Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

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

doi:10.1002/bit.27345

Cited by 15 publications

(19 citation statements)

References 68 publications

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“…Taking into account this particle size, a (2.4 ± 0.1) × 10 3 monomers/nm 3 ratio was calculated, interestingly sharing the stoichiometry observed in VLPs produced by TGE in HEK293. The total number of diffracting particles was also measured for insect-based production platforms and it was observed that Sf9 and High Five cells produced significantly more (p < 0.0001) extracellular vesicles than HEK293 platforms observed by NTA, being High Five cells the production platform producing the highest number of extracellular vesicles, in agreement with previous works(González-Domínguez et al, 2020b;Puente-Massaguer et al, 2018;Puente-Massaguer et al, 2020;…”

supporting

confidence: 89%

Characterization of HIV‐1 virus‐like particles and determination of Gag stoichiometry for different production platforms

Lavado‐García

Jorge

Boix-Besora

et al. 2021

Biotech & Bioengineering

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The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Viruslike particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, massspectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

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supporting

confidence: 89%

Characterization of HIV‐1 virus‐like particles and determination of Gag stoichiometry for different production platforms

Lavado‐García

Jorge

Boix-Besora

et al. 2021

Biotech & Bioengineering

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“…Cell membrane (red, CellMask™) colocalization with Gag-eGFP was visualized as yellow regions in the cell membrane of both cell lines, hence indicating that VLP budding was taking place. The latter was confirmed using a cutting-edge strategy based on the combination of super-resolution (Hyvolution2) and high-speed acquisition (Resonant, Leica) (30). Using this approach, the living VLP formation process and budding to the extracellular space could be recorded in both infected cell lines (Fig.…”

Section: Cell Growth and Study Of Bv Infectionmentioning

confidence: 76%

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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“…SRFM was performed with a TCS SP8 confocal microscope equipped with Huygens deconvolution suite embedded via a direct interface with LAS X software and GPU arrays (Leica Microsystems, Wetzlar, Germany) at Servei d'Anatomia Patològica from Hospital Sant Joan de Déu (Esplugues de Llobregat, Barcelona, Spain), as previously described [24]. A summary of the protocol is depicted in Figure 4A.…”

Section: Super-resolution Fluorescence Microscopy (Srfm)mentioning

confidence: 99%

“…Deconvolution was performed with the SVI Huygens Professional program and the best resolution strategy (Scientific Volume Imaging B.V., Hilversum, the Netherlands). HIV-1 Gag-eGFP VLP concentration was calculated based on the division of particle number by 3D image volume as previously described [24]. Briefly, direct quantification was performed on deposited HIV-1 Gag-eGFP VLP samples with 23 × 23 × 3 µm in xyz from a total loaded volume of 50 µL distributed in 24 × 60 mm under the cover glass and a total height of 34 µm.…”

Section: Super-resolution Fluorescence Microscopy (Srfm)mentioning

confidence: 99%

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

Puente‐Massaguer

Cervera

Gòdia

2020

Viruses

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Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants.

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Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

Cited by 15 publications

References 68 publications

Characterization of HIV‐1 virus‐like particles and determination of Gag stoichiometry for different production platforms

Characterization of HIV‐1 virus‐like particles and determination of Gag stoichiometry for different production platforms

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

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

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