Developement of live attenuated anti-Shigella vaccine based on molecular pathogenesis studies

细菌性痢疾由志贺氏菌引起，是世界上，特别是发展中国家严重肠道疾病的一个重要原因。因此，迫切需要有效的疫苗。几种有希望的候选疫苗正在进行不同程度的调查，但到目前为止还没有一种疫苗可用于公共卫生目的。近年来，志贺氏菌致病的遗传和分子基础以及宿主对细菌毒力因子的反应被阐明，这为构建基于合理策略的减毒活疫苗提供了可能。在感染过程的某个已知步骤所需的基因可以选择性地突变，以破坏与肠道上皮细胞的相互作用和/或在一般的肠道组织内存活。在对志贺氏菌致病特性进行遗传学和分子生物学研究的基础上，我们构建了福氏2a志贺氏菌Thya双突变株，并对其作为细菌性痢疾减毒活疫苗的候选疫苗进行了评价。病毒是扩散到邻近的上皮细胞所必需的，而Thya是入侵细菌繁殖所必需的。这些突变是通过插入Tn10失活，然后对四环素敏感的衍生物进行反选择而引入的。这种双重突变体完全保护了豚鼠免受角结膜炎的侵袭。给食蟹猴灌胃给药也是安全的，细菌学、组织病理学和免疫学检查表明，它对同源野生型福氏志贺氏菌株的攻击提供了不完全但非常显著的保护。

Bacillary dysentery, caused by Shigella, is a significant cause of serious enteric illness in the world, particularly in developing countries. Efficacious vaccines are thus urgently needed. Several promising vaccine candidates are at various levels of investigations, but to date none is available for public health purposes. Recently genetic and molecular basis of the pathogenicity of Shigella and host responses to the bacterial virulence factors have been elucidated, that have made it possible to construct a live attenuated vaccine based on rational strategies. The genes required at a certain known step of infection process can be selectively mutated to impair the interaction with intestinal epithelial cells and/or survive within intestinal tissues in general. Based on the genetic and molecular studies on Shigella pathogenic properties, we have constructed and evaluated a virG thyA double mutant of S.flexneri serotype 2a for utilization as a candidate live attenuated oral vaccine against bacillary dysentery. virG is required for spreading into adjacent epithelial cells and thyA essential for the multiplication of the invading bacteria. These mutations were introduced by insertional inactivation with Tn10 followed by counterselection for a tetracycline-sensitive derivative. This double mutant completely protected guinea pigs from provoking keratoconjunctivitis. It also appeared safe when administered intragastrically to cynomolgus monkeys and gave an incomplete but highly significant protection against a challenge by the homologous wild type shigella flexneri strain as judged by bacteriological, histopathological and immunological examinations.