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Background: There are an estimated 1·3-4·0 million cases of cholera and 20 000-140 000 cholera-related deaths worldwide each year. The rice-based cholera toxin B subunit (CTB) vaccine, MucoRice-CTB, is an oral candidate vaccine that does not require a cold chain, has shown efficacy in animal models, and could be of benefit in places where there is a paucity of medical infrastructure. We aim to assess the safety, tolerability, and immunogenicity of MucoRice-CTB in humans., Methods: We did a double-blind, randomised, placebo-controlled, dose-escalation, phase 1 study at one centre in Tokyo, Japan. Eligible participants were healthy adult men with measurable serum and faecal antibodies against CTB at screening. Participants were excluded if they had allergy to rice; history of cholera or travellers' diarrhoea; poorly controlled constipation; abnormal results on hepatic, renal, or haematological screening tests; use of any over-the-counter drugs within 7 days before first administration; inability to use a medically acceptable means of contraception; or other reasons by medical judgment of the investigator. Three dose cohorts of participants were randomly assigned by block to receive oral MucoRice-CTB (1 g, 3 g, or 6 g) or placebo (1 g, 3 g, or 6 g), once every 2 weeks for 8 weeks (for a total of 4 doses). The dose groups were performed sequentially, and each dose cohort was completed before the higher dose cohort began. All medical staff, participants, and most trial staff were masked to treatment allocation. The primary outcomes were safety and tolerability, measured by 12-lead electrocardiogram; vital signs; haematology, biochemistry, and urinalysis; rice protein-specific serum IgE antibody concentration; and monitoring of adverse events. Participants were assessed at baseline and at 1, 2, 4, 6, 8, and 16 weeks after the first administration of vaccine or placebo. The safety analysis set included all participants enrolled in the trial who received at least one dose of the study drug or placebo and were compliant with good clinical practice. The full analysis population included all participants enrolled in the trial who received at least one dose of the study drug and for whom any data were obtained after the start of study drug administration. Meta-genomic analysis of study participants was performed using bacterial DNA from faecal samples before vaccination. This trial is registered with UMIN.ac.jp, UMIN000018001., Findings: Between June 23, 2015, and May 31, 2016, 226 participants were recruited and assessed for eligibility. 166 participants were excluded based on health condition or schedule. We then randomly selected 60 male volunteers aged 20-40 years who were enrolled and assigned to MucoRice-CTB (10 participants assigned to 1 g, 10 participants assigned to 3 g, and 10 participants assigned to 6 g), or placebo (10 participants assigned to 1 g, 10 participants assigned to 3 g, and 10 participants assigned to 6 g). All participants received at least one dose of study drug or placebo and were included in the safety analyses. Two participants given MucoRice-CTB 3 g and one participant given MucoRice-CTB 6 g were lost to follow-up and excluded from the efficacy analysis. Serum CTB-specific IgG and IgA antibody concentrations in participants who received 6 g MucoRice-CTB increased significantly in both a time-dependent and dose-dependent manner compared with those in the placebo groups (p for interaction=0·002 for IgG, p=0·004 for IgA). Genome analysis of subjects' faeces before vaccination revealed that compared to non-responders, responders had a gut microbiota of higher diversity with the presence of Escherichia coli and Shigella spp. 28 (93%) of 30 participants who received MucoRice-CTB at any dose had at least one adverse event during the study period, compared with 30 (100%) of 30 participants given placebo. Grade 3 or higher adverse events were reported in four participants in the MucoRice-CTB group (5 events) and four participants in the placebo group (10 events). The most common serious adverse event was haemoglobin decreased (2 events in 2 participants in the pooled MucoRice-CTB group, 2 events in 2 participants in the placebo group; all grade 3)., Interpretation: Participants given MucoRice-CTB showed increased CTB-specific serum IgG and IgA antibody concentrations without inducing serious adverse events, indicating that MucoRice-CTB could be a safe and potent vaccine to prevent diarrhoeal disease. MucoRice-CTB induced neutralising antibodies against diarrhoeal toxins in a gut microbiota-dependent manner. A similar phase 1 trial will be done with participants of other ethnicities to substantiate our findings., Funding: Translational Research Acceleration Network Program of Japan Agency for Medical Research and Development; Ministry of Education, Culture, Sports, Science and Technology, Japan; Science and Technology Research Partnership for Sustainable Development; Grant-in-Aid for Scientific Research (S) (18H05280) (to H K) from the Japan Society for the Promotion of Science (JSPS); Grant-in-Aid for Young Scientists (B) (16K16144) (to Y K) from JSPS; Grant-in-Aid for Young Scientists (18K18148) (to Y K) from JSPS; Grant from International Joint Usage/Research Center (K3002), the Institute of Medical Science, University of Tokyo., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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.

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.

Background: We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker–free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. Results: We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. Conclusions: All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Rice-expressed cholera toxin B (CTB) subunit is a cold-chain-free oral vaccine that effectively induces enterotoxin-neutralising immunity. We created another rice-based vaccine, MucoRice, expressing nontoxic double-mutant cholera toxin (dmCT) with CTA and CTB subunits. Western-blot analysis suggested that MucoRice-dmCT had the shape of a multicomponent vaccine. Oral administration of MucoRice-dmCT induced CTB- but not CTA-specific serum IgG and mucosal IgA antibodies, generating protective immunity against cholera toxin without inducing rice-protein-specific antibody responses. The potency of MucoRice-dmCT was equal to that of MucoRice-CTB vaccine. MucoRice has the potential to be used as a safe multicomponent vaccine expression system. [Copyright &y& Elsevier]