Viral constructs used to enhance T-cell priming during vaccination

Abstract

The present invention provides a viral-based expression vector containing an immune checkpoint inhibitor insert for use as an effective adjuvant in enhancing T cell priming to antigens in a host during a vaccination regimen. In particular, the compositions described herein are novel recombinant modified vaccinia Ankara (MVA) viral constructs encoding one or more peptides that are expressed in a multimeric structure upon administration and then cleaved and secreted from cells. Such peptides can downregulate immune checkpoint pathways, for example, by inhibiting the activation of programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), or another immune checkpoint regulator, or a combination thereof. When used in concert with the administration of an antigen during a vaccination strategy, the MVA viral constructs expressing immune checkpoints can enhance the expression of antigen-specific CD8 + This results in significantly improved T cell proliferation, increased antigenic responses, and improved vaccination efficacy.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims the interests of U.S. Provisional Patent Application No. 63 / 144,834, filed on 2 February 2021. This application in its entirety constitutes a part of this specification by reference for all purposes.

[0002] The present invention provides a virus-based expression vector comprising a nucleic acid insert encoding an immune checkpoint inhibitor, which is used as an effective adjuvant in enhancing T-cell priming against an antigen in a host during a vaccination regimen. In particular, the composition described herein is a novel recombinant modified vaccinia ankara (MVA) virus construct encoding an immune checkpoint inhibitor peptide that, upon administration, is expressed in a multimeric structure and subsequently cleaved and secreted from cells.

[0003] [References] The contents of the text file, named "19101-014WO1_SEQ_TXT," created on February 1, 2022, and measuring 564KB, constitute part of this specification by quoting its entire contents. [Background technology]

[0004] Vaccines are considered one of the most important advances in modern medicine, significantly improving quality of life by mitigating or eliminating many serious infectious diseases. Vaccines have been developed against a wide range of human pathogens, including, for example, bacterial toxins (e.g., tetanus toxin and diphtheria toxin), acute viral pathogens (e.g., measles virus, mumps virus, rubella virus), latent or chronic viral pathogens (e.g., varicella-zoster virus [VZV] and human papillomavirus [HPV], respectively), respiratory pathogens (e.g., influenza virus, Bordetella pertussis virus), and enteric pathogens (e.g., poliovirus, Salmonella typhi virus). Most approved vaccines can be classified as attenuated live vaccines, non-replicating whole-particle vaccines (including virus-like particles, i.e., VLPs), and subunit vaccines.

[0005] However, successful vaccine development requires robust and sustained protective immunity consisting of both humoral and cellular immune responses. Both elements of immunity are essential for effectively eliminating pathogens. While progress has been made in the development of vaccines against many pathogens, robust and durable protective T-cell immunity, particularly CD8, is crucial. + The inability to induce a T-cell response is a major obstacle and a primary reason for the failure of many vaccine development efforts, particularly against intracellular pathogens (see, for example, Non-Patent Document 1).

[0006] One strategy to overcome these inherent limitations is to identify and use immunogenic adjuvants, and considerable research has been devoted to evaluating the effects of putative adjuvants on innate immune activation and adaptive immune responses to model antigens and potential vaccines (see, for example, Non-Patent Documents 2, 3, and 4).

[0007] For example, alpha-hydrogels are distinctive aluminum hydroxide adjuvants that are currently included in several FDA-approved vaccines. Alpha-hydrogels provide a depot effect, resulting in a slower release of the antigen in vivo, thus extending antigen exposure, which may or may not contribute to adjuvant activity (Non-Patent Literature 5). Furthermore, alpha-hydrogels have been shown to activate inflammasomes, which may contribute to the immunogenicity of alpha-hydrogel-based vaccines (Non-Patent Literature 6).

[0008] PolyICLC is a double-stranded RNA stabilized by poly-L-lysine in carboxymethylcellulose (Non-Patent Literature 7). PolyICLC signals via Toll-like receptor-3 (TLR3) and potentially the melanoma differentiation-associated protein 5 (MDA5) receptor, inducing a potent type I IFN response, which biases the immune response towards a Th1 profile response (Non-Patent Literature 8). PolyICLC is in numerous clinical trials for both therapeutic and vaccine purposes (Non-Patent Literature 9).

[0009] CpG oligodeoxynucleotides (i.e., CpG ODNs) are short, single-stranded synthetic DNA molecules containing a cytosine triphosphate deoxynucleotide ("C") followed by a guanine triphosphate deoxynucleotide ("G"). "p" refers to a phosphodiester linkage between consecutive nucleotides, although ODNs may instead have a modified phosphorothioate (PS) skeleton. When these CpG motifs are unmethylated, they act as immunostimulants and have also been investigated as adjuvants (Non-Patent Literature 10).

[0010] MPL is a TLR4 agonist and the active ingredient in the GSK adjuvant AS04 (Non-Patent Literature 11). MPL has been shown to be very effective as an adjuvant, especially when combined with aluminum-based adjuvants or nanoparticle formulations such as alpha-hydrogels (Non-Patent Literature 12).

[0011] Other well-known adjuvants include alum-based adjuvants, oil-based adjuvants, Freund's adjuvants, specol, Ribi adjuvants, Mycobacterium vaccae, immunostimulatory complexes (ISCOMS), MF-59, SBAS-2, SBAS-4, detox B SE (Enhanzyn®), Lipid A mimetic RC-529, aminoalkylglucosaminide 4-phosphate (AGP), CRX-527, monophosphoryl lipid A (e.g., MPL-SE), detoxified saponin derivatives (e.g., QS-21, QS7), escin, digitonin, gypsophila saponins, and quinoa saponins (see, for example, Non-Patent Document 13).

[0012] Despite significant advancements in adjuvant formulation and use, the majority of adjuvants are designed to generate innate inflammatory danger signals. While these danger signals are essential for innate immune activation, including antigen presentation and cytokine production, their direct effect on T cell priming is limited (Non-Patent Literature 14, Non-Patent Literature 15), and most vaccination strategies using common adjuvants focus on long-term memory CD8 + It is not possible to induce T cells (Non-Patent Literature 16). This is particularly true during vaccination for chronic infections and cancer, where immunomodulatory strategies are needed to enhance the T cell response necessary to overcome the immunosuppressive microenvironment.

[0013] One such strategy involved downmodulating immune checkpoint inhibitor receptors such as programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1). For example, PD-1 functions in regulating the threshold, intensity, and duration of the T cell response to antigen presentation (Non-Patent Literature 17). PD1 is rapidly upmodulated upon activation of naive T cells, which is necessary to minimize host damage from uncontrolled inflammation during and after infection (Non-Patent Literature 18). In non-human primates, immunization with a combination of a SIVgag adenovirus-based vaccine and an anti-PD1 monoclonal antibody significantly increased the peak of the Gag-specific T cell response (Non-Patent Literature 19).

[0014] While monoclonal antibody (mAb)-based checkpoint inhibitors developed to treat cancer can effectively restore immune function, they are not immediately useful in the field of infectious disease vaccinology. Due to their long serum half-lives, anti-PD1 monoclonal antibodies are unacceptable as prophylactic vaccine adjuvants because they can cause severe immune-related adverse events (irAEs) and potentially lead to autoimmune diseases (Non-Patent Literature 20, Non-Patent Literature 21).

[0015] Therefore, there is a need for improved methods of using immune checkpoint inhibitors in vaccination strategies that provide safe and effective immunization. [Prior art documents] [Non-patent literature]

[0016] [Non-Patent Document 1] Seder et al., "Vaccines against intracellular infections requiring cellular immunity," Nature. 2000 Aug 17;406(6797):793-8 [Non-Patent Document 2] "Development of a Molecular Adjuvant to Enhance Antigen-Specific CD8+ T Cell Responses," by Halbroth et al., Sci Rep. 2018 Oct 9;8(1):15020. [Non-Patent Document 3] "Delta inulin-based adjuvants promote the generation of polyfunctional CD4+ T cell responses and protection against Mycobacterium tuberculosis infection." (Sci Rep. 2017 Aug 17;7(1):8582) by Counoupas et al. [Non-Patent Document 4] “Intracellular Pathogens: Host Immunity and Microbial Persistence Strategies.” by Thakur et al. Immunol Res. 2019 Apr 14;2019:1356540 [Non-Patent Document 5] Hutchison et al., "Antigen depot is not required for alum adjuvantity." FASEB J. 2012;26:1272-1279 [Non-Patent Document 6] Guven et al., "Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway." PLoS One. 2013;8:e74445 [Non-Patent Document 7] "A modified polyriboinosinic-polyribocytidylic acid complex that induces interferon in primates," by Levy et al., J. Infect. Dis. 1975;132:434-439. [Non-Patent Document 8] "Cutting edge: polyinosinic:polycytidylic acid boosts the generation of memory CD8 T cells through melanoma differentiation-associated protein 5 expressed in stromal cells," by Wang et al., J. Immunol. 2010;184:2751-2755. [Non-Patent Document 9] "Vaccine adjuvant uses of poly-ic and derivatives," by Martins et al., Expert Rev. Vaccines. 2015;14:447-459. [Non-Patent Document 10] "Identification of a novel cpg DNA class and motif that optimally stimulates B cell and plasmacytoid dendritic cell functions," by Marshall et al., J. Leukoc. Biol. 2003;73:781-792. [Non-Patent Document 11] "Comparative humoral and cellular immunogenicity and safety of human papillomavirus (HPV)-16 / 18 AS04-adjuvanted vaccine and HPV-6 / 11 / 16 / 18 vaccine in healthy women aged 18-45 years: follow-up through month 48 in a Phase III randomized study." Hum. Vaccines Immunother. 2014;10:3455-3465. [Non-Patent Document 12] "The immunobiology of Toll-Like receptor 4 agonists: from endotoxin tolerance to immunoadjuvants," by Bohannon et al., Shock. 2013;40:451-462. [Non-Patent Document 13] "Adjuvants for Human Vaccines," by Alving et al., Curr Opin Immunol. 2012 Jun; 24(3): 310-315. [Non-Patent Document 14] "Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes," by Powell et al., Clin Exp Vaccine Res. 2015 Jan;4(1):23-45. [Non-Patent Document 15] "Comparative Safety of Vaccine Adjuvants: A Summary of Current Evidence and Future Needs," by Petrovsky N., Drug Saf. 2015 Nov;38(11):1059-74. [Non-Patent Document 16] "Beyond Adjuvants: Immunomodulation strategies to enhance T cell immunity," by Kamphorst et al., Vaccine. 2015 Jun 8; 33(0 2): B21-B28. [Non-Patent Document 17] "A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application," by Okazaki et al., Nat Immunol. 2013 Dec;14(12):1212-8. [Non-Patent Document 18] "Role of PD-1 during effector CD8 T cell differentiation," by Ahn et al., PNAS 2018 May 1;115(18):4749-4754. [Non-Patent Document 19] Finnefrock et al., "PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination," J Immunol. 2009 Jan 15;182(2):980-7 [Non-Patent Document 20] "Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates." by Brahmer et al. J Clin Oncol. 2010 Jul 1;28(19):3167-75 [Non-Patent Document 21] "Safety, activity, and immune correlates of anti-PD-1 antibody in cancer," by Topalian et al., N Engl J Med. 2012 Jun 28;366(26):2443-54. [Overview of the project]

[0017] This specification provides compositions comprising a recombinant modified vaccinia ankara (rMVA) virus vector for use as an adjuvant or vaccine in immunization protocols in a host such as a human. The rMVA is constructed to express at high concentrations peptides (immune checkpoint inhibitor peptides) that can inhibit one or more immune checkpoint pathways. In some embodiments, the immune checkpoint inhibitor peptides are expressed from a polycistronic multimeric nucleic acid insert and secreted from cells.

[0018] When the PD-1 inhibitor peptide (LD01-SEQ ID NO: 1) is administered in combination with an adenovirus-based or radiation-sporozoite-based prophylactic malaria vaccine, antigen-specific CD8 receptor activity is increased in immunocompetent mice. + It has been previously shown that T cell proliferation is enhanced (see Phares et al., "A peptide-based PD1 antagonist enhances T-cell priming and efficacy of a prophylactic malaria vaccine and promotes survival in a lethal malaria model." Front. Immunol. 11, 1377 (2020) (this is incorporated herein by reference)). As shown herein, the natural tropism of MVA viral vectors includes specialized antigen-presenting cells such as dendritic cells that can migrate to influx region lymph nodes and spread throughout the body. Therefore, it has now been found that using MVA as a delivery carrier to express immune checkpoint inhibitors offers significant advantages in vaccination strategies. By expressing sufficient and high doses of therapeutic-level immune checkpoint inhibitors in the lymph node environment, for example in polycistronic multimeric structures, during host antigen exposure, CD8 +It is believed that T cell priming is significantly enhanced. As shown in the following examples, when used in conjunction with antigen administration during vaccination strategies, the rMVA virus construct expressing immune checkpoints is thought to enhance antigen-specific CD8 compared to, for example, the unprocessed administration of such immune checkpoint inhibitor peptides. + This results in a significant improvement in T cell proliferation, increased antigen response, and improved vaccine efficacy, representing a substantial improvement over conventional adjuvant strategies.

[0019] In one embodiment, the Specified Reference Intake of a Viral Genetic Vector (rMVA) is provided, comprising a heterologous polycistronic nucleic acid insert encoding one or more chimeric polypeptides, e.g., one, two, three, four, five, six, seven, eight, nine, or ten or more chimeric polypeptides, wherein each chimeric polypeptide comprises a secretion signal peptide and an immune checkpoint inhibitor peptide. In some embodiments, the rMVA viral vector comprises a heterologous nucleic acid insert encoding two or more chimeric polypeptides, wherein the two or more chimeric polypeptides are expressed from a single heterologous polycistronic nucleic acid insert, and each of the nucleic acid sequences encoding the two or more chimeric polypeptides is operably linked in the polycistronic nucleic acid sequence. In some embodiments, the rMVA comprises two or more heterologous polycistronic inserts, e.g., two, three, or four or more polycistronic inserts. In some embodiments, the population of chimeric polypeptides expressed from the rMVA consists of two or more different immune checkpoint inhibitor peptides. In some embodiments, rMVA further encodes one or more antigenic peptides that, when expressed by rMVA, can induce sufficient immunogenicity to provide or enhance protective immunity against infectious agents. In some embodiments, rMVA, when expressed by rMVA, can induce an immune response in the host that improves one or more symptoms or conditions of a disorder, such as infectious disease or cancer.

[0020] In some embodiments, each chimeric polypeptide comprising a secretory signaling peptide and an immune checkpoint inhibitor peptide encoded by a polycistronic nucleic acid insert includes a peptide sequence that can be cleaved in or after translation, ligated to the C-terminus of the immune checkpoint inhibitor peptide. When the secretory immune checkpoint inhibitor peptide is inserted in a multimeric structure, the inclusion of a cleavable peptide allows each chimeric polypeptide in the multimer to be expressed as a monomer in translation (e.g., through a nascent chain separation event), or, in alternative embodiments, to be cleaved into monomers after translation, or a combination of both. In some embodiments, the chimeric polypeptide encoded by the most 3' nucleic acid lacks a cleavable peptide sequence.

[0021] In some embodiments, as used herein, an rMVA viral vector comprising a heterologous nucleic acid insert encoding a polypeptide, wherein the polypeptide is sequence (M)(secretion signal peptide-immune checkpoint inhibitor peptide) x An rMVA viral vector is provided, comprising (where x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine).

[0022] In some embodiments, as used herein, an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, wherein the polypeptide is a tandem repeat sequence (M) (secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x An rMVA viral vector is provided comprising (where x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, Figures 1A and 1B). In some embodiments, as herein, two or more polypeptides in a tandem repeat sequence and an additional polypeptide in a tandem repeat sequence fused to the C-terminus of the last polypeptide ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)x (Secretory signal peptide-immune checkpoint inhibitor peptide)) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M is methionine), an rMVA virus vector containing a heterologous polycistronic nucleic acid insert is provided (see, for example, FIGS. 2A and 2B).

[0023] In some embodiments, the rMVA virus vector contains a polycistronic nucleic acid insert encoding two or more polypeptides, where the polypeptides are the tandem repeat sequences described herein, such as ((M)(Secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretory signal peptide-immune checkpoint inhibitor peptide)) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, M = methionine, and the sequence encoding the first polypeptide is oriented in the 5'→3' direction), a first polypeptide tandem repeat sequence, and ((M)(Secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretory signal peptide-immune checkpoint inhibitor peptide)) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, M = methionine, and the sequence encoding the second polypeptide is oriented in the 3'→5' direction), a second polypeptide tandem repeat sequence, where each cistron contains a poxvirus promoter capable of initiating transcription. In some embodiments, x = 3, 4, 5, or 6.

[0024] As provided herein, rMVA can be used as an adjuvant to enhance the immunogenicity of one or more concurrently administered antigens during a vaccination protocol. Immunomodulatory activity, which often prevents the development of antigenicity sufficient to induce immunity, can be downregulated by locally expressing one or more immune checkpoint inhibitor peptides that can downmodulate one or more checkpoint inhibitor pathways. In certain embodiments, immune checkpoint inhibitor peptides include, but are not limited to, programmed cell death protein-1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-3 (TIM-3), T cell activation V domain Ig suppressor (VISTA), B7 homolog protein (B7), B7 homolog 3 protein (B7-H3), B7 homolog 4 protein (B7-H4), B7 homolog 5 protein (B7-H5), OX-40 (OX-40), OX-40 ligand (OX-40L), and glucocorticoid-inducible TNF The activity of immune checkpoint pathways mediated by receptor proteins selected from R-related protein (GITR), CD137, CD40, B and T lymphocyte attenuators (BTLA), herpesvirus entry mediator (HVEM), galactin-9 (GAL9), killer cell immunoglobulin-like receptor (KIR), natural killer cell receptor 2B4 (2B4), CD160, checkpoint kinase 1 (CHK1), checkpoint kinase 2 (CHK2), adenosine A2a receptor (A2aR), T cell immune receptor having Ig and ITIM domains (TIGIT), inducible T cell costimulator (ICOS), inducible T cell costimulator ligand (ICOS-L), or combinations thereof can be inhibited. In some embodiments, immune checkpoint inhibitor peptides can inhibit PD-1. In some embodiments, immune checkpoint inhibitor peptides can inhibit PD-L1.In some embodiments, immune checkpoint inhibitor peptides can inhibit CTLA-4. In some embodiments, immune checkpoint inhibitor peptides can inhibit PD-1, PD-L1, or CTLA-4, or a combination thereof. In some embodiments, immune checkpoint inhibitor peptides can inhibit both PD-1 and CTLA-4.

[0025] In some embodiments, the immune checkpoint inhibitor peptide is selected from the peptides listed in Table 1, or their homologs, derivatives, or fragments. In some embodiments, the immune checkpoint inhibitor peptide is selected from peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 56, or peptides having an amino acid sequence identical to them by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the immune checkpoint inhibitor peptide is selected from peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5, or peptides having an amino acid sequence identical to them by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the immune checkpoint inhibitor peptide is selected from peptides having the amino acid sequence of SEQ ID NO: 1 (CRRTSTGQISTLRVNITAPLSQ), or peptides having an amino acid sequence identical to it by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the immune checkpoint inhibitor peptide is selected from a peptide having the amino acid sequence of SEQ ID NO: 5 (STGQISTLRVNITAPLSQ), or from amino acids having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide is selected from a peptide having the amino acid sequence of SEQ ID NO: 6 (STGQISTLAVNITAPLSQ), or from amino acids having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0026] In some embodiments provided herein, each immune checkpoint inhibitor peptide expressed by an rMVA is fused at its N-terminus to a secretory signal peptide, where the rMVA expresses two or more immune checkpoint inhibitor peptides and is fused at its C-terminus to one or more cleavable peptides. The secretory signal peptide enables the immune checkpoint inhibitor peptides to translocate into the endoplasmic reticulum (ER). Following translation of the growing peptide chain and simultaneous insertion into the ER lumen, the signal peptidase cleaves the signal peptide from the immune checkpoint inhibitor peptide, and the immune checkpoint inhibitor is secreted (see, for example, Figures 3A, 3B, and 3C). The secretory signal peptide used herein may be any suitable signal peptide that enables the secretion of immune checkpoint inhibitor peptides. The secretory signal peptides used in the present invention are known in the art (see, for example, Kober et al., "Optimized signal peptides for the development of high expressing CHO cell lines," Biotechnol Bioengin. 2013;110:1164-1173 (which is incorporated herein by reference)). In some embodiments, the secretory signal peptide is a short peptide having a length between approximately 15 and 30 amino acids derived from a native human efflux protein. In some embodiments, the secretory signal is a secretory signal selected from the secretory signals in Table 2 (SEQ ID NOs. 57 to 90), or their homologs, derivatives, or fragments. In some embodiments, the secretory signal peptide is, for example, but not limited to, human growth factors, human cytokines, interleukin-1, interleukin-2, human immunoglobulin kappa light chain, trypsinogen, serum albumin, prolactin, tissue plasminogen activator, alkaline phosphatase, or other suitable secretory signal sequences described herein, or derived from them.In some embodiments, the secretion signal peptide is derived from human tissue plasminogen activator. In some embodiments, the secretion signal peptide is derived from human tissue plasminogen activator containing the amino acid sequence DAMKRGLCCVLLLCGAVFVSPSQ (SEQ ID NO: 65), or from a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% thereto. In some embodiments, the secretion signal peptide is derived from human tissue plasminogen activator containing the amino acid sequence DAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGAR (SEQ ID NO: 66), or from a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% thereto. In some embodiments, the secretion signal peptide of the first polypeptide encoded by the polycistronic nucleic acid insert further comprises the starting amino acid methionine (M).

[0027] In some embodiments, one or more immune checkpoint inhibitor chimeric polypeptides include one or more peptide sequences fused to the C-terminus of an immune checkpoint inhibitor peptide, which can be cleaved during or after translation of a polycistronic nucleic acid, or in combination thereof (see, for example, Figures 3A, 3B, and 3C). In some embodiments, the most C-terminal immune checkpoint inhibitor chimeric polypeptide does not contain a cleavable peptide. In some embodiments, the cleavable peptide can be cleaved by a proprotein convertase enzyme, including, for example, furin or a furin-like proprotein convertase, but are not limited to these. In some embodiments, the cleavable peptide sequence includes a basic amino acid target sequence (typically RX(R / K)R) (where X = any amino acid) (SEQ ID NO: 91). In some embodiments, the cleavable peptide sequence includes a basic amino acid target sequence (typically RX(R / K)R) (where X = R, K, or H) (SEQ ID NO: 92). In some embodiments, the cleavable peptide sequence is RAKR (SEQ ID NO: 93). In some embodiments, the cleavable peptide sequence is RRRR (SEQ ID NO: 94). In some embodiments, the cleavable peptide is RKRR (SEQ ID NO: 95). In some embodiments, the cleavable peptide is RRKR (SEQ ID NO: 96). In some embodiments, the cleavable peptide is RKKR (SEQ ID NO: 97). By including a cleavable peptide sequence in each of the covalently linked chimeric polypeptides, the multimeric polypeptide expressed during translation of the polycistronic nucleic acid insert can be processed into monomeric chimeric polypeptides via a cleavage mechanism after translation. This allows each chimeric polypeptide, containing an immune checkpoint inhibitor peptide, to be secreted from the cell and function to downmodulate an undesirable immune checkpoint pathway (see, for example, Figure 3A).

[0028] In some embodiments, each chimeric polypeptide comprises one or more peptide sequences fused to the C-terminus of an immune checkpoint inhibitor peptide that can induce ribozyme skipping during translation of a polycistronic nucleic acid. Ribosome “skipping” is an alternative translation mechanism in which a particular peptide sequence prevents the covalent linking of newly inserted amino acids by ribosomes, but translation still continues. This results in polyprotein “cleavage” by induction of ribosome skipping. In some embodiments, the peptide capable of inducing ribosome skipping is a cis-acting hydrolase element peptide (CHYSEL). In some embodiments, the CHYSEL sequence comprises a non-conserved sequence of amino acids with a strong α-helix orientation, followed by a consensus sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98), where ribosome skipping cleavage occurs between the G sequence and the P sequence. In some embodiments, the CHYSEL sequence comprises DVEENPGP (SEQ ID NO: 99). In some embodiments, the CHYSEL peptide sequence is a sequence selected from the sequences in Table 4, or a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the CHYSEL peptide sequence is an amino acid sequence selected from SEQ ID NOs. 100 to 122, or a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the CHYSEL peptide sequence is an amino acid sequence selected from SEQ ID NOs. 118 to 122, or a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the CHYSEL peptide sequence includes GSGATNFSLLKQAGDVEENPGP (SEQ ID NOs. 120), or a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of it.By including peptide sequences that induce ribosome skipping, numerous chimeric polypeptides encoded by polycistronic nucleic acid inserts are expressed as monomers, which are then secreted from cells and function to downmodulate unwanted immune checkpoint pathways (see, for example, Figure 3B).

[0029] In some embodiments, the cleavable peptide sequence includes two or more sequences that can be cleaved by different mechanisms, for example, a cleavable peptide sequence that can be cleaved after translation of a polycistronic nucleic acid and a peptide sequence that can induce ribozyme skipping during translation of a polycistronic nucleic acid. By providing cleavable peptide sequences that follow a number of cleavage methods, the efficiency of monomer formation from polycistronic nucleic acids can be improved. In some embodiments, the immune checkpoint inhibitor peptide is fused at its C-terminus to a furin-cleavable peptide sequence, for example, the peptide sequence RX(R / K)R (where X = any amino acid) (SEQ ID NO: 91), and fused at the C-terminus of the furin-cleavable peptide sequence is a CHYSEL peptide sequence including, for example, D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98). For example, by including a furin-cleavable peptide sequence such as RAKR (SEQ ID NO: 93) fused to the N-terminus of the CHYSEL peptide sequence between each chimeric polypeptide, the transcribed polycistronic nucleic acid undergoes ribozyme skipping during translation, resulting in the production of monomeric chimeric polypeptides. After post-translational processing and furin peptide cleavage, all but the arginine (R) and alanine (A) residues of the furin-cleavable sequence remain at the C-terminus of the immune checkpoint inhibitor peptide, thus limiting the potential interference of extra amino acid sequences with the function of the immune checkpoint inhibitor peptide (see, for example, Figure 3C). In alternative embodiments, the use of furin-cleavable peptides such as RRRR (SEQ ID NO: 94), RKRR (SEQ ID NO: 95), or RRKR (SEQ ID NO: 96) results in the complete removal of the furin-cleavable sequence from the C-terminus of the immune checkpoint inhibitor peptide, leaving no residual amino acids. In some embodiments, the hybrid cleavage sequence is RAKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 123), or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.In some embodiments, the hybrid cleavage sequence is RRRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 124), or a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% thereof. In some embodiments, the hybrid cleavage sequence is RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 125), or a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% thereof. In some embodiments, the hybrid cleavage sequence is RRKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 126), or a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% thereof. In some embodiments, the hybrid cleavage sequence is RKKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 127), or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0030] In some embodiments, the rMVA viral vector includes a heteropolycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence selected from SEQ ID NOs. 309 to 340, or SEQ ID NOs. 341 to 348. In some embodiments, the rMVA viral vector includes a heteropolycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NOs. 325 to 340, or SEQ ID NOs. 345 to 348. In some embodiments, the rMVA viral vector includes a heteropolycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NOs. 325. In some embodiments, the rMVA viral vector includes a heteropolycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NOs. 329. In some embodiments, the rMVA viral vector includes a heteropolycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NOs. 333. In some embodiments, the rMVA viral vector includes a heteropolycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NOs. 337.

[0031] Transcription of the nucleic acid insert may be initiated by one or more promoters compatible with an MVA viral vector, located 5' of the first start codon of the first coding sequence contained within the nucleic acid and operably ligated thereto. Suitable promoters compatible with poxvirus expression vectors are known in the art and are not limited to pmH5, p11, pSyn, pHyb, or any other suitable MVA promoter sequence. In some embodiments, the promoter is a natural promoter for the ORF of MVA. In some embodiments, the promoter is selected from the promoters in Table 7, or nucleic acids having sequences identical to them by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the promoter sequence is selected from SEQ ID NOs. 128 to SEQ ID NOs. 308, or nucleic acid sequences identical to them by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the promoter sequence is selected from SEQ ID NOs: 130 to 132, or nucleic acid sequences that are at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter sequence is SEQ ID NO: 130, or a nucleic acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0032] In some embodiments in which multiple immune checkpoint inhibitor peptides are expressed, the chimeric polypeptide is transcribed as a single transcript; therefore, the polycistronic nucleic acid insert includes one or more termination signals (e.g., stop codons such as TAA, TAG, or TGA, or combinations or repetitions thereof) following only the ORF sequence of the last chimeric polypeptide. Upon transcription, the multiple chimeric polypeptides produce a single transcript, which is then translated. After post-translational processing, multiple monomeric chimeric polypeptides are generated.

[0033] The rMVA viral construct provided by the present invention can be used as an adjuvant to treat or prevent infectious diseases or cancer in a subject, or to induce an immune response to infectious diseases or cancer. In some embodiments, the rMVA viral construct is administered to a subject requiring administration, such as a human, in a prophylactic vaccination protocol to prevent infectious diseases, for example, in the initial vaccination stage, the booster vaccination stage, or both the initial vaccination stage and the booster vaccination stage. In alternative embodiments, the rMVA viral construct is administered to a subject requiring administration, such as a human, in a therapeutic approach including a vaccination protocol to treat, for example, cancer. Thus, the rMVA viral construct can be administered in conjunction with one or more antigens intended to induce an immune response to an antigenic target to induce partial or complete immunization in a subject requiring induction.

[0034] Therefore, the rMVA of the present invention can be administered together with one or more antigens targeting infectious diseases or cancer. Examples of antigens and antigen delivery carriers that can be used together with rMVA as an adjuvant include antigenic proteins, polypeptides, or peptides or fragments thereof, nucleic acids encoding one or more antigens, such as mRNA or DNA, polysaccharides, or conjugates of polysaccharides and proteins, glycolipids, such as gangliosides, toxoids, subunits (e.g., subunits of viruses, bacteria, fungi, amoebas, parasites, etc.), virus-like particles, live viruses, split viruses, attenuated viruses, inactivated viruses, enveloped viruses, viral vectors expressing one or more antigens, tumor-associated antigens, or any combination thereof.

[0035] In a particular embodiment, the present invention provides a method for preventing or treating an infectious disease in a subject requiring prevention, treatment, or induction, or for inducing an immune response thereto, comprising administering an effective amount of the rMVA of the present invention in combination with one or more antigens or antigen-expressing vectors in a prophylactic or therapeutic effective amount, alternately or in conjunction therewith, wherein the rMVA enhances immunity against the targeted infectious disease.

[0036] In some embodiments, the target infections are viral infections including but not limited to double-stranded DNA viruses, including but not limited to adenoviruses, herpesviruses, and poxviruses; single-stranded DNA viruses, including but not limited to parvoviruses; double-stranded RNA viruses, including but not limited to reoviruses; positive-sense single-stranded RNA viruses, including but not limited to coronaviruses such as SARS-CoV-2, picornaviruses, and togaviruses; negative-sense single-stranded RNA viruses, including but not limited to orthomyxoviruses and rhabdoviruses; single-stranded RNA retroviruses, including but not limited to retroviruses; or double-stranded DNA retroviruses, including but not limited to hepadnaviruses. In some embodiments, the target viruses include adenovirus, avian influenza virus, coxsackievirus, cytomegalovirus, dengue virus, Ebola virus, Epstein-Barr virus, equine encephalitis virus, flavivirus, hepadnavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus, human immunodeficiency virus, human papillomavirus, influenza virus, Japanese encephalitis virus, JC virus, measles virus (measles morbillivirus), Marburg virus, Middle East respiratory syndrome coronavirus, mumps sulfura virus, orthomyxovirus, papillomavirus, parainfluenza virus, parvovirus, picornavirus, poliovirus, poxvirus, rabies virus, reovirus, respiratory syncytial virus, retrovirus, rhabdovirus, rhinovirus, Rift Valley fever virus, rotavirus, rubella virus, and measles virus (rubeola). These include coronaviruses, severe acute respiratory syndrome coronavirus type 1, severe acute respiratory syndrome coronavirus type 2, smallpox virus, toga virus, swine influenza virus, varicella-zoster virus, varicella-zoster virus, varicella-major varicella virus, varicella-minor varicella virus, and yellow fever virus.

[0037] In some embodiments, the target infections include several species of the genus Borrelia, Bacillus anthraces, Borrelia burgdorferi, Bordetella pertussis, and Campylobacter jejuni; several species of the genus Chlamydia, Chlamydial psittaci, and Chlamydial trachomatis; several species of the genus Clostridium, Clostridium tetani, Clostridium botulinum, and Clostridium perfringens. * Listeria interrogans), several species of the genus Listeria, Listeria monocytogenes (ListeriaMycobacterium monocytogenes, several species of the genus Mycobacterium, Mycobacterium tuberculosis, Mycobacterium leprae, several species of the genus Mycoplasma, Mycoplasma pneumoniae, several species of the genus Neisseria, Neisseria meningitidis, Neisseria gonorrhoeae, several species of the genus Pneumococcus, several species of the genus Pseudomonas, Pseudomonas aeruginosa, several species of the genus Salmonella, Salmonella ciffii Streptococcus typhi), Salmonella enterica, several species of the genus Streptococcus, several species of the genus Rickettsia, Rickettsia ricketsii, Rickettsia typhi, several species of the genus Shigella, several species of the genus Staphylococcus, Staphylococcus aureus, several species of the genus Streptococcus, Streptococcus pneumoniae, Streptococcus pyrogenes, Streptococcus mutans Treponema mutans), several species of the genus Treponema, Treponema pallidum, several species of the genus VibrioThis category includes, but is not limited to, *Vibrio cholerae* and *Yersinia pestis*.

[0038] In some embodiments, the target infections include multiple species of the genus Aspergillus, multiple species of the genus Candida, Candida albicans, Candida tropicalis, multiple species of the genus Cryptococcus, Cryptococcus neoformans, Entamoeba histolytica, Histoplasma capsulatum, multiple species of the genus Leishmania, Nocardia asteroides, Plasmodium falciparum, Toxoplasma gondii, and Trichomonas vaginalis. This is a fungal infection that includes, but is not limited to, fungi of the genus Toxoplasma (vaginalis), several species of Toxoplasma, Trypanosoma brucei, Schistosoma mansoni, several species of Fusarium, and several species of Trichophyton.

[0039] In some embodiments, the target infections include, but are not limited to, parasites from the genera Plasmodium, Toxoplasma, Entamoeba, Babesia, Trypanosoma, Leshmania, Pneumocystis, Trichomonas, Giardia, and Schisostoma.

[0040] In some embodiments, a method for preventing or treating cancer in a subject requiring prevention, treatment, or induction, or for inducing an immune response thereto, comprising administering an effective amount of the rMVA of the present invention in combination with a prophylactic or therapeutic effective amount of one or more tumor-associated antigens or vectors expressing tumor-associated antigens, alternately or in conjunction with them, wherein the rMVA enhances immunity against cancer. In some embodiments, tumor-associated antigens (TAAs) are, but are not limited to, carcinoembryonic TAAs typically expressed only in fetal tissue and cancerous somatic cells, oncoviral TAAs typically encoded by tumorigenic transforming viruses, overexpressed / accumulated TAAs typically expressed in both normal and neoplastic tissues, with significantly increased expression levels in neoplasms, oncological-testicular TAAs typically expressed only in cancer cells and adult reproductive tissues such as the testes and placenta, lineage-specific TAAs typically expressed primarily by a single cancer histological type, mutated TAAs typically expressed only by cancer as a result of gene mutation or transcriptional changes, post-translational modified TAAs typically having tumor-associated changes such as glycosylation, and idiotype TAAs typically in which tumor cells express a specific "chronotype," i.e., highly polymorphic genes similar to those in B-cell and T-cell lymphoma / leukemia resulting from clonal abnormalities.In some embodiments, TAA is expressed as Wilms tumor protein (WT1), melanoma antigen preferentially expressed in tumors (PRAME), survivin, cancer / testicular antigen 1 (NY-ESO-1), melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4), proteinase 3 (Pr3), cyclin A1, synovial sarcoma X2 (SSX2) showing high homology, neutrophil elastase (NE), mucin 1 (MUC1), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), epithelial tumor antigen (ETA), tyrosinase, and ras. The abnormal product is selected from abnormal products, abnormal products of p53, Epstein-Barr virus initial antigen (EA), latent membrane protein 1 (LMP1), and latent membrane protein 2 (LMP2), gangliosides such as GM1b, GD1c, GM3, GM2, GM1a, GD1a, GT1a, GD3, GD2, GD1b, GT1b, GQ1b, GT3, GT2, GT1c, GQ1c, and GP1c, and ganglioside derivatives such as 9-O-Ac-GD3, 9-O-Ac-GD2, 5-N-de-GM3, N-glycolylGM3, NeuGcGM3, and fucosyl-GM1, or combinations thereof.

[0041] In some embodiments, the antigen is derived from the amino acid sequences of SEQ ID NOs: 349 to 394.

[0042] In alternative embodiments, the rMVA viral vector of the present invention can be further constructed to encode and express one or more antigenic peptides, in addition to its ability to express a number of immune checkpoint inhibitor peptides. The one or more antigenic peptides may be encoded on one or more separate nucleic acid inserts, or, in alternative embodiments, the one or more antigenic peptides may be encoded on the same polycistronic nucleic acid insert as the number of immune checkpoint inhibitor peptides. In some embodiments, as used herein, an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, wherein the polypeptide is ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) xAn rMVA viral vector is provided comprising (antigenic peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigenic peptide is contained in a chimeric polypeptide comprising a secretion signal peptide fused to the N-terminus of the antigenic peptide, for example ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretionary signal peptide-antigenic peptide))(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, Figures 4A and 4B). In some embodiments, the antigenic peptide is also provided such that two or more antigenic peptides are encoded in a polycistronic nucleic acid insert, and each chimeric polypeptide is separated by a cleavable peptide as described herein. In some embodiments, the antigenic peptide is contained in a chimeric polypeptide comprising a secretion signal peptide fused to the N-terminus of the antigenic peptide and a cleavable peptide fused to the C-terminus of the antigenic peptide, for example ((M)(Secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretory signaling peptide - Antigenic peptide - Cleavable peptide) y )(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigen-containing chimeric polypeptide fused to the C-terminus of the last antigen-containing chimeric polypeptide does not contain a cleavable sequence, for example ((M)(secretionary signaling peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretory signaling peptide - Antigenic peptide - Cleavable peptide) x(Secretionary signal peptide - Antigenic peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigenic peptide contained in a chimeric polypeptide comprising a secretion signal peptide fused to the N-terminus of the antigenic peptide and a cleavable peptide fused to the C-terminus of the antigenic peptide may be oriented in a polycistronic nucleic acid insert such that the nucleic acid encoding the antigen-containing chimeric polypeptide is located at 5' of the immune checkpoint inhibitor peptide-containing chimeric polypeptide, for example ((M)(Secretionary signal peptide - Antigenic peptide - Cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x ), or alternatively ((M)(secretory signaling peptide-antigenic peptide-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x (Secretionary signal peptide - Immune checkpoint inhibitor peptide) (where y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine). In some embodiments, the antigenic peptide includes its native secretory signal peptide. In alternative peptides, the secretory signal peptide is not derived from the antigen, but rather from a different protein, synthetic secretory signal, or consensus secretory signal peptide. In some embodiments, the antigenic peptide is selected from SEQ ID NOs: 349 to 394.

[0043] In some embodiments, the antigenic peptide encoded by the polycistronic nucleic acid insert in the rMVA is contained in a chimeric polypeptide comprising a viral glycoprotein signal sequence fused to the N-terminus of the antigenic peptide and a viral glycoprotein transmembrane domain fused to the C-terminus of the antigenic peptide, and the rMVA is further constructed to encode a viral matrix protein, where, upon translational cleavage of the antigen-containing chimeric peptide, the viral matrix protein and the antigen-viral glycoprotein chimeric polypeptide can form non-infectious virus-like particles (VLPs). In some embodiments, as used herein, an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, wherein the polypeptide is ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x An rMVA viral vector is provided comprising (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain))(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, Figures 5A and 5B). In some embodiments, the antigenic peptide is contained in a chimeric polypeptide comprising a viral glycoprotein signal sequence fused to the N-terminus of the antigenic peptide, a viral glycoprotein transmembrane domain fused to the C-terminus of the antigenic peptide, and a cleavable peptide fused to the C-terminus of the viral glycoprotein transmembrane domain, for example ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) x )(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigen-containing chimeric polypeptide fused to the C-terminus of the last antigen-containing chimeric polypeptide does not contain a cleavable sequence, for example ((M)(secretionary signaling peptide-immune checkpoint inhibitor peptide-cleavable peptide) x(Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine). In some embodiments, (glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (where y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10) may be oriented in the polycistronic nucleic acid insert such that the nucleic acid encoding the antigen-containing chimeric polypeptide is located at the 5' position of the immune checkpoint inhibitor peptide-containing chimeric polypeptide, for example ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x ), or alternatively, ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x (Secretionary signal peptide-immune checkpoint inhibitor peptide))(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine). In further embodiments, the polycistronic nucleic acid insert of rMVA further encodes a viral matrix protein, for example ((M)(Secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide)(viral matrix protein))(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, Figures 6A and 6B). In alternative embodiments, the coding sequences for both the antigen-containing chimeric polypeptide and the viral matrix protein are included in one or more copies in the polycistronic nucleic acid, for example ((M)(secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (Viral matrix protein - cleavable peptide) y )(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine). In some embodiments, the most C-terminal viral matrix protein lacks a cleavable peptide, for example ((M)(secretory signaling peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) x (Viral matrix protein - cleavable peptide) y (Viral matrix protein))(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine). In some embodiments, ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Viral matrix protein - cleavable peptide) y)(where y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) may be oriented in a polycistronic nucleic acid insert so as to be located at 5' of an immune checkpoint inhibitor peptide-containing chimeric polypeptide, for example ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Viral matrix protein - cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x ), or alternatively, ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Viral matrix protein - cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x (Secretionary signal peptide - Immune checkpoint inhibitor peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine). In some embodiments, the innate secretory signal from the antigen is replaced by a viral glycoprotein signal peptide. In some embodiments, the antigenic peptide is selected from SEQ ID NOs: 349 to 394.

[0044] The generation of virus-like particles containing target antigens is particularly suitable for use in vaccine strategies against enveloped viruses because they can induce both potent and sustained humoral and cellular immune responses. See, for example, Salvato et al., "A Single Dose of Modified Vaccinia Ankara Expressing Lassa Virus-like Particles Protects Mice from Lethal Intra-cerebral Virus Challenge," Pathogens (2019) 8:133. Suitable glycoproteins and matrix proteins for use in the production of antigen-containing VLPs include, but are not limited to, those derived from the filoviridae family, e.g., Marburg virus, Ebola virus, or Sudan virus; the retroviridae family, e.g., human immunodeficiency virus type 1 (HIV-1); the arenaviridae family, e.g., Lassa virus; and the flaviviridae family, e.g., dengue virus and Zika virus. In certain embodiments, the glycoprotein and matrix protein are derived from Marburg virus (MARV). In certain embodiments, the glycoprotein is derived from the GP protein of MARV (Genbank accession number AFV31202.1). The amino acid sequence of the GP protein of MARV is shown as SEQ ID NO: 395 in Table 10 below.In certain embodiments, the GPS domain of MARV comprises amino acids 2-19 of the glycoprotein (WTTCFFISLILIQGIKTL) (SEQ ID NO: 396, which may be encoded by an MVA-optimized nucleic acid sequence, for example, SEQ ID NO: 397), and the GPTM domain comprises amino acid sequences 644-673 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG) (SEQ ID NO: 398, which may be encoded by an MVA-optimized nucleic acid sequence, for example, SEQ ID NO: 399). In some embodiments, the GPS signal of MARV further comprises methionine as the first amino acid.

[0045] The VP40 amino acid sequence of MARV is available under GenBank accession number JX458834 and is provided as SEQ ID NO: 400 in Table 10 below, or as a nucleic acid sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical thereto. In some embodiments, the VP40 signal of MARV further includes methionine as the first amino acid.

[0046] In some embodiments, the rMVA antigenic peptide encoded by a polycistronic nucleic acid insert in the rMVA is contained in a chimeric polypeptide comprising a viral glycoprotein signal sequence fused to the N-terminus of the antigenic peptide and a viral glycoprotein transmembrane domain fused to the C-terminus of the antigenic peptide, wherein the rMVA is further constructed to encode a viral matrix protein, and upon translational cleavage of the antigen-containing chimeric peptide, the viral matrix protein and the antigen-viral glycoprotein chimeric polypeptide can form non-infectious virus-like particles (VLPs).

[0047] In an alternative embodiment, the rMVA viral vector of the present invention is further constructed to encode and express one or more antigenic peptides, in addition to its ability to express multiple immune checkpoint inhibitor peptides, where the one or more antigenic peptides are encoded on one or more separate nucleic acid inserts.

[0048] In some embodiments, the herein comprises one or more heterogeneous nucleic acid inserts encoding one or more chimeric polypeptides, each chimeric polypeptide being ((M)(immune checkpoint inhibitor peptide) x A recombinant modified vaccinia ankara (rMVA) virus vector is provided, comprising ((M)(immune checkpoint inhibitor peptide)) (where x = 1 to 10 and M is methionine), wherein the heterogeneous nucleic acid insert is under the control of the vaccinia virus promoter. In certain embodiments, the herein comprises one or more heterogeneous nucleic acid inserts encoding one or more chimeric polypeptides, each chimeric polypeptide being ((M)(immune checkpoint inhibitor peptide) x A recombinant modified vaccinia ankara (rMVA) virus vector is provided, comprising ((M)(immune checkpoint inhibitor peptide) (where x = 1 to 10, the immune checkpoint inhibitor comprises SEQ ID NO: 1, and M is methionine), wherein the heterogeneous nucleic acid insert is under the control of the vaccinia virus promoter. In certain embodiments, the herein comprises one or more heterogeneous nucleic acid inserts encoding one or more chimeric polypeptides, each chimeric polypeptide comprising ((M)(immune checkpoint inhibitor peptide) x A recombinant modified vaccinia ankara (rMVA) virus vector is provided, comprising (where x = 1 to 10, the immune checkpoint inhibitor comprises SEQ ID NO: 5, and M is methionine), wherein the heterologous nucleic acid insert is under the control of the vaccinia virus promoter.

[0049] In some embodiments, the Specified Viral Vector Provided is a recombinant modified vaccinia ankara (rMVA) viral vector comprising: i) a first nucleic acid sequence encoding a chimeric amino acid sequence including (a) an extracellular fragment of MUC-1, (b) a transmembrane domain of the Marburg virus (MARV) glycoprotein (GP), and (c) an intracellular fragment of MUC-1; ii) a second nucleic acid sequence encoding the VP40 matrix protein of MARV; and iii) a third nucleic acid sequence encoding one or more immune checkpoint inhibitor peptides, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are under the control of a vaccinia virus promoter, and when expressed, the chimeric amino acid sequence and the VP40 matrix protein can assemble together to form a virus-like particle (VLP). In certain embodiments, the Specified Viral Vector Provided is a recombinant modified vaccinia ankara (rMVA) virus vector comprising i) a first nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 402, ii) a second nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 404, and iii) a third nucleic acid sequence encoding one or more immune checkpoint inhibitor peptides, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are under the control of a vaccinia virus promoter, and when expressed, the chimeric amino acid sequence and VP40 matrix protein can assemble together to form a virus-like particle (VLP). In certain embodiments, the Specified Viral Vector Provided is a recombinant modified vaccinia ankara (rMVA) virus vector comprising: i) a first nucleic acid sequence encoding a chimeric amino acid sequence comprising the amino acid sequence of SEQ ID NO: 403; ii) a second nucleic acid sequence encoding the VP40 matrix protein of MARV comprising the amino acid sequence of SEQ ID NO: 405; and iii) a third nucleic acid sequence encoding one or more immune checkpoint inhibitor peptides, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are under the control of a vaccinia virus promoter, and when expressed, the chimeric amino acid sequence and the VP40 matrix protein can assemble together to form a virus-like particle (VLP).

[0050] In one embodiment, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are inserted into one or more deletion sites of an MVA selected from I, II, III, IV, V, or VI.

[0051] In another embodiment, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are inserted into the MVA at a natural deletion site, a modified natural deletion site, or between essential or non-essential MVA genes.

[0052] In another embodiment, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are inserted into the same natural deletion site, a modified natural deletion site, or between the same essential or non-essential MVA genes.

[0053] In another embodiment, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are inserted into different native deletion sites, different modified deletion sites, or between different essential or non-essential MVA genes.

[0054] In another embodiment, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are inserted between two essential, highly conserved MVA genes, and the matrix protein sequence is inserted into the reconstructed and modified deletion III.

[0055] In another embodiment, the first nucleic acid sequence is inserted between I8R and G1L of the MVA gene, the second nucleic acid sequence is inserted between A50R and B1R of the MVA gene at the reconstructed and modified deletion site III, and the third nucleic acid sequence is inserted between A5R and A6L of the two essential MVA genes.

[0056] In another embodiment, the vaccinia virus promoter is a nucleic acid sequence selected from SEQ ID NOs. 128 to 308.

[0057] In another embodiment, the vaccinia virus promoter is SEQ ID NO: 130, or a nucleic acid sequence that is 95% identical thereto.

[0058] In some embodiments, the MUC-1 nucleic acid sequence is provided as SEQ ID NO: 403, or as a nucleic acid sequence identical to it by 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the Marburg VP40 nucleic acid sequence is provided as SEQ ID NO: 404, or as a nucleic acid sequence identical to it by 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the 5xLD01 nucleic acid sequence is provided as SEQ ID NO: 408, or as a nucleic acid sequence identical to it by 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the 5xLD10 nucleic acid sequence is provided as SEQ ID NO: 409, or as a nucleic acid sequence identical to it by 70%, 75%, 80%, 85%, 90%, 95% or more.

[0059] Furthermore, this specification provides not only a shuttle vector containing a polycistronic nucleic acid sequence to be inserted into the MVA described herein, but also an isolated nucleic acid sequence containing the polycistronic nucleic acid sequence insert described herein. In addition, this specification provides cells containing the rMVA viral vector described herein. [Brief explanation of the drawing]

[0060] [Figure 1A] An exemplary diagram of an exemplary recombinant MVA viral vector open reading frame (ORF) of a polycistronic nucleic acid insert encoding multiple chimeric polypeptides is shown, where each chimeric polypeptide comprises a secretion signal peptide, an immune checkpoint inhibitor peptide fused to the C-terminus of the signal peptide, and a cleavable peptide fused to the C-terminus of the immune checkpoint inhibitor peptide. The polycistronic nucleic acid insert can encode 2 to 10 or more chimeric polypeptides, each containing methionine as its first amino acid. [Figure 1B]An exemplary diagram of an exemplary recombinant MVA viral vector containing a polycistronic nucleic acid insert encoding multiple chimeric polypeptides is shown, where each chimeric polypeptide comprises a secretory signal peptide (SP), an immune checkpoint inhibitor peptide (ICIP) fused to the C-terminus of the secretory signal peptide, and a cleavable peptide (cleavage sequence) fused to the C-terminus of the immune checkpoint inhibitor peptide. As exemplify, a promoter capable of initiating transcription of the ORF of MVA (e.g., the mH5 promoter (pmH5)) is operably ligated to the nucleic acid encoding the multiple chimeric polypeptides. The insert may contain a translation initiation sequence, e.g., a Kozak sequence, before the start codon of the ORF of the most 5' end chimeric polypeptide. As exemplify, a stop codon is present at 3' of the ORF of the last chimeric polypeptide. [Figure 2A] An exemplary diagram of the open reading frame (ORF) of an exemplary recombinant MVA viral vector of a polycistronic nucleic acid insert encoding multiple chimeric polypeptides is shown, where all chimeric polypeptides, except for the most C-terminal chimeric polypeptide lacking a cleavable peptide, include a secretory signal peptide (SP), an immune checkpoint inhibitor peptide fused to the C-terminus of the signal peptide, and a cleavable peptide fused to the C-terminus of the immune checkpoint inhibitor peptide. The polycistronic nucleic acid insert can encode 2 to 10 or more chimeric polypeptides, each containing methionine as its first amino acid. [Figure 2B]An exemplary diagram of an exemplary recombinant MVA viral vector containing a polycistronic nucleic acid insert encoding multiple chimeric polypeptides is shown, where each chimeric polypeptide comprises a secretory signal peptide (SP), an immune checkpoint inhibitor peptide (ICIP) fused to the C-terminus of the secretory signal peptide, and a cleavable peptide (cleavage sequence) fused to the C-terminus of the immune checkpoint inhibitor peptide, except for the most C-terminal chimeric polypeptide lacking a cleavable peptide. As exemplified, a promoter capable of initiating transcription of the ORF of MVA (e.g., the mH5 promoter (pmH5)) is operably ligated to the nucleic acid encoding the multiple chimeric polypeptides. The insert may contain a translation initiation sequence, e.g., a Kozak sequence, before the start codon of the ORF of the most 5' chimeric polypeptide. As exemplified, a stop codon is present at 3' of the ORF of the last chimeric polypeptide. [Figure 3A-3C]This figure shows exemplary schematic diagrams of the translational processing of various expressed chimeric polypeptides encoded by the polycistronic nucleic acid insert of the present invention. In Figure 3A, the chimeric polypeptide encodes a cleavable peptide sequence, such as a furin cleavage sequence or furin-like cleavage sequence, which is cleaved post-translation of the polycistronic nucleic acid transcript. Furthermore, during or after translation, the secretory signal peptide fused to the immune checkpoint inhibitor peptide is also cleaved, and the resulting monomeric immune checkpoint inhibitor peptide is subsequently secreted from the cell. In Figure 3B, the chimeric polypeptide encodes a cleavable peptide sequence that induces ribosome skipping, such as a CHYSEL cleavage sequence, where the polyprotein undergoes cleavage concurrently with translation, resulting in the generation of a monomeric immune checkpoint inhibitor peptide during translation. Post-translation or during translation, the chimeric polypeptide undergoes further cleavage of the secreted signal peptide, and the resulting monomeric immune checkpoint inhibitor peptide is subsequently secreted from the cell. In Figure 3C, the chimeric polypeptide encodes both a furin-cleavage sequence or furin-like cleavage sequence and a CHYSEL sequence, e.g., RAKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 123). During translation, ribosome skipping is induced at the glycine (G) and proline (P) amino acids at the C-terminus of the CHYSEL sequence, where the polyprotein undergoes cleavage concurrently with translation, resulting in the formation of a monomeric immune checkpoint inhibitor peptide during translation. The monomeric immune checkpoint inhibitor peptide undergoes further processing during or after translation, where the secreted signal peptide is cleaved. Furthermore, post-translation, the furin peptide sequence or furin-like peptide sequence is cleaved, resulting in a monomeric immune checkpoint inhibitor peptide containing only the arginine (R) and alanine (A) residues of the furin-cleavage sequence or furin-like cleavage sequence, reducing the potential for interference by immune checkpoint inhibitor peptides. [Figure 4A]An exemplary diagram of an exemplary recombinant MVA viral vector open reading frame (ORF) of a polycistronic nucleic acid insert is shown, encoding a chimeric polypeptide containing a secretory signal peptide, an immune checkpoint inhibitor peptide fused to the C-terminus of the signal peptide, a cleavable peptide fused to the C-terminus of the immune checkpoint inhibitor peptide, and a chimeric polypeptide containing a signal peptide fused to an antigenic peptide, where the antigen-containing chimeric polypeptide is fused to the most C-terminal immune checkpoint inhibitor-containing chimeric peptide. The polycistronic nucleic acid insert can encode one to more than ten immune checkpoint inhibitor-containing chimeric peptides, each containing methionine as its first amino acid. This same general concept above can be applied to any of the constructs provided herein that include cleavable sequences. [Figure 4B] An exemplary diagram of an exemplary recombinant MVA viral vector is shown, which includes a polycistronic nucleic acid insert encoding a chimeric polypeptide comprising a secretory signal peptide (SP), an immune checkpoint inhibitor peptide (ICIP) fused to the C-terminus of the signal peptide, and a cleavable peptide (cleavage sequence) fused to the C-terminus of the immune checkpoint inhibitor peptide, and an antigen-containing chimeric polypeptide comprising a secretory signal peptide (SP) fused to an antigenic peptide (antigen), wherein the antigen-containing chimeric polypeptide is fused to the most C-terminal immune checkpoint inhibitor-containing chimeric peptide. As exemplified, a promoter capable of initiating transcription of the ORF of MVA (e.g., the mH5 promoter (pmH5)) is operably ligated to the nucleic acid encoding the multiple chimeric polypeptides. The insert may include a translation initiation sequence, e.g., a Kozak sequence, before the start codon of the ORF of the most 5' chimeric polypeptide. As exemplified, a stop codon is present at 3' of the ORF of the last chimeric polypeptide. [Figure 5A]An exemplary diagram of a recombinant MVA viral vector open reading frame (ORF) of a polycistronic nucleic acid insert is shown, encoding a secretory signal peptide, a chimeric polypeptide containing a tandem repeat of an immune checkpoint inhibitor peptide fused to the C-terminus of the signal peptide, and a cleavable peptide fused to the C-terminus of the immune checkpoint inhibitor peptide, and an antigen-containing chimeric polypeptide containing a viral glycoprotein signal peptide fused to an antigenic peptide, the antigenic peptide being fused to the transmembrane domain of the viral glycoprotein, where the antigen-containing chimeric polypeptide is fused to the most C-terminal immune checkpoint inhibitor-containing chimeric peptide. The polycistronic nucleic acid insert can encode one to more than ten immune checkpoint inhibitor-containing chimeric polypeptides, each containing methionine as its first amino acid. [Figure 5B] An exemplary diagram of an exemplary recombinant MVA viral vector is shown, which includes a polycistronic nucleic acid insert encoding a multi-parametric chimeric polypeptide comprising a secretory signal peptide (SP), an immune checkpoint inhibitor peptide (ICIP) fused to the C-terminus of the signal peptide, and a cleavable peptide (cleavage sequence) fused to the C-terminus of the immune checkpoint inhibitor peptide, and an antigen-containing chimeric polypeptide comprising a viral glycoprotein signal peptide (GPSP) fused to an antigenic peptide (antigen), the antigenic peptide (antigen) being fused to the transmembrane domain of the viral glycoprotein transmembrane domain (GPTM), which is fused to the most C-terminal immune checkpoint inhibitor-containing chimeric peptide. As illustrated, a promoter capable of initiating transcription of the ORF of MVA (e.g., the mH5 promoter (pmH5)) is operably linked to the polycistronic nucleic acid encoding the multi-parametric chimeric polypeptide. The insert may include a translation initiation sequence, e.g., a Kozak sequence, before the start codon of the ORF of the most 5' chimeric polypeptide. As illustrated, a stop codon exists at the 3' position of the ORF of the last polypeptide. [Figure 6A]An exemplary diagram of an exemplary recombinant MVA viral vector open reading frame (ORF) of a polycistronic nucleic acid insert is shown, encoding a chimeric polypeptide containing a tandem repeat of a secretory signal peptide, an immune checkpoint inhibitor peptide fused to the C-terminus of the signal peptide, and a cleavable peptide fused to the C-terminus of the immune checkpoint inhibitor peptide, and an antigen-containing chimeric polypeptide containing a viral glycoprotein signal peptide fused to an antigenic peptide, the antigenic peptide being fused to the transmembrane domain of the viral glycoprotein and further fused to a cleavable peptide, where the antigen-containing chimeric polypeptide is fused to the most C-terminal immune checkpoint inhibitor-containing chimeric polypeptide, and further containing a viral matrix protein, the viral matrix protein being fused to the C-terminus of the cleavable peptide of the antigen-containing chimeric polypeptide. The polycistronic nucleic acid insert can encode 1 to 10 or more immune checkpoint inhibitor-containing chimeric polypeptides, the first amino acid being methionine. [Figure 6B]An exemplary diagram of an exemplary recombinant MVA viral vector is shown, which includes a polycistronic nucleic acid insert encoding a polycistronic nucleic acid insert comprising a secretory signal peptide (SP), an immune checkpoint inhibitor peptide (ICIP) fused to the C-terminus of the signal peptide, a cleavable peptide (cleavage sequence) fused to the C-terminus of the immune checkpoint inhibitor peptide, and an antigen-containing polycistronic nucleic acid insert encoding an antigen-containing polycistronic polypeptide comprising a viral glycoprotein signal peptide (GPSP) fused to an antigenic peptide (antigen), the antigen-containing polycistronic peptide (antigen) fused to the transmembrane domain of a viral glycoprotein transmembrane domain (GPTM) fused to the cleavable peptide, the antigen-containing polycistronic polypeptide fused to the most C-terminal immune checkpoint inhibitor-containing polycistronic polypeptide, and further comprising a viral matrix protein, the viral matrix protein fused to the C-terminus of the cleavable peptide of the antigen-containing polycistronic polypeptide. As illustrated, a promoter capable of initiating transcription of the ORF of MVA (e.g., the mH5 promoter (pmH5)) is operably linked to the polycistronic nucleic acid encoding the polycistronic polypeptide. The insert may contain a translation initiation sequence, such as a Kozak sequence, before the start codon of the ORF of the chimeric polypeptide at the 5' end. As illustrated, a stop codon is present at 3' of the viral matrix protein ORF. [Figure 7] These are schematic diagrams of the MVA-5X.LD01 and MVA-5X.LD10 vectors, showing the design of peptide sequences inserted into the MVA genome between two essential genes under the control of an MVA-specific promoter. Prior to the LD01 and LD10 sequences are signal sequences that route and secrete the peptide, followed by cleavage sites that separate the repeating peptide. The secretion signal, peptide sequence, and cleavage sites are repeated five times before transcription terminates at a stop codon. [Figure 8]This figure shows the production of LD01 and LD10 by MVA-infected cells. (Figure 8A) DF-1 cells were infected with MVA-5X.LD01, MVA-5X.LD10, or parental MVA. Two days after infection, the cells were fixed, permeabilized, and stained with antibodies specific to LD01 and LD10. The results show that the peptides were detected within the cells. LD01-positive and LD10-positive cells were stained as shown. Micrographs are shown at 20x magnification. (Figure 8B) DF-1 cells were infected with MVA-5X.LD01, MVA-5X.LD10, or parental MVA. Two days after infection, the supernatant was collected, concentrated, and scattered on a membrane with chemically synthesized peptide (LD01), and probed with antibodies specific to LD01 and LD10. The results show that the peptides were secreted from infected cells. [Figure 9]This figure (Figures 9A and 9B) shows that delivery of LD01 or LD10 via a viral vector enhances the proliferation of vaccine-induced antigen-specific CD8+ T cells. Immunogenicity was assessed 12 days after AdPyCS immunization by measuring the number of PyCS-specific IFN-γ secreting CD8+ T cells in the spleen using ELISpot assay (Figure 9A) and flow cytometry (Figure 9B) after stimulation with the H-2kD-restricted CD8 epitope SYVPSAEQI (SEQ ID NO: 406). Immediately after vaccination, a 100 μg dose of LD01 or LD10da was administered via SC. For viral vectors, 107 TCID50 MVA-5X.LD01, MVA-5X.LD10, or parental MVA were injected via SC after vaccination. Data are expressed as mean ± SEM. Data from one of two independent experiments are shown. Significant differences between AdPyCS monotherapy mice and treated mice were determined using unpaired two-sided t-tests and are indicated by **(p<0.001), ***(p<0.0005), and ****(p<0.0001). In Figure 9A, the x-axis represents AdPyCS monotherapy mice and treated mice, and the y-axis represents the number of IFN-γ spots per 1 × 10⁶ splenocytes as measured by count. In Figure 9B, the x-axis represents AdPyCS monotherapy mice and treated mice, and the y-axis represents the number of IFN-γ CB8T cells in total CB8T cells as measured by percentage. [Figure 10] This figure shows PCR gels of LD10 inserts, MUC-1 inserts, and VP40 inserts amplified from DNA samples of DF-1 cells infected with the MVA-VLP-MUC-1-LD10 virus. Parental MVA (negative control), plasmids containing the LD10 insert, MUC-1 insert, or VP40 insert (positive control), or DF1 cells infected with the MVA-VLP-MUC-1-LD10 recombinant virus were collected for viral DNA. Insert integrity was confirmed by PCR analysis. [Figure 11]This shows the expected PCR fragment sizes of LD10 inserts, MUC-1 inserts, and VP40 inserts collected from DF-1 cells infected with the MVA-VLP-MUC-1-LD10 virus. The expected fragment sizes were consistent with the band sizes on the PCR gel. [Figure 12] This figure shows the expression of recombinant MUC-1 protein in DF-1 cells infected with MVA-VLP-MUC-1-LD10. DF1 cells were infected with either parental modified vaccinia ankara (pMVA) or MVA encoding VLP-MUC-1-LD10. Uninfected cells were included as a negative control. Cell lysates and supernatants were collected for protein analysis and analyzed by immunoblotting. Probing of the membrane with MUC-1 antibody (mouse monoclonal VU4H5, Santa Cruz number sc-7313, 1:200) revealed a protein band of approximately 63 kDa in the MVS-VLP-MUC-1-LD10 lysate sample. [Figure 13] This figure shows the expression of recombinant VP40 protein in DF-1 cells infected with MVA-VLP-MUC-1-LD10. DF1 cells were infected with either parental modified vaccinia ankara (pMVA) or MVA encoding VLP-MUC-1-LD10. Uninfected cells were included as a negative control. Cell lysates and supernatants were collected for protein analysis and analyzed by immunoblotting. When membranes were probed with VP40 antibody, a protein band of approximately 32 kDa was labeled in the MVS-VLP-MUC-1-LD10 supernatant and lysate samples. [Figure 14]This figure shows the expression of recombinant LD10 protein in DF-1 cells infected with MVA-VLP-MUC-1-LD10. DF1 cells were transfected with either parental modified vaccinia ankara (pMVA) or MVA encoding VLP-MUC-1-LD10. Uninfected cells were included as a negative control. Cell lysates were collected for protein analysis and applied to a nitrocellulose membrane using a dot blotting apparatus. 20 micrograms of LD10 peptide were also loaded onto the membrane as a positive control for the LD10 antibody. When the membranes were probed with the LD10 antibody, signals were observed in both the MVA-VLP-MUC-1-LD10 peptide sample and the LD10 peptide sample. [Figure 15] This figure shows the percentage of MUC-1 positive plaques after infecting DF-1 cells with various amounts of recombinant MVA-VLP-MUC-1-LD10 virus. In a 6-well plate, DF1 cells were infected with 30 plaque-forming units (PFUs) and 60 PFUs of MVA-VLP-MUC-1-LD10 virus in three wells, respectively. All wells were probed with MUC-1 antibody, and the number of MUC-1 positive plaques was counted. After washing the wells, the cells were probed again with MVA antibody, and the number of MVA positive plaques was counted. The percentage of MUC-1 positive plaques relative to the number of MVA positive plaques is shown to calculate vaccine purity. The number of positive plaques per individual replica is shown at the bottom of the figure. [Figure 16]This figure shows the percentage of VP40-positive plaques after infecting DF-1 cells with various amounts of recombinant MVA-VLP-MUC-1-LD10 virus. In a 6-well plate, DF1 cells were infected with 30 plaque-forming units (PFUs) and 60 PFUs of MVA-VLP-MUC-1-LD10 virus in three wells, respectively. All wells were probed with MUC-1 antibody, and the number of VP40-positive plaques was counted. After washing the wells, the cells were probed again with MVA antibody, and the number of MVA-positive plaques was counted. The percentage of VP40-positive plaques relative to the number of MVA-positive plaques is shown to calculate vaccine purity. The number of positive plaques per individual replica is shown at the bottom of the figure. [Modes for carrying out the invention]

[0061] definition Where a term is provided in the singular form, the inventors also intend aspects of the invention described by the plural form of that term. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless the context clearly indicates otherwise, for example, “a peptide” or “a “chimeric polypeptide” includes multiple peptides or multiple chimeric polypeptides. Thus, for example, a reference to “method” includes one or more methods and / or steps of the type described herein, and / or this will be apparent to those skilled in the art by reading this disclosure.

[0062] As used herein, the term “adjuvant” means the use of rMVA as described herein to enhance the immunogenicity of one or more antigens.

[0063] The term "antigen" refers to a substance or molecule, such as a protein, or a fragment thereof, such as a peptide, that can induce an immune response.

[0064] As used herein, "chimeric" or "fused" refers to the covalent linking of peptides or proteins that do not exist in nature, resulting in a hybrid polypeptide. Translation of the chimeric or fused polypeptides described herein imparts functional properties derived from each of the fused peptides or proteins.

[0065] As used herein, "coding sequence," "coding nucleic acid," or "coding nucleic acid sequence" means a nucleic acid (RNA or DNA molecule) comprising an amino acid sequence, for example, a nucleotide sequence encoding a polyprotein, polypeptide, protein, peptide, or fragment thereof. The coding sequence may further include start and end signals operably linked to a regulatory element comprising a promoter and a polyadenylation signal that can direct the expression of the nucleic acid in human or mammalian cells to which it is administered.

[0066] The term "conservative amino acid substitution" refers to the substitution of a native amino acid residue with a non-native amino acid residue that has little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and does not result in substantially altered immunogenicity. For example, these may be substitutions within the following groups: valine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and corresponding modifications to the coding nucleotide) can produce polypeptides with similar functional and chemical characteristics to those of the parent polypeptide.

[0067] In relation to polypeptides or proteins, the term "deletion" refers to the removal of a codon for one or more amino acid residues from a polypeptide or protein sequence, with both regions joined together. In relation to nucleic acids, the term "deletion" refers to the removal of one or more bases from a nucleic acid sequence, with both regions joined together.

[0068] In relation to proteinaceous substances, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least two consecutive amino acid residues, at least five consecutive amino acid residues, at least ten consecutive amino acid residues, at least fifteen consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues. In one embodiment, the fragment constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference polypeptide. In one embodiment, a fragment of the full-length protein retains the activity of the full-length protein. In another embodiment, a fragment of the full-length protein does not retain the activity of the full-length protein.

[0069] In relation to nucleic acids, the term “fragment” refers to a nucleic acid containing a nucleic acid sequence of at least two consecutive nucleotides, at least five consecutive nucleotides, at least ten consecutive nucleotides, at least fifteen consecutive nucleotides, at least 20 consecutive nucleotides, at least 25 consecutive nucleotides, at least 30 consecutive nucleotides, at least 35 consecutive nucleotides, at least 40 consecutive nucleotides, at least 50 consecutive nucleotides, at least 60 consecutive nucleotides, at least 70 consecutive nucleotides, at least 80 consecutive nucleotides, at least 90 consecutive nucleotides, at least 100 consecutive nucleotides, at least 125 consecutive nucleotides, at least 150 consecutive nucleotides, at least 175 consecutive nucleotides, at least 200 consecutive nucleotides, at least 250 consecutive nucleotides, at least 300 consecutive nucleotides, at least 350 consecutive nucleotides, or at least 380 consecutive nucleotides that encode a peptide, polypeptide, or protein. In one embodiment, the fragment comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid sequence. In a preferred embodiment, the nucleic acid fragment encodes a peptide or polypeptide that retains the activity of the full-length protein. In another embodiment, the fragment encodes a peptide or polypeptide that does not retain the activity of the full-length protein.

[0070] As used herein, the term “heterogeneous sequence” means any nucleic acid, protein, polypeptide, or peptide sequence that does not normally and naturally associate with another nucleic acid, protein, polypeptide, or peptide sequence of interest.

[0071] As used herein, the term “heteronucleotide insert” refers to any nucleic acid sequence inserted into or intended to be inserted into a recombinant vector described herein. A heteronucleotide insert may refer only to a sequence encoding a gene product, or it may refer to a sequence containing a promoter, a sequence encoding a gene product (e.g., a chimeric polypeptide of a secretory signaling peptide-immune checkpoint inhibitor peptide), and any regulatory sequences associated with or operably linked thereto.

[0072] The term "homopolymer stretch" refers to a sequence containing at least four identical nucleotides, e.g., GGGG or TTTTTTT, that are not interrupted by any other nucleotides.

[0073] When used in the context of nucleic acid sequences, terms such as “percent identical,” “percent homologous,” or “percent similar” refer to two sequences being identical if their residues are compared and aligned to the greatest extent possible. The length of the sequence identity comparison may extend to the full length of the sequence, or alternatively, to a fragment of at least about 50 to 2500 nucleotides. Similarly, the terms “percent identical,” “percent homologous,” or “percent similar” can be readily determined for the amino acid sequence over the full length of a protein or fragment thereof. Appropriately, the fragment may be at least about 8 amino acids long and up to about 7500 amino acids long. Examples of appropriate fragments are provided herein. In general, “identity,” “homology,” or “similarity” is determined based on an “aligned” sequence. An “aligned” sequence or “alignment” refers to a number of nucleic acid sequences or protein (amino acid) sequences, often involving corrections for missing or additional bases or amino acids compared to a reference sequence. Alignment can be performed using any of the various publicly available or commercially available multiple sequence alignment programs. Examples of such programs include "Clustal Omega," "Clustal W," "CAP Sequence Assembly," "MAP," and "MEME," which are accessible via web servers on the internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility can also be used. There are also numerous algorithms known in the art that can be used to measure the identity of nucleotide sequences, including those included in the programs mentioned above. As another example, polynucleotide sequences can be compared using the Fasta® program in GCG version 6.1. Fasta® provides alignment and percentage sequence identity of the region with the highest overlap between the query sequence and the search sequence.For example, the percentage sequence identity between nucleic acid sequences can be determined using Fasta®, with its default parameters (word size of 6 and NOPAM coefficients for the scoring matrix) provided in GCG version 6.1 (which is part of this specification by reference). For amino acid sequences, multiple sequence alignment programs are also available, such as the "Clustal Omega," "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box" programs. Generally, these programs are used with their default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can use other algorithms or computer programs that provide at least the level of identity or alignment provided by the referenced algorithms and programs. See, for example, JD Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

[0074] The term "induces an immune response" means eliciting a humoral response (e.g., antibody production) or a cellular response (e.g., T cell activation) to one or more antigenic proteins or fragments expressed by rMVA in a subject administered with rMVA, or both humoral and cellular responses.

[0075] The term "modified vaccinia Ankara," or "MVA," typically refers to a highly attenuated vaccinia virus strain, or its variants or derivatives, developed by Dr. Anton Mayr through serial passage in chick embryo fibroblasts. MVA is outlined in Mayr, A. et al. 1975 Infection 3:6-14. The genomic sequences of MVA and various variants are described, for example, under GenBank accession numbers AY603355, U94848, and DQ983238. In some embodiments, the MVAs provided herein can be obtained synthetically, for example, via chemically synthesized plasmids, and can be reconstituted into full-length genomic MVA sequences in host cells, for example, as described in U.S. Patent Publication No. 2018 / 0251736, U.S. Patent Publication No. 2021 / 0230560, and International Publication No. 2021 / 158565 (each, respectively, forming part of this specification by reference).

[0076] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked to one another. A single-stranded description also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the single-stranded description. Many variants of a nucleic acid can be used for the same purposes as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0077] Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic and cDNA), RNA, or hybrids, and may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0078] As used herein, “operably linked” means that the expression of a gene is under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in the art, variations in this distance can be adjusted without loss of promoter function.

[0079] As used herein, "peptide," "protein," "polypeptide," or "polyprotein" may mean a linked sequence of amino acids, and may be natural, synthetic, modified, or a combination of natural and synthetic.

[0080] As used herein, “promoter” means a synthetic or naturally occurring molecule that can confer, activate, or enhance the transcription of nucleic acids within a cell. A promoter may include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also include a distal enhancer or repressor element, which may be located thousands of base pairs from the transcription start site.

[0081] The terms “prevent,” “prevention,” and “prevention” refer to the prevention of the onset or development of a particular condition (e.g., an infectious disease), or the prevention of the recurrence, onset, or development of one or more symptoms of a condition in a subject resulting from the application of a treatment or combination of treatments.

[0082] The term "preventive effective dose" refers to an amount of a composition (e.g., the target antigenic composition and / or rMVA described herein) that is sufficient to prevent the onset, recurrence, or development of a particular condition or its symptoms (e.g., a viral infection) or related symptoms, or to enhance or improve the preventive effect of another treatment(s).

[0083] In relation to viral vectors, the term "recombinant" refers to a vector (e.g., a viral genome) that has been manipulated in vitro, for example, by using recombinant nucleic acid technology to express a heterologous viral nucleic acid sequence.

[0084] The term "regulatory sequence" and "regulatory sequences" collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRESs"), enhancers, etc., which collectively provide for the transcription and translation of coding sequences. Not all of these regulatory sequences are always necessary as long as the selected gene can be transcribed and translated.

[0085] The term "shuttle vector" refers to a genetic vector (e.g., a DNA plasmid) that is useful for transferring genetic material from one host system to another. A shuttle vector can replicate independently (without the presence of any other vectors) in at least one host (e.g., Escherichia coli). In relation to the construction of MVA vectors, a shuttle vector is typically a DNA plasmid that can be manipulated in Escherichia coli and then introduced into cultured cells infected with the MVA vector, resulting in the generation of a new recombinant MVA vector, for example, via homologous recombination.

[0086] The term "silent mutation" refers to a change in the nucleotide sequence that does not cause a change in the primary structure of the protein encoded by the nucleotide sequence, for example, a change from AAA (which encodes lysine) to AAG (which also encodes lysine).

[0087] The “host,” “patient,” or “subject” being treated is typically a human patient, but it should be understood that the methods described herein are also effective for other animals, such as mammals. More specifically, the term patient may include, but is not limited to, animals used in assays, such as animals used in preclinical studies, including not only mice, rats, monkeys, dogs, pigs, and rabbits, but also domestic pigs (swimming pigs and hogs), ruminants, horses, poultry, cats, cattle, mice, dogs, etc. The determination of such “at-risk” subjects can be made by any objective or subjective determination based on diagnostic tests or opinions of the subject or healthcare provider (e.g., genetic testing, enzyme or protein markers, marker history, etc.).

[0088] The term "synonymous codon" refers to the use of codons with different nucleic acid sequences that code for the same amino acid, such as AAA and AAG (both coding for lysine). Codon optimization involves changing the codons in a protein to the synonymous codons most frequently used by the vector or host cell.

[0089] The term "therapeutic effective dose" means the amount of a composition (e.g., an antigenic composition and / or recombinant MVA vector or pharmaceutical composition) that, when administered to a subject, is sufficient to have an effect on such treatment or prevention of a disorder, such as an infection or cancer.

[0090] The terms “to treat” or “to treat” refer to the elimination or control of a disorder, or a reduction or improvement in the progression, severity, and / or duration of a disorder or one or more symptoms caused by that disorder, resulting from the application of one or more treatments.

[0091] The term "vaccine" refers to a substance used to induce an immune response and confer immunity after the substance has been administered to a subject. Such immunity may include cellular or humoral immune responses that occur when the subject is exposed to an immunogen after vaccine administration.

[0092] The term "virus-like particle," or "VLP," refers to structures that resemble viruses but are not infectious because they do not contain viral genetic material.

[0093] In the enumeration of numerical ranges as described herein, each number in between is explicitly intended to be of a similar degree of precision. For example, in the range of 6 to 9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0094] Modified Vaccinia Ankara (MVA) virus vector In particular, modified vaccinia ankara (MVA) is used as a safe and potent viral vector vaccine against infectious diseases. MVA is a highly attenuated strain of vaccinia virus obtained by extensive serial passage in chicken embryonic fibroblasts (CEF) (Sutter G, Staib C., "Vaccinia vectors as candidate vaccines: the development of modified vaccinia virus Ankara for antigen delivery," Current Drug Targets-Infectious Disorders. 2003;3:263-71). MVA is distinguished by its significant attenuation, as demonstrated by reduced toxicity and decreased replication capacity in primate cells while maintaining good immunogenicity. Analysis of MVA viruses has determined genomic changes compared to the parental strain, the chaloalulois vaccinia virus ankara (CVA) strain. Six major deletions (deletion I, deletion II, deletion III, deletion IV, deletion V, and deletion VI) have been identified in the total 31,000 base pairs of genomic DNA (Meyer, H. et al. 1991 J Gen Virol 72: 1031-1038). The host cells for the resulting MVA virus are limited to avian cells. Therefore, the MVA vaccine can be produced on a large scale in chicken cell lines.

[0095] The viral vector compositions provided herein include the modified vaccinia ankara (MVA) strain of vaccinia virus. Modified vaccinia ankara (MVA) was produced by long-term serial passage of the vaccinia virus ankara strain (CVA) in chicken embryo fibroblasts (for an overview, see "Origin, Characterization and Use of Attenuated Vaccinia Strains" by Mayr A, et al., Infection 3: 6-14, 1975, Swiss Patent No. 568,392). The MVA virus is publicly available from the American Type Culture Collection as ATCC number VR-1508. MVA is distinguished by its significant attenuation, as demonstrated by reduced pathogenicity and reduced replication capacity in primate cells while maintaining good immunogenicity. MVA viruses have been analyzed to determine genomic alterations compared to the parent CVA strain. Six major deletions in the genomic DNA (deletions I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). The resulting MVA virus replicates only in avian cells.

[0096] In certain embodiments, the MVA used is the MVA available as ATCC VR-1566, which is a virus isolated by serial passage of the CVA (Ankara) strain in chick embryo fibroblasts (CEF) in Professor Anton Meyer's laboratory and subsequently submitted to the National Institutes of Health, where it was a plaque purified three times in CEF cells. VR-1566 was obtained by further limited passage of the stock received from the NIH in the SL-29 chicken embryo fibroblast line [ATCC CRL-1590].

[0097] In alternative embodiments, the MVA is obtained from an MVA having the genomic sequences described in GenBank accession numbers AY603355, U94848, and DQ983238. In some embodiments, the MVAs provided herein can be obtained synthetically, for example, via a chemically synthesized plasmid, and can be reconstituted into full-length genomic MVA sequences in a host cell, for example, as described in U.S. Patent Publication No. 2018 / 0251736, U.S. Patent Publication No. 2021 / 0230560, and International Publication No. 2021 / 158565 (each, respectively, forming part of this specification by reference).

[0098] The recombinant MVA (rMVA) viral vector of the present invention can be prepared by methods known in the art. For example, a DNA construct containing a heterologous polycistronic nucleic acid sequence as described herein may have MVA DNA sequences on both sides adjacent to a predetermined insertion site (e.g., between two conserved essential MVA genes such as I8R / G1L (see, e.g., U.S. Patent No. 9,133,478, which is entirely part of this specification by reference)), in a reconfigured and modified deletion III (see, e.g., U.S. Patent No. 9,133,480, which is entirely part of this specification by reference) or at other non-essential sites in the MVA genome), and homologous recombination is possible by introduction into MVA-infected cells. Once the DNA construct is introduced into eukaryotic cells and the foreign DNA is recombined with the viral DNA, the desired rMVA can be isolated in a manner known in itself, preferably using a marker. The inserted DNA construct may be linear or circular. Plasmids or polymerase chain reaction products are preferred. Such methods for preparing recombinant MVA vectors are described, for example, in U.S. Patent No. 9,133,478 (which is part of this specification by reference). The expression of a DNA sequence or gene requires the presence of regulatory sequences on the DNA necessary for the transcription of polycistronic nucleic acid sequences. Such regulatory sequences (called promoters) are known to those skilled in the art and are further described below, for example.DNA constructs can be introduced into MVA-infected cells by transfection, for example, by calcium phosphate precipitation (Graham et al. 1973 Virol 52:456-467, Wigler et al. 1979 Cell 16:777-785), by electroporation (Neumann et al. 1982 EMBO J. 1:841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101:482-492), using liposomes (Straubinger et al. 1983 Meth Enzymol 101:512-527), by spheroplasts (Schaffher 1980 PNAS USA 77:2163-2167), or by other methods known to those skilled in the art.

[0099] In some embodiments, the rMVA provided herein can be obtained synthetically, for example, via a chemically synthesized plasmid, and can be reconstituted into a full-length genome MVA sequence in a host cell, for example, as described in U.S. Patent Publication No. 2018 / 0251736, U.S. Patent Publication No. 2021 / 0230560, and International Publication No. 2021 / 158565 (each, respectively, forming part of this specification by reference).

[0100] As described above, the heterologous polycistronic nucleic acid sequence of the present invention can be inserted into any suitable site within the rMVA genome sequence. In some embodiments, the polycistronic nucleic acid sequence is inserted into an MVA vector at a native deletion site, a modified native deletion site, or between essential or non-essential MVA genes.

[0101] Immune checkpoint inhibitor peptides This specification provides compositions comprising a recombinant modified vaccinia ankara (rMVA) virus vector for use as an adjuvant or vaccine in immunization protocols in a host such as a human, wherein the rMVA is constructed to express at high concentrations peptides capable of inhibiting one or more immune checkpoint pathways (immune checkpoint inhibitor peptides). In some embodiments, the immune checkpoint inhibitor peptides are expressed from a polycistronic nucleic acid sequence containing a tandem repeat of immune checkpoint inhibitors that can be processed into monomers, secreted from cells, and enhance the immunogenicity of a target antigen. In some embodiments, rMVA is used as an adjuvant to enhance the immunogenicity of one or more co-administered antigens in a vaccination protocol. In some embodiments, rMVA further encodes one or more antigenic peptides and is used as an adjuvanted vaccine. By expressing two or more immune checkpoint inhibitor peptides that can downmodulate one or more checkpoint inhibitor pathways in high localized concentrations, immunomodulatory activity that often prevents the generation of antigenicity sufficient to induce immunity can be downmodulated.

[0102] In certain embodiments, immune checkpoint inhibitor peptides include, but are not limited to, programmed cell death protein-1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-3 (TIM-3), T cell activation V domain Ig suppressor (VISTA), B7 homolog protein (B7), B7 homolog 3 protein (B7-H3), B7 homolog 4 protein (B7-H4), B7 homolog 5 protein (B7-H5), OX-40 (OX-40), OX-40 ligand (OX-40L), and glucocorticoid-inducible TNF The activity of immune checkpoint pathways mediated by receptor proteins selected from R-related protein (GITR), CD137, CD40, B and T lymphocyte attenuators (BTLA), herpesvirus entry mediator (HVEM), galactin-9 (GAL9), killer cell immunoglobulin-like receptor (KIR), natural killer cell receptor 2B4 (2B4), CD160, checkpoint kinase 1 (CHK1), checkpoint kinase 2 (CHK2), adenosine A2a receptor (A2aR), T cell immune receptor having Ig and ITIM domains (TIGIT), inducible T cell costimulator (ICOS), inducible T cell costimulator ligand (ICOS-L), or combinations thereof can be inhibited. In some embodiments, immune checkpoint inhibitor peptides can inhibit PD-1. In some embodiments, immune checkpoint inhibitor peptides can inhibit PD-L1. In some embodiments, immune checkpoint inhibitor peptides can inhibit CTLA-4. In some embodiments, immune checkpoint inhibitor peptides can inhibit PD-1, PD-L1, or CTLA-4, or a combination thereof. In some embodiments, immune checkpoint inhibitor peptides can inhibit both PD-1 and CTLA-4.

[0103] In some embodiments, the immune checkpoint inhibitor is an inhibitor that can inhibit PD-1, PD-L1, CTLA4, LAG-3, TIM3, OX40, or a combination thereof. In some embodiments, the immune checkpoint inhibitor can inhibit PD-1 and CTLA4.

[0104] In some embodiments, the immune checkpoint inhibitor peptide is selected from the peptide sequences disclosed in Table 1, or fragments, homologs, or derivatives thereof. In some embodiments, the immune checkpoint inhibitor peptide is selected from the peptide sequences of SEQ ID NOs. 1 to 56, or peptides having amino acid sequences identical to them by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the immune checkpoint inhibitor peptide is selected from the peptide sequences of SEQ ID NOs. 1 to 15, or peptides having amino acid sequences identical to them by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO. 1, or a peptide having an amino acid sequence identical to it by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO. 2, or a peptide having an amino acid sequence identical to it by at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 3, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 4, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 5, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 6, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 7, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 8, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 9, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 10, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 11, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 12, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 13, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 14, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has the peptide sequence of SEQ ID NO: 15, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence selected from SEQ ID NOs: 16 to 56, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0105] [Table 1] TIFF0007911709000002.tif42170

[0106] The immune checkpoint inhibitors listed in Table 1, for example, SEQ ID NOs. 1 to 15 are listed in U.S. Patent Nos. 10,098,950, 10,799,555, and 10,799,581, and U.S. Patent Publication Nos. 2018 / 0071385, 2018 / 0185474, 2018 / 0200328, and 2018 / 0339044; SEQ ID NOs. 16 to 22 are listed in "Peptide Blocking of PD-1 / PD-L1 Interaction for Cancer Immunotherapy" by Li et al., Cancer Immunol Res February 1 2018 (6) (2) 178-188; and SEQ ID NOs. 23 to 26 are listed by Li et al. In "Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy" by al., J. Immunotherapy Cancer 7, 270 (2019), SEQ ID NOs. 27-31 are found in "D-1 and its ligands in T-cell immunity" by Keir et al., Curr Opin Immunol. 2007;19(3):309-14 and "Discovery of peptide inhibitors targeting human programmed death 1 (PD-1) receptor" by Li et al., Oncotarget. 2016;7(40):64967-64976, while SEQ ID NOs. 32-36 are found in Wang et al., Journal of Medicinal Chemistry 2019 62 (4), In 1715-1730, Sequence IDs 37-40 are from Xiao et al., ACS Appl. Mater.In Interfaces 2020, 12, 36, 40042-40051, Sequence IDs 41-42 are from "Rational design and development of a peptide inhibitor for the PD-1 / PD-L1 interaction" by Boohaker et al., and in Cancer Letters, 2018, 434, Pages 11-21, Sequence IDs 43-45 are from "Antigen-specific CD8..." by Zhai et al. + In "A novel cyclic peptide targeting LAG-3 for cancer immunotherapy by activating antigen-specific CD8+ T cell responses," Acta Pharmaceutica Sinica B, 2020, 10(6), Pages 1047-1060; 6, June 2020, Sequence IDs 46-56 were previously described in "The biologically functional identification of a novel TIM3-binding peptide P26 in vitro and in vivo" by Zhong et al., Cancer Chemother Pharmacol. 2020;86(6):783-792. All references are incorporated herein by reference.

[0107] Secretion signal peptides As provided herein, immune checkpoint inhibitor peptides expressed by rMVA are secreted from cells. In some embodiments, secretion can be achieved by including, where applicable, a native secretory signal associated with the immune checkpoint inhibitor peptide. In alternative embodiments, the immune checkpoint inhibitor peptide expressed by rMVA may be heterologous to the host or may not have appropriate secretory signaling to ensure secretion from host cells. For this reason, secretion of immune checkpoint inhibitor peptides can be achieved by expressing a chimeric polypeptide containing a secretory signaling peptide fused to the immune checkpoint inhibitor peptide.

[0108] During the translation of a chimeric polypeptide containing a secretory signal peptide and an immune checkpoint inhibitor peptide, the signal peptide is recognized upon exiting the ribosome, bound by a signal recognition particle (SRP), and translation is halted. The entire complex is transported to the outer surface of the endoplasmic reticulum (ER), where it binds to the SRP receptor, and the signal sequence is transferred to the translocon. Translation resumes while bound to the translocon, and the protein crosses the ER membrane into the lumen. At this point, the signal peptide is recognized and cleaved by a signal peptidase to produce an immune checkpoint inhibitor peptide, which is trafficked via the Golgi network and subsequently secreted from the cell via the classical secretory pathway.

[0109] Suitable secretory signals for use in the present invention may be naturally occurring secretory signals, consensus secretory signals (see, for example, U.S. Patent Application Publication 2010 / 0305002, which is part of this specification by reference), or synthetic secretory signals.

[0110] In some embodiments, the secretion signal is selected from the peptide sequences in Table 2, or their homologs, derivatives, or fragments. In some embodiments, the secretion signal has a peptide sequence selected from SEQ ID NOs. 57 to 90, or a peptide having an amino acid sequence identical to those of SEQ ID NOs. 57 to 90 by at least 85%, 90%, 95%, 97%, or 99%.

[0111] In some embodiments, the secretory signal is derived from the human tissue plasminogen activator (tPA) secretory signal, or its homolog, derivative, or fragment. In some embodiments, the secretory signal peptide has the peptide sequence of SEQ ID NO: 65, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the secretory signal peptide has the peptide sequence of SEQ ID NO: 66, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. The tPA secretory signal has been found to be a particularly suitable secretory signal for use in the present invention to further enhance the expression of immune checkpoint inhibitor peptides.

[0112] [Table 2] TIFF0007911709000004.tif22170

[0113] In some embodiments, the secretory signal peptide of the first polypeptide encoded by the polycistronic nucleic acid insert further comprises the starting amino acid methionine (M).

[0114] Cuttable array In addition to the secretory signaling peptide at the N-terminus of each immune checkpoint inhibitor peptide, the polypeptide may also include a self-cleaving peptide fused to the C-terminus of the immune checkpoint inhibitor peptide. By providing a self-cleaving peptide sequence fused to the C-terminus of an immune checkpoint inhibitor peptide, multiple immune checkpoint inhibitor peptides can be cleaved into multiple monomers during or after translation. Suitable cleavage sequences are known in the art (see, for example, Donnelly et al., "Analysis of the aphthovirus 2A / 2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'." J. Gen. Virol. 82, 1013-1025 (2001) (which, by reference, is in whole part herein)).

[0115] In some embodiments, one or more immune checkpoint inhibitor chimeric polypeptides include one or more peptide sequences fused to the C-terminus of an immune checkpoint inhibitor peptide that can be cleaved during or after translation of a polycistronic nucleic acid, or in combination thereof (see, for example, Figures 3A, 3B, and 3C). In some embodiments, the most C-terminal immune checkpoint inhibitor chimeric polypeptide does not contain a cleavable peptide.

[0116] In some embodiments, the cleavable peptide can be cleaved by a proprotein convertase enzyme, including, but not limited to, furin or furin-like proprotein convertase (Table 3). In some embodiments, the cleavable peptide sequence includes a basic amino acid target sequence (typically RX(R / K)R) (where X = any amino acid) (SEQ ID NO: 91). In some embodiments, the cleavable peptide sequence includes a basic amino acid target sequence (typically RX(R / K)R) (where X = R, K, or H) (SEQ ID NO: 92). In some embodiments, the cleavable peptide sequence is RAKR (SEQ ID NO: 93). In some embodiments, the cleavable peptide sequence is RRRR (SEQ ID NO: 94). In some embodiments, the cleavable peptide is RKRR (SEQ ID NO: 95). In some embodiments, the cleavable peptide is RRKR (SEQ ID NO: 96). In some embodiments, the cleavable peptide is RKKR (SEQ ID NO: 97). By including cleavable peptide sequences in each covalently linked chimeric polypeptide, the multimeric polypeptide expressed during translation of a polycistronic nucleic acid insert can be processed post-translation into monomeric chimeric polypeptides via a cleavage mechanism. This allows each chimeric polypeptide, containing an immune checkpoint inhibitor peptide, to be secreted from the cell and function to downmodulate unwanted immune checkpoint pathways (see, for example, Figure 3A).

[0117] [Table 3]

[0118] In some embodiments, each chimeric polypeptide comprises one or more peptide sequences fused to the C-terminus of an immune checkpoint inhibitor peptide that can induce ribozyme skipping during translation of a polycistronic nucleic acid. Ribosome “skipping” is an alternative translation mechanism in which a particular peptide sequence prevents the ribosome from covalently linking a newly inserted amino acid, but translation still continues. This results in polyprotein “cleavage” by induction of ribosome skipping (see, for example, Figure 3B).

[0119] In some embodiments, the peptide capable of inducing ribosome skipping is a cis-acting hydrolase element peptide (CHYSEL). In some embodiments, the CHYSEL sequence comprises a non-conserved amino acid sequence with a strong α-helix orientation, followed by a consensus sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98), where ribosome skipping cleavage occurs between the G sequence and the P sequence. In some embodiments, the CHYSEL sequence comprises DVEENPGP (SEQ ID NO: 99).

[0120] In some embodiments, the CHYSEL cleavage sequence is derived from one or more 2A self-processing peptides. The 2A sequence is an oligopeptide located between the P1 and P2 proteins in some members of the Viridae family, such as the Picornaviridae family, and can undergo autocleavage in eukaryotic cells to produce mature viral proteins P1 and P2 (Ahier et al., "Simultaneous expression of multiple proteins under a single promoter in Caenorhabditis elegans via a versatile 2A-based toolkit." Genetics. 2014;196:605-613; Luke et al., "Occurrence, function and evolutionary origins of '2A-like' sequences in virus genomes." J Gen Virol. 2008 Apr;89(Pt 4):1036-42; Doronina et al.) See "Dissection of a co-translational nascent chain separation event" by al., Biochem Soc Trans. 2008 Aug;36(Pt 4):712-6, and "A Model for Nonstoichiometric, Cotranslational Protein Scission in Eukaryotic Ribosomes" by Martin et al., Bioorganic Chemistry, Volume 27, Issue 1, February 1999, 55-79).The first 2A virus discovered was F2A (foot-and-mouth disease virus), and later E2A (equine rhinitis A virus), P2A (porcine tesiovirus-1 2A), and T2A (thosea asigna virus 2A) were also identified (Ryan et al., "Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence," The Journal of general virology. 1991;72(Pt 11):2727-2732; Szymczak et al., "Development of 2A peptide-based strategies in the design of multicistronic vectors," Expert opinion on biological therapy. 2005;5:627-638).

[0121] In some embodiments, the CHYSEL cleavage sequence is derived from one or more 2A self-processing peptides provided in Table 4, or from peptides having amino acid sequences identical to them by at least 85%, 90%, 95%, 97%, or 99%.

[0122] [Table 4]

[0123] In some embodiments, the cleavage sequence is an amino acid sequence derived from foot-and-mouth disease virus (FMDV), for example, VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 118), or a 2A cleavage sequence derived from a peptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the 2A cleavage sequence is a 2A cleavage sequence or 2A-like cleavage sequence selected from GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 119), GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 120), GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 121), or GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 122), or a peptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In certain embodiments, the 2A-like cleavage sequence is GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 120), or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0124] [Table 5]

[0125] In some embodiments, the cleavable peptide sequence includes two or more sequences that can be cleaved by different mechanisms, for example, a cleavable peptide sequence that can be cleaved after translation of a polycistronic nucleic acid and a peptide sequence that can induce ribozyme skipping during translation of a polycistronic nucleic acid. By providing cleavable peptide sequences that follow a number of cleavage schemes, the efficiency of monomer formation from polycistronic nucleic acids can be improved. In some embodiments, the immune checkpoint inhibitor peptide is fused at its C-terminus to a furin-cleavable peptide sequence, for example, the peptide sequence RX(R / K)R (where X = any amino acid) (SEQ ID NO: 91), and the peptide fused at the C-terminus of the furin-cleavable peptide sequence contains a CHYSEL peptide sequence, for example, the amino acid sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98). By including a furin-cleavable peptide sequence such as RAKR (SEQ ID NO: 93) fused to the N-terminus of the CHYSEL peptide sequence between each chimeric polypeptide, the transcribed polycistronic nucleic acid undergoes ribozyme skipping during translation, resulting in the production of monomeric chimeric polypeptides, with all but the arginine (R) and alanine (A) residues of the furin-cleavable sequence remaining at the C-terminus of the immune checkpoint inhibitor peptide, thus limiting the potential interference of the extra amino acid sequence to the function of the immune checkpoint inhibitor peptide (see, for example, Figure 3C). In an alternative embodiment including a furin-cleavable peptide sequence such as RRRR (SEQ ID NO: 94), RKRR (SEQ ID NO: 95), or RRKR (SEQ ID NO: 96) fused to the N-terminus of the CHYSEL peptide sequence between each chimeric polypeptide, the transcribed polycistronic nucleic acid undergoes ribozyme skipping during translation, resulting in the production of monomeric chimeric polypeptides, with the remaining furin-cleavable sequence and CHYSEL peptide sequence removed at the C-terminus of the immune checkpoint inhibitor peptide.

[0126] In some embodiments, the hybrid cleavable peptide sequence includes RAKR (SEQ ID NO: 93) fused to CHYSEL containing the amino acid sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98). In some embodiments, the hybrid cleavable peptide sequence includes RAKR (SEQ ID NO: 93) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 100 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RAKR (SEQ ID NO: 93) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RAKR (SEQ ID NO: 93) fused to the CHYSEL amino acid sequence of SEQ ID NO: 120, or a peptide having an amino acid sequence identical to it by at least 85%, 90%, 95%, 97%, or 99%. In a particular embodiment, the hybrid cleavable peptide is RAKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 123).

[0127] In some embodiments, the hybrid cleavable peptide sequence includes RRRR (SEQ ID NO: 94) fused to a CHYSEL containing the amino acid sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98). In some embodiments, the hybrid cleavable peptide sequence includes RRRR (SEQ ID NO: 94) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 100 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RRRR (SEQ ID NO: 93) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RRRR (SEQ ID NO: 94) fused to the CHYSEL amino acid sequence of SEQ ID NO: 120, or to a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In a particular embodiment, the hybrid cleavable peptide is RRRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 124).

[0128] In some embodiments, the hybrid cleavable peptide sequence includes RKRR (SEQ ID NO: 95) fused to CHYSEL containing the amino acid sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98). In some embodiments, the hybrid cleavable peptide sequence includes RKRR (SEQ ID NO: 95) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 100 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RKRR (SEQ ID NO: 95) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RKRR (SEQ ID NO: 95) fused to the CHYSEL amino acid sequence of SEQ ID NO: 120, or to a peptide having an amino acid sequence identical to it by at least 85%, 90%, 95%, 97%, or 99%. In a particular embodiment, the hybrid cleavable peptide is RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 125).

[0129] In some embodiments, the hybrid cleavable peptide sequence includes RRKR (SEQ ID NO: 96) fused to CHYSEL containing the amino acid sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98) (Table 6). In some embodiments, the hybrid cleavable peptide sequence includes RRKR (SEQ ID NO: 96) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 100 to 123, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RRKR (SEQ ID NO: 96) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RRKR (SEQ ID NO: 96) fused to the CHYSEL amino acid sequence of SEQ ID NO: 120, or a peptide having an amino acid sequence identical to it by at least 85%, 90%, 95%, 97%, or 99%. In certain embodiments, the hybrid cleavable peptide is RRKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 126).

[0130] In some embodiments, the hybrid cleavable peptide sequence includes RKKR (SEQ ID NO: 97) fused to CHYSEL containing the amino acid sequence D(V / I)EXNPGP (where X = any amino acid) (SEQ ID NO: 98). In some embodiments, the hybrid cleavable peptide sequence includes RKKR (SEQ ID NO: 97) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 100 to 123, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RKKR (SEQ ID NO: 97) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 122, or to a peptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the hybrid cleavable peptide sequence includes RKKR (SEQ ID NO: 97) fused to the CHYSEL amino acid sequence of SEQ ID NO: 120, or a peptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In certain embodiments, the hybrid cleavable peptide is RKKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 127).

[0131] [Table 6]

[0132] Regulatory array As provided herein, immune checkpoint inhibitor peptides are expressed from nucleic acid sequences inserted at appropriate locations within the MVA genome sequence. For the expression of nucleic acid inserts within the rMVA genome backbone, regulatory sequences such as promoters required for the transcription of polycistronic nucleic acids encoding polyproteins must be located in the 5' region of the nucleic acid insert, adjacent to the transcription start site. If the nucleic acid insert is a polycistronic nucleic acid encoding multiple proteins / peptides as a single polyprotein, one or more promoters may be located 5' relative to the transcription start site of the ORF encoding the most N-terminal polypeptide of the polyprotein.

[0133] Since MVA is a cytoplasmic virus, in some embodiments, suitable promoters include promoters derived from naturally occurring poxvirus promoters. Poxvirus genes, promoters, and transcription factors are classified into early, mid-stage, and late-stage classes according to the timing of their expression during poxvirus infection (see, for example, "Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes." by Assarsson et al., PNAS 2008;105(6):2140-2145, and "Genome-wide analysis of the 5' and 3' ends of vaccinia virus early mRNA delineates regulatory sequences of annotated and anomalous transcripts." by Yang Zet al., J Virol. 2011;85(12):5897-5909). In most mammalian cells (non-permissive cells), MVA replication stops after all stages of expression have occurred, at the point of virion aggregation.This supports the usefulness of all promoter classes, including late promoters that control transgene expression (Sancho et al., "The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis." J Virol. 2002;76(16):8318-8334; Geiben-Lynn et al., "Kinetics of recombinant adenovirus type 5, vaccinia virus, modified vaccinia ankara virus, and DNA antigen expression in vivo and the induction of memory T-lymphocyte responses." Clin Vaccine Immunol.) (2008;15(4):691-696). Some poxvirus promoters possess both early and late elements, allowing their open reading frames (ORFs) or recombinant antigens to be expressed in the early stages of viral infection and in the later stages after viral genome replication, respectively ("Vaccinia virus transcription" by Broyles SS, J Gen Virol. 2003;84(Pt 9):2293-2303).The poxvirus promoter can be used across strains (see "Comparative analysis of vaccinia virus promoter activity in fowlpox and vaccinia virus recombinants" by Prideaux et al., Virus Res. 1990;16(1):43-57, and "Regulation of foreign gene in fowlpox virus by a vaccinia virus promoter" by Tripathy et al., Avian Dis. 1990;34(1):218-220).

[0134] Such MVA promoter sequences are known to those skilled in the art, for example, the p11 promoter that drives the expression of the 11k protein encoded by the F17R ORF (Wittek et al., "Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome," J Virol. 1984;49(2):371-378), the p7.5 promoter (Cochran et al., "In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals," J Virol. 1985;54(1):30-37), and the pI1L promoter (Schmitt et al., "Sequence and transcriptional analysis of the vaccinia virus HindIII I fragment"). (virus HindIII I fragment.)" J Virol. 1988;62(6):1889-1897), pTK promoter (Weir and Moss, "Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase." Virology. 1987;158(1):206-210), pF7L promoter (Coupar et al.References include: "Effect of in vitro mutations in a vaccinia virus early promoter region monitored by herpes simplex virus thymidine kinase expression in recombinant vaccinia virus" (J Gen Virol. 1987;68(Pt 9):2299-2309), pH5 promoter ("Cloning and expression of foreign genes in vaccinia virus, using a host range selection system" by Perkus et al., J Virol. 1989;63(9):3829-3836), and short synthetic promoter pSyn ("Compact, synthetic, vaccinia virus early / late promoter for protein expression" by Chakrabarti et al., Biotechniques. 1997;23(6):1094-1097, "A synthetic vaccinia virus promoter with enhanced early and late activity." by Hammond et al. J Virol Methods. 1997;66(1):135-1380, pmH5 promoter (Wyatt et al.)The following are references to the following publications: "Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model," Vaccine. 1996;14(15):1451-1458; the pHyb promoter (Sancho et al., "The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis," J Virol. 2002;76(16):8318-8334); and the LEO promoter (Wyatt et al., "Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccine"). (Vaccinia virus Ankara HIV vaccines.) Vaccine. 2008;26(4):486-493), pB8 promoter (Orubu et al., "Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA." PLoS One.)Examples include the pF11 promoter (Orubu et al., "Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA," PLoS One. 2012;7(6):e40167). In some embodiments, the promoter is selected from one or more of pMH5, p11, pSyn, and pHyb, or a combination thereof.

[0135] In some embodiments, the promoter is the pH5 promoter AAAAAATGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAA (SEQ ID NO: 128), or a nucleic acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter is the pH5 promoter AAAAAATGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATT (SEQ ID NO: 129), or a nucleic acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0136] In some embodiments, the promoter is the modified pH5 promoter (pmH5) AAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAA (SEQ ID NO: 130), or a nucleic acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter is the modified pH5 promoter (pmH5) AAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATA (SEQ ID NO: 131), or a nucleic acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter is the modified pH5 promoter (pmH5)AAAAAATGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATA (SEQ ID NO: 132), or a nucleic acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0137] Additional vaccinia virus promoters that may be particularly suitable as promoters in the present invention include, for example, the innate promoter sequences shown in Table 7 below, or those derived from nucleic acid sequences that are at least 85%, 90%, 95%, 97%, or 99% identical thereto, where the nomenclature for the loci is based on the ORF nomenclature originally used for the WR and Copenhagen strains of the vaccine virus. In some embodiments, the promoter is selected from one or more of SEQ ID NOs: 133 to 308, or a combination thereof, or from nucleic acid sequences that are at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0138] [Table 7] TIFF0007911709000010.tif247170TIFF0007911709000011.tif247170TIFF00079117090 00012.tif247170TIFF0007911709000013.tif247170TIFF0007911709000014.tif110170

[0139] Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence (GCCACC / ATG).

[0140] Furthermore, the polycistronic nucleic acid sequence for insertion may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination or repeat thereof, at the 3' end of the nucleic acid sequence following the last amino acid coding sequence of the polypeptide. Additionally, the nucleic acid sequence may include a vaccinia virus termination sequence at the 3' end of the last stop codon of the polyprotein. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for constructing shuttle vectors that facilitate the insertion of coding sequences for immune checkpoint inhibitors.

[0141] antigenic target The rMVA viral construct provided by the present invention can be used as an adjuvant to treat or prevent infectious diseases or cancer in subjects. In some embodiments, the rMVA viral construct is administered to subjects in need, such as humans, in a prophylactic vaccination protocol, for example, in the initial vaccination stage, the booster vaccination stage, or both the initial vaccination stage and the booster vaccination stage, to prevent infectious diseases. In alternative embodiments, the rMVA viral construct is administered to subjects in need, such as humans, in a therapeutic approach including a vaccination protocol, to treat, for example, cancer. Thus, the rMVA viral construct can be administered in conjunction with one or more antigens intended to induce an immune response against an antigenic target to induce partial or complete immunization in subjects in need.

[0142] Therefore, the rMVA of the present invention can be administered together with one or more antigens targeting infectious diseases or cancer. Examples of antigens and antigen delivery carriers that can be used together with rMVA as an adjuvant include antigenic proteins, polypeptides, or peptides or fragments thereof, nucleic acids encoding one or more antigens, such as mRNA or DNA, polysaccharides, or conjugates of polysaccharides and proteins, glycolipids, such as gangliosides, toxoids, subunits (e.g., subunits of viruses, bacteria, fungi, amoebas, parasites, etc.), virus-like particles, live viruses, split viruses, attenuated viruses, inactivated viruses, enveloped viruses, viral vectors expressing one or more antigens, tumor-associated antigens, or any combination thereof.

[0143] In a particular embodiment, the present invention provides a method for preventing or treating an infectious disease in a subject requiring prevention or treatment, comprising administering an effective amount of the rMVA of the present invention in combination with one or more antigens or antigen-expressing vectors in a prophylactic or therapeutic effective amount, alternately or in conjunction with them, wherein the rMVA enhances immunity against the targeted infectious disease.

[0144] In some embodiments, the target infections are viral infections including but not limited to double-stranded DNA viruses, including but not limited to adenoviruses, herpesviruses, and poxviruses; single-stranded DNA viruses, including but not limited to parvoviruses; double-stranded RNA viruses, including but not limited to reoviruses; positive-sound single-stranded RNA viruses, including but not limited to coronaviruses, picornaviruses, and togaviruses; negative-sound single-stranded RNA viruses, including but not limited to orthomyxoviruses and rhabdoviruses; single-stranded RNA retroviruses, including but not limited to retroviruses; or double-stranded DNA retroviruses, including but not limited to hepadnaviruses. In some embodiments, the target viruses include adenovirus, avian influenza virus, coxsackievirus, cytomegalovirus, dengue virus, Ebola virus, Epstein-Barr virus, equine encephalitis virus, flavivirus, hepadnavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus, human immunodeficiency virus, human papillomavirus, influenza virus, Japanese encephalitis virus, JC virus, measles virus, Marburg virus, Middle East Respiratory Syndrome (MERS-CoV) coronavirus, and mumps sulfa virus. These include orthomyxovirus, papillomavirus, parainfluenza virus, parvovirus, picornavirus, poliovirus, poxvirus, rabies virus, reovirus, respiratory syncytial virus, retrovirus, rhabdovirus, rhinovirus, Rift Valley fever virus, rotavirus, rubella virus, measles virus, severe acute respiratory syndrome coronavirus type 1 (SARS-CoV), severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), smallpox virus, togavirus, swine influenza virus, varicella-zoster virus, varicella virus, varicella minor virus, and yellow fever virus.Examples of viruses that can be used as antigens include measles virus, mumps virus (mumpsulbra virus), rubella virus, varicella-zoster virus, or all four or any combination of three of these (e.g., measles virus, mumps virus, and rubella virus).

[0145] In some embodiments, the target infectious agents are Flaviviridae viruses, including infections caused by viruses of the genera Flavivirus and Pestivirus. Examples of Flavivirus infections include dengue fever, Kasanul Forest disease, Poissant disease, Wesselsbron disease, West Nile fever, yellow fever, Zika virus, Rio Bravo, Rocio, Negishi, and encephalitis, including California encephalitis, Central European encephalitis, Ilheus virus, Murray Valley encephalitis, St. Louis encephalitis, Japanese encephalitis type B, jumping disease, and Russian spring-rodents summer encephalitis. Examples of Pestivirus infections include livestock diseases, mainly swine fever in pigs, BVDV (bovine viral diarrhea virus) infection in cattle, or Border disease virus infection.

[0146] In some embodiments, the target infectious agent is an Alphavirus virus, such as Eastern Equine Encephalitis (EEE) virus, Venezuelan Equine Encephalitis (VEE) virus, Western Equine Encephalitis (WEE) virus, Everglades virus, Chikungunya virus, Mayarovirus, Ockervo virus, Onyonnyon virus, Ross River virus, Semryki Forest virus, or Sindbis virus (SINV).

[0147] In some embodiments, the target infectious agent is equine arteritis virus, bovine viral diarrhea virus (BVDV), Hog's cholera virus, or border disease virus. The only member of the rubivirus genus is rubella virus.

[0148] In some embodiments, the target infectious agents are filoviridae viruses such as Ebola virus and Marburg virus, paramyxoviridae viruses such as measles virus, mumps virus, Nipah virus, Hendra virus, respiratory syncytial virus (RSV), and Newcastle disease virus (NDV), rhabdoviridae viruses such as rabies virus, nyamiviridae viruses such as Niavirus, arenaviridae viruses such as Lassa virus, hantavirus, bunyaviridae viruses such as Crimean-Congo hemorrhagic fever virus, or ophioviridae viruses and orthomyxoviridae viruses such as influenza virus.

[0149] In one embodiment, the antigens include multiple species of the genus Borrelia, Bacillus ancilasis, Borrelia burgdorferi, Bordetella partsis, Campylobacter jejuni, multiple species of the genus Chlamydia, Chlamydia psittaci, Chlamydia trachomatis, multiple species of the genus Clostridium, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Corynebacterium diphtheriae, and multiple species of the genus Coxiella. Several species, multiple species of the genus Enterococcus, multiple species of the genus Ehrlichia, Escherichia coli, Francisella tularensis, multiple species of the genus Haemophilus, Haemophilus influenzae, Haemophilus parainfluenzae, multiple species of the genus Lactobacillus, multiple species of the genus Legionella, Legionella pneumophila, Leptospirosis interlogans, multiple species of the genus Listeria, Listeria monocytogenes, multiple species of the genus Mycobacterium, Mycobacterium Mycobacterium tubercurosis, Mycobacterium leprae, several species of Mycoplasma, Mycoplasma pneumoniae, several species of Neisseria, Neisseria meningitidis, Neisseria gonorea, several species of Pneumococcus, several species of Pseudomonas, Pseudomonas erginosa, several species of Salmonella, Salmonella ciffi, Salmonella enterica, several species of Streptococcus, several species of Rickettsia, Rickettsia The extracts are taken from one or more bacteria selected from Rickettsia, Rickettsia ciffii, several species of Sigella, several species of Staphylococcus, Staphylococcus aureus, several species of Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, several species of Treponema, Treponema parrhysm, several species of Vibrio, Vibrio cholerae, and Yersinia pestis. Such bacteria may be whole cells (e.g., living, attenuated, or inactivated), or polypeptides or polysaccharides of such bacteria.

[0150] In some embodiments, the target infectious agent is a bacterium. The target antigenic bacterial factor may be a polysaccharide-polypeptide antigen, such as a pneumococcus (e.g., S. pneumonia) polysaccharide (e.g., cell capsule sugars)-protein (e.g., diphtheria protein) conjugate. In some embodiments, the conjugate comprises S. pneumonia cell-capsulation sugars conjugated to a protein (e.g., diphtheria protein), for example, the cell capsule sugars are from seven serotypes of S. pneumoniae (4, 6B, 9V, 14, 18C, 19F, and 23F), which are conjugated with the diphtheria protein. In some embodiments, the conjugate comprises Streptococcus pneumoniae polysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F conjugated to proteins such as protein D derived from unclassifiable Haemophilus influenza, tetanus toxoid carrier protein, and / or diphtheria toxoid carrier protein. In some embodiments, the conjugate comprises Streptococcus pneumoniae capsular polysaccharides of serotypes 1, 3, 4, 5, 6a, 6b, 7f, 9v, 14, 18c, 23f, 19a, and 19f conjugated to diphtheria protein, for example, diphtheria crm197 protein.In some embodiments, one or more polysaccharide-protein conjugates are conjugated to one or more carrier proteins of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 11E, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 1 Contains capsular polysaccharides derived from at least one of 8A, 18B, 18C, 19F, 19A, 19B, 19C, 20A, 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 33E, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2, and CWPS3.

[0151] In some embodiments, the target infectious agent is one or more fungi selected from, for example, multiple species of the genera Aspergillus, Candida, Candida albicans, Candida tropicalis, multiple species of the genera Cryptococcus, Cryptococcus neoformans, Entamoeba historica, Histoplasma capsulatum, multiple species of the genera Leishmania, Nocardia asteroides, Plasmodium falsiparum, Toxoplasma gondii, Trichomonas vaginalis, multiple species of the genera Toxoplasma, Trypanosoma bursei, Cystosoma mansoni, multiple species of the genera Fusarium, and / or multiple species of the genera Trichophyton. Such fungi may be whole cells (e.g., living, attenuated, or inactivated), or polypeptides or polysaccharides of such fungi.

[0152] In some embodiments, the target infectious agent is one or more parasites selected from multiple species of the genera Plasmodium, Toxoplasma, Entomoeba, Babesia, Trypanosoma, Leishmania, Pneumocystis, Trichomonas, Giardia, and / or Cystosoma. Such parasitic antigens may be whole cells (e.g., living, attenuated, or inactivated) or polypeptides or polysaccharides of such parasites.

[0153] In some embodiments, the antigenic agent is encoded by a nucleic acid. For example, in some embodiments, the antigenic agent is encoded by a nucleic acid selected from DNA, RNA, mRNA, etc.

[0154] In some embodiments, the antigen is a toxoid. In some embodiments, the toxoid is diphtheria toxoid, tetanus toxoid, or a toxoid derived from C. difficile.

[0155] In certain embodiments, the target antigens include Ebola virus, e.g., the envelope glycoprotein of Ebola virus Zaire strain (e.g., UniProtKB-P87671(VGP_EBOEC)), the matrix protein VP40 of Ebola virus Zaire strain (e.g., UniProtKB-Q05128(VP40_EBOZM)), or the matrix protein of Ebola virus Sudan strain (e.g., UniProtKB-Q7T9D9(VGP_EBOSU)), Lassa virus, e.g., protein Z (e.g., UniProtKB-O73557(Z_LASSJ)), Zika virus, e.g., non-structural protein 1 (NSP-1), Marburg virus, e.g., Marburg virus glycoprotein (GenBank accession number A It is derived from FV31202.1), Marburg virus VP40 matrix protein (GenBank accession number JX458834), parasites of the genus Plasmodium, e.g., Plasmodium falsiparum, e.g., perisporozoite protein (CSP), male germ surface protein P230p (Pfs230 antigen), sporozoite microname protein (SPECT2) essential for cell translocation, or GTP-binding protein, putative antigen (GenBank accession number PF3D7_1462300), human immunodeficiency virus, e.g., Env protein, e.g., gp41, gp120, gp160, Gag protein, MA, CA, SP1, NC, SP2, P6, or Pol protein RT, RNase H, IN, PR.

[0156] In an alternative embodiment, the rMVA viral construct is administered to a subject requiring administration, such as a human, in a therapeutic setting including a vaccination protocol, for example, to treat cancer. Thus, the rMVA viral construct can be administered in conjunction with one or more antigens intended to induce an immune response against an antigenic target to induce partial or complete immunization in a subject requiring induction.

[0157] Antigens used in cancer immunotherapy are generally selected intentionally based on one of the following criteria: their uniqueness to tumor cells, higher expression in tumor cells compared to normal cells, or the potential for normal cells expressing the antigen to be adversely affected without posing a significant risk to normal cells or tissues. Tumor-associated antigens (TAAs) can be broadly classified into carcinoembryonic TAAs (typically expressed only in fetal tissues and cancerous somatic cells), oncoviral TAAs (encoded by tumorigenic transforming viruses), overexpressed / accumulated TAAs (expressed in both normal and neoplastic tissues, with significantly increased expression levels in neoplasms), cancer-testicular TAAs (expressed only in cancer cells and adult reproductive tissues such as the testes and placenta), lineage-restricted TAAs (primarily expressed by a single cancer histological type), mutated TAAs (expressed only by cancer as a result of gene mutations or transcriptional changes), post-translational modified TAAs (tumor-associated changes such as glycosylation), or idiotype TAAs (highly polymorphic genes similar to those in B-cell and T-cell lymphomas / leukemias, resulting from clonal abnormalities). TAAs are preferentially expressed by tumor cells, but are also frequently found in normal tissues. However, their expression differs from that in normal tissue in terms of the degree of their expression in tumors, the changes in their protein structure compared to normal counterparts, or their abnormal intracellular localization within malignant or tumor cells.

[0158] Examples of carcinoembryonic tumor-associated antigens include carcinoembryonic antigen (CEA), immature laminin receptor, and tumor-associated glycoprotein (TAG) 72. Examples of overexpressed / accumulated tumor-associated antigens include BING-4, calcium-activated chloride channel (CLCA) 2, cyclin A1, cyclin B1, 9D7, epithelial cell adhesion molecule (Ep-Cam), EphA3, Her2 / neu, telomerase, mesothelin, orphan tyrosine kinase receptor (ROR1), gastric cancer-associated protein tyrosine phosphatase 1 (SAP-1), and Survivin.

[0159] Examples of cancer-testicular antigens include the b melanoma antigen (BAGE) family, cancer-associated gene (CAGE) family, G antigen (GAGE) family, melanoma antigen (MAGE) family, sarcoma antigen (SAGE) family, and X antigen (XAGE) family, CT9, CT10, NY-ESO-1, L antigen (LAGE)1, melanoma antigen (PRAME) which is preferentially expressed in tumors, and synovial sarcoma X (SSX)2. Examples of lineage-specific tumor antigens include melanoma antigen-1 / 2 (Melan-A / MART-1 / 2) recognized by T cells, Gp100 / pmel17, tyrosine-related protein (TRP)1 and TRP2, P. polypeptide, melanocortin 1 receptor (MC1R), and prostate-specific antigen. Examples of mutated tumor antigens include β-catenin, breast cancer antigen (BRCA) 1 / 2, cyclin-dependent kinase (CDK) 4, chronic myeloid leukemia antigen (CML) 66, fibronectin, p53, Ras, and TGF-βRII. An example of a post-translational modified tumor antigen is mucin (MUC) 1. Examples of idiotype tumor antigens include immunoglobulin (Ig) and T cell receptor (TCR).

[0160] In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of CD19, CD20, CD22, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, ErbB3, or ErbB4, FBP, fetal acetylcholine receptor, HMW-MAA, IL-22R-alpha, IL-13R-alpha, kdr, kappa light chain, Lewis Y, MUC16 (CA-125), PSCA, NKG2D ligand, carcinoembryonic antigen, VEGF-R2, PSMA, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0161] Exemplary tumor antigens include, at a minimum, carcinoembryonic antigen (CEA) for colon cancer, CA-125 for ovarian cancer, MUC1 or epithelial tumor antigen (ETA) or CA15-3 for breast cancer, tyrosinase or melanoma-associated antigen (MAGE) for malignant melanoma, as well as ras, p53 abnormal products for various types of tumors, alpha-fetoprotein for liver cancer, ovarian cancer, or testicular cancer, the β-subunit of hCG for men with testicular cancer, prostate-specific antigen for prostate cancer, β2-microglobulin for multiple myeloma and certain lymphomas, CA19-9 for colorectal cancer, cholangiocarcinoma, and pancreatic cancer, chromogranin A for lung cancer and prostate cancer, melanoma, soft tissue sarcoma, and TA90 for breast cancer, colon cancer, and lung cancer. Examples of TAAs in this field are known, for example, in "Human Tumor Antigens and Cancer Immunotherapy" by N. Vigneron, BioMed Research International, vol. 2015, Article ID 948501, 17 pages, 2015. doi:10.1155 / 2015 / 948501; Ilyas et al., J Immunol. (2015) Dec 1; 195(11): 5117-5122; Coulie et al., Nature Reviews Cancer (2014) volume 14, pages 135-146; and Cheever et al., Clin Cancer Res. (2009) Sep 1;15(17):5323-37 (these are cited and their entirety constitutes part of this specification).

[0162] Examples of tumor viral TAAs include human papillomavirus (HPV) L1, E6, and E7; Epstein-Barr virus (EBV) Epstein-Barr nuclear antigen (EBNA) 1 and EBNA2; EBV capsid antigen (VCA) Igm or IgG; EBV initial antigen (EA); latent membrane protein (LMP) 1 and LMP2; hepatitis B surface antigen (HBsAg); hepatitis B e antigen (HBeAg); hepatitis B core antigen (HBcAg); hepatitis B x antigen (HBxAg); hepatitis C core antigen (HCV core Ag); human T-lymphotropic virus type 1 core antigen (HTLV-1 core antigen); HTLV-1 Tax antigen; HTLV-1 group-specific (Gag) antigen; HTLV-1 envelope (Env); H Examples include the protease antigen (Pro) of TLV-1, Tof of HTLV-1, Rof of HTLV-1, polymerase (Pro) antigen of HTLV-1, core antigen of human T lymphotropic virus type 2 (HTLV-2 core antigen), Tax antigen of HTLV-2, group-specific (Gag) antigen of HTLV-2, envelope (Env) of HTLV-2, protease antigen (Pro) of HTLV-2, Tof of HTLV-2, Rof of HTLV-2, polymerase (Pro) antigen of HTLV-2, latent-associated nuclear antigen (LANA), K8.1 of human herpesvirus-8 (HHV-8), large T antigen (LTAg) of Merkel cell polyomavirus, and small T antigen (sTAg) of Merkel cell polyomavirus.

[0163] Increased expression of certain types of glycolipids, such as gangliosides, is associated with enhanced tumor survival in certain types of cancer. Examples of gangliosides include GM1b, GD1c, GM3, GM2, GM1a, GD1a, GT1a, GD3, GD2, GD1b, GT1b, GQ1b, GT3, GT2, GT1c, GQ1c, and GP1c. Examples of ganglioside derivatives include 9-O-Ac-GD3, 9-O-Ac-GD2, 5-N-de-GM3, N-glycolylGM3, NeuGcGM3, and fucosyl-GM1. Exemplary gangliosides that are often present at higher levels in tumors, such as melanoma, small cell lung cancer, sarcoma, and neuroblastoma, include GD3, GM2, and GD2.

[0164] In addition to the TAAs described above, another class of TAAs is tumor-specific neoantigens, which arise through mutations that alter the amino acid coding sequence (non-synonymous somatic mutations). Some of these mutant peptides are expressed, processed, and presented on the cell surface and subsequently recognized by T cells. Since normal tissues do not possess these somatic mutations, neoantigen-specific T cells do not undergo central or peripheral immune tolerance and also lack the ability to induce the destruction of normal tissues. See, for example, Lu & Robins, "Cancer Immunotherapy Targeting Neoantigens," Seminars in Immunology, Volume 28, Issue 1, February 2016, Pages 22-27 (which is incorporated herein by reference).

[0165] In some embodiments, the TAA is specific to a carcinoembryonic TAA selected from the group consisting of carcinoembryonic antigen (CEA), immature laminin receptor, orphan tyrosine kinase receptor (ROR1), and tumor-associated glycoprotein (TAG) 72.

[0166] In some embodiments, the TAA is specific to an oncoviral TAA selected from the group consisting of human papillomavirus (HPV) E6 and E7, Epstein-Barr nuclear antigen (EBNA)1 and EBNA2, and latent membrane protein (LMP)1 and LMP2 of Epstein-Barr virus (EBV).

[0167] In some embodiments, TAA is specific to overexpressed / accumulated TAA selected from the group consisting of BING-4, calcium-activated chloride channel (CLCA)2, cyclin A1, cyclin B1, 9D7, epithelial cell adhesion molecule (Ep-Cam), EphA3, Her2 / neu, L1 cell adhesion molecule (L1-Cam), telomerase, mesothelin, gastric cancer-associated protein tyrosine phosphatase 1 (SAP-1), and survivorbin.

[0168] In some embodiments, TAA includes the b melanoma antigen (BAGE) family, cancer-associated gene (CAGE) family, G antigen (GAGE) family, melanoma antigen (MAGE) family, sarcoma antigen (SAGE) family and X antigen (XAGE) family, cutaneous T-cell lymphoma-associated antigen family (cTAGE), interleukin-13 receptor subunit α-1 (IL13RA), CT9, putative tumor antigen NA88-A, leucine zipper protein 4 (LUZP4), NY-ESO-1, L antigen (LAGE) 1, and It is specific to cancer-testis antigens selected from the group consisting of lycase antigen (HAGE), lipase I (LIPI), melanoma antigen (PRAME) which is preferentially expressed in tumors, synovial sarcoma X (SSX) family, nucleus-associated sperm protein (SPANX) family on the X chromosome, cancer / testis antigen 2 (CTAG2), calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein (CABYR), acrosin-binding protein (ACRBP), centrosome protein 55 (CEP55), and synaptonema complex protein 1 (SYCP1).

[0169] In some embodiments, TAAs are specific to lineage-limited tumor antigens selected from the group consisting of melanoma antigen-1 / 2 (Melan-A / MART-1 / 2), Gp100 / pmel17, tyrosinase, tyrosine-related protein (TRP)1 and TRP2, P. polypeptide, melanocortin 1 receptor (MC1R), and prostate-specific antigens, which are recognized by T cells.

[0170] In some embodiments, TAA is specific to a mutated TAA selected from the group consisting of β-catenin, breast cancer antigen (BRCA) 1 / 2, cyclin-dependent kinase (CDK) 4, chronic myeloid leukemia antigen (CML) 66, fibronectin, MART-2, p53, Ras, TGF-βRII, and partial epidermal growth factor (tEGFR).

[0171] In some embodiments, TAA is specific to post-translationally modified TAA mucin (MUC) 1.

[0172] In some embodiments, the TAA is specific to an idiotype TAA selected from the group consisting of immunoglobulin (Ig) and T cell receptor (TCR).

[0173] In some embodiments, TAAs are specific to BCMAs. In some embodiments, at least one T cell subpopulation is specific to BCMAs.

[0174] In some embodiments, TAA is specific to CS1.

[0175] In some embodiments, TAA is specific to XBP-1.

[0176] In some embodiments, TAA is specific to CD138.

[0177] In some embodiments, TAA is specific to WT1, PRAME, Survivin, NY-ESO-1, MAGE-A3, MAGE-A4, Pr3, Cyclin A1, SSX2, Neutrophil elastase (NE), HPV E6, HPV E7, EBV LMP1, EBV LMP2, EBV EBNA1, or EBV EBNA2.

[0178] In addition to the TAAs described above, another class of TAAs is tumor-specific neoantigens, which arise through mutations that alter the amino acid coding sequence (non-synonymous somatic mutations). Some of these mutant peptides are expressed, processed, and presented on the cell surface and subsequently recognized by T cells. Since normal tissues do not possess these somatic mutations, neoantigen-specific T cells do not undergo central or peripheral immune tolerance and also lack the ability to induce the destruction of normal tissues. See, for example, Lu & Robins, "Cancer Immunotherapy Targeting Neoantigens," Seminars in Immunology, Volume 28, Issue 1, February 2016, Pages 22-27 (which is incorporated herein by reference).

[0179] In certain embodiments, TAA is derived from mucin 1 (MUC1) (UniProtKB-P15941(MUC1_HUMAN)). In some embodiments, TAA is derived from cyclin B1 (UniProtKB-P14635(CCNB1_HUMAN)).

[0180] rMVA virus vector Provided herein are rMVA viral vectors comprising heterologous nucleic acid inserts encoding immune checkpoint inhibitors that may be secreted from cells.

[0181] In some embodiments, the rMVA viral vector comprises a heterologous nucleic acid insert encoding a polypeptide, wherein the polypeptide is (M)(secretory signal peptide-immune checkpoint inhibitor) x (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine).

[0182] In some embodiments, the rMVA viral vector comprises a heterologous polycistronic nucleic acid insert encoding a polypeptide, wherein the polypeptide is a tandem repeat sequence (M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (where x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, FIGS. 1A and 1B).

[0183] In some embodiments herein, a heterologous polycistronic nucleic acid insert encoding one or more polypeptides in a tandem repeat sequence and an additional polypeptide fused to the C-terminus of the last polypeptide in the tandem repeat sequence ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (secretory signal peptide-immune checkpoint inhibitor peptide)) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) is provided for an rMVA viral vector (see, for example, FIGS. 2A and 2B). In certain embodiments, the encoded polypeptide is (M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (where x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10), or in another embodiment, ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x(Secretory signal peptide-immune checkpoint inhibitor peptide)) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine), wherein the secretory signal peptide is selected from peptides having an amino acid sequence selected from SEQ ID NO: 57 to SEQ ID NO: 90, the immune checkpoint inhibitor peptide is selected from peptides having an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 56, and the cleavable peptide is selected from peptides having an amino acid sequence selected from SEQ ID NO: 91 to SEQ ID NO: 127. In some embodiments, the secretory signal peptide is selected from peptides having an amino acid sequence selected from SEQ ID NO: 65 and SEQ ID NO: 66, the immune checkpoint inhibitor peptide is selected from peptides having an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 5, and the cleavable peptide is selected from peptides having an amino acid sequence selected from SEQ ID NO: 93 to SEQ ID NO: 97, SEQ ID NO: 120, and SEQ ID NO: 123 to SEQ ID NO: 127.

[0184] In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, and the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1.

[0185] In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x = 2 to 10. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x ≥ 4. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x = 4, 5, or 6.

[0186] In some embodiments, the secretory signaling peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, and the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5.

[0187] In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x = 2 to 10. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x ≥ 4. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x = 4, 5, or 6.

[0188] In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing the amino acids listed in Table 8 below, or a polypeptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequences of SEQ ID NOs. 309 to 340 or SEQ ID NOs. 341 to 348, or a polypeptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NOs. 309, or a polypeptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of it. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NOs. 310, or a polypeptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of it. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 3110, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 312, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 313, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%.In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 314, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 315, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 316, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 317, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 318, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 319, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 320, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 321, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%.In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 322, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 323, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 324, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 325, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 326, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 327, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 328, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 329, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%.In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 330, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 331, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 332, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 333, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 334, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 335, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 336, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 337, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%.In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 338, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 339, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 340, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 341, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 342, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 343, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 344, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 345, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%.In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 346, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing amino acids selected from the amino acid sequence of SEQ ID NO: 347, or a polypeptide having an amino acid sequence identical to that of at least 85%, 90%, 95%, 97%, or 99%. In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide comprising amino acids selected from the amino acid sequence of SEQ ID NO: 348, or a polypeptide having an amino acid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0189] [Table 8] TIFF0007911709000016.tif247170TIFF0007911709000017.tif246170TIFF0007911709 000018.tif247170TIFF0007911709000019.tif246170TIFF0007911709000020.tif98170

[0190] As provided herein, polycistronic nucleic acid inserts encoding immune checkpoint inhibitor polypeptides described herein may be inserted into the MVA genome at any suitable location, for example, at native deletion sites, modified native deletion sites, at non-essential MVA genes, such as the MVA thymidine kinase locus, or in intergeneric regions between essential or non-essential MVA genes. Suitable insertion sites are described, for example, in U.S. Patent No. 6,998,252, U.S. Patent No. 9,133,478, Ober et al., “Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara.” J. Virol., Aug. 2002 (pg. 7713-7723), U.S. Patent No. 9,133,480, and U.S. Patent No. 8,288,125 (each of which is incorporated herein by reference).

[0191] In some embodiments, polycistronic nucleic acid inserts encoding the immune checkpoint inhibitor polypeptides described herein are inserted into native deletion sites, e.g., deletion sites selected from native deletion sites I, II, III, IV, V, or VI; modified native deletion sites, e.g., the reconstructed and modified deletion site III between A50R and B1R of the MVA gene (see, e.g., U.S. Patent No. 9,133,480); between non-essential MVA genes; between essential MVA genes, e.g., between I8R and G1L or between A5R and A6L; or into other suitable insertion sites at non-essential loci, e.g., the TK locus of MVA; or a combination thereof.

[0192] In an alternative embodiment, the rMVA viral vector of the present invention can be further constructed to encode and express one or more antigenic peptides, in addition to its ability to express a number of immune checkpoint inhibitor peptides. The one or more antigenic peptides may be encoded on one or more separate nucleic acid inserts, or, in an alternative embodiment, the one or more antigenic peptides may be encoded on the same polycistronic nucleic acid insert as the number of immune checkpoint inhibitor peptides.

[0193] In some embodiments, as used herein, an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, wherein the polypeptide is ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x An rMVA viral vector is provided comprising (antigenic peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigenic peptide is a chimeric polypeptide comprising a secretion signal peptide fused to the N-terminus of the antigenic peptide, for example ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretionary signal peptide-antigenic peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, Figures 4A and 4B). In some embodiments, the antigenic peptide is also provided such that two or more antigenic peptides are encoded in a polycistronic nucleic acid insert, and each chimeric polypeptide is separated by a cleavable peptide as described herein. In some embodiments, the antigenic peptide is a chimeric polypeptide comprising a secretion signal peptide fused to the N-terminus of the antigenic peptide and a cleavable peptide fused to the C-terminus of the antigenic peptide, for example, ((M)(Secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretory signaling peptide - Antigenic peptide - Cleavable peptide) y)(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigen-containing chimeric polypeptide fused to the C-terminus of the last antigen-containing chimeric polypeptide does not contain a cleavable sequence, for example ((M)(secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretory signaling peptide - Antigenic peptide - Cleavable peptide) x (Secretionary signal peptide - Antigenic peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigenic peptide contained in a chimeric polypeptide comprising a secretion signal peptide fused to the N-terminus of the antigenic peptide and a cleavable peptide fused to the C-terminus of the antigenic peptide may be oriented in a polycistronic nucleic acid insert such that the nucleic acid encoding the antigen-containing chimeric polypeptide is located at 5' of the immune checkpoint inhibitor peptide-containing chimeric polypeptide, for example ((M)(Secretionary signal peptide - Antigenic peptide - Cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x ), or alternatively ((M)(secretory signaling peptide-antigenic peptide-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x (Secretionary signaling peptide - immune checkpoint inhibitor peptide) (where y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine).

[0194] In some embodiments, the antigenic peptide is a peptide derived from an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, an antigen derived from a tumor-associated antigen or a factor described in the section titled “Antigenic Targets” above, which explicitly forms part of this section.

[0195] In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide containing the antigenic amino acids of Table 9 below, or a polypeptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them. In some embodiments, the polycistronic nucleic acid insert encodes an antigen containing amino acids derived from an amino acid sequence selected from SEQ ID NOs. 349 to 396, SEQ ID NOs. 398, SEQ ID NOs. 400, SEQ ID NOs. 402, or SEQ ID NOs. 405, or a fragment thereof, or a polypeptide having an amino acid sequence identical to at least 85%, 90%, 95%, 97%, or 99% of them.

[0196] [Table 9] TIFF0007911709000022.tif254170TIFF0007911709000023.tif253170TIFF0007911709000024.tif252170TIFF0007911709000025.tif252170 TIFF0007911709000026.tif252170TIFF0007911709000027.tif252170TIFF0007911709000028.tif252170TIFF0007911709000029.tif252170 TIFF0007911709000030.tif252170TIFF0007911709000031.tif252170TIFF0007911709000032.tif252170TIFF0007911709000033.tif252170 TIFF0007911709000034.tif252170TIFF0007911709000035.tif252170TIFF0007911709000036.tif252170TIFF0007911709000037.tif162170

[0197] In some embodiments, any of the above-mentioned SEQ ID NOs. 349 to 395, or SEQ ID NO. 401, further comprises the amino acid residue methionine (M) as the first amino acid residue.

[0198] In some embodiments, the antigenic insert is derived from a tumor-associated antigen. In some embodiments, the antigenic insert is derived from human mucin-1 or a fragment thereof. In some embodiments, the antigenic insert is derived from an amino acid sequence or fragment thereof selected from SEQ ID NO: 349, SEQ ID NOs. 358-364, or SEQ ID NO: 403, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0199] In some embodiments, the antigenic insert is derived from the human cyclin B1 protein or a fragment thereof. In some embodiments, the antigenic insert is an amino acid sequence selected from SEQ ID NO: 350, or a fragment thereof, or is derived from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0200] In some embodiments, the antigenic insert is derived from the hepatitis B virus protein or a fragment thereof. In some embodiments, the antigenic insert is an amino acid sequence selected from SEQ ID NOs: 351 to 354, or a fragment thereof, or is derived from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0201] In some embodiments, the antigenic insert is derived from a protein of a species of the genus Plasmodium or a fragment thereof. In some embodiments, the antigenic insert is an amino acid sequence selected from SEQ ID NOs: 355 to 357, or a fragment thereof, or is derived from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0202] In some embodiments, the antigenic insert is derived from the Lassa virus protein or a fragment thereof. In some embodiments, the antigenic insert is an amino acid sequence selected from SEQ ID NO: 365 or SEQ ID NO: 366, or a fragment thereof, or is derived from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0203] In some embodiments, the antigenic insert is derived from the Ebola virus protein or a fragment thereof. In some embodiments, the antigenic insert is derived from an amino acid sequence selected from SEQ ID NO: 367 or SEQ ID NO: 368, or a fragment thereof, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0204] In some embodiments, the antigenic insert is derived from the Zika virus protein or a fragment thereof. In some embodiments, the antigenic insert is derived from an amino acid sequence selected from SEQ ID NOs. 369 to 376, or a fragment thereof, or from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0205] In some embodiments, the antigenic insert is one or more SARS-CoV-2 proteins or polypeptides, such as spike (S) (NCBI reference sequence YP_009724390), membrane (M) (NCBI reference sequence YP_009724393), envelope (E) (NCBI reference sequence YP_009724392), nucleoside (N) (NCBI reference sequence YP_009724397), ORF1AB (NCBI reference sequence YP_009724 The antigenic insert is derived from a protein or peptide derived from one or more of the following: 389), ORF3a (NCBI reference sequence YP_009724391), ORF6 (NCBI reference sequence YP_009724394), ORF7a (NCBI reference sequence YP_009724395), ORF7b (NCBI reference sequence YP_009725318), ORF8 (NCBI reference sequence YP_009724396), or ORF10 (NCBI reference sequence YP_009725255). In certain embodiments, the antigenic insert is derived from the SARS-CoV-2 S protein or a variant thereof. In some embodiments, the S protein is expressed as a full-length protein and contains one or more amino acid substitutions compared to the NCBI reference sequence YP_009724390. In some embodiments, the S protein is derived from the amino acid sequence of SEQ ID NO: 377, or a fragment thereof, or from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the S protein is expressed as a full-length protein and contains one or more substitutions selected from K417T, E484K, or N501Y of SEQ ID NO: 377. In some embodiments, the S protein is expressed as a full-length protein and contains the following substitutions: K417T, E484K, and N501Y of SEQ ID NO: 377. In some embodiments, the rMVA contains a nucleic acid sequence encoding the S protein, further containing substitutions at L452R, T478K, or P681R of SEQ ID NO: 377, or a combination thereof. In some embodiments, the rMVA contains a nucleic acid sequence encoding the S protein, further containing substitutions at L452R, T478K, and P681R of SEQ ID NO: 377.In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions of N440K, S443A, G476S, E484R, and / or G502P in SEQ ID NO: 377, or combinations thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions of one or more of T19R, G142D, R158G, K417N, L452R, T478K, E484Q, D614G, P681R, D950N, E156del, F157del, N501Y, spike deletion 69-70del, spike deletion 144del, A570D, T716I, S982A, D1118H, P681H, L18F, D80A, D215G, 242-244del, R246I, K471N, E484K, A701V, N440K, S443A, G476S, E484R, and G502P in SEQ ID NO: 377, or any combination thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at T19R, T95I, G142D, E156del, F157del, R158G, L452R, T478K, D614G, P681R, and D950N in SEQ ID NO: 377. In some embodiments, the substitution is K417N. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at T19R, V70F, T95I, G142D, E156del, F157del, R158G, A222V, W258L, K417N, L452R, T478K, D614G, P681R, and D950N in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at N501Y, D614G, and P681H in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K, N501Y, D614G, and P681H in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at K417N, E484K, N501Y, D614G, and A701V in SEQ ID NO: 377.In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at K417T, E484K, N501Y, D614G, and H655Y in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R, T478K, D614G, and P681R in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K, D614G, and Q677H in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K, N501Y, D614G, and P681H in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R, E484Q, D614G, and P681R in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at S477N, E484K, D614G, and P681H in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at R346K, E484K, N501Y, D614G, and P681H in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452Q, F490S, and D614G in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R, E484Q, D614G, and P681R in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at Q414K, N450K, ins214TDR, and D614G in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at V367F, E484K, and Q613H in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R, N501Y, A653V, and H655Y in SEQ ID NO: 377.In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K, N501T, and H655Y in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R and D614G in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at P384L, K417N, E484K, N501Y, D614G, and A701V in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at K417N, E484K, N501Y, E516Q, D614G, and A701V in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R, N501Y, D614G, and P681H in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at S494P, N501Y, D614G, and P681H in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R, D614G, and Q677H in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K, D614G, N679K, and ins679GIAL in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K, D614G, and A701V in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R and D614G in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at S477N and D614G in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K, D614G, and P681H in SEQ ID NO: 377.In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at E484K and D614G in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at T478K and D614G in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at N439K, E484K, D614G, and P681H in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at D614G, E484K, H655Y, K417T, N501Y, and P681H in SEQ ID NO: 377. In some embodiments, rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at L452R, T478K, D614G, P681R, and K417N in SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein, further comprising substitutions at D614G, E484K, H655Y, N501Y, N679K, and Y449H in SEQ ID NO: 377.

[0206] In some embodiments, the S protein is expressed as a full-length protein and has a deletion of one or more spike protein amino acids H69, V70, or Y144 of SEQ ID NO: 377, or a combination thereof. In some embodiments, the S protein is expressed as a full-length protein and includes one or more substitutions selected from D614G, A570D, P681H, T716I, S982A, D1118H, K417N or K417T, D215G, A701V, L18F, R246I, Y453F, I692V, M1229I, N439K, A222V, S477N, or A376T of SEQ ID NO: 377, or a combination thereof. In some embodiments, the mutant is a SARS-CoV-2 virus having a deletion of the spike protein at amino acids 242-244 of SEQ ID NO: 377. In some embodiments, the S protein is expressed as a full-length protein and includes the following deletions and substitutions: deletions of amino acids 69 and 70 of SEQ ID NO: 377, deletion of amino acid Y144, amino acid substitution N501Y, amino acid substitution A570D, amino acid substitution D614G, amino acid substitution P681H, amino acid substitution T716I, amino acid substitution S982A, and amino acid substitution D1118H. In some embodiments, the S protein is expressed as a full-length protein and includes the following deletions and substitutions: deletions of N501Y, K417N or K417T, E484K, D80A, A701V, L18F, and amino acid deletions at amino acids 242-244 of SEQ ID NO: 377.In some embodiments, the S protein is expressed as a full-length protein with the following substitutions: D614G, D936Y, P1263L, L5F, N439K, R21I, D839Y, L54F, A879S, L18F, F1121L, R847K, L452R, T478I, A829T, Q675H, S477N, H49Y, T29I, G769V, G1124V, V1176F, K1073N, P479S ,S1252P,Y145 missing,E583D,R214L,A1020V,Q1208H,D215G,H146Y,S98F,T95I,G1219C,A846V,I197V,R102I,V367F,T572I,A1078S,A831V,P1162L,T73I,A845S,G1219V,H245Y,L8V,Q675R,S254F,V483A,Q677H,D138H,D80Y,M1237T,D1 146H, E654D, H655Y, S50L, S939F, S943P, G485R, Q613H, T76I, V341I, M153I, S221L, T859I, W258L, L242F, P681L, V2 89I, A520S, V1104L, V1228L, L176F, M1237I, T307I, T716I, L141, M1229I, A1087S, P26S, P330S, P384L, R765L, S940F , T323I, V826L, E1202Q, L1203F, L611F, V615I, A262S, A522V, A688V, A706V, A892S, E554D, Q836H, T1027I, T22I, A22 2V, A27S, A626V, C1247F, K1191N, M731I, P26L, S1147L, S1252F, S255F, V1264L, V308L, D80A, I670L, P251L, P631S,. *1274Q, A344S, A771S, A879T, D1084Y, D253G, H1101Y, L1200F, Q14H, Q239K, A623V, D215Y, E1150D, G476S, K7 7M, M177I, P812S, S704L, T51I, T547I, T791I, V1122L, Y145H, D574Y, G142D, G181V, I834T, N370S, P812L, S12 This includes one or more of F, T791P, V90F, W152L, A292S, A570V, A647S, A845V, D1163Y, G181R, L84I, L938F, P1143L, P809S, R78M, T1160I, V1133F, V213L, V615F, A831V, D839Y, D839N, D839E, S943P, P1263L, S13I, or V622F, and combinations thereof.

[0207] In some embodiments, the S protein is selected from the amino acid sequences of SEQ ID NOs. 377 to 384, or fragments thereof, or sequences that are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0208] In some embodiments, the stabilized S protein is expressed as a full-length protein and contains one or more substitutions selected from K417T, E484K, or N501Y of SEQ ID NO: 381. In some embodiments, the stabilized S protein is expressed as a full-length protein and contains the following substitutions: K417T, E484K, and N501Y of SEQ ID NO: 381. In some embodiments, the rMVA contains a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at L452R, T478K, or P681R of SEQ ID NO: 381, or combinations thereof. In some embodiments, the rMVA contains a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at L452R, T478K, and P681R of SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions of N440K, S443A, G476S, E484R, and / or G502P in SEQ ID NO: 381, or combinations thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions of one or more of T19R, G142D, R158G, K417N, L452R, T478K, E484Q, D614G, P681R, D950N, E156del, F157del, N501Y, spike deletion 69-70del, spike deletion 144del, A570D, T716I, S982A, D1118H, P681H, L18F, D80A, D215G, 242-244del, R246I, K471N, E484K, A701V, N440K, S443A, G476S, E484R, and G502P in SEQ ID NO: 381, or any combination thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further including substitutions at T19R, T95I, G142D, E156del, F157del, R158G, L452R, T478K, D614G, P681R, and D950N in SEQ ID NO: 381. In some embodiments, the substitution is K417N.In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at T19R, V70F, T95I, G142D, E156del, F157del, R158G, A222V, W258L, K417N, L452R, T478K, D614G, P681R, and D950N in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at N501Y, D614G, and P681H in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at E484K, N501Y, D614G, and P681H in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at K417N, E484K, N501Y, D614G, and A701V in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at K417T, E484K, N501Y, D614G, and H655Y in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, T478K, D614G, and P681R in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at E484K, D614G, and Q677H in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at E484K, N501Y, D614G, and P681H in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, E484Q, D614G, and P681R in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at S477N, E484K, D614G, and P681H in SEQ ID NO: 381.In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at R346K, E484K, N501Y, D614G, and P681H in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452Q, F490S, and D614G in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, E484Q, D614G, and P681R in SEQ ID NO: 8. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at Q414K, N450K, ins214TDR, and D614G in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at V367F, E484K, and Q613H in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, N501Y, A653V, and H655Y in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at E484K, N501T, and H655Y in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R and D614G in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at P384L, K417N, E484K, N501Y, D614G, and A701V in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at K417N, E484K, N501Y, E516Q, D614G, and A701V in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, N501Y, D614G, and P681H in SEQ ID NO: 381.In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at S494P, N501Y, D614G, and P681H in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, D614G, and Q677H in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at E484K, D614G, N679K, and ins679GIAL in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at E484K, D614G, and A701V in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, and D614G in SEQ ID NO: 8. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further including substitutions at S477N and D614G in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further including substitutions at E484K, D614G, and P681H in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further including substitutions at E484K and D614G in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further including substitutions at T478K and D614G in SEQ ID NO: 381. In some embodiments, rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further including substitutions at N439K, E484K, D614G, and P681H in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at D614G, E484K, H655Y, K417T, N501Y, and P681H in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at L452R, T478K, D614G, P681R, and K417N in SEQ ID NO: 381.In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions at D614G, E484K, H655Y, N501Y, N679K, and Y449H in SEQ ID NO: 381.

[0209] In some embodiments, the stabilized S protein is expressed as a full-length protein and has a deletion of one or more spike protein amino acids H69, V70, or Y144 of SEQ ID NO: 381, or a combination thereof. In some embodiments, the stabilized S protein is expressed as a full-length protein and includes one or more substitutions selected from D614G, A570D, P681H, T716I, S982A, D1118H, K417N or K417T, D215G, A701V, L18F, R246I, Y453F, I692V, M1229I, N439K, A222V, S477N, or A376T of SEQ ID NO: 1, or a combination thereof. In some embodiments, the mutant is a SARS-CoV-2 virus having a deletion of the spike protein at amino acids 242-244 of SEQ ID NO: 381. In some embodiments, the stabilized S protein is expressed as a full-length protein and contains the following deletions and substitutions: deletion of amino acids 69 and 70 of SEQ ID NO: 381, deletion of amino acid Y144, amino acid substitution N501Y, amino acid substitution A570D, amino acid substitution D614G, amino acid substitution P681H, amino acid substitution T716I, amino acid substitution S982A, and amino acid substitution D1118H. In some embodiments, the stabilized S protein is expressed as a full-length protein and contains the following deletions and substitutions: deletion of N501Y, K417N or K417T, E484K, D80A, A701V, L18F, and amino acid deletions at amino acids 242-244 of SEQ ID NO: 381. In some embodiments, the S protein is expressed as a full-length protein and has deletions of one or more spike protein amino acids H69, V70, or Y144 of SEQ ID NO: 381, or a combination thereof. In some embodiments, the S protein is expressed as a full-length protein and includes one or more substitutions selected from D614G, A570D, P681H, T716I, S982A, D1118H, K417N, K417T, D215G, A701V, L18F, R246I, Y453F, I692V, M1229I, N439K, A222V, S477N, or A376T of SEQ ID NO: 381, or a combination thereof.In some embodiments, the spike protein contains a deletion at amino acids 242-244 of SEQ ID NO: 381. In some embodiments, the S protein is expressed as a full-length protein and contains the following deletions and substitutions: deletions at amino acids 69 and 70 of SEQ ID NO: 381, deletion at amino acid Y144, amino acid substitution N501Y, amino acid substitution A570D, amino acid substitution D614G, amino acid substitution P681H, amino acid substitution T716I, amino acid substitution S982A, and amino acid substitution D1118H. In some embodiments, the S protein is expressed as a full-length protein and contains the following deletions and substitutions: N501Y, K417N or K417T, E484K, D80A, A701V, L18F, and amino acid deletions at amino acids 242-244 of SEQ ID NO: 381. The rMVA encodes a stabilized S protein further comprising substitutions at L452R, T478K, and P681R in SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at N440K, S443A, G476S, E484R, and / or G502P in SEQ ID NO: 381, or combinations thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein, further comprising substitutions of one or more of T19R, G142D, R158G, K417N, L452R, T478K, E484Q, D614G, P681R, D950N, E156del, F157del, N501Y, spike 69-70del, spike deletion 144del, A570D, T716I, S982A, D1118H, P681H, L18F, D80A, D215G, 242-244del, R246I, K471N, E484K, A701V, N440K, S443A, G476S, E484R, and G502P, or any combination thereof.

[0210] In some embodiments, the stabilized S protein is expressed as a full-length protein with the following substitutions: D614G, D936Y, P1263L, L5F, N439K, R21I, D839Y, L54F, A879S, L18F, F1121L, R847K, L452R, T478I, A829T, Q675H, S477N, H49Y, T29I, G769V, G1124V, V1176F, K1073N, P479S, S1252P, Y145 missing, E583D, R214L, A1020V, Q1208H, D215G, H146Y, S98F, T95I, G1219C, A846V, I197V, R102I, V367F, T572I, A1078S, A831V, P1162L, T73I, A845S, G1219V, H245Y, L8V, Q675R, S254F, V483A, Q677H, D138H, D80Y, M1237 T, D1146H, E654D, H655Y, S50L, S939F, S943P, G485R, Q613H, T76I, V341I, M153I, S221L, T859I, W258L, L242F, P681L , V289I, A520S, V1104L, V1228L, L176F, M1237I, T307I, T716I, L141, M1229I, A1087S, P26S, P330S, P384L, R765L, S94 0F, T323I, V826L, E1202Q, L1203F, L611F, V615I, A262S, A522V, A688V, A706V, A892S, E554D, Q836H, T1027I, T22I, A 222V, A27S, A626V, C1247F, K1191N, M731I, P26L, S1147L, S1252F, S255F, V1264L, V308L, D80A, I670L, P251L, P631S, *1274Q, A344S, A771S, A879T, D1084Y, D253G, H1101Y, L1200F, Q14H, Q239K, A623V, D215Y, E1150D, G476S, K7 7M, M177I, P812S, S704L, T51I, T547I, T791I, V1122L, Y145H, D574Y, G142D, G181V, I834T, N370S, P812L, S12 This includes one or more of F, T791P, V90F, W152L, A292S, A570V, A647S, A845V, D1163Y, G181R, L84I, L938F, P1143L, P809S, R78M, T1160I, V1133F, V213L, V615F, A831V, D839Y, D839N, D839E, S943P, P1263L, S13I, or V622F, and combinations thereof.

[0211] In some embodiments, the stabilized S protein is expressed as the full-length protein of SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, ​​SEQ ID NO: 383, or SEQ ID NO: 384, or as an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous to them.

[0212] SARS-CoV-2 is an enveloped, positive-sense single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). The viral particle contains RNA genetic material and structural proteins required for entry into host cells. Once inside the cell, the infectious RNA is used to encode structural proteins that make up the viral particle, non-structural proteins that induce viral assembly, transcription, replication, and host regulation, and accessory proteins whose functions have not yet been determined. The largest gene, ORF1ab, contains overlapping open reading frames that encode polyproteins PP1ab and PP1a. The polyprotein is cleaved to produce 16 non-structural proteins NSP1-NSP16. The production of the longer protein (PP1ab) or the shorter protein (PP1a) depends on a ribosome frameshift event -1. Proteins based on similarities with other coronaviruses include papain-like proteinase (NSP3), 3C-like proteinase (NSP5), RNA-dependent RNA polymerase (NSP12, RdRp), helicase (NSP13, HEL), endo-RNase (NSP15), 2'-O-ribose-methyltransferase (NSP16), and other non-structural proteins. A description of the various NSPs encoded by ORF1ab can be found, for example, in "Structural insights into SARS-CoV-2 proteins." by Arya et al., J Mol Biol. 2021 Jan 22; 433(2): 166725 (which is incorporated herein by reference). In some embodiments provided herein, the rMVA antigenic insert is derived from one or more SARS-CoV-2 proteins or polypeptides selected from SEQ ID NOs. 377 to 394.

[0213] In some embodiments, the antigenic insert is derived from the Marburg virus protein or a fragment thereof. In some embodiments, the antigenic insert is derived from an amino acid sequence or fragment thereof selected from SEQ ID NO: 395 or SEQ ID NO: 396, SEQ ID NO: 398 or SEQ ID NO: 400, or from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0214] In certain embodiments, the encoded polypeptide is ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) in various alternative embodiments. x (Antigenic peptide), ((M)(Secretory signaling peptide - immune checkpoint inhibitor peptide - cleavable peptide) x (Secretory signaling peptide - Antigenic peptide), ((M)(Secretory signaling peptide - Immune checkpoint inhibitor peptide - Cleavable peptide) x (Secretory signaling peptide - Antigenic peptide - Cleavable peptide) y ), ((M)(Secretionary signaling peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Secretory signaling peptide - Antigenic peptide - Cleavable peptide) xThe peptide comprises a (secretionary signal peptide - antigenic peptide) (where y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine), the secretionary signal peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 57 to 90, the immune checkpoint inhibitor peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 1 to 56, the cleavable peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 91 to 127, and the antigenic peptide is a peptide derived from an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a tumor-associated antigen. In some embodiments, the secretory signal peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 65 and 66, the immune checkpoint inhibitor peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 1 and 5, and the cleavable peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 93, 120, and 123. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NOs. 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NOs. 1, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NOs. 123, where x = 2 to 10. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NOs. 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NOs. 1, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NOs. 123, where x ≥ 4. In some embodiments, the secretory signaling peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x = 4, 5, or 6.In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x = 2 to 10. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x ≥ 4. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, and the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, where x = 4, 5, or 6. In some embodiments, the antigenic peptide is selected from SEQ ID NOs: 349 to 394.

[0215] In some embodiments, the antigenic peptide encoded by the polycistronic nucleic acid insert in the rMVA is contained in a chimeric polypeptide comprising a viral glycoprotein signal sequence fused to the N-terminus of the antigenic peptide and a viral glycoprotein transmembrane domain fused to the C-terminus of the antigenic peptide, and the rMVA is further constructed to encode a viral matrix protein, where, upon translational cleavage of the antigen-containing chimeric peptide, the viral matrix protein and the antigen-viral glycoprotein chimeric polypeptide can form non-infectious virus-like particles (VLPs). In some embodiments, as used herein, an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, wherein the polypeptide is ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) xAn rMVA viral vector is provided comprising (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain))(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, Figures 5A and 5B). In some embodiments, the antigenic peptide is contained in a chimeric polypeptide comprising a viral glycoprotein signal sequence fused to the N-terminus of the antigenic peptide, a viral glycoprotein transmembrane domain fused to the C-terminus of the antigenic peptide, and a cleavable peptide fused to the C-terminus of the viral glycoprotein transmembrane domain, for example ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y )(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the antigen-containing chimeric polypeptide fused to the C-terminus of the last antigen-containing chimeric polypeptide does not contain a cleavable sequence, for example ((M)(secretionary signaling peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y(where y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10) may be oriented in the polycistronic nucleic acid insert such that the nucleic acid encoding the antigen-containing chimeric polypeptide is located at the 5' position of the immune checkpoint inhibitor peptide-containing chimeric polypeptide, for example ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x ), or alternatively, ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x (Secretionary signal peptide-immune checkpoint inhibitor peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In further embodiments, the polycistronic nucleic acid insert of rMVA further encodes a viral matrix protein, for example ((M)(Secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide)(viral matrix protein))(where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) (see, for example, Figures 6A and 6B). In alternative embodiments, the coding sequences for both the antigen-containing chimeric polypeptide and the viral matrix protein are included in one or more copies in the polycistronic nucleic acid, for example ((M)(secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (Viral matrix protein - cleavable peptide) y)(Here, x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, the most C-terminal viral matrix protein lacks a cleavable peptide, for example ((M)(secretory signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x (glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) x (viral matrix protein - cleavable peptide) y (viral matrix protein))(Here, x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine). In some embodiments, ((glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (viral matrix protein - cleavable peptide) y )(Here, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, and M = methionine) may be oriented in a polycistronic nucleic acid insert such that these sequences are located 5' to the immune checkpoint inhibitor peptide-containing chimeric polypeptide, for example ((M)(glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (viral matrix protein - cleavable peptide) y (secretory signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x ), or alternatively ((M)(glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (viral matrix protein - cleavable peptide) y (secretory signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x(Secretionary signaling peptide - immune checkpoint inhibitor peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine).

[0216] In certain embodiments, the glycoprotein and matrix protein are derived from Marburg virus (MARV). In certain embodiments, the glycoprotein is derived from the GP protein of MARV (Genbank accession number AFV31202.1). The amino acid sequence of the GP protein of MARV is shown as SEQ ID NO: 395 in Table 10 below. In certain embodiments, the GPS domain of MARV contains amino acids 2 to 19 of the glycoprotein (WTTCFFISLILIQGIKTL) (SEQ ID NO: 396, which may be encoded by an MVA-optimized nucleic acid sequence, for example, SEQ ID NO: 397), and the GPTM domain contains amino acid sequences 644 to 673 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG) (SEQ ID NO: 398, which may be encoded by an MVA-optimized nucleic acid sequence, for example, SEQ ID NO: 399), or nucleic acid sequences that are 70%, 75%, 80%, 85%, 90%, 95% or more identical to them. In some embodiments, the GPS signal of MARV further includes methionine as the first amino acid.

[0217] The VP40 amino acid sequence of MARV is available under GenBank accession number JX458834 and is shown below in Table 10 as Sequence ID No. 400, which can be encoded by, for example, the MVA-optimized nucleic acid sequence of Sequence ID No. 401, or a nucleic acid sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical thereto. In some embodiments, the VP40 amino acid sequence of MARV further includes methionine as the first amino acid.

[0218] [Table 10]

[0219] In some embodiments, any of SEQ ID NOs. 395, 396, and 400 further comprises the amino acid residue methionine (M) as the first amino acid residue. In some embodiments, any of SEQ ID NOs. 397 and 401 further comprises the nucleic acid codon ATG as the first codon of the coding sequence. In certain embodiments, the encoded polypeptide is ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) in various alternative embodiments. x (Glycoprotein signal peptide - Antigenic peptide - Glycoprotein transmembrane domain)), ((M)(Secretionary signal peptide - Immune checkpoint inhibitor peptide - Cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) x ), ((M)(Secretionary signaling peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (Glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain)), ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x ), ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x (Secretory signaling peptide - immune checkpoint inhibitor peptide), ((M)(Secretory signaling peptide - immune checkpoint inhibitor peptide - cleavable peptide) x(Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) (viral matrix protein)), ((M) (secretionary signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) y (Viral matrix protein - cleavable peptide) y ), ((M)(Secretionary signaling peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Glycoprotein signal peptide - antigenic peptide - glycoprotein transmembrane domain - cleavable peptide) x (Viral matrix protein - cleavable peptide) y (Viral matrix protein), ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Viral matrix protein - cleavable peptide) y ), ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Viral matrix protein - cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) x ), or ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (Viral matrix protein - cleavable peptide) y (Secretory signaling peptides - immune checkpoint inhibitor peptides - cleavable peptides) xThe (secretionary signal peptide - immune checkpoint inhibitor peptide) (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, y = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10, and M = methionine) comprises, where the secretionary signal peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 57 to 90, the immune checkpoint inhibitor peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 1 to 56, the cleavable peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs. 91 to 127, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NOs. 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NOs. 398, the viral matrix protein is a peptide having the amino acid sequence of SEQ ID NOs. 400 if present, and the antigenic peptide is a peptide derived from an infectious agent, e.g., a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a tumor-associated antigen. In some embodiments, the antigenic peptide is selected from SEQ ID NOs: 349 to 394. In some embodiments, the secretory signal peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs: 65 and 66, the immune checkpoint inhibitor peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs: 1 and 5, the cleavable peptide is selected from peptides having amino acid sequences selected from SEQ ID NOs: 93, 120, and 123, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NOs: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NOs: 398, the viral matrix protein is a peptide having the amino acid sequence of SEQ ID NOs: 400 if present, and the antigenic peptide is a peptide derived from an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394.In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1, the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein, if present, is a peptide having the amino acid sequence of SEQ ID NO: 400, the antigenic peptide is an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a peptide derived from a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394, with x = 1 to 10. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1, the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein, if present, is a peptide having the amino acid sequence of SEQ ID NO: 400, and the antigenic peptide is a peptide derived from an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394, with x ≥ 4.In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 1, the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein is a peptide having the amino acid sequence of SEQ ID NO: 400 if present, the antigenic peptide is an infectious agent, e.g., a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a peptide derived from a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394, where x = 4, 5, or 6. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein, if present, is a peptide having the amino acid sequence of SEQ ID NO: 400, the antigenic peptide is an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a peptide derived from a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394, with x = 1 to 10.In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein, if present, is a peptide having the amino acid sequence of SEQ ID NO: 400, and the antigenic peptide is a peptide derived from an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a tumor-associated antigen, and the antigenic peptide is selected from SEQ ID NOs: 349 to 394, with x ≥ 4. In some embodiments, the secretory signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 66, the immune checkpoint inhibitor peptide is a peptide having the amino acid sequence of SEQ ID NO: 5, the cleavable peptide is a peptide having the amino acid sequence of SEQ ID NO: 123, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein is a peptide having the amino acid sequence of SEQ ID NO: 400 if present, the antigenic peptide is an infectious agent, e.g., a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a peptide derived from a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394, where x = 4, 5, or 6.In some embodiments, the encoded polypeptide comprises SEQ ID NO: 325 or SEQ ID NO: 333, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein, if present, is a peptide having the amino acid sequence of SEQ ID NO: 400, the antigenic peptide is a peptide derived from an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394. In some embodiments, the encoded polypeptide comprises SEQ ID NO: 329 or SEQ ID NO: 337, the glycoprotein signal peptide is a peptide having the amino acid sequence of SEQ ID NO: 396, the glycoprotein transmembrane domain is a peptide having the amino acid sequence of SEQ ID NO: 398, the viral matrix protein, if present, is a peptide having the amino acid sequence of SEQ ID NO: 400, the antigenic peptide is a peptide derived from an infectious agent, such as a virus, bacterium, parasite, fungus, or toxoid, or alternatively, a tumor-associated antigen, or the antigenic peptide is selected from SEQ ID NOs: 349 to 394.

[0220] In alternative embodiments, the rMVA viral vector of the present invention may be further constructed to encode and express one or more antigenic peptides encoded on one or more separate nucleic acid inserts, in addition to its ability to express a multitude of immune checkpoint inhibitor peptides. In some embodiments, the nucleic acid sequences encoding the multitude of immune checkpoint inhibitor peptides described herein are inserted into one locus of rMVA, and the one or more heterologous nucleic acid sequences encoding antigenic peptides are inserted into separate loci of rMVA. The one or more antigenic peptides may be derived from any of the targets described in the "Antigenic Targets" section, which together constitute part of this section for any purpose. In some embodiments, the antigenic peptides may be derived from any amino acid sequences selected from SEQ ID NOs. 349 to 396, SEQ ID NOs. 398, or 400, or fragments derived therefrom, or from amino acid sequences that are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. When inserted as a separate nucleic acid insert, the start codon encoding the amino acid residue methionine (M) may be included as the first residue of an antigen peptide derived from any of the amino acid sequences selected from SEQ ID NOs. 349 to 396, SEQ ID NOs. 398, or SEQ ID NOs. 400, or from a fragment derived therefrom, or from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0221] In certain embodiments, rMVA further encodes an antigenic peptide comprising a chimeric peptide containing the extracellular domain of an antigen and the transmembrane domain of a viral glycoprotein, in addition to the polycisronic nucleic acid encoding an immune checkpoint inhibitor polypeptide described herein, and further encodes a viral matrix protein, wherein the chimeric peptide and viral matrix protein, when expressed, can form virus-like particles (VLPs) in vivo. In some embodiments, the transmembrane domain of the viral glycoprotein is derived from the amino acids of SEQ ID NO: 398, or a fragment thereof, or from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the viral matrix protein is derived from, for example, the VP40 protein of the Marburg virus shown in SEQ ID NO: 404, or a fragment thereof, or from an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the rMVA encodes the amino acid sequence or fragment thereof of SEQ ID NO: 329, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; the amino acid sequence or fragment thereof of SEQ ID NO: 402, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and the amino acid sequence or fragment thereof of SEQ ID NO: 404, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0222] [Table 11] TIFF0007911709000040.tif31170

[0223] Array optimization One or more nucleic acid sequences containing polycistronic nucleic acid inserts of rMVA provided herein may be optimized for use in MVA vectors. Optimization includes codon optimization, which uses silent mutations to change selected codons from the native sequence to synonymous codons that are optimally expressed by the host vector system. Other types of optimization include using silent mutations to interrupt homopolymer stretches or transcriptional terminator motifs. Each of these optimization strategies can improve gene stability, transcript stability, or protein expression levels from the sequence. In exemplary embodiments, construct stabilization would involve reducing the number of homopolymer stretches in the heterologous DNA insert sequence. Silent mutations may provide something similar to a vaccinia termination signal.

[0224] In exemplary embodiments, the sequence is codon-optimized for expression in MVA, and sequences having more than 5 deoxyguanosine, more than 5 deoxycytidine, more than 5 deoxyadenosine, and more than 5 deoxythymidine sequences are interrupted by silent mutations to minimize expression loss due to frameshift mutations.

[0225] In particular, the nucleic acid for insertion can be optimized by codon-optimizing the original DNA sequence. For example, DNA sequences can be codon-optimized using "Invitrogen's GeneArt Gene software." To fully optimize the gene sequence, homopolymer sequences (G / C-rich or T / A-rich regions) are interrupted by silent mutations (sometimes multiple). MVA transcription terminators (T5NT(UUUUUNU)) are interrupted by silent mutations (sometimes multiple) within the range present in the nucleic acid insert sequence. Further optimizations may include, for example, the addition of Kozak sequences (GCCACC / ATG), the addition of a second stop codon, and the addition of vaccinia virus transcription terminators, specifically "TTTTTAT," or modified forms and / or combinations thereof.

[0226] Pharmaceutical composition The recombinant MVA virus vector of the present invention can be readily formulated, alone or in combination, as pharmaceutical compositions for veterinary or human use. The pharmaceutical composition may contain a pharmaceutically acceptable diluent, excipient, carrier, or adjuvant, or, in an alternative embodiment, one or more antigenic agents, such as antigens derived from infectious diseases, or, in an alternative embodiment, tumor-associated antigens.

[0227] In one embodiment, rMVA is used as an effective adjuvant to enhance immunogenicity against infectious agents and / or to treat infectious diseases, wherein rMVA comprises a polycistronic nucleic acid insert encoding at least two immune checkpoint inhibitor peptides as described herein. In an alternative embodiment, rMVA is used as an effective vaccine to enhance immunogenicity against infectious agents and / or to treat infectious diseases, wherein rMVA comprises a polycistronic nucleic acid insert encoding at least two immune checkpoint inhibitor peptides as described herein and one or more antigenic peptides.

[0228] As used herein, the term “pharmaceutically acceptable carrier” encompasses any standard pharmaceutically acceptable carrier, e.g., those suitable for parenteral administration, e.g., via intramuscular, intra-articular (in the articulation), intravenous, intradermal, intraperitoneal, and subcutaneous routes. Examples of such formulations include aqueous and non-aqueous isotonic sterile injection solutions containing antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the recipient to whom the formulation is intended, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. One exemplary pharmaceutically acceptable carrier is physiological saline. Carriers include excipients and diluents, which must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the patient being treated. Carriers may be inert or may have their own pharmaceutically beneficial properties. The amount of carrier used in combination with the compound is sufficient to provide a practical amount of the substance administered per unit dose of the compound.

[0229] Other physiologically acceptable diluents, excipients, carriers, or additional adjuvants and formulations thereof are known to those skilled in the art.

[0230] In some embodiments, additional adjuvants are used as further immune response enhancers. In various embodiments, additional immune response enhancers include aluminum-based adjuvants, oily adjuvants, Specol, RIBI, TiterMax, Montanide ISA50 or Montanide ISA 720, GM-CSF, nonionic block copolymer adjuvants, dimethyldioctadecylammonium bromide (DDA) adjuvants AS-1, AS-2, Ribi adjuvant adjuvants, QS21, Quil A, SAF (Syntex adjuvant in microfluidic form (SAF-m)), dimethyldioctadecylammonium bromide (DDA), and the human complement system adjuvant Mycobacterium vaccae (m. Selected from the group consisting of vaccae), ISCOMS, MF-59, SBAS-2, SBAS-4, Enhanzyn (trademark), RC-529, AGP, MPL-SE, QS7, escin; digitonin; gypsophila; and chenoa quinoa saponins.

[0231] The compositions used in the methods described herein can be administered by routes selected from, for example, parenteral, intramuscular, intra-arterial, intravascular, intravenous, intraperitoneal, subcutaneous, cutaneous, transdermal, ocular, inhalation, buccal, sublingual, perilingual, intranasal, topical, and oral administration. The preferred method of administration may vary depending on various factors (e.g., the components of the composition to be administered and the severity of the condition being treated). Formulations suitable for oral administration may consist of liquid solutions, such as an effective amount of the composition dissolved in a diluent (e.g., water, saline, or PEG-400), capsules, sachets, or tablets, each containing a predetermined amount of vaccine. The pharmaceutical composition may also be, for example, an aerosol formulation for inhalation into the bronchial passages. The aerosol formulation may be mixed with a pressurized, pharmaceutically acceptable propellant (e.g., dichlorodifluoromethane, propane, or nitrogen).

[0232] For the purposes of the present invention, pharmaceutical compositions suitable for delivering therapeutic agents or biologically active agents may include, for example, tablets, gel caps, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols. Any of these formulations can be prepared by methods well known and accepted in the art. See, for example, Remington: The Science and Practice of Pharmacy (21st ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006 (these are, by reference, part of this specification).

[0233] Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of vaccine dissolved in a diluent, such as water, saline, or PEG400; (b) capsules, sachets, or tablets (each containing a predetermined amount of vaccine as liquid, solid, granules, or gelatin); (c) suspensions in a suitable liquid; (d) suitable emulsions; and (e) polysaccharide polymers such as chitin. The vaccine may also be made into an aerosol formulation administered by inhalation, for example, into the bronchial passages, either alone or in combination with other suitable components. The aerosol formulation may be encapsulated in a pressurized, acceptable propellant, such as dichlorodifluoromethane, propane, or nitrogen.

[0234] Formulations suitable for rectal administration include, for example, suppositories, which consist of a vaccine comprising a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffinic hydrocarbons. Furthermore, gelatin rectal capsules consisting of a combination of the vaccine and a base comprising, for example, liquid triglycerides, polyethylene glycol, and paraffinic hydrocarbons can also be used. The vaccine of the present invention may also be administered co-administered with cytokines to further enhance immunogenicity. Cytokines may be administered by methods known to those skilled in the art, for example, as nucleic acid molecules in plasmid form, or as proteins or fusion proteins.

[0235] In addition to the active compound, the pharmaceutical formulation may contain other additives such as pH adjusting additives. Particularly useful pH adjusting agents include acids such as hydrochloric acid, bases, or buffers such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Furthermore, the formulation may contain antimicrobial preservatives. Useful antimicrobial preservatives include methylparaben, propylparaben, and benzyl alcohol. Antimicrobial preservatives are typically used when the formulation is placed in a vial designed for multiple doses. The pharmaceutical formulations described herein can be lyophilized using techniques well known in the art.

[0236] When aqueous suspensions and / or elixirs are desired for oral administration, the compositions of this disclosure can be combined not only with various sweeteners, flavorings, colorants, emulsifiers, and / or suspending agents, but also with diluents such as water, ethanol, propylene glycol, glycerin, and various similar combinations thereof.

[0237] In yet another embodiment, the pharmaceutical composition is provided as an injectable, stable, sterile formulation containing the rMVA described herein in unit dosage forms in a sealed container. The rMVA may be provided in the form of a lyophilized product that can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid formulation suitable for injection into a host.

[0238] The classes of carriers are not limited to, but include binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavorings, glidentifiers, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in two or more classes; for example, vegetable oil may be used as a lubricant in one formulation and as a diluent in another. A pharmaceutically acceptable carrier is one that, when administered in the amounts used in the corresponding pharmaceutical composition, does not cause any serious adverse reactions in the human body. Exemplary pharmaceutically acceptable carriers include sugars, starches, cellulose, tragacanth powder, malt, gelatin, talc, and vegetable oils. Any active ingredient that does not substantially interfere with the activity of the morphic form of the present invention or the pharmaceutical composition may be included in the pharmaceutical composition.

[0239] Formulations suitable for pulmonary administration can be delivered by a wide range of passive-powered and active-powered single / multi-dose dry powder inhalers (DPIs). The most commonly used devices for respiratory delivery include nebulizers, metered-dose inhalers, and dry powder inhalers. Several types of nebulizers are available, including jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. The selection of an appropriate lung delivery device depends on parameters such as the properties of the drug and its formulation, the site of action, and the pathophysiology of the lung.

[0240] In certain embodiments, the rMVA-containing pharmaceutical composition described herein is administered as a pharmaceutical composition comprising one or more excipients from the Handbook of Pharmaceutical Excipients, 9th edition (or earlier).

[0241] Non-limiting examples of pharmaceutically acceptable additional excipients include vegetable oils, animal oils, fish oils, or mineral oils. For example, medium-chain triglycerides, amaranth oil, apricot oil, apple oil, argan oil, artichoke oil, avocado oil, almond oil, acai berry extract, peanut oil, buffalo pumpkin oil, borage seed oil, borage oil, babassu oil, coconut oil, corn oil, cottonseed oil (cottonseed oil (cotton seed oil)), cashew oil, carob oil, coriander oil, camellia oil (camellia oil (Camellia oil)), cauliflower oil, Cape chestnut oil, blackcurrant oil, deer oil, evening primrose oil, grape syrup oila oil (hibiscus oil), grapeseed oil, gourd oil, hazelnut oil, hemp oil, kapok oil, krill oil, linseed oil, macadamia nut oil, Mongolian oil (Mongolia Oils include: Moringa oil, Marula oil, Meadowfoam oil, Mustard oil, Niger seed oil, Olive oil, Okrao oil (Hibiscus oil), Palm oil, Palm kernel oil, Peanut oil, Pecan oil, Pine oil, Pistachio oil, Pumpkin oil, Papaya oil, Perilla oil (Perilla oil), Poppy oil, Prune oil, Saw palmetto oil, Quinoa oil, Rapeseed oil, Rice germ oil, Rice bran oil, Rice oil, Rarelmanthia oil, Safflower oil (Safflower Oils selected from the group consisting of soybean oil, sesame oil, sunflower oil, thistle oil, tomato oil, wheat germ oil, walnut oil, watermelon oil, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), vitamin A oil, vitamin D oil, vitamin E oil, vitamin K oil, and their derivatives, and glycerophospholipids such as lecithin, and any combination thereof.

[0242] In certain embodiments, the excipient in the present invention may be a liquid at room temperature (e.g., fatty oil) or a solid at room temperature (e.g., fat).

[0243] How to use The composition of the present invention can be used as an adjuvant to enhance the immune response, or as a vaccine to induce an immune response.

[0244] In exemplary embodiments, the present invention provides an adjuvant used in a method for preventing an infectious disease in a subject requiring prophylaxis (e.g., a non-exposed subject), wherein the method comprises administering the composition of the present invention to the subject in combination with an effective amount of an antigenic agent. Alternatively, the present invention provides a vaccine used in a method for preventing an infectious disease in a subject requiring prophylaxis (e.g., a non-exposed subject), wherein the method comprises administering the composition of the present invention to the subject. The result of this method is that the subject is partially or completely immunized to the infectious disease.

[0245] In another exemplary embodiment, the present invention provides an adjuvant used in a method for treating a condition such as cancer in a subject requiring treatment, wherein the method comprises administering the composition of the present invention to the subject in combination with an effective amount of a tumor-associated antigenic agent. Alternatively, the present invention provides a vaccine used in a method for treating a condition such as cancer in a subject requiring treatment, wherein the method comprises administering the composition of the present invention to the subject.

[0246] In exemplary embodiments, the present invention provides an adjuvant used in a method for treating an infectious agent (e.g., an exposed subject, such as a subject who has recently been exposed but has not yet developed symptoms, or a subject who has recently been exposed and has only mild symptoms), wherein the method comprises administering the composition of the present invention to a subject in combination with a therapeutically effective amount of an antigenic agent targeting the infectious agent. In exemplary embodiments, the present invention provides a vaccine used in a method for treating an infectious agent (e.g., an exposed subject, such as a subject who has recently been exposed but has not yet developed symptoms, or a subject who has recently been exposed and has only mild symptoms), wherein the method comprises administering the composition of the present invention to a subject. The outcome of the treatment is that the subject has an improved therapeutic profile. The result is an improved therapeutic profile. In some cases, compared to an equivalent untreated control, treatment can improve the disorder or its symptoms by, for example, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, as measured by any standard technique. In some cases, treatment may result in inhibition of replication of the infectant, reduction of the titer or load of the infectant, or eradication or elimination of the infectant. In other embodiments, treatment may result in improvement of one or more symptoms of an infection, including any of the symptoms identified above. According to this embodiment, confirmation of treatment can be evaluated by detecting improvement or absence of symptoms.

[0247] Subjects treated according to the described method may be subjects diagnosed by a physician as having such a condition. The diagnosis may be made by any appropriate means. Subjects for whom the onset of infection has been prevented may or may not have received such a diagnosis. Those skilled in the art will understand that subjects treated according to the present invention may be identified using standard tests, or they may be identified as high-risk subjects without testing due to the presence of one or more risk factors (e.g., exposure to 2019-nCoV).

[0248] In other embodiments, treatment may result in a reduction or elimination of the subject's ability to transmit the infection to another uninfected subject. Confirmation of treatment according to this embodiment is generally evaluated using the same methods used to determine improvement in the disorder, however, a reduction in viral titer or viral load necessary to prevent transmission may differ from a reduction in viral titer or viral load necessary to improve the disorder.

[0249] In one embodiment, the present invention is a method for inducing an immune response in a subject (e.g., a human) by administering a recombinant MVA virus vector described herein, which encodes two or more immune checkpoint inhibitor peptides, in combination with an antigenic agent. The immune response may be a cellular immune response, a humoral immune response, or a combination thereof.

[0250] The composition can be administered, for example, by injection (e.g., intramuscular, intra-arterial, intravascular, intra-venous, intraperitoneal, or subcutaneous).

[0251] It will be understood that two or more routes of administration of the vaccine of the present invention can be used simultaneously or sequentially (e.g., booster doses). Furthermore, the adjuvant or vaccine of the present invention can be used in combination with conventional immunization approaches, such as using protein antigens, vaccinia viruses, and inactivated viruses as vaccines. Thus, in one embodiment, the vaccine of the present invention is administered to a subject (the subject receives the vaccine of the present invention as a "primary dose"), followed by the administration of a conventional vaccine (the subject receives the conventional vaccine as a "booster dose"). In another embodiment, the subject receives the conventional vaccine first, followed by the administration of the adjuvant or vaccine of the present invention. In yet another embodiment, the conventional vaccine and the adjuvant or vaccine of the present invention are administered simultaneously.

[0252] While not bound by any particular mechanism, it is believed that inoculation of the pharmaceutical compositions described herein induced the host's immune system to respond to the adjuvant or vaccine in combination with an antigenic agent by producing both secretory and serum antibodies specific to the infectious agent or tumor-associated antigen, and by generating a cell-mediated immune response specific to the target factor. As a result of vaccination, the host acquires at least partial or complete immunity to the target infection, or resistance to the development of moderate or severe illness caused by the target infection.

[0253] In some embodiments, the administration is a single dose. In some embodiments, the administration is repeated at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more than eight times.

[0254] In one embodiment, the administration is repeated twice.

[0255] In one embodiment, approximately 2 to 8 doses, approximately 4 to 8 doses, or approximately 6 to 8 doses are provided.

[0256] In one embodiment, intervals of approximately 1 to 4 weeks, 2 to 4 weeks, 3 to 4 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks are provided between administrations.

[0257] In one particular embodiment, a 4-week interval is used between two doses.

[0258] Dosage Adjuvants or vaccines combined with antigenic agents are administered in a manner appropriate to the formulation, in a dose that is therapeutically effective, immunogenic, and protective. The dose administered depends on the subject being treated, including, for example, the individual's immune system's ability to synthesize antibodies and, if necessary, its ability to produce a cell-mediated immune response. The exact amount of active ingredient required to be administered depends on the practitioner's judgment and may be monitored on a patient-by-patient basis. However, an appropriate dose range is readily determinable by those skilled in the art and is generally about 5.0 × 10⁻⁶ 6 TCID 50 ~5.0×10 9 TCID 50 This is within the range. The dosage may also depend, though not limited, on the route of administration, the patient's health condition and weight, and the properties of the formulation.

[0259] The pharmaceutical composition of the present invention is administered in a therapeutically effective amount that enhances the immunogenicity of a target antigen. The administered dose depends on the subject being treated (e.g., mode of administration and age, weight, immune system capacity, and overall health of the subject being treated). The composition is administered in an amount that provides a sufficient level of expression to enhance or induce an immune response without excessive adverse physiological effects. Preferably, the composition of the present invention is, for example, 1.0 × 10⁻⁶ 4 ~9.9×10 12 TCID 50 A viral vector, preferably 1.0 × 10⁻⁶. 5 TCID 50 ~1.0×10 11 TCID 50 PFU, more preferably 1.0 × 10 6 ~1.0×10 10 TCID 50 PFU, or most preferably 5.0 × 10 6 ~5.0×10 9 TCID 50 It is administered in the following dosage. The composition is, for example, at least 5.0 × 10 6 TCID 50 Virus vector (e.g., 1.0 × 10) 8 TCID 50It may contain a viral vector. A physician or researcher can determine the appropriate dose and administration regimen.

[0260] The composition of the above method is, for example, 1.0 × 10 4 ~9.9×10 12 TCID 50 A viral vector, preferably 1.0 × 10⁻⁶. 5 TCID 50 ~1.0×10 11 TCID 50 PFU, more preferably 1.0 × 10 6 ~1.0×10 10 TCID 50 PFU, or most preferably 5.0 × 10 6 ~5.0×10 9 TCID 50 The composition may include, for example, at least 5.0 × 10 6 TCID 50 Virus vector (e.g., 1.0 × 10) 8 TCID 50 This method may include a viral vector. This method may include, for example, administering the composition to a subject two or more times.

[0261] The term "effective dose" means the amount of composition administered to improve, inhibit or reverse a clinically relevant symptom of a subject's condition or disorder (e.g., improve, inhibit or reverse an arenavirus infection, or provide an effective immune response to the infection). Any improvement in the subject is considered sufficient to achieve treatment. Preferably, a sufficient dose to treat is an amount that prevents the onset or one or more symptoms, or an amount that reduces the severity of one or more symptoms of the target infection or cancer, or the duration of the subject's suffering therefrom (e.g., by at least 10%, 20%, or 30%, more preferably at least 50%, 60%, or 70%, most preferably at least 80%, 90%, 95%, 99%, or more, compared to a control subject not treated with the composition of the present invention).

[0262] In some cases, it may be desirable to combine the rMVA of the present invention with two or more infectious agents, particularly immunogenic compositions that induce a protective response against other viruses. For example, the adjuvant composition of the present invention can be administered simultaneously, separately, or sequentially with other gene-immunizing vaccines such as those for influenza (Ulmer, JB et al., Science 259:1745-1749 (1993), Raz, E. et al., PNAS (USA) 91:9519-9523 (1994)), malaria (Doolan, DL et al., J. Exp. Med. 183:1739-1746 (1996), Sedegah, M. et al., PNAS (USA) 91:9866-9870 (1994)), and tuberculosis (Tascon, RC et al., Nat.Med.2:888-892 (1996)).

[0263] Administration As used herein, the term “administer” refers to a method of giving a subject a dose of the pharmaceutical composition of the present invention. The compositions used in the methods described herein can be administered by routes selected from, for example, parenteral, cutaneous, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, rectal, topical, and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intra-arterial, intravascular, and intramuscular administration. Preferred administration methods may vary depending on various factors (e.g., the components of the composition to be administered and the severity of the condition being treated).

[0264] The pharmaceutical compositions of the present invention (e.g., adjuvants or vaccines) may be administered by any route known to those skilled in the art. Administration may be, for example, by intramuscular injection. The compositions used in the methods described herein may also be administered by routes selected from, for example, parenteral, cutaneous, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, rectal, topical, and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, and intramuscular administration. Preferred methods of administration may vary depending on various factors, such as the components of the composition being administered and the severity of the condition being treated.

[0265] Furthermore, a single or multiple doses of the composition of the present invention may be administered to a subject. For example, a subject particularly susceptible to the effects of a targeted antigenic agent may require multiple treatments to establish and / or maintain protection against a virus. The level of induced immunity provided by the pharmaceutical composition described herein can be monitored, for example, by measuring the amounts of neutralizing secretory antibodies and neutralizing serum antibodies. The dose may then be adjusted or repeated as necessary to maintain a desired level of protection against viral infection.

[0266] Embodiment In this specification, at least the following embodiments are provided: 1. A recombinant modified vaccinia ankara (rMVA) virus vector containing heterologous polycistronic nucleic acids, wherein the polycistronic nucleic acids are (M) (secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x An rMVA viral vector encoding (where x = 2 to 10 and M is methionine). 2. An rMVA viral vector comprising heterologous polycistronic nucleic acids, wherein the polycistronic nucleic acids are ((M)(secretionary signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) xAn rMVA viral vector encoding a (secretionary signal peptide-immune checkpoint inhibitor peptide) (where x = 1 to 10 and M is methionine). 3. The rMVA of Embodiment 1 or 2, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 56, or an amino acid sequence that is at least 95% identical thereto. 4. The rMVA of Embodiments 1 to 3, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 15, or an amino acid sequence that is at least 95% identical thereto. 5. The rMVA of Embodiments 1 to 4, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 5, or an amino acid sequence that is at least 95% identical thereto. 6. The rMVA of Embodiments 1 to 5, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 95% identical thereto. 7. The rMVA of Embodiments 1 to 5, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 95% identical thereto. 8. The rMVA of Embodiments 1 to 7, wherein the secretory signal peptide comprises an amino acid sequence selected from SEQ ID NOs. 57 to 90, or an amino acid sequence that is at least 95% identical thereto. 9. The rMVA of Embodiments 1 to 8, wherein the secretory signal peptide comprises an amino acid sequence selected from SEQ ID NO: 65, or an amino acid sequence that is at least 95% identical thereto. 10. The rMVA of Embodiments 1 to 8, wherein the secretory signal peptide comprises an amino acid sequence selected from SEQ ID NO: 66, or an amino acid sequence that is at least 95% identical thereto. 11. The rMVA of Embodiments 1 to 10, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs. 91 to 127, or an amino acid sequence that is at least 95% identical thereto. 12. The rMVA of Embodiments 1 to 11, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 93, SEQ ID NO: 120, and SEQ ID NO: 123, or an amino acid sequence that is at least 95% identical thereto. 13. The cleavable peptide comprises the rMVA of Embodiments 1 to 11, having the amino acid sequence RX(R / K)R (where X = any amino acid) (SEQ ID NO: 91). 14. The cleavable peptide is an rMVA of Embodiments 1 to 11, comprising the amino acid sequence RX(R / K)R (where X = R, K, or H) (SEQ ID NO: 92). 15. The cleavable peptide is RAKR (SEQ ID NO: 93), as of embodiments 1 to 12, which is the rMVA. 16. The cleavable peptide is RRRR (SEQ ID NO: 94), as of embodiments 1 to 11, which is the rMVA. 17. The cleavable peptide is RKRR (SEQ ID NO: 95), as of embodiments 1 to 11, which is the rMVA. 18. The cleavable peptide is RRKR (SEQ ID NO: 96), as of embodiments 1 to 11, which is the rMVA. 19. The cleavable peptide is RKKR (SEQ ID NO: 97), as of embodiments 1 to 11, which is the rMVA. 20. The rMVA of Embodiments 1 to 11, wherein the cleavable peptide has the amino acid sequence of SEQ ID NOs. 123 to 127, or an amino acid sequence that is at least 95% identical thereto. 21. The rMVA of Embodiments 1 to 12, wherein the cleavable peptide has the amino acid sequence of SEQ ID NO: 123, or at least 95% identical amino acids thereto. 22. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2, which encodes an amino acid sequence selected from SEQ ID NOs. 309 to 324, or an amino acid sequence that is at least 95% identical thereto. 23. The rMVA of embodiments 1 to 22, where x ≥ 4. 24. The rMVA of Embodiments 1 to 22, where x = 3, 4, or 5. 25. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2, which encodes an amino acid sequence selected from SEQ ID NOs. 325 to 340, or an amino acid sequence that is at least 95% identical thereto. 26. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2, which encodes an amino acid sequence selected from SEQ ID NOs. 341 to 344, or an amino acid sequence that is at least 95% identical thereto. 27. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2, which encodes an amino acid sequence selected from SEQ ID NOs. 345 to 348, or an amino acid sequence that is at least 95% identical thereto. 28. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2, which encodes the amino acid sequence of SEQ ID NO: 325, or an amino acid sequence that is at least 95% identical thereto. 29. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2 that encodes the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence that is at least 95% identical thereto. 30. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2 that encodes the amino acid sequence of SEQ ID NO: 333, or an amino acid sequence that is at least 95% identical thereto. 31. The polycistronic nucleic acid is an rMVA of Embodiment 1 or 2 that encodes the amino acid sequence of SEQ ID NO: 337, or an amino acid sequence that is at least 95% identical thereto. 32. The polycistronic nucleic acid further comprises the rMVA of Embodiments 1 to 31, which encodes an antigenic peptide. 33. The rMVA of Embodiment 32, wherein the antigenic peptide is derived from the group consisting of infectious factors and tumor-associated antigens. 34. The rMVA of Embodiment 33, wherein the infectious agent is a virus, bacteria, fungus, parasite, or amoeba. 35. The rMVA of Embodiment 34, wherein the virus is selected from the group consisting of adenovirus, herpesvirus, poxvirus, single-stranded DNA virus, parvovirus, double-stranded RNA virus, reovirus, positive-positive single-stranded RNA virus, coronavirus, picornavirus, togavirus, negative-positive single-stranded RNA virus, orthomyxovirus, rhabdovirus, single-stranded RNA retrovirus, double-stranded DNA retrovirus, flaviviridae virus, alphavirus virus, filoviridae virus, paramyxoviridae virus, rhabdoviridae virus, nyamiviridae virus, arenaviridae virus, bunyaviridae virus, or ophioviridae virus, and orthomyxoviridae virus. 36. The antigenic peptides include Ebola virus, Ebola virus envelope glycoprotein, Ebola virus matrix protein VP40, Lassa virus, Lassa virus protein Z, Zika virus, Zika virus nonstructural protein 1 (NSP-1), Marburg virus, Marburg virus glycoprotein, Marburg VP40 matrix protein, parasites of Plasmodium species, Plasmodium falciparum, perisporozoite proteins (CSPs) of Plasmodium species, and male germ surface protein P230p (Pfs) of Plasmodium species. rMVA of Embodiment 32, derived from 230 antigens, sporozoite microname proteins of Plasmodium species essential for cell translocation (SPECT2), GTP-binding proteins of Plasmodium species, putative antigens, human immunodeficiency virus, HIV Env protein, HIV gp41, HIV gp120, HIV gp160, HIV Gag protein, HIV MA, HIV CA, HIV SP1, HIV NC, HIV SP2, HIV P6, HIV Pol protein, HIV RT, HIV RNase H, HIV IN, and HIV PR, or fragments thereof. 37. The antigenic peptide is SARS-CoV-2, SARS-CoV-2 full-length S protein Wuhan strain, SARS-CoV-2 S protein having the substitutions K417T, E484K, and N501Y, SARS-CoV-2 full-length S protein delta mutant, SARS-CoV-2 full-length S protein delta mutant plus, SARS-CoV-2 full-length S protein stabilized by two proline substitutions, SARS-CoV-2 full-length stabilized S protein, SARS-CoV-2 full-length stabilized S protein having the substitutions K417T, E484K, and N501Y, SARS-CoV-2 full-length stabilized S protein delta mutant, SARS-CoV-2 full-length stabilized S protein delta mutant plus, S The amino acid sequences of the ARS-CoV-2 E protein, SARS-CoV-2 M protein, SARS-CoV-2 PP1ab polyprotein, SARS-CoV-2 PP1a polyprotein (Wuhan Hu1), SARS-CoV-2 NSP1-NSP3 amino acid sequences (Wuhan Hu1), SARS-CoV-2 NSP4-NSP11 amino acid sequences (Wuhan Hu1), SARS-CoV-2 ORF1b polyprotein NSP12-NSP16 amino acid sequences (Wuhan Hu1), SARS-CoV-2 NSP12 amino acid sequence (Wuhan Hu1), SARS-CoV-2 NSP13-NSP14 amino acid sequences (Wuhan Hu1), SARS-CoV-2 NSP15-NSP16 amino acid sequences (Wuhan Hu1), and MUC-1. rMVA of Embodiment 32, derived from the amino acid sequence of MARV's GPTM, the amino acid sequence of Marburg virus's VP40, and the MUC-1-ECD-MARVTM-ICD sequence, or fragments thereof. 38. The tumor-associated antigen is a carcinoembryonic tumor-associated antigen, a tumor-viral tumor-associated antigen, an overexpressed / accumulated tumor-associated antigen, a cancer-testicular tumor-associated antigen, a lineage-specific tumor-associated antigen, a mutated tumor-associated antigen, or an idiotype tumor-associated antigen, or a rMVA of Embodiment 33 derived from fragments thereof. 39. The tumor-associated antigens include the b melanoma antigen (BAGE) family, cancer-associated gene (CAGE) family, G antigen (GAGE) family, melanoma antigen (MAGE) family, sarcoma antigen (SAGE) family, and X antigen (XAGE) family, CT9, CT10, NY-ESO-1, L antigen (LAGE)1, melanoma antigen (PRAME) preferentially expressed in tumors, and synovial sarcoma X (SSX)2, and melanoma antigen-1 / 2 (Me) recognized by T cells. rMVA of Embodiment 33, derived from lan-A / MART-1 / 2), Gp100 / pmel17, tyrosine-related protein (TRP)1 and TRP2, P. polypeptide, melanocortin 1 receptor (MC1R), and prostate-specific antigen, β-catenin, breast cancer antigen (BRCA)1 / 2, cyclin-dependent kinase (CDK)4, chronic myeloid leukemia antigen (CML)66, fibronectin, p53, Ras, or TGF-βRII, or fragments thereof. 40. The antigenic peptide is the rMVA of Embodiment 32, derived from mucin 1 or a fragment thereof. 41. The rMVA of Embodiment 40, wherein the mucin 1 is encoded by the nucleic acid sequence of Sequence ID No. 402, or a nucleic acid sequence that is at least 95% identical thereto. 42. The method of Embodiment 40, wherein the mucin 1 comprises the amino acid sequence of Sequence ID No. 349, or an amino acid sequence that is at least 95% identical thereto. 43. The rMVA of Embodiment 40, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence that is at least 95% identical thereto. 44. The rMVA of Embodiment 40, wherein the mucin 1 comprises an extracellular domain fragment of human mucin 1. 45. The extracellular domain fragment of human mucin 1 is selected from SEQ ID NOs. 358 to 361, or from amino acid sequences that are at least 95% identical thereto, according to Embodiment 44, the rMVA. 46. ​​The rMVA of Embodiment 40, wherein the mucin 1 comprises an intracellular domain fragment of human mucin 1. 47. The rMVA of Embodiment 46, wherein the intracellular domain fragment of human mucin 1 comprises the amino acid sequence of Sequence ID No. 362, or an amino acid sequence that is at least 95% identical thereto. 48. The method of Embodiment 40, wherein the mucin 1 is selected from the amino acid sequence of SEQ ID NO: 363 or SEQ ID NO: 364, or at least 95% identical thereto. 49. The method of Embodiment 48, wherein the mucin 1 comprises the amino acid sequence of Sequence ID No. 363, or an amino acid sequence that is at least 95% identical thereto. 50. The method of Embodiment 48, wherein the mucin 1 comprises the amino acid sequence of Sequence ID No. 364, or an amino acid sequence that is at least 95% identical thereto. 51. The rMVA of Embodiment 32, wherein the antigenic peptide is derived from an amino acid sequence selected from SEQ ID NOs. 349 to 357, or from an amino acid sequence that is at least 95% identical thereto. 52. The rMVA of Embodiment 32, wherein the antigenic peptide is derived from an amino acid sequence selected from SEQ ID NOs. 358 to 394, or from an amino acid sequence that is at least 95% identical thereto. 53. The antigenic peptide is an rMVA of Embodiment 51 or 52, which is derived from an amino acid sequence selected from SEQ ID NO: 350, SEQ ID NO: 354, SEQ ID NO: 356, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 377, or SEQ ID NO: 379, or from an amino acid sequence that is at least 95% identical thereto. 54. The antigenic peptide is the rMVA of Embodiments 32-53, which includes a secretory signal. 55. The rMVA of Embodiment 54, wherein the secretory signal is fused to the N-terminus of the antigenic peptide. 56. The rMVA of Embodiment 55, wherein the secretion signal is selected from the amino acid sequences of SEQ ID NOs. 57 to 90, or amino acid sequences that are at least 95% identical thereto. 57. The secretory signal comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that is at least 95% identical thereto, according to Embodiment 56, the rMVA. 58. The rMVA of Embodiment 56, wherein the secretion signal comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that is at least 95% identical thereto. 59. The polycistronic nucleic acid is inserted between two essential, highly conserved MVA genes, as in the rMVA of Embodiments 1 to 58. 60. The polycistronic nucleic acid is an rMVA of Embodiments 1 to 58, in which the polycistronic nucleic acid is inserted into a natural deletion site. 61. The rMVA of Embodiments 1 to 58, wherein the polycistronic nucleic acid is inserted into the MVA at a site selected from between I8R and G1L of the MVA gene, between A50R and B1R of the MVA gene at the reconstructed and modified deletion site III, or between A5 and A6L of the MVA gene. 62. The rMVA of Embodiments 1 to 58, wherein the polycistronic nucleic acid is inserted into the rMVA at a site selected between I8R and G1L of the MVA gene. 63. The rMVA of Embodiments 1 to 58, wherein the polycistronic nucleic acid is inserted into the rMVA at a site selected from between A50R and B1R of the MVA gene in the reconstructed and modified deletion site III. 64. The rMVA of Embodiments 1 to 58, wherein the polycistronic nucleic acid is inserted into the rMVA at a site selected between A5 and A6L of the MVA gene. 65. The nucleic acid encoding the amino acid sequence of the antigenic peptide is the rMVA of embodiments 32-64, located in the open reading frame downstream of the methionine (M) start codon. 66. A method for increasing an immune response to a target antigen in a patient, comprising administering to the patient an effective amount of an rMVA viral vector of Embodiments 1 to 65, wherein the patient has been administered or is administered an effective amount of the target antigen. 67. The method of Embodiment 66, wherein the rMVA viral vector is administered simultaneously with or after the administration of the target antigen. 68. The method of Embodiment 66 or 67, wherein the target antigen is selected from the group consisting of infectious agents and tumor-associated antigens. 69. The method of Embodiment 68, wherein the infectious agent is a virus, bacteria, fungi, parasites, or amoebas. 70. The method of Embodiment 69, wherein the virus is selected from the group consisting of adenovirus, herpesvirus, poxvirus, single-stranded DNA virus, parvovirus, double-stranded RNA virus, reovirus, positive-positive single-stranded RNA virus, coronavirus, picornavirus, togavirus, negative-positive single-stranded RNA virus, orthomyxovirus, rhabdovirus, single-stranded RNA retrovirus, double-stranded DNA retrovirus, flaviviridae virus, alphavirus virus, filoviridae virus, paramyxoviridae virus, rhabdoviridae virus, nyamiviridae virus, arenaviridae virus, bunyaviridae virus, or ophioviridae virus, and orthomyxoviridae virus. 71. The target antigens include Ebola virus, Ebola virus envelope glycoprotein, Ebola virus matrix protein VP40, Lassa virus, Lassa virus protein Z, Zika virus, Zika virus nonstructural protein 1 (NSP-1), Marburg virus, Marburg virus glycoprotein, Marburg VP40 matrix protein, parasites of Plasmodium species, Plasmodium falciparum, perisporozoite proteins (CSP) of Plasmodium species, and male germ surface protein P230p (Pfs230) of Plasmodium species. Methods of Embodiments 66 or 67, derived from the antigen, sporozoite microname protein (SPECT2) of Plasmodium species essential for cell translocation, GTP-binding protein of Plasmodium species, putative antigen, human immunodeficiency virus, HIV Env protein, HIV gp41, HIV gp120, HIV gp160, HIV Gag protein, HIV MA, HIV CA, HIV SP1, HIV NC, HIV SP2, HIV P6, HIV Pol protein, HIV RT, HIV RNase H, HIV IN, and HIV PR, or fragments thereof. 72. The target antigens include SARS-CoV-2, SARS-CoV-2 full-length S protein Wuhan strain, SARS-CoV-2 S protein with K417T, E484K, and N501Y substitutions, SARS-CoV-2 full-length S protein delta mutant, SARS-CoV-2 full-length S protein delta mutant plus, SARS-CoV-2 full-length S protein stabilized by two proline substitutions, SARS-CoV-2 full-length stabilized S protein, SARS-CoV-2 full-length stabilized S protein with K417T, E484K, and N501Y substitutions, SARS-CoV-2 full-length stabilized S protein delta mutant, SARS-CoV-2 full-length stabilized S protein delta mutant plus, SARS-CoV-2 E protein, S The method of Embodiment 66 or 67, derived from the group consisting of the M protein of ARS-CoV2, the amino acid sequence of the PP1ab polyprotein of SARS-CoV2, the amino acid sequence of the PP1a polyprotein of SARS-CoV2 (Wuhan Hu1), the amino acid sequences of NSP1 to NSP3 of SARS-CoV2 (Wuhan Hu1), the amino acid sequences of NSP4 to NSP11 of SARS-CoV2 (Wuhan Hu1), the amino acid sequences of NSP12 to NSP16 of the ORF1b polyprotein of SARS-CoV2 (Wuhan Hu1), the amino acid sequence of NSP12 of SARS-CoV2 (Wuhan Hu1), the amino acid sequences of NSP13 to NSP14 of SARS-CoV2 (Wuhan Hu1), and the amino acid sequences of NSP15 to NSP16 of SARS-CoV2 (Wuhan Hu1), or fragments thereof. 73. The method of Embodiment 68, wherein the tumor-associated antigen is derived from carcinoembryonic tumor-associated antigen, oncoviral tumor-associated antigen, overexpressed / accumulated tumor-associated antigen, cancer-testicular tumor-associated antigen, lineage-specific tumor-associated antigen, mutated tumor-associated antigen, or idiotype tumor-associated antigen, or fragments thereof. 74. The tumor-associated antigens include the b melanoma antigen (BAGE) family, cancer-associated gene (CAGE) family, G antigen (GAGE) family, melanoma antigen (MAGE) family, sarcoma antigen (SAGE) family, and X antigen (XAGE) family, CT9, CT10, NY-ESO-1, L antigen (LAGE)1, melanoma antigen (PRAME) preferentially expressed in tumors, and synovial sarcoma X (SSX)2, and melanoma antigen-1 / 2 (M) recognized by T cells. The method of Embodiment 68, derived from elan-A / MART-1 / 2), Gp100 / pmel17, tyrosine-related protein (TRP)1 and TRP2, P. polypeptide, melanocortin 1 receptor (MC1R), and prostate-specific antigen, β-catenin, breast cancer antigen (BRCA)1 / 2, cyclin-dependent kinase (CDK)4, chronic myeloid leukemia antigen (CML)66, fibronectin, p53, Ras, or TGF-βRII, or fragments thereof. 75. The method of Embodiment 66 or 67, wherein the target antigen is derived from mucin 1 or a fragment thereof. 76. The method of Embodiment 75, wherein the mucin 1 is encoded by the nucleic acid sequence of Sequence ID No. 402, or a nucleic acid sequence that is at least 95% identical thereto. 77. The method of Embodiment 75, wherein the mucin 1 comprises the amino acid sequence of Sequence ID No. 349, or an amino acid sequence that is at least 95% identical thereto. 78. The method of Embodiment 75, wherein the mucin 1 comprises the amino acid sequence of Sequence ID No. 403, or an amino acid sequence that is at least 95% identical thereto. 79. The method of Embodiment 75, wherein the mucin 1 comprises an extracellular domain fragment of human mucin 1. 80. The method of Embodiment 79, wherein the extracellular domain fragment of human mucin 1 is selected from SEQ ID NOs. 358 to 361, or from amino acid sequences that are at least 95% identical thereto. 81. The method of Embodiment 75, wherein the mucin 1 comprises an intracellular domain fragment of human mucin 1. 82. The method of Embodiment 81, wherein the intracellular domain fragment of human mucin 1 comprises the amino acid sequence of Sequence ID No. 362, or an amino acid sequence that is at least 95% identical thereto. 83. The method of Embodiment 75, wherein the mucin 1 is selected from the amino acid sequence of SEQ ID NO: 363 or SEQ ID NO: 364, or at least 95% identical thereto. 84. The method of Embodiment 83, wherein the mucin 1 comprises the amino acid sequence of Sequence ID No. 363, or an amino acid sequence that is at least 95% identical thereto. 85. The method of Embodiment 83, wherein the mucin 1 comprises the amino acid sequence of Sequence ID No. 364, or an amino acid sequence that is at least 95% identical thereto. 86. The method of Embodiment 66 or 67, wherein the target antigen is derived from an amino acid sequence selected from SEQ ID NOs. 349 to 357, or from an amino acid sequence that is at least 95% identical thereto. 87. The method of Embodiment 66 or 67, wherein the target antigen is derived from an amino acid sequence selected from SEQ ID NOs. 358 to 394, or from an amino acid sequence that is at least 95% identical thereto. 88. The method of Embodiment 66 or 67, wherein the target antigen is derived from an amino acid sequence selected from SEQ ID NO: 350, SEQ ID NO: 354, SEQ ID NO: 356, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 377, or SEQ ID NO: 379, or from an amino acid sequence that is at least 95% identical thereto. 89. An rMVA viral vector comprising heterologous polycistronic nucleic acids, wherein the polycistronic nucleic acids are (M) (secretionary signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x An rMVA viral vector encoding (secretionary signal peptide-antigenic peptide) (where x = 1 to 10 and M is methionine). 90. An rMVA viral vector comprising heterologous polycistronic nucleic acids, wherein the polycistronic nucleic acids are (M) (secretionary signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x An rMVA viral vector encoding (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane peptide) (where x = 1 to 10 and M is methionine). 91. An rMVA viral vector comprising heterologous polycistronic nucleic acids, wherein the polycistronic nucleic acids are (M) (secretionary signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x An rMVA viral vector encoding (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane peptide-cleavable peptide)(viral matrix protein) (where x = 1 to 10 and M is methionine). 92. Recombinant modified vaccinia ankara (rMVA) virus vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, wherein the polypeptide is ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x An rMVA viral vector containing (antigenic peptide) (where x = 1 to 10 and M is methionine). 93. The rMVA of Embodiments 89-92, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 56, or an amino acid sequence that is at least 95% identical thereto. 94. The rMVA of Embodiments 89 to 93, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 15, or an amino acid sequence that is at least 95% identical thereto. 95. The rMVA of Embodiments 89-94, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 5, or an amino acid sequence that is at least 95% identical thereto. 96. The rMVA of Embodiments 89-95, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 95% identical thereto. 97. The rMVA of Embodiments 89-95, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 95% identical thereto. 98. The rMVA of Embodiments 89 to 97, wherein the secretory signal peptide comprises an amino acid sequence selected from SEQ ID NOs. 57 to 90, or an amino acid sequence that is at least 95% identical thereto. 99. The rMVA of Embodiments 89-98, wherein the secretory signal peptide comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that is at least 95% identical thereto. 100. The rMVA of Embodiments 89-98, wherein the secretory signal peptide comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that is at least 95% identical thereto. 101. The rMVA of Embodiments 89-100, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs. 91-126, or an amino acid sequence that is at least 95% identical thereto. 102. The rMVA of Embodiments 89-101, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 93, SEQ ID NO: 120, and SEQ ID NO: 123. 103. The cleavable peptide co...

Claims

1. An rMVA viral vector comprising a heterologous polycistronic nucleic acid encoding ((M)(secretion signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)x(secretion signal peptide-immune checkpoint inhibitor peptide)), x = 1 to 10, M is methionine, and the immune checkpoint inhibitor peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 15 or an amino acid sequence that is at least 90% identical thereto. rMVA virus vector.

2. The rMVA virus vector according to claim 1, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO:

1.

3. The rMVA virus vector according to claim 1, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO:

5.

4. The rMVA viral vector according to claim 1, wherein the secretion signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 57 to 90, or an amino acid sequence that is at least 85% identical thereto.

5. The rMVA viral vector according to claim 4, wherein the secreted signal peptide comprises the amino acid sequence of SEQ ID NO:

65.

6. The rMVA viral vector according to claim 4, wherein the secreted signal peptide comprises the amino acid sequence of SEQ ID NO:

66.

7. The rMVA viral vector according to claim 1, wherein the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 91 to 97.

8. The rMVA viral vector according to claim 1, wherein the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 100 to 122, or an amino acid sequence that is at least 95% identical thereto.

9. The rMVA viral vector according to claim 1, wherein the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 123 to 127, or an amino acid sequence that is at least 95% identical thereto.

10. The rMVA viral vector according to claim 8, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

120.

11. The rMVA viral vector according to claim 9, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

123.

12. The rMVA virus vector according to claim 1, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs. 317 to 324, or an amino acid sequence that is at least 95% identical thereto.

13. The rMVA virus vector according to claim 1, wherein x = 3, 4, or 5.

14. The rMVA viral vector according to claim 1, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs. 329 to 332 and SEQ ID NOs. 337 to 340, or an amino acid sequence that is at least 95% identical thereto.

15. The rMVA viral vector according to claim 1, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs. 343 to 344, or an amino acid sequence that is at least 95% identical thereto.

16. The rMVA viral vector according to claim 1, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from SEQ ID NOs. 347 to 348, or an amino acid sequence that is at least 95% identical thereto.

17. The rMVA viral vector according to claim 14, wherein the polycistronic nucleic acid encodes the amino acid sequence of SEQ ID NO:

329.

18. The rMVA viral vector according to claim 14, wherein the polycistronic nucleic acid encodes the amino acid sequence of SEQ ID NO:

337.

19. The rMVA viral vector according to claim 1, wherein the polycistronic nucleic acid further encodes an antigenic peptide.

20. The rMVA viral vector according to claim 7, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

93.

21. The rMVA viral vector according to claim 1, wherein the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 98 to 99.

22. The rMVA virus vector according to claim 1, wherein the polycistronic nucleic acid comprises the nucleic acid sequence of sequence number 408.

23. The rMVA virus vector according to claim 1, wherein the polycistronic nucleic acid comprises the nucleic acid sequence of sequence number 409.

24. The rMVA virus vector according to claim 13, wherein x = 4.

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