Abstract
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.
Highlights
Virus-like particles (VLPs) are considered a promising platform in the field of vaccine development.Nowadays, there are several licensed VLP-based vaccines, such as Cervarix®, Gardasil®, Hecolin®or Porcilis PCV® and more than 100 candidates are undergoing clinical trials [1]
VLPs were observed as spherical electrodense structures surrounded by a bright corona that might correspond to structured Gag-eGFP monomers surrounded by the lipid membrane [36,45]
extracellular vesicles (EVs) were observed as less electrodense nanoparticles and that could be identified in conditioned medium samples (Supplementary materials S1) [46]
Summary
Virus-like particles (VLPs) are considered a promising platform in the field of vaccine development.Nowadays, there are several licensed VLP-based vaccines, such as Cervarix® , Gardasil® , Hecolin®or Porcilis PCV® and more than 100 candidates are undergoing clinical trials [1]. Virus-like particles (VLPs) are considered a promising platform in the field of vaccine development. There are several licensed VLP-based vaccines, such as Cervarix® , Gardasil® , Hecolin®. Porcilis PCV® and more than 100 candidates are undergoing clinical trials [1]. Their success as immunogens lies on their ability to mimic native viruses without containing a viral genome. Their highly organized and repetitive antigen structure has shown effective cellular and humoral immune responses [2]. Advances in the field of bioengineering have widened their possible applications; VLP technology accepts several modifications including encapsulation, chemical conjugation or genetic engineering. VLPs can be pseudotyped or used either as DNA or drug nanocarriers [1,3]
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