Characterization and quantitation of fluorescent Gag virus-like particles
© Gutiérrez-Granados et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
Upon expression, the Gag polyprotein of HIV-1 spontaneously assembles giving rise to enveloped virus-like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines. In order to develop robust VLP manufacturing processes, the availability of simple, fast and reliable quantitation tools is crucial. Traditionally, commercial p24 ELISA kits are used to estimate Gag VLP concentrations. However, this quantitation technique is time-consuming, laborious, costly and prone to methodological variability. Reporter proteins are frequently used during process development to allow a straightforward monitoring and quantitation of labeled products. This alternative was evaluated in the present work by using a Gag-GFP fusion construct.
Materials and methods
Generation of fluorescent VLPs was carried out by transient transfection of HEK 293 suspension cells with a plasmid coding for Gag fused to GFP (NIH AIDS Reagent Program). VLP budding from producer cells was visualized by electron microscopy (JEM-1400, Jeol) and confocal fluorescence microscopy (Fluoview® FV1000, Olympus, Japan). A purified standard of Gag-GFP VLP material was obtained by ultracentrifugation through a sucrose cushion and fully characterized. SDS-PAGE, Western blot, size-exclusion chromatography (SEC), nanoparticle tracking analysis (NTA, NanoSight®, UK) and transmission electron microscopy (TEM) were used for VLP characterization. The standard VLP material was used for the development and validation of a Gag-GFP VLP quantitation technique based on fluorescence. Viral particle titers estimated using this method were compared with those obtained by p24 ELISA (Innotest®, Innogenetics, Belgium), densitometry, TEM and NTA.
A purified Gag-GFP VLP standard material was obtained by harvesting VLPs from cell culture supernatants of transfected HEK 293 cells by low speed centrifugation followed by VLP pelleting through a 30% sucrose cushion. The purity of the standard material was analyzed by SEC. The SEC chromatogram showed a single peak eluting in the column void volume (V0 = 44 mL) as determined by UV and fluorescence analyses of collected fractions (Figure 1D). The A260/A280 ratio was 1.24 which is consistent with reported ratios for purified retroviral particles . The standard VLP material was further characterized using different techniques. Particle morphology was analyzed by TEM. Roughly spherical viral particles surrounded by a lipid envelope and containing an electro-dense core could be observed (Figure 1C). The mean VLP diameter according to TEM analysis was determined to be 141 ± 22 nm (n = 100), which is the expected size of Gag-GFP VLPs as they resemble immature HIV particles that are larger than wild-type HIV-1 virions . NTA analyses of the standard material showed that the most frequent particle size value (statistical mode) was 149 ± 5 nm, which is consistent with our TEM results. SDS-PAGE analysis of the standard VLP material (Figure 1E) was performed. Approximately, 65% of the total protein loaded in the gel corresponded to Gag-GFP (Figure 1E, full arrow), the major HIV-1 VLP structural protein. The other minor bands should correspond to cellular proteins derived from host cells as retroviral particles are known to promiscuously incorporate a significant amount of host proteins [3, 4]. A Gag-GFP band of the expected molecular weight (~81 kDa) was specifically detected using an anti-p24 mAb by Western blot analysis (Figure 1E, full arrow). The presence of a Gag-GFP fragment (Figure 1E, empty arrow), representing only 5% of the total Gag-GFP loaded, was also observed in the gel.
Comparison between the fluorescence-based quantitation method and the p24 ELISA assay
p24 ELISA assay
Gag-GFP fusion protein
Gag-GFP fusion protein
7 to 1000 RFU (10 to 3600 ng of p24/mL)
10 to 300 pg of p24/mL
Limit of detection
10 ng/mL of p24
10 pg/mL of p24
Time (96 samples)
Price (96 samples)
Due to the flexibility of the retrovirus particle assembly process, fluorescently tagged Gag VLPs can be easily generated by expressing Gag as a fusion construct with GFP. Although fluorescently labeled Gag has mainly been used to study retrovirus replication in living cells, this attractive feature is exploited in our laboratory to facilitate the monitoring and quantitation of Gag VLPs. A purified standard VLP material was obtained and fully characterized. VLPs in the standard material showed to be of the expected size, morphology and with a composition consistent with immature HIV-1 particles. A fast, reliable and cost-effective quantitation method based on fluorescence was developed and validated using the standard VLP material. The fluorescence-based quantification method should facilitate the development and optimization of bioprocessing strategies for Gag-based VLPs.
We would like to thank Dr. Amine Kamen (National Research Council of Canada) for helpful discussions about this project and for kindly providing the cGMP compliant HEK 293SF-3F6 cell line. The pGag-GFP plasmid was obtained through the NIH AIDS reagent program (Cat #11468). The contribution of Dr. Julià Blanco and Dr. Jorge Carrillo (IrsiCaixa, Spain) to this work is greatly appreciated. This project was financially supported by MINECO-SEIDI, reference BIO2012-31251.
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