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.
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The authors would like to thank Dr. Paula Alves (Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal) and Dr. Nick Berrow (Institute for Research in Biomedicine, Barcelona, Spain) for providing the High Five and Sf9 cell lines, respectively. Marta Martínez-Calle developed the BV-GageGFP. Ángel Calvache and Jorge Fomaro (Beckman Coulter) facilitated the access to the CytoFlex LX flow cytometer. The support of Martí de Cabo and Mónica Roldán from Servei de Microscòpia of Universitat Autònoma de Barcelona (UAB) with the cryo-TEM and confocal microscopy, respectively, and Paolo Saccardo from Plataforma de Producción de Proteínas, CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) of UAB with baculovirus titration are appreciated. The research group is recognized as 2017 SGR 898 by Generalitat de Catalunya.
Eduard Puente-Massaguer is a recipient of an FPU grant from Ministerio de Educación, Cultura y Deporte of Spain (FPU15/03577).
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Electronic supplementary material
Time lapse of High Five cells infected with BV-GageGFP recorded during 6.5 min. Cell membrane was stained with CellMaskTM (red) and cell nucleus with Hoechst (blue). Green dots in the cell membrane and in the supernatant correspond to GageGFP VLPs. (AVI 39171 kb)
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Puente-Massaguer, E., Lecina, M. & Gòdia, F. Integrating nanoparticle quantification and statistical design of experiments for efficient HIV-1 virus-like particle production in High Five cells. Appl Microbiol Biotechnol (2020). https://doi.org/10.1007/s00253-019-10319-x
- High Five cells
- Virus-like particle
- Response surface methodology
- Multiple-criteria decision analysis