The nature of enveloped virus-like particles (VLPs) has triggered high interest in their application to different research fields, including vaccine development. The baculovirus expression vector system (BEVS) has been used as an efficient platform for obtaining large amounts of these complex nanoparticles. To date, most of the studies dealing with VLP production by recombinant baculovirus infection utilize indirect detection or quantification techniques that hinder the appropriate characterization of the process and product. Here, we propose the application of cutting-edge quantification methodologies in combination with advanced statistical designs to exploit the full potential of the High Five/BEVS as a platform to produce HIV-1 Gag VLPs. The synergies between CCI, MOI, and TOH were studied using a response surface methodology approach on four different response functions: baculovirus infection, VLP production, VLP assembly, and VLP productivity. TOH and MOI proved to be the major influencing factors in contrast with previous reported data. Interestingly, a remarkable competition between Gag VLP production and non-assembled Gag was detected. Also, the use of nanoparticle tracking analysis and flow virometry revealed the existence of remarkable quantities of extracellular vesicles. The different responses of the study were combined to determine two global optimum conditions, one aiming to maximize the VLP titer (quantity) and the second aiming to find a compromise between VLP yield and the ratio of assembled VLPs (quality). This study provides a valuable approach to optimize VLP production and demonstrates that the High Five/BEVS can support mass production of Gag VLPs and potentially other complex nanoparticles.

中文翻译：

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集成纳米颗粒定量和实验统计设计，可在高五细胞中高效生产HIV-1病毒样颗粒。

包膜病毒样颗粒（VLP）的性质引起了人们对其应用到包括疫苗开发在内的不同研究领域的高度兴趣。杆状病毒表达载体系统（BEVS）已被用作获得大量这些复杂纳米颗粒的有效平台。迄今为止，有关重组杆状病毒感染VLP产生的大多数研究都利用了间接检测或定量技术，这些技术阻碍了过程和产物的适当表征。在这里，我们提出将先进的量化方法与先进的统计设计相结合的应用，以充分利用High Five / BEVS作为生产HIV-1 Gag VLP的平台的全部潜力。CCI，MOI，使用响应面方法学方法对TOH和TOH进行了四种不同的响应功能研究：杆状病毒感染，VLP产生，VLP装配和VLP生产率。与先前报道的数据相比，TOH和MOI被证明是主要的影响因素。有趣的是，检测到Gag VLP生产与未组装的Gag之间存在明显的竞争。同样，使用纳米颗粒跟踪分析和流式病毒分析显示存在大量的细胞外囊泡。结合研究的不同反应，确定了两个全局最佳条件，一个旨在最大化VLP效价（数量），第二个旨在寻找VLP产量与组装VLP的比例（质量）之间的折衷。