inoculated in each quadriceps with 10 g of each DNA vector and, as control, equal amounts of either void or pVAX1-Nefmut vector

inoculated in each quadriceps with 10 g of each DNA vector and, as control, equal amounts of either void or pVAX1-Nefmut vector. 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 Nefmut 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 Nefmut-based fusion proteins can be proposed for new anti-SARS-CoV-2 vaccine strategies. kinases (PAK)-2 activation [32,33]. Furthermore, we observed that the efficiency of Nefmut incorporation AG-490 into EVs is maintained even when a foreign protein is fused to its C-terminus [31,32,34,35,36,37]. AG-490 When DNA vectors expressing Nefmut-based fusion proteins are intramuscularly (i.m.) injected in mice, high amounts of the fusion protein are packed into EVs while not altering their spontaneous release from muscle tissue. Nefmut-fused antigens AG-490 released inside muscle-derived-EVs are then internalized by antigen-presenting cells (APCs), which, in turn, cross-present EV content to activate antigen-specific CD8+ T cells. These in vivo engineered EVs are assumed to freely circulate into the body, thus having the potential to reach distal tissues. We already documented that they act as an effective vaccine by eliciting potent antigen-specific CTL responses [31,32,37]. Both effectiveness and flexibility of this vaccine platform have been demonstrated with an array of viral products of various origins and sizes, including but not limited to: Human Papilloma Virus (HPV)16-E6 and -E7; Ebola Virus VP24, VP40, and NP; Hepatitis C Virus NS3; West Nile Virus NS3; and Crimean-Congo Hemorrhagic Fever NP [31,37,38]. Of note, in our hands very low to undetectable antigen-specific CD8+ T immune responses were observed when animals were injected with DNA vectors expressing the antigen open reading frame (ORF) devoid of the Nefmut sequences [31,37]. The immune response elicited through the Nefmut-based platform essentially involves the CTL function in the absence of detectable antibody response [32,37,38]. Here, we tested three SARS-CoV-2 structural antigens, namely spike (S), membrane (M), and nucleocapsid (N) proteins in the context of the Nefmut system. The immunogenicity of DNA vectors expressing each SARS-CoV-2 protein fused with Nefmut and injected in mice either alone or in combination was evaluated in both spleens and lung airways. 2. Materials and Methods 2.1. DNA Vector Synthesis ORFs coding for Nefmut fused with S1, S2, M, or N SARS-CoV-2 proteins were cloned into pVAX1 plasmid (Thermo Fisher, Waltham, MA, USA), i.e., a vector approved by FDA for use in humans. To obtain the pVAX1 vector expressing Nefmut, its ORF was cloned into Nhe I and EcoR I sites. To recover vectors expressing Nefmut-based fusion products, an intermediate vector referred to as pVAX1/Nefmutfusion was produced. Here, the whole Nefmut ORF deprived of its stop codon was followed by a sequence coding a GPGP linker including a unique Apa I restriction site. In this way, sequences comprising the Apa I site at their 5 end, and the Pme I one at their 3 end were fused in frame with Nefmut ORF. Stop codons of SARS-CoV-2-related sequences were preceded by sequences coding for a DYKDDDK epitope tag (flag-tag). SARS-CoV-2 sequences were optimized for expression in human cells through GeneSmart software from Genescript. All these vectors were synthesized by Explora Biotech. The pTargeT vector expressing the Nefmut/HPV16-E7 fusion protein was already described [38]. 2.2. Cell Cultures and Transfection Human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) were grown in DMEM (Gibco, Thermo Fisher, Waltham, MA) plus 10% heat-inactivated fetal calf serum (FCS, Gibco, Thermo Fisher, Waltham, MA). Transfection assays were performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA)-based method. 2.3. EV Isolation Cells transfected with vectors expressing the Nefmut-based fusion proteins were washed IL1R 24 h later, and reseeded in medium supplemented with.