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

由志贺氏菌引起的细菌性痢疾是世界上严重肠道疾病的一个重要原因，特别是在发展中国家。因此迫切需要有效的疫苗。几种有希望的候选疫苗正处于不同程度的调查中，但迄今为止没有一种疫苗可用于公共卫生目的。近年来，志贺氏菌致病性的遗传和分子基础以及宿主对细菌毒力因子的反应已被阐明，这使得基于合理策略构建志贺氏菌减毒活疫苗成为可能。在感染过程的某个已知步骤中所需的基因可以选择性突变以破坏与肠上皮细胞的相互作用和/或在肠道组织中存活。在对志贺氏菌致病特性进行遗传和分子研究的基础上，我们构建并评价了福氏沙门氏菌血清型2a的virG - thyA双突变体，以作为拮抗细菌性痢疾减毒口服活疫苗候选株。virG是扩散到邻近上皮细胞所必需的，也是入侵细菌繁殖的必要条件。这些突变是通过Tn10的插入失活引入的，随后是对四环素敏感衍生物的反选择。这种双突变体完全保护豚鼠免于引发角膜结膜炎。经细菌学、组织病理学和免疫学检查，该疫苗对食蟹猴的胃内注射也是安全的，并且对同源野生型福氏志贺氏菌菌株的攻击具有不完全但高度显著的保护作用。

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.