Viral constructs used in enhancing 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 TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 144,834, filed February 2, 2021, the entirety of which is incorporated by reference herein for all purposes.

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

[0003] [Incorporated by reference] The content of the text file is named "19101-014WO1_SEQ_TXT", was created on February 1, 2022, is 564KB in size, and is incorporated herein by reference in its entirety. [Background technology]

[0004] Vaccines are considered one of the most important advances in modern medicine and have greatly improved the quality of life by reducing or eliminating many serious infectious diseases. Vaccines have been developed against a wide range of human pathogens, including 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 of the approved vaccines can be classified as live attenuated vaccines, nonreplicating whole particle vaccines (including virus-like particles, or VLPs), and subunit vaccines.

[0005] However, successful vaccine development requires strong and durable protective immunity, consisting of humoral and cellular immune responses. Both components of immunity are essential for effective pathogen elimination. While progress has been made in developing vaccines against many pathogens, strong and durable protective T cell immunity, especially CD8 + The inability to induce T cell responses has been a major obstacle and is the main reason for the failure of many vaccine development efforts, especially against intracellular pathogens (see, e.g., Non-Patent Document 1).

[0006] One strategy to overcome these inherent obstacles is to identify and use adjuvants that enhance immunogenicity, and considerable research has been devoted to evaluating the impact of putative adjuvants on innate immune activation and adaptive immune responses to model antigens and potential vaccines (see, e.g., J. Immunol. 2003, 144: 1111-1112, 1112-1113, 1113-1114).

[0007] For example, Alhydrogel is a well-characterized aluminum hydroxide adjuvant that is currently included in several FDA-approved vaccines. Alhydrogel confers a depot effect in which antigens are released more slowly in vivo, resulting in extended antigen exposure that may or may not contribute to adjuvanticity (Non-Patent Document 5). Furthermore, Alhydrogel has been shown to activate inflammasomes, which may contribute to the immunogenicity of Alhydrogel-based vaccines (Non-Patent Document 6).

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

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

[0010] MPL is a TLR4 agonist and the active component of the GSK adjuvant AS04 (Non-Patent Document 11). MPL has been shown to be highly effective as an adjuvant, especially when combined with aluminum-based adjuvants such as Alhydrogel or nanoparticle formulations (Non-Patent Document 12).

[0011] Other well-known adjuvants include alum-based adjuvants, oil-based adjuvants, Freund's adjuvant, specol, Ribi adjuvant, mycobacterium vaccae, immune stimulating complexes (ISCOMS), MF-59, SBAS-2, SBAS-4, detox B SE (Enhanzyn™), lipid A mimetic RC-529, aminoalkyl glucosaminide 4-phosphate (AGP), CRX-527, monophosphoryl lipid A (e.g., MPL-SE), detoxified saponin derivatives (e.g., QS-21, QS7), escin, digitonin, gypsophila saponin, and Chenopodium quinoa saponin (see, e.g., Non-Patent Document 13).

[0012] Despite significant advances in adjuvant formulation and use, the majority of adjuvants are designed to generate innate inflammatory danger signals. These danger signals are essential for innate immune activation, including antigen presentation and cytokine production, but their direct effect on T cell priming is limited (Non-Patent Document 14, Non-Patent Document 15), and most vaccination strategies using common adjuvants target long-term memory CD8 + (Non-Patent Document 16) This is especially true during vaccination against chronic infections and cancer, which require immunomodulatory strategies to enhance the T cell responses necessary to overcome the immunosuppressive microenvironment.

[0013] One such strategy has been to down-regulate 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, strength, and duration of T cell responses to antigen presentation (Non-Patent Document 17). PD1 is rapidly up-regulated upon activation of naive T cells, which is required to minimize damage to the host caused by uncontrolled inflammation during and after infection (Non-Patent Document 18). In non-human primates, immunization with a combination of an SIVgag adenovirus-based vaccine and an anti-PD1 monoclonal antibody significantly increased the peak of Gag-specific T cell responses (Non-Patent Document 19).

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

[0015] Thus, there is a need for improved methods of using immune checkpoint inhibitors in vaccination strategies that result in 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] Halbroth et al., "Development of a Molecular Adjuvant to Enhance Antigen-Specific CD8+T Cell Responses." Sci Rep. 2018 Oct 9;8(1):15020 [Non-Patent Document 3] Counoupas et al., "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 [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 adjuvanticity." 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] Levy et al., "A modified polyriboinosinic-polyribocytidylic acid complex that induces interferon in primates." J. Infect. Dis. 1975;132:434-439 [Non-Patent Document 8] Wang et al., "Cutting edge: polyinosinic:polycytidylic acid boosts the generation of memory CD8 T cells through melanoma differentiation-associated protein 5 expressed in stromal cells." J. Immunol. 2010;184:2751-2755 [Non-Patent Document 9] Martins et al., "Vaccine adjuvant uses of poly-ic and derivatives." Expert Rev. Vaccines. 2015;14:447-459 [Non-Patent Document 10] Marshall et al., "Identification of a novel cpg DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions." J. Leukoc. Biol. 2003;73:781-792 [Non-Patent Document 11] Einstein et al., "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] Bohannon et al., "The immunobiology of Toll-Like receptor 4 agonists: from endotoxin tolerance to immunoadjuvants." Shock. 2013;40:451-462 [Non-Patent Document 13] Alving et al., "Adjuvants for Human Vaccines." Curr Opin Immunol. 2012 Jun; 24(3): 310-315 [Non-Patent Document 14] Powell et al., "Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes." Clin Exp Vaccine Res. 2015 Jan;4(1):23-45 [Non-Patent Document 15] Petrovsky N., "Comparative Safety of Vaccine Adjuvants: A Summary of Current Evidence and Future Needs." Drug Saf. 2015 Nov;38(11):1059-74 [Non-Patent Document 16] Kamphorst et al., "Beyond Adjuvants: Immunomodulation strategies to enhance T cell immunity." Vaccine. 2015 Jun 8; 33(0 2): B21-B28 [Non-Patent Document 17] Okazaki et al., "A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application." Nat Immunol. 2013 Dec;14(12):1212-8 [Non-Patent Document 18] Ahn et al., "Role of PD-1 during effector CD8 T cell differentiation." 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] Brahmer et al., "Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates." J Clin Oncol. 2010 Jul 1;28(19):3167-75 [Non-Patent Document 21] Topalian et al., "Safety, activity, and immune correlates of anti-PD-1 antibody in cancer." N Engl J Med. 2012 Jun 28;366(26):2443-54 Summary of the Invention

[0017] Provided herein are compositions comprising recombinant modified vaccinia Ankara (rMVA) viral vectors for use as adjuvants or vaccines during immunization protocols in hosts, such as humans. The rMVA is engineered to express high concentrations of 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 polycistronic multimeric nucleic acid inserts and secreted from the cell.

[0018] The use of a PD-1 inhibitor peptide (LD01 - SEQ ID NO:1) administered in combination with an adenovirus-based or irradiated sporozoite-based prophylactic malaria vaccine increased antigen-specific CD8 expression in immune-competent mice. + It has previously been shown that a peptide-based PD1 antagonist enhances T-cell priming and efficacy of a prophylactic malaria vaccine and promotes survival in a lethal malaria model., Phares et al., Front. Immunol. 11, 1377 (2020), incorporated herein by reference. As shown herein, because the natural tropism of MVA viral vectors includes professional antigen-presenting cells such as dendritic cells that can migrate to draining lymph nodes and spread systemically, it has now been found that expressing immune checkpoint inhibitors using MVA as a delivery vehicle offers significant advantages during vaccination strategies. By expressing sufficient and large therapeutic levels of immune checkpoint inhibitors in the lymph node environment, for example in polycistronic multimeric structures, during host antigen exposure, it is possible to enhance the expression of CD8 +It is believed that T cell priming is significantly enhanced. As shown in the Examples below, when used in concert with the administration of an antigen during a vaccination strategy, rMVA viral constructs expressing immune checkpoints can, for example, enhance antigen-specific CD8 T cell priming compared to the administration of such immune checkpoint inhibitor peptides alone. + This results in significantly improved T cell proliferation, increased antigen responses and improved vaccination efficacy, providing a significant improvement over prior art adjuvant strategies.

[0019] In one aspect, provided herein is an rMVA viral vector 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, each chimeric polypeptide comprising a secretory 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 are 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 is comprised of two or more different immune checkpoint inhibitor peptides. In some embodiments, the rMVA further encodes one or more antigenic peptides that, when expressed by the rMVA, are capable of inducing sufficient immunogenicity to provide or enhance protective immunity against an infectious agent, hi some embodiments, the rMVA is capable of inducing an immune response in a host that, when expressed by the rMVA, ameliorates one or more symptoms or pathology of a disorder, e.g., an infectious disease or cancer.

[0020] In some embodiments, each of the chimeric polypeptides comprising a secretory signal peptide and an immune checkpoint inhibitor peptide encoded by the polycistronic nucleic acid insert comprises a peptide sequence that can be cleaved during or after translation linked to the C-terminus of the immune checkpoint inhibitor peptide. When the secreted immune checkpoint inhibitor peptide is inserted in a multimeric structure, the inclusion of a cleavable peptide allows each chimeric polypeptide of the multimer to be expressed as a monomer during translation (e.g., through a translational nascent strand 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 3'-most nucleic acid lacks a cleavable peptide sequence.

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

[0022] In some embodiments, provided herein is an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, the polypeptide comprising a tandem repeat sequence (M) (secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x (wherein x=2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine) (see, e.g., FIG. 1A and FIG. 1B). In some embodiments, provided herein is a rMVA viral vector comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)) encoding two or more polypeptides in a tandem repeat sequence and an additional polypeptide in the tandem repeat sequence fused to the C-terminus of the last polypeptide.x Provided are rMVA viral vectors comprising a heterologous polycistronic nucleic acid insert (secretory signal peptide-immune checkpoint inhibitor peptide), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M is methionine (see, e.g., Figures 2A and 2B).

[0023] In some embodiments, the rMVA viral vector comprises a polycistronic nucleic acid insert encoding two or more polypeptides, where the polypeptides include a tandem repeat sequence described herein, such as ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x a first polypeptide tandem repeat sequence comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, M=methionine, and the sequence encoding the first polypeptide is oriented in a 5' to 3' direction; x and a second polypeptide tandem repeat sequence comprising: (secretory signal peptide-immune checkpoint inhibitor peptide), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, M=methionine, and the sequence encoding the second polypeptide is oriented in a 3' to 5' direction, where each cistron comprises 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 co-administered antigens during vaccination protocols.By expressing one or more immune checkpoint inhibitor peptides locally in large amounts, which can down-regulate one or more checkpoint inhibitor pathways, it can down-regulate the immune-modulating activity that often prevents the development of sufficient antigenicity to induce immunity. In certain embodiments, the immune checkpoint inhibitor peptide is selected from the group consisting of, but not limited to, programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), programmed 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), V domain Ig suppressor of T cell activation (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), glucocorticoid-induced TNF The immune checkpoint inhibitor peptide may inhibit activity of an immune checkpoint pathway mediated by a receptor protein selected from R-associated protein (GITR), CD137, CD40, B- and T-lymphocyte attenuator (BTLA), herpes virus 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 immunoreceptor with Ig and ITIM domains (TIGIT), inducible T cell costimulator (ICOS), inducible T cell costimulator ligand (ICOS-L), or a combination thereof. In some embodiments, the immune checkpoint inhibitor peptide may inhibit PD-1. In some embodiments, the immune checkpoint inhibitor peptide may inhibit PD-L1.In some embodiments, the immune checkpoint inhibitor peptide can inhibit CTLA-4. In some embodiments, the immune checkpoint inhibitor peptide can inhibit PD-1, PD-L1, or CTLA-4, or a combination thereof. In some embodiments, the immune checkpoint inhibitor peptide 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 homologs, derivatives, or fragments thereof. In some embodiments, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-SEQ ID NO:56, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-SEQ ID NO:5, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence of SEQ ID NO:1 (CRRTSTGQISTLRVNITAPLSQ), or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence of SEQ ID NO:5 (STGQISTLRVNITAPLSQ), or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence of SEQ ID NO:6 (STGQISTLAVNITAPLSQ), or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0026] In some embodiments provided herein, each of the immune checkpoint inhibitor peptides expressed by the rMVA is fused at its N-terminus to a secretory signal peptide, where the rMVA expresses two or more immune checkpoint inhibitor peptides fused at its C-terminus to one or more cleavable peptides. The secretory signal peptide allows the immune checkpoint inhibitor peptide to be translocated to the endoplasmic reticulum (ER). After co-translational insertion of the growing peptide chain into the ER lumen, the signal peptide is cleaved from the immune checkpoint inhibitor peptide by a signal peptidase, and the immune checkpoint inhibitor is secreted (see, e.g., Figures 3A, 3B, and 3C). The secretory signal peptide used herein can be any suitable signal peptide that allows the secretion of the immune checkpoint inhibitor peptide. Secretory signal peptides for use 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, incorporated herein by reference). In some embodiments, the secretory signal peptide is a short peptide having a length of between about 15 and 30 amino acids derived from a native human excretory protein. In some embodiments, the secretory signal is a secretory signal selected from the secretory signals in Table 2 (SEQ ID NO: 57 to SEQ ID NO: 90), or a homologue, derivative, or fragment thereof. In some embodiments, the secretory signal peptide is or is derived from a suitable secretory signal sequence, such as, but not limited to, a human growth factor, a human cytokine, interleukin-1, interleukin-2, human immunoglobulin kappa light chain, trypsinogen, serum albumin, prolactin, tissue plasminogen activator, alkaline phosphatase, or other suitable secretory signal sequence described herein.In some embodiments, the secretory signal peptide is derived from human tissue plasminogen activator. In some embodiments, the secretory signal peptide is derived from human tissue plasminogen activator comprising the amino acid sequence DAMKRGLCCVLLLCGAVFVSPSQ (SEQ ID NO: 65), or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the secretory signal peptide is derived from human tissue plasminogen activator comprising the amino acid sequence DAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGAR (SEQ ID NO: 66), or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the secretory signal peptide of the first polypeptide encoded by the polycistronic nucleic acid insert further comprises an initial amino acid methionine (M).

[0027] In some embodiments, one or more of the immune checkpoint inhibitor chimeric polypeptides include one or more peptide sequences fused to the C-terminus of the immune checkpoint inhibitor peptide that can be cleaved during or after translation of the polycistronic nucleic acid, or a combination thereof (see, e.g., FIG. 3A, FIG. 3B, and FIG. 3C). In some embodiments, the most C-terminal immune checkpoint inhibitor chimeric polypeptide does not include a cleavable peptide. In some embodiments, the cleavable peptide can be cleaved by a proprotein convertase enzyme, including, for example, but not limited to, furin or a furin-like proprotein convertase. 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 post-translationally processed into monomeric chimeric polypeptides through a cleavage mechanism. This allows each chimeric polypeptide containing an immune checkpoint inhibitor peptide to be secreted from the cell and function to downregulate undesirable immune checkpoint pathways (see, e.g., FIG. 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 capable of inducing ribozyme skipping during translation of a polycistronic nucleic acid. Ribosomal "skipping" is an alternative mechanism of translation in which certain peptide sequences prevent the ribosome from covalently ligating a newly inserted amino acid, but translation still continues. Induction of ribosomal skipping thus results in the "cleavage" of the polyprotein. In some embodiments, the peptide capable of inducing ribosomal skipping is a cis-acting hydrolase element peptide (CHYSEL). In some embodiments, the CHYSEL sequence comprises a non-conserved sequence of amino acids with strong alpha-helical propensity, followed by the consensus sequence D(V / I)EXNPGP (where X=any amino acid) (SEQ ID NO: 98), where ribosomal skipping cleavage occurs between the G and P sequences. 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 at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the CHYSEL peptide sequence is an amino acid sequence selected from SEQ ID NO: 100-SEQ ID NO: 122, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the CHYSEL peptide sequence is an amino acid sequence selected from SEQ ID NO: 118-SEQ ID NO: 122, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the CHYSEL peptide sequence comprises GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 120), or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.By including a peptide sequence that induces ribosome skipping, multiple chimeric polypeptides encoded by the polycistronic nucleic acid inserts are expressed as monomers, which are then secreted from the cell and function to downregulate undesirable immune checkpoint pathways (see, e.g., Figure 3B).

[0029] In some embodiments, the cleavable peptide sequence comprises two or more sequences that can be cleaved by different mechanisms, e.g., a cleavable peptide sequence that can be cleaved after translation of the polycistronic nucleic acid and a peptide sequence that can induce ribozyme skipping during translation of the polycistronic nucleic acid. By providing a cleavable peptide sequence that is amenable to multiple cleavage schemes, the efficiency of monomer formation from the polycistronic nucleic acid can be improved. In some embodiments, the immune checkpoint inhibitor peptide is fused at its C-terminus to a furin-cleavable peptide sequence, e.g., the peptide sequence RX(R / K)R (where X=any amino acid) (SEQ ID NO: 91), and fused to the C-terminus of the furin-cleavable peptide sequence is a CHYSEL peptide sequence that includes, e.g., 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, and after post-translational processing and cleavage of the furin peptide, all but the arginine (R) and alanine (A) residues of the furin cleavage sequence remain at the C-terminus of the immune checkpoint inhibitor peptide, limiting potential interference of the extra amino acid sequence with the function of the immune checkpoint inhibitor peptide (see, e.g., FIG. 3C). In alternative embodiments, the use of the furin-cleavable peptides RRRR (SEQ ID NO: 94), RKRR (SEQ ID NO: 95), or RRKR (SEQ ID NO: 96) results in the removal of the complete furin cleavage 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 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 at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavage sequence is RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 125), or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavage sequence is RRKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 126), or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavage sequence is RKKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 127), or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0030] In some embodiments, the rMVA viral vector comprises a heterologous polycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence selected from SEQ ID NO:309-340, or SEQ ID NO:341-348. In some embodiments, the rMVA viral vector comprises a heterologous polycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence selected from SEQ ID NO:325-340, or SEQ ID NO:345-348. In some embodiments, the rMVA viral vector comprises a heterologous polycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NO:325. In some embodiments, the rMVA viral vector comprises a heterologous polycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NO:329. In some embodiments, the rMVA viral vector comprises a heterologous polycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NO:333. In some embodiments, the rMVA viral vector comprises a heterologous polycistronic nucleic acid insert encoding a polypeptide having an amino acid sequence of SEQ ID NO:337.

[0031] Transcription of the nucleic acid insert may be initiated by one or more promoters compatible with MVA viral vectors operably linked thereto and located 5' to the first start codon of the first coding sequence contained within the nucleic acid. Suitable promoters compatible with poxvirus expression vectors are known in the art and include, but are not limited to, pmH5, p11, pSyn, pHyb, or any other suitable MVA promoter sequence. In some embodiments, the promoter is the native promoter for the MVA ORF. In some embodiments, the promoter is selected from the promoters in Table 7, or a nucleic acid having a sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter sequence is selected from SEQ ID NO: 128 to SEQ ID NO: 308, or a nucleic acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter sequence is selected from SEQ ID NO: 130 to SEQ ID NO: 132, or a nucleic acid sequence 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 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 polypeptides are transcribed as a single transcript, so that the polycistronic nucleic acid insert contains one or more termination signals (e.g., a stop codon such as TAA, TAG, or TGA, or combinations or repeats thereof) following only the ORF sequence of the last chimeric polypeptide. When transcribed, the multiple chimeric polypeptides give rise to a single transcript, which is then translated. After post-translational processing, multiple monomeric chimeric polypeptides are generated.

[0033] The provided rMVA virus constructs of the present invention can be used as adjuvants to treat or prevent infectious diseases or cancer in a subject, or to induce an immune response against infectious diseases or cancer. In some embodiments, the rMVA virus constructs are administered to a subject in need thereof, e.g., a human, in a prophylactic vaccination protocol to prevent infectious diseases, e.g., in a prime step, a boost step, or both a prime step and a boost step. In alternative embodiments, the rMVA virus constructs are administered to a subject in need thereof, e.g., a human, in a therapeutic regimen, including a vaccination protocol to treat cancer. Thus, the rMVA virus constructs can be administered in concert with one or more antigens intended to induce an immune response against an antigenic target, to induce partial or complete immunization in a subject in need thereof.

[0034] Thus, the rMVA of the present invention can be administered with one or more antigens targeting infectious diseases or cancer. Examples of antigens and antigen delivery vehicles with which the rMVA can be used as an adjuvant include antigenic proteins, polypeptides, or peptides, or fragments thereof, nucleic acids, such as mRNA or DNA, encoding one or more antigens, polysaccharides or polysaccharide-protein conjugates, 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 aspect, the present invention provides a method for preventing or treating, or inducing an immune response against, an infectious disease in a subject in need thereof, comprising administering an effective amount of an rMVA of the present invention in combination, alternating or linked with a prophylactically or therapeutically effective amount of one or more antigens or vectors expressing antigens, wherein the rMVA enhances immunity against the targeted infectious disease.

[0036] In some embodiments, the target infection is a viral infection, including but not limited to a double-stranded DNA virus, including but not limited to adenovirus, herpes virus, and poxvirus; a single-stranded DNA virus, including but not limited to parvovirus; a double-stranded RNA virus, including but not limited to reovirus; a coronavirus, e.g., SARS-CoV2; a positive single-stranded RNA virus, including but not limited to picornavirus, and togavirus; a negative single-stranded RNA virus, including but not limited to orthomyxovirus and rhabdovirus; a single-stranded RNA retrovirus, including but not limited to retrovirus; or a double-stranded DNA retrovirus, including but not limited to hepadnavirus. In some embodiments, the target virus is an adenovirus, an avian influenza virus, a coxsackievirus, a cytomegalovirus, a dengue virus, an Ebola virus, an Epstein-Barr virus, an equine encephalitis virus, a flavivirus, a hepadnavirus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a hepatitis D virus, a hepatitis E virus, a herpes simplex virus, a human immunodeficiency virus, a human papillomavirus, an influenza virus, a Japanese encephalitis virus, a JC virus, a measles morbillivirus, a Marburg virus, a Middle East respiratory syndrome coronavirus, a mumps rubra virus, an orthomyxovirus, a papillomavirus, a parainfluenza virus, a parvovirus, a picornavirus, a poliovirus, a poxvirus, a rabies virus, a reovirus, a respiratory syncytial virus, a retrovirus, a rhabdovirus, a rhinovirus, a Rift Valley fever virus, a rotavirus, a rubella virus, a measles virus, a rubella virus, a measles virus, a mumps virus, a rubella virus, a mumps ... virus), severe acute respiratory syndrome coronavirus type 1, severe acute respiratory syndrome coronavirus type 2, smallpox virus, togavirus, swine influenza virus, varicella-zoster virus, variola major virus, variola minor virus, and yellow fever virus.

[0037] In some embodiments, the target infection is selected from the group consisting of Borrelia species, Bacillus anthraces, Borrelia burgdorferi, Bordetella pertussis, Campylobacter jejuni, Chlamydia species, Chlamydial psittaci, Chlamydial trachomatis, Clostridium species, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, and the like. perfringens, Corynebacterium diphtheriae, Coxiella species, Enterococcus species, Erlichia species, Escherichia coli, Francisella tularensis, Haemophilus species, Haemophilus influenzae, Haemophilus parainfluenzae, Lactobacillus species, Legionella species, Legionella pneumophila, Leptospirosis interrogans interrogans, Listeria species, Listeria monocytogenesmonocytogenes, Mycobacterium species, Mycobacterium tuberculosis, Mycobacterium leprae, Mycoplasma species, Mycoplasma pneumoniae, Neisseria species, Neisseria meningitidis, Neisseria gonorrhoeae, Pneumococcus species, Pseudomonas species, Pseudomonas aeruginosa, Salmonella species, Salmonella typhi typhi, Salmonella enterica, Streptococcus species, Rickettsia species, Rickettsia ricketsii, Rickettsia typhi, Shigella species, Staphylococcus species, Staphylococcus aureus, Streptococcus species, Streptococcus pneumoniae, Streptococcus pyrogenes, Streptococcus mutans mutans, Treponema species, Treponema pallidum, Vibrio speciesThe bacteria include, but are not limited to, Bacillus subtilis species, Vibrio cholerae, and Yersinia pestis.

[0038] In some embodiments, the target infection is selected from the group consisting of Aspergillus species, Candida species, Candida albicans, Candida tropicalis, Cryptococcus species, Cryptococcus neoformans, Entamoeba histolytica, Histoplasma capsulatum, Leishmania species, Nocardia asteroides, Plasmodium falciparum, Toxoplasma gondii, Trichomonas vaginalis, and the like. Fungal infections include, but are not limited to, fungal infections caused by the fungi Toxoplasma species, Trypanosoma brucei, Schistosoma mansoni, Fusarium species, and Trichophyton species.

[0039] In some embodiments, the targeted infectious disease is a parasite, including but not limited to, a parasite from the following genera: Plasmodium species, Toxoplasma species, Entamoeba species, Babesia species, Trypanosoma species, Leshmania species, Pneumocystis species, Trichomonas species, Giardia species, and Schisostoma species.

[0040] In some embodiments, a method for preventing or treating, or inducing an immune response against, cancer in a subject in need thereof, comprising administering an effective amount of an rMVA of the present invention in combination, alternating or in conjunction with a prophylactically or therapeutically effective amount of one or more tumor-associated antigens or vectors expressing tumor-associated antigens, wherein the rMVA enhances immunity against the cancer. In some embodiments, the tumor associated antigens (TAA) include, but are not limited to, oncofetal TAAs, which are typically expressed only in fetal tissues and cancerous somatic cells; oncoviral TAAs, which are typically encoded by oncogenic transforming viruses; overexpressed / accumulated TAAs, which are typically expressed by both normal and neoplastic tissues, with expression levels greatly elevated in neoplasms; cancer-testicular TAAs, which are typically expressed only by cancer cells and adult reproductive tissues such as testis and placenta; lineage-restricted TAAs, which are typically expressed primarily by a single cancer tissue type; mutated TAAs, which are typically expressed only by cancers as a result of genetic mutations or transcriptional changes; post-translationally modified TAAs, which typically have tumor-associated changes in glycosylation, etc.; and idiotypic TAAs, which are highly polymorphic genes similar to B-cell, T-cell lymphomas / leukemias that typically result from clonal abnormalities in which tumor cells express a particular "clonotype," i.e., a clonal abnormality.In some embodiments, the TAA is selected from the group consisting of Wilms tumor protein (WT1), melanoma antigen preferentially expressed in tumors (PRAME), survivin, cancer / testis antigen 1 (NY-ESO-1), melanoma associated antigen 3 (MAGE-A3), melanoma associated antigen 4 (MAGE-A4), proteinase 3 (Pr3), cyclin A1, highly homologous synovial sarcoma X2 (SSX2), neutrophil elastase (NE), mucin 1 (MUC1), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), epithelial tumor antigen (ETA), tyrosinase, ras. aberrant products, aberrant products of p53, Epstein-Barr virus early 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-glycolyl GM3, NeuGcGM3, and fucosyl-GM1, or combinations thereof.

[0041] In some embodiments, the antigen is derived from the amino acid sequence of SEQ ID NO:349-SEQ ID NO:394.

[0042] In alternative embodiments, the rMVA viral vectors of the present invention, in addition to being capable of expressing multiple immune checkpoint inhibitor peptides, can be further constructed to encode and express one or more antigenic peptides. The one or more antigenic peptides can be encoded on one or more separate nucleic acid inserts, or in alternative embodiments, the one or more antigenic peptides are encoded on the same polycistronic nucleic acid insert as multiple immune checkpoint inhibitor peptides. Provided herein in some embodiments is an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, the polypeptide comprising the sequence of ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) xProvided is a rMVA viral vector comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine. In some embodiments, the antigenic peptide is comprised in a chimeric polypeptide that includes a secretory signal peptide fused to the N-terminus of the antigenic peptide, e.g., ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)). x (secretory signal peptide-antigenic peptide) (where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine) (see, e.g., Figures 4A and 4B). In some embodiments, antigenic peptides are also provided where two or more antigenic peptides are encoded in a polycistronic nucleic acid insert, with each chimeric polypeptide separated by a cleavable peptide as described herein. In some embodiments, the antigenic peptide is comprised in a chimeric polypeptide comprising a secretory signal peptide fused to the N-terminus of the antigenic peptide and a cleavable peptide fused to the C-terminus of the antigenic peptide, e.g., ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (secretory signal peptide-antigenic peptide-cleavable peptide) y ) (where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 comprise a cleavable sequence, e.g., ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (secretory signal peptide-antigenic peptide-cleavable peptide) x(secretory signal peptide-antigenic peptide)), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine. In some embodiments, the antigenic peptide included in the chimeric polypeptide comprising a secretory 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 5' to the immune checkpoint inhibitor peptide-containing chimeric polypeptide, e.g., ((M)(secretory signal peptide-antigenic peptide-cleavable peptide)). y (Secretory signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x ), or alternatively ((M) (secretory signal peptide-antigenic peptide-cleavable peptide) y (Secretory signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x (secretory signal peptide-immune checkpoint inhibitor peptide) where y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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, a synthetic secretory signal, or a consensus secretory signal peptide. In some embodiments, the antigenic peptide is selected from SEQ ID NO: 349 to SEQ ID NO: 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 are capable of forming a non-infectious virus-like particle (VLP). In some embodiments, provided herein is an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, the polypeptide comprising: ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x Provided are rMVA viral vectors comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine (see, e.g., Figures 5A and 5B). In some embodiments, the antigenic peptide is comprised 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, e.g., ((M)(secretory 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 more 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, e.g., ((M)(secretory signal 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 more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine). In some embodiments, the (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-cleavable peptide) y (where y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) may be oriented such that in a polycistronic nucleic acid insert, the nucleic acid encoding the antigen-containing chimeric polypeptide is located 5' to the immune checkpoint inhibitor peptide-containing chimeric polypeptide, e.g., ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-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 (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 more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine. In still further embodiments, the polycistronic nucleic acid insert of the rMVA further encodes a viral matrix protein, such as ((M)(secretory 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 more than 10 and M=methionine (see, e.g., Figures 6A and 6B). In alternative embodiments, the coding sequences for both the antigen-containing chimeric polypeptide and the viral matrix protein are contained in one or more copies in the polycistronic nucleic acid, such as ((M)(secretory 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 more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine). In some embodiments, the most C-terminal viral matrix protein lacks a cleavable peptide, e.g., ((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)) (where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 more 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, e.g., ((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 (secretory signal peptide-immune checkpoint inhibitor peptide) where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine. In some embodiments, the native secretory signal from the antigen is replaced with a viral glycoprotein signal peptide. In some embodiments, the antigenic peptide is selected from SEQ ID NO:349 to SEQ ID NO:394.

[0044] The generation of virus-like particles containing target antigens is particularly suitable for use in vaccine strategies against enveloped viruses, as they can induce both strong and sustained humoral and cellular immune responses. See, e.g., 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. Glycoproteins and matrix proteins suitable for use in generating antigen-containing VLPs include, but are not limited to, those derived from Filoviridae, such as Marburg virus, Ebola virus, or Sudan virus, Retroviridae, such as Human Immunodeficiency Virus Type 1 (HIV-1), Arenaviridaea, such as Lassa virus, Flaviviridae, such as 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 MARV GP protein (Genbank Accession No. AFV31202.1). The amino acid sequence of MARV GP protein is shown in Table 10 below as SEQ ID NO: 395.In certain embodiments, the MARV GPS domain comprises amino acids 2 to 19 of the glycoprotein (WTTCFFISLILIQGIKTL) (SEQ ID NO: 396, which may be encoded by, e.g., an MVA-optimized nucleic acid sequence of SEQ ID NO: 397), and the GPTM domain comprises amino acid sequence 644 to 673 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG) (SEQ ID NO: 398, which may be encoded by, e.g., an MVA-optimized nucleic acid sequence of SEQ ID NO: 399). In some embodiments, the MARV GPS signal further comprises a methionine as the first amino acid.

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

[0046] In some embodiments, the rMVA 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 are capable of forming a non-infectious virus-like particle (VLP).

[0047] In alternative embodiments, the rMVA viral vectors of the present invention, in addition to being capable of expressing multiple immune checkpoint inhibitor peptides, are further constructed to encode and express one or more antigenic peptides, where the one or more antigenic peptides are encoded on one or more separate nucleic acid inserts.

[0048] In some embodiments, the present invention relates to a method for the preparation of a chimeric polypeptide comprising administering to a patient ... x ) (where x=1-10 and M is methionine), wherein the heterologous nucleic acid insert is under the control of a vaccinia virus promoter. In certain aspects, provided herein is a viral vector comprising one or more heterologous nucleic acid inserts encoding one or more chimeric polypeptides, each chimeric polypeptide comprising ((M)(immune checkpoint inhibitor peptide) x ) (where x=1-10, the immune checkpoint inhibitor comprises SEQ ID NO:1, and M is methionine), wherein the heterologous nucleic acid insert is under the control of a vaccinia virus promoter. In certain aspects, provided herein is a viral vector comprising one or more heterologous nucleic acid inserts encoding one or more chimeric polypeptides, each chimeric polypeptide comprising ((M)(immune checkpoint inhibitor peptide) x ) (wherein x=1-10, the immune checkpoint inhibitor comprises SEQ ID NO:5, and M is methionine), wherein the heterologous nucleic acid insert is under the control of a vaccinia virus promoter.

[0049] In some aspects, provided herein is a recombinant modified vaccinia Ankara (rMVA) viral vector comprising: i) a first nucleic acid sequence encoding a chimeric amino acid sequence comprising (a) an extracellular fragment of MUC-1, (b) a transmembrane domain of the glycoprotein (GP) of Marburg virus (MARV), and (c) an intracellular fragment of MUC-1; ii) a second nucleic acid sequence encoding a 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 viral 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 a particular aspect, provided herein is a recombinant modified Vaccinia Ankara (rMVA) viral 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 viral promoter, and upon expression, the chimeric amino acid sequence and VP40 matrix protein can assemble together to form a virus-like particle (VLP). In a particular aspect, provided herein is a recombinant modified vaccinia Ankara (rMVA) viral 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 a 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 viral 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 the 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 naturally occurring deletion site, an engineered naturally occurring 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 naturally occurring deletion site, an engineered naturally occurring 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 naturally occurring deletion sites, different engineered 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 the two essential, highly conserved MVA genes and the matrix protein sequence is inserted into the reconstructed modified deletion III.

[0055] In another embodiment, a first nucleic acid sequence is inserted between I8R and G1L of the MVA gene, a second nucleic acid sequence is inserted between A50R and B1R of the MVA gene at the reconstructed modified deletion site III, and a 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 NO:128 to SEQ ID NO:308.

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

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

[0059] Also provided herein are shuttle vectors comprising the polycistronic nucleic acid sequences described herein for insertion into the MVA, as well as isolated nucleic acid sequences comprising the polycistronic nucleic acid sequence inserts described herein. Further provided herein are cells comprising the rMVA viral vectors described herein. [Brief description of the drawings]

[0060] [Figure 1A] 1 shows an exemplary diagram of an exemplary recombinant MVA viral vector of an open reading frame (ORF) of a polycistronic nucleic acid insert encoding multiple chimeric polypeptides, where each chimeric polypeptide comprises 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. The polycistronic nucleic acid insert can encode from 2 to 10 or more chimeric polypeptides, each of which comprises a methionine as its first amino acid. [Figure 1B]1 shows an exemplary diagram of an exemplary recombinant MVA virus vector containing a polycistronic nucleic acid insert encoding multiple chimeric polypeptides, 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 illustrated, a promoter capable of initiating transcription of the ORF of MVA (e.g., mH5 promoter (pmH5)) is operably linked 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 5'-most chimeric polypeptide. As illustrated, there is a stop codon 3' of the ORF of the last chimeric polypeptide. [Figure 2A] 1 shows an exemplary diagram of an exemplary recombinant MVA viral vector of the open reading frame (ORF) of a polycistronic nucleic acid insert encoding multiple chimeric polypeptides, where all chimeric polypeptides, except the most C-terminal chimeric polypeptide lacking a cleavable peptide, contain 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 from 2 to 10 or more chimeric polypeptides, each of which contains a methionine as its first amino acid. [Figure 2B]1 shows an exemplary diagram of an exemplary recombinant MVA virus vector containing a polycistronic nucleic acid insert encoding multiple chimeric polypeptides, where each chimeric polypeptide, except for the most C-terminal chimeric polypeptide lacking a cleavable peptide, contains 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 illustrated, a promoter capable of initiating transcription of the ORF of MVA (e.g., mH5 promoter (pmH5)) is operably linked 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 illustrated, there is a stop codon 3' of the ORF of the last chimeric polypeptide. [Figure 3A-3C]3A shows an exemplary schematic diagram of translational processing of various expressed chimeric polypeptides encoded by the polycistronic nucleic acid inserts of the present invention. In FIG. 3A, the chimeric polypeptide encodes a cleavable peptide sequence, such as a furin cleavage sequence or a furin-like cleavage sequence, which is cleaved after translation of the polycistronic nucleic acid transcript. In addition, 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 FIG. 3B, the chimeric polypeptide encodes a cleavable peptide sequence, such as a CHYSEL cleavage sequence, which induces ribosome skipping, where the polyprotein undergoes co-translational cleavage, resulting in the production of a monomeric immune checkpoint inhibitor peptide during translation. After or during translation, the chimeric polypeptide undergoes further cleavage of the secretory signal peptide, and the resulting monomeric immune checkpoint inhibitor peptide is subsequently secreted from the cell. In FIG. 3C, the chimeric polypeptide encodes multiple cleavable peptide sequences, such as both a furin cleavage sequence or a furin-like cleavage sequence and a CHYSEL sequence, such as 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 co-translational cleavage, resulting in the production of a monomeric immune checkpoint inhibitor peptide during translation. The monomeric immune checkpoint inhibitor peptide undergoes further processing during or after translation, where the secretory signal peptide is cleaved. Furthermore, the furin peptide sequence or the furin-like peptide sequence is cleaved post-translationally, resulting in a monomeric immune checkpoint inhibitor peptide that contains only the arginine (R) and alanine (A) residues of the furin cleavage sequence or the furin-like cleavage sequence, reducing the possibility of interference by the immune checkpoint inhibitor peptide. [Figure 4A]1 shows an exemplary diagram of an exemplary recombinant MVA viral vector of an open reading frame (ORF) of a polycistronic nucleic acid insert encoding multiple chimeric polypeptides comprising tandem repeats 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 a chimeric polypeptide comprising a signal peptide fused to an antigenic peptide, with the antigen-containing chimeric polypeptide fused to the most C-terminal immune checkpoint inhibitor-containing chimeric peptide. The polycistronic nucleic acid insert can encode one to ten or more immune checkpoint inhibitor-containing chimeric peptides, including a methionine as its first amino acid. This same general concept above can be applied to any of the constructs provided herein that include a cleavable sequence. [Figure 4B] 1 shows an exemplary diagram of an exemplary recombinant MVA viral vector containing a polycistronic nucleic acid insert encoding multiple chimeric polypeptides containing a tandem repeat of 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 containing a secretory signal peptide (SP) fused to an antigenic peptide (antigen), the antigen-containing chimeric polypeptide being 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., mH5 promoter (pmH5)) is operably linked to the nucleic acid encoding 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 illustrated, there is a stop codon 3' of the ORF of the last chimeric polypeptide. [Figure 5A]1 shows an exemplary diagram of an exemplary recombinant MVA viral vector of an open reading frame (ORF) of a polycistronic nucleic acid insert encoding multiple chimeric polypeptides comprising tandem repeats 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 comprising 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 from 1 to 10 or more immune checkpoint inhibitor-containing chimeric polypeptides and includes a methionine as its first amino acid. [Figure 5B] 1 shows an exemplary diagram of an exemplary recombinant MVA viral vector containing a polycistronic nucleic acid insert encoding multiple chimeric polypeptides containing a secretory signal peptide (SP), a tandem repeat of 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 containing a viral glycoprotein signal peptide (GPSP) fused to an antigenic peptide (antigen), which is fused to the transmembrane domain of a 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., mH5 promoter (pmH5)) is operably linked to the polycistronic nucleic acid encoding 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 illustrated, there is a stop codon 3' to the ORF of the last polypeptide. [Figure 6A]1 shows an exemplary diagram of an exemplary recombinant MVA viral vector of an open reading frame (ORF) of a polycistronic nucleic acid insert encoding multiple chimeric polypeptides comprising tandem repeats 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 comprising a viral glycoprotein signal peptide fused to an antigenic peptide, the antigenic peptide being fused to a 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 peptide and further comprises a viral matrix protein, where the viral matrix protein is fused to the C-terminus of the cleavable peptide of the antigen-containing chimeric polypeptide. The polycistronic nucleic acid insert can encode from 1 to 10 or more immune checkpoint inhibitor-containing chimeric polypeptides and includes a methionine as its first amino acid. [Figure 6B]1 shows an exemplary diagram of an exemplary recombinant MVA viral vector comprising a polycistronic nucleic acid insert encoding multiple chimeric polypeptides 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 a viral glycoprotein transmembrane domain (GPTM) fused to a cleavable peptide, where the antigen-containing chimeric polypeptide is fused to the most C-terminal immune checkpoint inhibitor-containing chimeric peptide, and further comprising a viral matrix protein, where the viral matrix protein is fused to the C-terminus of the cleavable peptide of the antigen-containing chimeric 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 multiple chimeric polypeptides. The insert may contain a translation initiation sequence, e.g., a Kozak sequence, prior to the start codon of the 5'-most chimeric polypeptide ORF. As illustrated, there is a stop codon 3' to the viral matrix protein ORF. [Figure 7] Schematic diagram of the MVA-5X.LD01 and MVA-5X.LD10 vectors showing the design of the peptide sequence inserted into the MVA genome between two essential genes under the control of an MVA-specific promoter. The LD01 and LD10 sequences are preceded by a signal sequence that routes and secretes the peptide, and are followed by a cleavage site that separates the repeated peptides. After five repeats of the secretion signal, peptide sequence, and cleavage site, transcription terminates with a stop codon. [Figure 8]Figure 8A 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, cells were fixed, permeabilized, and stained with antibodies specific for LD01 and LD10. The results show that the peptides are detected intracellularly. LD01- and LD10-positive cells were stained as indicated. Photomicrographs 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, supernatants were collected, concentrated, and spotted onto membranes with chemically synthesized peptide (LD01) and probed with antibodies specific for LD01 and LD10. The results show that the peptides are secreted from infected cells. [Figure 9]9A and 9B show that delivery of LD01 or LD10 via viral vector enhances vaccine-induced antigen-specific CD8+ T cell proliferation. (FIG. 9A and FIG. 9B) Immunogenicity was assessed 12 days after AdPyCS immunization by measuring the number of splenic PyCS-specific IFN-γ-secreting CD8+ T cells using an ELISpot assay (FIG. 9A) and flow cytometry (FIG. 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 SC. For viral vectors, 107 TCID50 of MVA-5X.LD01, MVA-5X.LD10, or parental MVA was injected SC after vaccination. Data are presented as mean ± SEM. Data from one of two independent experiments are shown. Significant differences between AdPyCS alone and treated mice were determined using unpaired two-tailed t-tests and are indicated by ** (p<0.001), *** (p<0.0005), and **** (p<0.0001). For Figure 9A, the x-axis is AdPyCS alone and treated mice, and the y-axis is the number of IFN-γ spots per 1x106 splenocytes measured in counts. For Figure 9B, the x-axis is AdPyCS alone and treated mice, and the y-axis is the number of IFN-γ CB8T cells within total CB8T cells measured in percentage. [Figure 10] Figure 1 shows a PCR gel of LD10, MUC-1, and VP40 inserts amplified from DNA samples of DF-1 cells infected with MVA-VLP-MUC-1-LD10 virus. DF1 cells infected with parental MVA (negative control), plasmids carrying the LD10, MUC-1, or VP40 inserts (positive control), or MVA-VLP-MUC-1-LD10 recombinant virus were harvested for viral DNA. Insert integrity was confirmed by PCR analysis. [Figure 11]Expected PCR fragment sizes of LD10 insert, MUC-1 insert, and VP40 insert collected from DF-1 cells infected with MVA-VLP-MUC-1-LD10 virus are shown. The expected fragment sizes matched the PCR gel band sizes. [Figure 12] Figure 1 shows the expression of recombinant MUC-1 protein in DF-1 cells infected with MVA-VLP-MUC-1-LD10. DF1 cells were infected with 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 harvested for protein and analyzed by immunoblotting. Membranes were probed with MUC-1 antibody (mouse monoclonal VU4H5, Santa Cruz no. sc-7313, 1:200), which labeled a protein band of approximately 63 kDa in the MVS-VLP-MUC-1-LD10 lysate sample. [Figure 13] Figure 1 shows the expression of recombinant VP40 protein in DF-1 cells infected with MVA-VLP-MUC-1-LD10. DF1 cells were infected with 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 harvested for protein and analyzed by immunoblotting. Membranes were probed with VP40 antibody, which labeled a protein band of approximately 32 kDa in MVS-VLP-MUC-1-LD10 supernatant and lysate samples. [Figure 14]Figure 1 shows the expression of recombinant LD10 protein in DF-1 cells infected with MVA-VLP-MUC-1-LD10. DF1 cells were transfected with parental modified vaccinia Ankara (pMVA) or MVA encoding VLP-MUC-1-LD10. Uninfected cells were included as a negative control. Cell lysates were harvested for protein and applied to nitrocellulose membranes using a dot blot apparatus. As a positive control for the LD10 antibody, 20 micrograms of LD10 peptide was also loaded onto the membrane. The membrane was probed with LD10 antibody, showing a signal in the MVA-VLP-MUC-1-LD10 peptide sample and the LD10 peptide sample. [Figure 15] Figure 1 shows the percentage of MUC-1 positive plaques after infection of 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 (PFU) and 60 PFU of MVA-VLP-MUC-1-LD10 virus, respectively, in triplicate wells. All wells were probed with MUC-1 antibody and the number of MUC-1 positive plaques was counted. The wells were then washed and then probed again with MVA antibody and the number of MVA positive plaques was counted. To calculate the purity of the vaccine, the percentage of MUC-1 positive plaques relative to the number of MVA positive plaques is shown. The number of positive plaques for each individual replicate is shown at the bottom of the figure. [Figure 16]Figure 1 shows the percentage of VP40-positive plaques after infection of 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 (PFU) and 60 PFU of MVA-VLP-MUC-1-LD10 virus, respectively, in triplicate wells. All wells were probed with MUC-1 antibody and the number of VP40-positive plaques was counted. The wells were then washed and then probed again with MVA antibody and the number of MVA-positive plaques was counted. To calculate the purity of the vaccine, the percentage of VP40-positive plaques relative to the number of MVA-positive plaques is shown. The number of positive plaques per individual replicate is shown at the bottom of the figure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] definition Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise, e.g., "a peptide" or "a "chimeric polypeptide" includes a plurality of peptides or a plurality of chimeric polypeptides. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0062] As used herein, the term "adjuvant" refers to the use of a 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, e.g., a peptide, that is capable of inducing an immune response.

[0064] As used herein, "chimeric" or "fused" refers to the covalent linkage of non-naturally occurring peptides or proteins resulting in a hybrid polypeptide. Translation of the chimeric or fused polypeptides described herein imparts functional properties derived from each of the respective fused peptides or proteins.

[0065] As used herein, "coding sequence" or "nucleic acid encoding" or "nucleic acid sequence encoding" and the like refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes an amino acid sequence, e.g., a polyprotein, polypeptide, protein, peptide, or fragments thereof. The coding sequence can further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in human or mammalian cells to which the nucleic acid is administered.

[0066] The term "conservative amino acid substitution" refers to the substitution of a natural amino acid residue with a non-natural 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 can 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 the corresponding modifications to the encoding nucleotides) can produce polypeptides with functional and chemical characteristics similar to those of the parent polypeptide.

[0067] The term "deletion" in reference to a polypeptide or protein refers to the removal of a codon for one or more amino acid residues from a polypeptide or protein sequence, with the flanking regions joined together. The term "deletion" in reference to a nucleic acid refers to the removal of one or more bases from a nucleic acid sequence, with the flanking regions joined together.

[0068] The term "fragment" in the context of a proteinaceous agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of a peptide, polypeptide, or protein. In one embodiment, a fragment constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference polypeptide. In one embodiment, a fragment of a full-length protein retains an activity of the full-length protein. In another embodiment, a fragment of a full-length protein does not retain an activity of the full-length protein.

[0069] The term "fragment", in the context of a nucleic acid, refers to a nucleic acid comprising a nucleic acid sequence of at least 2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, at least 15 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, at least 60 contiguous nucleotides, at least 70 contiguous nucleotides, at least 80 contiguous nucleotides, at least 90 contiguous nucleotides, at least 100 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 contiguous nucleotides, at least 175 contiguous nucleotides, at least 200 contiguous nucleotides, at least 250 contiguous nucleotides, at least 300 contiguous nucleotides, at least 350 contiguous nucleotides, or at least 380 contiguous nucleotides of a nucleic acid sequence encoding a peptide, polypeptide, or protein. In one embodiment, a fragment comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of a reference nucleic acid sequence. In a preferred embodiment, a fragment of a nucleic acid encodes a peptide or polypeptide that retains an activity of the full-length protein. In another embodiment, a fragment encodes a peptide or polypeptide that does not retain an activity of the full-length protein.

[0070] As used herein, the phrase "heterologous sequence" refers to any nucleic acid, protein, polypeptide or peptide sequence that is not normally associated in nature with another nucleic acid or protein, polypeptide or peptide sequence of interest.

[0071] As used herein, the phrase "heterologous nucleic acid insert" refers to any nucleic acid sequence that has been inserted or is to be inserted into a recombinant vector described herein. Heterologous nucleic acid insert may refer to only the sequence that encodes a gene product, or may refer to a sequence that includes a promoter, a sequence that encodes a gene product (e.g., a secretory signal peptide-immune checkpoint inhibitor peptide chimeric polypeptide), and any regulatory sequences associated or operably linked thereto.

[0072] The term "homopolymer stretch" refers to a sequence comprising at least four identical nucleotides, e.g., GGGG or TTTTTTT, uninterrupted by any other nucleotides.

[0073] The terms "percent identical", "percent homology", or "percent similarity", etc., when used in the context of nucleic acid sequences, refer to comparing residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may span the full length of the sequences, or alternatively a fragment of at least about 50 to 2500 nucleotides. Similarly, the terms "percent identical", "percent homology", or "percent similarity" may be readily determined for amino acid sequences spanning the full length of a protein or fragment thereof. Suitably, the fragment is at least about 8 amino acids in length and may be up to about 7500 amino acids. Examples of suitable fragments are described herein. In general, "identity", "homology", or "similarity" is determined with reference to an "aligned" sequence. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, and often includes correction for missing or additional bases or amino acids compared to a reference sequence. The alignment may be performed using any of a variety of 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 through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility is used. There are also many 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 Fasta™, a program in GCG version 6.1. Fasta™ provides alignments of the regions of highest overlap between the query sequence and the search sequence and percent sequence identity.For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (word size of 6 and NOPAM coefficient for scoring matrix) provided in GCG version 6.1 (herein incorporated by reference). For amino acid sequences, multiple sequence alignment programs are also available, such as the "Clustal Omega" program, the "Clustal X" program, the "MAP" program, the "PIMA" program, the "MSA" program, the "BLOCKMAKER" program, the "MEME" program, and the "Match-Box" program. Generally, all of these programs are used with default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as 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 "inducing an immune response" means eliciting a humoral response (e.g., production of antibodies) or a cellular response (e.g., activation of T cells), or both a humoral response and a cellular response, to one or more antigenic proteins or fragments thereof expressed by the rMVA in a subject to which the rMVA is administered.

[0075] The term "modified vaccinia Ankara," or "MVA," generally refers to a highly attenuated vaccinia virus strain developed by Dr. Anton Mayr through serial passage in chick embryo fibroblast cells, or to mutants or derivatives thereof. MVA is reviewed in Mayr, A. et al. 1975 Infection 3:6-14. The genomic sequences of MVA and various mutants are described, for example, in GenBank accession numbers AY603355, U94848, and DQ983238. In some embodiments, the MVA provided herein can be obtained synthetically, for example via a chemically synthesized plasmid, and can be reconstituted into a full-length genomic MVA sequence in a host cell, for example, as described in U.S. Patent Application Publication No. 2018 / 0251736, U.S. Patent Application Publication No. 2021 / 0230560, and International Publication No. WO 2021 / 158565 (each of which is incorporated herein by reference).

[0076] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose 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 a probe that hybridizes under stringent hybridization conditions.

[0077] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA (both genomic and cDNA), RNA, or hybrids, and can contain combinations of deoxyribonucleotides and ribonucleotides, and 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 the promoter to which it is spatially connected. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

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

[0080] As used herein, a "promoter" refers to a synthetic or naturally occurring molecule that can confer, activate or enhance transcription of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0081] The terms "prevent," "preventing," and "prevention" refer to inhibiting the occurrence or development of a condition (e.g., an infectious disease), or preventing the recurrence, onset, or occurrence of one or more symptoms of a condition in a subject resulting from the administration of a therapeutic agent or combination of therapeutic agents.

[0082] The term "prophylactically effective amount" refers to an amount of a composition (e.g., a targeted antigenic composition and / or rMVA described herein) that is sufficient to result in the prevention of the occurrence, recurrence or onset of a condition or a symptom thereof (e.g., a viral infection) or a symptom associated therewith, or to enhance or improve the prophylactic effect(s) of another therapy.

[0083] The term "recombinant" with reference to viral vectors means a vector (eg, a viral genome) that has been manipulated in vitro, eg, to express heterologous viral nucleic acid sequences, using recombinant nucleic acid techniques.

[0084] The terms "regulatory sequence" and "regulatory sequences" refer collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence. Not all of these control sequences need always be present so long as the selected gene is capable of being 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 is capable of replicating alone (without the presence of any other vector) in at least one host (e.g., E. coli). In the context of constructing MVA vectors, a shuttle vector is typically a DNA plasmid that can be manipulated in E. coli and then introduced into cultured cells infected with the MVA vector, resulting in the generation of new recombinant MVA vectors, for example, via homologous recombination.

[0086] The term "silent mutation" refers to a change in a nucleotide sequence that does not result in a change in the primary structure of the protein encoded by the nucleotide sequence, e.g., from AAA (encoding lysine) to AAG (also encoding lysine).

[0087] The "host," "patient," or "subject" to be treated is typically a human patient, although it should be understood that the methods described herein are effective on other animals, such as mammals. More specifically, the term patient can include animals used in assays, such as animals used in preclinical trials, including, but not limited to, mice, rats, monkeys, dogs, pigs, and rabbits, as well as domestic pigs (pigs and hogs), ruminants, horses, poultry, cats, cows, rats, dogs, etc. The determination of those "at risk" subjects can be made by any objective or subjective determination by the subject or the health care provider's diagnostic test or opinion (e.g., genetic testing, enzyme or protein markers, marker history, etc.).

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

[0089] The term "therapeutically effective amount" means an amount of a composition (e.g., an antigenic composition and / or a recombinant MVA vector or a pharmaceutical composition) that, when administered to a subject to treat or prevent a disorder, e.g., an infectious disease or cancer, is sufficient to affect such treatment or prevention of the disorder.

[0090] The term "treating" or "treat" refers to the eradication or control of a disorder, the reduction or amelioration of the progression, severity, and / or duration of a disorder or one or more symptoms caused by the disorder, resulting from the administration of one or more therapeutic modalities.

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

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

[0093] With respect to the recitation of numerical ranges herein, each intervening numerical value is expressly contemplated with the same degree of precision, for example, in the range of 6 to 9, the numerical values ​​7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numerical values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0094] Modified vaccinia Ankara (MVA) virus vector In particular, modified vaccinia Ankara (MVA) has been 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 embryo fibroblasts (CEFs) (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 its reduced virulence and reduced replicative capacity in primate cells, while maintaining good immunogenicity. The MVA virus has been analyzed to determine the genomic changes relative to the parental strain, chorioallantoic vaccinia virus Ankara (CVA). Six major deletions (deletion I, deletion II, deletion III, deletion IV, deletion V, and deletion VI) of genomic DNA totaling 31,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72: 1031-1038). The host cell of the resulting MVA virus is restricted to avian cells. Therefore, MVA vaccines can be produced on a large scale in chicken cell lines.

[0095] The viral vector compositions provided herein include the vaccinia virus strain Modified Vaccinia Ankara (MVA). Modified Vaccinia Ankara (MVA) was generated by long-term serial passage of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblast cells (for review, see Mayr A, et al., "Origin, properties, and uses of attenuated vaccinia strains," Infection 3: 6-14, 1975, Swiss Patent No. 568,392). MVA virus is publicly available from the American Type Culture Collection under ATCC number VR-1508. MVA is distinguished by its significant attenuation, as demonstrated by its reduced pathogenicity and reduced replicative capacity in primate cells, while maintaining good immunogenicity. MVA virus has been analyzed to determine genomic changes relative 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 is restricted in host cell replication to avian cells.

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

[0097] In alternative embodiments, the MVA is obtained from an MVA having a genomic sequence set forth in GenBank Accession Nos. AY603355, U94848, and DQ983238. In some embodiments, the MVA provided herein can be obtained synthetically, for example, via a chemically synthesized plasmid, and can be reconstituted into a full-length genomic MVA sequence in a host cell, for example, as described in U.S. Patent Application Publication No. 2018 / 0251736, U.S. Patent Application Publication No. 2021 / 0230560, and International Publication No. WO 2021 / 158565, each of which is incorporated herein by reference.

[0098] The construction of the recombinant MVA (rMVA) viral vector of the present invention can be prepared by methods known in the art. For example, a DNA construct comprising a heterologous polycistronic nucleic acid sequence as described herein may be flanked by DNA sequences of MVA flanking a predetermined insertion site (e.g., between two conserved essential MVA genes such as I8R / G1L (see, e.g., U.S. Pat. No. 9,133,478, which is incorporated herein by reference in its entirety), in a reconstructed and modified deletion III (see, e.g., U.S. Pat. No. 9,133,480, which is incorporated herein by reference in its entirety), or at other non-essential sites in the MVA genome), allowing homologous recombination upon introduction into a cell infected with MVA. Once the DNA construct has been introduced into a eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired rMVA in a manner known per se, preferably with the aid of a marker. The DNA construct to be inserted may be linear or circular. A plasmid or a polymerase chain reaction product is preferred. Such methods for producing recombinant MVA vectors are described, for example, in U.S. Patent No. 9,133,478, which is incorporated herein by reference. Expression of a DNA sequence or gene requires the presence on the DNA of regulatory sequences required for transcription of the polycistronic nucleic acid sequence. Such regulatory sequences (called promoters) are known to those skilled in the art and include, for example, those further described below.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 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 genomic MVA sequence in a host cell, for example, as described in U.S. Patent Application Publication No. 2018 / 0251736, U.S. Patent Application Publication No. 2021 / 0230560, and International Publication No. WO 2021 / 158565 (each of which is incorporated herein by reference).

[0100] As noted above, the heterologous polycistronic nucleic acid sequences of the invention can be inserted at any suitable site within the rMVA genome sequence, hi some embodiments, the polycistronic nucleic acid sequences are inserted into the MVA vector at a naturally occurring deletion site, an engineered naturally occurring deletion site, or between essential or non-essential MVA genes.

[0101] Immune Checkpoint Inhibitor Peptides Provided herein are compositions comprising recombinant modified vaccinia Ankara (rMVA) viral vectors for use as adjuvants or vaccines during immunization protocols in hosts, such as humans, where the rMVA is constructed to express high concentrations of 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 polycistronic nucleic acid sequences that contain tandem repeats of immune checkpoint inhibitors that can be processed into monomers and secreted from cells to enhance the immunogenicity of the targeted antigen. In some embodiments, the rMVA is used as an adjuvant to enhance the immunogenicity of one or more co-administered antigens during vaccination protocols. In some embodiments, the rMVA further encodes one or more antigenic peptides and is used as an adjuvanted vaccine. By expressing two or more immune checkpoint inhibitor peptides locally and in large amounts that can downregulate one or more checkpoint inhibitor pathways, immunomodulatory activities that often prevent the development of sufficient antigenicity to induce immunity can be downregulated.

[0102] In certain embodiments, the immune checkpoint inhibitor peptide is selected from the group consisting of, but not limited to, programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), programmed 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), V domain Ig suppressor of T cell activation (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), glucocorticoid-induced TNF The immune checkpoint inhibitor peptide may inhibit the activity of an immune checkpoint pathway mediated by a receptor protein selected from R-associated protein (GITR), CD137, CD40, B- and T-lymphocyte attenuator (BTLA), herpes virus 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 immunoreceptor with Ig and ITIM domains (TIGIT), inducible T cell costimulator (ICOS), inducible T cell costimulator ligand (ICOS-L), or a combination thereof. In some embodiments, the immune checkpoint inhibitor peptide may inhibit PD-1. In some embodiments, the immune checkpoint inhibitor peptide may inhibit PD-L1. In some embodiments, the immune checkpoint inhibitor peptide may inhibit CTLA-4. In some embodiments, the immune checkpoint inhibitor peptide can inhibit PD-1, PD-L1, or CTLA-4, or a combination thereof, hi some embodiments, the immune checkpoint inhibitor peptide 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 is capable of inhibiting 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 NO:1 to SEQ ID NO:56, or peptides having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide is selected from the peptide sequences of SEQ ID NO:1 to SEQ ID NO:15, or peptides having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO:1, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO:2, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 3, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 4, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 5, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 6, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 7, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 8, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 9, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 10, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 11, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 12, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 13, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 14, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the immune checkpoint inhibitor peptide has a peptide sequence of SEQ ID NO: 15, or a peptide having an amino acid sequence 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 NO: 16-56, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0105] [Table 1] TIFF2024505274000003.tif42170

[0106] The immune checkpoint inhibitors in Table 1, for example, SEQ ID NO: 1 to SEQ ID NO: 15, are described in U.S. Pat. Nos. 10,098,950, 10,799,555, and 10,799,581, as well as U.S. Patent Application Publication Nos. 2018 / 0071385, 2018 / 0185474, 2018 / 0200328, and 2018 / 0339044; SEQ ID NO: 16 to SEQ ID NO: 22, are described in Li et al., "Peptide Blocking of PD-1 / PD-L1 Interaction for Cancer Immunotherapy," Cancer Immunol Res February 1, 2018 (6) (2) 178-188; SEQ ID NO: 23 to SEQ ID NO: 26, are described in Liu et al. al., "Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy," J. Immunotherapy Cancer 7, 270 (2019), SEQ ID NOs: 27 to 31 are the sequences of the peptides in Keir et al., "D-1 and its ligands in T-cell immunity," Curr Opin Immunol. 2007;19(3):309-14, and Li et al., "Discovery of peptide inhibitors targeting human programmed death 1 (PD-1) receptor," Oncotarget. 2016;7(40):64967-64976, and SEQ ID NOs: 32 to 36 are the sequences of the peptides in Wang et al., Journal of Medicinal Chemistry 2019 62 (4), 1715-1730, SEQ ID NO: 37 to SEQ ID NO: 40 are the same as those in Xiao et al., ACS Appl. Mater.Interfaces 2020, 12, 36, 40042-40051, SEQ ID NO: 41 to SEQ ID NO: 42 are from Boohaker et al., "Rational design and development of a peptide inhibitor for the PD-1 / PD-L1 interaction," Cancer Letters, 2018, 434, Pages 11-21, SEQ ID NO: 43 to SEQ ID NO: 45 are from Zhai et al., "Antigen-specific CD8. + "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, and SEQ ID NOs: 46 to 56 have been previously described in "The biologically functional identification of a novel TIM3-binding peptide P26 in vitro and in vivo," Cancer Chemother Pharmacol. 2020;86(6):783-792, by Zhong et al. All references are incorporated herein by reference.

[0107] Secretory signal peptide As provided herein, the immune checkpoint inhibitor peptide expressed by the rMVA is secreted from the cell. In some embodiments, secretion can be achieved by including a natural secretion signal associated with the immune checkpoint inhibitor peptide, if applicable. In alternative embodiments, the immune checkpoint inhibitor peptide expressed by the rMVA may be heterologous to the host or may not have the appropriate secretion signaling to ensure secretion from the host cell. Thus, secretion of the immune checkpoint inhibitor peptide can be achieved by expressing a chimeric polypeptide that includes a secretion signal peptide fused to the immune checkpoint inhibitor peptide.

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

[0109] Secretion signals suitable for use in the present invention can be naturally occurring secretion signals, consensus secretion signals (see, for example, US Patent Application Publication No. 2010 / 0305002, which is incorporated herein by reference), or synthetic secretion signals.

[0110] In some embodiments, the secretion signal is selected from the peptide sequences in Table 2, or homologs, derivatives, or fragments thereof. In some embodiments, the secretion signal comprises a peptide sequence selected from SEQ ID NO:57 to SEQ ID NO:90, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

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

[0112] [Table 2] TIFF2024505274000005.tif22170

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

[0114] Cleavable sequence In addition to the secretory signal peptide at the N-terminus of each immune checkpoint inhibitor peptide, the polypeptide may also include an autocleaving peptide fused to the C-terminus of the immune checkpoint inhibitor peptide. By providing an autocleaving peptide sequence fused to the C-terminus of the 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), incorporated herein by reference in its entirety).

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

[0116] In some embodiments, the cleavable peptide may be cleaved by a proprotein convertase enzyme, including, for example, but not limited to, Furin or a Furin-like proprotein convertase (Table 3). In some embodiments, the cleavable peptide sequence comprises 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 comprises 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 post-translationally processed into monomeric chimeric polypeptides through a cleavage mechanism, allowing each chimeric polypeptide containing an immune checkpoint inhibitor peptide to be secreted from the cell and function to downregulate undesirable immune checkpoint pathways (see, e.g., FIG. 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 mechanism of translation in which certain peptide sequences prevent the ribosome from covalently linking the newly inserted amino acid, but translation still continues. This leads to the "cleavage" of the polyprotein by inducing ribosome skipping (see, for example, FIG. 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 sequence of amino acids with strong alpha-helical propensity followed by the consensus sequence D(V / I)EXNPGP (where X=any amino acid) (SEQ ID NO:98), where ribosome skipping cleavage occurs between the G and P sequences. 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. 2A sequences are oligopeptides located between the P1 and P2 proteins in some members of virus families, e.g., the Picornaviridae, that can undergo self-cleavage in eukaryotic cells to generate the 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., "Simultaneous expression of multiple proteins under a single promoter in Caenorhabditis elegans via a versatile 2A-based toolkit." Genetics. 2014;196:605-613; al., "Dissection of a co-translational nascent chain separation event." Biochem Soc Trans. 2008 Aug;36(Pt 4):712-6; Martin et al., "A Model for Nonstoichiometric, Cotranslational Protein Scission in Eukaryotic Ribosomes." Bioorganic Chemistry, Volume 27, Issue 1, February 1999, 55-79.The first 2A discovered was F2A (foot-and-mouth disease virus), and later E2A (equine rhinitis A virus), P2A (porcine teschovirus-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 a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the CHYSEL cleavage sequence is derived from one or more 2A self-processing peptides having an amino acid sequence selected from SEQ ID NO:100 to SEQ ID NO:117, or a peptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0122] [Table 4]

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

[0124] [Table 5]

[0125] In some embodiments, the cleavable peptide sequence comprises two or more sequences that can be cleaved by different mechanisms, for example, a cleavable peptide sequence that can be cleaved after translation of the polycistronic nucleic acid and a peptide sequence that can induce ribozyme skipping during translation of the polycistronic nucleic acid. By providing a cleavable peptide sequence that is amenable to multiple cleavage schemes, the efficiency of monomer formation from the polycistronic nucleic acid 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 (wherein X=any amino acid) (SEQ ID NO:91), and fused to the C-terminus of the furin-cleavable peptide sequence is a peptide that includes a CHYSEL peptide sequence, for example, the amino acid sequence D(V / I)EXNPGP (wherein 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 a monomeric chimeric polypeptide, and all but the arginine (R) and alanine (A) residues of the furin cleavage sequence remain at the C-terminus of the immune checkpoint inhibitor peptide, limiting the potential interference of extra amino acid sequences with the function of the immune checkpoint inhibitor peptide (see, for example, FIG. 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 a monomeric chimeric polypeptide, and the remaining furin cleavage sequence and CHYSEL peptide sequence are removed at the C-terminus of the immune checkpoint inhibitor peptide.

[0126] In some embodiments, the hybrid cleavable peptide sequence comprises RAKR (SEQ ID NO: 93) fused to CHYSEL comprising 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 comprises RAKR (SEQ ID NO: 93) fused to a peptide having a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 100 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RAKR (SEQ ID NO: 93) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RAKR (SEQ ID NO:93) fused to a peptide having the CHYSEL amino acid sequence of amino acids SEQ ID NO: 120, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In certain embodiments, the hybrid cleavable peptide is RAKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:123).

[0127] In some embodiments, the hybrid cleavable peptide sequence comprises RRRR (SEQ ID NO: 94) fused to CHYSEL comprising 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 comprises RRRR (SEQ ID NO: 94) fused to a peptide having a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 100 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RRRR (SEQ ID NO: 93) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RRRR (SEQ ID NO:94) fused to a peptide having the CHYSEL amino acid sequence of amino acids SEQ ID NO: 120, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In certain embodiments, the hybrid cleavable peptide is RRRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:124).

[0128] In some embodiments, the hybrid cleavable peptide sequence comprises RKRR (SEQ ID NO: 95) fused to CHYSEL comprising 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 comprises RKRR (SEQ ID NO: 95) fused to a peptide having a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 100 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RKRR (SEQ ID NO: 95) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RKRR (SEQ ID NO:95) fused to a peptide having the CHYSEL amino acid sequence of amino acids of SEQ ID NO: 120, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In certain embodiments, the hybrid cleavable peptide is RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:125).

[0129] In some embodiments, the hybrid cleavable peptide sequence comprises RRKR (SEQ ID NO: 96) fused to CHYSEL comprising 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 comprises RRKR (SEQ ID NO: 96) fused to a peptide having a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 100 to SEQ ID NO: 123, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RRKR (SEQ ID NO: 96) fused to a peptide having a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RRKR (SEQ ID NO:96) fused to a peptide having the CHYSEL amino acid sequence of amino acids SEQ ID NO: 120, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In certain embodiments, the hybrid cleavable peptide is RRKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:126).

[0130] In some embodiments, the hybrid cleavable peptide sequence comprises RKKR (SEQ ID NO: 97) fused to CHYSEL comprising 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 comprises RKKR (SEQ ID NO: 97) fused to a peptide having a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 100 to SEQ ID NO: 123, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RKKR (SEQ ID NO: 97) fused to a CHYSEL amino acid sequence selected from the group consisting of SEQ ID NO: 118 to SEQ ID NO: 122, or an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the hybrid cleavable peptide sequence comprises RKKR (SEQ ID NO:97) fused to a peptide having the CHYSEL amino acid sequence of amino acids SEQ ID NO: 120, or an amino acid sequence 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 Sequences As provided herein, immune checkpoint inhibitor peptides are expressed from nucleic acid sequences inserted into the appropriate position in the MVA genome sequence. For expression of nucleic acid inserts in the rMVA genome backbone, regulatory sequences such as promoters required for 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 that initiates transcription. When the nucleic acid insert is a polycistronic nucleic acid that encodes multiple proteins / peptides as a single polyprotein, one or more promoters can be located 5' to the transcription start site of the ORF that encodes the most N-terminal polypeptide of the polyprotein.

[0133] Since MVA is a cytoplasmic virus, in some embodiments, suitable promoters include those derived from naturally occurring poxvirus promoters. Poxvirus genes, promoters, and transcription factors are classified into early, middle, and late classes depending on the timing of their expression during poxvirus infection (see, e.g., Assarsson et al., "Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes." PNAS 2008;105(6):2140-2145; Yang Zet al., "Genome-wide analysis of the 5' and 3' ends of vaccinia virus early mRNAs delineates regulatory sequences of annotated and anomalous transcripts." J Virol. 2011;85(12):5897-5909). Replication of MVA in most mammalian cells (non-permissive cells) is arrested upon assembly of progeny virions after all stages of expression have been carried out.This supports the utility of all promoter classes, including late promoters, to 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 contain both early and late elements, allowing their open reading frames (ORFs) or recombinant antigens to be expressed early during viral infection and late after viral genome replication, respectively (Broyles SS, Vaccinia virus transcription. J Gen Virol. 2003;84(Pt 9):2293-2303).Poxvirus promoters can be used across strains (see Prideaux et al., "Comparative analysis of vaccinia virus promoter activity in fowlpox and vaccinia virus recombinants." Virus Res. 1990;16(1):43-57; Tripathy et al., "Regulation of foreign gene in fowlpox virus by a vaccinia virus promoter." Avian Dis. 1990;34(1):218-220).

[0134] Such MVA promoter sequences are known to those skilled in the art and include, for example, the p11 promoter driving expression of the 11k protein encoded by the ORF of F17R (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), the pI1L promoter (Schmitt et al., "Sequence and transcriptional analysis of the vaccinia virus HindIII I fragment." J. Virol. 1986;10(1):30-37), and the p21 promoter (Schmitt et al., "Sequence and transcriptional analysis of the vaccinia virus HindIII I fragment." J. Virol. 1987;10(1):30-37). 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."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 (Perkus et al., "Cloning and expression of foreign genes in vaccinia virus, using a host range selection system." J Virol. 1989;63(9):3829-3836), and the short synthetic promoter pSyn (Chakrabarti et al., "Compact, synthetic, vaccinia virus early / late promoter for protein expression." Biotechniques. 1997;23(6):1094-1097, Hammond et al., "A synthetic vaccinia virus promoter with enhanced early and late activity." J Virol Methods. 1997;66(1):135-1380), pmH5 promoter (Wyatt et al."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), the LEO promoter (Wyatt et al., "Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccine." J Virol. 2002;76(16):8318-8334), 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.2012;7(6):e40167), and 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, 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 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 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 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 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 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, those derived from the native promoter sequences shown in Table 7 below, or nucleic acid sequences at least 85%, 90%, 95%, 97%, or 99% identical thereto, where the nomenclature for the locus is based on the ORF nomenclature originally used for the WR and Copenhagen strains of vaccinia virus. In some embodiments, the promoter is selected from one or more of SEQ ID NO: 133 to SEQ ID NO: 308, or a combination thereof, or a nucleic acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0138] [Table 7] TIFF2024505274000011.tif247170TIFF2024505274000012.tif247170TIFF20245052740 00013.tif247170TIFF2024505274000014.tif247170TIFF2024505274000015.tif110170

[0139] Additionally, the nucleic acid sequence for insertion may further comprise an appropriate translation initiation sequence, such as, for example, the Kozak consensus sequence (GCCACC / ATG).

[0140] Additionally, the polycistronic nucleic acid sequence for insertion may include a suitable stop codon, such as TAA, TAG, or TGA, or combinations or repeats 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 3' of the last stop codon of the polyprotein. Additionally, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating shuttle vectors that facilitate insertion of the coding sequence for the immune checkpoint inhibitor.

[0141] antigenic target The provided rMVA virus constructs of the present invention can be used as adjuvants to treat or prevent infectious diseases or cancer in a subject. In some embodiments, the rMVA virus constructs are administered to a subject in need thereof, e.g., a human, in a prophylactic vaccination protocol, e.g., in a prime step, a boost step, or both a prime step and a boost step, to prevent infectious diseases. In alternative embodiments, the rMVA virus constructs are administered to a subject in need thereof, e.g., a human, in a therapeutic regimen, including a vaccination protocol, to treat, e.g., cancer. Thus, the rMVA virus constructs can be administered in concert with one or more antigens intended to induce an immune response against an antigenic target, to induce partial or complete immunization in a subject in need thereof.

[0142] Thus, the rMVA of the present invention can be administered with one or more antigens targeting infectious diseases or cancer. Examples of antigens and antigen delivery vehicles with which the rMVA can be used as an adjuvant include antigenic proteins, polypeptides, or peptides, or fragments thereof, nucleic acids, such as mRNA or DNA, encoding one or more antigens, polysaccharides or polysaccharide-protein conjugates, 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 aspect, the present invention provides a method for preventing or treating an infectious disease in a subject in need of prevention or treatment, comprising administering an effective amount of an rMVA of the present invention in combination, alternating or linked with a prophylactically or therapeutically effective amount of one or more antigens or vectors expressing antigens, wherein the rMVA enhances immunity against the targeted infectious disease.

[0144] In some embodiments, the target infection is a viral infection, including but not limited to a double-stranded DNA virus, including but not limited to adenovirus, herpes virus, and poxvirus; a single-stranded DNA virus, including but not limited to parvovirus; a double-stranded RNA virus, including but not limited to reovirus; a positive single-stranded RNA virus, including but not limited to coronavirus, picornavirus, and togavirus; a negative single-stranded RNA virus, including but not limited to orthomyxovirus and rhabdovirus; a single-stranded RNA retrovirus, including but not limited to retrovirus; or a double-stranded DNA retrovirus, including but not limited to hepadnavirus. In some embodiments, the target virus is an adenovirus, an avian influenza virus, a coxsackie virus, a cytomegalovirus, a dengue virus, an Ebola virus, an Epstein-Barr virus, an equine encephalitis virus, a flavivirus, a hepadnavirus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a hepatitis D virus, a hepatitis E virus, a herpes simplex virus, a human immunodeficiency virus, a human papilloma virus, an influenza virus, a Japanese encephalitis virus, a JC virus, a measles virus, a Marburg virus, a Middle East respiratory syndrome (MERS-CoV) coronavirus, a mumps rubra virus, , orthomyxoviruses, papillomaviruses, parainfluenza viruses, parvoviruses, picornaviruses, polioviruses, poxviruses, rabies virus, reoviruses, respiratory syncytial viruses, retroviruses, rhabdoviruses, rhinoviruses, Rift Valley fever virus, rotaviruses, rubella virus, measles virus, severe acute respiratory syndrome coronavirus 1 (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), smallpox virus, togaviruses, swine influenza virus, varicella-zoster virus, variola major virus, variola minor virus, and yellow fever virus.Examples of viruses that can be used as antigens include measles virus, mumps virus (mumps rubra virus), rubella virus, varicella zoster virus, or a combination of all four or three thereof (e.g., measles virus, mumps virus, and rubella virus).

[0145] In some embodiments, the target infectious agent is a Flaviviridae virus, including infections caused by viruses of the genera Flavivirus and Pestivirus. Flavivirus infections include dengue fever, Kyasanur Forest disease, Powassan disease, Wesselsbron disease, West Nile fever, yellow fever, Zika virus, Rio bravo, Rocio, Negishi, and encephalitis, including California encephalitis, Central European encephalitis, Irvine virus, Murray Valley encephalitis, St. Louis encephalitis, Japanese encephalitis type B, louping disease, and Russian spring-rodents summer encephalitis. Pestivirus infections include diseases of livestock, including swine fever, primarily 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, Mayaro virus, Ockelbo virus, O'Nyong-Nyong virus, Ross River virus, Semliki Forest virus, or Sindbis virus (SINV).

[0147] In some embodiments, the target infectious agent is Equine Arteritis Virus, Bovine Viral Diarrhea Virus (BVDV), Hog Cholera Virus, or Border Disease Virus. The only member of the Rubivirus genus is the Rubella virus.

[0148] In some embodiments, the target infectious agent is a Filoviridae virus, such as Ebola virus and Marburg virus; a Paramyxoviridae virus, such as Measles virus, Mumps virus, Nipah virus, Hendra virus, Respiratory Syncytial Virus (RSV), and Newcastle Disease Virus (NDV); a Rhabdoviridae virus, such as Rabies virus; a Niyamiviridae virus, such as Nia virus; a Arenaviridae virus, such as Lassa virus; a Bunyaviridae virus, such as Hantavirus and Crimean-Congo hemorrhagic fever virus; or an Ophioviridae or Orthomyxoviridae virus, such as Influenza virus.

[0149] In one embodiment, the antigen is selected from Borrelia species, Bacillus anthracis, Borrelia burgdorferi, Bordetella pertsis, Campylobacter jejuni, Chlamydia species, Chlamydia psittaci, Chlamydia trachomatis, Clostridium species, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Corynebacterium diphtheriae, Coxiella species, Multiple species, Enterococcus multiple species, Ehrlichia multiple species, Escherichia coli, Francisella tularensis, Multiple species of Haemophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Multiple species of Lactobacillus, Multiple species of Legionella, Legionella pneumophila, Leptospirosis interrogans, Multiple species of Listeria, Listeria monocytogenes, Multiple species of Mycobacterium, Multiple species of Mycobacterium Mycobacterium tuberculosis, Mycobacterium leprae, Mycoplasma spp., Mycoplasma pneumoniae, Neisseria spp., Neisseria meningitidis, Neisseria gonorrhoeae, Pneumococcus spp., Pseudomonas spp., Pseudomonas aeruginosa, Salmonella spp., Salmonella typhi, Salmonella enterica, Streptococcus spp., Rickettsia spp., Rickettsia The antibody may be derived from one or more bacteria selected from the group consisting of Bacillus rickettsii, Rickettsia typhi, Shigella spp., Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Treponema spp., Treponema pallidum, Vibrio spp., Vibrio cholerae, and Yersinia pestis. Such bacteria may be whole cells (e.g., live, attenuated, or inactivated), or polypeptides or polysaccharides of such bacteria.

[0150] In some embodiments, the targeted infectious agent is a bacterium. The targeted antigenic bacterial agent may be a polysaccharide-polypeptide antigen, such as a pneumococcal (e.g. S. pneumonia) polysaccharide (e.g. cell capsular saccharide)-protein (e.g. diphtheria protein) conjugate. In some embodiments, the conjugate comprises a cell-trapping saccharide of S. pneumoniae conjugated to a protein (e.g. diphtheria protein), for example the cell capsular saccharide is of seven serotypes of S. pneumoniae bacteria (4, 6B, 9V, 14, 18C, 19F, and 23F) which is conjugated to a diphtheria protein. In some embodiments, the conjugate comprises pneumococcal polysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F conjugated to a protein such as protein D from nontypeable Haemophilus influenza, tetanus toxoid carrier protein, and / or diphtheria toxoid carrier protein. In some embodiments, the conjugate comprises Streptococcus pneumoniae capsular polysaccharide conjugated to a diphtheria protein, e.g., capsular polysaccharides of Streptococcus pneumoniae types 1, 3, 4, 5, 6a, 6b, 7f, 9v, 14, 18c, 23f, 19a, and 19f conjugated to a protein such as diphtheria crm197 protein.In some embodiments, one or more of the polysaccharide-protein conjugates are selected from the group consisting 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, 19A, 20A, 21A, 22A, 23A, 24A, 25A, 26A, 27A, 28A, 29A, 30A, 31A, 32A, 33A, 34A, 35A, 36A, 37A, 38A, 39A, 40A, 41A, 42A, 43A, 44A, 45A, 46A, 47A, 48A, 49A, 50A, 51A, 52A, 53A, 54A, 55A, 56A, 57A, 58A, 59A, 60A, 61A, 62A, 63A, 64A, 65A, 66A, 67A, 68A, 69A, 70A, 71A, 72A, 73A, 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, CWPS3.

[0151] In some embodiments, the target infectious agent is a fungus, such as, but not limited to, one or more fungi selected from Aspergillus spp., Candida spp., Candida albicans, Candida tropicalis, Cryptococcus spp., Cryptococcus neoformans, Entamoeba histolytica, Histoplasma capsulatum, Leishmania spp., Nocardia asteroides, Plasmodium falciparum, Toxoplasma gondii, Trichomonas vaginalis, Toxoplasma spp., Trypanosoma brucei, Schistosoma mansoni, Fusarium spp., and / or Trichophyton spp. Such fungi can be whole cells (e.g., live, attenuated, or inactivated), or polypeptides or polysaccharides of such fungi.

[0152] In some embodiments, the targeted infectious agent is one or more parasites selected from Plasmodium species, Toxoplasma species, Entamoeba species, Babesia species, Trypanosoma species, Leishmania species, Pneumocystis species, Trichomonas species, Giardia species, and / or Schistosoma species. Such parasite antigens can be whole cells (e.g., live, 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, and the like.

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

[0155] In certain embodiments, the targeted antigen is an antigen selected from the group consisting of Ebola virus, e.g., the envelope glycoprotein of the Ebola virus Zaire strain (e.g., UniProtKB-P87671 (VGP_EBOEC)), the matrix protein VP40 of the Ebola virus Zaire strain (e.g., UniProtKB-Q05128 (VP40_EBOZM)), or the matrix protein of the 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., nonstructural protein 1 (NSP-1), Marburg virus, e.g., Marburg virus glycoprotein (GenBank Accession No. A FV31202.1), Marburg virus VP40 matrix protein (GenBank Accession No. JX458834), parasites of Plasmodium species, e.g., Plasmodium falciparum, e.g., circumsporozoite protein (CSP), male gametocyte surface protein P230p (Pfs230 antigen), sporozoite microneme protein essential for cell traversal (SPECT2), or GTP-binding protein, putative antigen (GenBank Accession No. PF3D7_1462300), human immunodeficiency virus, e.g., Env proteins, e.g., gp41, gp120, gp160, Gag proteins, MA, CA, SP1, NC, SP2, P6, or derived from Pol proteins RT, RNase H, IN, PR.

[0156] In an alternative embodiment, the rMVA viral construct is administered to a subject in need thereof, e.g., a human, in a therapeutic regimen including a vaccination protocol, e.g., to treat cancer. Thus, the rMVA viral construct can be administered in concert with one or more antigens intended to induce an immune response against an antigenic target, to induce partial or complete immunization in a subject in need thereof.

[0157] Antigens used in cancer immunotherapy are generally purposefully selected based on either their uniqueness to tumor cells, their higher expression in tumor cells compared to normal cells, or the potential for normal cells with antigen expression to be adversely affected without significant risk to normal cells or tissues. Tumor-associated antigens (TAA) can be broadly classified as oncofetal TAAs (typically expressed only in fetal tissues and cancerous somatic cells), oncoviral TAAs (encoded by oncogenic transforming viruses), overexpressed / accumulated TAAs (expressed by both normal and neoplastic tissues, with expression levels greatly elevated in neoplasms), cancer-testicular TAAs (expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted TAAs (expressed primarily by a single cancer tissue type), mutated TAAs (expressed only by cancers as a result of gene mutations or transcriptional changes), post-translationally modified TAAs (tumor-associated changes in glycosylation, etc.), or idiotypic TAAs (highly polymorphic genes similar to B-cell, T-cell lymphomas / leukemias in which tumor cells express a particular "clonotype," i.e., resulting from clonal abnormalities). TAAs are preferentially expressed by tumor cells, but are also often found in normal tissues. However, their expression differs from that of normal tissues in terms of their degree of expression in tumors, changes in their protein structure compared to their normal counterparts, or their abnormal subcellular 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-testis antigens include the b-melanoma antigen (BAGE) family, the cancer associated gene (CAGE) family, the G antigen (GAGE) family, the melanoma antigen (MAGE) family, the sarcoma antigen (SAGE) family, and the X antigen (XAGE) family, CT9, CT10, NY-ESO-1, L antigen (LAGE)1, melanoma antigen preferentially expressed in tumors (PRAME), and synovial sarcoma X (SSX)2. Examples of lineage-restricted tumor antigens include melanoma antigen-1 / 2 (Melan-A / MART-1 / 2), 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-translationally modified tumor antigen is mucin (MUC)1. Examples of idiotypic tumor antigens include immunoglobulins (Ig) and T cell receptors (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, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, 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 least the following: carcinoembryonic antigen (CEA) for intestinal 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, and ras for various types of tumors, abnormal products of p53, alpha-fetoprotein for liver, ovarian, or testicular cancer, beta-subunit of hCG for men with testicular cancer, prostate specific antigen for prostate cancer, beta2-microglobulin for multiple myeloma and in some lymphomas, CA19-9 for colorectal, bile duct, and pancreatic cancer, chromogranin A for lung and prostate cancer, TA90 for melanoma, soft tissue sarcoma, and breast, colon, and lung cancer. Examples of TAAs are known in the art, for example, in N. Vigneron, "Human Tumor Antigens and Cancer Immunotherapy," 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; Cheever et al., Clin Cancer Res. (2009) Sep 1;15(17):5323-37, which are incorporated herein by reference in their entireties.

[0162] Examples of oncoviral TAAs include L1, E6, and E7 of human papillomavirus (HPV), Epstein-Barr nuclear antigen (EBNA) 1 and EBNA2 of Epstein-Barr virus (EBV), EBV viral capsid antigen (VCA) Igm or IgG, EBV early 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), HCV-1 IgG, ... These include TLV-1 protease antigen (Pro), HTLV-1 Tof, HTLV-1 Rof, HTLV-1 polymerase (Pro) antigen, human T-lymphotropic virus type 2 core antigen (HTLV-2 core antigen), HTLV-2 Tax antigen, HTLV-2 group-specific (Gag) antigen, HTLV-2 envelope (Env), HTLV-2 protease antigen (Pro), HTLV-2 Tof, HTLV-2 Rof, HTLV-2 polymerase (Pro) antigen, latency-associated nuclear antigen (LANA), human herpesvirus-8 (HHV-8) K8.1, Merkel cell polyomavirus large T antigen (LTAg), and Merkel cell polyomavirus small T antigen (sTAg).

[0163] Elevated expression of certain types of glycolipids, such as gangliosides, is associated with the promotion of tumor survival in certain types of cancer. Examples of gangliosides include, for example, GM1b, GD1c, GM3, GM2, GM1a, GD1a, GT1a, GD3, GD2, GD1b, GT1b, GQ1b, GT3, GT2, GT1c, GQ1c, and GP1c. Examples of ganglioside derivatives include, for example, 9-O-Ac-GD3, 9-O-Ac-GD2, 5-N-de-GM3, N-glycolyl GM3, 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 above TAAs, another class of TAAs are tumor-specific neoantigens, which arise through mutations that alter the amino acid coding sequence (nonsynonymous somatic mutations). Some of these mutant peptides are expressed, processed, and presented on the cell surface, and are subsequently recognized by T cells. Because normal tissues do not have these somatic mutations, neoantigen-specific T cells do not undergo central and peripheral immune tolerance, and also lack the ability to induce 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, incorporated herein by reference.

[0165] In some embodiments, the TAA is specific for an oncofetal 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 for an oncoviral TAA selected from the group consisting of E6 and E7 of human papillomavirus (HPV), Epstein-Barr nuclear antigen (EBNA) 1 and EBNA2, latent membrane protein (LMP) 1 and LMP2 of Epstein-Barr virus (EBV).

[0167] In some embodiments, the TAA is specific for an 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 survivin.

[0168] In some embodiments, the TAA is selected from the group consisting of 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 alpha-1 (IL13RA), CT9, putative tumor antigen NA88-A, leucine zipper protein 4 (LUZP4), NY-ESO-1, L antigen (LAGE) 1, hepatoma antigen (HAGE) 2, hepatoma antigen (HAGE) 3, hepatoma antigen (HAGE) 4, hepatoma antigen (HAGE) 5, hepatoma antigen (HAGE) 6, hepatoma antigen (HAGE) 7, hepatoma antigen (HAGE) 8, hepatoma antigen (HAGE) 9, hepatoma antigen (HAGE) 10, hepatoma antigen (HAGE) 11, hepatoma antigen (HAGE) 12, hepatoma antigen (HAGE) 13, hepatoma antigen (HAGE) 14, hepatoma antigen (HAGE) 15, hepatoma antigen (HAGE) 16, hepatoma antigen (HAGE) 17, hepatoma antigen (HAGE) 18, hepatoma antigen (HAGE) 19, hepatoma antigen (HAGE) 20, hepatoma antigen (HAGE) 21, hepatoma antigen (HAGE) 22, hepatoma antigen (HAGE) 23, hepatoma antigen (HAGE) 24, hepatoma antigen (HAGE) 25, hepatoma antigen (HAGE) 26, hepatoma antigen (HAGE) 27, hepatoma antigen (HAGE) 28, hepatoma antigen (HAGE) 29, hepatoma antigen (HAGE) 30, hepatoma antigen (HAGE) 31, hepatoma antigen ( The antigen is specific for a cancer-testis antigen selected from the group consisting of hepatic sarcoma antigen (HAGE), lipase I (LIPI), melanoma antigen preferentially expressed in tumors (PRAME), synovial sarcoma X (SSX) family, sperm protein associated with nucleus on chromosome X (SPANX) family, cancer / testis antigen 2 (CTAG2), calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein (CABYR), acrosin-binding protein (ACRBP), centrosomal protein 55 (CEP55), and synaptonemal complex protein 1 (SYCP1).

[0169] In some embodiments, the TAA is specific for a lineage-restricted tumor antigen 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 antigen recognized by T cells.

[0170] In some embodiments, the TAA is specific for 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 receptor (tEGFR).

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

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

[0173] In some embodiments, the TAA is specific for BCMA. In some embodiments, at least one T cell subpopulation is specific for BCMA.

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

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

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

[0177] In some embodiments, the TAA is specific for 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 above TAAs, another class of TAAs are tumor-specific neoantigens, which arise through mutations that alter the amino acid coding sequence (nonsynonymous somatic mutations). Some of these mutant peptides are expressed, processed, and presented on the cell surface, and are subsequently recognized by T cells. Because normal tissues do not have these somatic mutations, neoantigen-specific T cells do not undergo central and peripheral immune tolerance, and also lack the ability to induce 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, incorporated herein by reference.

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

[0180] rMVA viral vector Provided herein is a rMVA viral vector comprising a heterologous nucleic acid insert encoding an immune checkpoint inhibitor that can be secreted from a cell.

[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 comprises 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 more than 10 and M=methionine) (see, e.g., Figures 1A and 1B).

[0183] In some embodiments, a nucleic acid sequence encoding one or more of the polypeptides in the 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)) is provided herein. x Provided are rMVA viral vectors that contain a heterologous polycistronic nucleic acid insert (where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine) (see, e.g., FIG. 2A and FIG. 2B). In certain embodiments, the encoded polypeptide is (M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x (wherein x=2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 more than 10 and M=methionine), where the secretory signal peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:57-90, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:1-56, and the cleavable peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:91-127. In some embodiments, the secretory signal peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:65 and SEQ ID NO:66, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:5, and the cleavable peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:93-97, SEQ ID NO:120, and SEQ ID NO:123-127.

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

[0189] [Table 8] TIFF2024505274000017.tif247170TIFF2024505274000018.tif246170TIFF2024505274 000019.tif247170TIFF2024505274000020.tif246170TIFF2024505274000021.tif98170

[0190] As provided herein, a polycistronic nucleic acid insert encoding an immune checkpoint inhibitor polypeptide as described herein can be inserted into the MVA genome at any suitable location, for example, at a naturally occurring deletion site, an engineered naturally occurring deletion site, in a non-essential MVA gene, such as the MVA thymidine kinase locus, or in an intergenic region between essential or non-essential MVA genes. Suitable insertion sites are described, for example, in U.S. Pat. No. 6,998,252, U.S. Pat. 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. Pat. No. 9,133,480, and U.S. Pat. No. 8,288,125, each of which is incorporated herein by reference.

[0191] In some embodiments, a polycistronic nucleic acid insert encoding an immune checkpoint inhibitor polypeptide described herein is inserted into a naturally occurring deletion site, e.g., a deletion site selected from naturally occurring deletion sites I, II, III, IV, V, or VI, a modified naturally occurring deletion site, e.g., the reconstructed modified deletion III site between A50R and B1R of the MVA gene (see, e.g., U.S. Pat. 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 other suitable insertion site in a non-essential locus, e.g., the TK locus of MVA, or a combination thereof.

[0192] In alternative embodiments, the rMVA viral vectors of the present invention, in addition to being capable of expressing multiple immune checkpoint inhibitor peptides, can further be constructed to encode and express one or more antigenic peptides. The one or more antigenic peptides can be encoded on one or more separate nucleic acid inserts, or in alternative embodiments, the one or more antigenic peptides are encoded on the same polycistronic nucleic acid insert as multiple immune checkpoint inhibitor peptides.

[0193] In some embodiments, provided herein is an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, the polypeptide comprising the sequence of ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x Provided is a rMVA viral vector comprising: ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine. In some embodiments, the antigenic peptide is a chimeric polypeptide comprising a secretory signal peptide fused to the N-terminus of the antigenic peptide, such as ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x (secretory signal peptide-antigenic peptide) (where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine) (see, e.g., Figures 4A and 4B). In some embodiments, antigenic peptides are also provided where two or more antigenic peptides are encoded in a polycistronic nucleic acid insert, with each chimeric polypeptide separated by a cleavable peptide as described herein. In some embodiments, the antigenic peptide is a chimeric polypeptide comprising a secretory signal peptide fused to the N-terminus of the antigenic peptide and a cleavable peptide fused to the C-terminus of the antigenic peptide, such as ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (secretory signal peptide-antigenic peptide-cleavable peptide) y) (where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 include a cleavable sequence, e.g., ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (secretory signal peptide-antigenic peptide-cleavable peptide) x (secretory signal peptide-antigenic peptide)), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine. In some embodiments, the antigenic peptide included in the chimeric polypeptide comprising a secretory 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 5' to the immune checkpoint inhibitor peptide-containing chimeric polypeptide, e.g., ((M)(secretory signal peptide-antigenic peptide-cleavable peptide)). y (Secretory signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x ), or alternatively ((M) (secretory signal peptide-antigenic peptide-cleavable peptide) y (Secretory signal peptide - immune checkpoint inhibitor peptide - cleavable peptide) x (secretory signal peptide-immune checkpoint inhibitor peptide)), where y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 an agent described above in the section entitled "Antigenic Targets," which is expressly formed part of this section.

[0195] In some embodiments, the polycistronic nucleic acid insert encodes a polypeptide comprising an antigenic amino acid sequence in Table 9 below, or a polypeptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the polycistronic nucleic acid insert encodes an antigen comprising amino acids derived from an amino acid sequence selected from SEQ ID NO:349 to SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, or SEQ ID NO:405, or a fragment thereof, or a polypeptide having an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0196] [Table 9] TIFF2024505274000023.tif254170TIFF2024505274000024.tif253170TIFF2024505274000025.tif252170TIFF2024505274000026.tif252170 TIFF2024505274000027.tif252170TIFF2024505274000028.tif252170TIFF2024505274000029.tif252170TIFF2024505274000030.tif252170 TIFF2024505274000031.tif252170TIFF2024505274000032.tif252170TIFF2024505274000033.tif252170TIFF2024505274000034.tif252170 TIFF2024505274000035.tif252170TIFF2024505274000036.tif252170TIFF2024505274000037.tif252170TIFF2024505274000038.tif162170

[0197] In some embodiments, any of SEQ ID NOs: 349 to 395, or SEQ ID NO: 401 above 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 selected from SEQ ID NO: 349, SEQ ID NO: 358 to SEQ ID NO: 364, or SEQ ID NO: 403, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

[0200] In some embodiments, the antigenic insert is derived from a Hepatitis B virus protein, or a fragment thereof. In some embodiments, the antigenic insert is derived from an amino acid sequence selected from SEQ ID NO: 351 to SEQ ID NO: 354, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0201] In some embodiments, the antigenic insert is derived from a Plasmodium species protein, or a fragment thereof. In some embodiments, the antigenic insert is derived from an amino acid sequence selected from SEQ ID NO: 355 to SEQ ID NO: 357, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

[0203] In some embodiments, the antigenic insert is derived from an Ebola virus protein or a fragment thereof, hi 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 at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0204] In some embodiments, the antigenic insert is derived from a Zika virus protein or a fragment thereof, hi some embodiments, the antigenic insert is derived from an amino acid sequence selected from SEQ ID NO: 369-376, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0205] In some embodiments, the antigenic insert comprises one or more proteins or polypeptides of SARS-CoV-2, 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_009724398), or ribosomal protein (RI) (NCBI Reference Sequence YP_009724399). 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 S protein of SARS-CoV2, 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 an amino acid sequence 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 includes 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 includes the following substitutions: K417T, E484K, and N501Y of SEQ ID NO:377. In some embodiments, the rMVA includes a nucleic acid sequence encoding an S protein further including a substitution at L452R, T478K, or P681R of SEQ ID NO:377, or a combination thereof. In some embodiments, the rMVA includes a nucleic acid sequence encoding an S protein further including a substitution at L452R, T478K, and P681R of SEQ ID NO:377.In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein that further comprises substitutions at N440K, S443A, G476S, E484R, and / or G502P of SEQ ID NO:377, or combinations thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein that further comprises one or more substitutions 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 of 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 of 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 of 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 of 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 of 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 of SEQ ID NO: 377.In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at K417T, E484K, N501Y, D614G, and H655Y of SEQ ID NO: 377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at L452R, T478K, D614G, and P681R of 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 Q677H of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at L452R, N501Y, D614G, and P681H of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at S494P, N501Y, D614G, and P681H of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at L452R, D614G, and Q677H of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at E484K, D614G, N679K, and ins679GIAL of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at E484K, D614G, N679K, and ins679GIAL of 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 of 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 of 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 of SEQ ID NO: 377.In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at E484K and D614G of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at T478K and D614G of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at N439K, E484K, D614G, and P681H of 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, K417T, N501Y, and P681H of SEQ ID NO:377. In some embodiments, the rMVA comprises a nucleic acid sequence encoding an S protein further comprising substitutions at L452R, T478K, D614G, P681R, and K417N of 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 of 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 strain is a SARS-CoV2 virus having a deletion of the spike protein from amino acid 242 to amino acid 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: a deletion of amino acid 69 and amino acid 70, a 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 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: N501Y, K417N or K417T, E484K, D80A, A701V, L18F, and an amino acid deletion at amino acids 242 to 244 of SEQ ID NO: 377.In some embodiments, the S protein is expressed as a full-length protein and contains 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 in SEQ ID NO:377. , S1252P, Y145 deletion, 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 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 SEQ ID NO: 377 to SEQ ID NO: 384, or a fragment thereof, or an amino acid sequence 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 comprises 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 comprises the following substitutions: K417T, E484K, and N501Y of SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein that further comprises a substitution with L452R, T478K, or P681R of SEQ ID NO: 381, or a combination thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein that further comprises a substitution with L452R, T478K, and P681R of SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein that further comprises substitutions at N440K, S443A, G476S, E484R, and / or G502P of SEQ ID NO:381, or combinations thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein that further comprises one or more substitutions 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 of SEQ ID NO:381, or any combination thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at T19R, T95I, G142D, E156del, F157del, R158G, L452R, T478K, D614G, P681R, and D950N of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of SEQ ID NO: 381.In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at R346K, E484K, N501Y, D614G, and P681H of SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at L452Q, F490S, and D614G of 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 of SEQ ID NO: 8. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at Q414K, N450K, ins214TDR, and D614G of 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 of 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 of 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 of 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 of 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 of 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 of 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 of SEQ ID NO: 381.In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at S494P, N501Y, D614G, and P681H of SEQ ID NO:381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at L452R, D614G, and Q677H of 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, N679K, and ins679GIAL of 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 A701V of 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 of SEQ ID NO:8. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at S477N and D614G of 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 P681H of SEQ ID NO:381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at E484K and D614G of SEQ ID NO:381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at T478K and D614G of SEQ ID NO:381. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein further comprising substitutions at N439K, E484K, D614G, and P681H of 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 of 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 of 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 of 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 contains 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 strain is a SARS-CoV2 virus having a spike protein deletion from amino acid 242 to amino acid 244 of SEQ ID NO: 381. In some embodiments, the stabilized S protein is expressed as a full-length protein and comprises the following deletions and substitutions: a deletion of amino acid 69 and amino acid 70, a 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 of SEQ ID NO: 381. In some embodiments, the stabilized S protein is expressed as a full-length protein and comprises the following deletions and substitutions: N501Y, K417N or K417T, E484K, D80A, A701V, L18F, and an amino acid deletion at amino acids 242 to 244 of SEQ ID NO: 381. 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: 381, or a combination thereof. In some embodiments, the S protein is expressed as a full-length protein and contains 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 comprises a deletion at amino acid 242 to amino acid 244 of SEQ ID NO: 381. In some embodiments, the S protein is expressed as a full-length protein and comprises the following deletions and substitutions: a deletion of amino acid 69 and amino acid 70 of SEQ ID NO: 381, a 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 comprises the following deletions and substitutions: N501Y, K417N or K417T, E484K, D80A, A701V, L18F, and an amino acid deletion at amino acid 242 to amino acid 244 of SEQ ID NO: 381. The rMVA encodes a stabilized S protein that further comprises substitutions at L452R, T478K, and P681R of SEQ ID NO: 381. In some embodiments, the rMVA comprises a nucleic acid sequence that encodes a stabilized S protein that further comprises substitutions at N440K, S443A, G476S, E484R, and / or G502P of SEQ ID NO: 381, or combinations thereof. In some embodiments, the rMVA comprises a nucleic acid sequence encoding a stabilized S protein that further comprises one or more substitutions 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 of SEQ ID NO:381, or any combination thereof.

[0210] In some embodiments, the stabilized S protein is expressed as a full-length protein and contains 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 in SEQ ID NO: 381, P479S, S1252P, Y145 deletion, 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 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 an amino acid sequence 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto.

[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 host cell invasion. Once inside the cell, the infectious RNA is used to encode the structural proteins that make up the viral particle, the nonstructural proteins that guide viral assembly, transcription, replication, and host control, and accessory proteins of undefined function. The largest gene, ORF1ab, contains overlapping open reading frames that encode the polyproteins PP1ab and PP1a. The polyprotein is cleaved to generate 16 nonstructural proteins, NSP1 to NSP16. The production of the longer protein (PP1ab) or the shorter protein (PP1a) depends on a -1 ribosomal frameshift event. Proteins based on similarity to other coronaviruses include papain-like proteinase protein (NSP3), 3C-like proteinase (NSP5), RNA-dependent RNA polymerase (NSP12, RdRp), helicase (NSP13, HEL), endoRNase (NSP15), 2'-O-ribose-methyltransferase (NSP16), and other nonstructural proteins. A description of the various NSPs encoded by ORF1ab can be found, for example, in Arya et al., "Structural insights into SARS-CoV-2 proteins." J Mol Biol. 2021 Jan 22; 433(2): 166725, 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 NO:377 to SEQ ID NO:394.

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

[0214] In certain embodiments, the encoded polypeptide comprises a sequence similar to that described above, but in various alternative embodiments, the sequence may be selected from the group consisting of ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (Antigenic peptide), ((M) (Secretory signal peptide-Immune checkpoint inhibitor peptide-Cleaving peptide) x (Secretory signal peptide-antigenic peptide), ((M) (Secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (secretory signal peptide-antigenic peptide-cleavable peptide) y ), ((M) (secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x (secretory signal peptide-antigenic peptide-cleavable peptide) x(secretory signal peptide-antigenic peptide) (where y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine), wherein the secretory signal peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:57 to SEQ ID NO:90, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:56, the cleavable peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:91 to SEQ ID NO:127, and the antigenic peptide is a peptide derived from an infectious agent, e.g., a virus, a bacterium, a parasite, a fungus, or a toxoid, or alternatively a tumor-associated antigen. In some embodiments, the secretory signal peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:65 and SEQ ID NO:66, the immune checkpoint inhibitor peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:5, and the cleavable peptide is selected from a peptide having an amino acid sequence selected from SEQ ID NO:93, SEQ ID NO:120, and SEQ ID NO:123. In some embodiments, the secretory signal peptide is a peptide having an amino acid sequence of SEQ ID NO:66, the immune checkpoint inhibitor peptide is a peptide having an amino acid sequence of SEQ ID NO:1, and the cleavable peptide is a peptide having an amino acid sequence of SEQ ID NO:123, where x=2 to 10. In some embodiments, the secretory signal peptide is a peptide having an amino acid sequence of SEQ ID NO:66, the immune checkpoint inhibitor peptide is a peptide having an amino acid sequence of SEQ ID NO:1, and the cleavable peptide is a peptide having an 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.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 or more. 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 are capable of forming a non-infectious virus-like particle (VLP). Provided herein in some embodiments is an rMVA viral vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, the polypeptide comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) xProvided are rMVA viral vectors comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 and M=methionine (see, e.g., Figures 5A and 5B). In some embodiments, the antigenic peptide is comprised 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, e.g., ((M)(secretory 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 more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 comprise a cleavable sequence, e.g., ((M)(secretory signal 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 more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 more than 10) may be oriented such that in a polycistronic nucleic acid insert, the nucleic acid encoding the antigen-containing chimeric polypeptide is located 5' to the immune checkpoint inhibitor peptide-containing chimeric polypeptide, e.g., ((M)(glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane domain-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 (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 more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine. In yet a further embodiment, the polycistronic nucleic acid insert of the rMVA further encodes a viral matrix protein, such as ((M)(secretory 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 more than 10 and M=methionine (see, e.g., Figures 6A and 6B). In an alternative embodiment, the coding sequences for both the antigen-containing chimeric polypeptide and the viral matrix protein are contained in one or more copies in the polycistronic nucleic acid, such as ((M)(secretory 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 more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine). In some embodiments, the most C-terminal viral matrix protein lacks a cleavable peptide, e.g., ((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)) (where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, and M=methionine). In some embodiments, the (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 more 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, e.g., ((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(secretory signal peptide-immune checkpoint inhibitor peptide)), where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, y=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 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 No. AFV31202.1). The amino acid sequence of the GP protein of MARV is set forth in Table 10 below as SEQ ID NO: 395. In certain embodiments, the GPS domain of MARV comprises amino acids 2 to 19 of the glycoprotein (WTTCFFISLILIQGIKTL) (SEQ ID NO: 396, which may be encoded, for example, by the MVA-optimized nucleic acid sequence of SEQ ID NO: 397), and the GPTM domain comprises amino acid sequence 644 to 673 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG) (SEQ ID NO: 398, which may be encoded, for example, by the MVA-optimized nucleic acid sequence of SEQ ID NO: 399), or a nucleic acid sequence 70%, 75%, 80%, 85%, 90%, 95% or more identical thereto. In some embodiments, the MARV GPS signal further comprises a methionine as the first amino acid.

[0217] The MARV VP40 amino acid sequence is available at GenBank Accession No. JX458834, and is set forth below in Table 10 as SEQ ID NO: 400, which may be encoded by, for example, the MVA-optimized nucleic acid sequence of SEQ ID NO: 401, or a nucleic acid sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more identical thereto. In some embodiments, the MARV VP40 amino acid sequence further comprises a methionine as the first amino acid.

[0218] [Table 10]

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

[0220] In alternative embodiments, the rMVA viral vectors of the invention, in addition to being capable of expressing multiple immune checkpoint inhibitor peptides, may further be constructed to encode and express one or more antigenic peptides encoded on one or more separate nucleic acid inserts. In some embodiments, a nucleic acid sequence encoding multiple immune checkpoint inhibitor peptides described herein is inserted into one locus of the rMVA, and one or more heterologous nucleic acid sequences encoding antigenic peptides are inserted into separate loci of the rMVA. The one or more antigenic peptides can be derived from any of the targets described in the "Antigenic Targets" section, which are incorporated in their entirety for all purposes. In some embodiments, the antigenic peptide is derived from any of the amino acid sequences selected from SEQ ID NO:349-SEQ ID NO:396, SEQ ID NO:398, or SEQ ID NO:400, or a fragment derived therefrom, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. When inserted as a separate nucleic acid insert, an initiation codon encoding the amino acid residue methionine (M) may be included as the first residue of an antigenic peptide derived from any of the amino acid sequences selected from SEQ ID NO:349 to SEQ ID NO:396, SEQ ID NO:398, or SEQ ID NO:400, or a fragment derived therefrom, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0221] In certain embodiments, the rMVA, in addition to the polycistronic nucleic acid encoding the immune checkpoint inhibitor polypeptide described herein, further encodes an antigenic peptide comprising a chimeric peptide comprising an extracellular domain of an antigen and a transmembrane domain of a viral glycoprotein, and further encodes a viral matrix protein, wherein the chimeric peptide and the viral matrix protein, when expressed, are capable of forming a virus-like particle (VLP) in vivo. In some embodiments, the transmembrane domain of the viral glycoprotein is derived from an amino acid sequence of SEQ ID NO: 398, or a fragment thereof, or an amino acid sequence 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 Marburg virus as set forth in SEQ ID NO: 404, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the rMVA encodes the amino acid sequence of SEQ ID NO: 329, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, the amino acid sequence of SEQ ID NO: 402, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 404, or a fragment thereof, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0222] [Table 11] TIFF2024505274000041.tif31170

[0223] Array Optimization One or more nucleic acid sequences comprising the polycistronic nucleic acid insert of the rMVA provided herein can be optimized for use in the MVA vector. 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 homopolymeric stretches or transcription terminator motifs. Each of these optimization strategies can improve gene stability, improve transcript stability, or improve protein expression levels from the sequence. In an exemplary embodiment, to stabilize the construct, the number of homopolymeric stretches in the heterologous DNA insert sequence will be reduced. Silent mutations can provide a similarity to the vaccinia termination signal.

[0224] In an exemplary embodiment, the sequence is codon-optimized for expression in MVA, with sequences having runs of more than five deoxyguanosines, more than five deoxycytidines, more than five deoxyadenosines, and more than five deoxythymidines interrupted by silent mutations to minimize loss of expression due to frameshift mutations.

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

[0226] Pharmaceutical Compositions The recombinant MVA viral vectors of the invention, alone or in combination, are readily formulated into pharmaceutical compositions for veterinary or human use, which may include a pharma- ceutical acceptable diluent, excipient, carrier, or adjuvant, or in alternative embodiments, one or more antigenic agents, such as antigens derived from infectious diseases, or in alternative embodiments, tumor-associated antigens.

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

[0228] The phrase "pharmaceutically acceptable carrier" as used herein includes any of the standard pharmaceutical carriers, such as those suitable for parenteral administration, for example, by intramuscular, intraarticular (in a joint), intravenous, intradermal, intraperitoneal, and subcutaneous routes. Examples of such formulations include aqueous and non-aqueous isotonic sterile injection solutions that contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, 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 pharmaceutical benefits of their own. The amount of carrier used in combination with the compound is sufficient to provide a practical amount of the substance to be administered per unit dose of the compound.

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

[0230] In some embodiments, an additional adjuvant is used as a further immune response enhancer. In various embodiments, the additional immune response enhancer is an alum-based adjuvant, an oil-based adjuvant, Specol, RIBI, TiterMax, Montanide ISA50 or Montanide ISA 720, GM-CSF, a non-ionic block copolymer-based adjuvant, dimethyldioctadecylammonium bromide (DDA)-based adjuvant AS-1, AS-2, a Ribi adjuvant-based adjuvant, QS21, Quil A, SAF (a microfluidized form of Syntex adjuvant (SAF-m)), dimethyldioctadecylammonium bromide (DDA), a human complement system adjuvant Mycobacterium vaccae (m. vaccae, ISCOMS, MF-59, SBAS-2, SBAS-4, Enhanzyn™, RC-529, AGP, MPL-SE, QS7, escin; digitonin; Gypsophila; and Chenopodium quinoa saponin.

[0231] The compositions utilized in the methods described herein can be administered by a route selected from, for example, parenteral, intramuscular, intraarterial, intravascular, intravenous, intraperitoneal, subcutaneous, dermal, transdermal, ocular, inhalation, buccal, sublingual, perilingual, intranasal, topical, and oral administration. The preferred method of administration can vary depending on a variety of factors, such as the components of the composition being administered and the severity of the condition being treated. Formulations suitable for oral administration can consist of a liquid solution, 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 can also be an aerosol formulation, for example, for inhalation into the bronchial passages. The aerosol formulation can be mixed with a pressurized, pharma- ceutically acceptable propellant (e.g., dichlorodifluoromethane, propane, or nitrogen).

[0232] For the purposes of the present invention, pharmaceutical compositions suitable for delivering therapeutic or biologically active agents may include, for example, tablets, gelcaps, 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, each of which is incorporated herein by reference.

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

[0234] Suitable preparations for rectal administration include, for example, suppositories, which consist of the vaccine with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, gelatin rectal capsules can be used, which consist of a combination of the vaccine with a base that includes, for example, liquid triglycerides, polyethylene glycol, and paraffin hydrocarbons. The vaccine of the present invention can also be co-administered with cytokines to further enhance immunogenicity. Cytokines can be administered by methods known to those skilled in the art, for example, as nucleic acid molecules in the form of plasmids, 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. In addition, the formulation may contain an antimicrobial preservative. 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 the present disclosure may be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents, and / or suspending agents, as well as 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 comprising the rMVA described herein in a unit dosage form in a sealed container. The rMVA may be provided in the form of a lyophilizate that can be reconstituted with a suitable pharma- ceutically acceptable carrier to form a liquid formulation suitable for injection into a host.

[0238] Classes of carriers include, but are not limited to, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavorings, glidants, glidants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example, vegetable oils may be used as lubricants in some formulations and as diluents in other formulations. A pharmaceutically acceptable carrier is one that does not cause any severe adverse reactions in humans when administered in the amount used in the corresponding pharmaceutical composition. Exemplary pharmaceutically acceptable carriers include sugars, starches, cellulose, powdered tragacanth, malt, gelatin, talc, and vegetable oils. Any active agent that does not substantially interfere with the activity of the morphic form or pharmaceutical composition of the present invention may be included in the pharmaceutical composition.

[0239] Formulations suitable for pulmonary administration can be delivered by a wide range of passive respiratory 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 pulmonary delivery device depends on parameters such as the nature of the drug and its formulation, the site of action, and the pathophysiology of the lungs.

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

[0241] Additional non-limiting examples of pharma- ceutically acceptable 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, babassu oil, coconut oil, corn oil, cottonseed oil, cashew oil, carob oil, coriander 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 oil), 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 seed oil, prune oil, saw palmetto oil, quinoa oil, rapeseed oil, rice germ oil, rice bran oil, rice oil, rarelman cheer oil, safflower oil (safflower oil 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 derivatives thereof, and glycerophospholipids such as lecithin, and any combination thereof.

[0242] In certain embodiments, excipients herein can be liquid at room temperature (such as fatty oils) or solid at room temperature (such as fats).

[0243] How to use The compositions of the invention can be used as adjuvants to enhance the immune response or as vaccines to induce an immune response.

[0244] In an exemplary embodiment, the invention provides an adjuvant for use in a method of preventing an infectious disease in a subject in need of prevention (e.g., an unexposed subject), the method comprising administering to the subject a composition of the invention in combination with an effective amount of an antigenic agent. Alternatively, the invention provides a vaccine for use in a method of preventing an infectious disease in a subject in need of prevention (e.g., an unexposed subject), the method comprising administering to the subject a composition of the invention. The result of this method is that the subject is partially or fully immunized against the infectious disease.

[0245] In other exemplary embodiments, the invention provides an adjuvant for use in a method of treating a condition, such as cancer, in a subject in need of treatment, the method comprising administering to the subject a composition of the invention in combination with an effective amount of a tumor-associated antigenic agent.Alternatively, the invention provides a vaccine for use in a method of treating a condition, such as cancer, in a subject in need of treatment, the method comprising administering to the subject a composition of the invention.

[0246] In an exemplary embodiment, the invention provides an adjuvant for use in a method of treating an infectious agent (e.g., an exposed subject, such as a subject who has been recently exposed but is not yet symptomatic, or a subject who has been recently exposed and is only mildly symptomatic), the method comprising administering to the subject a composition of the invention in combination with a therapeutically effective amount of an antigenic agent that targets the infectious agent. In an exemplary embodiment, the invention provides a vaccine for use in a method of treating an infectious agent (e.g., an exposed subject, such as a subject who has been recently exposed but is not yet symptomatic, or a subject who has been recently exposed and is only mildly symptomatic), the method comprising administering to the subject a composition of the invention. The outcome of the treatment is that the subject has an improved therapeutic profile. The outcome is an improved therapeutic profile. In some cases, 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, compared to an equivalent untreated control. In some cases, treatment can result in inhibition of replication of the infectious agent, reduction in the titer or load of the infectious agent, eradication or elimination of the infectious agent. In other embodiments, treatment can result in improvement of one or more symptoms of the 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] The subject to be treated according to the described method may be a subject diagnosed by a physician as having such a condition. Diagnosis may be performed by any suitable means. The subject in whom the development of an infectious disease is prevented may or may not have been so diagnosed. Those skilled in the art will understand that the subject to be treated according to the present invention may have been identified using standard tests or may have been identified as a high-risk subject without testing due to the presence of one or more risk factors (e.g., exposure to 2019-nCoV, etc.).

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

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

[0250] The compositions can be administered, for example, by injection (eg, intramuscular, intraarterial, intravascular, intravenous, intraperitoneal, or subcutaneous).

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

[0252] Without being bound by any particular mechanism, it is believed that upon inoculation with the pharmaceutical compositions described herein, the host's immune system responds to the antigenic agent combined with the adjuvant, or vaccine, by producing both secretory and serum antibodies specific for the infectious agent or tumor-associated antigen, and by generating a cell-mediated immune response specific for the targeted agent. As a result of vaccination, the host becomes at least partially or completely immune to the targeted infectious disease, or is resistant to developing moderate or severe disease caused by the targeted infectious disease.

[0253] In some embodiments, the administration is once. In some embodiments, the administration is repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or more than 8 times.

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

[0255] In one embodiment, about 2-8, about 4-8, or about 6-8 administrations are provided.

[0256] In one embodiment, there is an interval of about 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 between doses.

[0257] In one particular embodiment, an interval of four weeks between two doses is used.

[0258] Dosage The adjuvant or vaccine combined with the antigenic agent is administered in a manner compatible with the dosage formulation, and in such amounts as will be therapeutically effective, immunogenic and protective. The amount administered will depend on the subject being treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and, if necessary, the capacity to generate a cell-mediated immune response. The precise amount of active ingredient required to be administered depends on the judgment of the practitioner and can be monitored on a patient-by-patient basis. However, suitable dosage ranges can be readily determined by those skilled in the art, and generally range from about 5.0×10 6 TCID 50 ~5.0×10 9 TCID 50 The dosage may also depend, but is not limited to, on the route of administration, the health condition and weight of the patient, and the nature of the formulation.

[0259] The pharmaceutical compositions of the present invention are administered in a therapeutically effective amount that enhances the immunogenicity of the targeted antigen. The dosage administered will depend on the subject being treated (e.g., the mode of administration and the age, weight, immune system capacity, and general health of the subject being treated). The compositions are administered in an amount that provides a sufficient level of expression to enhance or elicit an immune response without undue adverse physiological effects. Preferably, the compositions of the present invention are administered in an amount that provides a sufficient level of expression to enhance or elicit an immune response without undue adverse physiological effects, e.g., 1.0×10 4 ~9.9×10 12 TCID 50 of viral vector, preferably 1.0 × 10 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 x 10 6 ~5.0×10 9 TCID 50 The composition is administered at a dose of, for example, at least 5.0×10 6 TCID 50 of viral vector (e.g., 1.0 × 10 8 TCID 50A physician or researcher can determine the appropriate amount and administration regimen.

[0260] The composition of the above method may be, for example, 1.0×10 4 ~9.9×10 12 TCID 50 of viral vector, preferably 1.0 × 10 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 x 10 6 ~5.0×10 9 TCID 50 The composition may, for example, comprise at least 5.0×10 6 TCID 50 of viral vector (e.g., 1.0 × 10 8 TCID 50 The method can include, for example, administering the composition to a subject two or more times.

[0261] The term "effective amount" refers to the amount of a composition administered to ameliorate, inhibit or ameliorate a symptom of a condition or disorder in a subject in a clinically relevant manner (e.g., ameliorate, inhibit or ameliorate infection by an arenavirus or provide an effective immune response to an infection). Any improvement in the subject is considered sufficient to achieve treatment. Preferably, an amount sufficient to treat is an amount that prevents the occurrence or one or more symptoms, or reduces the severity of or the length of time that the subject suffers from one or more symptoms of the targeted infection or cancer (e.g., by at least 10%, 20%, or 30%, more preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, 95%, 99%, or more relative to a control subject not treated with a composition of the invention).

[0262] In some instances, it may be desirable to combine the rMVA of the present invention with immunogenic compositions that induce protective responses against more than one infectious agent, particularly other viruses. For example, the adjuvant compositions of the present invention can be administered simultaneously, separately, or sequentially with other genetic immunization 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 The term "administering" as used herein refers to a method of giving a dosage of the pharmaceutical composition of the present invention to a subject. The compositions utilized in the methods described herein can be administered, for example, by a route selected from parenteral, dermal, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, rectal, topical, and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intraarterial, intravascular, and intramuscular administration. The preferred method of administration can vary depending on a variety of factors, such as the components of the composition being administered and the severity of the condition being treated.

[0264] Administration of the pharmaceutical compositions (e.g., adjuvants or vaccines) of the present invention can be by any route known to those of skill in the art. Administration can be, for example, by intramuscular injection. The compositions utilized in the methods described herein can also be administered by a route selected from, for example, parenteral, dermal, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, rectal, topical administration, and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, and intramuscular administration. The preferred method of administration can vary depending on a variety of 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 given to the subject. For example, a subject who is particularly susceptible to the targeted antigenic agent may require multiple treatments to establish and / or maintain protection against the virus. The level of induced immunity provided by the pharmaceutical composition described herein can be monitored, for example, by measuring the amount of neutralizing secretory antibodies and neutralizing serum antibodies. Dosages can then be adjusted or repeated as necessary to maintain the desired level of protection against viral infection.

[0266] Embodiment At least the following embodiments are provided herein: 1. A recombinant modified vaccinia Ankara (rMVA) virus vector comprising a heterologous polycistronic nucleic acid, said polycistronic nucleic acid comprising (M) (secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x (wherein x=2-10 and M is methionine). 2. A rMVA viral vector comprising a heterologous polycistronic nucleic acid, the polycistronic nucleic acid comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide) x(secretory signal peptide-immune checkpoint inhibitor peptide) (where x=1-10 and M is methionine), an rMVA viral vector encoding the nucleotide sequence of 3. The rMVA of embodiment 1 or 2, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 56, or an amino acid sequence 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 NO: 1 to SEQ ID NO: 15, or an amino acid sequence 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 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 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 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 NO: 57 to SEQ ID NO: 90, or an amino acid sequence 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 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 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 NO: 91 to SEQ ID NO: 127, or an amino acid sequence 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 at least 95% identical thereto. 13. The rMVA of embodiments 1-11, wherein the cleavable peptide comprises the amino acid sequence RX(R / K)R, where X=any amino acid (SEQ ID NO:91). 14. The rMVA of embodiments 1 to 11, wherein the cleavable peptide comprises the amino acid sequence RX(R / K)R, where X=R, K, or H (SEQ ID NO: 92). 15. The rMVA of embodiments 1 to 12, wherein the cleavable peptide is RAKR (sequence number 93). 16. The rMVA of embodiments 1 to 11, wherein the cleavable peptide is RRRR (sequence number 94). 17. The rMVA of embodiments 1 to 11, wherein the cleavable peptide is RKRR (sequence number 95). 18. The rMVA of embodiments 1 to 11, wherein the cleavable peptide is RRKR (sequence number 96). 19. The rMVA of embodiments 1 to 11, wherein the cleavable peptide is RKKR (sequence number 97). 20. The rMVA of embodiments 1 to 11, wherein the cleavable peptide is an amino acid sequence of SEQ ID NO: 123 to SEQ ID NO: 127, or an amino acid sequence 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 an amino acid sequence at least 95% identical thereto. 22. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from SEQ ID NO: 309 to SEQ ID NO: 324, or an amino acid sequence at least 95% identical thereto. 23. The rMVA of embodiments 1 to 22, wherein x≧4. 24. The rMVA of embodiments 1 to 22, wherein x=3, 4, or 5. 25. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from SEQ ID NO: 325 to SEQ ID NO: 340, or an amino acid sequence at least 95% identical thereto. 26. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from SEQ ID NO: 341 to SEQ ID NO: 344, or an amino acid sequence at least 95% identical thereto. 27. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from SEQ ID NO: 345 to SEQ ID NO: 348, or an amino acid sequence at least 95% identical thereto. 28. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes an amino acid sequence of SEQ ID NO: 325, or an amino acid sequence at least 95% identical thereto. 29. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes an amino acid sequence of SEQ ID NO: 329, or an amino acid sequence at least 95% identical thereto. 30. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes an amino acid sequence of SEQ ID NO: 333, or an amino acid sequence at least 95% identical thereto. 31. The rMVA of embodiment 1 or 2, wherein the polycistronic nucleic acid encodes the amino acid sequence of SEQ ID NO: 337, or an amino acid sequence at least 95% identical thereto. 32. The rMVA of embodiments 1 to 31, wherein the polycistronic nucleic acid further encodes an antigenic peptide. 33. The rMVA of embodiment 32, wherein the antigenic peptide is derived from the group consisting of infectious agents and tumor-associated antigens. 34. The rMVA of embodiment 33, wherein the infectious agent is a virus, bacterium, 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 single-stranded RNA virus, coronavirus, picornavirus, togavirus, negative 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 peptide is selected from the group consisting of Ebola virus, envelope glycoprotein of Ebola virus, matrix protein VP40 of Ebola virus, Lassa virus, protein Z of Lassa virus, Zika virus, nonstructural protein 1 (NSP-1) of Zika virus, Marburg virus, glycoprotein of Marburg virus, matrix protein VP40 of Marburg, parasite of Plasmodium species, Plasmodium falciparum, circumsporozoite protein (CSP) of Plasmodium species, male gamete surface protein P230p (Pfs 230 antigen), Plasmodium spp. sporozoite microneme protein essential for cell traversal (SPECT2), Plasmodium spp. GTP-binding protein, 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. 37. The antigenic peptide is selected from the group consisting of SARS-CoV2, full-length S protein Wuhan strain of SARS-CoV2, S protein of SARS-CoV2 with substitutions K417T, E484K, and N501Y, full-length S protein delta mutant of SARS-CoV2, full-length S protein delta mutant of SARS-CoV2 plus, full-length S protein of SARS-CoV2 stabilized by two proline substitutions, full-length stabilized S protein of SARS-CoV2, full-length stabilized S protein of SARS-CoV2 with substitutions K417T, E484K, and N501Y, full-length stabilized S protein delta mutant of SARS-CoV2, full-length stabilized S protein delta mutant of SARS-CoV2 plus, S E protein of ARS-CoV2, M protein of SARS-CoV2, amino acid sequence of PP1ab polyprotein of SARS-CoV2, amino acid sequence of PP1a polyprotein of SARS-CoV2 (Wuhan Hu1), amino acid sequence of NSP1 to NSP3 of SARS-CoV2 (Wuhan Hu1), amino acid sequence of NSP4 to NSP11 of SARS-CoV2 (Wuhan Hu1), amino acid sequence of NSP12 to NSP16 of ORF1b polyprotein of SARS-CoV2 (Wuhan Hu1), amino acid sequence of NSP12 of SARS-CoV2 (Wuhan Hu1), amino acid sequence of NSP13 to NSP14 of SARS-CoV2 (Wuhan Hu1), amino acid sequence of NSP15 to NSP16 of SARS-CoV2 (Wuhan Hu1), MUC-1 33. The rMVA of embodiment 32, which is derived from the group consisting of the amino acid sequence of MARV GPTM, the amino acid sequence of Marburg virus VP40, and the MUC-1-ECD-MARVTM-ICD sequence, or a fragment thereof. 38. The rMVA of embodiment 33, wherein the tumor-associated antigen is derived from a carcinoembryonic tumor-associated antigen, a tumor viral tumor-associated antigen, an overexpressed / accumulated tumor-associated antigen, a cancer-testis tumor-associated antigen, a lineage-restricted tumor-associated antigen, a mutated tumor-associated antigen, or an idiotypic tumor-associated antigen, or a fragment thereof. 39. The tumor-associated antigens include 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 preferentially expressed in tumors (PRAME), and synovial sarcoma X (SSX)2, melanoma antigen-1 / 2 recognized by T cells (Me lan-A / MART-1 / 2), Gp100 / pmel17, tyrosine-related protein (TRP) 1 and TRP2, P. polypeptide, melanocortin 1 receptor (MC1R), and prostate specific antigen, beta-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 rMVA of embodiment 32, wherein the antigenic peptide is derived from mucin 1 or a fragment thereof. 41. The rMVA of embodiment 40, wherein the mucin 1 is encoded by a nucleic acid sequence of SEQ ID NO: 402, or a nucleic acid sequence at least 95% identical thereto. 42. The method of embodiment 40, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 349, or an amino acid sequence 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 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 rMVA of embodiment 44, wherein the extracellular domain fragment of human mucin 1 is selected from SEQ ID NO: 358 to SEQ ID NO: 361, or an amino acid sequence at least 95% identical thereto. 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 SEQ ID NO: 362, or an amino acid sequence at least 95% identical thereto. 48. The method of embodiment 40, wherein said mucin 1 is selected from SEQ ID NO: 363 or SEQ ID NO: 364, or an amino acid sequence at least 95% identical thereto. 49. The method of embodiment 48, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 363, or an amino acid sequence at least 95% identical thereto. 50. The method of embodiment 48, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 364, or an amino acid sequence 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 NO: 349 to SEQ ID NO: 357, or an amino acid sequence 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 NO: 358 to SEQ ID NO: 394, or an amino acid sequence at least 95% identical thereto. 53. The rMVA of embodiment 51 or 52, wherein the antigenic peptide 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, SEQ ID NO: 379, or an amino acid sequence at least 95% identical thereto. 54. An rMVA of embodiments 32 to 53, wherein the antigenic peptide comprises a secretory signal. 55. The rMVA of embodiment 54, wherein the secretion 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 NO: 57 to SEQ ID NO: 90, or amino acid sequences at least 95% identical thereto. 57. The rMVA of embodiment 56, wherein the secretion signal comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence at least 95% identical thereto. 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 at least 95% identical thereto. 59. The rMVA of embodiments 1 to 58, wherein the polycistronic nucleic acid is inserted between two essential, highly conserved MVA genes. 60. The rMVA of embodiments 1 to 58, wherein the polycistronic nucleic acid is inserted at a naturally occurring deletion site. 61. An 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. An 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. An rMVA according to any one of embodiments 1 to 58, wherein the polycistronic nucleic acid is inserted into the rMVA at a site selected between A50R and B1R of the MVA gene at the reconstructed modified deletion site III. 64. An 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 rMVA of embodiments 32 to 64, wherein the nucleic acid encoding the amino acid sequence of the antigenic peptide is in an open reading frame downstream of a 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 has been administered an effective amount of the target antigen. 67. The method of embodiment 66, wherein the rMVA viral vector is administered simultaneously with administration of the target antigen or after administration of the target antigen. 68. The method of embodiment 66 or 67, wherein the target antigen is selected from the group consisting of an infectious agent and a tumor-associated antigen. 69. The method of embodiment 68, wherein the infectious agent is a virus, bacterium, fungus, parasite, or amoeba. 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 single-stranded RNA virus, coronavirus, picornavirus, togavirus, negative 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 antigen is selected from the group consisting of Ebola virus, envelope glycoprotein of Ebola virus, matrix protein VP40 of Ebola virus, Lassa virus, protein Z of Lassa virus, Zika virus, nonstructural protein 1 (NSP-1) of Zika virus, Marburg virus, glycoprotein of Marburg virus, matrix protein VP40 of Marburg, Plasmodium species parasite, Plasmodium falciparum, circumsporozoite protein (CSP) of Plasmodium species, male gamete surface protein P230p (Pfs230p) of Plasmodium species, 68. The method of embodiment 66 or 67, wherein the putative antigen is derived from a human immunodeficiency virus, an HIV Env protein, an HIV gp41, an HIV gp120, an HIV gp160, an HIV Gag protein, an HIV MA, an HIV CA, an HIV SP1, an HIV NC, an HIV SP2, an HIV P6, an HIV Pol protein, an HIV RT, an HIV RNase H, an HIV IN, and an HIV PR, or a fragment thereof. 72. The target antigen is SARS-CoV2, full-length S protein Wuhan strain of SARS-CoV2, S protein of SARS-CoV2 with substitutions K417T, E484K, and N501Y, full-length S protein delta mutant of SARS-CoV2, full-length S protein delta mutant of SARS-CoV2 plus, full-length S protein of SARS-CoV2 stabilized by two proline substitutions, full-length stabilized S protein of SARS-CoV2, full-length stabilized S protein of SARS-CoV2 with substitutions K417T, E484K, and N501Y, full-length stabilized S protein delta mutant of SARS-CoV2, full-length stabilized S protein delta mutant of SARS-CoV2 plus, E protein of SARS-CoV2, S 68. The method of embodiment 66 or 67, wherein the amino acid sequence is 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 sequence of NSP1 to NSP3 of SARS-CoV2 (Wuhan Hu1), the amino acid sequence of NSP4 to NSP11 of SARS-CoV2 (Wuhan Hu1), the amino acid sequence of the polyprotein NSP12 to NSP16 of ORF1b of SARS-CoV2 (Wuhan Hu1), the amino acid sequence of NSP12 of SARS-CoV2 (Wuhan Hu1), the amino acid sequence of NSP13 to NSP14 of SARS-CoV2 (Wuhan Hu1), and the amino acid sequence of NSP15 to NSP16 of SARS-CoV2 (Wuhan Hu1), or a fragment thereof. 73. The method of embodiment 68, wherein the tumor-associated antigen is derived from a carcinoembryonic tumor-associated antigen, a tumor viral tumor-associated antigen, an overexpressed / accumulated tumor-associated antigen, a cancer-testis tumor-associated antigen, a lineage-restricted tumor-associated antigen, a mutated tumor-associated antigen, or an idiotypic tumor-associated antigen, or a fragment thereof. 74. The tumor-associated antigens include 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 preferentially expressed in tumors (PRAME), and synovial sarcoma X (SSX)2, melanoma antigen-1 / 2 (MAGE) recognized by T cells. 69. The method of embodiment 68, wherein the polypeptide is derived from a polypeptide selected from the group consisting of elan-A / MART-1 / 2), Gp100 / pmel17, tyrosine-related protein (TRP) 1 and TRP2, P. polypeptide, melanocortin 1 receptor (MC1R), and prostate specific antigen, beta-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 a nucleic acid sequence of SEQ ID NO: 402, or a nucleic acid sequence at least 95% identical thereto. 77. The method of embodiment 75, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 349, or an amino acid sequence at least 95% identical thereto. 78. The method of embodiment 75, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence 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 NO: 358 to SEQ ID NO: 361, or an amino acid sequence 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 SEQ ID NO: 362, or an amino acid sequence at least 95% identical thereto. 83. The method of embodiment 75, wherein said mucin 1 is selected from SEQ ID NO: 363 or SEQ ID NO: 364, or an amino acid sequence at least 95% identical thereto. 84. The method of embodiment 83, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 363, or an amino acid sequence at least 95% identical thereto. 85. The method of embodiment 83, wherein the mucin 1 comprises the amino acid sequence of SEQ ID NO: 364, or an amino acid sequence 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 NO: 349 to SEQ ID NO: 357, or an amino acid sequence 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 NO: 358 to SEQ ID NO: 394, or an amino acid sequence 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, SEQ ID NO: 379, or an amino acid sequence at least 95% identical thereto. 89. A rMVA viral vector comprising a heterologous polycistronic nucleic acid, the polycistronic nucleic acid comprising (M) (secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x (secretory signal peptide-antigenic peptide), where x=1-10 and M is methionine. 90. A rMVA viral vector comprising a heterologous polycistronic nucleic acid, the polycistronic nucleic acid comprising (M) (secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane peptide), where x=1-10 and M is methionine. 91. A rMVA viral vector comprising a heterologous polycistronic nucleic acid, the polycistronic nucleic acid comprising (M) (secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x A rMVA viral vector encoding (glycoprotein signal peptide-antigenic peptide-glycoprotein transmembrane peptide-cleavable peptide)(viral matrix protein), where x=1-10 and M is methionine. 92. A recombinant modified vaccinia Ankara (rMVA) virus vector comprising a heterologous polycistronic nucleic acid insert encoding a polypeptide, the polypeptide comprising ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide). x (antigenic peptide)), where x=1 to 10 and M is methionine. 93. An rMVA of embodiments 89 to 92, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 56, or an amino acid sequence at least 95% identical thereto. 94. An rMVA of embodiments 89 to 93, wherein the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 15, or an amino acid sequence at least 95% identical thereto. 95. An rMVA of embodiments 89 to 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 at least 95% identical thereto. 96. An rMVA of embodiments 89 to 95, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 95% identical thereto. 97. An rMVA of embodiments 89 to 95, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence at least 95% identical thereto. 98. An rMVA of embodiments 89 to 97, wherein the secretory signal peptide comprises an amino acid sequence selected from SEQ ID NO: 57 to SEQ ID NO: 90, or an amino acid sequence at least 95% identical thereto. 99. An rMVA of embodiments 89 to 98, wherein the secretory signal peptide comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence at least 95% identical thereto. 100. An rMVA of embodiments 89 to 98, wherein the secretory signal peptide comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence at least 95% identical thereto. 101. The rMVA of embodiments 89 to 100, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 91 to SEQ ID NO: 126, or an amino acid sequence at least 95% identical thereto. 102. The rMVA of embodiments 89 to 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 rMVA of embodiments 89 to 101, wherein the cleavable peptide comprises the amino acid sequence RX(R / K)R, where X=any amino acid (SEQ ID NO:91). 104. The rMVA of embodiments 89 to 101, wherein the cleavable peptide comprises the amino acid sequence RX(R / K)R, where X=R, K, or H (sequence number 92). 105. The rMVA of embodiments 89 to 102, wherein the cleavable peptide is RAKR (sequence number 93). 106. The rMVA of embodiments 89 to 101, wherein the cleavable peptide is RRRR (sequence number 94). 107. The rMVA of embodiments 89 to 101, wherein the cleavable peptide is RKRR (sequence number 95). 108. The rMVA of embodiments 89 to 101, wherein the cleavable peptide is RRKR (sequence num...

Claims

1. (M) a rMVA viral vector comprising a heterologous nucleic acid insert encoding a polypeptide comprising (secretory signal peptide-immune checkpoint inhibitor peptide)x, x=1-10 and M is methionine; The immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-56, or an amino acid sequence at least 95% identical thereto; rMVA viral vector.

2. The rMVA viral vector described in claim 1, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO:

1.

3. The rMVA viral vector described in claim 1, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO:

5.

4. A rMVA viral vector comprising a heterologous polycistronic nucleic acid encoding ((M)(secretory signal peptide-immune checkpoint inhibitor peptide-cleavable peptide)x(secretory signal peptide-immune checkpoint inhibitor peptide)), x=1-10, M is methionine, and the immune checkpoint inhibitor peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-56, or an amino acid sequence at least 95% identical thereto; rMVA viral vector.

5. The rMVA viral vector of claim 4, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO:

1.

6. The rMVA viral vector of claim 4, wherein the immune checkpoint inhibitor peptide comprises the amino acid sequence of SEQ ID NO:

5.

7. 5. The rMVA viral vector of claim 4, wherein the secretory signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57 to SEQ ID NO:90, or an amino acid sequence at least 95% identical thereto.

8. The rMVA viral vector of claim 7, wherein the secretory signal peptide comprises the amino acid sequence of SEQ ID NO:

65.

9. The rMVA viral vector of claim 7, wherein the secretory signal peptide comprises the amino acid sequence of SEQ ID NO:

66.

10. 5. The rMVA viral vector of claim 4, wherein the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:91 to SEQ ID NO:127, or an amino acid sequence at least 95% identical thereto.

11. The rMVA viral vector of claim 10, wherein the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:93, SEQ ID NO:120, and SEQ ID NO:123, or an amino acid sequence at least 95% identical thereto.

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

93.

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

120.

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

123.

15. The rMVA viral vector of claim 4, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 309 to SEQ ID NO: 324, or an amino acid sequence at least 95% identical thereto.

16. The rMVA viral vector of claim 4, wherein x=3, 4, or 5.

17. The rMVA viral vector of claim 4, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 325 to SEQ ID NO: 340, or an amino acid sequence at least 95% identical thereto.

18. The rMVA viral vector of claim 4, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 341 to SEQ ID NO: 344, or an amino acid sequence at least 95% identical thereto.

19. 5. The rMVA viral vector of claim 4, wherein the polycistronic nucleic acid encodes an amino acid sequence selected from SEQ ID NO: 345 to SEQ ID NO: 348, or an amino acid sequence at least 95% identical thereto.

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

325.

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

329.

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

333.

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

337.

24. The rMVA viral vector of claim 4 , wherein the polycistronic nucleic acid further encodes an antigenic peptide.

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