Mammalian cell line for the production of modified vaccinia virus Ankara (MVA)

Abstract

The present invention relates to a mammalian non-human cell line, specifically a Chinese hamster ovary (CHO) cell line, in which the genes are modified to express CP77, K1L and / or SPI-1, which are host range genes of poxviruses not expressed by MVA, and to the use of such cell lines in the replication of MVA.

Description

Technical Field

[0001] The present invention relates to vaccines using viral vectors. More specifically, the present invention relates to mammalian cell substrates for the replication of modified vaccinia virus Ankara (MVA). In particular, the present invention relates to mammalian cell lines other than human cells, specifically, Chinese hamster ovary (CHO) cells whose genes have been modified to express the host range genes of poxviruses that are not expressed by MVA. The present invention further relates to the production of such cell lines and their use in the production of MVA-based vaccines. The present invention also relates to recombinant MVA that expresses the host range genes of poxviruses that are not endogenously expressed by MVA.

Background Art

[0002] Modified vaccinia virus Ankara (MVA) is a highly attenuated subspecies of vaccinia virus (Orthopoxvirus genus). The strain MVA-BN® (Bavarian Nordic) has been approved for use as a safe human vaccine against smallpox and monkeypox (1 - 3). Furthermore, MVA is widely used as a safe viral vector for the production of recombinant vaccines against infectious diseases and cancer (3 - 5).

[0003] MVA was obtained by serially passaging the ancestral chorioallantoic vaccinia virus Ankara (CVA) more than 500 times in chicken embryo fibroblasts (CEF or CEF cells) (3, 6). During passage, multiple mutations and deletions accumulated in the viral genome. Compared to the parental CVA, MVA lacks six large genomic fragments and has acquired multiple mutations and small deletions throughout its genome (7 - 9). As a result, MVA has lost its replication ability in most mammalian cells, and only a few cell lines are known to support productive viral replication. Thus, MVA is characterized by different viral host range restrictions compared to the parental CVA and other members of the Orthopoxvirus.

[0004] To date, the only cell substrate approved for MVA-based vaccine production is chicken embryo fibroblast (CEF) cells (10, 11). Primary CEF cells are well-established cell substrates for MVA production, but preparing them from developing chicken eggs is labor-intensive and time-consuming. Moreover, the procedure is prone to contamination. None of these are ideal for a stable and efficient production process, especially for vaccine production on an industrial scale under the strict requirements of Good Manufacturing Practice (GMP).

[0005] Therefore, alternative cell substrates for MVA replication are needed.

[0006] U.S. Patent No. 5,830,688 (issued in 1998) describes the identification of sequences involved in the growth of vaccinia virus in Chinese hamster ovary (CHO) cells, which, when introduced into vaccinia virus (which cannot essentially grow in CHO cells), enables the growth of vaccinia in this cell line.

[0007] However, replication deficiency in most mammalian cell lines is an important safety feature of MVA from the perspective of its use for human vaccines. Therefore, genetic modification to enable MVA to replicate in mammalian cells is not an option.

[0008] Rather, this is considered to involve expressing one or more poxvirus host range genes that can support the replication of MVA in cell lines that are otherwise non-permissive. If the appropriate host range genes are expressed, the modified cells should acquire the ability to assist in replicating MVA to an acceptable titer.

[0009] The genetic basis of the host range restriction of MVA is not well understood, and in most cell lines, the causative poxvirus genes have remained largely uncharacterized. At least, the six major genomic deletions in MVA (associated with CVA) do not alone account for the strong attenuation and highly restricted host range properties of MVA (12). This indicates that the host range restriction of MVA is likely to be a synergistic effect involving not only the six major deletions but also other smaller gene deletions and mutations present throughout the genome (12).

[0010] Among the well-defined host range genes of orthopoxviruses, the cowpox virus (CPXV or CWPX virus) CP77 gene is known to promote the replication of vaccinia virus (VACV) and ectromelia virus in Chinese hamster ovary (CHO) cells and RK13 (rabbit kidney-derived) cells (13 - 17). Genetically engineered CHO cell lines expressing the CP77 gene and the VACV-derived D13L gene were generated for the production of replication-deficient recombinant VACV (18). Expression of the CP77 gene has also been shown to expand the host range of MVA by alleviating inhibition prior to late protein synthesis in CHO cells (19). However, CP77 alone may not be sufficient for high-titer replication of MVA in cells that are otherwise non-permissive.

[0011] In addition to CP77, MVA is lacking at least three other genes, namely K1L, SPI-1 (C12L), and C9L, which may also contribute to the host range of orthopoxviruses in different cell lines (20 - 25). K1L is a VACV host range gene known to be involved in the negative regulation of the NF-κB signaling pathway and can restore the replication of MVA in RK13 cells (21, 26). It has also been reported that the K1L gene is involved in the replication of VACV in RK13 cells and that CP77 can complement the effect of the K1L gene in this cell line

[18] .

[0012] Serine protease inhibitor 1 (SPI-1) was first discovered in rabbitpox virus and has been shown to act as an apoptosis inhibitor (22, 27). The SPI-1 deletion mutant of VACV cannot replicate efficiently in primary human keratinocytes or human lung cancer cells (A549) (28). On the other hand, SPI-1 has been identified as a host range factor of MVA (29). Furthermore, A549 cells co-expressing the recently identified host range gene, namely C16L / B22R, together with the SPI-1 gene were found to be permissive to MVA (30).

[0013] The vaccinia C9L gene has been shown to act as an inhibitor of the host immune response by antagonizing the action of interferon early in the viral replication cycle (23).

[0014] The expression of host range genes CP77, K1L, SPI-1, and C9L in MVA and other orthopoxvirus members is summarized in Table 1 below. Furthermore, cell lines that do not support the replication of host range gene-deficient viruses are listed in Table 1.

Table 1

Summary of the Invention

[0015] It is an object of the present invention to provide a cell substrate for the replication of MVA.

[0016] The object of the present invention is solved by providing CHO cells that express a combination of orthopoxvirus host range genes not expressed by MVA. In particular, the present invention is defined by the appended claims, as well as by the following aspects and their embodiments.

[0017] In a first aspect, the present invention provides cells of a continuous cell line, the genes of which have been modified to express CP77 and K1L, which are orthopoxvirus host range genes.

[0018] In certain aspects, the present invention provides cells of a continuous passage cell line, wherein the genes are modified to express CP77, K1L, and SPI-1, which are host range genes of poxviruses.

[0019] In another aspect, the present invention provides the use of the cells according to the present invention for the replication of MVA.

[0020] In yet another aspect, the present invention provides the use of the cells according to the present invention in the production of a vaccine comprising MVA.

[0021] In yet another aspect, the present invention provides a vaccine comprising MVA, wherein the MVA is prepared using the cells according to the present invention.

[0022] In yet another aspect, the present invention provides a method for producing the cells according to the present invention, comprising the following: (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous passage cell line, wherein the nucleic acid comprises (i) CP77, which is a host range gene of poxviruses, operably linked to a promoter, or (ii) K1L, which is a host range gene of poxviruses, operably linked to a promoter, or (iii) CP77 and K1L, which are host range genes of poxviruses, each operably linked to a promoter, (b) introducing the nucleic acid (i) and (ii) obtained in step (a), or the nucleic acid (iii) obtained in step (a) into the cells, and (c) selecting a cell population or clone expressing CP77 and K1L, which are host range genes of poxviruses.

[0023] In yet another aspect, the present invention provides a method for producing the cells according to the present invention, comprising the following: (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous passage cell line, wherein the nucleic acid comprises (i) CP77, a host range gene of poxvirus operably linked to a promoter, or (ii) K1L, a host range gene of poxvirus operably linked to a promoter, or (iii) SPI-1, a host range gene of poxvirus operably linked to a promoter, or (iv) a step comprising CP77, K1L and SPI-1, each a host range gene of poxvirus operably linked to a promoter, (b) a step of introducing into a cell the nucleic acids (i), (ii) and (iii) obtained in step (a), or the nucleic acid (iv) obtained in step (a), and (c) a step of selecting a cell population or clone expressing CP77, K1L and SPI-1, host range genes of poxvirus.

[0024] In yet another aspect, the present invention provides a cell produced by the method according to the present invention.

[0025] In yet another aspect, the present invention provides MVA replicated using the cell according to the present invention.

[0026] In yet another aspect, the present invention provides the use of CP77 and K1L, host range genes of poxvirus, to enable a cultured cell to express CP77 and K1L.

[0027] In yet another aspect, the present invention provides the use of CP77, K1L and SPI-1, host range genes of poxvirus, to enable a cell to express CP77, K1L and SPI-1.

[0028] In yet another aspect, the present invention provides MVA comprising CP77 and K1L, host range genes of poxvirus.

[0029] In yet another aspect, the present invention provides a MVA comprising the poxvirus host range genes CP77, K1L, and SPI-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0030]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

[0031] SEQ ID NO: 1 shows the amino acid sequence encoded by the CP77 gene. SEQ ID NO: 2 shows the nucleic acid sequence of the CP77 gene. SEQ ID NO: 3 shows the amino acid sequence encoded by the K1L gene. SEQ ID NO: 4 shows the nucleic acid sequence of the K1L gene. SEQ ID NO: 5 shows the amino acid sequence encoded by the mRFP1-CP77 gene. SEQ ID NO: 6 shows the nucleic acid sequence of the mRFP1-CP77 gene. SEQ ID NO: 7 shows the amino acid sequence encoded by the K1L-V5 gene. SEQ ID NO: 8 shows the nucleic acid sequence of the K1L-V5 gene. SEQ ID NO: 9 shows the amino acid sequence encoded by the SPI-1 gene. SEQ ID NO: 10 shows the nucleic acid sequence of the SPI-1 gene. SEQ ID NO: 11 shows the amino acid sequence encoded by the C9L gene. SEQ ID NO: 12 shows the nucleic acid sequence of the C9L gene. Note: The nucleotide sequences of mRFP1-CP77, K1L-V5, and SPI-1 have been codon-optimized for expression in human cells. The C9L gene was amplified by PCR from VACV-WR genomic DNA. SEQ ID NO: 13 shows the nucleic acid sequence of the PrH5m promoter. SEQ ID NO: 14 shows the nucleic acid sequence of the Pr1328 promoter. SEQ ID NO: 15 shows the nucleic acid sequence of the Pr13.5 promoter. SEQ ID NO: 16 shows the nucleic acid sequence of the forward primer for GFP. SEQ ID NO: 17 shows the nucleic acid sequence of the reverse primer for GFP. SEQ ID NO: 18 shows the nucleic acid sequence of the forward primer for CP77. SEQ ID NO: 19 shows the nucleic acid sequence of the reverse primer for CP77. SEQ ID NO: 20 shows the nucleic acid sequence of the forward primer for K1L. SEQ ID NO: 21 shows the nucleic acid sequence of the reverse primer for K1L. SEQ ID NO: 22 shows the nucleic acid sequence of the forward primer for SPI-1. SEQ ID NO: 23 shows the nucleic acid sequence of the reverse primer for SPI-1. SEQ ID NO: 24 shows the nucleic acid sequence of the forward primer for C9L. SEQ ID NO: 25 shows the nucleic acid sequence of the reverse primer for C9L.

DETAILED DESCRIPTION OF THE INVENTION

[0032] This specification reports on the expansion of the host range of MVA by introducing genes of the host range of poxvirus into the virus genome. The host range genes CP77, K1L, SPI-1, and C9L were sequentially inserted into MVA, and the MVA recombinants produced in each step were screened for their ability to infect and replicate in continuous cell lines.

[0033] Among the cell lines tested, only CHO cells and RK13 cells (neither of which is permissive to MVA) enabled the replication of MVA recombinants expressing at least the CP77 gene.

[0034] As shown in the case of CHO cells, the insertion of the K1L gene into an MVA recombinant already encoding CP77 increased the replicative ability of the recombinant, and the insertion of an additional SPI-1 gene further enhanced its replicative ability. The latter step, i.e., the insertion of the SPI-1 gene in addition to the CP77 and K1L genes, even brought about the most significant improvement in the replicative ability of MVA within the stepwise insertion procedure of host range genes. In contrast, the insertion of the C9L gene in addition to the CP77, K1L, and SPI-1 genes did not lead to further improvement.

[0035] Notably, MVA recombinants expressing the CP77, K1L, and SPI-1 genes replicated in CHO cells to a titer comparable to that of wild-type MVA grown in primary CEF cells, its standard substrate. Based on this, CHO cells genetically modified to co-express CP77, K1L, and optionally SPI-1 of the poxvirus host range genes were regarded as an excellent substrate for MVA replication.

[0036] CHO are continuous subculturing non-human cell lines widely used in the biopharmaceutical industry for the production of biologics. They can be grown as suspension cultures in synthetic growth media to high cell densities within industrial-scale bioreactors. Thus, the ability to use CHO cells as an alternative to primary CEF cells for MVA replication represents a significant advancement in the production of MVA-based vaccines.

[0037] Definitions As used herein, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a nucleic acid" includes one or more nucleic acid sequences.

[0038] As used herein, the connective "and / or" between multiple recited elements is understood to encompass both individual choices and combinations of choices. For example, when two elements are connected by "and / or", in the first alternative, it refers to the first element being applicable without using the second element. In the second alternative, it refers to the second element being applicable without using the first element. In the third alternative, it refers to the first element and the second element being applicable together. Any one of these alternatives falls within the scope of its meaning and is thus understood to meet the requirements of the term "and / or" as used herein. The applicability of two or more of the alternatives simultaneously also falls within the scope of its meaning and is thus understood to meet the requirements of the term "and / or".

[0039] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted as including the recited integer or step or group of integers or steps but not as excluding any other integer or step or group of integers or steps. When used in connection with an aspect or embodiment of the present invention, the term "comprising" is amendable and accordingly may be replaced with the term "containing" or "including" or, when used herein, may be replaced with the term "having". Similarly, any of the foregoing terms (comprising, containing, including, having), when used in connection with an aspect or embodiment in the description of the present invention, in any case, includes the terms "consisting of" or "consisting essentially of", each of which represents a different specific legal meaning by jurisdiction.

[0040] As used herein, "consisting of" excludes any element, step, or component not specified in the claims. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claims.

[0041] As used herein, the term "MVA recombinant" refers to MVA having one or more inserted poxvirus host range genes that are not endogenously expressed in native MVA, and additionally the EGFP gene. Similarly, "CVA recombinant" refers to CVA having an inserted poxvirus host range gene that is not expressed in native CVA, and additionally the EGFP gene.

[0042] As used herein, the terms "natural MVA" or "natural CVA" refer to MVA and CVA, respectively, in which the genes have not been modified. The term "not endogenously expressed" refers to genes that are not expressed by or contained in natural MVA or CVA.

[0043] The terms "wild-type MVA" (MVA-wt) or "wild-type CVA" (CVA-wt) refer to MVA and CVA, respectively, that contain the EGFP gene and are each used as a control for recombinant MVA or recombinant CVA.

[0044] The "host range" of a virus is the range of cell types that the virus can infect. The term "host range gene" refers to the genetic basis of the virus's host range.

[0045] The terms "primary cell culture" or "primary culture cell" refer to cultured cells at an early stage after the cells have been isolated from tissue. The opposite is a continuously passaged cell line or an immortalized cell line. The term "continuously passaged cell line" refers to cells that can be continuously propagated in culture.

[0046] The term "cell line in a permissive state" refers to cells that allow a virus to replicate. The term "permissivity" refers to the ability of cells to allow a virus to replicate.

[0047] The term "cell substrate" refers to cells that allow the replication of a virus (here, MVA).

[0048] The term "reproduction" refers to the replication or propagation of MVA.

[0049] As used herein, the expression "cells genetically modified to express" means that, without the modification and before the modification, the cells were unable to express the gene. Abbreviations BAC bacterial artificial chromosome (bacterial artificial chromosome) CEF chicken embryo fibroblast (chicken embryo fibroblast) CHO cell Chinese hamster ovary cell (Chinese hamster ovary cell) CPXV, CWPX cowpox virus (cowpox virus) CVA chorioallantois vaccinia virus Ankara (chorioallantois vaccinia virus Ankara) CVA-C CVA-wt encoding mRFP1-CP77 CVA-wt CVA wild type encoding EGFP EGFP enhanced green fluorescent protein (enhanced green fluorescent protein) mRFP1 mono red fluorescent protein (mono red fluorescent protein) MVA Modified Vaccinia Virus Ankara (Modified Vaccinia Virus Ankara) MVA-BN (registered trademark) MVA-Bavarian Nordic,[[]] Owned by Bavarian Nordic MVA-C MVA-wt encoding mRFP1-CP77 MVA-CK MVA-wt encoding mRFP1-CP77 and K1L-V5 MVA-wt MVA-CKS MVA-wt encoding mRFP1-CP77, K1L-V5 and SPI-1 MVA-wt encoding MVA-CKS-C9 MVA-wt encoding mRFP1-CP77, K1L-V5, SPI-1, and C9L MVA-KS encoding K1L and MVA-wt encoding SPI-1 MVA-BN® BAC encoding MVA-wt EGFP MVA reconstituted from the clone ORF open reading frame (Open reading frame) RT-PCT reverse transcription polymerase chain reaction (Reverse transcription polymerase chain reaction) VACV vaccinia virus (vaccinia virus) VACV-WR vaccinia virus strain Western Reserve (Vaccinia virus strain Western Reserve)

[0050] Embodiment In one aspect, the present invention provides cells of a continuously passaged cell line capable of expressing CP77 and K1L, which are host range genes of poxvirus.

[0051] In one aspect, the present invention provides cells of a continuously passaged cell line whose genes are modified to express CP77 and K1L, which are host range genes of poxvirus.

[0052] In one embodiment, the genome of the cell comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3.

[0053] In one embodiment, the genome of the cell comprises the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 4.

[0054] In certain aspects, the present invention provides cells of a continuously passaged cell line capable of expressing CP77, K1L and SPI-1, which are host range genes of poxvirus.

[0055] In certain embodiments, the invention provides cells of a continuously passaged cell line whose genes are modified to express CP77, K1L, and SPI-1, which are host range genes of poxvirus.

[0056] In one embodiment, the genome of the cell comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9.

[0057] In one embodiment, the genome of the cell comprises the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 4, and the nucleotide sequence of SEQ ID NO: 10.

[0058] In one embodiment, the cell is unable to express the host range gene without genetic modification and prior to genetic modification.

[0059] In one embodiment, the cell is infected with MVA.

[0060] In one embodiment of all aspects, the cell is a cultured cell or a non-primary cell.

[0061] In one embodiment of all aspects, the cell is not CEF, DF-1, or quail cells, or is a non-avian cell.

[0062] In one embodiment of all aspects, the cell line is a mammalian cell line, preferably a cell line of a non-human mammal.

[0063] In one embodiment of all aspects, the cell is a CHO cell.

[0064] In another aspect, the invention provides the use of the cells according to the invention for the replication of MVA.

[0065] In another aspect, the invention provides the use of the cells according to the invention in the manufacture of a vaccine comprising MVA.

[0066] In yet another aspect, the present invention provides a vaccine comprising MVA, wherein the MVA or the vaccine is prepared using the cells according to the present invention.

[0067] In yet another aspect, the present invention provides a method for producing the cells according to the present invention, comprising the following (a) preparing a nucleic acid suitable for gene introduction into cells of a continuous cell line, wherein the nucleic acid (i) is CP77, a host range gene of poxvirus operably linked to a promoter, or (ii) is K1L, a host range gene of poxvirus operably linked to a promoter, or (iii) comprises CP77 and K1L, host range genes of poxvirus, each operably linked to a promoter, (b) introducing the nucleic acid (i) and (ii) obtained in step (a), or the nucleic acid (iii) obtained in step (a) into the cells, and (c) selecting a cell population or clone expressing CP77 and K1L, host range genes of poxvirus.

[0068] In yet another aspect, the present invention provides a method for producing the cells according to the present invention, comprising the following (a) preparing a nucleic acid suitable for gene introduction into cells of a continuous cell line, wherein the nucleic acid (i) is CP77, a host range gene of poxvirus operably linked to a promoter, or (ii) is K1L, a host range gene of poxvirus operably linked to a promoter, or (iii) is SPI-1, a host range gene of poxvirus operably linked to a promoter, or (iv) comprises CP77, K1L and SPI-1, host range genes of poxvirus, each operably linked to a promoter, (b) Introducing the nucleic acids (i), (ii), and (iii) obtained in step (a), or the nucleic acid (iv) obtained in step (a) into the cell, and (c) Selecting a cell population or clone that expresses the poxvirus host range genes CP77, K1L, and SPI-1.

[0069] In one embodiment of the method, the nucleic acid suitable for gene transfer is a vector or plasmid.

[0070] In one embodiment of the method, the promoter functionally linked to the host range gene is active in the cell into which the nucleic acid obtained in step (a) is introduced.

[0071] In one embodiment of the method, the promoter functionally linked to the host range gene is a eukaryotic promoter.

[0072] In one embodiment of the method, the nucleic acid obtained in step (a) contains a polyadenylation signal.

[0073] In yet another aspect, the present invention provides a cell produced by the method according to the present invention.

[0074] In yet another aspect, the present invention provides MVA replicated using the cell according to the present invention.

[0075] In yet another aspect, the present invention provides the use of CP77 and K1L, which are poxvirus host range genes, to make a cell, preferably a cell of a continuous cell line, capable of expressing the CP77 and K1L genes.

[0076] In yet another aspect, the present invention provides the use of CP77 and K1L, which are poxvirus host range genes, to make a cell, preferably a cell of a continuous cell line, capable of enabling the replication of MVA in the cell.

[0077] In one embodiment of use, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 are used to enable a cell to be able to express the CP77 and K1L genes.

[0078] In one embodiment of use, the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 4 are used to enable a cell to be able to express the CP77 and K1L genes.

[0079] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77, K1L and SPI-1 to enable a cell, preferably a cell of a continuous cell line, to be able to express the CP77, K1L and SPI-1 genes.

[0080] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77, K1L and SPI-1 to enable a cell, preferably a cell of a continuous cell line, to be able to enable the replication of MVA within that cell.

[0081] In one embodiment of use, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid of SEQ ID NO: 9 are used to enable a cell to be able to express the CP77, K1L and SPI-1 genes.

[0082] In one embodiment of use, the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 10 are used to enable a cell to be able to express the CP77, K1L and SPI-1 genes.

[0083] In yet another aspect, the present invention provides MVA expressing the poxvirus host range genes CP77 and K1L.

[0084] In one embodiment of the MVA, the MVA comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3.

[0085] In one embodiment of the MVA, the MVA comprises the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 4.

[0086] In yet another aspect, the present invention provides an MVA that expresses mRFP1-CP77 and K1L-V5, which are host range genes of poxvirus.

[0087] In one embodiment of the MVA, the MVA comprises an mRFP1-CP77 fusion gene, and preferably, the CP77 gene is fused to the C-terminus of RFP via a flexible linker.

[0088] In one embodiment of the MVA, the MVA comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7.

[0089] In one embodiment of the MVA, the MVA comprises the nucleotide sequence of SEQ ID NO: 6 and the nucleotide sequence of SEQ ID NO: 8.

[0090] In yet another aspect, the present invention provides an MVA that expresses CP77, K1L, and SPI-1, which are host range genes of poxvirus.

[0091] In one embodiment of the MVA, the MVA comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7, and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9.

[0092] In one embodiment of the MVA, the MVA comprises the nucleotide sequence of SEQ ID NO: 6, the nucleotide sequence of SEQ ID NO: 8, and the nucleotide sequence of SEQ ID NO: 10.

[0093] In one embodiment of MVA, the gene of the host region is operably linked to a promoter selected from the group consisting of PrH5m, Pr1328, and Pr13.5.

[0094] In one embodiment of MVA, MVA comprises a PrH5m promoter operably linked to the mRFP1-CP77 or CP77 gene.

[0095] In one embodiment of MVA, MVA comprises a Pr1328 promoter operably linked to the K1L-V5 or K1L gene.

[0096] In one embodiment, MVA comprises a Pr13.5 promoter operably linked to the SPI-1 host region gene.

[0097] In one embodiment of MVA, the insertion site of the mRFP1-CP77 or CP77 gene is the intergenic region (IGR44 / 45) between MVA44L and MVA45L of the ORF.

[0098] In one embodiment of MVA, the insertion site of the K1L-V5 or K1L gene is between MVA88R and MVA89L of the ORF.

[0099] In one embodiment of MVA, the insertion site of the SPI-1 host region gene is between MVA88R and MVA89L of the ORF.

[0100] In one embodiment of MVA, MVA comprises an expression cassette comprising the mRFP1-CP77 or CP77 gene operably linked to the PrH5m promoter, and the expression cassette is inserted into the insertion sites MVA44L and MVA45L (IGR44 / 45).

[0101] In one embodiment of MVA, MVA comprises an expression cassette comprising the K1L-V5 or K1L gene operably linked to the Pr1328 promoter, and the expression cassette is inserted into the insertion sites MVA88R and MVA89L.

[0102] In one embodiment of the MVA, the MVA comprises an expression cassette comprising an SPI-1 host range gene operably linked to a Pr13.5 promoter, and the expression cassette is inserted into insertion sites MVA88R and MVA89L.

[0103] Embodiments related to MVA In one embodiment, the recombinant MVA is made from an MVA selected from the group consisting of MVA-572, MVA-575, MVA-I721, NIH clone 1, and MVA-BN, preferably from MVA-BN or a derivative thereof.

[0104] MVA-572 was deposited on January 27, 1994 as ECACC V94012707, MVA-575 was deposited on December 7, 2000 as ECACC V00120707, MVA-I721 is referred to in Suter et al. Vaccine 2009, 27:7442-7450, NIH clone 1 was deposited on March 27, 2003 as ATCC® PTA-5095, and MVA-BN was deposited on August 30, 2000 with the European Collection of Cell Cultures (ECACC) under the number V00083008.

[0105] In one embodiment, the recombinant MVA is a recombinant MVA-BN or a recombinant MVA-BN derivative.

[0106] Detailed description Modified vaccinia virus Ankara (MVA) In the past, MVA was generated by serially passaging the Ankara strain of vaccinia virus (CVA) 516 times in chicken embryo fibroblasts (see Mayr et al. 1975 for a review). This virus was renamed from CVA to MVA at passage 516 in view of its substantially altered properties. MVA was further passaged up to passage 570. As a result of these long-term passages, the genome of the resulting MVA virus has a deletion of approximately 31 kilobases of its genomic sequence, and therefore, it has been explained that its host range for replication in avian cells is very restricted (Meyer et al. 1991). The resulting MVA has been shown to be highly non-pathogenic in various animal models compared to a fully replication-competent starting material (Mayr and Danner 1978).

[0107] MVAs useful in the practice of the present invention include MVA-572 (deposited on January 27, 1994 as ECACC V94012707), MVA-575 (deposited on December 7, 2000 as ECACC V00120707), MVA-I721 (referenced in Suter et al. 2009), NIH clone 1 (deposited on March 27, 2003 as ATCC® PTA-5095), and MVA-BN (deposited on August 30, 2000 with the European Collection of Cell Cultures (ECACC) under the number V00083008).

[0108] More preferably, MVAs used according to the present invention include MVA-BN and MVA-BN derivatives. MVA-BN is described in WO02 / 042480. "MVA-BN derivative" refers to any virus that exhibits essentially the same replication properties as MVA-BN as described herein but shows differences in one or more parts of their genomes.

[0109] MVA-BN and MVA-BN derivatives are replication-incompetent, which means they cannot replicate by reproductive means in vivo and in vitro. More specifically, in vitro, MVA-BN or MVA-BN derivatives can replicate by reproductive means in chicken embryo fibroblasts (CEF), but are described as unable to replicate by reproductive means in the human keratinocyte cell line HaCat (Boukamp et al 1988), the human osteosarcoma cell line 143B (ECACC deposit number 91112502), the human fetal kidney cell line 293 (ECACC deposit number 85120602), and the human cervical adenocarcinoma cell line HeLa (ATCC deposit number CCL-2). In addition, MVA-BN or MVA-BN derivatives have a viral amplification rate in Hela cells and HaCaT cell lines that is at least one-half, more preferably one-third, of MVA-575. Tests and assays regarding these properties of MVA-BN and MVA-BN derivatives are described in WO02 / 42480 and WO03 / 048184.

[0110] As noted above, the term "unable to replicate by reproductive means" in human cell lines in vitro is described, for example, in WO02 / 42480, which also teaches a method for obtaining MVA with the desired properties as described above. This term applies to viruses with a viral amplification rate of less than 1 in vitro 4 days after infection, using the assays described in WO02 / 42480 or US6,761,893.

[0111] Exemplary of recombinant MVA viruses Different methods may be applied to generate the recombinant MVA described in this specification. The DNA sequence to be inserted into the virus can be placed in an E. coli plasmid construct in which DNA homologous to a section of poxvirus DNA has been inserted. Alternatively, the DNA sequence to be inserted can be ligated to a promoter. This promoter-gene conjugate can be placed within a plasmid construct such that the promoter-gene conjugate is flanked at both ends by DNA homologous to the DNA sequence adjacent to a poxvirus DNA region containing non-essential loci. The resulting plasmid construct can be amplified by growth in E. coli bacteria and isolated. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into a cell culture, such as chicken embryo fibroblasts (CEF), at the same time as the culture is infected with MVA. Recombination between the homologous MVA viral DNA and the viral genome within the plasmid can generate an MVA that is modified by the presence of the foreign DNA sequence, namely the nucleotide sequence encoding the SARS-CoV-2 antigen, respectively.

[0112] According to a preferred embodiment, MVA virus can infect cells suitable for cell culture, such as CEF cells. Subsequently, the infected cells can be transfected with a first plasmid vector containing one or more foreign or heterologous gene(s), such as one of the nucleic acids provided herein, preferably under the transcriptional control of a poxvirus expression control element. As described above, the plasmid vector also includes sequences capable of inducing the insertion of foreign sequences into selected portions of the MVA virus genome. Optionally, the plasmid vector also contains a cassette containing a marker gene and / or a selectable gene operably linked to a poxvirus promoter. The use of the selection cassette or the marker cassette simplifies the identification and isolation of the produced recombinant MVA. However, recombinant poxviruses can also be identified by PCR techniques. Thereafter, additional cells can be infected with the recombinant MVA obtained as described above, and transfected with a second vector containing a second foreign or heterologous gene(s). For the sake of caution, this gene should be introduced into a different insertion site of the poxvirus genome, and in the second vector, the poxvirus-homologous sequences that induce the integration of the second foreign gene(s) into the poxvirus genome are different. After homologous recombination has occurred, recombinant viruses containing two or more foreign or heterologous genes can be isolated. When introducing additional foreign genes into the recombinant virus, the steps of infection and transfection can be repeated by using the recombinant virus isolated in the previous infection step and by using an additional vector containing an additional foreign gene(s) for transfection. There are numerous other techniques known for producing recombinant MVA.

[0113] In the practice of the present invention, unless otherwise specified, the prior art of immunology, molecular biology, microbiology, cell biology, and recombinant techniques is used, all of which are within the skills of those skilled in the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Current Protocols in Molecular Biology, Ausubel FM, et al., eds, 1987, Methods in Enzymology (Academic Press, Inc.) series, PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995, Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988.

Example

[0114] The following examples serve to further illustrate the present disclosure. They should not be understood as limiting the invention, the scope of which is determined by the appended claims.

[0115] Example 1: Preparation of Recombinant Vaccinia Virus 1.1 Recombinant MVA and CVA An MVA recombinant encoding a poxvirus host range gene that is not endogenous to MVA (see Table 2 below) and a modified MVA designated "wild type" were derived from MVA-BN®. CVA, as the parental strain of MVA, was used for comparison with a CVA recombinant encoding CP77 (which is not endogenous to CVA) and "wild type" CVA.

[0116] MVA and CVA were reconstituted from bacterial artificial chromosome (BAC) clones constructed from MVA-BN (registered trademark) and characterized as previously described (12). Both the BAC clones of MVA and CVA contained a bicistronic expression cassette under the control of a strong synthetic early / late pS promoter, which cassette encoded a neomycin-phosphotransferase selection marker (NPT II). The clones further contained the gene for an internal ribosome entry site (IRES) and a highly sensitive green fluorescent protein (EGFP) reporter protein.

[0117] Recombinant MVA and CVA were reconstituted from BAC clones encoding EGFP that had been modified to contain the host-range gene of interest. MVA and CVA reconstituted from unmodified BAC clones encoding EGFP were designated "wild-type", i.e., "MVA-wt" and "CVA-wt", respectively. No significant difference was observed between the titers obtained from recombinant MVA and MVA-wt upon preparation of the virus stocks.

[0118] 1.2 Cell culture Primary CEF cells were prepared from 11-day-old embryonated chicken eggs (VALO BioMedia GmbH) and cultured in VP-SFM medium (Gibco).

[0119] CHO cells were obtained from ATCC (CCL-61, CHO-K1) and cultured in Ham's F-12 nutrient mixture medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS).

[0120] BHK-21 cells (obtained from ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco / Thermos Fisher Scientific) supplemented with 10% FCS (Pan Biotech).

[0121] 1.3 Production of recombinant CVA A recombinant CVA encoding CP77 (CVA-C) was prepared by homologous recombination. To facilitate detection, the CP77 gene was fused to the C-terminus of monomeric red fluorescent protein (mRFP1) via a flexible linker.

[0122] A plasmid pMISC564 containing a homology flank for homologous recombination between positions 44 and 45 of the MVA open reading frame and an mRFP1-CP77 fusion gene induced by the PrH5m promoter was synthesized by Invitrogen GeneArt (Thermo Fisher Scientific). pBN564 was linearized using SacI and NheI and transfected into monolayer CHO cells infected with CVA-wt using Fugene HD (Roche Diagnostics). Forty-eight hours after transfection, the cells and supernatant were harvested and sonicated with a cup sonicator. To remove CVA-wt from the virus preparation, recombinant CVA containing the mRFP1-CP77 fusion gene was selected by passage in CHO cells. After four passages in CHO cells, total DNA was extracted from the infected cells using the NucleoSpin Blood Mini kit (Macherey-Nagel) and screened by PCR to confirm the absence of unwanted CVA-wt DNA and the presence of the desired recombinant CVA-C.

[0123] 1.4 Preparation of Recombinant MVA MVA recombinants encoding the host-range genes of interest are summarized in Table 2 below.

[0124]

Table 2

[0125] The design of the MVA recombinants, i.e., the expression cassette having the host-range gene and the promoter and the insertion site of the expression cassette, is shown in Figure 1.

[0126] 1.4.1 Preparation of MVA-BAC Clones An MVA recombinant was prepared using the BAC-λ Red recombination technique.

[0127] All BAC clones were grown in E. coli strain MDS42 containing plasmid pKD46. BAC DNA was extracted using the NucleoBond® Xtra BAC kit (Macherey-Nagel). The host-range gene of interest, induced by the selectable poxvirus promoter, was inserted into the MVA BAC clone using the λ Red recombination system described previously (12, 31, 32). Briefly, a counter-selectable rpsL / neo cassette carrying a positive selection marker and a negative selection marker flanked by homology arms was generated by PCR. The rpsL / neo cassette was electroporated into E. coli strain MDS42 carrying MVA BAC and plasmid pKD46 (33). Plasmid pKD46, which encodes the proteins for λ Red recombination, was provided by B. L. Wanner

[27] . Using the λ Red recombination proteins expressed from pKD46, the rpsL / neo cassette was inserted into the corresponding insertion site induced by homologous sequences in the PCR fragment. In a second recombination step, the rpsl / neo cassette was replaced with a fragment encoding the host-range gene(s) (in plasmid pMISC564 or pMISC576). Plasmid pMISC564, containing flanks for homologous recombination and the mRFP1-CP77 gene under the control of the PrH5m promoter, was synthesized by GeneArt (ThermoFisher Scientific). To monitor the spread of the recombinant virus in cell substrates, CP77 was fused to the C-terminus of mRFP1 using a glycine-serine (GS) linker, as previously described for the GFP-CP77 fusion protein (34). Plasmid pMISC576, containing flanks for homologous recombination, the K1L-V5 gene under the control of the Pr1328 promoter, and the SPI-1 gene induced by the Pr13.5 promoter, was synthesized by GeneArt (ThermoFisher Scientific). Since an antibody against K1L was not available, a V5 tag was added to the C-terminus of the K1L gene by a GS linker to detect the K1L protein.Since C9L is disrupted in MVA and CVA, the C9L gene was repaired in situ in MVA-CKS to generate MVA-CKS-C9. Briefly, the disrupted C9L gene in the MVA-CKS BAC clone was replaced with the rpsL / neo cassette. In a second recombination step, the rpsL / neo cassette was replaced with a C9L DNA sequence amplified by PCR using the genomic DNA of VACV-WR (obtained from ATCC) as a template. The success of recombination in each step was confirmed by PCR and sequencing.

[0128] 1.4.2 Reconstitution of Infectious Recombinant MVA Infectious MVA recombinants were reconstituted from the respective MVA-BAC clones as previously described (12). Briefly, BHK-21 cells seeded in 6-well plates were transfected with 3 μg of MVA-BAC DNA using Fugene HD (Promega), and 60 minutes later, infected with the helper virus, Shope fibroma virus (SFV) (obtained from ATCC). Cells were monitored for EGFP expression and harvested 3 days after transfection. Cell lysates were used to infect CEF cells, and three rounds of cell passage were performed to remove the helper virus SFV, which cannot grow in CEF cells. Total DNA was extracted from the infected cells and used for detection of residual SFV by PCR means.

[0129] 1.5 Analysis of Host Range Gene Expression The expression of the gene inserted into CVA or MVA was analyzed using reverse transcription-PCR (RT-PCR). For RNA extraction and analysis, CEF cells in 6-well plates were infected with CVA-wt, CVA-C, MVA-wt, or the MVA recombinants MVA-C, MVA-CK, MVA-CKS, MVA-CKS-C9, or MVA-KS.

[0130] Forty-eight hours post-infection, the supernatant was removed, the cells were scraped, and resuspended in 350 μl of RTL lysis buffer. The cell lysate was homogenized using a QIAshredder column (Qiagen) and genomic DNA was removed using a gDNA eliminator column (Qiagen). The flow-through RNA from the genomic DNA eliminator column was purified using an RNeasy Plus mini kit (Qiagen) according to the manufacturer's instructions. RNA was eluted from the RNeasy spin column using 50 μl of RNase-free water. Residual viral and cellular DNA in the purified samples was digested with Turbo DNase (Ambion / Life Technologies), and then the DNase was inactivated with EDTA (Sigma-Aldrich) to a final concentration of 15 mM. Reverse transcription of a 3 μl sample of the isolated RNA was performed using an OneTaq RT-PCR kit (NEB) according to the manufacturer's instructions. Samples without reverse transcriptase (RT) were set as negative controls. A total of 2 μl of the RT reaction mixture was used to detect the transcription of genes in different host ranges using OneTaq Hot Start 2× master mix (NEB) according to the manufacturer's instructions. Detection of the EGFP transcript was performed as a positive control. The primers used for gene detection and the sizes of the expected PCR products are listed in Table 3.

[0131]

Table 3

[0132] As shown in Figure 2, transcripts from all inserted host-range genes (i.e., the mRFP1-CP77 fusion gene as well as the genes for K1L-V5, SPI-1, and C9L) were detected by RT-PCR and were thus expressed by the recombinants of MVA and CVA. Detection of the EGFP transcript in samples treated with reverse transcriptase was used as a control. In the case of C9L, only truncated defective transcripts were detected, which could be due to the primers targeting the remaining sequences of the defective C9L transcripts (7).

[0133] Example 2: Replication of Recombinant Vaccinia Virus in CHO Cells Regarding the influence of the inserted host-range gene on the replication characteristics of the MVA recombinants, it was examined in CHO cells as an example of a non-permissive mammalian cell line.

[0134] 2.1 Cell Culture The CHO cell culture and the preparation of CEF cells were as described in the above brief description (see Example 1.1).

[0135] 2.2 Virus Replication in CHO Cells For the analysis of virus replication and spread, confluent CHO cell monolayers in 6-well culture plates were washed once with 500 μl of DMEM without FCS and then infected with 1×10 5 TCID 50 per cell at 0.1 TCID 50 . After 60 minutes at 37 °C, the cells were washed once with DMEM and further incubated with 2 ml of DMEM containing 2% FCS. In the case of infection, CHO cells were incubated at 37 °C with 2 ml of F-12 Ham's medium containing 2% FCS. The spread of the virus was determined by detecting EGFP using a fluorescence microscope 72 hours after infection.

[0136] As shown in Figure 3, detectable expression of the EGFP and mRFP1 of the virus reporter gene was not observed in CHO cells infected with CVA-wt. In contrast, CHO cells infected with CVA-C showed extensive EGFP and mRFP1 signals as well as a prominent cytopathic effect (CPE), indicating that the latter is a fully permissive cell line. Clearly, the mRP1-CP77 fusion gene in CVA-C expressed a protein that was fully functional in supporting the replication of MVA in CHO cells.

[0137] As further shown in Figure 3, CHO cells were not permissive to MVA-wt. In contrast, signals of mRFP1 and EGFP as well as CPE were particularly observed in MVA-CKS, and were less pronounced in MVA-C and MVA-CK. However, the replication of MVA-KS in CHO cells was not better than that of MVAS-wt.

[0138] 2.3 Replication ability of MVA recombinants in CHO cells The replication ability, which is a measure of how fast the virus replicates, was examined.

[0139] CHO cells were infected as described above (see Example 2.2). Cells and supernatants were harvested, sonicated to release the virus, and titrated on CEF cells according to the TCID 50 method (35). Statistical analysis was performed using GraphPad PRISM (GraphPad Software, San Diego, USA).

[0140] As shown in Figure 4, all MVA recombinants expressing the CP77 gene replicated in CHO cells, and their replication was in the following order: MVA-CKS = MVA-CKS-9 > MVA-CK > MVA-C.

[0141] MVA expressing only CP77 (MVA-C) replicated in CHO cells at a titer at least 1 log higher than the titer produced by MVA-wt. The titer produced in CHO cells infected with MVA-CKS was approximately 3 logs higher than that of MVA-C. The strongest stage of titer improvement was about 2 digits and was observed in MVA-CKS compared to MVA-CK.

[0142] Therefore, the insertion of poxvirus host range genes into MVA, namely, mRFP1-CP77 (MVA-C), followed by the addition of K1L-V5 (MVA-CK), and then the addition of SPI-1 (MVA-CKS), resulted in a marked stepwise improvement in the replication ability of MVA in CHO cells.

[0143] Interestingly, in addition to the CP77, K1L and SPI-1 genes, the insertion of the C9L gene (MVA-CKS-C9) did not result in a further increase in virus titer compared to the titer of MVA-CKA. The virus titer of MVA-KS was even lower than the titer measured with MVA-wt.

[0144] Notably, the titer obtained with MVA-CKS in CHO cells was equivalent to the titer routinely obtained with MVA-wt in primary CEF cells.

[0145] In conclusion, the CP77 gene is required for MVA replication in CHO cells, but K1-L and SPI-1 in combination with CP77 allow the MVA recombinants to replicate in the order known for the replication of native MVA in CEF cells.

[0146] 2.4 Growth of MVA recombinants in CHO cells The growth kinetics of MVA-CKS in CHO cells were also analyzed.

[0147] Briefly, CHO cells were infected with MVA-wt or MVA-CKS and cultured as described above (see Example 2.2). MVA was harvested at different times and titrated on CEF cells as described above (see Example 2.3).

[0148] As shown in Figure 5, MVA-CKS replicated efficiently in CHO cells within 24 hours after infection and reached the maximum virus titer after 48 - 72 hours. Figure 4 also shows that MVA-wt cannot replicate in CHO cells.

[0149] Example 3: Replication of recombinant vaccinia virus in additional cells 3.1 Cell culture CEF cells were prepared as described above (see Example 1.2).

[0150] All cell lines were obtained from the American Type Culture Collection (ATCC) or the European Collection of Authenticated Cell Cultures (ECACC).

[0151] CHO cells were cultured as described in the above simple description (see Example 1.2).

[0152] Cell lines other than CHO cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco / Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Pan Biotech).

[0153] 3.2 Virus replication in HEK293 cells, RK13 cells and chicken cells Virus replication in HEK293 cells and RK13 cells was analyzed by fluorescence microscopy as described above (see Example 2.2).

[0154] As shown in Figure 6, human fetal kidney HEK293 cells were not permissive to MVA-wt but were permissive to CVA-wt. However, none of the MVA recombinants replicated in HEK293 cells.

[0155] In contrast, as shown in Figure 7, RK13 cells were permissive to CVA-wt and all MVA recombinants but not to MVA-wt. K1L is a host range gene of VACV known to restore MVA replication in RK13 cells (21), and since the host range function of K1L can be complemented by CP77 in RK13 cells (17), here RK13 cells were used as a control cell substrate to confirm the host range function of the fusion protein of K1L-V5 and mRFP1-CP77. The result that RK13 cells were permissive to MVA-C and MVA-KS confirmed the host range function of K1L-V5 and mRFP1-CP77.

[0156] Furthermore, the viral replication of MVA-CKS and MVA-wt was analyzed as described above with respect to the virus titer produced by cells derived from chicken (see Example 2.3).

[0157] Cells of primary CEF cells and the chicken fibroblast DF-1 cell line were permissive to both MVA-wt and MVA-CKS, and no significant difference was found between MVA-CKS and MVA-wt in their ability to infect CEF cells and DF-1 cells.

[0158] Example 4: Preparation of CHO cells permissive to MVA Based on the finding that MVA-CKS replicated in CHO cells (see Example 2 above), the generation of CHO cells stably expressing the poxvirus host range genes CP77, K1L, and SPI-1 was considered. Methods for generating CHO cell lines stably expressing transgenes are available (e.g., 18).

[0159] 4.1 Cloning of plasmids containing CP77, K1L, SPI-1, and NPT II genes Plasmids containing three host range genes CP77, K1L, and SPI-1, respectively, induced by different promoters (CMV promoter, β-globin promoter, and β-actin promoter) for gene expression in mammalian cells were prepared. The plasmids further contained an NPT II-IRES-EGFP cassette and a reporter gene induced by the SV40 promoter as a selection marker.

[0160] 4.2 Transfection of CHO cells and analysis of clones CHO cells (ATCC CCL-61) seeded in a 6-well plate containing F-12 Ham medium supplemented with 10% FCS were transfected with 1 μg of linearized plasmid using FuGENE® HD transfection reagent (Promega) according to the manufacturer's instructions. After 24 hours, the medium was replaced with fresh medium containing the selection antibiotic (G418). Cell lines were established by single cell sorting followed by further expansion using the selection antibiotic. Clones were analyzed by detecting transcription of the transgene by RT-PCR. Positive clones were further tested by infection with MVA. The virus was harvested and titrated 48 hours after infection.

[0161] Final observations: Throughout the body of this specification several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety. To the extent that the incorporated material conflicts with or is inconsistent with this specification, this specification shall control over any such material. No admission is made that any description in this specification constitutes prior art to the claimed invention.

[0162] References 1. Stickl H, Hochstein-Mintzel V, Mayr A, Huber HC, Schafer H, Holzner A. 1974. [MVA vaccination against smallpox: clinical tests with an attenuated live vaccinia virus strain (MVA) (Author's translation)]. Dtsch Med Wochenschr 99:2386-92. 2. Kennedy JS, Greenberg RN. 2009. IMVAMUNE: modified vaccinia Ankara strain as an attenuated smallpox vaccine. Expert Rev Vaccines 8:13-24. 3. Volz A,Sutter G.2017.Modified Vaccinia Virus Ankara:History,Value in Basic Research,and Current Perspectives for Vaccine Development.Adv Virus Res 97:187-243. 4. Gomez CE,Perdiguero B,Garcia-Arriaza J,Esteban M.2013.Clinical applications of attenuated MVA poxvirus strain.Expert Rev Vaccines 12:1395-416. 5. Sutter G,Moss B.1992.Nonreplicating vaccinia vector efficiently expresses recombinant genes.Proc Natl Acad Sci U S A 89:10847-51. 6. Mayr A,Hochstein-Mintzel V,Stickl H.1975.Abstammung,Eigenschaften und Verwendung des attenuierten Vaccinia-Stammes MVA.Infection 3:6-14. 7. Meisinger-Henschel C,Schmidt M,Lukassen S,Linke B,Krause L,Konietzny S,Goesmann A,Howley P,Chaplin P,Suter M,Hausmann J.2007.Genomic sequence of chorioallantois vaccinia virus Ankara,the ancestor of modified vaccinia virus Ankara.J Gen Virol 88:3249-59. 8. Antoine G, Scheiflinger F, Dorner F, Falkner FG. 1998. The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 244:365-96. 9. Meyer H, Sutter G, Mayr A. 1991. Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J Gen Virol 72(Pt 5):1031-8. 10. Carroll MW, Moss B. 1997. Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238:198-211. 11. Blanchard TJ, Alcami A, Andrea P, Smith GL. 1998. Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. J Gen Virol 79(Pt 5):1159-67. 12. Meisinger-Henschel C,Spath M,Lukassen S,Wolferstatter M,Kachelriess H,Baur K,Dirmeier U,Wagner M,Chaplin P,Suter M,Hausmann J.2010.Introduction of the six major genomic deletions of modified vaccinia virus Ankara(MVA)into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice.J Virol 84:9907-19. 13. Spehner D,Gillard S,Drillien R,Kirn A.1988.A cowpox virus gene required for multiplication in Chinese hamster ovary cells.J Virol 62:1297-304. 14. Perkus ME,Goebel SJ,Davis SW,Johnson GP,Limbach K,Norton EK,Paoletti E.1990.Vaccinia virus host range genes.Virology 179:276-286. 15. Ramsey-Ewing A,Moss B.1995.Restriction of vaccinia virus replication in CHO cells occurs at the stage of viral intermediate protein synthesis.Virology 206:984-93. 16. Shchelkunov SN,Safronov PF,Totmenin AV,Petrov NA,Ryazankina OI,Gutorov VV,Kotwal GJ.1998.The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins.Virology 243:432-60. 17. Ramsey-Ewing AL,Moss B.1996.Complementation of a vaccinia virus host-range K1L gene deletion by the nonhomologous CP77 gene.Virology 222:75-86. 18. Eldi P,Cooper TH,Liu L,Prow NA,Diener KR,Howley PM,Suhrbier A,Hayball JD.2017.Production of a Chikungunya Vaccine Using a CHO Cell and Attenuated Viral-Based Platform Technology.Mol Ther 25:2332-2344. 19. Lynch HE, Ray CA, Oie KL, Pollara JJ, Petty IT, Sadler AJ, Williams BR, Pickup DJ. 2009. Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication. Virology 391:177-86. 20. McFadden G. 2005. Poxvirus tropism. Nat Rev Microbiol 3:201-13. 21. Sutter G, Ramsey-Ewing A, Rosales R, Moss B. 1994. Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant. J Virol 68:4109-16. 22. Ali AN, Turner PC, Brooks MA, Moyer RW. 1994. The SPI-1 gene of rabbitpox virus determines host range and is required for hemorrhagic pock formation. Virology 202:305-14. 23. Liu R, Moss B. 2018. Vaccinia Virus C9 Ankyrin Repeat / F-Box Protein Is a Newly Identified Antagonist of the Type I Interferon-Induced Antiviral State. J Virol 92. 24. Werden SJ, Rahman MM, McFadden G. 2008. Poxvirus host range genes. Adv Virus Res 71:135 - 71. 25. Bratke KA, McLysaght A, Rothenburg S. 2013. A survey of host range genes in poxvirus genomes. Infection, Genetics and Evolution. 26. Shisler JL, Jin XL. 2004. The vaccinia virus K1L gene product inhibits host NF - kappaB activation by preventing IkappaBalpha degradation. J Virol 78:3553 - 60. 27. Brooks MA, Ali AN, Turner PC, Moyer RW. 1995. A rabbitpox virus serpin gene controls host range by inhibiting apoptosis in restrictive cells. J Virol 69:7688 - 98. 28. Shisler JL, Isaacs SN, Moss B. 1999. Vaccinia virus serpin - 1 deletion mutant exhibits a host range defect characterized by low levels of intermediate and late mRNAs. Virology 262:298 - 311. 29. Liu R, Mendez-Rios JD, Peng C, Xiao W, Weisberg AS, Wyatt LS, Moss B. 2019. SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells. PLoS Pathog 15: e1007710. 30. Peng C, Moss B. 2020. Repair of a previously uncharacterized second host-range gene contributes to full replication of modified vaccinia virus Ankara (MVA) in human cells. Proc Natl Acad Sci U S A 117: 3759-3767. 31. Wang S, Zhao Y, Leiby M, Zhu J. 2009. A new positive / negative selection scheme for precise BAC recombineering. Mol Biotechnol 42: 110-6. 32. Reyrat JM, Pelicic V, Gicquel B, Rappuoli R. 1998. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect Immun 66: 4011-7. 33. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640-5. 34. Hsiao JC,Chao CC,Young MJ,Chang YT,Cho EC,Chang W.2006.A poxvirus host range protein,CP77,binds to a cellular protein,HMG20A,and regulates its dissociation from the vaccinia virus genome in CHO-K1 cells.J Virol 80:7714-28. 35. Staib C,Drexler I,Sutter G.2004.Construction and isolation of recombinant MVA.Methods Mol Biol 269:77-100.

[0163] Sequence The amino acid sequence encoded by the CP77 gene of SEQ ID NO:1 MFDYLENEEVALDELKQMLRDRDPNDTRNQFKNNALHAYLFNEHCNNVEVVKLLLDSGTNPLHKNWRQLTPLGEYTNSRHGKVNKDIAMVLLEATGYSNINDFNIFTYMKSKNVDIDLIKVLVEHGFDFSVKCEKHHSVIENYVMTDDPVPEIIDLFIENGCSVIYEDEDDEYGYAYEEYHSQNDDYQPRNCGTVLHLYIISHLYSESDSRSCVNPEVVKCLINHGINPSSIDKNYCTALQYYIKSSHIDIDIVKLLMKGIDNTAYSYIDDLTCCTRGIMADYLNSDYRYNKDVDLDLVKLFLENGKPHGIMCSIVPLWRNDKETISLILKTMNSDVLQHILIEYITFSDIDISLVEYMLEYGAVVNKEAIHGYFKNINIDSYTMKYLLKKEGGDAVNHLDDGEIPIGHLCKSNYGRYNFYTDTYRQGFRDMSYACPILSTINICLPYLKDINMIDKRGETLLHKAVRYNKQSLVSLLLESGSDVNIRSNNGYTCIAIAINESRNIELLNMLLCHKPTLDCVIDSLREISNIVDNAYAIKQCIRYAMIIDDCISSKIPESISKHYNDYIDICNQELNEMKKIIVGGNTMFSLIFTDHGAKIIHRYANNPELRAYYESKQNKIYVEVYDIISNAIVKHNKIHKNIESVDDNTYISNLPYTIKYKIFEQQ

[0164] Nucleic acid sequence of the array number 2 CP77 gene

[0165] Amino acid sequence encoded by the array number 3 K1L gene MDLSRINTWKSKQLKSFLSSKDAFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLENEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKN

[0166] Nucleic acid sequence of the array number 4 K1L gene ATGGACCTGAGCCGGATCAACACCTGGAAGTCCAAGCAGCTGAAGTCCTTCCTGAGCAGCAAGGACGCCTTCAAGGCCGATGTGCACGGACACAGCGCCCTGTACTATGCCATTGCCGACAACAACGTGCGGCTCGTGTGCACCCTTCTGAATGCCGGCGCTCTGAAGAACCTGCTGGAAAACGAGTTCCCTCTGCACCAGGCCGCCACACTGGAAGATACCAAGATCGTGAAGATTCTGCTGTTCAGCGGCCTGGACGACAGCCAGTTCGACGACAAGGGAAACACCGCTCTGTACTACGCCGTGGACAGCGGCAATATGCAGACCGTGAAGCTGTTCGTGAAGAAAAACTGGCGGCTGATGTTCTACGGCAAGACCGGATGGAAAACCAGCTTCTACCACGCCGTGATGCTGAACGATGTGTCTATCGTGTCCTACTTCCTGTCTGAGATCCCCAGCACCTTCGACCTGGCCATCCTGCTGAGCTGCATCCACATCACCATCAAGAACGGCCACGTGGACATGATGATCCTGCTGCTGGACTACATGACCAGCACCAACACCAACAACAGCCTGCTGTTTATCCCCGACATCAAGCTGGCCATCGACAACAAGGACATCGAGATGCTGCAGGCCCTGTTTAAGTACGACATCAACATCTACAGCGCCAACCTCGAGAACGTCCTGCTGGACGATGCCGAGATCGCCAAGATGATCATTGAGAAGCACGTCGAGTACAAGAGCGACAGCTACACCAAGGACCTGGACATTGTGAAGAACAACAAGCTGGACGAGATCATCAGCAAGAACAAAGAACTGCGGCTTATGTACGTGAACTGCGTGAAAAAGAACTGA

[0167] Amino acid sequence encoded by the SEQ ID NO:5 mRFP1-CP77 gene MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGAGGGSGGGGSGGGGSFDYLENEEVALDELKQMLRDRDPNDTRNQFKNNALHAYLFNEHCNNVEVVKLLLDSGTNPLHKNWRQLTPLGEYTNSRHGKVNKDIAMVLLEATGYSNINDFNIFTYMKSKNVDIDLIKVLVEHGFDFSVKCEKHHSVIENYVMTDDPVPEIIDLFIENGCSVIYEDEDDEYGYAYEEYHSQNDDYQPRNCGTVLHLYIISHLYSESDSRSCVNPEVVKCLINHGINPSSIDKNYCTALQYYIKSSHIDIDIVKLLMKGIDNTAYSYIDDLTCCTRGIMADYLNSDYRYNKDVDLDLVKLFLENGKPHGIMCSIVPLWRNDKETISLILKTMNSDVLQHILIEYITFSDIDISLVEYMLEYGAVVNKEAIHGYFKNINIDSYTMKYLLKKEGGDAVNHLDDGEIPIGHLCKSNYGRYNFYTDTYRQGFRDMSYACPILSTINICLPYLKDINMIDKRGETLLHKAVRYNKQSLVSLLLESGSDVNIRSNNGYTCIAIAINESRNIELLNMLLCHKPTLDCVIDSLREISNIVDNAYAIKQCIRYAMIIDDCISSKIPESISKHYNDYIDICNQELNEMKKIIVGGNTMFSLIFTDHGAKIIHRYANNPELRAYYESKQNKIYVEVYDIISNAIVKHNKIHKNIESVDDNTYISNLPYTIKYKIFEQQ

[0168] Nucleic acid sequence of the SEQ ID NO: 6 mRFP1-CP77 gene

[0169] Amino acid sequence encoded by the array number 7 K1L-V5 gene MDLSRINTWKSKQLKSFLSSKDAFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLENEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKNGGSGKPIPNPLLGLDST

[0170] Nucleic acid sequence of the array number 8 K1L-V5 gene ATGGACCTGAGCCGGATCAACACCTGGAAGTCCAAGCAGCTGAAGTCCTTCCTGAGCAGCAAGGACGCCTTCAAGGCCGATGTGCACGGACACAGCGCCCTGTACTATGCCATTGCCGACAACAACGTGCGGCTCGTGTGCACCCTTCTGAATGCCGGCGCTCTGAAGAACCTGCTGGAAAACGAGTTCCCTCTGCACCAGGCCGCCACACTGGAAGATACCAAGATCGTGAAGATTCTGCTGTTCAGCGGCCTGGACGACAGCCAGTTCGACGACAAGGGAAACACCGCTCTGTACTACGCCGTGGACAGCGGCAATATGCAGACCGTGAAGCTGTTCGTGAAGAAAAACTGGCGGCTGATGTTCTACGGCAAGACCGGATGGAAAACCAGCTTCTACCACGCCGTGATGCTGAACGATGTGTCTATCGTGTCCTACTTCCTGTCTGAGATCCCCAGCACCTTCGACCTGGCCATCCTGCTGAGCTGCATCCACATCACCATCAAGAACGGCCACGTGGACATGATGATCCTGCTGCTGGACTACATGACCAGCACCAACACCAACAACAGCCTGCTGTTTATCCCCGACATCAAGCTGGCCATCGACAACAAGGACATCGAGATGCTGCAGGCCCTGTTTAAGTACGACATCAACATCTACAGCGCCAACCTCGAGAACGTCCTGCTGGACGATGCCGAGATCGCCAAGATGATCATTGAGAAGCACGTCGAGTACAAGAGCGACAGCTACACCAAGGACCTGGACATTGTGAAGAACAACAAGCTGGACGAGATCATCAGCAAGAACAAAGAACTGCGGCTTATGTACGTGAACTGCGTGAAAAAGAACGGCGGCAGCGGCAAGCCCATTCCTAATCCACTGCTGGGCCTCGACAGCACCTGA

[0171] Amino acid sequence encoded by SEQ ID NO:9 SPI-1 gene MGGSDIFKELILKHTDENVLISPVSILSTLSILNHGAAGSTAEQLSKYIENMNENTPDDNNDMDVDIPYCATLATANKIYGSDSIEFHASFLQKIKDDFQTVNFNNANQTKELINEWVKTMTNGKINSLLTSPLSINTRMTVVSAVHFKAMWKYPFSKHLTYTDKFYISKNIVTSVDMMVSTENNLQYVHINELFGGFSIIDIPYEGNSSMVIILPDDIEGIYNIEKNITDEKFKKWCGMLSTKSIDLYMPKFKVEMTEPYNLVPILENLGLTNIFGYYADFSKMCNETITVEKFLHTTFIDVNEEYTEASAVTGVFMTNFSMVYRTKVYINHPFMYMIKDNTGRILFIGKYCYPQ

[0172] Nucleic acid sequence of SEQ ID NO:10 SPI-1 gene

[0173] Amino acid sequence encoded by the array number 11 C9L gene MVNDKILYDSCKTFNIDASSAQSLIESGANPLYEYDGETPLKAYVTKKNNNIKNDVVILLLSSVDYKNINDFDIFEYLCSDNIDIDLLKLLISKGIEINSIKNGINIVEKYATTSNPNVDVFKLLLDKGIPTCSNIQYGYKIKIEQIRRAGEYYNWDDELDDYDYDYTTDYDDRMGKTVLYYYIITRSQDGYATSLDVINYLISHKKEMRYYTYREHTTLYYYLDKCDIKREIFDALFDSNYSGHELMNILSNYLRKQFRKKNHKIDNYIVDQLLFDRDTFYILELCNSLRNNILISTILKRYTDSIQDLLLEYVSYHTVYINVIKCMIDEGATLYRFKHINKYFQKFGNRDPKVVEYILKNGNLVVDNDNDDNLINIMPLFPTFSMRELDVLSILKLCKPYIDDINKIDKHGCSILYHCIKSHSVSLVEWLIDNGADINIITKYGFTCITICVILADKYIPEIAELYIKILEIILSKLPTIECIKKTVDYLDDHRYLFIGGNNKSLLKICIKYFILVDYKYTCSMYPSYIEFITDCEKEIADMRQIKINGTDMLTVMYMLNKPTKKRYVNNPIFTDWANKQYKFYNQIIYNANKLIEQSKKIDDMIEEVSIDDNRLSTLPLEIRHLIFSYAFL

[0174] Nucleic acid sequence of the array number 12 C9L gene

[0175] Array No. 13 PrH5m promoter TAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAATTTCATTATCGCGATATCCGTTAAGTTTGTATCGTA

[0176] Array No. 14 Pr1328 promoter TATATTATTAAGTGTGGTGTTTGGTCGATGTAAAATTTTTGTCGATAAAAATTAAAAAATAACTTAATTTATTATTGATCTCGTGTGTACAACCGAAATC

[0177] Array No. 15 Pr13.5 promoter TAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTATTGCTCTTGTGACTAGAGACTTTAGTTAAGGTACTGTAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTA

[0178] Array No. 16 EGFP forward primer CAGCTCGTCCATGCCGAGAG

[0179] Array No. 17 EGFP forward primer TGAAGTTCATCTGCACCACC

[0180] Array No. 18 CP77 forward primer TGAACCCTGAGGTGGTCAAGTGC

[0181] Array No. 19 CP77 reverse primer CGATCAGGATGTGCTGGAGCAC

[0182] Array No. 20 K1L forward primer CGATGTGCACGGACACAGC

[0183] SEQ ID NO: 21 K1L Reverse Primer GCTGGTCATGTAGTCCAGCAGC

[0184] SEQ ID NO: 22 SPI-1 Forward Primer CGATCAGGATGTGCTGGAGCAC

[0185] SEQ ID NO: 23 SPI-1 Reverse Primer GTACACCTTGGTCCGGTACACC

[0186] SEQ ID NO: 24 C9L Forward Primer CATGCTACATGTGTACTTATAATCGAC

[0187] SEQ ID NO: 25 C9L Reverse Primer GCCGTATATTGATGATATAAACAAAATAG

Claims

1. Cells of a serially passaged cell line, wherein the genes have been modified to express CP77 and K1L, which are host domain genes of a poxvirus.

2. The cell according to claim 1, wherein the genome comprises CP77 and K1L, which are host region genes of a poxvirus, and preferably comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

3.

3. The cell according to claim 1, wherein the cell has been genetically modified to express CP77, K1L, and SPI-1, which are host region genes of a poxvirus.

4. The cell according to claim 3, wherein the genome comprises CP77, K1L and SPI-1, which are host region genes of a poxvirus, and preferably comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

9.

5. The cells according to any one of claims 1 to 4, wherein the cells are from a mammalian cell line other than human, preferably Chinese hamster ovary (CHO) cells.

6. A cell according to any one of claims 1 to 4, which is infected with modified vaccinia virus ankara (MVA).

7. The cell according to claim 5, which is infected with modified vaccinia virus ankara (MVA).

8. Use of the cells according to any one of claims 1 to 4 for replication of modified vaccinia virus ankara (MVA).

9. Use of the cells according to claim 5 for replication of modified vaccinia virus ankara (MVA).

10. Use of cells according to any one of claims 1 to 4 in the manufacture of a vaccine containing modified vaccinia virus ankara (MVA).

11. Use of the cells according to claim 5 in the manufacture of a vaccine comprising modified vaccinia virus ankara (MVA).

12. A vaccine comprising a modified vaccinia virus ankara (MVA), wherein the MVA is prepared using cells according to any one of claims 1 to 4.

13. A vaccine comprising a modified vaccinia virus ankara (MVA), wherein the MVA is prepared using the cells described in claim 5.

14. below, (a) A step of preparing nucleic acids suitable for gene transfer into cells of a serially passaged cell line, wherein the nucleic acid is (i) CP77, a host region gene of the poxvirus that is functionally linked to the promoter, or (ii) K1L, a host region gene of the poxvirus that is functionally linked to the promoter, or (iii) The step comprising CP77 and K1L, which are host region genes of a poxvirus, each functionally linked to a promoter, (b) A step of introducing nucleic acids (i) and (ii) obtained in step (a), or nucleic acid (iii) obtained in step (a), into the cells, and (c) A step of selecting a cell population or clone that expresses CP77 and K1L, which are host region genes of the poxvirus. A method for producing the cells according to claim 1 or 2, including the method described in claim 1 or 2.

15. The following, (a) A step of preparing nucleic acids suitable for gene transfer into cells of a serially passaged cell line, wherein the nucleic acid is (i) CP77, a host region gene of the poxvirus that is functionally linked to the promoter, or (ii) K1L, a host region gene of the poxvirus that is functionally linked to the promoter, or (iii) The step comprising CP77 and K1L, which are host region genes of a poxvirus, each functionally linked to a promoter, (b) A step of introducing nucleic acids (i) and (ii) obtained in step (a), or nucleic acid (iii) obtained in step (a), into the cells, and (c) A step of selecting a cell population or clone that expresses CP77 and K1L, which are host region genes of the poxvirus. A method for producing the cells described in claim 5, including the method described in claim 5.

16. below, (a) A step of preparing nucleic acids suitable for gene transfer into cells of a serially passaged cell line, wherein the nucleic acid is (i) CP77, a host region gene of the poxvirus that is functionally linked to the promoter, or (ii) K1L, a host region gene of the poxvirus that is functionally linked to the promoter, or (iii) SPI-1, a poxvirus host domain gene functionally linked to the promoter, or (iv) The process comprising CP77, K1L, and SPI-1, which are host domain genes of a poxvirus, each functionally linked to a promoter, (b) A step of introducing nucleic acids (i), (ii), and (iii) obtained in step (a), or nucleic acid (iv) obtained in step (a), into the cells, and (c) A step of selecting a cell population or clone that expresses the host region genes of the poxvirus CP77, K1L and SPI-1, A method for producing the cells according to claim 3 or 4, including the method described in claim 3 or 4.

17. The following, (a) A step of preparing nucleic acids suitable for gene transfer into cells of a serially passaged cell line, wherein the nucleic acid is (i) CP77, a host region gene of the poxvirus that is functionally linked to the promoter, or (ii) K1L, a host region gene of the poxvirus that is functionally linked to the promoter, or (iii) SPI-1, a poxvirus host domain gene functionally linked to the promoter, or (iv) The process comprising CP77, K1L, and SPI-1, which are host domain genes of a poxvirus, each functionally linked to a promoter, (b) A step of introducing nucleic acids (i), (ii), and (iii) obtained in step (a), or nucleic acid (iv) obtained in step (a), into the cells, and (c) A step of selecting a cell population or clone that expresses the host region genes of the poxvirus CP77, K1L and SPI-1, A method for producing the cells described in claim 5, including the method described in claim 5.

18. Modified vaccinia virus ankara (MVA), replicated using cells according to any one of claims 1 to 4.

19. Modified vaccinia virus ankara (MVA) replicated using the cells described in Claim 5.

20. The use of nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 3, which are host domain genes of the poxvirus, to enable the cell to express the CP77 and K1L genes.

21. The use according to claim 20, further comprising using SPI-1, which is a poxvirus host region gene, to enable the cell to express the CP77, K1L, and SPI-1 genes, preferably further comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

9.

22. The use according to claim 20 or 21, wherein the cells are cells of a mammalian cell line other than human, preferably Chinese hamster ovary (CHO) cells.

23. A modified vaccinia virus ankara (MVA) comprising CP77 and K1L, which are host region genes of the poxvirus, and preferably comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

3.

24. The MVA according to claim 23, further comprising SPI-1, which is a host region gene of a poxvirus, and preferably further comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9.

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