Department of Bioengineering, McGill University, Montréal, Québec, Canada; Department of Chemical Engineering, École Polytechnique de Montréal, Montréal, Québec, Canada; Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
Vaccine. 2019 Nov 8;37(47):7100-7107. doi: 10.1016/j.vaccine.2019.07.057. Epub 2019 Jul 26.
One of the concerns associated with the use of influenza virus-like particles (VLPs) as vaccine candidate or delivery system is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out not only the viral antigenic proteins but also host proteins. In addition, the intrinsic nature of cells to produce membrane derived vesicles or extracellular vesicles (EVs), which have similar size to the VLPs, makes VLP purification process challenging. To further characterize these particles and identify proteins that are unique to each population, comparative proteomic analyses were completed to ultimately provide guidance for rational design of separation protocols. The VLPs were produced in suspension and serum free media by transient transfection of an inducible clone of a Human Embryonic Kidney (HEK-293SF) cells expressing HA and NA (H1N1/A/Puerto Rico/8/34), with a plasmid containing the gag gene of HIV-1 fused to GFP. EVs were produced independently from the non-transformed HEK-293SF cell line as a control for comparative studies. Both preparations were characterized for total nucleic acids and protein concentrations and extensively analyzed by nanoLC-MS/MS for their protein compositions. The proteomic analyses showed that aside from the recombinant VLP proteins, nucleolin was the most abundant host cell protein uniquely identified within VLPs (considering the MASCOT score value) while lactotransferrin and heat shock protein 90 were the most abundant proteins in EVs. Overall, this comparative study identifies potential target proteins as specific markers to guide VLP purification and discusses the biogenesis of enveloped particles released in HEK-293 cell suspension cultures emphasizing on the biological functions of host cell proteins identified.
作为候选疫苗或输送系统，使用流感病毒样颗粒 (VLPs) 引起的关注之一是其异质组成。包膜 VLPs 在出芽部位摄取宿主细胞膜，不仅携带病毒抗原蛋白，还携带宿主蛋白。此外，细胞产生膜衍生小泡或细胞外囊泡 (EVs) 的内在性质，这些囊泡具有与 VLPs 相似的大小，使得 VLP 纯化过程具有挑战性。为了进一步表征这些颗粒并鉴定每种群体特有的蛋白质，进行了比较蛋白质组学分析，最终为分离方案的合理设计提供了指导。VLPs 通过瞬时转染表达 HA 和 NA (H1N1/A/Puerto Rico/8/34) 的可诱导克隆的人胚肾 (HEK-293SF) 细胞在悬浮液和无血清培养基中产生，带有包含 HIV-1 gag 基因融合 GFP 的质粒。EVs 由未转化的 HEK-293SF 细胞系独立产生，作为比较研究的对照。对两种制剂的总核酸和蛋白质浓度进行了表征，并通过纳升液相色谱-串联质谱 (nanoLC-MS/MS) 对其蛋白质组成进行了广泛分析。蛋白质组学分析表明，除了重组 VLP 蛋白外，核仁素是唯一在 VLPs 中鉴定出的最丰富的宿主细胞蛋白（考虑到 MASCOT 评分值），而乳转铁蛋白和热休克蛋白 90 是 EVs 中最丰富的蛋白质。总体而言，这项比较研究确定了潜在的靶蛋白作为特定标志物，以指导 VLP 纯化，并讨论了在 HEK-293 细胞悬浮培养物中释放的包膜颗粒的生物发生，强调了鉴定的宿主细胞蛋白的生物学功能。
