Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

利用噬菌体展示文库(来自感染幼虫L3和成年雌性)和已知可溶性人类受体的筛选，我们已经鉴定、克隆和鉴定了与人IL-5R、IL-10R和IL-13R结合的不同分子。我们称之为BM-IL5RBP的分子，马来丝虫IL5RBP(IL5RBP)以一种保持其与人类受体结合能力的方式高水平表达。针对预测免疫原肽的抗体抑制RBM-IL5BP与其人受体的结合，并已被用来(通过免疫EM)将BM-IL5RBP定位于感染期幼虫的表面。RBM-IL5BP本身不能延长人嗜酸性粒细胞的存活，但确实抑制了人IL5延长嗜酸性粒细胞存活的能力。 马来芽胞杆菌热休克蛋白12.6(BmHSP12.6)已被鉴定为与人IL-10R结合的分子。BmHSP12.6的结构分析表明，它有一个高度保守的α-晶体蛋白中心结构域，这是其他小分子热休克蛋白(HSPs)的特征，但因为。短的N-末端结构域和位于晶体蛋白结构域两侧的异常小的C-末端结构域似乎属于一类新的小HSPs。重组BmHSP12.6以剂量依赖的方式与人IL-10受体结合，并抑制人IL-10与其受体的结合。RBmHSP12.6在体外也能促进MC/9肥大细胞的生长和增殖，与huIL10相似，提示这种寄生虫可以主动合成一种可能调节人类免疫反应的分子。 有趣的是，我们还鉴定了马来丝虫L3的一种分泌产物，它对人嗜酸性粒细胞具有趋化作用。这种分泌产物通过G偶联受体CXCR3产生信号。 我们用分子方法明确地证明了恒温曼氏杆菌(Mansonella perstans，MP)含有内共生菌Wolbachia。利用已知的扩增其他丝虫沃尔巴克氏菌16S核糖体DNA的引物，发现了一条相同的1393bp条带，序列分析表明，沃尔巴克氏菌16S rDNA与其他丝虫线虫的16S rDNA序列最相似。通过系统发育树分析，沃尔巴克氏菌与其他沃尔巴克氏菌16S rDNA的亲缘关系显示，沃尔巴克氏菌与其寄生虫宿主的5S rRNA几乎相同。从含MP的全血中提取的蛋白中也含有沃尔巴克氏菌表面蛋白(WSP)。在MP的治疗试验之前，建立了MP沃尔巴克氏菌的定量方法，不仅用来证明微丝虫数量和沃尔巴克氏菌拷贝数之间的关系，而且还用来证明抗生素对清除MP沃尔巴克氏菌的效果。 作为许多工作者联盟的一部分，我们参与了过去一年发表的丝虫病基因组草稿的注释。 一个单独的蛋白质组学分析也已经完成，使用质谱仪与整个阶段特定的胰酶消化相结合。不仅已经完成了成年雄性、成年雌性、微丝虫和感染性幼虫的阶段特异性蛋白质组，而且我们还完成了沃尔巴克氏布鲁氏菌蛋白质组和排泄/分泌蛋白质组。这些分析已经提供了明确的证据，证明那些编码“假设蛋白质”的基因是真实的，并且已经识别了所有预测的开放阅读框架中的近70%。此外，分泌组分析揭示了意想不到的分泌蛋白，其中许多具有宿主免疫调节特性。 目前正在扩大对丝虫感染和类圆线虫的保护性免疫。用辐照幼虫免疫的小鼠和对SS感染有免疫力的人的血清进行亲和纯化，鉴定出6个候选抗原已在大肠杆菌、酵母和杆状病毒中表达。用一种来自大肠杆菌的重组蛋白进行测试，在小鼠模型中提供了50%的保护作用。 利用多重荧光素酶免疫印迹试验(LIP)已开发出对圆线虫、钉螺和Loa Loa感染的快速诊断。由于LOA感染需要更定量的诊断，因此通过构建新的LOA文库和进行EST生成，已经进行了新的诊断靶点的识别。已确定5个候选诊断基因，并将其克隆到适合于LIPs的载体中。

Using phage display libraries (from infective larvae L3 and adult females) Brugia malayi and screens with known soluble human receptors, we have identified, cloned and characterized distinct molecules that bind to the human IL-5R, IL-10R, and IL-13R. The molecule we term, Bm-IL5RBP Brugia malayi IL5Rbinding protein(IL5Rbp) has been expressed at high levels in a manner that retains its ability to bind to the human receptor. Antibodies raised to predicted immunogenic peptides inhibit the binding of rBm-IL5BP to its human receptor and have been used to localize (by immuno-EM) Bm-IL5RBP to the surface of the infective stage larvae. The rBm-IL5BP does not itself prolong the survival of human eosinophils, but does inhibit the ability of human IL5 to prolong eosinophil survival. B. malayi HSP12.6 (BmHSP12.6)has been identified and characterized as a molecule that binds to the human IL-10R. Structural analyses of BmHSP12.6 showed that it has a highly conserved alpha-crystallin central domain that is characteristic of other small heat shock proteins (HSPs) but because of . short N-terminal domain and an unusually small C-terminal domain flanking the crystallin domain appears to belong to a novel class of small HSPs. Recombinant BmHSP12.6 binds to huIL10R in a dose dependent fashion and inhibits the binding of human IL-10 (huIL10) to its receptor. rBmHSP12.6 also enhanced the growth and proliferation of MC/9 mast cells in vitro similar to huIL10, suggesting that this parasite actively synthesizes a moleclule that may modulate the human immune response. Interestingly, we have also identified a secreted product from Brugia malayi L3 that is chemotactic for human eosinophils. This secreted products signals through the G-coupled receptor CXCR3. We used a molecular approach to show definitively that the fialrial parasite, Mansonella perstans (Mp) contains the endosymbiont, Wolbachia. Using primers known to amplify the 16S ribosomal DNA of other filarial Wolbachiae, an identical 1393bp band was found that on sequence analysis demonstrated a single consensus sequence for Mp Wolbachia 16S rDNA that was most similar to Wolbachia sequences from other filarial nematodes.Phylogenetic dendrograms, examining the relationship of the Mp Wolbachia to other Wolbachia 16S rDNA, showed that the Wolbachia tracked almost identically to the 5S rRNA of their parasite host. Wolbachia surface protein (WSP) was also demonstrated in protein extracted from Mp-containing whole blood. In advance of a treatment trial of Mp, a method for the quantitation of Mp Wolbachia was developed and used to demonstrate not only a relationship between microfilarial numbers and Wolbachia copy numbers, but also to demonstrate the effect of antibiotic on ridding Mp of Wolbachia. As part of a consortium of many workers, we have been a part of the annotation of the filarial genome published in draft from this past year. A separate proteomic analysis has also been completed using mass spectrospcopy in tandem with whole stage-specific tryptic digestion. Not only has the stage specific proteomes been completed for adult males, adult females, microfilariae, and infective larvae, but we have also completed the Brugia Wolbachia proteome and the excretory/secretory proteome. These analyses have provided clear evidence that those genes encoding "hypothetical proteins" are real and have also identified close to 70% of all predicted open reading frames. Further the secretome analyses have revealed unexpected secreted proteins many of which have host-immunomodulatory properties. An expanded approach to protective immunity in both filarial infections and in strongyloides is underway. Antigens affinity purified using sera from either mice immunized with irradiated larvae that show close to 100% protection to challenge infection with S. strongyloides or humans demonstrating immunity to Ss infection have identified 6 candidates that have been expressed in E. coli, yeast and baculovirus. Testing with one E. coli-derived recombinant has provided 50% protection in a mouse model. Rapid diagnostics have been developed for Strongyloides, Onchocerca and Loa loa infections using mutiplexed luciferase immunoprecitation assays (LIPS). Because Loa loa infections are in need of a more quantitative diagnostic, identification of new diagnostic targets have been undertaken by constructing new Loa cDNA libraries and performing EST generation. 5 candidate diagnostics have been identified and cloned into vectors suitable for LIPS.