Mammalian cell lines for production of improved vaccinia virus ankara (MVA)

Abstract

The present invention relates to a mammalian non-human cell line, in particular a Chinese hamster ovary (CHO) cell, genetically modified to express poxvirus host range genes CP77, K1L and / or SPI-1, which are not expressed in MVA, and to the use of said cell line in MVA propagation.

Description

Technical Field

[0001] The present invention relates to the field of vaccines based on viral vectors. More specifically, the present invention relates to mammalian cell substrates for propagating Modified Vaccinia Ankara Virus (MVA). Specifically, the present invention relates to a mammalian non-human cell line, in particular Chinese Hamster Ovary (CHO) cells, which are genetically modified to express a poxvirus host range gene that is not expressed in MVA. The present invention also relates to the generation of such cell lines and their use in the production of vaccines based on MVA. The present invention also relates to recombinant MVA, which expresses a poxvirus host range gene that is not expressed essentially in MVA. Background Art

[0002] Modified vaccinia virus Ankara (MVA) is a highly attenuated subspecies of vaccinia virus (genus Orthopoxvirus). The virus strain (Bavarian Nordic virus strain) has been approved for use as a safe human vaccine against smallpox and monkeypox (1-3). In addition, 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 derived by serially passaged the ancestral chorioallantoic vaccinia virus Ankara (CVA) for more than 500 times in chicken embryo fibroblasts (CEF or CEF cells) (3,6). During the passage process, multiple mutations and deletions accumulated in the viral genome. Compared with the parental CVA, MVA lacks six large genomic segments and has acquired multiple mutations and small deletions throughout the genome (7-9). As a result, MVA has lost the ability to replicate in most mammalian cells, and only a few cell lines are known to support efficient viral replication. Therefore, compared with the parental CVA and other members of the orthopoxvirus family, MVA is characterized by a significantly restricted viral host range.

[0004] To date, the only cell substrate approved for MVA-based vaccine production is CEF cells (10, 11). Although primary CEF cells are a mature cell substrate for MVA production, their preparation from embryonic eggs is laborious and time-consuming. In addition, the process is susceptible to contamination. All of this is not ideal for a stable and efficient production process, especially for industrial-scale and strictly Good Manufacturing Practice (GMP) vaccine production.

[0005] Therefore, alternative cell substrates are needed for the propagation of MVA.

[0006] U.S. Patent 5,830,688 (1998) describes the identification of a sequence that is involved in the propagation of vaccinia virus in Chinese hamster ovary (CHO) cells and, when transferred into vaccinia virus (which is intrinsically incapable of propagation in CHO cells), allows the propagation of vaccinia virus in this cell line.

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

[0008] Instead, it is thought to express one or more poxvirus host range genes that are capable of supporting MVA replication in otherwise non-susceptible cell lines. If the appropriate host range genes are expressed, the modified cells should be able to support MVA replication to acceptable titers.

[0009] The genetic basis for the host range restriction of MVA is not yet clear, and for most cell lines, the responsible poxvirus genes remain largely unknown. At least, the six major genomic deletions in MVA (associated with CVA) are not the only ones responsible for the strongly attenuated and highly restricted host range of MVA (12). This suggests that the host range restriction of MVA is likely a synergistic effect involving not only the six major deletions but also other smaller genetic deletions and mutations present throughout the genome (12).

[0010] Among the well-defined host range genes of orthopoxviruses, the CP77 gene of vaccinia virus (CPXV or CWPX virus) is known to promote the replication of vaccinia virus (VACV) and mousepox virus in Chinese hamster ovary (CHO) and RK13 (from rabbit kidney) cells (13-17). A genetically engineered CHO cell line expressing CP77 and the VACV-derived D13L gene was generated for the production of replication-defective recombinant VACV (18). Expression of the CP77 gene has also been shown to expand the host range of MVA by alleviating a block prior to late protein synthesis in CHO cells. However, CP77 alone may not be sufficient to achieve high-titer MVA replication in otherwise nonpermissive cells.

[0011] In addition to CP77, MVA lacks 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 that is 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 responsible for the replication of VACV in RK13 cells, and CP77 can complement the function of the K1L gene in this cell line

[18] .

[0012] The host range function of serine protease inhibitor 1 (SPI-1) was first discovered in rabbit poxvirus and has been shown to act as an inhibitor of apoptosis (22,27). VACV SPI-1 deletion mutants are unable to replicate efficiently in primary human keratinocytes or human lung cancer cells (A549) (28). SPI-1 has also been identified as a host range factor for MVA (29). Furthermore, A549 cells co-expressing a recently identified host range gene (i.e., C16L / B22R) with the SPI-1 gene have been shown to be permissive for MVA (30).

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

[0014] Table 1 below summarizes the expression of host range genes CP77, K1L, SPI-1 and C9L in MVA and other members of the orthopoxviruses. In addition, Table 1 lists cell lines that do not support replication of host range gene-deficient viruses.

[0015] Table 1: Left: Poxvirus host range genes. Middle: Intrinsic expression of host range genes in orthopoxviruses (+ / -). Right: Cultured cells are unable to compensate for defects in host range genes.

[0016]

[0017] Abbreviations: COP = vaccinia virus Copenhagen; WR = vaccinia virus Western Reserve; CPXV = cowpox virus; IFNβ = interferon β. Summary of the invention

[0018] An object of the present invention is to provide a cell substrate for propagating MVA.

[0019] The object of the present invention is solved by providing a CHO cell expressing a combination of poxvirus host range genes not expressed by MVA.In particular, the present invention is defined by the appended claims and the following aspects and embodiments thereof.

[0020] In a first aspect, the present invention provides cells of a continuous cell line, the cells being genetically modified to express the poxvirus host range genes CP77 and K1L.

[0021] In a specific aspect, the invention provides cells of a continuous cell line, which are genetically modified to express the poxvirus host range genes CP77, K1L and SPI-1.

[0022] In another aspect, the invention provides the use of a cell according to the invention for the propagation of MVA.

[0023] In a further aspect, the invention provides the use of a cell according to the invention in the production of a vaccine comprising MVA.

[0024] In another aspect, the invention provides a vaccine comprising MVA prepared using a cell according to the invention.

[0025] In a further aspect, the present invention provides a method for producing a cell according to the present invention, the method comprising the following steps:

[0026] (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous cell line, the nucleic acid comprising:

[0027] (i) the poxvirus host range gene CP77 operably linked to a promoter; or

[0028] (ii) the poxvirus host range gene K1L operably linked to a promoter; or

[0029] (iii) poxvirus host range genes CP77 and K1L, each operably linked to a promoter;

[0030] (b) introducing the nucleic acids (i) and (ii) obtained in step (a), or the nucleic acid (iii) obtained in step (a) into the cell; and

[0031] (c) Selecting cell populations or clones expressing the poxvirus host range genes CP77 and K1L.

[0032] In a further aspect, the present invention provides a method for producing a cell according to the present invention, the method comprising the following steps:

[0033] (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous cell line, the nucleic acid comprising:

[0034] (i) a poxvirus host range gene CP77 operably linked to a promoter; or

[0035] (ii) the poxvirus host range gene K1L operably linked to a promoter; or

[0036] (iii) the poxvirus host range gene SPI-1 operably linked to a promoter; or

[0037] (iv) poxvirus host range genes CP77, K1L and SPI-1, each operably linked to a promoter;

[0038] (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

[0039] (c) Selecting cell populations or clones expressing the poxvirus host range genes CP77, K1L and SPI-1.

[0040] In a further aspect, the invention provides a cell produced by a method according to the invention.

[0041] In a further aspect, the invention provides an MVA propagated using a cell according to the invention.

[0042] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77 and K1L for enabling cultured cells to express CP77 and K1L.

[0043] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77, K1L and SPI-1 for enabling cultured cells to express CP77, K1L and SPI-1.

[0044] In yet another aspect, the present invention provides an MVA comprising the poxvirus host range genes CP77 and K1L.

[0045] In yet another aspect, the present invention provides an MVA comprising the poxvirus host range genes CP77, K1L and SPI-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic illustration of the design of a vaccinia virus (MVA or CVA) with one or more host range genes inserted into its genome.

[0047] C = CP77, K = K1L-V5, S = SPI-1, C9 = C9L; mRFP1-CP77 = mRFP1-CP77 fusion gene. Promoter: PrH5m, Pr1328, Pr13.5, pS. Insertion site: 059-060 (CVA), 44L-45L (MVA), 88R-89L (MVA).

[0048] CVA-wt and MVA-wt were reconstructed from BAC clones, each containing a bicistronic expression cassette under the control of a strong synthetic early / late pS promoter encoding an NPT II-IRES-EGFP cassette. CVA-C was generated by inserting the mRFP-CP77 gene into CVA-wt using homologous recombination. To generate MVA recombinants, host range genes of interest were inserted into MVA BAC clones using λred recombination technology.

[0049] Figure 2 Transcription of the inserted host range gene by vaccinia virus grown in CEF cells (analyzed by RT-PCR) is shown.

[0050] Upper row: recombinant MVA or CVA compared to the corresponding wild type (MVA-wt and CVA-wt, respectively). RT = reverse transcriptase; "+" = sample treated with RT; "-" = control sample not treated with RT.

[0051] Left lane: size of target gene and related PCR products. Right lane: size of bands in 100 bp DNA ladder (New England Biolabs).

[0052] Figure 3 Microscopic pictures (red and green fluorescence, phase contrast) of CHO cells infected with MVA (wild-type wt or recombinant) or CVA (wild-type wt or recombinant) are shown. Pictures were taken after infection with vaccinia virus and incubation of cells for 72 hours. Upper row: MVA-wt and MVA recombinant, CVA-wt and CVA recombinant; presence ("+") or absence ("-") of the EGFP (enhanced green fluorescent protein) gene and the host range genes CP77 (coupled to mRFP1, a single red fluorescent protein), K1L-V5 and SPI-1.

[0053] Figure 4 The replication capacity of MVA (wild-type wt or recombinant) in CHO cells is shown. Infected cells and supernatants were harvested after infection with MVA and incubation for 48 hours. For the abbreviations of the host range genes inserted into MVA (x-axis), see Figure 1 Legend for .

[0054] Figure 5 Shown are the growth kinetics of MVA encoding the host range genes CP77, K1L, and SPI-1 ("MVA-CKS") and wild-type ("MVA-wt") in CHO cells.

[0055] Figure 6Shown are microscopic images (red and green fluorescence, phase contrast) of HEK293 cells infected with MVA (wild-type wt or recombinant) or CVA (wild-type wt) 72 hours after infection.

[0056] Figure 7 Shown are microscopic images (red and green fluorescence, phase contrast) of RK13 cells infected with MVA (wild-type wt or recombinant) or CVA (wild-type wt) at 72 hours post infection.

[0057] A brief description of the sequence

[0058] SEQ ID NO: 1 describes the amino acid sequence encoded by the CP77 gene.

[0059] SEQ ID NO: 2 describes the nucleic acid sequence of the CP77 gene.

[0060] SEQ ID NO: 3 describes the amino acid sequence encoded by the K1L gene.

[0061] SEQ ID NO:4 describes the nucleic acid sequence of the K1L gene.

[0062] SEQ ID NO:5 describes the amino acid sequence encoded by the mRFP1-CP77 gene.

[0063] SEQ ID NO:6 describes the nucleic acid sequence of the mRFP1-CP77 gene.

[0064] SEQ ID NO:7 describes the amino acid sequence encoded by the K1L-V5 gene.

[0065] SEQ ID NO:8 describes the nucleic acid sequence of the K1L-V5 gene.

[0066] SEQ ID NO:9 describes the amino acid sequence encoded by the SPI-1 gene.

[0067] SEQ ID NO: 10 describes the nucleic acid sequence of the SPI-1 gene.

[0068] SEQ ID NO: 11 describes the amino acid sequence encoded by the C9L gene.

[0069] SEQ ID NO: 12 describes the nucleic acid sequence of the C9L gene.

[0070] Note: The nucleotide sequences of mRFP1-CP77, K1L-V5, and SPI-1 were codon modified for expression in human cells. The C9L gene was PCR amplified from VACV-WR genomic DNA.

[0071] SEQ ID NO: 13 describes the nucleic acid sequence of the PrH5m promoter.

[0072] SEQ ID NO: 14 describes the nucleic acid sequence of the Pr1328 promoter.

[0073] SEQ ID NO: 15 describes the nucleic acid sequence of the Pr13.5 promoter.

[0074] SEQ ID NO: 16 describes the nucleic acid sequence of the GFP forward primer.

[0075] SEQ ID NO: 17 depicts the nucleic acid sequence of the GFP reverse primer.

[0076] SEQ ID NO: 18 depicts the nucleic acid sequence of the CP77 forward primer.

[0077] SEQ ID NO: 19 depicts the nucleic acid sequence of the CP77 reverse primer.

[0078] SEQ ID NO:20 depicts the nucleic acid sequence of the K1L forward primer.

[0079] SEQ ID NO:21 depicts the nucleic acid sequence of the K1L reverse primer.

[0080] SEQ ID NO:22 describes the nucleic acid sequence of the SPI-1 forward primer.

[0081] SEQ ID NO:23 describes the nucleic acid sequence of the SPI-1 reverse primer.

[0082] SEQ ID NO:24 describes the nucleic acid sequence of the C9L forward primer.

[0083] SEQ ID NO:25 depicts the nucleic acid sequence of the C9L reverse primer. DETAILED DESCRIPTION

[0084] Here, we report the expansion of the host range of MVA by introducing poxvirus host range genes into the viral genome. The host range genes CP77, K1L, SPI-1, and C9L were sequentially inserted into MVA, and the MVA recombinants generated at each step were screened for their ability to infect and replicate in continuous cell lines.

[0085] Among the cell lines tested, only CHO and RK13 cells (both non-permissive for MVA) allowed the replication of MVA recombinants expressing at least the CP77 gene.

[0086] As shown for CHO cells, the insertion of the K1L gene into the MVA recombinant already encoding CP77 enhanced the replication capacity of the recombinant, while the additional insertion of the SPI-1 gene further enhanced its replication capacity. The latter step, i.e. the insertion of the SPI-1 gene in addition to the CP77 and K1L genes, even resulted in the most significant improvement in the replication capacity of MVA in the stepwise host range gene insertion procedure. In contrast, the insertion of the C9L gene in addition to the CP77, K1L and SPI-1 genes did not lead to further improvements.

[0087] It is noteworthy that MVA recombinants expressing the CP77, K1L and SPI-1 genes replicated in CHO cells to titers comparable to wild-type MVA grown in a standard substrate, i.e., primary CEF cells. On this basis, CHO cells genetically modified to co-express the poxvirus host range genes CP77, K1L and optionally SPI-1 are considered to be excellent substrates for MVA propagation.

[0088] CHO is a continuous non-human cell line widely used in the biopharmaceutical industry for the manufacture of biologics. It can be grown to high cell densities in chemically defined growth media as suspension cultures in industrial-scale bioreactors. Therefore, the possibility of using CHO cells instead of primary CEF cells to replicate MVA provides a significant advance in the production of MVA-based vaccines.

[0089] definition

[0090] It must be noted that, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid sequence" includes one or more nucleic acid sequences.

[0091] As used herein, the connection term "and / or" between multiple listed elements is understood to include separate and combined options. For example, where two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" used herein. The simultaneous applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0092] Throughout the specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are to be understood as including the stated integer or step or group of integers or steps but not excluding any other integer or step or group of integers or steps. The term "comprises" when used in the context of one aspect or embodiment in the description of the present invention may be modified and thus replaced by the terms "containing" or "including", or by the term "having" when used herein. Similarly, any of the foregoing terms (comprise, contain, include, have), whenever used in the context of one aspect or embodiment in the description of the present invention, include the terms "consisting of" or "consisting essentially of", each of which means a specific legal meaning depending on jurisdiction.

[0093] When used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0094] As used herein, the term "MVA recombinant" refers to MVA into which one or more poxvirus host range genes not expressed intrinsically in native MVA and an additional EGFP gene have been inserted. Similarly, "CVA recombinant" refers to CVA into which poxvirus host range genes not expressed in native CVA and an additional EGFP gene have been inserted.

[0095] As used herein, the term "native MVA" or "native CVA" refers to MVA and CVA, respectively, that have not been genetically modified. The term "non-intrinsically expressed" refers to a gene that is not expressed or contained in a native MVA or CVA.

[0096] 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 used as controls for recombinant MVA or CVA, respectively.

[0097] The "host range" of a virus is the range of cell types that the virus is able to infect. The term "host range gene" refers to the genetic basis of a virus' host range.

[0098] The term "primary cell culture" or "primary cultured cells" refers to the early stage of cell culture after the cells have been isolated from tissue. In contrast are continuous or immortalized cell lines. The term "continuous cell line" refers to cells that can be propagated continuously in culture.

[0099] The term "permissive cell line" refers to cells that allow viral replication. The term "permissiveness" refers to the ability of a cell to allow viral replication.

[0100] The term "cellular substrate" refers to cells that allow the virus (here MVA) to multiply.

[0101] The term "reproduction" refers to the replication or spread of MVA.

[0102] As used herein, the phrase "cell is genetically modified to express" means that in the absence of the modification and before the modification, the cell is not capable of expressing the gene.

[0103] abbreviation

[0104] BAC bacterial artificial chromosome

[0105] CEF Chicken embryonic fibroblasts

[0106] CHO cells Chinese Hamster Ovary cells

[0107] CPXV, CWPX vaccinia virus

[0108] CVA Chorioallantoic vaccinia virus Ankara

[0109] CVA-C CVA-wt encoding mRFP1-CP77

[0110] CVA-wt CVA wild type encoding EGFP

[0111] EGFP enhanced green fluorescent protein

[0112] mRFP1 Single red fluorescent protein

[0113] MVA Modified vaccinia virus Ankara

[0114] MVA - Bavarian Nordic, Bavarian Nordic's proprietary strain

[0115] MVA-C MVA-wt encoding mRFP1-CP77

[0116] MVA-CK MVA-wt encoding mRFP1-CP77 and K1L-V5

[0117] MVA-CKS MVA-wt encoding mRFP1-CP77, K1L-V5 and SPI-1

[0118] MVA-CKS-C9 encodes mRFP1-CP77, K1L-V5, SPI-1, and C9L MVA-wt

[0119] MVA-KS encodes K1L and SPI-1 MVA-wt

[0120] MVA-wt is stained with EGFP Reconstruction of MVA from BAC clones

[0121] ORF open reading frame

[0122] RT-PCT reverse transcription polymerase chain reaction

[0123] VACV Vaccinia virus

[0124] VACV-WR Vaccinia virus Western Reserve strain

[0125] Implementation

[0126] In one aspect, the invention provides cells of a continuous cell line capable of expressing the poxvirus host range genes CP77 and K1L.

[0127] In one aspect, the invention provides cells of a continuous cell line that are genetically modified to express the poxvirus host range genes CP77 and K1L.

[0128] 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.

[0129] 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.

[0130] In a specific aspect, the present invention provides cells of a continuous cell line capable of expressing the poxvirus host range genes CP77, K1L and SPI-1.

[0131] In a specific aspect, the invention provides cells of a continuous cell line, which are genetically modified to express the poxvirus host range genes CP77, K1L and SPI-1.

[0132] 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.

[0133] 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.

[0134] In one embodiment, the cell is incapable of expressing a host range gene in the absence of, or prior to, the genetic modification.

[0135] In one embodiment, the cell is infected with MVA.

[0136] In one embodiment of all aspects the cell is a cultured cell or a non-primary cell.

[0137] In one embodiment of all aspects the cell is not a CEF, DF-1 or quail cell, or is a non-avian cell.

[0138] In one embodiment of all aspects the cell line is a mammalian cell line, preferably a non-human mammalian cell line.

[0139] In one embodiment of all aspects the cell is a CHO cell.

[0140] In another aspect, the invention provides the use of a cell according to the invention for the propagation of MVA.

[0141] In another aspect, the invention provides the use of a cell according to the invention in the production of a vaccine comprising MVA.

[0142] In another aspect, the invention provides a vaccine comprising MVA, said MVA or vaccine being prepared using a cell according to the invention.

[0143] In a further aspect, the present invention provides a method for producing a cell according to the present invention, the method comprising the following steps:

[0144] (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous cell line, the nucleic acid comprising:

[0145] (i) a poxvirus host range gene CP77 operably linked to a promoter; or

[0146] (ii) the poxvirus host range gene K1L operably linked to a promoter; or

[0147] (iii) poxvirus host range genes CP77 and K1L, each operably linked to a promoter;

[0148] (b) introducing the nucleic acids (i) and (ii) obtained in step (a), or the nucleic acid (iii) obtained in step (a) into the cell; and

[0149] (c) Selecting cell populations or clones expressing the poxvirus host range genes CP77 and K1L.

[0150] In a further aspect, the present invention provides a method for producing a cell according to the present invention, the method comprising the following steps:

[0151] (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous cell line, the nucleic acid comprising:

[0152] (i) a poxvirus host range gene CP77 operably linked to a promoter; or

[0153] (ii) the poxvirus host range gene K1L operably linked to a promoter; or

[0154] (iii) the poxvirus host range gene SPI-1 operably linked to a promoter; or

[0155] (iv) poxvirus host range genes CP77, K1L and SPI-1, each operably linked to a promoter;

[0156] (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

[0157] (c) Selecting cell populations or clones expressing the poxvirus host range genes CP77, K1L and SPI-1.

[0158] In one embodiment of the method, the nucleic acid suitable for gene transfer is a vector or a plasmid.

[0159] In one embodiment of the method, the promoter operably linked to the host range gene is a promoter active in the cell into which the nucleic acid obtained in step (a) is transferred.

[0160] In one embodiment of the method, the promoter operably linked to the host range gene is a eukaryotic promoter.

[0161] In one embodiment of the method, the nucleic acid obtained in step (a) comprises a polyadenylation signal.

[0162] In a further aspect, the invention provides a cell produced by a method according to the invention.

[0163] In a further aspect, the invention provides an MVA propagated using a cell according to the invention.

[0164] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77 and K1L for enabling cells (preferably cells of a continuous cell line) to express the CP77 and K1L genes.

[0165] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77 and K1L for rendering cells, preferably cells of a continuous cell line, permissive for the propagation of MVA in said cells.

[0166] In one embodiment of the 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 express the CP77 and K1L genes.

[0167] In one embodiment of the 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 express the CP77 and K1L genes.

[0168] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77, K1L and SPI-1 for enabling cells (preferably cells of a continuous cell line) to express the CP77, K1L and SPI-1 genes.

[0169] In yet another aspect, the present invention provides the use of the poxvirus host range genes CP77, K1L and SPI-1 for rendering cells, preferably cells of a continuous cell line, permissive for the propagation of MVA in said cells.

[0170] In one embodiment of the use, 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 are used to enable cells to express CP77, K1L, and SPI-1 genes.

[0171] In one embodiment of the 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 express the CP77, K1L and SPI-1 genes.

[0172] In yet another aspect, the present invention provides an MVA expressing the poxvirus host range genes CP77 and K1L.

[0173] 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.

[0174] 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.

[0175] In yet another aspect, the present invention provides an MVA expressing the poxvirus host range genes mRFP1-CP 77 and K1L-V5.

[0176] In one embodiment of the MVA, the MVA comprises an mRFP1-CP77 fusion gene, preferably wherein the CP77 gene is fused to the C-terminus of RFP via a flexible linker.

[0177] 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.

[0178] 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.

[0179] In yet another aspect, the present invention provides an MVA expressing the poxvirus host range genes CP77, K1L and SPI-1.

[0180] 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.

[0181] 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.

[0182] In one embodiment of the MVA, the host range gene is operably linked to a promoter selected from the group consisting of PrH5m, Pr1328, and Pr13.5.

[0183] In one embodiment of the MVA, the MVA comprises the mRFP1-CP77 or CP77 gene operably linked to the PrH5m promoter.

[0184] In one embodiment of the MVA, the MVA comprises a K1L-V5 or K1L gene operably linked to a Pr1328 promoter.

[0185] In one embodiment, the MVA comprises the SPI-1 host range gene operably linked to a Pr13.5 promoter.

[0186] In one embodiment of MVA, the insertion site of the mRFP1-CP77 or CP77 gene is the intergenic region between ORFs MVA44L and MVA45L (IGR 44 / 45).

[0187] In one embodiment of MVA, the insertion site of the K1L-V5 or K1L gene is located between ORFs MVA88R and MVA89L.

[0188] In one embodiment of MVA, the insertion site for the SPI-1 host range gene is located between ORFs MVA88R and MVA89L.

[0189] In one embodiment of the MVA, the MVA comprises an expression cassette comprising the mRFP1-CP77 or CP77 gene operably linked to the PrH5m promoter, the expression cassette being inserted into the insertion sites MVA44L and MVA45L (IGR 44 / 45).

[0190] In one embodiment of the MVA, the MVA comprises an expression cassette comprising the K1L-V5 or K1L gene operably linked to the Pr1328 promoter, the expression cassette being inserted into the insertion sites MVA88R and MVA89L.

[0191] In one embodiment of the MVA, the MVA comprises an expression cassette comprising the SPI-1 host range gene operably linked to a Pr13.5 promoter, the expression cassette being inserted into insertion sites MVA88R and MVA89L.

[0192] MVA-related implementation plans

[0193] In one embodiment, the recombinant MVA is produced 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.

[0194] MVA-572 was deposited on January 27, 1994 as ECACC V94012707; MVA-575 was deposited on December 7, 2000 as ECACC V00120707; MVA-I721 was cited in Suter et al. Vaccine 2009, 27:7442-7450; NIH clone 1 was deposited on March 27, 2003 as PTA-5095 deposited; and MVA-BN has been deposited with the European Collection of Cell Cultures (ECACC) on August 30, 2000 under the number V00083008.

[0195] In one embodiment, the recombinant MVA is recombinant MVA-BN or a recombinant MVA-BN derivative.

[0196] Further explanation

[0197] Modified vaccinia virus Ankara (MVA)

[0198] In the past, MVA was produced in chicken embryonic fibroblasts (CVA) of the Ankara vaccinia virus strain by 516 serial passages (reviewed in Mayr et al. 1975). The virus was renamed from CVA to MVA at passage 516 to indicate that its properties had undergone a major change. MVA was further passaged until the number of passages exceeded 570. As a result of these long-term passages, the genome of the resulting MVA virus lacked approximately 31 kb of genomic sequence and was therefore described as a host cell that was highly restricted to replication in avian cells (Meyer et al. 1991). The MVA thus produced was shown to be significantly avirulent compared to the fully replicable starting material in a variety of animal models (Mayr and Danner 1978).

[0199] 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 (cited in Suter et al. 2009), NIH clone 1 (deposited on March 27, 2003 as PTA-5095 deposited) and MVA-BN (deposited with the European Collection of Cell Cultures (ECACC) on August 30, 2000 under the number V00083008).

[0200] More preferably, the MVA used according to the present invention includes MVA-BN and MVA-BN derivatives. MVA-BN has been described in WO 02 / 042480. As used herein, "MVA-BN derivative" refers to any virus that exhibits substantially the same replication characteristics as MVA-BN but exhibits differences in one or more parts of its genome.

[0201] MVA-BN and MVA-BN derivatives are not able to replicate, meaning that they are not able to replicate reproductively in vivo and in vitro. More specifically, in vitro, MVA-BN or MVA-BN derivatives have been described as being able to replicate reproductively in chicken embryo fibroblasts (CEF), but not in the human keratinocyte cell line HaCat (Boukamp et al. 1988), the human bone osteosarcoma cell line 143B (ECACC deposit number 91112502), the human embryonic kidney cell line 293 (ECACC deposit number 85120602) and the human cervical adenocarcinoma cell line HeLa (ATCC deposit number CCL-2). In addition, the viral amplification rate of MVA-BN or MVA-BN derivatives in Hela cells and HaCaT cell lines is at least two times smaller than that of MVA-575, and more preferably three times smaller. Testing and analysis of these properties of MVA-BN and MVA-BN derivatives are described in WO 02 / 42480 and WO 03 / 048184.

[0202] The term "incapable of reproductive replication" in vitro in human cell lines as described above is for example described in WO02 / 42480, which also teaches how to obtain MVAs with the desired properties as described above. The term applies to viruses having an in vitro viral amplification rate of less than 1 4 days after infection using the assays described in WO02 / 42480 or US 6,761,893.

[0203] Exemplary Generation of Recombinant MVA Viruses

[0204] For the production of recombinant MVA as described herein, different methods can be applied. The DNA sequence to be inserted into the virus can be placed in an E. coli plasmid construct, into which DNA homologous to a poxvirus DNA fragment is inserted. In addition, the DNA sequence to be inserted can be connected to a promoter. The promoter-gene linkage can be positioned in the plasmid construct so that the two ends of the promoter-gene linkage are flanked by DNA homologous to the DNA sequence, which is flanked by a poxvirus DNA region containing a non-essential locus. The resulting plasmid construct can be amplified and isolated by propagation in E. coli. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into, for example, a chicken embryo fibroblast (CEF) cell culture, and the culture is infected with MVA at the same time. The recombination between the homologous MVA viral DNA in the plasmid and the viral genome can produce an MVA modified by the presence of an exogenous DNA sequence, i.e., a nucleotide sequence encoding a SARS-CoV-2 antigen.

[0205] According to a preferred embodiment, cells of a suitable cell culture, such as CEF cells, can be infected with the MVA virus. The infected cells can then be transfected with a first plasmid vector comprising one or more exogenous or heterologous genes, such as one or more nucleic acids provided herein; preferably under the transcriptional control of a poxvirus expression control element. As described above, the plasmid vector also includes a sequence capable of directing the insertion of an exogenous sequence into a selected portion of the MVA viral genome. Optionally, the plasmid vector also contains a box comprising a marker and / or selection gene operably linked to a poxvirus promoter. The use of a selection or marker box simplifies the identification and isolation of the recombinant MVA produced. However, the recombinant poxvirus can also be identified by PCR technology. Subsequently, another cell can be infected with the recombinant MVA obtained as described above and transfected with a second vector comprising one or more second exogenous or heterologous genes. If this gene is introduced into a different insertion site of the MVA genome, the second vector also differs in the poxvirus homologous sequence that directs the integration of one or more second exogenous genes into the poxvirus genome. After homologous recombination occurs, a recombinant virus containing two or more foreign or heterologous genes can be isolated. To introduce additional foreign genes into the recombinant virus, the infection and transfection steps can be repeated by infecting with the recombinant virus isolated in the previous step and by transfecting with another vector containing another foreign gene or genes. A number of other techniques are known for producing recombinant MVA.

[0206] If not otherwise indicated, the practice of the present invention will employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant technology, which are within the technical scope of 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., 1987; Series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, 1988.

[0207] Example

[0208] The following examples are used to further illustrate the present invention. They should not be construed as limiting the present invention, the scope of the present invention being determined by the appended claims.

[0209] Example 1: Preparation of recombinant vaccinia virus

[0210] 1.1 Recombinant MVA and CVA

[0211] MVA recombinants (see Table 2 below) and modified MVA (referred to as "wild type") that encode poxvirus host range genes not present in MVA are derived from CVA was used as the MVA parent strain for comparison: a CVA recombinant encoding CP77 (natively absent in CVA) and "wild-type" CVA.

[0212] MVA and CVA are determined by The bacterial artificial chromosome (BAC) clones constructed were reconstructed and characterized as previously described (12). Both the MVA and CVA BAC clones contained a bicistronic expression cassette encoding a neomycin phosphotransferase selection marker (NPT II) under the control of a strong synthetic early / late pS promoter. The clones also contained genes for an internal ribosome entry site (IRES) and an enhanced green fluorescent protein (EGFP) reporter protein.

[0213] Recombinant MVA and CVA were reconstructed from BAC clones encoding EGFP that had been modified to contain the host range gene of interest. MVA and CVA reconstructed from unmodified BAC clones encoding EGFP were referred to as "wild type," "MVA-wt" and "CVA-wt," respectively. No significant differences were observed between the titers obtained for recombinant MVA and MVA-wt during the production of viral stocks.

[0214] 1.2 Cell culture

[0215] Primary CEF cells were prepared from 11-day-old embryonated chicken eggs (VALO BioMedia GmbH) and cultured in VP-SFM medium (Gibco).

[0216] CHO cells were obtained from ATCC (CCL-61, CHO-K1) and cultured in Nutrient Cocktail F-12 Ham's Medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS).

[0217] BHK-21 cells (obtained from ATCC) were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco / Thermos Fisher Scientific) supplemented with 10% FCS (Pan Biotech).

[0218] 1.3 Generation of recombinant CVA

[0219] Recombinant CVA encoding CP77 ("CVA-C") was prepared by homologous recombination. To facilitate detection, the CP77 gene was fused to the C-terminus of single red fluorescent protein (mRFP1) via a flexible linker.

[0220] Invitrogen GeneArt (Thermo Fisher Scientific) synthesized plasmid pMISC564, which contains the flanks of homologous recombination between MVA open reading frames 44 and 45, and the mRFP1-CP77 fusion gene driven by the PrH5m promoter. pBN564 was linearized with SacI and NheI and transfected into CHO cell monolayers infected with CVA-wt using Fugene HD (Roche Diagnostics). 48 hours after transfection, cells and supernatants were harvested and sonicated with a cup sonicator. In order to eliminate CVA-wt from the viral 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 infected cells using the NucleoSpin Blood Mini Kit (Macherey-Nagel) and screened by PCR to verify the absence of unwanted CVA-wt DNA and the presence of the desired recombinant CVA-C.

[0221] 1.4 Production of recombinant MVA

[0222] MVA recombinants encoding host range genes of interest are summarized in Table 2 below.

[0223] Table 2: Inserted recombinant MVA and host range genes.

[0224] Recombinant MVA Host range gene MVA-C mRFP1-CP77 MVA-CK mRFP1-CP77+K1L-V5 MVA-CKS mRFP1-CP77+K1L-V5+SPI-1 MVA-CKS-C9 mRFP1-CP77+K1L-V5+SPI-1+C9L MVA-KS K1L-V5+SPI-1

[0225] The design of the MVA recombinant, i.e. the expression cassette with the host range gene and the promoter and the insertion site of the cassette, is as follows Figure 1 shown.

[0226] 1.4.1 Preparation of MVA-BAC clones

[0227] The MVA recombinant was prepared using BAC-λRed recombination technology.

[0228] All BAC clones were propagated in E. coli strain MDS42 containing plasmid pKD46. BAC DNA was extracted using the Xtra BAC kit (Macherey-Nagel). The host range gene of interest driven by the selected poxvirus promoter was inserted into the MVA BAC clone using the λRed recombination system as previously described (12, 31, 32). Briefly, a reverse selectable rpsL / neo cassette was generated by PCR with a positive and a negative selection marker flanked by homology arms. The rpsL / neo cassette was electroporated into E. coli strain MDS42 carrying the MVA BAC and plasmid pKD46 (33). Plasmid pKD46 encoding the protein for λRed recombination was provided by BL Wanner

[27] . Using the λRed recombinant protein expressed by pKD46, the rpsL / neo cassette was inserted into the corresponding insertion site in the PCR fragment guided by the homologous sequence. In the second recombination step, the rpsl / neo cassette was replaced by a fragment encoding the host range gene (in plasmid pMISC564 or pMISC576). Plasmid pMISC564 was synthesized by GeneArt (ThermoFisher Scientific), which contains homologous recombination flanks and the mRFP1-CP77 gene controlled by the PrH5m promoter. To monitor the spread of the recombinant virus in the cell substrate, CP77 was fused to the C-terminus of mRFP1 via a glycine-serine (GS) linker, similar to the previously described GFP-CP77 fusion protein (34). Plasmid pMISC576 containing homologous recombination flanks, the K1L-V5 gene under the control of the Pr1328 promoter, and the SPI-1 gene driven by the Pr13.5 promoter was synthesized by GeneArt (ThermoFisher Scientific). Since there is no available antibody against K1L, a V5 tag was added to the C-terminus of the K1L gene via a GS linker for detection of the K1L protein. Since C9L in MVA and CVA is truncated, the C9L gene was repaired in situ in MVA-CKS to produce MVA-CKS-C9. In brief, the truncated C9L gene in the MVA-CKS BAC clone was replaced by the rpsL / neo cassette. In the second recombination step, the rpsL / neo cassette was replaced by the C9L DNA sequence, which was amplified by PCR using VACV-WR genomic DNA as a template (VACV-WR is from ATCC). The successful recombination of each step was confirmed by PCR and sequencing.

[0229] 1.4.2 Reconstruction of infectious recombinant MVA

[0230] Infectious MVA recombinants were reconstructed by MVA-BAC clones constructed separately, as previously described (12). In brief, BHK-21 cells seeded in 6-well plates were transfected with 3 μg MVA-BAC DNA using Fugene HD (Promega) and infected with the helper virus Shope fibroid virus (SFV) (obtained from ATCC) after 60 minutes. The EGFP expression of the cells was monitored and the cells were harvested 3 days after transfection. CEF cells were infected with cell lysate and three cell passages were performed to remove the helper virus SFV that could not be propagated in CEF cells. Total DNA was extracted from infected cells and used to detect residual (contamination) SFV by PCR.

[0231] 1.5 Host range gene expression analysis

[0232] The expression of genes 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 MVA recombinants MVA-C, MVA-CK, MVA-CKS, MVA-CKS-C9, or MVA-KS.

[0233] 48 hours after infection, the supernatant was removed, and the cells were scraped and resuspended in 350 μl RTL lysis buffer. The cell lysate was homogenized using a QIAshredder column (Qiagen), and genomic DNA was removed using a gDNA elimination column (Qiagen). RNA in the effluent of the genomic DNA elimination column was purified using an RNeasy Plus mini kit (Qiagen) according to the manufacturer's instructions. RNA was eluted from the RNeasy spin column with 50 μl of RNase-free water. The remaining viral and cellular DNA in the purified sample was digested with Turbo DNase (Ambion / Life Technologies), and the DNase was then inactivated with EDTA (Sigma-Aldrich) to a final concentration of 15 mM. 3 μl of the isolated RNA sample was reverse transcribed using the OneTaq RT-PCR kit (NEB) according to the manufacturer's instructions. The sample without reverse transcriptase (RT) was set as a negative control. A total of 2 μl of RT reaction mixture was used to detect the transcription of different host range genes using OneTaq Hot Start 2X Master Mix (NEB) according to the manufacturer's instructions. Detection of EGFP transcripts was performed as a positive control. Table 3 lists the primers used to detect genes and the size of the expected PCR products.

[0234] Table 3:Primers and expected product size for RT-PCR detection of mRNA

[0235]

[0236] like Figure 2 As shown, transcripts of all inserted host range genes (i.e., the mRFP1-CP77 fusion gene as well as genes for K1L-V5, SPI-1, and C9L) were detected by RT-PCR and thus expressed by MVA and CVA recombinants. Detection of EGFP transcripts in samples treated with reverse transcriptase served as a control. For C9L, only a truncated defective transcript was detected, which was possible because the primers were directed against residual sequences of the defective C9L transcript (7).

[0237] Example 2: Replication of recombinant vaccinia virus in CHO cells

[0238] Using CHO cells as an example of a non-permissive mammalian cell line, the effect of the inserted host range gene on the replication characteristics of MVA recombinants was studied.

[0239] 2.1 Cell culture

[0240] CHO cell culture and CEF cell preparation were as briefly described above (see Example 1.1).

[0241] 2.2 Virus replication in CHO cells

[0242] To analyze viral replication and spread, 1 × 10 cells / mL of 500 μl DMEM without FCS were used. 5 TCID 50 At 0.1 TCID per cell 50 Confluent CHO cell monolayers in 6-well culture plates were infected with 50 μg of 5 ...

[0243] like Figure 3 As shown, no detectable expression of the viral reporter genes EGFP and mRFP1 was observed in CHO cells infected with CVA-wt. In contrast, CHO cells infected with CVA-C displayed widespread EGFP and mRFP1 signals as well as a significant cytopathic effect (CPE), the latter indicating a fully permissive cell line. Apparently, the mRP1-CP77 fusion gene in CVA-C expresses a protein that is fully functional in supporting MVA replication in CHO cells.

[0244] like Figure 3 As further shown, CHO cells are not permissive for MVA-wt. In contrast, mRFP1 and EGFP signals as well as CPE were specifically observed in MVA-CKS and to a lesser extent in MVA-C and MVA-CK. However, MVA-KS replicated no better in CHO cells than in MVA-wt.

[0245] 2.3 Replication ability of MVA recombinant in CHO cells

[0246] The replication capacity, a measure of how quickly the virus replicates, was studied.

[0247] Infection of CHO cells was performed as described above (see Example 2.2). Cells and supernatants were harvested, sonicated to release the virus, and quantified according to TCID 50 Methods Titrations were performed on CEF cells (35). Statistical analysis was performed using GraphPad PRISM (GraphPad Software, San Diego, USA).

[0248] like Figure 4 As shown, all MVA recombinants expressing the CP77 gene replicated in CHO cells and replicated in the following order: MVA-CKS=MVA-CKS-9>MVA-CK>MVA-C.

[0249] MVA expressing only CP77 (MVA-C) replicated in CHO cells with titers at least 1 log higher than those produced by MVA-wt. CHO cells infected with MVA-CKS produced titers nearly 3 logs higher than those produced by MVA-C. The greatest increase in titer, approximately two orders of magnitude, was observed with MVA-CKS compared to MVA-CK.

[0250] Thus, insertion of the poxvirus host range gene into MVA, namely mRFP1-CP77 (MV AC), followed by addition of K1L-V5 (MVA-CK) and subsequently SPI-1 (MVA-CK S), resulted in a significant stepwise increase in the replication capacity of MVA in CHO cells.

[0251] Interestingly, the insertion of the C9L gene in addition to the CP77, K1L and SPI-1 genes (MVA-CKS-C9) did not further increase the viral titer compared to MVA-CKA. The viral titer of MVA-KS was even lower than that measured for MVA-wt.

[0252] Notably, the titers of MVA-CKS obtained in CHO cells were comparable to those obtained with MVA-wt in primary CEF cells.

[0253] In summary, the CP77 gene is essential for MVA replication in CHO cells, but the combination of K1-L and SPI-1 with CP77 enables the MVA recombinant to replicate in the order of natural MVA replication in CEF cells.

[0254] 2.4 Growth of recombinant MVA in CHO cells

[0255] The growth kinetics of MVA-CKS in CHO cells were also analyzed.

[0256] 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).

[0257] like Figure 5 As shown, MVA-CKS replicated efficiently in CHO cells within 24 h after infection and reached maximum viral titers between 48 and 72 h. Figure 4 It was also shown that MVA-wt was unable to replicate in CHO cells.

[0258] Example 3: Replication of recombinant vaccinia virus in other cells

[0259] 3.1 Cell culture

[0260] The preparation of CEF cells was as described above (see Example 1.2).

[0261] All cell lines were obtained from the American Type Culture Collection (ATCC) or the European Collection of Authenticated Cell Cultures (ECACC).

[0262] CHO cells were cultured as described above (see Example 1.2).

[0263] Cell lines except CHO cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco / Thermos Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Pan Biotech).

[0264] 3.2 Virus replication in HEK293, RK13, and chicken cells

[0265] Viral replication in HEK293 and RK13 cells was analyzed by fluorescence microscopy as described above (see Example 2.2).

[0266] like Figure 6 As shown, human embryonic kidney HEK293 cells were non-permissive for MVA-wt but permissive for CVA-wt. However, all MVA recombinants failed to replicate in HEK293 cells.

[0267] In contrast, Figure 7 As shown, RK13 cells are permissive for CVA-wt and all MVA recombinants, but not for MVA-wt. Since K1L is a VACV host range gene known to restore the replication of MVA in RK13 cells (21), and the host range function of K1L can be complemented by CP77 in RK13 cells (17), RK13 cells were used here as a control cell substrate to verify the host range function of K1L-V5 and mRFP1-CP77 fusion proteins. The results that RK13 cells are permissive for MVA-C and MVA-KS confirm the host range function of K1L-V5 and mRFP1-CP77.

[0268] In addition, viral replication of MVA-CKS and MVA-wt was analyzed based on virus titers produced in chicken-derived cells as described above (see Example 2.3).

[0269] Primary CEF cells and cells of the chicken fibroblast DF-1 cell line were both permissive for MVA-wt and MVA-CKS, and no significant differences were found between MVA-CKS and MVA-wt in their ability to infect CEF and DF-1 cells.

[0270] Example 4: Preparation of MVA-permissive CHO cells

[0271] Based on the discovery that MVA-CKS replicates in CHO cells (see Example 2 above), it is contemplated to generate CHO cells that stably express the poxvirus host range genes CP77, K1L, and SPI-1. Methods for generating CHO cell lines that stably express transgenes are available (e.g., 18).

[0272] 4.1 Cloning of plasmids containing CP77, K1L, SPI-1 and NPT II genes

[0273] A plasmid containing three host range genes CP77, K1L and SPI-1 driven by different promoters (CMV promoter, β-globin promoter and β-actin promoter, respectively) was prepared for gene expression in mammalian cells. The plasmid also contained an NPT II-IRES-EGFP cassette driven by the SV40 promoter as a selection marker and reporter gene.

[0274] 4.2 CHO cell transfection and clonal analysis

[0275] According to the manufacturer's instructions, use HD transfection reagent (Promega) was used to transfect CHO cells (ATCC CCL-61) seeded in 6-well plates containing F-12 Ham medium supplemented with 10% FCS. After 24 hours, the medium was replaced with fresh medium containing antibiotic selection (G418). The cell line was established by single cell sorting and then further amplified by antibiotic selection. Clones were analyzed by RT-PCR detection of transgene transcription. Positive clones were further tested by infection with MVA. Viruses were harvested 48 hours after infection and titrated.

[0276] Final Note: Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, this specification supersedes any such material. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

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[0305] 28.Shisler JL, lsaacs SN.Moss B.1999.Vaccinia virus serpin-1 deletionmutant exhibits a host range defect characterized by low levels ofintermediate and late mRNAs.Virology 262:298-311.

[0306] 29.Liu R,Mendez-Rios JD,Peng C,Xiao W,Weisberg AS,Wyatt LS,MossB.2019.SPI-1 is a missing host-range factor required for replication of theattenuated modified vaccinia Ankara(MVA)vaccine vector in human cells.PLoSPathog 15:e1007710.

[0307] 30.Peng C,Moss B.2020.Repair of a previously uncharacterized secondhost-range gene contributes to full replication of modified vaccinia virusAnkara(MVA)in human cells.Proc Natl Acad Sci U S A 117:3759-3767.

[0308] 31.Wang S,Zhao Y,Leiby M,Zhu J.2009.A new positive / negative selectionscheme for precise BAC recombineering.Mol Biotechnol 42:110-6.

[0309] 32.Reyrat JM,Pelicic V,Gicquel B,RappuoliR.1998.Counterselectablemarkers:untapped tools for bacterial genetics and pathogenesis.Infect Immun66:4011-7.

[0310] 33.Datsenko KA,Wanner BL.2000.One-step inactivation of chromosomalgenes in Escherichia coli K-12using PCR products.Proc Natl Acad Sci U S A 97:6640-5.

[0311] 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.

[0312] 35. Staib C, Drexler L, Sutter G. 2004. Construction and isolation of recombinant MVA. Methods Mol Biol 269: 77-100.

[0313] sequence

[0314] SEQ ID NO: 1 Amino acid sequence encoded by CP77 gene

[0315] MFDYLENEEVALDELKQMLRDRDPNDTRNQFKNNALHAYLFNEHCNNVEVVKLLLDSGTNPLHKNWRQLTPLGEYTNSRHGKVNKDIAMVLLEATGYSNINDFNIFTYMKSKNVDIDLIKVLVEHGFDFSVKCEKHHSVIENYVMTDDPVPEIIDLFIENGCSVIYEDEDDEYGYAYEEYHSQNDDYQPRNCGTVLHLYIISHLYSESDSRSCVNPEVVKCLINHGINPSSIDKNYCTALQYYIKSSHIDIDIVKLLMKGIDNTAYSYIDDLTCCTRGIMADYLNSDYRYNKDVDLDLVKLFLENGKPHGIMCSIVPLWRNDKETISLILKTMNSDVLQHILIEYITFSDIDISLVEYMLEYGAVVNKEAIHGYFKNINIDSYTMKYLLKKEGGDAVNHLDDGEIPIGHLCKSNYGRYNFYTDTYRQGFRDMSYACPILSTINICLPYLKDINMIDKRGETLLHKAVRYNKQSLVSLLLESGSDVNIRSNNGYTCIAIAINESRNIELLNMLLCHKPTLDCVIDSLREISNIVDNAYAIKQCIRYAMIIDDCISSKIPESISKHYNDYIDICNQELNEMKKIIVGGNTMFSLIFTDHGAKIIHRYANNPELRAYYESKQNKIYVEVYDIISNAIVKHNKIHKNIESVDDNTYISNLPYTIKYKIFEQQ

[0316] Nucleic acid sequence of SEQ ID NO:2 CP77 gene

[0317]

[0318] Amino acid sequence encoded by SEQ ID NO:3 K1L gene

[0319] MDLSRINTWKSKQLKSFLSSKDAFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLENEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKN

[0320] Nucleic acid sequence of SEQ ID NO:4 K1L gene

[0321] ATGGACCTGAGCCGGATCAACACCTGGAAGTCCAAGCAGCTGAAGTCCTTCCTGAGCAGCAAGGACGCCTTCAAGGCCGATGTGCACGGACACAGCGCCCTGTACTATGCCATTGCCGACAACAACGTGCGGCTCGTGTGCACCCTTCTGAATGCCGGCGCTCTGAAGAACCTGCTGGAAAACGAGTTCCCTCTGCACCAGGCCGCCACACTGGAAGATACCAAGATCGTGAAGATTCTGCTGTTCAGCGGCCTGGACGACAGCCAGTTCGACGACAAGGGAAACACCGCTCTGTACTACGCCGTGGACAGCGGCAATATGCAGACCGTGAAGCTGTTCGTGAAGAAAAACTGGCGGCTGATGTTCTACGGCAAGACCGGATGGAAAACCAGCTTCTACCACGCCGTGATGCTGAACGATGTGTCTATCGTGTCCTACTTCCTGTCTGAGATCCCCAGCACCTTCGACCTGGCCATCCTGCTGAGCTGCATCCACATCACCATCAAGAACGGCCACGTGGACATGATGATCCTGCTGCTGGACTACATGACCAGCACCAACACCAACAACAGCCTGCTGTTTATCCCCGACATCAAGCTGGCCATCGACAACAAGGACATCGAGATGCTGCAGGCCCTGTTTAAGTACGACATCAACATCTACAGCGCCAACCTCGAGAACGTCCTGCTGGACGATGCCGAGATCGCCAAGATGATCATTGAGAAGCACGTCGAGTACAAGAGCGACAGCTACACCAAGGACCTGGACATTGTGAAGAACAACAAGCTGGACGAGATCATCAGCAAGAACAAAGAACTGCGGCTTATGTACGTGAACTGCGTGAAAAAGAACTGA

[0322] Amino acid sequence encoded by SEQ ID NO:5 mRFP1-CP77 gene

[0323] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGAGGGSGGGGSGGGGSFDYLENEEVALDELKQMLRDRDPNDTRNQFKNNALHAYLFNEHCNNVEVVKLLLDSGTNPLHKNWRQLTPLGEYTNSRHGKVNKDIAMVLLEATGYSNINDFNIFTYMKSKNVDIDLIKVLVEHGFDFSVKCEKHHSVIENYVMTDDPVPEIIDLFIENGCSVIYEDEDDEYGYAYEEYHSQNDDYQPRNCGTVLHLYIISHLYSESDSRSCVNPEVVKCLINHGINPSSIDKNYCTALQYYIKSSHIDIDIVKLLMKGIDNTAYSYIDDLTCCTRGIMADYLNSDYRYNKDVDLDLVKLFLENGKPHGIMCSIVPLWRNDKETISLILKTMNSDVLQHILIEYITFSDIDISLVEYMLEYGAVVNKEAIHGYFKNINIDSYTMKYLLKKEGGDAVNHLDDGEIPIGHLCKSNYGRYNFYTDTYRQGFRDMSYACPILSTINICLPYLKDINMIDKRGETLLHKAVRYNKQSLVSLLLESGSDVNIRSNNGYTCIAIAINESRNIELLNMLLCHKPTLDCVIDSLREISNIVDNAYAIKQCIRYAMIIDDCISSKIPESISKHYNDYIDICNQELNEMKKIIVGGNTMFSLIFTDHGAKIIHRYANNPELRAYYESKQNKIYVEVYDIISNAIVKHNKIHKNIESVDDNTYISNLPYTIKYKIFEQQ

[0324] SEQ ID NO:6 Nucleic acid sequence of mRFP1-CP77 gene

[0325]

[0326] Amino acid sequence encoded by SEQ ID NO:7 K1L-V5 gene

[0327] MDLSRINTWKSKQLKSFLSSKDAFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLENEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKNGGSGKPIPNPLLGLDST

[0328] Nucleic acid sequence of SEQ ID NO:8 K1L-V5 gene

[0329] ATGGACCTGAGCCGGATCAACACCTGGAAGTCCAAGCAGCTGAAGTCCTTCCTGAGCAGCAAGGACGCCTTCAAGGCCGATGTGCACGGACACAGCGCCCTGTACTATGCCATTGCCGACAACAACGTGCGGCTCGTGTGCACCCTTCTGAATGCCGGCGCTCTGAAGAACCTGCTGGAAAACGAGTTCCCTCTGCACCAGGCCGCCACACTGGAAGATACCAAGATCGTGAAGATTCTGCTGTTCAGCGGCCTGGACGACAGCCAGTTCGACGACAAGGGAAACACCGCTCTGTACTACGCCGTGGACAGCGGCAATATGCAGACCGTGAAGCTGTTCGTGAAGAAAAACTGGCGGCTGATGTTCTACGGCAAGACCGGATGGAAAACCAGCTTCTACCACGCCGTGATGCTGAACGATGTGTCTATCGTGTCCTACTTCCTGTCTGAGATCCCCAGCACCTTCGACCTGGCCATCCTGCTGAGCTGCATCCACATCACCATCAAGAACGGCCACGTGGACATGATGATCCTGCTGCTGGACTACATGACCAGCACCAACACCAACAACAGCCTGCTGTTTATCCCCGACATCAAGCTGGCCATCGACAACAAGGACATCGAGATGCTGCAGGCCCTGTTTAAGTACGACATCAACATCTACAGCGCCAACCTCGAGAACGTCCTGCTGGACGATGCCGAGATCGCCAAGATGATCATTGAGAAGCACGTCGAGTACAAGAGCGACAGCTACACCAAGGACCTGGACATTGTGAAGAACAACAAGCTGGACGAGATCATCAGCAAGAACAAAGAACTGCGGCTTATGTACGTGAACTGCGTGAAAAAGAACGGCGGCAGCGGCAAGCCCATTCCTAATCCACTGCTGGGCCTCGACAGCACCTGA

[0330] SEQ ID NO:9 Amino acid sequence encoded by SPI-1 gene

[0331] MGGSDIFKELILKHTDENVLISPVSILSTLSILNHGAAGSTAEQLSKYIENMNENTPDDNNDMDVDIPYCATLATANKIYGSDSIEFHASFLQKIKDDFQTVNFNNANQTKELINEWVKTMTNGKINSLLTSPLSINTRMTVVSAVHFKAMWKYPFSKHLTYTDKFYISKNIVTSVDMMVSTENNLQYVHINELFGGFSIIDIPYEGNSSMVIILPDDIEGIYNIEKNITDEKFKKWCGMLSTKSIDLYMPKFKVEMTEPYNLVPILENLGLTNIFGYYADFSKMCNETITVEKFLHTTFIDVNEEYTEASAVTGVFMTNFSMVYRTKVYINHPFMYMIKDNTGRILFIGKYCYPQ

[0332] SEQ ID NO:10 Nucleic acid sequence of SPI-1 gene

[0333]

[0334] Amino acid sequence encoded by SEQ ID NO:11 C9L gene

[0335] MVNDKILYDSCKTFNIDASSAQSLIESGANPLYEYDGETPLKAYVTKKNNNIKNDVVILLLSSVDYKNINDFDIFEYLCSDNIDIDLLKLLISKGIEINSIKNGINIVEKYATTSNPNVDVFKLLLDKGIPTCSNIQYGYKIKIEQIRRAGEYYNWDDELDDYDYDYTTDYDDRMGKTVLYYYIITRSQDGYATSLDVINYLISHKKEMRYYTYREHTTLYYYLDKCDIKREIFDALFDSNYSGHELMNILSNYLRKQFRKKNHKIDNYIVDQLLFDRDTFYILELCNSLRNNILISTILKRYTDSIQDLLLEYVSYHTVYINVIKCMIDEGATLYRFKHINKYFQKFGNRDPKVVEYILKNGNLVVDNDNDDNLINIMPLFPTFSMRELDVLSILKLCKPYIDDINKIDKHGCSILYHCIKSHSVSLVEWLIDNGADINIITKYGFTCITICVILADKYIPEIAELYIKILEIILSKLPTIECIKKTVDYLDDHRYLFIGGNNKSLLKICIKYFILVDYKYTCSMYPSYIEFITDCEKEIADMRQIKINGTDMLTVMYMLNKPTKKRYVNNPIFTDWANKQYKFYNQIIYNANKLIEQSKKIDDMIEEVSIDDNRLSTLPLEIRHLIFSYAFL

[0336] Nucleic acid sequence of SEQ ID NO:12 C9L gene

[0337]

[0338] SEQ ID NO: 13 PrH5m promoter

[0339] TAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAATTTCATTATCGCGATATCCGTTAAGTTTGTATCGTA

[0340] SEQ ID NO:14 Pr1328 promoter

[0341] TATATTATTAAGTGTGGTGTTTGGTCGATGTAAAATTTTTGTCGATAAAAATTAAAAAATAACTTAATTTATTATTGATCTCGTGTGTACAACCGAAATC

[0342] SEQ ID NO:15 Pr13.5 promoter

[0343] TAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTATTGCTCTTGTGACTAGAGACTTTAGTTAAGGTACTGTAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTA

[0344] SEQ ID NO: 16 EGFP forward primer

[0345] CAGCTCGTCCATGCCGAGAG

[0346] SEQ ID-NO: 17 EGFP reverse primer

[0347] TGAAGTTCATCTGCACCACC

[0348] SEQ ID NO: 18CP77 forward primer

[0349] TGAACCCTGAGGTGGTCAAGTGC

[0350] SEQ ID NO: 19CP77 reverse primer

[0351] CGATCAGGATGTGCTGGAGCAC

[0352] SEQ ID NO: 20K1L forward primer

[0353] CGATGTGCACGGACACAGC

[0354] SEQ ID NO:21K1L reverse primer

[0355] GCTGGTCATGTAGTCCAGCAGC

[0356] SEQ ID NO:22SPI-1 forward primer

[0357] CGATCAGGATGTGCTGGAGCAC

[0358] SEQ ID NO:23 SPI-1 reverse primer

[0359] GTACACCTTGGTCCGGTACACC

[0360] SEQ ID NO:24C9L forward primer

[0361] CATGCTACATGTGTACTTATAATCGAC

[0362] SEQ ID NO: 25C9L reverse primer

[0363] GCCGTATATTGATGATATAAACAAAATAG

Claims

1. A continuous cell line of cells genetically modified to express the poxvirus host range genes CP77 and K1L.

2. The cell according to claim 1, wherein the genome of the cell comprises the poxvirus host range genes CP77 and K1L, 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 of claim 1, which is genetically modified to express the poxvirus host range genes CP77, K1L and SPI-1.

4. The cell according to claim 3, wherein the genome of the cell comprises the poxvirus host range genes CP77, K1L and SPI-1, 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 cell according to any one of claims 1 to 4, which is a cell of a non-human mammalian cell line, preferably a Chinese Hamster Ovary (CHO) cell.

6. The cell of any one of claims 1 to 5, which is infected with Modified Vaccinia Virus Ankara (MVA).

7. Use of the cell according to any one of claims 1 to 6 for propagating Modified Vaccinia Ankara Virus (MVA).

8. Use of a cell according to any one of claims 1 to 6 for the production of a vaccine comprising a modified vaccinia virus Ankara (MVA).

9. A vaccine comprising modified vaccinia virus Ankara (MVA) prepared using the cell of any one of claims 1 to 6.

10. A method for producing a cell as described in any one of claims 1, 2, 5 and 6, the method comprising the steps of: (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous cell line, the nucleic acid comprising: (i) the poxvirus host range gene CP77 operably linked to a promoter; or (ii) the poxvirus host range gene K1L operably linked to a promoter; or (iii) poxvirus host range genes CP77 and K1L, each operably linked to a promoter; (b) introducing the nucleic acids (i) and (ii) obtained in step (a), or the nucleic acid (iii) obtained in step (a) into the cell; and (c) Selecting cell populations or clones expressing the poxvirus host range genes CP77 and K1L.

11. A method for producing a cell as claimed in any one of claims 3 to 6, the method comprising the steps of: (a) preparing a nucleic acid suitable for gene transfer into cells of a continuous cell line, the nucleic acid comprising: (i) the poxvirus host range gene CP77 operably linked to a promoter; or (ii) the poxvirus host range gene K1L operably linked to a promoter; or (iii) the poxvirus host range gene SPI-1 operably linked to a promoter; or (iv) poxvirus host range genes CP77, K1L and SPI-1, 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 cell populations or clones expressing the poxvirus host range genes CP77, K1L and SPI-1.

12. A modified vaccinia virus Ankara (MVA) propagated using the cell of any one of claims 1 to 6.

13. Use of poxvirus host range genes CP77 and K1L, preferably use of 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, for enabling cells to express CP77 and K1L genes.

14. The use according to claim 13, further using the poxvirus host range gene SPI-1, preferably further using a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9, for enabling the cell to express CP77, K1L and SPI-1 genes.

15. The use according to claim 13 or 14, wherein the cell is a cell of a non-human mammalian cell line, preferably a Chinese Hamster Ovary (CHO) cell.

16. A modified vaccinia virus Ankara (MVA), comprising the poxvirus host range genes CP77 and K1L, 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.

17. The MVA of claim 16, further comprising the poxvirus host range gene SPI-1, preferably further comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9.

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