MOLECULAR DEFINITION OF FILARIAL AND RELATED NONFILARIAL GENES AND PROTEINS

利用对盘尾丝虫感染推定免疫的个体血清，筛选了cDNA表达文库，并将四种最有希望的（作为疫苗靶点）置于表达载体（酵母、基于细菌的表达系统）中，并对重组蛋白进行了表征。这包括一种丝状1,6二磷酸醛缩酶，它不仅在动物中诱导强烈的免疫反应，而且在假定的免疫个体中也是T细胞的靶标。该分子已在小型动物模型中显示可诱导保护性免疫，并正准备在大型动物（牛和非人灵长类动物）中进行疫苗试验。这种分子的单克隆抗体也被动地将保护性免疫反应传递给受容动物。在淋巴丝虫病中诱导保护性免疫的丝虫病寄生虫幼虫阶段的分子识别和表征也仍然是我们工作的重要组成部分。利用基于t细胞和抗体的新型筛选技术，我们从班氏乌氏杆菌幼虫cDNA表达文库中鉴定出12个疫苗靶点。其中三种已经过表达，每一种都被置于基于dna的疫苗结构中。测试正在进行中。淋巴丝虫病诊断抗原的研制也在进行中。为此，构建了成虫雌性寄生虫cDNA表达文库，并与合适的血清进行了差异筛选。超过25个单独的重组已被确定为潜在的诊断性重组，用大量明确定义的血清进行表达和广泛测试，已经确定了四种重组，似乎是淋巴丝虫病个体的特异性重组。序列分析表明，其中3/4与hmg样蛋白同源；其中一个是独特的，与各种数据库中的任何蛋白质都不同源。这种独特的多肽，称为Wb1.2和其中一种hmg样蛋白，Wb5.4，在几种不同的原核表达载体中高水平表达。然后，部分纯化的蛋白作为免疫印迹检测的基础，使用具有良好特征的血清进行免疫印迹检测，这些血清来自bancroffti W.感染（n=36）和B. malayi感染（n=9），以及来自非淋巴丝虫病感染（n=27）和非丝虫病寄生虫感染（n=36）。正常个体（n=15）无寄生虫。单独或联合使用这些重组蛋白的灵敏度在65%到98%之间，特异性在89%到100%之间。此外，已经合成了一组重叠肽，并利用它们来绘制这两种蛋白的b细胞表位，希望利用基于肽的检测方法来诊断未治愈丝虫病。对Loa Loa诊断抗原的定义也采取了类似的策略。迄今为止，已经确定了四种候选诊断性重组，并正在进行实地试验。-丝虫病，盘尾丝虫病，淋巴丝虫病，Loa Loa，疫苗，bancrofti， B. malayi，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