How Do Anti-SARS-CoV-2 mRNA Vaccines Protect from Severe Disease?

COVID-19 pathogenesis develops in two phases. First, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 spreads within the epithelial cells of the mucosa of upper and, possibly, lower respiratory tracts. While the virus dissemination can be controlled by an emerging adaptive host immune response, if the virus diffuses to the pulmonary alveoli, a potentially lethal mechanism can arise in the second phase. It consists of an uncontrolled burst of cytokines/inflammatory factors (i.e., cytokine storm), leading to the insurgence of respiratory symptoms and, consequently, multi-organ failures. Messenger (m)RNA-based vaccines represent the most innovative approach in terms of prophylaxis against SARS-CoV-2-induced disease. The cumulating data indicate that the response to mRNA vaccines is basically ineffective to counteract the viral replication in the upper respiratory tracts, while showing efficacy in containing the development of severe disease. Considering that the antiviral immunity elicited by intramuscularly delivered mRNA vaccines is expected to show similar quantitative and qualitative features in upper and lower respiratory tracts, the different outcomes appear surprising and deserve accurate consideration. In this review, a still unexplored mechanism accounting for the mRNA vaccine effect against severe disease is proposed. Based on well-established experimental evidence, a possible inhibitory effect on alveolar macrophages as a consequence of the diffusion of the extracellular and/or cell-associated Spike protein can be envisioned as a key event counteracting the cytokine storm. This benefit, however, may be associated with defects in the immune functions of macrophages in other tissues whose possible consequences deserve careful evaluation.

HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases.

The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.

Exploiting Manipulated Small Extracellular Vesicles to Subvert Immunosuppression at the Tumor Microenvironment through Mannose Receptor/CD206 Targeting.

Immunosuppression at tumor microenvironment (TME) is one of the major obstacles to be overcome for an effective therapeutic intervention against solid tumors. Tumor-associated macrophages (TAMs) comprise a sub-population that plays multiple pro-tumoral roles in tumor development including general immunosuppression, which can be identified in terms of high expression of mannose receptor (MR or CD206). Immunosuppressive TAMs, like other macrophage sub-populations, display functional plasticity that allows them to be re-programmed to inflammatory macrophages. In order to mitigate immunosuppression at the TME, several efforts are ongoing to effectively re-educate pro-tumoral TAMs. Extracellular vesicles (EVs), released by both normal and tumor cells types, are emerging as key mediators of the cell to cell communication and have been shown to have a role in the modulation of immune responses in the TME. Recent studies demonstrated the enrichment of high mannose glycans on the surface of small EVs (sEVs), a subtype of EVs of endosomal origin of 30-150 nm in diameter. This characteristic renders sEVs an ideal tool for the delivery of therapeutic molecules into MR/CD206-expressing TAMs. In this review, we report the most recent literature data highlighting the critical role of TAMs in tumor development, as well as the experimental evidences that has emerged from the biochemical characterization of sEV membranes. In addition, we propose an original way to target immunosuppressive TAMs at the TME by endogenously engineered sEVs for a new therapeutic approach against solid tumors.

Development of a novel human phage display-derived anti-LAG3 scFv antibody targeting CD8+ T lymphocyte exhaustion.

BACKGROUND: Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. RESULTS: We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γ release by both ELISA and ELISPOT assays. CONCLUSIONS: Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment.

Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir.

Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4+ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4+ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4+ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4+ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4+ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4+ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

The Multifaceted Functions of Exosomes in Health and Disease: An Overview.

Exosomes are extracellular vesicles of 50-150 nm in diameter secreted by basically all cell types. They mediate micro-communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Research on exosomes is a rapidly growing field, however many aspects of their biogenesis and functions still await a complete clarification. In our review we summarize most recent findings regarding biogenesis, structure, and functions of exosomes. In addition, an overview regarding the role of exosomes in both infectious and non-infectious diseases is provided. Finally, the use of exosomes as biomarkers and delivery tools for therapeutic molecules is addressed. Considering the body of literature data, exosomes have to be considered key components of the intercellular communication in both health and disease.

Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3.

An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abacavir (ABC), 2',3'-dideoxyinosine (DDI), lamivudine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegravir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37 °C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37 °C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti-HIV activity, as assayed by this method, was EFV > 3TC > AZT > NVP > DDI > ABC and ETR > RPV > RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the optimized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quantitative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.

Latent HIV-1 is activated by exosomes from cells infected with either replication-competent or defective HIV-1.

BACKGROUND: Completion of HIV life cycle in CD4(+) T lymphocytes needs cell activation. We recently reported that treatment of resting CD4(+) T lymphocytes with exosomes produced by HIV-1 infected cells induces cell activation and susceptibility to HIV replication. Here, we present data regarding the effects of these exosomes on cells latently infected with HIV-1. RESULTS: HIV-1 latently infecting U937-derived U1 cells was activated upon challenge with exosomes purified from the supernatant of U937 cells chronically infected with HIV-1. This effect was no more detectable when exosomes from cells infected with HIV-1 strains either nef-deleted or expressing a functionally defective Nef were used, indicating that Nef is the viral determinant of exosome-induced HIV-1 activation. Treatment with either TAPI-2, i.e., a specific inhibitor of the pro-TNFα-processing ADAM17 enzyme, or anti-TNFα Abs abolished HIV-1 activation. Hence, similar to what previously demonstrated for the exosome-mediated activation of uninfected CD4(+) T lymphocytes, the Nef-ADAM17-TNFα axis is part of the mechanism of latent HIV-1 activation. It is noteworthy that these observations have been reproduced using: (1) primary CD4(+) T lymphocytes latently infected with HIV-1; (2) exosomes from both primary CD4(+) T lymphocytes and macrophages acutely infected with HIV-1; (3) co-cultures of HIV-1 acutely infected CD4(+) T lymphocytes and autologous lymphocytes latently infected with HIV-1, and (4) exosomes from cells expressing a defective HIV-1. CONCLUSIONS: Our results strongly suggest that latent HIV-1 can be activated by TNFα released by cells upon ingestion of exosomes released by infected cells, and that this effect depends on the activity of exosome-associated ADAM17. These pieces of evidence shed new light on the mechanism of HIV reactivation in latent reservoirs, and might also be relevant to design new therapeutic interventions focused on HIV eradication.

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation.

BACKGROUND: Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. RESULTS: CCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses. CONCLUSIONS: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.

Exosomes from human immunodeficiency virus type 1 (HIV-1)-infected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a Nef- and ADAM17-dependent mechanism.

UNLABELLED: Resting CD4+ T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4+ T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4+ T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4+ T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the 62EEEE65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-α) in its mature form, associates with exosomes from HIV-1-infected cells, and plays a key role in the HIV-1 replication in quiescent CD4+ T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4+ T lymphocytes. TNF-α is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-α antibodies blocked the HIV-1 replication. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4+ T lymphocytes, thus stimulating viral spread. IMPORTANCE: Overall, our findings support the idea that HIV evolved to usurp the exosome-based intercellular communication network to favor its spread in infected hosts.

Cell activation and HIV-1 replication in unstimulated CD4+ T lymphocytes ingesting exosomes from cells expressing defective HIV-1.

BACKGROUND: A relevant burden of defective HIV-1 genomes populates PBMCs from HIV-1 infected patients, especially during HAART treatment. These viral genomes, although unable to codify for infectious viral particles, can express viral proteins which may affect functions of host cells as well as bystander ones. Cells expressing defective HIV-1 have a lifespan longer than that of cells producing infectious particles. Hence, their interaction with other cell types, including resting lymphocytes, is expected to occur frequently in tissues where HIV actively replicates. We investigated the effects of the expression of a prototype of functionally defective HIV-1 on bystander, unstimulated CD4+ T lymphocytes. RESULTS: We observed that unstimulated human primary CD4+ T lymphocytes were activated and became permissive for HIV-1 replication when co-cultivated with cells expressing a functionally defective HIV-1 (F12/Hut-78 cells). This effect depended on the presence in F12/Hut-78 supernatants of nanovesicles we identified as exosomes. By inspecting the underlying mechanism, we found that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNF-α in its mature form, associated with exosomes from F12/Hut-78 cells, and played a key role in the HIV-1 replication in unstimulated CD4+ T lymphocytes. In fact, the treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in unstimulated CD4+ T lymphocytes. TNF-α appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-α or its receptors blocked the HIV-1 replication. Finally, we found that the expression of NefF12 in exosome-producing cells was sufficient to induce the susceptibility to HIV-1 infection in unstimulated CD4+ T lymphocytes. CONCLUSIONS: Exosomes from cells expressing a functionally defective mutant can induce cell activation and HIV-1 susceptibility in unstimulated CD4+ T lymphocytes. This evidence highlights the relevance for AIDS pathogenesis of the expression of viral products from defective HIV-1 genomes.

Sequences within RNA coding for HIV-1 Gag p17 are efficiently targeted to exosomes.

HIV budding requires the interaction with cell factors involved in the biogenesis of exosomes. This implies the possibility that viral products undergo exosome incorporation. While this has been already described for both Gag and Nef HIV-1 proteins, no conclusive results on HIV genome have been produced so far. Here, we report that unspliced, but not single or double spliced, HIV-1 RNA species are incorporated in exosomes. Deletion mutant analysis indicated that the presence of a stretch of sequences within the 5' end of the Gag p17 open reading frame is sufficient for HIV-1 RNA exosome incorporation. These sequences were found associating with exosomes also out of the HIV-1 context, thus indicating that the diversion towards the vesicular compartment can occur without need of additional HIV-1 sequences. Finally, the incorporation of genomic HIV-1 RNA in exosomes significantly increased when producer cells express HIV-1 defective for viral genome packaging. Manipulating infected cells to favour the selective incorporation in exosomes of genomic HIV-1 RNA might have therapeutic implications.

The Limitations of Current T Cell-Driven Anticancer Immunotherapies Can Be Overcome with an Original Extracellular-Vesicle-Based Vaccine Strategy.

The emergence of tumors associated with defects in immune surveillance often involve the impairment of key functions of T lymphocytes. Therefore, several anticancer immunotherapies have focused on the induction/strengthening of the tumor-specific activity of T cells. In particular, strategies based on immune checkpoint inhibitors, CAR-T cells, and mRNA vaccines share a common goal of inducing/recovering an effective antitumor cytotoxic activity, often resulting in either exhausted or absent in patients' lymphocytes. In many instances, these approaches have been met with success, becoming part of current clinic protocols. However, the most practiced strategies sometimes also pay significant tolls in terms of adverse events, a lack of target specificity, tumor escape, and unsustainable costs. Hence, new antitumor immunotherapies facing at least some of these issues need to be explored. In this perspective article, the characteristics of a novel CD8+ T cell-specific anticancer vaccine strategy based on in vivo-engineered extracellular vesicles are described. How this approach can be exploited to overcome at least some of the limitations of current antitumor immunotherapies is also discussed.

SARS-CoV-2-Specific CD8+ T-Cells in Blood but Not in the Lungs of Vaccinated K18-hACE2 Mice after Infection.

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.

Antiviral effect of SARS-CoV-2 N-specific CD8+ T cells induced in lungs by engineered extracellular vesicles.

Induction of effective immunity in the lungs should be a requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 Nucleocapsid (N) protein induced immunity in the lungs of K18-hACE2 transgenic mice, which then can survive the lethal virus infection. However, nothing is known about the ability of the N-specific CD8+ T cell immunity in controlling viral replication in the lungs, a major pathogenic signature of severe disease in humans. To fill the gap, we investigated the immunity generated in the lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8+ T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in the lungs were evaluated. Three weeks after the second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to the control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8+ T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting, and associated with the persistence of N-specific CD8+ T-resident memory lymphocytes. In view of the quite low mutation rate of the N protein, the present vaccine strategy has the potential to control the replication of all emerging variants.

Perturbed replication induced genome wide or at common fragile sites is differently managed in the absence of WRN.

The Werner syndrome protein (WRN) is a member of the RecQ helicase family. Loss of WRN results in a human disease, the Werner syndrome (WS), characterized by high genomic instability, elevated cancer risk and premature aging. WRN is crucial for the recovery of stalled replication forks and possesses both helicase and exonuclease enzymatic activities of uncertain biological significance. Previous work revealed that WRN promotes formation of MUS81-dependent double strand breaks (DSBs) at HU-induced stalled forks, allowing replication restart at the expense of chromosome stability. Here, using cells expressing the helicase- or exonuclease-dead WRN mutant, we show that both activities of WRN are required to prevent MUS81-dependent breakage after HU-induced replication arrest. Moreover, we provide evidence that, in WS cells, DSBs generated by MUS81 do not require RAD51 activity for their formation. Surprisingly, when replication is specifically perturbed at common fragile sites (CFS) by aphidicolin, WRN limits accumulation of ssDNA gaps and no MUS81-dependent DSBs are detected. However, in both cases, RAD51 is essential to ensure viability of WS cells, although by different mechanisms. Thus, the role of WRN in response to perturbation of replication along CFS is functionally distinct from that carried out at stalled forks genome wide. Our results contribute to unveil two different mechanisms used by the cell to overcome the absence of WRN.

Biological and Immune Responses to Current Anti-SARS-CoV-2 mRNA Vaccines beyond Anti-Spike Antibody Production.

Several vaccine strategies are now available to fight the current SARS-CoV-2 pandemic. Those based on the administration of lipid-complexed messenger(m)RNA molecules represent the last frontiers in terms of technology innovation. mRNA molecules coding for the SARS-CoV-2 Spike protein are intramuscularly injected, thereby entering cells by virtue of their encapsulation into synthetic lipid nanovesicles. mRNA-targeted cells express the Spike protein on their plasma membrane in a way that it can be sensed by the immune system, which reacts generating anti-Spike antibodies. Although this class of vaccines appears as the most effective against SARS-CoV-2 infection and disease, their safety and efficiency are challenged by several factors included, but not limited to the following: emergence of viral variants, lack of adequate pharmacokinetics/pharmacodynamics studies, inability to protect oral mucosa from infection, and antibody waning. Emergence of viral variants can be a consequence of mass vaccination carried out in a pandemic time using suboptimal vaccines against an RNA virus. On the other hand, understanding the remainder flaws could be of some help in designing next generation anti-SARS-CoV-2 vaccines. In this commentary, issues regarding the fate of injected mRNA, the tissue distribution of the induced antiviral antibodies, and the generation of memory B cells are discussed. Careful evaluation of both experimental and clinical observations on these key aspects should be taken into account before planning booster administration, vaccination to non-at-risk population, and social restrictions.

Extracellular Vesicles and Their Use as Vehicles of Immunogens.

Healthy cells constitutively release lipid bilayered vesicles of different sizes and recognizing different biogenesis, collectively referred to as extracellular vesicles (EVs). EVs can be distinguished in exosomes and microvesicles. Biological and biomedical research on EVs is an emerging field that is rapidly growing. Many EV features including biogenesis, cell uptake, and functions still require unambiguous elucidation. Nevertheless, it has been well established that EVs are involved in communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Here, we summarize most recent findings regarding biogenesis, structure, and functions of both exosomes and microvesicles. In addition, the use of EVs as delivery tools to induce CD8+ T cell immunity is addressed compared to current designs exploiting enveloped viral vectors and virus-like particles. Finally, we describe a both safe and original approach conceived for the induction of strong CTL immunity against antigens uploaded in EVs constitutively released by muscle cells.

Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein.

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

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 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 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 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.