假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具
Summary
假型病毒 （PV） 是复制缺陷的病毒粒子，用于在比处理真实病毒更安全的条件下研究宿主-病毒相互作用。这里介绍的是一个详细的方案，显示了如何使用 SARS-CoV-2 PV 来测试 COVID-19 疫苗接种后患者血清的中和能力。
Abstract
假型病毒 （PV） 是一种分子工具，可用于研究宿主-病毒相互作用和测试血清样本的中和能力，此外，它们在基因治疗中更广为人知的用途是递送目标基因。PV 具有复制缺陷，因为病毒基因组被分成不同的质粒，这些质粒未掺入 PV 中。这种安全且多功能的系统允许在生物安全 2 级实验室中使用 PV。在这里，我们提出了一种基于此处提到的三种质粒生产慢病毒 PV 的通用方法：（1） 携带监测感染所需的报告基因的骨架质粒;（2）包装质粒携带产生PV所需的所有结构蛋白的基因;（3）包膜表面糖蛋白表达质粒，决定病毒嗜性并介导病毒进入宿主细胞。在这项工作中，SARS-CoV-2 Spike 是用于生产非复制性 SARS-CoV-2 假型慢病毒的包膜糖蛋白。
简而言之，使用标准方法将包装细胞 （HEK293T） 与三种不同的质粒共转染。48小时后，收获含有PV的上清液，过滤，并在-80°C下储存。 通过研究感染后 48 小时靶细胞系中报告基因（荧光素酶）的表达来测试 SARS-CoV-2 PV 的感染性。相对发光单位 （RLU） 的值越高，感染/转导率越高。此外，将传染性PV添加到连续稀释的血清样品中，以研究假病毒进入靶细胞的中和过程，以RLU强度的降低来衡量：较低的值对应于高中和活性。
Introduction
假型病毒 （PV） 是微生物学中用于研究宿主-病毒和病原体-病原体相互作用的分子工具 1,2,3,4。PV 由内部部分（保护病毒基因组的病毒核心）和外部部分（定义向性5 的病毒表面的包膜糖蛋白）组成。假病毒在靶细胞中是复制不合格的，因为它不包含产生新病毒颗粒的所有遗传信息。这种特殊特征的组合使PV成为野生型病毒的安全替代品。另一方面，野生型病毒具有高致病性，不能在 BSL 2 实验室中用于分析6。
PV的传染性可以通过报告基因来监测，报告基因通常编码荧光蛋白（GFP、RFP、YFP）或产生化学发光产物的酶（荧光素酶）。它包含在用于 PV 生产的质粒之一中，并掺入假病毒7 的基因组中。
目前存在几种类型的 PV 核心，包括基于 HIV-1 基因组的慢病毒衍生颗粒。与其他平台相比，基于 HIV-1 的 PV 的最大优势在于它们在靶细胞基因组中的内在整合过程8。尽管HIV-1是一种高度传染性的病毒，并且是艾滋病的病原体，但由于多年来进行了广泛的优化步骤，这些慢病毒载体可以安全使用。通过引入第二代慢病毒载体实现了最佳安全条件，其中病毒基因在不影响转导能力的情况下被耗尽9。第 3 代和第 4 代有助于提高慢病毒载体处理的安全性，将病毒基因组进一步分裂为单独的质粒10,11。最新一代的PV通常用于生产用于基因治疗的慢病毒载体。
PV可用于研究病毒和宿主细胞之间的相互作用，包括生产和感染阶段。PV 特别用于假病毒中和测定 （PVNA）。PVNA 经过广泛验证，可通过靶向 PV 包膜上的病毒糖蛋白来评估血清或血浆的中和潜力12,13。中和活性，表示为抑制浓度 50 （IC50），定义为血清/血浆的稀释，阻断 50% 的病毒颗粒进入14。在该方案中，我们描述了 PVNA 的设置，以测试在接受加强疫苗剂量之前和之后收集的血清中针对严重急性呼吸系统综合症 – 冠状病毒 2 （SARS-CoV-2） 的抗体活性。
Protocol
本协议已获得维罗纳大学伦理委员会的批准并遵循其指导方针（批准协议编号 1538）。从参与研究的人类受试者那里获得知情书面同意。从正在接种抗SARS-CoV-2疫苗的医护人员（HCW）志愿者身上采集全血样本。将这些样品收集在含有抗凝剂的塑料管中，以便随后分离血清15。 以下所有过程必须在 2 类生物罩中执行，在无菌条件下工作。必须小心处理病毒，所有…
Representative Results
该协议描述了 SARS-CoV-2 PV 的产生以及这些 PV 的下游应用，以分析接受抗 COVID-19 疫苗接种的受试者血清/血浆的中和活性17。此外，该协议可用于产生每个 SARS-CoV-2 关注变体 （VOC） 的假型，以测试中和反应的演变。尽管该协议有助于研究 COVID-19 疫苗接种后的体液免疫反应，但它可以适应于轻松测试不同血清/血浆对不同病毒的中和作用 13,18,19。<sup class="xr…
Discussion
尽管使用野生型病毒模拟实际感染，但慢病毒 PV 是研究与病毒进入和感染相关的机制的更安全选择，而无需处理致病病毒所需的严格安全要求 4,20,21。PV 由复制缺陷的病毒核心组成，该核心被致病病毒的表面包膜糖蛋白包围，这是研究的目的。
基于 HIV-1 的 PV 是使用最广泛的平台之一，这些平台已在该协?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢卫生保健工作者志愿者的贡献。该项目得到了意大利 MUR 2023/2027 卓越部的支持。AR 和 DZ 得到了 PRIN2022（欧盟资助;下一代欧盟）
Materials
| 0.45 μm filter | SARSTEDT | 83 1826 | |
| 6-well plate | SARSTEDT | 83 3920 | |
| 96-well plate | SARSTEDT | 8,33,924 | |
| Amicon Ultra-15 Centrifugal Filter Units | Merck | 10403892 | |
| Black Opaque 96-well Microplate | Perkin Elmer | 60005270 | |
| Dulbecco's Modified Eagle Medium | SIGMA-ALDRICH | D6546 – 500ML | |
| Dulbecco's phosphate buffered saline (PBS 1x) | AUROGENE | AU-L0615-500 | |
| Foetal Bovine Serum | AUROGENE | AU-S1810-500 | |
| Graphpad Prism version 7 | graphpad dotmatics | NA | In the manuscript, we replace the commercial name with 'data analysis program' |
| HEK293T cells | ATCC | CRL-3216 | |
| HEK293T/ACE2 cells | ATCC | CRL-3216 | HEK293T has been transduced to overexpress ACE2 with a lentiviral vector. |
| L-glutamine | AUROGENE | AU-X0550-100 | |
| Luminometer – Victor3 | Perkin Elmer | HH35000500 | In the manuscript, we replace the commercial name with 'luminometer' |
| Opti-MEM | Thermo Fisher Scientific | 11058021 | In the manuscript, we replace the commercial name with 'reduced serum medium' |
| p8.91 packaging plasmid | Di Genova et al., 2021 | A kind gift from Prof. Nigel Temperton (ref 16.) | |
| pCSFLW reporter plasmid | Di Genova et al., 2021 | A kind gift from Prof. Nigel Temperton (ref 16.) | |
| Penicillin/streptomycin | AUROGENE | AU-L0022-100 | |
| Polyethylenimine, branched (PEI) (25 kDa) | SIGMA-ALDRICH | 408727 | |
| RRL.sin.cPPT.SFFV/Ace2.IRES-puro.WPRE (MT126) | Addgene | 145839 | This plasmid was used to generate HEK293Tcells/ACE2 |
| SARS-CoV-2 Spike expressing plasmid | Addgene | pGBW-m4137382 | |
| steadylite plus Reporter Gene Assay System | Perkin Elmer | 6066759 | In the manuscript, we replaced the commercial name with 'luciferase reading reagent' |
| Trypsin EDTA 1x | AUROGENE | AU-L0949-100 |
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Cite This Article
Fantoni, T., Bissoli, M., Stefani, C., Voi, M., Dabija, A., Casula, R., Minafra, D. L., da Fonseca Palmeira, J., Argañaraz, E. R., Mayora-Neto, M., Temperton, N. J., Zipeto, D., Ruggiero, A. Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay. J. Vis. Exp. (201), e65658, doi:10.3791/65658 (2023).