Viral vector constructs expressing genetic adjuvants for activation of CD40 and STING pathways

Abstract

The present invention provides a viral vector for use as a gene immunotherapy agent, including preventive and therapeutic vaccines and compositions that enhance cellular immune responses and innate immune responses. The vector is particularly suitable for treating or preventing cancer and infectious diseases. The vector includes a lentiviral vector encoding one or more antigens and an adjuvant composition, and optionally may encode one or more soluble and secreted checkpoint inhibitor molecules. The adjuvant includes latent membrane protein 1 (LMP1) from Epstein-Barr virus, and a fusion protein including LMP1, wherein the intracytoplasmic domain of the LMP1 has been replaced by human IPS1 or a variant thereof that can activate the STING pathway. The sequence encoded by the vector is codon-optimized for human expression.

Description

Background Art

[0001] Standardized vaccine strategies based on the induction of antibody-triggered immune responses have enabled the eradication or near-eradication of many previously lethal infectious diseases, such as smallpox, polio, and tetanus. However, these classic human vaccines are poorly effective or unsafe for use against other infectious diseases, such as HIV and hepatitis, as well as 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 pathogens themselves. Nucleic acid vaccines, especially 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 create effective vaccines. Classical vaccines usually require adjuvants, such as aluminum salts, to achieve optimal effects, but conventional adjuvants are generally weak in enhancing cellular immune responses. Several strategies have been proposed to improve the quality and magnitude of cellular immune responses triggered by viral vectors. A new class of genetic adjuvants has been developed to improve the cellular immune responses induced by vector-based immunotherapy. Genetic adjuvants consist of DNA sequences that encode immunomodulator molecules.

[0004] Cluster of differentiation 40 (CD40) is a membrane protein that is present on a variety of cells, particularly antigen presenting cells such as dendritic cells (DC). CD40 is essential for the initiation and progression of cellular and humoral adaptive immunity, and is involved in processes such as the maturation of DCs, the production of cytokines, the isotype switching of antibodies, the development of memory B cells, and the formation of germinal centers. The activation of CD40 requires its aggregation in the membrane so that its signaling domain in the cytoplasm forms a supramolecular signaling complex, which then activates different pro-inflammatory signaling pathways. The aggregation of CD40 is triggered by a polymeric form of its ligand (CD40 ligand or CD40L) or an anti-CD40 antibody, which must be arranged on nearby cells by binding to Fc receptors. The mRNA form of the CD40 ligand has been used as an adjuvant in vaccines that trigger cellular immune responses (e.g., Argos Therapeutics AGS003, TriMix).

[0005] Stone et al. (WO2013 / 0039942) disclose the use of gene adjuvants that induce cellular immune responses that mimic activated CD40 receptors. In this method, a nucleic acid vaccine encodes latent membrane protein 1 (LMP1) of Epstein-Barr virus. The results show that when full-length LMP1 is expressed in various forms (e.g., plasmids, mRNA, viruses, and vectors), it spontaneously forms clusters that mimic activated CD40L and its adjuvant effect. For example:

[0006] (i) by infecting macrophages in vitro with HIV-1 expressing LMP1 to stimulate their production of immunostimulatory cytokines, including IL-8, MIP-1β, IL1-β, IL-6, IL-12p70, and TNFα (without any production of IL-10, an immunosuppressive cytokine);

[0007] (ii) by in vitro infection of human dendritic cells with HIV-1 expressing LMP1 to stimulate their production of stimulatory cytokines, including IL-8, IL-1β, TNF-α, IL-6, and IL-12p70;

[0008] (iii) stimulation of human dendritic cells by single-cycle SIV (scSIV) expressing LMP1 to produce stimulatory cytokines, including IL-8, IL1-β, IL-6, IL-12p70, and TNFα;

[0009] (iv) In addition to immune stimulation, HIV-1-LMP1 and scSIV-LMP1 also self-adjuvant in vitro by enhancing the antigen presentation function of dendritic cells to induce the proliferation of HIV- and SIV-antigen-specific T cells;

[0010] (v) HIV-LMPI stimulates DCs and macrophages in vitro to upregulate immunologically important cell surface co-stimulatory molecules such as CD40, CD80, and CD83, as well as migration signals such as CCR-7; and

[0011] (vi) Mice were injected intramuscularly three times every two weeks with a mixture of plasmids encoding LMP1 and a melanoma-specific antigen (gp100) to prevent their tumor growth.

[0012] Stone et al. (WO2014 / 039961) disclose the use of gene adjuvants, which 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 a fusion protein in addition to a transgene encoding a marker protein or antigen, wherein the fusion protein includes a transmembrane portion of the LMP1 protein, wherein the intracytoplasmic domain has been replaced by an immune effector or adapter protein such as an IPS1 protein. Activation of the IFN-β-activated stimulator (IPS1, also known as MAVS, VISA or Cardif) produces an effective T cell response through the STING (stimulator of interferon genes) pathway. When expressed in cells, the transmembrane domain of LMP1 spontaneously forms clusters, which allow IPS1 to aggregate into intracytoplasmic clusters, thereby activating the STING pathway. It has been shown that in mouse macrophages, the transmembrane domain of LMP1 fused to full-length murine IPS1 is able to induce the secretion of IFNα, IFNβ and IL-6, and also induces the expression of maturation markers (CD40 and CCR7) and activation markers (CD80 and CD86).

[0013] Self-adjuvanting vaccines are needed to induce strong cellular immune responses to break immune tolerance found in diseases such as cancer, HIV, and other unmet medical needs. SUMMARY OF THE INVENTION

[0015] The technology of the present invention provides a viral vector encoding a gene adjuvant for improving immune response, in particular improving cell-mediated immune response, such as immune response to cancer or infection, and provides a method of using the viral vector. The antigen and adjuvant constructs of the present technology enhance the immune response through an activation process that simultaneously mimics the activation of CD40 and activates the STING pathway. The construct sequence has been optimized for use in human subjects.

[0016] One aspect of the technology is a viral vector comprising (i) a transgene encoding one or more marker proteins, antigens, epitopes or a combination thereof, (ii) full-length latent membrane protein 1 (LMP1) of Epstein-Barr virus, which is codon-optimized for human expression, and (iii) a transgene encoding a fusion protein comprising a transmembrane portion of latent membrane protein 1 (LMP1) of Epstein-Barr virus, wherein the intracytoplasmic domain has been replaced by human IPS1 or a variant thereof capable of activating the STING pathway. Optionally, the vector further comprises (iv) a nucleic acid sequence encoding one or more soluble and secreted immune checkpoint inhibitor molecules or one or more soluble immunomodulator molecules. In a preferred embodiment, the viral vector is a lentiviral vector. In some embodiments, the vector comprises a functional lentiviral integrase protein and can therefore be integrated into the genome of the cell transduced therewith.

[0017] Another aspect of the technology is a viral vector comprising (i) a transgene encoding one or more marker proteins, antigens, epitopes or a combination thereof, (ii) a fusion protein comprising an intracytoplasmic domain fused to the transmembrane domain of latent membrane protein 1 (LMP1) of Epstein-Barr virus, which is either (a) a wild-type LMP1 intracytoplasmic domain fused to human IPS1 or a variant thereof capable of activating the STING pathway (e.g., hIPS1 delta TM, or hIPS1 delta TM delta PR or reverse hIPS1, or reverse hIPS1 delta TM), or (b) human IPS1 or a variant thereof capable of activating the STING pathway (e.g., hIPS1 delta TM, or hIPS1 delta TM delta PR or reverse hIPS1, or reverse hIPS1 delta TM) fused to a wild-type LMP1 intracytoplasmic domain. Optionally, the vector further comprises (iii) a nucleic acid sequence encoding one or more soluble and secretory immune checkpoint inhibitor molecules or soluble immunomodulator molecules. 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 a cell it transduces.

[0018] The antigen may be a tumor antigen, a viral antigen or a microbial antigen. The vector encodes multiple antigens or selected epitopes of 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 nonstructural protein, hemagglutinin and combinations thereof. In certain embodiments, the vector further encodes at least one immune checkpoint inhibitor molecule or soluble immunomodulator molecule, such as an anti-CTLA-4 molecule, a PD1 blocker, a PDL1 blocker or a combination thereof.

[0019] In certain embodiments, the viral vector includes more than one nucleic acid sequence. In some such embodiments, the first nucleic acid sequence encodes one or more marker proteins, antigens, epitopes, or combinations thereof; the second nucleic acid sequence encodes the full-length latent membrane protein 1 (LMP1) of Epstein-Barr virus, which is codon-optimized for human expression, and the third nucleic acid sequence encodes a fusion protein, which includes the transmembrane portion of the latent membrane protein 1 (LMP1) of Epstein-Barr virus, wherein the intracytoplasmic domain of the LMP1 has been replaced by human IPS1 or a variant thereof that can activate the STING pathway; optionally, the fourth nucleic acid sequence encodes one or more immune checkpoint inhibitor molecules ("anti-checkpoint") or soluble immunomodulator molecules. Preferably, the first nucleic acid sequence and the second nucleic acid sequence, and the second nucleic acid sequence and the third nucleic acid sequence and the fourth 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 can 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 (eg, a 2A peptide) or an internal ribosome entry site (IRES).

[0020] In other embodiments, the viral vector includes more than one nucleic acid sequence. In some such embodiments, the first nucleic acid sequence encodes one or more marker proteins, antigens, epitopes, or combinations thereof; the second nucleic acid sequence encodes the full-length latent membrane protein 1 (LMP1) of Epstein-Barr virus fused to the intracytoplasmic domain of human IPS1 or its variant capable of activating the STING pathway (the resulting fusion protein has been codon-optimized for human expression), and the third nucleic acid sequence encodes the full-length latent membrane protein 1 (LMP1) of Epstein-Barr virus, which has been codon-optimized for human expression; and optionally, the fourth nucleic acid sequence encodes one or more immune checkpoint inhibitor molecules ("anti-checkpoint") or soluble immunomodulator molecules. 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 can 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 (eg, a 2A peptide) or an internal ribosome entry site (IRES).

[0021] Another aspect of the technology is an immunotherapeutic formulation comprising a viral vector for preventing or treating a disease or condition in a subject. In a preferred embodiment, the disease or condition is cancer or infection.

[0022] Another aspect of the technology is a method of inducing an immune response against cancer or infection in a subject, the method comprising administering a viral vector or immunotherapeutic formulation to a subject in need thereof. In some embodiments, administering a viral vector to a subject vaccinates the subject against cancer or infection.

[0023] In some embodiments, the cancer is selected from the group consisting of: melanoma, glioma, prostate cancer, breast cancer, cervical cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, ovarian cancer, sarcoma and pancreatic cancer. In some embodiments, the cancer has the tumor antigens listed above. In some embodiments, the cancer is sensitive to anti-checkpoints. 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 the viral or microbial antigens listed above. In some embodiments, the infectious disease is sensitive to anti-checkpoints.

[0024] The present technology can also be summarized by the following list of implementations.

[0025] 1. A viral vector comprising a first nucleic acid sequence encoding an antigen or an antigenic epitope, a second nucleic acid sequence encoding the full-length latent membrane protein 1 (LMP1) of Epstein-Barr virus, and a third nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises the transmembrane portion of LMP1 and IPS1 replacing the intracytoplasmic domain of LMP1 or a variant thereof capable of activating the STING pathway, wherein the coding sequence of the vector is codon-optimized for human expression, and wherein the second nucleic acid sequence and the third nucleic acid sequence are connected after the first nucleic acid sequence in any order.

[0026] 2. A viral vector, comprising a first nucleic acid sequence encoding an antigen or an antigenic epitope; a second nucleic acid sequence encoding a fusion of the full-length latent membrane protein 1 (LMP1) of Epstein-Barr virus and the intracytoplasmic domain of human IPS1 or a variant thereof capable of activating the STING pathway (the resulting fusion protein has been codon-optimized for human expression); or the second nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises the transmembrane portion of the latent membrane protein 1 (LMP1) of Epstein-Barr virus, human IPS1 or a variant thereof capable of activating the STING pathway replacing the intracytoplasmic domain of LMP1, and the intracytoplasmic domain of LMP1 (the resulting fusion protein has been codon-optimized for human expression).

[0027] 3. The viral vector according to embodiment 1 or 2, wherein the vector is a lentiviral vector.

[0028] 4. In the viral vector according to any of the preceding embodiments, the first nucleic acid sequence encodes a fusion protein comprising two or more antigens or two or more antigenic epitopes.

[0029] 5. The viral vector as described in any of the preceding embodiments, the second nucleic acid sequence as described in embodiment 1 or 2, or the third nucleic acid sequence as described in embodiment 1, comprises a sequence selected from the group consisting of SEQ ID NO.1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 and SEQ ID NO:23.

[0030] 6. A viral vector as described in any of the preceding embodiments, wherein the vector further comprises a nucleic acid sequence encoding a soluble or secretory immune checkpoint inhibitor molecule or a soluble immunomodulator molecule.

[0031] 7. In the viral vector as described in Embodiment 6, the soluble immune checkpoint inhibitor molecule or 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 a combination thereof.

[0032] 8. The viral vector according to any of the preceding embodiments, further comprising a functional lentiviral integrase protein, wherein the vector is self-inactivating.

[0033] 9. A viral vector as described in any of the preceding embodiments, 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 nonstructural protein, hemagglutinin, and combinations thereof.

[0034] 10. An immunotherapeutic preparation for preventing or treating cancer or infection in a subject, the preparation comprising the viral vector according to any one of embodiments 1-9.

[0035] 11. A method for inducing or enhancing an immune response against cancer or an infectious disease in a subject, the method comprising administering the viral vector described in any one of embodiments 1-9 or the immunotherapeutic preparation described in embodiment 10 to a subject in need thereof, thereby inducing or enhancing an immune response against the cancer or infectious disease in the subject.

[0036] 12. The method of embodiment 11, wherein the method 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, renal cancer, lung cancer, lymphoma and pancreatic cancer.

[0037] 13. The method of embodiment 11, wherein the method 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

[0038] Figure 1 A schematic diagram of the secondary structure of the full-length LMP1 protein is shown.

[0039] Figure 2A schematic diagram of the secondary structure of a truncated LMP1 protein in which the intracytoplasmic signaling domain has been removed is shown.

[0040] FIG3A shows a schematic diagram of the IPS1 protein, and FIG3B shows its orientation in the mitochondrial membrane.

[0041] Figure 4 A schematic diagram of the LPM1-IPS1 fusion protein is shown.

[0042] Figure 5 A schematic diagram showing the secondary structure of the LPM1-IPS1 fusion protein that should be produced when expressed in the sequence described in WO2014 / 039961 is shown.

[0043] Figure 6 Schematic diagram showing the secondary structure of the LPM1-IPS1 fusion protein with the IPS1 transmembrane domain removed.

[0044] Figure 7 Shown is the structure of the LPM1-reverse IPS1 fusion protein in which the IPS transmembrane domain is removed and the caspase recruitment domain (CARD) and proline-rich (PRO) domain are in the opposite orientation.

[0045] Figures 8A-8B Schematic representations of several molecular constructs are shown. Fig. 8A A construct is shown, which includes (a) a promoter (human ubiquitin protein, Ubi); (b) a reporter gene (e.g., green fluorescent protein) or, optionally, one or more antigen genes; (c) a gene for full-length LMP1; (d) a gene for the fusion protein LMP1-IPS1 (the fusion protein may include human IPS1 or a functional variant thereof having STING enhancing activity); and (e) a gene encoding a soluble and secreted immune checkpoint inhibitor or a soluble and secreted immunomodulator molecule. Figure 8B A construct is shown, which includes (a) a promoter (human ubiquitin protein, Ubi); (b) a reporter gene (such as green fluorescent protein) or, optionally, one or more antigen genes; (c) a gene for full-length LMP1; (d) a gene for the fusion protein LMP1-IPS1 (the fusion protein may include human IPS1 or a functional variant thereof with STING enhancing activity); (e) a gene encoding a soluble and secreted immune checkpoint inhibitor or a soluble and secreted immunomodulator molecule.

[0046] Fig. 9A and 9B A single molecule adjuvant example of this technology is shown. Fig. 9AAs shown, the adjuvant is a fusion of LMP1 (deltaTM) with the cytoplasmic signaling domain of LMP1, which in turn is fused to the hIPS1 cytoplasmic signaling domain (STING activator) or a functional equivalent thereof. Fig. 9B Shows something like Fig. 9A However, the two cytoplasmic signaling domains are presented in reverse order.

[0047] Fig.10 Viral vector constructs featuring unimolecular adjuvants are shown.

[0048] Figures 11A-11B Shown are the expression levels of GFP transgene in human dendritic cells and macrophages transduced by lentiviral vectors. Fig.11A Shown is GFP transgene expression in human dendritic cells 96 hours after transduction with lentiviral constructs. Fig. 11B Shown is GFP transgene expression in human macrophages 96 hours after transduction with the lentiviral construct.

[0049] Figures 12A-12D The activation and maturation of human dendritic cells and macrophages induced by lentiviral vectors in vitro are shown. Fig. 12A Shown is the panel of cytokines upregulated in human dendritic cells 96 hours after transduction with lentiviral constructs. Fig. 12B Shown are the panel of markers upregulated in human GFP-positive-dendritic cells 96 hours after transduction with lentiviral constructs (expression normalized to GFP). Fig. 12C Shown is the panel of upregulated cytokines in human macrophages 96 h after transduction with lentiviral constructs. Fig.12D Shown is a panel of markers upregulated in GFP-positive human macrophages 96 hours after transduction with lentiviral constructs (expression normalized to GFP). (expression normalized to GFP). DETAILED DESCRIPTION OF THE INVENTION

[0051] The present technology provides a viral vector construct for expressing a gene adjuvant used in an immunotherapy product and a method for using the vector. The vector construct can improve the quality and intensity of immune responses, such as those against cancer or infection, and is particularly suitable for inducing and / or enhancing cell-mediated immune responses. The vector construct of the present technology is particularly effective in enhancing immune responses because the construct leads to the expression of the intracytoplasmic signaling domain of LMP1 from EBV and the activation of the CD40-like pathway promoted by activation, thereby mediating the activation of specific cell-mediated immune responses, as well as the expression of the LMP1-IPS1 fusion protein and the activation of the STING pathway promoted by activation, thereby activating the innate immune response. The activation of the CD-40-like pathway or the STING pathway can be mediated by the aggregation of the LMP1 transmembrane domain that activates the intracytoplasmic signaling domain.

[0052] The present technology describes the purposes of a single vector construct including an antigen cassette and a gene adjuvant. Compared with the simultaneous injection of two vectors (one encoding antigen and one encoding adjuvant), the use of a single product will simplify development (including industrial, regulatory and clinical aspects) and improve the effectiveness and safety of treatment. Utilizing this unique construct, cells expressing antigenic expression cassettes will constitutively benefit from the expression of adjuvants, thereby improving the intensity and quality of the immune response triggered. Transduced cells will be quickly eliminated by the immune response, which reduces the risk of any long-term and undesirable expression of genetic sequences, which may be an important consideration for regulatory agencies. In addition, compared with injecting two different vectors, only producing and injecting a vector will be more cost-effective.

[0053] The viral vector constructs of the present technology are organized according to one of two different strategies. In the first strategy, the vector contains two independent adjuvant expression cassettes, one encoding the full-length LMP1 protein and the other encoding a fusion protein containing LMP1, whose intracytoplasmic domain is replaced by human IPS1 or a variant thereof that activates the STING pathway. Under this strategy, the vector contains one or more nucleic acid sequences encoding: (i) full-length EBV LMP1 protein, which has been codon-optimized for human expression, (ii) EBV LMP1 protein, whose intracytoplasmic domain is replaced by human IPS1 or a variant thereof that can activate the STING pathway, and (iii) one or more antigens.

[0054] In the second strategy, the vector contains a single adjuvant expression cassette encoding (i) the intracytoplasmic domain of human IPS1 or a variant thereof capable of activating the STING pathway fused to full-length LMP1, or (ii) a fusion protein comprising a transmembrane portion of LMP1 in which the intracytoplasmic domain has been replaced by human IPS1 or a variant thereof capable of activating the STING pathway fused to the intracytoplasmic domain of LMP1. In addition, the vector encodes one or more antigens.

[0055] In a typical embodiment, the technology activates an immune response by the aggregation of two or more full-length LMP1 proteins in the cell membrane, as well as the aggregation of two or more truncated LMP1 proteins in the cell membrane (whose original intracytoplasmic signaling domain is missing), and / or the aggregation of two or more IPS1 intracytoplasmic signaling domains fused to truncated LMP1 proteins.

[0056] Following direct injection, the introduction of nucleic acid sequences and subsequent protein expression can occur in any type of cell, but preferably in skeletal muscle cells or immune cells. This technology can be used for traditional preventive or therapeutic vaccines against cancer and infectious diseases, as well as cell-based therapies such as dendritic cell therapy. In the experiments described herein, it is expected that viral vectors significantly enhance immune responses and protect or treat infections and cancers.

[0057] "Vector" refers to a molecule that includes a nucleic acid sequence encoding at least a portion 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. Vectors may contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of operably linked coding sequences in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0058] "Construct" can be any type of engineered nucleic acid encoding a gene product, wherein some or all of the nucleic acid coding sequence can be transcribed. The transcript is usually translated into protein, but not necessarily. In certain embodiments, expression includes transcription of the gene and translation of mRNA into the gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding the gene of interest.

[0059] As used in the present invention, "vaccine" includes all preventive and therapeutic vaccines. "Adjuvant" can be any molecule or composition that activates or enhances the immune response to an antigen. An adjuvant can enhance the efficacy of a vaccine by assisting in changing the immune response to a specific type of immune system cell. An adjuvant can be an immunostimulant that triggers the activation of antigen presenting cells such as dendritic cells, macrophages, and B cells. An adjuvant is also understood to provide a "danger" signal, indicating that the immune system should enter a state of alert. An adjuvant 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, the immune response may not proceed, or may be converted into ineffective immunity or tolerance. Adjuvants are usually required for effective preventive or therapeutic vaccines, or for inducing anti-tumor immune responses. A "genetic adjuvant" is an adjuvant provided in the form of a nucleic acid, which is expressed by a target cell to produce a molecule that acts as an adjuvant.

[0060] Antigen presenting cell (APC) is any of a variety of cells that can display, obtain or present at least one antigen or antigen fragment on its cell surface. Generally, the term "antigen presenting cell" can refer to any cell that helps enhance the immune response (i.e., T cells or B cell arms from the immune system) to antigens or antigen compositions to achieve the technical goal. Those skilled in the art can define such cells using the methods disclosed in the present invention and the art. As understood by those of ordinary skill in the art, and in certain embodiments used in the present invention, cells that usually or preferentially display or present antigens to immune cells with class II major histocompatibility molecules or complexes are "antigen presenting cells". In some aspects, cells (e.g., APC) can be fused with another cell, such as recombinant cells or tumor cells expressing desired antigens. Methods for preparing the fission of two or more cells are well known in the art. In some cases, the immune cells to which antigen presenting cells display or present antigens are CD4+T cells or CD8+T cells. Other molecules expressed on APC or other immune cells can help or improve the enhancement of immune response. Secreted or soluble molecules, such as cytokines and adjuvants, can also help or enhance the immune response to antigens. Dendritic cells (DC) are antigen presenting cells present in vivo, in vitro, in vitro or in a host or subject, or can be derived from hematopoietic stem cells or monocytes. Dendritic cells and their precursors can be separated from a variety of lymphoid organs, such as spleen, lymph nodes and bone marrow and peripheral blood. DC has a unique morphology, and thin sheets (lamellar pseudopodia) extend in multiple directions from the dendritic cell body. Typically, dendritic cells express a high level of major histocompatibility complex (MHC) and costimulatory molecules (such as B7-1 and B7-2). Dendritic cells can induce the antigen-specific differentiation of T cells in vitro, and can start primary T cells in vitro and in vivo to respond.

[0061] The term "immune response" refers to the induction of antibodies and / or immune cell-mediated responses specifically directed against 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 composition of the attacked organism, the chemical composition and configuration of the antigen or allergen or drug or biological agent, and the mode and cycle 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 by interacting with antigens or allergens or other cells of the immune system, the release of cytokines, and the responsiveness to these cytokines. Immune responses are generally divided into two major categories - humoral and cell-mediated. The humoral component of the immune response includes the production of antibodies specific to the antigen or allergen or drug or biological agent. The cell-mediated component includes the production of delayed hypersensitivity reactions and cytotoxic effector cells directed against the antigen or allergen.

[0062] 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 (CTL). 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 molecules.

[0063] The term "LMP1 gene" refers to a nucleic acid sequence encoding a natural Epstein-Barr virus LMP1, such as a natural Epstein-Barr virus LMP1 gene; a nucleic acid having a sequence that can transcribe LMP1 cDNA; and / or variants and homologs of the aforementioned alleles. An exemplary nucleic acid sequence of LMP1 is GenBank Accession No. M58153.1. The term includes double-stranded DNA, single-stranded DNA, and RNA.

[0064] 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 showing the functional activity of the native LMP1 protein. The "functional activity" of a protein is any activity associated with the physiological function of the protein. LMP1 consists of an N-terminal transmembrane region connected to a C-terminal cell signaling region, which is similar to the CD40 receptor on immune cells. In addition to anchoring LMP1 into the membrane, the N-terminus of LMP1 self-aggregates and causes aggregation of LMP1 or any protein connected 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 No. AAA66330.1.

[0065] Latent membrane protein-1 (LMP1) is a gene in Epstein-Barr virus (EBV). Its N-terminus consists of 6 consecutive transmembrane domains, which anchor the protein in the membrane. Figure 1 The structure of the LMP1 protein is shown, showing the transmembrane domain 101 and the intracytoplasmic 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 a cluster in the cell membrane and thereby clusters the intracytoplasmic domains to which it is linked by peptide bonds into a single polypeptide chain. In this sense, LMP1 is referred to as "constitutively active." Similarly, a fusion protein that connects the 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 the extraction of a ligand from the receptor.

[0066] Interferon-stimulating factor-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 mitochondria and, once activated, forms aggregates (i.e., multimers). IPS1 is also found in peroxisomes and mitochondrial-associated membranes. IPS1 also contains a caspase recruitment domain (CARD), which is essential for downstream protein-protein interactions, and three TRAF interaction motifs (TIMs), two of which are included in the N-terminal proline-rich region and the third is located in the C-terminal region. The membrane localization of IPS1 may be important for its activity, as removal of the transmembrane domain inhibits IPS1-mediated antiviral responses. 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, leading to IPS1 multimerization and activation. The activated IPS1 complex then recruits IKK and TBK1 / IKKi complexes, triggering a signaling cascade that leads to the activation of transcription factors NF-κB and IRF3. NF-κB and IRF3 bind to and activate the interferon promoter, resulting in an effective cell-mediated immune response by producing type 1 interferons. RIG-1 activation also activates the STING pathway, further enhancing the cell-mediated antiviral immune response. In the present technology, fusion of IPS1 to the N-terminal domain of LMP1 promotes the aggregation and activation of LMP1-IPS1, which mimics the activation caused by dsRNA.

[0067] 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 the Epstein-Barr virus, wherein the intracytoplasmic 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 by aggregation (i.e., multimerization) of two or more LMP1 proteins.

[0068] The viral vector can be any type of suitable vector, such as an expression vector or a plasmid. In a preferred embodiment, the vector is a lentiviral vector. A lentiviral vector is a modified lentivirus, such as from human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV). The modified lentiviral vector has reduced pathogenicity. The vector can also be modified to introduce a beneficial therapeutic effect. The lentiviral vector itself is non-toxic, and unlike other retroviruses, lentiviruses are able to transduce non-dividing cells, particularly dendritic cells, allowing antigens to be presented through endogenous pathways.

[0069] The lentiviral vector may include 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 includes a recombinant DNA molecule and associated viral proteins to form particles. The lentiviral vector particles may contain single-stranded or double-stranded nucleic acid molecules.

[0070] In a preferred embodiment, the lentiviral vector has 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 show genetic mutations that hinder the ability of lentiviral vector particles to integrate into the host genome. The terms "transfection" and "transduction" refer to the process of introducing exogenous DNA sequences into eukaryotic host cells. Transfection is the non-viral delivery of nucleic acids (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 acids by viruses or viral vectors, wherein nucleic acids are typical DNA for DNA viruses and typical RNA for RNA viruses.

[0071] In some embodiments, the lentiviral vector is self-inactivating and does not contain an enhancer. The self-inactivating lentiviral vector has a modification in the U3 (ΔU3) region of the 3'LTR that renders the vector incapable of replication in the host cell. The U3 region encodes binding sites essential for basal promoter activity and viral replication, and elimination of these binding sites results in almost complete inactivation of viral replication.

[0072] Numerous factors can affect the efficacy of viral vectors, even after successful transduction and optionally integration into the host genome: gene expression and translation; protein folding, transport and turnover; and cell-to-cell interactions, to name a few. These factors depend, among other things, on the nucleic acid sequence encoded by the vector. Preferred DNA sequences for implementing the present technology include modifications of native sequences intended to improve viral vector efficacy and efficiency. These modifications include: codon optimization for use in humans; removal of the first methionine of the IPS1 sequence in the fusion protein; removal of the IPS1 transmembrane domain and the proline-rich domain, and use of a reversed 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 the activity of the protein.

[0073] The viral vector of the present technology encodes one or more antigens. The term "antigen" as used in the present invention refers to a molecule that triggers 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. Therefore, the technician recognizes that any macromolecule, including almost all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. Those skilled in the art recognize that any DNA containing a nucleotide sequence or a partial nucleotide sequence of a pathogenic genome or a gene or gene fragment of a protein that triggers an immune response will lead to the synthesis of an antigen. In addition, those skilled in the art recognize that the present technology is not limited to the use of a complete nucleic acid sequence of a gene or genome. The present technology includes, but is not limited to, the use of more than one gene or partial nucleic acid sequence of a genome, and its nucleic acid sequence is arranged in various combinations to trigger a desired immune response.

[0074] The antigen can be any desired antigen that enhances 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 proteins gag, env, pol, tat, rev, nef, reverse transcriptase, and other HIV components. E6 and E7 proteins from human papillomavirus are also suitable antigens. In addition, the EBNA1 antigen from herpes simplex virus is suitable. Other viral antigens used 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 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 measles virus fusion protein and other measles virus components; rubella virus antigens such as 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 RSV fusion protein, M2 protein and other respiratory syncytial virus antigen components; Herpes simplex virus antigens such as immediate early protein, 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 protein E, ME, ME-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 protein; and Ebola virus envelope protein. For other examples of viral antigens, see Fundamental Virology, Second Edition, eds. Knipe, DMand, Howley PM (Lippincott Williams & Wilkins, New York, 2001). In addition, the present invention also discloses bacterial antigens.Bacterial antigens that can be used in the compositions and methods of the present technology include, but are not limited to, pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertussis adhesin, FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diphtheria bacterial antigens such as diphtheria toxin or toxoid and other diphtheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components; Streptococcus bacterial antigens such as M protein and other Streptococcus bacterial antigen components; Staphylococcus bacterial antigens such as IsdA, IsdB, SdrD and SdrE; Gram-negative bacilli bacterial antigens such as lipopolysaccharide, flagellin and other Gram-negative bacterial antigen components; tuberculosis bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components. Mycobacterium bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), 30kDa major secretory protein, antigen 85A, ESAT-6 and other mycobacterium antigen components; Helicobacter pylori bacterial antigen components; Pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharide and other pneumococcal bacterial antigen components; Haemophilus influenzae bacterial antigens such as capsular polysaccharide and other Haemophilus influenzae bacterial antigen components; Anthrax bacterial antigens such as anthrax protective antigen, anthrax lethal factor and other anthrax bacterial antigen components; F1 and V proteins from Yersinia pestis; Rickettsia bacterial antigens such as outer membrane proteins (romps) and other Rickettsia bacterial antigen components. The bacterial antigens described herein also include any other bacteria, mycobacteria, mycoplasma, rickettsia or chlamydia 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 antigens such as SAG-1, p30 and other Toxoplasma 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 related 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 Candida, Aspergillus species, Bacillus species, Histoplasma, Coccidium species, Malassezia furfur and others, Exophiala werneckii and others, Piedraia hortai and others, Trichosporum beigelii and others, Microsporum species, Trichophyton species, Epidermophyton species, Sporothrix schenckii and others, Fonsecaea pedrosoi and others, Wangiella dermatitidis and others, Pseudallescheria boydii and others, Madurella grisea and other species, Rhizopus species, Absidia species, and Mucor species. Examples of prion disease antigens include PrP, β-amyloid and other prion-related proteins.

[0075] In addition to the above-mentioned infectious and parasitic media, another area where it is desirable to enhance immunogenicity to non-infectious media is the field of inflammatory and autoimmune diseases, neurodegenerative diseases, and proliferative diseases, including but not limited to cancer, in which cells expressing cancer antigens are ideally 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 mutant, papillomavirus antigen, ganglioside or other carbohydrate-containing components of melanoma or other tumor cells. The present technology anticipates that antigens from any type of tumor cell can be used in the compositions and methods of the present invention. 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 inducing tolerance of the present technology include, but are not limited to, myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein in multiple sclerosis and CII collagen in rheumatoid arthritis.

[0076] The antigen may be part of an infectious agent such as HIV-1, EBV, HBV, influenza virus, SARS virus, pox virus, malaria or HSV, as non-limiting examples, where a vaccine that mobilizes strong T cell mediated immunity (via dendritic cells) is desired.

[0077] The term "cancer" as used herein is defined as an excessive proliferation of cells whose distinctive characteristics - loss of normal control - result in unregulated growth, lack of differentiation, invasion of local tissues 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, gum 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.

[0078] The term "tumor" means a cell or cell mass of at least one new form of tissue growth, in particular in the form of a spontaneous, autonomous and irreversible, more or less de-inhibited overgrowth of an 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 host organism's regulatory mechanisms is not effective, for example, melanoma or carcinoma. Tumor antigens include not only antigens present in or on the surface of the malignant cells themselves, but also antigens present on the matrix supporting tissue of the tumor, including endothelial cells and other vascular components. In a related aspect, "neoplastic" refers to an abnormal new growth, which is therefore the same as a tumor and can be benign or malignant. In addition, such tumors will include cell proliferation disorders.

[0079] The lentiviral vector of the present technology also includes a nucleic acid sequence encoding one or more adjuvants. In one embodiment, the DNA sequence encoding the full-length LMP1 codon-optimized for human use (LMP1 CO) includes SEQ ID NO.1 (as shown below). The amino acid sequence of the encoded full-length LMP1 is shown below in SEQ ID NO:2.

[0080]

[0081] MDLDLERGPPPGPRRPGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRHSDEH HHDDSLPHPQQATDDSSNQSDSNSNEGRHLLLVSGAGDGPPLCSQNLGAPGGGPNNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHDGIDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO:2)

[0082] The effective control gene adjuvant is provided by a truncated form of LMP1 (LMP1_CO delta IC) with a missing intracytoplasmic signaling domain. The DNA sequence of this form (codons optimized for expression in human cells) is shown below as SEQ ID NO: 3, and the encoded amino acid sequence is shown in SEQ ID NO: 4. By comparing the response to expression of SEQ ID NO: 1 (including the signaling domain) with the response to expression of SEQ ID NO: 3 (missing the signaling domain), the function of the signaling domain can be revealed.

[0083] (SEQ ID NO:3)

[0084] MDLDLERGPPPGPRRPGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQR(SEQ ID NO:4)

[0085] The preferred adjuvant is a fusion protein LMP1 (delta IC) hIPS1, which contains LMP1 from Epstein-Barr virus without the intracytoplasmic region, fused to full-length 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 sequence of the fusion protein are as follows:

[0086] DNA sequence

[0087]

[0088] Protein sequence

[0089] (SEQ ID NO:6)

[0090] Another preferred adjuvant is a fusion protein LMP1 (delta IC) hIPS1 (delta TM), which contains LMP1 from Epstein-Barr virus without the intracytoplasmic region, fused to amino acids 2-439 of human IPS1 without the transmembrane region. 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 as follows:

[0091] DNA sequence

[0092]

[0093] Protein sequence

[0094] (SEQ ID NO:8)

[0095] Another preferred adjuvant is a fusion protein LMP1 (delta IC) hIPS1 (delta-TM delta-Pro), which contains LMP1 from Epstein-Barr virus without the intracytoplasmic region, fused to amino acids 2-93 of human IPS1 (truncated IPS1, C-terminal proline-rich domain and transmembrane domain are both missing). 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 as follows:

[0096] DNA sequence

[0097]

[0098] Protein sequence

[0099] (SEQ ID NO: 10)

[0100] Another preferred adjuvant is a fusion protein LMP1 (delta IC) reverse hIPS1 (delta TM), which contains LMP1 from Epstein-Barr virus without the intracytoplasmic region and the 2-439 amino acids of human IPS1 fused (truncated IPS1, in which the transmembrane domain is removed and presented in reverse amino acid order, i.e., from 439 to 2, the C-terminus to the N-terminus direction of natural IPS1). In the fusion protein, the first amino acid (methionine) of human IPS1 (encoded by natural direct DNA) is removed. The fusion protein is codon-optimized for human use. The DNA and encoded amino acid sequences of the fusion protein are as follows:

[0101] DNA sequence:

[0102]

[0103] Protein sequence

[0104] (SEQ ID NO:12)

[0105] Another preferred adjuvant is a fusion protein LMP1 hIPS1 (delta TM), which contains the full-length LMP1 from Epstein-Barr virus fused to amino acids 2-513 of human IPS1 (truncated hIPS1 with the C-terminal transmembrane domain removed. 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:

[0106] DNA sequence

[0107]

[0108] Protein sequence

[0109] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRHSDEHHHDDSLPHPQQATDDSSNQSDSNSNEGRHLLLVSGAGDGPPLCSQNLGAPGGGPNNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHDGIDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYDPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSDRPPDPLEPPSLPAERPGPPTPAAAHSIPYNSCREKEPSYPMPVQETQAPESPGENSEQALQTLSPRAIPRNPDGGPLESSSDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRASRLPGPTGSVVSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLPSKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPINSTRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSSENRGLGSELSKPGVLASQVDSPFSGCFEDLAISASTSLGMGPCHGPEENEYKSEGTFGIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSP(SEQ ID NO:14)

[0110] The highlighted portions of SEQ ID NOs: 13 and 14 represent the proline-rich domains.

[0111] Another preferred adjuvant is a fusion protein LMP1 hIPS1 (delta Pro Delta TM), which contains the full-length LMP1 from Epstein-Barr virus, fused to amino acids 2-462 of a modified human IPS1 that has had its proline-rich domain and transmembrane domain removed. 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 as follows:

[0112] DNA sequence

[0113] (SEQ ID NO:15)

[0114] Protein sequence

[0115] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRHSDEHHHDDSLPHPQQATDDSSNQSDSNSNEGRHLLLVSGAGDGPPLCSQNLGAPGGGPNNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHDGIDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYDPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSDRGENSEQALQTLSPRAIPRNPDGGPLESSSDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRASRLPGPTGSVVSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLPSKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPINSTRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSSENRGLGSELSKPGVLASQVDSPFSGCFEDLAISASTSLGMGPCHGPEENEYKSEGTFGIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSP (SEQ ID NO:16)

[0116] Another preferred adjuvant is the fusion protein LMP1 hIPS1 delta TM (Rev), which contains the full-length LMP1 from Epstein-Barr virus, fused to the amino acids 2-514 of the human IPS1 sequence (wherein the transmembrane domain has been removed) in reverse order (i.e., from the C-terminus to the N-terminus of the native sequence). In the fusion protein, the first amino acid (methionine, encoded by the original direct human DNA) and the TM domain of human IPS1 are removed. The fusion protein is codon-optimized for human use. The DNA and encoded amino acid sequences of the fusion protein are as follows:

[0117] DNA sequence

[0118]

[0119] Protein sequence

[0120] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRHSDEHHHDDSLPHPQQATDDSSNQSDSNSNEGRHLLLVSGAGDGPPLCSQNLGAPGGGPNNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHDGIDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYDPSPRHCPVEREQFKRDAQPRPGGDPDAPPGPNGELLQISPNEAVHIGFTGESKYENEEPGHCPGMGLSTSASIALDEFCGSFPSDVQSALVGPKSLESGLGRNESSSDLWASSGGTAGAPTPAAPTEENRSSGDTPVTSASVKTLVMSTPVKSPVMGARTSNIPLKSPAPNTLANSPVAGPPKSSTPLKSPVTSVSAPNAPVKLAVTNVPMLTTPVKSPLSNAPAEAGSSCIIPEAQDSEAGQKGEAAGASALGPSSSSFSTGTSVVSGTPGPLRSARPTSRALPQFSVSPSVPGRSPTLSSTAGATHTSGLETDQEQHGSSTLPSLAALDSSSELPGGDPNRPIARPSLTQLAQESNEGPSEPAQTEQVPMPYSPEKERCSNYPISHAAAPTPPGPREAPLSPPELPDPPRDSTRPQYSQYVSAVEDALDVLECGRLAAIFYEVWGPRRQLTNFLHWLTDRNGSLTCTARLRDQDRATLCPLYPLIEVVDVNCFNSFNRCIYKYTKDEAFPM(SEQ ID NO:18)

[0121] The highlighted portions of SEQ ID NOs: 17 and 18 represent the proline-rich domains.

[0122] Another preferred adjuvant is a fusion protein LMP1 (delta IC) hIPS1 (delta TM) LMP1 (cyt), which contains a truncated LMP1 sequence (missing the intracytoplasmic domain) and is fused to human IPS1 that lacks the transmembrane domain, and then fused to the LMP1 intracytoplasmic 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 the fusion protein are as follows:

[0123] DNA sequence

[0124]

[0125] Protein sequence

[0126] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSDRPPDPLEPPSLPAERPGPPTPAAAHSIPYNSCREKEPSYPMPVQETQAPESPGENSEQALQTLSPRAIPRNPDGGPLESSSDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRASRLPGPTGSVVSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLPSKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPINSTRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSSENRGLGSELSKPGVLASQVDSPFSGCFEDLAISASTSLGMGPCHGPEENEYKSEGTFGIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSPHSDEHHHDDSLPHPQQATDDSSNQSDSNSNEGRHLLLVSGAGDGPPLCSQNLGAPGGGPNNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHDGIDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO:20)

[0127] The highlighted portions of SEQ ID NOs: 19 and 20 represent the proline-rich domains.

[0128] Another preferred adjuvant is a fusion protein LMP1 (delta IC) hIPS1 (delta TM Pro) LMP1 (cyt), which contains a truncated LMP1 sequence (lacking the intracytoplasmic domain) and is fused to human IPS1 lacking a transmembrane domain and a proline-rich domain, and then fused to the LMP1 intracytoplasmic 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 the fusion protein are as follows:

[0129] DNA sequence

[0130]

[0131] Protein sequence

[0132] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSDRGENSEQALQTLSPRAIPRNPDGGPLESSSDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRASRLPGPTGSVVSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLPSKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPINSTRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSSENRGLGSELSKPGVLASQVDSPFSGCFEDLAISASTSLGMGPCHGPEENEYKSEGTFGIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSPHSDEHHHDDSLPHPQQATDDSSNQSDSNSNEGRHLLLVSGAGDGPPLCSQNLGAPGGGPNNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHDGIDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD

[0133] (SEQ ID NO:22)

[0134] Another preferred adjuvant is a fusion protein LMP1 (delta IC) hIPS1 (delta TM Rev) LMP1 (cyt), which contains a truncated LMP1 sequence (missing the intracytoplasmic domain) and is fused to the human IPS1 sequence presented in the reverse order (i.e., from the C-terminus to the N-terminus of the native sequence, excluding the TM domain), and then fused to the LMP1 intracytoplasmic 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 the fusion protein are shown below:

[0135] DNA sequence

[0136]

[0137] Protein sequence

[0138] MDLDLERGPPGPRRPPRGPPLSSSIGLALLLLLLALLFWLYIIMSNWTGGALLVLYAFALMLVIIILIIFIFRRDLLCPLGALCLLLLMITLLLIALWNLHGQALYLGIVLFIFGCLLVLGLWIYLLEILWRLGATIWQLLAFFLAFFLDIILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRPSPRHCPVEREQFKRDAQPRPGGDPDAPPGPNGELLQISPNEAVHIGFTGESKYENEEPGHCPGMGLSTSASIALDEFCGSFPSDVQSALVGPKSLESGLGRNESSSDLWASSGGTAGAPTPAAPTEENRSSGDTPVTSASVKTLVMSTPVKSPVMGARTSNIPLKSPAPNTLANSPVAGPPKSSTPLKSPVTSVSAPNAPVKLAVTNVPMLTTPVKSPLSNAPAEAGSSCIIPEAQDSEAGQKGEAAGASALGPSSSSFSTGTSVVSGTPGPLRSARPTSRALPQFSVSPSVPGRSPTLSSTAGATHTSGLETDQEQHGSSTLPSLAALDSSSELPGGDPNRPIARPSLTQLAQESNEGPSEPAQTEQVPMPYSPEKERCSNYPISHAAAPTPPGPREAPLSPPELPDPPRDSTRPQYSQYVSAVEDALDVLECGRLAAIFYEVWGPRRQLTNFLHWLTDRNGSLTCTARLRDQDRATLCPLYPLIEVVDVNCFNSFNRCIYKYTKDEAFPMHSDEHHHDDSLPHPQQATDDSSNQSDSNSNEGRHLLLVSGAGDGPPLCSQNLGAPGGGPNNGPQDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHNPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHDGIDPHLPTLLLGTSGSGGDDDDPHGPVQLSYYD(SEQ ID NO:24)

[0139] The highlighted portions of SEQ ID NOs: 23 and 24 represent the proline-rich domains.

[0140] In a preferred embodiment, immune checkpoint inhibitor molecules or soluble immunomodulator molecules will be encoded in the viral vector to enhance the immune response against the tumor. Immune checkpoint inhibitor molecules can be, but are not limited to, anti-CTLA-4 molecules, PD1 blockers, and PDL1 blockers. Immune checkpoint inhibitor molecules can be proteins, such as antibodies, or soluble forms of anti-checkpoints.

[0141] In certain embodiments, the viral vector may include more than one expression cassette. In some embodiments, the viral vector particle may include more than one nucleic acid molecule, such as two or three nucleic acid molecules, which may be delivered alone or operably connected. 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 immune checkpoint inhibitor molecule different from the second nucleic acid molecule encoding.

[0142] In one aspect, the technology is an immunotherapeutic formulation for preventing or treating a disease or condition in a subject. The vaccine includes a therapeutically effective amount of a viral vector. The disease can be any disease that requires vaccination against a pharmaceutical agent, such as cancer or infection.

[0143] In another aspect, the technology is a method for inducing or enhancing an immune response against cancer or infection in a subject. The method comprises administering a therapeutically effective amount of a viral vector or an immunotherapeutic formulation to a subject in need thereof. DETAILED DESCRIPTION

[0144] Example 1. Molecular constructs.

[0145] According to the bimolecular adjuvant strategy, a vector containing the following elements is constructed: (a) a promoter, preferably a human ubiquitin promoter; (b) a reporter gene (e.g., green fluorescent protein) or, optionally, one or more antigens fused in a single transgene; (c) an IRES followed by a first adjuvant gene (i.e., LMP1 or LMP1 CO); (d) an IRES followed by a second adjuvant gene (i.e., LMP1-IPS1 fusion protein); (e) optionally, an IRES followed by one or more genes encoding soluble and secreted immune checkpoint inhibitors or soluble immunomodulator molecules (see, e.g., Figures 8A-8B ). Generally, the sequences are preferably in the order described above (particularly, the order of (c) and (d) may be reversed), but the genes may be located in the vector in any other suitable order. Control vectors having some but not all of the above regions were also constructed.

[0146] According to the single molecule adjuvant strategy, a vector containing the following elements is constructed: (a) a promoter, preferably a human ubiquitin promoter; (b) a reporter gene (e.g., green fluorescent protein) or, optionally, one or more antigens fused in a single transgene; (c) an IRES followed by an adjuvant fusion protein gene (i.e., LMP1 lacking the intracytoplasmic signaling domain is fused to hIPS1 or its functional equivalent (e.g., hIPS1 delta TM), which is further fused to the intracytoplasmic signaling domain of LMP1; or LMP1 lacking the intracytoplasmic signaling domain is fused to hIPS1 or its functional equivalent, which is further fused to the intracytoplasmic signaling domain of LMP1; and (d) optionally, an IRES followed by one or more genes encoding soluble and secreted immune checkpoint inhibitors or soluble immunomodulator molecules (see, e.g., Figures 8A-8B ). Generally, the sequence is preferably in the order described above, but the genes can be located in the vector in any other suitable order. Control vectors with some but not all of the above regions are also constructed.

[0147] Example 2. Production of viral vectors.

[0148] Lentiviral vectors were produced by transient calcium phosphate transfection of HEK 293T cell line as described by Nasri et al. (2014). HEK 293T cells were cultured at 1.6 × 10 8 Each cell culture vessel was inoculated in 250 mL of complete medium in a two-chamber cell culture vessel (Cell Stack, Corning) and kept in an incubator with a humidified atmosphere of 5% CO2 at 37°C for 24 hours to allow cell adhesion. For each vector production, each cell culture vessel was transfected as follows. Lentivirus 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. The DNA mixture was then added dropwise to 12.1 mL of 37°C preheated HBS 2X, pH = 7.1, and the resulting 24.2 mL of precipitate was added to the cell culture medium after incubation at room temperature for 30 minutes. The transfected cells were incubated at 37°C, 5% CO2. 24 hours after transfection, the medium was replaced with 210 mL of harvest medium without serum and phenol red, and the viral supernatant was harvested after another 24 hours and clarified by centrifugation at 2500 rpm for 5 minutes. The harvested clarified bodies (210 mL) were treated with DNase I for 30 minutes in the presence of MgCl2 to cleave any residual DNA and concentrated by centrifugation at 22000 rpm, 4°C for 1 hour. The vector pellets were resuspended in 70 μl Tris-trehalose (50 mM), combined in 1.5 mL microtubes, divided into 50 μL aliquots, frozen and stored at ≤-70°C.

[0149] The adjuvanted vectors gave slightly lower yields compared to the GFP vectors, certainly due to the presence of the longer DNA expression cassette. However, for all adjuvanted constructs, the titers were at least 10 9 TU / mL range and was found to be consistent across different production campaigns. No issues were observed that would affect industrial bioproduction.

[0150] Example 3. In vitro effects of dual adjuvant lentiviral vectors on activation of CD40L and STING pathways

[0151] Fresh human dendritic cells and macrophages were obtained from healthy human donors (leukocytes) by density gradient. CD14+ monocytes were purified from PBMC 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 using a published method. 10% of the culture medium was replaced after 3 days to supplement cytokines, and cells were harvested after a total of 6 days of culture using a non-enzymatic cell dissociation solution. DCs were then re-plated in complete RPMI culture medium + 4 μg / ml polybrene + lentiviral constructs (MOI of 15) + GM-CSF and IL-4. After 2 hours, 700 μl of complete RPMI culture medium + GM-CSF / IL-4 were 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 activation marker expression.

[0152] CD14+ monocytes were differentiated into M1 or M2 macrophages with GM-CSF (M1) or M-CSF (M2). After 3 days, 10% of the culture medium was replaced to supplement cytokines, and the cells were harvested after a total of 6 days of culture using a non-enzymatic cell dissociation solution, and then the macrophages were combined at a ratio of 1:1. The M1 / M2 macrophages were then re-seeded in 300μl complete RPMI+4μg / ml polybrene+lentiviral construct (MOI of 15)+M-CSF). After 2 hours, 700μl complete RPMI+M-CSF was added, and the cells were cultured for a total of 96 hours. Additional control wells were stimulated for a total of 96 hours with IFN-γ and LPS (M1) or IL-13 and IL-4 (M2) as positive controls for activation marker expression.

[0153] Human DCs and macrophages were transduced with lentiviral vectors containing the expression cassette at an MOI of 15 as follows:

[0154] Construct 1: GFP-IRES-LMP1(dIC)-IPS1(dTM)-LMP1(IC)

[0155] Construct 2: GFP-IRES-LMP1(dIC)-IPS1(dTM dPro)-LMP1(IC)

[0156] Construct 3: GFP-IRES-LMP1(dIC)-IPS1(dTM Rev)-LMP1(IC)

[0157] Construct 4: GFP-IRES-LMP1-IPS1 (dTM)

[0158] Construct 5: GFP-IRES-LMP1-IPS1 (dTM dPro)

[0159] Construct 6: GFP-IRES-LMP1-IPS1 (dTM Rev)

[0160] Control construct 1: GFP

[0161] Control construct 2: GFP-IRES-LMP1 (dIC)

[0162] See also Figures 8A-8B For illustration of adjuvant constructs, and see Fig.10 For illustration of control constructs.

[0163] The proliferation of dendritic cells and macrophages was quantified after 24 h of culture. 3 H-TdR pulses and overnight culture were followed by harvesting and determination of radioactive thymidine incorporation by a standard scintillation counter. Proliferation was slightly reduced using the adjuvanted vector compared to the GFP vector, most likely due to the presence of a longer DNA expression cassette. Viability of transduced cells was determined by staining with a fixable viability dye prior to analysis using a BD FACS Canto System flow cytometer, as described previously. Although slight differences were observed with the adjuvanted vector, no significant toxicity was found.

[0164] The expression of GFP in cells transduced with each construct was determined by measuring fluorescence using an Attune NxT flow cytometer after 96 hours of culture. Fig.11A (dendritic cells) and 11B (macrophages). The percentage of live cells and GFP-positive cells was determined by gating the exclusion of debris / live cells / single cells. Three independent experiments were performed using PBMCs isolated from different donors. The data in the graphs represent the average of replicates from a representative experiment. The results are shown in Fig.11AAs shown in 11A (dendritic cells) and 11B (macrophages), slight differences were observed between the adjuvanted vectors for both cell types, but significant expression of GFP / transgene was observed in all IRES constructs. Removal of the Pro domain in the constructs resulted in increased expression of the GFP transgene in constructs 2 and 5, most likely due to the presence of a shorter DNA cassette. Reversal of the IPS1 CARD and PRO domains strongly reduced expression of the GFP transgene, as observed in constructs 3 and 6.

[0165] The activation and maturation of dendritic cells and macrophages induced by lentiviral vectors were evaluated by measuring the expression of surface markers and assessing their cytokine and chemokine release profiles. To determine the level of lentiviral integration and dendritic cell / macrophage activation, cells were harvested after 96 hours of culture, 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 for 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 culture, the production of IFN-α and IFN-β in the culture supernatant and the immunostimulatory cytokines IL-8, IL-1beta, TNF-α, IL-6 and IL-12p70 was measured by Luminex analysis using the Bioplex200 system with high-throughput fluidics (BioRad) to evaluate the activation of the STING pathway and the CD40 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. The graphical data represent the average of replicates of representative experiments. The results are shown in Fig. 12A (dendritic cells, cytokines), 12B (dendritic cells, membrane markers), 12C (macrophages, cytokines), and 12D (macrophages, membrane markers).

[0166] For transduced dendritic cells, the results of GFP-positive cell expression of surface markers showed that the IRES construct upregulated the expression of the following immune activation markers: MHCII (upregulation was observed in constructs 1, 2, 4, and 5); CD40 (significantly increased in constructs 1, 2, 3, and 6); CD83 (2-fold increase in constructs 1, 3, 4, and 5), CD80 / 86 (slightly upregulated in constructs 1, 2, 3, 4, and 6). Consistent with the upregulation of these activation markers, the increase in cytokine expression was as follows: pro-inflammatory IL-6 was expressed in constructs 1 and 2; pro-inflammatory TNF-α was significantly increased in constructs 1, 2, and 5; IL-12 was significantly increased in constructs 1 and 5. The level of anti-inflammatory IL-10 was not affected in any of the constructs evaluated.

[0167] Similarly, results of GFP-positive cell expression markers in transduced macrophages showed that the IRES constructs upregulated the expression of immune activation markers: MHCII was induced in constructs 1, 2, 4, and 5; CD83 was increased 2-fold in constructs 1 and 6; CD80 / 86 was increased in constructs 1 and 2. Consistent with the upregulation of these activation markers, the increase in cytokine expression was as follows: pro-inflammatory IL-1β was increased 4-fold in construct 1 and lower than that in constructs 3, 4, and 6; the levels of pro-inflammatory IL-6 were significantly increased in constructs 1, 2, 4, and 5; and the pro-inflammatory TNF-α was increased 4-fold in constructs 1 and 2. The levels of anti-inflammatory IL-10 were not affected in any of the constructs evaluated.

[0168] In conclusion, removal of the IPS1 transmembrane domain and reversing the orientation of the IPS1 CARD and PRO domains did not show any immunostimulatory effect. Removal of the IPS1 transmembrane domain increased the activity of the adjuvant, while the orientation of LMP1 and IPS1 had no significant effect on the adjuvant effect of the IRES construct.

[0169] Example 4. Using single or multiple antibodies via dual adjuvanted LMP1-IPS1 (CD40L and STING) lentiviral vectors In vivo immunogenicity in healthy mice treated with the original drug showed excellent immunogenicity.

[0170] Healthy mice were treated differently with viral vectors containing the expression cassette as described in Example 1. Experiments were performed to compare the immune response when antigen and adjuvant (i.e., CD40L and STING pathway) were expressed alone or together after two administrations (prime + boost). 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 mice, which allowed the detection and quantification of antigen-specific immune cells, such as CD4 + 、CD8 +Treatment with dual-adjuvanted lentiviral vectors encoding antigen and LMP1-IPS1 fusion is expected to increase specific immunogenicity compared with single-adjuvanted lentiviral vectors, or when expressing only the membrane domain of LMP1.

[0171] Example 5. In vivo immunogenicity in a specific tumor mouse model including multiple antigens and CD40L and Superior efficacy of dual-adjuvant lentiviral vectors in combination with STING pathway activators.

[0172] As described in Example 1, a mouse model of a specific tumor is treated with a viral vector containing an expression cassette. Mice are divided into different treatment groups according to vector type and construct, dose and number of injections (primary immunization + booster injection). In vivo efficacy and immunogenicity are evaluated by detection of tumor growth rate, survival rate, CD4+ and CD8+ and memory T cell specific antigens according to FACS analysis of mouse blood biomarkers (IFN-γ and various interleukins). It is expected that the double adjuvanted lentiviral vector encoding the indicated specific antigen induces the most effective and lasting immune response in all experimental groups, thereby inducing higher survival rate and / or lower tumor growth in the treated mouse group.

[0173] Example 6. In vivo immunogenicity in a mouse model of specific anti-checkpoint sensitive tumors shows that Superior efficacy of dual-adjuvanted lentiviral vectors for multiple antigen and anti-checkpoint combinations.

[0174] Mouse models of specific tumors were treated with viral vectors containing expression cassettes and soluble and secreted forms of one or more anti-checkpoint molecules as described in Example 1. Mice were divided into different treatment groups according to vector construct, dose and number of injections (prime + booster injection). Tumor growth rate, survival rate and CD4 were detected based on FACS analysis of mouse blood biomarkers (IFN-γ and various interleukins). + 、CD8 + In vivo efficacy and immunogenicity will be assessed by detection of specific antigens and memory T cells. Dual-adjuvanted lentiviral vectors encoding indicated specific antigens and anti-checkpoint molecules are expected to induce the most potent and durable immune responses in all experimental groups.

[0175] This application claims priority to U.S. Provisional Application No. 62 / 426,860, filed on November 28, 2016, the entire contents of which are incorporated herein by reference.

[0176] As used herein, "consisting essentially of" allows for the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation of the term "comprising / including" in the present invention, particularly in the description of components of a composition or in the description of elements of a device, can be interchanged with "consisting essentially of" or "consisting of".

[0177] While the invention has been described in conjunction with certain preferred embodiments, upon reading the foregoing description, one of ordinary skill will be able to make various changes, substitutions, and other alterations to the compositions and methods described herein.

[0178] References

[0179] Barry,M.et al.Role of endogenous endonucleases and tissue site intransfection and CpG-mediated immune activation after naked DNA injection

[0180] Hum Gene Ther,10(15)(1999),pp.2461–2480

[0181] McNamara,M.et al.RNA-Based Vaccines in Cancer Immunotherapy.J ImmunolRes.2015;2015:794528.

[0182] 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 Bio <120> Viral vector constructs expressing genetic adjuvants for activation of CD40 and STING pathways <130> P19111934WP <150> US 62 / 426,860 <151> 2016-11-28 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 1158 <212> DNA <213> Epstein-Barr virus <400> 1 atggatctgg acctggaaag 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 gggaatcgtg 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 cacagcgacg aacaccacca tgatgacagc 600 ctgcctcatc ctcagcaggc caccgacgat agcagcaacc agagcgacag caacagcaac 660 gagggcagac atctgctgct ggtgtctggt gctggcgacg gacctcctct gtgttctcaa 720 aatcttggcg cccctggcgg cggaccaaac aatggacctc aggaccccga caacaccgac 780 gacaatggcc ctcaagatcc tgataatacc gatgacaacg gcccacacga ccctctgcct 840 caagacccag ataacacaga cgataacggt ccacaagatc cggacaatac tgacgataat 900 ggaccccacg atccactgcc tcacaaccct agcgatagcg ccggaaatga tggcggacct 960 ccacagctga ccgaggaagt ggaaaacaaa ggcggagatc agggccctcc tctgatgacc 1020 gatggcggag gtggacactc tcacgattct ggccacgacg gcatcgaccc tcatctgcct 1080 acactgctgc tcggcacatc tggctctggc ggcgacgatg atgatcctca tggacctgtg 1140 cagctgagct actacgac 1158 <210> 2 <211> 386 <212> PRT <213> Epstein - Barr virus <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 His Ser 180 185 190 Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr 195 200 205 Asp Asp Ser Ser Asn Gln Ser Asp Ser Asn Ser Asn Glu Gly Arg His 210 215 220 Leu Leu Leu Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln 225 230 235 240 Asn Leu Gly Ala Pro Gly Gly Gly Pro Asn Asn Gly Pro Gln Asp Pro 245 250 255 Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp 260 265 270 Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp 275 280 285 Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp 290 295 300 Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro 305 310 315 320 Pro Gln Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly Pro 325 330 335 Pro Leu Met Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly His 340 345 350 Asp Gly Ile Asp Pro His Leu Pro Thr Leu Leu Leu Gly Thr Ser Gly 355 360 365 Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser Tyr 370 375 380 Tyr Asp 385 <210> 3 <211> 570 <212> DNA <213> Epstein - Barr virus <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 570 <210> 4 <211> 190 <212> PRT <213> Epstein - Barr virus <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 180 185 190 <210> 5 <211> 2187 <212> DNA <213> Artificial sequence <220> <223> Fusion protein of LMP1 from Epstein-Barr virus and ISP1 from Homo sapiens <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 cggcctcctg atcctctcga acctccatct 900 ctgcccgccg aaagacctgg acctcctaca ccagctgccg ctcacagcat cccttacaac 960 agctgcagag agaaagaacc tagctacccc atgcctgtgc aagagacaca ggccccagaa 1020 agccctggcg agaatagcga acaggctctg cagaacactga gccccagagc cattcctaga 1080 aaccctgatg gcggccctct ggaaagctct agtgatctgg ccgctctgtc ccctctgaca 1140 agctctggac accaagagca ggataccgag ctgggcagca cacatacagc cggcgctaca 1200 agcagcctga cacctttag 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 agctcccaac cagctctaag 1560 ccacctggcg ccgtgccatc taacgccctg acaaatcctg ctccaagcaa gctgcccatc 1620 aactccacaa gagccggcat ggtgccctct aaggtgccca catctatggt gctgaccaag 1680 gtgtccgcca gcaccgtgcc aacagatggc agctccagaa acgaggaac ccctgccgct cctactcctg ctggcgctac aggcggatct tctgcttggc tggatagcag cagcgagaac agaggcctgg gcagcgagct ttctaaacct ggcgtgctgg cttcccaggt ggacagccca ttttccggct gctttgagga cctggctatc agcgcctcta caagcctcgg catgggacct 1920 tgtcacggcc ccgaggaaa cgagtacaag agcgagggca ccttcggcat ccacgtggcc gagaatccta gcatccaact gctggaaggc aaccccggac ctccagctga tccagatggc ggaccaagac ctcaggccga cagaaagttc caagagcgcg aggtgccctg ccacagacct tctccaggtg ctctgtggct gcaggttgca gtgacaggcg tcctggtggt tacactgctc gtggtcctgt atagcggcg gctgcac 2187 <210> 6 <211> 729 <212> PRT <213> Artificial sequence <220> <223> If you want to have a specific LMP1 user interface, you need to create an ISP1 user interface <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 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> 7 <211> 2106 <212> DNA <213> Artificial sequence <220> <223> Fusion protein of LMP1 from Epstein-Barr virus and ISP1 from Homo sapiens <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 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 cagaacactga gccccagagc cattcctaga 1080 aaccctgatg gcggccctct ggaaagctct agtgatctgg ccgctctgtc ccctctgaca 1140 agctctggac accaagagca ggataccgag ctgggcagca cacatacagc cggcgctaca 1200 agcagcctga cacctttag 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 agctcccaac cagctctaag 1560 ccacctggcg ccgtgccatc taacgccctg acaaatcctg ctccaagcaa gctgcccatc 1620 aactccacaa gagccggcat ggtgccctct aaggtgccca catctatggt gctgaccaag gtgtccgcca gcaccgtgcc aacagatggc agctccagaa acgaggaac ccctgccgct cctactcctg ctggcgctac aggcggatct tctgcttggc tggatagcag cagcgagaac agaggcctgg gcagcgagct ttctaaacct ggcgtgctgg cttcccaggt ggacagccca ttttccggct gctttgagga cctggctatc agcgcctcta caagcctcgg catgggacct 1920 tgtcacggcc ccgaggaaa cgagtacaag agcgagggca ccttcggcat ccacgtggcc gagaatccta gcatccaact gctggaaggc aaccccggac ctccagctga tccagatggc ggaccaagac ctcaggccga cagaaagttc caagagcgcg aggtgccctg ccacagacct tctcca 2106 <210> 8 <211> 702 <212> PRT <213> Artificial sequence <220> <223> If you want to have a specific LMP1 user interface, you need to create an ISP1 user interface <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 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> 9 <211> 1953 <212> DNA <213> Artificial sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens-derived ISP1 <400> 9 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> 10 <211> 651 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens ISP1 <400> 10 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> 11 <211> 2109 <212> DNA <213> Artificial sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens ISP1 <400> 11 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 cccagcccca gacactgccc cgtggagaga 600 gagcagttca agagagacgc ccagcccaga cccggcggcg accccgacgc cccccccggc 660 cccaacggcg agctgctgca gatcagcccc aacgaggccg tgcacatcgg cttcaccggc 720 gagagcaagt acgagaacga ggagcccggc cactgccccg gcatgggcct gagcaccagc 780 gccagcatcg ccctggacga gttctgcggc agcttcccca gcgacgtgca gagcgccctg 840 gtgggcccca agagcctgga gagcggcctg ggcagaaacg agagcagcag cgacctgtgg 900 gccagcagcg gcggcaccgc cggcgccccc acccccgccg cccccaccga ggagaacaga 960 agcagcggcg acacccccgt gaccagcgcc agcgtgaaga ccctggtgat gagcaccccc 1020 gtgaagagcc ccgtgatggg cgccagaacc agcaacatcc ccctgaagag ccccgccccc 1080 aacaccctgg ccaacagccc cgtggccggc ccccccaaga gcagcacccc cctgaagagc 1140 cccgtgacca gcgtgagcgc ccccaacgcc cccgtgaagc tggccgtgac caacgtgccc 1200 atgctgacca cccccgtgaa gagccccctg agcaacgccc ccgccgaggc cggcagcagc 1260 tgcatcatcc ccgaggccca ggacagcgag gccggccaga agggcgaggc cgccggcgcc 1320 agcgccctgg gccccagcag cagcagcttc agcaccggca ccagcgtggt gagcggcacc 1380 cccggccccc tgagaagcgc cagacccacc agcagagccc tgccccagtt cagcgtgagc 1440 cccagcgtgc ccggcagaag ccccaccctg agcagcaccg ccggcgccac ccacaccagc 1500 ggcctggaga ccgaccagga gcagcacggc agcagcaccc tgcccagcct ggccgccctg 1560 gacagcagca gcgagctgcc cggcggcgac cccaacagac ccatcgccag acccagcctg 1620 acccagctgg cccaggagag caacgagggc cccagcgagc ccgcccagac cgagcaggtg 1680. cccatgccct acagccccga gaaggagaga tgcagcaact accccatcag ccacgccgcc gcccccaccc cccccggccc cagagaggcc cccctgagcc cccccgagct gcccgacccc 1800. cccagagaca gcaccagacc ccagtacagc cagtacgtga gcgccgtgga ggacgccctg gacgtgctgg agtgcggcag actggccgcc atcttctacg aggtgtgggg ccccagaaga cagctgacca acttcctgca ctggctgacc gacagaaacg gcagcctgac ctgcaccgcc agactgagag accaggacag agccaccctg tgccccctgt accccctgat cgaggtggtg gacgtgaact gcttcaacag cttcaacaga tgcatctaca agtacaccaa ggacgaggcc ttccccatg 2109 <210> 12 <211> 703 <212> PRT <213> Artificial sequence <220> <223> Even if the LMP1 enables an ISP1 encryption cycle <400> 12 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 <210> 13 <211> 2694 <212> DNA <213> Artificial sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens ISP1 <400> 13 atggatctgg acctggaaag 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 gggaatcgtg 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 cacagcgacg aacaccacca tgatgacagc 600 ctgcctcatc ctcagcaggc caccgacgat agcagcaacc agagcgacag caacagcaac 660 gagggcagac atctgctgct ggtgtctggt gctggcgacg gacctcctct gtgttctcaa 720 aatcttggcg cccctggcgg cggaccaaac aatggacctc aggaccccga caacaccgac 780 gacaatggcc ctcaagatcc tgataatacc gatgacaacg gcccacacga ccctctgcct 840 caagacccag ataacacaga cgataacggt ccacaagatc cggacaatac tgacgataat 900 ggaccccacg atccactgcc tcacaaccct agcgatagcg ccggaaatga tggcggacct 960 ccacagctga ccgaggaagt ggaaaacaaa ggcggagatc agggccctcc tctgatgacc 1020 gatggcggag gtggacactc tcacgattct ggccacgacg gcatcgaccc tcatctgcct 1080 acactgctgc tcggcacatc tggctctggc ggcgacgatg atgatcctca tggacctgtg 1140 cagctgagct actacgaccc tttcgccgag gacaagacct acaagtacat ctgccggaac 1200 ttcagcaact tctgcaacgt ggacgtggtg gaaattctgc cctacctgcc ttgcctgacc 1260 gccagagatc aggacagact gagagccaca tgtaccctga gcggcaacag agacacactg 1320 tggcacctgt tcaacaccct gcagagaagg cctggctgggg tcgagtactt tatcgccgct 1380 ctgagaggct gcgagctggt cgatctggct gatgaagtgg ccagcgtgta ccagagctac 1440 cagcctagaa ccagcgaccg gcctcctgat cctctcgaac ctccatctct gcccgccgaa 1500 agacctggac ctcctacacc agctgccgct cacagcatcc cttaacacag ctgcagagag 1560 aaagaaccta gctacccat gcctgtgcaa gagacacagg ccccagaaag ccctggcgag 1620 aatagcgaac aggctctgca gacactgagc cccagagcca ttcctagaaa ccctgatggc 1680 ggccctctgg aaagctctag tgatctggcc gctctgtccc ctctgacaag ctctggacac 1740 caagagcagg ataccgagct gggcagcaca catacagccg gcgctacaag cagcctgaca 1800 ccttctagag gccccgtgtc tcccagcgtg tcatttcagc ctctggccag gtctacccct 1860 agggcttcta gactgcctgg accaacaggc agcgtggtgt ctaccggcac aagcttcagc 1920 tctagctctc ctggactggc tagtgccggt gccgctgagg gaaaacaagg cgccgaatct 1980 gatcaggccg agcctatcat ctgtagcagc ggagcagaag cccctgccaa tagcctgcct 2040 agcaaggtgc caaccacact gatgcccgtg aacacagtgg ccctgaaggt gccagctaat 2100 cctgcctccg tgtccaccgt gccttctaag ctgccaacca gctctaagcc acctggcgcc 2160 gtgccatcta acgccctgac aaatcctgct ccaagcaagc tgcccatcaa ctccacaaga 2220 gccggcatgg tgccctctaa ggtgcccaca tctatggtgc tgaccaaggt gtccgccagc 2280 accgtgccaa cagatggcag ctccagaaac gaggaaaccc ctgccgctcc tactcctgct 2340 ggcgctacag gcggatcttc tgcttggctg gatagcagca gcgagaacag aggcctgggc 2400 agcgagcttt ctaaacctgg cgtgctggct tcccaggtgg acagcccatt ttccggctgc 2460 tttgaggacc tggctatcag cgcctctaca agcctcggca tgggaccttg tcacggcccc 2520 gaggaaaacg agtacaagag cgagggcacc ttcggcatcc acgtggccga gaatcctagc 2580 atccaactgc tggaaggcaa ccccggacct ccagctgatc cagatggcgg accaagacct 2640 caggccgaca gaaagttcca agagcgcgag gtgccctgcc acagaccttc tcca 2694 <210> 14 <211> 898 <212> PRT <213> Artificial sequence <220> <223> Connect the LMP1 to the ISP1 connection <400> 14 Met Asp Leu Asp Leu Glu Arg Gly 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 His Ser 180 185 190 Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr 195 200 205 Asp Asp Ser Ser Asn Gln Ser Asp Ser Asn Ser Asn Glu Gly Arg His 210 215 220 Leu Leu Leu Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln 225 230 235 240 Asn Leu Gly Ala Pro Gly Gly Gly Pro Asn Asn Gly Pro Gln Asp Pro 245 250 255 Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp 260 265 270 Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp 275 280 285 Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp 290 295 300 Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro 305 310 315 320 Pro Gln Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly Pro 325 330 335 Pro Leu Met Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly His 340 345 350 Asp Gly Ile Asp Pro His Leu Pro Thr Leu Leu Leu Gly Thr Ser Gly 355 360 365 Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser Tyr 370 375 380 Tyr Asp Pro Phe Ala Glu Asp Lys Thr Tyr Lys Tyr Ile Cys Arg Asn 385 390 395 400 Phe Ser Asn Phe Cys Asn Val Asp Val Val Glu Ile Leu Pro Tyr Leu 405 410 415 Pro Cys Leu Thr Ala Arg Asp Gln Asp Arg Leu Arg Ala Thr Cys Thr 420 425 430 Leu Ser Gly Asn Arg Asp Thr Leu Trp His Leu Phe Asn Thr Leu Gln 435 440 445 Arg Arg Pro Gly Trp Val Glu Tyr Phe Ile Ala Ala Leu Arg Gly Cys 450 455 460 Glu Leu Val Asp Leu Ala Asp Glu Val Ala Ser Val Tyr Gln Ser Tyr 465 470 475 480 Gln Pro Arg Thr Ser Asp Arg Pro Pro Asp Pro Leu Glu Pro Pro Ser 485 490 495 Leu Pro Ala Glu Arg Pro Gly Pro Pro Thr Pro Ala Ala Ala His Ser 500 505 510 Ile Pro Tyr Asn Ser Cys Arg Glu Lys Glu Pro Ser Tyr Pro Met Pro 515 520 525 Val Gln Glu Thr Gln Ala Pro Glu Ser Pro Gly Glu Asn Ser Glu Gln 530 535 540 Ala Leu Gln Thr Leu Ser Pro Arg Ala Ile Pro Arg Asn Pro Asp Gly 545 550 555 560 Gly Pro Leu Glu Ser Ser Ser Asp Leu Ala Ala Leu Ser Pro Leu Thr 565 570 575 Ser Ser Gly His Gln Glu Gln Asp Thr Glu Leu Gly Ser Thr His Thr 580 585 590 Wing Gly Wing Thr Ser Ser Leu Thr Pro Ser Arg Gly Pro Val Ser Pro 595 600 605 Ser Val Ser Phe Gln Pro Leu Ala Arg Ser Thr Pro Arg Ala Ser Arg 610 615 620 Leu Pro Gly Pro Thr Gly Ser Val Val Ser Thr Gly Thr Ser Phe Ser 625 630 635 640 Ser Ser Ser Pro Gly Leu Ala Ser Ala Gly Ala Ala Glu Gly Lys Gln 645 650 655 Gly Ala Glu Ser Asp Gln Ala Glu Pro Ile Ile Cys Ser Ser Gly Ala 660 665 670 Glu Ala Pro Ala Asn Ser Leu Pro Ser Lys Val Pro Thr Thr Leu Met 675 680 685 Pro Val Asn Thr Val Ala Leu Lys Val Pro Ala Asn Pro Ala Ser Val 690 695 700 Ser Thr Val Pro Ser Lys Leu Pro Thr Ser Ser Lys Pro Pro Gly Ala 705 710 715 720 Val Pro Ser Asn Ala Leu Thr Asn Pro Ala Pro Ser Lys Leu Pro Ile 725 730 735 Asn Ser Thr Arg Ala Gly Met Val Pro Ser Lys Val Pro Thr Ser Met 740 745 750 Val Leu Thr Lys Val Ser Ala Ser Thr Val Pro Thr Asp Gly Ser Ser 755 760 765 Arg Asn Glu Glu Thr Pro Ala Ala Pro Thr Pro Ala Gly Ala Thr Gly 770 775 780 Gly Ser Ser Ala Trp Leu Asp Ser Ser Ser Glu Asn Arg Gly Leu Gly 785 790 795 800 Ser Glu Leu Ser Lys Pro Gly Val Leu Ala Ser Gln Val Asp Ser Pro 805 810 815 Phe Ser Gly Cys Phe Glu Asp Leu Ala Ile Ser Ala Ser Thr Ser Leu 820 825 830 Gly Met Gly Pro Cys His Gly Pro Glu Glu Asn Glu Tyr Lys Ser Glu 835 840 845 Gly Thr Phe Gly Ile His Val Ala Glu Asn Pro Ser Ile Gln Leu Leu 850 855 860 Glu Gly Asn Pro Gly Pro Pro Ala Asp Pro Asp Gly Gly Pro Arg Pro 865 870 875 880 Gln Ala Asp Arg Lys Phe Gln Glu Arg Glu Val Pro Cys His Arg Pro 885 890 895 Ser Pro <210> 15 <211> 2541 <212> DNA <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens IPS1 <400> 15 atggatctgg acctggaaag 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 gggaatcgtg 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 cacagcgacg aacaccacca tgatgacagc 600 ctgcctcatc ctcagcaggc caccgacgat agcagcaacc agagcgacag caacagcaac 660 gagggcagac atctgctgct ggtgtctggt gctggcgacg gacctcctct gtgttctcaa 720 aatcttggcg cccctggcgg cggaccaaac aatggacctc aggaccccga caacaccgac 780 gacaatggcc ctcaagatcc tgataatacc gatgacaacg gcccacacga ccctctgcct 840 caagacccag ataacacaga cgataacggt ccacaagatc cggacaatac tgacgataat 900 ggaccccacg atccactgcc tcacaaccct agcgatagcg ccggaaatga tggcggacct 960 ccacagctga ccgaggaagt ggaaaacaaa ggcggagatc agggccctcc tctgatgacc 1020 gatggcggag gtggacactc tcacgattct ggccacgacg gcatcgaccc tcatctgcct 1080 acactgctgc tcggcacatc tggctctggc ggcgacgatg atgatcctca tggacctgtg 1140 cagctgagct actacgaccc tttcgccgag gacaagacct acaagtacat ctgccggaac 1200 ttcagcaact tctgcaacgt ggacgtggtg gaaattctgc cctacctgcc ttgcctgacc 1260 gccagagatc aggacagact gagagccaca tgtaccctga gcggcaacag agacacactg 1320 tggcacctgt tcaacaccct gcagagaagg cctggctggg tcgagtactt tatcgccgct 1380 ctgagaggct gcgagctggt cgatctggct gatgaagtgg ccagcgtgta ccagagctac 1440 cagcctagaa ccagcgaccg gggcgagaat agcgaacagg ctctgcagac actgagcccc 1500 agagccattc ctagaaaccc tgatggcggc cctctggaaa gctctagtga tctggccgct 1560 ctgtcccctc tgacaagctc tggacaccaa gagcaggata ccgagctggg cagcacacat 1620 acagccggcg ctacaagcag cctgacacct tctagaggcc ccgtgtctcc cagcgtgtca 1680 tttcagcctc tggccaggtc tacccctagg gcttctagac tgcctggacc aacaggcagc 1740 gtggtgtcta ccggcacaag cttcagctct agctctcctg gactggctag tgccggtgcc 1800 gctgagggaa aacaaggcgc cgaatctgat caggccgagc ctatcatctg tagcagcgga 1860 gcagaagccc ctgccaatag cctgcctagc aaggtgccaa ccacactgat gcccgtgaac 1920 acagtggccc tgaaggtgcc agctaatcct gcctccgtgt ccaccgtgcc ttctaagctg 1980 ccaaccagct ctaagccacc tggcgccgtg ccatctaacg ccctgacaaa tcctgctcca 2040 agcaagctgc ccatcaactc cacaagagcc ggcatggtgc cctctaaggt gcccacatct 2100 atggtgctga ccaaggtgtc cgccagcacc gtgccaacag atggcagctc cagaaacgag 2160 gaaacccctg ccgctcctac tcctgctggc gctacaggcg gatcttctgc ttggctggat 2220 agcagcagcg agaacagagg cctgggcagc gagctttcta aacctggcgt gctggcttcc 2280 caggtggaca gcccattttc cggctgcttt gaggacctgg ctatcagcgc ctctacaagc 2340 ctcggcatgg gaccttgtca cggccccgag gaaaacgagt acaagagcga gggcaccttc 2400 ggcatccacg tggccgagaa tcctagcatc caactgctgg aaggcaaccc cggacctcca 2460 gctgatccag atggcggacc aagacctcag gccgacagaa agttccaaga gcgcgaggtg 2520 ccctgccaca gaccttctcc a 2541 <210> 16 <211> 847 <212> PRT <213> Artificial sequence <220> <223> Epstein-Barr virus LMP1 and Homo sapiens ISP1 fusion protein <400> 16 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 His Ser 180 185 190 Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr 195 200 205 Asp Asp Ser Ser Asn Gln Ser Asp Ser Asn Ser Asn Glu Gly Arg His 210 215 220 Leu Leu Leu Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln 225 230 235 240 Asn Leu Gly Ala Pro Gly Gly Gly Pro Asn Asn Gly Pro Gln Asp Pro 245 250 255 Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp 260 265 270 Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp 275 280 285 Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp 290 295 300 Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro 305 310 315 320 Pro Gln Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly Pro 325 330 335 Pro Leu Met Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly His 340 345 350 Asp Gly Ile Asp Pro His Leu Pro Thr Leu Leu Leu Gly Thr Ser Gly 355 360 365 Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser Tyr 370 375 380 Tyr Asp Pro Phe Ala Glu Asp Lys Thr Tyr Lys Tyr Ile Cys Arg Asn 385 390 395 400 Phe Ser Asn Phe Cys Asn Val Asp Val Val Glu Ile Leu Pro Tyr Leu 405 410 415 Pro Cys Leu Thr Ala Arg Asp Gln Asp Arg Leu Arg Ala Thr Cys Thr 420 425 430 Leu Ser Gly Asn Arg Asp Thr Leu Trp His Leu Phe Asn Thr Leu Gln 435 440 445 Arg Arg Pro Gly Trp Val Glu Tyr Phe Ile Ala Ala Leu Arg Gly Cys 450 455 460 Glu Leu Val Asp Leu Ala Asp Glu Val Ala Ser Val Tyr Gln Ser Tyr 465 470 475 480 Gln Pro Arg Thr Ser Asp Arg Gly Glu Asn Ser Glu Gln Ala Leu Gln 485 490 495 Thr Leu Ser Pro Arg Ala Ile Pro Arg Asn Pro Asp Gly Gly Pro Leu 500 505 510 Glu Ser Ser Ser Asp Leu Ala Ala Leu Ser Pro Leu Thr Ser Ser Gly 515 520 525 His Gln Glu Gln Asp Thr Glu Leu Gly Ser Thr His Thr Ala Gly Ala 530 535 540 Thr Ser Ser Leu Thr Pro Ser Arg Gly Pro Val Ser Pro Ser Val Ser 545 550 555 560 Phe Gln Pro Leu Ala Arg Ser Thr Pro Arg Ala Ser Arg Leu Pro Gly 565 570 575 Pro Thr Gly Ser Val Val Ser Thr Gly Thr Ser Phe Ser Ser Ser Ser 580 585 590 Pro Gly Leu Ala Ser Ala Gly Ala Ala Glu Gly Lys Gln Gly Ala Glu 595 600 605 Ser Asp Gln Ala Glu Pro Ile Ile Cys Ser Ser Gly Ala Glu Ala Pro 610 615 620 Wing Asn Ser Leu Pro Ser Lys Val Pro Thr Thr Leu Met Pro Val Asn 625 630 635 640 Thr Val Ala Leu Lys Val Pro Ala Asn Pro Ala Ser Val Ser Thr Val 645 650 655 Pro Ser Lys Leu Pro Thr Ser Ser Lys Pro Pro Gly Ala Val Pro Ser 660 665 670 Asn Ala Leu Thr Asn Pro Ala Pro Ser Lys Leu Pro Ile Asn Ser Thr 675 680 685 Arg Ala Gly Met Val Pro Ser Lys Val Pro Thr Ser Met Val Leu Thr 690 695 700 Lys Val Ser Ala Ser Thr Val Pro Thr Asp Gly Ser Arg Asn Glu 705 710 715 720 Glu Thr Pro Ala Ala Pro Thr Pro Ala Gly Ala Thr Gly Gly Ser Ser 725 730 735 Wing Trp Leu Asp Ser Ser Glu Asn Arg Gly Leu Gly Ser Glu Leu 740 745 750 Ser Lys Pro Gly Val Leu Ala Ser Gln Val Asp Ser Pro Phe Ser Gly 755 760 765 Cys Phe Glu Asp Leu Ala Ile Ser Ala Ser Thr Ser Leu Gly Met Gly 770 775 780 Pro Cys His Gly Pro Glu Glu Asn Glu Tyr Lys Ser Glu Gly Thr Phe 785 790 795 800 Gly Ile His Val Ala Glu Asn Pro Ser Ile Gln Leu Leu Glu Gly Asn 805 810 815 Pro Gly Pro Pro Ala Asp Pro Asp Gly Gly Pro Arg Pro Gln Ala Asp 820 825 830 Arg Lys Phe Gln Glu Arg Glu Val Pro Cys His Arg Pro Ser Pro 835 840 845 <210> 17 <211> 2697 <212> DNA <213> Artificial sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens ISP1 <400> 17 atggatctgg acctggaaag 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 gggaatcgtg 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 cacagcgacg aacaccacca tgatgacagc 600 ctgcctcatc ctcagcaggc caccgacgat agcagcaacc agagcgacag caacagcaac 660 gagggcagac atctgctgct ggtgtctggt gctggcgacg gacctcctct gtgttctcaa 720 aatcttggcg cccctggcgg cggaccaaac aatggacctc aggaccccga caacaccgac 780 gacaatggcc ctcaagatcc tgataatacc gatgacaacg gcccacacga ccctctgcct 840 caagacccag ataacacaga cgataacggt ccacaagatc cggacaatac tgacgataat 900 ggaccccacg atccactgcc tcacaaccct agcgatagcg ccggaaatga tggcggacct 960 ccacagctga ccgaggaagt ggaaaacaaa ggcggagatc agggccctcc tctgatgacc 1020 gatggcggag gtggacactc tcacgattct ggccacgacg gcatcgaccc tcatctgcct 1080 acactgctgc tcggcacatc tggctctggc ggcgacgatg atgatcctca tggacctgtg 1140 cagctgagct actacgaccc ttctccaaga cactgcccag tggaaagaga gcagttcaag 1200 agggacgccc agcctagacc tggcggagat cctgatgctc cacctggacc aaatggcgag 1260 ctgctgcaga tcagccctaa tgaggccgtg cacatcggct tcaccggcga gtctaagtac 1320 gagaacgagg aacccggcca ctgtcctggc atgggccttt ctacatctgc ctctatcgcc 1380 ctggacgagt tctgcggcag ctttccatct gatgtgcagt ctgccctcgt gggccctaag 1440 tctctggaat ctggcctggg cagaaacgag agcagctccg atctgtgggc tagctctggt 1500 ggaacagctg gcgctcctac accagccgct cctaccgaag agaatagaag cagcggcgac 1560 acccctgtga caagcgcctc tgtgaaaacc ctggtcatga gcaccccagt gaagtcccca 1620 gtgatgggcg ccagaacctc caacattccc ctgaagtctc ccgctcctaa cacactggcc 1680 aactctccag tggctggccc tcctaagtct agcacccctc tgaaaagccc cgtgacctct 1740 gtgtctgccc ctaacgctcc tgtgaaactg gccgtgacca acgtgcccat gctgaccaca 1800 cctgtgaaat ccccactgag caatgcccct gccgaggccg gaagctcttg tatcattccc 1860 gaggctcagg atagcgaggc tggccaaaaa ggcgaagctg caggcgcttc tgctctgggc 1920 cctagctcta gctcttttag caccggcacc agcgtggtgt ctggcacacc aggacctctg 1980 agaagcgcca gacctacctc tagagccctg cctcagttta gcgtgtcccc tagtgtgcct 2040 ggcagaagcc ctacactgtc tagtacagcc ggcgctacac acaccagcgg actggaaaca 2100 2160 gaactgccag gcggcgaccc caatagacct atcgctagac ctagcctgac acagctggcc 2220 caagagagca atgagggccc ttctgagcct gctcagaccg aacaggtgcc aatgccttac 2280 agccccgaga aagagcggtg cagcaactac cctatcagcc atgccgctgc tcccacacct 2340 cctggtccaa gagaagctcc tctgagccct cctgagctgc ccgatcctcc aagatagc 2400 accagacctc agtactccca gtacgtgtcc gccgtggaag atgccctgga tgtgctggaa 2460 tgtggcagac tggccgccat cttctacgaa gtgtggggcc ctagaaggca gctgaccaac 2520 tttctgcact ggctgaccga cagaaacggc agcctgacat gtaccgccag actgagagat 2580 caggaccggg ccacactgtg ccctctgtat cctctgatcg aggtggtgga cgtgaactgc 2640 ttcaacagct tcaaccggtg catctacaag tacaccaagg acgaggcttt ccctatg 2697 <210> 18 <211> 899 <212> PRT <213> Artificial sequence <220> <223> Connect the LMP1 to the ISP1 connection <400> 18 Met Asp Leu Asp Leu Glu Arg Gly 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 His Ser 180 185 190 Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr 195 200 205 Asp Asp Ser Ser Asn Gln Ser Asp Ser Asn Ser Asn Glu Gly Arg His 210 215 220 Leu Leu Leu Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln 225 230 235 240 Asn Leu Gly Ala Pro Gly Gly Gly Pro Asn Asn Gly Pro Gln Asp Pro 245 250 255 Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp 260 265 270 Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp 275 280 285 Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp 290 295 300 Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro 305 310 315 320 Pro Gln Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly Pro 325 330 335 Pro Leu Met Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly His 340 345 350 Asp Gly Ile Asp Pro His Leu Pro Thr Leu Leu Leu Gly Thr Ser Gly 355 360 365 Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser Tyr 370 375 380 Tyr Asp Pro Ser Pro Arg His Cys Pro Val Glu Arg Glu Gln Phe Lys 385 390 395 400 Arg Asp Ala Gln Pro Arg Pro Gly Gly Asp Pro Asp Ala Pro Pro Gly 405 410 415 Pro Asn Gly Glu Leu Leu Gln Ile Ser Pro Asn Glu Ala Val His Ile 420 425 430 Gly Phe Thr Gly Glu Ser Lys Tyr Glu Asn Glu Glu Pro Gly His Cys 435 440 445 Pro Gly Met Gly Leu Ser Thr Ser Ala Ser Ile Ala Leu Asp Glu Phe 450 455 460 Cys Gly Ser Phe Pro Ser Asp Val Gln Ser Ala Leu Val Gly Pro Lys 465 470 475 480 Ser Leu Glu Ser Gly Leu Gly Arg Asn Glu Ser Ser Ser Asp Leu Trp 485 490 495 Ala Ser Ser Gly Gly Thr Ala Gly Ala Pro Thr Pro Ala Ala Pro Thr 500 505 510 Glu Glu Asn Arg Ser Ser Gly Asp Thr Pro Val Thr Ser Ala Ser Val 515 520 525 Lys Thr Leu Val Met Ser Thr Pro Val Lys Ser Pro Val Met Gly Ala 530 535 540 Arg Thr Ser Asn Ile Pro Leu Lys Ser Pro Ala Pro Asn Thr Leu Ala 545 550 555 560 Asn Ser Pro Val Ala Gly Pro Pro Lys Ser Ser Thr Pro Leu Lys Ser 565 570 575 Pro Val Thr Ser Val Ser Ala Pro Asn Ala Pro Val Lys Leu Ala Val 580 585 590 Thr Asn Val Pro Met Leu Thr Thr Pro Val Lys Ser Pro Leu Ser Asn 595 600 605 Ala Pro Ala Glu Ala Gly Ser Ser Cys Ile Ile Pro Glu Ala Gln Asp 610 615 620 Ser Glu Ala Gly Gln Lys Gly Glu Ala Ala Gly Ala Ser Ala Leu Gly 625 630 635 640 Pro Ser Ser Ser Ser Phe Ser Thr Gly Thr Ser Val Val Ser Gly Thr 645 650 655 Pro Gly Pro Leu Arg Ser Ala Arg Pro Thr Ser Arg Ala Leu Pro Gln 660 665 670 Phe Ser Val Ser Pro Ser Val Pro Gly Arg Ser Pro Thr Leu Ser Ser 675 680 685 Thr Ala Gly Ala Thr His Thr Ser Gly Leu Glu Thr Asp Gln Glu Gln 690 695 700 His Gly Ser Ser Thr Leu Pro Ser Leu Ala Ala Leu Asp Ser Ser Ser 705 710 715 720 Glu Leu Pro Gly Gly Asp Pro Asn Arg Pro Ile Ala Arg Pro Ser Leu 725 730 735 Thr Gln Leu Ala Gln Glu Ser Asn Glu Gly Pro Ser Glu Pro Ala Gln 740 745 750 Thr Glu Gln Val Pro Met Pro Tyr Ser Pro Glu Lys Glu Arg Cys Ser 755 760 765 Asn Tyr Pro Ile Ser His Ala Ala Ala Pro Thr Pro Pro Gly Pro Arg 770 775 780 Glu Ala Pro Leu Ser Pro Pro Glu Leu Pro Asp Pro Pro Arg Asp Ser 785 790 795 800 Thr Arg Pro Gln Tyr Ser Gln Tyr Val Ser Ala Val Glu Asp Ala Leu 805 810 815 Asp Val Leu Glu Cys Gly Arg Leu Ala Ala Ile Phe Tyr Glu Val Trp 820 825 830 Gly Pro Arg Arg Gln Leu Thr Asn Phe Leu His Trp Leu Thr Asp Arg 835 840 845 Asn Gly Ser Leu Thr Cys Thr Ala Arg Leu Arg Asp Gln Asp Arg Ala 850 855 860 Thr Leu Cys Pro Leu Tyr Pro Leu Ile Glu Val Val Asp Val Asn Cys 865 870 875 880 Phe Asn Ser Phe Asn Arg Cys Ile Tyr Lys Tyr Thr Lys Asp Glu Ala 885 890 895 Phe Pro Met <210> 19 <211> 2694 <212> DNA <213> Artificial sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens ISP1 <400> 19 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 aggagaagac ctacaagtac 600 atctgccgga acttcagcaa cttctgcaac gtggacgtgg tggaaattct gccctacctg 660 ccttgcctga ccgccagaga tcaggagaga ctgagagcca catgtaccct gagcggcaac 720 agacacac tgtggcacct gttcaacacc ctgcagaagaa ggcctggctg ggtcgagtac 780 tttatcgccg ctctgagagg ctgcgagctg gtcgatctgg ctgatgaagt ggccagccgtg 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 cagaacactga 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 gctgggaaggc aaccccggac ctccagctga tccagatggc 2040 ggaccaagac ctcaggccga cagaaagttc caagagcgcg aggtgccctg ccacagacct 2100 tctccacaca gcgacgaaca ccaccatgat gacagcctgc ctcatcctca gcaggccacc 2160 gacgatagca gcaaccagag cgacagcaac agcaacgagg gcagacatct gctgctggtg 2220 tctggtgctg gcgacggacc tcctctgtgt tctcaaaatc ttggcgcccc tggcggcgga 2280 ccaaacaatg gacctcagga ccccgacaac accgacgaca atggccctca agatcctgat 2340 aataccgatg aaacggccc acacgaccct ctgcctcaag acccagataa cacagacgat 2400 aacggtccac aagatccgga caatactgac gataatggac cccacgatcc actgcctcac 2460 aaccctagcg atagcgccgg aaatgatggc ggacctccac agctgaccga ggaagtggaa 2520 aacaaaggcg gagatcaggg ccctcctctg atgaccgatg gcggaggtgg acactctcac 2580 gattctggcc acgacggcat cgaccctcat ctgcctacac tgctgctcgg cacatctggc 2640 tctggcggcg acgatgatga tcctcatgga cctgtgcagc tgagctacta cgac 2694 <210> 20 <211> 898 <212> PRT <213> Artificial sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens ISP1 <400> 20 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 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 His Ser 690 695 700 Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr 705 710 715 720 Asp Asp Ser Ser Asn Gln Ser Asp Ser Asn Ser Asn Glu Gly Arg His 725 730 735 Leu Leu Leu Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln 740 745 750 Asn Leu Gly Ala Pro Gly Gly Gly Pro Asn Asn Gly Pro Gln Asp Pro 755 760 765 Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp 770 775 780 Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp 785 790 795 800 Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp 805 810 815 Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro 820 825 830 Pro Gln Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly Pro 835 840 845 Pro Leu Met Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly His 850 855 860 Asp Gly Ile Asp Pro His Leu Pro Thr Leu Leu Leu Gly Thr Ser Gly 865 870 875 880 Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser Tyr 885 890 895 Tyr Asp <210> 21 <211> 2541 <212> DNA <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens ISP1 fusion protein <400> 21 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 ccacacagcg acgaacacca ccatgatgac 1980 agcctgcctc atcctcagca ggccaccgac gatagcagca accagagcga cagcaacagc 2040 aacgagggca gacatctgct gctggtgtct ggtgctggcg acggacctcc tctgtgttct 2100 caaaatcttg gcgcccctgg cggcggacca aacaatggac ctcaggaccc cgacaacacc 2160 gacgacaatg gccctcaaga tcctgataat accgatgaca acggcccaca cgaccctctg 2220 cctcaagacc cagataacac agacgataac ggtccacaag atccggacaa tactgacgat 2280 aatggacccc acgatccact gcctcacaac cctagcgata gcgccggaaa tgatggcgga 2340 cctccacagc tgaccgagga agtggaaaac aaaggcggag atcagggccc tcctctgatg 2400 accgatggcg gaggtggaca ctctcacgat tctggccacg acggcatcga ccctcatctg 2460 cctacactgc tgctcggcac atctggctct ggcggcgacg atgatgatcc tcatggacct 2520 gtgcagctga gctactacga c 2541 <210> 22 <211> 847 <212> PRT <213> Artificial sequence <220> <223> Epstein-Barr virus LMP1 and Homo sapiens ISP1 fusion protein <400> 22 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 His Ser Asp Glu His 645 650 655 His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala Thr Asp Asp Ser 660 665 670 Ser Asn Gln Ser Asp Ser Asn Ser Asn Glu Gly Arg His Leu Leu Leu 675 680 685 Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser Gln Asn Leu Gly 690 695 700 Ala Pro Gly Gly Gly Pro Asn Asn Gly Pro Gln Asp Pro Asp Asn Thr 705 710 715 720 Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro 725 730 735 His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro 740 745 750 Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp Pro Leu Pro 755 760 765 His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro Pro Gln Leu 770 775 780 Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly Pro Pro Leu Met 785 790 795 800 Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly His Asp Gly Ile 805 810 815 Asp Pro His Leu Pro Thr Leu Leu Leu Gly Thr Ser Gly Ser Gly Gly 820 825 830 Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser Tyr Tyr Asp 835 840 845 <210> 23 <211> 2697 <212> DNA <213> Artificial sequence <220> <223> Epstein - Barr virus LMP1 and Homo sapiens ISP1 fusion protein <400> 23 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 agtggagaaga 600 gagcagttca agagggacgc ccagcctaga cctggcggag atcctgatgc tccacctgga 660 ccaaatggcg agctgctgca gatcagccct aatgaggccg tgcacatcgg cttcaccggc 720 gagtctaagt agagaacga ggaacccggc cactgtcctg gcatgggcct ttctacatct 780 gcctctatcg ccctggacga gttctgcggc agctttccat ctgatgtgca gtctgccctc 840 gtgggcccta agtctctgga atctggcctg ggcagaaacg agagcagctc cgatctgtgg 900 gctagctctg gtggaacagc tggcgctcct acaccagccg ctcctaccga agaataga 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 ttccctatgc acagcgacga acaccaccat gatgacagcc tgcctcatcc tcagcaggcc 2160 accgacgata gcagcaacca gagcgacagc aacagcaacg agggcagaca tctgctgctg 2220 gtgtctggtg ctggcgacgg acctcctctg tgttctcaaa atcttggcgc ccctggcggc 2280 ggaccaaaca atggacctca ggaccccgac aacaccgacg acaatggccc tcaagatcct 2340 gataataccg atgacaacgg cccacacgac cctctgcctc aagacccaga taacacagac 2400 gataacggtc cacaagatcc ggacaatact gacgataatg gaccccacga tccactgcct 2460 cacaacccta gcgatagcgc cggaaatgat ggcggacctc cacagctgac cgaggaagtg 2520 gaaaacaaag gcggagatca gggccctcct ctgatgaccg atggcggagg tggacactct 2580 cacgattctg gccacgacgg catcgaccct catctgccta cactgctgct cggcacatct 2640 ggctctggcg gcgacgatga tgatcctcat ggacctgtgc agctgagcta ctacgac 2697 <210> twenty four <211> 899 <212> PRT <213> Artificial sequence <220> <223> Fusion protein of Epstein-Barr virus LMP1 and Homo sapiens ISP1 <400> twenty four 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 His 690 695 700 Ser Asp Glu His His His Asp Asp Ser Leu Pro His Pro Gln Gln Ala 705 710 715 720 Thr Asp Asp Ser Ser Asn Gln Ser Asp Ser Asn Ser Asn Glu Gly Arg 725 730 735 His Leu Leu Leu Val Ser Gly Ala Gly Asp Gly Pro Pro Leu Cys Ser 740 745 750 Gln Asn Leu Gly Ala Pro Gly Gly Gly Pro Asn Asn Gly Pro Gln Asp 755 760 765 Pro Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp 770 775 780 Asp Asn Gly Pro His Asp Pro Leu Pro Gln Asp Pro Asp Asn Thr Asp 785 790 795 800 Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His 805 810 815 Asp Pro Leu Pro His Asn Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly 820 825 830 Pro Pro Gln Leu Thr Glu Glu Val Glu Asn Lys Gly Gly Asp Gln Gly 835 840 845 Pro Pro Leu Met Thr Asp Gly Gly Gly Gly His Ser His Asp Ser Gly 850 855 860 His Asp Gly Ile Asp Pro His Leu Pro Thr Leu Leu Leu Gly Thr Ser 865 870 875 880 Gly Ser Gly Gly Asp Asp Asp Asp Pro His Gly Pro Val Gln Leu Ser 885 890 895 Tyr Tyr Asp

Claims

1. A viral vector, comprising a first nucleic acid sequence encoding an antigen or an antigenic epitope; a second nucleic acid sequence encoding the full-length latent membrane protein 1 (LMP1) of Epstein-Barr virus; and a third nucleic acid sequence encoding a fusion protein comprising a transmembrane portion of LMP1, wherein the intracytoplasmic domain of LMP1 is replaced by IPS1; wherein the coding sequence of the vector is codon-optimized for human expression, wherein the second nucleic acid sequence and the third nucleic acid sequence are connected in any order after the first nucleic acid sequence; the vector further comprises a nucleic acid sequence encoding a soluble immune checkpoint inhibitor molecule; the soluble immune checkpoint inhibitor molecule is selected from the group consisting of CTLA-4, PD-1, PDL-1, LAG-3, TIM 3, B7-H3, ICOS, IDO, CD47, B7-H4, TIGIT, CD160 and a combination thereof; The IPS1 lacks a transmembrane domain and a proline-rich domain; in, The second nucleic acid sequence is shown as SEQ ID NO:3, and the third nucleic acid sequence is shown as SEQ ID NO:

9.

2. The viral vector according to claim 1, characterized in that The vector is a lentiviral vector.

3. The viral vector according to claim 1, characterized in that 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, characterized in that The vector also includes a functional lentiviral integrase protein, wherein the vector is self-inactivating.

5. The viral vector according to claim 1, characterized in that 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 nonstructural protein, hemagglutinin, and combinations thereof.

6. An immunotherapeutic formulation for treating cancer or infection in a subject, the formulation comprising the viral vector of claim 1.

7. Use of the viral vector according to claim 1 or the preparation according to claim 6 in the preparation of a medicament for inducing or enhancing an immune response against cancer or an infectious disease in a subject, wherein the medicament induces or enhances an immune response against the cancer or infectious disease in a subject; in, The cancer is selected from the group consisting of melanoma, glioma, prostate cancer, breast cancer, cervical cancer, colorectal cancer, renal cancer, lung cancer, lymphoma, ovarian cancer, sarcoma and pancreatic cancer; wherein the cancer is indicated by a tumor antigen of the cancer; and the infectious disease is indicated by a viral or microbial antigen of the infectious disease; 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.

Images