Grup d'Enginyeria Cel·lular i Bioprocessos, Department of Chemical, Biological and Environmental Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, Barcelona, Spain.
Laboratory of Cardiovascular Proteomics, Vascular Physiopathology area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, Madrid, Spain.
Biotechnol Bioeng. 2021 Jul;118(7):2660-2675. doi: 10.1002/bit.27786. Epub 2021 Apr 23.
The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.
开发新疫苗技术的重要性，以应对全球病毒爆发的多功能平台，已经在最近的严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）大流行中得到了证明。病毒样颗粒（VLPs）是一种高度免疫原性、安全且强大的方法，可以基于多种疫苗候选物。特别是 HIV-1 Gag VLPs 是一个灵活的系统，由一个被脂质双层包围的 Gag 核心组成，可以对其进行修饰以呈现多种类型的膜蛋白或针对多种疾病的抗原，如流感、登革热、西尼罗河病毒或人乳头瘤病毒，在这些疾病中已经被证明是成功的。产生的 VLPs 的大小分布和结构特征取决于用于生产它们的细胞系。在这项研究中，我们建立了一种 Gag 蛋白核心的分析方法，并阐明了 HIV-1 VLPs 中 Gag 化学计量的当前可变性取决于基于细胞的生产平台，直接确定每个 VLP 中的 Gag 分子数。已经验证了三种 Gag 肽来使用平行反应监测定量单体数量，这是一种准确且快速的基于质谱的方法，可用于评估所产生的 Gag VLPs 的质量，而与使用的细胞系无关。对于 HEK293，每个 VLP 平均获得 3617 ± 17 个单体，在不同平台之间有很大差异，包括哺乳动物和昆虫细胞。这在大规模定量和质量控制方法中提供了一个关键优势，以加速新的重组疫苗生产技术。
