Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.
Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.
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DOI：
10.1016/j.antiviral.2015.11.012
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发表时间：
2016-02
影响因子：
7.6
通讯作者：
Holtsberg FW
中科院分区：
文献类型：
--
作者：
Vu H;Shulenin S;Grolla A;Audet J;He S;Kobinger G;Unfer RC;Warfield KL;Aman MJ;Holtsberg FW
西非埃博拉病毒病（EVD）爆发规模空前，引起全世界对这种致命病毒传播的担忧。最近在非人灵长类动物 (NHP) 中的研究结果表明，抗体可以预防埃博拉病毒病。然而，抗体反应在疫苗介导的保护中的作用尚未完全了解。为了解决这些问题，需要进行定量血清学测定来测量对关键埃博拉病毒 (EBOV) 蛋白的抗体反应。使用参考检测抗体的血清学酶联免疫吸附测定 (ELISA) 的开发是为了标准化已接种疫苗的 NHP 或暴露于 EBOV 或使用 EBOV 疫苗免疫的人体中抗体水平的定量。生产了关键试剂来支持血清学 ELISA 的开发。重组 EBOV 基质蛋白 (VP40) 在大肠杆菌中表达并纯化。从哺乳动物细胞系统中表达并纯化了糖蛋白 (GP) 的两种变体，即缺乏跨膜结构域的胞外域 (GPΔTM) 和缺乏粘蛋白样结构域的工程化 GP (GPΔmuc)。使用这些蛋白质，开发并优化了三种 ELISA 方法，以提高重现性和稳健性，包括关键试剂的稳定性测试。该测定用于确定 NHP 疫苗研究中针对 VP40、GPΔTM 和 GPΔmuc 的抗体反应，该研究使用表达 GP、VP40 和核蛋白的 EBOV 病毒样颗粒 (VLP) 疫苗。此外，这些 ELISA 还用于成功检测 EBOV 感染者的人血清中对 VP40、GPΔTM 和 GPΔmuc 的抗体反应。
The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA’s), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in E. coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals.