Construction of SARS-CoV-2 pseudovirus and its application in measuring the neutralization activity of serum antibodies

Abstract

<p><strong>Objective</strong> To construct SARS-CoV-2 pseudovirus, optimize its preparation protocol, and apply it to the evaluation of antibody neutralization activity. <strong>Methods</strong> The optimized sequence of spike (S) gene of SARS-CoV-2 was synthesized, the pseudovirus titers were measured, and the expressed S protein was then detected by Western blot. Finally, quantitative ELISA was used to measure the serum IgG antibody titers in recipients who had received either one or two doses of COVID-19 inactivated vaccine. Meanwhile, the sera were tested for their reactivity with the pseudovirus using neutralization tests. <strong>Results</strong> S gene was confirmed to have been successfully cloned into the vector, generating the pcDNA3.1-S plasmid. Co-transfection of pNL4-3.Luc.R-E- and pcDNA3.1-S at a ratio of 2∶1 could lead to higher packing efficacy and pseudovirus titers. Expression of the S protein was verified by Western blot. Moreover, this SARS-CoV-2 pseudovirus showed a broad host infectivity in Vero, Huh7.5, A549-hACE2 and 293T-hACE2 cells, with the highest relative luciferase unit (RLU) in 293T-hACE2. Comparing the IgG levels measured by ELISA in sera collected from COVID-19 vaccine recipients, we observed a higher titer in those who received two doses of inactivated vaccine (S/CO=10.27±3.33), measured one week after the second shot. However, the IgG level significantly dropped （ S/CO=2.36±2.25） half year post-vaccination. Amongst the serum samples tested, one with an S/CO of 10.32 could successfully inhibit the infection of SARS-CoV-2 pseudovirus in 293T-hACE2 cells at a dilution of 1/1 066. <strong>Conclusion</strong> We have established a method for preparing the SARS-CoV-2 pseudovirus, which can be used for detection of the neutralizing antibodies and the evaluation of humoral immune response post-vaccination.<br></p>

目的 构建新型冠状病毒（SARS-CoV-2）假病毒，优化假病毒制备条件并应用于中和抗体活性检测。 方法 优化合成 SARS-CoV-2 刺突蛋白（S）基因，进行假病毒包装；通过 Western blot 检测 S 基因的表达；应用定量 ELI‐SA 检测接种新冠灭活疫苗后血清中新型冠状病毒 IgG 抗体的滴度，同时应用假病毒中和实验对接种新冠疫苗后血清中和抗体活性进行评价。结果 成功构建了整合 SARS-CoV-2 S 基因的表达载体 pcDNA3.1-S，确定 pNL4-3.Luc.R-E-∶pcDNA3.1-S 最佳转染比例为 2∶1，可成功包装出高滴度的假病毒粒子。Western blot 结果表明 S 蛋白已成功地在假病毒中进行表达。SARS-CoV-2 假病毒可感染 Vero、Huh7.5、A549-hACE2 和 293T-hACE2 等 4 种靶细胞，表明所构建的假病毒具有感染活力，且具有广泛的宿主范围，其中 293T-hACE2 细胞感染假病毒后相对其他细胞株可检测出更高的萤火虫荧光素酶活性。ELISA 检测接种新冠疫苗后收集的血清，结果表明接种第二针灭活疫苗一周后可检测出较高血清 IgG 滴度（S/CO=10.27±3.33），半年后血清 IgG 滴度降低（S/CO=2.36±2.25）；假病毒中和实验结果表明 1 例 IgG 阳性（S/CO=10.32）免疫后血清可有效抑制 SARS-CoV-2 假病毒的感染活力，中和效价达到 1/1 066。结论 成功构建 SARS-CoV-2 假病毒，该假病毒可用于后续有关 SARS-CoV-2 中和抗体活性检测和疫苗接种后体液免疫效果的评价研究。