Texas Children's Hospital Center for Vaccine Development, Houston, TX 77030, USA.
Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Viruses. 2024 Sep 20;16(9):1492. doi: 10.3390/v16091492.
Over the past three years, new SARS-CoV-2 variants have continuously emerged, evolving to a point where an immune response against the original vaccine no longer provided optimal protection against these new strains. During this time, high-throughput neutralization assays based on pseudoviruses have become a valuable tool for assessing the efficacy of new vaccines, screening updated vaccine candidates against emerging variants, and testing the efficacy of new therapeutics such as monoclonal antibodies. Lentiviral vectors derived from HIV-1 are popular for developing pseudo and chimeric viruses due to their ease of use, stability, and long-term transgene expression. However, the HIV-based platform has lower transduction rates for pseudotyping coronavirus spike proteins than other pseudovirus platforms, necessitating more optimized methods. As the SARS-CoV-2 virus evolved, we produced over 18 variants of the spike protein for pseudotyping with an HIV-based vector, optimizing experimental parameters for their production and transduction. In this article, we present key parameters that were assessed to improve such technology, including (a) the timing and method of collection of pseudovirus supernatant; (b) the timing of host cell transduction; (c) cell culture media replenishment after pseudovirus adsorption; and (d) the centrifugation (spinoculation) parameters of the host cell+ pseudovirus mix, towards improved transduction. Additionally, we found that, for some pseudoviruses, the addition of a cationic polymer (polybrene) to the culture medium improved the transduction process. These findings were applicable across variant spike pseudoviruses that include not only SARS-CoV-2 variants, but also SARS, MERS, Alpha Coronavirus (NL-63), and bat-like coronaviruses. In summary, we present improvements in transduction efficiency, which can broaden the dynamic range of the pseudovirus titration and neutralization assays.
在过去的三年中，不断有新的 SARS-CoV-2 变体出现，这些变体进化到了对原始疫苗的免疫反应不再能为这些新菌株提供最佳保护的程度。在此期间，基于假病毒的高通量中和测定已成为评估新疫苗效力、筛选针对新出现变体的更新疫苗候选物以及测试新疗法（如单克隆抗体）疗效的宝贵工具。源自 HIV-1 的慢病毒载体因其易于使用、稳定性和长期转基因表达而成为开发假病毒和嵌合病毒的热门选择。然而，基于 HIV 的平台对冠状病毒刺突蛋白进行假型化的转导率低于其他假病毒平台，因此需要更优化的方法。随着 SARS-CoV-2 病毒的进化，我们用基于 HIV 的载体对超过 18 种刺突蛋白变体进行了假型化，优化了其生产和转导的实验参数。在本文中，我们介绍了评估提高该技术的关键参数，包括：（a）收集假病毒上清液的时间和方法；（b）宿主细胞转导的时间；（c）假病毒吸附后细胞培养物的补充；以及（d）宿主细胞+假病毒混合物的离心（旋转感染）参数，以提高转导效率。此外，我们发现对于某些假病毒，在培养基中添加阳离子聚合物（聚凝胺）可以改善转导过程。这些发现适用于包括 SARS-CoV-2 变体在内的各种变体刺突假病毒，以及 SARS、MERS、甲型冠状病毒（NL-63）和类似蝙蝠的冠状病毒。总之，我们提出了转导效率的改进，可以拓宽假病毒滴定和中和测定的动态范围。
