Engineered retrovirus-like dendritic cell derived extracellular vesicles

Abstract

The current disclosure is directed to an immunogenic composition comprising engineered extracellular vesicles with enhanced immunity, methods of making engineered extracellular vesicles with enhanced immunity, a method of slowing or stopping a disease in a subject through the use of the disclosed immunogenic composition, and a method of immunizing a subject through administering the disclosed immunogenic composition.

Description

ENGINEERED RETROVIRUS-LIKE DENDRITIC CELL DERIVED EXTRACELLULAR VESICLES CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 563,016, filed March 8, 2024, and U.S. Provisional Patent Application No.63 / 713,311, filed October 29, 2024, the contents of which are incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The sequence listing in XML, named as 42955WO_10879_03_PC_SequenceListing.xml of 46,920 bytes, created on March 5, 2025, and submitted via Patent Center, is incorporated herein by reference. BACKGROUND

[0003] Dendritic cells (DCs) are of bone marrow origin and express a high level of major histocompatibility complex (MHC) molecules, including MHC class I and MHC class II molecules. DCs are most potent among the antigen-presenting cells and are believed to be crucial for the initiation of a primary T-cell response to foreign antigens. DC vaccines capitalize on the inherent ability of these cells to initiate and modulate immune responses. By loading these cells with tumor-specific antigens and subsequently reintroducing them into the patient, they can potentially direct the immune system to recognize and combat cancer cells. This approach offers specificity in targeting the individual's cancer and the ability to present multiple tumor-associated antigens, eliciting a broad immune response. DC vaccines can stimulate both helper and cytotoxic T-cells, potentially overcoming tumor evasion tactics such as MHC molecule downregulation or immunosuppressive environments. The emergence of DC-derived extracellular vesicles (DEVs) has introduced a new dimension to this therapeutic strategy. DEVs facilitate long-range communication between the tumor microenvironment (TME) and lymphoid organs. DEVs are adept at traveling from DCs that have infiltrated tumors to distant sites, where they present tumor antigens not only to T cells but also modulate other immune cells, enhancing the body's ability to detect and respond to cancer cells throughout. Reflecting their parental DCs' molecular profile, DEVs have demonstrated the capability to robustly activate CD8+ and CD4+ T cell responses through both direct and indirect antigen presentation. Activated DEVs employ direct antigen presentation via MHC-I and MHC-II molecules, and indirect modalities byinfluencing adjacent DCs through mechanisms such as vesicle internalization and cross-dressing. As autologous structures, DEVs excel in antigen delivery, with reduced immunogenicity risks compared to synthetic agents. Their inherent nature facilitates precise tumor antigen presentation, making them prime candidates for targeted immunotherapy.

[0004] Despite these promising features, challenges remain in achieving potent and consistent functionality. Early clinical trials of dendritic cell-derived exosome (DEX) vaccines have demonstrated safety and tolerability, yet robust T cell activation remains inconsistent, particularly in tumors like melanoma and non-small cell lung cancer (NSCLC). Limited T cell infiltration and the immunosuppressive tumor microenvironment hinder their efficacy, despite their potential for presenting tumor antigens. Ongoing efforts aim to refine DEVs to induce stronger and more sustained immune responses across diverse patient populations.

[0005] Activity-regulated cytoskeleton-associated protein (Arc) emerges as a fascinating regulator in immune modulation, particularly within the domain of DC antigen presentation. Originally found in migratory DCs within the skin, Arc plays a pivotal role in augmenting DCs’ response to inflammation. Furthermore, its critical roles in facilitating optimal T cell activation in cancers and autoimmune diseases highlight its significance. Intriguingly, Arc is a retrotransposon gene encoding a protein that self-assembles into virus-like capsids, which subsequently exit host cells in the form of EVs. These endogenous virus-like vesicles (VLVs) are vehicles of long-range intercellular communication and molecular transfer. Drawing parallels to its retro-viral Gag protein counterparts, Arc engages envelop proteins (ENVs) and other membrane-associated proteins in its assembly to interact with T cell and B cell receptors. SUMMARY

[0006] The current disclosure is directed to an immunogenic composition comprising engineered extracellular vesicles with enhanced immunity, methods of making engineered extracellular vesicles with enhanced immunity, a method of slowing or stopping a disease in a subject through the use of the disclosed immunogenic composition, and a method of immunizing a subject through administering the disclosed immunogenic composition.

[0007] One aspect of the current disclosure is directed to an immunogenic composition comprising an engineered extracellular vesicle (EV), the EV comprising: at least one group-specific antigen (Gag) homolog; at least one major histocompatibility complex (MHC) molecule; andat least one antigen of interest; and wherein the engineered EV possesses antigen presenting characteristic.

[0008] In some embodiments, the at least one Gag homologue is overexpressed. In some embodiments, the EV provides enhanced antigen presentation. In some embodiments, the at least one Gag homologue has a similar protein structure to human Arc. In some embodiments, the Gag homologue is a retroviral Arc homologue. In some embodiments, the at least one Gag homologue is selected from the group consisting of human Arc, mouse Arc, rat Arc, drosophila Arc, PEG10, GAK5, GAK6, GAK7, GAK8, GAK9, GAK19, GAK21, GAK24, and GAK113.

[0009] In some embodiments, the immunogenic composition further comprises an RNA stabilizer. In some embodiments, the stabilizer is a 5’ untranslated region (UTR) of a long terminal repeat (LTR) retroviral homologue. In some embodiments, the 5’ UTR LTR retroviral homologue is selected from the group consisting of: 5’ UTR Arc, PEG105’ UTR, RTL15’ UTR, and GAK105’ UTR. In some embodiments, the stabilizer is a 5’ UTR Arc sequence. In some embodiments, the 5’ UTR Arc sequence is selected from the group consisting of: human 5’ UTR Arc, mouse 5’ UTR Arc, rat 5’ UTR Arc, and drosophila 5’ UTR dArc. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1.

[0010] In some embodiments, the MHC molecule is an MHC-I molecule. In some embodiments, the MHC molecule is an MHC-II molecule.

[0011] In some embodiments, the antigen of interest is selected from the group consisting of: Tumor Associated Antigens, autoimmune disease antigens, neoantigens, and infectious disease antigens. In some embodiments, the antigen of interest is an antigen from an extracellular vesicle (AEV) derived from an antigen EV forming cell. In some embodiments, the antigen EV forming cell is a cancer cell or a cell of precancerous tissue. In some embodiments, the antigen EV forming cell is an induced pluripotent stem cell.

[0012] In some embodiments, the antigen is a peptide fragment of a protein selected from the group consisting of: CXorf61 (Chromosome X Open Reading Frame 61); CBX2 (Chromobox Protein Homologue2); PLAC1 (Placenta-Specific 1); CLDN6 (Claudin 6); SPANX (Sperm Protein Associated with the Nucleus on the X chromosome); MAGEA3 (Melanoma-Associated Antigen A3); TPTE (Transmembrane Phosphatase with Tensin Homology); ACTL8 (Actin-Like Protein 8); ANKRD30A (Ankyrin Repeat Domain 30A); CDKN2A (Cyclin-Dependent KinaseInhibitor 2A); MAD2L1 (Mitotic Arrest Deficient 2 Like 1); MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); SUNC1 (Sad1 and UNC84 Domain Containing 1); MAGEA10 (Melanoma-Associated Antigen A10); LRRN1 (Leucine- Rich Repeat Neuronal 1); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO- 1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1): 7490; CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); CA125 (Cancer Antigen 125); CA19-9 (Cancer Antigen 19-9); CA15-3 (Cancer Antigen 15-3); CA27.29 (Cancer Antigen 27.29); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); GPC3 (Glypican 3); CTAG1B (Cancer / Testis Antigen 1B); CTAG2 (Cancer / Testis Antigen 2); CTAG3 (Cancer / Testis Antigen 3); CTAG4 (Cancer / Testis Antigen 4); CTAG5 (Cancer / Testis Antigen 5); CTAG6 (Cancer / Testis Antigen 6); CTAG7 (Cancer / Testis Antigen 7); CTAG8 (Cancer / Testis Antigen 8); BAGE (B melanoma Antigen); GAGE1 (G antigen 1); GAGE2 (G antigen 2); GAGE3 (G antigen 3); GAGE4 (G antigen 4); GAGE5 (G antigen 5); GAGE6 (G antigen 6); GAGE7 (G antigen 7); GAGE8 (G antigen 8); PRAME (Preferentially Expressed Antigen in Melanoma); BIRC5 (Baculoviral IAP Repeat Containing 5); MUC16 (Mucin 16, Cell Surface Associated); SPAG9 (Sperm Associated Antigen 9); CAGE1 (Cancer Antigen 1); SSX1 (Synovial Sarcoma, X Breakpoint 1); SSX2 (Synovial Sarcoma, X Breakpoint 2); SSX3 (Synovial Sarcoma, X Breakpoint 3); SSX4 (Synovial Sarcoma, X Breakpoint 4); SSX5 (Synovial Sarcoma, X Breakpoint 5); SSX6 (Synovial Sarcoma, X Breakpoint 6); SSX7 (Synovial Sarcoma, X Breakpoint 7); SSX8 (Synovial Sarcoma, X Breakpoint 8); GAGE9 (G antigen 9); GAGE10 (G antigen 10); GAGE11 (G antigen 11); GAGE12 (G antigen 12); GAGE13 (G antigen 13); GAGE14 (G antigen 14); GAGE15 (G antigen 15); GAGE16 (G antigen 16); GAGE17 (G antigen 17); GAGE18 (G antigen 18); GAGE19 (G antigen 19); GAGE20 (G antigen 20); GAGE21 (G antigen 21); GAGE22 (G antigen 22); GAGE23 (G antigen 23); GAGE24 (G antigen 24); GAGE25 (G antigen 25); GAGE26 (G antigen 26); GAGE27 (G antigen 27); GAGE28 (G antigen 28); GAGE29 (G antigen 29); GAGE30 (G antigen 30); GAGE31 (G antigen 31); GAGE32 (G antigen 32);GAGE33 (G antigen 33); GAGE34 (G antigen 34); GAGE35 (G antigen 35); GAGE36 (G antigen 36); GAGE37 (G antigen 37); GAGE38 (G antigen 38); GAGE39 (G antigen 39); GAGE40 (G antigen 40); GAGE41 (G antigen 41); GAGE42 (G antigen 42); GAGE43 (G antigen 43); GAGE44 (G antigen 44); GAGE45 (G antigen 45); GAGE46 (G antigen 46); GAGE47 (G antigen 47); GAGE48 (G antigen 48); GAGE49 (G antigen 49); GAGE50 (G antigen 50); GAGE51 (G antigen 51); GAGE52 (G antigen 52); GAGE53 (G antigen 53); GAGE54 (G antigen 54); GAGE55 (G antigen 55); Nid1 (P14543); Pdia3 (P30101); Atic (P31939); Ephx1 (P07099); Ppp1cb (P62140); Plec (Q15149); Abcf1 (Q8NE71); Trim25 (Q14258); Ube2z (Q9H832); Ufd1 (Q92890); Srp54 (P61011); Osbpl9 (Q96SU4); Serbp1 (Q8NC51); Pon1 (P27169); Pdk1 (Q15118); Qars1 (P47897); Fgg (P02679); Fkbp4 (Q02790); Map4 (P27816); Usp9x (Q93008); Acsl1 (P33121); Mtrex (P42285); Prdx1 (Q06830); Gpi (P06744); Srpra (P08240); Slc3a2 (P08195); Gsr (P00390); Erp44 (Q9BS26); Epb41 (P11171); Lgals8 (O00214); Hsph1 (Q92598); Vps26a (O75436); Mars1 (P56192); Trim50 (Q86XT4); Snrpe (P62304); Armt1 (Q9H993); Ppip5k2 (O43314); Tbcd (Q9BTW9); Ehd1 (Q9H4M9); Ddi2 (Q5TDH0); Actbl2 (Q562R1); Osbpl9 (Q96SU4); Rps6ka1 (E9PGT3); Snrnp200 (O75643); Eif3l (Q9Y262); Ap2m1 (Q96CW1); Ephx1 (P07099); Clec2d (Q9UHP7); Abhd14b (Q96IU4); H6pd (O95479); Rpl27 (P61353); Lrp1 (Q94CK9); and Itih2 (P19823).

[0013] In some embodiments, the antigen is selected from the group consisting of: MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); SUNC1 (Sad1 and UNC84 Domain Containing 1); MAGEA10 (Melanoma-Associated Antigen A10); LRRN1 (Leucine-Rich Repeat Neuronal 1); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1); CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); CA125 (Cancer Antigen 125); CA19-9 (Cancer Antigen 19-9); CA15-3 (Cancer Antigen 15-3); CA27.29 (Cancer Antigen 27.29); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); GPC3 (Glypican 3); CTAG1B (Cancer / Testis Antigen 1B); CTAG2 (Cancer / Testis Antigen 2); CTAG3 (Cancer / Testis Antigen 3); CTAG4(Cancer / Testis Antigen 4); CTAG5 (Cancer / Testis Antigen 5); CTAG6 (Cancer / Testis Antigen 6); CTAG7 (Cancer / Testis Antigen 7); CTAG8 (Cancer / Testis Antigen 8); and BAGE (B melanoma Antigen).

[0014] In some embodiments, the antigen is selected from the group consisting of: MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); MAGEA10 (Melanoma-Associated Antigen A10); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1); CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); and BAGE (B melanoma Antigen). In some embodiments, the antigen is selected from the group consisting of: Tpd52l2 (Tumor protein D54, O43399), Tradd (Tumor necrosis factor receptor type 1-associated DEATH domain protein, Q15628), Tpt1 (Translationally-controlled tumor protein, P13693), Serpinf1 (Pigment epithelium-derived factor, P36955), Ceacam1 (Cell adhesion molecule CEACAM1, P13688), Pcna (Proliferating cell nuclear antigen, P12004), Mageb4 (Melanoma-associated antigen B4, O15481), or Bst2 (Bone marrow stromal antigen 2, Q10589).

[0015] In some embodiments, the EV is derived from a cell that is an engineered cell. In some embodiments, the cell is engineered to overexpress the at least one Gag homolog. In some embodiments, the cell is an antigen presenting cell (APC). In some embodiments, the APC is a dendritic cell (DC). In some embodiments, the APC or DC is a human cell. In some embodiments, the EVs are generated through donor cells. In some embodiments, the donor cells are APCs. In some embodiments, the donor APCs are DCs. In some embodiments, the donor is a patient in need of the immunogenic composition. In some embodiments, the donor cells are HLA matched to a patient in need of the immunogenic composition.

[0016] Another aspect of the current disclosure is directed to a method for producing cell- derived extracellular vesicles (EVs) with enhanced antigen presentation, the method comprising:A) transfecting cells with a nucleic acid encoding a virus-like capsid of a Gag homologue; B) contacting the transfected cells with a target antigen, wherein the target antigen is presented via MHC complexes on the membrane of the transfected cells; and C) collecting EVs generated from the transfected cells wherein the collected EVs present the target antigen.

[0017] In some embodiments, the target antigen is from isolated extracellular vesicles derived from cells harboring the target antigen and the isolated extracellular vesicles inherently comprise the target antigen. In some embodiments, the cells are also transfected with a stabilizer wherein the stabilizer is a 5’ untranslated region (UTR) of a long terminal repeat (LTR) retroviral homologue. In some embodiments, the LTR retroviral homologue is an Arc and the stabilizer is a 5’ UTR Arc sequence.

[0018] In some embodiments, the method further comprises concentrating the EVs collected in step C. In some embodiments, the concentration is performed through tangential flow filtration and / or ultrafiltration.

[0019] In some embodiments, the cells are donor cells. In some embodiments, the cells are autologous cells. In some embodiments, the cells are antigen presenting cells (APCs). In some embodiments, the APCs are dendritic cells.

[0020] A further aspect of the current disclosure is directed to a method of slowing or stopping the progression of a disease in a subject, the method comprising administering the immunogenic composition disclosed herein thereby inducing an immune response that slows or stops the progression of the disease.

[0021] Another aspect of the current disclosure is directed to a method of immunizing a mammalian subject against a disease, the method comprising administering the immunogenic composition as described herein to the mammalian subject. In some embodiments, the immunogenic composition is administered more than once. In some embodiments, there is a set period of time between each administration of the immunogenic composition.

[0022] In some embodiments, the disease is selected from the group consisting of: cancers of organs and tissues, autoimmune diseases, and neurodegenerative diseases. In some embodiments, the cancer is selected from the group consisting of Breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer (melanoma), skin cancer (non-melanoma),bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, liver cancer, kidney cancer (renal cell carcinoma), brain cancer (glioblastoma), leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, soft tissue sarcoma, bone sarcoma, esophageal cancer, stomach cancer, head and neck cancer, testicular cancer, vulvar cancer, penile cancer, gallbladder cancer, bile duct cancer, adrenal gland cancer, small intestine cancer, anal cancer, neuroendocrine tumors, mesothelioma, Merkel cell carcinoma, gastrointestinal stromal tumor (GIST), carcinoid tumors, choriocarcinoma, pleomorphic carcinoma, adenoid cystic carcinoma, salivary gland tumors, thymoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, liposarcoma, leiomyosarcoma, chondrosarcoma, Ewing sarcoma, rhabdomyosarcoma, fibrosarcoma, dermatofibrosarcoma protuberans, synovial sarcoma, alveolar soft part sarcoma, clear cell sarcoma, epithelioid sarcoma, desmoid tumors, ovarian germ cell tumors, osteosarcoma, chordoma, glioma, astrocytoma, medulloblastoma, meningioma, pituitary tumors, craniopharyngioma, neuroblastoma, retinoblastoma, Wilms tumor (nephroblastoma), adrenocortical carcinoma, mesenchymal chondrosarcoma, giant cell tumor of bone, fibrous dysplasia, desmoplastic small round cell tumor, extraskeletal myxoid chondrosarcoma, Kaposi sarcoma, mycosis fungoides, Sézary syndrome, Castleman disease, Langerhans cell histiocytosis, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), plasma cell neoplasms (multiple myeloma), non-Hodgkin lymphoma (follicular lymphoma), non-Hodgkin lymphoma (diffuse large B-cell lymphoma), non-Hodgkin lymphoma (mantle cell lymphoma), non-Hodgkin lymphoma (peripheral T-cell lymphoma), fibromatosis (desmoid tumor), neurofibromatosis, gliomatosis cerebri, MALT lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large cell lymphoma, extranodal NK / T-cell lymphoma, acinic cell carcinoma, adult T-cell leukemia / lymphoma, aggressive NK-cell leukemia, basal cell carcinoma, basaloid squamous cell carcinoma, Burkitt lymphoma, carcinoid syndrome, carcinoma in situ, chondroblastoma, clear cell adenocarcinoma, clear cell carcinoma, clear cell renal cell carcinoma, cystadenocarcinoma, embryonal carcinoma, endometrioid adenocarcinoma, ependymoma, epithelial-myoepithelial carcinoma, extranodal marginal zone lymphoma, follicular adenocarcinoma, follicular carcinoma, follicular dendritic cell sarcoma, gastric adenocarcinoma, gastrointestinal carcinoid tumor, giant cell tumor of tendon sheath, granular celltumor, granulocytic sarcoma, granulosa cell tumor, hemangioblastoma, hemangiopericytoma, hepatocellular carcinoma, Hurthle cell carcinoma, inflammatory myofibroblastic tumor, inflammatory pseudotumor, intraductal papillary mucinous neoplasm (IPMN), invasive lobular carcinoma, Krukenberg tumor, Langerhans cell sarcoma, leiomyoma, leiomyosarcoma, lipomatous tumor, low-grade serous carcinoma, lymphoepithelial carcinoma, malignant fibrous histiocytoma (MFH), malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant triton tumor, medullary carcinoma, medullary thyroid carcinoma, Merkel cell polyomavirus-associated carcinoma, Merkel cell polyomavirus-negative carcinoma, metastatic squamous neck cancer with occult primary, micropapillary carcinoma, mixed ductal-lobular carcinoma, mucoepidermoid carcinoma, myoepithelial carcinoma, myxofibrosarcoma, myxoid chondrosarcoma, myxoid liposarcoma, nasopharyngeal carcinoma, neurofibroma, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor, neurofibromatosis type 2-associated tumors, neurogenic sarcoma, nevoid basal cell carcinoma syndrome, nodular malignant melanoma, non-small cell lung carcinoma, noninvasive papillary urothelial carcinoma, oncocytoma, oropharyngeal carcinoma, ossifying fibroma, osteoblastoma, ovarian serous carcinoma, Paget disease, papillary adenocarcinoma, papillary carcinoma, papillary renal cell carcinoma, papillary serous carcinoma, parathyroid carcinoma, parathyroid adenoma, Peutz- Jeghers syndrome-associated tumors, phyllodes tumor, pilomatricoma, plasmacytoma, pleomorphic adenoma, and plexiform neurofibroma. In some embodiments, the cancer is selected from the group consisting of the cancer is selected from the group consisting of breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer (melanoma), skin cancer (non- melanoma), bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, liver cancer, kidney cancer (renal cell carcinoma), brain cancer (glioblastoma), leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, soft tissue sarcoma, bone sarcoma, esophageal cancer, stomach cancer, head and neck cancer, testicular cancer, vulvar cancer, penile cancer, gallbladder cancer, bile duct cancer, adrenal gland cancer, small intestine cancer, anal cancer, neuroendocrine tumors, mesothelioma, Merkel cell carcinoma, gastrointestinal stromal tumor (GIST), carcinoid tumors, choriocarcinoma, pleomorphic carcinoma, adenoid cystic carcinoma, salivary gland tumors, thymoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, liposarcoma, leiomyosarcoma, chondrosarcoma, Ewing sarcoma, rhabdomyosarcoma, fibrosarcoma, dermatofibrosarcoma protuberans, synovialsarcoma, alveolar soft part sarcoma, clear cell sarcoma, epithelioid sarcoma, desmoid tumors, ovarian germ cell tumors, osteosarcoma, chordoma, glioma, astrocytoma, medulloblastoma, meningioma, pituitary tumors, craniopharyngioma, neuroblastoma, and retinoblastoma. In some embodiments, the cancer is selected from the group consisting of Breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer (melanoma), skin cancer (non-melanoma), bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, liver cancer, kidney cancer (renal cell carcinoma), brain cancer (glioblastoma), leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, multiple myeloma, soft tissue sarcoma, bone sarcoma, esophageal cancer, stomach cancer, head and neck cancer, testicular cancer, vulvar cancer, penile cancer, gallbladder cancer, bile duct cancer, adrenal gland cancer, small intestine cancer, anal cancer, neuroendocrine tumors, and mesothelioma. In some embodiments, the cancer is breast cancer or melanoma.

[0023] In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), polymyalgia rheumatica, Goodpasture syndrome, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, vitiligo, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Wegener's granulomatosis (granulomatosis with polyangiitis), microscopic polyangiitis, Behçet's disease, Takayasu's arteritis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), IgA nephropathy (Berger's disease), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), antiphospholipid syndrome, autoimmune hepatitis, mixed connective tissue disease (MCTD), autoimmune pancreatitis, pernicious anemia, lichen sclerosus, relapsing polychondritis, pustular psoriasis, eosinophilic esophagitis, autoimmune thyroid disease, autoimmune encephalitis, autoimmune polyendocrine syndromes (APS-1 to APS-21), autoimmune polyendocrine syndrome type 1 (APS-1), autoimmune polyendocrine syndrome type 2 (APS-2), autoimmune polyendocrine syndrome type 3 (APS-3), autoimmune polyendocrine syndrome type 4 (APS-4), autoimmune polyendocrine syndrome type 5 (APS-5), autoimmune polyendocrine syndrome type 6 (APS-6), autoimmune polyendocrine syndrometype 7 (APS-7), autoimmune polyendocrine syndrome type 8 (APS-8), autoimmune polyendocrine syndrome type 9 (APS-9), autoimmune polyendocrine syndrome type 10 (APS- 10), autoimmune polyendocrine syndrome type 11 (APS-11), autoimmune polyendocrine syndrome type 12 (APS-12), autoimmune polyendocrine syndrome type 13 (APS-13), autoimmune polyendocrine syndrome type 14 (APS-14), autoimmune polyendocrine syndrome type 15 (APS-15), autoimmune polyendocrine syndrome type 16 (APS-16), autoimmune polyendocrine syndrome type 17 (APS-17), autoimmune polyendocrine syndrome type 18 (APS- 18), autoimmune polyendocrine syndrome type 19 (APS-19), and autoimmune polyendocrine syndrome type 20 (APS-20). In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), polymyalgia rheumatica, Goodpasture syndrome, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, vitiligo, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Wegener's granulomatosis (granulomatosis with polyangiitis), microscopic polyangiitis, Behçet's disease, Takayasu's arteritis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), IgA nephropathy (Berger's disease). In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), and polymyalgia rheumatica. In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, and inflammatory bowel disease (Crohn's disease and ulcerative colitis).

[0024] In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia, Lewy body dementia, Pick'sdisease, Frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration, Creutzfeldt-Jakob disease, Wilson's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Charcot-Marie-Tooth disease, hereditary spastic paraplegia, Krabbe disease, Batten disease (neuronal ceroid lipofuscinoses), Niemann-Pick disease, Gaucher disease, Tay-Sachs disease, Canavan disease, Alexander disease, metachromatic leukodystrophy, Pelizaeus-Merzbacher disease, Sanfilippo syndrome, mucopolysaccharidoses, Rett syndrome, fragile X-associated tremor / ataxia syndrome, myotonic dystrophy, Huntington's disease-like syndromes, spinal and bulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, Perry syndrome, and prion diseases. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia, Lewy body dementia, Pick's disease, Frontotemporal dementia (FTD). In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD).

[0025] In some embodiments, the infectious disease includes HIV / AIDS, Influenza, coronaviruses, tuberculosis, malaria, hepatitis (A, B, C, D, and E), dengue fever, Zika virus, Ebola virus disease, cholera, typhoid fever, measles, chickenpox, shingles, polio, rabies, meningitis (bacterial, viral, and fungal), pneumonia (bacterial and viral), Lyme disease, syphilis, gonorrhea, chlamydia, HPV (human papillomavirus), herpes (HSV-1 and HSV-2), candidiasis, trichomoniasis, giardiasis, cryptosporidiosis, leishmaniasis, schistosomiasis, filariasis, onchocerciasis, trachoma, tetanus, pertussis, anthrax, bubonic plague, tularemia, brucellosis, leprosy, listeriosis, toxoplasmosis, Q fever, rocky mountain spotted fever, yellow fever, West Nile virus, Japanese encephalitis, Rift Valley fever, Hantavirus pulmonary syndrome, Nipah virus infection, and Middle East respiratory syndrome (MERS). In some embodiments, the infectious disease is selected from the group consisting of HIV / AIDS, Influenza, coronaviruses, tuberculosis, Lyme disease, HPV (human papillomavirus), and Middle East respiratory syndrome (MERS). In some embodiments, the infectious disease is selected from the group consisting of HIV / AIDS, Influenza, and coronavirus.BRIEF DESCRIPTION OF DRAWINGS

[0026] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

[0027] FIG.1A-I. Arc VLVs respond to stimuli in APCs, present antigens, target secondary lymphoid organs, and directly interact with T cells. (A) Arc mRNA expression in EVs upon stimulation: This panel shows the qPCR results indicating the increase in Arc mRNA expression in EVs post-stimulation with various agents (LPS, Ova, Lipofectamine, Xfect). The graph is presented as purple bars aligned with the right Y-axis. (B) A schematic diagram demonstrates the genetic modification of the DC2.4 cell line to overexpress Arc for enhanced Arc EV production. (C) Venn diagram showing the overlap of proteins identified by MS with those listed in established EV protein databases such as ExoCarta and Evpedia, highlighting the extensive commonality in protein content. (D) Heat map plotting label-free MS quantification of protein abundance in Ova+ engineered VLVs compared to control DEVs. It shows consistent expression levels of EV markers and an increase in microvesicle-associated proteins, vesicle trafficking and cell adhesion-related proteins, suggesting enhanced vesicle functionalities. (E) Heat map showing the presence and upregulation of many proteins involved in MHC-I and MHC-II antigen processing pathways. (F) Mass spectrometry analysis revealed a substantial and significant increase in the abundance of Ova peptide presented by MHC-I, while no significant changes were observed in the presentation of MHC-II / Ova. Data represents mean ± standard error of the mean (SEM) from two to four readings. (G) The IVIS results showcase the distribution of Arc+ EVs in spleen six hours post-injection in mice. A significant enrichment of Arc+ EVs in the spleen is highlighted (left Y-axis), indicating efficient trafficking to secondary immune organs. Data represents mean ± SEM, N=2. (H) Spleen enrichment of Arc+ EVs: A closer look at the spleen's cryo-sectioned samples, showing the preferential binding of Arc+ EVs to splenocytes. (I) Represents the Arc+ DEV distribution in spleen tissue showing the variation in CD4 and CD8 expression levels.

[0028] FIG.2A-C. Characterization of engineered DEVs. (A) Donor cell RNA-liposome transfection efficiency: Arc and A5U-GFP transfected primary BM-DC / MΦs. (B) Nanoparticle tracking analysis of DEVs: This panel presents representative NTA results, showcasing the size distribution of EVs. The histogram illustrates the concentrations of EVs, comparing sampleswith and without the Arc capsid and A5U stabilizer. The plot provides insights into the size uniformity and particle concentration across different EV preparations. (C) Western blot analysis for EV markers: Western blot results demonstrate the presence of common EV markers in our samples. This panel verifies the EV nature of our preparations by confirming the expression of established extracellular vesicle proteins.

[0029] FIG.3A-B. Analysis of Arc+ DEV distribution in spleen tissue. FIG.3A-B provides an expanded view of spleen tissue, offering a broader perspective than FIG.1I by illustrating the widespread presence of both Arc+ cells and Arc+ EVs within the spleen. (A) Shows magnified regions of spleen with CD4 expression. (B) Represents spleen tissue showing CD8 expression. The detailed zoom-in regions emphasize variations in CD4 and CD8 expression levels among different cell populations, allowing for a finer examination of the interaction dynamics between Arc+ EVs with varying degrees of CD4 and CD8 expression.

[0030] FIG.4A-F. Presenting a model antigen Ova, Arc VLVs induce robust, balanced cellular and sustained humoral immune response. In panels A-C, mice were injected with antigen+ DEVs one to three days before the collection of organs in various independent experiments. (A) RT-qPCR analysis of immune-related gene expression: Arc+ groups exhibited upregulated expression of key genes including IFNγ, IL-2, IL-4, IL-10, and CD86. Each group was represented by four technical replicates of samples derived from a total of 10 lymph nodes. Mean ± SD; N = 4 technical replicates representative of three independent experiments. (B) Flow cytometry analysis revealed a significant increase in both CD3+ (i) and CD4+ (ii) splenocytes from mice receiving Arc+ DEVs on day 1 post-injection, with no corresponding increase in CD8+ T cells at this time. By day 3 post-injection, there was a noticeable decline in CD4+ T cells, contrasted by an upregulation of CD8+ T cells (iii). Day 1: N = 3, 4, or 2 biological replicates as shown. Day 3: N = 3 technical replicates. (C) Immunohistochemistry staining of CD4 in spleen cryosections showed increased numbers of CD4+ T cells in mice treated with eraVLVs. Long-term effect: (D) Schedule of multiple Arc+ DEV administrations and sample collections: the timeline illustrates the regimen used to evaluate the potential of Arc+ DEVs in generating a sustained immune response. (E) ELISA of blood serum samples showed a robust IgG response in mice receiving Arc+ DEVs, compared to those receiving control DEVs. The graph quantifies the IgG titers, highlighting the superior and sustaining immunogenic potency of Arc+ DEVs. Mean±SD, based on six biological replicates in Ova+ / Arc+ andOva+ / Arc- groups, two biological replicates in the DEV- group, and three biological replicates in the Ova- DEV group. (F) Long-term immunological memory post-booster dose: ELISpot analysis was conducted three days after administering a booster dose two months following the initial treatments. (i) ELISpot images display the results of the ELISpot assay, visually representing the immune cell activation following the booster dose. (ii) Quantification of spot counts. Mean ± SD; N= 3 technical replicates.

[0031] FIG.5A-B. (A) Flow cytometry analysis of T cell populations in splenocytes extracted from C57BL6 mice treated with WT or Arc KO DEVs presenting the model Ova antigen. (B) Despite similar proportions of T cell populations, the total numbers of CD4+ and CD8+ T cells, as well as pre-effector CD4+ and CD8+ T cells, were significantly upregulated, while memory T cells remained unchanged. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by Student’s T test.

[0032] FIG.6A-B. Functional characterization of Arc in APC functionality and T cell activation (A) Morphological differences between primary BM-DC / MΦs from Arc KO mice and WT mice were visualized on day 6 of differentiation. Arc KO cells appeared smaller and exhibited less uniform morphology compared to WT cells. (B) RT-qPCR analysis was conducted to evaluate the expression levels of immune activation markers, including CD86, IFN-γ, and IL6, in primary BM-DC / MΦ post-stimulation with B16 / F10 melanoma cell-derived EVs. Three sample groups were compared: WT non-transfected, Arc+ transfected, and Arc KO BM-DC / MΦ cells. The results revealed higher expression levels of CD86, IFN-γ, and IL6 in Arc+ transfected cells compared to WT non-transfected cells, indicating a positive regulatory role of Arc in immune activation. Interestingly, Arc KO cells also showed low-level compensatory expression of these genes upon B16 / F10 EVs stimulation.

[0033] FIG.7A-J. Arc plays essential roles in immune activation and memory T cell generation. (A) Primary BM-DC / MΦs from both WT and Arc KO (AGN) mice were differentiated. WT BM-DC / MΦs were either transfected with rArc (Group 1) or left non- transfected (Group 2), while Arc KO BM-DC / MΦs formed Group 3. All groups were co- incubated with B16 / F10 EVs to prepare vaccines, which were administered to WT mice. Cells from all sample groups (Arc+ WT, WT, and Arc- KO) were stimulated with B16 / F10 melanoma EVs. (B) Expression of mArc and SynA in differentiated and B16-stimulated BM-DC / MΦs wasanalyzed by qPCR, which showed a substantial increase of both genes in Arc-overexpressing donor cells. (C) C57BL / 6J mice were injected with EVs from all three groups stimulated by B16- F10 EVs. On day 8 post injection, splenocytes were collected and restimulated with B16-F10 EVs for 6 hours. (D) Flow cytometry analysis revealed enhanced CD4+ / CD44+ / IFNγ+ memory T cell populations in mice treated with Arc+ DEVs, indicating memory T cell generation. (E) An increase in activated and memory T cell populations, including both CD62L- effector cells and CD62L+ central memory cells, was observed in response to Arc+ DEVs, as quantified in (F). (G) Visualization of splenocyte and melanoma cell interactions post-vaccination with DEVs. Isolated splenocytes from vaccinated mice were stained with CSFE (displayed as red in bright field images i-iii and yellow in fluorescent image v), and co-incubated with B16-F10 melanoma cells stained with NucSpot 650 (purple in fluorescent image v). 24 hours later, non-adherent cells in the supernatant medium were removed, highlighting increased interactions between melanoma cells and splenocytes (image iv) from the Arc-transfected WT group. (H) To track the uptake of B16-F10 EVs by DCs, B16-F10 DEVs and VLVs were labeled with CMDR, along with a PBS only negative control. (i-iv) Fluorescence imaging conducted on the donor DC culture illustrates the efficient uptake of CMDR-labeled B16-F10 EVs by the DCs, providing visual evidence of the successful incorporation of melanoma antigens into the DCs. (iv) As a control, CMDR was added to EV-absent DPBS, undergoing the same ultrafiltration purification steps as B16-F10 EVs. This CMDR-treated DPBS was then introduced to DCs, serving as a negative control to ensure the specificity of method. (v) The purity of the collected DEV vaccine samples was ascertained through CMDR fluorescence intensity readings, confirming that the DEVs used in subsequent experiments were free from B16-F10 EVs. Mean±SD, based on three biological replicates. The observed fluorescence in the DEVs is attributed to CMDR dyes incorporated onto the plasma membrane of DCs upon B16-F10 EV uptake, which were subsequently integrated into the DEVs. This step was crucial in ensuring the specificity and accuracy of the vaccine efficacy tests. (I) Three days post-administration, flow cytometry analysis showed increased proliferation of CD3+, CD4+, and CD8+ T cells, as well as CD4+ and CD8+ pre-effector T cells in mice treated with B16+ VLVs, with quantification shown in (J).

[0034] FIG.8. qPCR analysis of splenocytes and lymphocytes collected 3 days after IV injection of various DEVs and VLVs showed upregulated levels of IL2 and IL4 in B16+ DEVs and VLVs.

[0035] FIG.9A-H. DC-derived eraVLV vaccines effectively prevent tumor growth. (A) This panel depicts the timeline and dosage schedule of B16+ DEV vaccines administered to wild-type C57BL6 mice, alongside the initial injection of melanoma cells and rechallenge with both melanoma and breast cancer cells. Tumor measurements were conducted using micro-CT scanning and double-blind caliper measurements by multiple independent researchers to ensure data precision and reliability. (B) Mice treated with B16higheraVLV vaccines exhibited a significant survival advantage (60%) compared toB16highWT DEV control (37.5%) and B16lowWT DEV control (33.3%) 80 days post the initial injection of B16-F10 cells. DEV-negative control mice were euthanized on days 13-17 due to tumor volume exceeding 1500 mm3. N = 12 (DEV-), 12 (B16lowDEV+), 16 (B16highDEV+), 20 (B16high eraVLV+) tumors, with data collected from three independent experiments. **** P<0.0001. (C) The graph illustrates daily tumor size measurements starting five days post-injection of B16 melanoma cells. A marked reduction in tumor size is observed in the Arc+ group compared to the control group. N = 12 (DEV-), 12 (B16lowDEV+), 16 (B16highDEV+), 12 (B16higheraVLV), with data collected from three independent experiments. Mean±SEM. ****P < 0.0001. (D) In one of the three independent experiments, on day 17 post-B16-F10 cell injection, the DEV-negative control mouse with the slowest tumor progression had two tumors exceeding size limitations. It was euthanized along with one mouse from each other sample group with the largest tumors for size comparison, tissue sectioning, and histochemistry and immunohistochemistry staining. N= 2 (DEV-), 12 (B16lowDEV+), 16 (B16highDEV+), 12 (B16higheraVLV). Mean±SEM. (E) H and E staining showed that Arc+ DEV vaccines resulted in not only smaller, but also more disrupted tumors, likely due to increased T cell infiltration, as indicated by CD3 staining. (F) Survival following challenge with melanoma cells and rechallenge with both melanoma and breast cancer cells. (i) In one of three independent experiments, long-term survivor mice were rechallenged with B16-F10 melanoma and EO771 breast cancer cells on day 132. A second batch of DEV- negative controls was introduced as members of the first batch succumbed. (ii) Median survival times were as follows: 13.5 days for the first batch of DEV-negative control (NC1); 14 days for the second batch of DEV-negative control (NC2); 26.5 days for B16lowDEV control; 22.5 days for B16highDEV control; and 154 days for the B16higheraVLV group. (G) Visual tumor size comparison at euthanasia between control and vaccinated mice. (H) Graph illustrating that control mice (DEV-) exhibited rapid melanoma progression with correspondingly larger tumorsat death. Vaccinated mice (B16+ DEV+), protected against melanoma, survived longer, allowing breast cancer tumors to grow larger over an extended period, highlighting the differential impact of the B16+ DEV vaccine on melanoma versus breast cancer.

[0036] FIG.10. Extraction of spleens from the same tumor-bearing mice, followed by sectioning and staining for CD8, revealed abundant cytotoxic T cells present in the spleen in the B16highWT DEV control group, whereas less cytotoxic T cells were observed in the spleen of the B16highArc+ DEV group.

[0037] FIG.11A-F. DC-derived eraVLV vaccines enhance behavioral wellness of treated mice and engage T cells via endogenous virus-like envelop proteins. (A) In addition to monitoring tumor growth, the behavioral well-being of treated mice was assessed, including locomotion and cognitive capacities using a novel object test. The movement tracks (orange) provide a visual representation of the paths taken by the mice within the test chamber. The heat map shows the density of movement within different areas, indicating the regions where the mice spent the most time. Besides the negative control mice, which are from new cancer cell injections, subjects in all experimental groups studied here are treated survivors after two rounds of tumor rechallenge. (B-D) B16highvaccinated survivor mice exhibited increased average speed (B), total travel distance (C), and number of entries into the novel object zone (D), compared to control mice. The eraVLV group exhibited the highest level of locomotion activity. N = 3-5 in each sample group. Mean ± SEM. (E) MS label-free quantification enabled the identification and quantification of EV membrane proteins. The heat map provides a comparative analysis of protein expression levels across different sample groups. The heat map shows an upregulation of endogenous ENVs in OVA-stimulated eraVLVs, compared to both the OVA- WT DEV control and the Arc+ / A5U- DEV control. (F) Structural comparison of the MLV envelope (bottom) and AlphaFold prediction of ENV1 (top). MS analysis revealed a high degree of sequence similarity in the extracellular receptor-binding domain between MLV ENV and a specific sequence enriched in Ova+ eraVLVs (ENV1 in E). A beta-strand sequence within this domain was identified as 100% conserved, highlighting significant structural conservation critical for receptor interaction.

[0038] FIG.12. Locomotion and cognitive assessment using a novel object test. FIG.12 presents the detailed movement tracks of mice from different sample groups within the testchamber in different independent experiments. The orange tracks visualize the specific paths taken by the mice, while the heat map indicates the density of movement and highlights the regions where the mice spent the most time. Besides the negative control mice, which are from new cancer cell injections, subjects in all experimental groups studied here are vaccinated survivors that survived two rounds of tumor cell rechallenge. All treated mice seem to perform similarly in this test.

[0039] FIG.13A-M. Proteomic and immunogenic profiling of melanoma antigen-containing vesicles (A) Venn diagram compares the proteins identified in the melanoma+ and Ova+ DC- derived vesicles with those annotated in the ExoCarta and EVpedia databases. (B) Label-free MS quantification shows an increase in the abundance of several tumor-associated proteins in VLVs and a decrease in CD47 levels. (C) (i) Venn diagram presents proteins that show significant changes in expression between each pair of comparisons: B16+ VLV versus B16+ DEV, B16+ DEV versus B16- DEV, and B16+ VLV versus B16- DEV. Volcano plots represent the proteomic profiling of DC-derived vesicles. The x-axis indicates the Log2 (Fold Change) and the y-axis represents the negative logarithm (base 10) of the adjusted p-value. Each plot compares different conditions: (ii) B16+ DEV vs B16- DEV, (iii) B16+ VLV vs B16+ DEV, and (iv) B16+ VLV vs B16- DEV. Red points above the threshold line indicate proteins with statistically significant changes in expression. (D) Gene ontology (GO) enrichment analysis of extracellular vesicle (EV) proteins (using the G database, GO: Biological Process) reveals the primary biological processes in which the identified proteins are involved. (i) Significantly upregulated proteins in B16+ VLVs compared to B16- DEVs. (ii) Significantly upregulated proteins in B16+ VLVs compared to B16+ DEVs. (iii) Significantly upregulated proteins in B16+ DEVs compared to B16- DEVs. (E) Comparative analysis with the IEDB for mouse melanoma epitopes reveals significant overlap. (F) Heat map displaying normalized Log10(Fold Change) values for 46 shared epitopes between B16+ DC2.4 vesicles and the melanoma database. Many epitopes show upregulation in B16+ VLVs compared to DEVs. (H) Unique epitopes in DC2.4 are primarily upregulated in B16+ VLVs compared to DEVs. (I) Shared epitopes between immortal and primary DEVs exhibit relatively stable expression levels. (J) Analysis of shared 46 epitopes reveals GO: cellular component (GO:CC) term “melanosome” being highly enriched. (K)Viral-related processes were identified as "TRUE," indicating statistically enriched processes with high confidence. (L) Many of these proteins involved inviral processes were increased in B16+ VLVs. (M) IEDB ANN4.0 binding prediction tool results for MHC binding, with upregulated peptides in DC2.4 B16+ VLVs showing IC50 values below 500, indicating strong binding affinity to H-2-Kb or H-2-Db molecules.

[0040] FIG.14. Venn diagram compares the proteins identified in our samples with those annotated in the ExoCarta, Vesiclepedia, and EVpedia databases.

[0041] FIG.15A-C. Graphical abstract. (A) Depiction of DCs engineered to overexpress the virus-like capsid protein Arc, along with a stabilizer RNA motif, exposing the DCs to EVs derived from unmodified cancer cells to act as tumor antigens. The engineered DCs substantially increased the production of endogenous retroviral Arc VLVs (or eraVLV), enhancing DEVs' antigen presentation capabilities, leading to sustained immune responses against specific antigens. eraVLVs proficiently present B16-F10 melanoma antigens to T-cells in the secondary lymphoid creating effector cytotoxic T-cells against the cancer cells. (B) Shows immunization timeline for two experimental groups EVs derived from wild-type DCs, and DCs were exposed to a higher dose of B16-F10 antigen EVs (B16-F10highDEV+) and EVs from DCs overexpressing Arc, with a stabilizer motif introduced to enhance the Arc capsid stability (B16- F10higheraVLV+). 4 immunizations were given at 28 days, 21 days, 14 days, and 7 days before challenge with melanoma cells. Median survival rate was 132 days after two sequential challenges, first with melanoma cells and the second with a combination of melanoma and breast cancer cells. (C) Graphical representation of the probability of survival. DEV-NC-1 (Negative Control 1) did not receive any EVs and served as the initial negative control. These mice were exposed to the first challenge only and succumbed shortly thereafter, necessitating the use of a second batch of negative controls. DEV-NC-2 (Negative Control 2): Like DEV-NC-1, this group did not receive any EVs. These controls were introduced to evaluate survival after the second challenge with both melanoma and breast cancer cells, alongside survivors from vaccinated experimental groups. B16-F10lowDEV+ are EVs derived from wild-type DCs, which were exposed to a lower dose of B16-F10 melanoma antigen EVs. This group tested the efficacy of a baseline level of antigen presentation. B16-F10highDEV+: EVs derived from wild-type DCs, and DCs were exposed to a higher dose of B16-F10 antigen EVs. This group was used to assess the impact of increased antigen load on efficacy. B16-F10higheraVLV+ are EVs from DCs overexpressing Arc, with a stabilizer motif introduced to enhance the Arc capsid stability. This sample contained a higher percentage of eraVLVs among total DEVs. eraVLVs presentingmelanoma antigens led to a 6.84-fold increase in median survival compared to traditional DEV vaccine. DETAILED DESCRIPTION

[0042] The current disclosure is directed to an immunogenic composition, methods of making extracellular vesicles with enhanced immunity, a method of slowing or stopping a disease in a subject through the use of the disclosed immunogenic composition, and a method of immunizing a subject through administering the disclosed immunogenic composition.

[0043] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0044] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value).

[0045] In the description that follows, certain conventions will be followed as regards to the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art. In practicing the present disclosure, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology are used, which are within the skill of the art. These techniques are described in greater detail in, for example, Molecular Cloning: a Laboratory Manual 4th edition, J.F. Sambrook and D.W. Russell, ed. Cold Spring Harbor Laboratory Press 2012; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001). The contents of these references and other references containing standard protocols,widely known to and relied upon by those of skill in the art, including manufacturers’ instructions are hereby incorporated by reference as part of the disclosure.

[0046] One aspect of the current disclosure is directed to an immunogenic composition comprising an extracellular vesicle (EV), the EV comprising: at least one group-specific antigen (Gag) homolog; at least one major histocompatibility complex (MHC) molecule; and at least one antigen of interest; and wherein the EV possesses antigen presenting characteristic.

[0047] As used herein, “immunogenic” refers to the ability to induce an immune response against an antigen being presented.

[0048] The terms "extracellular vesicle (EV)" or “vesicle” as used herein, refer to a cell-derived vesicle or a synthetic vesicle-like structure which functions as a cell-derived vesicle. Cell- derived vesicles are generated through a combination of endocytotic and exocytotic events that result in the encapsulation of various biomolecules. All prokaryotic and eukaryotic cells release EVs as part of their normal physiology and during acquired abnormalities. While EVs can be broadly divided into two categories, ectosomes and exosomes, the terms “ectosomes,” “exosomes,” and “EVs” may be used interchangeably for purposes of this disclosure. Ectosomes are vesicles that pinch off the surface of the plasma membrane via outward budding, and include microvesicles, microparticles, and large vesicles in the size range of ~50 nm to 1μm in diameter. Exosomes are EVs with a size range of ~40 to 160 nm (average ~100 nm) in diameter with an endosomal origin. Such encapsulation may protect a therapeutic nucleic acid from enzymatic degradation or other environmental stresses (e.g., ionic strength, pH etc.). The association of proteins with an EV provides stability in both extracellular and intracellular environments as well as facilitates a cell-targeting mechanism for cell-cell communication.

[0049] As used herein, “engineered extracellular vesicle” refers to an EV derived from an engineered cell. The engineered cell is manipulated in order to overexpress a gag homologue. In some embodiments, EVs may be created in prokaryotes, eukaryotes, or viruses. In some embodiments, engineered EVs are made in yeast, bacteria, virus, protists, or other types of cells, whether they be unicellular organisms or multicellular organisms, or non-living items which contain DNA.

[0050] EVs are produced by many different types of cells, including immune cells such as B lymphocytes, T lymphocytes, dendritic cells (DCs) and most cells. EVs are also produced, for example, by glioma cells, platelets, reticulocytes, neurons, intestinal epithelial cells and tumor cells. EVs for use in the disclosed compositions and methods can be derived from any suitable cell, including the cells identified above. As used herein, “derived” refers to the cell from which the EV was released, i.e. the parent cell of the EV.

[0051] In some embodiments, EVs are derived from antigen presenting cells (APCs). An antigen-presenting cell (APC) or accessory cell is a cell that displays an antigen bound by major histocompatibility complex (MHC) proteins on its surface; this process is known as antigen presentation. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0052] Almost all cell types can present antigens in some way. APCs are found in a variety of tissue types. Dedicated antigen-presenting cells, including macrophages, B cells and dendritic cells, present foreign antigens to helper T cells, while virus-infected cells (or cancer cells) can present antigens originating inside the cell to cytotoxic T cells. In addition to the MHC family of proteins, antigen presentation relies on other specialized signaling molecules on the surfaces of both APCs and T cells.

[0053] APC and DC derived EVs can be identified by specific surface markers and molecular signatures as known in the art. Some of these surface proteins include MHC-I, MHC-II, CD86, CD80, and CD40, which are function in antigen presentation. DC-derived EVs specifically express CD11c, CD83, and CD209 (DC-SIGN). Proteomic analysis conducted herein showed that APC / DC-derived EVs contain heat shock proteins (HSP70, HSP90), cytokines (IL-6, IL-10, TNF-α), and co-stimulatory molecules that reflect their immune-modulating role. While many of these markers are not exclusive to DCs or APCs, the source cell (parent cell) of an EV should be able to be determined when a combination of enough markers are identified. Methods known in the art are used for such analysis. Some non-limiting examples of commonly used methods to characterize EVs include flow cytometry, proteomic analysis, and RNA profiling. As such, identification of an EVs cellular type of origin is possible even without prior knowledge of their production method.

[0054] As used herein, homologue refers to proteins that are similar to a reference protein in both structure and function. Being similar in structure means being similar in shape (3D proteinfolding) and / or amino acid sequence. In some embodiments, proteins are homologous if they share about 80% or greater sequence identity. In some embodiments, proteins are homologous if they share about 85% or greater sequence identity. In some embodiments, proteins are homologous if they share about 90% or greater sequence identity. In some embodiments, proteins are homologous if they share about 91% or greater sequence identity. In some embodiments, proteins are homologous if they share about 92% or greater sequence identity. In some embodiments, proteins are homologous if they share about 93% or greater sequence identity. In some embodiments, proteins are homologous if they share about 94% or greater sequence identity. In some embodiments, proteins are homologous if they share about 95% or greater sequence identity. In some embodiments, proteins are homologous if they share about 96% or greater sequence identity. In some embodiments, proteins are homologous if they share about 97% or greater sequence identity. In some embodiments, proteins are homologous if they share about 98% or greater sequence identity. In some embodiments, proteins are homologous if they share about 99% sequence identity.

[0055] As such, “Gag homologue” or “Group-specific antigen (Gag) homologue” refers to proteins that are similar to Gag proteins in both structure and function. Being similar in structure means to have common sequence similarity with Gag gene (nucleic acid sequence) or Gag protein (amino acid). Gag proteins are found in retroviruses and retrotransposons. Gag homologs can be found in humans and fruit flies.

[0056] In some embodiments, the EV is derived from a cell (parent cell) and that cell is an engineered cell. In some embodiments, the parent cell is engineered to overexpress the at least one Gag homolog. As used herein, “overexpress” refers to an increase in the number of copies of a gene (also called amplification), or an increase in the amount of a protein. In some embodiments, the engineered parent cell is an antigen presenting cell (APC). In some embodiments, the engineered parent APC cell is a dendritic cell (DC). In some embodiments, the at least one Gag homologue is overexpressed.

[0057] In some embodiments, the EV provides enhanced antigen presentation. By “enhanced” antigen presentation, as used herein, it means that the engineered EV disclosed herein (comprising at least one Gag homolog) has an increased ability to stimulate or activate T cell proliferative responses as compared to an EV without the at least one endogenous gag homologue. For example, enhanced antigen presentation is relative to non-engineered EVs orEVs derived from cells without an overexpression of the Gag homolog. The ability of a DEV to stimulate or activate T cell proliferative responses can be evaluated by assays known in the art, e.g., as described measuring Ova peptides presented by MHC-I and MHC-II, where an increase in the level of Ova peptides presented by MHC-I while consistent presentation of Ova peptides in MHC-II represents enhanced antigen presentation. In some embodiments, the extent of enhancement may be at least 10%. In some embodiments, the extent of enhancement may be at least 20%. In some embodiments, the extent of enhancement may be at least 30%. In some embodiments, the extent of enhancement may be at least 40%. In some embodiments, the extent of enhancement may be at least 50%. In some embodiments, the extent of enhancement may be at least 75%, or greater. In some embodiments, the extent of enhancement may be presented in fold increase. In some embodiments, the extent of enhancement may be at least a 1-fold increase. In some embodiments, the extent of enhancement may be at least a 1.5-fold increase. In some embodiments, the extent of enhancement may be at least a 2-fold increase. In some embodiments, the extent of enhancement may be at least a 2.5-fold increase. In some embodiments, the extent of enhancement may be at least a 3-fold increase or more.

[0058] In some embodiments, the at least one Gag homologue has a similar protein structure to human Arc. In some embodiments, the Gag homologue shares 80% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 85% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 90% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares an 91% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 92% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 93% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 94% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 95% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 96% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 97% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 98% sequence identity with the amino acid sequenceset out in SEQ ID NO: 2. In some embodiments, the Gag homologue shares 99% sequence identity with the amino acid sequence set out in SEQ ID NO: 2. In some embodiments, the Gag homologue is a retroviral Arc homologue. In some embodiments, the at least one Gag homologue is selected from the group consisting of human Arc, mouse Arc, rat Arc, drosophila Arc, PEG10, GAK5, GAK6, GAK7, GAK8, GAK9, GAK19, GAK21, GAK24, and GAK113.

[0059] In some embodiments, the immunogenic composition further comprises an RNA stabilizer. The RNA stabilizer functions to prevent RNA degradation by inactivating RNases and preserving RNA integrity. In some embodiments, the stabilizer is a 5’ untranslated region (UTR) of a long terminal repeat (LTR) retroviral homologue. In some embodiments, the 5’ UTR LTR retroviral homologue is selected from the group consisting of: 5’ UTR Arc, PEG105’ UTR, RTL15’ UTR, and GAK105’ UTR. In some embodiments, the stabilizer is a 5’ UTR Arc sequence. In some embodiments, the 5’ UTR Arc sequence is selected from the group consisting of: human 5’ UTR Arc, mouse 5’ UTR Arc, rat 5’ UTR Arc, and drosophila 5’ UTR dArc.

[0060] In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 91% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 92% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 93% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 94% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 97% sequence identityto the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 5’ UTR Arc sequence comprises a nucleotide sequence set forth in SEQ ID NO: 1.

[0061] The major histocompatibility complex (MHC) is a large locus on vertebrate DNA containing a set of closely linked polymorphic genes that code for cell surface proteins essential for the adaptive immune system. These cell surface proteins are called MHC molecules. MHC molecules bind peptide fragments derived from pathogens and display them on the cell surface for recognition by the appropriate T cells. MHC class I molecules are found on the surface of most nucleated cells, including epithelial cells. MHC class I molecules present peptides from within the cell to the immune system playing a role in antivirus and antitumor defenses. MHC class I presents peptides to cytotoxic CD8+ T cells. MHC class II molecules are found on antigen-presenting cells, including dendritic cells, macrophages, and B cells. MHC class II molecules present peptides that come from outside the cell to the immune system and plays a role in effective antibody responses. MHC class II presents peptides to helper CD4+ T cells. MHC molecules bind to both the T cell receptor and a CD4 / CD8 co-receptor on T lymphocytes. The antigen epitope held in the peptide-binding groove of the MHC molecule interacts with the variable Ig-Like domain of the T cell receptor (TCR) triggering T-cell activation. However, the MHC molecules can act as antigens themselves causing an immune response in the subject. Human cells express six MHC class I alleles (one HLA-A, -B, and -C allele from each parent) and six to eight MHC class II alleles (one HLA -DP and -DQ, and one or two HLA-DR from each parent, and combinations of these). The variation of MHC is high in the human population where at least 300 alleles for HLA-A genes, 620 alleles for HLA-B, 400 alleles for HLA-DR, and 90 alleles for HLA-DQ. In some embodiments, the MHC molecule is an MHC-I molecule. In some embodiments, the MHC molecule is an MHC-II molecule.

[0062] The term “HLA-type” refers to the complement of HLA antigens present on the cells of a subject.

[0063] In some embodiments, the antigen of interest is selected from the group consisting of: Tumor Associated Antigens, autoimmune disease antigens, neoantigens, and infectious diseaseantigens. In some embodiments, the antigen of interest is an antigen from an extracellular vesicle (AEV) derived from an antigen EV forming cell. In some embodiments, the antigen EV forming cell is a cancer cell or a cell of precancerous tissue. In some embodiments, the antigen EV forming cell is an induced pluripotent stem cell.

[0064] In some embodiments, the antigen is a peptide fragment of a protein selected from the group consisting of: CXorf61 (Chromosome X Open Reading Frame 61); CBX2 (Chromobox Protein Homologue 2); PLAC1 (Placenta-Specific 1); CLDN6 (Claudin 6); SPANX (Sperm Protein Associated with the Nucleus on the X chromosome); MAGEA3 (Melanoma-Associated Antigen A3); TPTE (Transmembrane Phosphatase with Tensin Homology); ACTL8 (Actin-Like Protein 8); ANKRD30A (Ankyrin Repeat Domain 30A); CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A); MAD2L1 (Mitotic Arrest Deficient 2 Like 1); MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); SUNC1 (Sad1 and UNC84 Domain Containing 1); MAGEA10 (Melanoma-Associated Antigen A10); LRRN1 (Leucine- Rich Repeat Neuronal 1); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO- 1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1): 7490; CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); CA125 (Cancer Antigen 125); CA19-9 (Cancer Antigen 19-9); CA15-3 (Cancer Antigen 15-3); CA27.29 (Cancer Antigen 27.29); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); GPC3 (Glypican 3); CTAG1B (Cancer / Testis Antigen 1B); CTAG2 (Cancer / Testis Antigen 2); CTAG3 (Cancer / Testis Antigen 3); CTAG4 (Cancer / Testis Antigen 4); CTAG5 (Cancer / Testis Antigen 5); CTAG6 (Cancer / Testis Antigen 6); CTAG7 (Cancer / Testis Antigen 7); CTAG8 (Cancer / Testis Antigen 8); BAGE (B melanoma Antigen); GAGE1 (G antigen 1); GAGE2 (G antigen 2); GAGE3 (G antigen 3); GAGE4 (G antigen 4); GAGE5 (G antigen 5); GAGE6 (G antigen 6); GAGE7 (G antigen 7); GAGE8 (G antigen 8); PRAME (Preferentially Expressed Antigen in Melanoma); BIRC5 (Baculoviral IAP Repeat Containing 5); MUC16 (Mucin 16, Cell Surface Associated); SPAG9 (Sperm Associated Antigen 9); CAGE1 (Cancer Antigen 1); SSX1 (Synovial Sarcoma, X Breakpoint 1); SSX2 (Synovial Sarcoma, XBreakpoint 2); SSX3 (Synovial Sarcoma, X Breakpoint 3); SSX4 (Synovial Sarcoma, X Breakpoint 4); SSX5 (Synovial Sarcoma, X Breakpoint 5); SSX6 (Synovial Sarcoma, X Breakpoint 6); SSX7 (Synovial Sarcoma, X Breakpoint 7); SSX8 (Synovial Sarcoma, X Breakpoint 8); GAGE9 (G antigen 9); GAGE10 (G antigen 10); GAGE11 (G antigen 11); GAGE12 (G antigen 12); GAGE13 (G antigen 13); GAGE14 (G antigen 14); GAGE15 (G antigen 15); GAGE16 (G antigen 16); GAGE17 (G antigen 17); GAGE18 (G antigen 18); GAGE19 (G antigen 19); GAGE20 (G antigen 20); GAGE21 (G antigen 21); GAGE22 (G antigen 22); GAGE23 (G antigen 23); GAGE24 (G antigen 24); GAGE25 (G antigen 25); GAGE26 (G antigen 26); GAGE27 (G antigen 27); GAGE28 (G antigen 28); GAGE29 (G antigen 29); GAGE30 (G antigen 30); GAGE31 (G antigen 31); GAGE32 (G antigen 32); GAGE33 (G antigen 33); GAGE34 (G antigen 34); GAGE35 (G antigen 35); GAGE36 (G antigen 36); GAGE37 (G antigen 37); GAGE38 (G antigen 38); GAGE39 (G antigen 39); GAGE40 (G antigen 40); GAGE41 (G antigen 41); GAGE42 (G antigen 42); GAGE43 (G antigen 43); GAGE44 (G antigen 44); GAGE45 (G antigen 45); GAGE46 (G antigen 46); GAGE47 (G antigen 47); GAGE48 (G antigen 48); GAGE49 (G antigen 49); GAGE50 (G antigen 50); GAGE51 (G antigen 51); GAGE52 (G antigen 52); GAGE53 (G antigen 53); GAGE54 (G antigen 54); GAGE55 (G antigen 55); Nid1 (P14543); Pdia3 (P30101); Atic (P31939); Ephx1 (P07099); Ppp1cb (P62140); Plec (Q15149); Abcf1 (Q8NE71); Trim25 (Q14258); Ube2z (Q9H832); Ufd1 (Q92890); Srp54 (P61011); Osbpl9 (Q96SU4); Serbp1 (Q8NC51); Pon1 (P27169); Pdk1 (Q15118); Qars1 (P47897); Fgg (P02679); Fkbp4 (Q02790); Map4 (P27816); Usp9x (Q93008); Acsl1 (P33121); Mtrex (P42285); Prdx1 (Q06830); Gpi (P06744); Srpra (P08240); Slc3a2 (P08195); Gsr (P00390); Erp44 (Q9BS26); Epb41 (P11171); Lgals8 (O00214); Hsph1 (Q92598); Vps26a (O75436); Mars1 (P56192); Trim50 (Q86XT4); Snrpe (P62304); Armt1 (Q9H993); Ppip5k2 (O43314); Tbcd (Q9BTW9); Ehd1 (Q9H4M9); Ddi2 (Q5TDH0); Actbl2 (Q562R1); Osbpl9 (Q96SU4); Rps6ka1 (E9PGT3); Snrnp200 (O75643); Eif3l (Q9Y262); Ap2m1 (Q96CW1); Ephx1 (P07099); Clec2d (Q9UHP7); Abhd14b (Q96IU4); H6pd (O95479); Rpl27 (P61353); Lrp1 (Q94CK9); and Itih2 (P19823).

[0065] In some embodiments, the antigen is selected from the group consisting of: MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); SUNC1 (Sad1 and UNC84 Domain Containing 1); MAGEA10 (Melanoma-Associated Antigen A10); LRRN1 (Leucine-Rich Repeat Neuronal 1); MAGEA9 (Melanoma-Associated Antigen A9);HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1); CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); CA125 (Cancer Antigen 125); CA19-9 (Cancer Antigen 19-9); CA15-3 (Cancer Antigen 15-3); CA27.29 (Cancer Antigen 27.29); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); GPC3 (Glypican 3); CTAG1B (Cancer / Testis Antigen 1B); CTAG2 (Cancer / Testis Antigen 2); CTAG3 (Cancer / Testis Antigen 3); CTAG4 (Cancer / Testis Antigen 4); CTAG5 (Cancer / Testis Antigen 5); CTAG6 (Cancer / Testis Antigen 6); CTAG7 (Cancer / Testis Antigen 7); CTAG8 (Cancer / Testis Antigen 8); and BAGE (B melanoma Antigen).

[0066] In some embodiments, the antigen is selected from the group consisting of: MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); MAGEA10 (Melanoma-Associated Antigen A10); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1); CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); and BAGE (B melanoma Antigen). In some embodiments, the antigen is selected from the group consisting of: Tpd52l2 (Tumor protein D54, O43399), Tradd (Tumor necrosis factor receptor type 1-associated DEATH domain protein, Q15628), Tpt1 (Translationally-controlled tumor protein, P13693), Serpinf1 (Pigment epithelium-derived factor, P36955), Ceacam1 (Cell adhesion molecule CEACAM1, P13688), Pcna (Proliferating cell nuclear antigen, P12004), Mageb4 (Melanoma-associated antigen B4, O15481), or Bst2 (Bone marrow stromal antigen 2, Q10589).

[0067] In some embodiments, the EV is derived from a cell (parent cell) and that cell is an engineered cell. In some embodiments, the parent cell is engineered to overexpress the at least one Gag homolog. As used herein, “overexpress” refers to an increase in the number of copies of a gene (also called amplification), or an increase in the amount of a protein. In some embodiments, the engineered parent cell is an antigen presenting cell (APC). In some embodiments, the engineered parent APC cell is a dendritic cell (DC). In some embodiments, the EVs are generated through donor cells. In some embodiments, the donor cells are APCs. In some embodiments, the donor APCs are DCs. In some embodiments, the donor is a patient in need of the immunogenic composition. In some embodiments, the donor cells are HLA matched to a patient in need of the immunogenic composition.

[0068] Another aspect of the current disclosure is directed to a method for producing cell- derived extracellular vesicles (EVs) with enhanced antigen presentation, the method comprising: A) transfecting cells with a nucleic acid encoding a virus-like capsid of a Gag homologue; B) contacting the transfected cells with a target antigen, wherein the target antigen is presented via MHC complexes on the membrane of the transfected cells; and C) collecting EVs generated from the transfected cells which present the target antigen.

[0069] As used herein, “transfection”, “transfected”, or “transfecting” refers to the transfer of genetic material into a cell. This genetic transfer can occur through methods known in the art, including, but not limited to, physical methods such as direct micro injection, biolistic particle delivery, electroporation, sonoporation, and laser-based optical transfection; and chemical methods, such as calcium phosphate, cationic polymer, lipofection, fugene, or dendrimer transfection. In some embodiments, the transfer of nucleic acids into to the cells takes place through polyethyleneimine (PEI) complexation, electroporation, cationic lipids complexation, lipid nanoparticle-mediated delivery, microinjection, or through the use of adenoviral vectors.

[0070] In some embodiments, the target antigen is from isolated extracellular vesicles derived from cells harboring the target antigen and the isolated extracellular vesicles inherently comprise the target antigen.

[0071] In some embodiments, the cells are also transfected with a stabilizer wherein the stabilizer is a 5’ untranslated region (UTR) of a long terminal repeat (LTR) retroviral homologue.In some embodiments, the LTR retroviral homologue is an Arc and the stabilizer is a 5’ UTR Arc sequence.

[0072] In some embodiments, EVs are created in prokaryotes, eukaryotes, or viruses. In some embodiments, the engineered EVs are made in yeasts, bacteria, viruses, protists, or other types of cells, whether they be unicellular organisms, multicellular organisms, or non-living items which contain DNA. In some embodiments, the method of making EVs further comprises using cells that are selected from neural cells, epithelial cells, endothelial cells, hematopoietic cells, connective tissue cells, muscle cells, bone cells, cartilage cells, germline cells, adipocytes, stem cells, self-derived ex vivo differentiated cells, iPSC-derived ex vivo differentiated cells, cancer cells, and combinations thereof. As any cell produces extracellular vesicles, any cell can be used in embodiments of making extracellular vesicles of the disclosure. In further embodiments, the cells are leukocytes. In some embodiments, the cells are self-derived ex vivo differentiated leukocytes. In some embodiments, the cells are self-derived ex vivo differentiated monocytes, macrophages, dendritic cells, or combinations thereof. In some embodiments, the cells are iPSC- derived ex vivo differentiated leukocytes. In some embodiments, the cells are iPSC-derived ex vivo differentiated monocytes, macrophages, dendritic cells, or combinations thereof.

[0073] EVs are generated by cells through a combination of endocytotic and exocytotic events that result in the budding off and encapsulation of various biomolecules. The majority of EVs are derived from multivesicular bodies (MVBs) that are formed by intracellular lysosomal particles, called endosomes. Other EVs are generated through a cell shedding microvesicles. Other EVs are left behind by migrating cells (these are called migrasomes) or dividing cells (midbody remnants). EVs are released into the extracellular matrix through the fusion of the outer membrane of the MVBs with the membrane of source / parent cell.

[0074] In some embodiments, the method further comprises concentrating the EVs collected in step C. In some embodiments, the concentration is performed through tangential flow filtration and / or ultrafiltration. EVs produced from cells can be collected from the culture medium by any suitable method. Typically, a preparation of EVs can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of these methods. For example, EVs can be prepared by differential centrifugation, that is low speed (<20000 g) centrifugation to pellet larger particles followed by high speed (> 100000 g) centrifugation to pellet exosomes,size filtration with appropriate filters (for example, 0.22μm filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods.

[0075] In some embodiments, the cells are donor cells. In some embodiments, the cells are autologous cells. In some embodiments, the cells are antigen presenting cells (APCs). In some embodiments, the APCs are dendritic cells.

[0076] A further aspect of the current disclosure is directed to a method of slowing or stopping the progression of a disease in a subject, the method comprising administering the immunogenic composition disclosed herein, thereby inducing an immune response that slows or stops the progression of the disease. As used herein, “disease progression” refers to the worsening or advancement of a disease over time. Disease progression can be measured in different ways, depending on the specific disease and the patient's condition. In general, disease progression describes the gradual deterioration of a subject’s condition, characterized by the severity of symptoms, spread of the disease, the subject’s functional abilities, and / or through measurable indicators such as known biomarkers. Slowing the progression of a disease refers to delaying the growth / spread of the disease, symptom severity, and decline of the subject’s functional abilities.

[0077] The disclosed immunogenic composition may be administered to a subject by any suitable means. Administration to a human or animal subject may be selected from parenteral, intramuscular, intracerebral, intravascular, subcutaneous, or transdermal administration. In some embodiments, the method of delivery is by injection. Preferably the injection is intramuscular or intravascular (e.g. intravenous). A physician skilled in the art will be able to determine the required route of administration for each patient in need of therapy.

[0078] Another aspect of the current disclosure is directed to a method of immunizing a mammalian subject against a disease, the method comprising administering the immunogenic composition as described herein to the mammalian subject. In some embodiments, the immunogenic composition is administered more than once. In some embodiments, there is a set period of time between each administration of the immunogenic composition.

[0079] In some embodiments, the disease is selected from the group consisting of: cancers of organs and tissues, autoimmune diseases, and neurodegenerative diseases.

[0080] In some embodiments, the cancer is selected from the group consisting of Breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer (melanoma), skin cancer (non- melanoma), bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer,liver cancer, kidney cancer (renal cell carcinoma), brain cancer (glioblastoma), leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, soft tissue sarcoma, bone sarcoma, esophageal cancer, stomach cancer, head and neck cancer, testicular cancer, vulvar cancer, penile cancer, gallbladder cancer, bile duct cancer, adrenal gland cancer, small intestine cancer, anal cancer, neuroendocrine tumors, mesothelioma, Merkel cell carcinoma, gastrointestinal stromal tumor (GIST), carcinoid tumors, choriocarcinoma, pleomorphic carcinoma, adenoid cystic carcinoma, salivary gland tumors, thymoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, liposarcoma, leiomyosarcoma, chondrosarcoma, Ewing sarcoma, rhabdomyosarcoma, fibrosarcoma, dermatofibrosarcoma protuberans, synovial sarcoma, alveolar soft part sarcoma, clear cell sarcoma, epithelioid sarcoma, desmoid tumors, ovarian germ cell tumors, osteosarcoma, chordoma, glioma, astrocytoma, medulloblastoma, meningioma, pituitary tumors, craniopharyngioma, neuroblastoma, retinoblastoma, Wilms tumor (nephroblastoma), adrenocortical carcinoma, mesenchymal chondrosarcoma, giant cell tumor of bone, fibrous dysplasia, desmoplastic small round cell tumor, extraskeletal myxoid chondrosarcoma, Kaposi sarcoma, mycosis fungoides, Sézary syndrome, Castleman disease, Langerhans cell histiocytosis, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), plasma cell neoplasms (multiple myeloma), non-Hodgkin lymphoma (follicular lymphoma), non-Hodgkin lymphoma (diffuse large B-cell lymphoma), non-Hodgkin lymphoma (mantle cell lymphoma), non-Hodgkin lymphoma (peripheral T-cell lymphoma), fibromatosis (desmoid tumor), neurofibromatosis, gliomatosis cerebri, MALT lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large cell lymphoma, extranodal NK / T-cell lymphoma, acinic cell carcinoma, adult T-cell leukemia / lymphoma, aggressive NK-cell leukemia, basal cell carcinoma, basaloid squamous cell carcinoma, Burkitt lymphoma, carcinoid syndrome, carcinoma in situ, chondroblastoma, clear cell adenocarcinoma, clear cell carcinoma, clear cell renal cell carcinoma, cystadenocarcinoma, embryonal carcinoma, endometrioid adenocarcinoma, ependymoma, epithelial-myoepithelial carcinoma, extranodal marginal zone lymphoma, follicular adenocarcinoma, follicular carcinoma, follicular dendritic cell sarcoma, gastric adenocarcinoma, gastrointestinal carcinoid tumor, giant cell tumor of tendon sheath, granular cell tumor, granulocytic sarcoma, granulosa cell tumor, hemangioblastoma, hemangiopericytoma,hepatocellular carcinoma, Hurthle cell carcinoma, inflammatory myofibroblastic tumor, inflammatory pseudotumor, intraductal papillary mucinous neoplasm (IPMN), invasive lobular carcinoma, Krukenberg tumor, Langerhans cell sarcoma, leiomyoma, leiomyosarcoma, lipomatous tumor, low-grade serous carcinoma, lymphoepithelial carcinoma, malignant fibrous histiocytoma (MFH), malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant triton tumor, medullary carcinoma, medullary thyroid carcinoma, Merkel cell polyomavirus-associated carcinoma, Merkel cell polyomavirus-negative carcinoma, metastatic squamous neck cancer with occult primary, micropapillary carcinoma, mixed ductal-lobular carcinoma, mucoepidermoid carcinoma, myoepithelial carcinoma, myxofibrosarcoma, myxoid chondrosarcoma, myxoid liposarcoma, nasopharyngeal carcinoma, neurofibroma, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor, neurofibromatosis type 2-associated tumors, neurogenic sarcoma, nevoid basal cell carcinoma syndrome, nodular malignant melanoma, non-small cell lung carcinoma, noninvasive papillary urothelial carcinoma, oncocytoma, oropharyngeal carcinoma, ossifying fibroma, osteoblastoma, ovarian serous carcinoma, Paget disease, papillary adenocarcinoma, papillary carcinoma, papillary renal cell carcinoma, papillary serous carcinoma, parathyroid carcinoma, parathyroid adenoma, Peutz- Jeghers syndrome-associated tumors, phyllodes tumor, pilomatricoma, plasmacytoma, pleomorphic adenoma, and plexiform neurofibroma.

[0081] In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), polymyalgia rheumatica, Goodpasture syndrome, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, vitiligo, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Wegener's granulomatosis (granulomatosis with polyangiitis), microscopic polyangiitis, Behçet's disease, Takayasu's arteritis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), IgA nephropathy (Berger's disease), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), antiphospholipid syndrome, autoimmune hepatitis, mixed connective tissue disease(MCTD), autoimmune pancreatitis, pernicious anemia, lichen sclerosus, relapsing polychondritis, pustular psoriasis, eosinophilic esophagitis, autoimmune thyroid disease, autoimmune encephalitis, autoimmune polyendocrine syndromes (APS-1 to APS-21), autoimmune polyendocrine syndrome type 1 (APS-1), autoimmune polyendocrine syndrome type 2 (APS-2), autoimmune polyendocrine syndrome type 3 (APS-3), autoimmune polyendocrine syndrome type 4 (APS-4), autoimmune polyendocrine syndrome type 5 (APS-5), autoimmune polyendocrine syndrome type 6 (APS-6), autoimmune polyendocrine syndrome type 7 (APS-7), autoimmune polyendocrine syndrome type 8 (APS-8), autoimmune polyendocrine syndrome type 9 (APS-9), autoimmune polyendocrine syndrome type 10 (APS- 10), autoimmune polyendocrine syndrome type 11 (APS-11), autoimmune polyendocrine syndrome type 12 (APS-12), autoimmune polyendocrine syndrome type 13 (APS-13), autoimmune polyendocrine syndrome type 14 (APS-14), autoimmune polyendocrine syndrome type 15 (APS-15), autoimmune polyendocrine syndrome type 16 (APS-16), autoimmune polyendocrine syndrome type 17 (APS-17), autoimmune polyendocrine syndrome type 18 (APS- 18), autoimmune polyendocrine syndrome type 19 (APS-19), and autoimmune polyendocrine syndrome type 20 (APS-20). In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), polymyalgia rheumatica, Goodpasture syndrome, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, vitiligo, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Wegener's granulomatosis (granulomatosis with polyangiitis), microscopic polyangiitis, Behçet's disease, Takayasu's arteritis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), IgA nephropathy (Berger's disease). In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis,Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), and polymyalgia rheumatica. In some embodiments, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, and inflammatory bowel disease (Crohn's disease and ulcerative colitis).

[0082] In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia, Lewy body dementia, Pick's disease, Frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration, Creutzfeldt-Jakob disease, Wilson's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Charcot-Marie-Tooth disease, hereditary spastic paraplegia, Krabbe disease, Batten disease (neuronal ceroid lipofuscinoses), Niemann-Pick disease, Gaucher disease, Tay-Sachs disease, Canavan disease, Alexander disease, metachromatic leukodystrophy, Pelizaeus-Merzbacher disease, Sanfilippo syndrome, mucopolysaccharidoses, Rett syndrome, fragile X-associated tremor / ataxia syndrome, myotonic dystrophy, Huntington's disease-like syndromes, spinal and bulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, Perry syndrome, and prion diseases. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia, Lewy body dementia, Pick's disease, Frontotemporal dementia (FTD). In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD).

[0083] In some embodiments, the infectious disease includes HIV / AIDS, Influenza, coronaviruses, tuberculosis, malaria, hepatitis (A, B, C, D, and E), dengue fever, Zika virus, Ebola virus disease, cholera, typhoid fever, measles, chickenpox, shingles, polio, rabies, meningitis (bacterial, viral, and fungal), pneumonia (bacterial and viral), Lyme disease, syphilis, gonorrhea, chlamydia, HPV (human papillomavirus), herpes (HSV-1 and HSV-2), candidiasis, trichomoniasis, giardiasis, cryptosporidiosis, leishmaniasis, schistosomiasis, filariasis, onchocerciasis, trachoma, tetanus, pertussis, anthrax, bubonic plague, tularemia, brucellosis, leprosy, listeriosis, toxoplasmosis, Q fever, rocky mountain spotted fever, yellow fever, WestNile virus, Japanese encephalitis, Rift Valley fever, Hantavirus pulmonary syndrome, Nipah virus infection, and Middle East respiratory syndrome (MERS). In some embodiments, the infectious disease is selected from the group consisting of HIV / AIDS, Influenza, coronaviruses, tuberculosis, Lyme disease, HPV (human papillomavirus), and Middle East respiratory syndrome (MERS). In some embodiments, the infectious disease is selected from the group consisting of HIV / AIDS, Influenza, and coronavirus. Examples

[0084] Example 1. Arc VLVs respond to stimuli in APCs, present antigens, target secondary lymphoid organs, and directly interact with T cells.

[0085] To investigate the role of Arc VLVs in mediating long-range intercellular communication between immune cells, we first studied how Arc naturally responds to external stimulation in APCs, particularly in DCs and macrophages (MΦs). Extracted and differentiated from mice, primary bone marrow-derived DCs and MΦ (BM-DC / MΦ) were exposed to various stimuli including lipopolysaccharide (LPS), Ovalbumin (Ova), and transfection reagents (Lipofectamine and Xfect). Reverse transcription quantitative PCR (RT-qPCR) analysis revealed a rapid increase in Arc mRNA expression half an hour post-stimulation (FIG.1A, purple bars, right Y axis). EVs collected from the supernatant medium 6 hours post-stimulation showed an elevated level of extracellular Arc proteins, as quantified using a fluorescent Arc-specific antibody and an epifluorescence plate reader (FIG.1A, black bars, left Y axis). These findings suggest that DCs and MΦs initiate Arc expression and secrete Arc VLVs in response to various stimuli, indicating a cirtical role of Arc VLVs following APC activation

[0086] In our study of the composition and functionality of DC-derived endogenous VLVs, we utilized the murine DC2.4 cell line. Simply overexpressing the Arc protein was insufficient for increasing a significant production of Arc-VLVs, and we therefore complemented the overexpression with a stabilizing RNA motif—the 5' UTR of the Arc gene (A5U). Employing liposome-mediated transfection of DNA plasmids and RNA transcripts encoding the rat Arc (rArc) alongside A5U (FIG.2A), we substantially increased the production of engineered retrotransponson Arc VLVs (eraVLVs). We used rArc for easy differentiation from mouse Arc in in vivo models and human Arc in in vitro models. Employing the Ova model antigen, which wasdirectly added to the culture medium, the control and engineered DC2.4 cells were enabled to process and present Ova peptides via MHC complexes on their surfaces, subsequently secreting Ova-presenting vesicles. Subsequently, the control DEVs and eraVLVs were purified using tangential flow filtration (TFF) and ultrafiltration. Subsequent nanoparticle tracking analysis (NTA) identified a distinct subpopulation of larger vesicles within the engineered vesicles, resembling retrovirus homologues that bud directly from the plasma membrane (FIG.2B). The composition of these vesicles was further validated through Western blot analysis of general EV markers (FIG.2C).

[0087] Mass spectrometry (MS) analysis provided a detailed protein profile of the vesicles, showing significant overlap with documented EV proteins from databases such as ExoCarta and Evpedia (FIG.1C). The G:Profiler database identified several TRUE Gene Ontology Biological Process (GO:BP) terms associated with these proteins, which emphasize the vesicles' capabilities in immune-related functions (FIG.1D). These include organonitrogen compound metabolic process, establishment of localization in cell, regulation of protein polymerization, antigen processing and presentation, inositol biosynthetic process and protein homooligomerization, all of which are crucial for the effective transport, processing, and presentation of antigens. Other identified functions include involvement in viral processes and T cell mediated cytotoxicity, highlighting the veiscles’ potential in activating immune responses. Additionally, proteins linked to cell-substrate adhesion indicate impacts on vesicle migration and target cell interaction. Label-free MS quantification highlighted that in Ova+ eraVLVs compared to control DEVs, the monocyte lineage marker, CD14, and EV markers including tetraspanins CD9 and CD63, and transmembrane glycoproteins Lamp1 and Lamp2, along with several cytoskeletal proteins (Actr2, Actr3, Arpc2, Arpc3, Capza2) and other membrane-associated proteins (YWHAE, Myl6, Capns1), maintained stable expression levels (FIG.1D). Conversely, there was a significant increase in the expression of microvesicle-associated proteins such as Anxa6, Anxa1, Anxa4, Anxa5, Anxa3, and Rab2a, structural proteins like Actbl2 and Tubb3, alongside cell trafficking and adhesion-related proteins such as CD44, CD147 and Dsg1b, suggests improvements in vesicle exocytosis, endocytosis, stability, cellular interactions, and potential immunomodulatory functions (FIG.1E). The abundance of more proteins associated with cell adhesion were analyzed (FIG.1F). Specifically, increased Vimentin enhances eraVLV structural stability, aiding transport and cell interactions, while elevated ICAM1 and CD44 improve adhesion to andinteraction with recipient cells, particularly enhancing immune cell activation and homing to hyaluronic acid-rich tissues like lymphoid areas and inflamed sites. The upregulation of small GTPases such as Rap1b, Rac1, and Cdc42 enhances vesicle mobility and interaction capabilities. Conversely, decreased integrins may reduce EV adhesiveness to the vascular wall, potentially extending their circulation half-life, allowing for prolonged systemic presence and improved delivery efficiency. Furthermore, proteins involved in both MHC-I and MHC-II antigen processing pathways (FIG.1F, ii-iii), as well as membrane proteins crucial for T cell interaction and activation were upregulated in Arc+ vesciles (FIG.1F, iv). Importantly, we observed an increase of the MHC-I Ova peptide sequence SIINFEKL (SEQ ID NO: 33) on eraVLVs (FIG. 1G), whereas the MHC-II Ova peptide sequence ISQAVHAAHAEINEAGR (SEQ ID NO: 34) in eraVLVs was found to be comparable to non-transfected control DEVs. The observed changes in the protein composition of Arc+ vesicles collectively indicate that they acquire a spectrum of immune-modulating membrane proteins from DCs, tailored for effective long-range inter- cellular communication and immune activation and preferentially enhancing MHC-I mediated antigen presentation.

[0088] To further investigate the roles of eraVLVs in vivo, we systemically administered both control and engineered vesicles presenting Ova peptides to study their distribution and interactions with target organs and cells. Labeled with a lipid membrane dye (Cell Mask Deep Red, or CMDR) for tracking, equal quantities from each sample group, including EV- control (DPBS), Ova- DEVs, Ova+ DEVs, Ova+ / A5U+ DEVs, Ova+ / Arc+ DEVs, and Ova+ / Arc+ / A5U+ DEVs (or Ova+ eraVLVs), were administered intravenously into C57BL6 mice. Six hours post-injection, the spleens were harvested and analyzed using an in vivo imaging system (IVIS), demonstrating a substantial enrichment of Arc+ vesicles (FIG.1G). This highlights the efficient trafficking of eraVLVs to a key secondary lymphoid organ, implying their potential in effectively reaching sites critical for initiating T cell responses. Further analysis of immunohistochemically-stained spleen cryosections revealed Arc VLVs binding to spleen cells (FIG.1H & FIG.3). So far, we demonstrated that APCs initiate Arc expression and secrete Arc VLVs in response to various stimuli, and that these vesicles effciently traffic to secondary lymphoid organs to interact with T cells, highlighting their critical roles in APC functionality and therapeutic relevance in immunotherapies.

[0089] Example 2. Presenting a model antigen Ova, Arc VLVs induce robust, balanced cellular and sustained humoral immune response.

[0090] To further investigate the immunological impact of eraVLVs at the transcriptional level, we analyzed immune response markers using reverse transcription quantitative PCR (RT-qPCR). This was performed twenty-four hours after the systemic administration of eraVLVs and control DEVs, extracting total mRNA from the lymph nodes and spleens of mice. Notably, in the Arc+ groups, there was an upregulation of a broad spectrum of immune-related genes compared to the Arc- Ova+ DEVs (FIG.4A). Key upregulated genes included IFNγ, a crucial pro-inflammatory cytokine in anti-tumor immunity produced by NK and T cells; IL-2, vital for T cell proliferation and an essential factor in anti-cancer responses; IL-4 and IL-10, which contribute to a balanced immune response, crucial for effective cancer immunotherapy; and CD86, a co-stimulatory molecule critical for T cell activation. This gene expression profile suggests the capability of eraVLVs in eliciting a robust yet balanced immune response against specific antigens.

[0091] Subsequently, we assessed the impact of eraVLVs on T cell proliferation and activation, comparing it to those of control DEVs. Twenty-four hours following systemic administration of these vesicles, we harvested primary splenocytes from mice for flow cytometry analysis. Notably, the Arc+ groups showed increased proliferation of CD3+ T cells and CD4+ T cells (FIG.4Bi-ii, and 2C). CD3+ T cells encompass all T cell subsets, while CD4+ T cells, the helper T cells, are pivotal in driving the immune response. In contrast, no increase in CD8+ T cells, the cytotoxic T cells, was observed within the 24-hour post-injection window (FIG.4Biii). However, three days after IV injection, we not only observed a significant increase in total CD8+ T cells (FIG.4Biii) but also in the subset of CD8+ / CD44+ / CD62L+ effector T cells (FIG.5). In additional experiments using wildtype C57BL / 6 primary DCs as donor to produce Ova- presenting vesicles, three days post injection of these vesicles, splenocytes extracted also showed an upregulation in the total numbers of CD4+ and CD8+ T cells, as well as pre-effector CD4+ and CD8+ T cells (FIG.6C). The proliferation of these cells suggests enhanced immune activation, potentially attributable to the efficient antigen presentation and immune-modulatory capabilities of the eraVLVs. This delayed response in CD8+ T cell proliferation underscores the multi-phased nature of the immune response elicited by Arc+ DEVs, initially involving CD4+ T cell activation followed by a subsequent activation of CD8+ T cells, crucial for direct cellular responses against tumor cells.

[0092] We also explored whether multiple administrations of Ova+ eraVLVs could generate a sustained humoral immune response. When administered repeatedly over four weeks (FIG.4D), eraVLVs elicited a robust IgG response, indicative of a potent and enduring adaptive immune response, with significantly superior immunogenic potency compared to control DEVs (FIG. 4F). Additionally, the Ova+ eraVLVs were observed to create long-term immunological memory. A booster dose administered two months after the initial four doses, prior to ELISpot analysis, yielded several key findings: 1) activation of CD8+ T cells, 2) an effective booster response, 3) potential cytotoxic activity, 4) long-term immunological memory, and 5) preference to the MHC-I response (FIG.4F). The specific response to MHC-I / Ova peptide suggests effective antigen presentation through MHC class I molecules to CD8+ T cells. The detection of IFNγ, predominantly produced by activated CD8+ T cells, underscores their activation and potential transformation into cytotoxic T lymphocytes, crucial in cancer vaccine strategies. The booster response months after the initial doses indicates the successful establishment of long- term immunological memory. The specificity of the response to the MHC-I / Ova peptide, as opposed to the MHC-II peptide, implies a more pronounced CD8+ T cell response. In summary, these findings suggest that the eraVLV vaccine can induce a long-term T cell immune response, reactivated by a booster dose.

[0093] Example 3. Arc plays essential roles in immune activation and memory T cell generation.

[0094] To further understand the role of the Arc protein in APC-driven T cell activation, particularly within the context of cancer antigen presentation, we conducted studies using EVs derived from Arc knockout APCs. As illustrated in FIG.7A, primary DCs and MΦs were differentiated from bone marrow GM-CSF / IL4 cultures of both Arc+ wildype (WT) and Arc- / - knockout (KO) mice. We observed obvious differences in morphology between primary BM- DC / MΦ cells from Arc KO mice and those from WT mice suggesting that Arc plays essential roles in the development and functionality of APCs (FIG.6A). These cells were then transfected for a 16-hour period and stimulated with EVs derived from B16-F10 melanoma cells in three distinct sample groups: (1) a wild-type (WT) untransfected control group, serving as a baseline comparison; (2) a WT group transfected with rArc and rA5U, overexpressing eraVLVs; and (3) an Arc knockout (Arc- / - KO) untransfected group, which lacks Arc-containing VLVs. This experimental setup was designed to assess the impact of Arc presence or absence on the cellularresponse to melanoma-derived EVs, thereby exploring the role of Arc in immune cell modulation and cancer antigen presentation. After an additional 24-hour culture in fresh serum-free medium, the cells from each of the three groups were harvested, and total RNA was extracted for RT- qPCR analysis, quantifying the expression of various immune-related genes, as well as endogenous Gag virus-like components including the Arc capsid and the envelope protein Syncytin A (SynA). The results revealed that in the rArc+ rA5U+ transfected cells, mouse Arc mRNA levels were substantially upregulated compared to the WT untransfected control group, and this upregulation was accompanied by an increase in SynA expression (FIG.7B). In contrast, in the Arc- / - KO group, there was no observed increase in mArc or mSynA expression post-stimulation. These suggest that endogenous virus-like components respond to antigenic stimulation in APCs and the capsid and envelope proteins are integral to each other's functionality. Moreover, qPCR showed an increase in CD86, interferon-gamma (IFN-γ), and IL6 expression in the Arc+ transfected group compared to both the WT untransfected control and Arc- / - KO cells, suggesting that Arc may act as a positive regulator of immune activation in APCs (FIG.6B). We noticed a low level of upregulation of these genes in the Arc- / - KO cells compared to untransfected WT cells (FIG.6B), suggesting that there may be compensatory mechanisms or alternative pathways involved in the absence of Arc, albeit to a lesser extent. Altogether, these findings indicate that the Arc protein is essential for the activation of APCs, and that endogenous virus-like envelope proteins, such as SynA, may also contribute to this process, working synergistically with the capsid components.

[0095] Meanwhile, we collected the three groups of EVs from primary BM-DC / MΦ cultures mentioned above and injected them systemically into C57BL / 6J WT mice. Following intravenous injection, spleens were collected from treated mice on day 8, and splenocytes were extracted (FIG.7C). These splenocytes were then restimulated with B16-F10 EVs for 6 hours, followed by flow cytometry analysis of various immune cell markers. Notably, splenocytes extracted from mice treated with WT DEVs, both transfected with rArc or untransfected, exhibited enhanced responsiveness to B16-F10 EVs during restimulation in vitro, shown by an increased proportion of CD4+ / CD44+ / IFNγ+ memory Th1 cells (FIG.7D). In contrast, splenocytes from mice treated with Arc- / - DEVs failed to demonstrate this enhanced T cell response. These findings imply that Arc+ DEVs have the capacity to induce the generation of memory T cells capable of recognizing and responding to B16-F10 melanoma EVs. In addition,we also demonstrated an upregulation of various actviated and memory T cell populations (CD4+ / CD44+ and CD8+ / CD44+), including both the CD62L- subpopulation that are poised to exert effector functions at sites of inflammation or infection, and the CD62L+ subpopulation with the ability to home to secondary lymphoid organs and contribute to long-term immune surveillance and memory responses (FIG.7E-F). Subsequent visualization of the interactions between splenocytes and B16-F10 melanoma cells post-vaccination showed increased interactions between adherent splenocytes and melanoma cells, highlighting the memorization of melanoma by the immune cells, particularly in the Arc+ group (FIG.7G). Overall, these findings not only confirm the critical role of Arc VLVs in antigen presentation and T cell activation but also imply the potential of eraVLVs as powerful immunogenic agents capable of eliciting robust, long-term, and specific T-cell responses against tumor antigens.

[0096] Building on this potential, we next explored the application of eraVLVs within the context of cancer vaccines. One significant advantage of our vaccine system is its ability to create highly personalized and adaptive cancer treatments tailored for individual patients' progressing antigens, without the need for prior knowledge of specific neoantigen sequences. To evaluate the practical efficacy of the eraVLV vaccine, we applied our approach in an unmodified orthotopic mouse melanoma model using B16-F10 cells. To produce eraVLV melanoma vaccines, we transferred the supernatant medium of B16-F10 cells cultured in serum-free conditions to engineered DCs. This supernatant medium, in addition to containing a broad spectrum of secreted antigenic proteins, also includes tumor-derived EVs, which are effective cancer antigens carrying diverse tumor-associated molecules. We first verified the uptake of B16-F10 EVs by DCs. The uptake of B16-F10 EVs by DCs was visualized, using CMDR labeling and fluorescence imaging (FIG.7Hi-iv), demonstrating the capability of DCs to capture and process cancer antigens from tumor-derived vesicles. Additionally, to ensure the purity of our vaccine, we conducted thorough checks to confirm that the collected DEVs did not contain B16-F10 EVs (FIG.7Hv). Overall, the use of comprehensive tumor-derived antigens in the VLV vaccine highlights our potential to adapt to the unique and evolving antigenic landscape of an individual’s cancer, offering personalized, dynamic and effective treatments.

[0097] Three days following the administration of B16-F10 DEVs into WT mice, we analyzed the induced immune response. We collected splenocytes and lymphocytes for flow cytometry and qPCR analyses. The flow cytometry results revealed that B16-F10+ eraVLVs led to asubstantial increase in the proliferation of several T cell types: CD3+, CD4+, CD8+ T cells, as well as CD4+ and CD8+ pre-effector T cells (FIG.7I-J). This increased proliferation across a broad spectrum of T cells indicated a robust activation of the immune system. Complementing these findings, our qPCR analysis corroborated the flow cytometry results and aligned with our previous observations in the Ova antigen model. We noted increased levels of key cytokines such as IFNγ, IL2, and IL4 in (FIG.8), signifying a robust immune response and T cell activation. The elevation of these cytokines is indicative of an active and multifaceted immune response, involving both helper and cytotoxic T cell activation. Altogether, these findings indicate the strong potential of eraVLVs as effective cancer vaccines. The ability of eraVLVs to induce the proliferation of various T cell subsets, coupled with a balanced cytokine response, highlights their potential as a therapeutic tool in cancer immunotherapy.

[0098] Example 4. DC-derived eraVLV vaccines effectively prevent tumor growth.

[0099] In the final phase of our study, we injected four doses of B16-F10+ DEV vaccines into WT mice and, five days later, administered B16-F10 melanoma cells to these mice to assess the preventive efficacy of the eraVLV vaccines (FIG.9A). The unbiased data collection was ensured through double-blind caliper measurements conducted by multiple researchers, complemented with micro-CT scanning to further enhance precision (FIG.9A). Survival analysis revealed a substantial and significant advantage for mice treated with eraVLV vaccines, with a 60% survival rate compared to 37.5% for WT DEVs made with a high dose of B16-F10 EV antigens (B16highDEV, with a ratio of donorB16:recipientDC2.4 = 2:1) and 33.3% for B16lowDEV control (B16:DC2.4 = 1:1) groups (FIG.9B). In comparison, DEV-negative control mice were euthanized between days 13-17 due to tumor volumes exceeding 1500 mm3. Comparative tumor size analysis showcased a significant reduction in tumor growth in the Arc+ group, with daily measurements revealing a clear trend starting from five days post-B16 melanoma cell injection (FIG.9C). By day 17 post-injection of B16-F10 cells, 8 out of 10 tumors in the DEV-negative control mice had already surpassed the predefined size limit and were collected on earlier days. The two tumors with the slowest progression were collected on day 17, as shown in FIG.9D. In contrast, most sites in other sample groups remained tumor-free. For these groups, the largest tumor, in most cases the only tumor present, was extracted for size comparison and further analyzed through tissue sectioning and both histochemical and immunohistochemical staining (FIG.9D). Histology examination further demonstrated the efficacy of B16higheraVLV vaccines,showing tumor destruction along with increased CD3+ T cell infiltration compared to the B16highDEVs (FIG.9E). Supporting these findings, analysis of tumor-bearing mice revealed differences in immune cell localization between groups. Specifically, a higher number of CD8+ cytotoxic T cells were observed remaining in the spleen in the DEV control group. In contrast, the eraVLV group displayed markedly fewer CD8+ cytotoxic T cells in the spleen, implying an active recruitment of cytotoxic T cells from the spleen to the tumor site (FIG.10). This provides one explanation for the enhanced efficacy of the eraVLV treatment in directing immune responses directly to the tumor environment.

[0100] In our rechallenge experiment, we intradermally injected each mouse with B16-F10 melanoma cells on one flank and subcutaneously with EO771 breast cancer cells on the other. After the initial cohort succumbed, we introduced a second batch of DEV-negative control. The eraVLV vaccine made against melanoma, effectively preventing not only melanoma but also breast cancer, substantially extending survival and achieving up to an 11-fold increase in median survival compared to the DEV-negative control groups, and nearly 7 times longer than the B16highDEV control (FIG.9F). DC2.4 cells, immortalized murine DCs created by transducing bone marrow isolates from C57BL / 6 mice with retroviral vectors expressing GM-CSF along with the myc and raf oncogenes, may present certain oncogenic antigens via their EVs, potentially enhancing the universality of vaccinations. Moreover, the presence of shared tumor antigens between melanoma and breast cancer, such as MAGE proteins and WT1, may further contribute to the protective efficacy of our eraVLV vaccine across multiple cancer types, supporting its potential as a broad-spectrum therapy. Nonetheless, despite the potential broad coverage, we also observed preferential protection against melanoma overall. The untreated negative control mice reached euthanasia criteria quickly due to large fast-growing melanoma tumors, when the less aggressive breast cancer tumors remained small (FIG.9G-H). However, vaccination with B16-F10+ DEVs and VLVs showed substantial protection against melanoma, extending the survival periods of treated mice significantly (FIG.9F), allowing them more time to grow larger breast cancer tumors. Eventually, these vaccinated mice reached euthanasia criteria with significantly larger breast cancer tumors (FIG.9G-H). These findings indicate that DC-derived eraVLV vaccines provide targeted protection against specific tumor types, yet their efficacy is so pronounced that they confer a broad defense against tumor progression.

[0101] Example 5. DC-derived eraVLV vaccines enhance behavioral wellness of treated mice and engage T cells via endogenous virus-like envelop proteins.

[0102] In addition to monitoring tumor growth of vaccinated tumor-bearing mice, we assessed their locomotive and cognitive wellness using a novel object behavior test (FIG.11A). Mice treated with B16highDEV vaccines exhibited enhanced exploratory behavior in the novel object behavior test, displaying higher frequencies of interaction with the novel object (FIG.11A & FIG.12). They showed increased speed (FIG.11B), travel distance (FIG.11C), and number of entries into the novel object zone (FIG.11D). In contrast, mice in the DEV- negative control group tended to avoid the novel object zone. This suggests that eraVLVs not only inhibit tumor growth but also have positive effects on cognitive function and overall wellness in treated mice.

[0103] In our further exploration of the underlying mechanisms that enhance the efficacy of eraVLV vaccines, we found that Arc can recruit endogenous ENV proteins, which may bind T cell receptors, in a manner reminiscent of retroviral ENVs. A label-free MS quantitative analysis uncovered an upregulation of endogenous virus-like ENVs on eraVLVs stimulated with Ova, in comparison to Ova- DEVs and the Ova+ Arc+ A5U- DEVs. MS identified a high-degree similarity in the sequence between the extracellular receptor-binding domains of the murine leukemia virus (MLV) ENV with a specific sequence found enriched in Ova+ eraVLVs (FIG. 11E, ENV1), with one beta-sheet sequence within this domain found to be 100% conserved (FIG.11F). In addition to this sequence, an extracellular peptide sequence from the endogenous porcine ENV was also increased in eraVLVs (FIG.11E, ENV2). The natural targets of MLV include B cells and T cells. The conservation between retroviral ENVs and Arc-associated DEV membrane proteins suggests that eraVLV vaccines may be exploiting similar pathways used by retroviruses to engage and activate immune cells. Importantly, the upregulation of ENVs was observed exclusively in eraVLVs when both the rArc capsid protein and rArc 5' UTR were co- introduced, suggesting that the 5' UTR of Arc plays crucial roles in ENV recruitment. Moreover, the upregulation of several membrane proteins involved in leukocyte adhesion and immune surveillance in Arc+ DEVs suggests complex interactions between VLV components and immune activation pathways. Such molecular mimicry opens new avenues for understanding how DEV vaccines can be fine-tuned to harness virus-like properties for improved vaccine efficacy against diseases like cancer.

[0104] Next, proteomic profiling of melanoma antigen-presenting vesicles was carried out through MS. Proteins identified in our melanoma dataset were first compared with our Ova dataset, the ExoCarta, Vesiclepedia, and EVpedia EV databases, showcasing both new and previously documented EV proteins (FIG.13A and 14). We first identified several proteins overexpressed in B16+ VLVs compared to DEVs, including Mageb4, Serpinf1, Tradd, Pcna, Ceacam1, and Bst2, all of which play key roles in tumor biology and immune regulation (FIG. 13B). Mageb4 (melanoma antigen family B4) contributes to tumor immunogenicity and is commonly expressed in cancer cells but restricted in normal tissues, making it an attractive target for cancer immunotherapy. Tradd (tumor necrosis factor receptor type 1-associated DEATH domain protein) is involved in apoptosis and immune signaling through the TNF receptor pathway, potentially supporting immune responses against tumors. Tpt1 (translationally- controlled tumor protein) and Tpd52l2 (tumor protein D54) are linked to cell proliferation and survival, both of which are often upregulated in tumors to promote growth. Pcna (proliferating cell nuclear antigen), essential for DNA repair and cell cycle progression, is typically overexpressed in tumors, supporting rapid cell proliferation. Ceacam1 (carcinoembryonic antigen-related cell adhesion molecule 1) is associated with cell adhesion and immune modulation, with overexpression linked to tumor growth and immune escape. Bst2 (bone marrow stromal antigen 2), or tetherin, has roles in immune signaling and is upregulated in various tumors, where it may contribute to modulating immune responses. Interestingly, CD47 is downregulated in VLVs (FIG.13B). By reducing CD47, VLVs may engage T cells more actively and avoid sending any inhibitory signals that could suppress T cell activation, leading to a more effective anti-tumor response. Together, the modulation of proteins related to immune evasion, cell survival, and proliferation, suggests VLV’s relevance in cancer treatment.

[0105] Example 6. Proteomic and immunogenic profiling of melanoma antigen-containing vesicles.

[0106] To study the impact of adding B16 melanoma antigens and virus-like components, we compared three experimental groups: B16- DEVs, B16+ DEVs, and B16+ eraVLVs, derived from an immortal DC line, DC2.4 and primary BM-DC / M (FIG.13C). To identify the biological processes associated with proteins significantly upregulated in each group, UniProt accession numbers of the upregulated proteins in each comparison pair were inputted into the G:Profiler database to conduct Gene Ontology (GO) enrichment analysis, using the GO: Biological Process(GO:BP) category to retrieve enriched biological processes (FIG.13D). First, the GO terms for the B16+ VLV over B16- DEV comparison include similar terms identified in both B16+ VLV over B16+ DEV and B16+ DEV over B16- DEV comparisons. This makes sense given that the B16+ VLV condition builds upon both the addition of B16 melanoma antigens to DEVs and further enhancement with the Arc and A5U components. Second, the enrichment of specific biological processes in B16+ eraVLVs compared to B16- DEVs like response to virus and cytoplasmic pattern recognition signaling pathways indicates that eraVLVs may leverage viral mimicry to enhance immune detection and response (FIG.13Di). The enrichment in organonitrogen compound metabolism and non-membrane-bounded organelle assembly likely reflects metabolic adaptations facilitating the antigen processing and presentation. Notably, the inclusion of pathways related to multicellular organism development and regeneration in the eraVLVs could indicate that these vesicles support cellular communication and tissue- regenerative processes in the tumor microenvironment, potentially priming it for immunogenic interactions. Moreover, comparing B16+ eraVLVs to B16+ DEVs, the most significantly enriched GO:BP is "response to virus,", agreeing with the addition of virus-like components (FIG.13Dii). Beyond viral responses, other enriched pathways in B16+ eraVLVs, such as positive regulation of gene expression and TNF production, indicate active inflammatory signaling that favors immune cell recruitment and activation. Processes like peptide metabolism, ribosome assembly, and protein localization further support enhanced protein processing and trafficking, potentially improving the efficiency of antigen presentation and immune cell priming. Collectively, these findings suggest that B16+ eraVLVs not only present antigens but also actively promote an inflammatory microenvironment that mimics viral infection, amplifying immune activation against the tumor antigens. Lastly, for B16+ DEVs relative to B16- DEVs, upregulated pathways, including blood coagulation, TGFβ regulation, and MAPK cascade modulation, suggest that the inclusion of melanoma antigens may enhance DEVs’ capacity in migration, signaling, and inter-cellular communication (FIG.13Diii). The upregulation of processes like response to metal ions and the acute phase response aligns well with the fact that B16+ DCs’ response to tumor-associated antigens. These suggest that melanoma antigens potentially prime the immune system to recognize tumors, and adding virus-like components further boosts anti-viral pathways and promotes immune activation beyond basic antigen presentation, enhancing immune cell recruitment and activation.

[0107] Zooming in on specific protein targets, the comparative analysis between B16+ and B16- DEVs revealed an upregulation of proteins crucial for endocytosis, such as Igf2r, Lcat, Fgb, Lum, Thbs1, and Fn1, suggesting efficient uptake and processing of melanoma-derived EV antigens. Proteins involved in the regulation of proteolysis, peptidase activity, cross-presentation of soluble exogenous antigens, such as Ctnnb1, Psmb1, Klkb1, F2, Psmb2, Hgfac, Psmb10, Fgb, Gsk3a, Ufd1, Serpinf1, Gsn, Mug1, Thbs1, Itih2, Fn1, Fbln1, Serpinc1, Pzp, and Itih4, were also highlighted, priming DCs for effective antigen presentation. Additionally, the increased presence of immune activators like C1qtnf3 and Serpinc1, alongside metabolic enzymes (Mat1a, Aldob), indicates a strategic adaptive response designed to optimize immune interactions and meet the metabolic demands associated with processing complex EV antigens. On the other hand, the proteins upregulated in eraVLVs, such as Gbp2, Iigp1, and Ifit1, are indicative of a robust antiviral response, suggesting that endogenous viral-like components mimic viral infection in DCs. We observed enhanced interferon signaling by Stat1, Stat2, Stat3, Eif2ak2, Trim25, and Oasl1, suggesting cytokine-mediated immune activation. Key proteins like Hmox1 and Gclm indicate increased oxidative stress management, vital for protecting cells during immune responses. Moreover, proteins such as Sqstm1, Serpinc1 and Serpinf1 illustrate cellular responses to stress and regulation of inflammation, critical for maintaining homeostasis and effective immune surveillance.

[0108] To assess the immunogenic relevance of the peptides identified from B16+ DEVs and eraVLVs, we performed a comparative analysis with the Immune Epitope Database (IEDB) for mouse melanoma epitopes. This comparison identified peptides in B16+ vesicles aligning with known immunogenic sequences, helping to verify their potential to activate immune responses specific to melanoma (FIG.13E). Specifically, hundreds of overlapping proteins were identified, with 16 of which significantly upregulated in B16+ DC2.4 eraVLVs compared to B16- DC2.4 DEVs. Further comparison using specific peptide sequences revealed 46 epitopes overlapping between B16+ DC2.4 vesicles and the melanoma database (FIG.13F-G). Many of these epitopes are shared with vesicles from primary donor cells, indicating common antigenic features across different DC types. Notably, the epitopes unique to DC2.4 EVs are mostly upregulated in B16+ eraVLVs compared to DEVs (FIG.13H), suggesting enhanced immunogenic potential of eraVLVs in presenting melanoma antigens. In contrast, the shared epitopes that are also present in primary DEVs showed mostly unchanged expression levels (FIG.13I). Further analysis of theshared 46 epitopes identified significant GO terms related to cellular compartments and biological processes relevant to melanoma and VLVs. The GO term “melanosome” was highly enriched, with proteins associated with this organelle showing elevated abundance levels in B16+ DC2.4 eraVLVs (FIG.13J). This suggests that the VLVs may effectively present melanoma-specific antigens. Additionally, several important GO terms linked to viral process were identified, including virion assembly, modulation by host of viral processes, and regulation of centrosome duplication (FIG.13K). Other processes like amide metabolic process and ubiquitin-independent protein catabolic process via the multivesicular body sorting pathway were also enriched. The upregulation of these proteins in eraVLVs hints at a viral mimicry mechanism, which enhances the immunogenic potential of DEVs by promoting efficient antigen presentation and immune activation (FIG.13L). Lastly, we entered the MS-identified peptide sequences into the IEDB’s ANN 4.0 prediction tool to evaluate their potential for MHC binding. FIG.13M shows peptides with IC50 values below 500, indicating strong binding affinity, which are upregulated in DC2.4-derived B16+ eraVLVs compared to DEVs and are predicted to bind to H-2-Kb and H-2-Db molecules. Although we found only 10 exact matches, many of the strong- binding candidates are partial sequences within longer peptide chains, likely acting as precursors to the epitopes to be presented. Since this MS analysis did not involve MHC pull-downs, it included all proteins within the vesicles, encompassing both lumenal and membrane-bound proteins rather than exclusively those bound to MHC molecules. Overall, these results highlight that the VLVs not only present melanoma-relevant antigens but may also leverage viral-like processes to boost their effectiveness.

[0109] In conclusion, our study highlights the critical biological function of Arc VLVs in the immune system, underscoring how DCs have incorporated ancient virus components to enhance their functionality. Through our investigation, we first demonstrated that Arc is not only required for but also promotes APC activation and T cell responses. Next, we showed the indispensable role of Arc VLVs in enhancing DCs' antigen presentation capabilities using an Ova antigen model. Notably, the Ova+ eraVLVs were found to activate robust and balanced immune responses rapidly and effectively. Moreover, when administered multiple times, these vaccines generated sustained humoral immune responses and established long-term immunological memory, demonstrating their efficacy in not just triggering, but also maintaining, an effective immune response. Furthermore, our findings indicate that eraVLVs can proficiently process andpresent B16-F10 melanoma antigens. Notably, the B16+ eraVLV vaccines have shown remarkable efficacy in preventing melanoma development in our in vivo mouse models. We also discovered that Arc recruits endogenous ENV proteins to engage T cells, closely resembling the function of retroviral ENVs. Through extensive protein mapping, we revealed that endogenous virus-like components act as built-in adjuvants, enhancing antiviral and broader immune activation while presenting antigens. Our study uncovers a crucial mechanism underlying the long-range interaction between DCs and T cells, advancing vaccine design and development strategies to combat various diseases.

[0110] Example 7: General Methods and Materials

[0111] Biological resources: An Arc plasmid (pGEX-6p1-GST-ArcFL, Addgene plasmid #119877) and a GFP plasmid (pcDNA3-EGFP, Addgene plasmid #13031) were purchased from Addgene. The rat Arc 5’ UTR sequence was synthesized by Genscript. Molecular cloning techniques (PCR amplification, restriction enzyme digestion and Gibson assembly) were used to generate constructs pCDNA3-rArc and pCDNA3-rA5U-GFP. Correct sequences of all plasmids have been verified by whole plasmid sequencing by PlasmidSaurus. We regularly sequence our plasmids to ensure their sequence integrity.

[0112] In vitro transcription of DNA plasmids and RNA transcripts: DNA plasmids were transfected into donor cells using lipofectamine LTX with PLUS reagent, while primary cells had RNA transfection via lipofectamine mMessenger Max. These plasmids were linearized by PCR or enzyme digestion for in vitro transcription using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB), with pseudo-UTPs. The last two nucleotides of the T7 promoter in the DNA templates for RNA IVT were converted to AG. After transcription, DNase removed residual DNA and RNAs were purified with the Monarch Kit (NEB). RNA quality was checked via gel electrophoresis followed by a fragment bioanalyzer.

[0113] Cells and cell culture: Stable cell line culture: DC2.4 and B16-F10 cells were obtained from EMD Millipore and ATCC respectively and stored in liquid nitrogen. EO771 breast cancer cells were a gift from Cornell Weiss lab. Cells were maintained according to ATCC guidelines and transfected using liposomes. No antibiotics were used in stable cell line cultures. For in vivo tests, we matched donor strains of cells with corresponding recipient mice (DC2.4 with C57BL / 6J for example). Primary BM GM-CSF / IL-4 cultures (BM-DC / MΦ): Primary bone marrow cells were extracted from wildtype mice (C57BL / 6J) after disinfection with 70%ethanol. The femurs and tibiae were dissected and soaked in HBSS (Hank's Balanced Salt Solution, Gibco) or RPMI-1640 medium (ATCC) supplemented with 1% FBS (Gibco). Both ends of the bone were cut open with surgical scissors, and a 25G needle (with a 20-mL syringe) was inserted into the bone cavity to rinse the BM cells out of the femur, whereas a 27G needle was used for the tibia. A total volume of 20 mL complete RPMI-1640 medium was used to slowly (dropwise) flush out BM cells from each femur and a total volume of 10mL was used for each tibia. The cell suspension was passed through 70 µm cell strainer and centrifuged at 180X g for 10 min. The cell pellet was resuspended with ice cold eBioscience red blood cell (RBC) lysis buffer and incubated on ice for 5 minutes to lyse RBCs. Following a second centrifugation, the supernatant was discarded, and the pelleted cells were rinsed with complete medium and collected. 10 × 106collected cells were cultured in each tissue culture-treated T25 flask in 5 ml of complete medium (ATCC RPMI-1640 supplemented with 2-mercaptoethanol (Invitrogen), 10% fetal bovine serum (Gibco), Pen-Strep, murine GM-CSF (20 ng / ml, Peprotech) and IL-4 (5 ng / ml, Peprotech). Half of the medium was removed on day 2 and replaced by fresh warm medium supplemented with 40 ng / ml GMCSF and 10 ng / ml IL-4. The culture medium was entirely discarded on day 3 and replaced by fresh warm medium with 20 ng / ml GM-CSF and 5 ng / ml IL-4. On day 6, all cells in the culture including adherent, suspension, and loosely attached cells, harvested by gentle washing with warm DPBS, were pooled and used as the source of leukocyte EVs.

[0114] EV production: Donor cells underwent transfection, followed by EV isolation and characterization. Thorough titration and time-lapse experiments optimized EV production. Transfection methods: Stable cell lines were transfected via Lipofectamine LTX with PLUS reagent DNA or lipofectamine mMessenger Max RNA transfection. Liposome-based RNA transfection (lipofectamine mMessenger Max) was used to transfect primary cells. Monitoring transfection efficacy: In addition to stabilizing Arc VLVs, A5U-GFP also served as a fluorescent reporter to closely track transfection efficacy within donor cells. To minimize background in the control, rigorous DNase and RNase treatments were essential. Optimal production time and consistency: Depending on donor cell confluency, the time to achieve peak transfection efficacy and thus EV production fluctuate. Given the inherent variability in primary cells across different batches, we utilized live cell imaging to monitor GFP expression in donor cells. EVs were produced in a serum-free OptiMEM culture medium or Macrophage Serum Free Medium toenhance the yield of production. EVs were freshly prepared before both in vivo and in vitro experiments, as long-term storage was found to reduce their integrity and functionality.

[0115] Preparation of tumor EVs: B16-F10 melanoma cell culture: B16-F10 cells were cultured in DMEM supplemented with 10% FBS. EV labeling: B16 / F10 cell derived EVs were labeled with a plasma membrane dye, Cell Mask Deep Red (CMDR), for tracking purposes. 1:5000 of the dye was added to 1mL of EV samples, for a half-an-hour incubation at room temperature covered from light. A DPBS sample group was always included as mock-staining negative control that undergoes the same staining and washing steps. DPBS washes using ultrafiltration were applied to remove free dyes until the DPBS control washed clear (typically five to six washes).

[0116] Fluorescent NTA: The NanoSight NS300 detects nanovesicles with stable fluorophores using a 488 nm or 532 nm laser. We apply green and red filters to spot fluorescent signals. A negative control helps adjust the camera to exclude non-fluorescent signals. In fluorescent mode, individual particle movement helps determine particle size and concentration, as larger particles move slower than smaller ones. In light scatter mode, we measure the total particle count. We use specific fluorescent labels to identify different types of EVs, including general EV markers, Arc, or plasma membrane dyes.

[0117] Mouse experiments: Mice were housed at Cornell University's CARE facility. All experiments adhered to Cornell's IACUC protocol (#2020-0037 and #2023-0101). Vaccine administration: Four doses of B16+ DEVs were intravenously injected (retro-orbital injection into alternating eyes each week) into wild-type C57BL / 6J mice (>=8-week-old).

[0118] Microscopy: Epifluorescence live-cell imaging: Plated cells were imaged with a Cytation 7 imager (BioTek) during various stages. Using Gen5 software, DAPI staining helped count and assess cell brightness compared to controls. Each test involved four replicates, capturing around 20,000 cells. EVs were diluted for precision, and DNA / RNA transfection was optimized using live-cell imagery. Confocal microscopy: Confocal Laser Scanning Microscopy (CLSM) was used for high-resolution imaging. We utilized a Zeiss LSM 800 confocal scanning laser microscope, equipped with both 5X and 20X air objectives, a 10X water objective, and a 60X oil objective, to acquire images. For data analysis, we used ZEISS ZEN (blue) and ImageJ. ICC and IHC: Immunocytochemistry (ICC) and immunohistochemistry (IHC) were used to visualize specific protein markers in EVs, cells, and tissues. Samples were either PFA-fixed cells, thincryo-sections, or thick tissue sections. We employed antibodies like NeuN-AlexaFluor647 and GFP-AlexaFluor488, among others. Stained samples were preserved with ProLong™ Gold Antifade Mountant with DAPI for thick sections and PVA DABCO for thin ones. HCR FISH: We used the hybridization chain reaction fluorescent in situ hybridization (HCR FISH) technique to visualize specific mRNAs within our engineered EVs and cells. We created DNA probes matching the target mRNA sequences and labelled them with fluorescent markers. After treating the EV, cell, and tissue samples with these probes, fluorescence imaging was applied to analyse the distribution of specific mRNAs within the samples. IVIS (in vivo imaging system) spectrum: Before imaging organs with the Perkin Elmer IVIS machine, we do a trans-cardiac perfusion. Due to strong autofluorescence from whole organs, we use negative controls for adjustment. Data analysis used Aura and ImageJ.

[0119] Tumor measurement and imaging: Manual tumor size measurement: Tumor measurements commenced five days after the injection of B16-F10 melanoma cells. Measurements were continued daily thereafter to capture the progressive changes in tumor size throughout the experimental period, using digital calipers. To minimize bias and ensure the reliability of data, tumor measurements were performed in a double-blinded manner by a group of five researchers (independent measurements). This approach involved concealing the treatment groups from the individuals performing the measurements, thereby preventing potential observer bias. Micro-CT scan: Micro-computed tomography (micro-CT) scans were conducted to enable non-invasive and high-resolution imaging of tumor growth in live animals. Prior to scanning, animals were anesthetized to minimize motion artifacts during image acquisition. Micro-CT scans were performed using specialized imaging equipment, which generated cross-sectional images of the entire body, including tumor-bearing regions. The acquired micro-CT images were subjected to detailed analysis using dedicated imaging software. This software allowed for the precise delineation and measurement of tumor volumes based on differences in tissue density. Tumor volumes were quantified by outlining the tumor boundaries on each cross-sectional image and summing the volumes across all slices. This rigorous analysis facilitated accurate assessment of tumor growth kinetics and treatment efficacy over time.

[0120] Analysis of immune response: Cell collection: Following treatment, mice were euthanized, and spleens and lymph nodes were collected aseptically. Splenocytes were isolated by mechanically disrupting the spleen tissue and passing it through a cell strainer (70 μm) toobtain a single-cell suspension. Red blood cells were lysed using the RBC lysis buffer, and the remaining splenocytes were washed and resuspended in optiMEM or flow cytometry buffer for downstream analysis. Lymphocytes were harvested from lymph nodes by mechanical disruption and filtration through cell strainers (70 μm and then 40 μm) to obtain a single-cell suspension. The cells were then washed and resuspended in a suitable buffer for subsequent analysis. Flow cytometry: Prior to analysis, cells were stained with a panel of fluorescently labeled antibodies targeting specific cell surface markers. In addition to CD45 and live / dead stain, our panel included antibodies against CD3, CD4, and CD8, which are commonly used to identify T cell subsets. Additionally, other markers relevant to T cell proliferation and activation were included in the staining panel to provide comprehensive characterization of the immune response, including CD44, CD62L, INFgamma. Stained cells were analyzed using a flow cytometer (Novocyte quanteon 4025), which detects and quantifies the fluorescence emitted by individual cells as they pass through a laser beam. Data acquisition was performed using specialized Novocyte software, with the data analyzed using FlowJo to categorize the cell populations.

[0121] ELISA: At time points of 3, 4, 6, 9, and 13 weeks after the first dose, blood samples were collected by completing cheek blood draws. These samples were analyzed using enzyme-linked immunosorbent assays (ELISAs) to determine the protein levels of relevant antibodies (IgG, IgM, and IgA). This involves coating 96 well plates with ova protein, blocking them with a blocking solution (SuperBlock), adding samples at various concentrations, adding secondary antibody, developing the plates using TMB substrate, stopping the reaction using sulfuric acid, and imaging the plates on a Cytation imaging machine.

[0122] qPCR: Total RNA was extracted from harvested cells using a Trizol / BCP. cDNA synthesis: 1-2 μg of total RNA was then reverse transcribed into cDNA using Applied Biosystem High-Capacity cDNA Reverse Transcription Kit. Primers: Resulting cDNA was prepared for qPCR using PowerUp SYBRgreen Master Mix (Thermo Fisher Scientific) in a 96-well plate with primers against various cytokines and virus-like components. The specificity of primers was confirmed by a melt curve analysis. Thermal cycling conditions: The qPCR machine was programmed with an initial denaturation step, followed by 40 cycles of denaturation, annealing, and extension. Data analysis using the standard curve method: Expression differences were gauged using the standard curve method. A standard DNA sample was diluted and analysed. From its linear equation, Ct values of test samples were determined and transformed to match thestandard sample's dilution. Differences were determined by comparing the test's fold-change to the average control values. Data analysis using the ΔCt method: Ct values for samples were noted for SynA and a housekeeping gene. The difference (ΔCt) between these values was calculated for each sample. For comparing SynA expression in LPS-treated versus untreated cells, the ΔΔCt method was used. Fold change was derived from the formula 2^(-ΔΔCt). This data was then analyzed for statistical significance.

[0123] ELISpot assay: Following the booster dose administration, an Enzyme-Linked Immunospot (ELISpot) assay was performed to assess T cell activation and memory response elicited by the treatment. This highly sensitive assay allows for the detection and enumeration of individual cytokine-secreting cells at the single-cell level.96-well plates were coated with antibodies specific to IFN-γ. Treated cells were then added to the wells and incubated under conditions conducive to cytokine secretion (Ova or B16 / F10 EV coincubation). Upon cytokine secretion by activated T cells, cytokine molecules were captured by the immobilized antibodies, resulting in the formation of visible spots at the locations of cytokine-secreting cells. After incubation, the plates were washed to remove unbound cells and cytokines, followed by the addition of detection antibodies and a substrate solution. This led to the development of colored spots corresponding to individual cytokine-secreting cells. The number of spots in each well, representing the frequency of cytokine-secreting cells, was quantified using an automated ELISpot reader. The magnitude and quality of T cell responses were assessed based on the frequency and distribution of cytokine-secreting cells. A higher number of spots indicated a more robust T cell response, while the pattern of cytokine secretion provided insights into the functionality and memory status of T cells.

[0124] Assessment of the interaction between activated splenocytes and cancer cells: BMDCs (bone marrow dendritic cells) were isolated from WT and Arc Arc KO mice. One group of the WT BMDCs were transfected with rArc- and rA5U- plasmids using lipofectamine RNA transfection reagent. The (1) transfected Arc-overexpressing BMDCs, (2) non-transfected WT BMDCs (also containing endogenous mArc), along with (3) Arc KO BMDCs, were then cultured with B16 / F10 melanoma-derived EVs, which served as melanoma antigens for DEV vaccine preparation. C57BL / 6 mice were injected with DEVs of these three groups. Eight days post- vaccination, splenocytes were collected from the vaccinated mice and labeled with CFSE dye for tracking purposes. The labeled splenocytes were co-incubated with B16 / F10 melanoma cells,pre-stained with NucSpot 650 dye. After 24 hours of co-incubation, the supernatant medium containing suspension splenocytes was removed, B16 / F10 cells were washed thoroughly, and fluorescence imaging was performed to assess the interaction between splenocytes and melanoma cells.

[0125] Mass spectrometry analysis: Protein Isolation: Proteins associated with Arc DEVs were isolated from the culture supernatant of genetically modified DCs overexpressing the Arc protein. The culture supernatant was collected and subjected to centrifugation to remove cellular debris, followed by TFF and ultrafiltration to isolate and concentrate the EVs. The isolated EVs were lysed using an IP lysis buffer containing detergents and protease inhibitors. This lysis step involved one freeze-thaw cycle and vortexes while incubating on ice to disrupt the EV membrane, releasing the protein cargo into solution. The lysate was then centrifuged to remove insoluble debris, and the supernatant containing the solubilized proteins was collected for downstream processing. The concentration of solubilized proteins was determined using a protein quantification assay (Pierce 660). This step ensured that equal amounts of protein were loaded onto the subsequent steps of the analysis, enabling accurate and reproducible results. To prepare the proteins for electrophoresis, the solubilized protein sample was denatured and reduced. Denaturation involved heating the sample in the presence of denaturing agents sodium dodecyl sulfate (SDS), which disrupted the protein's secondary and tertiary structures, while reduction involved the addition of reducing agents (5% beta-mercaptoethanol) to break disulfide bonds.

[0126] SDS-PAGE Electrophoresis: The denatured and reduced proteins were separated by size using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein sample was loaded into wells of an acrylamide gel and subjected to an electric field, causing the proteins to migrate through the gel matrix based on their molecular weight. This step allowed for the separation of proteins according to their size. Following electrophoresis, the protein bands were visualized using staining methods (SimplyBlue or silver staining). These staining techniques allowed for the detection of protein bands within the gel, which appeared as distinct bands against a clear background. In-gel trypsin digestion: The gel bands from the lanes of interest were excised, cut into ~2 mm cubes and subjected to in-gel digestion. The excised gel pieces were washed / incubated at room temperature consecutively with 150-400μL deionized water for 5 minutes, followed by 150-400 μl 50mM ammonium bicarbonate in water / 50%acetonitrile (ACN) for 10 minutes and finally 150-400µl 100% ACN for 5 minutes. The dehydrated gel pieces were dried in a speed vacuum (SpeedVac SC110 Thermo Savant, Milford, MA) and reduced with 50-250μL of 10mM DTT (dithiothreitol) (w / v) in 100mM ammonium bicarbonate in water for 1 hour at 60°C, then alkylated by adding 50-250μL of 55mM iodoacetamide in 100mM ammonium bicarbonate (w / v) and incubation at room temperature, in the dark, for 45 minutes. Wash steps were repeated as described above. The gel pieces were dried in a speed vacuum and rehydrated with 40 - 120 μl trypsin (Promega Sequencing Grade) at 10ng / μl (w / v) in 50mM ammonium bicarbonate / 10% ACN on ice for 20 minutes, topped with 10-50 μl 50 mM ammonium bicarbonate in water, and incubated at 37°C for 16 hours. The digestion was stopped by addition of 50-200 μl 2% formic acid (FA) in water, incubated at room temperature for 10 minutes and the supernatant transferred to a clean polypropylene low-bind microfuge tube. The gel pieces were further extracted twice by adding 100-400μl of 50% ACN / 5% FA and vortexing at 1500 rpm for 10 minutes followed by sonication for 5 minutes, and once by adding 100-400μl of 90% ACN / 5% FA with incubation at room temperature for 5 minutes. All supernatants were combined in the corresponding microfuge tube, dried in a speed vacuum, and redried from 100 μl water. The final sample was reconstituted in 2% ACN / 0.5% FA and filtered through a 0.22µm cellulose acetate spin filter (Corning Costar Spin-X) prior to nanoLC-MS / MS analysis. Protein identification by nano LC / MS / MS analysis: The analysis was carried out using an Orbitrap FusionTMTribridTM(Thermo-Fisher Scientific, San Jose, CA) mass spectrometer equipped with a nanospray Flex Ion Source, and coupled with a Dionex UltiMate 3000 RSLCnano system (Thermo, Sunnyvale, CA). The peptide samples (10 μL) were injected onto a PepMap C-18 RP viper trapping column (5 μm, 100 µm i.d x 20 mm) at 20 μL / min flow rate for rapid sample loading and then separated on a PepMap C-18 RP nano column (2 μm, 75 µm x 25 cm) at 35 °C. The tryptic peptides were eluted in a 90-min gradient of 5% to 35% ACN in 0.1% formic acid at 300 nL / min, followed by an 8-min ramping to 90% ACN-0.1% FA and an 8-min hold at 90% ACN-0.1% FA. The column was re-equilibrated with 0.1% FA for 25 min prior to the next run. The Orbitrap Fusion was operated in positive ion mode with spray voltage set at 1.2 kV and source temperature at 275°C. External calibration for FT, IT and quadrupole mass analyzers was performed. In data-dependent acquisition (DDA) analysis, the instrument was operated using FT mass analyzer in MS scan to select precursor ions followed by 3 second “Top Speed” data-dependent CID ion trap MS / MS scans at 1.6 m / z quadrupole isolation forprecursor peptides with multiple charged ions above a threshold ion count of 10,000 and normalized collision energy of 30%. MS survey scans at a resolving power of 120,000 (fwhm at m / z 200), for the mass range of m / z 300-1600. Dynamic exclusion parameters were set at 50 s of exclusion duration with ±10 ppm exclusion mass width. All data were acquired under Xcalibur 4.4 operation software (Thermo-Fisher Scientific). Data analysis: The DDA raw files with MS and MS / MS were subjected to database searches using Proteome Discoverer (PD) 2.4 software (Thermo Fisher Scientific, Bremen, Germany) with the Sequest HT algorithm. The PD 2.4 processing workflow containing an additional node of Minora Feature Detector for precursor ion-based quantification was used for protein identification and relative quantitation of identified peptides and their modified forms. The database search was conducted against Mus musculus NCBI ref database which contains 28230 sequences and the EVs database with added chicken ovalbumin customer shared with us which contains 74 sequences. The peptide precursor tolerance was set to 10 ppm and fragment ion tolerance was set to 0.6 Da. Oxidation of M, deamidation of N and Q were specified as dynamic modifications of amino acid residues; protein N-terminal acetylation, M-loss and M-loss plus acetylation were set as a variable modification; carbamidomethyl C was specified as a static modification. Only high confidence peptides defined by Sequest HT with a 1% FDR by Percolator were considered for confident peptide identification. Relative quantitation of identified proteins between the paired groups was determined by the Label Free Quantitation (LFQ) workflow in PD 2.4. After retention time alignment for each of the identified peptides across samples, the precursor abundance intensity for each peptide identified by MS / MS in each sample were automatically determined and their unique and razor peptides for each protein in each sample were summed and used for calculating the protein abundance by PD 2.5 software.

[0127] Melanoma LC-MS / MS Analysis: A NanoElute LC system coupled to a timsTOF Pro (Bruker Daltonics, Germany) via a CaptiveSpray source was employed for the analysis. Samples (100 ng) were injected onto an in-house packed column (75 mm x 15 cm, 1.9 µm ReproSil-Pur C18 particles, Dr. Maisch GmbH, Germany) maintained at 40 °C. The mobile phases consisted of buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile). A 21- minute gradient was applied, starting with 2% buffer B and increasing to 30% over 17.8 minutes, followed by a rapid increase to 95% buffer B by 18.3 minutes, and held for an additional 2.4 minutes. Mass spectrometry data were acquired using a data-independent acquisition parallelaccumulation-serial fragmentation (diaPASEF) method with 16 m / z and ion mobility windows. The electrospray voltage was set at 1.5 kV, with the ion transfer tube maintained at 180 °C. Full MS scans were conducted over an m / z range of 100–1700. Collision energy was linearly ramped from 20 eV at 1 / K0 = 0.6 V·s / cm² to 59 eV at 1 / K0 = 1.6 V·s / cm². Data were processed using DIANN version 1.8 software (Demichev et al., 2020), with default settings for peptide and protein identification and quantification from diaPASEF data. An in-house spectral library was utilized, generated from the UniProt-SwissProt Homo sapiens database (Taxon ID 9606, downloaded on 01 / 20 / 2023, containing 20,404 entries). Cysteine carbamidomethylation was specified as a fixed modification, while methionine oxidation and acetylation were considered variable modifications. The false discovery rate (FDR) was controlled to be below 1% at both the peptide and protein levels.

[0128] Ethical compliance: All procedures involving animals were reviewed and approved by the institutional animal care and use committee (IACUC), adhering to ethical guidelines.

[0129] Statistical analysis: Experimental data are shown as mean ± SD using dot plots or bar graphs. Statistical significance was gauged using the Student's T-test or two-way ANOVA, with GraphPad Prism 9 Software. A p-value < 0.05 was considered significant. In vitro: Multiple areas were chosen randomly from four wells for imaging. Each view contained tens of thousands of cells, analyzed for parameters like fluorescence intensity. Background was corrected using control values. Experiments were repeated at least three times for consistency. In vivo: A power analysis determined a group size of five mice would provide adequate statistical power for the study. The sCargo+ / Arc‒ group showed slight elevations in neuronal cargo mRNA levels compared to other controls. Across experiments, a p-value < 0.05 indicated statistical significance.SEQUENCES: SEQ ID NO: 1 Human 5’ UTR arc sequence (NCBI Gene ID #23237): Nucleic Acid Sequence Homo sapiens GCAGAGCTCGGGCGCGGCGTCCTCCCTCCGCAGCAGCCGAGCCGGACCTGCCTCCC CGGGCGTGCTCCGCCGGCCCCGCCGCCGGCCCGCAGCGACAGACAGGCGCTCCCCG CAGCTCCGCACGGGACCCAGGCCGCCGGACCCCAGCGCCGGACCACCCTCTGTCCG CCCCGAGGAGTTTGCCGCCTGCCGGAGCACCTGCGCACAG SEQ ID NO: 2 Human Arc amino (NCBI Gene ID #23237) Amino Acid Homo sapiens MELDHRTSGGLHAYPGPRGGQVAKPNVILQIGKCRAEMLEHVRRTHRHLLAEVSKQVE RELKGLHRSVGKLESNLDGYVPTSDSQRWKKSIKACLCRCQETIANLERWVKREMHVW REVFYRLERWADRLESTGGKYPVGSESARHTVSVGVGGPESYCHEADGYDYTVSPYAI TPPPAAGELPGQEPAEAQQYQPWVPGEDGQPSPGVDTQIFEDPREFLSHLEEYLRQVGG SEEYWLSQIQNHMNGPAKKWWEFKQGSVKNWVEFKKEFLQYSEGTLSREAIQRELDLP QKQGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLRHPLPKTLEQLIQ RGMEVQDDLEQAAEPAGPHLPVEDEAETLTPAPNSESVASDRTQPE SEQ ID NO: 3 Mouse Arc (NCBI Gene ID #11838) Mus musculus Amino Acid MELDHMTTGGLHAYPAPRGGPAAKPNVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVE RELKGLHRSVGKLENNLDGYVPTGDSQRWKKSIKACLCRCQETIANLERWVKREMHV WREVFYRLERWADRLESMGGKYPVGSEPARHTVSVGVGGPEPYCQEADGYDYTVSPY AITPPPAAGELPEQESVEAQQYQSWGPGEDGQPSPGVDTQIFEDPREFLSHLEEYLRQVG GSEEYWLSQIQNHMNGPAKKWWEFKQGSVKNWVEFKKEFLQYSEGTLSREAIQRELELPQKQGEPLDQFLWRKRDLYQTLYVDAEEEEIIQYVVGTLQPKLKRFLRHPLPKTLEQLIQ RGMEVQDGLEQAAEPSGTPLPTEDETEALTPALTSESVASDRTQPE SEQ ID NO: 4 Rat Arc (NCBI Gene ID #54323) Rattus norvegicus Amino Acid MELDHMTTGGLHAYPAPRGGPAAKPNVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVE RELKGLHRSVGKLENNLDGYVPTGDSQRWKKSIKACLCRCQETIANLERWVKREMHV WREVFYRLERWADRLESMGGKYPVGSEPARHTVSVGVGGPEPYCQEADGYDYTVSPY AITPPPAAGELPEQESVGAQQYQSWVPGEDGQPSPGVDTQIFEDPREFLSHLEEYLRQVG GSEEYWLSQIQNHMNGPAKKWWEFKQGSVKNWVEFKKEFLQYSEGTLSREAIQRELDL PQKQGEPLDQFLWRKRDLYQTLYVDAEEEEIIQYVVGTLQPKFKRFLRHPLPKTLEQLIQ RGMEVQDGLEQAAEPSVTPLPTEDETEALTPALTSESVASDRTQPE SEQ ID NO: 5 Drosophila Arc (NCBI Gene ID #36595) Drosophila melanogaster Amino Acid MAQLTQMTNEQLRELIEAVRAAAVGAAGSAAAAGGADASRGKGNFSACTHSFGGTRD HDVVEEFIGNIETYKDVEGISDENALKGISLLFYGMASTWWQGVRKEATTWKEAIALIR EHFSPTKPAYQIYMEFFQNKQDDHDPIDTFVIQKRALLAQLPSGRHDEETELDLLFGLLNI KYRKHISRHSVHTFKDLLEQGRIIEHNNQEDEEQLATAKNTRGSKRTTRCTYCSFRGHTF DNCRKRQKDRQEEQHEE SEQ ID NO: 6 Human Retrotransposon-derived PEG10 Homo sapiens Amino Acid MTERRRDELSEEINNLREKVMKQSEENNNLQSQVQKLTEENTTLREQVEPTPEDEDDDI ELRGAAAAAAPPPPIEEECPEDLPEKFDGNPDMLAPFMAQCQIFMEKSTRDFSVDRVRVCFVTSMMTGRAARWASAKLERSHYLMHNYPAFMMEMKHVFEDPQRREVAKRKIRRL RQGMGSVIDYSNAFQMIAQDLDWNEPALIDQYHEGLSDHIQEELSHLEVAKSLSALIGQ CIHIERRLARAAAARKPRSPPRALVLPHIASHHQVDPTEPVGGARMRLTQEEKERRRKL NLCLYCGTGGHYADNCPAKASKSSPAGKLPGPAVEGPSATGPEIIRSPQDDASSPHLQV MLQIHLPGRHTLFVRAMIDSGASGNFIDHEYVAQNGIPLRIKDWPILVEAIDGRPIASGPV VHETHDLIVDLGDHREVLSFDVTQSPFFPVVLGVRWLSTHDPNITWSTRSIVFDSEYCRY HCRMYSPIPPSLPPPAPQPPLYYPVDGYRVYQPVRYYYVQNVYTPVDEHVYPDHRLVDP HIEMIPGAHSIPSGHVYSLSEPEMAALRDFVARNVKDGLITPTIAPNGAQVLQVKRGWK LQVSYDCRAPNNFTIQNQYPRLSIPNLEDQAHLATYTEFVPQIPGYQTYPTYAAYPTYPV GFAWYPVGRDGQGRSLYVPVMITWNPHWYRQPPVPQYPPPQPPPPPPPPPPPPSYSTL SEQ ID NO: 7 GAK5_Human Endogenous retrovirus group K member 5 Gag Homo sapiens Amino Acid MGQTKSKTKSKYASYLSFIKILLKRGGVRVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGEELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTKEDSVSVSDAPGSCVIDCNEKTG RKSQKETESLHCEYVTEPVMAQSTQNVDYNQLQGVIYPETLKLEGKGPELVGPSESKPR GPSPLPAGQVPVTLQPQTQVKENKTQPPVAYQYWPPAELQYLPPPESQYGYPGMPPALQ GRAPYPQPPTVRLNPTASRSGQGGTLHAVIDEARKQGDLEAWRFLVILQLVQAGEETQV GAPARAETRCEPFTMKMLKDIKEGVKQYGSNSPYIRTLLDSIAHGNRLTPYDWESLAKS SLSSSQYLQFKTWWIDGVQEQVRKNQATKPTVNIDADQLLGTGPNWSTINQQSVMQNE AIEQVRAICLRAWGKIQDPGTAFPINSIRQGSKEPYPDFVARLQDAAQKSITDDNARKVI VELMAYENANPECQSAIKPLKGKVPAGVDVITEYVKACDGIGGAMHKAMLMAQAMR GLTLGGQVRTFGKKCYNCGQIGHLKRSCPVLNKQNIINQAITAKNKKPSGLCPKCGKGK HWANQCHSKFDKDGQPLSGNRKRGQPQAPQQTGAFPVQLFVPQGFQGQQPLQKIPPLQ GVSQLQQSNSCPAPQQAAPQ SEQ ID NO: 8 GAK6_Human Endogenous retrovirus group K member 6 Gag Homo sapiensAmino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSVSVSDAPGSCIIDCNENTRK KSQKETEGLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGPELVGPSESKPRG TSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAPQ GRAPYPQPPTRRLNPTAPPSRQGSKLHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQEG EPPTVEARYKSFSIKKLKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKSSL SPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAIE QVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARKVIV ELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAITGVV LGGQVRTFGRKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKHWA SQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLP QYNNCPPPQAAVQQ SEQ ID NO: 9 GAK7_Human Endogenous retrovirus group K member 7 Gag Homo sapiens Amino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEKDSVSVSDALGSCIIDCNENTR KKSQKETEGLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGPELVGPSESKPR GTSHLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAP QGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQE GEPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKS SLSPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNE AIEQVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARK VIVELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAIT GVVLGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGK HWASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQEQQPPLSQVFQGI SQLPQYNNCPPPQAAVQQSEQ ID NO: 10 GAK8_Human Endogenous retrovirus group K member 8 Gag Homo sapiens Amino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSISVSDAPGSCLIDCNENTRK KSQKETESLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGPELVGPSESKPRG TSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAPQ GREPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQEG EPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKSS LSPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAI EQVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARKVI VELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAITGV VLGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKHW ASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQDNNPHCPKCFRE SEQ ID NO: 11 GAK9_Human Endogenous retrovirus group K member 9 Gag Homo sapiens Amino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSISVSDAPGSGIIDCNEKTRK KSQKETESLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGPELVGPSESKPRG TSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAPQ GRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQEG EPPTVEARYKSFSIKILKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKSSL SPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAIE QVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARKVIV ELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAITGVV LGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKHWASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLP QYNNCPPPQVAVQQ SEQ ID NO: 12 GAK19_Human Endogenous retrovirus group K member 19 Gag Homo sapiens Amino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSVSVSDAPGSCIIDCNENTRK KSQKETESLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKVPELVGPSESKPRG TSRLPAGQVPVTLQPQTQVKENKTQPPVAYQYWPPAELQYRPPLESQYGYPGMPPAPQ GRAPYPQPPTRRLNPTAPPSRRGSELHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQEG EPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKSS LSPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAI EQVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIAIEKARKVIV ELMAYENPNPECQSAIKPLKGKVPAGSDVISEYVKACDGMGGAMHKAMLMAQAITGV VLGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKHW ASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPHGFQGQQPPLSQVFQGISQL PQYNNCPPPQAAVQQ SEQ ID NO: 13 GAK21_Human Endogenous retrovirus group K member 21 Gag Homo sapiens Amino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSISVSDAPGSCIIDCNENTRK KSQKETEGLHCEYAAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGPELVGPSESKPRG TSPLPAGQVPVTLQPQTQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAPQ GRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVMLEPMPPGEGAQEG EPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKSS LLPSQFLQFKTWWIDGVQEQVQRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAIEQVRAICLRAWEKIQDPGSTCPSFNTVRQSSKEPYPDFVARLQDVAQKSIADEKARKVIV ELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAITGVV LGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKHWA SQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLP QYNNCPPPQAAVQQ SEQ ID NO: 14 GAK24_Human Endogenous retrovirus group K member 24 Gag Homo sapiens Amino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSVSVSDAPGSCLIDCNEKTR KKSQKETESLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGPELVGPSESKPR GTSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAP QGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQE GEPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMRTLLDSIAYGHRLIPYDWEILAKS SLSPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNE AIEQVRAICLRAWEKIQDPGSACPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARK VIVELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAIT GVVLGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGK HWASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGI SQLPQYNNCPLPQAAVQQ SEQ ID NO: 15 GAK113_Human Endogenous retrovirus group K member 113 Gag Homo sapiens Amino Acid MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDW KRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSVSVSDAPGSCIIDCNEKTRK KSQKETESLHCEYVAEPVMAQSTQNADYNQLQEVIYPETLKLEGKGPELMGPSESKPRG TSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYQPPPESQYGYPGMPPAPQGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVTLELMPPGEGAQEG EPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKSS LSPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAI EQVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARKVI VELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGMGGAMHKAMLMAQAITG VVLGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKH WASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGIS QLPQYNNCPPPQAAVQQ SEQ ID NO: 16 ENK9_Human Endogenous retrovirus group K member 9 Env Homo sapiens Amino Acid MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLT QLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTNWAYVPFPPLIRA VTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPICLGRAPGCLMPA VQNWLVEVPIVSPICRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKES KNTEVLVWEECVANSAVILQNNEFGTIIDWTPQGQFYHNCSGQTQSCPSAQVSPAVDSD LTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIISPVSGPEHPELWRLTVASHHIRIWSG NQTLETRDRKPFYTVDLNSSLTLPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCID STFNWQHRILLVRAREGVWIPVSMDRPWEASPSIHILTEVLKGVLNRSKRFIFTLIAVIMG LIAVTATAAVAGVALHSSVQSVNFVNDGQKNSTRLWNSQSSIDQKLANQINDLRQTVI WMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISK LKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLV CRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV SEQ ID NO: 17 ENK18_Human Endogenous retrovirus group K member 18 Env Homo sapiens Amino AcidMVTPVTWMDNPIEVYVNDSVWVPGPTDDRCPAKPEEEGMMINISIGYHYPPICLGRAPG CLMPAVQNWLVEVPTVSPNSRFTYHMVSGMSLRPRVNCLQDFSYQRSLKFRPKGKTCP KEIPKGSKNTEVLVWEECVANSVVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQV SPAVDSDLTESLDKHKHKKLQSFYLWEWEEKGISTPRPKIISPVSGPEHPELWRLTVASH HIRIWSGNQTLETRYRKPFYTIDLNSILTVPLQSCVKPPYMLVVGNIVIKPASQTITCENCR LFTCIDSTFNWQHRILLVRAREGMWIPVSTDRPWEASPSIHILTEILKGVLNRSKRFIFTLI AVIMGLIAVTATAAVAGVALHSSVQSVNFVNYWQKNSTRLWNSQSSIDQKLASQINDL RQTVIWMGDRLMTLEHHFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNL TLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWIKTIRSTMIINLILIVVCL FCLLLVCRCTQQLRRDSDIENGP SEQ ID NO: 18 ENK19_Human Endogenous retrovirus group K member 19 Env Homo sapiens Amino Acid MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLT QLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPLIRA VTWMDNPIEVYVNDSVWVPGPTDDHCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMP AVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSFKFRPKGKPCPKEIPK ESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVD SDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIISPVSGPEHPELWRLTVASHHIRIWS GNQTLETRDRKPFYTVDLNSSVTVPLQSCIKPPYMLVVGNIVIKPDSQTITCENCRLLTCI DSTFNWQHRILLVRAREGVWIPVSMDRPWETSPSIHTLTEVLKGVLNRSKRFIFTLIAVI MGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKLANQINDLRQT VIWMGDRLMSLEHRFQLQCDWNTSDFSITPQIYNESEHHWDMVRRHLQGREDNLTLDI SKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLL LVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV SEQ ID NO: 19 ENK21_Human Endogenous retrovirus group K member 21 Env Homo sapiensAmino Acid MHPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEQMKLPSTKKAEPPTWAQLKKLTQ LATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTNWAYVPFPPLIRAV TWMDNPIEVYVNDSVWVHGPIDDRCPAKPEEEGMMINISIGYHYPPICLGRAPGCLMPA VQNWLVEVPTVSPISRFTYNMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKES KNTEVLVWEECVANSVVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSD LTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIISPVSGPEHPELWRLTVASHHIRIWSG NQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCID STFNWQHRILLVRAREGVWIPVSMDRPWEASPSIHILTEVLKGVLNRSKRFIFTLIAVIMG LIAVTAMAAVAGVALHSFVQSVNFVNDWQKNSTRLWNSQSSIDQKLANQINDLRQTVI WMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISK LKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLV CRCTQQLRRDSDHRERAMMTMVVLSKRKGGNVGKSKRDQIVTVSV SEQ ID NO: 20 VPK10_Human Endogenous retrovirus group K member 10 Pro Homo sapiens Amino Acid WASQVSENRPVCKAIIQGKQFEGLVDTGADVSIIALNQWPKNWPKQKAVTGLVGIGTAS EVYQSMEILHCLGPDNQESTVQPMITSIPLNLWGRDLLQQWGAEITMPAPLYSPTSQKIM TKMGYIPGKGLGKNEDGIKVPVEAKINQEREGIGYPF SEQ ID NO: 21 SYCY1_Human Syncytin-1 Homo sapiens Amino Acid MALPYHIFLFTVLLPSFTLTAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLSKGTPT FTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQTGMSDG GGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLFNTTLTGLH EVSAQNPTNCWICLPLNFRPYVSIPVPEQWNNFSTEINTTSVLVGPLVSNLEITHTSNLTC VKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCLNGSSESMCFLSFLVPPMTIYTEQDLYSYVISKPRNKRVPILPFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDME RVADSLVTLQDQLNSLAAVVLQNRRALDLLTAERGGTCLFLGEECCYYVNQSGIVTEK VKEIRDRIQRRAEELRNTGPWGLLSQWMPWILPFLGPLAAIILLLLFGPCIFNLLVNFVSS RIEAVKLQMEPKMQSKTKIYRRPLDRPASPRSDVNDIKGTPPEEISAAQPLLRPNSAGSS SEQ ID NO: 22 SYCY2_Human Syncytin-2 Homo sapiens Amino Acid MGLLLLVLILTPSLAAYRHPDFPLLEKAQQLLQSTGSPYSTNCWLCTSSSTETPGTAYPA SPREWTSIEAELHISYRWDPNLKGLMRPANSLLSTVKQDFPDIRQKPPIFGPIFTNINLMGI APICVMAKRKNGTNVGTLPSTVCNVTFTVDSNQQTYQTYTHNQFRHQPRFPKPPNITFP QGTLLDKSSRFCQGRPSSCSTRNFWFRPADYNQCLQISNLSSTAEWVLLDQTRNSLFWE NKTKGANQSQTPCVQVLAGMTIATSYLGISAVSEFFGTSLTPLFHFHISTCLKTQGAFYIC GQSIHQCLPSNWTGTCTIGYVTPDIFIAPGNLSLPIPIYGNSPLPRVRRAIHFIPLLAGLGIL AGTGTGIAGITKASLTYSQLSKEIANNIDTMAKALTTMQEQIDSLAAVVLQNRRGLDML TAAQGGICLALDEKCCFWVNQSGKVQDNIRQLLNQASSLRERATQGWLNWEGTWKWF SWVLPLTGPLVSLLLLLLFGPCLLNLITQFVSSRLQAIKLQTNLSAGRHPRNIQESPF SEQ ID NO: 23 ENK6_Human Endogenous retrovirus group K member 6 Env Homo sapiens Amino Acid MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLT QLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPLIRA VTWMDNPTEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYHYPPICLGRAPGCLMP AVQNWLVEVPTVSPICRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPK ESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVD SDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIVSPVSGPEHPELWRLTVASHHIRIW SGNQTLETRDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTC IDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKLANQINDLRQT VIWMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDI SKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLL LVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV SEQ ID NO: 24 ENK7_HumanEndogenous retrovirus group K member 7 Env Homo sapiens Amino Acid MVTPVTWMDNPIEIYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGC LMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKE IPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPA VDSDLTESLDKHKHKKLQSFYPWEWGEKRISTPRPKIVSPVSGPEHPELWRLTVASHHIR IWSGNQTLETRDCKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLL SCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIA VIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKLANQINDLR QTVIWMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTL DISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFC LLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV SEQ ID NO: 25 ENK8_Human Endogenous retrovirus group K member 8 Env Homo sapiens Amino Acid MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLT QLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAVANYTNWAYVPFPPLIRA VTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMP AVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPK ESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVD SDLTESLDKHKHKKLQSFYPWEWGEKRISTPRPKIVSPVSGPEHPELWRLTVASHHIRIW SGNQTLETRDRKPFYTVDLNSSLTLPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVI MGLIAVTATAAVAGVALHSSVQSVNFVNDGQKNSTRLWNSQSSIDQKLANQINDLRQT VIWMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNDSEHHWDMVRRHLQGREDNLTLDI SKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLL LVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV SEQ ID NO: 26 Mouse 5’ UTR arc (NCBI Gene ID #11838): Mus musculus Nucleic Acid AGAGCTCAAGCGAGTTCTCCCGCAGCCGCAGTCTCTGGGCCTCTCTAGCTTCAGCGG CGACGAGCCTGCCACACTCGCTAAGCTCCTCCGGCACCGCACACCTGCCACTGCCGC TGCAGCCGCCGGCTCTGCTCCCTTCCGGCTTCTGCCTCAGAGGAGTTCTTAGCCTGTT CGGAGCCGCAGCACCGACGACCAG SEQ ID NO: 27 Rat 5’ UTR arc (NCBI Gene ID #54323) Rattus norvegicus Nucleic Acid AGTGCTCTGGCGAGTAGTCCTCCCTCAGCCGCAGTCTCTGGGCCTCTTCAGCTTGAG CGGCGGCGAGCCTGCTACACTCGCTAAGCTCCTCCGGCACCGCGCACTTGCCACTGC CACTGCCGCTTCGCGCCCGCTGCAGCCGCCGGCTCTGAATCCTTCTGGCTTCCGCCTC AGAGGAGTTCTTAGCCTGTCCCGAACCGTAACCCCGGCGAGCAG SEQ ID NO: 28 Drosophila 5’ UTR dArc1 (NCBI Gene ID #36595) Drosophila melanogaster Nucleic Acid TGTTCAGTTCAAATCACCGGCCGCATTCGCTACACTGGCTTTGTCCGCCGACTGAACC AAGATTAATTTGATCACCTAACCTCACACAGCAGCGAAASEQ ID NO: 29 Human PEG105' UTR (NCBI Gene ID #23089) Homo sapiens Nucleic Acid CTCCTCGGTGCAACCTATATAAGGCTCACAGTCTGCGCTCCTGGTACACGCGCTTCA ACTTCGGTTGGTGTGTGTCGAAGAAACCTGACTGCGCCCTGAGGAGAACAGCGGAG AAGGTCCACCGAGCCTGGCGAAAGGTCCGCTGAGCGGGCTGTCGTCCGGAGCCACT CCGGGCTGCGGAGCACCCAGTGGAGACCGCGCCTGGCTCAGGTGTGGGACCCCATC CTTCCTGTCTTCGCAGAGGAGTCCTCGCGTGAAATAAGCGGGTTTTGAAAACAAAAA AAAGAAGGAGTGGAAGAGGGGGCCAGGATCCAGGCCTCCATCCCCACAGAAGTGA AGCTACAGCTGGGAGGTCTCCTCCCACCCCAACCGTCACCCTGGGTCCCGACTGCCC ACCTCCTCCTCCTCCCCCTCCCCCCAACAACAACAACAACAACAACTCCAAGCACAC CGGCCATAAGAGTGCGTGTGTCCCCAAC SEQ ID NO: 30 Human RTL15' UTR (NCBI Gene ID #388015) Homo sapiens Nucleic Acid ACACCCCCCCAGCACTCGGCACAGCCTCGCTGAGCAGCCTTCAGCAGCAGGGTGTG GTGGGGAGCCTCGGAGAGTCTGGGGTTCCATCCTGGCCCAGCCTCACACCAGCTGA GAGACGACCGACTGAGCTTCGAGGACAGGAAGCCACCGGCATCACTAAGCCACCGG CATCACTTCATCCCCAGCCTCACGCTTGGGACTGGGCCCGGGGAAGCAAGCAGCGA GGATCGAGTGGCACAGGACGGGAGGAGATCCACTTGAACGCTTGCAGCCAAGGTTC TGATCTCCACGGTCCCAGCTACTGACTGGACGCCATCACAACCTTACCAATCTTCAG AATACACTCCTTTCCATCCGACGAA SEQ ID NO: 31 Human GAK105'UTR (NCBI Gene ID #100616101) Homo sapiens Nucleic Acid GAAAATTTCACATACAACAGGAATTCCTTATAATTCCCAAGGACAGGCCATAGTTGAAAGAACTAATAGAACACTCAAAACTCAATTAGTTAAACAAAAAGAAGGGGGAGAC AGTAAGGAGTGTACCACTCCTCAG SEQ ID NO: 32 HHLA2_Human HERV-H LTR-associating protein 2 Homo sapiens Amino Acid MKAQTALSFFLILITSLSGSQGIFPLAFFIYVPMNEQIVIGRLDEDIILPSSFERGSEVVIHW KYQDSYKVHSYYKGSDHLESQDPRYANRTSLFYNEIQNGNASLFFRRVSLLDEGIYTCY VGTAIQVITNKVVLKVGVFLTPVMKYEKRNTNSFLICSVLSVYPRPIITWKMDNTPISEN NMEETGSLDSFSINSPLNITGSNSSYECTIENSLLKQTWTGRWTMKDGLHKMQSEHVSLS CQPVNDYFSPNQDFKVTWSRMKSGTFSVLAYYLSSSQNTIINESRFSWNKELINQSDFSM NLMDLNLSDSGEYLCNISSDEYTLLTIHTVHVEPSQETASHNKGLWILVPSAILAAFLLIW SVKCCRAQLEARRSRHPADGAQQERCCVPPGERCPSAPDNGEENVPLSGKV SEQ ID NO: 33 MHC-I Ova Peptide Engineered Amino Acid SIINFEKL SEQ ID NO: 34 MHC-II Ova Peptide Engineered Amino Acid ISQAVHAAHAEINEAGR

Claims

WHAT IS CLAIMED IS:

1. An immunogenic composition comprising an engineered extracellular vesicle (EV), the EV comprising: at least one group-specific antigen (Gag) homolog; at least one major histocompatibility complex (MHC) molecule; and at least one antigen of interest; and wherein the EV possesses antigen presenting characteristic.

2. The immunogenic composition of claim 1, wherein the at least one Gag homologue is overexpressed.

3. The immunogenic composition of claim 1 or 2, wherein the EV provides enhanced antigen presentation.

4. The immunogenic composition of any one of claims 1-3, wherein the at least one Gag homologue has a similar protein structure to human Arc.

5. The immunogenic composition of any one of claims 1-4, wherein the Gag homologue is a retroviral Arc homologue.

6. The immunogenic composition of claims 1-5, wherein the at least one Gag homologue is selected from the group consisting of human Arc, mouse Arc, rat Arc, drosophila Arc, PEG10, GAK5, GAK6, GAK7, GAK8, GAK9, GAK19, GAK21, GAK24, and GAK113.

7. The immunogenic composition of any one of the previous claims, further comprising an RNA stabilizer.

8. The immunogenic composition of claim 7, wherein the stabilizer is a 5’ untranslated region (UTR) of a long terminal repeat (LTR) retroviral homologue.

9. The immunogenic composition of claim 8, wherein the 5’ UTR LTR retroviral homologue is selected from the group consisting of: 5’ UTR Arc, PEG105’ UTR, RTL15’ UTR, and GAK105’ UTR.

10. The immunogenic composition of claim 9, wherein the stabilizer is a 5’ UTR Arc sequence.

11. The immunogenic composition of claim 10, wherein the 5’ UTR Arc sequence is selected from the group consisting of: human 5’ UTR Arc, mouse 5’ UTR Arc, rat 5’ UTR Arc, drosophila 5’ UTR dArc, 12. The immunogenic composition of claim 11, wherein the 5’ UTR Arc sequence comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO:

1.

13. The immunogenic composition of any one of the previous claims, wherein the MHC molecule is an MHC-I molecule.

14. The immunogenic composition of any one of claims 1-12, wherein the MHC molecule is an MHC-II molecule.

15. The immunogenic composition of any one of the previous claims, wherein the antigen of interest is selected from the group consisting of: Tumor Associated Antigens, autoimmune disease antigens, neoantigens, and infectious disease antigens.

16. The immunogenic composition of any one of the previous claims, wherein the antigen of interest is an antigen from an extracellular vesicle (AEV) derived from an antigen EV forming cell.

17. The immunogenic composition of claim 16, wherein the antigen EV forming cell is a cancer cell or a cell of precancerous tissue.

18. The immunogenic composition of claim 17, wherein the antigen EV forming cell is an induced pluripotent stem cell.

19. The immunogenic composition of claim 15, wherein the antigen is a peptide fragment of a protein selected from the group consisting of: CXorf61 (Chromosome X Open Reading Frame 61); CBX2 (Chromobox Protein Homologue 2); PLAC1 (Placenta-Specific 1); CLDN6 (Claudin 6); SPANX (Sperm Protein Associated with the Nucleus on the X chromosome); MAGEA3 (Melanoma-Associated Antigen A3); TPTE (Transmembrane Phosphatase with Tensin Homology); ACTL8 (Actin-Like Protein 8); ANKRD30A (Ankyrin Repeat Domain 30A); CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A); MAD2L1 (Mitotic Arrest Deficient 2 Like 1); MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); SUNC1 (Sad1 and UNC84 Domain Containing 1); MAGEA10 (Melanoma-Associated Antigen A10); LRRN1 (Leucine-Rich Repeat Neuronal 1); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto- Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1): 7490; CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); CA125 (Cancer Antigen 125); CA19-9 (Cancer Antigen 19-9); CA15-3 (Cancer Antigen 15-3); CA27.29 (Cancer Antigen 27.29); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); GPC3 (Glypican 3); CTAG1B (Cancer / Testis Antigen 1B); CTAG2 (Cancer / Testis Antigen 2); CTAG3 (Cancer / Testis Antigen 3); CTAG4 (Cancer / Testis Antigen 4); CTAG5 (Cancer / Testis Antigen 5); CTAG6 (Cancer / Testis Antigen 6); CTAG7 (Cancer / Testis Antigen 7); CTAG8 (Cancer / Testis Antigen 8); BAGE (B melanoma Antigen); GAGE1 (G antigen 1); GAGE2 (G antigen 2); GAGE3 (G antigen 3); GAGE4 (G antigen 4); GAGE5 (G antigen 5); GAGE6 (G antigen 6); GAGE7 (G antigen 7); GAGE8 (G antigen 8); PRAME (Preferentially Expressed Antigen in Melanoma); BIRC5 (Baculoviral IAP Repeat Containing 5); MUC16 (Mucin 16, Cell Surface Associated); SPAG9 (Sperm Associated Antigen 9); CAGE1 (Cancer Antigen 1); SSX1 (Synovial Sarcoma, X Breakpoint 1); SSX2 (Synovial Sarcoma, X Breakpoint 2); SSX3 (Synovial Sarcoma, XBreakpoint 3); SSX4 (Synovial Sarcoma, X Breakpoint 4); SSX5 (Synovial Sarcoma, X Breakpoint 5); SSX6 (Synovial Sarcoma, X Breakpoint 6); SSX7 (Synovial Sarcoma, X Breakpoint 7); SSX8 (Synovial Sarcoma, X Breakpoint 8); GAGE9 (G antigen 9); GAGE10 (G antigen 10); GAGE11 (G antigen 11); GAGE12 (G antigen 12); GAGE13 (G antigen 13); GAGE14 (G antigen 14); GAGE15 (G antigen 15); GAGE16 (G antigen 16); GAGE17 (G antigen 17); GAGE18 (G antigen 18); GAGE19 (G antigen 19); GAGE20 (G antigen 20); GAGE21 (G antigen 21); GAGE22 (G antigen 22); GAGE23 (G antigen 23); GAGE24 (G antigen 24); GAGE25 (G antigen 25); GAGE26 (G antigen 26); GAGE27 (G antigen 27); GAGE28 (G antigen 28); GAGE29 (G antigen 29); GAGE30 (G antigen 30); GAGE31 (G antigen 31); GAGE32 (G antigen 32); GAGE33 (G antigen 33); GAGE34 (G antigen 34); GAGE35 (G antigen 35); GAGE36 (G antigen 36); GAGE37 (G antigen 37); GAGE38 (G antigen 38); GAGE39 (G antigen 39); GAGE40 (G antigen 40); GAGE41 (G antigen 41); GAGE42 (G antigen 42); GAGE43 (G antigen 43); GAGE44 (G antigen 44); GAGE45 (G antigen 45); GAGE46 (G antigen 46); GAGE47 (G antigen 47); GAGE48 (G antigen 48); GAGE49 (G antigen 49); GAGE50 (G antigen 50); GAGE51 (G antigen 51); GAGE52 (G antigen 52); GAGE53 (G antigen 53); GAGE54 (G antigen 54); GAGE55 (G antigen 55); Nid1 (P14543); Pdia3 (P30101); Atic (P31939); Ephx1 (P07099); Ppp1cb (P62140); Plec (Q15149); Abcf1 (Q8NE71); Trim25 (Q14258); Ube2z (Q9H832); Ufd1 (Q92890); Srp54 (P61011); Osbpl9 (Q96SU4); Serbp1 (Q8NC51); Pon1 (P27169); Pdk1 (Q15118); Qars1 (P47897); Fgg (P02679); Fkbp4 (Q02790); Map4 (P27816); Usp9x (Q93008); Acsl1 (P33121); Mtrex (P42285); Prdx1 (Q06830); Gpi (P06744); Srpra (P08240); Slc3a2 (P08195); Gsr (P00390); Erp44 (Q9BS26); Epb41 (P11171); Lgals8 (O00214); Hsph1 (Q92598); Vps26a (O75436); Mars1 (P56192); Trim50 (Q86XT4); Snrpe (P62304); Armt1 (Q9H993); Ppip5k2 (O43314); Tbcd (Q9BTW9); Ehd1 (Q9H4M9); Ddi2 (Q5TDH0); Actbl2 (Q562R1); Osbpl9 (Q96SU4); Rps6ka1 (E9PGT3); Snrnp200 (O75643); Eif3l (Q9Y262); Ap2m1 (Q96CW1); Ephx1 (P07099); Clec2d (Q9UHP7); Abhd14b (Q96IU4); H6pd (O95479); Rpl27 (P61353); Lrp1 (Q94CK9); and Itih2 (P19823).

20. The immunogenic composition of claim 19, wherein the antigen is selected from the group consisting of: MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma-Associated Antigen A5); SUNC1 (Sad1 and UNC84 Domain Containing 1); MAGEA10 (Melanoma-Associated Antigen A10); LRRN1 (Leucine-Rich Repeat Neuronal 1); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1); CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); CA125 (Cancer Antigen 125); CA19-9 (Cancer Antigen 19-9); CA15-3 (Cancer Antigen 15-3); CA27.29 (Cancer Antigen 27.29); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY- BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); GPC3 (Glypican 3); CTAG1B (Cancer / Testis Antigen 1B); CTAG2 (Cancer / Testis Antigen 2); CTAG3 (Cancer / Testis Antigen 3); CTAG4 (Cancer / Testis Antigen 4); CTAG5 (Cancer / Testis Antigen 5); CTAG6 (Cancer / Testis Antigen 6); CTAG7 (Cancer / Testis Antigen 7); CTAG8 (Cancer / Testis Antigen 8); and BAGE (B melanoma Antigen)..

21. The immunogenic composition of claim 19, wherein the antigen is selected from the group consisting of: MAGEA4 (Melanoma-Associated Antigen A4); MAGEA5 (Melanoma- Associated Antigen A5); MAGEA10 (Melanoma-Associated Antigen A10); MAGEA9 (Melanoma-Associated Antigen A9); HER2 / neu (ERBB2); EGFR (Epidermal Growth Factor Receptor); KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog); TP53 (Tumor Protein P53); BRAF (B-Raf Proto-Oncogene, Serine / Threonine Kinase); MUC1 (Mucin 1, Cell Surface Associated); PSA (Prostate Specific Antigen); NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1 Antigen); WT1 (Wilms Tumor 1); CEA (Carcinoembryonic Antigen); PSMA (Prostate-Specific Membrane Antigen); MAGE-A1 (Melanoma-Associated Antigen A1); MAGE-A12 (Melanoma-Associated Antigen A12); NY-BR-1 (New York Breast Cancer Antigen 1); WT1-AS (WT1 Antisense RNA); and BAGE (B melanoma Antigen)..

22. The immunogenic composition of claim 19, wherein the antigen is selected from the group consisting of: Tpd52l2 (Tumor protein D54, O43399), Tradd (Tumor necrosis factor receptor type 1-associated DEATH domain protein, Q15628), Tpt1 (Translationally-controlledtumor protein, P13693), Serpinf1 (Pigment epithelium-derived factor, P36955), Ceacam1 (Cell adhesion molecule CEACAM1, P13688), Pcna (Proliferating cell nuclear antigen, P12004), Mageb4 (Melanoma-associated antigen B4, O15481), or Bst2 (Bone marrow stromal antigen 2, Q10589).

23. The immunogenic composition of any one of the previous claims, wherein the EV is derived from a cell that is an engineered cell.

24. The immunogenic composition of claim 23, wherein the cell is an antigen presenting cell (APC).

25. The immunogenic composition of claim 24, wherein the APC is a dendritic cell (DC).

26. The immunogenic composition of claim 24 or 25, wherein the APC or DC is a human cell.

27. The immunogenic composition of any one of the previous claims, wherein the EVs are generated through donor cells.

28. The immunogenic composition of claim 27, wherein the donor cells are APCs.

29. The immunogenic composition of claim 28, wherein the donor APCs are DCs.

30. The immunogenic composition of any one of claims 27, 28, and 29, wherein the donor is a patient in need of the immunogenic composition.

31. The immunogenic composition of any one of claims 27, 28, and 29, wherein the donor cells are HLA matched to a patient in need of the immunogenic composition.

32. A method for producing cell-derived extracellular vesicles (EVs) with enhanced antigen presentation, the method comprising: A) transfecting cells with a nucleic acid encoding a virus-like capsid of a Gag homologue;B) contacting the transfected cells with a target antigen, to permit the target antigen to be presented via MHC complexes on the membrane of the transfected cells; and C) collecting EVs generated from the transfected cells, wherein the collected EVs present the target antigen.

33. The method of claim 32, wherein the target antigen is from isolated extracellular vesicles derived from cells harboring the target antigen and the isolated extracellular vesicles inherently comprise the target antigen.

34. The method of claim 32 or 33, wherein the cells are also transfected with a stabilizer wherein the stabilizer is a 5’ untranslated region (UTR) of a long terminal repeat (LTR) retroviral homologue.

35. The method of claim 34, wherein the LTR retroviral homologue is an Arc and the stabilizer is a 5’ UTR Arc sequence.

36. The method of any one of claims 32-35, further comprising concentrating the EVs collected in step C.

37. The method of claim 36, wherein the concentration is performed through tangential flow filtration and / or ultrafiltration.

38. The method of any one of claims 32-37, wherein the cells are donor cells.

39. The method of any one of claims 32-38, wherein the cells are autologous cells.

40. The method of any one of claims 32-39, wherein the cells are antigen presenting cells (APCs).

41. The method of claim 40, wherein the APCs are dendritic cells.

42. A method of slowing or stopping the progression of a disease in a subject, the method comprising administering the immunogenic composition of any one of claims 1-31 to the subject, thereby inducing an immune response that slows or stops the progression of the disease.

43. A method of immunizing a mammalian subject against a disease, the method comprising administering the immunogenic composition of any one of claims 1-31 to the mammalian subject.

44. The method of either claim 42 or 43, wherein the immunogenic composition is administered more than once.

45. The method of claim 44, wherein there is a set period of time between each administration of the immunogenic composition.

46. The method of any one of claims 32-45, wherein the disease is selected from the group consisting of: cancers of organs and tissues, autoimmune diseases, and neurodegenerative diseases.

47. The method of claim 46, wherein the cancer is selected from the group consisting of Breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer (melanoma), skin cancer (non-melanoma), bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, liver cancer, kidney cancer (renal cell carcinoma), brain cancer (glioblastoma), leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, soft tissue sarcoma, bone sarcoma, esophageal cancer, stomach cancer, head and neck cancer, testicular cancer, vulvar cancer, penile cancer, gallbladder cancer, bile duct cancer, adrenal gland cancer, small intestine cancer, anal cancer, neuroendocrine tumors, mesothelioma, Merkel cell carcinoma, gastrointestinal stromal tumor (GIST), carcinoid tumors, choriocarcinoma, pleomorphic carcinoma, adenoid cystic carcinoma, salivary gland tumors, thymoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, liposarcoma, leiomyosarcoma, chondrosarcoma, Ewing sarcoma, rhabdomyosarcoma, fibrosarcoma, dermatofibrosarcoma protuberans, synovial sarcoma, alveolar soft part sarcoma, clear cell sarcoma, epithelioid sarcoma, desmoid tumors, ovarian germ cell tumors, osteosarcoma, chordoma, glioma, astrocytoma, medulloblastoma, meningioma, pituitary tumors, craniopharyngioma, neuroblastoma, retinoblastoma, Wilms tumor (nephroblastoma), adrenocortical carcinoma, mesenchymal chondrosarcoma, giant cell tumor of bone, fibrous dysplasia, desmoplastic small round cell tumor, extraskeletal myxoid chondrosarcoma, Kaposi sarcoma, mycosis fungoides, Sézary syndrome, Castleman disease, Langerhans cell histiocytosis, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), plasma cell neoplasms (multiple myeloma), non-Hodgkin lymphoma (follicular lymphoma), non-Hodgkin lymphoma (diffuse large B-cell lymphoma), non-Hodgkin lymphoma (mantle cell lymphoma), non-Hodgkin lymphoma (peripheral T-cell lymphoma), fibromatosis (desmoid tumor), neurofibromatosis, gliomatosis cerebri, MALT lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large cell lymphoma, extranodal NK / T-cell lymphoma, acinic cell carcinoma, adult T-cell leukemia / lymphoma, aggressive NK-cell leukemia, basal cell carcinoma, basaloid squamous cell carcinoma, Burkitt lymphoma, carcinoid syndrome, carcinoma in situ, chondroblastoma, clear cell adenocarcinoma, clear cell carcinoma, clear cell renal cell carcinoma, cystadenocarcinoma, embryonal carcinoma, endometrioid adenocarcinoma, ependymoma, epithelial-myoepithelial carcinoma, extranodal marginal zone lymphoma, follicular adenocarcinoma, follicular carcinoma, follicular dendritic cell sarcoma, gastric adenocarcinoma, gastrointestinal carcinoid tumor, giant cell tumor of tendon sheath, granular cell tumor, granulocytic sarcoma, granulosa cell tumor, hemangioblastoma, hemangiopericytoma, hepatocellular carcinoma, Hurthle cell carcinoma, inflammatory myofibroblastic tumor, inflammatory pseudotumor, intraductal papillary mucinous neoplasm (IPMN), invasive lobular carcinoma, Krukenberg tumor, Langerhans cell sarcoma, leiomyoma, leiomyosarcoma, lipomatous tumor, low-grade serous carcinoma, lymphoepithelial carcinoma, malignant fibrous histiocytoma (MFH), malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant triton tumor, medullary carcinoma, medullary thyroid carcinoma, Merkel cell polyomavirus-associated carcinoma, Merkel cell polyomavirus-negative carcinoma, metastatic squamous neck cancer with occult primary, micropapillary carcinoma, mixed ductal-lobular carcinoma, mucoepidermoid carcinoma, myoepithelial carcinoma, myxofibrosarcoma, myxoid chondrosarcoma, myxoid liposarcoma, nasopharyngeal carcinoma, neurofibroma, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor, neurofibromatosis type 2-associated tumors, neurogenic sarcoma, nevoid basal cell carcinoma syndrome, nodular malignant melanoma, non-small cell lung carcinoma, noninvasive papillary urothelial carcinoma, oncocytoma, oropharyngeal carcinoma, ossifying fibroma, osteoblastoma, ovarian serous carcinoma, Paget disease, papillary adenocarcinoma, papillary carcinoma, papillary renal cell carcinoma, papillary serous carcinoma, parathyroid carcinoma, parathyroid adenoma, Peutz-Jeghers syndrome-associated tumors, phyllodes tumor, pilomatricoma, plasmacytoma, pleomorphic adenoma, and plexiform neurofibroma.

48. The method of claim 47, wherein the cancer is selected from the group consisting of Breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer (melanoma), skin cancer (non-melanoma), bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, liver cancer, kidney cancer (renal cell carcinoma), brain cancer (glioblastoma), leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, soft tissue sarcoma, bone sarcoma, esophageal cancer, stomach cancer, head and neck cancer, testicular cancer, vulvar cancer, penile cancer, gallbladder cancer, bile duct cancer, adrenal gland cancer, small intestine cancer, anal cancer, neuroendocrine tumors, mesothelioma, Merkel cell carcinoma, gastrointestinal stromal tumor (GIST), carcinoid tumors, choriocarcinoma, pleomorphic carcinoma, adenoid cystic carcinoma, salivary gland tumors, thymoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, liposarcoma, leiomyosarcoma, chondrosarcoma, Ewing sarcoma, rhabdomyosarcoma, fibrosarcoma, dermatofibrosarcoma protuberans, synovial sarcoma, alveolar soft part sarcoma, clear cell sarcoma, epithelioid sarcoma, desmoid tumors, ovarian germ cell tumors, osteosarcoma, chordoma, glioma, astrocytoma, medulloblastoma, meningioma, pituitary tumors, craniopharyngioma, neuroblastoma, and retinoblastoma..

49. The method of claim 47, wherein the cancer is selected from the group consisting of Breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer (melanoma), skin cancer (non-melanoma), bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, liver cancer, kidney cancer (renal cell carcinoma), brain cancer (glioblastoma), leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, soft tissue sarcoma, bone sarcoma, esophageal cancer, stomach cancer, head and neck cancer, testicular cancer, vulvar cancer, penile cancer, gallbladder cancer, bile duct cancer, adrenal gland cancer, small intestine cancer, anal cancer, neuroendocrine tumors, and mesothelioma.

50. The method of claim 47, wherein the cancer is selected from the group consisting of melanomas and breast cancers.

51. The method of claim 46, wherein the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis,Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), polymyalgia rheumatica, Goodpasture syndrome, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, vitiligo, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Wegener's granulomatosis (granulomatosis with polyangiitis), microscopic polyangiitis, Behçet's disease, Takayasu's arteritis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), IgA nephropathy (Berger's disease), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), antiphospholipid syndrome, autoimmune hepatitis, mixed connective tissue disease (MCTD), autoimmune pancreatitis, pernicious anemia, lichen sclerosus, relapsing polychondritis, pustular psoriasis, eosinophilic esophagitis, autoimmune thyroid disease, autoimmune encephalitis, autoimmune polyendocrine syndromes (APS-1 to APS-21), autoimmune polyendocrine syndrome type 1 (APS-1), autoimmune polyendocrine syndrome type 2 (APS-2), autoimmune polyendocrine syndrome type 3 (APS-3), autoimmune polyendocrine syndrome type 4 (APS-4), autoimmune polyendocrine syndrome type 5 (APS-5), autoimmune polyendocrine syndrome type 6 (APS-6), autoimmune polyendocrine syndrome type 7 (APS-7), autoimmune polyendocrine syndrome type 8 (APS-8), autoimmune polyendocrine syndrome type 9 (APS-9), autoimmune polyendocrine syndrome type 10 (APS- 10), autoimmune polyendocrine syndrome type 11 (APS-11), autoimmune polyendocrine syndrome type 12 (APS-12), autoimmune polyendocrine syndrome type 13 (APS-13), autoimmune polyendocrine syndrome type 14 (APS-14), autoimmune polyendocrine syndrome type 15 (APS-15), autoimmune polyendocrine syndrome type 16 (APS-16), autoimmune polyendocrine syndrome type 17 (APS-17), autoimmune polyendocrine syndrome type 18 (APS- 18), autoimmune polyendocrine syndrome type 19 (APS-19), and autoimmune polyendocrine syndrome type 20 (APS-20).

52. The method of claim 51, wherein the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis,ankylosing spondylitis, scleroderma (systemic sclerosis), polymyalgia rheumatica, Goodpasture syndrome, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, vitiligo, idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Wegener's granulomatosis (granulomatosis with polyangiitis), microscopic polyangiitis, Behçet's disease, Takayasu's arteritis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), IgA nephropathy (Berger's disease).

53. The method of claim 51, wherein the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, Sjögren's syndrome, dermatomyositis, polymyositis, polyarteritis nodosa, giant cell arteritis, Takayasu arteritis, ankylosing spondylitis, scleroderma (systemic sclerosis), and polymyalgia rheumatica.

54. The method of claim 51, wherein the autoimmune disease is selected from the group consisting of Rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, and inflammatory bowel disease (Crohn's disease and ulcerative colitis).

55. The method of claim 46, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia, Lewy body dementia, Pick's disease, Frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration, Creutzfeldt-Jakob disease, Wilson's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Charcot-Marie-Tooth disease, hereditary spastic paraplegia, Krabbe disease, Batten disease (neuronal ceroid lipofuscinoses), Niemann-Pick disease, Gaucher disease, Tay-Sachs disease, Canavan disease, Alexander disease, metachromatic leukodystrophy, Pelizaeus-Merzbacher disease, Sanfilippo syndrome, mucopolysaccharidoses, Rett syndrome, fragile X-associated tremor / ataxia syndrome, myotonic dystrophy, Huntington's disease-like syndromes, spinal and bulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, Perry syndrome, and prion diseases.

56. The method of claim 55, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia, Lewy body dementia, Pick's disease, Frontotemporal dementia (FTD).

57. The method of claim 55, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD).

58. The method of claim 46, wherein the infectious disease includes HIV / AIDS, Influenza, coronaviruses, tuberculosis, malaria, hepatitis (A, B, C, D, and E), dengue fever, Zika virus, Ebola virus disease, cholera, typhoid fever, measles, chickenpox, shingles, polio, rabies, meningitis (bacterial, viral, and fungal), pneumonia (bacterial and viral), Lyme disease, syphilis, gonorrhea, chlamydia, HPV (human papillomavirus), herpes (HSV-1 and HSV-2), candidiasis, trichomoniasis, giardiasis, cryptosporidiosis, leishmaniasis, schistosomiasis, filariasis, onchocerciasis, trachoma, tetanus, pertussis, anthrax, bubonic plague, tularemia, brucellosis, leprosy, listeriosis, toxoplasmosis, Q fever, rocky mountain spotted fever, yellow fever, West Nile virus, Japanese encephalitis, Rift Valley fever, Hantavirus pulmonary syndrome, Nipah virus infection, and Middle East respiratory syndrome (MERS).

59. The method of claim 58, wherein the infectious disease is selected from the group consisting of HIV / AIDS, Influenza, coronaviruses, tuberculosis, Lyme disease, HPV (human papillomavirus), and Middle East respiratory syndrome (MERS)..

60. The method of claim 59, wherein the infectious disease is selected from the group consisting of HIV / AIDS, Influenza, and coronavirus.