Hauke Caitlyn A, Taylor Ronald K
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.
PLoS One. 2017 Apr 6;12(4):e0175170. doi: 10.1371/journal.pone.0175170. eCollection 2017.
The use of whole cell killed (WCK) oral cholera vaccines is an important strategy for cholera prevention in endemic areas. To overcome current vaccine limitations, we engineered strains of V. cholerae to be non-toxigenic and to express the protective protein colonization factor, toxin-coregulated pilus (TCP), under scale-up conditions potentially amenable to vaccine production. Two V. cholerae clinical strains were selected and their cholera toxin genes deleted. The tcp operon was placed under control of a rhamnose-inducible promoter. Production and stability of TCP were assessed under various conditions. The strains lack detectable cholera toxin production. The addition of 0.1% rhamnose to the growth medium induced robust production of TCP and TcpA antigen. The strains produced intact TCP in larger growth volumes (1 L), and pili appeared stable during heat-killing or acid treatment of the bacterial cultures. To date, no WCK cholera vaccines have included TCP. We have constructed putative strains of V. cholerae for use in a vaccine that produce high levels of stable TCP antigen, which has not previously been achieved.
使用全细胞灭活（WCK）口服霍乱疫苗是流行地区预防霍乱的一项重要策略。为克服当前疫苗的局限性，我们对霍乱弧菌菌株进行改造，使其不产毒素，并在可能适合疫苗生产的放大培养条件下表达保护性蛋白定居因子——毒素协同菌毛（TCP）。选择了两株霍乱弧菌临床菌株，并删除了它们的霍乱毒素基因。将tcp操纵子置于鼠李糖诱导型启动子的控制之下。在各种条件下评估TCP的产生和稳定性。这些菌株检测不到霍乱毒素的产生。向生长培养基中添加0.1%的鼠李糖可诱导TCP和TcpA抗原的大量产生。这些菌株在更大的培养体积（1升）中产生完整的TCP，并且在对细菌培养物进行热灭活或酸处理期间菌毛似乎是稳定的。迄今为止，尚无WCK霍乱疫苗包含TCP。我们构建了用于疫苗的假定霍乱弧菌菌株，这些菌株可产生高水平的稳定TCP抗原，而这是以前未曾实现的。
