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

Abstract

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 Nefmut, 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 Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro‐produced EVs has supported the uploading of N protein when fused with truncated Nefmut. 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.