Characterization of Candidate Live Oral Salmonella typhi Vaccine Strains Harboring Defined Mutations in aroA, aroC, and htrA

The properties of two candidate Salmonella typhi-based live oral typhoid vaccine strains, BRD691 (S. typhi Ty2 harboring mutations in aroA and aroC) and BRD1116 (S. typhi Ty2 harboring mutations in aroA, aroC, and htrA), were compared in a number of in vitro and in vivo assays. BRD1116 exhibited an increased susceptibility to oxidative stress compared with BRD691, but both strains were equally resistant to heat shock. Both strains showed a similar ability to invade Caco-2 and HT-29 epithelial cells and U937 macrophage-like cells, but BRD1116 was less efficient at surviving in epithelial cells than BRD691. BRD1116 and BRD691 were equally susceptible to intracellular killing within U937 cells. Similar findings were demonstrated in vivo, with BRD1116 being less able to survive and translocate to secondary sites of infection when inoculated into the lumen of human intestinal xenografts in SCID mice. However, translocation of BRD1116 to spleens and livers in SCID mice occurred as efficiently as that of BRD691 when inoculated intraperitonally. The ability of BRD1116 to increase the secretion of interleukin-8 following infection of HT-29 epithelial cells was comparable to that of BRD691. Therefore, loss of the HtrA protease in S. typhi does not seem to alter its ability to invade epithelial cells or macrophages or to induce proinflammatory cytokines such as IL-8 but significantly reduces intracellular survival in human intestinal epithelial cells in vitro and in vivo.

本研究对两株基于伤寒沙门菌（Salmonella typhi）的口服活伤寒疫苗候选菌株的生物学特性开展了对比分析：分别为携带aroA与aroC基因突变的伤寒沙门菌Ty2菌株BRD691，以及携带aroA、aroC及htrA基因突变的伤寒沙门菌Ty2菌株BRD1116。实验通过多组体外与体内检测体系展开，结果显示，相较于BRD691，BRD1116对氧化应激的敏感性显著升高，但二者对热休克的抗性水平无明显差异。两菌株侵袭Caco-2、HT-29上皮细胞与U937巨噬细胞样细胞的能力相近，但BRD1116在上皮细胞内的存活效率低于BRD691；而二者在U937细胞内的胞内杀伤敏感性并无显著区别。体内实验得到了一致的结果：当将菌株接种至重症联合免疫缺陷小鼠（SCID mice）的人肠道异种移植肠腔时，BRD1116的存活能力及向继发感染部位移位的能力均弱于BRD691。但若采用腹腔接种途径，BRD1116与BRD691向小鼠脾脏及肝脏的移位效率并无显著差异。在感染HT-29上皮细胞后，BRD1116诱导白细胞介素-8（interleukin-8, IL-8）分泌的能力与BRD691相当。综上，伤寒沙门菌中HtrA蛋白酶的缺失似乎并不会改变其侵袭上皮细胞与巨噬细胞的能力，亦不会影响其诱导IL-8等促炎细胞因子的能力，但可显著降低其在体外及体内人肠道上皮细胞内的胞内存活能力。