Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Puente‐Massaguer, Eduard; Cervera, Laura; Gòdia, Francesc

doi:10.3390/v12020223

Cited by 34 publications

(38 citation statements)

References 60 publications

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“…Around a 10-fold increase in the quantification of these nanoparticles was observed in NTA over flow virometry. Despite the fact that flow virometry is a faster technique, the lack of sensitivity in the small nanoparticle size range, known as "swarm effect", could explain the differences observed in this study (38).…”

Section: Discussioncontrasting

confidence: 55%

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

et al. 2020

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Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag‐based virus‐like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 cells. Cryogenic transmission electron microscopy (cryo‐TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion‐derived BV in High Five cells, whereas Sf9 cells evidenced a larger proportion of the budded virus phenotype (23‐fold). Initial evaluation of the VLP production process using spectrofluorometry showed that higher levels of the Gag‐eGFP polyprotein were obtained in High Five cells (3.6‐fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 cells were 1.7‐ and 1.5‐fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five‐derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry

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Section: Discussioncontrasting

confidence: 55%

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

et al. 2020

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“…Several studies indicate that the lower nanoparticle levels measured by flow virometry could be the consequence of detecting several nanoparticles as a single larger particle, a phenomenon known as swarm effect [39]. However, it is not clear whether these differences can be fully attributed to this event or to the non-detection of nanoparticles that are below the flow cytometer detection threshold [40].…”

Section: Transferability Of Vlp Production To Bioreactormentioning

confidence: 99%

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

et al. 2020

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High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.

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“…Flow virometry experiments were performed with a CytoFLEX LX (Beckman Coulter Inc., Brea, CA, USA) with a Violet SSC (VSSC) 405 nm filter, as reported previously. 39 The instrument was standardized using Megamix-Plus SSC and FSC fluorescent polystyrene beads (0.1, 0.16, 0.20, 0.24, and 0.5 μm; Biocytex, Marseille, France) as a quality control tool. The threshold of the trigger signal (VSSC) was manually adjusted to 1500, and gains were set as 95, 9, and 115 for FSC, VSSC, and B525-FITC lasers, respectively.…”

Section: Experimental Sectionmentioning

confidence: 99%

Molecular Characterization of the Coproduced Extracellular Vesicles in HEK293 during Virus-Like Particle Production

et al. 2020

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Vaccine therapies based on virus-like particles (VLPs) are currently in the spotlight due to their potential for generating high immunogenic responses while presenting fewer side effects than conventional vaccines. These self-assembled nanostructures resemble the native conformation of the virus but lack genetic material. They are becoming a promising platform for vaccine candidates against several diseases due to the ability of modifying their membrane with antigens from different viruses. The coproduction of extracellular vesicles (EVs) when producing VLPs is a key phenomenon currently still under study. In order to characterize this extracellular environment, a quantitative proteomics approach has been carried out. Three conditions were studied: non-transfected, transfected with an empty plasmid as control, and transfected with a plasmid coding for HIV-1 Gag polyprotein. A shift in EV biogenesis has been detected upon transfection, changing the production from large to small EVs. Another remarkable trait found was the presence of DNA being secreted within vesicles smaller than 200 nm. Studying the protein profile of these biological nanocarriers, it was observed that EVs were reflecting an overall energy homeostasis disruption via mitochondrial protein deregulation. Also, immunomodulatory proteins like ITGB1, ENO3, and PRDX5 were identified and quantified in VLP and EV fractions. These findings provide insight on the nature of the VLP extracellular environment defining the characteristics and protein profile of EVs, with potential to develop new downstream separation strategies or using them as adjuvants in viral therapies.

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Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Cited by 34 publications

References 60 publications

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

Molecular Characterization of the Coproduced Extracellular Vesicles in HEK293 during Virus-Like Particle Production

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