WO2020236925A1

WO2020236925A1 - Mammalian cell for producing modified vaccinia ankara (mva) virus

Info

Publication number: WO2020236925A1
Application number: PCT/US2020/033788
Authority: WO
Inventors: Bernard Moss; Linda S. Wyatt; Ruikang LIU; Jorge MENDEZ-RIOS; Chen Peng; Gilad SIVAN; Shira GLUSHAKOW-SMITH
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 2019-05-23
Filing date: 2020-05-20
Publication date: 2020-11-26
Anticipated expiration: 2021-11-23

Abstract

The present invention relates to modified animal cells comprising a vaccinia virus gene encoding a protein having the activity of a vaccinia virus C12, C16, or C17 protein, and/or that is deficient in an activity that inhibits replication of MVA virus, such as ZAP activity. Also provided are altered MVA viruses that encode a protein comprising the activity of vaccinia virus C12 protein, a vaccinia virus C16 protein, or a vaccinia virus C17 protein, or that comprise a mutation in an MVA ORF encoding a decapping protein, such as ORF D9 or D10. Such viruses have an extended host range (i.e., (HRE MVA viruses). Also provided are methods of producing modified cells and HRE MVA viruses of the disclosure, as well as methods of producing progeny MVA virus in modified and unmodified animal cells. Also provided are methods of using MVA viruses of the disclosure to vaccinate individuals.

Description

MAMMALIAN CELL FOR PRODUCING MODIFIED

VACCINIA ANKARA (MV A) VIRUS

TECHNICAL FIELD

The disclosure relates methods of producing modified Vaccinia Ankara (MV A) virus in mammalian cells.

BACKGROUND

Vaccinia virus (VACV) has been developed as a live recombinant expression vector that is widely used for making candidate vaccines against unrelated pathogens. Although VACV was successfully used as a smallpox vaccine, concerns regarding safety with regard to the creation of new vaccines led to interest in more attenuated poxvirus vectors including fowlpox virus, canarypox virus, and recombinant VACV strains in which one or more genes were deleted selectively or by blind passaging. One such attenuated strain, modified vaccinia virus Ankara (MV A), was produced by passaging the parental chorioallantois vaccinia virus (CVA) strain more than 500 times in chicken embryo fibroblasts (CEF) for the purpose of producing a safe smallpox vaccine. Initial analysis of the MVA genome revealed six major deletions compared to the parent virus. These large deletions as well as numerous additional genetic changes were confirmed by genome sequencing. Notwithstanding the loss of considerable genetic material and the consequent inability to efficiently produce infectious virus in most mammalian cells, MVA retains the ability to express viral as well as recombinant proteins regulated by VACV promoters in non-permissive cells at levels comparable to replicating VACV and to induce both humoral and cellular immune responses. These beneficial features propelled the use of MVA for development of numerous candidate vaccines, some of which are in clinical trials.

While MVA virus is shows promise as a vaccine platform, challenges remain. In particular, in order to produce enough vaccine doses for a large-scale immunization campaign, large bulks of tissue culture cells are required. Yet, as discussed above, the range of cells that support high-titer growth of MVA virus is limited. While MVA virus grows well on CEF cells, and while the use of CEF cells is well established in vaccine manufacturing, the production of large bulks of such cells is cumbersome. Primary CEF cells are prepared from embryonated eggs, and are not typically amplified following their isolation from the embryonated eggs. Thus, producing large bulks of CEF cells requires obtaining, incubating and processing millions of embryonated eggs. Additionally, while CEF cells may be cryopreserved for use a later time point, cryopreservation impacts the quality of the cells. Therefore, especially in the context of pandemic preparedness, continuous cell lines that allow for efficient MVA propagation, would be beneficial.

Despite extensive testing of candidate MVA vaccines in humans, the basis for the host-restriction of MVA remains unknown. The large number of deletions, truncations and mutations that occurred during the long passage history of MVA in CEF severely complicates efforts to determine those changes important for its host-range defect. Indeed, a comparison of MVA with its parent CVA revealed 71 orthologous ORFs predicted to encode identical gene products, whereas the remaining 124 ORFs encode gene products with amino acid changes, insertions or deletions. One attempt to investigate the genetic changes responsible for the replication defect consisted of deleting DNA sequences corresponding to the six major deletions of MVA from the genome of the parental CVA. Remarkably, the loss or truncation of 31 open reading frames (ORFS) totaling- 25 kbp of DNA from the parental virus was insufficient to produce the host-range phenotype of MVA, leading to the conclusion that the major determinants lie outside of these deletions. However, the details of the sever host range restriction of MVA remain largely unknown.

It is clear that the severely restricted host rage of MVA virus is an impediment to the efficient and practical use of MVA virus as a vaccine platform. There is a need for an expanded range of cells that are easily maintained culture, in culture, and that allow for the production of high titers of infectious MVA virus. The present invention addresses this need by providing methods of modifying cells and/or MVA viruses in a way that allows for the growth of MVA virus in cells that were previously considered unsuitable for such purpose.

SUMMARY

One aspect of this disclosure provides a recombinant cell comprising a

heterologous gene encoding a heterologous protein comprising the activity of at least one poxvirus protein selected from a vaccinia virus C12L protein, a vaccinia virus C16L protein, a vaccinia virus C17L protein. The at least one heterologous gene may be operatively linked to a promoter recognized by an RNA polymerase of the animal cell.

This recombinant animal cell may be a mammalian cell, a bird cell, a fish cell, an amphibian cell, or a reptilian cell. This recombinant animal cell may be a primate cell, a monkey cell, a human cell, a mouse cell, a hamster cell, or a rabbit cell. For example, the recombinant animal cell may be a human A549 cell, a human MRC-5 cell, a HeLa cell, or a monkey BSC-1 cell. Any of these recombinant animal cells may be derived from an animal cell that has reduced permissiveness or is non-permissive for MVA replication.

In these recombinant animal cells, the heterologous gene may be a homologue of a poxvirus ORF selected from a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, and a vaccinia virus C17L ORF. The heterologous gene may comprise a nucleic acid sequence at least 80% identical to a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, or a vaccinia virus C17L ORF. The heterologous gene may comprise a nucleic acid sequence at least 80% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 100% identical to SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO:5.

In these recombinant animal cells, the heterologous protein may comprise an amino acid sequence at least 80% identical to an amino acid sequence encoded by at least one of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, or a vaccinia virus C17L ORF. The heterologous protein may comprise an amino acid sequence at least 80% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 100% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In these recombinant animal cells, the heterologous gene may encode a protein that has the activity of a protein selected from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

In these recombinant animal cells, the promoter to which the heterologous gene may be optionally linked may be a promoter that is not naturally associated with a homologue of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, or a vaccinia virus C17L ORF, or with a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, or a vaccinia virus C17L ORF. For example, the promoter may be a promoter normally associated with a mammalian gene, or a promoter from the genome of a mammalian virus. For example, the promoter may be a cytomegalovirus promoter, an sv40 promoter, a polyomavirus promoter, a papillomavirus promoter, a herpesvirus promoter, an adenovirus promoter, an adeno-associated virus promoter, or a retrovirus promoter.

These recombinant animal cells are preferably not infected with a poxvirus.

Further, these recombinant animal cells may lack a homologue of a vaccinia virus Cl 1L ORF, a vaccinia virus C13L ORF, and/or a vaccinia virus C14L ORF. These recombinant animal cells may lack a vaccinia virus Cl 1L ORF, a vaccinia virus C13L ORF, and/or a vaccinia virus C14L ORF.

In these recombinant animal cells, the heterologous gene may be present on a plasmid of a cosmid in the recombinant animal cell. Alternatively or additionally, the heterologous gene may be inserted into the genome of the recombinant animal cell. These recombinant animal cells may be deficient in at least one activity that inhibits the replication of modified vaccinia Ankara (MV A) virus. For example, the deficiency may be due to a decrease in the level of the at least one activity, and/or the deficiency may be due to a reduction in the level of a protein that comprises the activity. The deficiency may be in the level of at least one activity associated with a mammalian ZAP protein. For example, the recombinant cell may have a decreased level of ZAP protein or the recombinant animal cell may not produce ZAP protein.

Another aspect of this disclosure provides a method to produce MVA virus by contacting an MVA virus with these recombinant animal cells and incubating the contacted cell under conditions suitable for replication of MVA virus, thereby producing MVA virus.

Another aspect of this disclosure provides a method to produce an altered MVA virus that replicates in animal cells that are non-permissive for the replication of unaltered MVA virus, by inserting into the genome of an MVA virus a gene encoding a protein comprising the activity of at least one protein encoded by a vaccinia virus ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF. Another aspect of this disclosure provides an altered MVA virus produced using these methods, wherein the genome of the altered MVA virus comprises a gene encoding a protein comprising the activity of a protein encoded by at least one vaccinia virus ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF. In these methods, the altered MVA virus may lack a vaccinia virus C13L ORF, a vaccinia virus C16L ORF, a vaccinia virus C17L IORF, or homologues thereof. In these altered MVA viruses, the gene may comprise a nucleic acid sequence at least 80% identical to a vaccinia virus ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF. For example, the gene may comprise a nucleic acid sequence at least 80% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 100% identical identical to SEQ ID NO: l, SEQ ID NO:3, or SEQ ID NO:5. In these altered MVA viruses, the gene may encode a protein comprising an amino acid sequence at least 80% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 100% identical to a vaccinia virus protein selected from the group consisting of vaccinia virus C121 protein, vaccinia virus C16L protein, and vaccinia virus C17L protein. In these methods and altered MVA viruses, the gene may encode a protein comprising an amino acid sequence at least 80% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 100% identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

In these methods and altered MVA viruses, the gene may be inserted into a non- essential ORF in the MVA virus genome. In these methods and altered MVA viruses, the gene may be inserted into an IGR in the MVA virus genome. In these methods and altered MVA viruses, the gene may be functionally linked to a poxvirus promoter. In these methods and altered MVA viruses, the gene may be functionally linked to a promoter that is recognized by an RNA polymerase from an animal cell.

Another aspect of this disclosure provides a method to produce an altered MVA virus that produces titers of virus in non-permissive cells, or in MVA-restrictive cells, that are higher than the titers produced by an unaltered MVA virus in the same type of non- permissive or MVA-restrictive cell, the method comprising introducing one or more mutations into an MVA ORF encoding a poxvirus decapping enzyme. Another aspect of this disclosure provides an altered MVA virus produced using these methods, wherein the genome of the altered MVA virus comprises one or more mutations in an MVA virus ORF encoding a poxvirus decapping enzyme. In these aspects, the MVA ORFs encoding a decapping enzyme may be MVA D9 ORF or D10. In these methods and altered MVA viruses, the introduced one or more mutations may result in substitution mutations in the encoded protein. In certain aspects, the substitutions mutations may be conservative substitutions. In these methods and altered MVA viruses, introduction of one or more mutations into the MVA virus D10 ORF may result in a substitution mutation at a position corresponding to C25, A226, and/or H233 of SEQ ID NO:7. In certain aspects, introduction of one or more mutations into the MVA virus D10 ORF may result in the encoded mutated protein having a substitution mutation at a position corresponding to C25, A226, and/or H233 of SEQ ID NO:7. In certain aspects, introduction of one or more mutations into the MVA virus D10 ORF may result in the encoded mutated protein having a substitution mutation at a position selected from C25, A226, and/or H233 of SEQ ID NO:7. In these methods and altered MVA viruses, the encoded mutated protein may comprise one or more mutations selected from the group consisting of substitution of the amino acid corresponding to C25 of SEQ ID NO:7 with a tyrosine, substitution of the amino acid corresponding to A226 of SEQ ID NO:7 with a threonine, and substitution of the amino acid corresponding to H233 of SEQ ID NO:7 with a tyrosine. In these methods, introduction of one or more mutations into an MVA virus ORF encoding a poxvirus decapping enzyme results in an altered MVA virus that produces titers in MVA-restrictive cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X) higher than the titer produced by non-altered MVA virus in the same type of MVA-restrictive cell. In certain aspects, the MVA-restrictive cells may BS- C-l cells (BSC-1 cells), HeLa cells, or A549 cells. In certain aspects, introduction of one or more mutations into an MVA virus ORF encoding a poxvirus decapping enzyme results in an altered MVA virus that produces titers in BS-C-1 cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X) higher than the titer produced by non-altered MVA virus in BS-C-1 cells.

In certain aspects of the disclosure, methods of the disclosure may be combined to produce an altered MVA virus comprising more than one alteration (mutation) of the disclosure. In certain aspects, a method of the disclosure comprises producing an altered MVA virus that produces titers of virus in non-permissive, or in MVA-restrictive cells, that are higher than the titers produced by an unaltered MVA virus in the same type of non-permissive or MVA restrictive cell, the method comprising 1) inserting into the genome of an MVA virus a gene encoding a protein comprising the activity of a protein encoded by at least one vaccinia virus ORF selected from the group consisting of vaccinia virus Cl 21 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF; and 2) introducing one or more mutations into an MVA ORF encoding a poxvirus decapping enzyme. Another aspect of this disclosure provides an altered MVA virus comprising more than one alteration (mutation) of the disclosure. In certain aspects, an altered MVA virus of the disclosure comprises an insertion into the MVA genome of a gene encoding a protein comprising the activity of a protein encoded by at least one vaccinia virus ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF; and 2) one or more mutations into an MVA ORF encoding a poxvirus decapping enzyme. It should be understood that the limitations and definitions relating to methods and altered viruses comprising individual alterations also apply to the altered viruses, and methods of making same, comprising the multiple alterations.

In these methods and altered MVA viruses, the altered MVA virus may comprise a heterologous nucleic acid molecule encoding an immunogenic protein or a therapeutic agent. The therapeutic agent may be a tumor-suppressor protein, a pro-apototic protein, a cytokine, a lymphokine, a monokine, a growth factor, an enzyme, a hormone, an immunomodulatory protein, a cytotoxic peptide, a suicide protein, a cytotoxin, a pro-drug, or a therapeutic RNA.

Another aspect of this disclosure provides a method to produce progeny altered MVA virus, by contacting these altered MVA viruses with an animal cell and incubating the contacted cell under conditions suitable for replication of the contacted altered MVA virus. In these methods, the animal cell may be non-permissive, or have reduced permissiveness, for replication of wild-type MVA virus. In these methods, the animal cell may be deficient in at least one activity that inhibits replication of MVA virus. In these methods, the deficiency may be a reduction in the level of MVA virus inhibitory activity. In these methods, the deficiency may be a reduction in the level of a protein comprising the MVA virus inhibitory activity. In these methods, the deficiency may be due to a lack of virus inhibitory activity, and/or due to a lack of a protein comprising the MVA virus inhibitory activity, and/or due to the presence of a functional RNA molecule in the cell, wherein the functional RNA molecule affects the level of the MVA virus inhibitory activity. The deficiency may be in the level of at least one activity associated with a mammalian ZAP protein. In these methods, the deficiency may be a reduction in the level of ZAP protein or the deficiency is a lack at least one activity associated with the ZAP protein, or the deficiency is a lack ZAP protein.

Another aspect of this disclosure provides a method of producing progeny MVA virus, by contacting MVA virus with a cell that is deficient in at least one activity that inhibits replication of MVA virus. In these methods, the deficiency is a reduction in the level of MVA virus inhibitory activity, or the deficiency may be a reduction in the level of a protein comprising the MVA virus inhibitory activity, or the deficiency is a lack of virus inhibitory activity, or the deficiency may be due to a lack of a protein comprising the MVA virus inhibitory activity, or the deficiency may be in the level of at least one activity associated with a mammalian ZAP protein. For example, the deficiency may be a reduction in the level of ZAP protein, or the deficiency may be a lack of at least one activity associated with the ZAP protein, or the lack or absence of the ZAP protein, or the deficiency may be due to the presence of a functional RNA molecule in the cell, wherein the functional RNA molecule affects the level of the MVA virus inhibitory activity. In any of these methods, the cell may be a tumor cell.

Another aspect of this disclosure provides a method of treating a tumor cell by contacting the tumor cell with an MVA virus. In these methods, the tumor cell may be deficient in at least one activity that inhibits MVA replication. In these methods, the cell may be deficient in an activity associated with ZAP protein. In these methods, the cell may be deficient in ZAP protein. In these methods, the MVA virus may be a wild-type MVA virus. In these methods, the MVA virus may be an altered MVA virus. In these methods, the tumor cell may be in culture, or the tumor cell may be in an individual.

This disclosure also provides a system for producing MVA virus, including a population of the recombinant animal cells and an MVA virus. Such systems may further include a solid substrate comprising the population of mammalian cells. This disclosure also provides a kit including a mammalian cell and an altered MVA virus of this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the morphogenesis defects of WRASPI-l and MVA. A549 cells infected with VACV WR (panels A and B), WRASPI-l (panels C and D) or MVA (panels E and F) were analyzed by transmission electron microscopy. Abbreviations: MV, mature virion; WV, wrapped virion; C, crescent; IV, immature virion; D, dense virion.

Magnification shown below each panel.

FIGS. 2A and 2B show the requirement of SPI-1 for spread of MVA in human cells. FIG. 2A shows the effect of addition of SPI-1 gene to MVA. CEF, BS-C-1, HeLa, A549 and MRC-5 cells in 12-well plates were infected with MVA or MVA-SPI-1 at MOI of 0.001, 0.01 and 0.1 and overlaid with methylcellulose. After 48 h, the overlay was removed and cells were stained with antibody to VACV. FIG. 2B shows the effect of SPI- 1 gene deletion on v51.2 spread. CEF, BS-C-1, HeLa, A549 and MRC-5 cells in 12-well plates were infected with v51.2 or v51.2ASPI at MOI of 0.001, 0.01 and 0.1 and overlaid with methylcellulose. After 48 h, the overlay was removed and cells were stained with antibody to VACV.

FIG. 3 shows the effect of SPI-1 gene insertions and deletions on spread of additional HRE MVAs. CEF and MRC-5 cells in 12-well plates were infected with the indicated viruses at MOI of 0.001, 0.01, 0.1 and overlaid with methylcellulose. After 48 h, the overlay was removed and cells were stained with antibody to VACV.

FIGS. 4A and 4B show the effect of SPI-1 gene insertions and deletions on yields of recombinant MVAs. FIG. 4A shows the effect on MRC-5 cells infected in triplicate with indicated viruses at a MOI of 0.001 for 48 h. Virus titers were determined in duplicate on CEF. Error bars indicate SEM. FIG. 4B shows the effect on MRC-5 cells infected as in FIG. 4A with MVA-SPI-1 or independently isolated MVA recombinants containing F322A or T309R mutations in the SPI-1 ORF. FIGS. 5A-5E show replication of recombinant MV A, VACV WR, and RPXV in parental or SPI-1 -expressing MRC-5 and A549 cells. FIGS. 5A and 5C show expression of SPI-1. MRC-5 (FIG. 5A) and A549 (FIG. 5C) cells infected with retroviruses that express 2xMyc-SPI-l or control retroviruses and selected by antibiotic resistance. Western blots with antibody to the Myc tag are shown. FIGS. 5B and 5D shows replication of MV A, MVA-SPI-1, v51.2, v51.2ASPI-l at MOI of 0.01 in control and SPI-1 expressing MRC-5 (FIG. 5B) and A549 (FIG. 5D) cells. FIG. 5E shows A549 and MRC-5 cells infected at a MOI of 0.01 in triplicate with VACV WR expressing GFP without (WR- GFP) or with a deletion of SPI-1 (WRA SPI-1 -GFP) and RPXV expressing GFP without (RPXV-GFP) or with a deletion of SPI-1 (RPXVASPI-l-GFP). In FIGS. 5B, 5D, and 5E the cells were infected in triplicate for 48 h and titers were determined in duplicate on CEF. Error bars represent SEM and fold difference in titers are indicated.

FIGS. 6A and 6B shows multiple alignment of genome sequences of the left ends of MV A and HRE MVAs. FIG. 6A shows the alignment of left 80,351 bp. Arrows indicate the lengths and directions of ORFs. Yellow, ORFs common to MVA; Green, ORFs added to MVA by recombination dl, dV and dll indicate the sites of three of the large deletions in MVA. The corresponding Hindlll fragments are shown below the alignments. FIG. 6B shows the alignment of the left 10,000 bp of MVA and v51.2. The names of the ORFs follow the Copenhagen nomenclature.

FIGS. 7A and 7B show the effects of deletions of additional ORFs in v51.2 on virus spread. CEF, A549, and MRC-5 cells grown in 12-well plates were infected with the indicated viruses at MOI of 0.001, 0.01, 0.1 and overlaid with methylcellulose. After 48 h, the overlay was removed and cells were stained with antibody to VACV.

FIG. 8A shows Western blotting analysis of ZAP expression in cell lysates collected from A549 and MRC5 cells. GAPDH is also probed for loading control. FIG. 8B shows the increase in replication of MVA A549, HeLa, and MRC-5 cells transfected with individual siRNAs targeting ZAP.

FIG. 9 shows the inactivation of ZAP in A549 cells causes enhanced replication of MVA as well as a strain of MVA (47.1) that was adapted to grow in monkey BS-C-1 cells.

FIG. 10 shows electron microscopy photographs demonstrating enhanced assembly of MVA virions.

FIG. 11 shows that MVA-expressing SPI-1 replicates better in ZAP knock-out A549 cells than in A549 cells. Cells were infected with 3 or 0.01 PFU per cell as indicated. FIG. 12 shows expression of SPI-1 in ZAP knock out cells enhances MVA replication more than either alone. The indicated cells, grown in 24-well plates, were infected with MVA, 47.1 or 51.2 (all cushion purified) at an MOI of 0.01. Viruses collected at 48hpi and titered on CEF cells.

FIG. 13 A shows Western blotting analyses performed with A549 and HCT116 cell lysates using antibodies to human ZAP or GAPDH (as a loading control), demonstrating that human cancer cell line HCT116 is ZAP-deficient. FIG. 13B shows virus titer for A549, A549-ZAP-KO, and HCT116 cells infected with MVA. Viruses were collected at 48 hpi and tittered on CEF (chicken embryo fibroblasts) cells. Error bars indicate standard deviation. FIG. 13C shows virus titer for A549, A549-ZAP-KO, and HCT116 cells infected with MVA expressing SPI-1. Viruses were collected at 48 hpi and tittered on CEF (chicken embryo fibroblasts) cells, showing that MVA + SPI1 can only replicate in ZAP- deficient human cell lines. Error bars indicate standard deviation.

FIG. 14 shows virus titer for A549 and A549-ZAP-KO cells infected with recombinant viruses MVA+C16+C17 or MVA+SPI1+C16+C17 generated by cloning C16 and C17 from vaccinia virus-Copenhagen and inserted into MVA or MVA+SPI1 between 18 and G1 with their natural promoters. Viruses were collected at 48 hpi and tittered on CEF (chicken embryo fibroblasts) cells. Error bars indicate standard deviation.

FIG. 15 shows plaques formed by serial passage of MVA virus on BS-C-1 monolayers. The different frames show the plaques formed at different passage (P) numbers.

FIG. 16 shows the titers of virus produced in BS-C-1 cells following infection with unadapted MVA virus (MVA 1, MVA 2, and MV A3), or MVA virus adapted to grow in BS-C-1 cells by passaging 10 times in BS-C-1 cells (#1 MVA 1, #1 MVA 2, #1 MVA 3, #2 MVA 1, #2 MVA 2, #2 MVA 3). Checkered bars show titers at 3 hours post infection. Black bars show titers 48 hours post-infection.

FIG. 17 shows the sequence of the amino acid sequence encoded by MVA virus D10 ORF. The location of three different mutations that result in increased titers of MVA virus in BS-C-1 cells are indicated with arrows.

FIG. 18 shows the titers of virus resulting from infection of various cell types with unmodified MVA virus (MVA), or MVA viruses having mutations at various locations in in the protein encoded by the D10 ORF (A226T, H233Y, and C26Y).

FIG. 19 shows the titers of virus resulting from infection of BS-C-1 cells with

MVA viruses having mutations in the protein encoded by their D10 ORFs. A26T is a virus having the alanine at position 26 replaced with a threonine. H233 Y is a virus having the alanine at position 233 replaced with a tyrosine. C25Y is a virus having the cysteine at position 25 replaced with a tyrosine.

DETAILED DESCRIPTION

The present disclosure generally relates to the production of modified vaccinia

Ankara (MV A) virus, and in particular, to methods for expanding the range of cells (also referred to herein as host range) that may be used to produce MVA virus. More specifically, the disclosure relates to the modification of a non-permissive animal cell such that the modified animal cell becomes permissive for MVA replication, and thus, supports the replication of MVA virus. The disclosure also relates to the modification of an animal cell that has reduced permissiveness (i.e., is restrictive) for MVA virus replication, wherein the modification increases the titer of infectious virus produced by an MVA- infected modified animal cell, relative to the titer of virus obtained infection with MVA virus of an unmodified cell of the same type. The disclosure also relates to an MVA virus that has been altered so that it is able to replicate in an animal cell that is non-permissive for replication of unaltered (i.e., wild-type) MVA virus. Such altered MVA viruses may also produce titers of infectious virus in cells having reduced permissiveness (i.e., MVA- restrictive cells,) that are higher than the titer of infectious virus produced by infection of such MVA-restrictive cells by unaltered MVA virus. The invention disclosed herein is based on two key discoveries made by the inventors. The inventors have identified certain orthopoxvirus open-reading frames (ORFs), missing from the MVA virus genome, that when present in an MVA-infected cell, allow MVA virus to replicate in animal cells that are normally non-permissive for MVA virus replication, or that act to increase the titer of infectious virus obtained from infection of an MVA-restricted cell with MVA virus. The inventors have also discovered that a reduction in the level of, or an absence of, certain cellular activities (e.g., protein associated activities), allows MVA virus to replicate in normally non-permissive cells, and increases the titer of infectious virus obtained from

MVA infection of cells having reduced permissiveness. In addition, the inventors have discovered that altering the genome of an MVA virus so that 1) it comprises one or more poxvirus ORFs that are normally absent from the MVA genome, and/or 2) it comprises one or more mutations in an ORF encoding a decapping enzyme, allows the altered MVA virus to replicate in normally non-permissive cells, and/or allows he altered MV virus to produce titers of virus in MVA-restrictive cells that are higher than the titers produce by unaltered MVA virus in the same type of MVA-restrictive cell. Accordingly, the present invention may generally be practiced by modifying an animal cell that is non-permissive, or that has reduced permissiveness (i.e., is restrictive for MV A), for MVA replication, so that the modified animal cell 1) comprises at least one poxvirus ORF that is not present in the MVA virus genome, the at least one poxvirus ORF being operationally linked to a promoter other than its natural promoter, wherein the at least one poxvirus ORF renders modified animal cell permissive for MVA virus replication, or increases the tire of infectious virus obtained ; and/or, 2) is deficient in at least one cellular activity that inhibits the ability of MVA virus to replicate. Such a modified cell may be used to produce high titers of MVA virus. The invention may also generally be practiced by modifying a MVA virus to comprise 1) at least one poxvirus ORF that is normally missing from the MVA virus genome, wherein the at least one poxvirus ORF renders the modified MVA virus capable of replicating to high titers in an animal cell that normally has reduced permissiveness, or that is non-permissive, for MVA virus replication; and/or 2) at least one mutation in an MVA ORF encoding a decapping enzyme.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive

terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

One embodiment of the invention is a modified, animal cell comprising at least one heterologous gene encoding a heterologous protein comprising the activity of at least one poxvirus protein encoded by an open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L, and a vaccinia virus C17L ORF, wherein the at least one heterologous gene is operatively linked to a promoter recognized by an RNA polymerase of the animal cell.

As used herein, a modified cell refers to a cell that has been altered by the hand of man, so that the cell contains heterologous DNA (e.g., the heterologous gene encoding the heterologous protein), and/or the cell contains mutations in its genome. Modified cells of the present invention are modified animal cells. In certain aspects, the animal cell is a mammalian cell, a bird cell, a fish cell, an amphibian cell, or a reptilian cell. In certain aspects, the animal cell is a primate cell, a monkey cell, a human cell, a mouse cell, a hamster cell, or a rabbit cell. In one aspect, the animal cell may be a tissue culture cell. Examples of animal cells useful for practicing the invention include, but are not limited to, human A549 cells, human MRC-5 cells, HeLa cells, and monkey BSC-1 cells.

Animal cells used to produce modified cells of the invention may, prior to modification, be non-permissive for MVA virus replication, or they may have reduced permissiveness for MVA virus replication. The poxvirus life cycle is known to involve several stages including binding of the virus to the cell, entry of the virus into the cell, uncoating of the virus, expression of early genes, expression of intermediate gene, expression of late genes, genome replication, assembly of IMV, and formation of CEV particles. In a cell that is non-permissive for MVA virus replication, or that has reduced permissiveness, the MVA virus may fail, or have a significantly reduced ability, to pass through any stage of virus replication. For example, in at least some non-permissive cell lines, the host cell restriction of MVA is believed to be associated with a late block in the assembly of viral particles.

As used herein, a cell that is non-permissive for MVA virus replication is one that when contacted with a wild-type MVA virus, and incubated under conditions suitable for replication of MVA virus, produces a titer of progeny virus particles that is less than 5% the titer of progeny virus particles produced when same wild-type MVA virus is contacted with chicken embryo fibroblast (CEF) cells. As used herein, a wild-type MVA virus is an MVA virus that has not been modified by the hand of man. In certain aspects, a non- permissive cell may fail to produce any progeny virus (i.e., a non-detectable amount of infectious virus) when contacted with a wild-type MVA virus that is capable of producing a high titer (e.g., at least 10⁵ or at least 10⁶ infectious virus particles/ milliliter) of progeny virus when contacted with CEF cells. Examples of cells that are non-permissive for MVA virus include, but are not limited to, monkey transformed B (M1B) cells, HeLa cells, SK- 29 cells, HEK 293 cells, LC-5 cells, LC-5 cells, and C8166 cells.

As used herein, a cell that has reduced permissiveness for MVA is one in which wild-type MVA virus is capable or replicating (i.e., producing progeny virus), but in which the ability of the wild-type MVA virus to replicate is reduced relative to the ability of wild-type MVA virus to replicate in CEF cells. Such cells may also be referred to as MVA restrictive cells, MVA-restricted cells, and the like, in that while the MVA virus is capable of replicating in the cell, it is restricted in its ability to produce high titers of MVA virus. Thus, the amount of infectious MVA progeny virus obtained from infection of cells having reduced permissiveness (MVA restrictive cells), is significantly less than the amount of MVA progeny virus obtained from infection of CEF cells. For example, the titer of progeny MVA virus obtained using cells having reduced permissiveness, may be less than 25% of the titer of progeny virus particles obtained from infection of CEF cells with the same MVA virus. The titer of progeny MVA virus obtained from cells having reduced permissiveness (i.e., MVA-restrictive cells) may be at least 1 log less, at least 2 logs less, or at least 3 logs less than the titer of progeny MVA virus obtained from infection of chick embryo fibroblasts (CEFs). Examples of cells having reduced permissiveness for MVA virus include, but are not limited to, baby hamster kidney (BHK) cells, and monkey kidney (MK) cells. In certain aspects, a permissive cell may refer to a cell useful for efficiently producing an amount of virus suitable for use as a vaccine. In one aspect, a permissive cell, when infected with wild-type MVA virus, produces a titer of at least 10⁶, or at least 10⁷, MVA infectious virus particles per ml using standard culture techniques known in the art.

As used herein, the term“heterologous”, when used with reference to molecules such as proteins and nucleic acid molecules, indicates that the molecule to which it refers is from an organism that is different than the organism in which the molecule is present in the invention. For example, poxvirus nucleic acid molecules and proteins are not normally produced by, or found in, an uninfected animal cell and thus, such nucleic acid molecules and proteins are considered heterologous to the animal cell. Similarly, nucleic acid molecules encoding mammalian proteins are not normally found in the MVA genome and thus, such nucleic acid molecules would be considered heterologous to MVA virus.

Heterologous nucleic acid molecules and proteins are generally present in viruses and animal cells of the invention as a result of recombinant DNA technology using techniques well known in the art.

As stated previously, the at least one heterologous protein present in modified animal cells of the invention comprises at least one activity of at least one poxvirus protein encoded by an open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF (represented by SEQ ID NO: 1, encoding SEQ ID NO:2), a vaccinia virus C16L ORF (represented by SEQ ID NO:3, encoding SEQ ID NO:4), and a vaccinia virus C17L ORF (represented by SEQ ID NO:5, encoding SEQ ID NO:6). For ease of discussion, the protein encoded by the vaccinia virus C12L ORF may be referred to as the C12L or C12 protein (or the serine protease inhibitor 1 (SPI-1) protein); the protein encoded by the vaccinia virus C16L ORF may be referred to as the C16L or C16 protein; and the protein encoded by the vaccinia virus C17L ORF may be referred to as the C17L or C17 protein. A heterologous protein that comprises the activity of the C12L protein, the C16L protein or the C17L protein, means the heterologous protein comprises at least one functional activity of the C12L protein, the C16L protein or the C17L protein. For example, the functional activity may be enzymatic activity, inhibitory activity, structural activity, or immunogenic activity. Thus, it will be understood by the skilled artisan that the activity of the heterologous protein may substitute for the activity of the vaccinia virus C12L protein, the vaccinia virus C16L protein, or the vaccinia virus C17L protein. To further illustrate, it is known that the vaccinia virus C12L protein (SPI-1) is able to inhibit caspases 1 and 8. Thus, a heterologous protein of the invention may comprise the ability to inhibit caspase 1 and/or 8.

As used herein, a“gene” refers to a nucleotide sequence that encodes an amino acid sequence (e.g., protein or peptide). A gene may or may not include regulatory sequences, such as operator and promoter sequences. According to this disclosure, a gene may or may not comprise introns. Thus, it should be appreciated that, as used herein, the term gene may encompass open reading frames (ORFs). The term“ORF” refers to a nucleic acid sequence (or polynucleotide sequence) that encodes an amino acid sequence, but which lacks introns. Thus, the entire sequence of an ORF, with the possible exception of the final termination codon, encodes an amino acid sequence. It should be understood that in the context of this disclosure, the terms gene and ORF may be used

interchangeably.

In certain aspects of the invention, the at least one heterologous gene may be a homologue of a poxvirus ORF. In these aspects, the heterologous gene may be a homologue of a poxvirus open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L, and a vaccinia virus C17L ORF, wherein the at least one heterologous gene is operatively linked to a promoter recognized by an RNA polymerase of the animal cell. In one aspect, the heterologous gene may be a homologue of a vaccinia virus C12L ORF. In one aspect, the heterologous gene may be a homologue of a vaccinia virus C16L ORF. In one aspect, the heterologous gene may be a homologue of a vaccinia virus C17L ORF.

In certain aspects of the invention, the at least one heterologous gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to a vaccinia virus ORF selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, and a vaccinia virus C17L ORF. In one aspect, the at least one heterologous gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO: l, SEQ ID NO:3, or SEQ ID NO: 5. In these aspects, the at least one heterologous gene may encode a protein retaining the activity of a protein comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

In certain aspects of the invention, the at least one heterologous gene may encode a heterologous protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to a vaccinia virus protein selected from the group consisting of a vaccinia virus C12L protein, a vaccinia virus C16L protein, and a vaccinia virus C17L protein. In one aspect of the invention, the at least one heterologous gene may encode a heterologous protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In these aspects, the encoded protein may retain the activity of a protein comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

As stated above, the at least one heterologous gene may be operatively linked to a promoter recognized by an RNA polymerase of the animal cell. As used herein, the term “functionally linked” refers to two or more nucleic acids sequences, or partial sequences, which are positioned so that they functionally interact to perform their intended functions. For example, a promoter is functionally linked to a gene or ORF (e.g. a coding sequence) if it is able to direct RNA polymerase-mediated transcription of the linked gene or ORF in the cis position. Although a functionally linked promoter is generally located upstream of the coding sequence, the transcription of which it promotes, it does not necessarily have to be close to it. Any promoter may be used to direct expression of the functionally linked gene, as long as the promoter is not the natural promoter associated with a homologue of a vaccinia virus C12L, C16L, or C17L ORF, and as long as the promoter is recognized by an RNA polymerase in the animal cell. A natural promoter associated with a vaccinia virus C12L, C16L, or C17L homologue is the promoter sequence associated with the vaccinia virus C12L, C16L, or C17L homologue in the viral genome, and that drives expression of the vaccinia virus C12L, C16L, or C17L homologue. Thus, the promoter is not a vaccinia virus C12L promoter, a vaccinia virus C16L promoter, or a C17L promoter. In one aspect, the promoter may be the promoter associated with a mammalian gene. Examples of such promoters include, but are not limited to, the promoter for the gene encoding elongation factor la (EFla), the promoter for the gene encoding cytokeratin 18 (K18), the promoter for the gene encoding cytokeratin 19 (K19), the promoter for the gene encoding kallikrein (Kail), and the promoter for the gene encoding amylase (AMY). In one aspect, the promoter may be obtained from a mammalian virus. Examples of such viruses include, but are not limited to, a cytomegalovirus promoter, an SV40 promoter, a polyomavirus promoter, a papillomavirus promoter, a herpesvirus promoter, an adenovirus promoter, or a retrovirus promoter. The promoter may be a constitutive promoter or an inducible promoter.

As discussed previously, the gene encoding the heterologous protein may be heterologous to the animal cell. That is, it is not normally present in the unmodified animal cell. In addition, the heterologous gene is functionally linked to a promoter that is not the natural promoter associated with a homologue of a vaccinia virus C12L, C16L, or C17L ORF. Thus, it should be understood that a nucleic acid molecule comprising the heterologous gene functionally linked to a promoter other than the heterologous gene’s natural promoter, may be a recombinant nucleic acid molecule that is introduced into the animal cell by the hand of man. It will also be understood by those skilled in the art that such a construct is not introduced by introducing wild-type poxvirus DNA into the cell (e.g., by infection with a wild-type poxvirus), since such a virus would contain a C12L, C16L, and/or C17 ORF functionally linked to the ORFs natural promoter. Thus, in these aspects, the modified animal cell is not infected with a wild-type poxvirus. In one aspect, the modified animal cell lacks a homologue of a vaccinia virus Cl 1L ORF, a vaccine virus C13L ORF, and/or a vaccinia virus C14L ORF. In one aspect, the heterologous gene is present on a plasmid or a cosmid in the modified animal cell. In one aspect, the heterologous gene is inserted in the genome of the modified animal cell.

It is known in the art that animal cells may contain molecules (proteins, nucleic acid molecules, etc.) that inhibit replication of poxviruses within the animal cell. Without being bound by theory, such molecules may inhibit MVA virus replication by interacting with MVA virus molecules (e.g., proteins, nucleic acid molecules, etc.) or they may inhibit

MVA virus replication by interacting other cellular molecules, thereby altering the intracellular environment so that it is unfavorable for MVA replication. It will be understood by those skilled in the art that a reduction in the level of, or the elimination of, such proteins, or a reduction in the level of, or the elimination of, an activity (e.g., enzymatic activity), possessed by such proteins, may result in an increase in the amount of poxvirus produced in the animal cell. Thus, in certain aspects of the invention, the animal cell may be deficient in at least one activity that inhibits replication of modified vaccinia

Ankara (MVA) virus. As used in this context, the term“deficient” means the animal cell either has a reduced level of the molecule that inhibits MVA virus replication, has a reduced level of an activity associated with the molecule that inhibits MVA replication, or completely lacks (i.e., fails to produce) the molecule, or an activity associated therewith.

In one aspect, the deficiency may be a reduction in the level of the molecule that inhibits MVA virus replication. Such reduction may be due to a mutation in a gene encoding the inhibitory molecule, or in an element that controls transcription of the gene (e.g., enhancer, promoter, etc.). In one aspect, the animal cell may completely lack (i.e., fails to produce) the inhibitory molecule. In one aspect, the deficiency may be a reduction in the level of activity possessed by the molecule that inhibits MVA virus replication. Such reduction in the level of activity may be due to a mutation in the gene encoding a protein possessing the inhibitory activity. The mutation may be an inactivating mutation such that the inhibitory molecule lacks any inhibitory activity. In one aspect, the deficiency may be due to the animal cell lacking at least a portion of the gene encoding the inhibitory molecule.

In one aspect, the deficiency in the level of a molecule that inhibits MVA virus replication may be due to the presence of a functional RNA molecule (e.g., siRNA, micro RNA) in the cell, wherein the functional RNA molecule affects the level of the inhibitory molecule present in the cell. The functional RNA may prevent transcription of a gene encoding the inhibitory molecule. The functional RNA may prevent translation of an mRNA molecule encoding the inhibitory molecule. The functional RNA may increase degradation of an mRNA molecule encoding the inhibitory molecule.

One example of an inhibitory molecule that inhibits the replication of MVA virus is zinc finger CCCH-type antiviral protein 1 (aka, ZAP protein) encoded by the

ZC3HAV1 gene (Entrez Gene ID No. 56829). Thus, in one aspect of the invention, the animal cell may have a deficiency in the level of ZAP protein. In one aspect, the animal cell may have a deficiency in the level of at least one activity associated with ZAP. In one aspect, the animal cell may have a mutation in a gene encoding the ZAP protein. The mutation may cause a reduction in the level of ZAP in the animal cell. The mutation may cause a reduction in the level of at least one ZAP-associated activity. The mutation may affect transcription of translation of a mRNA encoding the ZAP protein. The mutation may result in the animal cell completely lacking ZAP protein.

Modified animal cells of the disclosure allow MVA virus replication so that such cells may be used to produce high-titer stocks of MVA virus. Thus, one embodiment of the invention is a method to produce MVA virus, comprising contacting an MVA virus with a modified animal cell of the disclosure, and incubating the contacted cell under conditions suitable for replication of MVA virus, thereby producing MVA virus. In one aspect, the modified, animal cell comprises at least one heterologous gene encoding a heterologous protein comprising the activity of at least one poxvirus protein encoded by an open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L, and a vaccinia virus C17L ORF. In certain aspects, the at least one heterologous gene is operatively linked to a promoter recognized by an RNA polymerase of the animal cell. The animal cell may be a mammalian cell, a bird cell, a fish cell, an amphibian cell, or a reptilian cell. The animal cell may be a primate cell, a monkey cell, a human cell, a mouse cell, a hamster cell, or a rabbit cell. The animal cell may be a tissue culture cell. The animal cell may be an immortalized tissue culture cell. Examples of animal cells useful for practicing the invention include, but are not limited to, human A549 cells, human MRC-5 cells, HeLa cells, and monkey BSC-1 cells. In one aspect of the method, prior to modification, the animal cell may have been non-permissive for MVA virus replication, or the animal cell may have had reduced permissiveness for MVA virus replication.

In one aspect of the method, the at least one heterologous gene may be a homologue of a poxvirus ORF. The at least one heterologous gene may be a homologue of a poxvirus open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L, and a vaccinia virus C17L ORF. The at least one heterologous gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to a vaccinia virus ORF selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, and a vaccinia virus C17L ORF. The at least one heterologous gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO: l, SEQ ID NO:3, or SEQ ID NO:5. The at least one heterologous gene may encode a heterologous protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to a vaccinia virus protein selected from the group consisting of a vaccinia virus C12L protein, a vaccinia virus C16L protein, and a vaccinia virus C17L protein. The at least one heterologous gene may encode a heterologous protein

comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In these aspects, the encoded protein may retain the activity of a protein comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In one aspect, the promoter is not the natural promoter associated with a homologue of a vaccinia virus C12L, C16L, or C17L ORF, and as long as the promoter is recognized by an RNA polymerase in the animal cell. The promoter is not a vaccinia virus C12L promoter, a vaccinia virus C16L promoter, or a C17L promoter. The promoter may be a mammalian promoter (i.e., a promoter associated with a mammalian gene). Examples of such promoters include, but are not limited to, the promoter for the gene encoding elongation factor la (EFla), the promoter for the gene encoding cytokeratin 18 (K18), the promoter for the gene encoding cytokeratin 19 (K19), the promoter for the gene encoding kallikrein (Kail), and the promoter for the gene encoding amylase (AMY). The promoter may be obtained from a mammalian virus. The promoter may be a cytomegalovirus promoter, an SV40 promoter, a polyomavirus promoter, a papillomavirus promoter, a herpesvirus promoter, an adenovirus promoter, or a retrovirus promoter. The promoter may be a constitutive promoter or an inducible promoter.

In one aspect, the modified animal cell may lack a homologue of a vaccinia virus Cl 1L ORF, a vaccine virus C13L ORF, and/or a vaccinia virus C14L ORF. The heterologous gene may be present on a plasmid or a cosmid in the modified animal cell. The heterologous gene may be inserted in the genome of the modified animal cell.

In one aspect of the method, the animal cell may be deficient in at least one activity that inhibits replication of modified vaccinia Ankara (MV A) virus. The deficiency may be a reduction in the level of the molecule that inhibits MVA virus replication. The reduction in activity may be due to a mutation in a gene encoding the inhibitory molecule, or in an element that controls transcription of the gene (e.g., enhancer, promoter, etc.). The animal cell may completely lack (i.e., fail to produce) the inhibitory molecule. In one aspect of the method, the deficiency may be a reduction in the level of activity possessed by the molecule that inhibits MVA virus replication. Such reduction in the level of activity may be due to a mutation in the gene encoding a protein possessing the inhibitory activity. The mutation may be an inactivating mutation such that the inhibitory molecule lacks any inhibitory activity. The deficiency may be due to the animal cell lacking at least a portion of the gene encoding the inhibitory molecule.

In one aspect of the method, the deficiency in the level of a molecule that inhibits

MVA virus replication may be due to the presence of a functional RNA molecule (e.g., siRNA, micro RNA) in the cell, wherein the functional RNA molecule affects the level of the inhibitory molecule present in the cell. The functional RNA may prevent transcription of a gene encoding the inhibitory molecule. The functional RNA may prevent translation of an mRNA molecule encoding the inhibitory molecule. The functional RNA may increase degradation of an mRNA molecule encoding the inhibitory molecule.

In one aspect of the invention, the animal cell may have a deficiency in the level of ZAP protein. In one aspect, the animal cell may have a deficiency in the level of at least one activity associated with ZAP. In one aspect, the animal cell may have a mutation in a gene encoding the ZAP protein. The mutation may cause a reduction in the level of ZAP in the animal cell. The mutation may cause a reduction in the level of at least one ZAP- associated activity. The mutation may affect transcription of translation of an mRNA molecule encoding the ZAP protein. The mutation may result in the animal cell completely lacking ZAP protein.

It is known in the art that the genome of MV A is missing all or part of homologues of vaccinia virus ORFS C12L, Cl 6, and C17L. The inventors have discovered that introducing one or more of these ORFS into the MVA virus genome to produce a recombinant MVA virus, results in the recombinant MVA virus being able to replicate to high titer in cells that are non-permissive, or that have reduced permissiveness, for MVA virus. Thus, one embodiment of the invention is a method to produce an altered MVA virus particle that is capable of replicating in animal cells that are non-permissive for unaltered (i.e., wild-type) MVA virus, the method comprising inserting into the genome of a an MVA virus, a gene encoding a protein comprising the activity of at least one poxvirus protein encoded by an open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L, and a vaccinia virus C17L ORF, wherein the gene is functionally linked to a promoter. One embodiment of the invention is an altered, modified vaccinia Ankara (MVA) virus, comprising a gene encoding a protein comprising the activity of at least one poxvirus protein encoded by an open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L, and a vaccinia virus C17L ORF, wherein the gene is functionally linked to a promoter. Such a virus can replicate in cells that are non-permissive for replication of wild-type MVA virus and thus, such a virus has an extended host range. Accordingly, such a virus may be referred to as a host range extended (HRE) MVA virus. In these embodiments, the altered MVA virus (i.e., the HRE MVA virus) was created by inserting the gene encoding the protein comprising the activity of the at least one poxvirus protein into the genome of MVA virus. In these embodiments, the altered MVA virus may not contain a homologue of a vaccinia virus C13L ORF or a C14L ORF. In these embodiments, the inserted gene may be a homologue of a poxvirus open reading frame (ORF) selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L, and a vaccinia virus C17L ORF. In certain aspects, the inserted gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to a vaccinia virus ORF selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, and a vaccinia virus C17L ORF. The inserted gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In these aspects, the inserted gene encodes a protein retaining the activity of a protein comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

In these embodiments, the inserted gene may encode a protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to a vaccinia virus protein selected from the group consisting of a vaccinia virus C12L protein, a vaccinia virus C16L protein, and a vaccinia virus C17L protein. The inserted gene may encode a heterologous protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In these aspects, the encoded protein retains the activity of a protein comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

In one aspect, the gene is inserted into a non-essential ORF in the MVA virus. The gene may be inserted into a non-essential intergenic region between two ORFs in the

MVA genome. As used herein an“intergenic region” (IGR) means a nucleic acid sequence between the closest terminal codons of adjacent ORFs, that does not contain encode a protein. IGR sequences lie outside the stop codons of adjacent ORFs and thus do not encode any portion of the protein encoded by the adjacent ORFs. As used herein, a

“nonessential ORF” or“non-essential IGR” is an ORF, or IGR, that when interrupted in a poxvirus (e.g., by insertion of another nucleic acid sequence) has no, or almost no, effect on the ability of the poxvirus to produce progeny virus. That is, interruption of a non- essential ORF or IGR reduces the titer of progeny poxvirus by less than 10%, less than

15%, or less than 20%, relative to the titer of virus obtained from a poxvirus of the same species in which the non-essential ORF or IGR is not interrupted. Interruption of a non- essential ORF or IGR reduces the titer of progeny poxvirus by less than half a log (0.5 x 10^L1) relative to the titer of virus obtained from a poxvirus of the same species in which the non-essential ORF or IGR is not interrupted. Consequently, an“essential ORF’ or “essential IGR” is a nucleic acid sequence that, when interrupted, results in the poxvirus comprising the interrupted sequence to have a decrease in titer of progeny virus of at least 10%, at least 15%, or at least 20%, relative to the same species of poxvirus in which the ORF or IGR has not been interrupted. In certain aspects, an“essential ORF’ or“essential IGR” is a nucleic acid sequence that, when interrupted, results in the poxvirus comprising the interrupted sequence to have a decrease in titer of progeny virus of at least 10%, less than 15%, or less than 20%, relative to the same species of poxvirus in which the ORF or IGR has not been interrupted. It is known that MVA virus contains six deletion regions, I- VI. In one aspect, the gene may be inserted in a deletion region of the MVA genome.

As noted above, the gene inserted into the MVA genome is functionally linked to a promoter. The promoter may be a poxvirus promoter, which may be an early promoter, an intermediate promoter, a late promoter, or a synthetic promoter (e.g., mH5). The promoter may be a promoter that is recognized by an RNA polymerase from an animal cell.

The inventors have also discovered that mutations in an MVA ORF encoding a poxvirus decapping enzyme, allows the virus to replicate to high titers in cells that are normally non-permissive, or that normally have reduced permissiveness for MVA virus.

Thus, one embodiment of the invention is a method to produce an altered MVA virus particle that is capable of replicating to high titers in normally non-permissive cells, or that is capable of replicating to higher titers than an unaltered MVA virus in normally restrictive cells, the method comprising introducing one or more mutations into an MVA virus ORF encoding a poxvirus decapping enzyme. Examples of MVA ORFs that encode a decapping enzyme include, but are not limited to, MVA virus ORFs D9 and D10. In one embodiment, the MVA virus ORF into which a mutation is made may be MVA virus D9

ORF or MVA virus D10 ORF. The introduced one or more mutations may result in substitution mutations in the encoded protein, and optionally the substitutions mutations may be conservative substitutions. Introduction of one or more mutations into the MVA virus D10 ORF may result in a substitution mutation at a position corresponding to C25,

A226, and/or H233 of SEQ ID NO:7. Similarly, introduction of one or more mutations into the MVA virus D10 ORF may result in the encoded mutated protein having a substitution mutation at a position corresponding to C25, A226, and/or H233 of SEQ ID

NO:7. Introduction of one or more mutations into the MVA virus D10 ORF may result in the encoded mutated protein having a substitution mutation at position C25, A226, and/or H233 of SEQ ID NO:7. The encoded mutated protein may comprise one or more mutations selected from the group consisting of substitution of the amino acid

corresponding to C25 of SEQ ID NO: 7 with a tyrosine, substitution of the amino acid corresponding to A226 of SEQ ID NO:7 with a threonine, and substitution of the amino acid corresponding to H233 of SEQ ID NO: 7 with a tyrosine. The encoded mutated protein may comprise one or more mutations selected from the group consisting of substitution of C25 of SEQ ID NO:7 with a tyrosine, substitution of A226 of SEQ ID NO:7 with a threonine, and substitution of H233 of SEQ ID NO:7 with a tyrosine. The encoded mutated protein may comprise a mutation selected from the group consisting of C25Y, A226T, and H233Y.

As discussed, mutations in MVA virus decapping proteins may allow the mutated MVA virus to replicate in cells that are normally non-permissive for MVA virus replication. Such mutations may also allow the mutated MVA virus to produce titers of virus in MVA virus -restrictive cells that are higher than the titers produced by non- mutated MVA virus in the same type of MVA virus - restrictive cell. In certain embodiments, the decapping protein mutations described herein result in the mutated MVA virus producing titers in MVA virus -restrictive cells that are at least one log (10X) or at least two logs (100X) higher than the titer produced by non-mutated MVA virus in the same type of MVA-restrictive cell. Examples of such MVA-restrictive cells include BS-C-1 cells (BSC-1 cells), HeLa cells, and A549 cells. In certain embodiments, the decapping protein mutations described herein result in the mutated MVA virus producing titers in BS-C-1 cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X) higher than the titer produced by non-mutated MVA virus in BS-C-1 cells. In certain embodiments, a mutation in MVA virus ORF D10 at a position corresponding to C25, A226, and/or H233 of SEQ ID NO:7, results in the mutated MVA virus producing titers in BS-C-1 cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X), higher than the titer produced by non-mutated MVA virus in BS-C-1 cells. In certain embodiments, a mutation in MVA virus D10 ORF selected from the group consisting of substitution of the amino acid corresponding to C25 of SEQ ID NO:7 with a tyrosine, substitution of the amino acid corresponding to A226 of SEQ ID NO:7 with a threonine, and substitution of the amino acid corresponding to H233 of SEQ ID NO:7 with a tyrosine, results in the mutated MVA virus producing titers in BS-

C-1 cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X), higher than the titer produced by non-mutated MVA virus in BS-C-1 cells. In certain embodiments, mutation of the MVA virus D10 ORF so that the encoded protein comprises at least one mutation selected from C25Y, A226T, and H233Y, results in the mutated MVA virus producing titers in BS-C-1 cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X), higher than the titer produced by non-mutated MVA virus in BS-C-1 cells.

One embodiment of the invention is an altered modified vaccinia Ankara (MVA) virus comprising a mutation in an ORF encoding a decapping enzyme, wherein the altered MVA virus is capable of replicating to high titers in normally non-permissive cells, or is capable of replicating to higher titers than an unaltered MVA virus in normally restrictive cells. The MVA virus ORF encoding the decapping enzyme may be MVA virus D9 ORF or MVA virus D10 ORF. The introduced mutation may result in substitution mutations in the encoded protein. The substitutions mutation may be conservative substitutions. The substitution mutation may be at a position corresponding to C25, A226, and/or H233 of SEQ ID NO:7. The mutated protein may comprise one or more mutations selected from the group consisting of substitution of the amino acid corresponding to C25 of SEQ ID NO:7 with a tyrosine, substitution of the amino acid corresponding to A226 of SEQ ID NO:7 with a threonine, and substitution of the amino acid corresponding to H233 of SEQ ID NO:7 with a tyrosine. The mutated protein may comprise one or more mutations selected from the group consisting of substitution of C25 of SEQ ID NO:7 with a tyrosine, substitution of A226 of SEQ ID NO:7 with a threonine, and substitution of H233 of SEQ ID NO:7 with a tyrosine. The encoded mutated protein may comprise a mutation selected from the group consisting of C25Y, A226T, and H233Y. The altered MVA virus may produce titers in MVA virus -restrictive cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X), higher than the titer produced by non- mutated MVA virus in the same type of MVA-restrictive cell. The MVA virus -restrictive cells may include BS-C-1 cells (BSC-1 cells), HeLa cells, and A549 cells. The altered MVA virus may produce titers in BS-C-1 cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X), higher than the titer produced by non-mutated MVA virus in BS-C-1 cells.

It should be appreciated that altered viruses of the disclosure may comprise any combination of the alterations described herein. For example, an altered MVA virus may comprise 1) one or more inserted genes, each inserted gene encoding a protein comprising the activity of a protein encoded by vaccinia virus C12L ORF, vaccinia virus C16L ORF, or vaccinia virus C17L ORF, (as described supra); and 2) a mutated form of an ORF encoding a decapping enzyme (e.g., D9 or D10).

Altered MVA viruses of the disclosure may be used to elicit an immune response to an infectious organism, or to treat an individual for a disease. Thus, in one aspect, an altered MVA virus of the disclosure comprises a gene encoding an immunogenic protein that elicits an immune response against a microorganism, wherein the microorganism is not a poxvirus. As used herein, the term immunogenic refers to the ability of a specific protein, or a specific region thereof, to elicit an immune response to the specific protein, or to a protein comprising an amino acid sequence having a high degree of identity with the specific, immunogenic protein. According to this disclosure, two polypeptides having a high degree of identity comprise contiguous amino acid sequences that are at least 80% identical, at least 85% identical, at least 87% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical. The encoded \immunogenic protein may be selected from the group consisting of a viral protein and a bacterial protein. The encoded heterologous immunogenic protein may be from a virus selected from the group consisting of adenoviruses, herpesviruses, papilloma viruses, polyomaviruses, hepadnaviruses, parvoviruses, astroviruses, caliciviruses, picornaviruses, coronaviruses, flaviviruses, togaviruses, hepeviruses, retroviruses, orthomyxoviruses, arenaviruses, bunyaviruses, filoviruses, paramyxoviruses,

rhabdoviruses, reoviruses, and poxviruses.

In one aspect, an altered MVA virus of the disclosure comprises a gene encoding a therapeutic agent that is capable of treating a disease. Examples of such therapeutic agents include, but are not limited to, a tumor-suppressor protein, a pro-apototic protein, a cytokine, a lymphokine, a monokine, a growth factor, an enzyme, a hormone, an immunomodulatory protein, a cytotoxic peptide, a suicide protein, a cytotoxin, a pro-drug, and a therapeutic RNA.

One embodiment of the invention is a method to produce progeny altered (or mutant) modified vaccinia Ankara (MVA) virus, comprising contacting an altered (or mutant) MVA virus of the disclosure with an animal cell. In this embodiment, the altered

MVA virus was created by inserting into the genome of an MVA virus, a gene encoding a protein comprising the activity of vaccinia virus C12L protein, vaccinia virus C16 L protein, or vaccinia virus C17L protein, wherein the inserted gee is functionally linked to a promoter. The animal cell may be permissive for MVA virus replication, non-permissive for MVA virus replication, or have reduced permissiveness for MV virus.

In one aspect, the altered MVA virus may not contain a homologue of a vaccinia virus C13L ORF or a C14L ORF. The inserted gene may be a homologue of vaccinia virus C12L ORF, vaccinia virus C16 ORFL, or vaccinia virus C17L ORF. The inserted gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to vaccinia virus C12L ORF, vaccinia virus C16L ORF, or vaccinia virus C17L ORF. The inserted gene may comprise a nucleic acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO: l, SEQ ID NO:3, or SEQ ID NO: 5. The inserted gene may encode a protein retaining the activity of a protein comprising SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. The inserted gene may encode a protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to vaccinia virus C12L protein, vaccinia virus C16L protein, or vaccinia virus C17L protein. The inserted gene may encode a protein comprising an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at last 95% identical, at least 97% identical, at least 99% identical, or 100% identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. The gene may be inserted into a non- essential ORF or IGR in the MVA virus. The gene may be inserted in a deletion region of the MVA genome. The functionally linked promoter may be a poxvirus promoter, such as an early promoter, an intermediate promoter, a late promoter, or a synthetic promoter (e.g., mH5). The functionally linked promoter may be a promoter that is recognized by an RNA polymerase from an animal cell. The functionally linked promoter may be a promoter from an animal cell.

The altered modified vaccinia Ankara (MVA) virus may comprise a mutation in an MVA ORF encoding a decapping enzyme, wherein the altered MVA virus is capable of replicating to high titers in normally non-permissive cells, or is capable of replicating to higher titers than an unaltered MVA virus in normally restrictive cells. The MVA ORF encoding the decapping enzyme may be MVA virus D9 ORF or MVA virus DIOORF.

The introduced mutation may result in substitution mutations in the encoded protein. The substitutions mutation may be a conservative substitution. The substitution mutation may be at a position corresponding to C25, A226, and/or H233 of SEQ ID NO:7. The mutated protein may comprise one or more mutations selected from the group consisting of substitution of the amino acid corresponding to C25 of SEQ ID NO:7 with a tyrosine, substitution of the amino acid corresponding to A226 of SEQ ID NO:7 with a threonine, and substitution of the amino acid corresponding to H233 of SEQ ID NO:7 with a tyrosine. The mutated protein may comprise one or more mutations selected from the group consisting of substitution of C25 of SEQ ID NO:7 with a tyrosine, substitution of A226 of SEQ ID NO:7 with a threonine, and substitution of H233 of SEQ ID NO:7 with a tyrosine. The encoded mutated protein may comprise a mutation selected from the group consisting of C25Y, A226T, and H233Y. The altered MVA virus may produce titers in MVA-restrictive cells that are at least one log (10X) or at least two logs (100X) higher than the titer produced by non-mutated MVA virus in the same type of MVA-restrictive cell. The MVA virus -restrictive cells may include BS-C-1 cells (BSC-1 cells), HeLa cells, and A549 cells. The altered MVA virus may produce titers in BS-C-1 cells that are at least one log (10X), at least two logs (100X), or at least three logs (1000X), than the titer produced by non-mutated MVA virus in BS-C-1 cells.

It should be appreciated that altered viruses of the disclosure may comprise combinations of the alterations described herein. For example, an altered MVA virus may comprise 1) one or more inserted genes, each inserted gene encoding a protein comprising the activity of a protein encoded by vaccinia virus C12L ORF, vaccinia virus C16L ORF, or vaccinia virus C17L ORF, (as described supra); and 2) a mutated form of an MVA ORF encoding a decapping enzyme (e.g., D9 or D10).

In one aspect, the altered MVA virus comprises a gene encoding an immunogenic protein that elicits an immune response against a microorganism, wherein the

microorganism is not a poxvirus. The encoded immunogenic protein may be a viral protein or a bacterial protein.

In one aspect, an altered MVA virus of the disclosure comprises a gene encoding a therapeutic agent that is capable of treating a disease. Examples of such therapeutic agents have been disclosed herein.

In one aspect of the method, the animal cell may be deficient in at least one activity that inhibits replication of modified vaccinia Ankara (MVA) virus. The deficiency may be a reduction in the level of the molecule that inhibits MVA virus replication. The reduction in activity may be due to a mutation in a gene encoding the inhibitory molecule, or in an element that controls transcription of the gene (e.g., enhancer, promoter, etc.). The animal cell may completely lack (i.e., fail to produce) the inhibitory molecule. In one aspect of the method, the deficiency may be a reduction in the level of activity possessed by the molecule that inhibits MVA virus replication, which may be due to a mutation in the gene encoding a protein possessing the inhibitory activity. The mutation may be an inactivating mutation such that the inhibitory molecule lacks any inhibitory activity. The deficiency may be due to the animal cell lacking at least a portion of the gene encoding the inhibitory molecule.

In one aspect of the method, the deficiency in the level of a molecule that inhibits MVA virus replication may be due to the presence of a functional RNA molecule (e.g., siRNA, micro RNA) in the cell, wherein the functional RNA molecule affects the level of the inhibitory molecule present in the cell. The functional RNA may prevent transcription of a gene encoding the inhibitory molecule. The functional RNA may prevent translation of an mRNA molecule encoding the inhibitory molecule. The functional RNA may increase degradation of an mRNA molecule encoding the inhibitory molecule.

In another aspect of the method, the deficiency is due to an alteration in the gene that inhibits MVA virus replication.

One embodiment of the invention is a method of producing progeny MVA virus comprising contacting MVA virus with an animal cell that is deficient in at least one activity that inhibits replication of modified vaccinia Ankara (MVA) virus. The deficiency may be a reduction in the level of a molecule comprising the inhibitory activity.

The reduction in activity may be due to a mutation in a gene encoding the molecule comprising the MVA virus inhibitory activity, or in an element that controls transcription of the gene (e.g., enhancer, promoter, etc.). The animal cell may completely lack (i.e., fail to produce) the inhibitory molecule. In one aspect of the method, the deficiency may be a reduction in the level of activity possessed by the molecule that inhibits MVA virus replication, which may be due to a mutation in the gene encoding a protein possessing the inhibitory activity. The mutation may be an inactivating mutation such that the inhibitory molecule lacks any inhibitory activity. The deficiency may be due to the animal cell lacking at least a portion of the gene encoding the inhibitory molecule.

It is known in the art that many tumor cells have reduced levels of, or completely lack, ZAP protein. Thus in one aspect, the animal cell may be a tumor cell. The tumor cell may be in culture or it may be in an individual.

One embodiment of the invention is a method of treating tumor cells comprising contacting the tumor cells with an MVA virus. In one aspect, the MVA virus may be a wild-type MVA virus. In one aspect, the MVA virus may be an altered MVA virus (a HRE MVA virus) of the disclosure.

One embodiment of the invention is a method of treating tumor cells in an individual, comprising administering to the individual an MVA virus, such that the MVA virus contacts the tumor cells. In one aspect, the MVA virus may be a wild-type MVA virus. In one aspect, the MVA virus may be an altered MVA virus of the disclosure.

One embodiment of the invention is a system for producing MVA virus, the system comprising a modified animal cell of the disclosure, and an MVA virus. In one aspect, the MVA virus may be a wild-type MVA virus. The MVA virus may be an altered MVA virus of the disclosure. In one aspect, the system comprises a solid substrate comprising the modified animal cell. This disclosure also includes kits suitable for producing compositions comprising MVA viruses of this disclosure. Kits may include, for example, modified cells of this disclosure, nucleic acid molecules for constructing modified animal cells, and/or altered MVA viruses of this disclosure. Kits may also comprise associated components, such as, but not limited to, proteins, enzymes, cell culture media, buffers, labels, containers, vials, syringes, instructions for using the kit and the like.

EXAMPLES

The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, and temperature is in degrees Celsius. Standard abbreviations are used.

The following methods and materials were used to conduct the studies described in Examples 1-11, below:

Cells: A549 cells (ATCC CCL-185) were grown in Dulbecco’s modified Eagle’s medium/F-12

(Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 2 mM L-glutamine, 100 units of penicillin, and 100 pg of streptomycin per ml (Quality Biologicals). Primary CEF prepared from 10-day old fertile eggs (Charles River), BS-C-1 (ATCC CCL-26) and MRC-5 (ATCC CCL-171) cells were grown in minimum essential medium with Earle’s balanced salts (EMEM) supplemented with 10% FBS, 2 mM L- glutamine, 100 units of penicillin, and 100 pg of streptomycin per ml. HeLa cells were grown in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 units of penicillin, and 100 pg of streptomycin per ml.

Antibodies: Rabbit antibody to VACV strain WR was described previously (Davies DH, et ak, J Virol. 2008; 82(2):652-63); c-Myc antibody (9E10) conjugated to horse radish peroxidase (HRP) (catalog number sc-40 HRP) was from Santa Cruz

Biotechnology; and rabbit anti-actin was from Sigma-Aldrich.

Viruses: WRASPI-l was derived from the Western Reserve (WR) strain of VACV

(ATCC VR-1354) and was described previously (Panda, et ak, Proc Natl Acad Sci USA. 2017; 114(14):3720-5). A panel of human replication-competent recombinant MVAs (rMVAs) (51.1, 51.2, 44.1, and 44/47.1) with segments of added VACV DNA of various lengths was described (Wyatt LS, et al., Virology. 1998; 251 :334-487). RPXV and VACV WR SPI-1 deletion mutants were described previously (Panda, et al., supra).

Modified viruses were constructed by homologous recombination using

fluorescent reporter genes. To generate MVA-SPI-1, a C12L DNA segment was introduced into the genome of MVA at the deletion III site by inserting the DNA fragment downstream of PI 1 VACV promoter driven GFP in pLW44-derived vector (Bisht H, et al., Proc Natl Acad Sci USA. 2004; 101 :6641-6). The MVA-SPI-1 F322A and MVA-SPI- 1 T309R were constructed by mutating the Phe322 into Ala and Thr309 into Arg using Q5 Site- Directed Mutagenesis Kit (New England Biolabs).

C12L genes from rMVA (51.1, 51.2, 44.1, and 44/47.1) were deleted by homologous recombination with a PCR product containing the PI 1 VACV promoter- driven GFP gene flanked by sequences on either side of C12L. Fluorescent plaques were identified and cloned by repeated plaque isolation. Similarly, C10L and Cl 1R were deleted by replacing the corresponding gene with PI 1 promoter-driven mCherry. Red plaques were picked and purified by repeated isolation. To generate vAC12/Cl 1 and vAC 12/C 10, fluorescent foci that expressed both GFP and mCherry were picked and plaque purified. A similar strategy was adopted to delete the C15L, C16L, and C17L from v51.2AC12. The recombinant viruses were PCR amplified and sequenced to confirm the identities.

Homologous recombination was carried out by infecting CEF cells with 1 PFU/cell of virus, followed by transfection with assembled PCR products using Lipofectamine 2000 (Thermo Fisher). After 24 h, cells were harvested and lysed by three freeze-thaw cycles. The lysates were diluted 10-fold and used to infect CEF cell monolayers. Fluorescent recombinant plaques were distinguished from the parental plaques and clonally purified five times. The purities of the recombinant viruses were confirmed by PCR amplification and sequencing of the modified region. MVA and recombinant viruses were propagated in CEF cells.

Virus yield determination: CEF cells were grown in 12-well plates and infected with 0.001 or 0.01 PFU/cell of rMVA in MEM supplemented with 2.5% FBS for 2 h. The cells were washed extensively with the same medium, incubated at 37°C, and harvested at 48 h after infection. Harvested cells were lysed by 3 freeze-thaw cycles, and virus titers were determined by plaque assay on CEF monolayers. Plaque assay and immunostaining: Vims samples were disrupted in a chilled water bath sonicator with two 30-s periods of vibration, followed by 10-fold serial dilutions in EMEM supplemented with 2.5% FBS. Diluted viruses were distributed onto CEF monolayers. After adsorption for 2 h, the medium was aspirated and replaced with medium containing 2.5% FBS and 0.5% methylcellulose. After 48 or 72 h, infected cells were fixed with methanol-acetone (1 : 1), washed with tap water, and incubated with rabbit anti-VACV antibody (1 :2,000 dilution) for 1 h. The cells were washed again with tap water and incubated with a 1 :3,000 dilution of protein A conjugated with peroxidase (Thermo Scientific) for 1 h. The cells were washed and incubated with the substrate dianisidine saturated in ethanol for 5 min. After color formation, the dianisidine solution was removed, and the cells were washed in tap water.

Construction of the 2xMyc-SPI-l cell lines: A549 and MRC-5 cells expressing the 2xMyc tagged SPI-1 protein were created using retroviral transduction. A eukaryotic codon-optimized SPI-1 ORF with an N-terminal 2xMyc tag (2xMyc-SPI-l) was cloned into pQCXIP (Clontech) to generate pQCXIP-2xMyc- SPI-1. Retrovirus particles were produced by co-transfecting pQCXIP or pQCXIP-2xMyc-SPI-l (transfer plasmid), pMLV-Gag-Pol (packaging plasmid), and pVSV-G (VSV-G envelope plasmid) into 293T cells using Lipofectamine 2000. A549 and MRC-5 cells were infected with the

retroviruses in the presence of 5 pg/ml polybrene (Sigma-Aldrich). The cells were subcultured and passaged several times in selection medium containing 1 pg/ml of puromycin (Sigma-Aldrich). The expression of SPI-1 protein was determined by Western blotting using HRP-conjugated anti-Myc antibody (9E10).

Western blotting: Cells were harvested, washed, and lysed in Lysis buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitor) on wet ice for 30 min with frequent agitation. Cell lysates were cleared by centrifugation at 13,000 xg for 10 min at 4°C; the proteins were resolved on 4 to 12% NuPAGE Bis-Tris gels (Thermo Fisher) and transferred to a nitrocellulose membrane with an iBlot2 system (Thermo Fisher). The membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBS) for 1 h, washed with TBS with 0.1% Tween 20 (TBST), and then incubated with the primary antibody in 5% nonfat milk in TBST overnight at 4°C. The membrane was washed with TBST and incubated with the secondary antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch) in TBST with 5% nonfat milk for 1 h. After the membrane was washed, the bound proteins were detected with SuperSignal West Dura substrates (Thermo Scientific). Transmission electron microscopy: The cells were fixed, dehydrated and embedded in Embed 812 resin (Electron Microscopy Sciences, Hatfield, PA) as described previously (Maruri-Avidal L, et al., J Virol. 2011; 85(6):2504-l 1). Specimens were viewed with a FEI Tecnai Spirit transmission electron microscope (FEI, Hillsboro).

Genome sequencing: Libraries for 454 pyrosequencing were made using Rapid

Library Preparation Method Manual (October 2009) GS FLX Titanium Series (Roche) and Paired End Library Preparation Method Manual, 3kb Span (October 2009) GS FLX Titanium Series. Each library was processed using emPCR Method, Manual Lib-L MV (October 2009) in separate emulsion reactions. The paired-end sample was loaded on a single lane and the fragment sample was loaded in two lanes of an 8-region 454 GS FLX Titanium sequencing run. Genome assembly and gap closure was performed as previously described (Liu B, et al., J Virol. 2018; 92(7).).

Accession numbers: Genome sequences of rMVA 44.1, 44/47.1, 47.1 and 51.2 were deposited as GenBank Accession Numbers MK314710, MK314711, MK314712 and MK314713, respectively. The GenBank Accession Number MG663594 for rMVA51.1 was previously published (Liu, et al., supra).

Example 1. Comparison of the morphogenesis block of MV A and a VACV WR SPI-1 deletion mutant

Although numerous genes were deleted or truncated during the long passage history of MV A, the only ones with known human host-range function are C12L encoding SPI-1 and K1L. Table 1 summarizes previous PCR data confirming the absence of C12L DNA and truncation of K1L in MVA.

Table 1. Correlation of C12L and K1L with host range in human and rabbit cells^a

aAbility to replicate better than MVA (+) or equivalent to MVA (-). ^Viruses are named according to the cosmid or cosmids used for marker rescue and the decimals indicate separate clones. Data are summarized from Wyatt et al. (Virology. 1998; 251:334-42).

Strikingly, C12L was detected by PCR in all HRE MVAs that were able to replicate in human cells, whereas the presence of full-length K1L correlated with replication only in rabbit kidney cells. The correlation of C12L and replication in human cells focused our attention on SPI-1 as a missing host-range factor for MVA. Unlike most orthopoxvirus host-range mutants, which exhibit blocks in gene expression, the earliest recognized defect in MVA replication occurs during morphogenesis. Interestingly, the second exception to the general rule for the predominance of impaired gene expression is the morphogenesis defect of SPI-1 deletion mutants of rabbitpox virus and the WR strain of VAC V in non-permissive cells. The possibility that the absence of the SPI-1 gene contributes to the host-range defect of MVA persuaded us to compare their morphogenesis blocks. Human A549 cells that had been infected for 24 h with VACV WR, a WR SPI-1 deletion mutant WRVSPI- l or MVA were prepared for transmission electron microscopy. In the cells infected with WR (FIG. 1, panel A), there was a predominance of brick-shaped mature virions (MVs) and some wrapped or partially wrapped virions (WVs) as well as crescents (C) and immature virions (IV s). The cells infected with WRVSPI- l (FIG. 1, panel C) had many aberrant particles with the spherical shape and dimensions of IVs but with dense unstructured interiors, which are referred to as dense virions (DVs). Many spherical DVs were also present in the cells infected with MVA (FIG. 1, panel E). Higher magnification confirmed the similar appearances of the DVs in the cells infected with MVA and the WRVSPI- l (FIG. 1, panels D and F) and the more mature morphology of MVs in the cells infected with WR (FIG. 1, panel B). Thus, the impairment in

morphogenesis occurred at a similar stage in non-permissive cells infected with MVA and WRVSPI- l . Nevertheless, this similarity only provided suggestive evidence of related defects. Example 2. Restoration of the SPI-1 gene increases MVA replication in human cells

The presence of the C12L ORF in HRE MVAs that replicate in human cells and the similarity in the morphogenesis block of MVA and other SPI-1 deletion mutants led us to investigate whether the introduction of SPI-1 into the MVA genome would have a discernible effect on replication in human cells. DNA containing the C12L ORF encoding SPI-1 regulated by the early/late mH5 promoter together with the green fluorescent protein (GFP) ORF regulated by the pi 1 late promoter was inserted into the site of deletion III located near the right end of MVA so as not to interrupt or alter additional genes.

Permissive CEF were used for infection and transfection and the recombinant virus, named MVA-SPI-1, was clonally isolated by several rounds of picking fluorescent foci. PCR and Sanger sequencing were performed to confirm insertion of the complete C12L ORF.

The effect of the C12L ORF addition was assessed by virus spread in several cell lines with a range of virus multiplicities of MVA or MVA-SPI-1. MVA is less cytopathic than other strains of VAC V and does not form regular shaped plaques under semisolid medium that can be easily discerned by staining with crystal violet or neutral red.

Consequently, the irregular foci formed were visualized by immunostaining with antibody to VACV. In permissive CEF, the two viruses formed foci of similar number, size and staining intensity best seen at a MOI of 0.001 before confluence occurred (FIG. 2A). In monkey BSC-1 and human MRC-5 cells, MVA-SPI-1 foci were larger and exhibited more intense staining relative to MVA, which could be seen at all MOI (FIG. 2A). MVA-SPI-1 exhibited a less pronounced increase in staining relative to MVA in HeLa and A549 cells, which was best seen at a MOI of 0.1 (FIG. 2A).

Example 3. Deletion of the SPI-1 gene from HRE MVAs reduces replication in human cells

To complement the results of addition of the SPI-1 gene to MVA, we deleted the gene from the HRE MVAs. This was accomplished by homologous recombination with DNA containing the GFP ORF regulated by the pi 1 late promoter within C12 flanking sequences. Recombination was carried out in CEF and the virus in fluorescent foci were clonally purified by repeated isolations. The loss of the C12L gene was confirmed by PCR and Sanger sequencing. The effect of the gene deletion from v51.2 (v51.2ASPI-l) was determined by infecting cells with 0.001 to 0.1 PFU per cell. There was no discernible effect of the gene deletion in CEF and only a slight effect in BS-C-1 cells, whereas in HeLa, A549 and MRC-5 cells the foci formed by v51.2ASPI-l stained less intensely than those formed by the parent virus v51.2 (FIG. 2B). However, the deleterious effect of SPI-1 deletion from v51.2 was most dramatic on MRC-5 cells just as addition of SPI-1 to MVA was most effective on MRC-5 cells. However, comparison of FIG. 2A and B suggested that loss of SPI-1 by v51.2 had a greater impact than gain of SPI-1 by MVA in A549 cells.

We also compared the effects of SPI-1 deletions on the other independently isolated HRE MVAs (v51.1, v44.1 and v44/47.1). Although deletion of the C12L ORF had no discernible effect in CEF cells, in each case the staining intensity of foci was reduced in MRC-5 cells, which was most clearly seen at the lowest MOI (FIG. 3).

Example 4. Effects of addition and deletion of SPI-1 on virus yields

Virus yields were determined at 48 h after inoculating MRC-5 cells with modified MVAs at a multiplicity of infection (MOI) of 0.001 in order to quantify the effects of SPI- 1 on replication and spread. Addition of the C12L ORF to MVA increased the yield by 160-fold (p=0.0006) and deletion of C12L from v51.2, v51.1, v44.1 and v44/47.1 reduced the yields by approximately 400-fold in each case (p<0.01) (FIG. 4A). However, addition of C12L did not increase the yield to the levels of the HRE viruses, which all have C12L, nor did deletion of C12L from the latter viruses reduce the yield to the level of MVA. Therefore, we concluded that absence of SPI-1 strongly contributes to the host-range defect of MVA but is not the sole factor responsible.

Serine protease inhibitor activity of RPXV SPI-1 was suggested by the formation of a stable complex with cathepsin G in vitro, which was prevented by mutation of the phenylalanine to alanine in the putative reactive loop. Furthermore, when the

phenylalanine to alanine mutation was introduced into the RPXV genome, the host range was similar to that of an SPI-1 deletion mutant in A549 cells. However, we found that recombinant MVAs containing SPI-1 with or without the reactive loop mutation (F322A) or a control mutation outside of the loop (T309R) enhanced MVA spread similarly in MRC-5 cells (FIG. 4B) suggesting that SPI-1 may have more than one mode of function. Example 5. Effects of ectopic expression of SPI-1 on host range

We have shown that expression of SPI-1 by MVA and HRE MVAs enhanced replication in human cells. However, viral genome alterations could have unanticipated effects. To circumvent this potential problem, we determined the effect of trans expression of SPI-1 on MVA replication. The C12L ORF with a 2xMyc tag regulated by the CMV promoter was introduced into A549 and MRC-5 cells by transduction with a retroviral vector. Expression of SPI-1 was demonstrated by Western blotting (FIGS. 5A, 5C). We would expect that ectopic expression of SPI-1 would have little or no effect on the replication of MVA-SPI-1 and v51.2 as they already express SPI-1, whereas there would be enhancement of MVA and v51.2ASPI-l. This is precisely what occurred in MRC-5 cells, with increases of approximately 6- and 11-fold for MVA and v51.2ASPI-l, respectively (FIG. 5B). Ectopic expression of SPI-1 in A549 cells also increased replication of v51.2ASPI-l (FIG. 5D) but had little or no effect on replication of MVA.

The latter result was consistent with the small enhancement of MVA-SPI-1 spread compared to MVA in A549 cells (FIG. 2A). Thus, both trans- and cis-expression of SPI-1 have similar effects on host range.

Example 6. VACV WR and RPXV exhibit a requirement for SPI-1 in MRC-5 cells similar to MVA

We were curious to determine whether the greater effects of addition and deletion of SPI-1 in MRC-5 cells compared to A549 cells was specific for MVA. To our knowledge, A549 is the only human cell line in which the effect of SPI-1 deletion had been tested for either RPXV or VACV WR. For comparison, A549 and MRC-5 cells were infected with VACV WR and RPXV SPI-1 deletion mutants and the parental viruses. In A549 cells, deletion of SPI-1 reduced the spread of VACV WR and RPXV by 15-and 640- fold respectively, whereas in MRC-5 cells the reductions were 39- and 12,000 respectively (FIG. 5E). Thus, not only was RPXV more dependent than VACV WR on SPI-1, but the requirement was greatly increased in MRC-5 cells compared to A549 cells for both viruses. We conclude that viral as well as cellular genetic backgrounds determine the degree of dependency on SPI-1 for replication. Example 7. Whole genome sequences of EIRE MV As and effects of deletion of additional genes

The v51.2, v51.1, v44.1 and v44/47.1 HRE MVAs each replicated more than 3 logs higher than the parental MVA (FIG. 4A). However, even after deletion of C12L they still replicated at least one log higher than MVA suggesting the presence of one or more additional host range genes. The entire genomes of the recombinant HRE MVAs were sequenced in order to identify additional genes that might contribute to the alleviation of the host-range defect and were deposited in GenBank. In the multiple alignments depicted in FIG. 6 A, the ORFs derived from the partially overlapping cosmids used for marker rescue are colored green and the ORFs retained from MVA are colored yellow. Inserted DNA was detected near the left ends of v51.1, v51.2, v44.1 and v44/47.1, consistent with the cosmids used for their generation. No DNA was inserted into the left end of v47.1, which acquired the ability to replicate in monkey but not human cells likely due to a spontaneous mutation. The left end deletions I, V and II in MVA are annotated. Repair of deletion I, which included insertion of C12L, occurred in each of the recombinant viruses able to replicate in human cells, whereas repair of deletions II and V only occurred in v51.1 and v44/47.1 and therefore were not essential for replication although the F5L gene affects plaque morphology. Furthermore, the genetic changes in v51.2, which were concentrated around deletion I, were present in each of the HRE MVAs capable of replicating in human cells (FIGS. 6A, 6B), suggesting that they included the minimal set of potential host range genes. Our strategy was to delete these genes from v51.2ASPI-l to see if that further reduced virus spread in MRC-5 and A549 cells but not CEF.

The C15L, C16L and C17L ORFs of v51.2ASPI-l, which are truncated or absent from MVA, were individually replaced with mCherry regulated by the pi 1 promoter. MVA, v51.2, v51.2ASPI-l, v51.2ASPI-lAC15, v51.2ASPI-lAC16 and v51.2ASPI-lAC17 viruses replicated equally well in permissive CEF. In A549 and MRC-5 cells, the replication of v51.2 was diminished to the same extent by deletion of SPI-1 alone and deletion of both SPI-1 and either C15L, C16L, or C17L (FIG. 7A). Although C10L and Cl 1R are present in MVA, there are sequence differences in the homologs of the HRE MVAs that potentially could affect host range. However, deletion of C10L or Cl 1R from v51.2 or v51.2ASPI had no effect on virus spread in CEF or MRC-5 cells (FIG. 7B), even though Cl 1R is a growth factor and has been shown to enhance VACV spread under some conditions. Thus, we did not identify an additional gene in v51.2 that significantly impaired replication in MRC-5 cells.

Example 8. ZAP is a Human Restriction Factor for MVA

A human genome-wide RNAi screen identified ZC3HAV1 (ZAP) as a putative restriction factor for MVA.

We determined that MRC5 cells express low level of ZAP. Codon-optimized vaccinia virus SPI-1 was stably expressed in A549 cells or MRC-5 cells by retrovirus transduction, and cell lysates were collected and used for Western blotting analysis to detect ZAP expression (FIG. 8A; GAPDH is also probed for loading control). The low level of ZAP in MRC-5 cells compared to A549 cells explains why C12 is sufficient for MRC-5 but not A549 cells

Replication of MVA was increased 1 to 2 logs in human A549, HeLa, and MRC-5 cells transfected with individual siRNAs targeting ZAP (FIG. 8B). Furthermore, inactivation of ZAP in A549 cells by CRISPR/Cas9 mutagenesis enhanced replication of MVA as well as a strain of MVA (47.1) that was adapted to grow in monkey BS-C-1 cells (FIG. 9). Enhanced assembly of MVA virions was demonstrated by electron microscopy (FIG. 10). Insertion of the SPI-1 gene into MVA enhances replication, particularly in MRC-5 cells, and to a lesser degree in A549 cells. We also showed that MVA expressing SPI-1 replicates better in ZAP knock-out A549 cells than in A549 cells, indicating that SPI-1 insertion into MVA and ZAP knock-out in A549 cells have additive roles in enhancing MVA replication in human cells and likely work by different mechanisms (FIG. 11). Importantly, we also showed that expression of SPI-1 in ZAP knock out cells enhances MVA replication more than either one alone (FIG. 12).

Example 9. ZAP -Deficient Human cancer cell line HCT116 is permissive for replication of MVA and MVA-expressing SPI-1

A549 and HCT116 cells were harvested and Western blotting analyses were performed with the cell lysates using antibody to human ZAP or GAPDH (FIG. 13 A) and the results demonstrated that human cancer cell line HCT116 is ZAP-deficient.

A549, A549-ZAP-KO and HCT116 cells were infected with MVA at 0.01

PFU/cell for 2 hours and washed 2 times with PBS to remove unabsorbed virus particles. Viruses were collected at 48 hpi and tittered on CEF (chicken embryo fibroblasts) cells (FIG. 13B) and the results demonstrate that MVA has enhanced replication in this ZAP- deficient human cancer cell line.

A549, A549-ZAP-KO, and HCT116 cells were infected with MVA expressing SPI-1 at 0.01 PFU/cell for 2 hours and washed 2 times with PBS to remove unabsorbed virus particles. Viruses were collected at 48 hpi and tittered on CEF (chicken embryo fibroblasts) cells (FIG. 13C). These results demonstrate that MVA-expressing SPI-1 also has enhanced replication in this ZAP-deficient human cancer cell line.

Example 10. Addition of vaccinia virus C16 and C17 enhances virus replication in human A549 cells and A549 ZAP KO cells

VACV C16 and C17 were inserted into MVA or MVA+SPI1 between ORFs 18 and G1 with their natural promoters to generate recombinant viruses MVA+C16+C17 or MVA+SPI1+C16+C17, respectively. A549 and A549-ZAP-KO cells were then infected with the indicated viruses at 0.01 PFU/cell for 2 hours and washed 2 times with PBS to remove unabsorbed virus particles. Viruses were collected at 48 hpi and tittered on CEF (chicken embryo fibroblasts) cells (FIG. 14). The results demonstrate that the addition of vaccinia virus ZAP inhibitors C16 and C17 allows the virus to spread in ZAP-competent human cells.

Example 11. Isolation of spontaneous mutants with enhanced replication in BS-C-1 cells.

The level of MVA replication is known to vary in different cell lines. While MVA titers in Syrian hamster (BHK-21) cells approach those obtained in chicken embryo fibroblast (CEF) cells, most other mammalian cells tested, including human cells, are non- permissive, although MVA makes very tiny plaques in African green monkey BS-C-1 cells. This replication defect was mapped to the left end of the MVA genome by marker rescue using large DNA fragments cloned into cosmids from a replicative competent VACV. Following cosmid transfections, screening for large plaque-forming virus was performed on BS-C-1 cells. After plaque purification, seven of eight virus isolates were found to also replicate in three human cell lines tested, whereas only one (v47.1) replicated in BS-C-1 cells. Genome sequencing revealed that each of the seven human replication competent viruses had repaired deletion I in the MVA genome. Further studies demonstrated that C12L and C16L together were sufficient to fully restore MVA replication in human cells. In contrast, v47.1, the virus with enhanced replication in BS-C- 1 cells, but which was still restricted in human cells did not have DNA insertions, although some sequence differences from MVA were noted.

The genome sequence of v47.1 was compared with genome sequence of MVA. Two specific sequence differences were observed. One was a deletion extending from nucleotide 6,178 to 8,104 containing fragmented ORFs. However, when this sequence was deleted from MVA by homologous recombination, replication in BS-C-1 cells was not enhanced (data not shown). Moreover, v47.1 was still able to replicate in BS-C-1 cells when this deletion was repaired by homologous recombination. The second sequence difference between v47.1 and MVA was one nucleotide change in the D10L open reading frame (ORF) resulting in change of cysteine 25 to tyrosine, which initially seemed unlikely to confer enhanced replication.

Since no added DNA was detected in the v47.1 genome, it was considered that an adaptive mutation might have arisen spontaneously. To test this hypothesis, MVA was passaged in BS-C-1 cells to see whether large plaque forming viruses would form without co-transfection of poxvirus DNA. Two independent passages were made by infecting monolayers at a multiplicity of 0.01 PFU per cell. After 72 hours, the cells were harvested, and the infections were repeated for a total of 10 rounds. After each round, samples were analyzed by staining with antiserum to VACV to determine plaque phenotype on BS-C-1 cells. By the second round, larger plaques, many of which appeared to have holes in their centers, began to appear and by the sixth round of passaging, such plaques were predominant (Fig. 15). After the tenth round of passaging, virus from three plaques from each passage was cloned by three successive plaque isolations on BS-C-1 cells, and then virus stocks were made in BS-C-1 cells. The parental MVA was also plaque purified in CEF to obtain three clones. Each of the BS-C-1 cell adapted viruses reached titers about 2 logs higher than non-adapted MVA (Fig. 16).

Genome sequencing: Four of the cloned viruses were partially purified by sedimentation through a cushion of sucrose and DNA was extracted, purified and analyzed by short read Illumina sequencing and de novo assembly. Each of the clones from passage 1 had a single nucleotide substitution in the D10 gene resulting in alanine to threonine mutation at amino acid 226 and each of the clones from passage 2 had a histidine to tyrosine mutation at amino acid 233. In contrast to the C-terminal proximal mutations in the newly isolated viruses, the D10 mutation in v47.1 was near the N-terminus at amino acid 25. The locations of the mutations in D10 were distant from the active site MutT motif GX5EX7REUXEEXGU, where“U” represents one of the bulky hydrophobic amino acids isoleucine, leucine, or valine and“X” is any amino acid (Fig. 17).

The replication of viruses with each of the D10 mutations was strongly enhanced in BS-C-1 cells and slightly enhanced in human HeLa and A549 cells compared to MVA (Fig. 18).

Construction of MVA with single nucleotide mutations by recombination.

The occurrence of D10 mutations in each of the BS-C-1 adapted MVA strains strongly suggested that they were responsible for the enhanced replication. However, some of the mutants also had additional mutations elsewhere in the genome. To confirm that the D10 mutations were sufficient for enhanced replication in BS-C-1 cells, a high frequency recombination strategy was employed, in which a PCR product of -1,000 bp containing a single nucleotide change was transfected into BS-C-1 cells infected with 1 PFU per cell of MVA. Following infection/transfection, lysates were prepared, and plaque assayed in BS- C-1 cells. Approximately 20-30% of the plaques were larger than those produced by MVA suggesting that the D10 mutations were responsible for the plaque phenotype. To confirm this, individual plaques were picked and re-plaqued three times in BS-C-1 cells to obtain clonal purity. Whole genome sequencing confirmed that most of the viruses had only the expected D10 mutation. Replication of two clones of each with only the expected mutation was determined in BS-C-1 cells. The titers of each mutant increased between 24 and 48 h reaching levels about 2 logs higher than MVA (Fig.19). These results confirmed that single mutations in D10, far from the catalytic site, enhanced replication of MVA in BS-C-1 cells.

The following non-exclusive list provides some exemplary embodiments of the invention.

1. A recombinant, animal cell comprising a heterologous gene encoding a heterologous protein comprising the activity of at least one poxvirus protein selected from the group consisting of a vaccinia virus C12L protein, a vaccinia virus C16L protein, a vaccinia virus C17L protein, wherein the at least one heterologous gene is operatively linked to a promoter recognized by an RNA polymerase of the animal cell.

2. The recombinant animal cell of 1, wherein the cell is a mammalian cell, a bird cell, a fish cell, an amphibian cell, or a reptilian cell.

3. The recombinant animal cell of 1 or 2, wherein the cell is a primate cell, a monkey cell, a human cell, a mouse cell, a hamster cell, or a rabbit cell. 4. The recombinant animal cell of any one of 1-3, wherein the cell is a human A549 cell, a human MRC-5 cell, a HeLa cell, or a monkey BSC-1 cell.

5. The recombinant, animal cell of any one of 1-4, wherein the recombinant, animal cell is derived from a natural animal cell that has reduced permissiveness for MVA replication.

6. The recombinant, animal cell of any one of 1-4, wherein the recombinant, animal cell is derived from a natural animal cell that is non-permissive for MVA replication.

7. The recombinant animal cell of any one of 1-6, wherein the heterologous gene is a homologue of a poxvirus ORF selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, and a vaccinia virus C17L ORF.

8. The recombinant animal cell of any one of 1-7, wherein the heterologous gene comprise a nucleic acid sequence at least 80% identical to a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, or a vaccinia virus C17L ORF.

9. The recombinant animal cell of any one of 1-8, wherein the heterologous gene comprise a nucleic acid sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO:5.

10. The recombinant animal cell of any one of 1-9, wherein the heterologous protein comprises an amino acid sequence at least 80% identical to a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, or a vaccinia virus C17L ORF.

11. The recombinant animal cell of any one of 1-10, wherein the heterologous protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

12. The recombinant animal cell of any one of 8, 9, 10, or 11, wherein the heterologous gene encodes a protein comprising the activity of a protein consisting of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

13. The recombinant animal cell of any one of 1-12, wherein the promoter is not the natural promoter associated with a homologue of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, or a vaccinia virus C17L ORF.

14. The recombinant animal cell of any one of 1-13, wherein the promoter is a promoter normally associated with a mammalian gene.

15. The recombinant animal cell of any one of 1-14, wherein the promoter is from the genome of a mammalian virus. 16. The recombinant animal cell of any one of 1-15, wherein the promoter is a cytomegalovirus promoter, an sv40 promoter, a polyomavirus promoter, a papillomavirus promoter, a herpesvirus promoter, an adenovirus promoter, an adeno-associated virus promoter, or a retrovirus promoter.

17. The recombinant animal cell of any one of 1-16, wherein the recombinant animal cell is not infected with a poxvirus.

18. The recombinant animal cell of any one of 1-17, wherein the recombinant animal cell lacks a homologue of a vaccinia virus Cl 1L ORF, a vaccinia virus C13L ORF, and/or a vaccinia virus C14L ORF.

19. The recombinant animal cell of any one of 1-18, wherein the heterologous gene is present on a plasmid of a cosmid in the recombinant animal cell.

20. The recombinant animal cell of any one of 1-18, wherein the heterologous gene is inserted into the genome of the recombinant animal cell.

21. The recombinant, animal cell of any one of 1-20, wherein the recombinant animal cell is deficient in at least one activity that inhibits the replication of modified vaccinia Ankara (MV A) virus.

22. The recombinant animal cell of 21, wherein the deficiency in the at least one activity is due to a decrease in the level of the at least one activity.

23. The recombinant animal cell of 21 or 22, wherein the deficiency in the at least one activity is due to a reduction in the level of a protein that comprises the activity.

24. The recombinant animal cell 21, wherein the recombinant animal cell lacks the at least one activity.

25. The recombinant animal cell of 21, wherein the deficiency is in the level of at least one activity associated with a mammalian ZAP protein.

26. The recombinant animal cell of 25, wherein the recombinant cell has a decreased level of ZAP protein.

27. The recombinant animal cell of 25, wherein the recombinant animal cell lacks at least one activity associated with the ZAP protein.

28. The recombinant animal cell of 25, wherein the recombinant animal cell fails to produce ZAP protein.

29. A method to produce MVA virus, comprising contacting an MVA virus with the recombinant animal cell of any one of 1-28, and incubating the contacted cell under conditions suitable for replication of MVA virus, thereby producing MVA virus. 30. A method to produce an altered MVA virus that replicates in animal cells that are restrictive or non-permissive for the replication of unaltered MVA virus, the method comprising:

a) inserting into the genome of an MVA virus a gene encoding a protein comprising the activity of at least one vaccinia virus ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF; or,

b) introducing a mutation in the D10 ORF of an MVA virus;

thereby creating the altered MV virus.

31. An altered MVA virus produced using the method of 30, wherein the genome of the altered MVA virus comprises:

a) a gene encoding a protein comprising the activity of at least one vaccinia virus

ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus

C16L ORF, and vaccinia virus C17L ORF; or,

b) a mutation in the D10 ORF of the altered MVA virus, wherein the mutation allows the altered MVA virus to replicate in animal cells that are restrictive or non- permissive for replication of unaltered MVA virus .

32. The altered MVA virus of 31, wherein the altered MVA virus lacks a vaccinia virus C13L ORF, a vaccinia virus C16L ORF, a vaccinia virus C17L IORF, or homologues thereof.

33. The method of 30 or the altered MVA virus of 31 or 32, wherein the gene comprise a nucleic acid sequence at least 80% identical to a vaccinia virus ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF.

34. The method of 30 or the altered MVA virus of 31, 32 or 33, wherein the gene comprise a nucleic acid sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO:5.

35. The method of 30 or the altered MVA virus of any one of 31-34, wherein the gene encodes a protein comprising an amino acid sequence at least 80% identical to a vaccinia virus protein selected from the group consisting of vaccinia virus C121 protein, vaccinia virus C16L protein, and vaccinia virus C17L protein.

36. The method of 30 or the altered MVA virus of any one of 31-35, wherein the gene encodes a protein comprising an amino acid sequence at least 80% identical to

SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. 37. The method of 30 or the altered MVA virus of any one of 31-36, wherein the gene is inserted into a non-essential ORF in the MVA virus genome.

38. The method of 30 or the altered MVA virus of any one of 31-37, wherein the gene is inserted into an IGR in the MVA virus genome.

39. The method of 30 or the altered MVA virus of any one of 31-38, wherein the gene is functionally linked to a poxvirus promoter.

40. The method of 30 or the altered MVA virus of any one of 31-39, wherein the mutation in the MVA D10 ORF is a substitution mutation.

41. The method of 30 or the altered MVA virus of any one of 31-39, wherein the mutation in the MVA D10 ORF is introduced at an amino acid position corresponding to an amino acid position selected from the group consisting of C25, A226, and H233 of SEQ ID NO:7.

42. The method of 30 or the altered MVA virus of any one of 31-39, wherein the mutation is selected from the group consisting:

a) substitution of the amino acid corresponding to C25 of SEQ ID NO:7 with a tyrosine;

b) substitution of the amino acid corresponding to A226 of SEQ ID NO: 7 with a threonine; and,

c) substitution of the amino acid corresponding to H233 of SEQ ID NO: 7 with a tyrosine.

43. The method of 30 or the altered MVA virus of any one of 31-42, wherein the altered MVA virus comprises a heterologous nucleic acid sequence encoding an immunogenic protein or a therapeutic agent.

44. The method of 43, or the altered virus of any one of 31-43, wherein the heterologous nucleic acid sequence is functionally linked to a promoter that is recognized by an MVA or a cellular RNA polymerase.

45. The method of 43 or 44, wherein the therapeutic agent is a tumor- suppressor protein, a pro-apototic protein, a cytokine, a lymphokine, a monokine, a growth factor, an enzyme, a hormone, an immunomodulatory protein, a cytotoxic peptide, a suicide protein, a cytotoxin, a pro-drug, or a therapeutic RNA.

46. A method to produce progeny altered MVA virus, comprising contacting the altered MVA virus of any one of 31-45 with an animal cell, and incubating the contacted cell under conditions suitable for replication of the contacted altered MVA virus.

47. The method of 46, wherein the animal cell is non-permissive, or has reduced permissiveness, for replication of wild-type MVA virus.

48. The method of 46, wherein the animal cell is deficient in at least one activity that inhibits replication of MVA virus.

49. The method of 48, wherein the deficiency is a reduction in the level of MVA virus inhibitory activity.

50. The method of 48 or 49, wherein the deficiency is a reduction in the level of a protein comprising the MVA virus inhibitory activity.

51. The method of 48 or 49, wherein the deficiency is a lack of virus inhibitory activity.

52. The method of 48 - 51, wherein the deficiency is due to a lack of a protein comprising the MVA virus inhibitory activity.

53. The method of any one of 48-51 wherein the deficiency is due to the presence of a functional RNA molecule in the cell, wherein the functional RNA molecule affects the level of the MVA virus inhibitory activity.

54. The method of any one of 48-53, wherein the deficiency is in the level of at least one activity associated with a mammalian ZAP protein.

55. The method of any one of 48-54, wherein the deficiency is a reduction in the level of ZAP protein.

56. The method of any one of 48-54, wherein the deficiency is a lack at least one activity associated with the ZAP protein.

57. The method of any one of 48-56, wherein the deficiency is a lack of ZAP protein.

58. A method of producing progeny MVA virus, comprising contacting MVA virus with a cell that is deficient in at least one activity that inhibits replication of MVA virus.

59. The method of 58 wherein the deficiency is a reduction in the level of MVA virus inhibitory activity.

60. The method of 58 or 59, wherein the deficiency is a reduction in the level of a protein comprising the MVA virus inhibitory activity.

61. The method of 60, wherein the deficiency is a lack of virus inhibitory activity. 62. The method of 60 or 61, wherein the deficiency is due to a lack of a protein comprising the MV A virus inhibitory activity.

63. The method of any one of 58-62, wherein the deficiency is in the level of at least one activity associated with a mammalian ZAP protein.

64. The method of any one of 58-63, wherein the deficiency is a reduction in the level of ZAP protein.

65. The method of any one of 58-64, wherein the deficiency is a lack at least one activity associated with the ZAP protein.

66. The method of any one of 58-65, wherein the deficiency is a lack ZAP protein.

67. The method of any one of 58-65, wherein the cell is a tumor cell.

68. The method of any one of 58-6465 wherein the deficiency is due to the presence of a functional RNA molecule in the cell, wherein the functional RNA molecule affects the level of the MVA virus inhibitory activity.

69. A method of treating a tumor cell, comprising contacting the tumor cell with an MVA virus.

70. The method of 69, wherein the tumor cell is deficient in at least one activity that inhibits MVA replication.

71. The method of 69 or 70, wherein the cell is deficient in an activity associated with ZAP protein.

72. The method of any of 69-71, wherein the tumor cell is deficient in ZAP protein.

73. The method of any one of 69-72, wherein the MVA virus is a wild-type MVA virus.

74. The method of any one of 69-72, wherein the MVA virus is the altered MVA virus of any one of 31-43.

75. The method of any one of 69-74, wherein the tumor cell is in culture.

76. The method of any one of 69-74, wherein the tumor cell is in an individual.

77. A system for producing MVA virus, comprising a population of the recombinant, animal cell of any one of 1-28, and an MVA virus.

78. The system of 77, comprising a solid substrate comprising the population of mammalian cells. 79. A kit comprising the mammalian cell of any one of 1-28, the altered MVA virus of any one of 31-45, or the system of 77 or 78.

Claims

What is claimed:

1. A recombinant, animal cell comprising a heterologous gene encoding a

heterologous protein comprising the activity of at least one poxvirus protein selected from the group consisting of a vaccinia virus C12L protein, a vaccinia virus C16L protein, a vaccinia virus C17L protein, wherein the at least one heterologous gene is operatively linked to a promoter recognized by an RNA polymerase of the animal cell.

2. The recombinant animal cell of claim 1, wherein the cell is selected from the group consisting of a human cell, a mouse cell, a hamster cell, or a rabbit cell, a mammalian cell, a primate cell, a monkey cell, an A549 cell, an MRC-5 cell, a HeLa cell, a BSC-1 cell, a bird cell, a fish cell, an amphibian cell, or a reptilian cell.

3. The recombinant animal cell of any one of claims 1 or 2 wherein the heterologous gene is a homologue of a poxvirus ORF selected from the group consisting of a vaccinia virus C12L ORF, a vaccinia virus C16L ORF, and a vaccinia virus C17L ORF.

4. The recombinant animal cell of any one of claims 1-3, wherein the heterologous gene is inserted into the genome of the recombinant animal cell, or wherein the heterologous gene is present on a plasmid or a cosmid in the recombinant animal cell.

5. The recombinant, animal cell of any one of claims 1-4, wherein the recombinant animal cell is deficient in at least one activity that inhibits the replication of modified vaccinia Ankara (MV A) virus.

6. The recombinant animal cell of claim 5, wherein the deficiency is in at least one activity associated with a mammalian ZAP protein.

7. A method to produce MVA virus, comprising contacting an MVA virus with the recombinant animal cell of any one of claims 1-6, and incubating the contacted cell under conditions suitable for replication of MVA virus, thereby producing MVA virus.

8. A method to produce an altered MVA virus that replicates in animal cells that are restrictive or non-permissive for the replication of unaltered MVA virus, the method comprising:

a) inserting into the genome of an MVA virus a gene encoding a protein comprising the activity of a protein encoded by at least one vaccinia virus ORF selected from the group consisting of vaccinia virus C121 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF; or, b) introducing a mutation in an MVA virus ORF that encodes a decapping protein; thereby producing the altered MVA virus.

9. An altered MVA virus produced using the method of claim 8, wherein the genome of the altered MVA virus comprises at least one of:

a) a gene encoding a protein comprising the activity of a protein encoded by at least one vaccinia virus ORF selected from the group consisting of vaccinia virus Cl 21 ORF, vaccinia virus C16L ORF, and vaccinia virus C17L ORF; or, b) a mutation in an MVA virus ORF that encodes a decapping protein , wherein the mutation allows the altered MVA virus to replicate in animal cells that are restrictive for unaltered MVA virus, or wherein the mutation allows the altered MVA virus to produce titers that are higher than the titers produced by unaltered MVA virus in the same type of MVA virus-restrictive cell.

10. The altered MVA virus of claim 9, wherein the altered MVA virus lacks a vaccinia virus C13L ORF, a vaccinia virus Cl 1L ORF, a vaccinia virus C14L IORF, or homologues thereof.

11. The method of claim 8 or the altered MVA virus of claim 9 or 10 wherein the ORF that encodes a decapping protein is MVA virus ORF D9 or D10.

12. The method of claim 8 or 11, or the altered MVA virus of any one of claims 9-11, wherein the altered MVA virus comprises a heterologous nucleic acid sequence encoding an immunogenic protein or a therapeutic agent.

13. A method of producing progeny MVA virus, comprising contacting MVA virus with a cell that is deficient in at least one activity that inhibits replication of MVA virus.

14. The method of claim 13, wherein the deficiency is in the level of at least one activity associated with a mammalian ZAP protein.

15. A method of treating a tumor cell, comprising contacting the tumor cell with an MVA virus.

16. A system for producing MVA virus, comprising a population of the recombinant, animal cell of any one of claims 1-6, and an MVA virus.

17. A kit comprising the mammalian cell of any one of claims 1-6, the altered MVA virus of any one of claims 9-12, or the system of claim 16.