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

Lavado-García, Jesús; Jorge, Inmaculada; Boix-Besora, Arnau; Vázquez, Jesús; Gòdia, Francesc; Cervera, Laura

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent SARS-CoV-2 pandemic. Virus-like 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 are 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 HPV, 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 work we established an analytical method of characterization for the Gag protein core and clarify 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 (PRM), an accurate and fast, mass-spectrometry-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 large scale to accelerate new recombinant vaccine production technologies. This article is protected by copyright. All rights reserved.