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

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.