding for the Crp3 peptide (including the KDEL ER retention signal at the C terminus) was ligated to the 2.3 kb GluB-1, 1.5 kb GluC, or 1.8 kb 10 kD prolamin endosperm-specific promoter containing the individual signal peptide, and these three expression cassettes were inserted into a binary vector by use of the multisite Gateway system, resulting in pC, which was then introduced into the rice genome. Twenty-four independent transgenic rice plants containing Crp3 were obtained and the Crp3 peptide was strongly accumulated in seeds (80 and 72 lg/grain for lines 1 and 8), and was detected as a visible band on CBB-stained SDS-PAGE gels (Fig. 3c). This result indicates that the Crp3 peptide can be highly produced in rice seeds, as is the 7Crp peptide (Supplementary Table 2). Many JCP patients have specific IgEs to pollen allergens of both Japanese cypress and Japanese cedar. The Cry j 1 and Cry j 2 major pollen allergens from Japanese cedar are approximately 80 and 74 % identical with Cha o 1 and Cha o 2, respectively, from cypress (Suzuki et al. 1996; Mori et al. 1999). Dominant T-cell epitopes have been identified in the Cha o 1 and Cha o 2 sequences (Sone et al. 2005, 2009). Notably, several T-cell epitopes are identical in these cedar and cypress pollen allergens (Cry j 1 vs. Cha o 1) whereas some epitopes are unique to cypress pollen allergens. Therefore, if T-cell epitopes common to the Cha o and Cry j allergens and specific T-cell epitopes unique to the Cha o allergen are selected and expressed together in transgenic rice seeds, these seeds should be highly applicable as edible vaccines for patients suffering from seasonal Japanese cedar and cypress pollinosis. Four T-cell epitopes (at positions 11–30, 211–230, 251–270, and 331–350) in the Cha o1 sequence are common to Cry j 1, and four T-cell epitopes (at positions 61–80, 71–90, 311–330, and 321–340) are unique to Cha o 1. On the other hand, three T-cell epitopes (at positions 41–61, 141–170, and 350–390) are characteristic of Cha o 2 (Sone et al. 2005, 2009) (Supplementary Table 1).
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A
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Fig. 2 The amino acid sequences of 12Crp, Crp3, and 6Chao. The major T-cell epitopes derived from Cry j 1, Cry j 2, Cha o 1, and Cha o 2 are indicated below the corresponding sequences. Amino acid position numbers of individual T-cell epitope in the
mature Cry j 1 and Cry j 2 are in accordance with those determined by Sone et al. (1994) and Namba et al. (1994), and position numbers in parentis are in accordance with the Cry j 2 sequence determined by Komiyama et al. (1994)
We selected six unique T-cell epitopes from the Cha o 1 and Cha o 2 sequences, which were linked together to generate the 6Chao hybrid peptide. The 6Chao peptide is composed of 154 amino acids, and its predicted molecular mass is 17,033 Da (Fig. 2c). When the 6Chao peptide was produced in transgenic
rice seeds under the control of the 2.3 kb GluB-1 promoter containing its signal peptide (plasmid pD), this peptide could not be detected among any of the 23 transgenic rice lines assayed by CBB staining after SDS-PAGE (Fig. 3d). Accumulation levels were 7.2 and 6.6 lg/grain for lines 1 and 8, respectively.
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Fig. 3 Expression of T-cell epitope peptides in transgenic rice seeds. Total proteins were extracted with 600 ll urea–SDS buffer from non-transgenic O. sativa cv. Kita-ake (NT) and transgenic rice seeds containing 7Crp, 12Crp, Crp3, and 6Chao, and 2 ll of each sample was loaded on to 12 % SDS-PAGE, and the gels (left panels) were CBB-stained. The accumulated T-cell epitope peptides (right panels) were immune-detected with the
corresponding antibodies. Molecular markers are shown on the left. Open and solid arrowheads and the solid arrow indicate the 6Chao, 7Crp, and 12Crp bands, respectively, and the asterisk indicates Crp3. Dimers of 7Crp, 12Crp, and Crp3 are indicated by open arrows, and arabic numerals represent transgenic line numbers. For 12Crp, lines 11 and 45 are from pB1 and pB2, respectively
The 6Chao peptide expression cassette, together with the 7Crp expression cassette or the 7Crp and Crp3 expression cassettes, was introduced into a binary vector by use of the Gateway system, resulting plasmid pE and pF, respectively, as shown in Fig. 1. We generated 24 independent transgenic rice plants expressing 6Chao and 7Crp double peptides, and another 24 independent plants expressing 6Chao, 7Crp, and Cryp3 triple peptides (Fig. 3e, f). As shown in Fig. 3f, the 6Chao peptide was detected as a weak band by CBB staining, when expressed together with the 7Crp and Crp3 peptides. The 7Crp, Crp3, and 6Chao hybrid peptides accumulated at relatively high levels (32.4, 55.0, and 12.0 lg/grain for 7Crp, Crp3, and 6Chao, respectively) in transgenic rice seeds containing the triple peptides (Supplementary Table 2). Therefore, many T-cell epitopes accumulated in rice seeds, and are expected to act as tolerogens to induce immune tolerance against the Japanese cedar and cypress pollen allergens when patients are fed these transgenic rice seeds.
Resistance to pepsin digestion in transgenic rice seeds accumulating T-cell epitopes We previously reported that 7Crp peptide predominantly accumulates in ER-derived PBs (PB-Is) via an interaction with Cys-rich prolamins at their disulfide bonds (Takaiwa et al. 2009). For oral-peptide vaccines, resistance to digestion by enzymes in the gastrointestinal tract and to harsh acidic conditions in the stomach is critical for the delivery of these vaccines to GALT to induce immune tolerance (Takaiwa 2011). When transgenic rice seeds accumulating Crp3, 6Chao, and 7Crp/Crp3/6Chao were subjected to in-vitro digestion with pepsin, there was no significant difference in the time required for complete digestion compared with rice seeds containing 7Crp. Notably, as shown in Fig. 4, resistance of the 6Chao and Crp3 peptides in rice seeds to pepsin digestion was higher than that of the endogenous glutelins deposited in storage vacuole-derived PBs (PB-IIs), although the Cys-rich 13 kD prolamin (RM1)
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Fig. 4 In-vitro digestibility of transgenic rice seed powder containing 7Crp (pA) and 6Chao/Crp3/7Crp (pF). Seed powder (5 mg) was added to the reaction mixture containing 0.1 % pepsin and incubated at 37 °C for 0, 2, 5, 15, 30, 60, 120, or 180 min. Total proteins in each reaction mixture were extracted
with urea–SDS buffer and examined by immunoblotting with the corresponding antibodies. RM1 and GluA denote Cys-rich 13 kD prolamin and glutelin A, respectively. Two bands in the GluA panel represent glutelin A precursor and its acidic subunit
deposited in ER-derived PB (PB-I) in the same transgenic rice seeds was much more resistant to pepsin digestion. Specifically, the hybrid peptides were completely digested with pepsin within 30 min, whereas 15 min was required for complete digestion of glutelins. These results suggest that these hybrid peptides (comprising several T-cell epitopes) are deposited into ER-derived PBs,
which is also true for the 7Crp peptide (Takaiwa et al. 2009). The interaction via disulfide bonds is especially critical for the 6Chao peptide, because no 6Chao peptide could be extracted before removal of the Cysrich prolamins. This finding also supports the notion that these peptides, composed of T-cell epitopes from Japanese cedar and cypress pollen allergens, are mainly deposited as aggregates in ER-derived PBs (PB-Is) through interactions with Cys-rich prolamins via disulfide bonds.
Interaction of T-cell epitope peptides with cysteine-rich prolamins The Crp3 and 6Chao peptides contain four and two cystein residues, respectively. We previously showed by immune-electron-microscopy that the 7Crp peptide is predominantly targeted to PB-I (Takaiwa et al. 2009). In this study we found that the 7Crp peptide could be extracted only after removal of Cys-rich prolamins, which suggests there is an interaction between Cys-rich prolamins, for example 10 kD, 16 kD, and Cys-rich 13 kD prolamins, within PB-Is. We then examined whether there was any interaction between the Cys-rich prolamins and the 6Chao or Crp3 peptide. As shown in Fig. 5, when seed proteins were extracted with different extraction buffers in a step-wise manner, these peptides could be extracted only after removal of various endogenous Cys-rich prolamins with 60 % propanol containing 2 % 2-ME,
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Discussion PIT using major T-cell epitopes is a safe and effective treatment strategy, without side effects, because T-cell epitopes are unable to cross-link specific IgE molecule on mast cells and basophils, which leads to anaphylaxis shock, and they cannot activate effecter cells (Larche 2007). Furthermore, high-dose administration of these peptides beginning at the initial stage of treatment shortens the treatment period. However, characterizing T-cell epitopes from the target antigens is a prerequisite for use of PIT. Furthermore, PIT is not applicable to all patients suffering from allergies, because T-cell epitopes differ among humans as a result of genetic diversity. To compensate for this genetic diversity, several major T-cell epitopes
Transgenic Res (2014) 23:573–584 Fig. 5 SDS-PAGE and immune-blot analyses of peptides Crp3, 6Chao, and 7Crp extracted with 1 % lactic acid after preextraction with different solvents. A Crp3 (pC), B 6Cha o (pD), and C Crp3/ 6Cha o/7Crp (pF). T total seed proteins; plus and minus signs indicate with and without pre-extraction, respectively. The upper panel gels are CBB-stained and molecular markers are indicated on the left
derived from the causative antigen sequences can be linked together to yield a hybrid peptide. However, because it is difficult to cover this amount of genetic diversity perfectly, it may be necessary to use many T-cell epitopes to increase coverage for applicable patients. The 7Crp and Cry-consensus human T-cell epitope hybrid peptide derived from cedar pollen allergens Cry j 1 and Cry j 2 manifests proliferative responses to approximately 90 % of PBMCs from JCP patients (Sone et al. 1998; Hirahara et al. 2001). Four T-cell epitopes used in this hybrid epitope peptide are fundamentally identical with each other. To enhance coverage, we included two Cry j 1 T-cell epitopes used in the Cry-consensus peptide and five additional major T-cell epitopes in the new hybrid peptide, Crp3, produced in this study. The sequence common to the 7Crp and Crp3 peptides comprises 235 amino acids, which accounts for approximately 31 % of the total Cry j 1 and Cry j 2 sequences (742 amino acids). On the other hand, 70–80 % of Japanese cedar pollen patients are cross-reactive to Japanese cypress pollen allergens, as diagnosed by the radio allergosorbent test (RAST) or by skin testing (Yasueda et al. 2001). This may be because the major allergens of these pollens are highly homologous (75–80 %) with each other at the amino acid level. Major T-cell
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A
B
C
epitopes in these allergens are well characterized, as shown in Supplementary Table 1. Some major T-cell epitopes in cedar and cypress allergens are identical; unique T-cell epitopes characteristic of the cypress allergens Cha o 1 and Cha o 2 have also been well characterized (Sone et al. 2005, 2009). T-cells from JCP patients recognize Cry j 1 p211–230 and also respond to Cha o 1 p209–228 (Oono et al. 2000), because the minimum peptide sequences (p213–230) of the cross-reacting T-cell epitopes in Cry j 1 and Cha o 1 are identical. There are also three additional common T-cell epitopes (at p11–30, p251–270, and p331–350) between Cha o 1 and Cry j 1 (Sone et al. 2005); thus common antigenicity at the T-cell level between Japanese cedar and cypress pollen allergens may be because of the existence of identical T-cell epitopes, leading to cross-reactivity. Indeed, cedar pollinosis patients continue to have allergic symptoms even after the end of the cedar pollen season, which is followed by the cypress pollen season. Therefore, transgenic rice seeds containing the common and unique T-cell epitopes derived from both the major cedar and cypress pollen allergens at levels of 0.6–2.75 mg/g mature seed for individual hybrid peptides (32.4, 55.0, and 12.0 lg/grain for 7Crp, Crp3 and 6Chao, respectively) are expected to be
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applicable to these patients as oral tolerogens to induce immune tolerance against the causative antigens. It is notable that levels of accumulation of these hybrid peptides were quite different, irrespective of use of the same promoter for their expression. Because lower levels of the 6Chao peptide than of the 7Crp or 3Crp peptides were observed, even when they were introduced into a single same binary vector and used as independent binary vectors, accumulation levels of individual hybrid peptide products may depend on their inherent structural and functional properties. Furthermore, when the 3Crp gene was expressed under the control of three different endosperm-specific promoters, its level of accumulation increased compared with that directed by one endosperm-specific promoter. This finding suggests that expression by different promoters in the same cell may result in greater accumulation. Because the hybrid T-cell epitope peptides generated in this study are specialized for PIT of Japanese cedar and cypress pollinosis among humans it is difficult to evaluate the efficacy of these treatments by use of animal models. Notably, one epitope (Cry j 1 p211–230) in the 7Crp peptide is identical to a major T-cell epitope in the Cry j 1 sequence found in B10.S mice (Yoshitomi et al. 2002). When transgenic rice seeds accumulating 7Crp peptide were fed orally to B10.S mice, allergenspecific T-cell proliferation and IgE accumulation were significantly suppressed compared with those fed nontransgenic rice seeds, indicating that immune tolerance against Cry j 1 can be induced by oral administration of transgenic seeds containing the specific T-cell epitope (Takagi et al. 2005b). Moreover, when the Cryconsensus peptide consisting of six major T-cell epitopes containing Cry j 1 p211–230 was injected subcutaneously into B10.S mice immunized with Cry j 1, reduction of the number of sneezes and of infiltration of eosinophils into nasal tissue, and down-regulation of the specific IgE and Th2-type cytokines IL-4 and IL-5, were observed (Tsunematsu et al. 2007). These results indicate that a major T-cell epitope can function as a safe tolerogen in place of native antigen (Cry j 1), which can cause side effects. Transgenic rice seeds containing T-cell epitopes derived from Japanese cedar and/or cypress pollen allergens have been developed as an oral tolerogen to induce immune tolerance. When T-cell epitope peptides are administered orally, resistance to digestive enzymes in the gastrointestinal tract is highly critical
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for efficacy. In our previous experiments, deposition of the target antigen into ER-derived PBs (PB-I) resulted in a requirement of approximately 33 % less transgenic rice seed for the suppression of allergenspecific IgE compared with antigen deposited into protein-storage vacuoles (PB-IIs), indicating the importance of having a deposition site with greater resistance to digestive enzymes (Takagi et al. 2010). Thus, it is important to examine whether the synthesized hybrid T-cell epitope peptides are bioencapsulated in BP-I or PB-II. The resistance to pepsin digestion of the Crp3 and 6Chao hybrid peptides produced in transgenic rice seeds was similar to that of the 7Crp peptide, as shown in Fig. 4. The greater resistance of the hybrid epitope peptide compared with that of endogenous glutelins may be attributed to their different intracellular location sites, because glutelins are deposited into protein storage vacuoles (PB-IIs). Ligation of the KDEL ER retention signal to these peptides at the C termini may result in preferential sorting into ER-derived PBs (PBIs) when the peptides are produced as secretory proteins in rice seeds. We previously showed by immuneelectron microscopy observation that the 7Crp peptide is predominantly deposited into ER-derived PBs (Takaiwa et al. 2009). This selective trafficking pattern and stable deposition are affected by the interaction of this peptide with cysteine-rich prolamins via disulfide bonds, because most of the 7Crp peptide could be extracted after removal of Cys-rich prolamins. By use of step-wise extraction experiments we demonstrated that the Crp3 and 6Chao peptides interact with Cys-rich prolamins via disulfide bonds (Fig. 5). These findings suggest that these hybrid epitope peptides may accumulate in ER-derived PBs (PB-Is) via formation of aggregates between these peptides and Cys-rich prolamins, although immune-electron microscopy observation is not reported for this study, because of the poor quality of the antibodies. The high resistance of hybrid peptides to pepsin compared with endogenous glutelins also supports the notion that hybrid peptides are deposited into PB-I. These results suggest that, unlike 7Crp, Crp3 and 6Chao bioencapsulated in PBs may be efficiently delivered to GALT without being severely degraded. A recent example of the efficacy of peptide vaccine against pollinosis involves immunotherapy for JCP using sublingual application of 7Crp peptide to JCP allergy patients (Yamanaka et al. 2009). This
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treatment resulted in up-regulation of IL-10 produced by Tr1 cells. The subgroup with increased levels of iTreg cells (IL-10 producing Tr1 cells) had lower clinical symptom scores than the subgroup patients with lower levels of Tr1 cells, who did not receive this PIT. In the current study we developed transgenic rice seeds containing many T-cell epitope peptides derived from Japanese cedar and cypress allergens, which my be suitable for cedar and cypress pollen allergy patients. In the near future it will be necessary to examine (in a clinical study) whether the rice seedbased peptide allergy vaccine produced in this study is effective when administered orally. Conclusion We have generated a rice seed-based peptide vaccine that accumulates most major T-cell epitopes from Japanese cedar and cypress pollen allergens by expressing hybrid peptides composed of T-cell epitopes under the control of endosperm-specific promoters. Large amounts of hybrid peptides accumulated in PBs and were resistant to digestive enzymes. Acknowledgments We thank Ms M. Utsuno, Y. Ikemoto, K. Miyashita, and Y. Yajima for technical assistance. This work was supported by a grant from the Agri-Health Translational Research Project grant from the Ministry of Agriculture Forestry and Fisheries of Japan.
References Baba K, Nakae K (2008) Epidemiology of nasal allergy through Japan. Prog Med 28:2001–2012 (in Japanese) Frew AJ (2010) Allergen immunotherapy. J Allergy Clin Immunol 125(Suppl 2):S306–S313 Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17:282–286 Hashiguchi S, Hino K, Taniguchi Y, Kurimoto M, Fukuda K, Ohyama M, Fujiyoshi T, Sonoda S, Nishimura Y, Yamada G, Sugimura K (1996) Immunodominance of seven regions of a major allergen, Cry j 2, of Japanese cedar pollen for T-cell immunity. Allergy 51:621–632 Hirahara K, Tatsuta T, Takatori T, Otsuka M, Kirinaka H, Kawaguchi J, Serizawa N, Taniguchi Y, Saito S, Sakaguchi M, Inouye S, Shiraishi A (2001) Preclinical evaluation of an immunotherapeutic peptide comprising 7 T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen allergens. J Allergy Clin Immunol 108:94–100 Holgate S, Polosa R (2008) Treatment strategies for allergy and asthma. Nat Rev Immunol 8:218–230
583 Komiyama N, Sone T, Shimizu K, Morikubo K, Kino K (1994) cDNA cloning and expression of Cry j II, the second major allergen of Japanese cedar pollen. Biochem Biophys Res Commun 201:1021–1028 Larche M (2007) Peptide immunotherapy for allergic diseases. Allergy 62:325–331 Larche M, Akdis CA, Valenta R (2006) Immunological mechanisms of allergen-specific immunotherapy. Nat Rev Immunol 6:761–771 Masuyama K, Chikamatsu K, Ikegawa S, Matsuoka T, Takahashi G, Yamamoto T, Endo S (2009) Analysis of helper T cell responses to Cry j 1-derived peptides in patients with nasal allergy: candidate for peptide-based immunotherapy of Japanese cedar pollinosis. Allergol Int 58:63–70 Mori T, Yokoyama M, Komiyama N, Okano M, Kino K (1999) Purification, identification, and cDNA cloning of Cha o 2, the second major allergen of Japanese cypress pollen. Biochem Biophys Res Commun 263:166–171 Namba M, Kurose M, Torigoe K, Hino K, Taniguchi Y, Fukuda S, Usui M, Kurimoto M (1994) Molecular cloning of the second major allergen, Cry j II, from Japanese cedar pollen. FEBS Lett 353:124–128 Oono N, Ide T, Sakaguchi M, Inouye S, Saito S (2000) Common antigenicity between Japanese cedar (Cryptomeria japonica) pollen and Japanese cypress (Chamaecyparis obtuse) pollen, II. Determination of the cross-reacting T-cell epitope of Cry j 1 and Cha o 1 in mice. Immunology 99:630–634 Qu LQ, Takaiwa F (2004) Tissue specific expression and quantitative potential evaluation of seed storage component gene promoters in transgenic rice. Plant Biotech J 2:113–125 Sone T, Komiyama N, Shimizu K, Kusakabe T, Morikubo K, Kino K (1994) Cloning and sequencing of cDNA coding for Cry j I, a major allergen of Japanese cedar pollen. Biochem Biophys Res Commun 199:619–625 Sone T, Morikubo K, Miyahara M, Komiyama N, Shimizu K, Tsunoo H, Kino K (1998) T cell epitopes in Japanese cedar (Cryptomeria japonica) pollen allergens: choice of major T cell epitopes in Cry j 1 and Cry j 2 toward design of the peptide-based immunotherapeutics for the management of Japanese cedar pollinosis. J Immunol 161:448–457 Sone T, Dairiki K, Morikubo K, Shimizu K, Tsunoo H, Mori T, Kino K (2005) Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level. Clin Exp Allergy 35:664–671 Sone T, Dairiki K, Morikubo K, Shimizu K, Tsunoo H, Mori T, Kino K (2009) Recognition of T cell epitopes unique to Cha o 2, the major allergen in Japanese cypress pollen, in allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen. Allergol Int 58:237–245 Suzuki M, Komiyama N, Ithoh M, Itoh H, Sone T, Kino K, Takagi I, Ohta N (1996) Purification, characterization and molecular cloning of Cha o 1, a major allergen of Chamaecyparis obtuse (Japanese cypress) pollen. Mol Immunol 33:451–460 Tada Y, Utsumi S, Takaiwa F (2003) Foreign gene products can be enhanced by introduction into low storage protein mutants. Plant Biotech J 1:411–422
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584 Takagi H, Hiroi T, Yang L, Tada Y, Yuki Y, Takamura K, Ishimitsu R, Kawauchi H, Kiyono H, Takaiwa F (2005a) A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE response. Proc Natl Acad Sci USA 102:17525–17530 Takagi H, Saito S, Yang L, Nagasaka S, Nishizawa N, Takaiwa F (2005b) Oral immunotherapy against a pollen allergy using seed-based peptide vaccine. Plant Biotech J 3:521–533 Takagi H, Hirose S, Yasuda H, Takaiwa F (2006) Biochemical safety evaluation of transgenic rice seeds expressing T cell epitopes of Japanese cedar pollen allergens. J Agric Food Chem 54:9901–9905 Takagi H, Hiroi T, Hirose S, Yang L, Takaiwa F (2010) Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides. Peptides 31:1421–1425 Takaiwa F (2007) Transgenic rice seed as a nutriceutical delivery system. CAB Rev Perspect Agric Vet Sci Nutr Nat Resour 2:1–9 Takaiwa F (2011) Seed-based oral vaccines as allergen-specific immunotherapies. Hum Vaccine 7:357–366 Takaiwa F (2013) Update on the use of transgenic rice seeds in oral immunotherapy. Immunotherapy 5:301–312 Takaiwa F, Hirose S, Takagi H, Yang L, Wakasa Y (2009) Deposition of a recombinant peptide in ER-derived protein bodies by retention with cysteine-rich prolamins in transgenic rice seed. Planta 229:1147–1158 Tsunematsu M, Yamaji T, Kozutsumi D, Murakami R, Kimura S, Kino K (2007) Effect of Cry-consensus peptide, novel recombinant peptide for immunotherapy of Japanese cedar
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Transgenic Res (2014) 23:573–584 pollinosis, on an experimental allergic thinitis model in B10.S mice. Allergol Int 56:465–472 Valenta R, Ferreira F, Focke-Tejkl N, Linhart B, Niederberger V, Swoboda I, Vrtala S (2010) From allergen genes to allergy vaccines. Ann Rev Immunol 28:211–241 Wakasa Y, Takaiwa F (2013) The use of rice seeds to produce human pharmaceuticals for oral therapy. Biotechnol J 8:1133–1143 Wakasa Y, Yasuda H, Takaiwa F (2006) High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes. Plant Biotech J 4:499–510 Yamanaka K, Yuta A, Kaked M, Sasaki R, Kitagawa H, Gabazza E, Okubo K, Kurokawa I, Mizutani H (2009) Induction of IL-10-producing regulatory T cells with TCP diversity by epitope-specific immunotherapy in pollinosis. J Allergy Clin Immunol 124:842–845 Yang L, Hirose S, Takahashi H, Kawakatsu T, Takaiwa F (2012) Recombinant protein yield in rice seed is enhanced by specific suppression of endogenous seed proteins at the same deposit site. Plant Biotechnol J 10:1035–1045 Yasueda H, Saitou K, Sahashi N (2001) Relationship between pollen counts of Cryptomeria japonica and Cupressaceae and the severity of allergic symptoms. Allergol Int 50:133–142 Yoshitomi T, Hirahara K, Kawaguchi J, Serizawa N, Taniguchi Y, Saito S, Sakaguchi M, Inouye S, Shiraishi A (2002) Three T-cell determinants of Cry j 1 and Cry j2, the major Japanese cedar pollen antigens, retain their immunogenicity and tolerogenicity in a linked peptide. Immunology 107:517–522
