Strong SARS-CoV-2 N-Specific CD8 + T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice.

SARS-CoV-2-specific CD8 ⁺ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8 ⁺ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nef ^mut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8 ⁺ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8 ⁺ T lymphocytes induced by the immunization through the Nef ^mut -based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nef ^mut /S1 and Nef ^mut /N generated polyfunctional CD8 ⁺ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8 ⁺ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8 ⁺ T-resident memory cells in lungs, supporting the idea that the Nef ^mut -based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8 ⁺ T cell-based platform could be considered for a new combination prophylactic strategy.