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Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 enters the host by infecting nasal ciliated cells. Then, the virus can spread towards the oropharyngeal cavity and the pulmonary tissues. The antiviral adaptive immunity is promptly induced in response to the virus's detection, with virus-specific T-lymphocytes appearing before antiviral antibodies. Both the breadth and potency of antiviral CD8 + T-cell immunity have a key role in containing viral spread and disease severity. Current anti-SARS-CoV-2 vaccines do not impede the virus's replication in the upper respiratory tract, and there is consensus on the fact that the best potency of the antiviral immune response in both blood and the upper respiratory tract can be reached upon infection in vaccinees (i.e., breakthrough infection). However, whether the antiviral CD8 + T-cells developing in response to the breakthrough infection in the upper respiratory tract diffuse to the lungs is also still largely unknown. To fill the gap, we checked the CD8 + T-cell immunity elicited after infection of K18-hACE2 transgenic mice both at 3 weeks and 3 months after anti-spike vaccination. Virus-specific CD8 + T-cell immunity was monitored in both blood and the lungs before and after infection. By investigating the de novo generation of the CD8 + T-cells specific for SARS-CoV-2 viral proteins, we found that both membrane (M) and/or nucleocapsid (N)-specific CD8 + T-cells were induced at comparable levels in the blood of both unvaccinated and vaccinated mice. Conversely, N-specific CD8 + T-cells were readily found in the lungs of the control mice but were either rare or absent in those of vaccinated mice. These results support the idea that the hybrid cell immunity developing after asymptomatic/mild breakthrough infection strengthens the antiviral cell immunity in the lungs only marginally, implying that the direct exposition of viral antigens is required for the induction of an efficient antiviral cell immunity in the lungs.

The current battle against Severe Acute Respiratory Syndrome (SARS)-Coronavirus-2 benefits from the worldwide distribution of different vaccine formulations. All anti-SARS-CoV-2 vaccines in use are conceived to induce anti-Spike neutralizing antibodies. However, this strategy still has unresolved issues, the most relevant of which are: (i) the resistance to neutralizing antibodies of emerging SARS-CoV-2 variants and (ii) the waning of neutralizing antibodies. On the other hand, both pre-clinical evidence and clinical evidence support the idea that the immunity sustained by antigen-specific CD8 + T lymphocytes can complement and also surrogate the antiviral humoral immunity. As a distinctive feature, anti-SARS-CoV-2 CD8 + T-driven immunity maintains its efficacy even in the presence of viral protein mutations. In addition, on the basis of data obtained in survivors of the SARS-CoV epidemic, this immunity is expected to last for several years. In this review, both the mechanisms and role of CD8 + T-cell immunity in viral infections, particularly those induced by SARS-CoV and SARS-CoV-2, are analyzed. Moreover, a CD8 + T-cell-based vaccine platform relying on in vivo engineered extracellular vesicles is described. When applied to SARS-CoV-2, this strategy was proven to induce a strong immunogenicity, holding great promise for its translation into the clinic.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nef mut 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 mut -derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nef mut -based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nef mut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nef mut 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 mut 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 mut truncation does not influence stability, EV-anchoring, and CD8 + T cell immunogenicity of the fused antigen. Hence, the C-terminal deleted Nef mut may represent a safer alternative to the full-length isoform for vaccines in humans.

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.

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.

We recently described the induction of an efficient CD8⁺ T cell-mediated immune response against a tumor-associated antigen (TAA) uploaded in engineered exosomes used as an immunogen delivery tool. This immune response cleared tumor cells inoculated after immunization, and controlled the growth of tumors implanted before immunization. We looked for new protocols aimed at increasing the CD8⁺ T cell specific response to the antigen uploaded in engineered exosomes, assuming that an optimized CD8⁺ T cell immune response would correlate with a more effective depletion of tumor cells in the therapeutic setting. By considering HPV-E6 as a model of TAA, we found that the in vitro co-administration of engineered exosomes and ISCOMATRIX TM adjuvant, i.e., an adjuvant composed of purified ISCOPREP TM saponin, cholesterol, and phospholipids, led to a stronger antigen cross-presentation in both B- lymphoblastoid cell lines ( and monocyte-derived immature dendritic cells compared with that induced by the exosomes alone. Consistently, the co-inoculation in mice of ISCOMATRIX TM adjuvant and engineered exosomes induced a significant increase of TAA-specific CD8⁺ T cells compared to mice immunized with the exosomes alone. This result holds promise for effective usage of exosomes as well as alternative nanovesicles in anti-tumor therapeutic approaches., Competing Interests: The authors declare no conflict of interest.