MUCOSAL IMMUNE RESPONSES OF A RECOMBINANT CHLAMYDIA TRACHOMATIS MOMP PROTEIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。该项目的目标是推动全球努力，为预防沙眼衣原体生殖器感染开发有效的治疗方法。沙眼衣原体的主要外膜蛋白(MOMP)一直是开发有效的衣原体感染疫苗的目标。我们的假设是，重组(R)MOMP与粘膜佐剂结合将引起与天然MOMP抗原相当的免疫反应。本研究克隆、表达了沙眼衣原体MoPn Nigg II株的MOMP，并将其与修饰的霍乱毒素作为黏膜佐剂(霍乱毒素亚单位A2B)进行了基因融合。将rMOMP基因克隆到含有ctxA2B基因的载体pUAB084中。将重组表达载体转化大肠杆菌BL21(DE3)，异丙基-D-硫代半乳糖苷(IPTG)诱导表达rMOMP-CTB融合蛋白。用SDS-PAGE分析表达的宿主细胞中是否存在MOMP-CTB融合蛋白。用抗霍乱毒素多克隆抗体和MOMP蛋白进行免疫印迹和酶联免疫吸附试验，以证实MOMP-CTB融合蛋白的存在。GM1-ELISA法验证MOMP-CTB融合蛋白的正确结合。用rMOMP-CTB融合蛋白免疫6组BALB/c小鼠。用重组MOMP-CTB、活沙眼衣原体或PBS经鼻或阴道免疫小鼠。采集血清/阴道冲洗液，用酶联免疫吸附试验检测抗体效价。鼻腔注射rMOMP-CTB融合蛋白抗体效价最高。RMOMP鼻腔免疫小鼠血清中以IgA、IgG2a、IgG2b亚型最多见。与包括活沙眼衣原体在内的其他免疫接种相比，这些组的免疫球蛋白同型滴度显著更高。这些早期的研究支持这样的假设，即rMOMP蛋白与粘膜佐剂偶联可以刺激粘膜免疫反应。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to advance the global effort toward developing effective therapeutic approaches for the prevention of Chlamydia trachomatis genital infections. The major outer membrane protein (MOMP) of C. trachomatis has been the target of extensive efforts to develop efficacious vaccines against chlamydial infection. Our hypothesis is that recombinant (r) MOMP coupled with a mucosal adjuvant will elicit immune responses that are comparable with the native MOMP antigen. In this study, MOMP of C. trachomatis strain MoPn Nigg II was cloned, expressed and then genetically fused with modified cholera toxin as a mucosal adjuvant (cholera toxin subunit A2B). The rMOMP gene was amplified and cloned into the plasmid vector pUAB084, which contained the ctxA2B gene. The plasmids were used to transform E. coli BL21 (DE3) and rMOMP-CTB fusion protein was expressed by isopropyl-?-D-thiogalactoside (IPTG) induction. Fractions of the expression host cells were analyzed for the presence of the MOMP-CTB fusion protein by SDS-PAGE. Polyclonal antibodies to cholera toxin and the MOMP protein were used in western blot and ELISA to confirm the presence of the MOMP-CTB fusion protein. GM1-ELISA was also performed to confirm the proper association of MOMP-CTB fusion protein. The rMOMP-CTB fusion protein was used to immunize six groups of BALB/c mice. Groups of mice were immunized intranasally or intravaginally with recombinant MOMP-CTB, live C. trachomatis, or PBS. Serum/vaginal washes were collected and the antibody titers were determined by ELISA. rMOMP-CTB fusion protein administered intranasally provided the highest antibody titer. The IgA, IgG2a, IgG2b isotypes were most prevalent in serum collected from mice immunized intranasally with rMOMP. IgA isotype titers were significantly higher in these groups compared to other immunizations including live C. trachomatis. These early studies support the hypothesis that rMOMP protein coupled to a mucosal adjuvant can stimulate mucosal immune response