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Rice seed for delivery of vaccines to gut mucosal immune tissues

Rice seed for delivery of vaccines to gut mucosal immune tissues Introduction Plants provide a promising platform for the production of recombinant proteins, offering advantages over conventional fermentation systems that employ bacteria, yeast and mammalian cells in terms of scalability (agricultural scale because no specific facilities are required), safety (no contamination with mammalian pathogens such as viruses and prions) and cost‐effectiveness (Daniell et al ., ; Sharma and Sharma, ; Tiwari et al ., ; Twyman et al ., ). Plant production systems can be divided into those using stable transgenic plants generated by nuclear genome and plastid genome transformation and plant virus‐based or Agrobacterium ‐based transient expression platforms (Paul et al ., ). Each of production systems has advantages or disadvantages. When recombinant proteins are produced by transient expression system, robust yields of recombinant products can be obtained within a few weeks, but they have to be extracted and purified for use because tobacco is mainly used as production host. Plastid‐based expression system also gives rise to high‐level expressions of recombinant proteins without gene silencing and position effect due to the site‐specific homologous recombination and multicopy number of genome. Furthermore, there is little possibility of pollen‐mediated gene contamination due to the maternal transgene inheritance, but there is no post‐translation modification of products such as glycosylation (Daniell et al ., ). On the other hand, seed systems have the advantages of high productivity, stability and oral delivery (Lau and Sun, ; Stoger et al ., ; Takaiwa, ). Indeed, recombinant proteins stored in seeds are highly stable without degradation, even if stored at ambient temperatures for several years. Moreover, proteins can be produced at very high levels when they are expressed under the control of seed‐specific promoters. When orally administered, antigens stored in seed withstand proteolysis in the gastrointestinal tract (Nochi et al ., ; Takagi et al ., ). All approved biopharmaceutical proteins (biologics) produced to date are extracted and purified according to the specific guidelines and regulations of good manufacturing practice (GMP). The downstream extraction and purification steps are generally expensive, representing up to 80% of overall production cost (Kusnadi et al ., ; Wilken and Nkolov, ). Notably, the cost of processing and purification is approximately the same among the various production systems. However, if pharmaceutical proteins are produced in edible plants (crops), oral administration using crude or minimally processed products is feasible, thereby eliminating expensive downstream processing steps. However, one disadvantage of oral administration is that more than 100‐fold higher concentrations of oral antigens are required to achieve the same level of efficacy as with the parentally administered antigens. This disadvantage can be overcome by plant‐based products because the plant cell wall is resistant to harsh conditions and digestive enzymes. Pharmaceuticals produced in seeds are further fortified by two barriers comprising the rigid cell wall and protein bodies as bioencapsulated products, offering a suitable delivery vehicle to gut immune tissues. Oral vaccination has an additional benefit in that it can regulate both mucosal and systemic immune responses. Thus, seed‐based biopharmaceuticals are attractive as vaccines for infectious and allergic diseases. Advantage of rice seed as a production platform of pharmaceutical proteins Rice is a very important staple food that is consumed by nearly half of the global population. Rice seed is an efficient bioreactor for the production of pharmaceutical proteins due to its high biomass yield, low risk of gene flow due to self‐pollination, ease of transformation and convenience of scale‐up (Stoger et al ., ; Twyman et al ., ). Rice grain biomass yield is higher than many other cereals (wheat, barley, and rye) but is lower than maize. However, maize is an outcrossing plant, which can lead to problems of gene flow in open‐field cultivation. Marker‐free transgenic rice was easily obtained using the multi‐auto‐transformation (MAT)‐vector (Endo et al ., ) or Cre‐lox recombination system (Radhakrishnan and Srivastava, ), together with the Agrobacterium‐ mediated co‐transformation system by introducing two T‐DNAs (Komari et al ., ; Sripriya et al ., ). Use of such marker‐free systems may address the public's reluctance to accept transgenic plants and their products. Agricultural infrastructure for the cultivation, harvesting, processing and storage of rice is well established worldwide. In particular, the content of seed proteins in mature rice grains can be easily controlled by the dose of nitrogen fertilizer as nitrogen plays a key role in determining seed protein levels (Nishizawa et al ., ; Souza et al ., ). The complete genome sequence ( http://rgp.dna.affrc.go.jp/E/IRGSP/ ) and considerable genomic information regarding gene expression are available ( http://rapdb.dna.affrc.go.jp/ ). This genetic information can be readily applied to create the desired pharmaceutical products by genetic manipulation. Expression strategies for producing high amounts of recombinant proteins in rice seeds have been established in the last decade. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without purification (downstream processing) due to the absence of toxic compounds such as toxic metabolites or severe food allergens. The endosperm is an ideal bioreactor for the production of pharmaceutical proteins (Arcalis et al ., ; Ou et al ., ; Takaiwa et al ., ). Cereal endosperm is a specialized storage tissue, which makes up about 80%–90% of total seed weight, and in which starch, proteins and lipid are stored as nutrient resources for germinating seedling. About 80%–90% of cereal seed weight comprises starch (carbohydrate), with 6%–15% and 2%–5% being made up by storage proteins and lipid, respectively. This contrasts with the leguminous and oil seeds (soybean, pea, broad bean, rape, sunflower and Arabidopsis), which contain 20%–40% protein, 20%–60% starch and 5%–50% lipid. Seed storage proteins (SSPs) in dicotyledonous seeds are mainly stored in embryo or cotyledons, with the exception of some crops in which it occurs in the endosperm (tobacco or castor bean). Endosperm tissue consists of central starchy endosperm, subaleurone layer (SAL), aleurone layer (AL), basal endosperm transfer layer and embryo‐surrounding region (Figure ). Most SSPs are deposited in subaleurone and starchy endosperm cells. It is important to note that various types of recombinant proteins including artificial or toxic products can be highly and stably accumulated in the endosperm without having a detrimental effect on embryo development, thus providing an ideal production platform (bioreactor) (Peter and Stoger, ; Takaiwa, ). This can be achieved by protein deposition into the specialized storage compartments such as protein bodies (PBs) and protein storage vacuoles (PSVs) in the endosperm. When transgenic rice seed become desiccated during the process of maturation, the endosperm becomes dehydrated and has a reduced level of proteolytic activity, thus providing an ideal environment for storage of recombinant proteins. Rice endosperm tissue. Vertical (a, b) and transverse (c, d) sections of rice maturing seed at 15 days after flowering. AL, aleurone layer; BETL, basal endosperm transfer layer; CSE, central starchy endosperm; EM, embryo; ESR, embryo‐surrounding region; SAL, subaleurone layer. Seed storage proteins and their transport mechanism Seed storage proteins are deposited in specialized membrane‐bound storage organelles called PBs. There are two types of PBs, which differ in biogenesis in rice endosperm cells. One is ER‐derived PBs (PB‐I), with a size of 1–2 μm and a spherical structure, and the other is PSVs (designated as PB‐II), with a size of 2–4 μm and an irregular shaped structure (Krishnan et al ., ; Tanaka et al ., ). In the generation of ER‐derived PBs, hydrophobic prolamins form aggregates within the lumen of rough ER and bud off as a spherical organelle in the cytoplasm. Prolamins are grouped into three groups based on molecular size (10, 13, and 16 kDa), and the 13 kDa prolamin is further divided into two Cys‐rich and two Cys‐poor groups. At least six types of prolamins are highly and tightly packaged into PBs via disulphide bonds. PB formation starts as a core of 10 kDa prolamin, which is followed by synthesis of other prolamins (Nagamine et al ., ). They are arranged and organized in the order of Cys‐rich 10 kDa core, Cys‐poor 13 kDa inner layer, Cys‐rich 16 and 13 kDa middle layer, and Cys‐poor 13 kDa a outer layer in PB‐I (Saito et al ., ). Protein storage vacuoles contain distinct regions called matrix, crystalloid and globoid, which differ in electron density and coincide with the integrated proteins. Rice glutelin and 26 kDa globulin (α‐globulin) are incorporated in the crystalloid region and matrix of PSVs (PB‐II). Glutelin is the major SSP in rice, which accounts for 60%–80% of total seed protein. It shares homology with leguminous 11–12 S globulins such as soybean glycinin and pea legumin (Takaiwa et al ., ). Rice glutelin constitutes a multigene family classified into four groups (GluA, GluB, GluC and GluD) (Kawakatsu and Takaiwa, ). GluA and GluB are further divided into three and five classes respectively. By contrast, α‐globulin is encoded by a single copy and constitutes 5%–10% of total seed protein. Glutelins and α‐globulin are mainly deposited in PSVs by trafficking through the Golgi apparatus to the PSV via dense vesicles (DVs) after synthesis in the ER. In the case of storage proteins deposited in PSVs, Golgi‐dependent and Golgi‐independent pathways are involved in storage protein transport from the ER to the PSVs. Precursor‐accumulating vesicles (PACs) are implicated in the direct PSV pathway bypassing Golgi apparatus (Ibl and Stoger, ). The aggregation of storage proteins within the ER may be related to the direct transport to PSVs by PACs. PACs are observed in rice seed (Takahashi et al ., ). In the case of wheat, barley and oat seeds, their major prolamins (glutenins, hordeins and avenins) are initially deposited into ER‐derived PBs, which are subsequently sequestered to PSVs through the Golgi apparatus or directly via autophagy‐mediated transportation (Galili, ; Herman and Larkins, ; Tosi et al ., ). It is characteristic of rice endosperm cells that ER‐derived PBs and PSVs co‐exist within the same endosperm cell. Sorting mechanisms of prolamin and glutelin have been characterized. The transcripts are transported along the cytoskeleton to specific regions depending on cis‐acting RNA localization signals. Rice prolamin mRNAs are targeted to the PB‐ER that surrounds the PB‐Is, whereas rice glutelin mRNAs are localized in the cisternal ER (Okita and Choi, ). Selective distribution of sorted mRNA transcripts may play a role in efficient transport of prolamins and glutelins into PBs and PSVs. Sorting signals for proteins destined to PSVs have been characterized as N‐ and C‐terminal propeptides and internal regions of mature seed proteins and classified into C‐terminal vacuolar sorting signals (VSS), sequence‐specific VSS and physical structure VSS (Vitale and Hinz, ). However, such signals have not been identified in rice SSPs. Growing evidence indicates that post‐Golgi trafficking of storage proteins to the PSVs requires the retromer components (MAG1/VPS29, VPS35 and SNXs) involved in the recycling of vacuole sorting receptors (VSRs), the Rab family of small GTPases and their common guanine exchange factor (GEF) for specifying vesicular trafficking, and the SNARE complex for mediating membrane fusion between post‐Golgi compartments. Loss‐of‐function mutants of Rab5a, the small GTPase involved in vesicular membrane transport, exhibited Golgi‐derived PB and PSV formation, suggesting that OsRab5a is implicated in the intracellular transport of proglutelin from Golgi to the PSVs (Fukuda et al ., ; Wang et al ., ). Mutants of GEF also had disrupted transport of glutelin and globulin, followed by the formation of large dilated paramural bodies (Fukuda et al ., ). A regulatory complex between the small GTPase Rab5a and its GEF VPS9a was shown to be involved in the transport of proglutelins from the Golgi apparatus to PSVs through regulation of DV‐mediated post‐Golgi trafficking (Ren et al ., ). Transport of rice SSPs from the ER to PSVs depends on coat protein complex II (COPII). Sar 1 GTPase plays an essential role in the formation of COPII vesicles for ER to Golgi traffic. When this COPII‐mediated pathway was inhibited by suppression of OsSar1 expression, abnormal ER‐derived dense PBs containing glutelin precursor were generated by inhibition of SSP transport (Tian et al ., ). Tools to boost expression levels of recombinant proteins in rice seeds The expression levels of foreign recombinant proteins are primary determined by the level of transcription. Thus, the promoter used to drive the expression of the recombinant proteins (transgenes) is the most important element. To obtain expression in a particular tissue during a specific phase of development, several rice seed‐specific promoters were developed (Qu and Takaiwa, ; Qu et al ., ; Wu et al ., ). The advantage of this type of promoter is that it increases protein stability and avoids detrimental effects of accumulation of the recombinant proteins in vegetative plant tissues through expression in specific tissues in the transgenic plant. It is important to select the tissue‐specific promoter to maximize levels of recombinant proteins in the target harvest tissue. The seed storage tissue, endosperm, is a reasonable target because its function is to store nutrient resources for the germinating seedling. Targeting to a suitable subcellular location (organelle) combined with seed‐specific expression leads to high‐yield production of antigens because endosperm‐specific promoters differ in spatial and temporal characteristics. For example, α‐globulin and the glutelin GluD promoter is predominantly expressed in inner starchy endosperm, and most glutelin genes and prolamin genes are highly expressed in the subaleurone layer and outer peripheral region of the endosperm tissue, respectively (Kawakatsu et al ., ; Qu and Takaiwa, ; Wu et al ., ). The 18 kDa oleosin and embryo globulin ( REG2 ) promoters direct expression in the embryo and aleurone layer (Qu and Takaiwa, ). The basal endosperm transfer layer 1 ( BETL1 ) promoter confers specific expression at the basal endosperm transfer layer. In general, considering that seed proteins are mainly deposited in starchy endosperm cells and subaleurone cells in the rice endosperm tissue, recombinant proteins should be expressed in these cells under the control of these tissue‐specific promoters. High levels of expression can be obtained using the promoters for GluB‐1 and GluB‐4 glutelin and 10 and 16 kDa prolamin. The temporal expression pattern of these storage proteins during seed maturation differs. Expression of 10 and 16 kDa prolamin genes starts from 5 days after flowering (DAF), which is followed by expression of most glutelin genes and 13 kDa prolamin genes. Untranslated regions (UTRs) play an important role in the translation efficiency and stability of mRNAs. To stabilize the mRNA transcript, the full‐length 5′ UTR should be included. The flanking sequence around the ATG codon in monocot plants should be optimized as follows: (A/G)(A/C)C AUG GCG), which results in an increased rate of translation (Joshi et al ., ). 3′ UTR regions such as the GluB‐1 3′ UTR, which contains several poly(A) signals (AATAAA) that are involved in transcript (mRNA) stability and accurate termination, have to be considered to ensure high‐level expression of foreign proteins (Yang et al ., ). Codon optimization of sequences encoding recombinant proteins is critical to enhance the protein accumulation because it can affect the rate of protein production and mRNA stability (Gustafsson et al ., ). This is attributed to the presence of rare codons and signal sequences that affect mRNA stability. Rare codons, AU rich sequences and AUUUA repeats that are known to destabilize the transcript and splicing junction sequence should be modified without changing the amino acid sequence. It should be noted that codon usage (G+C content) of genes expressed in seeds differs from that in leaves (high GC rich), even in the same rice genes; that is, codon usage can differ among tissues. Codons in the target gene expressed in the rice endosperm should be optimized using codons frequently used in rice endosperm genes (Wakasa and Takaiwa, ). Because accumulation level, stability and post‐translational modification of recombinant proteins are largely dependent on intracellular localization, it is important to target protein expression to suitable locations by means of intracellular targeting signals (Hofbauer and Stoger, ; Khan et al ., ). In the case of seeds, a signal peptide leading to the secretory pathway is mandatory for stable accumulation of product. Generally, many pharmaceutical proteins are post‐translationally modified by glycosylation and, thus, may have to be synthesized as secretory proteins using an endomembrane system. Plant or native signal peptides should be ligated to the N‐terminus of the mature recombinant protein. Targeting signals leading to the desired subcellular compartments have been characterized in plant cells. For example, when the transit peptide is ligated to the N‐terminus, the recombinant protein is trafficked to the amyloplast (starch granule) in seed endosperm cells. Ligation of a KDEL (Lys‐Asp‐Glu‐Leu) sequence at the C‐terminus leads to ER retention, which acts as an ER retrieval signal. It has been reported using this strategy that recombinant protein yield was enhanced by 2‐ to 10‐fold in many tissues of a variety of plants (Conrad and Fiedler, ; Takagi et al ., ). Retaining the desired protein in the ER is a reasonable strategy because proteases are limited and sufficient amounts of chaperones for folding and assembling are present, providing a good environment for recombinant protein production (Oono et al ., ; Wakasa et al ., ). It is important to consider that the destination of recombinant proteins expressed as secretory proteins through an endomembrane system may be determined by interactions with the endogenous seed proteins or by its intrinsic physical properties. In particular, recombinant proteins with free cysteine residues interact with endogenous cysteine‐rich prolamins via disulphide bonds in ER lumen and are incorporated into accumulated in PBs (Takaiwa et al ., ). Such interaction sometimes prevents the proper interaction among the endogenous storage proteins, resulting in disruption of the ordered deposition of seed proteins and generation of aberrant PBs. Glycosylation patterns are also highly affected by subcellular localization (Arcalis et al ., ). Recombinant proteins are glycosylated along the secretory pathway as they move from the ER through Golgi to their final destination (Gomord et al ., ). Glycosylation enhances the physicochemical properties of a protein by promoting thermal resistance, protecting from proteolytic degradation and enhancing stability. Plants attach β1,2 xylose and α1,3 fucose residues to the site (Asn‐X‐Ser/Thr) of proteins as post‐translational N‐glycosylation, whereas mammals attach α1,6 fucose, β1,4 galactose and sialic acid residues (Gomord and Faye, ). Plant‐specific glycans are sometimes immunogenic (Garcia‐Casado et al ., ), and glycoengineering strategies were developed to avoid the addition of plant‐specific N glycans and to add human‐like glycans. Plant‐specific glycosyltransferases have been deleted or mutagenized by homologous recombination in rice (Ozawa et al ., ), while protein sialylation has been achieved by introducing the entire mammalian pathway for sialic acid synthesis in tobacco (Castilho et al ., ). Unfolded protein response is a bottleneck for high production of recombinant proteins Abundant accumulation of seed proteins is achieved through efficient packaging in ER‐localized PBs with the aid of chaperones and folding enzymes within the ER lumen. The folding and assembly of newly synthesized proteins is a complex process assessed by protein quality control mechanisms that involve many chaperones and folding enzymes. For deposition of recombinant protein into PBs, an array of chaperones, co‐chaperones, oxidoreductases, glucan chain modifying enzymes and lectins is involved in folding and assembly in a dynamic action, resulting in maintenance of ER homeostasis by the unfolded protein response (Schroder, ). Molecular chaperones and folding enzymes such as binding proteins (BiPs), protein disulphide isomerase (PDI) and calnexin are implicated in depositing high amounts of SSPs in PBs. For example, PDIL1‐1 is mainly localized on the dilated ER in rice endosperm cells. Mutation of this PDIL1‐1 (esp2) results in the formulation of abnormal PBs containing both proglutelins and prolamins in ER due to incorrect disulphide formation (Onda et al ., ; Takemoto et al ., ). On the other hand, PDIL2‐3 is exclusively localized on the surface of PB‐I. Knockdown of PDIL2‐3 inhibits the accumulation of Cys‐rich 10 kDa prolamin in the core (Onda et al ., ) of PB‐I, suggesting that PDIL2‐3 is involved in disulphide bond formation among prolamins in PB‐I. Extreme suppression or overexpression of major BiP1 in an endosperm‐specific manner had severe suppressive effects on the production of seed proteins accompanied by aberrant structural formation of PBs, whereas slight enhancement of BiP1 expression led to increased total storage protein levels (Wakasa et al ., ; Yasuda et al ., ). By contrast, major PDIL1‐1 had no apparent effect on accumulation of seed proteins, even when overexpressed in endosperm (Yasuda et al ., ). When foreign recombinant proteins accumulate at high levels in ER lumen as secretory proteins, ER stress is sometimes induced as the unfolded protein response (UPR) as a result of imbalance between the load of unfolded proteins and the ER folding capacity. Production levels of secretary proteins may be highly affected by the quality control machinery of ER. In plants, the UPR is mediated by at least two ER transmembrane sensors: activating transcription factor 6 (ATF6) homologues and inositol‐requiring enzyme 1 (Ire1) (Hetz, ; Howell, ; Iwata and Koizumi, ). In contrast to yeast and mammals, in plants there is no obvious homologue to PERK, which is implicated in attenuating general protein synthesis by the phosphorylation of the translation initiation factor eIF2a (Kamauchi et al ., ). ATF6 is a type II transmembrane transcription factor that senses ER stress via the C‐terminal luminal domain. Upon ER stress, rice orthologues of ATF6, OsbZIP39 and OsbZIP60 dissociate from BiP and are transported to the Golgi apparatus via specific trafficking with the COPII vesicles for proteolytic processing by serine proteases S1P and S2P (Takahashi et al ., ). The liberated N‐terminal cytoplasmic domain is transported into nuclei to activate UPR target genes. Inositol‐requiring enzyme 1 is a type I ER‐resident transmembrane protein with an ER luminal dimerization and a cytoplasmic domain with Ser/Thr kinase and endonuclease activities (Hetz et al ., ). ER stress allows IRE1 to autophosphorylate the kinase domain through dimerization and thereby activate the ribonuclease domain. The activated IRE1 mediates an unconventional cytoplasmic splicing of mRNA encoding transcription factor OsbZIP50 (rice orthologue of mammalian XPB‐I and yeast Hac I), leading to removal of the transmembrane domain as a result of frame shifting (Hayashi et al ., ). The activated OsbZIP50 is involved in up‐regulating the expression of ER quality control and ERAD‐related genes (various ER stress‐related genes). Ire1 RNase activity is also implicated in an mRNA degradation mechanism termed regulated IRE1‐dependent decay. This mechanism involves selective degradation of a subset of mRNAs coding for certain ER‐located proteins. Deletion of OsIRE1 RNase activity by site‐specific mutagenesis through homologous recombination resulted in relief of suppression of mRNA levels of secretory and membrane proteins independent of the OsbZIP50 pathway, whereas a lack of kinase activity induced by mutagenesis of the Ire1 kinase domain led to lethality in transgenic rice, indicating an essential function in cell fate (Wakasa et al ., ). Transcription of UPR genes is activated by binding of UPR transcription factors to the cis elements in their promoter. Three cis elements, URR element (UUPRE:TGACG‐GT), ER stress element‐I (ERSE‐I:CCAAT‐N9‐CCACG) and ERSE‐II (ATTGG‐N‐CCACG) were implicated in the ER stress response in mammalian cells (Kokame et al ., ; Wang et al ., ; Yoshida et al ., ). The consensus sequence of plant‐UPR element (P‐UPRE: ATTGGTCCACGTCATC) consisting of mammalian ERSE‐II and a UPRE element is conserved in plant‐UPR genes that are recognized by plant‐UPR transcription factors (Oh et al ., ). pUPRE‐II (5′‐GATGACGCGTAC‐3′) and pUPRE‐III (TCATCG) are also cis elements required for ER stress response‐related genes in plants (Hayashi et al ., ; Sun et al ., ). pUPRE‐II was reported to be bound by activated OsbZIP50 and OsbZIP60 and was involved in activation of UPR genes such as PDIL 2‐3 (Takahashi et al ., ). Expression of BiP4 and BiP5 is specifically induced under ER stress conditions, whereas BiP1, a major BiP protein, is constitutively expressed in various tissues and is up‐regulated in response to ER stress. Therefore, these BiPs can be used as ER stress markers (Wakasa et al ., ). Activation of the ER stress response is mainly mediated by the OsIRE1‐OsbZIP50 signal cascade. When proteins fail to fold into their native conformation, ER‐associated degradation (ERAD) machinery responsible for elimination of misfolded proteins is induced for protein quality control. The proteins are retrograde transported across the ER membrane back into the cytoplasm, where they are rapidly degraded by the ubiquitin‐proteasome system, in which they are degraded by 26S proteasome after marking by ubiquitination. The protein quality control system in the ER lumen plays a critical role as a checkpoint to determine whether expressed recombinant protein should be accumulated or degraded in plant cells. Enhancement of folding and assembly activities for various secretory proteins in ER lumen may lead to higher levels of accumulation of the desired recombinant protein in plant cells. Further study to understand the molecular mechanisms involved in the UPR will be required. Design of PBs as deposition space for high accumulation of recombinant proteins Suppression of some SSPs leads to higher accumulation of other types of SSPs by a proteome rebalancing mechanism that maintains the homeostasis of protein (nitrogen) levels in matured seed. Expression of recombinant protein in seed of low SSP mutants such as low glutelin lines LGC‐1, a123 resulted in about two‐fold higher yield than that of normal rice (Tada et al ., ). Moreover, reduction in some SSPs by silencing resulted in up‐regulation of other SSPs and foreign recombinant protein (Kawakatsu et al ., ; Shigemitsu et al ., ; Yuki et al., 2012; Yuki et al ., ). This mechanism is also conserved in other cereal and leguminous seeds (Goossens et al ., ; Schmidt and Herman, ; Wu and Messing, ). Thus, high‐level production of foreign recombinant proteins in transgenic seeds is expected to be obtained by suppression of endogenous SSPs. When expression of endogenous SSPs was suppressed by RNA interference technology to supply the deposition space for the desired recombinant protein, production yield of the desired protein was remarkably increased as a compensatory mechanism (Yang et al ., ). However, homeostasis of seed protein level by proteome rebalancing is not simple, as suppression of endogenous SSPs when using different subcellular compartments as a deposition site did not confer significant enhancement. This result suggests that relief of loading competition with the endogenous SSPs for deposition space leads to a high level of accumulation as a result of proteome rebalancing; that is, supply of vacant deposition space (PBs) for the desired recombinant protein through suppression of distinct SSPs is an ideal strategy for increasing the accumulation levels of recombinant proteins in seeds (Takaiwa, ). Production strategy for rice seed‐based vaccines When the recombinant proteins are produced as secretory proteins by ligating the signal peptide and the KDEL ER retention signal to the N‐ and C‐termini, they are deposited into various types of ER‐derived bodies, including PBs, that exhibit varying morphology based on their inherent physical properties (aggregation ability) and interaction with endogenous seed proteins through disulphide bonds. These differing types of PBs exhibited different resistance to digestive enzymes when assayed in vitro , which was attributed to the differing physical properties of the PBs in which the recombinant proteins were incorporated. On the other hand, some recombinant proteins failed to accumulate. In these cases, the proteins can be produced by expressing as parts of SSPs in a form of fusion protein. When recombinant proteins are fused with the C‐terminus of prolamins or inserted into the highly variable region of glutelins (C‐terminus of acidic subunit of glutelin) and then expressed under the control of endosperm‐specific promoters, fusion proteins accumulated as parts of native storage proteins are naturally deposited in PBs or PSVs. Taking advantage of these strategies, high amounts of recombinant proteins can be accumulated in PBs or PSVs in transgenic rice seed (Wakasa and Takaiwa, ). When digestibility and immunogenic activity (tolerance‐inducing capacity) were compared between ER‐derived PB (PB‐I)‐ and PSV (PB‐II)‐containing antigens, PB‐I localizing antigen exhibited greater immunogenicity than the PB‐II antigen (Takagi et al ., ). This difference is associated with the difference in resistance to gastrointestinal digestion enzymes, with proteins deposited in ER‐derived PB (PB‐I) being more resistant to digestive enzymes than those deposited in PSV (PB‐II). This physical difference may be related to the polymerized or aggregated formulation via Cys‐rich prolamins in ER‐derived PBs. Structure of GALT and immune reaction in the gastrointestinal tract The gastrointestinal tract containing GALT is a front line of defence against mucosal pathogens causing infectious diseases, but it is also involved in immune tolerance against foods and commensal bacteria. Homeostatic conditions in the gastrointestinal tract, with a balance between tolerance and sensitization, is maintained by the stringent regulatory mechanisms. The mucosal immune system consists of inductive and effector sites. In the gastrointestinal immune system, the Peyer's patches (PPs), isolated lymphoid follicle tissue (ILF) and the mesenteric lymph nodes (MLNs) act as inductive sites, while the lamina propria (LP) and intra‐epithelial lymphocytes (IEL) of the mucosal membrane constitute effective sites. Each PP is composed of B cell‐rich follicles (FO) surrounded by interfollicular regions (IFR) consisting of T cells (Figure ). Schematic representation of GALT. FAE, follicle‐associated epithelium; FO, follicle; GALT, gut‐associated lymphoid tissue; GC, germinal centre; IEL, intra‐epithelial lymphocyte; IFR, intrafollicular region; LP, lamina propria; M cell, microfold cell; MLN, mesenteric lymph node; SED, subepithelium domain; PP, Peyer's patch; B, B cell; T, T cell; P, plasma cell. Microfold cells (M cells) are specialized epithelial cells that predominantly reside in the follicle‐associated epithelium (FAE) overlying PPs. M cells in PPs have the ability to take up and transcytose antigens to antigen‐presenting cells (APCs). GALT contains an organized macro‐architecture of B and T lymphocyte zones that respond to the antigens presented by dendritic cells (DCs). DCs act as sentinels, taking up antigens and then migrating to the subepithelial dome at PPs or to the MLN to activate native T cells. Intestinal DCs are involved in the decision of tolerance vs. inflammation (Kunisawa et al ., ; Neutra and Kozlowski, ; Tsuji and Kosaka, ). When antigens are orally administered, the properties of the administered antigens can determine which type of immune reaction is induced (immune tolerance vs. immune induction). Protective immune responses are induced against potential pathogens. The local production and secretion of dimeric secretory immunoglobulin A (sIgA), which has function to neutralize pathogens, is characteristic of the mucosal adaptive immune response. The default responses to most nontoxic antigens such as dietary antigens are caused by mucosal immune tolerance that is favoured in intestinal immune tissue (Pabst and Mowat, ). The mucosal immune response in GALT is affected by the duration, dose and formulation of antigen (Chehade and Mayer, ; Mayer and Shao, ). High‐dose administration of antigen leads to deletion (apoptosis) or anergy of antigen‐specific T cells. On the other hand, repeated administration of low‐dose antigen over a long duration leads to immune suppression called ‘active suppression’, which is mediated by regulatory T cells (Dubois et al ., ; Weiner et al ., ). Immune tolerance is important in preventing allergic and autoimmune diseases, while it may pose an obstacle in the development of oral vaccines against pathogens. Uptake of antigens deposited in PBs Recognition and subsequent uptake of antigen is an essential function of the mucosal immune system. When vaccine antigens are delivered as a particle formulation smaller than 10 μm, such as PBs, to intestine immune tissues, these antigens are taken up by M cells of GALT. The APCs process the antigen and migrate within the PP to the T‐cell area and/or B‐cell follicles. Some PBs containing antigens may be also directly taken up by CX3CR1 + macrophages called ‘intra‐epithelial DCs’ in the LP, which extend their dendrites into the gut lumen (Pabst and Mowat, ). These latter DCs are a nonmigratory, gut‐resident population. The antigen taken up by intra‐epithelial DCs is passed to the neighbouring CD103 + DCs in the LP, which then migrate to the mesenteric lymph nodes (MLNs) for presentation to naïve T cells. It is important to examine whether ER‐derived PBs containing antigens are actually delivered to lymphoid tissues of GALT when orally administered to mice. It was demonstrated that BPs containing the antigen (shuffle Cry j 2)/GFP reporter hybrid were taken up in the gut (Wakasa et al ., ). Notably, fluorescence elicited from the GFP reporter could be detected in GALT tissue when rice seed powder or isolated crude PBs expressing the reporter were intragastrically administered. GFP fluorescence signals were detected at 8 h in both ileum and jejunum tissues of the small intestine and reached a peak at 12 h, disappearing after 18 h (Figure a–f). By contrast, no signal was detected in these tissues in mice fed nontransgenic rice. Furthermore, incorporation of PBs containing antigen was confirmed by immunoblotting analysis, in which antigen of the correct molecular weight could be detected in proteins extracted from peripheral gut tissues of mouse fed transgenic rice seed powder and prepared PBs (Figure g). These findings suggest that PBs may be taken up in intact or partial degraded form via M cells in the FAE and by DCs in PP. In a similar way, incorporation of PBs into PPs via M cells was shown by immune‐histochemical analysis with Ulex europaeus agglutinin (UEA‐1), which is a well‐known marker of murine M cells, when transgenic rice containing cholera toxin subunit B (CTB) that was deposited in PBs and PSVs, MucoRice‐CTB, was orally administered to mice (Nochi et al ., ). The CTBs deposited in PBs were taken up by UEA‐1‐positive M cells, but not columnar epithelial cells. Uptake of antigens by the intestinal tract. Balb/c mice were intragastrically administered with wild‐type rice seed powder, transgenic rice seed powder (GFP‐fused shuffled Cry j 2), wild‐type concentrated PB product or transgenic concentrated PB (deconstructed Cry j). (a) Ileum tissues before oral administration of rice seed powder expressing GFP‐fused shuffled Cry j 2. (b–d) Ileum tissue at 4, 8 and 12 h after oral administration of rice seed powder expressing GFP‐fused antigen. (e) Ileum tissue at 12 h after oral administration of nontransgenic rice. (f) Jejunum tissues at 12 h after oral administration of rice seed powder expressing GFP‐fused antigen. (g) Total proteins were extracted from ileum tissue at 2, 4, 6, 8, 13, 18 and 30 h after oral administration of transgenic concentrated PB product. F1 fragment derived from Cry j 1 was detected by immuneblotting using anti‐F1 antibody. (a–f) Adapted from Wakasa et al . ( ). Development of rice‐based oral vaccines as tools of immunotherapy Rice‐based oral vaccines have been developed for various allergic and infectious diseases (Azegami et al ., ; Takaiwa, ). Mucosal vaccines administered orally were shown to effectively induce antigen‐specific immune responses in both systemic and mucosal compartments (Brandtzaeg, ; Holmgren and Czerkinsky, ; Kwon et al ., ). For infectious disease, it has been shown that oral administration induced antigen‐specific IgA and IgG, which act as neutralizing antibodies and are involved in protection from invading pathogens (Marson et al ., ; Nochi et al ., ; Tokuhara et al ., ). A variety of vaccine antigens were expressed in rice seeds, including CTB (Nochi et al ., ; Yuki et al ., ), cholera toxin (Yuki et al ., ), infectious bursal disease virus immunogen VP2 (Wu et al ., ), hepatitis B virus surface antigen gene SS1 (Qian et al ., ), roundworm 14 and 16 kDa antigens (Matsumoto et al ., ; Nozoye et al ., ), surface glycoprotein F of Newcastle disease (Yang et al ., ), and amyloid β peptide (Nojima et al ., ; Oono et al ., ). MucoRice containing CTB has been extensively developed and is discussed in further detail below (Table ). Transgenic rice seeds as an edible vaccine for oral immunotherapy against various infectious, allergic and autoimmune diseases Target disease or pathogens Target antigen Promoter Expression level Localization References Vibrio cholerae Cholera toxin B subunit Glutelin GluB‐1 30 μg/grain PB‐I, PB‐II Nochi et al . ( ) Vibrio cholerae Modified cholera toxin B subunit (CTB/Q) 13 kDa prolamin 2.35 mg/g of seed Cell wall, cytoplasm Yuki et al . ( ) Vibrio cholerae Cholera toxin A and B subunits Glutelin GluB‐1 , GluB‐4 5 μg/grain NA Yuki et al . ( ) Infectious bursal disease virus VP2 of IBDV Glutelin Gt‐1 4.5% of total seed protein NA Wu et al . ( ) Newcasle disease virus Fusion(F) protein of NDV Glutelin Gt‐1 2.5–5.5 μg/g of seed NA Yang et al . ( ) Hepatitis B virus HBv surface protein and presurface1 Glutelin GluB‐4 31.5 ng/g of seed NA Qian et al . ( ) Arcaris suum 16 kDa protein of A. suum (As16) Glutelin GluB‐1 50 μg/grain NA Matsumoto et al . ( ) Arcaris suum 14 kDa protein of A. suum (As14) Glutelin GluB‐1 1.5 μg/grain NA Nozoye et al . ( ) Clostridium botulinum Heavy chain of botulinum type A neurotoxin 13 kDa prolamin 100 μg/grain Cytoplasm Yuki et al . ( ) Alzheimer's disease 2 × Amyloid β peptide (1–42) Glutelin GluB‐1 8 μg/grain PB‐I Oono et al . ( ) Alzheimer's disease Amyloid β peptide fused to GFP CaMV35S 400 μg/g of seed NA Nojima et al . ( ) Cedar pollen allergy T cell epitope, 7Crp Glutelin GluB‐1 60 μg/grain PB‐I Takagi et al . ( b ) Cedar pollen allergy T cell epitope, Crp3 Glutelin GluB‐1 80 μg/grain PB‐I Takaiwa and Yang ( ) Cedar pollen allergy Glutelin/Cry j 1 (3 fragment) shuffle Cry j 2 GluB‐1,GluB‐4 10 k, 16k prolamin 5–20 μg/grain PB‐I Wakasa et al . ( ) Crypsis pollen allergy T cell epitope, 6Chao Glutelin GluB‐4 12 μg/grain PB‐I Takaiwa and Yang ( ) Birch pollen allery Mutagenized Bet v 1, TPC7, TPC9 Glutelin GluB‐1 76–550 μg/grain TPC7 body Wang et al . ( ), Ogo et al . ( ) House dust mite allergy Der p 1fragment, P1(45–145) Glutelin GluB‐1 90 μg/grain PB‐I Suzuki et al . ( ) House dust mite allergy Cys mutagenized Der f 2 Glutelin GluB‐1 15–30 μg/grain Der f 2 body Yang et al . ( b ) Rheumatoid artheritis Type 2 collagen analogue glutelin/3 × APL6, APL7 CII256‐271 Glutelin GluB‐1 7–24.6 mg/g of seed PB‐II Iizuka et al . ( , b ) NA, not analyzed. CTB Cholera ( Vibrio cholerae ) affects the small intestine through its secreted cholera toxin (CT), which is composed of five receptor‐binding B subunits (CTB) surrounding a catalytic A subunit (Holmgren, ). CTB is potent mucosal immunogen and adjuvant. CTB as a transmucosal carrier offers an efficient oral delivery approach via specific receptor‐binding process which is achieved through a plasma membrane receptor GM1 ganglioside. Pentamer formation of the CTB fusion is critical for efficient oral delivery. The codon‐optimized CTB was expressed as secretory protein by ligating the signal peptide and KDEL ER retention signal at its N‐ and C‐termini under the control of the glutelin GluB‐1 promoter in transgenic rice seed. CTB was deposited in PBs at about 30 μg/grain (Nochi et al ., ). MucoRice‐CTB is not only stable at room temperature for several years at room temperature without immunogenicity, but also is protected from digestive enzymes. In a mouse study, MucoRice‐CTB was administered orally to animals, and specific immune responses and neutralizing activity in both systemic and mucosal compartments were detected. Interestingly, mice immunized with MucoRice‐CTB were protected from oral challenge with cholera toxin. In a similar study conducted in a non‐human primate model, cynomolgus macaques received orally administered MucoRice‐CTB (Nochi et al ., ). Animals had CT‐specific, neutralizing antibodies and high levels of systemic IgG and intestinal IgA antibodies. Importantly, cold chain‐free oral MucoRice‐CTB induced long‐lasting cross‐protective immunity against heat‐labile enterotoxin‐producing enterotoxigenic Escherichia coli in addition to CT‐producing Vibrio cholera (Tokuhara et al ., ). These results demonstrate that oral administration of a rice‐based vaccine provides a potent practical global strategy for the development of cold chain‐ and needle‐free vaccines that protect from gastrointestinal infection. Rice‐based oral vaccines for allergic and autoimmune diseases Administration of antigens by the oral route is prone to induce immune tolerance. Taking advantage of this property, several types of rice‐based vaccines against pollen and mite allergens and self‐components were developed. More than 30% of people in industrialized countries are afflicted with some kind of IgE‐mediated type I allergic disease (allergic rhinitis, asthma, food allergy, allergic conjunctivitis and venom allergy). Current treatments for allergic diseases include pharmacotherapy and allergen‐specific immunotherapy. The major treatment for allergic diseases is pharmacotherapy, which targets the release of chemical mediators and block specific receptors (Holgte and Polosa, ). This treatment can reduce the clinical symptoms but is not curative. Allergen‐specific immunotherapy is the only curative treatment that has a long‐lasting effect. It induces immune tolerance against the causative allergens by subcutaneous injections of increasing doses of natural crude allergens over a period of 3–5 years (Frew, ; Larche et al ., ). This treatment is accompanied by pain and sometimes side effects (anaphylactic shock). To overcome poor patient compliance, it is necessary to improve the safety of tolerogen therapy to enhance efficacy. In this review, we introduce four rice‐based allergy vaccines for Japanese cedar pollen allergy, birch pollen allergy, mite allergy and autoimmune disease. Japanese cedar pollinosis is the predominant seasonal allergic disease in Japan, afflicting over 26% of the Japanese population. Allergen immunotherapy using rice‐based allergy vaccines containing hypo‐allergens Genetic engineering allows the production of innovative allergy vaccines designed to reduce side effects and to increase clinical efficacy and treatment compliance. To reduce IgE reactivity (allergenicity), tertiary structure needs to be modified to reduce recognition by specific IgE. Destruction of allergen conformation by fragmentation, shuffling of the molecule, oligomerization or site‐specific mutagenesis of cysteine residues involved in disulphide bonds (cleavage of disulphide bonds) leads to drastic reduction in IgE binding activity (Linhart and Valenta, ; Valenta et al ., ). Allergens are presented to specific T cells on the surface of APCs as a ‘T‐cell epitope’ together with MHC class II. The T‐cell epitope is about ten amino acids in length and is generated after processing within the APC. Thus, a T‐cell epitope peptide is expected to act as an ideal immunogen lacking allergenicity. T‐cell epitope peptides used as tolerogens to induce immune tolerance can provide a safe treatment due to a lack of B‐cell epitopes for binding of specific IgE (Ali and Larche, ; Larche, ). Allergen‐specific immunotherapy (SIT) approaches using T‐cell epitope peptides and hypo‐allergens (modified allergens) are considered as the next generation of allergen‐specific immunotherapy. The goal of allergen modification (T‐cell epitopes and hypo‐allergens) is to minimize allergenicity (risk of anaphylaxis) while retaining immunogenicity (T‐cell reactivity), thus allowing high doses of antigen to be administered over a short time course. Furthermore, recombinant variants lacking allergenicity are ideal candidates for prophylactic vaccination due to the very low risk of sensitization to naturally occurring allergens. T‐cell epitope of Japanese cedar pollen allergens Clinical application of peptide immunotherapy using major T‐cell epitopes was achieved for cat, grass and house dust mite (HDM) allergy (Larche, ). Efficacy was observed in a much shorter time frame without side effect. To evaluate T‐cell epitope peptide accumulation in rice seed, mouse major T‐cell epitope peptides derived from major Japanese cedar pollen allergens Cry j 1 and Cry j 2 were inserted into highly variable regions of soya bean storage protein glycinin A1aB1b. The modified soya bean glycinin containing the two T‐cell epitope peptides was deposited in PB‐II of transgenic rice endosperm, with expression directed by the GluB‐1 promoter. Oral administration of transgenic rice seeds to cedar pollen‐immunized mice inhibited allergen‐specific IgE and Th‐2‐type cytokine responses. Furthermore, allergen‐induced serum histamine and sneezing responses were suppressed (Takagi et al ., ). Because of MHCII polymorphism in the population of allergic patients, combinations of several major T‐cell epitopes (hybrid peptides) restricted by different MHCII molecules have to be prepared. A hybrid peptide called ‘7Crp peptide’ consisting of seven T‐cell epitopes derived from the Cry j 1 and Cry j 2 allergens was created, which was predicted to be recognized by 92% of allergic patients (Hirahara et al ., ). When the 7Crp peptide was expressed in transgenic rice seed by ligating it with an N‐terminal signal peptide and a C‐terminal KDEL signal under the control of the glutelin GluB‐1 promoter, 7Crp was deposited predominantly into PB‐Is (Figure b) due to interaction with Cys‐rich prolamins (Takagi et al ., ; Takaiwa et al ., ). When orally fed to the allergic model mice, a reduction in allergen‐specific CD4 + T‐cell proliferation and the level of allergen‐specific IgE was observed compared with mice fed nontransgenic rice (Takagi et al ., ). Some patients are not expected to respond to the 7Crp peptide and, therefore, a more universal peptide vaccine, Crp3, with seven additional T‐cell epitopes was co‐expressed together with the 7Crp peptide in transgenic rice seed (Takaiwa and Yang, ). Electron microscopy of developing rice endosperm containing various antigens. Recombinant proteins are deposited as unique ER‐derived PBs depending on the physical properties of the proteins accumulated in the rice endosperm cells. Intracellular localization of recombinant proteins in developing rice endosperm cells was analysed by immunoelectron microscopy. (a) Nontransgenic wild‐type rice; (b) 7Crp: seven linked T‐cell epitope peptides derived from Japanese cedar pollen allergens Cry j 1 and Cry j 2; (c) deconstructed whole Cry j 1 and Cry j 2 by fragmentation and shuffling; (d) Der p 1 (45–145) fragment comprising amino acid positions 45–145 of group 1 house dust mite allergen Der p 1; (e) Der f 2 (‐Cys): full‐length sequence of the mutagenized group 2 house dust mite allergen Der f 2, in which all six cysteine residues were changed to serine residues; (f) Der f 2 (C8/119S): full‐length of the Der f 2, in which cysteine residues at positions 8 and 119 were changed to serine residues; (g) Der f 2 (8–119): full‐length of the Der f 2, in which cysteine residues at positions 21, 27, 73 and 78 were changed to serine residues; and (h) TPC7: birch major pollen allergen Bet v1 mutagenized by DNA shuffling. Scale bar represents 1 μm. PB: protein body. Starch: starch body. Figure modified from Takaiwa ( ). Deconstructed Cry j Cry j 1 was divided into three overlapping fragments to disrupt its tertiary structure. These fragments were inserted into the highly variable regions of the C‐terminus in the acidic subunit of GluA, GluB and GluC glutelin precursors (Wakasa et al ., ). By contrast, the tertiary structure of Cry j 2 was reconstructed in a form of tail‐to‐top inverse orientation by shuffling. Low allergenicity of these disrupted allergens was confirmed by dot‐blot IgE binding assay and the RBL‐2H3 basophil degranulation test. Four constructs (three Cry j 1 fragment/glutelin fusions and shuffled Cry j 2) were expressed under four different endosperm‐specific promoters in transgenic rice seed (Wakasa et al ., ). The expressed proteins accumulated in the endosperm at levels of 10–25 μg per grain and were specifically deposited in PB‐Is (Figure c). Oral administration of these transgenic rice seeds to cedar pollen‐immunized mice inhibited allergen‐induced IgE and IgG responses, Th2‐type cytokine synthesis and CD4 + T‐cell proliferation. In addition, the allergen‐induced sneezing response, serum histamine elevation and infiltration of eosinophils and total inflammatory cells in the nose and conjunctiva were attenuated (Fukuda et al ., ; Wakasa et al ., ). Der p 1 Approximately 45%–80% of patients with allergic asthma are sensitized to allergens from HDM of the genus Dermatophagoides , suggesting that HDM allergens are crucial for the development of bronchial asthma. The major HDM allergens are classified into group 1 (Der f 1 and Der p 1) and group 2 (Der f 2 and Der p 2). Group 1 allergens are heat‐labile acidic glycoproteins and are found in HDM faeces. These proteins have papain‐like cysteine protease activity. Most of the predominant Der p 1 T‐cell epitopes for both human and mouse T cells are located within the sequence p45–145 of the mature Der p 1 protein. The Der p 1 fragment was expressed as a secretory protein by fusing the glutelin signal and KDEL retention signal peptides under the control of the GluB‐1 endosperm promoter (Suzuki et al ., ). Using this system, the Der p 1 was mainly deposited in ER‐derived PB‐1 (Figure d). The therapeutic potential of transgenic rice containing this fragment was examined by oral administration in a murine model of asthma. Suppression of airway inflammation and reduction in serum antigen‐specific IgE level were observed in mice given the transgenic rice (Suzuki et al ., ). The production of Th2‐type cytokines and Der p 1‐specific CD4 + T‐cell proliferation were significantly reduced after oral vaccination with the transgenic rice. Allergen‐induced infiltration of eosinophils and neutrophils into the airways and bronchial hyper‐responsiveness were also inhibited by oral vaccination in this model. Der f 2 Group 2 HDM allergen is found in mite faeces. Der f 2 contains three disulphide bonds (Cys8–Cys119, Cys21–Cys27 and Cys73–Cys78), two of which (Cys8–Cys119 and Cys73–Cys78) are critical for its IgE binding capacity (Takai et al ., ). T‐cell epitopes of the group 2 allergens are distributed over the entire protein. Three Der f 2 derivatives were generated by mutagenizing cysteine residues: ΔC lacked all three disulphide bonds, C8/119S lacked the Cys8–Cys119 bond, and 8‐119C lacked the Cys21–Cys27 and Cys73–Cys78 bonds. Binding activity with HDM‐specific IgE was markedly decreased in ΔC, followed by C8/119S and 8‐119C. Three lines of transgenic rice expressing these Der f 2 derivatives were established. Der f 2 derivatives aggregated and formed a PB‐like structure, named Der f 2 body, which were distinguishable from PB‐I and PB‐II by their electron density and morphology (Figure e–g). Oral administration of Der f 2 transgenic rice containing C8/119S, 8‐119C or a mixture of both to Der f 2‐immunized mice inhibited allergen‐induced IgE and IgG responses (Yang et al ., ), suggesting the potential of this transgenic rice to treat HDM‐mediated allergic responses. Interestingly, IgE and IgG responses were not affected by Der f 2 transgenic rice containing ΔC. ΔC was water soluble and rapidly degraded by digestive enzymes in comparison with the other Der f 2 derivatives (Yang et al., ). These data strongly suggest that the digestibility of allergen is critical for the efficacy of oral IT using transgenic rice. Bet v 1 Bet v 1 is a major birch pollen allergen that belongs to the PR 10 protein group. More than 90% of birch pollen allergy patients are sensitive to Bet v1‐specific IgE. A versatile hypo‐allergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen‐specific immunotherapy. Hypo‐allergenic Bet v 1 tolerogens against birch pollen allergy were selected by DNA shuffling of 14 types of Fagales tree pollen allergens (Wallner et al ., ). The resulting chimeric molecules, TPC7 and TPC9, displayed low allergenicity and high immunogenicity compared with the native Bet v 1. Hypo‐allergens TPC7 and TPC9 and native Bet v 1 were expressed in transgenic rice seed as secretory proteins using the N‐terminal signal peptide and the C‐terminal KDEL tag under the control of an endosperm‐specific GluB‐1 promoter as an oral vaccine (Ogo et al ., ; Wang et al ., ). The products accumulated as glycoproteins with high‐mannose‐type N‐glycan, but without β1,2‐xylose or α1,3‐fucose. TPC7 was deposited as a novel, giant spherical ER‐derived protein body, >20 μm in diameter, which was referred to as the TPC7 body (Figure h). When transgenic rice containing hypo‐allergenic TPC7 and native Bet v 1 were orally administered to allergic model mice, infiltration of eosinophils and total inflammatory cells in the conjunctiva was decreased, resulting in a reduction in clinical symptom scores compared with mice fed nontransgenic rice. Rheumatoid arthritis Rheumatoid arthritis is an autoimmune disease associated with the recognition of self‐proteins expressed in arthritic joints. Parenteral administration of altered peptide ligands (APLs) of type II collagen (CII256‐271) can suppress the development of collagen‐induced arthritis (CIA) (Ohnishi et al ., ; Wakamatsu et al ., ). CII256‐271, APL6 and APL7 peptides were expressed as fusion proteins with the rice storage protein glutelin in the endosperm of transgenic rice seed. These transgene products successfully and stably accumulated at high levels (7–24 mg/g seeds) in PB‐II of mature seeds. The efficacy of these transgenic rice seeds in suppressing the development of CIA was examined by oral administration of the seeds to CIA model mice. Vaccination of sensitized mice with APL6 transgenic rice for 14 days significantly inhibited the development of arthritis (based on clinical score) and delayed disease onset during the early phase of arthritis. These effects were mediated by the induction of IL‐10 from CD4 + CD25 + T cells against CII antigen in splenocytes and inguinal lymph nodes (iLNs). Similarly, APL7 transgenic rice given for 14 days prior to immunization with CII prevented inflammation and erosion in the joints (Iizuka et al ., , b ). Future perspectives Human glucocerebrosidase for the treatment of Gaucher's disease known as taliglucerase alfa or Elelyso, which is produced in carrot cell culture, was approved for use in humans as the first plant‐made pharmaceutical in 2012 by Food and Drug Administration. At present, several pharmaceuticals products (HIV‐neutralizing monoclonal antibodies, human insulin, apolipoprotein Al, lactoferrin, etc.) derived from transgenic plants including seeds cultivated in open field have entered into human clinical trials, whereas several plant‐derived recombinant proteins for non‐biopharmaceuticals such as avidin and trypsin produced in maize seeds (ProdiGene), human albumin, lactoferrin and lysozyme produced in rice seeds (Ventria), cytokines and growth factors produced in barley seeds (ORF) are commercialized for diagnostics, research regent, cell culture supplement and cosmetic ingredient (Sack et al ., ). Different from the latter non‐biopharmaceuticals, it is critical that all of the biopharmaceutical products used for clinical trials have been purified according to the draft regulatory guidance of the GMP‐compliant manufacturing processes. Adaptation of intact or partially purified formulations as rice seed‐based oral vaccines remarkably decreases the production cost, as downstream purification processes, which represent up to 80% of overall production cost, are avoided. Moreover, oral administration permits the use of nonpurified vaccines (crude preparation of vaccines). It is important to note that oral vaccination elicits mucosal and humoral immune responses. As most pathogens invade the host through the mucosal route, protection at mucosal sites as a front line is thought to be the most effective means to prevent infectious diseases. On the other hand, antigen administration via the oral route is prone to invoke immune tolerance against the administered antigen, which can be exploited in the development of effective and safe oral allergy vaccines via allergen‐specific immune tolerance using safe tolerogens (hypo‐allergens and T‐cell epitopes). It is proposed that transgenic rice expressing antigens should be produced under the control of specific regulations and guidelines for practical use. Regulatory guidance for the production of recombinant pharmaceutical proteins in plants exists only as draft legislation, and this is based on the existing regulations for mammalian cells (Sabalza et al ., ). This legislation is inappropriate for applications involving whole plants. Rice seed‐based oral vaccines are a new form of medical drug that has to be manufactured according to GMP standards. Nonpurified crude biologics such as plant‐based edible vaccines have not yet been commercialized. The only approved poultry vaccine against Newcastle disease was produced from cultured transgenic tobacco cells and then purified. Presently, there are no guidelines or examples for developing (commercializing) such crude pharmaceutical proteins (Spök et al ., ). Before human clinical trials using transgenic rice grains and their crude extracts as oral vaccines are undertaken, it is necessary to establish production processes that can assure identical quality according to GMP guidelines (CPMP, , ; Fischer et al ., ). One concern is maintenance of equivalence of antigen in seeds because expression levels may vary among plants (seeds) and among generations when cultivated in open field under natural, but variable, environmental conditions. It is necessary to establish a production (cultivation) system in which seed quality (the content of the vaccine antigen accumulated in rice seeds) is kept constant by controlling the nitrogen fertilizer; such cultivation systems to control the desired seed protein level were established for rice in Japan. However, GMP regulations cannot be strictly applied to production processes of transgenic plants with cultivation in open fields. It may be reasonable to cultivate transgenic rice according to the guidelines of good agricultural practice (GAP) that conform to GMP‐like regulations. Standard operation protocols for transgenic rice cultivation during the process from sowing to harvesting have to be prepared. Of course, downstream processing of seed grains after harvesting has to be performed according to GMP conditions. Another major concern is gene transfer by pollen‐mediated outcrossing with any nontransgenic rice cultivated in the near vicinity. Although the percentage of outcrossing is very low for rice due to its capacity for self‐pollination and the short life of rice pollen, the possibility remains. To prevent gene transfer to nontransgenic rice, geographical isolation and separate plantings have to be established. It is also important to consider biological containment such as male sterility or cleistogamy (self‐fertilization without flower opening). Contamination may also occur by direct mixing during the harvest and distribution. Prevention of contamination in food and feed chains is strictly demanded by consumers and environmental organizations. It would be helpful to use discriminative markers such as colour‐coded rice to ensure traceability. Therefore, specific cultivation guidance and regulation for cultivation of plant‐made pharmaceuticals crops in open field has to be established with zero tolerance for contamination in food and feed chains in Japan. Acknowledgement This work was supported by a grant from the Agri‐Health Translational Research Project from the Ministry of Agriculture Forestry and Fisheries of Japan. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Biotechnology Journal Wiley

Rice seed for delivery of vaccines to gut mucosal immune tissues

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Copyright © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd
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10.1111/pbi.12423
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Abstract

Introduction Plants provide a promising platform for the production of recombinant proteins, offering advantages over conventional fermentation systems that employ bacteria, yeast and mammalian cells in terms of scalability (agricultural scale because no specific facilities are required), safety (no contamination with mammalian pathogens such as viruses and prions) and cost‐effectiveness (Daniell et al ., ; Sharma and Sharma, ; Tiwari et al ., ; Twyman et al ., ). Plant production systems can be divided into those using stable transgenic plants generated by nuclear genome and plastid genome transformation and plant virus‐based or Agrobacterium ‐based transient expression platforms (Paul et al ., ). Each of production systems has advantages or disadvantages. When recombinant proteins are produced by transient expression system, robust yields of recombinant products can be obtained within a few weeks, but they have to be extracted and purified for use because tobacco is mainly used as production host. Plastid‐based expression system also gives rise to high‐level expressions of recombinant proteins without gene silencing and position effect due to the site‐specific homologous recombination and multicopy number of genome. Furthermore, there is little possibility of pollen‐mediated gene contamination due to the maternal transgene inheritance, but there is no post‐translation modification of products such as glycosylation (Daniell et al ., ). On the other hand, seed systems have the advantages of high productivity, stability and oral delivery (Lau and Sun, ; Stoger et al ., ; Takaiwa, ). Indeed, recombinant proteins stored in seeds are highly stable without degradation, even if stored at ambient temperatures for several years. Moreover, proteins can be produced at very high levels when they are expressed under the control of seed‐specific promoters. When orally administered, antigens stored in seed withstand proteolysis in the gastrointestinal tract (Nochi et al ., ; Takagi et al ., ). All approved biopharmaceutical proteins (biologics) produced to date are extracted and purified according to the specific guidelines and regulations of good manufacturing practice (GMP). The downstream extraction and purification steps are generally expensive, representing up to 80% of overall production cost (Kusnadi et al ., ; Wilken and Nkolov, ). Notably, the cost of processing and purification is approximately the same among the various production systems. However, if pharmaceutical proteins are produced in edible plants (crops), oral administration using crude or minimally processed products is feasible, thereby eliminating expensive downstream processing steps. However, one disadvantage of oral administration is that more than 100‐fold higher concentrations of oral antigens are required to achieve the same level of efficacy as with the parentally administered antigens. This disadvantage can be overcome by plant‐based products because the plant cell wall is resistant to harsh conditions and digestive enzymes. Pharmaceuticals produced in seeds are further fortified by two barriers comprising the rigid cell wall and protein bodies as bioencapsulated products, offering a suitable delivery vehicle to gut immune tissues. Oral vaccination has an additional benefit in that it can regulate both mucosal and systemic immune responses. Thus, seed‐based biopharmaceuticals are attractive as vaccines for infectious and allergic diseases. Advantage of rice seed as a production platform of pharmaceutical proteins Rice is a very important staple food that is consumed by nearly half of the global population. Rice seed is an efficient bioreactor for the production of pharmaceutical proteins due to its high biomass yield, low risk of gene flow due to self‐pollination, ease of transformation and convenience of scale‐up (Stoger et al ., ; Twyman et al ., ). Rice grain biomass yield is higher than many other cereals (wheat, barley, and rye) but is lower than maize. However, maize is an outcrossing plant, which can lead to problems of gene flow in open‐field cultivation. Marker‐free transgenic rice was easily obtained using the multi‐auto‐transformation (MAT)‐vector (Endo et al ., ) or Cre‐lox recombination system (Radhakrishnan and Srivastava, ), together with the Agrobacterium‐ mediated co‐transformation system by introducing two T‐DNAs (Komari et al ., ; Sripriya et al ., ). Use of such marker‐free systems may address the public's reluctance to accept transgenic plants and their products. Agricultural infrastructure for the cultivation, harvesting, processing and storage of rice is well established worldwide. In particular, the content of seed proteins in mature rice grains can be easily controlled by the dose of nitrogen fertilizer as nitrogen plays a key role in determining seed protein levels (Nishizawa et al ., ; Souza et al ., ). The complete genome sequence ( http://rgp.dna.affrc.go.jp/E/IRGSP/ ) and considerable genomic information regarding gene expression are available ( http://rapdb.dna.affrc.go.jp/ ). This genetic information can be readily applied to create the desired pharmaceutical products by genetic manipulation. Expression strategies for producing high amounts of recombinant proteins in rice seeds have been established in the last decade. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without purification (downstream processing) due to the absence of toxic compounds such as toxic metabolites or severe food allergens. The endosperm is an ideal bioreactor for the production of pharmaceutical proteins (Arcalis et al ., ; Ou et al ., ; Takaiwa et al ., ). Cereal endosperm is a specialized storage tissue, which makes up about 80%–90% of total seed weight, and in which starch, proteins and lipid are stored as nutrient resources for germinating seedling. About 80%–90% of cereal seed weight comprises starch (carbohydrate), with 6%–15% and 2%–5% being made up by storage proteins and lipid, respectively. This contrasts with the leguminous and oil seeds (soybean, pea, broad bean, rape, sunflower and Arabidopsis), which contain 20%–40% protein, 20%–60% starch and 5%–50% lipid. Seed storage proteins (SSPs) in dicotyledonous seeds are mainly stored in embryo or cotyledons, with the exception of some crops in which it occurs in the endosperm (tobacco or castor bean). Endosperm tissue consists of central starchy endosperm, subaleurone layer (SAL), aleurone layer (AL), basal endosperm transfer layer and embryo‐surrounding region (Figure ). Most SSPs are deposited in subaleurone and starchy endosperm cells. It is important to note that various types of recombinant proteins including artificial or toxic products can be highly and stably accumulated in the endosperm without having a detrimental effect on embryo development, thus providing an ideal production platform (bioreactor) (Peter and Stoger, ; Takaiwa, ). This can be achieved by protein deposition into the specialized storage compartments such as protein bodies (PBs) and protein storage vacuoles (PSVs) in the endosperm. When transgenic rice seed become desiccated during the process of maturation, the endosperm becomes dehydrated and has a reduced level of proteolytic activity, thus providing an ideal environment for storage of recombinant proteins. Rice endosperm tissue. Vertical (a, b) and transverse (c, d) sections of rice maturing seed at 15 days after flowering. AL, aleurone layer; BETL, basal endosperm transfer layer; CSE, central starchy endosperm; EM, embryo; ESR, embryo‐surrounding region; SAL, subaleurone layer. Seed storage proteins and their transport mechanism Seed storage proteins are deposited in specialized membrane‐bound storage organelles called PBs. There are two types of PBs, which differ in biogenesis in rice endosperm cells. One is ER‐derived PBs (PB‐I), with a size of 1–2 μm and a spherical structure, and the other is PSVs (designated as PB‐II), with a size of 2–4 μm and an irregular shaped structure (Krishnan et al ., ; Tanaka et al ., ). In the generation of ER‐derived PBs, hydrophobic prolamins form aggregates within the lumen of rough ER and bud off as a spherical organelle in the cytoplasm. Prolamins are grouped into three groups based on molecular size (10, 13, and 16 kDa), and the 13 kDa prolamin is further divided into two Cys‐rich and two Cys‐poor groups. At least six types of prolamins are highly and tightly packaged into PBs via disulphide bonds. PB formation starts as a core of 10 kDa prolamin, which is followed by synthesis of other prolamins (Nagamine et al ., ). They are arranged and organized in the order of Cys‐rich 10 kDa core, Cys‐poor 13 kDa inner layer, Cys‐rich 16 and 13 kDa middle layer, and Cys‐poor 13 kDa a outer layer in PB‐I (Saito et al ., ). Protein storage vacuoles contain distinct regions called matrix, crystalloid and globoid, which differ in electron density and coincide with the integrated proteins. Rice glutelin and 26 kDa globulin (α‐globulin) are incorporated in the crystalloid region and matrix of PSVs (PB‐II). Glutelin is the major SSP in rice, which accounts for 60%–80% of total seed protein. It shares homology with leguminous 11–12 S globulins such as soybean glycinin and pea legumin (Takaiwa et al ., ). Rice glutelin constitutes a multigene family classified into four groups (GluA, GluB, GluC and GluD) (Kawakatsu and Takaiwa, ). GluA and GluB are further divided into three and five classes respectively. By contrast, α‐globulin is encoded by a single copy and constitutes 5%–10% of total seed protein. Glutelins and α‐globulin are mainly deposited in PSVs by trafficking through the Golgi apparatus to the PSV via dense vesicles (DVs) after synthesis in the ER. In the case of storage proteins deposited in PSVs, Golgi‐dependent and Golgi‐independent pathways are involved in storage protein transport from the ER to the PSVs. Precursor‐accumulating vesicles (PACs) are implicated in the direct PSV pathway bypassing Golgi apparatus (Ibl and Stoger, ). The aggregation of storage proteins within the ER may be related to the direct transport to PSVs by PACs. PACs are observed in rice seed (Takahashi et al ., ). In the case of wheat, barley and oat seeds, their major prolamins (glutenins, hordeins and avenins) are initially deposited into ER‐derived PBs, which are subsequently sequestered to PSVs through the Golgi apparatus or directly via autophagy‐mediated transportation (Galili, ; Herman and Larkins, ; Tosi et al ., ). It is characteristic of rice endosperm cells that ER‐derived PBs and PSVs co‐exist within the same endosperm cell. Sorting mechanisms of prolamin and glutelin have been characterized. The transcripts are transported along the cytoskeleton to specific regions depending on cis‐acting RNA localization signals. Rice prolamin mRNAs are targeted to the PB‐ER that surrounds the PB‐Is, whereas rice glutelin mRNAs are localized in the cisternal ER (Okita and Choi, ). Selective distribution of sorted mRNA transcripts may play a role in efficient transport of prolamins and glutelins into PBs and PSVs. Sorting signals for proteins destined to PSVs have been characterized as N‐ and C‐terminal propeptides and internal regions of mature seed proteins and classified into C‐terminal vacuolar sorting signals (VSS), sequence‐specific VSS and physical structure VSS (Vitale and Hinz, ). However, such signals have not been identified in rice SSPs. Growing evidence indicates that post‐Golgi trafficking of storage proteins to the PSVs requires the retromer components (MAG1/VPS29, VPS35 and SNXs) involved in the recycling of vacuole sorting receptors (VSRs), the Rab family of small GTPases and their common guanine exchange factor (GEF) for specifying vesicular trafficking, and the SNARE complex for mediating membrane fusion between post‐Golgi compartments. Loss‐of‐function mutants of Rab5a, the small GTPase involved in vesicular membrane transport, exhibited Golgi‐derived PB and PSV formation, suggesting that OsRab5a is implicated in the intracellular transport of proglutelin from Golgi to the PSVs (Fukuda et al ., ; Wang et al ., ). Mutants of GEF also had disrupted transport of glutelin and globulin, followed by the formation of large dilated paramural bodies (Fukuda et al ., ). A regulatory complex between the small GTPase Rab5a and its GEF VPS9a was shown to be involved in the transport of proglutelins from the Golgi apparatus to PSVs through regulation of DV‐mediated post‐Golgi trafficking (Ren et al ., ). Transport of rice SSPs from the ER to PSVs depends on coat protein complex II (COPII). Sar 1 GTPase plays an essential role in the formation of COPII vesicles for ER to Golgi traffic. When this COPII‐mediated pathway was inhibited by suppression of OsSar1 expression, abnormal ER‐derived dense PBs containing glutelin precursor were generated by inhibition of SSP transport (Tian et al ., ). Tools to boost expression levels of recombinant proteins in rice seeds The expression levels of foreign recombinant proteins are primary determined by the level of transcription. Thus, the promoter used to drive the expression of the recombinant proteins (transgenes) is the most important element. To obtain expression in a particular tissue during a specific phase of development, several rice seed‐specific promoters were developed (Qu and Takaiwa, ; Qu et al ., ; Wu et al ., ). The advantage of this type of promoter is that it increases protein stability and avoids detrimental effects of accumulation of the recombinant proteins in vegetative plant tissues through expression in specific tissues in the transgenic plant. It is important to select the tissue‐specific promoter to maximize levels of recombinant proteins in the target harvest tissue. The seed storage tissue, endosperm, is a reasonable target because its function is to store nutrient resources for the germinating seedling. Targeting to a suitable subcellular location (organelle) combined with seed‐specific expression leads to high‐yield production of antigens because endosperm‐specific promoters differ in spatial and temporal characteristics. For example, α‐globulin and the glutelin GluD promoter is predominantly expressed in inner starchy endosperm, and most glutelin genes and prolamin genes are highly expressed in the subaleurone layer and outer peripheral region of the endosperm tissue, respectively (Kawakatsu et al ., ; Qu and Takaiwa, ; Wu et al ., ). The 18 kDa oleosin and embryo globulin ( REG2 ) promoters direct expression in the embryo and aleurone layer (Qu and Takaiwa, ). The basal endosperm transfer layer 1 ( BETL1 ) promoter confers specific expression at the basal endosperm transfer layer. In general, considering that seed proteins are mainly deposited in starchy endosperm cells and subaleurone cells in the rice endosperm tissue, recombinant proteins should be expressed in these cells under the control of these tissue‐specific promoters. High levels of expression can be obtained using the promoters for GluB‐1 and GluB‐4 glutelin and 10 and 16 kDa prolamin. The temporal expression pattern of these storage proteins during seed maturation differs. Expression of 10 and 16 kDa prolamin genes starts from 5 days after flowering (DAF), which is followed by expression of most glutelin genes and 13 kDa prolamin genes. Untranslated regions (UTRs) play an important role in the translation efficiency and stability of mRNAs. To stabilize the mRNA transcript, the full‐length 5′ UTR should be included. The flanking sequence around the ATG codon in monocot plants should be optimized as follows: (A/G)(A/C)C AUG GCG), which results in an increased rate of translation (Joshi et al ., ). 3′ UTR regions such as the GluB‐1 3′ UTR, which contains several poly(A) signals (AATAAA) that are involved in transcript (mRNA) stability and accurate termination, have to be considered to ensure high‐level expression of foreign proteins (Yang et al ., ). Codon optimization of sequences encoding recombinant proteins is critical to enhance the protein accumulation because it can affect the rate of protein production and mRNA stability (Gustafsson et al ., ). This is attributed to the presence of rare codons and signal sequences that affect mRNA stability. Rare codons, AU rich sequences and AUUUA repeats that are known to destabilize the transcript and splicing junction sequence should be modified without changing the amino acid sequence. It should be noted that codon usage (G+C content) of genes expressed in seeds differs from that in leaves (high GC rich), even in the same rice genes; that is, codon usage can differ among tissues. Codons in the target gene expressed in the rice endosperm should be optimized using codons frequently used in rice endosperm genes (Wakasa and Takaiwa, ). Because accumulation level, stability and post‐translational modification of recombinant proteins are largely dependent on intracellular localization, it is important to target protein expression to suitable locations by means of intracellular targeting signals (Hofbauer and Stoger, ; Khan et al ., ). In the case of seeds, a signal peptide leading to the secretory pathway is mandatory for stable accumulation of product. Generally, many pharmaceutical proteins are post‐translationally modified by glycosylation and, thus, may have to be synthesized as secretory proteins using an endomembrane system. Plant or native signal peptides should be ligated to the N‐terminus of the mature recombinant protein. Targeting signals leading to the desired subcellular compartments have been characterized in plant cells. For example, when the transit peptide is ligated to the N‐terminus, the recombinant protein is trafficked to the amyloplast (starch granule) in seed endosperm cells. Ligation of a KDEL (Lys‐Asp‐Glu‐Leu) sequence at the C‐terminus leads to ER retention, which acts as an ER retrieval signal. It has been reported using this strategy that recombinant protein yield was enhanced by 2‐ to 10‐fold in many tissues of a variety of plants (Conrad and Fiedler, ; Takagi et al ., ). Retaining the desired protein in the ER is a reasonable strategy because proteases are limited and sufficient amounts of chaperones for folding and assembling are present, providing a good environment for recombinant protein production (Oono et al ., ; Wakasa et al ., ). It is important to consider that the destination of recombinant proteins expressed as secretory proteins through an endomembrane system may be determined by interactions with the endogenous seed proteins or by its intrinsic physical properties. In particular, recombinant proteins with free cysteine residues interact with endogenous cysteine‐rich prolamins via disulphide bonds in ER lumen and are incorporated into accumulated in PBs (Takaiwa et al ., ). Such interaction sometimes prevents the proper interaction among the endogenous storage proteins, resulting in disruption of the ordered deposition of seed proteins and generation of aberrant PBs. Glycosylation patterns are also highly affected by subcellular localization (Arcalis et al ., ). Recombinant proteins are glycosylated along the secretory pathway as they move from the ER through Golgi to their final destination (Gomord et al ., ). Glycosylation enhances the physicochemical properties of a protein by promoting thermal resistance, protecting from proteolytic degradation and enhancing stability. Plants attach β1,2 xylose and α1,3 fucose residues to the site (Asn‐X‐Ser/Thr) of proteins as post‐translational N‐glycosylation, whereas mammals attach α1,6 fucose, β1,4 galactose and sialic acid residues (Gomord and Faye, ). Plant‐specific glycans are sometimes immunogenic (Garcia‐Casado et al ., ), and glycoengineering strategies were developed to avoid the addition of plant‐specific N glycans and to add human‐like glycans. Plant‐specific glycosyltransferases have been deleted or mutagenized by homologous recombination in rice (Ozawa et al ., ), while protein sialylation has been achieved by introducing the entire mammalian pathway for sialic acid synthesis in tobacco (Castilho et al ., ). Unfolded protein response is a bottleneck for high production of recombinant proteins Abundant accumulation of seed proteins is achieved through efficient packaging in ER‐localized PBs with the aid of chaperones and folding enzymes within the ER lumen. The folding and assembly of newly synthesized proteins is a complex process assessed by protein quality control mechanisms that involve many chaperones and folding enzymes. For deposition of recombinant protein into PBs, an array of chaperones, co‐chaperones, oxidoreductases, glucan chain modifying enzymes and lectins is involved in folding and assembly in a dynamic action, resulting in maintenance of ER homeostasis by the unfolded protein response (Schroder, ). Molecular chaperones and folding enzymes such as binding proteins (BiPs), protein disulphide isomerase (PDI) and calnexin are implicated in depositing high amounts of SSPs in PBs. For example, PDIL1‐1 is mainly localized on the dilated ER in rice endosperm cells. Mutation of this PDIL1‐1 (esp2) results in the formulation of abnormal PBs containing both proglutelins and prolamins in ER due to incorrect disulphide formation (Onda et al ., ; Takemoto et al ., ). On the other hand, PDIL2‐3 is exclusively localized on the surface of PB‐I. Knockdown of PDIL2‐3 inhibits the accumulation of Cys‐rich 10 kDa prolamin in the core (Onda et al ., ) of PB‐I, suggesting that PDIL2‐3 is involved in disulphide bond formation among prolamins in PB‐I. Extreme suppression or overexpression of major BiP1 in an endosperm‐specific manner had severe suppressive effects on the production of seed proteins accompanied by aberrant structural formation of PBs, whereas slight enhancement of BiP1 expression led to increased total storage protein levels (Wakasa et al ., ; Yasuda et al ., ). By contrast, major PDIL1‐1 had no apparent effect on accumulation of seed proteins, even when overexpressed in endosperm (Yasuda et al ., ). When foreign recombinant proteins accumulate at high levels in ER lumen as secretory proteins, ER stress is sometimes induced as the unfolded protein response (UPR) as a result of imbalance between the load of unfolded proteins and the ER folding capacity. Production levels of secretary proteins may be highly affected by the quality control machinery of ER. In plants, the UPR is mediated by at least two ER transmembrane sensors: activating transcription factor 6 (ATF6) homologues and inositol‐requiring enzyme 1 (Ire1) (Hetz, ; Howell, ; Iwata and Koizumi, ). In contrast to yeast and mammals, in plants there is no obvious homologue to PERK, which is implicated in attenuating general protein synthesis by the phosphorylation of the translation initiation factor eIF2a (Kamauchi et al ., ). ATF6 is a type II transmembrane transcription factor that senses ER stress via the C‐terminal luminal domain. Upon ER stress, rice orthologues of ATF6, OsbZIP39 and OsbZIP60 dissociate from BiP and are transported to the Golgi apparatus via specific trafficking with the COPII vesicles for proteolytic processing by serine proteases S1P and S2P (Takahashi et al ., ). The liberated N‐terminal cytoplasmic domain is transported into nuclei to activate UPR target genes. Inositol‐requiring enzyme 1 is a type I ER‐resident transmembrane protein with an ER luminal dimerization and a cytoplasmic domain with Ser/Thr kinase and endonuclease activities (Hetz et al ., ). ER stress allows IRE1 to autophosphorylate the kinase domain through dimerization and thereby activate the ribonuclease domain. The activated IRE1 mediates an unconventional cytoplasmic splicing of mRNA encoding transcription factor OsbZIP50 (rice orthologue of mammalian XPB‐I and yeast Hac I), leading to removal of the transmembrane domain as a result of frame shifting (Hayashi et al ., ). The activated OsbZIP50 is involved in up‐regulating the expression of ER quality control and ERAD‐related genes (various ER stress‐related genes). Ire1 RNase activity is also implicated in an mRNA degradation mechanism termed regulated IRE1‐dependent decay. This mechanism involves selective degradation of a subset of mRNAs coding for certain ER‐located proteins. Deletion of OsIRE1 RNase activity by site‐specific mutagenesis through homologous recombination resulted in relief of suppression of mRNA levels of secretory and membrane proteins independent of the OsbZIP50 pathway, whereas a lack of kinase activity induced by mutagenesis of the Ire1 kinase domain led to lethality in transgenic rice, indicating an essential function in cell fate (Wakasa et al ., ). Transcription of UPR genes is activated by binding of UPR transcription factors to the cis elements in their promoter. Three cis elements, URR element (UUPRE:TGACG‐GT), ER stress element‐I (ERSE‐I:CCAAT‐N9‐CCACG) and ERSE‐II (ATTGG‐N‐CCACG) were implicated in the ER stress response in mammalian cells (Kokame et al ., ; Wang et al ., ; Yoshida et al ., ). The consensus sequence of plant‐UPR element (P‐UPRE: ATTGGTCCACGTCATC) consisting of mammalian ERSE‐II and a UPRE element is conserved in plant‐UPR genes that are recognized by plant‐UPR transcription factors (Oh et al ., ). pUPRE‐II (5′‐GATGACGCGTAC‐3′) and pUPRE‐III (TCATCG) are also cis elements required for ER stress response‐related genes in plants (Hayashi et al ., ; Sun et al ., ). pUPRE‐II was reported to be bound by activated OsbZIP50 and OsbZIP60 and was involved in activation of UPR genes such as PDIL 2‐3 (Takahashi et al ., ). Expression of BiP4 and BiP5 is specifically induced under ER stress conditions, whereas BiP1, a major BiP protein, is constitutively expressed in various tissues and is up‐regulated in response to ER stress. Therefore, these BiPs can be used as ER stress markers (Wakasa et al ., ). Activation of the ER stress response is mainly mediated by the OsIRE1‐OsbZIP50 signal cascade. When proteins fail to fold into their native conformation, ER‐associated degradation (ERAD) machinery responsible for elimination of misfolded proteins is induced for protein quality control. The proteins are retrograde transported across the ER membrane back into the cytoplasm, where they are rapidly degraded by the ubiquitin‐proteasome system, in which they are degraded by 26S proteasome after marking by ubiquitination. The protein quality control system in the ER lumen plays a critical role as a checkpoint to determine whether expressed recombinant protein should be accumulated or degraded in plant cells. Enhancement of folding and assembly activities for various secretory proteins in ER lumen may lead to higher levels of accumulation of the desired recombinant protein in plant cells. Further study to understand the molecular mechanisms involved in the UPR will be required. Design of PBs as deposition space for high accumulation of recombinant proteins Suppression of some SSPs leads to higher accumulation of other types of SSPs by a proteome rebalancing mechanism that maintains the homeostasis of protein (nitrogen) levels in matured seed. Expression of recombinant protein in seed of low SSP mutants such as low glutelin lines LGC‐1, a123 resulted in about two‐fold higher yield than that of normal rice (Tada et al ., ). Moreover, reduction in some SSPs by silencing resulted in up‐regulation of other SSPs and foreign recombinant protein (Kawakatsu et al ., ; Shigemitsu et al ., ; Yuki et al., 2012; Yuki et al ., ). This mechanism is also conserved in other cereal and leguminous seeds (Goossens et al ., ; Schmidt and Herman, ; Wu and Messing, ). Thus, high‐level production of foreign recombinant proteins in transgenic seeds is expected to be obtained by suppression of endogenous SSPs. When expression of endogenous SSPs was suppressed by RNA interference technology to supply the deposition space for the desired recombinant protein, production yield of the desired protein was remarkably increased as a compensatory mechanism (Yang et al ., ). However, homeostasis of seed protein level by proteome rebalancing is not simple, as suppression of endogenous SSPs when using different subcellular compartments as a deposition site did not confer significant enhancement. This result suggests that relief of loading competition with the endogenous SSPs for deposition space leads to a high level of accumulation as a result of proteome rebalancing; that is, supply of vacant deposition space (PBs) for the desired recombinant protein through suppression of distinct SSPs is an ideal strategy for increasing the accumulation levels of recombinant proteins in seeds (Takaiwa, ). Production strategy for rice seed‐based vaccines When the recombinant proteins are produced as secretory proteins by ligating the signal peptide and the KDEL ER retention signal to the N‐ and C‐termini, they are deposited into various types of ER‐derived bodies, including PBs, that exhibit varying morphology based on their inherent physical properties (aggregation ability) and interaction with endogenous seed proteins through disulphide bonds. These differing types of PBs exhibited different resistance to digestive enzymes when assayed in vitro , which was attributed to the differing physical properties of the PBs in which the recombinant proteins were incorporated. On the other hand, some recombinant proteins failed to accumulate. In these cases, the proteins can be produced by expressing as parts of SSPs in a form of fusion protein. When recombinant proteins are fused with the C‐terminus of prolamins or inserted into the highly variable region of glutelins (C‐terminus of acidic subunit of glutelin) and then expressed under the control of endosperm‐specific promoters, fusion proteins accumulated as parts of native storage proteins are naturally deposited in PBs or PSVs. Taking advantage of these strategies, high amounts of recombinant proteins can be accumulated in PBs or PSVs in transgenic rice seed (Wakasa and Takaiwa, ). When digestibility and immunogenic activity (tolerance‐inducing capacity) were compared between ER‐derived PB (PB‐I)‐ and PSV (PB‐II)‐containing antigens, PB‐I localizing antigen exhibited greater immunogenicity than the PB‐II antigen (Takagi et al ., ). This difference is associated with the difference in resistance to gastrointestinal digestion enzymes, with proteins deposited in ER‐derived PB (PB‐I) being more resistant to digestive enzymes than those deposited in PSV (PB‐II). This physical difference may be related to the polymerized or aggregated formulation via Cys‐rich prolamins in ER‐derived PBs. Structure of GALT and immune reaction in the gastrointestinal tract The gastrointestinal tract containing GALT is a front line of defence against mucosal pathogens causing infectious diseases, but it is also involved in immune tolerance against foods and commensal bacteria. Homeostatic conditions in the gastrointestinal tract, with a balance between tolerance and sensitization, is maintained by the stringent regulatory mechanisms. The mucosal immune system consists of inductive and effector sites. In the gastrointestinal immune system, the Peyer's patches (PPs), isolated lymphoid follicle tissue (ILF) and the mesenteric lymph nodes (MLNs) act as inductive sites, while the lamina propria (LP) and intra‐epithelial lymphocytes (IEL) of the mucosal membrane constitute effective sites. Each PP is composed of B cell‐rich follicles (FO) surrounded by interfollicular regions (IFR) consisting of T cells (Figure ). Schematic representation of GALT. FAE, follicle‐associated epithelium; FO, follicle; GALT, gut‐associated lymphoid tissue; GC, germinal centre; IEL, intra‐epithelial lymphocyte; IFR, intrafollicular region; LP, lamina propria; M cell, microfold cell; MLN, mesenteric lymph node; SED, subepithelium domain; PP, Peyer's patch; B, B cell; T, T cell; P, plasma cell. Microfold cells (M cells) are specialized epithelial cells that predominantly reside in the follicle‐associated epithelium (FAE) overlying PPs. M cells in PPs have the ability to take up and transcytose antigens to antigen‐presenting cells (APCs). GALT contains an organized macro‐architecture of B and T lymphocyte zones that respond to the antigens presented by dendritic cells (DCs). DCs act as sentinels, taking up antigens and then migrating to the subepithelial dome at PPs or to the MLN to activate native T cells. Intestinal DCs are involved in the decision of tolerance vs. inflammation (Kunisawa et al ., ; Neutra and Kozlowski, ; Tsuji and Kosaka, ). When antigens are orally administered, the properties of the administered antigens can determine which type of immune reaction is induced (immune tolerance vs. immune induction). Protective immune responses are induced against potential pathogens. The local production and secretion of dimeric secretory immunoglobulin A (sIgA), which has function to neutralize pathogens, is characteristic of the mucosal adaptive immune response. The default responses to most nontoxic antigens such as dietary antigens are caused by mucosal immune tolerance that is favoured in intestinal immune tissue (Pabst and Mowat, ). The mucosal immune response in GALT is affected by the duration, dose and formulation of antigen (Chehade and Mayer, ; Mayer and Shao, ). High‐dose administration of antigen leads to deletion (apoptosis) or anergy of antigen‐specific T cells. On the other hand, repeated administration of low‐dose antigen over a long duration leads to immune suppression called ‘active suppression’, which is mediated by regulatory T cells (Dubois et al ., ; Weiner et al ., ). Immune tolerance is important in preventing allergic and autoimmune diseases, while it may pose an obstacle in the development of oral vaccines against pathogens. Uptake of antigens deposited in PBs Recognition and subsequent uptake of antigen is an essential function of the mucosal immune system. When vaccine antigens are delivered as a particle formulation smaller than 10 μm, such as PBs, to intestine immune tissues, these antigens are taken up by M cells of GALT. The APCs process the antigen and migrate within the PP to the T‐cell area and/or B‐cell follicles. Some PBs containing antigens may be also directly taken up by CX3CR1 + macrophages called ‘intra‐epithelial DCs’ in the LP, which extend their dendrites into the gut lumen (Pabst and Mowat, ). These latter DCs are a nonmigratory, gut‐resident population. The antigen taken up by intra‐epithelial DCs is passed to the neighbouring CD103 + DCs in the LP, which then migrate to the mesenteric lymph nodes (MLNs) for presentation to naïve T cells. It is important to examine whether ER‐derived PBs containing antigens are actually delivered to lymphoid tissues of GALT when orally administered to mice. It was demonstrated that BPs containing the antigen (shuffle Cry j 2)/GFP reporter hybrid were taken up in the gut (Wakasa et al ., ). Notably, fluorescence elicited from the GFP reporter could be detected in GALT tissue when rice seed powder or isolated crude PBs expressing the reporter were intragastrically administered. GFP fluorescence signals were detected at 8 h in both ileum and jejunum tissues of the small intestine and reached a peak at 12 h, disappearing after 18 h (Figure a–f). By contrast, no signal was detected in these tissues in mice fed nontransgenic rice. Furthermore, incorporation of PBs containing antigen was confirmed by immunoblotting analysis, in which antigen of the correct molecular weight could be detected in proteins extracted from peripheral gut tissues of mouse fed transgenic rice seed powder and prepared PBs (Figure g). These findings suggest that PBs may be taken up in intact or partial degraded form via M cells in the FAE and by DCs in PP. In a similar way, incorporation of PBs into PPs via M cells was shown by immune‐histochemical analysis with Ulex europaeus agglutinin (UEA‐1), which is a well‐known marker of murine M cells, when transgenic rice containing cholera toxin subunit B (CTB) that was deposited in PBs and PSVs, MucoRice‐CTB, was orally administered to mice (Nochi et al ., ). The CTBs deposited in PBs were taken up by UEA‐1‐positive M cells, but not columnar epithelial cells. Uptake of antigens by the intestinal tract. Balb/c mice were intragastrically administered with wild‐type rice seed powder, transgenic rice seed powder (GFP‐fused shuffled Cry j 2), wild‐type concentrated PB product or transgenic concentrated PB (deconstructed Cry j). (a) Ileum tissues before oral administration of rice seed powder expressing GFP‐fused shuffled Cry j 2. (b–d) Ileum tissue at 4, 8 and 12 h after oral administration of rice seed powder expressing GFP‐fused antigen. (e) Ileum tissue at 12 h after oral administration of nontransgenic rice. (f) Jejunum tissues at 12 h after oral administration of rice seed powder expressing GFP‐fused antigen. (g) Total proteins were extracted from ileum tissue at 2, 4, 6, 8, 13, 18 and 30 h after oral administration of transgenic concentrated PB product. F1 fragment derived from Cry j 1 was detected by immuneblotting using anti‐F1 antibody. (a–f) Adapted from Wakasa et al . ( ). Development of rice‐based oral vaccines as tools of immunotherapy Rice‐based oral vaccines have been developed for various allergic and infectious diseases (Azegami et al ., ; Takaiwa, ). Mucosal vaccines administered orally were shown to effectively induce antigen‐specific immune responses in both systemic and mucosal compartments (Brandtzaeg, ; Holmgren and Czerkinsky, ; Kwon et al ., ). For infectious disease, it has been shown that oral administration induced antigen‐specific IgA and IgG, which act as neutralizing antibodies and are involved in protection from invading pathogens (Marson et al ., ; Nochi et al ., ; Tokuhara et al ., ). A variety of vaccine antigens were expressed in rice seeds, including CTB (Nochi et al ., ; Yuki et al ., ), cholera toxin (Yuki et al ., ), infectious bursal disease virus immunogen VP2 (Wu et al ., ), hepatitis B virus surface antigen gene SS1 (Qian et al ., ), roundworm 14 and 16 kDa antigens (Matsumoto et al ., ; Nozoye et al ., ), surface glycoprotein F of Newcastle disease (Yang et al ., ), and amyloid β peptide (Nojima et al ., ; Oono et al ., ). MucoRice containing CTB has been extensively developed and is discussed in further detail below (Table ). Transgenic rice seeds as an edible vaccine for oral immunotherapy against various infectious, allergic and autoimmune diseases Target disease or pathogens Target antigen Promoter Expression level Localization References Vibrio cholerae Cholera toxin B subunit Glutelin GluB‐1 30 μg/grain PB‐I, PB‐II Nochi et al . ( ) Vibrio cholerae Modified cholera toxin B subunit (CTB/Q) 13 kDa prolamin 2.35 mg/g of seed Cell wall, cytoplasm Yuki et al . ( ) Vibrio cholerae Cholera toxin A and B subunits Glutelin GluB‐1 , GluB‐4 5 μg/grain NA Yuki et al . ( ) Infectious bursal disease virus VP2 of IBDV Glutelin Gt‐1 4.5% of total seed protein NA Wu et al . ( ) Newcasle disease virus Fusion(F) protein of NDV Glutelin Gt‐1 2.5–5.5 μg/g of seed NA Yang et al . ( ) Hepatitis B virus HBv surface protein and presurface1 Glutelin GluB‐4 31.5 ng/g of seed NA Qian et al . ( ) Arcaris suum 16 kDa protein of A. suum (As16) Glutelin GluB‐1 50 μg/grain NA Matsumoto et al . ( ) Arcaris suum 14 kDa protein of A. suum (As14) Glutelin GluB‐1 1.5 μg/grain NA Nozoye et al . ( ) Clostridium botulinum Heavy chain of botulinum type A neurotoxin 13 kDa prolamin 100 μg/grain Cytoplasm Yuki et al . ( ) Alzheimer's disease 2 × Amyloid β peptide (1–42) Glutelin GluB‐1 8 μg/grain PB‐I Oono et al . ( ) Alzheimer's disease Amyloid β peptide fused to GFP CaMV35S 400 μg/g of seed NA Nojima et al . ( ) Cedar pollen allergy T cell epitope, 7Crp Glutelin GluB‐1 60 μg/grain PB‐I Takagi et al . ( b ) Cedar pollen allergy T cell epitope, Crp3 Glutelin GluB‐1 80 μg/grain PB‐I Takaiwa and Yang ( ) Cedar pollen allergy Glutelin/Cry j 1 (3 fragment) shuffle Cry j 2 GluB‐1,GluB‐4 10 k, 16k prolamin 5–20 μg/grain PB‐I Wakasa et al . ( ) Crypsis pollen allergy T cell epitope, 6Chao Glutelin GluB‐4 12 μg/grain PB‐I Takaiwa and Yang ( ) Birch pollen allery Mutagenized Bet v 1, TPC7, TPC9 Glutelin GluB‐1 76–550 μg/grain TPC7 body Wang et al . ( ), Ogo et al . ( ) House dust mite allergy Der p 1fragment, P1(45–145) Glutelin GluB‐1 90 μg/grain PB‐I Suzuki et al . ( ) House dust mite allergy Cys mutagenized Der f 2 Glutelin GluB‐1 15–30 μg/grain Der f 2 body Yang et al . ( b ) Rheumatoid artheritis Type 2 collagen analogue glutelin/3 × APL6, APL7 CII256‐271 Glutelin GluB‐1 7–24.6 mg/g of seed PB‐II Iizuka et al . ( , b ) NA, not analyzed. CTB Cholera ( Vibrio cholerae ) affects the small intestine through its secreted cholera toxin (CT), which is composed of five receptor‐binding B subunits (CTB) surrounding a catalytic A subunit (Holmgren, ). CTB is potent mucosal immunogen and adjuvant. CTB as a transmucosal carrier offers an efficient oral delivery approach via specific receptor‐binding process which is achieved through a plasma membrane receptor GM1 ganglioside. Pentamer formation of the CTB fusion is critical for efficient oral delivery. The codon‐optimized CTB was expressed as secretory protein by ligating the signal peptide and KDEL ER retention signal at its N‐ and C‐termini under the control of the glutelin GluB‐1 promoter in transgenic rice seed. CTB was deposited in PBs at about 30 μg/grain (Nochi et al ., ). MucoRice‐CTB is not only stable at room temperature for several years at room temperature without immunogenicity, but also is protected from digestive enzymes. In a mouse study, MucoRice‐CTB was administered orally to animals, and specific immune responses and neutralizing activity in both systemic and mucosal compartments were detected. Interestingly, mice immunized with MucoRice‐CTB were protected from oral challenge with cholera toxin. In a similar study conducted in a non‐human primate model, cynomolgus macaques received orally administered MucoRice‐CTB (Nochi et al ., ). Animals had CT‐specific, neutralizing antibodies and high levels of systemic IgG and intestinal IgA antibodies. Importantly, cold chain‐free oral MucoRice‐CTB induced long‐lasting cross‐protective immunity against heat‐labile enterotoxin‐producing enterotoxigenic Escherichia coli in addition to CT‐producing Vibrio cholera (Tokuhara et al ., ). These results demonstrate that oral administration of a rice‐based vaccine provides a potent practical global strategy for the development of cold chain‐ and needle‐free vaccines that protect from gastrointestinal infection. Rice‐based oral vaccines for allergic and autoimmune diseases Administration of antigens by the oral route is prone to induce immune tolerance. Taking advantage of this property, several types of rice‐based vaccines against pollen and mite allergens and self‐components were developed. More than 30% of people in industrialized countries are afflicted with some kind of IgE‐mediated type I allergic disease (allergic rhinitis, asthma, food allergy, allergic conjunctivitis and venom allergy). Current treatments for allergic diseases include pharmacotherapy and allergen‐specific immunotherapy. The major treatment for allergic diseases is pharmacotherapy, which targets the release of chemical mediators and block specific receptors (Holgte and Polosa, ). This treatment can reduce the clinical symptoms but is not curative. Allergen‐specific immunotherapy is the only curative treatment that has a long‐lasting effect. It induces immune tolerance against the causative allergens by subcutaneous injections of increasing doses of natural crude allergens over a period of 3–5 years (Frew, ; Larche et al ., ). This treatment is accompanied by pain and sometimes side effects (anaphylactic shock). To overcome poor patient compliance, it is necessary to improve the safety of tolerogen therapy to enhance efficacy. In this review, we introduce four rice‐based allergy vaccines for Japanese cedar pollen allergy, birch pollen allergy, mite allergy and autoimmune disease. Japanese cedar pollinosis is the predominant seasonal allergic disease in Japan, afflicting over 26% of the Japanese population. Allergen immunotherapy using rice‐based allergy vaccines containing hypo‐allergens Genetic engineering allows the production of innovative allergy vaccines designed to reduce side effects and to increase clinical efficacy and treatment compliance. To reduce IgE reactivity (allergenicity), tertiary structure needs to be modified to reduce recognition by specific IgE. Destruction of allergen conformation by fragmentation, shuffling of the molecule, oligomerization or site‐specific mutagenesis of cysteine residues involved in disulphide bonds (cleavage of disulphide bonds) leads to drastic reduction in IgE binding activity (Linhart and Valenta, ; Valenta et al ., ). Allergens are presented to specific T cells on the surface of APCs as a ‘T‐cell epitope’ together with MHC class II. The T‐cell epitope is about ten amino acids in length and is generated after processing within the APC. Thus, a T‐cell epitope peptide is expected to act as an ideal immunogen lacking allergenicity. T‐cell epitope peptides used as tolerogens to induce immune tolerance can provide a safe treatment due to a lack of B‐cell epitopes for binding of specific IgE (Ali and Larche, ; Larche, ). Allergen‐specific immunotherapy (SIT) approaches using T‐cell epitope peptides and hypo‐allergens (modified allergens) are considered as the next generation of allergen‐specific immunotherapy. The goal of allergen modification (T‐cell epitopes and hypo‐allergens) is to minimize allergenicity (risk of anaphylaxis) while retaining immunogenicity (T‐cell reactivity), thus allowing high doses of antigen to be administered over a short time course. Furthermore, recombinant variants lacking allergenicity are ideal candidates for prophylactic vaccination due to the very low risk of sensitization to naturally occurring allergens. T‐cell epitope of Japanese cedar pollen allergens Clinical application of peptide immunotherapy using major T‐cell epitopes was achieved for cat, grass and house dust mite (HDM) allergy (Larche, ). Efficacy was observed in a much shorter time frame without side effect. To evaluate T‐cell epitope peptide accumulation in rice seed, mouse major T‐cell epitope peptides derived from major Japanese cedar pollen allergens Cry j 1 and Cry j 2 were inserted into highly variable regions of soya bean storage protein glycinin A1aB1b. The modified soya bean glycinin containing the two T‐cell epitope peptides was deposited in PB‐II of transgenic rice endosperm, with expression directed by the GluB‐1 promoter. Oral administration of transgenic rice seeds to cedar pollen‐immunized mice inhibited allergen‐specific IgE and Th‐2‐type cytokine responses. Furthermore, allergen‐induced serum histamine and sneezing responses were suppressed (Takagi et al ., ). Because of MHCII polymorphism in the population of allergic patients, combinations of several major T‐cell epitopes (hybrid peptides) restricted by different MHCII molecules have to be prepared. A hybrid peptide called ‘7Crp peptide’ consisting of seven T‐cell epitopes derived from the Cry j 1 and Cry j 2 allergens was created, which was predicted to be recognized by 92% of allergic patients (Hirahara et al ., ). When the 7Crp peptide was expressed in transgenic rice seed by ligating it with an N‐terminal signal peptide and a C‐terminal KDEL signal under the control of the glutelin GluB‐1 promoter, 7Crp was deposited predominantly into PB‐Is (Figure b) due to interaction with Cys‐rich prolamins (Takagi et al ., ; Takaiwa et al ., ). When orally fed to the allergic model mice, a reduction in allergen‐specific CD4 + T‐cell proliferation and the level of allergen‐specific IgE was observed compared with mice fed nontransgenic rice (Takagi et al ., ). Some patients are not expected to respond to the 7Crp peptide and, therefore, a more universal peptide vaccine, Crp3, with seven additional T‐cell epitopes was co‐expressed together with the 7Crp peptide in transgenic rice seed (Takaiwa and Yang, ). Electron microscopy of developing rice endosperm containing various antigens. Recombinant proteins are deposited as unique ER‐derived PBs depending on the physical properties of the proteins accumulated in the rice endosperm cells. Intracellular localization of recombinant proteins in developing rice endosperm cells was analysed by immunoelectron microscopy. (a) Nontransgenic wild‐type rice; (b) 7Crp: seven linked T‐cell epitope peptides derived from Japanese cedar pollen allergens Cry j 1 and Cry j 2; (c) deconstructed whole Cry j 1 and Cry j 2 by fragmentation and shuffling; (d) Der p 1 (45–145) fragment comprising amino acid positions 45–145 of group 1 house dust mite allergen Der p 1; (e) Der f 2 (‐Cys): full‐length sequence of the mutagenized group 2 house dust mite allergen Der f 2, in which all six cysteine residues were changed to serine residues; (f) Der f 2 (C8/119S): full‐length of the Der f 2, in which cysteine residues at positions 8 and 119 were changed to serine residues; (g) Der f 2 (8–119): full‐length of the Der f 2, in which cysteine residues at positions 21, 27, 73 and 78 were changed to serine residues; and (h) TPC7: birch major pollen allergen Bet v1 mutagenized by DNA shuffling. Scale bar represents 1 μm. PB: protein body. Starch: starch body. Figure modified from Takaiwa ( ). Deconstructed Cry j Cry j 1 was divided into three overlapping fragments to disrupt its tertiary structure. These fragments were inserted into the highly variable regions of the C‐terminus in the acidic subunit of GluA, GluB and GluC glutelin precursors (Wakasa et al ., ). By contrast, the tertiary structure of Cry j 2 was reconstructed in a form of tail‐to‐top inverse orientation by shuffling. Low allergenicity of these disrupted allergens was confirmed by dot‐blot IgE binding assay and the RBL‐2H3 basophil degranulation test. Four constructs (three Cry j 1 fragment/glutelin fusions and shuffled Cry j 2) were expressed under four different endosperm‐specific promoters in transgenic rice seed (Wakasa et al ., ). The expressed proteins accumulated in the endosperm at levels of 10–25 μg per grain and were specifically deposited in PB‐Is (Figure c). Oral administration of these transgenic rice seeds to cedar pollen‐immunized mice inhibited allergen‐induced IgE and IgG responses, Th2‐type cytokine synthesis and CD4 + T‐cell proliferation. In addition, the allergen‐induced sneezing response, serum histamine elevation and infiltration of eosinophils and total inflammatory cells in the nose and conjunctiva were attenuated (Fukuda et al ., ; Wakasa et al ., ). Der p 1 Approximately 45%–80% of patients with allergic asthma are sensitized to allergens from HDM of the genus Dermatophagoides , suggesting that HDM allergens are crucial for the development of bronchial asthma. The major HDM allergens are classified into group 1 (Der f 1 and Der p 1) and group 2 (Der f 2 and Der p 2). Group 1 allergens are heat‐labile acidic glycoproteins and are found in HDM faeces. These proteins have papain‐like cysteine protease activity. Most of the predominant Der p 1 T‐cell epitopes for both human and mouse T cells are located within the sequence p45–145 of the mature Der p 1 protein. The Der p 1 fragment was expressed as a secretory protein by fusing the glutelin signal and KDEL retention signal peptides under the control of the GluB‐1 endosperm promoter (Suzuki et al ., ). Using this system, the Der p 1 was mainly deposited in ER‐derived PB‐1 (Figure d). The therapeutic potential of transgenic rice containing this fragment was examined by oral administration in a murine model of asthma. Suppression of airway inflammation and reduction in serum antigen‐specific IgE level were observed in mice given the transgenic rice (Suzuki et al ., ). The production of Th2‐type cytokines and Der p 1‐specific CD4 + T‐cell proliferation were significantly reduced after oral vaccination with the transgenic rice. Allergen‐induced infiltration of eosinophils and neutrophils into the airways and bronchial hyper‐responsiveness were also inhibited by oral vaccination in this model. Der f 2 Group 2 HDM allergen is found in mite faeces. Der f 2 contains three disulphide bonds (Cys8–Cys119, Cys21–Cys27 and Cys73–Cys78), two of which (Cys8–Cys119 and Cys73–Cys78) are critical for its IgE binding capacity (Takai et al ., ). T‐cell epitopes of the group 2 allergens are distributed over the entire protein. Three Der f 2 derivatives were generated by mutagenizing cysteine residues: ΔC lacked all three disulphide bonds, C8/119S lacked the Cys8–Cys119 bond, and 8‐119C lacked the Cys21–Cys27 and Cys73–Cys78 bonds. Binding activity with HDM‐specific IgE was markedly decreased in ΔC, followed by C8/119S and 8‐119C. Three lines of transgenic rice expressing these Der f 2 derivatives were established. Der f 2 derivatives aggregated and formed a PB‐like structure, named Der f 2 body, which were distinguishable from PB‐I and PB‐II by their electron density and morphology (Figure e–g). Oral administration of Der f 2 transgenic rice containing C8/119S, 8‐119C or a mixture of both to Der f 2‐immunized mice inhibited allergen‐induced IgE and IgG responses (Yang et al ., ), suggesting the potential of this transgenic rice to treat HDM‐mediated allergic responses. Interestingly, IgE and IgG responses were not affected by Der f 2 transgenic rice containing ΔC. ΔC was water soluble and rapidly degraded by digestive enzymes in comparison with the other Der f 2 derivatives (Yang et al., ). These data strongly suggest that the digestibility of allergen is critical for the efficacy of oral IT using transgenic rice. Bet v 1 Bet v 1 is a major birch pollen allergen that belongs to the PR 10 protein group. More than 90% of birch pollen allergy patients are sensitive to Bet v1‐specific IgE. A versatile hypo‐allergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen‐specific immunotherapy. Hypo‐allergenic Bet v 1 tolerogens against birch pollen allergy were selected by DNA shuffling of 14 types of Fagales tree pollen allergens (Wallner et al ., ). The resulting chimeric molecules, TPC7 and TPC9, displayed low allergenicity and high immunogenicity compared with the native Bet v 1. Hypo‐allergens TPC7 and TPC9 and native Bet v 1 were expressed in transgenic rice seed as secretory proteins using the N‐terminal signal peptide and the C‐terminal KDEL tag under the control of an endosperm‐specific GluB‐1 promoter as an oral vaccine (Ogo et al ., ; Wang et al ., ). The products accumulated as glycoproteins with high‐mannose‐type N‐glycan, but without β1,2‐xylose or α1,3‐fucose. TPC7 was deposited as a novel, giant spherical ER‐derived protein body, >20 μm in diameter, which was referred to as the TPC7 body (Figure h). When transgenic rice containing hypo‐allergenic TPC7 and native Bet v 1 were orally administered to allergic model mice, infiltration of eosinophils and total inflammatory cells in the conjunctiva was decreased, resulting in a reduction in clinical symptom scores compared with mice fed nontransgenic rice. Rheumatoid arthritis Rheumatoid arthritis is an autoimmune disease associated with the recognition of self‐proteins expressed in arthritic joints. Parenteral administration of altered peptide ligands (APLs) of type II collagen (CII256‐271) can suppress the development of collagen‐induced arthritis (CIA) (Ohnishi et al ., ; Wakamatsu et al ., ). CII256‐271, APL6 and APL7 peptides were expressed as fusion proteins with the rice storage protein glutelin in the endosperm of transgenic rice seed. These transgene products successfully and stably accumulated at high levels (7–24 mg/g seeds) in PB‐II of mature seeds. The efficacy of these transgenic rice seeds in suppressing the development of CIA was examined by oral administration of the seeds to CIA model mice. Vaccination of sensitized mice with APL6 transgenic rice for 14 days significantly inhibited the development of arthritis (based on clinical score) and delayed disease onset during the early phase of arthritis. These effects were mediated by the induction of IL‐10 from CD4 + CD25 + T cells against CII antigen in splenocytes and inguinal lymph nodes (iLNs). Similarly, APL7 transgenic rice given for 14 days prior to immunization with CII prevented inflammation and erosion in the joints (Iizuka et al ., , b ). Future perspectives Human glucocerebrosidase for the treatment of Gaucher's disease known as taliglucerase alfa or Elelyso, which is produced in carrot cell culture, was approved for use in humans as the first plant‐made pharmaceutical in 2012 by Food and Drug Administration. At present, several pharmaceuticals products (HIV‐neutralizing monoclonal antibodies, human insulin, apolipoprotein Al, lactoferrin, etc.) derived from transgenic plants including seeds cultivated in open field have entered into human clinical trials, whereas several plant‐derived recombinant proteins for non‐biopharmaceuticals such as avidin and trypsin produced in maize seeds (ProdiGene), human albumin, lactoferrin and lysozyme produced in rice seeds (Ventria), cytokines and growth factors produced in barley seeds (ORF) are commercialized for diagnostics, research regent, cell culture supplement and cosmetic ingredient (Sack et al ., ). Different from the latter non‐biopharmaceuticals, it is critical that all of the biopharmaceutical products used for clinical trials have been purified according to the draft regulatory guidance of the GMP‐compliant manufacturing processes. Adaptation of intact or partially purified formulations as rice seed‐based oral vaccines remarkably decreases the production cost, as downstream purification processes, which represent up to 80% of overall production cost, are avoided. Moreover, oral administration permits the use of nonpurified vaccines (crude preparation of vaccines). It is important to note that oral vaccination elicits mucosal and humoral immune responses. As most pathogens invade the host through the mucosal route, protection at mucosal sites as a front line is thought to be the most effective means to prevent infectious diseases. On the other hand, antigen administration via the oral route is prone to invoke immune tolerance against the administered antigen, which can be exploited in the development of effective and safe oral allergy vaccines via allergen‐specific immune tolerance using safe tolerogens (hypo‐allergens and T‐cell epitopes). It is proposed that transgenic rice expressing antigens should be produced under the control of specific regulations and guidelines for practical use. Regulatory guidance for the production of recombinant pharmaceutical proteins in plants exists only as draft legislation, and this is based on the existing regulations for mammalian cells (Sabalza et al ., ). This legislation is inappropriate for applications involving whole plants. Rice seed‐based oral vaccines are a new form of medical drug that has to be manufactured according to GMP standards. Nonpurified crude biologics such as plant‐based edible vaccines have not yet been commercialized. The only approved poultry vaccine against Newcastle disease was produced from cultured transgenic tobacco cells and then purified. Presently, there are no guidelines or examples for developing (commercializing) such crude pharmaceutical proteins (Spök et al ., ). Before human clinical trials using transgenic rice grains and their crude extracts as oral vaccines are undertaken, it is necessary to establish production processes that can assure identical quality according to GMP guidelines (CPMP, , ; Fischer et al ., ). One concern is maintenance of equivalence of antigen in seeds because expression levels may vary among plants (seeds) and among generations when cultivated in open field under natural, but variable, environmental conditions. It is necessary to establish a production (cultivation) system in which seed quality (the content of the vaccine antigen accumulated in rice seeds) is kept constant by controlling the nitrogen fertilizer; such cultivation systems to control the desired seed protein level were established for rice in Japan. However, GMP regulations cannot be strictly applied to production processes of transgenic plants with cultivation in open fields. It may be reasonable to cultivate transgenic rice according to the guidelines of good agricultural practice (GAP) that conform to GMP‐like regulations. Standard operation protocols for transgenic rice cultivation during the process from sowing to harvesting have to be prepared. Of course, downstream processing of seed grains after harvesting has to be performed according to GMP conditions. Another major concern is gene transfer by pollen‐mediated outcrossing with any nontransgenic rice cultivated in the near vicinity. Although the percentage of outcrossing is very low for rice due to its capacity for self‐pollination and the short life of rice pollen, the possibility remains. To prevent gene transfer to nontransgenic rice, geographical isolation and separate plantings have to be established. It is also important to consider biological containment such as male sterility or cleistogamy (self‐fertilization without flower opening). Contamination may also occur by direct mixing during the harvest and distribution. Prevention of contamination in food and feed chains is strictly demanded by consumers and environmental organizations. It would be helpful to use discriminative markers such as colour‐coded rice to ensure traceability. Therefore, specific cultivation guidance and regulation for cultivation of plant‐made pharmaceuticals crops in open field has to be established with zero tolerance for contamination in food and feed chains in Japan. Acknowledgement This work was supported by a grant from the Agri‐Health Translational Research Project from the Ministry of Agriculture Forestry and Fisheries of Japan.

Journal

Plant Biotechnology Journal – Wiley

Published: Oct 1, 2015

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