Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies.
Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies.
复制标题
DOI：
10.1128/msphere.01342-20
复制
发表时间：
2021-05-12
期刊：
影响因子：
4.8
通讯作者：
Kohara M
中科院分区：
文献类型：
--
作者：
Honda T;Gomi S;Yamane D;Yasui F;Yamamoto T;Munakata T;Itoh Y;Ogasawara K;Sanada T;Yamaji K;Yasutomi Y;Tsukiyama-Kohara K;Kohara M
流感病毒HA特异性抗体可通过血凝抑制（HI）试验、中和（NT）试验和酶联免疫吸附试验（ELISA）检测。然而，这些测定具有一些缺点，包括窄的动态范围和需要大量血清。抗体检测对于监测宿主对特定病原体抗原（Ags）的免疫应答和评估疫苗效力至关重要。荧光素酶免疫沉淀系统（LIPS）的开发用于灵敏地检测Ag特异性抗体在血清中从各种物种。在这项研究中，我们描述了NanoLIPS，一种基于NanoLuciferase（NLuc）的改进的LIPS检测，并采用该检测方法监测流感病毒感染或疫苗接种后的抗体反应。我们产生了用N-末端（N-NLuc-HA）或C-末端（C-NLuc-HA）NLuc报告基因标记的重组流感病毒血凝素（HA）蛋白。NLuc-HA产生的信噪比比采用重组HA-Gaussia princeps荧光素酶（GLuc）融合蛋白的LIPS测定高至少20倍。基于NanoLIPS的抗HA抗体检测产生了具有宽动态范围的高度可重复的结果。用表达流感病毒HA蛋白的重组牛痘病毒DIs株（rDIs-HA）接种的小鼠产生的针对C-NLuc-HA的抗体水平与rDIs-HA疫苗引起的保护作用显著相关。C-NLuc-HA经历具有天然构象的糖基化和组装以形成三聚体结构，并且通过检测HA的球状头部或茎域上存在的构象表位的单克隆抗体进行检测。因此，NanoLIPS适用于评价疫苗有效性。我们还表明，C-NLuc-HA适用于检测来自各种实验物种（包括小鼠、食蟹猴和树鼩）的血清中的HA特异性抗体。因此，基于NanoLIPS的HA检测提供了一种检测天然构象表位的简单且高灵敏度的方法，并可用于各种实验动物模型。重要性流感病毒HA特异性抗体可通过血凝抑制（HI）试验、中和（NT）试验和酶联免疫吸附试验（ELISA）检测。然而，这些测定具有一些缺点，包括窄的动态范围和需要大量血清。作为基于ELISA的方法的替代方法，开发了荧光素酶免疫沉淀系统（LIPS）。我们专注于纳米荧光素酶（NLuc），它具有小尺寸，更高的强度和更长的稳定性。在这项研究中，我们开发了一种技术上可行的和高灵敏度的方法，用于检测流感病毒特异性抗体，使用哺乳动物细胞中产生的NLuc-taged重组HA蛋白。当在哺乳动物细胞中表达时，具有C-末端NLuc延伸的HA（C-NLuc-HA）被糖基化并形成三聚体复合物。此外，C-NLuc-HA不仅被结合到球状头域的单克隆抗体识别，而且被结合到茎域的单克隆抗体识别。我们还证明了通过该测定获得的数据与动物模型中实验疫苗的保护相关。
Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. Antibody detection is crucial for monitoring host immune responses to specific pathogen antigens (Ags) and evaluating vaccine efficacies. The luciferase immunoprecipitation system (LIPS) was developed for sensitive detection of Ag-specific antibodies in sera from various species. In this study, we describe NanoLIPS, an improved LIPS assay based on NanoLuciferase (NLuc), and employ the assay for monitoring antibody responses following influenza virus infection or vaccination. We generated recombinant influenza virus hemagglutinin (HA) proteins tagged with N-terminal (N-NLuc-HA) or C-terminal (C-NLuc-HA) NLuc reporters. NLuc-HA yielded an at least 20-fold higher signal-to-noise ratio than did a LIPS assay employing a recombinant HA-Gaussia princeps luciferase (GLuc) fusion protein. NanoLIPS-based detection of anti-HA antibodies yielded highly reproducible results with a broad dynamic range. The levels of antibodies against C-NLuc-HA generated by mice vaccinated with recombinant vaccinia virus DIs strain expressing an influenza virus HA protein (rDIs-HA) was significantly correlated with the protective effect elicited by the rDIs-HA vaccine. C-NLuc-HA underwent glycosylation with native conformations and assembly to form a trimeric structure and was detected by monoclonal antibodies that detect conformational epitopes present on the globular head or stalk domain of HA. Therefore, NanoLIPS is applicable for evaluating vaccine efficacy. We also showed that C-NLuc-HA is applicable for detection of HA-specific antibodies in sera from various experimental species, including mouse, cynomolgus macaque, and tree shrew. Thus, NanoLIPS-based detection of HA offers a simple and high-sensitivity method that detects native conformational epitopes and can be used in various experimental animal models. IMPORTANCE Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. As an alternative to an ELISA-based method, luciferase immunoprecipitation system (LIPS) was developed. We focused on NanoLuciferase (NLuc), which has a small size, higher intensity, and longer stability. In this study, we developed a technically feasible and highly sensitive method for detecting influenza virus-specific antibodies using a NLuc-tagged recombinant HA protein produced in mammalian cells. HA with a C-terminal NLuc extension (C-NLuc-HA) was glycosylated and formed trimeric complexes when expressed in mammalian cells. Furthermore, C-NLuc-HA was recognized not only by monoclonal antibodies that bind to the globular head domain but also by those that bind to the stalk domain. We also demonstrated that the data obtained by this assay correlate with the protection of an experimental vaccine in animal models.