Simultaneous CD8⁺ T-Cell Immune Response against SARS-Cov-2 S, M, and N Induced by Endogenously Engineered Extracellular Vesicles in Both Spleen and Lungs

Most advanced vaccines against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 are designed to induce antibodies against spike (S) protein. Differently, we developed an original strategy to induce CD8⁺ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is a new vaccination approach based on intramuscular injection of DNA expression vectors coding for a biologically inactive HIV-1 Nef protein (Nef^mut) with an unusually high efficiency of incorporation into EVs, even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nef^mut-fused antigens released by muscle cells can freely circulate into the body and are internalized by antigen-presenting cells. Therefore, EV-associated antigens can be cross-presented to prime antigen-specific CD8⁺ T-cells. To apply this technology to a strategy of anti-SARS-CoV-2 vaccine, we designed DNA vectors expressing the products of fusion between Nef^mut and different viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. We provided evidence that all fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8⁺ T cell immunity became detectable in spleens and, most important, in lung airways. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lungs. Hence, DNA vectors expressing Nef^mut-based fusion proteins can be proposed for new anti-SARS-CoV-2 vaccine strategies.