Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8⁺ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nef^mut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nef^mut or a deletion mutant of Nef^mut referred to as Nef^mutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nef^mut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8⁺ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.