Yuki Yoshikazu, Kurokawa Shiho, Sugiura Kotomi, Kashima Koji, Maruyama Shinichi, Yamanoue Tomoyuki, Honma Ayaka, Mejima Mio, Takeyama Natsumi, Kuroda Masaharu, Kozuka-Hata Hiroko, Oyama Masaaki, Masumura Takehiro, Nakahashi-Ouchida Rika, Fujihashi Kohtaro, Hiraizumi Takashi, Goto Eiji, Kiyono Hiroshi
Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
R&D department, HanaVax Inc., Chiba, Japan.
Front Plant Sci. 2024 Mar 15;15:1342662. doi: 10.3389/fpls.2024.1342662. eCollection 2024.
We previously established the selection-marker-free rice-based oral cholera vaccine (MucoRice-CTB) line 51A for human use by -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 , 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 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 challenge study.
我们之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水稻口服霍乱疫苗（MucoRice-CTB）51A株系，并在日本和美国进行了一项双盲、随机、安慰剂对照的I期试验。尽管MucoRice-CTB 51A对健康的日本和美国受试者而言安全性可接受且耐受性良好，并诱导产生了能中和霍乱弧菌分泌的霍乱毒素的CTB特异性抗体，但由于MucoRice-CTB产量不足，我们在美国的试验中只能采用6克剂量组。由于MucoRice-CTB 51A在阳光下无法生长，我们重新检查了之前建立的无标记株系并选择了MucoRice-CTB 19A株系。对19A株系的Southern印迹分析显示 基因有单拷贝。我们对全基因组进行了重测序，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 19A株系的主种子库后，我们建立了一个配备LED照明的水培生产设施，以降低电力消耗并提高用于临床试验的产量。对MucoRice-CTB 19A的鸟枪法MS/MS蛋白质组学分析显示，α-淀粉酶/胰蛋白酶抑制剂样蛋白（主要的水稻过敏原）水平较低，这与51A株系的数据一致。我们还证明，MucoRice-CTB 19A具有高口服免疫原性，并能在小鼠中诱导针对霍乱毒素攻击的保护性免疫。这些结果表明，MucoRice-CTB 19A是一种适合用于人类I期和II期临床试验（包括 攻击研究）的口服霍乱疫苗候选物。
