Induction of cellular immunity by oral inoculation of attenuated Salmonella as a live vaccine vector

我们通过使用ClpXP或Lon蛋白酶破坏的肠沙门氏菌血清型鼠伤寒沙门氏菌（S. Typhimurium）突变体进行小鼠感染研究，评估应激反应蛋白酶基因缺失突变体作为口服活疫苗菌株抗沙门氏菌感染的候选菌株的有效性。在体外，ClpXP蛋白酶调节鞭毛合成，缺乏ClpXP的突变株表现出超鞭毛细菌细胞。另一方面，Lon蛋白酶负调控侵袭上皮细胞的功效和侵袭基因的表达。5周龄BALB/c小鼠口服5 × 10^8集落形成单位（CFU）的ClpXP-或lon -缺陷菌株，接种1周后在脾脏、肠系膜淋巴结、Peyer’s patches和盲肠中检测到10^3 ~ 10^4集落形成单位的细菌，然后各组织中的细菌逐渐减少。血清和胆汁中脂多糖特异性免疫球蛋白G （IgG）和分泌型IgA （S-IgA）分别在接种后第4周和至少12周显著升高。用ClpXP-或lon -缺陷菌株免疫可以保护小鼠免受鼠伤寒沙门氏菌毒力菌株的口服攻击。攻击后5天，被攻击的毒沙门氏菌和免疫的无毒沙门氏菌都从脾脏、肠系膜淋巴结、佩耶氏斑甚至盲肠中完全清除。这些数据表明，atp依赖性蛋白酶ClpXP或长链断裂沙门氏菌可用于开发活疫苗菌株。

We evaluated the efficacy of stress response protease gene-deleted mutants as candidates for live oral vaccine strains against Salmonella infection by performing infection studies in mice using a ClpXP or Lon protease-disrupted mutant of Salmonella enterica serovar Typhimurium (S. typhimurium). In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyper-flagellated bacterial cells. On the other hand, the Lon protease negatively regulates the efficacy to invade epithelial cells and the expression of invasion genes. When five-week-old BALB/c mice were orally administered with 5 x 10^8 colony-forming units (CFU) of the ClpXP- or Lon-deficient strain, bacteria were detected with 10^3 to 10^4 CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum one week after inoculation, and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretary IgA (S-IgA) were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the S. typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum five days after the challenge. These data indicate that the ATP-dependent protease ClpXP- or Lon-disrupted Salmonella can be useful in development a live vaccine strain.