The C-Terminal Domain of Nef Is Dispensable for the CD8 T Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nef fused at its C-terminus to viral and tumor antigens elicit a potent, effective, and anti-tolerogenic CD8 T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nef-derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nef-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nef retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nef to associate with EVs. The EV-anchoring function was also preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. Most important, the Nef C-terminal deletion did not affect levels, quality, and diffusion at distal sites of the antigen-specific CD8 T immunity. We concluded that the C-terminal Nef truncation does not influence stability, EV-anchoring, and CD8 T cell immunogenicity of the fused antigen. Hence, the C-terminal deleted Nef may represent a safer alternative to the full-length isoform for vaccines in humans.