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MucoRice-CTB line 19A, a new marker-free transgenic rice-based cholera vaccine produced in an LED-based hydroponic system.

ABSTRACT: 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.

SUBMITTER: Yuki Y

PROVIDER: S-EPMC10978600 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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MucoRice-CTB line 19A, a new marker-free transgenic rice-based cholera vaccine produced in an LED-based hydroponic system.

Yuki Yoshikazu Y Kurokawa Shiho S Sugiura Kotomi K Kashima Koji K Maruyama Shinichi S Yamanoue Tomoyuki T Honma Ayaka A Mejima Mio M Takeyama Natsumi N Kuroda Masaharu M Kozuka-Hata Hiroko H Oyama Masaaki M Masumura Takehiro T Nakahashi-Ouchida Rika R Fujihashi Kohtaro K Hiraizumi Takashi T Goto Eiji E Kiyono Hiroshi H

Frontiers in plant science 20240315

We previously established the selection-marker-free rice-based oral cholera vaccine (MucoRice-CTB) line 51A for human use by <i>Agrobacterium</i>-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 <i>Vibrio cholerae</i>, we were limited to ...[more]

PMID: 38559768

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Project description:BackgroundWe have developed a rice-based oral cholera vaccine named MucoRice-CTB (Cholera Toxin B-subunit) by using an Agrobacterium tumefaciens-mediated co-transformation system. To assess the genome-wide effects of this system on the rice genome, we compared the genomes of three selection marker-free MucoRice-CTB lines with those of two wild-type rice lines (Oryza sativa L. cv. Nipponbare). Mutation profiles of the transgenic and wild-type genomes were examined by next-generation sequencing (NGS).ResultsUsing paired-end short-read sequencing, a total of more than 300 million reads for each line were obtained and mapped onto the rice reference genome. The number and distribution of variants were similar in all five lines: the numbers of line-specific variants ranged from 524 to 842 and corresponding mutation rates ranged from 1.41 × 10(-6) per site to 2.28 × 10(-6) per site. The frequency of guanine-to-thymine and cytosine-to-adenine transversions was higher in MucoRice-CTB lines than in WT lines. The transition-to-transversion ratio was 1.12 in MucoRice-CTB lines and 1.65 in WT lines. Analysis of variant-sharing profiles showed that the variants common to all five lines were the most abundant, and the numbers of line-specific variant for all lines were similar. The numbers of non-synonymous amino acid substitutions in MucoRice-CTB lines (15 to 21) were slightly higher than those in WT lines (7 or 8), whereas the numbers of frame shifts were similar in all five lines.ConclusionsWe conclude that MucoRice-CTB and WT are almost identical at the genomic level and that genome-wide effects caused by the Agrobacterium-mediated transformation system for marker-free MucoRice-CTB lines were slight. The comparative whole-genome analyses between MucoRice-CTB and WT lines using NGS provides a reliable estimate of genome-wide differences. A similar approach may be applicable to other transgenic rice plants generated by using this Agrobacterium-mediated transformation system.

Dataset Information

MucoRice-CTB line 19A, a new marker-free transgenic rice-based cholera vaccine produced in an LED-based hydroponic system.

Publications

MucoRice-CTB line 19A, a new marker-free transgenic rice-based cholera vaccine produced in an LED-based hydroponic system.

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