Pısıl Yalçın, Shida Hisatoshi, Miura Tomoyuki
Laboratory of Primate Model, Research Center for Infectious Diseases, Institute for Frontier Life and Medical Science, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Graduate School of Human and Environmental Studies, Department of Interdisciplinary Environment, Dynamics of Natural Environment, Dynamics of Biological Environment, Kyoto University, Kyoto 606-8501, Japan.
Pathogens. 2021 Feb 2;10(2):153. doi: 10.3390/pathogens10020153.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic zoonotic virus that spreads rapidly. In this work, we improve the hitherto existing neutralization assay system to assess SARS-CoV-2 inhibitors using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein (LpVspike +) and angiotensin-converting enzyme 2 (ACE2)-transfected cat Crandell-Rees feline kidney (CRFK) cells as the host cell line. Our method was 10-fold more sensitive compared to the typical human embryonic kidney 293T (HEK293T) cell system, and it was successfully applied to quantify the titers of convalescent antisera and monoclonal anti-spike antibodies required for pseudo virus neutralization. The 50% inhibition dilution (ID50) of two human convalescent sera, SARS-CoV-2 immunoglobulin G (IgG) and SARS-CoV-2 immunoglobulin M (IgM), which were 1:350 (±1:20) and 1:1250 (±1:350), respectively. The 50% inhibitory concentration (IC50) of the IgG, IgM and immunoglobulin A (IgA) anti-SARS-CoV-2 monoclonal antibodies (mAbs) against LpVspike(+) were 0.45 (±0.1), 0.002 (±0.001) and 0.004 (±0.001) µg mL, respectively. We also found that reagents typically used to enhance infection were not effective in the CFRK system. This methodology is both efficient and safe; it can be employed by researchers to evaluate neutralizing monoclonal antibodies and contribute to the discovery of new antiviral inhibitors against SARS-CoV-2.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是一种高致病性人畜共患病毒，传播迅速。在这项工作中，我们改进了现有的中和试验系统，使用包被有SARS-CoV-2刺突蛋白的假型慢病毒（LpVspike +）和转染了血管紧张素转换酶2（ACE2）的猫源克兰德尔-里斯猫肾（CRFK）细胞作为宿主细胞系来评估SARS-CoV-2抑制剂。我们的方法比典型的人胚肾293T（HEK293T）细胞系统灵敏10倍，并成功应用于定量假病毒中和所需的康复期抗血清和抗刺突单克隆抗体的效价。两份人类康复期血清，即SARS-CoV-2免疫球蛋白G（IgG）和SARS-CoV-2免疫球蛋白M（IgM）的50%抑制稀释度（ID50）分别为1:350（±1:20）和1:1250（±1:350）。抗SARS-CoV-2单克隆抗体（mAb）的IgG、IgM和免疫球蛋白A（IgA）针对LpVspike(+)的50%抑制浓度（IC50）分别为0.45（±0.1）、0.002（±0.001）和0.004（±0.001）μg/mL。我们还发现，通常用于增强感染的试剂在CFRK系统中无效。这种方法既高效又安全；研究人员可以采用它来评估中和单克隆抗体，并有助于发现针对SARS-CoV-2的新型抗病毒抑制剂。
