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Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody
... The plant secondary metabolomics as an adjuvant has privilege over the chemical adjuvants based on the fact that certain plant species can have second metabolites that can exert certain medicinal properties (especially in traditional medicine approaches) and synergic effects to the plant oral vaccines [44,47]. Secondary metabolites such as Polysaccharides, Saponins, and Lectins have been used as plant-derived adjuvants in previous studies and have been reviewed in the article by Rosales-Mendoza et al. [14]. ...
The recombinant vaccinal proteins can provide a solution for prevention of the contagious diseases, especially in deprived countries. While using eukaryotic platforms such as E. coli lack the post-translational modifications for expression of the proteins and using mammalian cells and plant cells encounter a labor-intensive and continuous process of cell culture and the final product requires special transportation logistics, the plant seeds can provide a good solution for expression of the vaccinal proteins as an oral vaccine. Seeds contain a low amount of water content and can show a considerable bioencapsulation to protect the recombinant products from environmental degradation. However, for the expression of a recombinant protein, there are steps and considerations that should be considered. These considerations include the choice of an appropriate seed to be used as an expression platform (bioreactor), the choice of the promoter, adjustment of the codon preference, targeting the protein for in the cell and finally purification of the vaccinal protein in case of need. This article aims to describe the details of the recombinant vaccine production in the seed platform with the focus on oral delivery of the vaccine at a glance.
... Purified dried silica NPs are treated with aqueous solutions of proteins for 1 h. This allows the adsorption of protein in microporous silica mainly driven by non-electrostatic interactions (Clemments et al. 2015;Cárdenas et al. 2014) in comparison to the electrostatic interactions (Tu et al. 2016;Wu et al. 2013;Hartono et al. 2009), because the isoelectric points of both proteins are close to neutral pH (Cabra et al. 2005;Yuki et al. 2016), as well as both zein and rice proteins are predominant hydrophobic proteins. The extracted protein fractions thus obtained are analyzed to determine the molecular mass and their relative extracted amounts are presented in Fig. 6. ...
Monodisperse non-hybrid silica and hybrid colloidal silica of ≤ 200 nm decorated with small Au nanoparticles (NPs) were synthesized in a simple single-step method. Non-hybrid silica NPs were synthesized in the absence and presence of different twin tail cationic surfactants, while tiny Au NPs were grown under in situ reaction conditions on non-hybrid silica synthesized previously by using cationic dextran. Bio-applicability and cytotoxicity of both hybrid as well as non-hybrid silica NPs were tested by using them for the extraction of protein fractions from complex aqueous protein solutions and treating them with blood cells, respectively. Both non-hybrid and hybrid silica NPs demonstrate excellent ability to extract proteins fractions predominantly of relatively low molecular masses, i.e., ~ 80 kDa. Extraction preferences between both kinds of silica became prominent when predominantly hydrophobic proteins such zein and rice proteins were used rather than mainly polar protein like BSA. Applicability for more complex biological fluid like serum indicated the competitive extractions among strongly versus weakly bound proteins. With significant bearing in in vivo conditions, hybrid silica was potentially toxic towards the blood cells in comparison to non-hybrid silica. It stems from the collective interactions of silica as well as nanometallic surfaces of Au NPs to interact with the blood cells causing hemolysis and hence may not be the suitable vehicles for drug release in systemic circulation.
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We previously established the selection-marker-free rice-based oral cholera vaccine (MucoRice-CTB) line 51A for human use by Agrobacterium -mediated co-transformation and conducted a double-blind, randomized, placebo-controlled phase I trial in Japan and the United States. Although MucoRice-CTB 51A was acceptably safe and well tolerated by healthy Japanese and U.S. subjects and induced CTB-specific antibodies neutralizing cholera toxin secreted by Vibrio cholerae , we were limited to a 6-g cohort in the U.S. trial because of insufficient production of MucoRice-CTB. Since MucoRice-CTB 51A did not grow in sunlight, we re-examined the previously established marker-free lines and selected MucoRice-CTB line 19A. Southern blot analysis of line 19A showed a single copy of the CTB gene. We resequenced the whole genome and detected the transgene in an intergenic region in chromosome 1. After establishing a master seed bank of MucoRice-CTB line 19A, we established a hydroponic production facility with LED lighting to reduce electricity consumption and to increase production capacity for clinical trials. Shotgun MS/MS proteomics analysis of MucoRice-CTB 19A showed low levels of α-amylase/trypsin inhibitor-like proteins (major rice allergens), which was consistent with the data for line 51A. We also demonstrated that MucoRice-CTB 19A had high oral immunogenicity and induced protective immunity against cholera toxin challenge in mice. These results indicate that MucoRice-CTB 19A is a suitable oral cholera vaccine candidate for Phase I and II clinical trials in humans, including a V. cholerae challenge study.
Allergen Glb33 is an important allergen in rice that can cause allergic reactions such as asthma and atopic dermatitis. However, knowledge of the content in rice is sparse. In the present work, an absolute protein quantification method was established for allergen Glb33 in rice samples using liquid chromatography-tandem mass spectrometry. After extraction of allergen Glb33 from rice grains using salt solution, the isotope-labelled peptide internal standard was added to the extract, followed by enzymatic digestion with trypsin. The signature peptide and its isotope-labelled analogue from the tryptic hydrolysates of allergen Glb33 and the internal standard were detected by liquid chromatography-tandem mass spectrometry. The quantitative bias caused by tryptic efficiency and matrix effect was corrected by using two isotope-labelled standard peptides. The method exhibited good linearity in the range of 1–200 nM, with coefficients of determination (R2) > 0.998. A high sensitivity was observed, with a limit of quantification of 0.97 nM. Mean recoveries obtained from different rice matrices ranged from 82.7%–98.1% with precision < 8.5% in intra-day trials (n = 6), while mean recoveries were from 75.1%–107.4% with precision < 14.6% in inter-day trials (n = 14). The developed method was successfully applied to the analysis of allergen Glb33 in 24 different rice cultivars.
Infectious diarrhea in children can be life-threatening and imposes a large economic burden on healthcare systems, therefore more effective prophylactic and therapeutic drugs are needed urgently. Because most of the pathogens responsible for childhood diarrhea infect the gastrointestinal mucosa, providing protective immunity at the mucosal surface is an ideal way to control pathogen invasion and toxic activity. Mucosal (e.g., oral, nasal) vaccines are superior to systemic (subcutaneous or intramuscular) vaccination for conferring both mucosal and systemic pathogen-specific immune responses. Therefore, great efforts has been focused on the development of cost-effective mucosal vaccines for the past 50 years. Recent progress in plant genetic engineering has revolutonized the production of inexpensive and safe recombinant vaccine antigens. For example, rice plant biotechnology has facilitated the development of a cold-chain-free rice-based oral subunit vaccine against Vibrio cholerae. Furthermore, this technology has led to the creation of a rice-based oral antibody for prophyalaxis and treatment of rotavirus gastroenteritis. This review summarizes current perspectives regarding the mucosal immune system and the development of mucosal vaccines and therapeutic antibodies, particularly rice-based products, and discusses future prospects regarding mucosal vaccines for children.
Two types of proteinaceous particles were observed under the electron microscope in the starchy endosperm of rice seeds. One was spherical with lamellar structure (PB-I), while the other was stained homogeneously by osmium tetroxide and not lamellar structured (PB-II). Both types of proteinaceous particles were effectively condensed from the homogenate of developing rice endosperm by an aqueous polymer two-phase system using dextran-DEAE dextran-polyethylene glycol. Separation of both types was carried out by sucrose density gradient centrifugation. These proteinaceous particles were recovered at specific gravities of 1.27 and 1.29 for PB-I and PB-II, respectively. The protein composition of these particles and their solubility fractionation were examined. Prolamin appeared in the PB-I fraction, whereas PB-II was rich in glutelin and globulin.
Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.
GM crops have great potential to improve food quality, increase harvest yields and decrease dependency on certain chemical pesticides. Before entering the market their safety needs to be scrutinized. This includes a detailed analysis of allergenic risks, as the safety of allergic consumers has high priority. However, not all tests currently being applied to assessing allergenicity have a sound scientific basis. Recent events with transgenic crops reveal the fallacy of applying such tests to GM crops.
Rotavirus infection has been the leading cause of gastroenteritis among children in Taiwan. Studies have shown that 40% of hospitalization for acute gastroenteritis can be prevented through the use of vaccines, including a live, attenuated monovalent rotavirus vaccine and a pentavalent, human-bovine reassortant rotavirus vaccine. In 2009, the World Health Organization suggested that rotavirus vaccine should be included in all national immunization programs. This review summarizes issues and recommendations discussed during an expert meeting in Taiwan. The recommendations included: (1) rotavirus vaccine should be offered to all healthy infants (including those without contraindications, such as immunodeficiency) at an appropriate age; (2) either monovalent or pentavalent vaccine can be administered concurrently with routine injected vaccines; (3) the administration of rotavirus vaccine must be administered at least 2 weeks prior to oral polio vaccination; (4) the first vaccine dose for infants should be administered between age 6 weeks and age 14 weeks 6 days and the course should be completed by age 8 months 0 day; (5) pentavalent vaccines can be administered at 2 months, 4 months, and 6 months while monovalent vaccines can be taken at 2 months and 4 months; (6) a combined use of monovalent and pentavalent vaccine is justified only when the previous dose is unavailable or unknown; and (7) rotavirus vaccines may be given to premature infants, human immunodeficiency virus infected infants and infants who have received or are going to receive blood products.
Glioblastoma is one of the most aggressive tumors with poor prognosis. Although various studies have been performed so far, there are not effective treatments for patients with glioblastoma.
In order to systematically elucidate the aberrant signaling machinery activated in this malignant brain tumor, we investigated phosphoproteome dynamics of glioblastoma initiating cells using high-resolution nanoflow LC-MS/MS system in combination with SILAC technology. Through phosphopeptide enrichment by titanium dioxide beads, a total of 6,073 phosphopeptides from 2,282 phosphorylated proteins were identified based on the two peptide fragmentation methodologies of collision induced dissociation and higher-energy C-trap dissociation. The SILAC-based quantification described 516 up-regulated and 275 down-regulated phosphorylation sites upon epidermal growth factor stimulation, including the comprehensive status of the phosphorylation sites on stem cell markers such as nestin. Very intriguingly, our in-depth phosphoproteome analysis led to identification of novel phosphorylated molecules encoded by the undefined sequence regions of the human transcripts, one of which was regulated upon external stimulation in human glioblastoma initiating cells.
Our result unveils an expanded diversity of the regulatory phosphoproteome defined by the human transcriptome.
Signal transduction systems are known to widely regulate complex biological events such as cell proliferation and differentiation. Because phosphotyrosine-dependent networks play a key role in transmitting signals, a comprehensive and fine description of their dynamic behavior can lead us to systematically analyze the regulatory mechanisms that result in each biological effect. Here we established a mass spectrometry-based framework for analyzing tyrosine phosphoproteome dynamics through temporal network perturbation. A highly time-resolved description of the epidermal growth factor-dependent signaling pathways in human A431 cells revealed a global view of their multiphase network activation, comprising a spike signal transmission within 1 min of ligand stimulation followed by the prolonged activation of multiple Src-related molecules. Temporal perturbation of Src family kinases with the corresponding inhibitor PP2 in the prolonged activation phase led to the down-regulation of the molecules related to cell adhesion and receptor degradation, whereas the canonical cascades as well as the epidermal growth factor receptor relatively maintained their activities. Our methodology provides a system-wide view of the regulatory network clusters involved in signal transduction that is essential to refine the literature-based network structures for a systems biology analysis.
Cereal proteins are known to cause allergic reactions such as Baker's asthma and severe atopic dermatitis to certain populations. In rice allergy, proteins with molecular masses of 14-16, 26, 33, and 56 kDa have been demonstrated to be potentially allergenic. In this study, to identify and characterize the 33-kDa allergen, designated Glb33, this protein was first purified to homogeneity, and its cDNA clone was isolated. When expressed in Escherichia coli, the recombinant Glb33 was shown to be as reactive as the native Glb33 with mouse IgG and patients' IgE antibodies to Glb33. The Glb33 cDNA coded for a protein of 291 amino acids with two 120-amino acid residue repeats, and the amino acid sequence showed similarity to glyoxalase I from various organisms, including human, plant, yeast, and bacterium. As expected, both native Glb33 purified from rice seeds and the recombinant protein had glyoxalase I activity that catalyzes condensation of methylglyoxal and glutathione into S-lactoylglutathione. However, Glb33 had a higher sequence identity to the bacterial glyoxalase I rather than to known plant and yeast enzymes. Both the Glb33 transcript and the protein were detected not only in maturing seeds of rice but also in its stem and leaf. Taken all together, the rice allergen, Glb33, was identified to be a novel type of plant glyoxalase I that is expressed in various plant tissues, including maturing seeds.
To estimate the global illness and deaths caused by rotavirus disease, we reviewed studies published from 1986 to 2000 on deaths caused by diarrhea and on rotavirus infections in children. We assessed rotavirus-associated illness in three clinical settings (mild cases requiring home care alone, moderate cases requiring a clinic visit, and severe cases requiring hospitalization) and death rates in countries in different World Bank income groups. Each year, rotavirus causes approximately 111 million episodes of gastroenteritis requiring only home care, 25 million clinic visits, 2 million hospitalizations, and 352,000-592,000 deaths (median, 440,000 deaths) in children <5 years of age. By age 5, nearly every child will have an episode of rotavirus gastroenteritis, 1 in 5 will visit a clinic, 1 in 65 will be hospitalized, and approximately 1 in 293 will die. Children in the poorest countries account for 82% of rotavirus deaths. The tremendous incidence of rotavirus disease underscores the urgent need for interventions, such as vaccines, particularly to prevent childhood deaths in developing nations.
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 μg 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
GM crops have great potential to improve food quality, increase harvest yields and decrease dependency on certain chemical pesticides. Before entering the market their safety needs to be scrutinized. This includes a detailed analysis of allergenic risks, as the safety of allergic consumers has high priority. However, not all tests currently being applied to assessing allergenicity have a sound scientific basis. Recent events with transgenic crops reveal the fallacy of applying such tests to GM crops.
Two types of proteinaceous particles were observed under the electron microscope in the starchy endosperm of rice seeds. One was spherical with lamellar structure (PB-I), while the other was stained homogeneously by osmium tetroxide and not lamellar structured (PB-II). Both types of proteinaceous particles were effectively condensed from the homogenate of developing rice endosperm by an aqueous polymer two-phase system using dextran-DEAE dextran-polyethylene glycol. Separation of both types was carried out by sucrose density gradient centrifugation. These proteinaceous particles were recovered at specific gravities of 1.27 and 1.29 for PB-I and PB-II, respectively. The protein composition of these particles and their solubility fractionation were examined. Prolamin appeared in the PB-I fraction, whereas PB-II was rich in glutelin and globulin.
Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between non-transgenic (NT) and RBP rice seeds were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only 2 protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded 7 spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.
Since 2006, the availability of two new rotavirus vaccines has raised enthusiasm to consider the eventual control and elimination of severe rotavirus diarrhea through the global use of vaccines. Rotavirus remains the most severe cause of acute diarrhea in children worldwide responsible for several hundred thousands of deaths in low income countries and up to half of hospital admissions for diarrhea around the world. The new vaccines have been recommended by WHO for all infants and in more than 47 countries, their introduction into routine childhood immunization programs has led to a remarkable decline in hospital admissions and even deaths within 3 years of introduction. Challenges remain with issues of vaccine finance globally and the problem that these live oral vaccines perform less well in low income settings where they are needed most. Ongoing research that will accompany vaccine introduction might help address these issues of efficacy and new vaccines and novel financing schemes may both help make these vaccines universally available and affordable in the decade.
Key message:
RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.
& Aims Rotavirus infection is a leading cause of morbidity and mortality in children younger than 5 years of age. Current treatment options are limited. We assessed the efficacy of a llama-derived, heavy-chain antibody fragment called anti-rotavirus protein (ARP1), in modifying the severity and duration of diarrhea in male infants with rotavirus infection.
We performed a double-blind, placebo-controlled trial of 176 male infants (6-24 months old) with severe rotavirus-associated diarrhea at Dhaka Hospital, Bangladesh. The infants were randomly assigned to groups given oral ARP1 (15-30 mg/kg/day, n=88) or placebo (maltodextrin, n=88) for a maximum of 5 days. The primary outcomes were severity (stool output) and duration of diarrhea and fecal excretion of rotavirus. Secondary outcomes were intake of oral rehydration salt solution, severity of vomiting, and serum levels of rotavirus-specific IgA.
In infants with only rotavirus infection, total cumulative stool output was 305.47g/kg body weight among those given placebo (n= 63) and 237.03g/kg body weight among those given ARP1 (n=61) (a difference of 68.44 g/kg body weight or 22.5%; 95% confidence interval, 18.27-118.59 g/kg body weight; P=.0079). There was a significant reduction in rate of stool output (g/kg/d) in the ARP1 group compared with the placebo group (61%; P=0.002). ARP1 had no significant effect in infants with concomitant infections or on any other measured outcomes. No adverse events could be linked to ARP1.
In a placebo-controlled trial, ARP1 reduced stool output in male infants with severe rotavirus-associated diarrhea. Clinicaltrials.gov number: NCT01259765.
To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRice-CTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be up-reregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application.
Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use.
A gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called -globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band inEco RI,Hind III, andBam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.
Although rice (Oryza sativa) is one of the most common cereals produced and consumed around the world, there have been only a few reports on immediate hypersensitivity reactions after ingestion of rice. Few clinical studies on rice allergy in Asia have been reported concerning rhinitis, asthma and atopic dermatitis. In this case study, we identify allergens presumably responsible for anaphylaxis after ingestion of rice in a German patient.
Prick-to-prick tests, determination of specific IgE and the basophil activation test (BAT) were performed to confirm IgE-mediated allergy. IgE reactivity was further analyzed by immunoblotting of protein extracts from cooked commercial rice products. Rice allergens were purified, subjected to N-terminal sequencing and characterized by IgE binding and IgE inhibition assays using additional sera from 8 subjects with sensitization to rice and/or a history of hypersensitivity symptoms after rice ingestion.
Prick-to-prick tests were positive to raw and cooked rice (basmati rice and long-grain rice) and preparations of different rice extracts. Specific IgE against rice (f9) was 1.87 kU(A)/l. The BAT showed specific IgE-mediated activation of basophils after stimulation with rice extracts. Four IgE-reactive rice proteins with an apparent molecular weight of 49, 52, 56 and 98 kDa were identified. Interestingly, only binding to the 56-kDa glycoprotein was at least partially independent from cross-reactive carbohydrate determinants (CCD), whereas IgE binding to the other rice proteins was completely inhibited by pre-incubation with the CCD MUXF derived from bromelain.
Yet unidentified high-molecular-weight allergens from rice seeds, predominantly a 56-kDa glycoprotein, seem to be responsible for anaphylaxis after consumption of rice in a German patient.
A new set of plasmids for plant transgenic studies was developed. Its strong point is that independent gene cassettes are connected within one binary vector by the restriction endonuclease-based technique only. Using the set, two overexpressing cassettes and three RNA interference (RNAi) cassettes were successfully introduced into rice. Our plasmid set is useful for producing commercial transgenic plants, especially in the case of rice.
Rotavirus vaccine has proved effective for prevention of severe rotavirus gastroenteritis in infants in developed countries, but no efficacy studies have been done in developing countries in Asia. We assessed the clinical efficacy of live oral pentavalent rotavirus vaccine for prevention of severe rotavirus gastroenteritis in infants in Bangladesh and Vietnam.
In this multicentre, double-blind, placebo-controlled trial, undertaken in rural Matlab, Bangladesh, and urban and periurban Nha Trang, Vietnam, infants aged 4-12 weeks without symptoms of gastrointestinal disorders were randomly assigned (1:1) to receive three oral doses of pentavalent rotavirus vaccine 2 mL or placebo at around 6 weeks, 10 weeks, and 14 weeks of age, in conjunction with routine infant vaccines including oral poliovirus vaccine. Randomisation was done by computer-generated randomisation sequence in blocks of six. Episodes of gastroenteritis in infants who presented to study medical facilities were reported by clinical staff and from parent recollection. The primary endpoint was severe rotavirus gastroenteritis (Vesikari score >or=11) arising 14 days or more after the third dose of placebo or vaccine to end of study (March 31, 2009; around 21 months of age). Analysis was per protocol; infants who received scheduled doses of vaccine or placebo without intervening laboratory-confirmed naturally occurring rotavirus disease earlier than 14 days after the third dose and had complete clinical and laboratory results were included in the analysis. This study is registered with ClinicalTrials.gov, number NCT00362648.
2036 infants were randomly assigned to receive pentavalent rotavirus vaccine (n=1018) or placebo (n=1018). 991 infants assigned to pentavalent rotavirus vaccine and 978 assigned to placebo were included in the per-protocol analysis. Median follow up from 14 days after the third dose of placebo or vaccine until final disposition was 498 days (IQR 480-575). 38 cases of severe rotavirus gastroenteritis (Vesikari score >or=11) were reported during more than 1197 person-years of follow up in the vaccine group, compared with 71 cases in more than 1156 person years in the placebo group, resulting in a vaccine efficacy of 48.3% (95% CI 22.3-66.1) against severe disease (p=0.0005 for efficacy >0%) during nearly 2 years of follow-up. 25 (2.5%) of 1017 infants assigned to receive vaccine and 20 (2.0%) of 1018 assigned to receive placebo had a serious adverse event within 14 days of any dose. The most frequent serious adverse event was pneumonia (vaccine 12 [1.2%]; placebo 15 [1.5%]).
In infants in developing countries in Asia, pentavalent rotavirus vaccine is safe and efficacious against severe rotavirus gastroenteritis, and our results support expanded WHO recommendations to promote its global use.
PATH (GAVI Alliance grant) and Merck.
Rotavirus gastroenteritis causes many deaths in infants in sub-Saharan Africa. Because rotavirus vaccines have proven effective in developed countries but had not been tested in developing countries, we assessed efficacy of a pentavalent rotavirus vaccine against severe disease in Ghana, Kenya, and Mali between April, 2007, and March, 2009.
In our multicentre, double-blind, placebo-controlled trial, undertaken in rural areas of Ghana and Kenya and an urban area of Mali, we randomly assigned infants aged 4-12 weeks without symptoms of gastrointestinal disorders in a 1:1 ratio to receive three oral doses of pentavalent rotavirus vaccine 2 mL or placebo at around 6 weeks, 10 weeks, and 14 weeks of age. Infants with HIV infection were not excluded. Randomisation was done by computer-generated randomisation sequence in blocks of six. We obtained data for gastrointestinal symptoms from parents on presentation to health-care facilities and clinical data were obtained prospectively by clinicians. The primary endpoint was severe rotavirus gastroenteritis (Vesikari score >or=11), detected by enzyme immunoassay, arising 14 days or more after the third dose of placebo or vaccine to end of study (March 31, 2009; around 21 months of age). Analysis was per protocol; infants who received scheduled doses of vaccine or placebo without intervening laboratory-confirmed naturally occurring rotavirus disease earlier than 14 days after the third dose and had complete clinical and laboratory results were included in the analysis. This study is registered with ClinicalTrials.gov, number NCT00362648.
5468 infants were randomly assigned to receive pentavalent rotavirus vaccine (n=2733) or placebo (n=2735). 2357 infants assigned to vaccine and 2348 assigned to placebo were included in the per-protocol analysis. 79 cases of severe rotavirus gastroenteritis were reported in 2610.6 person-years in the vaccine group, compared with 129 cases in 2585.9 person-years in the placebo group, resulting in a vaccine efficacy against severe rotavirus gastroenteritis of 39.3% (95% CI 19.1-54.7, p=0.0003 for efficacy >0%). Median follow-up in both groups was 527 days starting 14 days after the third dose of vaccine or placebo was given. 42 (1.5%) of 2723 infants assigned to receive vaccine and 45 (1.7%) of 2724 infants assigned to receive placebo had a serious adverse event within 14 days of any dose. The most frequent serious adverse event was gastroenteritis (vaccine 17 [0.6%]; placebo 17 [0.6%]).
Pentavalent rotavirus vaccine is effective against severe rotavirus gastroenteritis in the first 2 years of life in African countries with high mortality in infants younger than 5 years. We support WHO's recommendation for adoption of rotavirus vaccine into national expanded programmes on immunisation in Africa.
PATH (GAVI Alliance grant) and Merck.
To produce crops that are more tolerant to stresses such as heat, cold, and salt, transgenic plants have been produced those express stress-associated proteins. In this study, we used immunoproteomic and two-dimensional difference gel electrophoresis (2D-DIGE) methods to investigate the allergenicity of transgenic potatoes expressing Arabidopsis DREB1A (dehydration responsive element-binding protein 1A), driven by the rd29A promoter or the 35S promoter. Immunoproteomic analysis using sera from potato-allergic patients revealed several immunoglobulin E (IgE)-binding protein spots. The patterns of protein binding were almost the same between transgenic and non-transgenic potatoes. The IgE-binding proteins in potato were identified as patatin precursors, a segment of serine protease inhibitor 2, and proteinase inhibitor II by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS/MS. 2D-DIGE analysis revealed several differences in protein expression between non-transgenic potato and transgenic potato; those showing increased expression in transgenic potatoes were identified as precursors of patatin, a major potato allergen, and those showing decreased expression in transgenic potatoes were identified as lipoxygenase and glycogen (starch) synthase. These results suggested that transgenic potatoes may express slightly higher levels of allergens, but their IgE-binding patterns were almost the same as those of control potatoes. Further research on changes in protein expressions in response to environmental factors is required to confirm whether the differences observed in this study are due to gene transfection, rather than environmental factors.
The 2D-DIGE (2-Dimensional Fluorescence Difference Gel Electrophoresis) method was applied to proteomic phenotyping of natural variants in 10 varieties of rice (Nipponbare, Koshihikari, Sasanishiki, Akitakomachi, Hitomebore, Hinohikari, Kasalath, Rexark, Bleiyo, Cho-ko) from the world rice collection (WRC) in the GenBank of the National Institute of Agrobiological Sciences (NIAS), Japan. Salt-soluble protein extracts of Nipponbare brown rice were labeled with Cy2 fluorochrome and used as an internal standard. Protein extracts from nine other rice varieties were labeled with Cy3 or Cy5 fluorochrome and applied to 2D-PAGE (13cm gel length) analysis. Approximately 700 spots of rice proteins were observed. Fluorescence intensities of each of these spots for the nine rice varieties were expressed as relative ratios to that of Nipponbare. Statistical analysis revealed the spot numbers of five Japanese rice varieties above threshold five (relative ratio of each variety to Nipponbare exceeded 5-fold or was less than 1/5) to be less than three, while those of four varieties from other countries were more than five (especially, Kasalath which was 29 and Bleiyo which was 23). The 2D-DIGE method seems to be useful for analyzing natural varieties of different cultivars and also for comparing the expression of allergen proteins.
Electron microscopy showed that the two types of protein bodies (PB) in starchy endosperms of rice were formed differently during the period of storage protein accumulation. Two routes for the transport of storage protein from the site of synthesis at the rough endoplasmic reticulum (RER) to the site of accumulation were also proposed. PB-I, bound by a single membrane to which ribosomes were attached, was thought to develop inside the cisternae of RER, while the PB-II membrane was thought to originate from the vacuole.
In the wheat germ cell-free translation system, storage protein-related polypeptides of developing rice endosperms, including a precursor of glutelin and putative precursors of prolamin, were directed by membrane-bound polysomes but not by free-polysomes. Immunoassay of the total translation products directed by a PB fraction showed that 46% were storage protein-related polypeptides.
Rice storage proteins (prolamin) that accumulate in PB-I appear to be synthesized by membrane-bound polysomes attached to PB-I or RER and to pass through the membrane into the lumen where they aggregate and are deposited. The proteins (glutelin and globulin) that accumulate in PB-II, however, seem to be synthesized by membrane-bound polysomes as a large precursor and to become sequestered into the cisternal space of RER, from where they are transferred to the vacuolar precursor of PB-II.
1006 patients with typical and atypical lesions of atopic dermatitis (AD) were analysed statistically. The clinical severity was closely correlated to serum IgE values and RAST (radio-allergosorbent test) positivity. The frequency of RAST-positive antigens was significantly correlated with serum IgE values (gamma = 0.712; p < 0.01). The analysis of multiple correlation between the clinical severity and each RAST score for mite, egg white and rice antigens suggested a strong contribution of rice allergy to the development of severe AD. 25 patients with severe AD and positive rice-RAST were treated by rice exclusion diet. The results were as follows: 9 cases remarkably responsive, 10 cases moderately responsive and 6 cases unresponsive. The rice-RAST titre decreased most remarkably in the 1st group. The wheat-RAST titre also decreased in the 1st, in spite of taking wheat foods every day, but increased in the 3rd. A probable role of rice allergy in severe AD in Japan is discussed.
Genomic and two novel cDNA clones for rice seed allergenic protein (RA) belonging to the alpha-amylase/trypsin inhibitor family were isolated and their nucleotide sequences determined. Ten cysteine residues deduced from nucleotide sequences were completely conserved among three cDNA clones including a clone, RA17, reported previously. One genomic clone, lambda 4, contained two RA genes, RAG1 and RAG2. Although RAG1 was cloned at the 5' portion only, two RA genes were arranged divergently. Nucleotide sequencing and DNA blotting analyses showed that RA are encoded by a multigene family consisting of at least four members. The transcriptional initiation site of RAG1 was localized at A, 26 bp upstream of the putative translational initiation codon, ATG, by the primer extension assay. The putative TATA box and CAAT box existed about 45 bp and 147 bp upstream of the transcription initiation site, respectively. A conserved sequence (ATGCAAAA) which was similar to the sequence (TGCAAAA) identified in rice glutelin promoters was observed in the 5' region of the two genes. In addition, RNA blotting analyses provided that RA genes specifically expressed in ripening seed and their transcripts accumulated maximally between 15 and 20 days after flowering.
Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres. Except in the case of 'heavy chain' disease, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains or cloned VH domains. Here we investigate the presence of considerable amounts of IgG-like material of M(r) 100K in the serum of the camel (Camelus dromedarius). These molecules are composed of heavy-chain dimers and are devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, a finding that calls into question the role of light chains in the camel. Camel heavy-chain IgGs lack CH1, which in one IgG class might be structurally replaced by an extended hinge. Heavy-chain IgGs are a feature of all camelids. These findings open new perspectives in the engineering of antibodies.
Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley alpha-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker's asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.
Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.
Proteomic analysis of complex protein mixtures using proteolytic digestion and liquid chromatography in combination with tandem mass spectrometry is a standard approach in biological studies. Data-dependent acquisition is used to automatically acquire tandem mass spectra of peptides eluting into the mass spectrometer. In more complicated mixtures, for example, whole cell lysates, data-dependent acquisition incompletely samples among the peptide ions present rather than acquiring tandem mass spectra for all ions available. We analyzed the sampling process and developed a statistical model to accurately predict the level of sampling expected for mixtures of a specific complexity. The model also predicts how many analyses are required for saturated sampling of a complex protein mixture. For a yeast-soluble cell lysate 10 analyses are required to reach a 95% saturation level on protein identifications based on our model. The statistical model also suggests a relationship between the level of sampling observed for a protein and the relative abundance of the protein in the mixture. We demonstrate a linear dynamic range over 2 orders of magnitude by using the number of spectra (spectral sampling) acquired for each protein.
In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90 degrees C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80 degrees C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80 degrees C. Interestingly, addition of the dye to the denatured VHH-R2 at 80 degrees C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibody-antigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications.
Rotavirus disease kills approximately half a million children annually in developing countries and accounts for one third of hospitalizations for diarrhea worldwide.1 In 1999, global efforts to control the tremendous health burden of gastroenteritis suffered an abrupt and unanticipated setback. The first licensed rotavirus vaccine (RotaShield) was withdrawn from the U.S. market less than a year after its introduction because it was associated with an uncommon but potentially life-threatening adverse event, intussusception, at an estimated rate of 1 incident per 10,000 vaccine recipients.2 (Figure) Debate ensued over the possible use of this vaccine in developing countries, where the health . . .
Recent studies show that the lipid transfer protein (LTP), the major Rosaceae allergen in patients not sensitized to birch pollen, is a largely cross-reacting allergen. Moreover, it is a potentially hazardous allergen due to its stability upon thermal treatment and pepsin digestion. The present study reports 3 cases of rice-induced anaphylaxis in LTP-allergic patients. In vitro inhibition studies, carried out using LTP purified from both rice and apple as well as whole peach extract, show that LTP was the relevant allergen in these patients and demonstrate the cross-reactivity between rice LTP and peach/apple LTP.
Sensitization to foods varies in different countries reflecting a possible interaction of genetic factors, cultural and dietary habits. Rice is a major food consumed world wide and needs evaluation for IgE mediated reactions. The present study was carried out to identify rice allergy in patients of rhinitis and asthma and identify the allergenic proteins in raw and cooked rice. Of 1200 patients screened using standard questionnaire, 165 presented with history of rice allergy. Of these, 20 (12.1%) patients demonstrated marked positive skin prick test (SPT) and 13 showed significantly raised specific IgE to rice compared to normal controls. Double blind placebo controlled food challenge (DBPCFC) confirmed rice allergy in 6/10 patients. Immunoblot with hypersensitive individual patients' sera showed 14-16, 33, 56 and 60 kDa proteins as major IgE-binding components in rice. Boiled rice retained four IgE reactive proteins of 16, 23, 33 and 53 kDa. In summary, IgE-mediated rice allergy affects 0.8% [(0.42-1.58) at 95% CI] of asthma and rhinitis cases. The subjects with severe SPT reactions (4 mm or above) and specific IgE, 6.9 ng/ml to rice demonstrated positive blinded food challenge with clinical symptoms.
Over the past decade, a series of experimental strategies for mass spectrometry based quantitative proteomics and corresponding computational methodology for the processing of the resulting data have been generated. We provide here an overview of the main quantification principles and available software solutions for the analysis of data generated by liquid chromatography coupled to mass spectrometry (LC-MS). Three conceptually different methods to perform quantitative LC-MS experiments have been introduced. In the first, quantification is achieved by spectral counting, in the second via differential stable isotopic labeling, and in the third by using the ion current in label-free LC-MS measurements. We discuss here advantages and challenges of each quantification approach and assess available software solutions with respect to their instrument compatibility and processing functionality. This review therefore serves as a starting point for researchers to choose an appropriate software solution for quantitative proteomic experiments based on their experimental and analytical requirements.
Proteomic and allergenomic analyses of rice and potato proteins
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- R Nakamura
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A 33-kDa allergen from rice (Oryza sativa L. Japonica). cDNA cloning, expression, and identification as a novel glyoxalase I Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies
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allergen from rice (Oryza sativa L. Japonica). cDNA cloning, expression, and
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A probable involvement of rice allergy in severe type of atopic dermatitis in Japan
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Arrowheads show ARP1 or RAG2 antigens
- Pb-Ii
- Cw
PB-II, protein body II; CW, cell wall. Arrowheads show ARP1 or
RAG2 antigens.
Proteomic analysis of known and candidate rice allergens between non-transgenic and transgenic plants
- R Satoh
- R Nakamura
- A Komatsu
- M Oshima
- R Teshima
- B S Shorrosh
- L Wen
- K C Zen
- J K Huang
- J S Pan
- M A Hermodson
- K Tanaka
- S Muthukrishnan
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Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection
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- S Kurokawa
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A 33-kDa allergen from rice (Oryza sativa L. Japonica). cDNA cloning, expression, and identification as a novel glyoxalase I
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- S G Schad
- S Scheurer
- A Conti
- S Vieths
- G Gross
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Usui, Y., Nakase, M., Hotta, H., Urisu, A., Aoki, N., Kitajima, K., et al., 2001. A 33-kDa
allergen from rice (Oryza sativa L. Japonica). cDNA cloning, expression, and
identification as a novel glyoxalase I. J. Biol. Chem. 276, 11376e11381.
van der Linden, R.H., Frenken, L.G., de Geus, B., Harmsen, M.M., Ruuls, R.C., Stok, W.,
et al., 1999. Comparison of physical chemical properties of llama VHH antibody
fragments and mouse monoclonal antibodies. Biochim. Biophys. Acta 1431,
37e46.
Proteomic and allergenomic analyses of rice and potato proteins
- Teshima