Viral vector construct for the expression of a gene adjuvant that activates the STING pathway

Abstract

The present invention provides viral vectors, which are used as gene immunotherapeutic agents, and the gene immunotherapeutic agents include prophylactic and therapeutic vaccines and compositions that enhance cellular immune responses. The vectors are particularly suitable for treating or preventing cancer and infectious diseases. The vectors include lentiviral vectors encoding one or more antigens and an adjuvant, and optionally may encode one or more soluble checkpoint inhibitor molecules. The adjuvant is a fusion protein including latent membrane protein 1 (LMP1) from Epstein-Barr virus, wherein the cytoplasmic domain has been replaced by human IPS1 or a variant thereof capable of activating the STING pathway. The sequences encoded by the vectors are codon-optimized for human expression.

Description

Background Art

[0001] Standardized vaccine strategies based on antibody-induced immune responses have been able to eradicate or nearly eradicate many previously fatal infectious diseases, such as smallpox, polio, and tetanus. However, the use of these classical human vaccines has been ineffective or unsafe for other infectious diseases (such as HIV and hepatitis) and non-infectious diseases (such as cancer).

[0002] A new generation of immunotherapeutic products designed to induce cellular immune responses can overcome the limitations of traditional vaccines by recognizing and killing cancer cells and infected cells rather than the pathogens themselves. Nucleic acid vaccines, particularly viral vectors, have shown great potential for clinical translation.

[0003] Cancer cells and many infectious agents have ways to evade the immune system, which makes it difficult to manufacture effective vaccines. Classical vaccines typically require adjuvants such as aluminum salts to achieve optimal effects, but conventional adjuvants are generally weak in enhancing cellular immune responses. Some strategies have been proposed to improve the quality and magnitude of cellular immune responses elicited by viral vectors. A new class of genetic adjuvants has been developed to improve cellular immune responses induced by vector-based immunotherapy. Genetic adjuvants consist of DNA sequences encoding immunomodulatory molecules.

[0004] Stone et al. (WO2014 / 039961) disclose the use of genetic adjuvants that induce the secretion of interferon α and β, thereby inducing the expression of interferon-stimulated genes. In this method, the nucleic acid vaccine optionally encodes, in addition to a transgene encoding a marker protein or antigen, a fusion protein that includes the transmembrane portion of the LMP1 protein, wherein the cytoplasmic domain has been replaced by an immune effector or adaptor protein such as the IPS1 protein. Activation of the IFN-β promoter stimulator (IPS1, also known as MAVS, VISA, or Cardif) via the STING (stimulator of interferon genes) pathway generates an effective T cell response. When expressed in cells, the transmembrane domain of LMP1 spontaneously forms clusters that allow IPS1 to aggregate into cytoplasmic clusters, activating the STING pathway. The transmembrane domain of LMP1 fused to full-length murine IPS1 has been shown to be capable of inducing the secretion of IFN-α, IFN-β, and IL-6 in murine macrophages, and also inducing the expression of maturation markers (CD40 and CCR7) and activation markers (CD80 and CD86).

[0005] There is a need for self-adjuvanting vaccines to induce strong cellular immune responses, which are necessary to break immune tolerance observed in diseases such as cancer, HIV, and other incurable indications. Summary of the Invention

[0007] The technology of the present invention provides viral vectors encoding gene adjuvants for improving immune responses, particularly cell-mediated immune responses, such as immune responses against cancer or infection, and provides methods of using such viral vectors. The antigen and adjuvant constructs of the technology of the present invention have been optimized for human subjects.

[0008] One aspect of the technology is a viral vector that includes (i) one or more transgenes encoding one or more marker proteins, antigens, epitopes, or combinations thereof, and (ii) a transgene encoding a fusion protein comprising the transmembrane portion of latent membrane protein 1 (LMP1) of Epstein Barr virus, wherein the cytoplasmic domain of the LMP1 has been replaced by human IPS1 or a variant thereof capable of activating the STING pathway. In a preferred embodiment, the viral vector is a lentiviral vector. In some embodiments, the vector includes a functional lentiviral integrase protein and can thus integrate into the genome of the cells it transduces.

[0009] The antigen can be a tumor antigen, a viral antigen, or a microbial antigen. The vector encodes multiple antigens or epitopes of selected one or more antigens. In certain embodiments, at least one antigen is selected from the group consisting of NY-ESO-1, mesothelin, PSA, MART-1, MART-2, Gp100, tyrosinase, p53, ras, MUC1, SAP-1, survivin, CEA, Ep-CAM, Her2, BRCA1 / 2, gag, reverse transcriptase, tat, circumsporozoite protein, HCV non-structural protein, hemagglutinin, and combinations thereof. In certain embodiments, the vector further encodes at least one immune checkpoint inhibitor molecule, such as an anti-CTLA-4 molecule, a PD-1 blocker, a PDL1 blocker, or combinations thereof.

[0010] In certain embodiments, the viral vector comprises more than one nucleic acid sequence. In some embodiments, the first nucleic acid sequence encodes one or more marker proteins, antigens, epitopes, or combinations thereof; the second nucleic acid sequence encodes a fusion protein comprising the transmembrane portion of latent membrane protein 1 (LMP1) of Epstein-Barr virus, wherein the cytoplasmic domain of the LMP1 has been replaced by human IPS1 or a variant thereof capable of activating the STING pathway; optionally, the third nucleic acid sequence encodes one or more immune checkpoint inhibitor molecules (“anti-checkpoint”). Preferably, the first nucleic acid sequence and the second nucleic acid sequence, and the second nucleic acid sequence and the third nucleic acid sequence are separated by a nucleic acid sequence encoding an internal ribosome entry site (IRES). The first nucleic acid sequence and the second nucleic acid sequence, and the second nucleic acid sequence and the third nucleic acid sequence may be separated by a nucleic acid sequence encoding a self-cleaving peptide (e.g., 2A peptide). The first nucleic acid sequence and the second nucleic acid sequence, and the second nucleic acid sequence and the third nucleic acid sequence may be separated by a nucleic acid sequence encoding a self-cleaving peptide (e.g., 2A peptide) or an internal ribosome entry site (IRES).

[0011] Another aspect of the technology is an immunotherapeutic formulation for preventing or treating a disease or disorder in a subject, comprising the viral vector. In a preferred embodiment, the disease or disorder is cancer or an infection.

[0012] Another aspect of the technology is a method of inducing an immune response against cancer or an infection in a subject, the method comprising administering the viral vector or the immunotherapeutic formulation to a subject in need thereof. In some embodiments, administering the viral vector to the subject vaccinates the subject against cancer or an infection.

[0013] In some embodiments, the cancer is selected from the group consisting of: melanoma, glioma, prostate cancer, ovarian cancer, breast cancer, cervical cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, sarcoma, and pancreatic cancer. In some embodiments, the cancer has a tumor antigen listed above. In some embodiments, the cancer is sensitive to anti-checkpoint. In some embodiments, the infectious disease is selected from the group consisting of: HIV / AIDS, hepatitis C, HPV, pneumonia, influenza, malaria, leishmaniasis, tuberculosis, Hansen's disease, rabies, dengue fever, Zika virus infection, Ebola virus infection, and schistosomiasis. In some embodiments, the infectious agent contains a virus or microbial antigen listed above. In some embodiments, the infectious disease is sensitive to anti-checkpoint.

[0014] The technology can also be summarized by the following list of examples.

[0015] 1. A viral vector, which comprises a first nucleic acid sequence encoding an antigen or an epitope and a second nucleic acid sequence encoding a fusion protein, the fusion protein comprising a transmembrane portion of latent membrane protein 1 (LMP1) of Epstein-Barr virus, the cytoplasmic domain of the LMP1 having been replaced by human IPS1 or a variant thereof, capable of activating the STING pathway, and the coding sequence of the vector being codon-optimized for human expression.

[0016] 2. The viral vector according to Example 1, wherein the vector is a lentiviral vector.

[0017] 3. The viral vector according to Example 1 or 2, wherein the first nucleic acid sequence encodes a fusion protein comprising two or more antigens or two or more epitopes.

[0018] 4. The viral vector according to any one of the foregoing examples, wherein the second nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NO.1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7.

[0019] 5. The viral vector according to any one of the foregoing examples, wherein the vector further comprises a third nucleic acid sequence encoding a soluble immune checkpoint inhibitor molecule or a soluble immunomodulatory molecule.

[0020] 6. The viral vector according to Example 5, wherein the soluble immune checkpoint inhibitor molecule or the soluble immunomodulatory molecule is selected from the group consisting of CTLA-4, PD-1, PDL-1, LAG-3, TIM 3, B7-H3, ICOS, IDO, 4-1BB, CD47, B7-H4, OX-40, TIGIT, CD160 and combinations thereof.

[0021] 7. The viral vector according to any one of the foregoing examples, wherein the vector further comprises a functional lentiviral integrase protein, and wherein the vector is self-inactivating.

[0022] 8. The viral vector according to any one of the foregoing examples, wherein the antigen is selected from the group consisting of NY-ESO-1, mesothelin, PSA, MART-1, MART-2, Gp100, tyrosinase, p53, ras, MUC1, SAP-1, survivin, CEA, Ep-CAM, Her2, BRCA1 / 2, gag, reverse transcriptase, tat, circumsporozoite protein, HCV non-structural protein, hemagglutinin and combinations thereof.

[0023] 9. An immunotherapeutic preparation for preventing or treating cancer or infection in a subject, the preparation comprising the viral vector according to any one of Examples 1-8.

[0024] 10. A method for inducing or enhancing an immune response against cancer or an infectious disease in a subject, the method comprising administering to the subject in need thereof a viral vector as described in any one of Examples 1-8 or an immunotherapeutic formulation as described in Example 9, thereby inducing or enhancing an immune response against the cancer or the infectious disease in the subject.

[0025] 11. The method according to Example 10, which induces or enhances an immune response against cancer, and the cancer is selected from the group consisting of: melanoma, glioma, prostate cancer, breast cancer, cervical cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, and pancreatic cancer.

[0026] 12. The method according to Example 10, which induces or enhances an immune response against an infectious disease, and the infectious disease is selected from the group consisting of: HIV / AIDS, hepatitis C, HPV, pneumonia, influenza, malaria, leishmaniasis, tuberculosis, Hansen's disease, rabies, dengue fever, Zika virus, Ebola virus, and schistosomiasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram showing the secondary structure of the LMP1 protein.

[0028] Figure 2 A schematic diagram showing the secondary structure of the truncated LMP1 protein, wherein the truncated LMP1 protein removes the cytoplasmic signaling domain.

[0029] Figure 3A A schematic diagram showing the IPS1 protein, Figure 3B showing its orientation in the mitochondrial membrane.

[0030] Figure 4 A schematic diagram showing the LPM1-IPS1 fusion protein.

[0031] Figure 5 A schematic diagram showing the secondary structure of the LPM1-IPS1 fusion protein that should be produced when expressed in the order described in WO2014 / 039961.

[0032] Figure 6 A schematic diagram showing the secondary structure of the LPM1-IPS1 fusion protein with the IPS1 transmembrane domain removed.

[0033] Figure 7 A schematic diagram showing the structure of the LPM1-reverse IPS1 fusion protein, wherein the IPS transmembrane domain is removed, and the caspase recruitment domain (CARD) and the proline-rich (PR) domain are in opposite directions.

[0034] Figures 8A - 8CShows schematic diagrams of several molecular constructs. Figure 8A Shows a lentiviral vector used as a control, in which the transgene is under the control of the human ubiquitin protein promoter. The transgene is a) GFP reporter gene, b) transmembrane domain of LMP1 protein, c) GFP reporter gene and transmembrane domain of LMP1 separated by an IRES sequence, and d) fusion of the LMP1 transmembrane domain with full-length human IPS1. Figure 8B Shows lentiviral vectors used to evaluate various adjuvants, in which the transgene is under the control of the human ubiquitin protein promoter. The transgene is a) GFP reporter gene and transmembrane domain of LMP1 fused with full-length human IPS1 protein separated by an IRES, b) GFP reporter gene and transmembrane domain of LMP1 fused with human IPS1 protein lacking the transmembrane domain separated by an IRES, c) GFP reporter gene and transmembrane domain of LMP1 fused with human IPS1 protein lacking the transmembrane domain and proline-rich domain separated by an IRES, and d) GFP reporter gene and transmembrane domain of LMP1 fused with human IPS1 protein lacking the transmembrane domain and with the CARD and Pro domains in reverse separated by an IRES. Figure 8C Shows Figure 8B the same construct, in which the adjuvant sequence is followed by an IRES and a checkpoint soluble molecule or a soluble immunomodulatory molecule.

[0035] Figures 9A - 9B Shows the expression levels of the GFP transgene in human dendritic cells and macrophages transduced with lentiviral vectors. Figure 9A Shows the GFP transgene expression in human dendritic cells 96 hours after transduction with a lentiviral construct. Figure 9B Shows the GFP transgene expression in human macrophages 96 hours after transduction with a lentiviral construct.

[0036] Figures 10A - 10D Shows the activation and maturation of human dendritic cells and macrophages induced by lentiviral vectors in vitro. Figure 10A Shows the group of cytokines upregulated in human dendritic cells 96 hours after transduction with a lentiviral construct. Figure 10B Shows the group of markers upregulated in GFP-positive human dendritic cells (expression normalized to GFP) 96 hours after transduction with a lentiviral construct. Figure 10C Shows the group of cytokines upregulated in human macrophages 96 hours after transduction with a lentiviral construct. Figure 10D Shows the group of markers upregulated in GFP-positive human macrophages (expression normalized to GFP) 96 hours after transduction with a lentiviral construct. Detailed Description of the Invention

[0038] The present technology provides viral vector constructs for expressing gene adjuvants used in immunotherapy products and methods of using such vectors. The vector constructs can improve the quality and intensity of immune responses, such as those against cancer or infection, and are particularly suitable for inducing and / or enhancing cell-mediated immune responses.

[0039] The present technology describes the use of a single vector construct comprising an antigen cassette and a gene adjuvant. Compared to injecting two vectors simultaneously (one encoding an antigen and one encoding an adjuvant), using a single product will simplify development (including industrial, regulatory, and clinical aspects) and improve the efficacy and safety of treatment. With this unique construct, cells expressing the antigenic expression cassette will constitutively benefit from the expression of the adjuvant, thereby enhancing the intensity and quality of the triggered immune response. Transduced cells will be rapidly eliminated by the immune response, which reduces the risk of any long-term and undesired expression of the gene sequence that may raise questions or concerns from regulatory agencies. Additionally, producing and injecting only one vector will be more cost-effective compared to injecting two different vectors.

[0040] The present technology includes one or more nucleic acid sequences encoding the EBV LMP1 protein, wherein the cytoplasmic domain of the EBV LMP1 protein has been replaced with human IPS1 or a variant thereof capable of activating the STING pathway and one or more antigens. In typical embodiments, the technology provides activation of the immune response through the aggregation of two or more LMP1 proteins in the cell membrane and / or the aggregation of two or more cytoplasmic signaling domains of IPS1. After direct injection, the introduction of the nucleic acid sequence and subsequent protein expression can occur in any type of cell, but preferably in immune cells. The technology can be used for traditional prophylactic or therapeutic vaccines against cancer and infectious diseases, as well as cell-based therapies such as dendritic cell therapy. In the experiments described in the present invention, it is expected that the viral vector significantly enhances the immune response and protects against or treats infection and cancer.

[0041] "Vector" refers to a molecule comprising a nucleic acid sequence encoding at least part of a gene product that can be transcribed. In some cases, the nucleic acid molecule is subsequently translated into a protein, polypeptide, or peptide. In other cases, such as in the production of antisense molecules, ribozymes, or aptamers, these sequences are not translated. A vector may contain a variety of control sequences, which refer to the nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to the control sequences governing transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0042] A "construct" can be any type of engineered nucleic acid encoding a gene product, where some or all of the nucleic acid coding sequences are capable of being transcribed. The transcript is typically translated into a protein, but this is not required. In some embodiments, expression includes transcription of the gene and translation of the mRNA into the gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest.

[0043] As used herein, "vaccine" includes all prophylactic and therapeutic vaccines.

[0044] An "adjuvant" can be any molecule or composition that activates or enhances the immune response against an antigen. Adjuvants can enhance the efficacy of a vaccine by assisting in altering the immune response to specific types of immune system cells. An adjuvant can be an immunostimulant that triggers the activation of antigen-presenting cells such as dendritic cells, macrophages, and B cells. Adjuvants are also understood to provide a "danger" signal indicating that the immune system should enter a state of alert. Adjuvants can act by promoting antigen presentation by antigen-presenting cells, by activating macrophages and lymphocytes, and / or by supporting the production of cytokines. Without an adjuvant, an immune response may not occur or may be converted into an ineffective immunity or tolerance. Adjuvants are generally required for effective prophylactic or therapeutic vaccines or for inducing anti-tumor immune responses. A "gene adjuvant" is an adjuvant provided in the form of nucleic acid that is expressed by target cells to produce a molecule that acts as an adjuvant.

[0045] An antigen-presenting cell (APC) is any one of a variety of cells that are capable of displaying, acquiring, or presenting at least one antigen or antigen fragment on their cell surface. Generally, the term "antigen-presenting cell" can refer to any cell that achieves this technical objective by helping to enhance an immune response against an antigen or antigen composition (i.e., the T-cell or B-cell arm of the immune system). Those skilled in the art can define such cells using the present invention and methods disclosed in the art. As understood by those of ordinary skill in the art and in certain embodiments used in the present invention, cells that typically or preferentially display or present antigens to immune cells using class II major histocompatibility molecules or complexes are "antigen-presenting cells". In some aspects, a cell (e.g., an APC) can be fused with another cell, such as a recombinant cell or a tumor cell expressing a desired antigen. Methods for preparing fissions of two or more cells are well known in the art. In some cases, the immune cells to which the antigen-presenting cell displays or presents an antigen are CD4+ T cells or CD8+ T cells. Other molecules expressed on the APC or other immune cells can help or improve the enhancement of the immune response. Secreted or soluble molecules, such as cytokines and adjuvants, can also help or enhance the immune response against an antigen. Dendritic cells (DCs) are antigen-presenting cells that are present in vivo, in vitro, ex vivo, or in a host or subject, or can be derived from hematopoietic stem cells or monocytes. Dendritic cells and their precursors can be isolated from a variety of lymphoid organs, such as the spleen, lymph nodes, as well as bone marrow and peripheral blood. DCs have a characteristic morphology, with thin sheets (lamellipodia) extending from the dendritic cell body in multiple directions. Generally, dendritic cells express high levels of major histocompatibility complex (MHC) and costimulatory molecules (e.g., B7-1 and B7-2). Dendritic cells can induce antigen-specific differentiation of T cells in vitro and are capable of initiating primary T cell responses in vitro and in vivo.

[0046] The term "immune response" refers to the induction of an antibody- and / or immune cell-mediated response that is specific to a single antigen or multiple antigens or allergens or drugs or biological agents. The induction of an immune response depends on many factors, including the immunogenic makeup of the organism under attack, the chemical composition and configuration of the antigen or allergen or drug or biological agent, and the mode and period of administration of the antigen or allergen or drug or biological agent. The immune response includes many aspects, some of which are manifested by cells of the immune system (e.g., B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immune system cells can participate in the immune response through interaction with antigens or allergens or other cells of the immune system, the release of cytokines, and responsiveness to these cytokines. The immune response is generally divided into two main categories - humoral-mediated and cell-mediated. The humoral component of the immune response includes the production of antibodies specific to an antigen or allergen or drug or biological agent. The cell-mediated component includes the production of delayed-type hypersensitivity and cytotoxic effector cells directed against an antigen or allergen.

[0047] Activation or stimulation of the immune system can be mediated by the activation of immune effector cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), and cytotoxic T lymphocytes (CTLs). It can be mediated by the activation and maturation of antigen-presenting cells such as dendritic cells. It can be mediated by blocking inhibitory pathways such as by inhibiting immune checkpoint inhibitors.

[0048] The term "LMP1 gene" refers to a nucleic acid sequence encoding the native Epstein-Barr virus LMP1, such as the native Epstein-Barr virus LMP1 gene; a nucleic acid having a sequence that can transcribe LMP1 cDNA; and / or variants and homologs of the foregoing alleles. An exemplary nucleic acid sequence of LMP1 is GenBank accession number M58153.1. The term includes double-stranded DNA, single-stranded DNA, and RNA.

[0049] The term "LMP1 protein" refers to the expression product of the LMP1 gene or a protein having at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity thereto and displaying the functional activity of the native LMP1 protein. "Functional activity" of a protein is any activity associated with the physiological function of the protein. LMP1 consists of an N-terminal transmembrane domain linked to a C-terminal cytoplasmic signaling region, which is similar to the CD40 receptor on immune cells. In addition to anchoring LMP1 to the membrane, the N-terminus of LMP1 self-aggregates and causes aggregation of LMP1 or any protein linked to the LMP1 N-terminal domain. The transmembrane (aggregation) domain of the LMP1 protein is amino acids 1-190 of the amino acid sequence listed in GenBank accession number AAA66330.1.

[0050] Latent membrane protein-1 (LMP1) is a gene in Epstein-Barr virus (EBV). Its N-terminus consists of 6 contiguous transmembrane domains that anchor the protein in the membrane. Figure 1 The structure of the LMP1 protein is shown, which shows transmembrane domain 101 and intracellular signaling domain 102. LMP1 does not require a ligand or antibody to initiate signaling through its cytoplasmic domain because its N-terminal transmembrane domain spontaneously forms clusters in the cell membrane and thereby clusters the intracellular domain linked to it by a peptide bond into a single polypeptide chain. In this sense, LMP1 is called "constitutively active". Similarly, a fusion protein that links an N-terminal transmembrane domain to a signaling domain that requires clustering to function can also be said to be "constitutively active" and no longer requires extraction of a ligand from a receptor.

[0051] Interferon promoter stimulator-1 (IPS1, also known as MAVS, VISA or Cardif) is a transmembrane mitochondrial protein associated with the STING pathway ("stimulator of interferon genes"; also known as TMEM173, MPYS, MITA and ERIS), which is important for the innate response to pathogen-derived nucleic acids in the cytoplasm. IPS1 contains a C-terminal transmembrane domain that anchors the protein to the outer membrane of the mitochondrion and, once activated, forms aggregates (i.e., multimers). IPS1 is also present in peroxisomes and mitochondria-associated membranes. IPS1 also contains a caspase recruitment domain (CARD), which is essential for downstream protein-protein interactions, and it also contains three TRAF interaction motifs (TIMs), two included in the N-terminal proline-rich region and the third located in the C-terminal region. The membrane localization of IPS1 may be important for its activity, as removal of the transmembrane domain inhibits the IPS1-mediated antiviral response. IPS1 functions as an adaptor protein for pathogen recognition receptors, such as retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), which patrol the cytoplasm for the presence of viral RNA. When double-stranded RNA binds to RLRs, they form a complex with IPS1 through their CARD domains, resulting in the multimerization and activation of IPS1. Then, the activated IPS1 complex recruits the IKK and TBK1 / IKKi complexes, thereby triggering a signaling cascade that leads to the activation of the transcription factors NF-κB and IRF3. NF-κB and IRF3 bind to and activate the interferon promoter, generating an effective cell-mediated immune response by producing type I interferons. RIG-1 activation also activates the STING pathway, further enhancing the cell-mediated antiviral immune response. In the present technology, the fusion of IPS1 with the LMP1 N-terminal domain promotes the aggregation and activation of LMP1-IPS1, which mimics the activation caused by dsRNA.

[0052] The viral vector of the present technology encodes one or more nucleic acid sequences capable of activating or enhancing the immune response of a subject. The nucleic acid encodes the latent membrane protein 1 (LMP1) of Epstein-Barr virus, wherein the cytoplasmic domain of the LMP1 has been replaced by human IPS1 or a variant thereof capable of activating the STING pathway. The LMP1 DNA sequence has been codon-optimized for expression in humans. The expression of the LMP1-IPS1 fusion protein provides activation of the immune response through the aggregation (i.e., multimerization) of two or more LMP1 proteins.

[0053] The viral vector can be any suitable type of vector, such as an expression vector or a plasmid. In a preferred embodiment, the vector is a lentiviral vector. Lentiviral vectors are modified lentiviruses, such as those derived from human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV). The modified lentiviral vectors have reduced pathogenicity. The vector can also be modified to introduce beneficial therapeutic effects. Lentiviral vectors are not toxic per se, and unlike other retroviruses, lentiviruses are able to transduce non-dividing cells, particularly dendritic cells, allowing antigen presentation via the endogenous pathway.

[0054] Lentiviral vectors can comprise RNA or DNA molecules. In some embodiments, the lentiviral vector is a recombinant DNA molecule, such as a plasmid. In some embodiments, the lentiviral vector comprises a recombinant DNA molecule and associated viral proteins to form particles. Lentiviral vector particles can contain single-stranded or double-stranded nucleic acid molecules.

[0055] In a preferred embodiment, lentiviral vectors have the ability to integrate into the genome of the transduced cell. In a preferred embodiment, they contain a functional integrase protein. Non-integrating vector particles exhibit gene mutations that impede the ability of lentiviral vector particles to integrate into the host genome. The terms "transfection" and "transduction" refer to the process of introducing an exogenous DNA sequence into a eukaryotic host cell. Transfection is the non-viral delivery of nucleic acid (DNA or RNA) and can be achieved by any of a variety of means, including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, polymer-mediated delivery, etc. Transduction refers to the delivery of nucleic acid by a virus or viral vector, where the nucleic acid is typically DNA for DNA viruses and RNA for RNA viruses.

[0056] In some embodiments, lentiviral vectors are self-inactivating and do not contain enhancers. Self-inactivating lentiviral vectors have a modification in the U3 (ΔU3) region of the 3' LTR such that the vector cannot replicate in the host cell. The U3 region encodes binding sites essential for basal promoter activity and viral replication, and the elimination of these binding sites results in near-complete inactivation of viral replication.

[0057] Numerous factors affect the efficacy of viral vectors, even after successful transduction and optionally integration into the host genome: gene expression and translation; protein folding, trafficking, and turnover; and cell-to-cell interactions, to name a few. These factors depend particularly on the nucleic acid sequences encoded by the vector. Preferred DNA sequences for practicing the present technique include modifications of native sequences designed to enhance the efficacy and efficiency of viral vectors. These modifications include: codon optimization for humans; removal of the first methionine of the IPS1 sequence in a fusion protein; removal of the IPS1 transmembrane domain and proline-rich domain, and use of the reverse IPS1 sequence. These modifications may affect the rate of transcription and / or translation, as well as the location of the protein in the cell and protein activity.

[0058] The viral vectors of the present technique encode one or more antigens. As used herein, the term "antigen" refers to a molecule that elicits an immune response. The immune response may involve antibody production, or activation of specific immunocompetent cells, or both. Antigens can be derived from organisms, subunits of proteins / antigens, killed or inactivated whole cells or lysates. Thus, one skilled in the art recognizes that any macromolecule, including nearly all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. One skilled in the art recognizes that any DNA containing a nucleotide sequence or partial nucleotide sequence of a pathogenic genome or a gene or gene fragment of a protein that elicits an immune response will result in the synthesis of an antigen. In addition, one skilled in the art recognizes that the present technique is not limited to the use of the complete nucleic acid sequence of a gene or genome. The present technique includes, but is not limited to, the use of partial nucleic acid sequences of more than one gene or genome, the nucleic acid sequences of which are arranged in various combinations to elicit the desired immune response.

[0059] The antigen can be any antigen that is desired to enhance the immune response. Such antigens include, but are not limited to, antigens from pathogens that cause infectious diseases that can elicit a protective immune response. For example, antigens from HIV include the proteins gag, env, pol, tat, rev, nef, reverse transcriptase, and other HIV components. The E6 and E7 proteins from human papillomavirus are also suitable antigens. In addition, the EBNA1 antigen from Epstein-Barr virus is suitable. Other viral antigens for use in the present technology are hepatitis virus antigens, such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis viruses such as hepatitis A, hepatitis B, and hepatitis C, viral components such as hepatitis C virus RNA; influenza virus antigens such as hemagglutinin, neuraminidase, nucleoprotein, M2, and other influenza virus components; measles virus antigens, such as the measles virus fusion protein and other measles virus components; rubella virus antigens such as the proteins E1 and E2 and other rubella virus components; rotavirus antigens such as VP7sc and other rotavirus components; cytomegalovirus antigens such as envelope glycoprotein B and other cytomegalovirus antigen components; respiratory syncytial virus antigens such as the RSV fusion protein, M2 protein, and other respiratory syncytial virus antigen components; herpes simplex virus antigens such as immediate early proteins, glycoprotein D, and other herpes simplex virus antigen components; varicella-zoster virus antigens, such as gpI, gpII, and other varicella-zoster virus antigen components; Japanese encephalitis virus antigens such as the protein E, M-E, M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis virus antigen components; rabies virus antigens such as rabies glycoprotein, rabies nucleoprotein, and other rabies virus antigen components; West Nile virus prM and E proteins; and Ebola virus envelope protein. See Fundamental Virology, Second Edition, eds. Knipe, D.M. and Howley P.M. (Lippincott Williams & Wilkins, New York, 2001) for other examples of viral antigens. In addition, the present invention also discloses bacterial antigens.Bacterial antigens useful in the compositions and methods of the present technology include, but are not limited to, Bordetella pertussis antigens such as pertussis toxin, filamentous hemagglutinin, Bordetella pertussis adhesin, FIM2, FIM3, adenylate cyclase, and other Bordetella pertussis antigen components; Corynebacterium diphtheriae antigens such as diphtheria toxin or toxoid and other Corynebacterium diphtheriae antigen components; Clostridium tetani antigens such as tetanus toxin or toxoid and other Clostridium tetani antigen components; Streptococcus bacteria antigens such as M protein and other Streptococcus bacteria antigen components; Staphylococcus bacteria antigens such as IsdA, IsdB, SdrD, and SdrE; Gram-negative bacillus bacteria antigens such as lipopolysaccharide, flagellin, and other Gram-negative bacteria antigen components; Mycobacterium tuberculosis bacteria antigens such as mycolic acid, heat shock protein 65 (HSP65), 30 kDa major secreted protein, antigen 85A, ESAT-6, and other mycobacterial antigen components; Helicobacter pylori bacteria antigen components; Streptococcus pneumoniae bacteria antigens such as pneumolysin, pneumococcal capsular polysaccharide, and other Streptococcus pneumoniae bacteria antigen components; Haemophilus influenzae bacteria antigens such as capsular polysaccharide and other Haemophilus influenzae bacteria antigen components; Bacillus anthracis bacteria antigens such as anthrax protective antigen, anthrax lethal factor, and other Bacillus anthracis bacteria antigen components; F1 and V proteins from Yersinia pestis; Rickettsia bacteria antigens such as outer membrane proteins (romps) and other Rickettsia bacteria antigen components. The bacterial antigens described herein also include any other bacterial, mycobacterial, mycoplasma, rickettsial, or chlamydial antigens. Examples of protozoan and other parasite antigens include, but are not limited to, Plasmodium falciparum antigens such as merozoite surface antigen, sporozoite surface antigen, circumsporozoite antigen, gametocyte / gamete surface antigen, blood stage antigen pf155 / RESA, and other Plasmodium antigen components; Toxoplasma gondii antigens such as SAG-1, p30, and other Toxoplasma gondii antigen components; Schistosoma antigens such as glutathione-S-transferase, paramyosin, and other Schistosoma antigen components; Leishmania and other Leishmania antigens such as gp63, lipophosphoglycan, and its associated proteins, and other Leishmania antigen components; and Trypanosoma cruzi antigens such as 75-77 kDa antigen, 56 kDa antigen, and other trypanosome antigen components.Examples of fungal antigens include, but are not limited to, those from the genera Candida, Aspergillus species, Bacillus species, Histoplasma, Coccidioides species, Malassezia furfur and other species, Exophiala werneckii and other species, Piedraia hortai and other species, Trichosporum beigelii and other species, Microsporum species, Trichophyton species, Epidermophyton species, Sporothrix schenckii and other species, Fonsecaea pedrosoi and other species, Wangiella dermatitidis and other species, Pseudallescheria boydii and other species, Madurella grisea and other species, Rhizopus species, Absidia species, and Mucor species. Examples of prion disease antigens include PrP, beta-amyloid protein, and other prion-related proteins.

[0060] In addition to the infectious and parasitic agents described above, another area where enhanced immunogenicity of non-infectious agents is desirable is in the area of inflammatory and autoimmune diseases, neurodegenerative diseases, and proliferative diseases, including but not limited to cancer, where cells expressing cancer antigens are desirably eliminated from the body. Tumor antigens that can be used in the compositions and methods of the present technology include, but are not limited to, prostate specific antigen (PSA), breast cancer, ovarian cancer, testicular cancer, melanoma, telomerase; multidrug resistance proteins such as P-glycoprotein; MAGE-1, alpha-fetoprotein, carcinoembryonic antigen, p53 mutants, papillomavirus antigens, gangliosides, or other carbohydrate-containing components of melanoma or other tumor cells. The present technology contemplates that antigens from any type of tumor cell can be used in the compositions and methods described herein. The antigen can be a cancer cell, or an immunogenic substance isolated from a cancer cell, such as a membrane protein. The MAGE family, including survivin and telomerase universal antigens and cancer-testis antigens. Antigens that have been shown to be involved in autoimmunity and can be used in the methods of the present technology to induce tolerance include, but are not limited to, myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein in multiple sclerosis and CII collagen in rheumatoid arthritis.

[0061] The antigen can be part of an infectious agent such as HIV-1, EBV, HBV, influenza virus, SARS virus, poxvirus, malaria or HSV, as non-limiting examples, for vaccines that require the mobilization of strong T cell-mediated immunity (through dendritic cells).

[0062] As used herein, the term "cancer" is defined as the excessive proliferation of cells, the unique trait of which - loss of normal control - results in unregulated growth, lack of differentiation, invasion of local tissue and metastasis. Examples include but are not limited to melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, leukemia, retinoblastoma, astrocytoma, glioblastoma, gingival cancer, tongue cancer, neuroblastoma, head cancer, neck cancer, breast cancer, pancreas, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, sarcoma or bladder cancer.

[0063] The term "tumor" denotes a cell or cell mass of at least one new form of tissue, in particular in the form of a more or less derepressed overgrowth that is spontaneous, autonomous and irreversible in endogenous tissue, the growth of which is usually associated with a more or less pronounced loss of specific cell and tissue functions. In terms of its growth, the inhibition of the cell or cell mass by its own or the regulatory mechanisms of the host organism is not effective, for example, melanoma or carcinoma. Tumor antigens include not only antigens present in or on the surface of malignant cells themselves, but also antigens present on the stromal support tissue of the tumor, including endothelial cells and other vascular components. In a related aspect, "neoplastic" refers to an abnormal new growth and thus is the same as a tumor and can be benign or malignant. In addition, such neoplasms will include cell proliferation disorders.

[0064] The lentiviral vector of the present technology further includes a nucleic acid sequence encoding one or more adjuvants. A preferred adjuvant is the fusion protein LMP1(delta)hIPS1, which contains LMP1 from Epstein-Barr virus, without the cytoplasmic region, and is fused to the full length of human IPS1. In the fusion protein, the first amino acid (methionine) of human IPS1 is removed. The fusion protein is codon-optimized for human use. The DNA and encoded amino acid sequences of the fusion protein are shown below:

[0065] DNA sequence

[0066]

[0067] Protein sequence

[0068] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSDRPPDPLEPPSLPAERPGPPTPAAAHSIPYNSCREKEPSYPMPVQETQAPESPGENSEQALQTLSPRAIPRNPDGGPLESSSDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRASRLPGPTGSVVSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLPSKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPINSTRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSSENRGLGSELSKPGVLASQVDSPFSGCFEDLAISASTSLGMGPCHGPEENEYKSEGTFGIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSPGALWLQVAVTGVLVVTLLVVLYRRRLH (SEQ ID NO:2)

[0069] Another preferred adjuvant is the fusion protein LMP1(deltaIC)hIPS1(deltaTM), which contains LMP1 from Epstein - Barr virus without the cytoplasmic domain, fused to amino acids 2 - 439 of human IPS1 without its transmembrane domain. In the fusion protein, the first amino acid (methionine) of human IPS1 is removed. The fusion protein is codon - optimized for human use. The DNA and encoded amino acid sequences of this fusion protein are shown below:

[0070] DNA sequence

[0071]

[0072] Protein sequence

[0073] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSDRPPDPLEPPSLPAERPGPPTPAAAHSIPYNSCREKEPSYPMPVQETQAPESPGENSEQALQTLSPRAIPRNPDGGPLESSSDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRASRLPGPTGSVVSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLPSKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPINSTRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSSENRGLGSELSKPGVLASQVDSPFSGCFEDLAISASTSLGMGPCHGPEENEYKSEGTFGIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSP(SEQ ID NO:4)

[0074] Another preferred adjuvant is the fusion protein LMP1(deltaIC)hIPS1(delta-TM delta-Pro), which contains LMP1 from Epstein-Barr virus without the cytoplasmic domain. It is fused with amino acids 2-93 of human IPS1 (truncated IPS1 with the C-terminal proline-rich domain and transmembrane domain deleted). In the fusion protein, the first amino acid (methionine) of human IPS1 is removed. The fusion protein is codon-optimized for human use. The DNA and encoded amino acid sequences of this fusion protein are shown below:

[0075] DNA sequence

[0076]

[0077] Protein sequence

[0078] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSDRGENSEQALQTLSPRAIPRNPDGGPLESSSDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRASRLPGPTGSVVSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLPSKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPINSTRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSSENRGLGSELSKPGVLASQVDSPFSGCFEDLAISASTSLGMGPCHGPEENEYKSEGTFGIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSP

[0079] (SEQ ID NO:6)

[0080] Another preferred adjuvant is the fusion protein LMP1(delta IC)hIPS1(reverse delta TM), which contains LMP1 from Epstein - Barr virus without the cytoplasmic region. It is fused with amino acids 2 - 439 of human IPS1 (truncated IPS1 with the transmembrane domain deleted and presented in reverse amino acid order, i.e., from position 439 to 2, in the direction from the C - terminus to the N - terminus of native IPS1). In the fusion protein, the first amino acid (methionine) of human IPS1 is removed. The fusion protein is codon - optimized for human use. The DNA and encoded amino acid sequences of this fusion protein are shown below:

[0081] DNA sequence:

[0082]

[0083] (SEQ ID NO:7)

[0084] Protein sequence:

[0085] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRPSPRHCPVEREQFKRDAQPRPGGDPDAPPGPNGELLQISPNEAVHIGFTGESKYENEEPGHCPGMGLSTSASIALDEFCGSFPSDVQSALVGPKSLESGLGRNESSSDLWASSGGTAGAPTPAAPTEENRSSGDTPVTSASVKTLVMSTPVKSPVMGARTSNIPLKSPAPNTLANSPVAGPPKSSTPLKSPVTSVSAPNAPVKLAVTNVPMLTTPVKSPLSNAPAEAGSSCIIPEAQDSEAGQKGEAAGASALGPSSSSFSTGTSVVSGTPGPLRSARPTSRALPQFSVSPSVPGRSPTLSSTAGATHTSGLETDQEQHGSSTLPSLAALDSSSELPGGDPNRPIARPSLTQLAQESNEGPSEPAQTEQVPMPYSPEKERCSNYPISHAAAPTPPGPREAPLSPPELPDPPRDSTRPQYSQYVSAVEDALDVLECGRLAAIFYEVWGPRRQLTNFLHWLTDRNGSLTCTARLRDQDRATLCPLYPLIEVVDVNCFNSFNRCIYKYTKDEAFPM(SEQ ID NO:8)

[0086] In a preferred embodiment, the immune checkpoint inhibitor molecule is encoded within a viral vector, enhancing the immune response against the tumor. The immune checkpoint inhibitor molecule can be, but is not limited to, an anti-CTLA-4 molecule, a PD1 blocker, and a PDL1 blocker. The immune checkpoint inhibitor molecule can be a protein, such as an antibody, or a soluble form of an anti-checkpoint.

[0087] In certain embodiments, the viral vector can include more than one expression cassette. In some embodiments, the viral vector particles can include more than one nucleic acid molecule, such as two or three nucleic acid molecules, which can be delivered separately or operably linked. In some embodiments, the second nucleic acid encodes an antigen and / or a soluble immune checkpoint inhibitor molecule or a soluble immunomodulator molecule. In some embodiments, the third nucleic acid encodes an antigen and / or an immune checkpoint inhibitor molecule different from that encoded by the second nucleic acid molecule.

[0088] In one aspect, the technology is an immunotherapeutic formulation for preventing or treating a disease or disorder in a subject. The vaccine includes a therapeutically effective amount of the viral vector. The disease can be any disease that requires an inoculant, such as cancer or an infection.

[0089] In another aspect, the technology is a method for inducing or enhancing an immune response against cancer or an infection in a subject. The method includes administering a therapeutically effective amount of the viral vector or the immunotherapeutic formulation to a subject in need thereof. Detailed Description

[0090] Example 1. Molecular Constructs.

[0091] Construct vectors to contain the following genetic elements: (a) a promoter, preferably the human ubiquitin protein promoter; (b) a reporter gene (such as green fluorescent protein), or one or more antigens fused into a single transgene; (c) an IRES, followed by an adjuvant gene (i.e., LMP1-IPS1CO or a functional variant thereof). Optionally, the vector can include (d) an IRES, followed by a soluble immune checkpoint inhibitor gene or a soluble immunomodulator gene ( Figure 8C ). Preferably, the sequences are in the above order, but the genes can be located in the vector in any other suitable order. Control vectors having some but not all of the above regions were also constructed.

[0092] Example 2. Generation of Viral Vectors.

[0093] As described by Nasri et al. (2014), lentiviral vectors are produced by transient calcium phosphate transfection of the HEK 293T cell line. The HEK 293T cells are seeded at 1.6×10 8Cells were seeded in 250 mL of complete medium in a two-chamber cell culture vessel (Cell Stack, Corning) and maintained in an incubator with a humid atmosphere of 5% CO2 at 37 °C for 24 hours to allow cell adhesion. For the production of each vector, each cell culture vessel was transfected as follows. The lentiviral backbone plasmid (235 μg), envelope-encoding plasmid (47 μg), and packaging plasmid (235 μg) were mixed with 8.6 mL of sterile distilled water and 3.0 mL of CaCl2. Then the DNA mixture was added dropwise to 12.1 mL of pre-warmed HBS 2X at 37 °C, pH = 7.1, and the resulting 24.2 mL precipitate was added to the cell medium after incubation at room temperature for 30 minutes. The transfected cells were incubated at 37 °C, 5% CO2. Twenty-four hours after transfection, the medium was replaced with 210 mL of serum-free and phenol red-free harvest medium, and after another 24 hours, the viral supernatant was harvested and clarified by centrifugation at 2500 rpm for 5 minutes. The harvested clarified material (210 mL) was treated with DNase I in the presence of MgCl2 for 30 minutes to lyse any residual DNA and concentrated by centrifugation at 22000 rpm, 4 °C for 1 hour. The vector pellet was resuspended in 70 μL of Tris-trehalose (50 mM), pooled in 1.5 mL microtubes, aliquoted into 50 μL portions, and frozen and stored at ≤ -70 °C. Compared to the GFP vector, the yield of the vector containing adjuvant was slightly lower, of course due to the presence of a longer DNA expression cassette. However, for all adjuvanted constructs, the titers were at least in the range of 109 TU / mL and were consistently obtained in different production runs. Therefore, there is no need to dispute the issue of future industrial bioproduction of these adjuvanted constructs.

[0094] Example 3. In Vitro Effects of Lentiviral Vectors Expressing LMP1 - IPS1

[0095] Fresh human dendritic cells and macrophages were obtained from healthy human donors (leukocyte concentrates) by density gradient. CD14+ monocytes were purified from PBMCs using a magnetic separation kit (positive selection) and plated in complete RPMI in 6-well plates. Monocytes were differentiated into dendritic cells using GM-CSF and IL-4 by a method that has been published. The medium was replaced with 10% to replenish cytokines after 3 days, and the cells were harvested after a total of 6 days of culture using a non-enzymatic cell dissociation solution. The DCs were then re-plated in complete RPMI medium + 4 μg / ml polybrene + lentiviral construct (MOI of 15) + GM-CSF and IL-4. After 2 hours, 700 μL of complete RPMI medium + GM-CSF / IL-4 was added, and the cells were cultured for a total of 96 hours. Additional control wells were stimulated with IFN-γ and LPS for 96 hours as a positive control for the expression of activation markers.

[0096] Differentiate CD14+ monocytes into M1 or M2 macrophages using GM-CSF (M1) or M-CSF (M2). Replace 10% of the medium after 3 days to replenish cytokines, and harvest the cells after culturing for a total of 6 days using a non-enzymatic cell dissociation solution. Then, combine the macrophages at a 1:1 ratio. Next, re-seed the M1 / M2 macrophages in 300 μl of complete RPMI + 4 μg / ml polybrene + lentiviral construct (MOI of 15) + M-CSF). After 2 hours, add 700 μl of complete RPMI + M-CSF, and culture the cells for a total of 96 hours. Stimulate additional control wells with IFN-γ and LPS (M1) or IL-13 and IL-4 (M2) for a total of 96 hours as a positive control for the expression of activation markers.

[0097] Transduce human DCs and macrophages with a lentiviral vector containing an expression cassette at an MOI of 15 as described below:

[0098] Construct 1: GFP-IRES-LMP1(dIC)hIPS1

[0099] Construct 2: GFP-IRES-LMP1(dIC)hIPS1(dTM)

[0100] Construct 3: GFP-IRES-LMP1(dIC)hIPS1(dTMdPro)

[0101] Construct 4: GFP-IRES-LMP1(dIC)hIPS1(dTMRev)

[0102] Control construct 1: GFP

[0103] Control construct 2: GFP + LMP1(dIC)

[0104] Control construct 3: Cells co-transduced with GFP and LMP1(dIC)hIPS1 (in separate vectors, each at an MOI of 15).

[0105] See Figure 8A For an illustration of the control constructs and Figure 8B Are adjuvanted constructs.

[0106] Quantify the proliferation of dendritic cells and macrophages after 24 hours of culture. Prepare samples in triplicate with 3H-TdR pulses were given and cultured overnight, then harvested and the incorporation of radioactive thymidine was measured by a standard scintillation counter. Compared to the GFP vector, the use of the adjuvanted vector slightly reduced proliferation, most likely due to the presence of a longer DNA expression cassette. As previously described, the viability of transduced cells was determined by staining with a fixable viability dye prior to analysis on a BD FACS Canto System flow cytometer. Although slight differences were observed with the adjuvanted vector, no significant toxicity was found.

[0107] Fluorescence was detected using an Attune NxT flow cytometer after 96 hours of culture, and GFP expression in cells transduced with each construct was measured, and the results are as Figure 9A (dendritic cells) and 9B (macrophages) shown. The percentages of live cells and GFP-positive cells were determined by gating on excluded debris / live cells / single cells. Three independent experiments were performed with PBMC isolated from different donors. Graph data represent the mean of replicates of a representative experiment. The results are as in Figure 9A (dendritic cells) and 9B (macrophages) shown. For both cell types, slight differences were observed between the adjuvanted vectors, and significant expression of GFP / transgene was observed in all IRES constructs. In the adjuvanted vectors, GFP / transgene expression increased with constructs 2 and 3, most likely due to the presence of a shorter DNA expression cassette. Removing the IPS1 transmembrane domain and reversing the orientation of the IPS1 CARD and PRO domains simultaneously did not result in improved GFP / transgene expression.

[0108] The activation and maturation of lentivirus vector-induced dendritic cells and macrophages were evaluated by measuring the expression of surface markers and assessing the release profiles of their cytokines and chemokines. To determine the levels of lentiviral integration and dendritic cell / macrophage activation, cells were harvested after 96 hours of culture and stained with a fixable viability dye and a panel of staining antibodies recognizing the following surface markers: CD25, CD40, CD69, CD80 / 86, CD83, CCR7, MHC I, and MHC II, and then analyzed using a BD FACS Canto System flow cytometer. Cell frequencies and geometric mean (Gmean) marker expression values were determined by gating on excluded debris / live cells / single cells. All expression levels were normalized to the expression of GFP. For dendritic cells and macrophages, after 96 hours of transduction, the production of IFN-α and IFN-β, as well as the immunostimulatory cytokines IL-8, IL-1beta, TNF-α, IL-6, and IL-12p70, in the culture supernatants was measured by Luminex analysis using a Bio-plex 200 system (BioRad) with high-throughput fluidics to evaluate the activation of the STING pathway. The production of the immunosuppressive cytokine IL-10 was measured as a control. Three independent experiments were performed using PBMCs isolated from different healthy donors. Graph data represent the repeated means of a representative experiment. The results are shown in Figure 10A (dendritic cells, cytokines), 10B (dendritic cells, membrane markers), 10C (macrophages, cytokines), and 10D (macrophages, membrane markers).

[0109] For transduced dendritic cells, the results of the expression of surface markers on GFP-positive cells showed that the IRES construct upregulated the expression of the following immune activation molecules: MHCII (constructs 1 and 2 were better); CD40 (increased 4- to 5-fold, especially constructs 1 and 4); CD80 / 86 (constructs 1, 2, and 4); CD83 (increased 3- to 4-fold in constructs 2 and 3, far better than control 3); and the CCR7 migration signal (constructs 1 and 2, higher than control 3). Consistent with the upregulation of these activation surface markers, the increase in cytokine expression was as follows: the pro-inflammatory IL-6 was stimulated in constructs 2 and control 3; the pro-inflammatory TNF-α increased in constructs 2 and 3 (better than controls 2 and 3); IL-12 increased in constructs 2 and 4 (better than controls 2 and 3). None of the evaluated constructs affected the level of the anti-inflammatory IL-10. The results of transduced dendritic cells indicated that the deletion of the IPS1 transmembrane domain increased the adjuvant activity; the deletion of the IPS1 transmembrane region and the proline (PRO)-rich domain slightly increased the adjuvant activity; and the deletion of the IPS1 transmembrane domain while reversing the orientation of the IPS1 CARD and PRO domains did not show any immunostimulatory effect.

[0110] For transduced macrophages, the expression results of surface markers of GFP-positive cells showed that the IRES construct upregulated the expression of the following immune activation molecules: CD83 was significantly increased in constructs 1 and 3, better than control 2; CD80 / 86 was increased in construct 2; and the early activation marker CD69 was induced at a level higher than control 3 in constructs 2 and 3. Consistent with the results observed in dendritic cells, the enhanced expression of activation markers was associated with an increase in cytokine expression as follows: the pro-inflammatory IL-1β was increased 4-fold in constructs 2 and 4, and also in control 3; the pro-inflammatory IL-6 was increased 4-fold in constructs 2 and 3, better than control 3; and the pro-inflammatory TNF-α was increased (better than control 3) in constructs 2 and 3. None of the constructs evaluated affected the level of anti-inflammatory IL-10.

[0111] In summary, the fusion of the LMP1 transmembrane region with the human IPS1 protein increased the adjuvant effect on dendritic cells and macrophages. In addition, the optimization of the construct (removing the transmembrane domain of the IPS1 protein, see Figure 8B ) increased the adjuvant activity.

[0112] When the transmembrane domain was removed and the proline-rich domain (PRO) was also removed, the adjuvant effect increased only slightly. The function of the PRO region could not be clearly described, but it may play a role in the conformation of the IPS1 protein.

[0113] Removing the IPS1 transmembrane domain while reversing the orientation of the IPS1 CARD and PRO domains showed a reduced immune-stimulating effect. This deletion may lead to an incorrect conformation of the protein and loss of activity.

[0114] Example 4. In Vivo Immunogenicity of Healthy Mice Treated with Single or Multiple Antigens is Demonstrated by the LMP1 - IPS1 Lentiviral Vector, Exhibiting Excellent Immunogenicity. Example 5. In Vivo Immunogenicity in a Specific Tumor Mouse Model Shows the Excellent Efficacy of a Lentiviral Vector Comprising Multiple Antigens and LMP1 - IPS1 as an Adjuvant.

[0115] Healthy mice were treated with different viral vectors containing an expression cassette encoding (a) a human ubiquitin protein promoter; (b) a tumor antigen; (c) the transmembrane domain of LMP1 (codons optimized for human expression) and a fusion protein of human IPS1 or its functional variant. Experiments were conducted to compare the immune responses after two administrations (prime + boost) when the antigen and adjuvant (i.e., the LMP1-IPS1 fusion) were expressed from different vectors vs when expressed from the same vector. Short-term (3 weeks) and long-term (3 months) in vivo immunogenicity assessments were performed by FACS analysis of biomarkers (IFN-γ and various interleukins) in the blood of the mice, which allowed the detection and quantification of antigen-specific immune cells, such as CD4+, CD8+, and memory T cells targeting the antigen present in the vector. Treatment with a lentiviral vector encoding the antigen and LMP1-IPS1 was expected to increase specific immunogenicity when compared to the same lentiviral vector without LMP1-IPS1 or expressing only the LMP1 membrane domain. In addition, a greater increase in immunogenicity was expected when the antigen and adjuvant were expressed from the same vector compared to expression from different vectors.

[0116] Example 6. In Vivo Immunogenicity in a Mouse Model of Specific Anti - Checkpoint - Sensitive Tumors Shows the Excellent Efficacy of a Lentiviral Vector Containing Multiple Antigens, Adjuvant, and Anti - Checkpoint Combinations. References

[0117] A mouse model of a specific tumor was treated with a lentiviral vector containing an expression cassette encoding (a) a human ubiquitin protein as a promoter; (b) a tumor-specific antigen; and a fusion protein of the transmembrane domain of LMP1 (codons optimized for human expression) and human IPS1 or its functional variant. The mice were divided into different treatment groups according to vector type and construct, dose, and number of injections. The vectors administered to the experimental groups (prime + boost injection) encoded: only the indicated specific antigen; only LMP1-IPS1 or the indicated specific antigen, and LMP1-IPS1 (or a variant with functional IPS1). The in vivo efficacy and immunogenicity were evaluated by detecting the specific antigen of CD4+, CD8+, and memory T cells through the tumor growth rate, survival rate, and FACS analysis of biomarkers (IFN-γ and various interleukins) in the blood of the mice. A lentiviral vector encoding the indicated specific antigen and the LMP1-IPS1 fusion protein was expected to induce the most effective and long-lasting immune response in all experimental groups, thereby inducing a higher survival rate and / or slower tumor growth in the treatment groups of the mice.

[0118] ​ ​

[0119] A mouse model of a specific tumor is treated with a lentiviral vector containing an expression cassette that encodes human ubiquitin protein as a promoter and at least one of the following: an indicator of a specific antigen, LMP1-IPS1, and soluble and secreted forms of one or more anti-checkpoint molecules. The mice are divided into different treatment groups according to the vector construct, dose, and number of injections. The vectors administered to the experimental groups (prime + boost) encode: only the indicator of a specific antigen; only LMP1-IPS1; only one or more soluble and secreted anti-checkpoint molecules; the indicator of a specific antigen and LMP1(deltaIC); the indicator of a specific antigen and LMP1(deltaIC), and one or more soluble and secreted anti-checkpoint molecules; the indicator of a specific antigen and LMP1-IPS1 (or a variant with functional IPS1); or the indicator of a specific antigen and one or more soluble and secreted anti-checkpoint molecules; or the indicator of a specific antigen, LMP1-IPS1 or a variant with functional IPS1, and one or more soluble and secreted anti-checkpoint molecules. In vivo efficacy and immunogenicity are evaluated by tumor growth rate, survival rate, and detection of specific antigens of CD4+, CD8+, and memory T cells using FACS analysis of mouse blood biomarkers (IFN-γ and various interleukins). Lentiviral vectors encoding an indicator of a specific antigen, LMP1-IPS1 (or a variant with functional IPS1), and anti-checkpoint molecules are expected to induce the most effective and long-lasting immune responses in all experimental groups.

[0120] This application claims priority to U.S. Provisional Application No. 62 / 426,855, filed on November 28, 2016, the entire content of which is incorporated herein by reference.

[0121] As used in the present invention, "consisting essentially of" allows for the inclusion of materials or steps that do not substantially affect the basic and novel features of the claim. Any recitation of the term "comprising" in the present invention, particularly in the description of the components of a composition or the elements of a device, may be interchanged with "consisting essentially of" or "consisting of".

[0122] ​

[0123] Barry, M. et al. Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection.

[0124] Hum Gene Ther,10(15)(1999),pp.2461–2480.

[0125] McNamara,M.et al.RNA-Based Vaccines in Cancer Immunotherapy.J ImmunolRes.2015;2015:794528.

[0126] Nasri et al.,Production,Purification and Titration of a Lentivirus-Based Vector for Gene Delivery Purposes,Cytotechnology 66,1031-8(2014). SEQUENCE LISTING <110> Olatinga AIO Biologics <120> Viral Vector Construct for Expression of a Gene Adjuvant for Activating the STING Pathway <130> P19111932WP <150> US 62 / 426,855 <151> 2016-11-28 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 2193 <212> DNA <213> Artificial sequence <220> <223> Fusion Protein of Epstein-Barr Virus LMP1 and Homo sapiens IP1 <400> 1 atggatctgg atctcgaaag aggacctcct ggacctagac ggcctcctag aggaccacct 60 ctgagcagct ctattggact ggccctgctg ctgcttctgc tggctctgct gttctggctg 120 tacatcatca tgagcaactg gaccggcgga gcactgctgg tgctgtatgc ctttgctctg 180 atgctggtca tcatcatcct gatcatcttc atcttccggc gggacctgct gtgtcctctg 240 Met Leu Val Ser Ile Ile Pro Asp Ile Leu Phe Ile Phe Arg Ala Asp Leu Leu Cys Ser Leu 240 ggagcacttt gtctgttgct gctgatgatc accctcctgc tgatcgccct gtggaacctg 300 Gly Ser Thr Phe Val Cys Cys Ala Ala Asp Asp Ile Thr Leu Leu Leu Ile Ala Leu Trp Asn Leu 300 catggacagg ccctgtatct gggcatcgtg ctgttcatct tcggctgcct gctggttctc 360 His Gly Asp Arg Pro Cys Ile Leu Gly Ile Val Leu Phe Ile Phe Gly Cys Pro Cys Gly Ser Ser 360 ggcctgtgga tctacctgct ggaaatcctt tggagactgg gcgccaccat ctggcagctg 420 Gly Pro Trp Ile Tyr Leu Leu Gly Asn Pro Phe Gly Asp Trp Ala Pro Ile Trp Gln Leu 420 ctggcctttt tcctggcctt ctttctggat atcatcctcc tcatcattgc cctgtacctg 480 Leu Ala Phe Phe Pro Gly Pro Phe Ser Gly Ile Ile Pro Ser Ile Ile Ala Pro Tyr Leu 480 cagcagaact ggtggaccct gctggtggat ctgctttggc tgctgctctt tctggccatc 540 Gln Gln Asn Gly Gly Thr Pro Leu Gly Gly Asp Leu Leu Gly Ala Ala Ala Ser Ser Gly Ile 540 ctgatttgga tgtactacca cggccagcgg cctttcgccg aggacaagac ctacaagtac 600 Leu Asp Phe Gly Cys Tyr Thr Thr Gly Gln Arg Ala Phe Arg Glu Asp Lys Thr Tyr Lys Tyr 600 atctgccgga acttcagcaa cttctgcaac gtggacgtgg tggaaattct gccctacctg 660 Ile Cys Gly Asn Phe Ser Asn Phe Cys Asn Val Asp Val Val Glu Ile Ser Ala Leu Leu 660 ccttgcctga ccgccagaga tcaggacaga ctgagagcca catgtaccct gagcggcaac 720 Pro Cys Pro Thr Ala Gln Arg Ile Arg Asp Arg Leu Ser Ala His Met Tyr Pro Glu Arg Asn 720 agagacacac tgtggcacct gttcaacacc ctgcagagaa ggcctggctg ggtcgagtac 780 Arg Asp Thr Cys Gly Thr Val Gln Thr Pro Gln Arg Lys Ala Trp Leu Gly Ser Glu Tyr 780 tttatcgccg ctctgagagg ctgcgagctg gtcgatctgg ctgatgaagt ggccagcgtg 840 Phe Ile Ala Ala Ser Glu Arg Ala Ala Glu Leu Val Asp Leu Ala Asp Glu Val Ala Gln Val 840 taccagagct accagcctag aaccagcgac cggcctcctg atcctctcga acctccatct 900 Tyr Gln Ser Tyr Gln Pro Arg Asn Gln Arg Thr Gly Pro Pro Ile Pro Ser Asn Pro His Ile 900 ctgcccgccg aaagacctgg acctcctaca ccagctgccg ctcacagcat cccttacaac 960 agctgcagag agaaagaacc tagctacccc atgcctgtgc aagagacaca ggccccagaa 1020 agccctggcg agaatagcga acaggctctg cagacactga gccccagagc cattcctaga 1080 aaccctgatg gcggccctct ggaaagctct agtgatctgg ccgctctgtc ccctctgaca 1140 agctctggac accaagagca ggataccgag ctgggcagca cacatacagc cggcgctaca 1200 agcagcctga caccttctag aggccccgtg tctcccagcg tgtcatttca gcctctggcc 1260 aggtctaccc ctagggcttc tagactgcct ggaccaacag gcagcgtggt gtctaccggc 1320 acaagcttca gctctagctc tcctggactg gctagtgccg gtgccgctga gggaaaacaa 1380 ggcgccgaat ctgatcaggc cgagcctatc atctgtagca gcggagcaga agcccctgcc 1440 aatagcctgc ctagcaaggt gccaaccaca ctgatgcccg tgaacacagt ggccctgaag 1500 gtgccagcta atcctgcctc cgtgtccacc gtgccttcta agctgccaac cagctctaag 1560 ccacctggcg ccgtgccatc taacgccctg acaaatcctg ctccaagcaa gctgcccatc 1620 aactccacaa gagccggcat ggtgccctct aaggtgccca catctatggt gctgaccaag 1680 gtgtccgcca gcaccgtgcc aacagatggc agctccagaa acgaggaaac ccctgccgct 1740 cctactcctg ctggcgctac aggcggatct tctgcttggc tggatagcag cagcgagaac 1800 agaggcctgg gcagcgagct ttctaaacct ggcgtgctgg cttcccaggt ggacagccca 1860 ttttccggct gctttgagga cctggctatc agcgcctcta caagcctcgg catgggacct 1920 tgtcacggcc ccgaggaaaa cgagtacaag agcgagggca ccttcggcat ccacgtggcc 1980 gagaatccta gcatccaact gctggaaggc aaccccggac ctccagctga tccagatggc 2040 ggaccaagac ctcaggccga cagaaagttc caagagcgcg aggtgccctg ccacagacct 2100 tctccaggtg ctctgtggct gcaggttgca gtgacaggcg tcctggtggt tacactgctc 2160 gtggtcctgt atagacggcg gctgcactga tga 2193 <210> 2 <211> 729 <212> PRT <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens IP1 fusion protein <400> 2 Met Asp Leu Asp Leu Glu Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro 1 5 10 15 Arg Gly Pro Pro Leu Ser Ser Ser Ile Gly Leu Ala Leu Leu Leu Leu 20 25 30 Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Ile Met Ser Asn Trp Thr 35 40 45 Gly Gly Ala Leu Leu Val Leu Tyr Ala Phe Ala Leu Met Leu Val Ile 50 55 60 Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu 65 70 75 80 Gly Ala Leu Cys Leu Leu Leu Leu Met Ile Thr Leu Leu Leu Ile Ala 85 90 95 Leu Trp Asn Leu His Gly Gln Ala Leu Tyr Leu Gly Ile Val Leu Phe 100 105 110 Ile Phe Gly Cys Leu Leu Val Leu Gly Leu Trp Ile Tyr Leu Leu Glu 115 120 125 Ile Leu Trp Arg Leu Gly Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe 130 135 140 Leu Ala Phe Phe Leu Asp Ile Ile Leu Leu Ile Ile Ala Leu Tyr Leu 145 150 155 160 Gln Gln Asn Trp Trp Thr Leu Leu Val Asp Leu Leu Trp Leu Leu Leu 165 170 175 Phe Leu Ala Ile Leu Ile Trp Met Tyr Tyr His Gly Gln Arg Pro Phe 180 185 190 Ala Glu Asp Lys Thr Tyr Lys Tyr Ile Cys Arg Asn Phe Ser Asn Phe 195 200 205 Cys Asn Val Asp Val Val Glu Ile Leu Pro Tyr Leu Pro Cys Leu Thr 210 215 220 Ala Arg Asp Gln Asp Arg Leu Arg Ala Thr Cys Thr Leu Ser Gly Asn 225 230 235 240 Arg Asp Thr Leu Trp His Leu Phe Asn Thr Leu Gln Arg Arg Pro Gly 245 250 255 Trp Val Glu Tyr Phe Ile Ala Ala Leu Arg Gly Cys Glu Leu Val Asp 260 265 270 Leu Ala Asp Glu Val Ala Ser Val Tyr Gln Ser Tyr Gln Pro Arg Thr 275 280 285 Ser Asp Arg Pro Pro Asp Pro Leu Glu Pro Pro Ser Leu Pro Ala Glu 290 295 300 Arg Pro Gly Pro Pro Thr Pro Ala Ala Ala His Ser Ile Pro Tyr Asn 305 310 315 320 Ser Cys Arg Glu Lys Glu Pro Ser Tyr Pro Met Pro Val Gln Glu Thr 325 330 335 Gln Ala Pro Glu Ser Pro Gly Glu Asn Ser Glu Gln Ala Leu Gln Thr 340 345 350 Leu Ser Pro Arg Ala Ile Pro Arg Asn Pro Asp Gly Gly Pro Leu Glu 355 360 365 Ser Ser Ser Asp Leu Ala Ala Leu Ser Pro Leu Thr Ser Ser Gly His 370 375 380 Gln Glu Gln Asp Thr Glu Leu Gly Ser Thr His Thr Ala Gly Ala Thr 385 390 395 400 Ser Ser Leu Thr Pro Ser Arg Gly Pro Val Ser Pro Ser Val Ser Phe 405 410 415 Gln Pro Leu Ala Arg Ser Thr Pro Arg Ala Ser Arg Leu Pro Gly Pro 420 425 430 Thr Gly Ser Val Val Ser Thr Gly Thr Ser Phe Ser Ser Ser Ser Pro 435 440 445 Gly Leu Ala Ser Ala Gly Ala Ala Glu Gly Lys Gln Gly Ala Glu Ser 450 455 460 Asp Gln Ala Glu Pro Ile Ile Cys Ser Ser Gly Ala Glu Ala Pro Ala 465 470 475 480 Asn Ser Leu Pro Ser Lys Val Pro Thr Thr Leu Met Pro Val Asn Thr 485 490 495 Val Ala Leu Lys Val Pro Ala Asn Pro Ala Ser Val Ser Thr Val Pro 500 505 510 Ser Lys Leu Pro Thr Ser Ser Lys Pro Pro Gly Ala Val Pro Ser Asn 515 520 525 Ala Leu Thr Asn Pro Ala Pro Ser Lys Leu Pro Ile Asn Ser Thr Arg 530 535 540 Ala Gly Met Val Pro Ser Lys Val Pro Thr Ser Met Val Leu Thr Lys 545 550 555 560 Val Ser Ala Ser Thr Val Pro Thr Asp Gly Ser Ser Arg Asn Glu Glu 565 570 575 Thr Pro Ala Ala Pro Thr Pro Ala Gly Ala Thr Gly Gly Ser Ser Ala 580 585 590 Trp Leu Asp Ser Ser Ser Glu Asn Arg Gly Leu Gly Ser Glu Leu Ser 595 600 605 Lys Pro Gly Val Leu Ala Ser Gln Val Asp Ser Pro Phe Ser Gly Cys 610 615 620 Phe Glu Asp Leu Ala Ile Ser Ala Ser Thr Ser Leu Gly Met Gly Pro 625 630 635 640 Cys His Gly Pro Glu Glu Asn Glu Tyr Lys Ser Glu Gly Thr Phe Gly 645 650 655 Ile His Val Ala Glu Asn Pro Ser Ile Gln Leu Leu Glu Gly Asn Pro 660 665 670 Gly Pro Pro Ala Asp Pro Asp Gly Gly Pro Arg Pro Gln Ala Asp Arg 675 680 685 Lys Phe Gln Glu Arg Glu Val Pro Cys His Arg Pro Ser Pro Gly Ala 690 695 700 Leu Trp Leu Gln Val Ala Val Thr Gly Val Leu Val Val Thr Leu Leu 705 710 715 720 Val Val Leu Tyr Arg Arg Arg Leu His 725 <210> 3 <211> 2106 <212> DNA <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens IP1 fusion protein <400> 3 atggatctgg atctcgaaag aggacctcct ggacctagac ggcctcctag aggaccacct 60 ctgagcagct ctattggact ggccctgctg ctgcttctgc tggctctgct gttctggctg 120 tacatcatca tgagcaactg gaccggcgga gcactgctgg tgctgtatgc ctttgctctg 180 atgctggtca tcatcatcct gatcatcttc atcttccggc gggacctgct gtgtcctctg 240 ggagcacttt gtctgttgct gctgatgatc accctcctgc tgatcgccct gtggaacctg 300 catggacagg ccctgtatct gggcatcgtg ctgttcatct tcggctgcct gctggttctc 360 ggcctgtgga tctacctgct ggaaatcctt tggagactgg gcgccaccat ctggcagctg 420 ctggcctttt tcctggcctt ctttctggat atcatcctcc tcatcattgc cctgtacctg 480 cagcagaact ggtggaccct gctggtggat ctgctttggc tgctgctctt tctggccatc 540 ctgatttgga tgtactacca cggccagcgg cctttcgccg aggacaagac ctacaagtac 600 atctgccgga acttcagcaa cttctgcaac gtggacgtgg tggaaattct gccctacctg 660 ccttgcctga ccgccagaga tcaggacaga ctgagagcca catgtaccct gagcggcaac 720 agagacacac tgtggcacct gttcaacacc ctgcagagaa ggcctggctg ggtcgagtac 780 tttatcgccg ctctgagagg ctgcgagctg gtcgatctgg ctgatgaagt ggccagcgtg 840 taccagagct accagcctag aaccagcgac cggcctcctg atcctctcga acctccatct 900 ctgcccgccg aaagacctgg acctcctaca ccagctgccg ctcacagcat cccttacaac 960 agctgcagag agaaagaacc tagctacccc atgcctgtgc aagagacaca ggccccagaa 1020 agccctggcg agaatagcga acaggctctg cagacactga gccccagagc cattcctaga 1080 aaccctgatg gcggccctct ggaaagctct agtgatctgg ccgctctgtc ccctctgaca 1140 agctctggac accaagagca ggataccgag ctgggcagca cacatacagc cggcgctaca 1200 agcagcctga caccttctag aggccccgtg tctcccagcg tgtcatttca gcctctggcc 1260 aggtctaccc ctagggcttc tagactgcct ggaccaacag gcagcgtggt gtctaccggc 1320 acaagcttca gctctagctc tcctggactg gctagtgccg gtgccgctga gggaaaacaa 1380 ggcgccgaat ctgatcaggc cgagcctatc atctgtagca gcggagcaga agcccctgcc 1440 aatagcctgc ctagcaaggt gccaaccaca ctgatgcccg tgaacacagt ggccctgaag 1500 gtgccagcta atcctgcctc cgtgtccacc gtgccttcta agctgccaac cagctctaag 1560 ccacctggcg ccgtgccatc taacgccctg acaaatcctg ctccaagcaa gctgcccatc 1620 aactccacaa gagccggcat ggtgccctct aaggtgccca catctatggt gctgaccaag 1680 gtgtccgcca gcaccgtgcc aacagatggc agctccagaa acgaggaaac ccctgccgct 1740 cctactcctg ctggcgctac aggcggatct tctgcttggc tggatagcag cagcgagaac 1800 agaggcctgg gcagcgagct ttctaaacct ggcgtgctgg cttcccaggt ggacagccca 1860 ttttccggct gctttgagga cctggctatc agcgcctcta caagcctcgg catgggacct 1920 tgtcacggcc ccgaggaaaa cgagtacaag agcgagggca ccttcggcat ccacgtggcc 1980 gagaatccta gcatccaact gctggaaggc aaccccggac ctccagctga tccagatggc 2040 ggaccaagac ctcaggccga cagaaagttc caagagcgcg aggtgccctg ccacagacct 2100 tctcca 2106 <210> 4 <211> 702 <212> PRT <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens IP1 fusion protein <400> 4 Met Asp Leu Asp Leu Glu Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro 1 5 10 15 Arg Gly Pro Pro Leu Ser Ser Ser Ile Gly Leu Ala Leu Leu Leu Leu 20 25 30 Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Ile Met Ser Asn Trp Thr 35 40 45 Gly Gly Ala Leu Leu Val Leu Tyr Ala Phe Ala Leu Met Leu Val Ile 50 55 60 Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu 65 70 75 80 Gly Ala Leu Cys Leu Leu Leu Leu Met Ile Thr Leu Leu Leu Ile Ala 85 90 95 Leu Trp Asn Leu His Gly Gln Ala Leu Tyr Leu Gly Ile Val Leu Phe 100 105 110 Ile Phe Gly Cys Leu Leu Val Leu Gly Leu Trp Ile Tyr Leu Leu Glu 115 120 125 Ile Leu Trp Arg Leu Gly Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe 130 135 140 Leu Ala Phe Phe Leu Asp Ile Ile Leu Leu Ile Ile Ala Leu Tyr Leu 145 150 155 160 Gln Gln Asn Trp Trp Thr Leu Leu Val Asp Leu Leu Trp Leu Leu Leu 165 170 175 Phe Leu Ala Ile Leu Ile Trp Met Tyr Tyr His Gly Gln Arg Pro Phe 180 185 190 Ala Glu Asp Lys Thr Tyr Lys Tyr Ile Cys Arg Asn Phe Ser Asn Phe 195 200 205 Cys Asn Val Asp Val Val Glu Ile Leu Pro Tyr Leu Pro Cys Leu Thr 210 215 220 Ala Arg Asp Gln Asp Arg Leu Arg Ala Thr Cys Thr Leu Ser Gly Asn 225 230 235 240 Arg Asp Thr Leu Trp His Leu Phe Asn Thr Leu Gln Arg Arg Pro Gly 245 250 255 Trp Val Glu Tyr Phe Ile Ala Ala Leu Arg Gly Cys Glu Leu Val Asp 260 265 270 Leu Ala Asp Glu Val Ala Ser Val Tyr Gln Ser Tyr Gln Pro Arg Thr 275 280 285 Ser Asp Arg Pro Pro Asp Pro Leu Glu Pro Pro Ser Leu Pro Ala Glu 290 295 300 Arg Pro Gly Pro Pro Thr Pro Ala Ala Ala His Ser Ile Pro Tyr Asn 305 310 315 320 Ser Cys Arg Glu Lys Glu Pro Ser Tyr Pro Met Pro Val Gln Glu Thr 325 330 335 Gln Ala Pro Glu Ser Pro Gly Glu Asn Ser Glu Gln Ala Leu Gln Thr 340 345 350 Leu Ser Pro Arg Ala Ile Pro Arg Asn Pro Asp Gly Gly Pro Leu Glu 355 360 365 Ser Ser Ser Asp Leu Ala Ala Leu Ser Pro Leu Thr Ser Ser Gly His 370 375 380 Gln Glu Gln Asp Thr Glu Leu Gly Ser Thr His Thr Ala Gly Ala Thr 385 390 395 400 Ser Ser Leu Thr Pro Ser Arg Gly Pro Val Ser Pro Ser Val Ser Phe 405 410 415 Gln Pro Leu Ala Arg Ser Thr Pro Arg Ala Ser Arg Leu Pro Gly Pro 420 425 430 Thr Gly Ser Val Val Ser Thr Gly Thr Ser Phe Ser Ser Ser Ser Pro 435 440 445 Gly Leu Ala Ser Ala Gly Ala Ala Glu Gly Lys Gln Gly Ala Glu Ser 450 455 460 Asp Gln Ala Glu Pro Ile Ile Cys Ser Ser Gly Ala Glu Ala Pro Ala 465 470 475 480 Asn Ser Leu Pro Ser Lys Val Pro Thr Thr Leu Met Pro Val Asn Thr 485 490 495 Val Ala Leu Lys Val Pro Ala Asn Pro Ala Ser Val Ser Thr Val Pro 500 505 510 Ser Lys Leu Pro Thr Ser Ser Lys Pro Pro Gly Ala Val Pro Ser Asn 515 520 525 Ala Leu Thr Asn Pro Ala Pro Ser Lys Leu Pro Ile Asn Ser Thr Arg 530 535 540 Ala Gly Met Val Pro Ser Lys Val Pro Thr Ser Met Val Leu Thr Lys 545 550 555 560 Val Ser Ala Ser Thr Val Pro Thr Asp Gly Ser Ser Arg Asn Glu Glu 565 570 575 Thr Pro Ala Ala Pro Thr Pro Ala Gly Ala Thr Gly Gly Ser Ser Ala 580 585 590 Trp Leu Asp Ser Ser Ser Glu Asn Arg Gly Leu Gly Ser Glu Leu Ser 595 600 605 Lys Pro Gly Val Leu Ala Ser Gln Val Asp Ser Pro Phe Ser Gly Cys 610 615 620 Phe Glu Asp Leu Ala Ile Ser Ala Ser Thr Ser Leu Gly Met Gly Pro 625 630 635 640 Cys His Gly Pro Glu Glu Asn Glu Tyr Lys Ser Glu Gly Thr Phe Gly 645 650 655 Ile His Val Ala Glu Asn Pro Ser Ile Gln Leu Leu Glu Gly Asn Pro 660 665 670 Gly Pro Pro Ala Asp Pro Asp Gly Gly Pro Arg Pro Gln Ala Asp Arg 675 680 685 Lys Phe Gln Glu Arg Glu Val Pro Cys His Arg Pro Ser Pro 690 695 700 <210> 5 <211> 1953 <212> DNA <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens IP1 fusion protein <400> 5 atggatctgg atctcgaaag aggacctcct ggacctagac ggcctcctag aggaccacct 60 ctgagcagct ctattggact ggccctgctg ctgcttctgc tggctctgct gttctggctg 120 tacatcatca tgagcaactg gaccggcgga gcactgctgg tgctgtatgc ctttgctctg 180 atgctggtca tcatcatcct gatcatcttc atcttccggc gggacctgct gtgtcctctg 240 ggagcacttt gtctgttgct gctgatgatc accctcctgc tgatcgccct gtggaacctg 300 catggacagg ccctgtatct gggcatcgtg ctgttcatct tcggctgcct gctggttctc 360 ggcctgtgga tctacctgct ggaaatcctt tggagactgg gcgccaccat ctggcagctg 420 ctggcctttt tcctggcctt ctttctggat atcatcctcc tcatcattgc cctgtacctg 480 cagcagaact ggtggaccct gctggtggat ctgctttggc tgctgctctt tctggccatc 540 ctgatttgga tgtactacca cggccagcgg cctttcgccg aggacaagac ctacaagtac 600 atctgccgga acttcagcaa cttctgcaac gtggacgtgg tggaaattct gccctacctg 660 ccttgcctga ccgccagaga tcaggacaga ctgagagcca catgtaccct gagcggcaac 720 agagacacac tgtggcacct gttcaacacc ctgcagagaa ggcctggctg ggtcgagtac 780 tttatcgccg ctctgagagg ctgcgagctg gtcgatctgg ctgatgaagt ggccagcgtg 840 taccagagct accagcctag aaccagcgac cggggcgaga atagcgaaca ggctctgcag 900 acactgagcc ccagagccat tcctagaaac cctgatggcg gccctctgga aagctctagt 960 gatctggccg ctctgtcccc tctgacaagc tctggacacc aagagcagga taccgagctg 1020 ggcagcacac atacagccgg cgctacaagc agcctgacac cttctagagg ccccgtgtct 1080 cccagcgtgt catttcagcc tctggccagg tctaccccta gggcttctag actgcctgga 1140 ccaacaggca gcgtggtgtc taccggcaca agcttcagct ctagctctcc tggactggct 1200 agtgccggtg ccgctgaggg aaaacaaggc gccgaatctg atcaggccga gcctatcatc 1260 tgtagcagcg gagcagaagc ccctgccaat agcctgccta gcaaggtgcc aaccacactg 1320 atgcccgtga acacagtggc cctgaaggtg ccagctaatc ctgcctccgt gtccaccgtg 1380 ccttctaagc tgccaaccag ctctaagcca cctggcgccg tgccatctaa cgccctgaca 1440 aatcctgctc caagcaagct gcccatcaac tccacaagag ccggcatggt gccctctaag 1500 gtgcccacat ctatggtgct gaccaaggtg tccgccagca ccgtgccaac agatggcagc 1560 tccagaaacg aggaaacccc tgccgctcct actcctgctg gcgctacagg cggatcttct 1620 gcttggctgg atagcagcag cgagaacaga ggcctgggca gcgagctttc taaacctggc 1680 gtgctggctt cccaggtgga cagcccattt tccggctgct ttgaggacct ggctatcagc 1740 gcctctacaa gcctcggcat gggaccttgt cacggccccg aggaaaacga gtacaagagc 1800 gagggcacct tcggcatcca cgtggccgag aatcctagca tccaactgct ggaaggcaac 1860 cccggacctc cagctgatcc agatggcgga ccaagacctc aggccgacag aaagttccaa 1920 gagcgcgagg tgccctgcca cagaccttct cca 1953 <210> 6 <211> 651 <212> PRT <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens IP1 fusion protein <400> 6 Met Asp Leu Asp Leu Glu Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro 1 5 10 15 Arg Gly Pro Pro Leu Ser Ser Ser Ile Gly Leu Ala Leu Leu Leu Leu 20 25 30 Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Ile Met Ser Asn Trp Thr 35 40 45 Gly Gly Ala Leu Leu Val Leu Tyr Ala Phe Ala Leu Met Leu Val Ile 50 55 60 Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu 65 70 75 80 Gly Ala Leu Cys Leu Leu Leu Leu Met Ile Thr Leu Leu Leu Ile Ala 85 90 95 Leu Trp Asn Leu His Gly Gln Ala Leu Tyr Leu Gly Ile Val Leu Phe 100 105 110 Ile Phe Gly Cys Leu Leu Val Leu Gly Leu Trp Ile Tyr Leu Leu Glu 115 120 125 Ile Leu Trp Arg Leu Gly Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe 130 135 140 Leu Ala Phe Phe Leu Asp Ile Ile Leu Leu Ile Ile Ala Leu Tyr Leu 145 150 155 160 Gln Gln Asn Trp Trp Thr Leu Leu Val Asp Leu Leu Trp Leu Leu Leu 165 170 175 Phe Leu Ala Ile Leu Ile Trp Met Tyr Tyr His Gly Gln Arg Pro Phe 180 185 190 Ala Glu Asp Lys Thr Tyr Lys Tyr Ile Cys Arg Asn Phe Ser Asn Phe 195 200 205 Cys Asn Val Asp Val Val Glu Ile Leu Pro Tyr Leu Pro Cys Leu Thr 210 215 220 Ala Arg Asp Gln Asp Arg Leu Arg Ala Thr Cys Thr Leu Ser Gly Asn 225 230 235 240 Arg Asp Thr Leu Trp His Leu Phe Asn Thr Leu Gln Arg Arg Pro Gly 245 250 255 Trp Val Glu Tyr Phe Ile Ala Ala Leu Arg Gly Cys Glu Leu Val Asp 260 265 270 Leu Ala Asp Glu Val Ala Ser Val Tyr Gln Ser Tyr Gln Pro Arg Thr 275 280 285 Ser Asp Arg Gly Glu Asn Ser Glu Gln Ala Leu Gln Thr Leu Ser Pro 290 295 300 Arg Ala Ile Pro Arg Asn Pro Asp Gly Gly Pro Leu Glu Ser Ser Ser 305 310 315 320 Asp Leu Ala Ala Leu Ser Pro Leu Thr Ser Ser Gly His Gln Glu Gln 325 330 335 Asp Thr Glu Leu Gly Ser Thr His Thr Ala Gly Ala Thr Ser Ser Leu 340 345 350 Thr Pro Ser Arg Gly Pro Val Ser Pro Ser Val Ser Phe Gln Pro Leu 355 360 365 Ala Arg Ser Thr Pro Arg Ala Ser Arg Leu Pro Gly Pro Thr Gly Ser 370 375 380 Val Val Ser Thr Gly Thr Ser Phe Ser Ser Ser Ser Pro Gly Leu Ala 385 390 395 400 Ser Ala Gly Ala Ala Glu Gly Lys Gln Gly Ala Glu Ser Asp Gln Ala 405 410 415 Glu Pro Ile Ile Cys Ser Ser Gly Ala Glu Ala Pro Ala Asn Ser Leu 420 425 430 Pro Ser Lys Val Pro Thr Thr Leu Met Pro Val Asn Thr Val Ala Leu 435 440 445 Lys Val Pro Ala Asn Pro Ala Ser Val Ser Thr Val Pro Ser Lys Leu 450 455 460 Pro Thr Ser Ser Lys Pro Pro Gly Ala Val Pro Ser Asn Ala Leu Thr 465 470 475 480 Asn Pro Ala Pro Ser Lys Leu Pro Ile Asn Ser Thr Arg Ala Gly Met 485 490 495 Val Pro Ser Lys Val Pro Thr Ser Met Val Leu Thr Lys Val Ser Ala 500 505 510 Ser Thr Val Pro Thr Asp Gly Ser Ser Arg Asn Glu Glu Thr Pro Ala 515 520 525 Ala Pro Thr Pro Ala Gly Ala Thr Gly Gly Ser Ser Ala Trp Leu Asp 530 535 540 Ser Ser Ser Glu Asn Arg Gly Leu Gly Ser Glu Leu Ser Lys Pro Gly 545 550 555 560 Val Leu Ala Ser Gln Val Asp Ser Pro Phe Ser Gly Cys Phe Glu Asp 565 570 575 Leu Ala Ile Ser Ala Ser Thr Ser Leu Gly Met Gly Pro Cys His Gly 580 585 590 Pro Glu Glu Asn Glu Tyr Lys Ser Glu Gly Thr Phe Gly Ile His Val 595 600 605 Ala Glu Asn Pro Ser Ile Gln Leu Leu Glu Gly Asn Pro Gly Pro Pro 610 615 620 Ala Asp Pro Asp Gly Gly Pro Arg Pro Gln Ala Asp Arg Lys Phe Gln 625 630 635 640 Glu Arg Glu Val Pro Cys His Arg Pro Ser Pro 645 650 <210> 7 <211> 2109 <212> DNA <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens IP1 fusion protein <400> 7 atggatctgg atctcgaaag aggacctcct ggacctagac ggcctcctag aggaccacct 60 ctgagcagct ctattggact ggccctgctg ctgcttctgc tggctctgct gttctggctg 120 tacatcatca tgagcaactg gaccggcgga gcactgctgg tgctgtatgc ctttgctctg 180 atgctggtca tcatcatcct gatcatcttc atcttccggc gggacctgct gtgtcctctg 240 ggagcacttt gtctgttgct gctgatgatc accctcctgc tgatcgccct gtggaacctg 300 catggacagg ccctgtatct gggcatcgtg ctgttcatct tcggctgcct gctggttctc 360 ggcctgtgga tctacctgct ggaaatcctt tggagactgg gcgccaccat ctggcagctg 420 ctggcctttt tcctggcctt ctttctggat atcatcctcc tcatcattgc cctgtacctg 480 cagcagaact ggtggaccct gctggtggat ctgctttggc tgctgctctt tctggccatc 540 ctgatttgga tgtactacca cggccagcgg ccttctccaa gacactgccc agtggaaaga 600 gagcagttca agagggacgc ccagcctaga cctggcggag atcctgatgc tccacctgga 660 ccaaatggcg agctgctgca gatcagccct aatgaggccg tgcacatcgg cttcaccggc 720 gagtctaagt acgagaacga ggaacccggc cactgtcctg gcatgggcct ttctacatct 780 gcctctatcg ccctggacga gttctgcggc agctttccat ctgatgtgca gtctgccctc 840 gtgggcccta agtctctgga atctggcctg ggcagaaacg agagcagctc cgatctgtgg 900 gctagctctg gtggaacagc tggcgctcct acaccagccg ctcctaccga agagaataga 960 agcagcggcg acacccctgt gacaagcgcc tctgtgaaaa ccctggtcat gagcacccca 1020 gtgaagtccc cagtgatggg cgccagaacc tccaacattc ccctgaagtc tcccgctcct 1080 aacacactgg ccaactctcc agtggctggc cctcctaagt ctagcacccc tctgaaaagc 1140 cccgtgacct ctgtgtctgc ccctaacgct cctgtgaaac tggccgtgac caacgtgccc 1200 atgctgacca cacctgtgaa atccccactg agcaatgccc ctgccgaggc cggaagctct 1260 tgtatcattc ccgaggctca ggatagcgag gctggccaaa aaggcgaagc tgcaggcgct 1320 tctgctctgg gccctagctc tagctctttt agcaccggca ccagcgtggt gtctggcaca 1380 ccaggacctc tgagaagcgc cagacctacc tctagagccc tgcctcagtt tagcgtgtcc 1440 cctagtgtgc ctggcagaag ccctacactg tctagtacag ccggcgctac acacaccagc 1500 ggactggaaa cagaccaaga acagcatggc agcagcaccc tgccttctct ggctgccctt 1560 gattctagca gcgaactgcc aggcggcgac cccaatagac ctatcgctag acctagcctg 1620 acacagctgg cccaagagag caatgagggc ccttctgagc ctgctcagac cgaacaggtg 1680 ccaatgcctt acagccccga gaaagagcgg tgcagcaact accctatcag ccatgccgct 1740 gctcccacac ctcctggtcc aagagaagct cctctgagcc ctcctgagct gcccgatcct 1800 ccaagagata gcaccagacc tcagtactcc cagtacgtgt ccgccgtgga agatgccctg 1860 gatgtgctgg aatgtggcag actggccgcc atcttctacg aagtgtgggg ccctagaagg 1920 cagctgacca actttctgca ctggctgacc gacagaaacg gcagcctgac atgtaccgcc 1980 agactgagag atcaggaccg ggccacactg tgccctctgt atcctctgat cgaggtggtg 2040 gacgtgaact gcttcaacag cttcaaccgg tgcatctaca agtacaccaa ggacgaggct 2100 ttccctatg 2109 <210> 8 <211> 703 <212> PRT <213> Artificial sequence <220> <223> Epstein-Barr virus LMP1 and Homo sapiens IP1 fusion protein <400> 8 Met Asp Leu Asp Leu Glu Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro 1 5 10 15 Arg Gly Pro Pro Leu Ser Ser Ser Ile Gly Leu Ala Leu Leu Leu Leu 20 25 30 Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Ile Met Ser Asn Trp Thr 35 40 45 Gly Gly Ala Leu Leu Val Leu Tyr Ala Phe Ala Leu Met Leu Val Ile 50 55 60 Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu 65 70 75 80 Gly Ala Leu Cys Leu Leu Leu Leu Met Ile Thr Leu Leu Leu Ile Ala 85 90 95 Leu Trp Asn Leu His Gly Gln Ala Leu Tyr Leu Gly Ile Val Leu Phe 100 105 110 Ile Phe Gly Cys Leu Leu Val Leu Gly Leu Trp Ile Tyr Leu Leu Glu 115 120 125 Ile Leu Trp Arg Leu Gly Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe 130 135 140 Leu Ala Phe Phe Leu Asp Ile Ile Leu Leu Ile Ile Ala Leu Tyr Leu 145 150 155 160 Gln Gln Asn Trp Trp Thr Leu Leu Val Asp Leu Leu Trp Leu Leu Leu 165 170 175 Phe Leu Ala Ile Leu Ile Trp Met Tyr Tyr His Gly Gln Arg Pro Ser 180 185 190 Pro Arg His Cys Pro Val Glu Arg Glu Gln Phe Lys Arg Asp Ala Gln 195 200 205 Pro Arg Pro Gly Gly Asp Pro Asp Ala Pro Pro Gly Pro Asn Gly Glu 210 215 220 Leu Leu Gln Ile Ser Pro Asn Glu Ala Val His Ile Gly Phe Thr Gly 225 230 235 240 Glu Ser Lys Tyr Glu Asn Glu Glu Pro Gly His Cys Pro Gly Met Gly 245 250 255 Leu Ser Thr Ser Ala Ser Ile Ala Leu Asp Glu Phe Cys Gly Ser Phe 260 265 270 Pro Ser Asp Val Gln Ser Ala Leu Val Gly Pro Lys Ser Leu Glu Ser 275 280 285 Gly Leu Gly Arg Asn Glu Ser Ser Ser Asp Leu Trp Ala Ser Ser Gly 290 295 300 Gly Thr Ala Gly Ala Pro Thr Pro Ala Ala Pro Thr Glu Glu Asn Arg 305 310 315 320 Ser Ser Gly Asp Thr Pro Val Thr Ser Ala Ser Val Lys Thr Leu Val 325 330 335 Met Ser Thr Pro Val Lys Ser Pro Val Met Gly Ala Arg Thr Ser Asn 340 345 350 Ile Pro Leu Lys Ser Pro Ala Pro Asn Thr Leu Ala Asn Ser Pro Val 355 360 365 Ala Gly Pro Pro Lys Ser Ser Thr Pro Leu Lys Ser Pro Val Thr Ser 370 375 380 Val Ser Ala Pro Asn Ala Pro Val Lys Leu Ala Val Thr Asn Val Pro 385 390 395 400 Met Leu Thr Thr Pro Val Lys Ser Pro Leu Ser Asn Ala Pro Ala Glu 405 410 415 Ala Gly Ser Ser Cys Ile Ile Pro Glu Ala Gln Asp Ser Glu Ala Gly 420 425 430 Gln Lys Gly Glu Ala Ala Gly Ala Ser Ala Leu Gly Pro Ser Ser Ser 435 440 445 Ser Phe Ser Thr Gly Thr Ser Val Val Ser Gly Thr Pro Gly Pro Leu 450 455 460 Arg Ser Ala Arg Pro Thr Ser Arg Ala Leu Pro Gln Phe Ser Val Ser 465 470 475 480 Pro Ser Val Pro Gly Arg Ser Pro Thr Leu Ser Ser Thr Ala Gly Ala 485 490 495 Thr His Thr Ser Gly Leu Glu Thr Asp Gln Glu Gln His Gly Ser Ser 500 505 510 Thr Leu Pro Ser Leu Ala Ala Leu Asp Ser Ser Ser Glu Leu Pro Gly 515 520 525 Gly Asp Pro Asn Arg Pro Ile Ala Arg Pro Ser Leu Thr Gln Leu Ala 530 535 540 Gln Glu Ser Asn Glu Gly Pro Ser Glu Pro Ala Gln Thr Glu Gln Val 545 550 555 560 Pro Met Pro Tyr Ser Pro Glu Lys Glu Arg Cys Ser Asn Tyr Pro Ile 565 570 575 Ser His Ala Ala Ala Pro Thr Pro Pro Gly Pro Arg Glu Ala Pro Leu 580 585 590 Ser Pro Pro Glu Leu Pro Asp Pro Pro Arg Asp Ser Thr Arg Pro Gln 595 600 605 Tyr Ser Gln Tyr Val Ser Ala Val Glu Asp Ala Leu Asp Val Leu Glu 610 615 620 Cys Gly Arg Leu Ala Ala Ile Phe Tyr Glu Val Trp Gly Pro Arg Arg 625 630 635 640 Gln Leu Thr Asn Phe Leu His Trp Leu Thr Asp Arg Asn Gly Ser Leu 645 650 655 Thr Cys Thr Ala Arg Leu Arg Asp Gln Asp Arg Ala Thr Leu Cys Pro 660 665 670 Leu Tyr Pro Leu Ile Glu Val Val Asp Val Asn Cys Phe Asn Ser Phe 675 680 685 Asn Arg Cys Ile Tyr Lys Tyr Thr Lys Asp Glu Ala Phe Pro Met 690 695 700

Claims

1. A viral vector comprising a first nucleic acid sequence encoding an antigen or antigenic epitope and a second nucleic acid sequence encoding a fusion protein, said fusion protein comprising a transmembrane portion of latent membrane protein 1 (LMP1) of Epstein-Barr virus, wherein the cytoplasmic domain of said LMP1 has been replaced by human IPS1 with a transmembrane domain deletion, wherein transduction of cells with said vector activates the STING pathway in the cells; wherein, The second nucleic acid sequence is the sequence shown in SEQ ID NO: 3 or SEQ ID NO:

5.

2. The viral vector according to claim 1, wherein, The vector is a lentiviral vector.

3. The viral vector according to claim 1, wherein, The first nucleic acid sequence encodes a fusion protein comprising two or more antigens or two or more antigenic epitopes.

4. The viral vector according to claim 1, wherein, The vector further comprises a third nucleic acid sequence encoding a soluble immune checkpoint inhibitor molecule or a soluble immunomodulator molecule.

5. The viral vector according to claim 4, wherein, The soluble immune checkpoint inhibitor molecule or the soluble immunomodulator molecule is selected from the group consisting of CTLA-4, PD-1, PDL-1, LAG-3, TIM 3, B7-H3, ICOS, IDO, 4-1BB, CD47, B7-H4, OX-40, TIGIT, CD160 and combinations thereof.

6. The viral vector according to claim 1, wherein, The vector further comprises a functional lentiviral integrase protein, wherein the vector is self-inactivating.

7. The viral vector according to claim 1, wherein, The antigen is selected from the group consisting of NY-ESO-1, mesothelin, PSA, MART-1, MART-2, Gp100, tyrosinase, p53, ras, MUC1, SAP-1, survivin, CEA, Ep-CAM, Her2, BRCA1 / 2, gag, reverse transcriptase, tat, circumsporozoite protein, HCV non-structural protein, hemagglutinin and combinations thereof.

8. An immunotherapeutic preparation for preventing or treating cancer or infection in a subject, said preparation comprising the viral vector according to claim 1.

9. Use of the viral vector according to claim 1 or the immunotherapeutic preparation according to claim 8 in the preparation of a medicament for inducing or enhancing an immune response against said cancer or infectious disease in a subject; wherein, Inducing or enhancing an immune response against cancer, and the cancer is selected from the group consisting of: melanoma, glioma, prostate cancer, breast cancer, cervical cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma and pancreatic cancer, the cancer being indicated by the tumor antigen of the cancer; or, Inducing or enhancing an immune response against an infectious disease, and the infectious disease is selected from the group consisting of: HIV / AIDS, hepatitis C, HPV, pneumonia, influenza, malaria, leishmaniasis, tuberculosis, Hansen's disease, rabies, dengue fever, Zika virus, Ebola virus and schistosomiasis, the infectious disease being indicated by the viral or microbial antigen of the infectious disease.

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