Recombinant poxviruses for cancer immunotherapy

Abstract

Disclosed herein are methods and compositions related to the treatment, prevention, and / or amelioration of cancer in a subject in need thereof. In particular, the present technology relates to the use of a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 virus, a MVAΔE5R-hFlt3L-OX40L virus (MQ710), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 virus (MQ832), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L virus (MQ953), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF virus (MQ954), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔNlL-hIL-7 virus (MQ955), or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors as an immunotherapeutic composition, in methods for treating a solid tumor wherein the solid tumor is resistant to immune checkpoint blockade inhibitor treatment, methods for treating a tumor in a subject in need thereof wherein the subject has a deficient adaptive immune system response, and methods for altering the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof.

Description

Atty. Dkt. No.115872-3093 RECOMBINANT POXVIRUSES FOR CANCER IMMUNOTHERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 585,478, filed September 26, 2023, the content of which is incorporated herein by reference in its entirety for any and all purposes. STATEMENT OF FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under CA008748, and CA280657 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The technology of the present disclosure relates generally to the fields of oncology, virology, and immunotherapy. In particular, the present technology relates to the use of poxviruses, including a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a heterologous nucleic acid molecule encoding OX40L (OX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding interleukin 12 (IL-12) (MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12), a MVAΔE5R-hFlt3L-OX40L virus (MQ710), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 virus (MQ832), a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors. In particular, the present technology relates to the use of a MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12 virus, a MVAΔE5R-hFlt3L-OX40L virus (MQ710), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 virus (MQ832), a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade 1 4873-2549-9626.1Atty. Dkt. No.115872-3093 inhibitors as an immunotherapeutic composition, in methods for treating a solid tumor, in methods for treating a solid tumor wherein the solid tumor is resistant to immune checkpoint blockade inhibitor treatment, methods for treating a tumor in a subject in need thereof wherein the subject has a deficient adaptive immune system response, and methods for altering the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof. BACKGROUND

[0004] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art.

[0005] Malignant tumors are inherently resistant to conventional therapies and present significant therapeutic challenges. Immunotherapy is an evolving area of research and an additional option for the treatment of certain types of cancers. The immunotherapy approach rests on the rationale that the immune system may be stimulated to identify tumor cells and target them for destruction. Despite presentation of antigens by cancer cells and the presence of immune cells that could potentially react against tumor cells, in many cases, the immune system is not activated or is affirmatively suppressed. Key to this phenomenon is the ability of tumors to protect themselves from immune response by coercing cells of the immune system to inhibit other cells of the immune system. Tumors develop a number of immunomodulatory mechanisms to evade antitumor immune responses. Thus, improved immunotherapeutic approaches are needed to enhance host antitumor immunity and target tumor cells for destruction. SUMMARY

[0006] In one aspect, the present disclosure provides, a method for treating a solid tumor in a subject in need thereof comprising administering to a tumor a composition comprising an effective amount of a modified vaccinia Ankara (MVA) virus genetically engineered to comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a heterologous nucleic acid molecule encoding OX40L, a heterologous nucleic acid molecule encoding hFlt3L, and a heterologous nucleic acid molecule encoding IL-12 (MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12), and a composition comprising an effective amount of hG-CSF. In some embodiments, the virus and the hG-CSF are administered to the subject by the same route or by different routes. In some embodiments, 2 4873-2549-9626.1Atty. Dkt. No.115872-3093 the virus is administered to the subject by intratumoral or intravenous injection or a simultaneous or sequential combination of intraturmoral and intravenous injection. In some embodiments, the hG-CSF is administered to the subject by intratumoral or intravenous injection or a simultaneous or sequential combination of intraturmoral and intravenous injection. In some embodiments, the virus is administered to the subject by intratumoral injection and the hG-CSF is administered intratumorally or systemically. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of one or more of anti-tumor neutrophils, macrophages, and / or monocytes and / or depletion of pro-tumor M2 macrophages and / or pro- tumor regulatory T cells. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils and macrophages. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils, macrophages, and monocytes. In some embodiments, the tumor is malignant peripheral nerve sheath tumors, melanoma, colon, breast, bladder, prostate carcinoma, sarcoma, ovarian, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, pancreatic cancer, malignant nerve sheath tumor, anaplastic thyroid cancer, or Extramammary Paget disease (EMPD). In some embodiments, the tumor is resistant to immune checkpoint blockade inhibitor treatment.

[0007] In one aspect, the present disclosure provides, a modified vaccinia Ankara (MVA) virus selected from the group consisting of: an MVA virus genetically engineered to 3 4873-2549-9626.1Atty. Dkt. No.115872-3093 comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a C12L gene (ΔC12L), a heterologous nucleic acid molecule encoding OX40L (OX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding IL-12 (IL-12) (MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L); an MVA virus genetically engineered to comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a mutant C12L gene (ΔC12L), a heterologous nucleic acid molecule encoding OX40L (OX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), a heterologous nucleic acid molecule encoding IL-12 (IL-12), and a heterologous nucleic acid molecule encoding human granulocyte colony-stimulating factor (hG-CSF) (MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF); an MVA virus genetically engineered to comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a mutant C12L gene (ΔC12L), a heterologous nucleic acid molecule encoding OX40L, a heterologous nucleic acid molecule encoding hFlt3L, a heterologous nucleic acid molecule encoding IL-12, a heterologous nucleic acid molecule encoding hG-CSF, and a heterologous nucleic acid molecule encoding human IL-7 (hIL-7) (MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7); and any combination thereof.

[0008] In one aspect, the present disclosure provides, an immunogenic composition comprising the virus of the preceding embodiment. In some embodiments, the immunogenic composition of further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of further comprises a pharmaceutically acceptable adjuvant.

[0009] In one aspect, the present disclosure provides, a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the virus of any of the preceding embodiments or the immunogenic composition of any one of the preceding embodiments. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some 4 4873-2549-9626.1Atty. Dkt. No.115872-3093 embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of one or more of stem-like T-cells, neutrophils, macrophages, and / or monocytes. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of stem- like T-cells. In some embodiments, the virus is MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199- IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7, or MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L further engineered to comprise a heterologous nucleic acid encoding hIL-7, or MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF further engineered to comprise a heterologous nucleic acid encoding hIL-7, or wherein the composition or immunogenic composition further comprises an effective amount of hIL-7. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils and macrophages. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils, macrophages, and monocytes. In some embodiments, the composition is administered to the subject by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is malignant peripheral nerve sheath tumors, melanoma, colon, breast, bladder, prostate carcinoma, sarcoma, ovarian, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, pancreatic cancer, malignant nerve sheath tumor, anaplastic thyroid cancer, Merkel cell carcinoma, or Extramammary Paget disease (EMPD).

[0010] In one aspect, the present disclosure provides, a method for inducing an immune response in a subject, the method comprising administering to the subject a composition comprising an effective amount of the virus of any of the preceding embodiments, or the immunogenic composition of any one of the preceding embodiments. In some 5 4873-2549-9626.1Atty. Dkt. No.115872-3093 embodiments, administration of the virus increases activation and / or expansion of one or more of stem-like T-cells, neutrophils, macrophages, and / or monocytes. In some embodiments, administration of the virus increases activation and / or expansion of stem-like T-cells. In some embodiments, the virus is MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L-hG-CSF-hIL-7, or MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L further engineered to comprise a heterologous nucleic acid encoding hIL-7, or MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF further engineered to comprise a heterologous nucleic acid encoding hIL-7, or wherein the composition or immunogenic composition further comprises an effective amount of hIL-7. In some embodiments, administration of the virus increases activation and / or expansion of neutrophils. In some embodiments, administration of the virus increases activation and / or expansion of neutrophils and macrophages. In some embodiments, administration of the virus increases activation and / or expansion of neutrophils, macrophages, and monocytes.

[0011] In one aspect, the present disclosure provides, a modified vaccinia virus comprising a deletion of the sequence encoding the 83 n-terminal amino acids of the E3L gene (ΔE3L83N), a mutation of the E5R gene (ΔE5R), a hFlt3L transgene, an mOX40L transgene, a mutation of the J2R gene, a mutation of the WR199 gene, a mutation of the WR200 gene, and a mIL-12 transgene (VACV-ΔE3L83N-ΔE5R-hFlt3L-mOX40L-ΔJ2R- ΔWR199-ΔWR200-mIL-12). In one aspect, the present disclosure provides, a method of treating a solid malignant tumor in a subject in need thereof, comprising administering an effective amount of the modified vaccinia virus of the preceding embodiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1A is a schematic diagram of the second-generation recombinant MVA construct MQ833. It is constructed based on MVA by inserting an expression cassette containing two transgenes encoding membrane-bound human Flt3L and murine OX40L linked by a P2A self-cleaving sequence into the E5R locus to generate the first-generation recombinant MVA (rMVA or MQ710; MVA∆E5R-hFlt3L-mOX40L). To further potentiate the antitumor efficacy of the virus, a second-generation recombinant MVA (MQ833) was made by removing two more viral immune evasion genes, E3L and WR199, and inserting the gene encoding interleukin 12 (IL-12) anchored to extracellular matrix to prevent systemic toxicity. FIG.1B is a schematic diagram of co-administration experiment. B2m KO B16-F10 melanoma (6 x 105) were implanted intradermally to the 6 4873-2549-9626.1Atty. Dkt. No.115872-3093 right flank of wild type C57BL / 6J mice. After tumors were established, they were injected with murine mMQ833 (MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12) (8 x 107pfu) and murine granulocyte colony stimulating factor (mG-CSF) (3 µg) or human granulocyte colony stimulating factor (hG-CSF) (3 µg). mMQ833 was injected twice three days apart. mG-CSF or hG-CSF were given intratumorally daily for 5 consecutive days. The injected tumors were harvested two days after the second dose of mMQ833. After obtaining single cell suspension, the tumor samples were stained with various markers, including CD45, CD11b, Ly6G, Ly6C, F4 / 80, CD68, and inducible nitric oxide synthase (iNOS) to evaluate neutrophil, macrophage, and monocyte recruitment and activation after either mMQ833 monotherapy or in combination of mG-CSF or hG-CSF. FIG.1C are flow cytometry analyses of iNOS+neutrophils (Ly6G+) in tumors treated with PBS, mMQ833, mMQ833 + mG-CSF, mMQ833 + hG-CSF. FIG.1D is a quantification of percentages of iNOS+neutrophils out of total neutrophils in tumors treated with PBS, mMQ833, mMQ833 + mG- CSF, mMQ833 + hG-CSF. FIG.1E are flow cytometry analyses of iNOS+macrophages (CD11b+) in tumors treated with PBS, mMQ833, mMQ833 + mG-CSF, mMQ833 + hG- CSF. FIG.1F is a quantification of percentages of iNOS+macrophages (CD11b+) out of total macrophages in tumors treated with PBS, mMQ833, mMQ833 + mG-CSF, mMQ833 + hG-CSF.

[0013] FIG.2A are flow cytometry analyses of inflammatory monocytes (Ly6C+) and neutrophils (Ly6G+) in tumors treated with PBS, mMQ833, mMQ833 + mG-CSF, mMQ833 + hG-CSF. FIG.2B are flow cytometry analyses of iNOS+monocytes (Ly6C+) in tumors treated with PBS, mMQ833, mMQ833 + mG-CSF, mMQ833 + hG-CSF. FIG. 2C is a quantification of percentages of iNOS+monocytes (Ly6C+) out of total monocytes in tumors treated with PBS, mMQ833, mMQ833 + mG-CSF, mMQ833 + hG-CSF.

[0014] FIG.3A are graphs showing individual tumor volumes over time of PRC2-wt or PRC2-loss SKP605 tumors and Kaplan-Meier survival curve of mice treated with IT MQ833 in the presence or absence of anti-CTLA-4 and anti-PD-1 antibodies. PBS was used as a control. SgCon: PRC2-wt SKP605; SgEed: PRC2-loss SKP605. FIGs.3B-3E: SKP605 sgCon and sgEed cells were infected by MQ833 at a MOI of 10. FIG.3B are graphs showing flow cytometric analysis of mOX40L and hFlt3L transgene expression. Cells and supernatant were collected at 16 hours post-infection (hpi). FIG.3C is a Western blot for indicated proteins and phosphorylated proteins. Cells were collected at 2, 4, 6, 8, 7 4873-2549-9626.1Atty. Dkt. No.115872-3093 and 16 hours post-infection (hpi). FIG.3D is a flow cytometric analysis of MHC1 and PD- L1 expression. Cells were collected after infection for 16 hours. FIG.3E are graphs showing mIL-12 p70 and mIFN-β expression. Cells were collected after infection for 16 hours. FIG.3F are graphs showing human IFN-β and IL-12p70 levels in the supernatants of human MQ833 (hMQ833)-infected human MPNST cells (M3).

[0015] FIG.4A is a schematic diagram of evaluating the population of neutrophils (Ly6G+CD11b+CD45+) with or without expression of inducible nitric oxide synthase (iNOS) in murine MPNST tumors SKP605 sgEed treated with intratumoral (IT) delivery of MQ833 or PBS. 1 x 106SKP605 sgEed tumor cells were implanted subcutaneously into the right flanks for C57BL / 6J mice. Once the tumors were established, they were treated with IT MQ833 or PBS twice three days apart. Tumors were harvested two days after the second injection. Tumor-infiltrating immune cells were analyzed. FIGs.4B and 4C are contour plots (FIG.4B) and a graph (FIG.4C) showing percentage of neutrophils among CD45+immune cells in SKP605 sgEed (PRC2-loss) tumors treated with IT MQ833. FIGs.4D and 4E are contour plots (FIG.4D) and a graph (FIG.4E) showing percentage of iNOS+cells among neutrophils in SKP605 sgEed (PRC2-loss) tumors treated with IT MQ833.

[0016] FIGs.5A and 5B are contour plots (FIG.5A) and graphs (FIG.5B) showing percentage of Granzyme B (Gzmb+) CD8+among CD8+T cells in SKP605 sgEed (PRC2- loss) tumors treated with IT MQ833 or PBS control. Granzyme B expression is a marker for T cell activation. FIGs.5C and 5D are contour plots (FIG.5C) and graph (FIG.5D) showing percentage of Tox+cells among CD8+T cells in SKP605 sgEed (PRC2-loss) tumors treated with IT MQ833. Tox expression is a marker for T cell exhaustion. FIGs. 5E and 5F are contour plots (FIG.5E) and graphs (FIG.5F) showing percentage of Granzyme B (Gzmb+) CD4+among CD4+T cells in SKP605 sgEed (PRC2-loss) tumors treated with IT MQ833 or PBS control. FIGs.5G and 5H are contour plots (FIG.5G) and a graph (FIG.5H) showing percentage of Tox+cells among CD4+T cells in SKP605 sgEed (PRC2-loss) tumors treated with IT MQ833 or PBS control. FIGs.5I and 5J are contour plots (FIG.5I) and a graph (FIG.5J) showing percentage of Foxp+cells among CD4+T cells in SKP605 sgEed (PRC2-loss) tumors treated with IT MQ833 or PBS control. Foxp+CD4+T cells are regulatory T cells.

[0017] FIG.6A is a schematic diagram of assessing the role of different immune cells in MQ833-induced antitumor immunity in SKP605 sgEed (PRC2-loss) tumor model. SKP605 8 4873-2549-9626.1Atty. Dkt. No.115872-3093 sgEed cells (1 x 106) were implanted subcutaneously into the right flanks of C57BL / 6J mice. Once the tumors are established, immune cell subsets were depleted by administering 200 μg monoclonal antibodies i.p. twice weekly starting 1 day prior to the first viral injection as indicated, continued until the animals either died, were euthanized, or were completely clear of tumors: anti-CD8-α for CD8+ T cells (clone 2.43, BioXCell), anti-CD4 for CD4+T cells (clone GK1.5, BioXCell), anti-NK1.1 for NK cells (clone PK136, BioXCell), and 500 μg of anti-Ly6G (clone 1A8, BioXCell) for neutrophil depletion.500 μg of anti-CSF1 (provided by Ming Li’s laboratory) were given every 5 days for macrophage depletion. Tumor sizes were measured, and mice survival were monitored. FIG.6B shows a Kaplan-Meier survival curve of SKP sgEed tumor-bearing mice treated with MQ833 virus in the presence or absence of depleting antibodies. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-10, *P<0.05, **P<0.01, ****P < 0.0001). FIG.6C are graphs showing tumor volumes of mice injected with PBS or MQ833 in the presence or absence of depleting antibodies including anti- NK1.1, anti-CD8, anti-CD4, anti-Ly6G, and anti-CSF1antibodies over days post treatment.

[0018] FIG.7A is a schematic diagram of the experimental schedule. 1x106of SKP605 sgCon or SKP605 sgEed cells were subcutaneously implanted into the right flank of WT orC57BL / 6J mice. Intratumoral virus treatments were given twice a week once the tumors were established. Tumor size and mice survival were monitored. FIG.7B shows Kaplan-Meier survival curve of MPNST tumor bearing Irf3- / -Irf7- / -mice treated with MQ833 virus. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test. (n=8-, *, P < 0.05). FIG.7C are graphs showing tumor volumes of Irf3- / -Irf7- / -mice injected with MQ833 or PBS over days post injection. FIG.7D is a schematic diagram of an in vivo therapeutic model to assess the role of adaptive immunity using Rag1- / -mice and type I IFN signaling using Stat2- / -mice in MQ833-induced antitumor immunity. SKP605 sgEed cells (1 x 106) were implanted subcutaneously into the right flanks of WT, Rag1- / -, or Stat2- / -C57BL / 6J mice. Once the tumors are established, they were treated with IT MQ833 injections twice weekly (8 x 107pfu per dose). Tumors sizes were measured twice weekly and mouse survival was monitored. FIG.7E shows a Kaplan-Meier survival curve of SKP sgEed tumor-bearing mice treated with MQ833 virus in WT, Rag1- / -, or Stat2- / -C57BL / 6J mice. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5, **P<0.01). FIG.7F are graphs showing tumor volumes of mice injected with MQ833 in WT, Rag1- / -, or Stat2- / -C57BL / 6J mice over days post treatment. 9 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0019] FIG.8A is a schematic diagram of the experimental schedule. 1x106of SKP605 sgCon or SKP605 sgEed cells were subcutaneously implanted into the right flank of C57BL / 6J mice. Once the tumors were established, IT MQ833 injection was given twice three days apart. Tumors were collected two days after the second injection and processed into single cell suspension, and CD45+cells were sorted and scRNA-seq analysis were performed. FIG.8B is a UMAP displaying different immune cell populations among CD45+cells from 4 samples combined following 10X Genomics scRNA-seq workflow (n = 33,201 cells). FIG.8C are plots of cell clusters separated by sample. FIG.8D is a stacked bar plot showing the percentage of each cluster across different samples. FIG.8E are expression plots of main cell types using their top marker genes. FIG.8F is a UMAP displaying subclustered CD45+CD3+cells from 4 samples combined following 10X Genomics scRNA-seq workflow (n = 5,943 cells). FIG.8G are expression plots of Cd4, Cd8a, Cd3d. FIG.8H are UMAP plots and FIG.8I is a stacked bar plot of cell clusters separated by sample. FIG.8J is a UMAP displaying subclustered neutrophils from 4 samples combined following 10X Genomics scRNA-seq workflow (n = 2,399 cells). FIG. 8K are UMAP plots of cell clusters separated by sample. FIG.8L are expression plots of Cxcr2, Nos2, Cd63, Cxcl10.

[0020] 1x106of SKP605 sgEed cells were subcutaneously implanted into the right flank of Ifnarfl / flor Cd4creIfnarfl / flC57BL / 6J mice. Intratumoral MQ833 treatments were given twice a week once the tumors were established. Tumor size and mice survival were monitored. FIG.9A shows a Kaplan-Meier survival curve of SKP605 sgCon tumor- bearing Cd4creIfnarfl / fland Ifnarfl / flC57BL / 6J mice treated with MQ833 virus. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-9, *P<0.05). FIG.9B are graphs showing tumor volumes of mice injected with MQ833 in CD4creIfnarfl / fland Ifnarfl / flC57BL / 6J mice over days post treatment. 1 x 106of MB49 cells were subcutaneously implanted into the right flank of Ifnarfl / fl, Foxp3creIfnarfl / fl, LyzcreIfnarfl / fl, CD64creIfnarfl / fl, and Cd4creIfnarfl / flC57BL / 6J mice. Intratumoral MQ833 treatments were given twice a week once the tumors were established. Tumor size and mice survival were monitored. FIG.9C shows Kaplan-Meier survival curves of MB49 tumor-bearing mice treated with MQ833 (n=8-14, **P<0.01, ****P < 0.0001). FIG.9D are graphs showing tumor volumes of Ifnarfl / fl, Foxp3creIfnarfl / fl, LyzcreIfnarfl / fl, CD64creIfnarfl / fl, and Cd4creIfnarfl / flC57BL / 6J mice treated with MQ833 over days post treatment. 10 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0021] FIG.10A is a schematic diagram of a recombinant oncolytic vaccinia OV-IL-12 with the same three transgenes as MQ833. It was constructed based on VACV∆E3L83N by inserting an expression cassette by inserting an expression cassette containing two transgenes encoding membrane-bound human Flt3L and murine OX40L linked by a P2A self-cleaving sequence into the E5R locus. To further potentiate the antitumor efficacy and enhance the safety of the virus, we further removed J2R, which encodes the thymidine kinase gene, and two more viral immune evasion genes (WR199 and WR200) and inserted the gene encoding interleukin 12 (IL-12) anchored to extracellular matrix to prevent systemic toxicity. WR199 encodes an ankyrin-repeat-containing protein with a F-box at the C-terminus. WR200 encodes a decoy receptor for type I IFNs. FIG.10B shows a Kaplan- Meier survival curve of B16-F10- tumor-bearing mice treated with OV-IL-12 virus. The mouse stains include WT, cGAS- / -, MDA5 / STING DKO, Rag1- / -, and Stat2- / -C57BL / 6J mice. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-9, *P<0.05, ***P<0.001). FIG.10C are graphs showing tumor volumes of mice injected with OV-IL- 12 or PBS in WT, cGAS- / -, MDA5 / STING DKO, Rag1- / -, and Stat2- / -C57BL / 6J mice over days post treatment. FIG.10D shows a Kaplan-Meier survival curve of B16-F10 tumor- bearing CD64creIfnarfl / fland Ifnarfl / flC57BL / 6J mice treated with OV-IL-12 virus or PBS. Survival data were analyzed by log-rank (Mantel-Cox) test (n=12-14, ****P<0.0001). FIG.10E are graphs showing tumor volumes of CD64creIfnarfl / fland Ifnarfl / flC57BL / 6J mice injected with OV-IL-12 or PBS over days post treatment. FIG.10F shows a Kaplan-Meier survival curve of MC38 tumor-bearing LyzcreIfnarfl / fland Ifnarfl / flC57BL / 6J mice treated with OV-IL-12 virus or PBS. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-8, *P<0.05). FIG.10G are graphs showing tumor volumes of LyzcreIfnarfl / fland C57BL / 6J mice injected with OV-IL-12 or PBS over days post treatment.

[0022] FIG.11A shows a Kaplan-Meier survival curve of B16-F10 tumor-bearing Foxp3creIfnarfl / fl, Ifnarfl / f, Foxp3creIfngrfl / flC57BL / 6J mice treated with OV-IL-12 virus or PBS. Survival data were analyzed by log-rank (Mantel-Cox) test (n=9-14, *P<0.05, ****P<0.0001). FIG.11B are graphs showing tumor volumes of Foxp3creIfnarfl / fl,Foxp3creIfngrfl / flC57BL / 6J mice injected with OV-IL-12 or PBS over days post treatment. FIG.11C shows a Kaplan-Meier survival curve of MC38 tumor-bearing Foxp3creIfnarfl / fl, Ifnarfl / f, Foxp3creIfngrfl / flC57BL / 6J mice treated with OV-IL-12 virus or PBS. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-13, *P<0.05, **P<0.01). FIG.11D are graphs showing tumor volumes of Foxp3creIfnarfl / fl, Ifnarfl / f, Foxp3creIfngrfl / flC57BL / 6J mice 11 4873-2549-9626.1Atty. Dkt. No.115872-3093 treated with IT OV-IL-12 or PBS over days post treatment. FIG.11E shows a Kaplan- Meier survival curve of B16-F10 tumor-bearing Foxp3creIfnarfl / fl, Ifnarfl / f, C57BL / 6J mice treated with OV-IL-12, MQ833, or PBS. Survival data were analyzed by log-rank (Mantel- Cox) test (n=4-13, *P<0.05, ***P<0.001). FIG.11F are graphs showing tumor volumes of Foxp3creIfnarfl / fl, Ifnarfl / fC57BL / 6J mice injected with OV-IL-12, MQ833, or PBS over days post treatment.

[0023] FIG.12A are dot plots showing hFlt3L and mOX40L expression on murine ovarian cancer MPB1-Luc cells infected with either OV-IL-12 or MQ833. MPB1-Luc (MYC;Trp53- / -; Brca1- / -) cells expressing luciferase were infected with either OV-IL-12 or MQ833 at a multiplicity of infection (MOI) of 10 for 16 h. Cells were stained with anti- hFlt3L and anti-mOX40L antibodies and subjected for flow cytometry analyses. FIG.12B are graphs showing the mIFN-β and mIL-12p70 levels in the supernatants of MPB1 or MPB1-luc cells infected with either OV-IL-12 or MQ833 after 16 h infection. FIG.12C are images of replicating OV-IL12 or non-replicating MQ833-infected COV644 cells. FIG. 12D is a schematic diagram of assessing antitumor effects of MQ833 vs OV-IL-12 in a MPB1-luc ovarian cancer model after intraperoitoneal implantation. C57BL / 6J mice were first implanated intraperitoneally with MPB1-Luc (2 x 106) cells for 8 days. MQ833 or OV- IL-12 were delivererd intraperitoneally twice weekly. In vivo bioluminescence imaging was performed, and mouse survival were monitored. FIG.12E shows a Kaplan-Meier survival curve of MPB1 tumor-bearing C57BL / 6J mice treated with OV-IL-12, MQ833, or PBS. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5, *P<0.05, **P<0.01). FIG.12F is a graph showing IVIS radiance of MPB1-luc bearing C57BL / 6J mice injected with OV-IL-12, MQ833, or PBS over days post treatment.

[0024] FIG.13A is a schematic diagram of an efficacy study in a SKP605 sgCon (PRC2- wt) murine malignant peripheral nerve sheath tumor evaluating the therapeutic potential of the combination of MQ833 with human G-CSF. SKP605 sgCon cells (1 x 106) were implanted subcutaneously into the right flanks of C57BL / 6J mice. Once the tumors are established, they were treated with IT MQ833 (8 x 107pfu), human G-CSF (3 µg), MQ833 + human G-CSF, or PBS twice weekly. Tumors were measured and mice survival were monitored. FIG.13B shows a Kaplan-Meier survival curve of SKP sgCon tumor-bearing mice treated with IT MQ833, human G-CSF, MQ833 + human G-CSF, or PBS. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-10, *P<0.05, ***P<0.001, ****P 12 4873-2549-9626.1Atty. Dkt. No.115872-3093 < 0.0001). FIG.13C are graphs showing tumor volumes of mice injected with PBS, MQ833, human G-CSF, MQ833 + human G-CSF over days post treatment.

[0025] FIG.14A is a schematic diagram of evaluating immune cell alterations in SKP605 sgCon (PRC2-wt) murine malignant peripheral nerve sheath tumor after treating with MQ833, human G-CSF, the combination of MQ833 with human G-CSF, or PBS. SKP605 sgCon cells (1 x 106) were implanted subcutaneously into the right flanks of C57BL / 6J mice. Once the tumors are established, they were treated with IT MQ833 (8 x 107pfu), human G-CSF (3 µg), MQ833 + human G-CSF, or PBS twice three days apart. Tumors were harvested two days post second injection. FIGs.14B-14D are contour plots (FIG. 14B) and graphs (FIG.14C and FIG.14D) showing percentages of iNOS+Ly6G+CD11b+neutrophils and CD11b levels (MFI) among tumor-infiltrating neutrophils cells in SKP605 sgCon (PRC2-loss) tumors treated with PBS, hG-CSF, MQ833, or MQ833 + hG-CSF. FIGs.14B and 14E are contour plots (FIG.14B) and a graph (FIG.14E) showing percentages of iNOS+Ly6G- Ly6C+monocytes among tumor-infiltrating monocytes cells in SKP605 sgCon (PRC2-loss) tumors treated with PBS, hG-CSF, MQ833, or MQ833 + hG- CSF.

[0026] FIGs.15A and 15B are graphs of mIFN-β and mIL-12p70 levels in the supernatants of MP (MYC;Trp53- / -), MP-luc (MP expressing luciferase), MPB1(MYC;Trp53- / -; Brca1- / -), and MPB1-luc (MPB1 expressing luciferase) infected with MQ833 at a MOI of 10 for 16 h. FIGs.15C and 15D are graphs of mIFN-β and mIL- 12p70 levels in the supernatants of TPEB (Tpo-Cre+ / p53fl / fl / eYFPhet / Braf-CAhet) cells infected with MQ833 at a MOI of 10 for 16 h. TPEB is a murine anaplastic thyroid cancer cell line with BrafV600E / WTTrp53- / -, syngeneic with C57BL / 6J mice. FIG.15E are graphs of mIFN-β levels in the supernatants of KPC2 and KPC3 infected with MQ833 at a MOI of 10 for 16 h. KPC2 and KPC3 are aggressive murine pancreatic ductal adenocarcimoma cell lines derived from genetically engineered mouse models (GEMM) (LSL-KrasG12D / +; LSL- Trp53R172H / +; Pdx-1-Cre). FIG.15F are graphs of hIFN-β levels in the supernatants of COV644 and TYK-nu infected with MQ833 at a MOI of 10 for 16 h. COV644 and TYK-nu are aggressive human high-grade serous ovarian carcinoma (HGSOC) cell lines.

[0027] FIG.16A is a schematic diagram of evaluating immune cell alterations in MP-luc murine malignant peripheral nerve sheath tumor after treating with MQ833, MQ833 plus human G-CSF, or PBS. MP-Luc (2 x 106cells) were implanted subcutaneously into the 13 4873-2549-9626.1Atty. Dkt. No.115872-3093 right flanks of C57BL / 6J mice. Once the tumors are established, they were treated with IT MQ833 (8 x 107pfu), MQ833 + human G-CSF (3 µg), or PBS twice three days apart. Tumors were harvested two days post second injection. FIGs.16B-16C are contour plots (FIG.16B) and a bar graphs (FIG.16C) showing percentages of iNOS+Ly6G+CD11b+neutrophils among tumor-infiltrating neutrophils cells in MP-Luc tumors treated with PBS, MQ833, or MQ833 + hG-CSF. FIGs.16D and 16E are contour plots (FIG.16D) and a bar graph (FIG.16E) showing percentages of iNOS+Ly6G- Ly6C+monocytes among tumor- infiltrating monocytes cells in MP-Luc tumors treated with PBS, MQ833, or MQ833 + hG- CSF.

[0028] FIGs.17A-17B are contour plots (FIG.17A) and a bar graphs (FIG.17B) showing percentages of Gzmb+CD8+T cells among tumor-infiltrating CD8+cells in MP- Luc tumors treated with PBS, MQ833, or MQ833 + hG-CSF. FIGs.17C-17D are contour plots (FIG.17C) and a bar graph (FIG.17D) showing percentages of KLRG1+CD8+T cells among tumor-infiltrating CD8+cells in MP-Luc tumors treated with PBS, MQ833, or MQ833 + hG-CSF. FIGs.17E-17F are contour plots (FIG.17E) and a bar graphs (FIG. 17F) showing percentages of Tox+CD8+T cells among tumor-infiltrating CD8+cells in MP-Luc tumors treated with PBS, MQ833, or MQ833 + hG-CSF. FIGs.17G-17H are contour plots (FIG.17G) and a bar graph (FIG.17H) showing percentages of PD-1+CD8+T cells among tumor-infiltrating CD8+cells in MP-Luc tumors treated with PBS, MQ833, or MQ833 + hG-CSF.

[0029] FIGs.18A-18B are contour plots (FIG.18A) and a bar graph (FIG.18B) showing percentages of Foxp3+CD4+T cells among tumor-infiltrating CD4+cells in MP-Luc tumors treated with PBS, MQ833, or MQ833 + hG-CSF. FIG.18C are bar graphs showing percentages of Gzmb+Foxp3-CD4+T cells, PD-1+Foxp3-CD4+T cells, Tim3+Foxp3-CD4+T cells among tumor-infiltrating Foxp3-CD4+T cells in MP-Luc tumors treated with PBS, MQ833, or MQ833 + hG-CSF.

[0030] FIG.19A is a schematic diagram of evaluation of CD8+T cells in the injected tumors after intraperitoneal administration of FTY720. B16-F10 (5 x 105) were implanted intradermally to the right flank of wild type C57BL / 6J mice. After tumors were established, they were injected with rMVA (MVA∆E5R-hFlt3L-mOX40L) (8 x 107pfu) twice three days apart. FTY720 was used to block T cell egress from lymphoid organs. Mice were injected with or without FTY720 (25 µg) intraperitoneally twice three days apart. The first 14 4873-2549-9626.1Atty. Dkt. No.115872-3093 FTY720 injection was given one day prior to virus injection. Two days after the second virus dose, tumors were harvested. Single-cell suspension of tumor cells was prepared by enzymatic digestion and stained with antibodies against CD45, CD3, CD8, and CD4. FIG. 19B is a graph showing percentages of CD8+T cells out of CD45+cells in the tumors treated with rMVA or PBS in the presence or absence of FTY720. FIG.19C is a graph showing the absolute numbers of CD8+T cells in the tumors treated with rMVA or PBS in the presence or absence of FTY720. Intratumoral injection of rMVA caused increased percentages of CD8+T cells out of CD45+immune cells. and the absolute numbers of tumor-infiltrating CD8+T cells. FTY720 treatment had no effect on the increase of CD8+T cells in the tumors in response to rMVA. These results indicate that intratumoral delivery of rMVA likely induced CD8+T cell proliferation in the tumors.

[0031] FIG.20A are flow cytometry dot plots showing that rMVA treatment resulted in pre-existing CD8+T cell activation in the tumors. Single-cell suspensions were restimulated with Cell Stimulation Cocktail (Thermo Fisher) and GolgiPlug (BD Biosciences) in complete RPMI for 6 h at 37°C. Cells were incubated with appropriate antibodies for surface labeling for 30 min at 4°C after staining dead cells with LIVE / DEAD™ Fixable Aqua Stain (Thermo Fisher). Cells were fixed and permeabilized using Foxp3 fixation and permeabilization kit (Thermo Fisher) for 1 hour at 4°C and then stained for IFNγ and TNFα. FIG.20B is a graph showing percentages of IFNγ+TNFα+CD8+T cells out of total CD8+T cells in the tumors treated with rMVA or PBS in the presence or absence of FTY720. Intratumoral injection of rMVA caused the activation of CD8+T cells, which was not affected by FTY720 treatment. This result indicates that rMVA treatment resulted in the activation of pre-existing CD8+T cells in the tumors.

[0032] FIG.21A is a schematic of isolating intratumoral CD8+T cells for single-cell RNA-seq and single-cell TCR-seq. B16-F10 (5 x 105) were implanted intradermally to the right flank of wild-type C57BL / 6J mice. After tumors were established, they were injected with rMVA (MVA∆E5R-hFlt3L-mOX40L) (8 x 107pfu) twice three days apart. FTY720 was used to block T cell egress from lymphoid organs. Mice were injected with or without FTY720 (25 µg) intraperitoneally twice three days apart. The first FTY720 injection was given one day prior to the virus injection. Two days after the second virus dose, tumors were harvested. Single-cell suspension of tumor cells was prepared by enzymatic digestion and stained with antibodies against CD45, CD3, and CD8. CD8+T cells were sorted by 15 4873-2549-9626.1Atty. Dkt. No.115872-3093 flow cytometry and scRNA-seq and sc-TCR-seq were performed on those cells. FIG.21B is a UMAP display of CD8+cell clusters from 3 samples following 10X Genomics scRNA- seq workflow (n = 14,473 cells). FIG.21C is a violin plot of selected genes across different samples. FIG.21D is a density plot of IL7R expression in TCF+clusters 0 and 1. scRNA-seq analyses of CD8+T cell populations identified a total of 6 clusters, including Tcf7+CD62L+Stem-like CD8+T cells (C1), Tcf7+CD62L- CD8+T cells (C0), Proliferating ToxintCD8+T cells (C2), Effector CD8 (C3), ToxintCD8+T cells (C4), and ToxhiCD8+ T cells (C5). Tcf7 expression is a hallmark of stem-like T cells. Il7r is highly expressed in the TCF7+CD8+T cell clusters (C1 and C0). These results indicate that intratumoral delivery of IL7 would be beneficial to expand stem-like CD8+T cells in the tumors.

[0033] FIG.22 is a schematic diagram of a working model for the combination of MQ833 plus human G-CSF for cancer immunotherapy. Mechanistically, IT MQ833- induced antitumor immunity requires the collaborative actions of activated neutrophils, macrophages, CD4+, and CD8+T cells. scRNA-seq analysis of MQ833-treated tumors revealed a large influx of neutrophils and their differentiation into distinct and heterogenous neutrophil populations upon IFN and other signals. Myeloid cell remodeling also involves the recruitment of monocytes into the injected tumors and differentiation into antitumor M1 macrophages, as well as the depletion of resident pro-tumor M2 macrophages. Together with the depletion of Tregs, and the expansion of ISG+CD4+T cells, effector CD8+T cells, and proliferating CD4+and CD8+T cells, the “immune-desert” tumors are destroyed by MQ833 monotherapy, or they are sensitized to ICB therapy. The combination of MQ833 with human G-CSF has generated superior antitumor effects compared with either agent alone. Human G-CSF promotes neutrophil recruitment and iNOS expression in neutrophils. Moreover, iNOS plays a crucial role in the antitumor effects induced by the combination of MQ833 and human G-CSF. These results provide further evidence that neutrophils and macrophages can be mobilized for antitumor activity.

[0034] FIG.23A is a schematic diagram of the third-generation recombinant MVA construct MQ954, which was engineered by inserting the human granulocyte colony- stimulating factor (hG-CSF) gene into the C12L locus of the second-generation virus MQ833 genome. The expression of hG-CSF is driven by the vaccinia synthetic early-late promoter P7.5, ensuring sustained production of the protein throughout the viral life cycle. The C-terminus of the human G-CSF was tagged with an extracellular matrix binding 16 4873-2549-9626.1Atty. Dkt. No.115872-3093 domain, allowing it to be anchored to the extracellular matrix within the tumor microenvironment, enhancing localized immune cell recruitment and activation. The C12L gene, which encodes IL-18 binding protein (IL-18BP), was deleted from the MQ833 genome. This deletion enhances the immunogenicity of the virus by preventing IL-18 suppression, thereby promoting a stronger Th1 immune response. FIG.23B are graphs showing mIL-12 p70, hG-CSF and mIFN-β expression in SKP605 sgCon and sgEed cells infected with MQ954 or mock infected. Cells were collected after infection for 16 hours. To compare the therapeutic effects of MQ833 and MQ954 in this murine tumor model, SKP605 sgCon cells (1 x 106) were implanted subcutaneously into the right flanks of C57BL / 6J mice. PRC2-wild type (SKP605 sgCon) tumors were found to be less responsive to MQ833 compared with PRC2-loss (SKP605 sgEed) tumors, and therefore was selected for this experiment. Once the tumors are established, they were treated with IT MQ833 or MQ954 (8 x 107pfu) or PBS twice weekly. Tumors were measured and mice survival were monitored. FIG.23C shows Kaplan-Meier survival curves of SKP sgCon tumor-bearing wild-type C57BL / 6J or Nos2- / -mice treated with IT MQ833, MQ954, or PBS. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-10, *P<0.05, ***P<0.001, ****P < 0.0001). FIG.23D are graphs showing tumor volumes of WT or Nos- / -mice treated with PBS, MQ833, MQ954 over days post treatment.

[0035] FIG.24A is a schematic diagram of an experimental schedule. 1x106of SKP605 sgCon or SKP605 sgEed cells were subcutaneously implanted into the right flank of C57BL / 6J mice. Once the tumors were established, IT MQ833 or MQ954 injection was given twice. Tumors were collected two days post the second injecton, and were processed into single cell suspension. CD45+cells were sorted and scRNA-seq analysis were performed. FIG.24B is a UMAP displaying different immune cell populations among CD45+cells from three combined samples isolated from SKP605 sgEed tumors following 10X Genomics scRNA-seq workflow (n = 28,112 cells) and their designations. FIG.24C is a UMAP displaying different immune cell populations among CD45+cells isolated from SKP605 sgEed tumors following 10X Genomics scRNA-seq workflow- plots of cell clusters separated by sample. FIG.24D is expression plot of Mrp8 (S100a8). FIG.24E is a UMAP displaying subclustered neutrophils (Mrp8 >0) and plots of neutrophil subclusters separated by sample. FIG.24F are expression plots of Cd74, Sell, Cxcr2, Nos2, Cd24a, Irf1, Isg15, Cxcl2, Ifngr1, Cd101, Cxcl10. 17 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0036] FIG.25A is a UMAP displaying subclustered monocytes and macrophages (clusters 0, 1, 3, 5, 7, and 17 in FIG.24B). FIG.25B is a UMAP displaying subclusters of monocytes and macrophages from six combined samples isolated from SKP605 sgCon and sgEed tumors treated with PBS, MQ833, or MQ954 following 10X Genomics scRNA-seq workflow and their designations. FIG.25C are plots of monocytes and macrophages subclusters separated by samples, including SKP605 sgCon and sgEed treated with PBS, MQ833, or MQ954. FIG.25D are expression plots of Trem2, Nos2, Isg15, Csf3r. FIG.25E is a heatmap describing the expression of selected differentially expressed genes in each myeloid cell cluster.

[0037] FIG.26A is a schematic diagram of an experimental schedule. 1 x 106of MB49 cells were subcutaneously implanted into the right flank of WT, Irf3- / -Irf7- / -, Rag1- / -, Stat2- / -, Ifnarfl / fl, LyzcreIfnarfl / fl, Mrp8creIfnarfl / fl, and Cd4creIfnarfl / flC57BL / 6J mice. Intratumoral rMVA, MQ833 or MQ954 treatments were given twice a week once the tumors were established. Tumor size and mice survival were monitored. FIG.26B shows Kaplan-Meier survival curves of MB49 tumor-bearing WT C57BL / 6J mice treated with indicated viruses, rMVA, MQ833, MQ954 (n=9-11,^*P<0.05, ***P<0.001, ****P < 0.0001). FIG.26C are graphs showing tumor volumes of WT C57BL / 6J mice treated with PBS, rMVA, MQ833, or MQ954 over days post treatment. FIG.26D shows Kaplan-Meier survival curves of MB49 tumor-bearing WT, Irf3- / -Irf7- / -and Stat2- / -mice treated with MQ954 (n=6- 10,^**P<0.01). FIG.26E are graphs showing tumor volumes of WT, Irf3- / -Irf7- / -and Stat2- / -C57BL / 6J mice treated with MQ954 over days post treatment. FIG.26F shows Kaplan- Meier survival curves of MB49 tumor-bearing Ifnarfl / fl, LyzcreIfnarfl / fl, and Cd4creIfnarfl / flmice treated with MQ954. Survival data were analyzed by log-rank (Mantel-Cox) test (n=9- 10,^*P<0.05). FIG.26G are graphs showing tumor volumes of Ifnarfl / fl, LyzcreIfnarfl / fl, and Cd4creIfnarfl / flmice C57BL / 6J mice treated with MQ954 over days post treatment. FIG. 26H shows Kaplan-Meier survival curves of MB49 tumor-bearing Ifnarfl / fl, Mrp8creIfnarfl / fl, WT, and Rag1- / -mice treated with MQ954. Survival data were analyzed by log-rank (Mantel-Cox) test (n=6-9,^*P<0.05; ****P < 0.0001). FIG.26I are graphs showing tumor volumes of Ifnarfl / fl, Mrp8creIfnarfl / fl, WT, and Rag1- / -C57BL / 6J mice treated with MQ954 over days post treatment.

[0038] FIG.27 is an illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 1) given by GenBank Accession No. U94848.1. 18 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0039] FIG.28 is a plasmid map of the pUC57-del186-pH5-huIL12 plasmid (SEQ ID NO: 2) (del186 also known as delWR199).

[0040] FIG.29 is a plasmid map of the P503 plasmid (SEQ ID NO: 3).

[0041] FIGs.30 and 31 are the plasmid maps of the pMA-MVA-ΔE5R-hFlt3L-OX40L vector (SEQ ID NO: 4; FIG.30) and the pMA-MVA∆E5R-hFlt3L-mOX40L vector (SEQ ID NO: 5; FIG.31).

[0042] FIGs.32 and 33 are illustrative, non-limiting plasmid maps for generation of MQ832.

[0043] FIG.34 is an illustrative, non-limiting plasmid design for deletion of WR199 with IL-12 sequence (murine) (SEQ ID NO: 26).

[0044] FIGs.35A, 35B, 36A, and 36B are illustrative, non-limiting plasmid maps for generation of viral constructs of the present technolgy when IL-7 is inserted into the C12L locus (FIG.35A), when G-CSF is inserted into the C12L locus (FIG.35B), when G-CSF is inserted into the N1L locus (FIG.36A), and when IL-7 is inserted into the N1L locus (FIG. 36B).

[0045] FIG.36C is a schematic diagram of engineering of MQ710, MQ832, MQ833, MQ953, MQ954, and MQ955.

[0046] FIG.37 is an illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 7) given by GenBank Accession No. AY603355. DETAILED DESCRIPTION

[0047] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. I. Definitions

[0048] The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs. 19 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0049] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like.

[0050] As used herein, the term “about” encompasses the range of experimental error that may occur in a measurement and will be clear to the skilled artisan.

[0051] As used herein, the term “adaptive immune response” or “adaptive immune system response” refers to an antigen-mediated response by the immune system of a subject. A subject has a “deficient adaptive immune system response” when the subject’s adaptive immune system does not respond to one or more antigens as an active adaptive immune system would (e.g., no T cell response or a diminished T cell response, or no B cell response or a diminished B cell response).

[0052] As used herein, the term “adjuvant” refers to a substance that enhances, augments, or potentiates the host’s immune response to antigens, including tumor antigens.

[0053] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intradermally, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally, or topically. Administration includes self-administration and the administration by another.

[0054] As used herein, the term “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the antigen is contained within a whole cell, such as in a tumor antigen-containing whole cell vaccine. In some embodiments, the target antigen encompasses cancer-related antigens or neoantigens and includes proteins or other molecules expressed by tumor or non-tumor cancers, such as molecules that are present in cancer cells but absent in non-cancer cells, and molecules that are up-regulated in cancer cells as compared to non-cancer cells.

[0055] As used herein, “attenuated,” as used in conjunction with a virus, refers to a virus having reduced virulence or pathogenicity as compared to a non-attenuated counterpart, yet 20 4873-2549-9626.1Atty. Dkt. No.115872-3093 is still viable or live. Typically, attenuation renders an infectious agent, such as a virus, less harmful or virulent to an infected subject compared to a non-attenuated virus. This is in contrast to a killed or completely inactivated virus.

[0056] As used herein, “conjoint administration” refers to administration of a second therapeutic modality in combination with one or more engineered poxviruses of the present technology. For example, human or murine granulocyte-colony stimulating factor (G-CSF) and / or an immune checkpoint blockade inhibitor administered in close temporal proximity with one or more engineered poxviruses of the present technology. For example, G-CSF, IL-7, and / or an immunce checkpoint blockade inhibitor can be administered simultaneously (i.e., concurrently) with one or more engineered poxviruses of the present technology (by intravenous or intratumoral injection when the engineered poxvirus of the present technology is administered intratumorally or systemically as stated above) or before or after the engineered poxvirus of the present technology administration. In some embodiments, if the engineered poxvirus of the present technology and the G-CSF, IL-7, and / or immune checkpoint blockade inhibitor are administered about 1 to about 7 days apart or even up to three weeks apart, this would still be within “close temporal proximity” as stated herein, therefore such administration will qualify as “conjoint.”

[0057] The term “corresponding wild-type strain” or “corresponding wild-type virus” is used herein to refer to the wild-type MVA from which the engineered MVA was derived. As used herein, a wild-type MVA strain or virus is a strain or virus that has not been engineered to disrupt or delete (knock out) a particular gene of interest and / or to express a heterologous nucleic acid. For example, in some embodiments, a wild-type MVA strain or virus is a strain or virus that has not been engineered to disrupt or delete (knock out) the E5R gene, E3L gene, and / or WR199 gene, and / or not engineered to express additional immunomodulatory proteins as described herein, such as Flt3L, OX40, and / or IL-12. The term “corresponding MVAΔE5R strain” or “corresponding MVAΔE5R virus” is used herein to refer to the MVA strain or virus having the same sequence as MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12 except for having an E5R deletion alone (i.e., an MVAΔE5R strain or virus that has not been engineered to disrupt or delete (knock out) the E3L and WR199 genes and that has not been engineered to express any of Flt3L, OX40, or IL-12 but that is otherwise identical to the corresponding MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12). 21 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0058] As used herein, the terms “delivering” and “contacting” refer to depositing the one or more engineered poxviruses of the present disclosure in the tumor microenvironment whether this is done by local administration to the tumor (intratumoral) or by, for example, intravenous route. The term focuses on engineered virus that reaches the tumor itself. In some embodiments, “delivering” is synonymous with administering, but it is used with a particular administration locale in mind, e.g., intratumoral.

[0059] The terms “disruption” and “mutation” and “mutant” are used interchangeably herein to refer to a detectable and heritable change in the genetic material. Mutations may include insertions, deletions, substitutions (e.g., transitions, transversion), transpositions, inversions, knockouts, and combinations thereof. Mutations may involve only a single nucleotide (e.g., a point mutation or a single nucleotide polymorphism) or multiple nucleotides. In some embodiments, mutations are silent, that is, no phenotypic effect of the mutation is detected. In other embodiments, the mutation causes a phenotypic change, for example, the expression level of the encoded product is altered, or the encoded product itself is altered. In some embodiments, a disruption or mutation may result in a disrupted gene with decreased levels of expression of a gene product (e.g., protein or RNA) as compared to the wild-type strain. In other embodiments, a disruption or mutation may result in an expressed protein with activity that is lower as compared to the activity of the expressed protein from the wild-type strain. As used herein, in some embodiments, a mutant gene may comprise, but is not limited to: a deletion of all or a portion of the gene; replacement of at least a portion of the gene with or insertion of one or more expression cassettes inserted into the gene locus, wherein the cassette comprises one or more heterologous nucleic acid molecules; and / or an insertion of one or more heterologous nucleic acid molecules into the gene.

[0060] As used herein, an “effective amount” or “therapeutically effective amount” refers to a sufficient amount of an agent, which, when administered at one or more dosages and for a period of time, is sufficient to provide a desired biological result in alleviating, curing, or palliating a disease. In the present disclosure, an effective amount of one or more engineered poxviruses of the present technology comprises an amount that (when administered for a suitable period of time and at a suitable frequency) reduces the number of cancer cells; or reduces the tumor size or eradicates the tumor; or inhibits (i.e., slows down or stops) cancer cell infiltration into peripheral organs; inhibits (i.e., slows down or 22 4873-2549-9626.1Atty. Dkt. No.115872-3093 stops) metastatic growth; inhibits (stabilizes or arrests) tumor growth; allows for treatment of the tumor; induces and promotes an immune response against the tumor; prevents cancer recurrence for a period of time in a subject in need thereof; or alters the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof. The TIME includes any immune cell or factor present in the tumor microenvironment, including, but not limited to, cytokines, myeloid cells, and / or lymphocytes. Alterations to the TIME may include, but are not limited to, increased proportions of pro-inflammatory cytokines, increased porportions of inflammatory monocytes and neutrophils, decreased proportions of M2-like macrophages, and / or increased proportion of polarized M1-like macrophages. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation in light of the present disclosure. Such determination may begin with amounts found effective in vitro and amounts found effective in animals. The therapeutically effective amount will be initially determined based on the concentration or concentrations found to confer a benefit to cells in culture. Effective amounts can be extrapolated from data within the cell culture and can be adjusted up or down based on factors such as detailed herein. Effective amounts of the viral constructs are generally within the range of about 105to about 1010plaque forming units (pfu), although a lower or higher dose may be administered. In some embodiments, the dosage is about 106-109pfu. In some embodiments, a unit dosage is administered in a volume within the range from 1 to 10 mL. The equivalence of pfu to virus particles can differ according to the specific pfu titration method used. Generally, pfu is equal to about 5 to 100 virus particles. A therapeutically effective amount of the viruses of the present technology can be administered in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration. For example, a therapeutically effective amount of the viruses in accordance with the present disclosure may vary according to factors such as the disease state, age, sex, weight, and general condition of the subject, and the potency of the viral constructs to elicit a desired immunological response in the particular subject for the particular cancer.

[0061] With particular reference to the viral-based immunostimulatory agents disclosed herein, an “effective amount” or “therapeutically effective amount” refers to an amount of a composition comprising one or more one or more engineered poxviruses of the present technology alone or in combination with an amount of a composition comprising human or murine G-CSF, or IL-7 sufficient to reduce, inhibit, or abrogate tumor cell growth, thereby 23 4873-2549-9626.1Atty. Dkt. No.115872-3093 reducing or eradicating the tumor, or sufficient to inhibit, reduce or abrogate metastatic spread either in vitro, ex vivo, or in a subject, or to elicit and promote an immune response against the tumor that will eventually result in one or more of metastatic spread reduction, inhibition, and / or abrogation as the case may be, or to preventing cancer recurrence for a period of time in a subject in need thereof, or to alter the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof. The reduction, inhibition, or eradication of tumor cell growth may be the result of necrosis, apoptosis, or an immune response, or a combination of two or more of the foregoing (however, the precipitation of apoptosis, for example, may not be due to the same factors as observed with oncolytic viruses). The amount that is therapeutically effective may vary depending on such factors as the particular virus used in the composition, the age and condition of the subject being treated, the extent of tumor formation, the presence or absence of other therapeutic modalities, and the like. Similarly, the dosage of the composition to be administered and the frequency of its administration will depend on a variety of factors, such as the potency of the active ingredient, the duration of its activity once administered, the route of administration, the size, age, sex, and physical condition of the subject, the risk of adverse reactions and the judgment of the medical practitioner. The compositions are administered in a variety of dosage forms, such as injectable solutions.

[0062] With particular reference to combination therapy with an immune checkpoint blockade inhibitor, an “effective amount” or “therapeutically effective amount” for an immune checkpoint blockade inhibitor means an amount of an immune checkpoint blockade inhibitor sufficient to reverse or reduce immune suppression in the tumor microenvironment and to activate or enhance host immunity in the subject being treated. Immune checkpoint blockade inhibitors include, but are not limited to, inhibitory antibodies against CD28 inhibitor such as CTLA-4 (cytotoxic T lymphocyte antigen 4) (e.g., ipilimumab), anti-PD-1 (programmed Death 1) inhibitory antibodies (e.g., nivolumab, pembrolizumab, pidilizumab, lambrolizumab), and anti-PD-L1 (Programmed death ligand 1) inhibitory antibodies (MPDL3280A, BMS-936559, MEDI4736, MSB 00107180), as well as inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T-cell immunoglobulin and mucin-3), B7-H3, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS- 986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, 24 4873-2549-9626.1Atty. Dkt. No.115872-3093 INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, or PDR001, and combinations thereof. Dosage ranges of the foregoing are known or readily within the skill in the art as several dosing clinical trials have been completed, making extrapolation to other agents possible.

[0063] In some embodiments, the tumor expresses the particular checkpoint, but in the context of the present technology, this is not strictly necessary as immune checkpoint blockade inhibitors block more generally immune suppressive mechanisms within the tumors, elicited by tumor cells, stromal cells, and tumor-infiltrating immune cells.

[0064] For example, the CTLA-4 inhibitor ipilimumab, when administered as adjuvant therapy after surgery in melanoma, can be administered at 1-2 mg / mL over 90 minutes for a total infusion amount of 3 mg / kg every three weeks for a total of 4 doses. This therapy is often accompanied by severe, even life-threatening, immune-mediated adverse reactions, which limits the tolerated dose as well as the cumulative amount that can be administered. It is anticipated that it will be possible to reduce the dose and / or cumulative amount of ipilimumab when it is administered conjointly with one or more engineered poxviruses of the present technology. In particular, in light of the experimental results set forth below, it is anticipated that it will be further possible to reduce the CTLA-4 inhibitor’s dose if it is administered directly to the tumor conjointly with one or both the foregoing MVA viruses. Accordingly, the amounts provided above for ipilimumab may be a starting point for determining the particular dosage and cumulative amount to be given to a patient in conjoint administration.

[0065] As another example, pembrolizumab is prescribed for administration as adjuvant therapy in melanoma, for example, diluted to 25 mg / mL. It can be administered at a dosage of 2 mg / kg over 30 minutes every three weeks. This may be a starting point for determining dosage and administration in the conjoint administration of one or more engineered poxviruses of the present technology.

[0066] Nivolumab could also serve as a starting point in determining the dosage and administration regimen of checkpoint inhibitors administered in combination with one or more engineered poxviruses of the present technology. Nivolumab can be prescribed for administration at 3 mg / kg as an intravenous infusion every two weeks, for example, or at a 25 4873-2549-9626.1Atty. Dkt. No.115872-3093 dose of 240 mg every two weeks, 360 mg every three weeks, or 480 mg every four weeks, for example, with the specific dose and infusion rate depending upon the tumor type.

[0067] Immune stimulating agents such as agonist antibodies have also been explored as immunotherapy for cancers. For example, anti-ICOS antibody binds to the extracellular domain of ICOS leading to the activation of ICOS signaling and T-cell activation. Anti- OX40 antibody can bind to OX40 and potentiate T-cell receptor signaling leading to T-cell activation, proliferation and survival. Other examples include agonist antibodies against 4- 1BB (CD137), and GITR.

[0068] The immune stimulating agonist antibodies can be used systemically in combination with intratumoral injection of one or more engineered poxviruses of the present technology. Alternatively, the immune stimulating agonist antibodies can be used conjointly with one or more engineered poxviruses of the present technology via intratumoral delivery either simultaneously (i.e., concurrently) or sequentially.

[0069] The terms “engineered” or “genetically engineered” are used herein to refer to an organism that has been manipulated to be genetically altered, modified, or changed, e.g., by disruption of the genome. For example, an “engineered modified vaccinia Ankara virus” refers to a modified vaccinia Ankara strain that has been manipulated to be genetically altered, modified, or changed. In the present context, “engineered” or “genetically engineered” includes recombinant modified vaccinia Ankara viruses.

[0070] The term “gene cassette” is used herein to refer to a DNA sequence encoding and capable of expressing one or more genes of interest (e.g., OX40L, Flt3L, IL-12, G-CSF, IL- 7, a selectable marker, or a combination thereof) that can be inserted between one or more selected restriction sites of a DNA sequence. The letter “m” preceding a gene or protein indicates that it is of murine origin. The letter “h” preceding a gene or protein indicates that it is of human origin. In the absence of an “h” or “m” preceding a gene or a protein, the gene or protein should be presumed to be either human or murine unless context indicates otherwise. In some embodiments, insertion of a gene cassette results in a disrupted gene. In some embodiments, disruption of the gene involves replacement of at least a portion of the gene with a gene cassette, which includes a nucleotide sequence encoding a gene of interest (e.g., OX40L, Flt3L, IL-12, G-CSF, IL-7, a selectable marker, or a combination thereof). 26 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0071] As used herein, “G-CSF,” “granulocyte-colony stimulating factor,” “mG-CSF,” or “hG-CSF” refer to the granulocyte-colony stumlating factor, either as a gene, gene product, or protein. G-CSF plays an important role in regulating the production and function of white blood cells, particularly neutrophils. For example, G-CSF primarily functions to stimulate the production of neutrophils in the bone marrow. G-CSF also helps neutrophils become more efficient in carrying out their immune defense functions. A non-limiting exemplary DNA sequence for G-CSF is (SEQ ID NO: 20) and a non-limiting exemplary amino acid sequence for G-CSF (SEQ ID NO: 21).

[0072] As used herein, “heterologous nucleic acid,” refers to a nucleic acid, DNA, or RNA, which has been introduced into a virus, and which is not a copy of a sequence naturally found in the virus into which it is introduced. Such heterologous nucleic acids may comprise segments that are a copy of a sequence that is naturally found in the virus into which it has been introduced.

[0073] As used herein, wherever a gene is described, the gene may be either human or murine such that the designation of human (h or hu) or murine (m or mu) may be used interchangeably and is not intended to be limiting. For example, where mG-CSF is described, hG-CSF may be substituted for hG-CSF in the described constructs, and vice versa. Additionally, where no designation of human or murine is used, the gene may be either human or murine.

[0074] As used herein, “IL-12,” “interleukin 12,” “mIL-12,” or “hIL-12” refer to interleukin 12, either as a gene, gene product, or protein. IL-12 belongs to a family of pro- inflammatory cytokines. IL-12 in particular is known, for example, to regulate T-cell and natural killer-cell responses, induce the production of interferon gamma, and promote differentiation of T helper cells. A non-limiting exemplary DNA sequence for IL-12 is (SEQ ID NO: 15) and a non-limiting exemplary amino acid sequence for IL-12 (SEQ ID NO: 16).

[0075] As used herein, “IL-7,” “interleukin 7,” “mIL-7,” or “hIL-7” refer to interleukin 7, either as a gene, gene product, or protein. IL-7 is known to promote the differentiation of hematopoietic stem cells into lymphoid progenitor cells and to promote the development of T cells and B cells. A non-limiting exemplary DNA sequence for IL-7 is (SEQ ID NO: 22) and a non-limiting exemplary amino acid sequence for IL-7 (SEQ ID NO: 23). 27 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0076] As used herein, “immune checkpoint inhibitor” or “immune checkpoint blocking agent” or “immune checkpoint blockade agent” or “immune checkpoint blockade inhibitor” or “ICB inhibitor” refers to molecules that completely or partially reduce, inhibit, interfere with, or modulate the activity of one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. Checkpoint proteins include, but are not limited to, CD28 receptor family members, CTLA-4 and its ligands CD80 and CD86; PD-1 and its ligands PD-L1 and PD-L2; LAG3, B7-H3, B7-H4, TIM3, ICOS, II DLBCL, BTLA or any combination of two or more of the foregoing. Non-limiting examples of immune checkpoint blockade inhibitors contemplated for use herein include, but are not limited to, inhibitory antibodies against CD28 inhibitor such as CTLA-4 (cytotoxic T lymphocyte antigen 4) (e.g., ipilimumab), anti-PD-1 (programmed Death 1) inhibitory antibodies (e.g., nivolumab, pembrolizumab, pidilizumab, lambrolizumab), and anti-PD-L1 (Programmed death ligand 1) inhibitory antibodies (MPDL3280A, BMS-936559, MEDI4736, MSB 00107180), as well as inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T-cell immunoglobulin and mucin-3), B7-H3, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP- 870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, or BTLA, PDR001, and combinations thereof.

[0077] As used herein, “immune response” refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, etc. An immune response may include a cellular response, such as a T-cell response that is an alteration (modulation, e.g., significant enhancement, stimulation, activation, impairment, or inhibition) of cellular, i.e., T-cell function. A T-cell response may include generation, proliferation or expansion, or stimulation of a particular type of T-cell, or subset of T-cells, for example, effector CD4+, CD4+helper, effector CD8+, CD8+cytotoxic, or natural killer (NK) cells. Such T-cell subsets may be identified by detecting one or more cell receptors or cell surface molecules (e.g., CD or cluster of differentiation molecules). A T-cell response may also include altered expression (statistically significant increase or decrease) of a cellular factor, such as 28 4873-2549-9626.1Atty. Dkt. No.115872-3093 a soluble mediator (e.g., a cytokine, lymphokine, cytokine binding protein, or interleukin) that influences the differentiation or proliferation of other cells. For example, Type I interferon (IFN-α / β) is a critical regulator of the innate immunity (Huber et al., Immunology 132(4):466-474 (2011)). Animal and human studies have shown a role for IFN-α / β in directly influencing the fate of both CD4+and CD8+T-cells during the initial phases of antigen recognition and anti-tumor immune response. IFN Type I is induced in response to activation of dendritic cells, in turn a sentinel of the innate immune system. An immune response may also include humoral (antibody) response.

[0078] The term “immunogenic composition” is used herein to refer to a composition that will elicit an immune response in a mammal that has been exposed to the composition. For example, in some embodiments, an immunogenic composition comprises an engineered poxvirus of the present technology, G-CSF, IL-7, an antigen, an adjuvant comprising any one or more of the foregoing engineered viruses, and / or an adjuvant comprising MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12, alone or in combination with immune checkpoint blockade inhibitors. As used herein, an immunogenic composition encompasses vaccines. In some embodiments, the immunogenic composition comprises a tumor antigen- containing whole cell vaccine (e.g., an irradiated whole cell vaccine).

[0079] As used herein, the term “inactivated MVA” refers to heat-inactivated MVA (Heat-iMVA) and / or UV-inactivated MVA which are infective, nonreplicative, and do not suppress IFN Type I production in infected DC cells. As used herein, the term “inactivated vaccinia virus” includes heat-inactivated vaccinia virus and / or UV-inactivated vaccinia virus. MVA or vaccinia virus inactivated by a combination of heat and UV radiation is also within the scope of the present disclosure.

[0080] As used herein, “Heat-inactivated MVA” (Heat-iMVA) and “Heat-inactivated vaccinia virus” refer to MVA and vaccinia virus, respectively, which have been exposed to heat treatment under conditions that do not destroy its immunogenicity or its ability to enter target cells (tumor cells) but remove residual replication ability of the virus as well as factors that inhibit the host’s immune response. An example of such conditions is exposure to a temperature within the range of about 50 to about 60˚C for a period of time of about an hour. Other times and temperatures can be determined by one of skill in the art. 29 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0081] As used herein, “UV-inactivated MVA” and “UV-inactivated vaccinia virus” refer to MVA and vaccinia virus, respectively, that have been inactivated by exposure to UV under conditions that do not destroy its immunogenicity or its ability to enter target cells (tumor cells) but remove residual replication ability of the virus. An example of such conditions, which can be useful in the present methods, is exposure to UV using, for example, a 365 nm UV bulb for a period of about 30 min to about 1 hour. Other limits of these conditions of UV wavelength and exposure can be determined by one of skill in the art.

[0082] A “knock out,” “knocked out gene,” or a “gene deletion” refers to a gene including a null mutation (e.g., the wild-type product encoded by the gene is not expressed, expressed at levels so low as to have no effect, or is non-functional). In some embodiments, the knocked out gene includes heterologous sequences (e.g., one or more gene cassettes comprising a heterologous nucleic acid sequence) or genetically engineered non-functional sequences of the gene itself, which renders the gene non-functional. In other embodiments, the knocked out gene is lacking a portion of the wild-type gene. For example, in some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 60% of the wild-type gene sequence is deleted. In other embodiments, the knocked out gene is lacking at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or at least about 100% of the wild- type gene sequence. In other embodiments, the knocked out gene may include up to 100% of the wild-type gene sequence (e.g., some portion of the wild-type gene sequence may be deleted) but also include one or more heterologous and / or non-functional nucleic acid sequences inserted therein.

[0083] As used herein, “metastasis” refers to the spread of cancer from its primary site to neighboring tissues or distal locations in the body. Cancer cells (including cancer stem cells) can break away from a primary tumor, penetrate lymphatic and blood vessels, circulate through the bloodstream, and grow in normal tissues elsewhere in the body. Metastasis is a sequential process, contingent on tumor cells (or cancer stem cells) breaking off from the primary tumor, traveling through the bloodstream or lymphatics, and stopping at a distant site. Once at another site, cancer cells re-penetrate through the blood vessels or lymphatic walls, continue to multiply, and eventually form a new tumor (metastatic tumor). In some embodiments, this new tumor is referred to as a metastatic (or secondary) tumor. 30 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0084] As used herein, “MVA” means “modified vaccinia Ankara” and refers to a highly attenuated strain of vaccinia derived from the Ankara strain and developed for use as a vaccine and vaccine adjuvant. The original MVA was isolated from the wild-type Ankara strain by successive passage through chicken embryonic cells. Treated thus, it lost about 15% of the genome of wild-type vaccinia including its ability to replicate efficiently in primate (including human) cells. (Mayr et al., Zentralbl Bakteriol B 167:375-390 (1978)). MVA is considered an appropriate candidate for development as a recombinant vector for gene or vaccination delivery against infectious diseases or tumors. (Verheust et al., Vaccine 30(16):2623-2632 (2012)). MVA has a genome of 178 kb in length and a sequence first disclosed in Antoine et al., Virol.244(2): 365-396 (1998). Illustrative, non-limiting sequences are disclosed in GenBank Accession No. U94848.1 (SEQ ID NO: 1) and GenBank Accession No. AY603355 (SEQ ID NO: 7). Clinical grade MVA is commercially and publicly available from Bavarian Nordic A / S Kvistgaard, Denmark. Additionally, MVA is available from ATCC, Rockville, MD, and from CMCN (Institut Pasteur Collection Nationale des Microorganismes) Paris, France.

[0085] The term “MVAΔE3L” means a deletion mutant of MVA which lacks a functional E3L gene and is infective but non-replicative and it is further impaired in its ability to evade the host’s immune system. It has been used as a vaccine vector to transfer tumor or viral antigens. The mutant MVA E3L knockout and its preparation have been described in U.S. Patent 7,049,145, for example.

[0086] The term “MVAΔE5R,” is used herein to refer to a modified vaccinia Ankara (MVA) mutant virus in which the E5R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “MVAΔE5R” includes a deletion mutant of MVA which lacks a functional E5R gene and is infective but non-replicative and it is further impaired in its ability to evade the host’s immune system. As used herein, “MVAΔE5R” encompasses a recombinant MVA virus that does not express a functional E5 protein. In some embodiments, the ΔE5R mutant includes a deletion of all or a majority of the E5R gene sequence. For example, as used herein, “MVAΔE5R” encompasses a recombinant MVA nucleic acid sequence, wherein all or a majority of the nucleic acid sequence corresponding to the position of E5R in the MVA genome (e.g., positions 44,179-45,175 set forth in GenBank Accession No. U94848.1 (SEQ ID NO: 1) or positions 38,432 to 39,385 set forth in GenBank Accession 31 4873-2549-9626.1Atty. Dkt. No.115872-3093 No. AY603355 (SEQ ID NO: 7)) is deleted. In some embodiments, all or a portion of the E5R gene is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes one or more specific genes of interest and / or a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MVAΔE5R virus encompasses a recombinant MVA virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, the MVAΔE5R viruses of the present technology contain no further heterologous genes and / or viral gene mutations other than those specifically referred to in the name of the virus.

[0087] As used herein, the terms “rMVA” or “MQ710” or “MVAΔE5R-hFlt3L-OX40L” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, and a mutation of the E5R gene. In some embodiments, both of the transgenes are inserted into the E5R locus. In some embodiments, Flt3L and OX40L are linked by a P2A self-cleaving sequence and their expression is driven by the vaccinia synthetic early-late promoter.

[0088] As used herein, the terms “rMVAΔE3L” or “MVAΔE3LΔE5R-hFlt3L-OX40L” refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, and a mutant E3L gene. In some embodiments, the rMVAΔE3L is made from an rMVA virus by engineering it to further comprise a mutant E3L gene. In some embodiments, the ΔE3L mutation is a deletion of the E3L gene. For example, as used herein, “MVAΔE3L” encompasses a recombinant MVA nucleic acid sequence, wherein all or a majority of the nucleic acid sequence corresponding to the position of E3L or MVA050L in the MVA genome (e.g., positions 42,697-43,269 set forth in GenBank Accession No. U94848.1 (SEQ ID NO: 1) or e.g., positions 36,907-37,479 set forth in GenBank Accession No. AY603355 (SEQ ID NO: 7)) is deleted.

[0089] As used herein, the terms “rMVAΔE3LΔWR199” or “MQ832” or “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, and also a mutant WR199 gene. In some embodiments, the rMVAΔE3LΔWR199 made from an rMVA virus by engineering it to further comprise both a mutant E3L gene and a mutant WR199 gene. Likewise, in some embodiments, the 32 4873-2549-9626.1Atty. Dkt. No.115872-3093 rMVAΔE3LΔWR199 is made from a rMVAΔE3L virus by engineering it to further coprise a mutant WR199 gene. In some embodiments, the mutation is a deletion of the E3L and WR199 genes. In some embodiments, ΔWR199 encompasses a recombinant MVA nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of WR199 or MVA186R in the MVA genome (e.g., position 158,399 to 160,143 of the sequence set forth in GenBank Accession No. AY603355 (SEQ ID NO: 7) or position 164,209-165,933 of the sequence set forth in GenBank Accession No. U94848.1 (SEQ ID NO: 1)) is deleted. In some embodiments, all or a portion of the WR199 gene is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes one or more specific genes of interest and / or a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry).

[0090] As used herein, the terms “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12” or “MVAΔE5R-hFlt3L-OX40L-ΔE3L-ΔWR199-IL-12” or “MQ833” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, and a third transgene, IL-12. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the MQ833 is an MQ832 virus that has been engineered to further comprise an IL-12 transgene. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g. RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 8) which anchors the IL-12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833.

[0091] As used herein, the terms “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L” or “MQ953” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, an IL-12 transgene, and a mutant C12L gene. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is 33 4873-2549-9626.1Atty. Dkt. No.115872-3093 inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g. RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 8) which anchors the IL-12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833, and the C12L gene is further mutated to generate MQ953. In some embodiments, the C12L gene is deleted. For example, all or a majority of the nucleic acid sequence corresponding to the position of C12L or MVA008L in the MVA genome (e.g., positions 13,052- 13,414, set forth in GenBank Accession No. U94848.1 (SEQ ID NO: 1) or positions e.g., positions 7262-7624 set forth in GenBank Accession No. AY603355 (SEQ ID NO: 7)) is deleted.

[0092] As used herein, the terms “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L-hG-CSF” or “MQ954” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, an IL-12 transgene, a mutant C12L gene, and a hG-CSF transgene, which may be inserted into the C12L locus. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g. RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 8) which anchors the IL-12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833, the C12L gene is further mutated to generate MQ953, and the G-CSF transgene is inserted into the C12L locus to generate MQ954. In some embodiments, the C12L gene is deleted. In some embodiments, the G-CSF transgene is inserted into the C12L locus. 34 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0093] As used herein, the terms “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L-hG-CSF-ΔN1L-hIL-7” or “MQ955” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, an IL-12 transgene, a mutant C12L gene, a G-CSF transgene, which may be inserted into the C12L locus, and a IL-7 transgene, which may be inserted into either the N1L locus (e.g., MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L-hG-CSF-ΔN1L-hIL-7), the N1L / N2L loci (e.g., MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1LΔN2L-hIL-7), or the C12L locus by linking hG-CSF and hIL-7 with a P2A sequence (e.g., MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199- IL-12ΔC12L-hG-CSF-hIL-7, in which case the virus may include a mutant N1L and / or N1L / N2L gene, e.g, MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-hIL- 7ΔN1L or MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-hIL- 7ΔN1LΔN2L). When the IL-7 transgene is inserted into the N1L and N2L locus, MQ955 further comprises a mutant N1L gene and a mutant N2L gene (ΔN1LΔN2L). In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g. RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 8) which anchors the IL-12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833, the C12L gene is further mutated to generate MQ953, the G-CSF transgene is inserted into C12L locus to generate MQ954, and the IL-7 transgene is inserted to generate MQ955. For example, in some embodiments, the IL-7 transgene may be inserted into the locus or loci of any one or more MVA genes that have been mutated (e.g., deleted). Illustrative, non-limiting examples of such loci into which IL-7 may be inserted may include the C12L locus, or the N1L, or the N1L and N2L loci. In some embodiments, the C12L gene is deleted. In some embodiments, the N1L gene is deleted. In some embodiments both the N1L and the N2L genes are deleted. In some embodiments, the G-CSF transgene is inserted into the C12L locus. In some embodiments, the IL-7 transgene is inserted into the C12L locus. In some embodiments, both the G-CSF and IL-7 transgenes are inserted into the C12L locus by 35 4873-2549-9626.1Atty. Dkt. No.115872-3093 linking the G-CSF and IL-7 transgenes with a P2A sequence. In some embodiments, the IL-7 transgene is inserted into the N1L locus or the N1L and N2L loci. In some embodiments, the G-CSF and IL-7 transgenes linked with P2A sequence are inserted into the N1L locus or N1L and N2L loci.

[0094] As used herein, “parenteral,” when used in the context of administration of a therapeutic substance or composition, includes any route of administration other than administration through the alimentary tract. Particularly relevant for the methods disclosed herein are intravenous (including, for example, through the hepatic portal vein for hepatic delivery), intratumoral, or intrathecal administration.

[0095] The terms “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” or “pharmaceutically acceptable adjuvant” refer to an excipient, diluent, carrier, and / or adjuvant useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient, diluent, carrier, and adjuvant that is acceptable for pharmaceutical use. “A pharmaceutically acceptable excipient, diluent, carrier and / or adjuvant” as used in the specification and claims includes one and more such excipients, diluents, carriers, and adjuvants.

[0096] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0097] As used herein, the term “recombinant” when used with reference, e.g., to a virus, or cell, or nucleic acid, or protein, or vector, indicates that the virus, cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a virus or cell so modified. Thus, for example, recombinant viruses or cells express genes that are not found within the native (non-recombinant) form of the virus or cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. 36 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0098] As used herein, “solid tumor” refers to all neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, except for hematologic cancers such as lymphomas, leukemias, and multiple myeloma. Examples of solid tumors include, but are not limited to: sarcomas including soft tissue sarcoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor and other bone tumors (e.g., osteosarcoma, malignant fibrous histiocytoma), Extramammary Paget Disease (EMPD), head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, brain / CNS tumors (e.g., astrocytoma, glioma, glioblastoma, childhood tumors, such as atypical teratoid / rhabdoid tumor, germ cell tumor, embryonal tumor, ependymoma) medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, malignant nerve sheath tumor, malignant peripheral nerve sheath tumor, anaplastic thyroid cancer, and retinoblastoma. Some of the most common solid tumors for which the compositions and methods of the present disclosure would be useful include: head-and-neck cancer, rectal adenocarcinoma, glioma, medulloblastoma, urothelial carcinoma, pancreatic adenocarcinoma, uterine (e.g., endometrial cancer, fallopian tube cancer) ovarian cancer, cervical cancer, prostate adenocarcinoma, non-small cell lung cancer (squamous and adenocarcinoma), small cell lung cancer, melanoma, breast carcinoma, bladder cancer, ductal carcinoma in situ, renal cell carcinoma, and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal, eye (e.g., melanoma, retinoblastoma), gallbladder, gastrointestinal, Wilms’ tumor, heart, head and neck, laryngeal and hypopharyngeal, oral (e.g., lip, mouth, salivary gland), nasopharyngeal, neuroblastoma, peritoneal, pituitary, Kaposi’s sarcoma, small intestine, stomach, testicular, thymus, thyroid, parathyroid, vaginal tumor, malignant nerve sheath tumor, malignant peripheral nerve sheath tumor, anaplastic thyroid cancer and the metastases of any of the foregoing. Any of 37 4873-2549-9626.1Atty. Dkt. No.115872-3093 the preceding types of tumors can, in some embodiments, be resistant to immune checkpoint blockade (ICB) inhibitor therapeutics. In some embodiments, the ICB inhibitor resistance is due to a deficiency in MHC-I antigen presentation. In some embodiments, the ICB inhibitor resistance is due to a mutation in the B2M gene and / or reduced B2M activity. The B2M protein plays an important role in MHC-I antigen presentation. In some embodiments, an ICB inhibitor-resistant tumor is resistant to CD8+T cell mediated immune responses.

[0099] As used herein, “resistant to immune checkpoint inhibitor,” “resistance to immune checkpoint blockade inhibitors,” or “resistance to immune checkpoint inhibitors,” or “resistance to immune checkpoint blockade inhibitors,” or “immune checkpoint blockade resistant” refers to a tumor that is resistant to immune checkpoint blockade (ICB) inhibitor treatment, wherein the ICB treatment does not elicit an immune response against the resistant tumor to the same degree as the ICB treatment would elicit against a non-resistant tumor of the same type. For example, a tumor that is resistant to immune checkpoint blockade (ICB) inhibitors may either increase or lack reduction in tumor size or volume, metastasis, nodal invasion, or progressive disease in spite of treatment with one or more ICB inhibitors. A tumor that is resistant to ICB inhibitors may exhibit no changes or diminished changes to the tumor immune microenvironment despite being treated with an ICB inhibitor. There are multiple possible mechanisms for resistance to immune checkpoint inhibitors, including, but not limited to, deficiency in MHC-I antigen presentation, mutations in the B2M gene, reduced B2M activity, loss-of-function of the histone-modifying polycomb repressive complex 2 (PRC2), inadequate tumor associated antigens and / or deficient antigen presentation thereof, inadequate cytokine expression, defficieny in cytokine co-stimulation, active immunosuppressive signaling pathways, or combinations thereof. A tumor that is resistant to ICB inhibitor treatment may comprise a mutation in the B2M gene and / or reduced B2M activity. A tumor that is resistant to ICB treatment may comprise a mutation in the EED and / or SUZ12 genes and / or reduced EED and / or SUZ12 activity. A tumor that is resistant to ICB inhibitor treatment may comprise mutations to one or more genes encoding proteins that are involved in the MHC-I antigen presentation process. Resistance to immune checkpoint inhibitors is a matter of degree, meaning that a tumor can be fully resistant or partially resistant to one or more immune checkpoint inhibitors. Resistance to immune checkpoint inhibitors protects tumors from the immune response. In some embodiments, resistance to immune checkpoint inhibitors 38 4873-2549-9626.1Atty. Dkt. No.115872-3093 protects a tumor from CD8+T cell mediated killing, meaning the tumor is resistant to CD8+T cell mediated immune responses. Tumors that are resistant to CD8+T cell mediated immune responses either do not elicit increased levels of CD8+T cells or are not responsive to increased levels of CD8+T cells. ICB resistant tumors include, but are not limited to, melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, malignant peripheral nerve sheath tumors, pancreatic cancer, malignant nerve sheath tumors, malignant peripheral nerve sheath tumors, anaplastic thyroid cancer, pancreatic cancer or Extramammary Paget disease (EMPD).

[0100] As used herein, “MHC-I antigen presentation” refers to the localization of major histocompatibility class (MHC) I proteins at the surface of a cell, wherein the MHC-I proteins form the MCH-I complex and present an antigenic peptide fragment for recognition by an immune cell. MHC-I antigen presentation is a step in the activation of CD8+T cells. “MHC-I antigen presentation deficiency” refers to a complete or partial loss of MHC-I antigen presentation capacity in a cell, cells, or tumor, and is a known mechanism for cancer immune evasion. Additionally, complete or partial loss of MHC-I antigen presentation is a mechanism for cancer resistance to immune checkpoint blockade inhibitor treatment. Cells and / or tumors are deficient in MHC-I antigen presentation when the cells and / or tumors possess diminished amounts of the MHC-I complex at the cell surface membrane and / or diminished amounts of antigen presenting MHC-I complexes at the cell surface membrane.

[0101] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably herein, and can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, “subject” means any animal (mammalian, human, or other) patient that can be afflicted with cancer and when thus afflicted is in need of treatment. In some embodiments, “subject” means human.

[0102] As used herein, a “synergistic therapeutic effect” in some embodiments reflects a greater-than-additive therapeutic effect that is produced by a combination of at least two agents, and which exceeds that which would otherwise result from the individual administration of the agents. In some embodiments, a “synergistic therapeutic effect” reflects an enhanced therapeutic effect that is produced by a combination of at least two agents relative to the individual administration of the agents. For example, lower doses of 39 4873-2549-9626.1Atty. Dkt. No.115872-3093 one or more agents may be used in treating a disease or disorder, resulting in increased therapeutic efficacy and decreased side-effects.

[0103] “Treating,” “treat,” “treated,” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission. In some embodiments, “inhibiting,” means reducing or slowing the growth of a tumor. In some embodiments, the inhibition of tumor growth may be, for example, by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In some embodiments, the inhibition may be complete.

[0104] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

[0105] As used herein, “tumor immunity” refers to one or more processes by which tumors evade recognition and clearance by the immune system. Thus, as a therapeutic concept, tumor immunity is “treated” when such evasion is attenuated or eliminated, and the tumors are recognized and attacked by the immune system (the latter being termed herein “anti-tumor immunity”). An example of tumor recognition is tumor binding, and examples of tumor attack are tumor reduction (in number, size, or both) and tumor clearance.

[0106] As used herein, “T-cell” refers to a thymus derived lymphocyte that participates in a variety of cell-mediated adaptive immune reactions. As used herein, “effector T-cell” includes helper, killer, and regulatory T-cells.

[0107] As used herein, “helper T-cell” refers to a CD4+T-cell; helper T-cells recognize antigen bound to MHC Class II molecules. There are at least two types of helper T-cells, Th1 and Th2, which produce different cytokines. 40 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0108] As used herein, “cytotoxic T-cell” refers to a T-cell that usually bears CD8 molecular markers on its surface (CD8+) and that functions in cell-mediated immunity by destroying a target T-cell having a specific antigenic molecule on its surface. Cytotoxic T- cells also release Granzyme, a serine protease that can enter target T-cells via the perforin- formed pore and induce apoptosis (cell death). Granzyme serves as a marker of cytotoxic phenotype. Other names for cytotoxic T-cell include CTL, cytolytic T-cell, cytolytic T lymphocyte, killer T-cell, or killer T lymphocyte. Targets of cytotoxic T-cells may include virus-infected cells, cells infected with bacterial or protozoal parasites, or cancer cells. Most cytotoxic T-cells have the protein CD8 present on their cell surfaces. CD8 is attracted to portions of the Class I MHC molecule. Typically, a cytotoxic T-cell is a CD8+cell.

[0109] As used herein, “tumor-infiltrating leukocytes” refers to white blood cells of a subject afflicted with a cancer (such as melanoma), that are resident in or otherwise have left the circulation (blood or lymphatic fluid) and have migrated into a tumor.

[0110] As used herein, “vector” includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively positioned,” “operatively linked,” “under control,” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term “expression vector or construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA) from a transcribed gene. A non-limiting example of a pCB-OX40L-gpt vector according to the present technology is set forth in SEQ ID NO: 9. A non-limiting example of a pUC57- hFlt3L-GFP vector according to the present technology is set forth in SEQ ID NO: 10. 41 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0111] The term “virulence” as used herein to refer to the relative ability of a pathogen to cause disease. The term “attenuated virulence” or “reduced virulence” is used herein to refer to a reduced relative ability of a pathogen to cause disease. II. Immune System and Cancer

[0112] Malignant tumors are inherently resistant to conventional therapies and present significant therapeutic challenges. Immunotherapy has become an evolving area of research and an additional option for the treatment of certain types of cancers. The immunotherapy approach rests on the rationale that the immune system may be stimulated to identify tumor cells and target them for destruction.

[0113] Numerous studies support the importance of the differential presence of immune system components in cancer progression (Jochems et al., Exp. Biol. Med.236(5):567–579 (2011)). Clinical data suggest that high densities of tumor-infiltrating lymphocytes are linked to improved clinical outcome (Mlecnik et al., Cancer Metastasis Rev.30:5–12, (2011)). The correlation between a robust lymphocyte infiltration and patient survival has been reported in various types of cancer, including melanoma, ovarian, head and neck, breast, bladder, urothelial, colorectal, lung, hepatocellular, gallbladder, and esophageal cancer (Angell et al., Current Opinion in Immunology 25:1-7, (2013)). Tumor immune infiltrates include macrophages, dendritic cells (DC), monocytes, neutrophils, natural killer (NK) cells, naïve and memory lymphocytes, B cells and effector T-cells (T lymphocytes), primarily responsible for the recognition of antigens expressed by tumor cells and subsequent destruction of the tumor cells by cytotoxic T-cells.

[0114] Despite presentation of antigens by cancer cells and the presence of immune cells that could potentially react against tumor cells, in many cases the immune system does not get activated or is affirmatively suppressed. Key to this phenomenon is the ability of tumors to protect themselves from immune response by coercing cells of the immune system to inhibit other cells of the immune system. Tumors develop a number of immunomodulatory mechanisms to evade antitumor immune responses. For example, tumor cells secrete immune inhibitory cytokines (such as TGF-β) or induce immune cells, such as CD4+T regulatory cells and macrophages, in tumor lesions to secrete these cytokines. Tumors also have the ability to bias CD4+T-cells to express the regulatory phenotype. The overall result is impaired T-cell responses and impaired induction of 42 4873-2549-9626.1Atty. Dkt. No.115872-3093 apoptosis or reduced anti-tumor immune capacity of CD8+cytotoxic T-cells. Additionally, tumor-associated altered expression of MHC class I on the surface of tumor cells makes them “invisible” to the immune response (Garrido et al. Cancer Immunol. Immunother. 59(10):1601–1606 (2010)). Inhibition of antigen-presenting functions and dendritic cell (DC) additionally contributes to the evasion of anti-tumor immunity (Gerlini et al. Am. J. Pathol.165(6):1853–1863 (2004)).

[0115] Moreover, the local immunosuppressive nature of the tumor microenvironment, along with immune editing, can lead to the escape of cancer cell subpopulations that do not express the target antigens. Thus, finding an approach that would promote the preservation and / or restoration of anti-tumor activities of the immune system would be of considerable therapeutic benefit.

[0116] Immune checkpoints have been implicated in the tumor-mediated downregulation of anti-tumor immunity and used as therapeutic targets. It has been demonstrated that T- cell dysfunction occurs concurrently with an induced expression of the inhibitory receptors, CTLA-4 and programmed death 1 polypeptide (PD-1), members of the CD28 family of receptors. PD-1 is an inhibitory member of the CD28 family of receptors that in addition to PD-1 includes CD28, CTLA-4, ICOS, and BTLA. However, while promise regarding the use of immunotherapy in the treatment of melanoma has been underscored by the clinical use and even regulatory approval of anti-CTLA-4 (ipilimumab) and anti-PD-1 drugs (e.g., pembrolizumab and nivolumab), the response of patients to these immunotherapies has been limited. Clinical trials, focused on blocking these inhibitory signals in T-cells (e.g., CTLA- 4, PD-1, and the ligand of PD-1, PD-L1), have shown that reversing T-cell suppression is critical for successful immunotherapy (Sharma et al., Science 348(6230):56-61 (2015); Topalian et al., Curr. Opin. Immunol.24(2):202-217 (2012)). These observations highlight the need for development of novel therapeutic approaches for harnessing the immune system against cancer.

[0117] Some cancers, including, but not limited to, melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head- and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, malignant peripheral nerve sheath tumors, pancreatic cancer, malignant nerve sheath tumors, malignant peripheral nerve sheath tumors, anaplastic thyroid cancer, pancreatic cancer or 43 4873-2549-9626.1Atty. Dkt. No.115872-3093 Extramammary Paget disease (EMPD), can be resistant to treatment with immune checkpoint blockade (ICB) inhibitors. Other cancers not specifically recited here can also be ICB inhibitor-resistant. In some embodiments, the ICB inhibitor resistance is due to a deficiency in MHC-I antigen presentation. In some embodiments, the ICB inhibitor resistance is due to a mutation in the B2M gene and / or reduced B2M activity. In some embodiments, the ICB inhibitor resistance is due to a mutation the the EED and / or SUZ12 genes and / or reduced EED and / or SUZ12 activity. The B2M protein plays an important role in MHC-I antigen presentation, and the EED and SUZ12 proteins play important roles in PRC-2 activity. In some embodiments, an ICB inhibitor-resistant tumor is resistant to CD8+T cell mediated immune responses. In some embodiments, a tumor is diagnosed as being resistant to treatment with ICB inhibitors due to lack of response to administration of one or more ICB inhibitors, such as, for example, either an increase or a lack of reduction in tumor size or volume, metastasis, nodal invasion, or progressive disease in spite of treatment with one or more ICB inhibitors. III. Modified Vaccinia Ankara (MVA) Virus

[0118] Poxviruses are in the forefront as oncolytic therapy for metastatic cancers (Kirn et al., Nature Review Cancer 9:64-71 (2009)). Poxviruses are well-suited as vectors to express multiple transgenes in cancer cells and thus to enhance therapeutic efficacy (Breitbach et al., Current pharmaceutical biotechnology 13:1768-1772 (2012)). Preclinical studies and clinical trials have demonstrated efficacy of using oncolytic vaccinia viruses and other poxviruses for treatment of advanced cancers refractory to conventional therapy (Park et al., Lacent Oncol.9:533-542 (2008); Kirn et al., PLoS Med 4:e353 (2007); Thorne et al., J. Clin. Invest.117:3350-3358 (2007)). The current oncolytic vaccinia strains in clinical trials (JX-594, for example) are replicative strains. They use wild-type vaccinia with deletion of thymidine kinase to enhance tumor selectivity, and with expression of transgenes such as granulocyte macrophage colony stimulating factor (GM-CSF) to stimulate immune responses (Breitbach et al., Curr. Pharm. Biotechnol.13:1768-1772 (2012)). Many studies have shown, however, that wild-type vaccinia has immune suppressive effects on antigen presenting cells (APCs) (Engelmayer et al., J. Immunol. 163:6762-6768 (1999); Jenne et al., Gene Therapy 7:1575-1583 (2000); P. Li et al., J. Immunol.175:6481-6488 (2005); Deng et al., J. Virol.80:9977-9987 (2006)), and thus adds to the immunosuppressive and immunoevasive effects of tumors themselves. 44 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0119] Modified Vaccinia Ankara (MVA) virus is a member of the Poxvirus family. MVA was generated by approximately 570 serial passages on chicken embryo fibroblasts (CEF) of the Ankara strain of vaccinia virus (CVA) (Mayr et al., Infection 3:6-14 (1975)). As a consequence of these long-term passages, the resulting MVA virus contains extensive genome deletions and is highly host cell restricted to avian cells (Meyer et al., J. Gen. Virol. 72:1031-1038 (1991)). It was shown in a variety of animal models that the resulting MVA is significantly avirulent (Mayr et al., Dev. Biol. Stand.41:225-34 (1978)).

[0120] The safety and immunogenicity of MVA has been extensively tested and documented in clinical trials, particularly against the human smallpox disease. These studies included over 120,000 individuals and have demonstrated excellent efficacy and safety in humans. Moreover, compared to other vaccinia based vaccines, MVA has weakened virulence (infectiousness) while it triggers a good specific immune response. Thus, MVA has been established as a safe vaccine vector, with the ability to induce a specific immune response.

[0121] Due to the above mentioned characteristics, MVA became an attractive candidate for the development of engineered MVA vectors, used for recombinant gene expression and vaccines. As a vaccine vector, MVA has been investigated against numerous pathological conditions, including HIV, tuberculosis and malaria, as well as cancer (Sutter et al., Curr. Drug Targets Infect. Disord.3:263–271(2003); Gomez et al., Curr. Gene Ther.8:97–120 (2008)).

[0122] It has been demonstrated that MVA infection of human monocyte-derived dendritic cells (DC) causes DC activation, characterized by the upregulation of co- stimulatory molecules and secretion of proinflammatory cytokines (Drillien et al., J. Gen. Virol.85:2167–2175 (2004)). In this respect, MVA differs from standard wild type vaccinia virus (WT-VAC), which fails to activate DCs. Dendritic cells can be classified into two main subtypes: conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). The former, especially the CD103+ / CD8α+subtype, are particularly adapted to cross-presenting antigens to T-cells; the latter are strong producers of Type I IFN.

[0123] Viral infection of human cells results in activation of an innate immune response (the first line of defense) mediated by type I interferons, notably interferon-alpha (α). This normally leads to activation of an immunological “cascade,” with recruitment and 45 4873-2549-9626.1Atty. Dkt. No.115872-3093 proliferation of activated T-cells (both CTL and helper) and eventually with antibody production. However, viruses express factors that dampen immune responses of the host. MVA is a better immunogen than WT-VAC and replicates poorly in mammalian cells. (See, e.g., Brandler et al., J. Virol.84:5314–5328 (2010)).

[0124] Illustrative, non-limiting MVA genome sequences are set forth in SEQ ID NO: 1 (GenBank Accession No. U94848.1) and SEQ ID NO: 7 (GenBank Accession No. AY603355). IV. OX40 ligand (OX40L)

[0125] The OX40 ligand (OX40L) and its binding partner, tumor necrosis factor receptor OX40, also known as CD134, are members of the TNFR / TNF superfamily. As used herein, the term OX40 can refer to human or murine OX40, likewise the term OX40L can refer to human or murine OX40L. OX40 is expressed on activated T cells, including regulatory T cells (Tregs) and effector T cells. OX40 plays an important role in T cell activation, survival, and differentiation. The OX40L-OX40 interaction provides survival and activation signals for T-cells expressing OX40. The binding of OX40 to its ligand (OX40L) can also modulate the suppressive function of Tregs. Specifically, OX40 signaling can reduce the suppressive activity of Tregs and may even convert some Tregs to a more effector-like phenotype. Given its role in modulating Treg function and its expression on activated T cells, OX40 has been a target for cancer immunotherapy. By targeting OX40, it might be possible to enhance antitumor immunity by promoting the function of effector T cells and inhibiting (or altering the function of) Tregs in the tumor microenvironment. The OX40L of the recombinant viruses of the present technology can be either huOX40L (or hOX40L) or muOX40L (or mOX40L).

[0126] Illustrative OX40L (huOX40L) nucleic acid (SEQ ID NO: 11) and polypeptide sequences (SEQ ID NO: 12) are provided below. huOX40L-ORF (SEQ ID NO:11): ATGGAAAGGG TCCAACCCCT GGAAGAGAAT GTGGGAAATG CAGCCAGGCC AAGATTCGAG AGGAACAAGC TATTGCTGGT GGCCTCTGTA ATTCAGGGAC TGGGGCTGCT CCTGTGCTTC ACCTACATCT GCCTGCACTT CTCTGCTCTT CAGGTATCAC ATCGGTATCC TCGAATTCAA AGTATCAAAG TACAATTTAC CGAATATAAG AAGGAGAAAG GTTTCATCCT CACTTCCCAA AAGGAGGATG AAATCATGAA GGTGCAGAAC AACTCAGTCA TCATCAACTG TGATGGGTTT TATCTCATCT CCCTGAAGGG CTACTTCTCC CAGGAAGTCA ACATTAGCCT TCATTACCAG AAGGATGAGG AGCCCCTCTT CCAACTGAAG AAGGTCAGGT CTGTCAACTC CTTGATGGTG GCCTCTCTGA CTTACAAAGA CAAAGTCTAC TTGAATGTGA CCACTGACAA TACCTCCCTG 46 4873-2549-9626.1Atty. Dkt. No.115872-3093 GATGACTTCC ATGTGAATGG CGGAGAACTG ATTCTTATCC ATCAAAATCC TGGTGAATTC TGTGTCCTTT GA huOX40L polypeptide (SEQ ID NO: 12) ME R V Q P L E E N V G N A A R P R F E R N K L L L V A S V I Q G L G L L L C F T Y I C L H F S A L Q V S H R Y P R I Q S I K V Q F T E Y K K E K G F I L T S Q K E D E I MK V Q N N S V I I N C D G F Y L I S L K G Y F S Q E V N I S L H Y Q K D E E P L F Q L K K V R S V N S L MV A S L T Y K D K V Y L N V T T D N T S L D D F H V N G G E L I L I H Q N P G E F C V L Stop

[0127] Illustrative murine OX40L (muOX40L) nucleic acid (SEQ ID NO: 13) and polypeptide sequences (SEQ ID NO: 14) are provided below. muOX40L-ORF (codon optimized)(SEQ ID NO: 13): ATGGAGGGCGAGGGGGTCCAGCCTCTGGACGAGAACCTCGAAAACGGGTCTCGCCCTCGCT TTAAATGGAAGAAGACTCTTAGGCTCGTTGTAAGCGGCATCAAGGGGGCCGGTATGTTGCT GTGCTTCATATATGTGTGTTTGCAACTTAGCTCTTCACCTGCAAAAGACCCCCCCATACAA CGCCTTCGGGGGGCTGTGACCCGCTGTGAAGATGGTCAATTGTTTATTTCTTCTTACAAGA ACGAGTATCAGACGATGGAAGTCCAGAATAACTCCGTAGTGATTAAGTGTGACGGACTGTA CATCATCTACTTGAAAGGATCTTTTTTCCAGGAGGTCAAAATTGACCTCCACTTCAGGGAG GATCACAACCCTATCTCAATCCCTATGTTGAACGACGGCAGAAGAATCGTCTTTACTGTAG TCGCTTCACTGGCCTTCAAGGATAAGGTGTACTTGACCGTAAACGCTCCTGATACCTTGTG CGAGCATTTGCAAATCAACGATGGAGAACTTATCGTTGTCCAACTCACACCAGGTTACTGT GCTCCTGAGGGCAGTTATCACAGTACAGTGAACCAAGTCCCACTGTGA muOX40L polypeptide (SEQ ID NO: 14):G S Y H S T V N Q V P L Stop V. Human Fms-like tyrosine kinase 3 ligand (hFlt3L)

[0128] Human Fms-like tyrosine kinase 3 ligand (hFlt3L), a type I transmembrane protein that stimulates the proliferation of bone marrow cells, was cloned in 1994 (Lyman et al., 1994). The use of hFlt3L has been explored in various preclinical and clinical settings including stem cell mobilization in preparation for bone marrow transplantation, cancer immunotherapy such as expansion of dendritic cells, as well as a vaccine adjuvant. Recombinant human Flt3L (rhuFlt3L) has been tested in more than 500 human subjects and is bioactive, safe, and well-tolerated. Much progress has been made in understanding the 47 4873-2549-9626.1Atty. Dkt. No.115872-3093 critical role of the growth factor Flt3L in the development of DC subsets, including CD8α+ / CD103+DCs and pDCs.

[0129] CD103+ / CD8α+DCs are required for spontaneous cross-priming of tumor antigen- specific CD8+T-cells. It has been reported that CD103+DCs are sparsely present within the tumors and they compete for tumor antigens with abundant tumor-associated macrophages. CD103+DCs are uniquely capable of stimulating naïve as well as activated CD8+T-cells and are critical for the success of adoptive T-cell therapy (Broz, et al. Cancer Cell, 26(5):638-52 (2014)). Spranger et al. reported that the activation of oncogenic signaling pathway WNT / β-catenin leads to reduction of CD103+DCs and anti-tumor T- cells within the tumors (Spranger et al., 2015). Intratumoral delivery of Flt3L-cultured bone marrow derived dendritic cells (BMDCs) leads to responsiveness to the combination of anti- CTLA-4 and anti-PD-L1 immunotherapy (Spranger et al., 2015). Systemic administration of Flt3L, a growth factor for CD103+DCs, and intratumor injection of poly I:C (TLR3 agonist) expanded and activated the CD103+DC populations within the tumors and overcame resistance or enhanced responsiveness to immunotherapy in a murine melanoma and MC38 colon cancer models.

[0130] The recent discovery of tumor neoantigens in various solid tumors indicates that solid tumors harbor unique neoantigens that usually differ from person to person (Castle et al., Cancer Res 72:1081-1091 (2012); Schumacher et al., Science 348:69-74 (2015)). The genetically engineered or recombinant viruses disclosed herein do not exert their activity by expressing tumor antigens. Intratumoral delivery of the present genetically engineered or recombinant MVA viruses allows efficient cross-presentation of tumor neoantigens and generation of anti-tumor adaptive immunity within the tumors (and also extending systemically), and therefore leads to “in situ cancer vaccination” utilizing tumor differentiation antigens and neoantigens expressed by the tumor cells in mounting an immune response against the tumor.

[0131] Despite the presence of neoantigens generated by somatic mutations within tumors, the functions of tumor antigen-specific T-cells are often held in check by multiple inhibitory mechanisms (Mellman et al., Nature 480, 480-489 (2011)). For example, the up- regulation of cytotoxic T lymphocyte antigen 4 (CTLA-4) on activated T-cells can compete with T-cell co-stimulator CD28 to interact with CD80 (B71) / CD86 (B7.2) on dendritic cells (DCs), and thereby inhibit T-cell activation and proliferation. CTLA-4 is also expressed on 48 4873-2549-9626.1Atty. Dkt. No.115872-3093 regulatory T (Treg) cells and plays an important role in mediating the inhibitory function of Tregs (Wing et al., Science 322:271-275 (2008); Peggs, et al., J. Exp. Med.206:1717-1725 (2009)). In addition, the expression of PD-L / PD-L2 on tumor cells can lead to the activation of the inhibitory receptor of the CD28 family, PD-1, leading to T-cell exhaustion. Immunotherapy utilizing antibodies against inhibitory receptors, such as CTLA-4 and programmed death 1 polypeptide (PD-1), have shown remarkable preclinical activities in animal studies and clinical responses in patients with metastatic cancers, and have been approved by the FDA for the treatment of metastatic melanoma, non-small cell lung cancer, as well as renal cell carcinoma (Leach et al., Science 271:1734-1746 (1996); Hodi et al., NEJM 363:711-723 (2010); Robert et al., NEJM 364:2517-2526 (2011); Topalian et al., Cancer Cell 27:450-461 (2012); Sharma et al., Science 348(6230):56-61 (2015)). A non- limiting exemplary amino acid sequence for hFlt3L (SEQ ID NO: 27) is provided below, and a non-limiting exemplary coding sequence for hFlt3L is provided in SEQ ID NO: 5: hFlt3L polypeptide (SEQ ID NO: 27): MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPV TVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCA FQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWS PRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSP QDLLLVEH VI. Interleukin 12 (IL-12)

[0132] Human interleukin 12 (IL-12) is a cytokine molecule that was independently identified by two investigators in 1989 and 1990. The use of IL-12 for its immunostimulatory properties has been heavily investigated, particularly in the cancer context. While systemic IL-12 treatment can result in a toxic, inflammatory response, multiple investigators have demonstrated that targeted treatment, via lipid nanoparticle delivery, for example, can suppress tumorigenesis without any undesirable toxic effects. Progress continues to be made in understanding the therapeutic potential of IL-12, as both a treatment and an adjuvant, in the anti-cancer context.

[0133] IL-12 consists of a p35 monomer and p40 monomer which are linked by disulfide bonds to form the biologically active p70 heterodimer. IL-12 functions by binding to the IL-12 receptor (IL-12R), which is predominantly expressed by natural killer (NK) cells and 49 4873-2549-9626.1Atty. Dkt. No.115872-3093 T cells. IL-12R activation results in a signaling cascade via the JAK-STAT pathway that eventually leads to an array of pro-immune responses, including the production of interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). As used herein, the term IL-12 can refer to human or murine IL-12. A non-limiting exemplary DNA sequence for hIL-12 (SEQ ID NO: 15) and a non-limiting exemplary amino acid sequence for hIL-12 (SEQ ID NO: 16) are provided below: Human IL-12 (amino acid sequence): MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEE DDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGI WSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTC GMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYST SFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEK MKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVR SATNFSLLKQAGDVEENPGPMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQS RNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCL ATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQII LDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTIN RVMGYLSSARRPKGRGKRRREKQRPTDCHL (SEQ ID NO: 16) Human IL-12 (DNA sequence): atgtgtccacaaaagctcaccatctcatggttcgctatagtattgctcgtttccccattgatggcaatgtgggagcttgaaaaagatgtgt atgtagtggaggttgactggactcctgatgcacccggagagacagtcaatttgacatgcgacacccccgaagaagatgatattacat ggacatccgaccaacggcacggggtcatcggatctgggaagaccttgacaattacagtgaaggagttcttggatgcaggacagta cacatgtcataaagggggcgagacactctcacattcacatctgctcctgcataaaaaggagaacggaatctggtccaccgaaatcct taagaatttcaagaacaaaacctttcttaagtgtgaggcccctaattattccggaagatttacatgcagctggttggtccagcgcaatat ggacctcaaatttaatatcaagtcttcttccagctccccagattctcgggcagtgacttgcggcatggcatccctctccgctgagaaag taacattggatcaacgagactacgagaagtactctgttagttgtcaagaggacgttacatgccctaccgcagaagagacattgccaat tgaattggcccttgaagcacgccagcagaataagtacgaaaattacagtacaagctttttcatccgagacataattaaacccgatcctc ctaagaatctccaaatgaaacctttgaagaatagccaagtggaagtttcatgggagtatccagattcctggtccacaccacattcctatt tctcccttaagttcttcgtcagaatccagaggaagaaagaaaagatgaaggaaaccgaggaaggctgcaaccagaagggcgccttt ctggtagagaaaaccagcactgaggttcagtgtaaggggggaaacgtgtgtgtgcaggcacaagatcgatactacaactcaagct gtagtaaatgggcctgcgtaccttgtcgggttcgatccagacgacgccggagacgagctaccaatttttccttgctcaagcaggcag gcgatgtggaggaaaacccagggccccttgacatgtgtcagagccggtacctccttttcctcgcaaccctggctctgcttaaccacct ctcactggctagggtaattcccgtatctgggcctgcccgatgcctcagccagagtcggaatctccttaagaccacagacgatatggta aaaacagcaagggagaaactcaaacattactcttgtacagcagaggacatcgatcatgaagacataacccgggaccagacctcaa cattgaaaacttgtctgccactggagcttcataagaacgagtcctgccttgccacacgagagacctctagcactacacgggggtcct gcctgcctccacagaaaacctccttgatgatgaccctgtgtctcggcagtatttacgaagatttgaagatgtaccaaacagagtttcag gccattaacgcagcattgcaaaaccataatcaccagcagataatccttgataagggtatgctggtagccatcgacgaacttatgcaat ctctgaatcataatggcgaaactctgcgacaaaagcccccagttggagaagccgacccctaccgagtcaagatgaaactctgcata ctcctgcatgccttttccacacgggttgttactatcaatcgagtcatggggtatctttcttcagcacggcgccctaaagggcgcggaaa acgccgccgggaaaaacaaagacctactgattgccatctgtga (SEQ ID NO: 15)

[0134] As used herein, the term IL-12 can refer to human or murine IL-12. Some of the genetically engineered or recombinant viruses of the present disclosure express a 50 4873-2549-9626.1Atty. Dkt. No.115872-3093 recombinant IL-12 which is capable of binding to the extracellular matrix, thus preventing a systemic delivery of IL-12 and the associated toxicity. Intratumoral delivery of the present genetically engineered or recombinant MVA viruses that comprise recombinant IL-12 may therefore allow for efficient, localized delivery of IL-12 to induce an immune response against the target tumor. An illustrative, non-limiting plasmid sequence used to generate engineered or recombinant MVA viruses comprising an IL-12 transgene and a deletion of the WR199 gene (also known as MVA186) include the sequence shown in Table 1. Table 1. pUC57-del186-pH5-huIL12 (FIG.25, SEQ ID NO: 2) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtttttttac tttaacgccc 421 ttgtatgttt ttacagagac tttatctaaa tttctagtgc ttgtatgtgt tataaatata 481 acgggatata gaactgaatc acctacctta gatacccaat tacattttat cagatccaga 541 taataaacaa attttgtcgc cctaactaat tctatattgt tatatatttt acaattggtt 601 atgatatcat gtaataactt ggagtctaac gcgcatcgtc gtacgtttat acaattgtga 661 tttagtgtag tatatctaca catgtatttt tccgcactat agtattctgg actagtgata 721 aaactatcgt tatatctgtc ttcaatgaac tcatcgagat attgctctct gtcatattca 51 4873-2549-9626.1Atty. Dkt. No.115872-3093 781 tacacctgca taaactttct agacatctta caatccgtgt tattttagga tcatatttac 841 atatttacgg gtatatcaaa gatgttagat tagttaatgg gaatcgtcta taataatgaa 901 tattaaacaa ttatatgagg acttttacca caaagcatca taaaactgca gaaaaattga 961 aaataaatac aaaggttctt gagggttgtg ttaaattgaa agcgagaaat aatcataaat 1021 aactggtcga gatgtgtcac cagcagctag tgatctcgtg gttctcccta gtatttctag 1081 cctctccgct agtagcgatc tgggagctaa aaaaggatgt ctacgtcgtc gagctagact 1141 ggtatccaga tgctccggga gaaatggtag tcctaacatg tgatacaccg gaagaggatg 1201 gaatcacctg gaccttggat caatcctccg aagtactagg atccggaaag accctaacca 1261 tccaagtcaa agaattcgga gatgcgggac agtacacctg tcacaaaggt ggagaagtcc 1321 tatcgcactc cctactacta ttgcacaaga aagaggacgg tatctggtcc accgatatcc 1381 taaaggatca gaaagagccg aagaacaaga ccttcctaag atgcgaagcg aagaactact 1441 ccggaagatt cacatgttgg tggctaacca caatctccac cgacctaacc ttctccgtca 1501 aatcttctag aggatccagt gatccgcagg gtgtaacatg tggtgctgct acattatctg 1561 cggagagagt cagaggagac aacaaagagt acgagtactc cgtcgagtgt caagaggatt 1621 ctgcttgtcc agctgcggaa gaatctctac cgatcgaagt aatggtagac gcggtccaca 1681 agttgaagta cgagaactac acctcctcgt tcttcatcag agacatcatc aaaccagatc 1741 cgccgaagaa tctacagcta aagccgctaa agaactccag acaggtcgaa gtatcttggg 1801 aatacccgga tacttggtct acaccgcact cctacttttc gttgaccttc tgtgtacaag 52 4873-2549-9626.1Atty. Dkt. No.115872-3093 1861 tccagggaaa gtccaagaga gagaagaagg acagagtctt taccgacaag acatccgcga 1921 ccgtcatctg tagaaagaac gcctctattt ctgtcagagc gcaggacaga tattactcct 1981 cgtcttggag tgaatgggcg tctgtaccat gttccagaag aagaagaaga agagcgacca 2041 acttctcgct attgaagcaa gcgggagatg tagaagaaaa tccgggaccg ttagatatgt 2101 gtccggcgag atctctacta ctagtcgcga cactagtcct actagaccat ctatctctag 2161 cgagaaattt gccagtagcg acaccagatc ctggaatgtt tccgtgtcta caccactcgc 2221 agaatctact aagagccgtg tctaacatgc tacagaaggc gagacagacc ttggaattct 2281 acccgtgtac ctccgaagaa atcgatcacg aggatatcac caaggacaag acctctacag 2341 tcgaagcttg tctaccgcta gagttgacca agaacgagtc ctgcctaaac tccagagaaa 2401 cctccttcat caccaacgga tcttgcctag cgtctagaaa gacctctttc atgatggcgc 2461 tatgcctatc ctctatctac gaggacctaa agatgtacca ggtcgaattc aagaccatga 2521 acgcgaagct actaatggac ccgaagagac agatcttctt ggaccagaat atgctagcgg 2581 tcatcgacga actaatgcag gcgctaaact tcaactctga aaccgtgccg cagaagtcca 2641 gtttagaaga accggatttc tacaagacca agatcaagct atgcatccta ctacacgcgt 2701 tcagaatcag agcggtcacc atcgatagag tcatgtctta cctaaacgcg tccagaagac 2761 cgaaaggaag aggaaagaga agaagagaaa agcaaagacc gaccgactgc catctatgag 2821 ttaacttgta tttttctcat gcgatgtgtg taaaaaaact gatattatat aaatatttta 2881 gtgccgtata atgaagatga cgatgaaaat gatggtacat atatatttcg tatcattatt 53 4873-2549-9626.1Atty. Dkt. No.115872-3093 2941 gttattgcta ttccacagtt acgccataga catcgaaaat gaaatcacag aattcttcaa 3001 taaaatgaga gatactctac cagctaaaga ctctaaatgg ttgaatccag catgtatgtt 3061 cggaggcaca atgaatgata tagccgctct aggagagcca ttcagcgcaa agtgtcctcc 3121 tattgaagac agtcttttat cgcacagata taaagactat gtggttaaat gggagaggct 3181 agaaaaaaat agacggcgac aggtttctaa taaacgtgtt aaacatggtg atttatggat 3241 agccaactat acatctaaat tcagtaaccg taggtatttg tgtaccgtaa ctacaaagaa 3301 tggtgactgt gttcagggta tagttagatc tcatattaaa aaacctcctt catgcattcc 3361 aaaaacatat gaactaggta ctcatgataa gtagcttggc gtaatcatgg tcatagctgt 3421 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 3481 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 3541 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 3601 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 3661 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 3721 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 3781 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 3841 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 3901 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 3961 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 54 4873-2549-9626.1Atty. Dkt. No.115872-3093 4021 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 4081 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 4141 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 4201 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 4261 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 4321 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 4381 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 4441 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 4501 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 4561 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 4621 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 4681 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 4741 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 4801 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 4861 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 4921 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 4981 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 5041 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 55 4873-2549-9626.1Atty. Dkt. No.115872-3093 5101 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 5161 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 5221 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 5281 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 5341 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 5401 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 5461 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 5521 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 5581 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c VII. E3L

[0135] Poxviruses are extraordinarily adept at evading and antagonizing multiple innate immune signaling pathways by encoding proteins that interdict the extracellular and intracellular components of those pathways (Seet et al. Annu. Rev. Immunol.21:377-423 (2003)). Chief among the poxvirus antagonists of intracellular innate immune signaling is the vaccinia virus duel Z-DNA and dsRNA-binding protein E3, also identified as MVA050L, which can inhibit the PKR and NF-κB pathways (Cheng et al., Proc. Natl. Acad. Sci. USA 89:4825-4829 (1992); Deng et al., J. Virol.80:9977-9987 (2006)) that would otherwise be activated by vaccinia virus infection. A mutant vaccinia virus lacking the E3L gene (∆E3L) has a restricted host range, is highly sensitive to IFN, and has greatly reduced virulence in animal models of lethal poxvirus infection (Beattie et al., Virus Genes 1289-94 (1996); Brandt et al., Virology 333263-270 (2004)). Recent studies have shown that infection of cultured cell lines with ∆E3L virus elicits proinflammatory responses that are masked during infection with wild-type vaccinia virus (Deng et al., J. Virol.80:9977- 9987 (2006); Langland et al. J. Virol.80:10083-10095). Infection of a mouse epidermal dendritic cell line with wild-type vaccinia virus attenuated proinflammatory responses to the 56 4873-2549-9626.1Atty. Dkt. No.115872-3093 TLR agonists lipopolysaccharide (LPS) and poly(I:C), an effect that was diminished by deletion of E3L. Moreover, infection of the dendritic cells with ∆E3L virus triggered NF- κB activation in the absence of exogenous agonists (Deng et al., J. Virol.80:9977-9987 (2006)). Whereas wild-type vaccinia virus infection of murine keratinocytes does not induce the production of proinflammatory cytokines and chemokines, infection with ∆E3L virus does induce the production of IFN-β, IL-6, CCL4 and CCL5 from murine keratinocytes, which is dependent on the cytosolic dsRNA-sensing pathway mediated by the mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytosolic RNA sensors RIG-I and MDA5) and the transcription factor IRF3 (Deng et al., J. Virol.82(21):10735– 10746 (2008)).

[0136] Deletion of E3L sensitizes vaccinia virus replication to IFN inhibition in permissive RK13 cells and results in a host range phenotype, whereby ∆E3L cannot replicate in HeLa or BSC40 cells (Chang et al., 1995). The C-terminal dsRNA-binding domain of E3 is responsible for the host range effects, whereas viruses with deletion of the N-terminal Z-DNA-binding domain are replication competent in HeLa and BSC40 cells (Brandt et al., 2001). An illustrative, non-limiting, plasmid sequence used to generate engineered or recombinant MVA viruses comprising a deletion of the E3L is shown in Table 2. Table 2. P503 P503 (FIG.29, SEQ ID NO: 3) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcaca ctatgtgata 57 4873-2549-9626.1Atty. Dkt. No.115872-3093 421 tgattaaggg tactagcggc accgaaaaag aattccttcg ctatctctta ttcggtataa 481 aatgcattaa gaaaggagta gaatacaata tagataaaat aaaggatgtg agttacaacg 541 attattttaa cgttctcgac gagaaataca atacaccgtg tcctaactgt aaaagtagga 601 atactacgcc gatgatgatt caaactagag ccgctgacga acctccacta gttagacatg 661 cgtgtagaga ttgcaaacaa cactttaagc ctcccaaatt tagagcattt cgcaatctta 721 atgttacaac gcaatcgata catgaaaaca aggaaataac agagattctt ccagataata 781 atccatctcc tccagaatct ccagaaccag catcacctat agatgacggg ttaatcagat 841 ccacattcga tagaaacgac gaaccaccag aggatgatga ataaaaaaat gataaaataa 901 attagtttta ttgctggttg tgttagttct ctctaactcg agttctagtt atcaataaca 961 gttagtagtt tagttataca ttgaatcata catattaatt tttttattga gatagattaa 1021 aaaatacaaa ttgtagtact attaacgcga ctagtatatt ctctaaagat gatatctgtc 1081 acagatattc gtagagcgtt tctagacaat gaatgccata ctatcacaaa agcgtttgga 1141 tatctgcacg aggacaaggc tatcgcatta attaaaatag gatttcatcc cacttatcta 1201 cccaaagtcc tttataataa tgttgtagaa ttcgttccag aaaaactata tctgtttaag 1261 ccaagaactg tagctccatt ggatttgata tctactataa caaaattaaa gaacgtggac 1321 aaatttgcct cacacataaa ttatcacaag aatagtatat tgataacagg agacaagtct 1381 ctaattgtta aatgtatgcc ttacatgatt atttcagatg atgatatacg attcataaga 1441 gaacagtttg ttggtacaaa ttctattgag tatattctat ccttcatcaa caaggaaagc 58 4873-2549-9626.1Atty. Dkt. No.115872-3093 1501 atatatagaa tgagttacat ctagatgcat tcgcgaggta catcggatcc cgggcccgtc 1561 gactgcagag gcctgcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga 1621 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 1681 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 1741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 1801 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 1861 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 1921 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 1981 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 2041 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 2101 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 2161 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 2221 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 2281 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 2341 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 2401 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 2461 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 2521 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 59 4873-2549-9626.1Atty. Dkt. No.115872-3093 2581 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 2641 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 2701 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 2761 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 2821 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 2881 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 2941 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 3001 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 3061 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 3121 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 3181 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 3241 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 3301 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 3361 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 3421 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 3481 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 3541 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 3601 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 60 4873-2549-9626.1Atty. Dkt. No.115872-3093 3661 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 3721 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 3781 ttaacctata aaaataggcg tatcacgagg ccctttcgtc VIII. E5R

[0137] The cytosolic DNA sensor cGAS plays an important role in detecting viral nucleic acid, which leads to type I IFN production. It has been shown that infection of conventional dendritic cells with modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia strain, induces IFN production via a cGAS / STING-dependent mechanism. However, MVA infection triggers cGAS degradation. It has been shown that vaccinia E5R is a dominant inhibitor of cGAS and is the key protein mediating cGAS degradation. MVA∆E5R induces much higher levels of type I IFN than MVA in multiple cell types, including bone marrow derived dendritic cells (BMDC), bone marrow-derived macrophages (BMDM), and skin primary fibroblasts. MVA∆E5R-mediated type I IFN production is dependent on cGAS. Furthermore, MVA∆E5R gains replication capability in cGAS- / -skin fibroblasts. As a vaccine vector, skin scarification or intradermal vaccination with MVA∆E5R-OVA leads to much higher OVA-specific CD8+T cell responses than MVA-OVA in vivo. Intratumoral injection of MVA∆E5R leads to stronger anti-tumor immune responses and better survival compared with MVA. E5R, also identified as MVA052R, is a key viral virulence factor targeting the cytosolic DNA sensor cGAS and thereby inhibits type I IFN production.

[0138] An illustrative full-length vaccinia virus E5R host range protein, given by GenBank Accession No. AAB59825.1 (SEQ ID NO: 17) is provided below. MLILTKVNIYMLIIVLWLYGYNFIISESQCPMINDDSFTLKRKYQIDSAESTIKMDKKRTK FQNRAKMVKEINQTIRAAQTHYETLKLGYIKFKRMIRTTTLEDIAPSIPNNQKTYKLFSDI SAIGKASRNPSKMVYALLLYMFPNLFGDDHRFIRYRMHPMSKIKHKIFSPFKLNLIRILVE ERFYNNECRSNKWRIIGTQVDKMLIAESDKYTIDARYNLKPMYRIKGKSEEDTLFIKQMVE QCVTSQELVEKVLKILFRDLFKSGEYKAYRYDDDVENGFIGLDTLKLNIVHDIVEPCMPVR RPVAKILCKEMVNKYFENPLHIIGKNLQECIDFVSE 61 4873-2549-9626.1Atty. Dkt. No.115872-3093 IX. WR199

[0139] WR199 is a vaccinia viral early gene which has been identified as a virulence associated gene encoding a host range factor. WR199, also identified as MVA186R, encodes an ankyrin-repeat / F-box protein that has been shown to be a cGAS inhibitor. X. N1L and N2L

[0140] Vaccinia N1L encodes a 14-kDa virulence factor. The crystal structure of N1 protein shows striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. N1 binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 in vitro. N1 protein has been shown as an inhibitor of IRF3 and NF-kB. The N1L gene in MVA, also identified as MVA020L, is truncated and non-functional. MVA with reconstituted N1L from vaccinia has reduced capacities to induce type I IFN in bone marrow derived dendritic cells. Vaccinia N2L encodes a 175 amino acid virulence factor that belong to the Bcl-2 family. N2 prevents Ifnb gene promoter activation by inhibiting IRF3. Deleting N2L, also identified as MVA021L, from MVA improves MVA as a vaccine vector. An illustrative, non-limiting, plasmid sequence used to generate engineered or recombinant MVA viruses comprising a deletion of N1L is shown in Table 3. XI. C12L

[0141] Vaccinia C12L encodes a virulence factor that has amino acid similarity to IL-18 binding protein. The recombinant vaccinia C12 protein can inhibit mouse IL-18 function in vitro. MVA C12L is also identified as MVA008L. An illustrative, non-limiting, plasmid sequence used to generate engineered or recombinant MVA viruses comprising a deletion of the C12L is shown in Table 3. In some embodiments, IL-7 is inserted into the C12L locus of MVA (e.g., MVAΔE3LΔ5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hIL-7). In some embodiments, G-CSF is inserted into the C12L locus of MVA. Table 3. N1L, C12L Deletion and IL-7, G-CSF Expression Plasmids pUC57-MVA-dN1LpLEO160-hG-CSF (SEQ ID NO: 18) (FIG.36A) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 62 4873-2549-9626.1Atty. Dkt. No.115872-3093 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ttaattacat gttgttttat 421 atatcgctac gaatttaaac agagaaatca gtttaggaaa aaaaaatatc tatctacatc 481 atcacgtctc tgtattctac gatagagtgc tactttaaga tgagacatat ccgtgtcatc 541 aaaaatatac tccattaaaa tgattattcc ggcagcgaac ttgatattgg atatatcaca 601 acctttgtta atatctacga caatagacag cagtcccatg gttccataaa cagtgagttt 661 atctttcttt gaagagatat tttgtagaga tcttataaaa ctgtcgaatg acatcgcatt 721 tatatcttta gctaaatcgt atatgttacc atcgtaatat ctaaccgcgt ctatcttaaa 781 cgtttccatc gctttaaaga cgtttccgat agatggtctc atttcatcag tcatactgag 841 ccaacaaata taatcgtgta taacatcttt gatagaatca gactctaaag aaaacgaatc 901 ggctttatta tacgcattca tgataaactt aatgaaaaat gtttttcgtt gtttaagttg 961 gatgaatagt atgtcttaat aattgttatt atttcattaa ttaatattta gtaacgagta 1021 cactctataa aaacgagaat gacataacta gttatcaaag tgtaggttac aagtgacaat 1081 ccgtcggacg ttgcttttca cgtcgccttt taccacgacc cttcggacgt ctaggttgag 1141 caagatgtct caatactcta taggatactt ccaagaaact ttgtagatga cttgctacta 1201 atacaccacc agctcttctt tgaaacgcac tagcaaatgc gggcatcgct ccctgtgtcg 1261 gttggagcgc aggtgccata cctagttctt ccatttgctg ccagatagta gtcgcgaaat 1321 cggcgacatc tagttggagg gtatctaggg ttggtcctag ttctggagaa attccctcta 1381 aggcttggag aagaccttga tacaagaata gtccggaatg caattgcgac aaacatcccg 1441 ccagttgcaa cgcttgggat gggcaggaag agagaggtgc ccaaggaatt cctaaagaat 1501 gacctagtag tactaattct tccggatggc atagtttata agtagcgcac tctgatacca 1561 atttttcttg tagagccgct ccgtcacctt gaatttttct aacctgctcc agacacttta 1621 gaagaaacga ctgtggtaaa gatgatgcag gtcccagagg tgtcgcttct tgtactgtcc 1681 acaaagcaga atgccataac aagagctgta atgccattaa tttcatcgga gactgagtag 1741 ccggtcctgc catctcgagg aattcggtac cccgggttcg aaatcgataa gcttggatcc 1801 ggagagctcc caacctcgag gaattcggta ccccgggttc gaaatcgata agcttggatc 1861 cggagagctc ccaacctcga gctagcgtct gaaacgagac gctaattagt gtatattttt 1921 tcaattttac cggatattta tattccaaaa aaaaaaaata aaaggcctga ttagttaaca 1981 actatttttt atgttaaatc aattagtaca ccgctatgtt taatacttat tcatatttta 2041 gtttttagga ttgagaatca atacaaaaat taatgcatca ttaattttag aaatacttag 2101 tttccacgta gtcaatgaaa catttgaact catcgtacag gacgttctcg tacaggacgt 2161 aactataaac cggtttatat ttgttcaaga tagatacaaa tccgataact ttttttacga 2221 attctacggg atccacttta aaagtgtcat accgggttct ttttattttt ttaaacagat 2281 taatggtgtg atgttgatta ggtcttttac gaatttgata tagaatagcg tttacatatt 2341 ctccataatg gtcaatcgcc atttgttcgt atgtcataaa ttctttaatt atatgacact 2401 gtgtattatt tagttcatcc ttgttcatca ttaggaatct atccaatatg gcaattatac 2461 tagaactata ggtgcgttgt atacacatat tgatgtgtct gtttatacaa tccatgctac 2521 taccttcggg taaaattgta gcatcatata ccatttctag tactttaggt tcattgttat 2581 ccattgcaga ggacgtcatg atcgcatcct aaaaaaatat attattttta tgttattttg 2641 ttaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 63 4873-2549-9626.1Atty. Dkt. No.115872-3093 2701 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 2761 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 2821 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 2881 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 2941 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 3001 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 3061 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 3121 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 3181 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 3241 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 3301 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 3361 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 3421 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 3481 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 3541 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 3601 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 3661 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 3721 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 3781 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 3841 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 3901 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 3961 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 4021 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 4081 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 4141 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 4201 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 4261 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 4321 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 4381 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 4441 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 4501 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 4561 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 4621 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 4681 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 4741 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 4801 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 4861 cgtatcacga ggccctttcg tc pUC57-MVA-ΔC12-hIL7 (SEQ ID NO: 19) (FIG.35A) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 64 4873-2549-9626.1Atty. Dkt. No.115872-3093 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgccggaagg gactatatga 421 ctaacttatc tagagataca ggaatacaac aatcaaaact tactgaaact gtcaaaaaaa 481 tagaaacata tatggtctat atatacacta caatttagtt attaattgga taaccgatgt 541 gattatcaat caatattaag aaggttggta aattggtaca tagctaataa tacctataca 601 cccaataata caacaaccat ttctgagttg gatatcatca aaatactgga taaatacgag 661 gacgtgtata gagtaagtaa agaaaaagaa tgtgaaattt gctatgaagt tgtttactca 721 aaacgataga tactttggtt tattggattc gtgtaatcat atattttgca taacatgcat 781 caatatatgg catagaacac gaagagaaac cggtgcgtcg gataattgtc ctatatgtcg 841 tacccgtttt agaaacataa caatgagcaa gttaactaat aaataaaaag tttaatttgt 901 tgacgacgta tgtcgttatt ttttctcgta taaaagatta atttgattct aatataatct 961 ttagtattgg ataaatatca attcaaatta attccattag attatatcat aaataaaaat 1021 agtagcacgc aaggctataa atgacagtca gttggacgtt gtttctcccg tcttctttta 1081 ccccttccct ttggtctacg atgttctttt gtgcccatca aaattttatt ccaacaagtt 1141 ttgatttcct gtaacaatct tttcaagaaa cacaaatcgt tcaatttttt ttgctccttc 1201 agtgacttat tttcttcaag ggattttgtc ggttgggctt ctcccaaagc agcgggtttc 1261 ctacccttaa cttgacctgt gcagttcagt aggatggtcg ttccttctga tactttcaac 1321 aaatgcaaat cgaaatctcc ggtggagttc atctttagaa attgtcttag cttccgcgct 1381 gcccgaaaaa ggaacatacc ctctttattg gcatcacata tatgtcgctt gaaaaaatta 1441 aactcattgt taagacaatt ggacccaatt tctttcatac tgtctaacag ttggtcaata 1501 gaaaccataa gcactgattc atactgtttt ccatcctttc cttcgatatc acaatcactg 1561 ctggcaacag gtagcagtac caaaattagc ggcggcaggc cgaagatata tctaaatgac 1621 acatgaaaca tttatgatct acttccttac cgtgcaataa attagaatat attttctact 1681 tttacgagaa attaattatt gtatttatta tttatgggtg aaaaacttac tataaaaagc 1741 gggtgggttt ggaattagtg aggcctcttg ccaaaatatc actagaaaaa atttatttat 1801 caattttaaa ggtataaaaa atacttattg ttgctcgaat attttgtatt tgatggtata 1861 cggaagatta gaaatgtagg tattatcatc aactgattct atggttttat gtattctatc 1921 atgtttcact attgcgttgg aaataatatc atatgcttcc acatatattt tattttgttt 1981 taactcataa tactcacgta attctggatt attgacatat ctatgaataa ttttagctcc 2041 atgatcagta aatattaatg agaacatagt attaccacct accattattt ttttcatctc 2101 attcaattct taattgcaaa gatctatata atcattatag cgttgactta tggactctgg 2161 aatcttagac gatgtacagt catctataat catggcatat ttaatacatt gttttatagc 2221 atagtcgtta tctacgatgt tagatatttc tctcaatgaa tcaatcacac aatctaatgt 2281 aggtttatga cataatagca ttttcagcag ttcaatgttt ttagattcgt tgatggcaat 2341 ggctatacat gtatatccgt tatttgatct aatgttgaca tctgaaccgg atagcttggc 2401 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 2461 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 65 4873-2549-9626.1Atty. Dkt. No.115872-3093 2521 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 2581 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 2641 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 2701 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 2761 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 2821 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 2881 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2941 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 3001 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 3061 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 3121 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 3181 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 3241 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 3301 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 3361 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 3421 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 3481 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 3541 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 3601 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 3661 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 3721 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 3781 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 3841 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3901 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3961 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 4021 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 4081 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 4141 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 4201 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 4261 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 4321 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 4381 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 4441 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 4501 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 4561 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 4621 gccctttcgt c pUC57-delC12L-hG-CSF (SEQ ID NO: 6) (FIG.35B) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 66 4873-2549-9626.1Atty. Dkt. No.115872-3093 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgccggaagg gactatatga 421 ctaacttatc tagagataca ggaatacaac aatcaaaact tactgaaact gtcaaaaaaa 481 tagaaacata tatggtctat atatacacta caatttagtt attaattgga taaccgatgt 541 gattatcaat caatattaag aaggttggta aattggtaca tagctaataa tacctataca 601 cccaataata caacaaccat ttctgagttg gatatcatca aaatactgga taaatacgag 661 gacgtgtata gagtaagtaa agaaaaagaa tgtgaaattt gctatgaagt tgtttactca 721 aaacgataga tactttggtt tattggattc gtgtaatcat atattttgca taacatgcat 781 caatatatgg catagaacac gaagagaaac cggtgcgtcg gataattgtc ctatatgtcg 841 tacccgtttt agaaacataa caatgagcaa gttaactaat aaataaaaag tttaatttgt 901 tgacgacgta tgtcgttatt ttttctcgta taaaagatta atttgattct aatataatct 961 ttagtattgg ataaatatca attcaaatta attccattag attatatcat aaataaaaat 1021 agtagcacgc aaggttacaa gtgacaatcc gtcggacgtt gcttttcacg tcgcctttta 1081 ccacgaccct tcggacgtct aggttgagca agatgtctca atactctata ggatacttcc 1141 aagaaacttt gtagatgact tgctactaat acaccaccag ctcttctttg aaacgcacta 1201 gcaaatgcgg gcatcgctcc ctgtgtcggt tggagcgcag gtgccatacc tagttcttcc 1261 atttgctgcc agatagtagt cgcgaaatcg gcgacatcta gttggagggt atctagggtt 1321 ggtcctagtt ctggagaaat tccctctaag gcttggagaa gaccttgata caagaatagt 1381 ccggaatgca attgcgacaa acatcccgcc agttgcaacg cttgggatgg gcaggaagag 1441 agaggtgccc aaggaattcc taaagaatga cctagtagta ctaattcttc cggatggcat 1501 agtttataag tagcgcactc tgataccaat ttttcttgta gagccgctcc gtcaccttga 1561 atttttctaa cctgctccag acactttaga agaaacgact gtggtaaaga tgatgcaggt 1621 cccagaggtg tcgcttcttg tactgtccac aaagcagaat gccataacaa gagctgtaat 1681 gccattaatt tcatcggaga ctgagtagcc ggtcctgcca tttatgatct acttccttac 1741 cgtgcaataa attagaatat attttctact tttacgagaa attaattatt gtatttatta 1801 tttatgggtg aaaaacttac tataaaaagc gggtgggttt ggaattagtg aggcctcttg 1861 ccaaaatatc actagaaaaa atttatttat caattttaaa ggtataaaaa atacttattg 1921 ttgctcgaat attttgtatt tgatggtata cggaagatta gaaatgtagg tattatcatc 1981 aactgattct atggttttat gtattctatc atgtttcact attgcgttgg aaataatatc 2041 atatgcttcc acatatattt tattttgttt taactcataa tactcacgta attctggatt 2101 attgacatat ctatgaataa ttttagctcc atgatcagta aatattaatg agaacatagt 2161 attaccacct accattattt ttttcatctc attcaattct taattgcaaa gatctatata 2221 atcattatag cgttgactta tggactctgg aatcttagac gatgtacagt catctataat 2281 catggcatat ttaatacatt gttttatagc atagtcgtta tctacgatgt tagatatttc 2341 tctcaatgaa tcaatcacac aatctaatgt aggtttatga cataatagca ttttcagcag 2401 ttcaatgttt ttagattcgt tgatggcaat ggctatacat gtatatccgt tatttgatct 2461 aatgttgaca tctgaaccgg atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 2521 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 67 4873-2549-9626.1Atty. Dkt. No.115872-3093 2581 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 2641 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 2701 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 2761 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 2821 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 2881 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 2941 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 3001 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 3061 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 3121 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 3181 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 3241 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 3301 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 3361 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 3421 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 3481 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 3541 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 3601 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 3661 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 3721 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 3781 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 3841 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 3901 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 3961 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 4021 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 4081 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 4141 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 4201 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 4261 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 4321 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 4381 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 4441 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 4501 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 4561 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 4621 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac 4681 attaacctat aaaaataggc gtatcacgag gccctttcgt c pUC57-MVA-delta-N1L-hIL-7-tGFP (SEQ ID NO: 28) (FIG.36B) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 68 4873-2549-9626.1Atty. Dkt. No.115872-3093 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgccgttaat tacatgttgt 421 tttatatatc gctacgaatt taaacagaga aatcagttta ggaaaaaaaa atatctatct 481 acatcatcac gtctctgtat tctacgatag agtgctactt taagatgaga catatccgtg 541 tcatcaaaaa tatactccat taaaatgatt attccggcag cgaacttgat attggatata 601 tcacaacctt tgttaatatc tacgacaata gacagcagtc ccatggttcc ataaacagtg 661 agtttatctt tctttgaaga gatattttgt agagatctta taaaactgtc gaatgacatc 721 gcatttatat ctttagctaa atcgtatatg ttaccatcgt aatatctaac cgcgtctatc 781 ttaaacgttt ccatcgcttt aaagacgttt ccgatagatg gtctcatttc atcagtcata 841 ctgagccaac aaatataatc gtgtataaca tctttgatag aatcagactc taaagaaaac 901 gaatcggctt tattatacgc attcatgata aacttaatga aaaatgtttt tcgttgttta 961 agttggatga atagtatgtc ttaataattg ttattatttc attaattaat atttagtaac 1021 gagtacactc tataaaaacg agaatgacat aactagttat caaagtgtga acttatctag 1081 agatggctat aaatgacagt cagttggacg ttgtttctcc cgtcttcttt taccccttcc 1141 ctttggtcta cgatgttctt ttgtgcccat caaaatttta ttccaacaag ttttgatttc 1201 ctgtaacaat cttttcaaga aacacaaatc gttcaatttt ttttgctcct tcagtgactt 1261 attttcttca agggattttg tcggttgggc ttctcccaaa gcagcgggtt tcctaccctt 1321 aacttgacct gtgcagttca gtaggatggt cgttccttct gatactttca acaaatgcaa 1381 atcgaaatct ccggtggagt tcatctttag aaattgtctt agcttccgcg ctgcccgaaa 1441 aaggaacata ccctctttat tggcatcaca tatatgtcgc ttgaaaaaat taaactcatt 1501 gttaagacaa ttggacccaa tttctttcat actgtctaac agttggtcaa tagaaaccat 1561 aagcactgat tcatactgtt ttccatcctt tccttcgata tcacaatcac tgctggcaac 1621 aggtagcagt accaaaatta gcggcggcag gccgaagata tatctaaatg acacatgaaa 1681 catttatgat ctacttcctt accgtgcaat aaattagaat atattttcta cttttacgag 1741 aaattaatta ttgtatttat tatttatggg tgaaaaactt actataaaaa gcgggtgggt 1801 ttggaattag tgaggttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa 1861 tcatgcgtaa aattgacgca tgggatcagc ttaaaaattg aaattttatt tttttttttt 1921 ggaatataaa taagctcgaa gtcgacatgg agagcgacga gagcggcctg cccgccatgg 1981 agatcgagtg ccgcatcacc ggcaccctga acggcgtgga gttcgagctg gtgggcggcg 2041 gagagggcac ccccgagcag ggccgcatga ccaacaagat gaagagcacc aaaggcgccc 2101 tgaccttcag cccctacctg ctgagccacg tgatgggcta cggcttctac cacttcggca 2161 cctaccccag cggctacgag aaccccttcc tgcacgccat caacaacggc ggctacacca 2221 acacccgcat cgagaagtac gaggacggcg gcgtgctgca cgtgagcttc agctaccgct 2281 acgaggccgg ccgcgtgatc ggcgacttca aggtgatggg caccggcttc cccgaggaca 2341 gcgtgatctt caccgacaag atcatccgca gcaacgccac cgtggagcac ctgcacccca 2401 tgggcgataa cgatctggat ggcagcttca cccgcacctt cagcctgcgc gacggcggct 2461 actacagctc cgtggtggac agccacatgc acttcaagag cgccatccac cccagcatcc 2521 tgcagaacgg gggccccatg ttcgccttcc gccgcgtgga ggaggatcac agcaacaccg 69 4873-2549-9626.1Atty. Dkt. No.115872-3093 2581 agctgggcat cgtggagtac cagcacgcct tcaagacccc ggatgcagat gccggtgaag 2641 aataaaggcc tgaaaccgga tagctgatta gttaacaact attttttatg ttaaatcaat 2701 tagtacaccg ctatgtttaa tacttattca tattttagtt tttaggattg agaatcaata 2761 caaaaattaa tgcatcatta attttagaaa tacttagttt ccacgtagtc aatgaaacat 2821 ttgaactcat cgtacaggac gttctcgtac aggacgtaac tataaaccgg tttatatttg 2881 ttcaagatag atacaaatcc gataactttt tttacgaatt ctacgggatc cactttaaaa 2941 gtgtcatacc gggttctttt tattttttta aacagattaa tggtgtgatg ttgattaggt 3001 cttttacgaa tttgatatag aatagcgttt acatattctc cataatggtc aatcgccatt 3061 tgttcgtatg tcataaattc tttaattata tgacactgtg tattatttag ttcatccttg 3121 ttcatcatta ggaatctatc caatatggca attatactag aactataggt gcgttgtata 3181 cacatattga tgtgtctgtt tatacaatcc atgctactac cttcgggtaa aattgtagca 3241 tcatatacca tttctagtac tttaggttca ttgttatcca ttgcagagga cgtcatgatc 3301 gcatcctaaa aaaatatatt atttttatgt tattttgtta agtggcgtaa tcatggtcat 3361 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 3421 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 3481 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 3541 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 3601 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3661 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3721 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 3781 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 3841 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 3901 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 3961 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4021 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4081 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 4141 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 4201 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 4261 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 4321 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 4381 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 4441 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 4501 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 4561 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 4621 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 4681 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 4741 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 4801 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 4861 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 4921 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 70 4873-2549-9626.1Atty. Dkt. No.115872-3093 4981 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 5041 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 5101 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 5161 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 5221 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 5281 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 5341 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 5401 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 5461 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 5521 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc XII. G-CSF

[0142] G-CSF or granulocyte-colony stumlating factor is known to stimulate bone marrow production of multiple immune cell types. A non-limiting exemplary DNA sequence for G-CSF is (SEQ ID NO: 20) and a non-limiting exemplary amino acid sequence for G-CSF (SEQ ID NO: 21). Illustrative, non-limiting, plasmid sequences used to generate engineered or recombinant MVA viruses comprising a G-CSF transgene are shown in Table 3 above. Human G-CSF (amino acid sequence): MAGPATQSPMKLMALQLLLWHSALWTVQEATPLGPASSLPQSFLLKCLEQVRKIQ GDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWALSSCPSQALQLAGCLSQLH SGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQG AMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQPRRPKGRGKRRREKQRP TDCHL* (SEQ ID NO: 21) Human G-CSF (DNA sequence): Atggcaggaccggctactcagtctccgatgaaattaatggcattacagctcttgttatggcattctgctttgtggacagtacaagaagc gacacctctgggacctgcatcatctttaccacagtcgtttcttctaaagtgtctggagcaggttagaaaaattcaaggtgacggagcgg ctctacaagaaaaattggtatcagagtgcgctacttataaactatgccatccggaagaattagtactactaggtcattctttaggaattcc ttgggcacctctctcttcctgcccatcccaagcgttgcaactggcgggatgtttgtcgcaattgcattccggactattcttgtatcaaggt cttctccaagccttagagggaatttctccagaactaggaccaaccctagataccctccaactagatgtcgccgatttcgcgactactat ctggcagcaaatggaagaactaggtatggcacctgcgctccaaccgacacagggagcgatgcccgcatttgctagtgcgtttcaaa gaagagctggtggtgtattagtagcaagtcatctacaaagtttcttggaagtatcctatagagtattgagacatcttgctcaacctagac gtccgaagggtcgtggtaaaaggcgacgtgaaaagcaacgtccgacggattgtcacttgtaa (SEQ ID NO: 20) 71 4873-2549-9626.1Atty. Dkt. No.115872-3093 XIII. IL-7

[0143] IL-7 or interleukin 7 belongs to a family of hematopoietic growth factors. IL-7 is known to promote the differentiation of hematopoietic stem cells into lymphoid progenitor cells and to promote the development of T cells and B cells. A non-limiting exemplary DNA sequence for IL-7 is (SEQ ID NO: 22) and a non-limiting exemplary amino acid sequence for IL-7 (SEQ ID NO: 23). Illustrative, non-limiting, plasmid sequences used to generate engineered or recombinant MVA viruses comprising an IL-7 transgene are shown in Table 3 above. Human IL-7 (amino acid sequence): MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSN CLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTIL LNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKI LMGTKEHRRPKGRGKRRREKQRPTDCHL* (SEQ ID NO: 23) Human IL-7 (DNA sequence): Atgtttcatgtgtcatttagatatatcttcggcctgccgccgctaattttggtactgctacctgttgccagcagtgattgtgatatcgaagg aaaggatggaaaacagtatgaatcagtgcttatggtttctattgaccaactgttagacagtatgaaagaaattgggtccaattgtcttaa caatgagtttaattttttcaagcgacatatatgtgatgccaataaagagggtatgttcctttttcgggcagcgcggaagctaagacaattt ctaaagatgaactccaccggagatttcgatttgcatttgttgaaagtatcagaaggaacgaccatcctactgaactgcacaggtcaagt taagggtaggaaacccgctgctttgggagaagcccaaccgacaaaatcccttgaagaaaataagtcactgaaggagcaaaaaaaa ttgaacgatttgtgtttcttgaaaagattgttacaggaaatcaaaacttgttggaataaaattttgatgggcacaaaagaacatcgtagac caaagggaaggggtaaaagaagacgggagaaacaacgtccaactgactgtcatttatag (SEQ ID NO: 22) XIV. Engineered Poxvirus Strains of the Present Technology

[0144] Illustrative, non-limiting examples of OX40L and hFlt3L expression constructs according to the present technology are shown in Table 4. Table 4. OX40L and hFlt3L expression constructs Exemplary nucleotide sequences for the open reading frames of the recombinant MVA constructs of the present technology. pCB-mOX40L-gpt vector nucleic acid sequence (SEQ ID NO: 9): CGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCTTTTTATCTGCGCGGTTAA CCGCCTTTTTATCCATCAGGTGATCTGTTTTTATTGTGGAGTCTAGATTATCACAGTGG GACTTGGTTCACTGTACTGTGATAACTGCCCTCAGGAGCACAGTAACCTGGTGTGAG TTGGACAACGATAAGTTCTCCATCGTTGATTTGCAAATGCTCGCACAAGGTATCAGG AGCGTTTACGGTCAAGTACACCTTATCCTTGAAGGCCAGTGAAGCGACTACAGTAAA 72 4873-2549-9626.1Atty. Dkt. No.115872-3093 GACGATTCTTCTGCCGTCGTTCAACATAGGGATTGAGATAGGGTTGTGATCCTCCCTG AAGTGGAGGTCAATTTTGACCTCCTGGAAAAAAGATCCTTTCAAGTAGATGATGTAC AGTCCGTCACACTTAATCACTACGGAGTTATTCTGGACTTCCATCGTCTGATACTCGT TCTTGTAAGAAGAAATAAACAATTGACCATCTTCACAGCGGGTCACAGCCCCCCGAA GGCGTTGTATGGGGGGGTCTTTTGCAGGTGAAGAGCTAAGTTGCAAACACACATATA TGAAGCACAGCAACATACCGGCCCCCTTGATGCCGCTTACAACGAGCCTAAGAGTCT TCTTCCATTTAAAGCGAGGGCGAGACCCGTTTTCGAGGTTCTCGTCCAGAGGCTGGA CCCCCTCGCCCTCCATGGTGGTGGCCTAGAATTCGATATCAAGCTCAGGCCTAGATCT GTCGACTTCGAGCTTATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTTAAGC TTTCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATA AATACAATAATTAATTTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTA AGGAAGTAGATCATAACTCGAGGAATTGGGGATCTCTATAATCTCGCGCAACCTATT TTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGGCTGGGACACTTCACATGAGC GAAAAATACATCGTCACCTGGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGC CGACTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTGGCGGTCTG GTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTCGTCATGTCGATACCGTTTGTA TTTCCAGCTACGATCACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAG GCGATGGCGAAGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGCGG TTGCGATTCGTGAAATGTATCCAAAAGCGCACTTTGTCACCATCTTCGCAAAACCGGC TGGTCGTCCGCTGGTTGATGACTATGTTGTTGATATCCCGCAAGATACCTGGATTGAA CAGCCGTGGGATATGGGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTAATCTTTTC AACGCCTGGCACTGCCGGGCGTTGTTCTTTTTAACTTCAGGCGGGTTACAATAGTTTC CAGTAAGTATTCTGGAGGCTGCATCCATGACACAGGCAAACCTGCGGATCCCAGCTT TTGTTCCCTTTAGTGAGGGTTAATTGCGCGCAGTTATAGTAGCCGCACTCGATGGGAC ATTTCAACGTAAACCGTTTAATAATATTTTGAATCTTATTCCATTATCTGAAATGGTG GTAAAACTAACTGCTGTGTGTATGAAATGCTTTAAGGAGGCTTCCTTTTCTAAACGAT TGGGTGAGGAAACCGAGATAGAAATAATAGGAGGTAATGATATGTATCAATCGGTG TGTAGAAAGTGTTACATCGACTCATAATATTATATTTTTTATCTAAAAAACTAAAAAT AAACATTGATTAAATTTTAATATAATACTTAAAAATGGATGTTGTGTCGTTAGATAAA CCGTTTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAGAAAGTGCA AATGAGGTCGCAAAAAAACTGCCGTATCAAGGACAGTTAAAACTATTACTAGGAGA ATTATTTTTTCTTAGTAAGTTACAGCGACACGGTATATTAGATGGTGCCACCGTAGTG TATATAGGATCTGCTCCCGGTACACATATACGTTATTTGAGAGATCATTTCTATAATT TAGGAGTGATCATCAAATGGATGCTAATTGACGGCCGCCATCATGATCCTATTTTAA ATGGATTGCGTGATGTGACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATC CATCAAAAAACAACTGCATCCTTCTAAGATTATTTTAATTTCTGATGTGAGATCCAAA CGAGGAGGAAATGAACCTAGTACGGCGGATTTACTAAGTAATTACGCTCTACAAAAT GTCATGATTAGTATTTTAAACCCCGTGGCGTCTAGTCTTAAATGGAGATGCCCGTTTC CAGATCAATGGATCAAGGACTTTTATATCCCACACGGTAATAAAATGTTACAACCTTT TGCTCCTTCATATTCAGCTGAAATGAGATTATTAAGTATTTATACCGGTGAGAACATG AGACTGACTCGGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGC ATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTC ACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTT TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTT AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCC 73 4873-2549-9626.1Atty. Dkt. No.115872-3093 CCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA TGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAG CCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTA TTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACG TTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAA AGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTA TCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCG ACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAAC TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCA AAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTA TTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG ACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGA GGCCCTTTCGTCTTCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATA AATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGA GACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTA CCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAG AAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACAT TTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGAGCTTTT GGGATCAATAAATGGATCACAACCAGTATCTCTTAACGATGTTCTTCGCAGATGATG ATTCATTTTTTAAGTATTTGGCTAGTCAAGATGATGAATCTTCATTATCTGATATATTG CAAATCACTCAATATGTAGCTAGACTTTCTGTTATTATTATTGATCCAATCAAAAAAT AAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACAGACAATTG ACAAAATTCACAGACTTTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTATTGAC AGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTTGATG CGATTCAAAAAAGAATCCTCTCTAGCTACCACCGCAATAGATCCTGTTAGATACATA GATCCTCGTCGCAATATCGCATTTTCTAACGTGATGGATATATTAAAGTCGAATAAAG TGAACAATAATTAATTCTTTATTGTCATCATGAACGGCGGACATATTCAGTTGATAAT CGGCCCCATGTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCA AATAGCTCAATATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAAC GGGACTATGGACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATG TGATCTCTTGGAATCAATTACAGATTTCTCCGTGATAGG pUC57-hFlt3L-GFP nucleic acid sequence (also referred to as pUC57-P501-GFP) (SEQ ID NO: 10): 1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG 51 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG 101 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG 151 CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA 201 CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT 251 CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA 351 ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT 401 CATATTACGA TGGTAACATA TACGATTTAG CTAAAGATAT AAATGCGATG 451 TCATTCGACA GTTTTATAAG ATCTCTACAA AATATCTCTT CAAAGAAAGA 501 TAAACTCACT GTTTATGGAA CCATGGGACT GCTGTCTATT GTCGTAGATA 551 TTAACAAAGG TTGTGATATA TCCAATATCA AGTTCGCTGC CGGAATAATC 601 ATTTTAATGG AGTATATTTT TGATGACACG GATATGTCTC ATCTTAAAGT 651 AGCACTCTAT CGTAGAATAC AGAGACGTGA TGATGTAGAT AGATATTTTT 701 TTTTCCTAAA CTGATTTCTC TGTTTAAATT CGTAGCGATA TATAAAACAA 751 CATGTAATTA ATTAATAAAC TTTAAGACAT GTGTGTTATA CTAAGATGGT 801 TGGCTTATTC CATAGTAGCT TGTGGAATTT ATAAACTTAT GATAGTAAAA 851 CTAGTACCCA ATATGTAAAG ATGAAAAAGT AAATTACTAT TAACGCCGTC 74 4873-2549-9626.1Atty. Dkt. No.115872-3093 901 GGTATTCGTT CATCCATTCA GTAAGCTTAA AAATTGAAAT TTTATTTTTT 951 TTTTTTGGAA TATAAATAAG CTCGAAGTCG ACGAATTCAT GACAGTGCTG 1001 GCGCCAGCCT GGAGCCCAAC AACCTATCTC CTCCTGCTGC TGCTGCTGAG 1051 CTCGGGACTC AGTGGGACCC AGGACTGCTC CTTCCAACAC AGCCCCATCT 1101 CCTCCGACTT CGCTGTCAAA ATCCGTGAGC TGTCTGACTA CCTGCTTCAA 1151 GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG 1201 GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA 1251 CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGGAG 1301 ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG 1351 CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC 1401 TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG 1451 GAGCTGCAGT GTCAGCCCGA CTCCTCAACC CTGCCACCCC CATGGAGTCC 1501 CCGGCCCCTG GAGGCCACAG CCCCGACAGC CCCGCAGCCC CCTCTGCTCC 1551 TCCTACTGCT GCTGCCCGTG GGCCTCCTGC TGCTGGCCGC TGCCTGGTGC 1601 CTGCACTGGC AGAGGACGCG GCGGAGGACA CCCCGCCCTG GGGAGCAGGT 1651 GCCCCCCGTC CCCAGTCCCC AGGACCTGCT GCTTGTGGAG CACTGACTCG 1701 AGTTTACTTG TACAGCTCGT CCATGCCGAG AGTGATCCCG GCGGCGGTCA 1751 CGAACTCCAG CAGGACCATG TGATCGCGCT TCTCGTTGGG GTCTTTGCTC 1801 AGGGCGGACT GGGTGCTCAG GTAGTGGTTG TCGGGCAGCA GCACGGGGCC 1851 GTCGCCGATG GGGGTGTTCT GCTGGTAGTG GTCGGCGAGC TGCACGCTGC 1901 CGTCCTCGAT GTTGTGGCGG ATCTTGAAGT TCACCTTGAT GCCGTTCTTC 1951 TGCTTGTCGG CCATGATATA GACGTTGTGG CTGTTGTAGT TGTACTCCAG 2001 CTTGTGCCCC AGGATGTTGC CGTCCTCCTT GAAGTCGATG CCCTTCAGCT 2051 CGATGCGGTT CACCAGGGTG TCGCCCTCGA ACTTCACCTC GGCGCGGGTC 2101 TTGTAGTTGC CGTCGTCCTT GAAGAAGATG GTGCGCTCCT GGACGTAGCC 2151 TTCGGGCATG GCGGACTTGA AGAAGTCGTG CTGCTTCATG TGGTCGGGGT 2201 AGCGGCTGAA GCACTGCACG CCGTAGGTCA GGGTGGTCAC GAGGGTGGGC 2251 CAGGGCACGG GCAGCTTGCC GGTGGTGCAG ATGAACTTCA GGGTCAGCTT 2301 GCCGTAGGTG GCATCGCCCT CGCCCTCGCC GGACACGCTG AACTTGTGGC 2351 CGTTTACGTC GCCGTCCAGC TCGACCAGGA TGGGCACCAC CCCGGTGAAC 2401 AGCTCCTCGC CCTTGCTCAC CATGGTACCA GGCCTAGATC TGTCGACTTC 2451 GAGCTTATTT ATATTCCAAA AAAAAAAAAT AAAATTTCAA TTTTTCTCGA 2501 GTATGAGTAT AGTGTTAAAT GACACTTACT AAATAGCCAA GGTGATTATT 2551 CGTATTTTTT TAAGGAGTAA CCATGTCCGC AATTAGATTT ATTGCATGTC 2601 TATATCTCAT TTCCATCTTC GGAAATTGTC ATGAGGATCC ATATTATCAA 2651 CCATTTGATA AATTAAACAT TACTCTAGAT ATATACACTT ATGAGGATCT 2701 AGTACCATAC ACCGTAGACA ATGACACAAC TTCTTTCGTT AAGATATACT 2751 TTAAAAATTT TTGGATTACG GTTATGACTA AATGGTGTGC TCCGTTTATT 2801 GATACCGTTA GCGTATACAC ATCTCATGAT AATCTGAATA TACAATTTTA 2851 TAGTAGGGAC GAATATGATA CACAAAGCGA GGATAAAATT TGTACCATTG 2901 ATGTTAAAGC ACGATGCAAA CATCTAACAA AACGAGAAGT TACAGTACAA 2951 CAAGAAGCCT ACAGATAATC TAGATGCATT CGCGAGGTAC CGAATCGGAT 3001 CCCGGGCCCG TCGACTGCAG AGGCCTGCAT GCAAGCTTGG CGTAATCATG 3051 GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA 3101 ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG 3151 AGCTAACTCA CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG 3201 AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG 3251 GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG 3301 CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT 3351 AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG 3401 CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG 3451 TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC 3501 AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC 3551 CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC 3601 GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG 3651 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG 3701 GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT 3751 AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC 3801 AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA 3851 CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGAACAGTA 3901 TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG 75 4873-2549-9626.1Atty. Dkt. No.115872-3093 3951 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG 4001 TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT 4051 TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA 4101 AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT 4151 TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT 4201 TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 4251 CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA 4301 CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG 4351 CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC 4401 CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC 4451 AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT 4501 AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC 4551 GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG 4601 TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT 4651 CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT 4701 GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT 4751 CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG 4801 CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA 4851 TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA 4901 AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT 4951 CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG 5001 GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA 5051 CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC 5101 ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA 5151 GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC 5201 CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG 5251 CGTATCACGA GGCCCTTTCG TC

[0145] An illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 1) given by GenBank Accession No. U94848.1 is provided in FIG.27. An illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 7) given by GenBank Accession No. AY603355 is provided in FIG.37. MQ710

[0146] The disclosure of the present technology relates to modified vaccinia Ankara (MVA) viruses referred to as “rMVA” or “MQ710” or “MVAΔE5R-hFlt3L-OX40L,” which may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, and a mutation of the E5R gene. In some embodiments, the present technology relates to an immunogenic composition comprising MQ710. In some embodiments, both of the transgenes are inserted into the E5R locus. In some embodiments, Flt3L and OX40L genes are linked by a P2A self-cleaving sequence and their expression is driven by the vaccinia synthetic early-late promoter. In some embodiments, plasmid constructs were used to create MQ710 via homologous recombination with the MVA genome. In some embodiments, illustrative, non-limiting, plasmid constructs used to create MQ710 include those shown in Table 5. The plasmid maps are shown in FIGs.30 and 31. 76 4873-2549-9626.1Atty. Dkt. No.115872-3093 Table 5. Exemplary nucleotide sequences for the MQ710 constructs of the present technology. pMA-MVA-ΔE5R-hFlt3L-OX40L vector nucleic acid sequence (SEQ ID NO: 4): 1 CTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC 61 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA 121 GATAGGGTTG AGTGGCCGCT ACAGGGCGCT CCCATTCGCC ATTCAGGCTG CGCAACTGTT 181 GGGAAGGGCG TTTCGGTGCG GGCCTCTTCG CTATTACGCC AGCTGGCGAA AGGGGGATGT 241 GCTGCAAGGC GATTAAGTTG GGTAACGCCA GGGTTTTCCC AGTCACGACG TTGTAAAACG 301 ACGGCCAGTG AGCGCGACGT AATACGACTC ACTATAGGGC GAATTGGCGG AAGGCCGTCA 361 AGGCCGCATT GGAGGTTCGT CAGCGGCTCT AGTTTGAATC ATCATCGGCG TAGTATTCCT 421 ACTTTTACAG TTAGGACACG GTGTATTGTA TTTCTCGTCG AGAACGTTAA AATAATCGTT 481 GTAACTCACA TCCTTTATTT TATCTATATT GTATTCTACT CCTTTCTTAA TGCATTTTAT 541 ACCGAATAAG AGATAGCGAA GGAATTCTTT TTCGGTGCCG CTAGTACCCT TAATCATATC 601 ACATAGTGTT TTATATTCCA AATTTGTGGC AATAGACGGT TTATTTCTAT ACGATAGTTT 661 GTTTCTGGAA TCCTTTGAGT ATTCTATACC AATATTATTC TTTGATTCGA ATTTAGTTTC 721 TTCGATATTA GATTTTGTAT TACCTATATT CTTGATGTAG TACTTTGATG ATTTTTCCAT 781 GGCCCATTCT ATTAAGTCTT CCAAGTTGGC ATCATCCACA TATTGTGATA GTAATTCTCG 841 GATATCAGTA GCGGCTACCG CCATTGATGT TTGTTCATTG GATGAGTAAC TACTAATGTA 901 TACATTTTCC ATTTATAACA CTTATGTATT AACTTTGTTC ATTTATATTT TTTCATTATT 961 ATGTTGATAT TAACAAAAGT GAATATATGG ATCCAAAAAT TGAAATTTTA TTTTTTTTTT 1021 TTGGAATATA AATAAGCTCG AAGTCGACGA ATTCATGACA GTACTAGCTC CAGCTTGGTC 1081 CCCGACAACA TACCTTCTAC TACTACTATT GCTATCCTCC GGACTATCTG GAACCCAGGA 1141 TTGCTCTTTT CAGCACTCTC CGATCTCGTC TGATTTCGCG GTTAAGATCA GAGAGCTATC 1201 CGACTACTTG CTACAGGATT ACCCAGTAAC CGTCGCGTCC AACCTACAAG ATGAAGAACT 1261 ATGTGGTGGA CTTTGGAGAC TAGTCCTAGC GCAAAGATGG ATGGAAAGAC TTAAGACCGT 1321 AGCGGGATCT AAGATGCAGG GACTACTAGA AAGAGTCAAC ACCGAGATCC ACTTCGTCAC 1381 AAAGTGTGCG TTTCAACCAC CACCGTCCTG TCTAAGATTC GTCCAGACAA ACATCTCCAG 1441 ACTACTACAA GAAACCTCCG AGCAGCTAGT AGCGCTAAAA CCGTGGATCA CAAGACAGAA 1501 CTTCTCGAGA TGTCTAGAGC TACAGTGTCA GCCGGATTCT TCTACATTAC CACCACCATG 1561 GTCACCAAGA CCACTAGAAG CTACAGCTCC AACTGCTCCA CAACCACCAT TGCTACTTTT 1621 GCTATTGCTA CCCGTCGGAT TGCTACTATT AGCTGCTGCT TGGTGTCTAC ACTGGCAGAG 77 4873-2549-9626.1Atty. Dkt. No.115872-3093 1681 AACTAGAAGA AGAACTCCAA GACCGGGAGA ACAAGTACCA CCAGTACCAT CTCCACAGGA 1741 CCTACTACTA GTCGAGCACA GAAGAAGAAG AAGATCGGGA GCGACCAACT TCTCGCTATT 1801 GAAACAAGCG GGAGATGTCG AAGAAAATCC GGGACCAATG GAAAGAGTAC AGCCGCTAGA 1861 AGAAAACGTA GGAAATGCGG CTAGACCGAG ATTCGAGAGA AACAAGCTAC TATTGGTCGC 1921 GTCCGTCATC CAAGGACTAG GATTGCTATT GTGCTTCACC TACATCTGCC TACACTTCTC 1981 CGCGCTACAA GTCTCTCATA GATACCCGAG AATCCAGTCC ATCAAGGTCC AGTTCACCGA 2041 GTACAAGAAA GAGAAGGGAT TCATCCTAAC CTCGCAGAAA GAGGACGAGA TCATGAAGGT 2101 CCAGAACAAC TCCGTCATCA TCAACTGCGA CGGATTCTAC CTAATCTCCC TAAAGGGATA 2161 CTTCTCCCAA GAAGTCAACA TCTCCTTGCA CTACCAGAAG GATGAGGAAC CGCTATTCCA 2221 GCTAAAGAAA GTCAGATCCG TCAACTCCCT AATGGTCGCC TCTCTAACGT ACAAGGACAA 2281 GGTCTACCTA AACGTCACCA CCGACAACAC ATCCCTAGAT GATTTCCACG TAAACGGTGG 2341 AGAGCTAATC CTAATCCATC AGAACCCGGG AGAGTTCTGT GTATTATAAG TTAACAGGCC 2401 TGCATTTCAT CTTTCTCCAA TACTAATTCA AATTGTTAAA TTAATAATGG ATAGTATAAA 2461 TAGTTATTAG TGATAAAATA GTAAAAATAA TTATTAGAAT AAGAGTGTAG TATCATAGAT 2521 AACTCTCTTC TATAAAAATG GATTTTATTC GTAGAAAGTA TCTTATATAC ACAGTAGAAA 2581 ATAATATAGA TTTTTTAAAG GATGATACAT TAAGTAAAGT AAACAATTTT ACCCTCAATC 2641 ATGTACTAGC TCTCAAGTAT CTAGTTAGCA ATTTTCCTCA ACACGTTATT ACTAAGGATG 2701 TATTAGCTAA TACCAATTTT TTTGTTTTCA TACATATGGT ACGATGTTGT AAAGTGTACG 2761 AAGCGGTTTT ACGACACGCA TTTGATGCAC CCACGTTGTA CGTTAAAGCA TTGACTAAGA 2821 ATTATTTATC GTTTAGTAAC GCAATACAAT CGTACAAGGA AACCGTGCAT AAACTAACAC 2881 AAGATGAAAA ATTTTTAGAG GTTGCCGAAT ACATGGACGA ATTAGGAGAA CTTATAGGCG 2941 TAAATTATGA CTTAGTTCTT AATCCATTAT TTCACGGAGG GGAACCCATC AAAGATATGG 3001 AAATCACTGG GCCTCATGGG CCTTCCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC 3061 GTGCCAGCTG CATTAACATG GTCATAGCTG TTTCCTTGCG TATTGGGCGC TCTCCGCTTC 3121 CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGGTAAAG CCTGGGGTGC CTAATGAGCA 3181 AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG 3241 CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG 3301 ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT 3361 CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT 3421 TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC 78 4873-2549-9626.1Atty. Dkt. No.115872-3093 3481 TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT 3541 GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT 3601 AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC 3661 TACACTAGAA GAACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA 3721 AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT 3781 TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT 3841 ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA 3901 TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA 3961 AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC 4021 TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT 4081 ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGAACCACGC 4141 TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT 4201 GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA 4261 AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG CATCGTGGTG 4321 TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT 4381 ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC 4441 AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT 4501 ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC 4561 TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG GGATAATACC 4621 GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA 4681 CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC 4741 TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA 4801 AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT 4861 TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA 4921 TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCAC pMA-MVA∆E5R-hFlt3L-mOX40L vector nucleic acid sequence (SEQ ID NO: 5): 1 CTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC 61 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA 121 GATAGGGTTG AGTGGCCGCT ACAGGGCGCT CCCATTCGCC ATTCAGGCTG CGCAACTGTT 181 GGGAAGGGCG TTTCGGTGCG GGCCTCTTCG CTATTACGCC AGCTGGCGAA AGGGGGATGT 241 GCTGCAAGGC GATTAAGTTG GGTAACGCCA GGGTTTTCCC AGTCACGACG TTGTAAAACG 79 4873-2549-9626.1Atty. Dkt. No.115872-3093 301 ACGGCCAGTG AGCGCGACGT AATACGACTC ACTATAGGGC GAATTGGCGG AAGGCCGTCA 361 AGGCCGCATT GGAGGTTCGT CAGCGGCTCT AGTTTGAATC ATCATCGGCG TAGTATTCCT 421 ACTTTTACAG TTAGGACACG GTGTATTGTA TTTCTCGTCG AGAACGTTAA AATAATCGTT 481 GTAACTCACA TCCTTTATTT TATCTATATT GTATTCTACT CCTTTCTTAA TGCATTTTAT 541 ACCGAATAAG AGATAGCGAA GGAATTCTTT TTCGGTGCCG CTAGTACCCT TAATCATATC 601 ACATAGTGTT TTATATTCCA AATTTGTGGC AATAGACGGT TTATTTCTAT ACGATAGTTT 661 GTTTCTGGAA TCCTTTGAGT ATTCTATACC AATATTATTC TTTGATTCGA ATTTAGTTTC 721 TTCGATATTA GATTTTGTAT TACCTATATT CTTGATGTAG TACTTTGATG ATTTTTCCAT 781 GGCCCATTCT ATTAAGTCTT CCAAGTTGGC ATCATCCACA TATTGTGATA GTAATTCTCG 841 GATATCAGTA GCGGCTACCG CCATTGATGT TTGTTCATTG GATGAGTAAC TACTAATGTA 901 TACATTTTCC ATTTATAACA CTTATGTATT AACTTTGTTC ATTTATATTT TTTCATTATT 961 ATGTTGATAT TAACAAAAGT GAATATATGG ATCCAAAAAT TGAAATTTTA TTTTTTTTTT 1021 TTGGAATATA AATAAGCTCG AAGTCGACGA ATTCATGACA GTACTAGCTC CAGCTTGGTC 1081 CCCGACAACA TACCTTCTAC TACTACTATT GCTATCCTCC GGACTATCTG GAACCCAGGA 1141 TTGCTCTTTT CAGCACTCTC CGATCTCGTC TGATTTCGCG GTTAAGATCA GAGAGCTATC 1201 CGACTACTTG CTACAGGATT ACCCAGTAAC CGTCGCGTCC AACCTACAAG ATGAAGAACT 1261 ATGTGGTGGA CTTTGGAGAC TAGTCCTAGC GCAAAGATGG ATGGAAAGAC TTAAGACCGT 1321 AGCGGGATCT AAGATGCAGG GACTACTAGA AAGAGTCAAC ACCGAGATCC ACTTCGTCAC 1381 AAAGTGTGCG TTTCAACCAC CACCGTCCTG TCTAAGATTC GTCCAGACAA ACATCTCCAG 1441 ACTACTACAA GAAACCTCCG AGCAGCTAGT AGCGCTAAAA CCGTGGATCA CAAGACAGAA 1501 CTTCTCGAGA TGTCTAGAGC TACAGTGTCA GCCGGATTCT TCTACATTAC CACCACCATG 1561 GTCACCAAGA CCACTAGAAG CTACAGCTCC AACTGCTCCA CAACCACCAT TGCTACTTTT 1621 GCTATTGCTA CCCGTCGGAT TGCTACTATT AGCTGCTGCT TGGTGTCTAC ACTGGCAGAG 1681 AACTAGAAGA AGAACTCCAA GACCGGGAGA ACAAGTACCA CCAGTACCAT CTCCACAGGA 1741 CCTACTACTA GTCGAGCACA GAAGAAGAAG AAGATCGGGA GCGACCAACT TCTCGCTATT 1801 GAAACAAGCG GGAGATGTCG AAGAAAATCC GGGACCAATG GAGGGCGAGG GGGTCCAGCC 1861 TCTGGACGAG AACCTCGAAA ACGGGTCTCG CCCTCGCTTT AAATGGAAGA AGACTCTTAG 1921 GCTCGTTGTA AGCGGCATCA AGGGGGCCGG TATGTTGCTG TGCTTCATAT ATGTGTGTTT 1981 GCAACTTAGC TCTTCACCTG CAAAAGACCC CCCCATACAA CGCCTTCGGG GGGCTGTGAC 2041 CCGCTGTGAA GATGGTCAAT TGTTTATTTC TTCTTACAAG AACGAGTATC AGACGATGGA 80 4873-2549-9626.1Atty. Dkt. No.115872-3093 2101 AGTCCAGAAT AACTCCGTAG TGATTAAGTG TGACGGACTG TACATCATCT ACTTGAAAGG 2161 ATCTTTTTTC CAGGAGGTCA AAATTGACCT CCACTTCAGG GAGGATCACA ACCCTATCTC 2221 AATCCCTATG TTGAACGACG GCAGAAGAAT CGTCTTTACT GTAGTCGCTT CACTGGCCTT 2281 CAAGGATAAG GTGTACTTGA CCGTAAACGC TCCTGATACC TTGTGCGAGC ATTTGCAAAT 2341 CAACGATGGA GAACTTATCG TTGTCCAACT CACACCAGGT TACTGTGCTC CTGAGGGCAG 2401 TTATCACAGT ACAGTGAACC AAGTCCCACT GTGAGTTAAC AGGCCTGCAT TTCATCTTTC 2461 TCCAATACTA ATTCAAATTG TTAAATTAAT AATGGATAGT ATAAATAGTT ATTAGTGATA 2521 AAATAGTAAA AATAATTATT AGAATAAGAG TGTAGTATCA TAGATAACTC TCTTCTATAA 2581 AAATGGATTT TATTCGTAGA AAGTATCTTA TATACACAGT AGAAAATAAT ATAGATTTTT 2641 TAAAGGATGA TACATTAAGT AAAGTAAACA ATTTTACCCT CAATCATGTA CTAGCTCTCA 2701 AGTATCTAGT TAGCAATTTT CCTCAACACG TTATTACTAA GGATGTATTA GCTAATACCA 2761 ATTTTTTTGT TTTCATACAT ATGGTACGAT GTTGTAAAGT GTACGAAGCG GTTTTACGAC 2821 ACGCATTTGA TGCACCCACG TTGTACGTTA AAGCATTGAC TAAGAATTAT TTATCGTTTA 2881 GTAACGCAAT ACAATCGTAC AAGGAAACCG TGCATAAACT AACACAAGAT GAAAAATTTT 2941 TAGAGGTTGC CGAATACATG GACGAATTAG GAGAACTTAT AGGCGTAAAT TATGACTTAG 3001 TTCTTAATCC ATTATTTCAC GGAGGGGAAC CCATCAAAGA TATGGAAATC ACTGGGCCTC 3061 ATGGGCCTTC CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA 3121 ACATGGTCAT AGCTGTTTCC TTGCGTATTG GGCGCTCTCC GCTTCCTCGC TCACTGACTC 3181 GCTGCGCTCG GTCGTTCGGG TAAAGCCTGG GGTGCCTAAT GAGCAAAAGG CCAGCAAAAG 3241 GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC 3301 GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA 3361 TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT 3421 ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC 3481 TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC 3541 CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA 3601 AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT 3661 GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGAACA 3721 GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT 3781 TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT 3841 ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT 81 4873-2549-9626.1Atty. Dkt. No.115872-3093 3901 CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC 3961 ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA 4021 ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA 4081 TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC 4141 TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGAAC CACGCTCACC GGCTCCAGAT 4201 TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA 4261 TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT 4321 AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT 4381 GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG 4441 TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC 4501 GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC 4561 GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG 4621 CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA 4681 ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA 4741 CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT 4801 TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG 4861 GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA 4921 AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT 4981 AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CAC MQ832

[0147] The disclosure of the present technology relates to modified vaccinia Ankara (MVA) viruses referred to as “rMVAΔE3LΔWR199” or “MQ832” or “MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199,” which may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant E5R gene, and a mutant WR199 gene. In some embodiments, the rMVAΔE3LΔWR199 is an rMVA or an rMVAΔE3L virus that has been engineered to further comprise both a mutant E3L gene and a mutant WR199 gene, or a mutant WR199 gene, respectively. In some embodiments, the mutation is a deletion of the E3L and WR199 genes. In some embodiments, the present technology relates to an immunogenic composition comprising MQ832. In some embodiments, plasmid constructs were used to 82 4873-2549-9626.1Atty. Dkt. No.115872-3093 create MQ832 via homologous recombination with the MVA genome. In some embodiments, illustrative, non-limiting, plasmid constructs used to create MQ832 include those shown in Table 6. Illustrative, non-limiting plasmids for engineering the E3L knockout are shown in FIG.32 (E3L_mcherryFRT_Kan (SEQ ID NO: 24)) and FIG.29 (P503 (SEQ ID NO: 3)). An illustrative, non-limiting plasmid for engineering the WR199 knockout is shown in Table 6 and in FIG.33 (WR199_FRT_mCherry (SEQ ID NO: 25)). Table 6. Exemplary nucleotide sequences for the MQ832 constructs of the present technology. E3L_mcherryFRT_Kan (SEQ ID NO: 24) 1 atagcgtccc taggacgaac tactgccatt aatatctcta ttatagcttc tggacataat 61 tcatctatta taccagaatt aatgggaact attccgtatc tatctaacat agttttaaga 121 aagtcagaat ctaagacctg atgttcatat attggttcat acatgaaatg atctctattg 181 atgatagtga ctatttcatt ctctgaaaat tggtaactca ttctatatat gctttccttg 241 ttgatgaagg atagaatata ctcaatagaa tttgtaccaa caaactgttc tcttatgaat 301 cgtatatcat catctgaaat aatcatgtaa ggcatacatt taacaattag agacttgtct 361 cctgttatca atatactatt cttgtgataa tttatgtgtg aggcaaattt gtccacgttc 421 tttaattttg ttatagtaga tatcaaatcc aatggagcta cagttcttgg cttaaacaga 481 tatagttttt ctggaacgaa ttctacaaca ttattataaa ggactttggg tagataagtg 541 ggatgaaatc ctattttaat taatgcgata gccttgtcct cgtgcagata tccaaacgct 601 tttgtgatag tatggcattc attgtctaga aacgctctac gaatatctgt gacagatatc 661 atctttagag aatatactag tcgcgttaat agtactacaa tttgtatttt ttaatctatc 721 tcaataaaaa aattaatatg tatgattcaa tgtataacta aactactaac tgttattgat 781 aactagaatc agaatctaat gatgacgtaa ccaagctagc gcggttaacc gcctttttat 841 ccatcaggtg atctgttttt attgtggagt ctagaactag tggatccccc gggctgcagg 901 aattcgatat caagctcagg cctagatctg tcgacttcga gcttatttat attccaaaaa 961 aaaaaaataa aatttcaatt tttaagcttt tgaagttcct atactttcta gagaatagga 1021 acttccacta attccaaacc cacccgcttt ttatagtaag tttttcaccc ataaataata 1081 aatacaataa ttaatttctc gtaaaagtag aaaatatatt ctaatttatt gcacggtaag 1141 gaagtagatc ataactcgag gaattgggga tctctataat ctcgcgcaac ctattttccc 1201 ctcgaacact ttttaagccg tagataaaca ggctgggaca cttcacacgc gtatggtgag 1261 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat 1321 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta 1381 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg 1441 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga 1501 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa 1561 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt 1621 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa 1681 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa 1741 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa 1801 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa 1861 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga 1921 gggccgccac tccaccggcg gcatggacga gctgtacaag taatgaagtt cctatacttt 1981 ctagagaata ggaacttccg gttaaccggt gatataggca accagcaata aaactaattt 2041 attttatcat ttttttattc atcatcctct ggtggttcgt cgtttctatc gaatgtggat 2101 ctgattaacc cgtcatctat aggtgatgct ggttctggag attctggagg agatggatta 2161 ttatctggaa gaatctctgt tatttccttg ttttcatgta tcgattgcgt tgtaacatta 83 4873-2549-9626.1Atty. Dkt. No.115872-3093 2221 agattgcgaa atgctctaaa tttgggaggc ttaaagtgtt gtttgcaatc tctacacgca 2281 tgtctaacta gtggaggttc gtcagcggct ctagtttgaa tcatcatcgg cgtagtattc 2341 ctacttttac agttaggaca cggtgtattg tatttctcgt cgagaacgtt aaaataatcg 2401 ttgtaactca catcctttat tttatctata ttgtattcta ctcctttctt aatgcatttt 2461 ataccgaata agagatagcg aaggaattct ttttcggtgc cgctagtacc cttaatcata 2521 tcacatagtg ttttatattc caaatttgtg gcaatagacg gtttatttct atacgatagt 2581 ttgtttctgg aatcctttga gtattctata ccaatattat tctttgattc gaatttagtt 2641 tcttcgatat tagattttgt attacctata ttcttgatgt agtactttga tgatttttcc 2701 atggcccatt ctatcacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat 2761 gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct 2821 tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg 2881 gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg 2941 gctttctcgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca agagacagga 3001 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg 3061 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc 3121 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt 3181 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt 3241 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc 3301 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc 3361 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac 3421 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag 3481 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag 3541 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat 3601 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg 3661 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa 3721 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc 3781 ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt cctgatgcgg 3841 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacaggtggc acttttcggg 3901 gaaatgtgcg cggaacccct attttcgttc cactgagcgt cagaccccgt agaaaagatc 3961 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 4021 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 4081 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 4141 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 4201 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 4261 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 4321 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 4381 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 4441 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 4501 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 4561 aacgccagca acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg 4621 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 4681 gataccgctc gccgcagccg aacgaccgag cgcagcgagt ca P503 (FIG.29, SEQ ID NO: 3) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 84 4873-2549-9626.1Atty. Dkt. No.115872-3093 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcaca ctatgtgata 421 tgattaaggg tactagcggc accgaaaaag aattccttcg ctatctctta ttcggtataa 481 aatgcattaa gaaaggagta gaatacaata tagataaaat aaaggatgtg agttacaacg 541 attattttaa cgttctcgac gagaaataca atacaccgtg tcctaactgt aaaagtagga 601 atactacgcc gatgatgatt caaactagag ccgctgacga acctccacta gttagacatg 661 cgtgtagaga ttgcaaacaa cactttaagc ctcccaaatt tagagcattt cgcaatctta 721 atgttacaac gcaatcgata catgaaaaca aggaaataac agagattctt ccagataata 781 atccatctcc tccagaatct ccagaaccag catcacctat agatgacggg ttaatcagat 841 ccacattcga tagaaacgac gaaccaccag aggatgatga ataaaaaaat gataaaataa 901 attagtttta ttgctggttg tgttagttct ctctaactcg agttctagtt atcaataaca 961 gttagtagtt tagttataca ttgaatcata catattaatt tttttattga gatagattaa 1021 aaaatacaaa ttgtagtact attaacgcga ctagtatatt ctctaaagat gatatctgtc 1081 acagatattc gtagagcgtt tctagacaat gaatgccata ctatcacaaa agcgtttgga 1141 tatctgcacg aggacaaggc tatcgcatta attaaaatag gatttcatcc cacttatcta 1201 cccaaagtcc tttataataa tgttgtagaa ttcgttccag aaaaactata tctgtttaag 1261 ccaagaactg tagctccatt ggatttgata tctactataa caaaattaaa gaacgtggac 1321 aaatttgcct cacacataaa ttatcacaag aatagtatat tgataacagg agacaagtct 1381 ctaattgtta aatgtatgcc ttacatgatt atttcagatg atgatatacg attcataaga 1441 gaacagtttg ttggtacaaa ttctattgag tatattctat ccttcatcaa caaggaaagc 1501 atatatagaa tgagttacat ctagatgcat tcgcgaggta catcggatcc cgggcccgtc 1561 gactgcagag gcctgcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga 1621 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 1681 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 1741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 1801 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 85 4873-2549-9626.1Atty. Dkt. No.115872-3093 1861 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 1921 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 1981 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 2041 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 2101 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 2161 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 2221 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 2281 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 2341 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 2401 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 2461 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 2521 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 2581 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 2641 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 2701 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 2761 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 2821 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 2881 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 2941 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 3001 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 3061 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 3121 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 3181 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 3241 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 3301 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 3361 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 3421 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 86 4873-2549-9626.1Atty. Dkt. No.115872-3093 3481 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 3541 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 3601 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 3661 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 3721 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 3781 ttaacctata aaaataggcg tatcacgagg ccctttcgtc WR199_FRT_mCherry (SEQ ID NO: 25): 1 cgtgaatgta tgttgttaca tttccatgtc aattgagttt ataagaattt ttatacatta 61 tcttccaaca aacaattgac gaacgtattg ctatgattaa ctcccacgat actatgcata 121 ttattaatca ttaacttgca gactatacct agtgctattt tgacatactc atgttcttgt 181 gtaattgcgg tatctatatt attaaagtac gtaaatctag ctatagtttt attatttaat 241 tttagataat ataccgtctc cttattttta aaaattgcca catcctttat taaatcatga 301 atgggaattt ctatgtcatc gttaatatat tgtgaacaac aagagcagat atctatagga 361 aagggtggaa tgcgatacat tgatctatgt agttttaaaa cacacgcgaa ctttgaagaa 421 tttatataaa tcattccatc gatacatcct tctatgttga catgtatata tccaggaatt 481 cttttattaa tgtcaggaaa tgtataaact aaaacattgc ccgaaagcgg tgcctctatc 541 tgcgttatat ccgttcttaa cttacaaaat gtaaccaata cctttgcatg acttgttttg 601 ttcggcaacg ttagtttaaa cttgacgaat ggattaatta caatagcatg atccgcgcat 661 ctattaagtt tttttacttt aacgcccttg tatgttttta cagagacttt atctaaattt 721 ctagtgcttg tatgtgttat aaatataacg ggatatagaa ctgaatcacc taccttagat 781 acccaattac attttatcag atccagataa taaacaaatt ttgtcgccct aactaattct 841 atattgttat atattttaca attggttatg atatcatgta ataacttgga gtctaacgcg 901 catcgtcgta cgtttataca attgtgattt agtgtagtat atctacacat gtatttttcc 961 gcactatagt attctggact agtgataaaa ctatcgttat atctgtcttc aatgaactca 1021 tcgagatatt gctctctgtc atattcatac acctgcataa actttctaga catcttacaa 1081 tccgtgttat tttaggatca tatttacata tttacgggta tatcaaagat gttagattag 1141 ttaatgggaa tcgtctataa taatgaatat taaacaatta tatgaggact ttaagctagc 1201 gcggttaacc gcctttttat ccatcaggtg atctgttttt attgtggagt ctagaactag 1261 tggatccccc gggctgcagg aattcgatat caagctcagg cctagatctg tcgacttcga 1321 gcttatttat attccaaaaa aaaaaaataa aatttcaatt tttaagcttt tgaagttcct 1381 atactttcta gagaatagga acttccacta attccaaacc cacccgcttt ttatagtaag 1441 tttttcaccc ataaataata aatacaataa ttaatttctc gtaaaagtag aaaatatatt 1501 ctaatttatt gcacggtaag gaagtagatc ataactcgag gaattgggga tctctataat 1561 ctcgcgcaac ctattttccc ctcgaacact ttttaagccg tagataaaca ggctgggaca 1621 cttcacacgc gtatggtgag caagggcgag gaggataaca tggccatcat caaggagttc 1681 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc 1741 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt 1801 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc 1861 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc 1921 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc 1981 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc 2041 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac 2101 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc 2161 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc 2221 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg 2281 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag 87 4873-2549-9626.1Atty. Dkt. No.115872-3093 2341 taatgaagtt cctatacttt ctagagaata ggaacttccg gttaaccggt gattgtattt 2401 ttctcatgcg atgtgtgtaa aaaaactgat attatataaa tattttagtg ccgtataatg 2461 aagatgacga tgaaaatgat ggtacatata tatttcgtat cattattgtt attgctattc 2521 cacagttacg ccatagacat cgaaaatgaa atcacagaat tcttcaataa aatgagagat 2581 actctaccag ctaaagactc taaatggttg aatccagcat gtatgttcgg aggcacaatg 2641 aatgatatag ccgctctagg agagccattc agcgcaaagt gtcctcctat tgaagacagt 2701 cttttatcgc acagatataa agactatgtg gttaaatggg agaggctaga aaaaaataga 2761 cggcgacagg tttctaataa acgtgttaaa catggtgatt tatggatagc caactataca 2821 tctaaattca gtaaccgtag gtatttgtgt accgtaacta caaagaatgg tgactgtgtt 2881 cagggtatag ttagatctca tattaaaaaa cctccttcat gcattccaaa aacatatgaa 2941 ctaggtactc atgataagta tggcatagac ttatactgtg gaattcttta cgcaaaacat 3001 tataataata taacttggta taaagataat aaggaaatta atatcgacga tattaagtat 3061 tcacaaacgg gaaagaaatt aattattcat aatccagagt tagaagatag tggaagatac 3121 aactgttacg ttcattacga cgacgttaga atcaagatgt aaaatactta cggttatacc 3181 gtcgcaagac cacaggttta aactaatact agatccaaaa atcaacgtaa cgataggaga 3241 acctgccaat ataacatgca ctgctgtgtc aacgtcatta ttgattgacg atgtactgat 3301 tgaatgggaa aatccatccg gatggcttat aggattcgat tttgatgtat actctgtttt 3361 aactagtaga ggcggtatca ccgaggcgac cttgtacttt gaaaatgtta ctgaagaata 3421 tataggtaat acatataaat gtcgtggaca caactattat tttgaaaaaa ccctacaact 3481 acagtagtat tggagtcctt ttcacgtaga aagccagtcc gcagaaacgg tgctgacccc 3541 ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc 3601 aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa 3661 gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa 3721 actggatggc tttctcgccg ccaaggatct gatggcgcag gggatcaagc tctgatcaag 3781 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 3841 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 3901 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 3961 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga 4021 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 4081 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 4141 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 4201 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 4261 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 4321 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 4381 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 4441 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 4501 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 4561 gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat tattaacgct tacaatttcc 4621 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata caggtggcac 4681 ttttcgggga aatgtgcgcg gaacccctat tttcgttcca ctgagcgtca gaccccgtag 4741 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 4801 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 4861 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 4921 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 4981 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 5041 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 5101 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 5161 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 5221 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 5281 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 88 4873-2549-9626.1Atty. Dkt. No.115872-3093 5341 tatggaaaaa cgccagcaac gcggcctttt tacggttcct gggcttttgc tggccttttg 5401 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 5461 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca MQ833

[0148] The disclosure of the present technology relates to modified vaccinia Ankara (MVA) viruses referred to as “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12” or “MVAΔE5R-hFlt3L-OX40L-ΔE3L-ΔWR199-IL-12” or “MQ833,” which may be used interchangeably and refer to a MVA mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E5R gene, a mutant E3L gene, a mutant WR199 gene, and a third transgene, IL-12. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the MQ833 is an MQ832 virus that has been engineered to further comprise an IL-12 transgene. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the present technology relates to an immunogenic composition comprising MQ833. In some embodiments, plasmid constructs were used to create MQ833 via homologous recombination with the MVA genome. In some embodiments, illustrative, non-limiting, plasmid constructs used to create MQ833 include those shown in Table 7. An illustrative, non-limiting plasmid design for deletion of WR199 (also known as MVA186) and insertion of IL-12 is shown in FIG.25 (SEQ ID NO: 2). An illustrative, non-limiting plasmid design for deletion of WR199 with IL-12 sequence (murine) is shown in Table 7 (Plasmid design for deletion of WR199 with IL-12 sequence (murine) (SEQ ID NO: 26). Table 7. Exemplary nucleotide sequences for the MQ833 constructs of the present technology. pUC57-del186-pH5-huIL12 (SEQ ID NO: 2) (FIG.28): 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 89 4873-2549-9626.1Atty. Dkt. No.115872-3093 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtttttttac tttaacgccc 421 ttgtatgttt ttacagagac tttatctaaa tttctagtgc ttgtatgtgt tataaatata 481 acgggatata gaactgaatc acctacctta gatacccaat tacattttat cagatccaga 541 taataaacaa attttgtcgc cctaactaat tctatattgt tatatatttt acaattggtt 601 atgatatcat gtaataactt ggagtctaac gcgcatcgtc gtacgtttat acaattgtga 661 tttagtgtag tatatctaca catgtatttt tccgcactat agtattctgg actagtgata 721 aaactatcgt tatatctgtc ttcaatgaac tcatcgagat attgctctct gtcatattca 781 tacacctgca taaactttct agacatctta caatccgtgt tattttagga tcatatttac 841 atatttacgg gtatatcaaa gatgttagat tagttaatgg gaatcgtcta taataatgaa 901 tattaaacaa ttatatgagg acttttacca caaagcatca taaaactgca gaaaaattga 961 aaataaatac aaaggttctt gagggttgtg ttaaattgaa agcgagaaat aatcataaat 1021 aactggtcga gatgtgtcac cagcagctag tgatctcgtg gttctcccta gtatttctag 1081 cctctccgct agtagcgatc tgggagctaa aaaaggatgt ctacgtcgtc gagctagact 1141 ggtatccaga tgctccggga gaaatggtag tcctaacatg tgatacaccg gaagaggatg 1201 gaatcacctg gaccttggat caatcctccg aagtactagg atccggaaag accctaacca 1261 tccaagtcaa agaattcgga gatgcgggac agtacacctg tcacaaaggt ggagaagtcc 1321 tatcgcactc cctactacta ttgcacaaga aagaggacgg tatctggtcc accgatatcc 1381 taaaggatca gaaagagccg aagaacaaga ccttcctaag atgcgaagcg aagaactact 1441 ccggaagatt cacatgttgg tggctaacca caatctccac cgacctaacc ttctccgtca 1501 aatcttctag aggatccagt gatccgcagg gtgtaacatg tggtgctgct acattatctg 1561 cggagagagt cagaggagac aacaaagagt acgagtactc cgtcgagtgt caagaggatt 1621 ctgcttgtcc agctgcggaa gaatctctac cgatcgaagt aatggtagac gcggtccaca 1681 agttgaagta cgagaactac acctcctcgt tcttcatcag agacatcatc aaaccagatc 1741 cgccgaagaa tctacagcta aagccgctaa agaactccag acaggtcgaa gtatcttggg 1801 aatacccgga tacttggtct acaccgcact cctacttttc gttgaccttc tgtgtacaag 90 4873-2549-9626.1Atty. Dkt. No.115872-3093 1861 tccagggaaa gtccaagaga gagaagaagg acagagtctt taccgacaag acatccgcga 1921 ccgtcatctg tagaaagaac gcctctattt ctgtcagagc gcaggacaga tattactcct 1981 cgtcttggag tgaatgggcg tctgtaccat gttccagaag aagaagaaga agagcgacca 2041 acttctcgct attgaagcaa gcgggagatg tagaagaaaa tccgggaccg ttagatatgt 2101 gtccggcgag atctctacta ctagtcgcga cactagtcct actagaccat ctatctctag 2161 cgagaaattt gccagtagcg acaccagatc ctggaatgtt tccgtgtcta caccactcgc 2221 agaatctact aagagccgtg tctaacatgc tacagaaggc gagacagacc ttggaattct 2281 acccgtgtac ctccgaagaa atcgatcacg aggatatcac caaggacaag acctctacag 2341 tcgaagcttg tctaccgcta gagttgacca agaacgagtc ctgcctaaac tccagagaaa 2401 cctccttcat caccaacgga tcttgcctag cgtctagaaa gacctctttc atgatggcgc 2461 tatgcctatc ctctatctac gaggacctaa agatgtacca ggtcgaattc aagaccatga 2521 acgcgaagct actaatggac ccgaagagac agatcttctt ggaccagaat atgctagcgg 2581 tcatcgacga actaatgcag gcgctaaact tcaactctga aaccgtgccg cagaagtcca 2641 gtttagaaga accggatttc tacaagacca agatcaagct atgcatccta ctacacgcgt 2701 tcagaatcag agcggtcacc atcgatagag tcatgtctta cctaaacgcg tccagaagac 2761 cgaaaggaag aggaaagaga agaagagaaa agcaaagacc gaccgactgc catctatgag 2821 ttaacttgta tttttctcat gcgatgtgtg taaaaaaact gatattatat aaatatttta 2881 gtgccgtata atgaagatga cgatgaaaat gatggtacat atatatttcg tatcattatt 2941 gttattgcta ttccacagtt acgccataga catcgaaaat gaaatcacag aattcttcaa 3001 taaaatgaga gatactctac cagctaaaga ctctaaatgg ttgaatccag catgtatgtt 3061 cggaggcaca atgaatgata tagccgctct aggagagcca ttcagcgcaa agtgtcctcc 3121 tattgaagac agtcttttat cgcacagata taaagactat gtggttaaat gggagaggct 3181 agaaaaaaat agacggcgac aggtttctaa taaacgtgtt aaacatggtg atttatggat 3241 agccaactat acatctaaat tcagtaaccg taggtatttg tgtaccgtaa ctacaaagaa 3301 tggtgactgt gttcagggta tagttagatc tcatattaaa aaacctcctt catgcattcc 3361 aaaaacatat gaactaggta ctcatgataa gtagcttggc gtaatcatgg tcatagctgt 3421 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 91 4873-2549-9626.1Atty. Dkt. No.115872-3093 3481 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 3541 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 3601 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 3661 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 3721 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 3781 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 3841 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 3901 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 3961 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 4021 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 4081 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 4141 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 4201 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 4261 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 4321 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 4381 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 4441 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 4501 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 4561 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 4621 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 4681 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 4741 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 4801 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 4861 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 4921 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 4981 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 5041 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 92 4873-2549-9626.1Atty. Dkt. No.115872-3093 5101 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 5161 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 5221 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 5281 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 5341 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 5401 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 5461 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 5521 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 5581 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c Plasmid design for deletion of WR199 with IL-12 sequence (murine) (SEQ ID NO: 26) (FIG.34): 1 cgtgaatgta tgttgttaca tttccatgtc aattgagttt ataagaattt ttatacatta 61 tcttccaaca aacaattgac gaacgtattg ctatgattaa ctcccacgat actatgcata 121 ttattaatca ttaacttgca gactatacct agtgctattt tgacatactc atgttcttgt 181 gtaattgcgg tatctatatt attaaagtac gtaaatctag ctatagtttt attatttaat 241 tttagataat ataccgtctc cttattttta aaaattgcca catcctttat taaatcatga 301 atgggaattt ctatgtcatc gttaatatat tgtgaacaac aagagcagat atctatagga 361 aagggtggaa tgcgatacat tgatctatgt agttttaaaa cacacgcgaa ctttgaagaa 421 tttatataaa tcattccatc gatacatcct tctatgttga catgtatata tccaggaatt 481 cttttattaa tgtcaggaaa tgtataaact aaaacattgc ccgaaagcgg tgcctctatc 541 tgcgttatat ccgttcttaa cttacaaaat gtaaccaata cctttgcatg acttgttttg 601 ttcggcaacg ttagtttaaa cttgacgaat ggattaatta caatagcatg atccgcgcat 661 ctattaagtt tttttacttt aacgcccttg tatgttttta cagagacttt atctaaattt 721 ctagtgcttg tatgtgttat aaatataacg ggatatagaa ctgaatcacc taccttagat 781 acccaattac attttatcag atccagataa taaacaaatt ttgtcgccct aactaattct 841 atattgttat atattttaca attggttatg atatcatgta ataacttgga gtctaacgcg 901 catcgtcgta cgtttataca attgtgattt agtgtagtat atctacacat gtatttttcc 961 gcactatagt attctggact agtgataaaa ctatcgttat atctgtcttc aatgaactca 1021 tcgagatatt gctctctgtc atattcatac acctgcataa actttctaga catcttacaa 1081 tccgtgttat tttaggatca tatttacata tttacgggta tatcaaagat gttagattag 1141 ttaatgggaa tcgtctataa taatgaatat taaacaatta tatgaggact ttaagctagc 1201 gcggttaacc gcctttttat ccatcaggtg atctgttttt attgtggagt ctagaactag 1261 tggatcacga attgttaact cacagatggc aatcagtagg tctttgtttt tcccggcggc 1321 gttttccgcg ccctttaggg cgccgtgctg aagaaagata ccccatgact cgattgatag 1381 taacaacccg tgtggaaaag gcatgcagga gtatgcagag tttcatcttg actcggtagg 1441 ggtcggcttc tccaactggg ggcttttgtc gcagagtttc gccattatga ttcagagatt 1501 gcataagttc gtcgatggct accagcatac ccttatcaag gattatctgc tggtgattat 1561 ggttttgcaa tgctgcgtta atggcctgaa actctgtttg gtacatcttc aaatcttcgt 1621 aaatactgcc gagacacagg gtcatcatca aggaggtttt ctgtggaggc aggcaggacc 1681 cccgtgtagt gctagaggtc tctcgtgtgg caaggcagga ctcgttctta tgaagctcca 1741 gtggcagaca agttttcaat gttgaggtct ggtcccgggt tatgtcttca tgatcgatgt 1801 cctctgctgt acaagagtaa tgtttgagtt tctcccttgc tgtttttacc atatcgtctg 1861 tggtcttaag gagattccga ctctggctga ggcatcgggc aggcccagat acgggaatta 93 4873-2549-9626.1Atty. Dkt. No.115872-3093 1921 ccctagccag tgagaggtgg ttaagcagag ccagggttgc gaggaaaagg aggtaccggc 1981 tctgacacat gtcaaggggc cctgggtttt cctccacatc gcctgcctgc ttgagcaagg 2041 aaaaattggt agctcgtctc cggcgtcgtc tggatcgaac ccgacaaggt acgcaggccc 2101 atttactaca gcttgagttg tagtatcgat cttgtgcctg cacacacacg tttcccccct 2161 tacactgaac ctcagtgctg gttttctcta ccagaaaggc gcccttctgg ttgcagcctt 2221 cctcggtttc cttcatcttt tctttcttcc tctggattct gacgaagaac ttaagggaga 2281 aataggaatg tggtgtggac caggaatctg gatactccca tgaaacttcc acttggctat 2341 tcttcaaagg tttcatttgg agattcttag gaggatcggg tttaattatg tctcggatga 2401 aaaagcttgt actgtaattt tcgtacttat tctgctggcg tgcttcaagg gccaattcaa 2461 ttggcaatgt ctcttctgcg gtagggcatg taacgtcctc ttgacaacta acagagtact 2521 tctcgtagtc tcgttgatcc aatgttactt tctcagcgga gagggatgcc atgccgcaag 2581 tcactgcccg agaatctggg gagctggaag aagacttgat attaaatttg aggtccatat 2641 tgcgctggac caaccagctg catgtaaatc ttccggaata attaggggcc tcacacttaa 2701 gaaaggtttt gttcttgaaa ttcttaagga tttcggtgga ccagattccg ttctcctttt 2761 tatgcaggag cagatgtgaa tgtgagagtg tctcgccccc tttatgacat gtgtactgtc 2821 ctgcatccaa gaactccttc actgtaattg tcaaggtctt cccagatccg atgaccccgt 2881 gccgttggtc ggatgtccat gtaatatcat cttcttcggg ggtgtcgcat gtcaaattga 2941 ctgtctctcc gggtgcatca ggagtccagt caacctccac tacatacaca tctttttcaa 3001 gctcccacat tgccatcaat ggggaaacga gcaatactat agcgaaccat gagatggtga 3061 gcttttgtgg acacatgaat tcgtcgactt cgagcttatt tatattccaa aaaaaaaaaa 3121 taaaatttca atttttaagc ttttgaagtt cctatacttt ctagagaata ggaacttcca 3181 ctaattccaa acccacccgc tttttatagt aagtttttca cccataaata ataaatacaa 3241 taattaattt ctcgtaaaag tagaaaatat attctaattt attgcacggt aaggaagtag 3301 atcataactc gaggaattgg ggatctctat aatctcgcgc aacctatttt cccctcgaac 3361 actttttaag ccgtagataa acaggctggg acacttcaca cgcgtatggt gagcaagggc 3421 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc 3481 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 3541 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc 3601 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc 3661 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 3721 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac 3781 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc 3841 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag 3901 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc 3961 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac 4021 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc 4081 cactccaccg gcggcatgga cgagctgtac aagtaatgaa gttcctatac tttctagaga 4141 ataggaactt ccggttaacc ggtgattgta tttttctcat gcgatgtgtg taaaaaaact 4201 gatattatat aaatatttta gtgccgtata atgaagatga cgatgaaaat gatggtacat 4261 atatatttcg tatcattatt gttattgcta ttccacagtt acgccataga catcgaaaat 4321 gaaatcacag aattcttcaa taaaatgaga gatactctac cagctaaaga ctctaaatgg 4381 ttgaatccag catgtatgtt cggaggcaca atgaatgata tagccgctct aggagagcca 4441 ttcagcgcaa agtgtcctcc tattgaagac agtcttttat cgcacagata taaagactat 4501 gtggttaaat gggagaggct agaaaaaaat agacggcgac aggtttctaa taaacgtgtt 4561 aaacatggtg atttatggat agccaactat acatctaaat tcagtaaccg taggtatttg 4621 tgtaccgtaa ctacaaagaa tggtgactgt gttcagggta tagttagatc tcatattaaa 4681 aaacctcctt catgcattcc aaaaacatat gaactaggta ctcatgataa gtatggcata 4741 gacttatact gtggaattct ttacgcaaaa cattataata atataacttg gtataaagat 4801 aataaggaaa ttaatatcga cgatattaag tattcacaaa cgggaaagaa attaattatt 4861 cataatccag agttagaaga tagtggaaga tacaactgtt acgttcatta cgacgacgtt 94 4873-2549-9626.1Atty. Dkt. No.115872-3093 4921 agaatcaaga tgtaaaatac ttacggttat accgtcgcaa gaccacaggt ttaaactaat 4981 actagatcca aaaatcaacg taacgatagg agaacctgcc aatataacat gcactgctgt 5041 gtcaacgtca ttattgattg acgatgtact gattgaatgg gaaaatccat ccggatggct 5101 tataggattc gattttgatg tatactctgt tttaactagt agaggcggta tcaccgaggc 5161 gaccttgtac tttgaaaatg ttactgaaga atatataggt aatacatata aatgtcgtgg 5221 acacaactat tattttgaaa aaaccctaca actacagtag tattggagtc cttttcacgt 5281 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct 5341 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 5401 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 5461 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttctcg ccgccaagga 5521 tctgatggcg caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga 5581 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 5641 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 5701 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag 5761 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 5821 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 5881 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 5941 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 6001 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 6061 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg 6121 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 6181 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 6241 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 6301 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 6361 agttcttctg aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc 6421 tgtgcggtat ttcacaccgc atacaggtgg cacttttcgg ggaaatgtgc gcggaacccc 6481 tattttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 6541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 6601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 6661 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 6721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 6781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 6841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 6901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 6961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 7021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 7081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 7141 ttttacggtt cctgggcttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 7201 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 7261 gaacgaccga gcgcagcgag tca

[0149] MQ833 is replication incompetent but, as demonstrated herein, can profoundly remodel the TIME to promote both innate and adaptive antitumor immunity. As demonstrated herein, MQ833-induced neutrophil recruitment and activation is independent of mature T and B cells, and IT MQ833 delayed tumor growth in subjects completely or partially lacking an adaptive immune system. Intratumoral (IT) delivery of MQ833 generates potent antitumor responses via CD8+ T cells, neutrophils, and M1-like 95 4873-2549-9626.1Atty. Dkt. No.115872-3093 macrophages, the nucleic acid-sensing pathways mediated by MDA5 / STING, and interferon feedback loop. IT MQ833 promotes the recruitment and activation of neutrophils and inflammatory monocytes into the injected tumors, depletion of M2-like macrophages, and expansion of M1-like macrophages, generating potent antitumor immunity against tumors resistant to ICB due to MHC-I antigen presentation and B2M deficiencies. IT administration of MQ833 demonstrated antitumor efficacy in B2m- / -tumors resistant to CD8+T cell killing. As demonstrated herein, IT MQ833 can be used to treat subjects with primary or acquired resistance to ICB. MQ833 is also efficacious in treating tumors in subjects lacking an adaptive immune response. MQ953

[0150] The disclosure of the present technology relates to modified vaccinia Ankara (MVA) viruses referred to as “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L” or “MQ953,” which may be used interchangeably and which refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, an IL-12 transgene, a mutant C12L gene. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g. RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 8) which anchors the IL-12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833, and the C12L gene is further mutated to generate MQ953. In some embodiments, the C12L gene is deleted. In some embodiments, illustrative, non-limiting, plasmid constructs for the mutation C12L is shown in Table 8. MQ954

[0151] The disclosure of the present technology relates to modified vaccinia Ankara (MVA) viruses referred to as “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L- hG-CSF” or “MQ954,” which may be used interchangeably and refer to a modified vaccinia 96 4873-2549-9626.1Atty. Dkt. No.115872-3093 Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, an IL-12 transgene, a mutant C12L gene, and a G-CSF transgene, which may be inserted into the C12L locus. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g. RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 8) which anchors the IL-12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833, the C12L gene is further mutated to generate MQ953, and the G-CSF transgene is inserted into the C12L gene locus to generate MQ954. In some embodiments, the C12L gene is deleted. In some embodiments, the G-CSF transgene is inserted into the C12L locus. In some embodiments, illustrative, non-limiting, plasmid constructs for the mutation of C12L and the insertion of the G-CSF transgene are shown in Table 9. Table 9: G-CSF insertion into N1L locus plasmid pUC57-MVA-ΔN1LpLEO160-hG-CSF (SEQ ID NO: 18) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ttaattacat gttgttttat 421 atatcgctac gaatttaaac agagaaatca gtttaggaaa aaaaaatatc tatctacatc 481 atcacgtctc tgtattctac gatagagtgc tactttaaga tgagacatat ccgtgtcatc 541 aaaaatatac tccattaaaa tgattattcc ggcagcgaac ttgatattgg atatatcaca 601 acctttgtta atatctacga caatagacag cagtcccatg gttccataaa cagtgagttt 661 atctttcttt gaagagatat tttgtagaga tcttataaaa ctgtcgaatg acatcgcatt 721 tatatcttta gctaaatcgt atatgttacc atcgtaatat ctaaccgcgt ctatcttaaa 781 cgtttccatc gctttaaaga cgtttccgat agatggtctc atttcatcag tcatactgag 841 ccaacaaata taatcgtgta taacatcttt gatagaatca gactctaaag aaaacgaatc 901 ggctttatta tacgcattca tgataaactt aatgaaaaat gtttttcgtt gtttaagttg 961 gatgaatagt atgtcttaat aattgttatt atttcattaa ttaatattta gtaacgagta 1021 cactctataa aaacgagaat gacataacta gttatcaaag tgtaggttac aagtgacaat 1081 ccgtcggacg ttgcttttca cgtcgccttt taccacgacc cttcggacgt ctaggttgag 1141 caagatgtct caatactcta taggatactt ccaagaaact ttgtagatga cttgctacta 1201 atacaccacc agctcttctt tgaaacgcac tagcaaatgc gggcatcgct ccctgtgtcg 1261 gttggagcgc aggtgccata cctagttctt ccatttgctg ccagatagta gtcgcgaaat 1321 cggcgacatc tagttggagg gtatctaggg ttggtcctag ttctggagaa attccctcta 1381 aggcttggag aagaccttga tacaagaata gtccggaatg caattgcgac aaacatcccg 97 4873-2549-9626.1Atty. Dkt. No.115872-3093 1441 ccagttgcaa cgcttgggat gggcaggaag agagaggtgc ccaaggaatt cctaaagaat 1501 gacctagtag tactaattct tccggatggc atagtttata agtagcgcac tctgatacca 1561 atttttcttg tagagccgct ccgtcacctt gaatttttct aacctgctcc agacacttta 1621 gaagaaacga ctgtggtaaa gatgatgcag gtcccagagg tgtcgcttct tgtactgtcc 1681 acaaagcaga atgccataac aagagctgta atgccattaa tttcatcgga gactgagtag 1741 ccggtcctgc catctcgagg aattcggtac cccgggttcg aaatcgataa gcttggatcc 1801 ggagagctcc caacctcgag gaattcggta ccccgggttc gaaatcgata agcttggatc 1861 cggagagctc ccaacctcga gctagcgtct gaaacgagac gctaattagt gtatattttt 1921 tcaattttac cggatattta tattccaaaa aaaaaaaata aaaggcctga ttagttaaca 1981 actatttttt atgttaaatc aattagtaca ccgctatgtt taatacttat tcatatttta 2041 gtttttagga ttgagaatca atacaaaaat taatgcatca ttaattttag aaatacttag 2101 tttccacgta gtcaatgaaa catttgaact catcgtacag gacgttctcg tacaggacgt 2161 aactataaac cggtttatat ttgttcaaga tagatacaaa tccgataact ttttttacga 2221 attctacggg atccacttta aaagtgtcat accgggttct ttttattttt ttaaacagat 2281 taatggtgtg atgttgatta ggtcttttac gaatttgata tagaatagcg tttacatatt 2341 ctccataatg gtcaatcgcc atttgttcgt atgtcataaa ttctttaatt atatgacact 2401 gtgtattatt tagttcatcc ttgttcatca ttaggaatct atccaatatg gcaattatac 2461 tagaactata ggtgcgttgt atacacatat tgatgtgtct gtttatacaa tccatgctac 2521 taccttcggg taaaattgta gcatcatata ccatttctag tactttaggt tcattgttat 2581 ccattgcaga ggacgtcatg atcgcatcct aaaaaaatat attattttta tgttattttg 2641 ttaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 2701 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 2761 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 2821 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 2881 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 2941 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 3001 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 3061 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 3121 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 3181 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 3241 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 3301 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 3361 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 3421 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 3481 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 3541 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 3601 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 3661 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 3721 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 3781 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 3841 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 3901 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 3961 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 4021 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 4081 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 4141 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 4201 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 4261 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 4321 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 4381 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 4441 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 4501 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 4561 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 4621 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 4681 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 4741 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 4801 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 4861 cgtatcacga ggccctttcg tc 98 4873-2549-9626.1Atty. Dkt. No.115872-3093 MQ955

[0152] The disclosure of the present technology relates to modified vaccinia Ankara (MVA) viruses referred to as “MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L- hG-CSF-ΔN1L-hIL-7” or “MQ955,” which may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, an IL-12 transgene, a mutant C12L gene, a G-CSF transgene, is inserted into the C12L locus, and a IL-7 transgene, which is inserted into the N1L locus. However, in some embodiments, the IL-7 transgene may be inserted into the N1L / N2L loci, or the C12L lcus by linking hG-CSF and hIL-7 with a P2A sequence. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g., RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 8) which anchors the IL-12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833, the C12L gene is mutated to generate MQ953, the G-CSF transgene is inserted into the C12L locus to generate MQ954, and the IL-7 transgene may be inserted to the C12L locus, the N1L locus, or the N1L and N2L loci to generate MQ955. For example, in some embodiments, the IL-7 transgene may be inserted into the locus or loci of any one or more MVA genes that have been mutated (e.g., deleted). Illustrative, non-limiting examples of such loci into which IL-7 may be inserted may include the C12L locus, the N1L locus, or the N1L and N2L loci. In some embodiments, the N1L gene is deleted. In some embodiments, the N1L and N2L genes are deleted. In some embodiments, the IL-7 transgene is inserted into the N1L locus or the N1L and N2L loci. In some embodiments, the IL-7 transgene is inserted into the C12L locus. In some embodiments, the G-CSF and IL-7 transgenes linked with P2A sequence are inserted into the N1L locus, the N1L and N2L loci, or the C12L locus. In some embodiments, illustrative, non-limiting, plasmid constructs for the the insertion of the IL-7 transgene include those shown in Table 10. 99 4873-2549-9626.1Atty. Dkt. No.115872-3093 Table 10: IL-7 insertion plasmid for inserting IL-7 into C12L locus pUC57-MVA-ΔC12L-hIL7 (SEQ ID NO: 19) (FIG.35A) 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgccggaagg gactatatga 421 ctaacttatc tagagataca ggaatacaac aatcaaaact tactgaaact gtcaaaaaaa 481 tagaaacata tatggtctat atatacacta caatttagtt attaattgga taaccgatgt 541 gattatcaat caatattaag aaggttggta aattggtaca tagctaataa tacctataca 601 cccaataata caacaaccat ttctgagttg gatatcatca aaatactgga taaatacgag 661 gacgtgtata gagtaagtaa agaaaaagaa tgtgaaattt gctatgaagt tgtttactca 721 aaacgataga tactttggtt tattggattc gtgtaatcat atattttgca taacatgcat 781 caatatatgg catagaacac gaagagaaac cggtgcgtcg gataattgtc ctatatgtcg 841 tacccgtttt agaaacataa caatgagcaa gttaactaat aaataaaaag tttaatttgt 901 tgacgacgta tgtcgttatt ttttctcgta taaaagatta atttgattct aatataatct 961 ttagtattgg ataaatatca attcaaatta attccattag attatatcat aaataaaaat 1021 agtagcacgc aaggctataa atgacagtca gttggacgtt gtttctcccg tcttctttta 1081 ccccttccct ttggtctacg atgttctttt gtgcccatca aaattttatt ccaacaagtt 1141 ttgatttcct gtaacaatct tttcaagaaa cacaaatcgt tcaatttttt ttgctccttc 1201 agtgacttat tttcttcaag ggattttgtc ggttgggctt ctcccaaagc agcgggtttc 1261 ctacccttaa cttgacctgt gcagttcagt aggatggtcg ttccttctga tactttcaac 1321 aaatgcaaat cgaaatctcc ggtggagttc atctttagaa attgtcttag cttccgcgct 1381 gcccgaaaaa ggaacatacc ctctttattg gcatcacata tatgtcgctt gaaaaaatta 1441 aactcattgt taagacaatt ggacccaatt tctttcatac tgtctaacag ttggtcaata 1501 gaaaccataa gcactgattc atactgtttt ccatcctttc cttcgatatc acaatcactg 1561 ctggcaacag gtagcagtac caaaattagc ggcggcaggc cgaagatata tctaaatgac 1621 acatgaaaca tttatgatct acttccttac cgtgcaataa attagaatat attttctact 1681 tttacgagaa attaattatt gtatttatta tttatgggtg aaaaacttac tataaaaagc 1741 gggtgggttt ggaattagtg aggcctcttg ccaaaatatc actagaaaaa atttatttat 1801 caattttaaa ggtataaaaa atacttattg ttgctcgaat attttgtatt tgatggtata 1861 cggaagatta gaaatgtagg tattatcatc aactgattct atggttttat gtattctatc 1921 atgtttcact attgcgttgg aaataatatc atatgcttcc acatatattt tattttgttt 1981 taactcataa tactcacgta attctggatt attgacatat ctatgaataa ttttagctcc 2041 atgatcagta aatattaatg agaacatagt attaccacct accattattt ttttcatctc 2101 attcaattct taattgcaaa gatctatata atcattatag cgttgactta tggactctgg 2161 aatcttagac gatgtacagt catctataat catggcatat ttaatacatt gttttatagc 2221 atagtcgtta tctacgatgt tagatatttc tctcaatgaa tcaatcacac aatctaatgt 2281 aggtttatga cataatagca ttttcagcag ttcaatgttt ttagattcgt tgatggcaat 2341 ggctatacat gtatatccgt tatttgatct aatgttgaca tctgaaccgg atagcttggc 2401 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 2461 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 2521 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 2581 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 2641 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 2701 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 2761 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 2821 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 2881 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2941 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 3001 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 3061 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 3121 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 3181 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 3241 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 3301 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 3361 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 3421 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 100 4873-2549-9626.1Atty. Dkt. No.115872-3093 3481 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 3541 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 3601 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 3661 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 3721 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 3781 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 3841 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3901 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3961 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 4021 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 4081 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 4141 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 4201 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 4261 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 4321 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 4381 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 4441 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 4501 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 4561 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 4621 gccctttcgt c OV-IL-12

[0153] The disclosure of the present technology relates to vaccinia viruses referred to as “VACV-ΔE3L83N-ΔE5R-hFlt3L-mOX40L-ΔJ2R-ΔWR199-ΔWR200-mIL-12” or “VACV-ΔE3L83N- hFlt3LmOX40LΔE5R--ΔJ2R- mIL-12ΔWR199ΔWR200” or “OV-IL- 12” which may be used interchangeably and refer to a modified vaccinia (VACV) mutant virus that has been engineered to comprise a deletion of the sequence encoding the 83 N terminus amino acids of the E3 gene (ΔE3L83N), a mutation of the E5R gene, an hFlt3L transgene, an mOX40L transgene, a mutation of the J2R gene, a mutation of the WR199 gene, a mutation of the WR200 gene, and an mIL-12 transgene. In some embodiments, the E5R mutation is a deletion. In some embodiments, the J2R mutation is a deletion. In some embodiments, the WR199 mutation is a deletion. In some embodiments, the WR200 mutation is a deletion. In some embodiments, the hFlt3L transgene, the mOX40L transgene, or some combination thereof, are inserted at the E5R locus. In some embodiments, the mIL-12 transgene is inserted at the WR199 and WR200 locus. Extensive discussion of the VACV virus genome and these transgenes can be found in WO 2020 / 056424, which is hereby incorporated by reference in it’s entirey. XV. Type IFN and the Cytosolic DNA-Sensing Pathway in Tumor Immunity

[0154] Type I IFN plays important roles in host antitumor immunity (Fuertes et al., Trends Immunol.34:67-73 (2013)). IFNAR1-deficent mice are more susceptible to developing tumors after implantation of tumor cells; spontaneous tumor-specific T-cell 101 4873-2549-9626.1Atty. Dkt. No.115872-3093 priming is also defective in IFNAR1-deficient mice (Diamond et al., J. Exp. Med. 208:1989-2003 (2011); Fuertes et al., J. Exp. Med.208:2005-2016 (2011)). More recent studies have shown that the cytosolic DNA-sensing pathway is important in the innate immune sensing of tumor-derived DNA, which leads to the development of antitumor CD8+T-cell immunity (Woo et al., Immunity 41:830-842 (2014)). This pathway also plays a role in radiation-induced antitumor immunity (Deng et al., Immunity 4: 843-852 (2014)). Although spontaneous anti-tumor T-cell responses can be detected in patients with cancers, cancers eventually overcome host antitumor immunity in most patients. Novel strategies to alter the tumor immune suppressive microenvironment would be beneficial for cancer therapy. XVI. Immune Response

[0155] In addition to induction of the immune response by up-regulation of particular immune system activities (such as antibody and / or cytokine production, or activation of cell mediated immunity), immune responses may also include suppression, attenuation, or any other down-regulation of detectable immunity, so as to reestablish homeostasis and prevent excessive damage to the host’s own organs and tissues. In some embodiments, an immune response that is induced according to the methods of the present disclosure generates effector T-cells (e.g., helper, killer, regulatory T-cells). In some embodiments, an immune response that is induced according to the methods of the present disclosure generates effector CD8+(antitumor cytotoxic CD8+) T-cells or activated T helper (TH) cells (e.g., effector CD4+T-cells), or both that can bring about directly or indirectly the death, or loss of the ability to propagate, of a tumor cell.

[0156] Induction of an immune response by the compositions and methods of the present disclosure may be determined by detecting any of a variety of well-known immunological parameters (Takaoka et al., Cancer Sci.94:405-11 (2003); Nagorsen et al., Crit. Rev. Immunol.22:449-62 (2002)). Induction of an immune response may therefore be established by any of a number of well-known assays, including immunological assays. Such assays include, but need not be limited to, in vivo, ex vivo, or in vitro determination of soluble immunoglobulins or antibodies; soluble mediators such as cytokines, chemokines, hormones, growth factors and the like as well as other soluble small peptide, carbohydrate, nucleotide and / or lipid mediators; cellular activation state changes as determined by altered functional or structural properties of cells of the immune system, for example cell 102 4873-2549-9626.1Atty. Dkt. No.115872-3093 proliferation, altered motility, altered intracellular cation gradient or concentration (such as calcium); phosphorylation or dephosphorylation of cellular polypeptides; induction of specialized activities such as specific gene expression or cytolytic behavior; cellular differentiation by cells of the immune system, including altered surface antigen expression profiles, or the onset of apoptosis (programmed cell death); or any other criterion by which the presence of an immune response may be detected. For example, cell surface markers that distinguish immune cell types may be detected by specific antibodies that bind to CD4+, CD8+, or NK cells. Other markers and cellular components that can be detected include but are not limited to interferon γ (IFN-γ), tumor necrosis factor (TNF), IFN-α, IFN-β (IFNB), IL-6, and CCL5. Common methods for detecting the immune response include, but are not limited to, flow cytometry, ELISA, immunohistochemistry. Procedures for performing these and similar assays are widely known and may be found, for example in Letkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, Current Protocols in Immunology, 1998). XVII. Pharmaceutical Compositions and Preparations of the Present Technology

[0157] Disclosed herein are pharmaceutical compositions comprising MVAΔE5R-hFlt3L- OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG- CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF- IL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors, that may contain a carrier or diluent, which can be a solvent or dispersion medium containing, for example, water, saline, Tris buffer, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be affected by various antibacterial and antifungal agents and preservatives, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars or sodium chloride, and buffering agents are included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, 103 4873-2549-9626.1Atty. Dkt. No.115872-3093 aluminum monostearate and gelatin or carrier molecules. Other excipients may include wetting or emulsifying agents. In general, excipients suitable for injectable preparations can be included as apparent to those skilled in the art.

[0158] Pharmaceutical compositions and preparations comprising MVAΔE5R-hFlt3L- OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG- CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF- ΔN1L-hIL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors, may be manufactured by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical viral compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate formulating virus preparations suitable for in vitro, in vivo, or ex vivo use. The compositions can be combined with one or more additional biologically active agents and may be formulated with a pharmaceutically acceptable carrier, diluent or excipient to generate pharmaceutical (including biologic) or veterinary compositions of the instant disclosure suitable for parenteral or intratumoral administration.

[0159] Many types of formulation are possible as is appreciated by those skilled in the art. The particular type chosen is dependent upon the route of administration chosen, as is well- recognized in the art. For example, systemic formulations will generally be designed for administration by injection, e.g., intravenous, as well as those designed for intratumoral delivery. In some embodiments, the systemic or intratumoral formulation is sterile.

[0160] Sterile injectable solutions are prepared by incorporating MVAΔE5R-hFlt3L- OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG- CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF- ΔN1L-hIL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors, in the required amount of the appropriate solvent with various other ingredients enumerated herein, as required, followed by suitable sterilization means. Generally, dispersions are prepared by incorporating the 104 4873-2549-9626.1Atty. Dkt. No.115872-3093 various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying techniques, which yield a powder of the virus plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0161] In some embodiments, the compositions of the present disclosure comprising MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors, may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hanks’s solution, Ringer’s solution, mannitol solutions or physiological saline buffer. In certain embodiments, any of the compositions comprising MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors, may contain formulator agents, such as suspending, stabilizing, penetrating or dispersing agents, buffers, lyoprotectants or preservatives such as polyethylene glycol, polysorbate 80, 1-dodecylhexahydro-2H-azepin-2-one (laurocapran), oleic acid, sodium citrate, Tris HCl, dextrose, propylene glycol, mannitol, polysorbate polyethylenesorbitan monolaurate (Tween®-20), isopropyl myristate, benzyl alcohol, isopropyl alcohol, ethanol sucrose, trehalose and other such generally known in the art may be used in any of the compositions of the instant disclosure. (Pramanick et al., Pharma Times 45(3):65-76 (2013)).

[0162] The biologic or pharmaceutical compositions of the present disclosure can be formulated to allow the virus contained therein to be available to infect tumor cells upon administration of the composition to a subject. The level of virus in serum, tumors, and if 105 4873-2549-9626.1Atty. Dkt. No.115872-3093 desired other tissues after administration can be monitored by various well-established techniques, such as antibody-based assays (e.g., ELISA, immunohistochemistry, etc.).

[0163] The engineered poxviruses of the present technology can be stored at -80°C with a titer of 3.5 x107pfu / mL formulated in about 10 mM Tris, 140 mM NaCl pH 7.7. For the preparation of vaccine shots, e.g., 102-108or 102-109viral particles can be lyophilized in 100 mL of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. Alternatively, the injectable preparations can be produced by stepwise freeze-drying of the engineered poxvirus in a formulation. This formulation can contain additional additives such as mannitol, dextran, sugar, glycine, lactose, or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers, or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and can be stored between 4°C and room temperature for several months. In some embodiments, the ampoule is stored at temperatures below -20°C.

[0164] For therapy, the lyophilisate can be dissolved in an aqueous solution, such as physiological saline or Tris buffer, and administered either systemically or intratumorally. The mode of administration, the dose, and the number of administrations can be optimized by those skilled in the art.

[0165] The pharmaceutical composition according to the present disclosure may comprise an additional adjuvant. As used herein, an “adjuvant” refers to a substance that enhances, augments, or potentiates the host’s immune response to tumor antigens. A typical adjuvant may be aluminum salts, such as aluminum hydroxide or aluminum phosphate, Quil A, bacterial cell wall peptidoglycans, virus-like particles, polysaccharides, toll-like receptors, nano-beads, etc. (Aguilar et al., Vaccine 25:3752-3762 (2007)). XVIII. Therapeutic Methods of the Present Technology

[0166] In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a heterologous nucleic acid molecule encoding OX40L (OX40L), a heterologous nucleic acid 106 4873-2549-9626.1Atty. Dkt. No.115872-3093 molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding IL-12 (IL-12) (MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12), a MVAΔE5R- hFlt3L-OX40L virus (MQ710), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 virus (MQ832), a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954) virus, a MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955) virus, or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVA, MVAΔE3L, or rMVA; and increased splenic production of effector T-cells as compared to the corresponding MVA, MVAΔE3L, or rMVA strain. In some embodiments, the subject is a human. In some embodiments, the composition of the present technology comprising MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors, is administered to the subject by intratumoral or intravenous injection or a simultaneous (i.e., concurrent) or sequential combination of intratumoral and intravenous injection. In one aspect the present disclosure provides a method for administering an adjuvant to a subject in need thereof, wherein the adjuvant comprises the composition of the present technology comprising MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and optionally G-CSF, IL-7, and / or one or more immune checkpoint blockade inhibitors. 107 4873-2549-9626.1Atty. Dkt. No.115872-3093 For examples of compositions comprising recombinant viruses for use as adjuvants and other relevant materials, see WO 2020 / 056424, which is incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0167] In some embodiments, the subject is diagnosed with a cancer such as melanoma, colon carcinoma, breast cancer, prostate cancer, sarcoma, soft-tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor and other bone tumors (e.g., osteosarcoma, malignant fibrous histiocytoma), leiomyosarcoma, rhabdomyosarcoma, pancreatic cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, brain / CNS tumors (e.g., astrocytoma, glioma, glioblastoma, childhood tumors, such as atypical teratoid / rhabdoid tumor, germ cell tumor, embryonal tumor, ependymoma) medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, head-and-neck cancer, rectal adenocarcinoma, glioma, urothelial carcinoma, uterine (e.g., endometrial cancer, fallopian tube cancer) non-small cell lung cancer (squamous and adenocarcinoma), ductal carcinoma in situ, and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal cancer, eye cancer (e.g., melanoma, retinoblastoma), gallbladder cancer, gastrointestinal cancer, heart cancer, laryngeal and hypopharyngeal cancer, oral cancer (e.g., lip, mouth, salivary gland), nasopharyngeal cancer, neuroblastoma, peritoneal cancer, pituitary cancer, Kaposi’s sarcoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, parathyroid cancer, malignant nerve sheath tumor, malignant peripheral nerve sheath tumor, anaplastic thyroid cancer vaginal tumor, and the metastases of any of the foregoing. In some embodiments, the cancer is a sarcoma.

[0168] In some embodiments, the subject is diagnosed with an immune checkpoint blockade inhibitor (ICB) resistant cancer. In some embodiments, the an immune checkpoint 108 4873-2549-9626.1Atty. Dkt. No.115872-3093 blockade inhibitor resistant cancer melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, malignant peripheral nerve sheath tumors, pancreatic cancer, malignant nerve sheath tumors, malignant peripheral nerve sheath tumors, anaplastic thyroid cancer, pancreatic cancer or Extramammary Paget disease (EMPD). Other cancers not expressly recited here can also be ICB inhibitor-resistant. In some embodiments, the ICB resistant cancer is a solid tumor. In some embodiments, the ICB resistant cancer is a sarcoma. In some embodiments, the cancer is ICB inhibitor resistant due to a deficiency in MHC-I antigen presentation. In some embodiments, the ICB inhibitor resistance is due to a mutation in the B2M gene and / or reduced B2M activity. The B2M protein plays an important role in MHC-I antigen presentation. In some embodiments, the ICB inhibitor resistance is due to loss-of-function of the histone- modifying polycomb repressive complex 2 (PRC2). In some embodiments, a tumor that is resistant to ICB treatment may comprise a mutation in the EED and / or SUZ12 genes and / or reduced EED and / or SUZ12 activity. In some embodiments, an ICB inhibitor-resistant tumor is resistant to CD8+T cell mediated immune responses. In some embodiments, a tumor is diagnosed as being resistant to treatment with ICB inhibitors due to lack of response to administration of one or more ICB inhibitors, such as, for example, either an increase or a lack of reduction in tumor size or volume, metastasis, nodal invasion, or progressive disease in spite of treatment with one or more ICB inhibitors. In some embodiments, the ICB resistant cancer comprises a solid tumor that is resistant to immune checkpoint blockade inhibitor treatment and / or wherein the tumor comprises tumor cells comprising loss or mutation of the beta-2-microglobulin (B2M) gene or are characterized by downregulation of B2M, and / or wherein the tumor is MHC-1 antigen presentation deficient. In some embodiments, engineered poxviruses of the present technology, i.e., MVAΔE5R- hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG- CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof, are not administered in combination with (i.e., separately, sequentially, or simultaneously with) any immune checkpoint blocking inhibitor. In some embodiments, a subject has previously received treatment with an immune checkpoint blockade inhibitor (e.g., has received at least one dose or in some 109 4873-2549-9626.1Atty. Dkt. No.115872-3093 cases has received a full course of treatment with an ICB inhibitor) prior to starting treatment with MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof. In some embodiments, the subject continues to receive treatment with an ICB inhibitor upon starting treatment with MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof. In other cases, treatment with an ICB inhibitor is discontinued upon starting treatment with MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof. In some cases, a subject is treated with MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof, and also with another therapy or treatment such as chemotherapy, hormonal treatment, surgery, or radiation. XIX. Combination Therapy

[0169] In some embodiments, the engineered poxviruses of the present technology (e.g., MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof) 110 4873-2549-9626.1Atty. Dkt. No.115872-3093 are combined or separately, sequentially, or simultaneously (i.e., concurrently) administered with G-CSF, IL-7, one or more anti-cancer agents, such as one or more chemotherapy agents, one or more hormonal agents, or one or more immune checkpoint blockade inhibitors. Thus, in some cases for treating an ICB inhibitor resistant cancer, the engineered poxviruses may be administered with G-CSF, IL-7, and / or one or more ICB inhibitors. In some embodiments, the combined administration of the engineered poxviruses of the present technology (e.g., MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof) with one or more immune checkpoint blockade inhibitors (such as anti-PD-1, anti-PD-L1, or anti-CTLA4 antibodies), IL-7, and / or G-CSF may result in a synergistic effect with respect to the treatment of solid tumors. Immune Checkpoint Blockade Inhibitors

[0170] In some embodiments, MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof is combined or separately, sequentially, or simultaneously (i.e., concurrently) administered with one or more immune checkpoint blockade (ICB) inhibitors. The one or more immune checkpoint blockade inhibitors may target any one or more of PD- 1 (programmed death 1), PD-L1 (programmed death ligand 1), or CTLA-4 (cytotoxic T lymphocyte antigen 4) (e.g., anti-huPD-1, anti-huPD-L1, or anti-huCTLA-4 antibodies).

[0171] In some embodiments, the one or more immune checkpoint blockade inhibitors are selected from ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, and durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T- cell immunoglobulin and mucin-3), B7-H3, B7-H4, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, 111 4873-2549-9626.1Atty. Dkt. No.115872-3093 Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS (inducible T-cell costimulatory), DLBCL (diffuse large B-cell lymphoma) inhibitors, BTLA (B and T lymphocyte attenuator), PDR001, and any combination thereof. Dosage ranges of the foregoing are known in or readily within the skill in the art as several dosing clinical trials have been completed, making extrapolation to other agents possible.

[0172] In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and one or more immune checkpoint blockade inhibitors may result in a synergistic effect. In some embodiments, the combination of MVAΔE5R- hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG- CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and one or more immune checkpoint blockade inhibitors may result in an enhanced anti-tumor effect. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and anti-PD-L1 may result in a synergistic effect with respect to the treatment of solid tumors. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and anti-PD-1 may result in a synergistic effect with respect to the treatment of solid tumors. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 112 4873-2549-9626.1Atty. Dkt. No.115872-3093 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and anti-CTLA-4 may result in a synergistic effect with respect to the treatment of solid tumors.

[0173] It has been reported that the sequential (i.e., serial) administration of anti-OX40 antibody followed by the immune checkpoint blockade inhibitor, anti-PD-1 antibody, improves the therapeutic efficacy of the combination, resulting in delayed tumor progression and, in some cases, complete tumor regression. (See, e.g., Shrimali et al., Cancer Immunol. Res.5(9): OF1-OF12 (2017); Messenheimer et al., Clin. Cancer Res. 23(20):6165-6177 (2017)). However, the same studies show that the simultaneous (i.e., concurrent) administration of anti-OX40 antibody and anti-PD-1 antibody negates the anti- tumor effects of OX40 antibody and results in poor treatment outcomes in mice. (See, Shrimali et al., (2017); Messenheimer et al., (2017)). In some embodiments, the combination of the viruses expressing the OX40L transgene of the present technology, e.g., MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and one or more immune checkpoint blockade inhibitors (e.g., anti-PD-L1 antibody, anti- PD-1 antibody, anti-CTLA-4 antibody) may result in an enhanced anti-tumor effect as compared to the combination of an immune checkpoint blockade inhibitor and anti-OX40 agonist antibody. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and one or more immune checkpoint blockade inhibitors (e.g., anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody) may result in a synergistic effect with respect to the treatment of solid tumors as compared to the combination of an immune checkpoint blockade inhibitor and anti-OX40 agonist antibody. 113 4873-2549-9626.1Atty. Dkt. No.115872-3093

[0174] In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof and one or more immune checkpoint blockade inhibitors may result in an enhanced anti-tumor effect against tumors that are resistant to immune checkpoint blockade inhibitor treatment. In some embodiments, the ICB resistant tumor is melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, malignant peripheral nerve sheath tumors, pancreatic cancer, malignant nerve sheath tumors, malignant peripheral nerve sheath tumors, anaplastic thyroid cancer, pancreatic cancer or Extramammary Paget disease (EMPD). G-CSF

[0175] In some embodiments, MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof is combined or separately, sequentially, or simultaneously (i.e., concurrently) administered with G-CSF.

[0176] Dosage ranges of the foregoing are known in or readily within the skill in the art as several dosing clinical trials have been completed, making extrapolation to other agents possible. For example, G-CSF is approved for therapeutic use in humans as lenograstim (Granocyte) and filgrastim (Neupogen, Zarzio, Nivestim, Accofil) and a pegylated-G-CSF is approved for therapeutic use in humans as long acting (pegylated) filgrastim (pegfilgrastim, Neulasta, Pelmeg, Ziextenco) and lipegfilgrastim (Lonquex). The dosage ranges and regimens of such agents are known to one of skill in the art.

[0177] In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- 114 4873-2549-9626.1Atty. Dkt. No.115872-3093 OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and G-CSF may result in a synergistic effect. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and G-CSF may result in an enhanced anti-tumor effect. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and G-CSF may result in a synergistic effect with respect to the treatment of solid tumors, including ICB resistant tumors. IL-7

[0178] In some embodiments, MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or a combination thereof is combined or separately, sequentially, or simultaneously (i.e., concurrently) administered with IL-7.

[0179] Dosage ranges of the foregoing are known in or readily within the skill in the art as several dosing clinical trials have been completed, making extrapolation to other agents possible. The dosage ranges and regimens of such agents are known to one of skill in the art.

[0180] In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- 115 4873-2549-9626.1Atty. Dkt. No.115872-3093 OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and IL-7 may result in a synergistic effect. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and IL-7 may result in an enhanced anti-tumor effect. In some embodiments, the combination of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof, and IL-7 may result in a synergistic effect with respect to the treatment of solid tumors, including ICB resistant tumors. XX. Kits Comprising Engineered Poxviruses of the Present Technology

[0181] The present disclosure provides for kits comprising one or more compositions comprising one or more of the engineered poxviruses, e.g., MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), described hereintogether with instructions for the administration of the engineered poxviruses to a subject to be treated. The instructions may indicate a dosage regimen for administering the composition or compositions as provided below. In some embodiments, the kits contain instructions for methods for treating a solid tumor wherein the solid tumor is resistant to immune checkpoint blockade inhibitor treatment, methods of preventing cancer recurrence for a period of time in a subject in need thereof, methods for treating a tumor in a subject in need thereof wherein the subject has a deficient adaptive 116 4873-2549-9626.1Atty. Dkt. No.115872-3093 immune system response, and methods for altering the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof.

[0182] In some embodiments, the kit may also comprise an additional therapeutic composition, such as, for example, a composition comprising G-CSF, IL-7, one or more chemotherapeutic agents, and / or immune checkpoint blockade inhibitors for conjoint administration with the engineered poxvirus, e.g., MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7 (MQ955), or combinations thereof. XXI. Effective Amount and Dosage of the Engineered Poxviruses of the Present Technology

[0183] In general, in some embodiments, the subject is administered a dosage of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12 (MQ833), MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L (MQ953), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF (MQ954), MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-IL-7 (MQ955), or combinations thereof in the range of about 106to about 1010plaque forming units (pfu), although a lower or higher dose may be administered. In some embodiments, the dosage ranges from about 102to about 1010pfu. In some embodiments, the dosage ranges from about 103to about 1010pfu. In some embodiments, the dosage ranges from about 104to about 1010pfu. In some embodiments, the dosage ranges from about 105to about 1010pfu. In some embodiments, the dosage ranges from about 106to about 1010pfu. In some embodiments, the dosage ranges from about 107to about 1010pfu. In some embodiments, the dosage ranges from about 108to about 1010pfu. In some embodiments, the dosage ranges from about 109to about 1010pfu. In some embodiments, dosage is about 107to about 109pfu. The equivalence of pfu to virus particles can differ according to the specific pfu titration method used. Generally, a pfu is equal to about 5 to 100 virus particles and 0.69 pfu is about 1 TCID50. A therapeutically effective amount of MVAΔE5R-hFlt3L-OX40L (MQ710), MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199 (MQ832), MVAΔE3LΔE5R-hFlt3L- 117 4873-2549-9626.1Atty. Dkt. No.115872-3093 OX40LΔWR19...

Claims

Atty. Dkt. No.115872-3093 CLAIMS WHAT IS CLAIMED IS:

1. A method for treating a solid tumor in a subject in need thereof comprising administering to a tumor a composition comprising an effective amount of a modified vaccinia Ankara (MVA) virus genetically engineered to comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a heterologous nucleic acid molecule encoding OX40L, a heterologous nucleic acid molecule encoding hFlt3L, and a heterologous nucleic acid molecule encoding IL-12 (MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12), and a composition comprising an effective amount of hG- CSF.

2. The method of claim 1, wherein the virus and the hG-CSF are administered to the subject by the same route or by different routes.

3. The method of claim 1, wherein the virus is administered to the subject by intratumoral or intravenous injection or a simultaneous or sequential combination of intraturmoral and intravenous injection.

4. The method of claim 1, wherein the hG-CSF is administered to the subject by intratumoral or intravenous injection or a simultaneous or sequential combination of intraturmoral and intravenous injection.

5. The method of claim 1, wherein the virus is administered to the subject by intratumoral injection and the hG-CSF is administered intratumorally or systemically.

6. The method of any one of claims 1-5, wherein the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject.

7. The method of claim 6, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of one or more of anti-tumor T 159 4873-2549-9626.1Atty. Dkt. No.115872-3093 cells, neutrophils, macrophages, and / or monocytes, and / or depletion of pro-tumor M2 macrophages and / or pro-tumor regulatory T cells.

8. The method of claim 6, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils.

9. The method of claim 6, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils and macrophages.

10. The method of claim 6, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils, macrophages, and monocytes.

11. The method of any one of claims 1-10, wherein the tumor is malignant peripheral nerve sheath tumors, melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, pancreatic cancer, malignant nerve sheath tumor, anaplastic thyroid cancer, or Extramammary Paget disease (EMPD).

12. The method of any one of claims 1-11, wherein the tumor is resistant to immune checkpoint blockade inhibitor treatment.

13. A modified vaccinia Ankara (MVA) virus selected from the group consisting of: an MVA virus genetically engineered to comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a mutant C12L gene (ΔC12L), a heterologous nucleic acid molecule encoding OX40L (OX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding interleukin 12 (IL-12) (MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL- 12ΔC12L); an MVA virus genetically engineered to comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a mutant C12L gene 160 4873-2549-9626.1Atty. Dkt. No.115872-3093 (ΔC12L), a heterologous nucleic acid molecule encoding OX40L (OX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), a heterologous nucleic acid molecule encoding interleukin 12 (IL-12), and a heterologous nucleic acid molecule encoding human granulocyte colony-stimulating factor (hG-CSF) (MVAΔE3LΔE5R- hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF); an MVA virus genetically engineered to comprise a mutant E3L gene (ΔE3L), a mutant E5R gene (∆E5R), a mutant WR199 gene (∆WR199), a mutant C12L gene (ΔC12L), a heterologous nucleic acid molecule encoding OX40L, a heterologous nucleic acid molecule encoding hFlt3L, a heterologous nucleic acid molecule encoding IL-12, a heterologous nucleic acid molecule encoding hG-CSF, and a heterologous nucleic acid molecule encoding human interleukin 7 (hIL-7) (MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7); and any combination thereof.

14. An immunogenic composition comprising the virus of claim 13.

15. The immunogenic composition of claim 14, further comprising a pharmaceutically acceptable carrier.

16. The immunogenic composition of claim 14 or claim 15, further comprising a pharmaceutically acceptable adjuvant.

17. A method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the virus of claim 13 or the immunogenic composition of any one of claims 14-16.

18. The method of claim 17, wherein the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject.

19. The method of claim 18, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the 161 4873-2549-9626.1Atty. Dkt. No.115872-3093 tumor in the subject comprises increasing activation and / or expansion of one or more of stem-like T-cells, neutrophils, macrophages, and / or monocytes.

20. The method of claim 19, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of stem-like T-cells.

21. The method of claim 20, wherein the virus is MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-ΔN1L-hIL-7, or MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L further engineered to comprise a heterologous nucleic acid encoding hIL-7, or MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF further engineered to comprise a heterologous nucleic acid encoding hIL-7, or wherein the composition or immunogenic composition further comprises an effective amount of hIL-7.

22. The method of claim 19, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils.

23. The method of claim 19, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils and macrophages.

24. The method of claim 19, wherein inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and / or expansion of anti-tumor neutrophils, macrophages, and monocytes.

25. The method of any one of claims 17-24, wherein the composition is administered to the subject by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection.

26. The method of any one of claims 17-25, wherein the tumor is malignant peripheral nerve sheath tumors, melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell 162 4873-2549-9626.1Atty. Dkt. No.115872-3093 carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, pancreatic cancer, malignant nerve sheath tumor, anaplastic thyroid cancer, Merkel cell carcinoma, or Extramammary Paget disease (EMPD).

27. A method for inducing an immune response in a subject, the method comprising administering to the subject a composition comprising an effective amount of the virus of claim 13, or the immunogenic composition of any one of claims 14-16.

28. The method of claim 27, wherein administration of the virus increases activation and / or expansion of one or more of stem-like T-cells, neutrophils, macrophages, and / or monocytes.

29. The method of claim 28, wherein administration of the virus increases activation and / or expansion of stem-like T-cells.

30. The method of claim 29, wherein the virus is MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L-hG-CSF-hIL-7, or MVAΔE3LΔE5R-hFlt3L- OX40LΔWR199-IL-12ΔC12L further engineered to comprise a heterologous nucleic acid encoding hIL-7, or MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-IL-12ΔC12L-hG-CSF further engineered to comprise a heterologous nucleic acid encoding hIL-7, or wherein the composition or immunogenic composition further comprises an effective amount of hIL-7.

31. The method of claim 28, wherein administration of the virus increases activation and / or expansion of neutrophils.

32. The method of claim 28, wherein administration of the virus increases activation and / or expansion of neutrophils and macrophages.

33. The method of claim 28, wherein administration of the virus increases activation and / or expansion of neutrophils, macrophages, and monocytes.

34. A modified vaccinia virus comprising a deletion of the sequence encoding the 83 n- terminal amino acids of the E3L gene (ΔE3L83N), a mutation of the E5R gene (ΔE5R), a hFlt3L transgene, an mOX40L transgene, a mutation of the J2R gene, a mutation of the WR199 gene, a mutation of the WR200 gene, and a mIL-12 transgene (VACV-ΔE3L83N- ΔE5R-hFlt3L-mOX40L-ΔJ2R-ΔWR199-ΔWR200-mIL-12). 163 4873-2549-9626.1Atty. Dkt. No.115872-3093 35. A method of treating a solid malignant tumor in a subject in need thereof, comprising administering an effective amount of the modified vaccinia virus of claim 34. 164 4873-2549-9626.1

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