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Transgenic Crop Research and Development Center

Development of rice-based edible allergic vaccine

Japanese cedar (Cryptomeria japonica) pollen is a potent seasonal aeroallergen that is

spread over most areas of Japan in the early spring. C. japonica pollen causes cedar

pollinosis with rhinitis, asthma and conjunctivitis as clinical symptoms. About 20% of

the Japanese population is currently afflicted, and more than half of the Japanese

population has specific circulating immunoglobulin E (IgE) for cedar pollen allergens.

Two major allergens, designated Cry j 1 and Cry j 2, have been isolated from

Japanese cedar pollen and characterized in detail. More than 90% of cedar pollinosis

patients have specific IgE to the allergens.

Allergen-specific immunotherapy is the only treatment that can provide a cure for

cedar pollinosis. Conventional allergen-specific immunotherapy has been conducted by

subcutaneous administration of increasing doses of allergen preparations (intact

allergen) throughout a period of 3 to 5 years. This treatment is associated with

side-effects such as anaphylactic shock due to the presence of IgE-binding activity, and

pain caused by inflammation. A safe, easy and convenient treatment would thus be a

boon to public health.

Peptide immunotherapy using dominant T-cell epitopes has been shown to be a safe

and effective treatment for the control of IgE-mediated allergic diseases because of the

absence of specific tertiary structure or B-cell epitopes recognized by specific IgE. We

have demonstrated that two rice-based edible vaccines expressing either mouse T-cell

epitopes or seven-linked human dominant T-cell epitopes (7Crp), derived from Cry j 1

and Cry j 2, have successfully inhibited allergen-specific Th2-medated IgE responses in


mouse models These results strongly support the clinical feasibility of allergen-derived

peptide expressed in rice seed, and indicate that rice seed-based peptide vaccines can be

used as a new allergen-specific immunotherapy for treatment of airway allergy.

However, T cell epitopes differ from each other dependent upon the genotypes

according to varieties of HLA, so that identification of major T cell epitopes is

inevitable for application to peptide immunotherapy. This process is drawback of

peptide immunotherapy.

Furthermore, the clinical use of a rice seed-based edible vaccine with low IgE binding

activity for humans and other mammals affected by cedar pollinosis requires the

accumulation of allergen in rice seed at a pharmacolocally appropriate level.

In order to further expand the application of seed-based allergen-specific

immunotherapy for controlling Japanese cedar pollinosis, we generated transgenic rice

plants that specifically express the entire T cell epitope Cry j 1 peptide in seeds. We

expressed Cry j 1 either as an independent gene cassette or as a fusion molecule as an

alternative to T-cell epitope peptides. Our preliminary results showed that partial- or

full-length Cry j 1 peptide were barely accumulated in the endosperm of transgenic rice

seed even under the control of the strong rice endosperm-specific GluB-1 promoter.

However, higher levels of accumulation were achieved by expressing Cry j 1 as a fusion

protein with rice glutelin. Three overlapping fragments covering the entire Cry j 1

region were inserted into the highly variable C terminal region of the GluA-2 acidic

subunit in the GluA-2 pro-glutelin precursor (Fig. 1). The highest accumulation level of

the fusion protein reached about 15% of total seed protein, but fusion protein precursors

containing Cry j 1 with an altered structure were not post-translationally processed into

mature forms and thus aggregated with Cys-rich prolamins in protein body I (PB-I) of
seed endosperm tissues.

Transgenic plants have been used as attractive bioreactors for the production of

recombinant proteins including pharmaceuticals and industrial enzymes. Transgenic

plant systems offer several advantages over conventional competing systems such as

microbial and mammalian cell culture systems with regard to lower production cost,

easy control of production scale and low risk of contamination of mammalian pathogens.

Choosing the ideal host plant or tissue for expression of recombinant proteins is an

important factor. We investigated the tissue and intracellular localization suitable for

production of artificial recombinant 7Crp peptide. This artificial peptide could be only

accumulated in the endosperm tissue of transgenic rice plants irrespective of high

amounts of transcripts in vegetative tissues such as leaf and stem, when it was expressed

under the control of constitutive promoters such as rice AGPase large subunit and

maizeubiquitin-1 promoters (Fig. 2). These results indicate that endosperm tissue is the

best production platform, when foreign recombinant proteins are expressed in

transgenic rice.
BamH1 BglII HindIII Nco 1Sac1 EcoR 1
pAg7 hpt CaMV35S P GluB -1 P SP GluB -1 T
LB RB

1 353
pJ1full Cry j1 full KDEL

1 195
pJ1N -half Cry j1 N -half KDEL

135 353
pJ1C -half Cry j1 C -half KDEL

1 144
Cry j1 F1 Cry j1 F1
126 257

Cry j1 F2 Cry j1 F2
231 353
Cry j1 F3
Cry j1 F3 Con V1-F1 V2-F1 V2-F2 V2-F3

1 253 283 477


GluA2 acidic subunit GluA2 basic subunit GluA2-V1
SmaI SmaI
1 144
pV1-F1 Cry j1 F1
1 187 283 477
GluA2 acidic subunit GluA2 basic subunit GluA2-V2
SmaI SmaI

1 144
pV2-F1 Cry j1 F1

126 257
pV2-F2 Cry j1 F2
231 353
pV2-F3 Cry j1 F3

Fig. 1. Expression of Cry j 1 fragments in transgenic rice seed.


Codon optimized Cry j 1 fragments were expressed directly or as fusion proteins with
rice glutelin GluA-2 under the control of the 2.3 kb glutelin GluB-1 promoter.

Wild Type Ubi-7Crp AGPase-7Crp Wild Type Ubi-7Crp AGPase-7Crp

S L Sm S L Sm S L Sm S L Sm S L Sm S L Sm

75 k

50 k
37 k

25 k

20 k

15 k

10 k

Fig 2. Expression of 7Crp peptide in various tissues of transgenic rice plants.


7Crp gene was expressed under the control of constitutive ubiquitin and AGPase
promoters. S: maturing seed, L: leaf, Sm:stem

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