Giovannelli, A. | Search bioRxiv

bioRxiv2022

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 Nefmut 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 Nefmut-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 Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses 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 Nefmut- 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.

microbiology↗

bioRxiv2021

Long-term antitumor CD8+ T cell immunity induced by endogenously engineered extracellular vesicles

We developed a novel approach to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is an innovative vaccination approach employing a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) that has lost the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nefmut traffics large amounts of antigens fused to it into EVs spontaneously released by cells expressing the Nefmut_based DNA vector. We previously provided evidence that the inoculation in mice of a DNA vector expressing the Nefmut/HPV16-E7 fusion protein induced an E7-specific CTL immune response as detected 2 weeks after the second immunization. In an effort to optimize the anti-HPV16 CD8+ T cell immune response, we found that the co-injection of DNA vectors expressing Nefmut fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that we observed in mice injected with the single vectors. When TC-1 cells, i.e., a tumor cell line co-expressing E6 and E7, were implanted before immunization, all mice survived until day 44, whereas no mice injected with either void or Nefmut_expressing vectors survived until day 32 after tumor implantation. A substantial part of mice (7 out of 12) cleared the tumor. When cured mice were re-challenged with a second sub cute implantation of TC-1 cells, and followed for additional 135 days, whereas none of them developed tumors. Both E6- and E7-specific CD8+ T immunity was still detectable at the end of the observation time. Hence, the immunity elicited by engineered EVs, besides curing already developed tumors, is strong enough to guarantee the resistance to additional tumor attack. This results is of relevance for therapy against both metastatic and relapsing tumors.

cancer biology↗