一种新型冠状病毒假病毒的制备及初步应用

Preparation and application of severe acute respiratory syndrome coronavirus 2 pseudoparticles

目的制备一种新型冠状病毒(SARS-CoV-2)假病毒,并将其应用于抗体中和能力检测和抗体广谱性评估。方法整合2种近期出现的SARS-CoV-2变异病毒株(20A.EU1和B.1.1.7)刺突(spike,S)蛋白的突变序列、以及对胞浆区肽段19个氨基酸进行局部缺失突变,构建突变型S蛋白表达质粒,转染293T细胞进行表达,用Western blot和流式细胞术检测确认S蛋白的表达。将S蛋白表达质粒与其它慢病毒包装质粒共转染293T细胞进行假病毒的包装,用包装好的假病毒分别感染293T-h ACE2细胞和Huh-7细胞,并进行假病毒的半数组织感染剂量(TCID_(50))检测。利用所制备的假病毒对1株抗S蛋白的单克隆抗体(YK-01)进行抗体的中和能力评价。结果Western blot和流式细胞术检测结果显示野生型和突变型S蛋白在293T细胞中表达水平一致,突变型S蛋白在细胞膜表面的分布量略高于野生型。相同的病毒包装条件下,所获得的突变型假病毒感染293T-hACE2细胞的TCID_(50)值为6.30×10^(6),野生型假病毒的TCID_(50)值为1.83×10^(6)。在Huh-7细胞中虽然能够观察到GFP阳性细胞,但GFP阳性细胞比例以及荧光强度均较低。对抗体的中和能力评价结果显示,从新冠肺炎的康复患者的血液筛选获得的YK-01抗体能够有效的中和2种假病毒,抑制假病毒对293T-h ACE2细胞的感染,该抗体对野生型假病毒的IC_(50)值为0.0614μg/ml,对突变型假病毒的IC_(50)值为0.3152μg/ml。结论成功制备了可替代20A.EU1和B.1.1.7变异毒株的假病毒,可用于针对SARS-CoV-2 S蛋白研发出的疫苗或抗体药物疫苗和抗体药物的筛选和相关性能的评价。

Pseudoviruses are useful virological tool for developing vaccine and therapeutics against infectious diseases.In this study,a pseudovirus-based neutralization assay had been established for evaluating antibodies neutralization potency and breadth against SARS-CoV-2 under biosafety level 2 condition.Key parameters of the assay had been optimized,including cell types,deletion and mutation of S protein gene to enhance viral packing titer and infection ability,by integrating mutated strains(20A.EU2 and B.1.1.7).The expression levels of mutated and wild-type spike S proteins in 293 T cells were similar and mainly on cell membrane.Under the same viral packaging condition,the titers(TCID_(50))of both pseudoviruses were similar as 6.30×10^(6)for mutant and 1.83×10^(6)for wild-tpye.GFP-positive cells could be observed in both pseudovirus-infected 293 T-hACE2 cells and Huh-7 cells,and the proportion of GFP-positive cells in 293 T-hACE2 cells was significantly high.By using the 293 T-hACE2 cells,the proportion of GFP-positive cells infected with the mutated pseudovirus was relatively higher as compared with the cells infected by the wild type.Using both wild-type and mutated pseudovirus systems,a novel monoclonal antibody named as YK-01 that we had isolated from a SARS-CoY-2 convalescent patient showed high neutralizing potency and breadth.The half effective concentration(IC_(50))of the antibody against mutated pseudovirus infection was 0.3152μg/ml,while the IC_(50)against wild type was 0.0614μg/ml.Overall,we have established a robust pseudo virus-based neutralization assay for SARS-CoV-2 testing and are glad to share materials and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.