MOLECULAR DEFINITION OF FILARIAL AND RELATED NONFILARIAL GENES AND PROTEINS

利用被认为对螺旋体丝虫感染具有免疫力的个体的血清，筛选了表达文库，并将四个最有希望的表达载体(酵母、基于细菌的表达系统)置于表达载体(酵母、细菌表达系统)中，并对重组蛋白进行了鉴定。这包括一种丝状的1，6-二磷酸缩醛酶，它不仅在动物中诱导强烈的免疫反应，而且也是假定免疫的个体T细胞的靶标。这种分子已被证明可以在小动物模型中诱导保护性免疫，并正在为在较大动物(奶牛和非人类灵长类动物)上进行疫苗试验做准备。针对该分子的单抗也被动地将保护性免疫反应传递给允许的动物。鉴定和表征那些来自丝虫幼虫阶段的分子在淋巴丝虫病中诱导保护性免疫也是我们工作的一大部分。利用新的基于T细胞和抗体的筛选技术，我们已经从班氏Wuchereria bancrofti幼虫的cDNA表达文库中鉴定出12个疫苗靶点。其中三个已经过度表达，每一个都被放置在基于DNA的疫苗构建中。目前正在进行检测，还开发了淋巴丝虫病的诊断抗原。为此，构建了成虫雌成虫表达文库，并用合适的血清进行了差异筛选。超过25个单独的重组体被确定为潜在的诊断，表达和大量定义明确的血清的广泛测试已经确定了四个重组体，它们似乎是淋巴丝虫病患者的特异性重组体。序列分析表明，3/4与HMG样蛋白相关和同源；1/4是独一无二的，与不同数据库中的任何蛋白质都不同源。Wb1.2和类HMG蛋白Wb5.4在几种不同的原核表达载体中得到了高效表达。然后使用部分纯化的蛋白作为免疫印迹分析的基础，使用来自班氏丝虫感染(n=36)和马来丝虫感染(n=9)以及来自非淋巴丝虫病感染(n=27)和非丝虫寄生虫感染(n=36)的个体的具有良好特征的血清。无寄生虫的正常人(n=15)也被研究。单独使用或联合使用这些重组子，灵敏度从65%到98%，特异度从89%到100%。此外，还合成了一组重叠的多肽，并将其用于绘制这两种蛋白的B细胞表位图，以期利用基于多肽的分析方法来诊断专利丝虫感染。LOA LOA诊断抗原的定义也采取了类似的策略。到目前为止，已经确定了四个候选诊断重组体，并正在进行现场测试。-丝虫病，盘尾丝虫病，淋巴丝虫病，LOA LOA，疫苗，班氏丝虫病，马来丝虫病，诊断，DNA免疫-人类受试者

Using sera from individuals identified as putatively immune to Onchocerca volvulus infections, cDNA expression libraries were been screened and the four most promising (as vaccine targets) have been placed in expression vectors (yeast, bacterial-based expression systems), and the recombinant proteins are being characterized. This includes a filarial 1,6 bis phosphate aldolase that not only induces a strong immune response in animals, but also is a target of T cells in putatively immune individuals. This molecule has been shown to induce protective immunity in small animal models and is being readied for vaccine trials in larger animals (cows and nonhuman primates). Monoclonal antibodies to this molecule also passively transfers a protective immune response to permissive animals.Identifying and characterizing those molecules from the larval stages of the filarial parasites that induce protective immunity in lymphatic filariasis has also remained a large part of our effort. Using novel T-cell-based and antibody-based screening techniques, we have identified 12 vaccine targets from Wuchereria bancrofti larval cDNA expression libraries. Three of these have been over-expressed and each has been placed in DNA-based vaccine constructs. Testing is underway.The development of diagnostic antigens for lymphatic filariasis has also been undertaken. To this end, a cDNA expression library from adult female parasites was constructed and differentially screened with appropriate sera. More than 25 individual recombinants have been identified as potentially diagnostic, and expression and extensive testing with large panels of well-defined sera has identified four recombinants that appeared to be specific for individuals with lymphatic filariasis. Sequence analysis indicated that 3/4 were interrelated and homologous to HMG-like proteins; one was unique and not homologous to any protein in the various databases. The unique polypeptide, termed Wb1.2 and one of the HMG-like proteins, Wb5.4, were expressed at high levels in several different prokaryotic expression vectors. Partially purified protein was then used as the basis for immunoblot assays using well- characterized sera from individuals with W. bancrofti infection (n=36) and B. malayi infection (n=9) as well as from individuals with nonlymphatic filarial infections (n=27), and nonfilarial parasitic infections (n=36). Normal individuals without parasites (n=15) were also studied. Using these recombinants alone or in combination gave sensitivities that ranged from 65 to 98% and specificities from 89 to 100%. In addition, sets of overlapping peptides have been synthesized and utilized to map the B-cell epitopes of these two proteins in the hopes of utilizing a peptide-based assay for diagnosis of patent filarial infection.A similar strategy has been taken for the definition of Loa loa diagnostic antigens. To date, four candidate diagnostic recombinants have been identified and are being field tested. - Filarial, onchocerciasis, lymphatic filariasis, Loa loa, vaccines, W. bancrofti, B. malayi, diagnosis, DNA immunization - Human Subjects