Roberts M, Li J, Bacon A, Chatfield S
Department of Veterinary Pathology, Glasgow University Veterinary School, Glasgow G61 1QH, United Kingdom.
Infect Immun. 1998 Jul;66(7):3080-7. doi: 10.1128/IAI.66.7.3080-3087.1998.
We have found the in vivo-regulated nirB promoter (PnirB) to be effective for directing expression of a number of antigens in salmonella in vivo. We wished to determine if other in vivo-regulated promoters have utility for antigen expression in salmonella and to compare the effectiveness of these promoters with that of PnirB. To this end, we have devised a scheme that allows the promoter element of the PnirB-fragment C plasmid pTETnir15 to be swapped with other promoters of interest. We demonstrate the usefulness of this system by replacing PnirB with PhtrA to create plasmid pTEThtrA1. htrA is a stress response gene that is required for virulence of salmonella in mice and survival within macrophages. Expression of fragment C in Salmonella typhimurium BRD509 (aroA aroD) harboring pTEThtrA1 (strain BRD937) correlated with growth temperature in vitro. A comparison was made of the immune responses to fragment C elicited in mice immunized orally with BRD937 or BRD847 (BRD509/pTETnir15) or subcutaneously with purified fragment C plus alhydrogel. High levels of anti-fragment C antibodies that persisted for at least 12 weeks were present in all groups of mice. Vaccination with BRD937 was the most effective means of immunization: the serum immunoglobulin G (IgG), IgA, and IgM anti-fragment C titers were higher in the BRD937-immunized mice throughout the duration of the study than in mice in the other groups. The kinetics of the serum anti-fragment C responses were different in different groups. The response was most rapid in the BRD937 group, with the titers almost at peak levels at 2 weeks postimmunization. Only the mice immunized with BRD937 or BRD847 developed an intestinal IgA response to fragment C. Again, the response was superior in the BRD937 group. The peak of the intestinal response was delayed with respect to the serum response. Analysis of the IgG subtype response to fragment C revealed a dominant IgG2a response in the salmonella-immunized mice, indicating a type 1 helper T-cell response to fragment C, whereas the major subtype in the group parenterally immunized with fragment C plus alhydrogel was IgG1. The IgG1/IgG2a ratio was much higher in sera of BRD937-immunized mice than in sera of BRD847-immunized mice. At 15 to 20 weeks after immunization, the mice immunized with BRD937 or BRD847 were solidly immune to tetanus toxin and salmonella. The immune responses to fragment C seen in mice immunized with BRD937 are the strongest we have observed and indicate that the htrA promoter may be very useful for expressing foreign antigens in salmonella vaccine strains.
我们发现体内调控的nirB启动子（PnirB）可有效指导多种抗原在沙门氏菌体内的表达。我们希望确定其他体内调控启动子是否可用于沙门氏菌中抗原的表达，并将这些启动子与PnirB的有效性进行比较。为此，我们设计了一种方案，使PnirB片段C质粒pTETnir15的启动子元件能够与其他感兴趣的启动子进行交换。我们通过用PhtrA替换PnirB来创建质粒pTEThtrA1，证明了该系统的实用性。htrA是一种应激反应基因，对于沙门氏菌在小鼠体内的毒力以及在巨噬细胞内存活是必需的。在携带pTEThtrA1的鼠伤寒沙门氏菌BRD509（aroA aroD）（菌株BRD937）中，片段C的表达与体外生长温度相关。对口服BRD937或BRD847（BRD509/pTETnir15）免疫的小鼠或皮下注射纯化的片段C加氢氧化铝凝胶免疫的小鼠中引发的针对片段C的免疫反应进行了比较。所有小鼠组中均存在持续至少12周的高水平抗片段C抗体。用BRD937进行疫苗接种是最有效的免疫方式：在整个研究期间，BRD937免疫小鼠中的血清免疫球蛋白G（IgG）、IgA和IgM抗片段C滴度均高于其他组的小鼠。不同组中血清抗片段C反应的动力学不同。BRD937组的反应最为迅速，免疫后2周滴度几乎达到峰值水平。只有用BRD937或BRD847免疫的小鼠对片段C产生肠道IgA反应。同样，BRD937组的反应更优。肠道反应的峰值相对于血清反应有所延迟。对片段C的IgG亚型反应分析显示，沙门氏菌免疫小鼠中主要为IgG2a反应，表明对片段C有1型辅助性T细胞反应，而在皮下注射片段C加氢氧化铝凝胶免疫的组中主要亚型为IgG1。BRD937免疫小鼠血清中的IgG1/IgG2a比值远高于BRD847免疫小鼠血清中的比值。免疫后15至20周，用BRD937或BRD847免疫的小鼠对破伤风毒素和沙门氏菌具有稳固的免疫力。在用BRD937免疫的小鼠中观察到的对片段C的免疫反应是我们所观察到的最强反应，表明htrA启动子可能对在沙门氏菌疫苗菌株中表达外源抗原非常有用。
