Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB) under the control of the endosperm-specific expression promoter 2.3-kb glutelin GluB-1 with codon usage optimization for expression in rice seed. An average of 30 g of CTB per seed was stored in the protein bodies, which are storage organelles in rice. When mucosally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches and induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for >1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they require neither refrigeration (cold-chain management) nor a needle, these rice-based mucosal vaccines offer a highly practical and cost-effective strategy for orally vaccinating large populations against mucosal infections, including those that may result from an act of bioterrorism. mucosal immunity ͉ protein body ͉ oral vaccine ͉ IgA ͉ cholera toxin B subunit

How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >1011 -fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a "shutter" that cytoplasmic factors might open or close to regulate iron release in vivo.Ferritins are vesicle-like assemblies of 24 polypeptide (4-helix bundle) subunits that concentrate iron in cells by directing the formation of a ferric mineral in the hollow protein interior (8 nm diameter) (1-3). Effective cellular iron concentrations Ͼ1011 times the solubility of the ferric ion are achieved by ferritins, which are found in microorganisms, plants, and animals. The complexity and the sophistication of the genetic regulation of the ferritins, involving both DNA and mRNA (4 -7), emphasize the central role of iron and ferritin in life. Rates of Fe(II) oxidation, translocation of Fe(II) and Fe(III) (1.0 -2.0 nm), and mineralization are all controlled by the protein (1, 2). Fe(II) release from ferritin following reduction of the mineral is slow and poorly understood (8, 9) but is important for the biosynthesis of iron-proteins, such as those required in respiration, photosynthesis, nitrogen fixation, and cell division, (1, 2) and as dietary iron (10). How and where the iron exits from ferritin in vivo is not known.We now show that localized unfolding in the assembled protein, at sites of cooperative subunit interactions, can increase the rate of exit of iron from ferritin. When conserved leucine 134 was replaced by proline (L134P), the protein assembled, oxidized Fe(II), and mineralized Fe(III), but the time for complete dissolution of mineral (480 iron) in vitro was greatly decreased (5 min compared with 150 min for the parent protein). X-ray diffraction studies of crystals of H-L134P ferritin showed a flexible region localized near the termini of two subunit helices (C, D), which form the interfaces of subunit trimers and a channel. The results indicate that iron can exit from ferritin at the trimer subunit junction. A possible mechanism for regulated iron release in vivo could be localized disorder in the assembled protein, enhanced by cytoplasmic changes with effects analogous to the effect of H-L134P. EXPERIMENTAL PROCEDURES Expression and Purification of Recombinant Ferritin Proteins-The coding sequence for H ferritin, H-L134P, K82Q, and H-L134P, R86Qwere obtained by the mutagenesis of PJD5F12L134P sequence (12) with a Chameleon TM double-stranded, site-directed mutagenesis kit (Stratagene). The oligonucleotides, 5F12P134L (5Ј-CACCTGTTCCTC-CAGGTATTCAGTCTCC-3Ј), 5F12K82Q (5Ј-CGCTCTGGTTTCTGGA-CATCCTGCAG-3Ј), and 5F12R86Q (5Ј-CCCCATTCATCCTGCTCTG-GTTTCTTGACATCC-3Ј), were used as the...

Peptide immunotherapy using multiple predominant allergen-specific T cell epitopes is a safe and promising strategy for the control of type I allergy. In this study, we developed transgenic rice plants expressing mouse dominant T cell epitope peptides of Cry j I and Cry j II allergens of Japanese cedar pollen as a fusion protein with the soybean seed storage protein glycinin. Under the control of the rice seed storage protein glutelin GluB-1 promoter, the fusion protein was specifically expressed and accumulated in seeds at a level of 0.5% of the total seed protein. Oral feeding to mice of transgenic rice seeds expressing the T cell epitope peptides of Cry j I and Cry j II before systemic challenge with total protein of cedar pollen inhibited the development of allergen-specific serum IgE and IgG antibody and CD4 ؉ T cell proliferative responses. The levels of allergen-specific CD4 ؉ T cell-derived allergy-associated T helper 2 cytokine production of IL-4, IL-5, and IL-13 and histamine release in serum were significantly decreased. Moreover, the development of pollen-induced clinical symptoms was inhibited in our experimental sneezing mouse model. These results indicate the potential of transgenic rice seeds in production and mucosal delivery of allergen-specific T cell epitope peptides for the induction of oral tolerance to pollen allergens.Japanese cedar pollinosis ͉ peptide immunotherapy ͉ seed-specific expression