Gag HIV-1 Virus-like Particles and Extracellular Vesicles Functionalization with Spike Epitopes of SARS-CoV-2 Using a Copper-Free Click Chemistry Approach.

Enveloped nanoparticles such as extracellular vesicles (EVs) and virus-like particles (VLPs) have emerged as promising nanocarriers capable of transporting bioactive molecules for drug delivery and vaccination. Optimized functionalization methodologies are required to increase the functionalization levels of these nanoparticles, enhancing their performance. Here, a bioorthogonal copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction has been optimized to functionalize human immunodeficiency virus type 1 (HIV-1) Gag-based VLPs and EVs. The optimization process has been carried out through reaction kinetics and design of experiments (DoE) using Cy5 as a reporter molecule. The functionalization of both VLPs and EVs has been studied using super-resolution fluorescence microscopy (SRFM), revealing remarkable differences between Gag-VLPs and coproduced EVs. EVs produced by mock transfection and cell growth have been functionalized achieving a mean of 3618.63 ± 48.91 and 6498.75 ± 352.71 Cy5 molecules covalently linked per particle (Cy5(cov)/particle), respectively. Different nanoparticles have been functionalized with two linear B-cell epitopes from the Spike protein of SARS-CoV-2, S(315-338) TSNFRVQPTESIVRFPNITNLCPF and S(648-663) GCLIGAEHVNNSYECD, and analyzed by an immunoassay with sera from COVID-19 patients. The obtained results validate the selected B-cell epitopes and highlight the potential of the optimized functionalization approach for the development of nanoparticle-based vaccines.