Curtiss R, Galan J E, Nakayama K, Kelly S M
Department of Biology, Washington University, St. Louis, MO 63130.
Res Microbiol. 1990 Sep-Oct;141(7-8):797-805. doi: 10.1016/0923-2508(90)90113-5.
Salmonella strains attenuated by various mutational alterations and expressing heterologous colonization and virulence antigens specified by cloned genes have begun to be widely used as vaccines for oral immunization to induce protective immunity against the pathogens supplying the genes for the colonization or virulence antigens. Problems associated with plasmid instability and/or poor expression of cloned gene products have frequently been encountered and regulatory agencies are now banning use of antibiotic resistance markers in live attenuated vaccine strains. We have therefore developed a balanced lethal host-vector system in which the chromosome of the attenuated vaccine strain contains a deletion mutation that impose a requirement for diaminopimelic acid (DAP), an essential constituent of the rigid layer of the cell wall of all Gram-negative and some Gram-positive microbes. The plasmid cloning vector contains the wild-type allele for this gene allowing the recombinant avirulent Salmonella to be independent of DAP. Since DAP is not prevalent in nature, especially in the animal host, essentially 100% of the surviving avirulent Salmonella recovered from an immunized animal host still contain the recombinant plasmid and express the foreign colonization or virulence antigen. Occasional loss of the plasmid renders the avirulent Salmonella DAP-requiring, which quickly results in DAPless death with lysis of the bacterium to release its antigenic contents, an occurrence which might further enhance the immune response to the foreign colonization or virulence antigen. We describe below strains of bacteria, recombinant vectors and the methods to make use of this system in a diversity of situations for development of live recombinant avirulent vaccines as well as for other potential applications.
通过各种突变改变减毒并表达由克隆基因指定的异源定植和毒力抗原的沙门氏菌菌株，已开始被广泛用作口服免疫疫苗，以诱导针对提供定植或毒力抗原基因的病原体的保护性免疫。经常遇到与质粒不稳定性和/或克隆基因产物表达不佳相关的问题，并且监管机构现在禁止在减毒活疫苗菌株中使用抗生素抗性标记。因此，我们开发了一种平衡致死宿主-载体系统，其中减毒疫苗菌株的染色体包含一个缺失突变，该突变要求二氨基庚二酸（DAP），DAP是所有革兰氏阴性菌和一些革兰氏阳性菌细胞壁刚性层的必需成分。质粒克隆载体包含该基因的野生型等位基因，使重组无毒沙门氏菌不依赖DAP。由于DAP在自然界中并不普遍，尤其是在动物宿主中，从免疫动物宿主中回收的基本上100%存活的无毒沙门氏菌仍然含有重组质粒并表达外源定植或毒力抗原。质粒偶尔丢失会使无毒沙门氏菌需要DAP，这会迅速导致无DAP死亡并伴有细菌裂解以释放其抗原成分，这种情况可能会进一步增强对外源定植或毒力抗原的免疫反应。我们在下面描述了细菌菌株、重组载体以及在各种情况下利用该系统开发减毒活重组疫苗以及其他潜在应用的方法。
