PDF(Pubmed)
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.
摘要：
我们先前通过农杆菌介导的共转化建立了用于人类的无选择标记的基于水稻的口服霍乱疫苗（MucoRice-CTB）系51A，并进行了双盲，随机化，日本和美国的安慰剂对照I期试验。尽管MucoRice-CTB51A对健康的日本和美国受试者具有可接受的安全性和良好的耐受性，并诱导了CTB特异性抗体中和霍乱弧菌分泌的霍乱毒素，在美国试验中,由于MucoRice-CTB产量不足,我们仅限于6-g队列.由于MucoRice-CTB51A在阳光下不生长，我们重新检查了先前建立的无标记品系，并选择了MucoRice-CTB品系19A。线19A的Southern印迹分析显示CTB基因的单拷贝。我们对整个基因组进行了重新测序，并在1号染色体的基因间区域检测到了转基因。在建立了MucoRice-CTB系19A的主种子库后，我们建立了具有LED照明的水培生产设施，以减少电力消耗并提高临床试验的生产能力。MucoRice-CTB19A的shot弹枪MS/MS蛋白质组学分析显示低水平的α-淀粉酶/胰蛋白酶抑制剂样蛋白（主要水稻过敏原），这与第51A行的数据一致。我们还证明了MucoRice-CTB19A具有高的口服免疫原性，并在小鼠中诱导了针对霍乱毒素攻击的保护性免疫。这些结果表明，MucoRice-CTB19A是适合人类I期和II期临床试验的口服霍乱疫苗候选物，包括霍乱弧菌挑战研究。
