Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs).
Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs).
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DOI：
10.1371/journal.pntd.0004271
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发表时间：
2015-12
影响因子：
3.8
通讯作者：
Smales CM
中科院分区：
文献类型：
--
作者：
Rooney B;Piening T;Büscher P;Rogé S;Smales CM
对昏睡病和其他由着丝质体引起的疾病的快速血清诊断测试的发展依赖于可负担得起的寄生虫特异性重组抗原的生产。在这里，我们描述了从冈比亚氏锥虫（T.b. gambiense）和相关物种链托利什曼原虫（L. tarentolae）中生产重组抗原，并将其诊断敏感性和特异性与目前用于针对一组人类血清的诊断试剂盒的天然抗原进行比较。根据目前现场使用的诊断结果和蛋白质组学分析发现的免疫诊断抗原的最新发现，选择了一些冈比亚锥虫蛋白抗原候选物在tarentolae中进行重组表达。其中，不变性表面糖蛋白65 （ISG65）、变异表面糖蛋白VSG LiTat 1.3和VSG LiTat 1.5的胞外结构域与c端组氨酸标签融合，并在培养的重组猪乳杆菌培养基中以可溶性蛋白形式表达。使用亲和层析法，从培养物中纯化平均10 mg/L的重组蛋白，随后与对照组昏睡病患者（即生活在HAT流行国家的无昏睡病患者）的一组血清进行测试。对172例冈比亚锥虫非洲人锥虫病（HAT）患者和119例对照血清的评价显示，两种重组VSG和rISG65片段的曲线下面积在0.97 ~ 0.98之间，比天然VSG LiTat 1.3和VSG LiTat 1.5的0.98 ~ 0.99具有很高的诊断潜力（统计学差异无统计学意义）。对78名罗得西亚锥虫HAT患者和100名对照者的血清的评估显示，rISG65具有可接受的诊断潜力，曲线下面积为0.83。这些结果表明，这些重组抗原的组合有潜力用于下一代快速血清诊断试验。此外，L. tarentolae表达系统能够简单、廉价和高效地生产用于诊断、疫苗和药物发现研究的重组动原体蛋白，而不依赖于动物来产生材料。非洲昏睡病和其他由着丝质体引起的疾病的快速血清诊断测试的发展，部分依赖于可负担得起的寄生虫特异性重组抗原的生产。昏睡病的大多数病例是由寄生虫布氏冈比亚锥虫（冈比亚锥虫）引起的，这种寄生虫在被受感染的采采蝇叮咬时传播。现有的测试依赖于利用寄生虫的提取物或使用在动物模型中培养的抗原。在这项研究中，我们已经证明，使用一种由类似于冈比亚锥虫的寄生虫设计的细胞培养系统可以产生重组抗原，这种抗原在快速诊断测试中与从实验室啮齿动物中生长的冈比亚锥虫寄生虫纯化的天然抗原一样有效。我们比较了我们重组生产的抗原与目前用于诊断试剂盒的天然抗原对一组人血清的诊断敏感性和特异性。对172例冈比亚锥虫患者和119例无昏睡病的对照血清的评价显示，两种重组抗原的诊断潜力非常高，反应与本地抗原无显著差异。这些结果表明，这些重组抗原的组合有潜力用于下一代快速血清诊断试验。
The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials. The development of rapid serodiagnostic tests for African sleeping sickness and other diseases caused by kinetoplastids relies in part on the affordable production of parasite-specific recombinant antigens. The majority of cases of sleeping sickness are caused by the parasite Trypanosoma brucei gambiense (T.b. gambiense) which is transmitted when bitten by an infected tsetse fly. Existing tests rely on the utilisation of extracts from the parasite or use antigens raised in animal models. In this study we have shown that using a cell culture system devised from a parasite similar to T.b. gambiense recombinant antigens can be produced that are as effective in rapid diagnostic tests as the native antigens purified from T.b. gambiense parasites grown in laboratory rodents. We compared the diagnostic sensitivity and specificity of the antigens we produced recombinantly to native antigens currently used in diagnostic kits against a panel of human sera. The evaluation on sera from 172 T.b. gambiense patients and from 119 controls without sleeping sickness showed very high diagnostic potential of two recombinant antigens where the response was not significantly different to that from the native antigens. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests.