MOLECULAR DEFINITION OF FILARIAL AND RELATED NONFILARIAL GENES AND PROTEINS

丝虫及相关非丝虫基因和蛋白质的分子定义

• 批准号: 6288864
• 负责人: THOMAS B NUTMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6288864
• 关键词: Filarioidae; Nematoda; Wuchereria; clinical research; enzyme linked immunosorbent assay; epitope mapping; filariasis; genetic library; helminthic antigen; human subject; immunity; microorganism genetics; molecular genetics; nucleic acid sequence; species difference

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

使用来自被确定为对盘尾丝虫感染具有推定免疫力的个体的血清，筛选了cDNA表达库，并将四个最有希望的（作为疫苗靶点）置于表达载体（酵母、基于细菌的表达系统）中，重组蛋白正在表征。这包括丝虫1，6二磷酸醛缩酶，其不仅在动物中诱导强烈的免疫应答，而且是脓毒症免疫个体中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，并且正在准备在较大动物（牛和非人类灵长类动物）中进行疫苗试验。针对该分子的单克隆抗体也被动地将保护性免疫应答传递给允许的动物。从丝虫幼虫阶段鉴定和表征在淋巴丝虫病中诱导保护性免疫的那些分子也仍然是我们努力的很大一部分。使用新的T细胞为基础的和抗体为基础的筛选技术，我们已经确定了12个疫苗的目标从班氏丝虫幼虫cDNA表达文库。其中三个已经过表达，并且每个都被置于基于DNA的疫苗构建体中。目前正在进行测试，并已着手研制淋巴丝虫病诊断抗原。为此，从成年女性寄生虫的cDNA表达文库的构建和差异筛选与适当的血清。超过25个单独的重组体已被确定为潜在的诊断，表达和广泛的测试与大面板的明确定义的血清已经确定了四个重组体，似乎是特定的个人与淋巴丝虫病。序列分析表明，3/4是相关的和同源的HMG样蛋白，一个是独特的，在各种数据库中的任何蛋白质都不同源。独特的多肽，称为Wb1.2和HMG样蛋白之一，Wb5.4，在几种不同的原核表达载体中以高水平表达。部分纯化的蛋白质然后被用作免疫印迹分析的基础，该免疫印迹分析使用来自W.班氏感染36例，B. B.马来人感染（n=9）以及非淋巴丝虫感染（n=27）和非丝虫寄生虫感染（n=36）的个体。还研究了无寄生虫的正常个体（n=15）。单独或组合使用这些重组体得到的灵敏度范围为65%至98%，特异性范围为89%至100%。此外，我们还合成了两组重叠肽，并利用它们绘制了B细胞抗原表位图，以期建立一种基于肽的丝虫病诊断方法，并将其应用于Loa loa诊断抗原的定义。到目前为止，已经确定了四种候选诊断重组体，并正在进行田间试验。- 丝虫病、盘尾丝虫病、淋巴丝虫病、罗阿罗阿丝虫病、疫苗、W.班克罗夫蒂湾，B.马来人，诊断，DNA免疫-人类受试者