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We recently described a cytotoxic CD8 + T lymphocyte (CTL) vaccine platform based on the intramuscular (i.m.) injection of DNA eukaryotic vectors expressing antigens of interest fused at the C-terminus of HIV-1 Nef mut , i.e., a functionally defective mutant that is incorporated at quite high levels into exosomes/extracellular vesicles (EVs). This system has been proven to elicit strong CTL immunity against a plethora of both viral and tumor antigens, as well as inhibit both transplantable and orthotopic tumors in mice. However, a number of open issues remain regarding the underlying mechanism. Here we provide evidence that hindering the uploading into EVs of Nef mut -derived products by removing the Nef mut N-terminal fatty acids leads to a dramatic reduction of the downstream antigen-specific CD8 + T-cell activation after i.m. injection of DNA vectors in mice. This result formally demonstrates that the generation of engineered EVs is part of the mechanism underlying the in vivo induced CD8 + T-cell immunogenicity. Gaining new insights on the EV-based vaccine platform can be relevant in view of its possible translation into the clinic to counteract both chronic and acute infections as well as tumors.

N-Terminal Fatty Acids of NEFMUT Are Required for the CD8+ T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles