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首页> 外文会议>Conference on vaccine technology VI >PROTEOMIC CHARACTERIZATION OF INFLUENZA H1N1 GAG VIRUS-LIKE PARTICLES AND EXTRACELLULAR VESICLES PRODUCED IN HEK-293SF
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PROTEOMIC CHARACTERIZATION OF INFLUENZA H1N1 GAG VIRUS-LIKE PARTICLES AND EXTRACELLULAR VESICLES PRODUCED IN HEK-293SF

机译：HEK-293SF中感染的H1N1流感病毒样颗粒和细胞外囊泡的蛋白质组学表征

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One of the major concerns associated with the use of influenza virus-like particles (VLPs) produced in cell culture as vaccine candidates is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out with them not only the viral antigenic proteins but also host cell proteins. In addition, the intrinsic nature of the cells to produce membrane derived vesicles which have similar size to the VLPs and can also contain the antigenic proteins, makes the VLP purification process challenging. Certainly, the expression system and the viral recombinant proteins employed will determine the nature of the proteins within the VLPs. To further characterize cell culture produced-influenza VLPs and contribute to enable their approval as vaccine candidates, the composition and biogenesis of VLPs need to be better understood. In this study we have characterized, by nanoscale liquid chromatography tandem mass spectrometry (n-LC-MS/MS), influenza H1N1 Gag-VLPs produced in human embryonic kidney cells adapted to serum-free medium (HEK-293SF). The cells stably express HA and NA, and the VLPs production occurs following transient transfection with a plasmid containing the gag gene of HIV-1 fused to GFP. Extracellular vesicles (EVs) produced by the unmodified HEK-293SF were also characterized by n-LC-MS/MS. A total of 73 host cell proteins were identified in the VLPs, whereas 98 were detected in the extracellular vesicles. From that, 32 host cell proteins were unique to VLPs while 41 proteins were found in both. Importantly, nucleolin was the most abundant host cell differential protein identified in VLPs while lactotransferrin and heat shock protein 90 were the most present in EVs. This study provides a detailed proteomic description of the VLPs and EVs produced in HEK-293SF as well as a critical discussion of the function of each protein incorporated in both nanoparticles species. The outcome of this research also sheds light on unique target proteins differentially identified either in VLPs and EVs that could potentially be exploited for the development of novel purification protocols to separate EVs from VLPs.
机译：与使用细胞培养中产生的流感病毒样颗粒（VLP）作为候选疫苗有关的主要问题之一是它们的异质组成。包膜的VLP在出芽部位吸收宿主细胞膜，不仅与病毒抗原蛋白结合，而且与宿主细胞蛋白结合。另外，细胞产生膜来源的囊泡的细胞的固有性质，膜囊泡具有与VLP相似的大小并且还可以包含抗原蛋白，这使得VLP纯化过程具有挑战性。当然，所使用的表达系统和病毒重组蛋白将决定VLP中蛋白质的性质。为了进一步表征细胞培养产生的流感病毒VLP并有助于使其成为疫苗候选者，需要更好地了解VLP的组成和生物发生。在这项研究中，我们已经通过纳米级液相色谱串联质谱（n-LC-MS / MS）表征了适应无血清培养基（HEK-293SF）的人胚肾细胞中产生的H1N1型流感病毒Gag-VLP。细胞稳定地表达HA和NA，并且在用含有与GFP融合的HIV-1的gag基因的质粒瞬时转染后，产生VLP。未修饰的HEK-293SF产生的细胞外囊泡（EV）也通过n-LC-MS / MS表征。在VLP中共鉴定出73种宿主细胞蛋白，而在细胞外囊泡中检出98种宿主细胞蛋白。因此，VLP特有32种宿主细胞蛋白，而两种蛋白中均发现41种蛋白。重要的是，核仁蛋白是在VLP中鉴定出的最丰富的宿主细胞差异蛋白，而乳运铁蛋白和热休克蛋白90在电动汽车中含量最高。这项研究提供了HEK-293SF中产生的VLP和EV的详细蛋白质组学描述，以及对两种纳米颗粒中每种蛋白质功能的重要讨论。这项研究的结果还揭示了在VLP和EV中差异鉴定的独特靶蛋白，这些靶蛋白可潜在地用于开发新型纯化方案以将EV与VLP分离。

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