Extracellular vesicle-based anti-HOXB7 CD8 T cell-specific vaccination strengthens antitumor effects induced by vaccination against Her2/neu.

We previously developed an innovative strategy to induce CD8 T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nef). Nef is very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells.

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.

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.

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 Nef, i.e.

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.

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 protein, i.

Long-Term Antitumor CD8 T Cell Immunity Induced by Endogenously Engineered Extracellular Vesicles.

We developed an innovative method to induce antigen-specific CD8 T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This approach employs a DNA vector expressing a mutated HIV-1 Nef protein (Nef) deprived of 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.

The C-Terminal Domain of Nef Is Dispensable for the CD8 T Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nef 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-derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nef-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nef retaining the EV-anchoring protein property.

Simultaneous CD8 T-Cell Immune Response against SARS-Cov-2 S, M, and N Induced by Endogenously Engineered Extracellular Vesicles in Both Spleen and Lungs.

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) with an unusually high efficiency of incorporation into EVs, even when foreign polypeptides are fused to its C-terminus.

KSHV G-protein coupled receptor vGPCR oncogenic signaling upregulation of Cyclooxygenase-2 expression mediates angiogenesis and tumorigenesis in Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) vGPCR is a constitutively active G protein-coupled receptor that subverts proliferative and inflammatory signaling pathways to induce cell transformation in Kaposi's sarcoma. Cyclooxygenase-2 (COX-2) is an inflammatory mediator that plays a key regulatory role in the activation of tumor angiogenesis. Using two different transformed mouse models and tumorigenic full KSHV genome-bearing cells, including KSHV-Bac16 based mutant system with a vGPCR deletion, we demostrate that vGPCR upregulates COX-2 expression and activity, signaling through selective MAPK cascades.

Engineered Extracellular Vesicles/Exosomes as a New Tool against Neurodegenerative Diseases.

Neurodegenerative diseases are commonly generated by intracellular accumulation of misfolded/aggregated mutated proteins. These abnormal protein aggregates impair the functions of mitochondria and induce oxidative stress, thereby resulting in neuronal cell death. In turn, neuronal damage induces chronic inflammation and neurodegeneration.

N-Terminal Fatty Acids of NEF Are Required for the CD8 T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

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, i.

Extracellular vesicle-mediated intercellular communication in HIV-1 infection and its role in the reservoir maintenance.

HIV-1 infection is efficiently controlled by combination anti-retroviral therapy (cART). However, despite preventing disease progression, cART does not eradicate virus infection which persists in a latent form for an individual's lifetime. The latent reservoir comprises memory CD4 T lymphocytes, macrophages, and dendritic cells; however, for the most part, the reservoir is generated by virus entry into activated CD4 T lymphocytes committed to return to a resting state, even though resting CD4 T lymphocytes can be latently infected as well.

The Intracellular Delivery Of Anti-HPV16 E7 scFvs Through Engineered Extracellular Vesicles Inhibits The Proliferation Of HPV-Infected Cells.

Purpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs).

Tumor cells endowed with professional antigen-presenting cell functions prime PBLs to generate antitumor CTLs.

Intrinsic genetic instability of tumor cells leads to continuous production of mutated proteins referred to as tumor-specific neoantigens. Generally, they are recognized as nonself products by the host immune system. However, an effective adaptive response clearing neoantigen-expressing cells is lost in tumor diseases.

DNA Vectors Generating Engineered Exosomes Potential CTL Vaccine Candidates Against AIDS, Hepatitis B, and Tumors.

Eukaryotic cells constitutively produce nanovesicles of 50-150 nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nef) with the antigen of interest.

Exosomes in Therapy: Engineering, Pharmacokinetics and Future Applications.

Background: Eukaryotic cells release vesicles of different sizes under both physiological and pathological conditions. On the basis of the respective biogenesis, extracellular vesicles are classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are considered tools for innovative therapeutic interventions, especially when engineered with effector molecules.

Engineered exosomes emerging from muscle cells break immune tolerance to HER2 in transgenic mice and induce antigen-specific CTLs upon challenge by human dendritic cells.

Unlabelled: We recently described a novel biotechnological platform for the production of unrestricted cytotoxic T lymphocyte (CTL) vaccines. It relies on in vivo engineering of exosomes, i.e.

An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.

Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, that is, Nef , the authors previously characterized for its high levels of incorporation in exosomes.

Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation.

We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nef exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nef fused with HPV E7.

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.

The CD8⁺ T Cell-Mediated Immunity Induced by HPV-E6 Uploaded in Engineered Exosomes Is Improved by ISCOMATRIX Adjuvant.

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.

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.

HIV-1 TAT and IMMUNE DYSREGULATION in AIDS PATHOGENESIS: a THERAPEUTIC TARGET.

The HIV-1 transactivator Tat protein plays a key role in AIDS pathogenesis. Besides the Tat role as activator of HIV-1 transcription, it exerts several important functions on infected and uninfected cells. In fact, HIV-1 Tat is released by infected cells and is taken up by neighboring cells.

HPV-E7 delivered by engineered exosomes elicits a protective CD8⁺ T cell-mediated immune response.

We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e.