A real-time and high-throughput neutralization test based on SARS-CoV-2 pseudovirus containing monomeric infrared fluorescent protein as reporter.
A real-time and high-throughput neutralization test based on SARS-CoV-2 pseudovirus containing monomeric infrared fluorescent protein as reporter.
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DOI：
10.1080/22221751.2021.1925163
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发表时间：
2021-12
影响因子：
13.2
通讯作者：
Wang WK
中科院分区：
文献类型：
--
作者：
Tsai WY;Ching LL;Hsieh SC;Melish ME;Nerurkar VR;Wang WK
SARS-CoV-2的中和抗体已被证明与动物和人类的保护、疾病严重程度、存活率和疫苗效力相关。随着不同国家的大规模疫苗接种持续进行，全球关注的新变异病毒不断激增，迫切需要一种便捷、经济、高通量的中和试验。传统的SARS-CoV-2中和试验繁琐、耗时，需要生物安全3级实验室。尽管最近报道了使用具有荧光素酶或绿色荧光蛋白报告基因的不同假病毒进行中和，但费力的步骤、测定间的变异性或高背景限制了它们的高通量潜力。在这项研究中，我们产生了包含单体红外荧光蛋白报告基因的基于慢病毒的假病毒，以开发中和试验。在产生的三种假病毒中发现了相似的嗜性、感染动力学和通过受体介导的内吞作用进入的机制。与假病毒D614相比，D614G突变的假病毒的脱落减少，颗粒上的S1蛋白密度增加。对假病毒D614或D614G的50%中和滴度与对活SARS-CoV-2的空斑减少中和滴度相关。48 - 72小时的周转时间、最小的自发荧光、一步图像定量、可扩展至384孔、顺序读出和流式细胞术双重定量支持其在非参考和生物安全2级实验室的高通量和多功能应用，特别是用于评估自然感染或不同疫苗接种的血清对新变体的中和敏感性，大流行病
Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the ongoing large-scale vaccination in different countries and continuous surge of new variants of global concerns, a convenient, cost-effective and high-throughput neutralization test is urgently needed. Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential. In this study we generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein reporter to develop neutralization assays. Similar tropism, infection kinetics and mechanism of entry through receptor-mediated endocytosis were found in the three pseudoviruses generated. Compared with pseudovirus D614, pseudovirus with D614G mutation had decreased shedding and higher density of S1 protein present on particles. The 50% neutralization titers to pseudoviruses D614 or D614G correlated with the plaque reduction neutralization titers to live SARS-CoV-2. The turn-around time of 48–72 h, minimal autofluorescence, one-step image quantification, expandable to 384-well, sequential readouts and dual quantifications by flow cytometry support its high-throughput and versatile applications at a non-reference and biosafety level 2 laboratory, in particular for assessing the neutralization sensitivity of new variants by sera from natural infection or different vaccinations during our fight against the pandemic.
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影响因子：
9.4
作者：
Addetia A;Crawford KHD;Dingens A;Zhu H;Roychoudhury P;Huang ML;Jerome KR;Bloom JD;Greninger AL
通讯作者：
Greninger AL
影响因子：
4.3
作者：
Hock, Andreas K.;Lee, Pearl;Vousden, Karen H.
通讯作者：
Vousden, Karen H.
影响因子：
15.3
作者：
Schmidt, Fabian;Weisblum, Yiska;Bieniasz, Paul D.
通讯作者：
Bieniasz, Paul D.
DOI：
10.1073/pnas.77.7.3884
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
ROSE, JK;WELCH, WJ;LING, NC
通讯作者：
LING, NC
影响因子：
64.5
作者：
Korber, Bette;Fischer, Will M.;Montefiori, David C.
通讯作者：
Montefiori, David C.