Downregulation of yield-reducing transgenes expressed by poxviruses

Abstract

The present invention relates to a recombinant poxvirus having a binding sequence for a transcriptional repressor protein, such as a tetracycline repressor, located between the poxvirus promoter and the open reading frame (ORF) of a yield-reducing transgenic gene, and to a transformed poxvirus producer cell expressing a corresponding repressor protein.

Description

Technology Field

[0001] The present invention relates to the field of viral vectors, particularly to viral vector-based vaccines. More specifically, the present invention relates to a recombinant poxvirus comprising a binding sequence of a transcriptional repressor, in particular a tetracycline repressor (TetR) family, most preferably a modified vaccinia virus Ankara (MVA), and to a transformed cell expressing a corresponding repressor. The present invention also relates to producing said recombinant poxvirus using the transformed cell as a poxvirus producer cell. Background Technology

[0002] A common problem associated with recombinant viral vectors for vaccine or gene therapy purposes is that the transgenic products expressed by these vectors can have a negative effect on the cellular processes of the vector-producing cells. This can ultimately lead to a decrease in the yield of a given recombinant viral vector (1). The yield reduction effect may be the result of the expression of a single or multiple transgenics, or the result of a combination of transgenic products that do not exhibit a yield reduction effect when expressed separately. The problem of yield-reducing transgenic products that reduce viral vector yield also involves poxviruses ( Poxviridae Orthofoxvirus genus belonging to ) Orthopoxvirus The vaccinia virus, the protozoan species of ) vaccinia virus It is related to vectors based on the Modified Vaccinia Virus Ankara (MVA), a replication-restricted variant of Vaccinia Virus Ankara derived from VACV. Attenuated replication has been observed, for example, in the HIV envelope gene expression MVA (2).

[0003] MVA-BN ®MVA is a well-characterized viral vector isolated from a modified vaccinia virus Ankara (MVA) virus stock. MVA is derived from the skin-derived VACV Ankara strain [Chorioallantois vaccinia virus Ankara (CVA)], which is a replicable vaccinia virus (3). Attenuated CVA-derived MVA was obtained through serial proliferation of CVA over more than 570 passages in primary chicken embryonic fibroblasts (CEF or CEF cells). This MVA was further passaged by Bavarian Nordic to form a more attenuated MVA strain, MVA-BN. ® MVA-BN was generated (4). ® It lacks approximately 15% of its genome compared to the progenitor CVA virus (a loss of 31 kb results in six major deletion sites). These deletions affect numerous virulence and host range genes, as well as type A inclusion genes. MVA-BN ® It can attach to and penetrate human cells, and highly efficiently express virus-encoded genes in infected human cells. However, the assembly and release of progeny viruses do not occur in human cells. Therefore, MVA-BN ® MVA-BN is an important versatile vaccine vector capable of efficiently expressing antigen-coding transgenes for use in vaccination approaches targeting diseases with unmet medical needs [e.g., Ebola virus disease (5)]. ® Preparations of and derivatives were administered to various types of animals, including immunodeficient individuals, and to over 10,500 human subjects in clinical studies, and no serious adverse events occurred.

[0004] MVA-BN ®Common producer cells for CEF-adapted MVA include primary CEF cells or several avian cell lines, including the serial chicken fibroblast DF-1. Mammalian BHK-21 cells (a clone derived from baby hamster kidney cells) are also acceptable, but produce MVA at a yield up to 10 times lower than CEF (15–17).

[0005] In algal producer cells, the reduction in viral yield of some MVA recombinants expressing yield-reducing transgenes can vary widely and can range from a significant decrease in viral yield to severe replication degradation, or even failure to produce recombinant MVAs expressing specific transgenes. Recombinant MVA replication degradation can be caused not only by a single transgene that significantly reduces yield, but also by a combination of multiple transgenes that may not appear to individually reduce yield during MVA-mediated expression. However, these minimal yield-reducing effects appear to aggregate or even synergize to significantly reduce MVA yield. Failure to produce recombinant MVAs containing specific transgenes is due to the selective disadvantage that the harmful transgene imposes on the replication process of the MVA recombinant, making replication so inefficient that it is impossible to successfully select and isolate them from the parental MVA background. Additionally, the genetic stability of the transgene insert or the genome of the viral vector expressing this transgene can be compromised by the expression of the harmful transgene during viral vector production (1, 2).

[0006] Therefore, an improved process is required to produce recombinant MVA containing the yield-reducing transgene.

[0007] The object of the present invention is to provide means and methods for producing a recombinant poxvirus containing a yield-reducing transgene at an increased virus yield, most preferably a recombinant modified vaccinia virus Ankara (MVA).

[0008] The fundamental problem of the present invention is solved by providing (1) a recombinant poxvirus having a binding sequence for a transcription repressor protein inserted between a yield-reducing transgenic gene and its promoter, most preferably a recombinant MVA, and (2) a transformed poxvirus producer cell expressing a transcription repressor protein, most preferably an MVA producer cell. When the recombinant poxvirus is proliferated in the transformed poxvirus producer cell, the expression of the transgenic gene is downregulated as a result of increased viral yield.

[0009] In particular, the present invention is defined by the appended claims and the following embodiments and examples thereof.

[0010] In one embodiment, the present invention provides a recombinant poxvirus, most preferably a recombinant MVA, comprising a nucleotide sequence comprising a viral yield reduction transgenic gene operably linked to a poxvirus promoter, wherein the nucleotide sequence further comprises a binding sequence to a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame (ORF) of the transgenic gene.

[0011] In another aspect, the present invention provides a transcription unit comprising a nucleotide sequence comprising a viral yield reduction transgenic gene connected to a poxvirus promoter in an operable state, wherein the nucleotide sequence further comprises a binding sequence to a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame [ORF] of the transgenic gene.

[0012] In an additional aspect, the present invention relates to a method for producing a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, wherein

[0013] (1) A step of providing a transcription unit comprising a nucleotide sequence comprising a viral yield reduction transgenic gene linked to a poxvirus promoter in an operable state, wherein the nucleotide sequence further comprises a binding sequence to a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame [ORF] of the transgenic gene,

[0014] (2) A step of inserting the transcription unit prepared in step (1) into the poxvirus genome, most preferably the MVA genome,

[0015] (3) A method is provided that includes the step of obtaining a recombinant poxvirus, most preferably a recombinant MVA.

[0016] In another aspect, the present invention provides a recombinant poxvirus produced by the method according to the present invention, most preferably a recombinant MVA.

[0017] In another aspect, the present invention provides a transgenic poxvirus producer cell that is genetically modified to express a transcription repressor protein, most preferably a transgenic MVA producer cell.

[0018] In another aspect, the present invention relates to a method for producing transformed poxvirus producer cells according to the present invention, most preferably transformed MVA producer cells, wherein

[0019] (1) A step of providing a poxvirus-allowing cell, most preferably an MVA-allowing cell,

[0020] (2) A step of providing a plasmid encoding a transcription repressor protein,

[0021] As long as step (3) can be achieved, step (2) can also occur prior to step (1),

[0022] (3) Step of transfecting the poxvirus-eating cells of Step (1), most preferably MVA-eating cells, with the plasmid of Step (2),

[0023] (4) A method is provided that includes the step of obtaining a transformed poxvirus producer cell, most preferably a transformed MVA producer cell.

[0024] In another aspect, the present invention provides a transformed poxvirus producer cell produced by the method according to the present invention, most preferably a transformed MVA producer cell.

[0025] In another aspect, the present invention provides a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, a transformed poxvirus producer cell according to the present invention for proliferating the recombinant MVA, most preferably a transformed MVA producer cell, preferably a use in the production of a poxvirus-based vaccine, most preferably an MVA-based vaccine.

[0026] In another aspect, the present invention provides a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, proliferated using a transformed poxvirus producer cell according to the present invention, most preferably a transformed MVA producer cell.

[0027] In another additional aspect, the present invention provides a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, and a transformed poxvirus producer cell according to the present invention, most preferably a transformed MVA producer cell, wherein a binding sequence for a transcription repressor protein contained in the recombinant poxvirus, most preferably a recombinant MVA, binds to a transcription repressor protein expressed by the transformed poxvirus producer cell, most preferably a transformed MVA producer cell, and / or conversely, that is, a transcription repressor protein expressed by the transformed poxvirus producer cell, most preferably a transformed MVA producer cell, may bind to a binding sequence for a transcription repressor protein contained in the recombinant poxvirus, most preferably a recombinant MVA.

[0028] In another additional aspect, the present invention provides a use for a recombinant poxvirus, most preferably a recombinant MVA according to the present invention, and a transformed poxvirus producer cell, most preferably a transformed MVA producer cell according to the present invention, for the purpose of proliferating a recombinant poxvirus, most preferably a recombinant MVA, in the production of a poxvirus-based vaccine, most preferably an MVA-based vaccine, wherein a binding sequence for a transcription repressor protein contained in the recombinant poxvirus, most preferably a recombinant MVA, binds to a transcription repressor protein expressed by the transformed poxvirus producer cell, most preferably a transformed MVA producer cell, and / or conversely, that is, a transcription repressor protein expressed by the transformed poxvirus producer cell, most preferably a transformed MVA producer cell, can bind to a binding sequence for a transcription repressor protein contained in the recombinant poxvirus, most preferably a recombinant MVA.

[0029] In another additional aspect, the present invention relates to a method for amplifying recombinant poxvirus, most preferably recombinant MVA, wherein

[0030] (1) A step of providing a recombinant poxvirus according to the present invention, most preferably a recombinant MVA,

[0031] (2) A step of providing a transformed cell according to the present invention,

[0032] As long as step (3) can be achieved, step (2) can also occur before step (1), and

[0033] A binding sequence for a transcription repressor protein contained in the recombinant poxvirus provided in step (1), most preferably in the recombinant MVA, can bind to and / or conversely to the transcription repressor protein expressed by the transformed poxvirus producer cell provided in step (2), most preferably in the transformed MVA producer cell, i.e., the transcription repressor protein expressed by the transformed poxvirus producer cell provided in step (2), most preferably in the transformed MVA producer cell, can bind to the binding sequence for a transcription repressor protein contained in the recombinant poxvirus provided in step (1), most preferably in the recombinant MVA.

[0034] (3) A step of infecting the transformed cells provided in step (2) with the recombinant poxvirus provided in step (1), most preferably recombinant MVA,

[0035] (4) A step of culturing the transfected cells of step (3) to proliferate recombinant poxvirus, most preferably recombinant MVA,

[0036] (5) A method is provided that includes the step of harvesting the recombinant poxvirus, most preferably the recombinant MVA, which was proliferated in step (4).

[0037] In another additional aspect, the present invention provides a pharmaceutical composition or vaccine comprising a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, and optionally further comprising a pharmaceutically acceptable carrier or excipient.

[0038] In another additional aspect, the present invention provides a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, for use as a medicine or vaccine, preferably for use in the treatment or prevention of a disease.

[0039] In another additional aspect, the present invention provides a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, for use in the treatment or prevention of infectious diseases or cancer.

[0040] In another additional aspect, the present invention provides a recombinant poxvirus according to the present invention for use in immunotherapy or gene therapy, most preferably a recombinant MVA.

[0041] In another additional aspect, the present invention provides a use of a recombinant poxvirus according to the present invention, most preferably a recombinant MVA, for the manufacture of a medicine or vaccine for use in the treatment or prevention of an infectious disease or cancer, or for the manufacture of a medicine or vaccine for use in immunotherapy or gene therapy.

[0042] In another additional aspect, the present invention provides a method for treating or preventing an infectious disease or cancer of a subject, comprising the step of administering to the subject a recombinant poxvirus according to the present invention, preferably a recombinant MVA.

[0043] In another additional aspect, the present invention provides a method of immunotherapy or gene therapy in a subject, comprising the step of administering to the subject a recombinant poxvirus according to the present invention, preferably a recombinant MVA.

[0044] In another aspect, the present invention provides a method or use of a transcription repressor protein and a corresponding binding sequence for downregulation of transgenic expression induced by a recombinant poxvirus, most preferably a recombinant MVA, primarily or completely late or mid-phase poxvirus promoter.

[0045] These aspects and embodiments thereof will be described in more detail in connection with the description of the present invention. Brief explanation of the drawing

[0046] Fig. 1 This represents the design of a transgene insert that encodes an EGFP or EBV-derived LMP1 / EBNA2 fusion protein and includes a Tet repressor (TetR) binding sequence 2xTetO2 between the promoter and the ORF of the transgene. Recombinant MVAs without the 2xTetO2 sequence (MVA-mBNbc440, MVA-res034, MVA-res057), those with the 2xTetO2 sequence linked to an EGFP transgene (MVA-res005), or those with the 2xTetO2 sequence linked to an LMP1 / EBNA2 transgene, under the control of a PrH5m promoter (MVA-res035) or a PrS promoter (MVA-res058). EGFP = Enhanced green fluorescent protein; 2xTetO2 = Two serial binding sites of version 2 of the Tet operon (=TetR binding) sequence; LMP1 / EBNA2 = Fusion protein of Epstein-Barr virus (EBV) latent protein 1 and EBV-derived EBV nuclear antigen 2; npt II = Neomycin phosphotransferase II; gpt=xanthine-guanine phospholiposyltransferase; mRFP=monomer red fluorescent protein; PrS=synthetic early / late (mostly late) promoter; PrH5m=early / late (mostly early) promoter; IRES=internal ribosomal entry site; IGR=intergenetic region. Number of MVA genes according to MVA genome annotation in the literature [Antoine et al., 1998(18)]. Fig. 2 This shows that the 2xTetO2 sequence inserted between the poxvirus promoter and the transposable gene ORF does not affect transposable gene expression by MVA. Cells specified as not expressing TetR (CCX.2C4, CEF, DF-1, HeLa) were seeded in 6-well plates the day before infection and infected with recombinant MVA-res005 or MVA-mBNbc440 with a multiplicity of infection (MOI) of 5 on day 0. Cells were harvested at 5 and 20 hours post-infection (pi) by accutase treatment, and EGFP fluorescence, indicating EGFP expression, was analyzed by flow cytometry. EGFP levels were determined by the geometric mean fluorescence intensity (GMFI) of EGFP-positive cells among mRFP-positive (i.e., infected) cells. Fig. 3 represents TetR expression in transformed DF-1 cell clones determined by immunoblot. Immunoblot results are presented for TetR expression by DF-1 clones #59 to #72 (clone #60 is shown in a separate blot). DF-1 cells were either transfected with 0.2 μg of a TetR-expressing plasmid ('wt DF-1 TetR transf') or untransfected ('wt DF-1'). DF-1 clones were selected for blasticidin resistance. Cell lysates of clones #59 to #72 were analyzed using anti-TetR antibodies. 'tetR' = 23 kDa. A band of approximately 21 kDa is a background band ('bkg') that is detectable in untreated DF-1 cells but undetectable after transient plasmid transfection. M = Marker. (*) A clone with only about 50–60% confluence at harvest. Fig. 4 This indicates TetR-mediated inhibition of PrS-induced EGFP expression by MVA-res005. DF1-TR59 cells were seeded in 6-well plates the day before infection, and from day (-1) throughout the experiment, they were treated with or not treated with doxycycline ('DOX') to enable EGFP expression (+DOX) or inhibit EGFP expression ('DOX untreated'). DF1-TR59 cells were infected with MVA-res005 at MOI 5 on day 0. Cells were harvested at the indicated time points after infection by accutase treatment, followed by fixation and permeation. MVA-res005-infected cells were analyzed for EGFP fluorescence by flow cytometry. Fig. 5 It shows that LMP1 / EBNA2 reduces the replication of MVA-res035 encoding LMP1 / EBNA2, which controls PrH5m-induced TetR in DF-1 cells that do not express TetR. Non-transformed DF-1 cells were seeded into 6-well plates the day before infection, and starting from day (-1), TetR was activated ('DOX-free') or inactivated ('+DOX') throughout the experiment by treating with or not treating with doxycycline ('DOX-free'). On day 0, at an MOI of 0.05, cells were infected three times per condition with BAC-derived reference MVA (MVA-mBNbc166; referred to as 'MVA-WT' in the figure) and MVA-res035. Cells were harvested on day 3 of pi, and lysates were inoculated into CEF cells using standard TCID 50 Titration was performed using the method. One titration was performed per well, and each bar represented the results of three biological replications. The total average yield of 2 ml of lysate per well is indicated. Fig. 6 ...represents the TetR-controlled PrH5m-induced expression of LMP1 / EBNA2 by MVA-res035. DF1-TR59 cells were seeded into 6-well plates the day before infection, and starting from day (-1), throughout the experiment, they were treated with doxycycline ('DOX') or not treated ('DOX untreated') to enable LMP1 / EBNA2 expression (+DOX) or inhibit LMP1 / EBNA2 expression (DOX untreated). DF1-TR59 cells were infected with MVA-res035 at MOI 5 on day 0. Cells were harvested at the time indicated on pi by accutase treatment, followed by fixation and permeation. MVA-res035-infected cells were stained with an EBNA2-specific antibody and cultured with an APC-coupled secondary antibody. Cells were analyzed using flow cytometry ( a ) Allophycocyanin (APC) fluorescence and ( b EGFP fluorescence was analyzed. The GMFI of APC is plotted as the GMFI of LMP1 / EBNA2. Fig. 7 ...represents the effect of TetR-controlled PrH5m-induced expression of LMP1 / EBNA2 on MVA-res035 replication. DF1-TR59 cells were seeded into 6-well plates the day before infection, and starting from day (-1), throughout the experiment, they were treated with doxycycline ('DOX') or not treated ('DOX-free') to enable LMP1 / EBNA2 expression (+DOX) or inhibit LMP1 / EBNA2 expression (no DOX). On day 0, at an MOI of 0.05, DF1-TR59 cells were infected three times per condition with either BAC-derived reference MVA (MVA-mBNbc166; referred to as 'MVA-WT' in the figure) or MVA-res035. Cells were harvested at the indicated time in pi, and lysates were placed on CEF cells at standard TCID 50 Titration was performed using the method. One titration was performed per well, and each data point represented three replicate results. The total average yield of 2 ml of lysate per well is displayed along with the inoculum titer for 0 days. Fig. 8It represents TetR-mediated regulation of early and late expression of EGFP in PrS early / late promoters. DF1-TR59 cells were seeded in 6-well plates the day before infection, and from day (-1) throughout the experiment, they were treated with doxycycline ('DOX') or not treated ('DOX untreated') to enable EGFP expression (+DOX) or inhibit EGFP expression induced by MVA-res005 ('DOX untreated'). DF1-TR59 cells were infected with MVA-res005 on day 0 at an MOI of 10. A subset of infected cell cultures were treated with 40 µg / ml cytosine arabinoside from 1 hour before the start of infection until the end of the infection period as indicated ('AraC treated', b , d ), at the same time, the infected cell cultures were kept untreated ('AraC untreated', a , c ). Cells were harvested at the indicated time points of pi by accutase treatment, then fixed and permeated. MVA-res005 infected cells were analyzed by flow cytometry using EGFP( a , b ) and mRFP( c , d Fluorescence was analyzed. The mRFP signal originated from MVA-encoded mRFP acting as a reference transgene. EGFP and mRFP expression levels are represented as GMFI of all cells within the cell gate ('living cell gate'), excluding fragments. Fig. 9 This represents the TetR-controlled PrS-induced expression of LMP1 / EBNA2 by MVA-res058. DF1-TR59 cells were seeded into 6-well plates the day before infection, and starting from day (-1), throughout the experiment, they were treated with doxycycline ('DOX') or not treated ('DOX untreated') to enable LMP1 / EBNA2 expression (+DOX) or inhibit LMP1 / EBNA2 expression (DOX untreated). DF1-TR59 cells were infected with MVA-res058 at MOI 5 on day 0. Cells were harvested at the indicated time points on pi by accutase treatment, followed by fixation and permeation. MVA-res058-infected cells were stained with an EBNA2-specific antibody and cultured with an APC-coupled secondary antibody. Cells were analyzed by flow cytometry using APC ( a ) and EGFP( b Fluorescence was analyzed. EGFP and EBNA2 / LMP1 expression levels are represented as GMFI of all cells in a living cell gate. The GMFI of APC is plotted as the GMFI of LMP1 / EBNA2. Fig. 10 ...represents the effect of TetR-controlled PrS-induced expression of LMP1 / EBNA2 on MVA-res058 replication. DF1-TR59 cells were seeded into 6-well plates the day before infection and, starting from day (-1), were treated with doxycycline ('DOX treatment') or not treated ('DOX non-treatment') throughout the experiment to enable LMP1 / EBNA2 expression (+DOX) or inhibit LMP1 / EBNA2 expression (DOX non-treatment). On day 0, at an MOI of 0.05, DF1-TR59 cells were infected three times per condition with either BAC-derived reference MVA (MVA-mBNbc166; referred to as 'MVA-WT' in the figure) or MVA-res058. Cells were harvested at the indicated time in pi, and the lysates were prepared using standard TCIDs with DF1-TR59 cells as the substrate. 50Titration was performed using the method. One titration was performed per well, and each data point represented three replicate results. The average total yield of 2 ml of lysate per well is displayed along with the inoculum titer at day 0. Background levels stained with anti-EBNA2 antibody were determined using cells infected with BAC-derived reference MVA, and the background LMP1 / EBNA2 signal in the live cell gate was subtracted from all GMFI values ​​for LMP1 / EBNA2 displayed. Fig. 11 This shows TetR expression by quail CCX.2C4-TR16 cell clones compared to sister clones. ( a CCX.2C4 clone TR16 cells (CCX.2C4-TR16) stably transfected with a TetR expression plasmid were maintained under 2 µg / ml blasticidin selection. Cells from the maternal CCX.2C4 wild-type ('2C4 wt') cell line and various clones were collected on the same day, lysed in Lamley buffer, and aliquots of cell lysates were immunoblotted using a TetR-specific antibody (TetR MW=23 kDa). Lysates from TetR-expressing DF1-TR59 cells were used as a reference. The upper portion of the same blot (molecule mass of protein greater than approximately 45 kDa) was developed with an anti-β-tubulin antibody (β-tubulin MW=55 kDa). It shows the β-tubulin-specific signal ('β-tubulin') used for normalization of sample loading, the TetR-specific signal ('tetR'), and the background signal ('bkg') revealed in cell lysates using anti-TetR antibody ( bStably transfected CCX.2C4-TR16 quail cells expressing TetR were infected with MVA-BN ('MVA') at an MOI of 5 or mock-infected for the indicated time. Cell lysates were analyzed for TetR and β-tubulin expression levels by semi-quantitative immunoblotting as described in (a) above. TetR and β-tubulin-specific signals were quantified using the ChemiDoc Touch system and ImageQuant software, and TetR expression levels are expressed in arbitrary units. The β-tubulin-specific signal was used to normalize the TetR signal values ​​shown in (b). Fig. 12 This represents the TetR-controlled PrS-induced expression of LMP1 / EBNA2 by MVA-res058 in CCX.2C4-TR16 cells. TetR-expressing CCX.2C4-TR16 cells were seeded into 12-well plates the day before infection, and starting from day (-1), throughout the experiment, they were treated with doxycycline ('DOX') or not treated ('DOX untreated') to allow LMP1 / EBNA2 expression (+DOX) or inhibit LMP1 / EBNA2 expression (DOX untreated). CCX.2C4-TR16 cells were seeded in MVA-res005( a ), MVA-res035( b ) or MVA-res058( cCells were doubly infected with () and infected with reference MVA (MVA-mBNbc166) in single wells on Day 0 at MOI 10. Cells were harvested at the indicated time points of pi by accutase treatment, followed by fixation and permeation. MVA-res035 or MVA-res058 infected cells were stained with an EBNA2-specific antibody and then inoculated with an APC-coupled secondary antibody. Cells were analyzed for APC and EGFP fluorescence by flow cytometry. EGFP, mRFP, and LMP1 / EBNA2 expression levels are represented as the GMFI of all cells in the live cell gate. For APC levels, background staining observed with the anti-LMP1 / EBNA2 antibody in cells infected with the reference MVA was subtracted from the GMFI of all MVA-res035 or MVA-res058 infected cells. The GMFI of APC is plotted as the GMFI of LMP1 / EBNA2. Fig. 13 ...represents the effect of TetR-controlled PrS-induced expression of LMP1 / EBNA2 on MVA-res058 replication in CCX.2C4-TR16 cells. TetR-expressing CCX.2C4-TR16 cells were seeded in 6-well plates the day before infection and, starting from day (-1), were treated with doxycycline ('DOX treatment') or not treated ('DOX non-treatment') throughout the experiment to enable LMP1 / EBNA2 expression (+DOX) or inhibit LMP1 / EBNA2 expression (DOX non-treatment). On day 0, at an MOI of 0.05, CCX.2C4-TR16 cells were infected three times per condition with either BAC-derived reference MVA (MVA-mBNbc166; referred to as 'MVA-WT' in the figure) or MVA-res058. Cells were harvested at the indicated times, and the lysates were prepared using standard TCIDs substrated with DF1-TR59 cells (also expressing TetR). 50Titration was performed using the method. One titration was performed per well, and each data point represented three replicate results. The total yield of 2 ml of lysate per well is displayed along with the inoculum titer at day 0. A brief explanation of the sequence Sequence No. 1 nucleic acid sequence of TetO2 Sequence No. 2 nucleic acid sequence of 2xTetO2 Sequence No. 3 Nucleic acid sequence of the TetR gene Sequence No. 4 Amino acid sequence encoded by the TetR gene Sequence No. 5 Nucleic acid sequence encoding the LMP1 / EBNA2 fusion protein Sequence number 6 Amino acid sequence of LMP1 / EBNA2 fusion protein Sequence No. 7 Nucleic acid sequence of the EGFP gene Sequence No. 8 Amino acid sequence encoded by the EGFP gene Sequence number 9 Nucleic acid sequence encoding the gpt-mRFP fusion protein Sequence number 10 nucleic acid encoding npt II Sequence number 11 PrS Promoter Sequence No. 12 PrH5m promoter Specific details for implementing the invention

[0047] The objective of the present invention was to improve the productivity of recombinant MVA containing a yield-reducing transfer gene.

[0048] To produce high-titer viral stocks of MVA constructs containing yield-reducing transgenes, it would be advantageous to downregulate the expression of these proteins during viral production. The inventors aimed to evaluate whether a circular tetracycline repressor (TetR), a protein of the tetracycline (Tet) repressor family (6), could be used to downregulate transgene expression during recombinant MVA production when expressed in MVA-producing cells. Although the TetR system is often used for the repression of nuclear transcription, TetR does not have a eukaryotic nuclear localization signal (NLS) and has a very high affinity for its DNA recognition sequence [tetracycline operon or operator (TetO)], so it must be available in the cytoplasm of eukaryotic cells in sufficient quantities to regulate MVA or other vaccinia viruses that replicate exclusively in the cytoplasm.

[0049] TetR functions as a homomer that binds to a short palindromic sequence of DNA, also known as the Tet operon sequence or TetR binding sequence (6-8). If this sequence is positioned between the promoter and the gene's open reading frame (ORF), the transcription of the gene will be blocked when TetR binds to this sequence. In this case, the addition of an 'inducer,' such as the antibiotic tetracycline or its more active derivative doxycycline (DOX), will lead to the binding of the inducer to the repressor, causing an allosteric reaction of the repressor. The three-dimensional structure of the repressor will change, causing a rapid decrease in affinity for the target sequence in DNA. Consequently, TetR will be released from DNA, and the transcription of the regulated gene can occur.

[0050] Original TetR technology, particularly fusion proteins of modified TetR derivatives and herpesvirus transcriptional activator proteins ('tet-on / tet-off' system), has been widely used to regulate gene expression in experimental systems and biotechnological applications. Conditional knockout mutants of replicated vaccinia virus strains were generated for the functional characterization of essential poxvirus genes by regulating natural VACV gene expression using not only TetR but also functionally similar lac repressors (9, 10). In most cases, recombinant or mutant vaccinia viruses themselves were used for TetR expression, which is undesirable for vaccine vectors intended for use in humans. Commercial human cell line T-REx with Tet operon sequence inserted into essential vaccinia virus genes TM -293 and T-REx TM - A system based on TetR expression by HeLa has been described as being able to regulate these essential vaccinia virus genes (11). TetR has also been used to reduce transgenic expression by adenovirus and vaccinia virus-based vectors. In the case of adenovirus vectors, the goal was to improve the genetic stability of adenovirus vectors expressing specific transgenic genes, and this study found that TetR-mediated inhibition of transgenic genes increases the yield of adenovirus vectors and promotes the production of recombinant adenoviruses with rapidly mutated transgenic genes without TetR regulation (1).

[0051] To regulate the expression of transgenic genes in recombinant vaccinia viruses, an indirect approach using a lac repressor system for regulating VACV-induced transgenic expression has been described, wherein the bacteriophage T7 RNA polymerase encoded by recombinant VACV induces the transcription of the T7 promoter-controlled transgenic gene in the same recombinant VACV (12, 13). However, this system requires the expression of T7 polymerase as well as the lac repressor in the recombinant VACV or MVA in addition to the transgenic gene expressed as a vaccine antigen or gene therapy protein. Furthermore, only early promoter-induced expression is supported in the described system (12, 13), and thus omits the potent late components of many poxvirus promoters used for transgenic expression, which produce a large amount of transgenic products advantageous as vaccine antigens or gene therapy proteins. The effect of cell expression TetR on late expression was observed on the expression of neutral transgenic genes (luciferase) by recombinant vaccinia virus in TetR expressing human HeLa cells (14).

[0052] Initial poxvirus gene expression is detectable within 15 to 30 minutes after VACV or MVA enters the producer cell and occurs prior to the replication of viral genomic DNA. In the case of VACV and MVA, initial expression can continue into the metaphase and late phases of gene expression, which begin with viral DNA replication at approximately 2 hours pi. Therefore, metaphase and late genes are also referred to as post-replication genes. Metaphase expression begins with DNA replication at 2 hours pi, followed by late expression as soon as the first late transcription factor encoded by metaphase genes is synthesized, whereas the metaphase transcription factor is encoded by the initial genes. Thus, the expression of each gene class at a specific timeframe depends on the synthesis of the gene class of the preceding timeframe; the initial transcription factor is encoded by the late genes, packaged into a virion, and initiates a new replication cycle upon viral entry into the cell. Late gene expression can persist throughout the fully late phase of the MVA infection cycle, from approximately 2 hours pi, when infected cells begin to die, to approximately 24 hours pi, and late genes are often expressed in high abundance. Most of the structural proteins of poxviruses required in large quantities for viral morphogenesis are encoded by late genes. Early, mid, or late expression of poxvirus genes is determined by poxvirus promoters, which exist in three corresponding types distinguished by the presence of specific sequence motifs that induce one of three distinct time-phase classes of poxvirus genes. Expression of a fourth class of genes, called the outpost, begins very early after infection, but since its expression is not a prerequisite for the expression of any other early poxvirus genes, these genes are defined as outposts based solely on the kinetics of their expression over time.

[0053] The expression of transgenic genes in poxvirus vectors depends on the poxvirus promoter belonging to the prodromal, early, meta, or late classes. Since poxviruses encode their own transcription machinery in the cytoplasm of infected cells, they cannot use cellular promoters that induce RNA polymerase II-mediated gene expression. The poxvirus promoters used to induce transgenic gene expression are often selected from a combined promoter class that initiates expression in the early as well as late stages of viral infection. The synthetic PrS promoter (24), designed to induce potent transgenic gene expression, is a typical example of this and is widely used in poxvirus vectors. Despite the presence of an optimized early promoter motif in the PrS promoter, transgenic gene expression under the control of this promoter occurs mainly in the late stages. However, for the best possible induction of the T cell response to the transgenic gene product, early, more preferably even prodromal expression of the transgenic gene is advantageous (28-30), and the early element must also be included. Therefore, the PrS promoter is a desirable promoter when both overall high expression and early expression initiation are required for superior T cell induction. Although the PrH5m promoter is generally considered to be an early / late promoter, when the replication of recombinant MVA expressing a transposable gene under the PrH5m promoter is stopped at the early stage by araC treatment, only a very small portion of transposable gene expression is blocked, indicating that PrH5m is primarily an early promoter with few late components.

[0054] Based on this, the approach to solving the fundamental problem of the present invention was to suppress yield-reducing transgenic expression during the proliferation of recombinant MVA, while simultaneously preserving the potential of recombinant MVA to induce a transgenic-specific immune response in vaccinated individuals.

[0055] To this end, a bacterial gene regulation system consisting of a transcriptional repressor protein, in this case the circular tetracycline repressor (TetR), and a TetR binding sequence (also called a tetracycline operon) placed between the promoter and the open reading frame [ORF] of the transposable gene to be downregulated was used.

[0056] Briefly, the present invention provides a recombinant MVA comprising (1) a TetR binding sequence inserted between a yield-reducing transgenic gene and its promoter, and (2) a transformed MVA producer cell expressing the corresponding TetR. When the recombinant MVA is grown on the MVA producer cell, TetR binds to its binding sequence and inhibits the expression of the yield-reducing transgenic gene. In contrast, when the recombinant MVA is administered to a vaccinated person, it does not affect transgenic gene expression because the TetR protein is absent in the recipient's body cells.

[0057] Here, it was demonstrated that both the transgenic chicken DF-1 and transgenic quail CCX.2C4 cell lines express a sufficient amount of TetR to efficiently downregulate the MVA expression transgenic gene bound to the TetR binding sequence.

[0058] In particular, TetR was effective in downregulating EGFP transgene expression induced by the late portion of the strong early / late PrS promoter, while expression in the early portion of the PrS promoter was not affected at all.

[0059] Consistent with the aforementioned findings, the expression of the potent yield-reducing transgenic gene encoding the LMP1 / EBNA2 fusion protein, primarily under the early PrH5m promoter, was transiently and moderately downregulated in TetR-expressing DF-1 cells, and only a transient increase in viral titer was achieved during the growth curve experiment.

[0060] Importantly, the expression of the LMP1 / EBNA2 transgene, which strongly reduces yield primarily in late-expressing PrS promoters, was significantly downregulated in TetR-expressing DF-1 and CCX.2C4 cells. Consequently, the insertion of the TetR binding sequence significantly increased the replication disorder of recombinant MVAs expressing the LMP1 / EBNA2 fusion protein, so that the replication efficiency of recombinant MVAs containing the LMP1 / EBNA2 transgene linked to the TetR binding sequence was equal to that of non-recombinant wild-type MVAs in TetR-expressing DF-1 and CCX.2C4 cells.

[0061] In addition, it was also proven that downregulating LMP1 / EBNA2 transgenes in TetR-expressing cells to a level greater than 50% of the doxycycline induction dose is unexpectedly effective in compensating for the negative effects of late-expressed yield-reducing transgenes on viral replication and increases viral yield by at least 10-fold, reaching a yield similar to that of wild-type MVA without any transgenes.

[0062] In conclusion, linking the binding sequence to TetR for the transgenic gene expressed by recombinant MVA was demonstrated to efficiently downregulate the expression of the transgenic gene in MVA-producing cells modified to express the corresponding TetR. Considering the reasons why the downregulation of the transgenic gene LMP1 / EBNA2 expression, which significantly reduces yield, was important but incomplete, and the very high activity of the poxvirus promoter used to obtain large amounts of transgenic gene products in vaccinated individuals, the yield of recombinant MVA containing the TetR-controlled LMP1 / EBNA2 transgenic gene stood out as a replication close to wild-type MVA with minimal degradation.

[0063] definition

[0064] It should be noted that singular nouns and the definite article 'the' as used herein include multiple referents unless the context clearly indicates otherwise. Therefore, for example, a reference to 'nucleic acid sequence' includes one or more nucleic acid sequences.

[0065] The connecting term 'and / or' between a plurality of described components as used herein is understood to include both individual options and combination options. For example, when two components are combined by 'and / or', the first option means the availability of the first component without the second component. The second option means the availability of the second component without the first component. The third option means the availability of the first component and the second component together. Any one of these options falls within that meaning and should be understood as satisfying the requirements of the term 'and / or' as used herein. The simultaneous availability of more than one of the options also falls within that meaning and should be understood as satisfying the requirements of the term 'and / or'.

[0066] Throughout this specification and the appended claims, unless otherwise required by the context, variations such as 'comprising', 'comprising', and 'including' will be understood to mean the inclusion of the mentioned characteristics but without the exclusion of any other characteristics. When used in the context of aspects or embodiments in the description of the invention, the term 'comprising' may be modified and thus replaced by the terms 'containing' or 'including', or, as used herein, replaced by the term 'having'. Similarly, whenever used herein in the context of aspects or embodiments in the description of the invention, any of the aforementioned terms (comprising, containing, including, having) include the term 'consisting of' or 'essentially consisting of', each indicating a specific legal meaning depending on the jurisdiction.

[0067] As used herein, “consisting of” excludes any feature, component, step, or component not mentioned in the claim. As used herein, “consisting of essentially” does not exclude a feature, material, or step that does not substantially affect the fundamental and novel characteristics of the claim.

[0068] The term 'poxvirus' refers to the family Poxviridae, which includes the two subfamilies of Chordopoxvirinae and Entomopoxvirinae. Viruses belonging to the Chordopoxvirinae infect vertebrates. Viruses belonging to the Entomopoxvirinae infect insects (i.e., invertebrates). The term 'poxvirus' also refers to members of any genus of the Chordopoxvirinae (e.g., avifoxvirus, caprifoxvirus, repolifoxvirus, molucifoxvirus, orthofoxvirus, parafoxvirus, sufoxvirus, and yatafoxvirus), including four of these that can infect humans (orthofoxvirus, parafoxvirus, yatafoxvirus, and molucifoxvirus). The term 'foxvirus' also refers to members of any genus of the family Entomofoxividae (e.g., alpha-entomofoxvirus, beta-entomofoxvirus, and gamma-entomofoxvirus). Abifoxviruses include canaryfoxvirus, fowlpoxvirus, mynafoxvirus, pigeonfoxvirus, and quailfoxvirus. Caprifoxviruses include sheepfoxvirus, goatfoxvirus, and lumpy skin virus. Repolifoxviruses include myxomavirus, Shope's fibromavirus (also known as rabbit fibromavirus), hare fibromavirus, and squirrel fibromavirus. Molucifoxviruses include infectious molluscum contagiosum virus. Orthofoxviruses include buffalofoxvirus, camelfoxvirus, cowpox virus, electromeliavirus, monkeyfoxvirus, raccoonfoxvirus, smallpox virus (also known as variola virus), and vaccinia virus. Parapox viruses include bovine papulosopathitis virus, ORF virus, New Zealand red deer parapox virus, and pseudopox virus.Suifox viruses include porcine smallpox virus. Yatafox viruses include tanafox virus and Yaba monkey tumor virus.

[0069] The term ‘vaccinia virus’ refers to both wild-type vaccinia virus and various subsequently isolated attenuated strains or isolates, such as vaccinia virus-Western Reserve, vaccinia virus Copenhagen, Dryvax (also known as vaccinia virus-Wyeth), ACAM2000, vaccinia virus Ankara (CVA), Modified Vaccinia Virus Ankara (MVA), and any of Bavaria Nordic’s MVA (MVA-BN®).

[0070] The term "recombinant poxvirus" as used herein refers to a poxvirus containing a nucleic acid sequence inserted into its genome that does not naturally exist in wild-type viruses (i.e., alien or heterozygous to poxviruses). Accordingly, a recombinant poxvirus refers to a poxvirus created by the artificial combination of two or more segments of nucleic acid sequences of synthetic or semi-synthetic origin linked to other nucleic acids in an arrangement that does not occur in nature or is not found in nature. A "recombinant poxvirus" is a genetically engineered or genetically modified poxvirus.

[0071] The term 'recombinant MVA' as used herein includes recombinant MVAs (e.g., derived from MVA-BN®) incorporated into the genome in the form of at least one recombinant nucleic acid, preferably a transcription unit. Recombinant MVAs may express heterologous polypeptides or proteins (antigens) upon induction of regulatory elements, e.g., promoters.

[0072] As used herein, the term 'transcription unit' basically comprises at least the coding sequence of the gene of interest, e.g., a transposable gene, and additionally a promoter and a termination factor linked thereto in an operable state.

[0073] As used herein with respect to promoters, the term 'operably linked' means that the promoter is positioned at a location capable of directing the transcription of the coding sequence of a gene of interest, for example, a transposable gene.

[0074] The term 'open reading frame' or 'ORF', for example, the ORF of a transgenic gene, refers to a nucleotide sequence located between the start codon and the stop codon.

[0075] As used herein, the term 'transgenic gene' refers to an alien or heterologous gene inserted into the genome of a poxvirus, e.g., MVA, by genetic engineering.

[0076] In contrast to 'viral yield reduction' transgenes, 'neutral' transgenes do not affect or have little effect on the replication of recombinant poxviruses carrying the transgene, e.g., recombinant MVA, and therefore do not reduce or have little effect on the viral yield when recombinant poxviruses, e.g., MVA, are replicated. EGFP is described herein as a circular 'neutral' transgene.

[0077] A 'viral yield reduction' or 'yield reduction' transgene is a transgene whose expression, or rather its expression product, e.g., the transgene protein, negatively affects the viral replication of a recombinant poxvirus, e.g., recombinant MVA, thereby reducing the viral yield. In this document, the LMP1 / EBNA2 fusion protein is described as a yield reduction transgene.

[0078] The term 'viral yield' or simply 'yield' refers to the production yield of recombinant poxvirus, e.g., recombinant MVA, that is, the difference in the amount of virus before and after the time of replication of recombinant poxvirus, e.g., recombinant MVA. As applied herein, viral yield is 50% of the tissue culture infection dose [tissue culture infection dose, TCID 10] per well. 50 It can be determined as ].

[0079] In the context of transgenic expression, the term 'downregulation' refers to a reduction or decrease in the amount of transgenic products. Such reduction or decrease occurs due to a decrease in the amount of transgenic mRNA or a decrease in the translation of transgenic mRNA. Alternatively, 'repression' or 'inhibition' of transgenic expression may be used.

[0080] The terms 'early' and 'late' as used herein to describe the promoter activity and kinetics of transgene expression during the infectious cycle of poxviruses, e.g., MVA, relate to the temporal sequence of gene expression observed for all poxviruses. Although early, meta, and late gene expression overlap, the temporal sequence of replication phases generally follows this pattern: pi 0–2 hours, the early phase; pi approximately 2 hours, the onset of meta and late gene expression; and very short intervals between the meta and late phases. The time between pi 2–4 hours marks the onset of the late phase, whereas pi 20 hours represents the late point during the late phase.

[0081] The PrS promoter is described as an 'early / late' promoter. Since most of the gene expression induced by PrS occurs in the late stage, such a promoter is also referred to as 'primarily late'.

[0082] Although the PrH5m promoter is described as an 'early / late' promoter, it is referred to herein as a 'primarily early' promoter because the major gene expression induced by this promoter occurs early in the infection cycle.

[0083] The term 'Tet operon' or 'Tet operator' refers to the nucleotide binding sequence to the tetracycline repressor (TetR), i.e., the TetR binding sequence.

[0084] The Tet operator or TetR binding sequence referred to as '2xTetO2' consists of two copies of the 19-nucleotide Tet operator 2 (TetO2) sequence separated by two base pair spacers (7, 8), 5'-TCCCTATCAGTGATAGAGA-3'. Thus, the '2xTetO2' sequence contains two copies of TetO2 arranged in series. Each 19-nucleotide TetO2 sequence serves as a binding site for two molecules of TetR.

[0085] When used in the context of a transcription repressor protein or a binding sequence to a transcription repressor protein, the term 'corresponding' means that the repressor protein can bind to its corresponding binding sequence, or that the binding sequence can bind to its corresponding repressor protein. For example, if the binding sequence contains the TetO2 sequence or consists of 2xTetO2, the circular TetR may represent the corresponding repressor protein.

[0086] abbreviation

[0087] araC Cytosine Arabinoside

[0088] CCX.2C4 attached quail CCX cell line, clone 2C4

[0089] CCX.E10 planktonic quail CCX cell line, clone E10

[0090] CEF chicken embryo fibroblasts

[0091] CMV Cytomegalovirus

[0092] DF-1 serial cell line derived from chicken embryonic fibroblasts

[0093] DOX doxycycline, a more stable tetracycline analogue

[0094] EBNA2 Epstein-Barr virus nuclear antigen 2

[0095] EBV Epstein-Barr virus

[0096] EGFP-enhanced green fluorescent protein

[0097] GMFI Geometric Mean Fluorescence Intensity

[0098] gpt xanthine-guanine phosphate ribosyltransferase

[0099] IGR intergene region

[0100] IRES internal ribosome entry site

[0101] LMP1 EBV-derived latent protein 1

[0102] min minutes

[0103] MOI infection multiplicity

[0104] mRFP monomeric red fluorescent protein

[0105] MVA variant vaccinia virus Ankara

[0106] MVA-BN MVA-BN ® (Bavarian Nordic)

[0107] MVA-wt MVA Wild Type

[0108] nptII Neomycin phosphotransferase II

[0109] After pi infection

[0110] ORF Open Decoding Frame

[0111] PrH5m modified early / late promoter, referred to here primarily as the early promoter.

[0112] PrS synthetic vaccinia virus early / late promoter

[0113] SFV (Schauf's Fibroma Virus)

[0114] TCID 50 50% of the tissue culture infection dose

[0115] Tet tetracycline

[0116] TetO2 Tet Operator 2

[0117] Tet repressor binding sequence containing two TetO2 sequences (separated by 2-nucleotide spacers) 2xTetO2

[0118] TetR tetracycline inhibitor

[0119] VACV Vaccinia Virus

[0120] embodiments

[0121] Aspects and embodiments relating to recombinant poxvirus

[0122] In one embodiment, a recombinant poxvirus is provided comprising a nucleotide sequence comprising a transposable gene, preferably a viral yield reduction transposable gene, which is operably linked to a poxvirus promoter, wherein the nucleotide sequence further comprises a binding sequence to a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame [ORF] of the transposable gene.

[0123] In one embodiment, a recombinant poxvirus is provided, comprising a transcription unit comprising a nucleotide sequence comprising a transposable gene, preferably a viral yield reduction transposable gene, which is operably linked to a poxvirus promoter, wherein the nucleotide sequence further comprises a binding sequence for a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame [ORF] of the transposable gene.

[0124] In one embodiment of a recombinant poxvirus, the viral yield reduction transgene is a transgene that reduces the yield of the recombinant poxvirus, preferably the production yield during the replication of the recombinant poxvirus.

[0125] In one embodiment of a recombinant poxvirus, the viral yield reduction transgene is a transgene that reduces the yield of the recombinant poxvirus compared to a poxvirus that does not contain a yield reduction transgene or a recombinant poxvirus.

[0126] In one embodiment of a recombinant poxvirus, the viral yield reduction transgene is a transgene that reduces the replication rate of the recombinant poxvirus, and preferably reduces the replication rate compared to a poxvirus or a recombinant poxvirus that does not contain the yield reduction transgene.

[0127] In one embodiment of a recombinant poxvirus, the viral yield reduction transgene is a transgene that reduces the yield or replication rate of the recombinant poxvirus by expression, and preferably reduces the yield or replication rate compared to a poxvirus or recombinant poxvirus that does not contain the yield reduction transgene.

[0128] In one embodiment of a recombinant poxvirus, the viral yield-reducing transgene is a transgene in which the expression product (e.g., mRNA, protein) reduces the yield or replication rate of the recombinant poxvirus, preferably reducing the yield or replication rate compared to the poxvirus or a recombinant poxvirus that does not contain the yield-reducing transgene.

[0129] In one embodiment of a recombinant poxvirus, a viral yield reduction transgenic gene reduces the yield or replication rate of the recombinant poxvirus by up to about 10%, 30%, 50%, 70%, 80%, 90%, or more than 90%.

[0130] In one embodiment, the recombinant poxvirus is derived from a member of the genus Abifoxvirus, Orthofoxvirus, or Parafoxvirus.

[0131] In one embodiment, a member of the genus Avivoxvirus is selected from the group consisting of canaryfoxvirus, fowlpoxvirus, mynafoxvirus, pigeonfoxvirus, and quailfoxvirus.

[0132] In one embodiment, a member of the genus Parapoxavian is selected from the group consisting of bovine papulostomatitis virus, ORF virus, New Zealand red deer parapoxia virus, and pseudopoxia virus.

[0133] In one embodiment, a member of the genus Orthofoxvirus is selected from the group consisting of Buffalofoxvirus, Camelfoxvirus, cowpox virus, Actromeliavirus, Monkeypox virus, Raccoonfoxvirus, Smallpox virus (Variola virus), or Vaccinia virus (VACV).

[0134] In a preferred embodiment, the recombinant poxvirus is a recombinant vaccinia virus.

[0135] In one embodiment, the recombinant vaccinia virus is derived from a wild-type vaccinia virus or an attenuated vaccinia virus lineage, preferably from vaccinia virus-Western Reserve, vaccinia virus Copenhagen, Dryvax (vaccinia virus Wyeth), ACAM2000, chorioalvicular vaccinia virus Ankara (CVA), and modified vaccinia virus Ankara (MVA).

[0136] In one embodiment, the recombinant poxvirus is derived from an attenuated poxvirus vector selected from the group consisting of ALVAC (canaryfox virus-based vector), NYVAC (vaccinia virus vector) and MVA.

[0137] In a particularly preferred embodiment, the recombinant poxvirus is a recombinant MVA.

[0138] In one embodiment, the recombinant poxvirus is a recombinant MVA, and a nucleotide sequence (i.e., a nucleotide sequence containing a transposable gene linked to a poxvirus promoter in an operable state, preferably a viral yield reduction transposable gene, further comprising a binding sequence for a transcription repressor protein, wherein the binding sequence is located between the poxvirus promoter and the open reading frame [ORF] of the transposable gene) is inserted into the MVA genome in an intergenic region [IGR] selected from the group consisting of IGR 44 / 45, 51 / 52, 64 / 65, 88 / 89 and 148 / 149.

[0139] In a preferred embodiment, the recombinant poxvirus is a recombinant MVA, and the IGR is an IGR 44 / 45 or an IGR 51 / 52, more preferably an IGR 51 / 52.

[0140] In one embodiment, the recombinant poxvirus is propagated using a poxvirus producer cell that expresses a transcription repressor protein capable of binding to a binding sequence (i.e., a binding sequence for a transcription repressor protein contained in the recombinant poxvirus).

[0141] In one embodiment, the recombinant poxvirus is propagated using a poxvirus producer cell that expresses a transcription repressor protein (i.e., a transcription repressor protein containing a sequence that binds to the recombinant poxvirus).

[0142] In one embodiment, the recombinant poxvirus is propagated using a transgenic poxvirus producer cell that has been genetically modified to express a transcription repressor protein (i.e., a transcription repressor protein containing a sequence that the recombinant poxvirus binds to).

[0143] In one embodiment, the recombinant poxvirus is a recombinant MVA derived from an MVA or an MVA derivative that has reproductive replication ability in chicken embryo fibroblast (CEF) cells in vitro or does not have reproductive replication ability in human keratinocyte cell line HaCaT, human bone osteosarcoma cell line 143B, human embryonic kidney cell line 293 and human cervical adenocarcinoma cell line HeLa.

[0144] In one embodiment, the recombinant poxvirus is MVA-BN, which was deposited with the European Collection of Animal Cell Cultures (ECACC) on August 30, 2000, under accession number V00083008. ® It is a recombinant MVA derived from.

[0145] Aspects and embodiments regarding the transfer unit

[0146] In one embodiment, a transcription unit is provided comprising a nucleotide sequence comprising a transposable gene, preferably a viral yield reduction transposable gene, which is operably linked to a poxvirus promoter, wherein the nucleotide sequence further comprises a binding sequence for a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame [ORF] of the transposable gene.

[0147] In one embodiment of the transcription unit, the virus yield reduction transgenic gene is a transgenic gene that reduces the yield of a recombinant poxvirus containing the transcription unit, and preferably reduces the production yield during the proliferation of the recombinant poxvirus.

[0148] In one embodiment of the transcription unit, the virus yield reduction transgene is a transgene that reduces the yield of a recombinant poxvirus containing a transcription unit compared to a poxvirus or recombinant poxvirus that does not contain a transcription unit.

[0149] In one embodiment of the transcription unit, the virus yield reduction transgenic gene is a transgenic gene that reduces the replication rate of a recombinant poxvirus containing the transcription unit, and preferably reduces the replication rate compared to a poxvirus or recombinant poxvirus that does not contain the transcription unit.

[0150] In one embodiment of the transcription unit, the viral yield reduction transgenic gene is a transgenic gene that reduces the yield or replication rate of a recombinant poxvirus containing the transcription unit by expression, and preferably reduces the yield or replication rate compared to a poxvirus not containing the transcription unit or a recombinant poxvirus.

[0151] In one embodiment of the transcription unit, the viral yield-reducing transgene is a transgene that reduces the yield or replication rate of a recombinant poxvirus containing the transcription unit, wherein the expression product (e.g., mRNA, protein) preferably reduces the yield or replication rate compared to a poxvirus or recombinant poxvirus that does not contain the transcription unit.

[0152] In one embodiment of the transcription unit, the viral yield reduction transgene reduces the yield or replication rate of the recombinant poxvirus containing the transcription unit by up to about 10%, 30%, 50%, 70%, 80%, 90%, or more than 90%.

[0153] In one embodiment, the transcription unit can be functionally inserted into the poxvirus genome or is inserted.

[0154] In one embodiment of the transcription unit, the recombinant poxvirus is preferably a recombinant vaccinia virus, and most preferably a recombinant modified vaccinia virus Ankara (MVA).

[0155] In one embodiment, the poxvirus is an MVA and the transcription unit can be functionally inserted into the MVA genome at an intergene region [IGR] selected from the group consisting of IGR 44 / 45, 51 / 52, 64 / 65, 88 / 89 and 148 / 149.

[0156] In a preferred embodiment, the poxvirus is MVA, and the IGR is IGR 44 / 45 or IGR 51 / 52, more preferably IGR 51 / 52.

[0157] In one embodiment, the poxvirus is an MVA and the transcription unit is functionally insertable or inserted into an MVA or MVA derivative that has reproductive replication ability in in vitro chicken embryonic fibroblast [CEF] cells but does not have reproductive replication ability in human keratinocyte cell line HaCaT, human bone osteosarcoma cell line 143B, human embryonic kidney cell line 293 and human cervical adenocarcinoma cell line HeLa.

[0158] In one embodiment, the poxvirus is MVA-BN, which was deposited with the European Animal Cell Culture Collection (ECACC) on August 30, 2000, under accession number V00083008. ® Or it can be functionally inserted into or is inserted into its derivatives.

[0159] An embodiment relating to a poxvirus promoter

[0160] The following embodiments may relate to any aspect of the present invention.

[0161] In one embodiment, the promoter is an early / late promoter or a mid-term promoter.

[0162] In a preferred embodiment, the poxvirus promoter is primarily or completely late promoter.

[0163] In one embodiment, the mainly or fully late promoter is selected from the group consisting of Pr11, Pr7.5, PrSSL, PrATI, and PrS promoters.

[0164] In a particularly preferred embodiment, the poxvirus promoter is primarily a late promoter.

[0165] In one embodiment, the late promoter is primarily PrS.

[0166] In another preferred embodiment, the expression of a transgenic gene included in a recombinant poxvirus or transcription unit, preferably a viral yield-reducing transgenic gene, is induced by a late portion of an early / late promoter linked to the transgenic gene in an operable state, e.g., a late portion of a PrS promoter.

[0167] In one embodiment, the foxvirus promoter is primarily an initial promoter.

[0168] In one embodiment, the initial promoter is mainly selected from the group consisting of the promoter long Pr13.5 / Pr13.5(29), PrHyb, Pr1328 and PrH5m, preferably PrH5m.

[0169] An embodiment relating to the binding sequence of a transcription repressor protein

[0170] The following embodiments may relate to any aspect of the present invention.

[0171] In one embodiment, the binding sequence to the transcription repressor protein can bind to the transcription repressor protein.

[0172] In one embodiment, the binding sequence may bind to a transcription repressor protein of the tetracycline repressor (TetR) or lactose repressor (LacR) family.

[0173] In one embodiment, the binding sequence may bind to a transcription repressor protein of the TetR family, preferably to TeR or a circular TetR, and, for example, E. coli [ E. coli Can be combined with ]

[0174] In one embodiment, the binding of the transcription repressor protein is a reversible binding.

[0175] In one embodiment, the binding sequence comprises two copies of the Tet operator 2 (TetO2) nucleotide sequence, wherein the TetO2 nucleotide sequence preferably follows SEQ ID NO. 1.

[0176] In one embodiment, the binding sequence comprises two copies of a Tet operator 2 (TetO2) nucleotide sequence arranged in series and separated by a 2-base spacer, wherein, preferably, the TetO2 nucleotide sequence follows SEQ ID NO. 1.

[0177] In one embodiment, the binding sequence to the transcription repressor protein comprises or consists of the nucleic acid sequence according to SEQ ID NO. 2.

[0178] In one embodiment, the amount of TetR protein produced in the transformed poxvirus producer cell is sufficient to downregulate transgene expression for at least 2, 4, 10, 20, 22, or 24 hours after infecting the transformed cell with a recombinant poxvirus containing a yield-reducing transgene linked to a binding sequence for the TetR protein.

[0179] Examples of transfer genes

[0180] The following embodiments may relate to any aspect of the present invention.

[0181] In one embodiment, the transgene encodes a protein or peptide, preferably a protein or peptide comprising one or more epitope determinants, more preferably a proteinic or peptide antigen.

[0182] In one embodiment, the transgenic gene encodes a protein or antigen or an antigenic portion thereof selected from the group consisting of viruses, bacteria, fungi, plants, parasites, non-human animals, and human proteins or antigens.

[0183] In one embodiment, the transgenic gene encodes a viral antigen or an antigenic portion thereof.

[0184] In one embodiment, the transgenic gene encodes a disease-related antigen or protein.

[0185] In one embodiment, the disease-related antigen or protein is a vaccine antigen or a gene therapy protein.

[0186] In one embodiment, the disease-related antigen is an alphavirus, adenovirus, coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus (CMV), dengue virus, Ebola virus, Epstein-Barr virus (EBV), eastern, western, or Venezuelan equine encephalitis virus (EEV), guanaritoh virus, herpes simplex virus-type 1 (HSV-1), herpes simplex virus-type 2 (HSV-2), human herpesvirus-type 8 (HHV-8), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (hepatitis E virus, HEV), human immunodeficiency virus (HIV), influenza virus, Zunin virus, Lassa virus, Machupo virus, Marburg virus, measles virus, human metapneumovirus, mumps virus, Norwalk virus, human papillomavirus (HPV), parainfluenza virus, parvovirus, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, rubella virus, savia virus, severe acute respiratory syndrome virus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus,It originates from a virus selected from a group consisting of MERS-CoV), varicella-zoster virus, varicella virus, West Nile virus, and yellow fever virus.

[0187] In one embodiment, the disease-related antigen is derived from the Epstein-Barr virus (EBV) or its antigenic portion, preferably selected from the group consisting of EBV proteins BLLF1a / b (gp350 / 220), BALF4 (gB, gp110), BXLF2 (gH, gp85), BKRF2 (gL, gp25), BZLF2 (gp42), BILF2 (gp78), BDLF3 (gp150), BBBRF3 (gM), BLRF1 (gN), BMRF2, EBNA1, EBNA2, EBNA3, LMP1, LMP2, BRLF1 or BZLF1.

[0188] In one embodiment, the transgene encodes an LMP1 / EBNA2 fusion protein.

[0189] In one embodiment, the transgenic gene encodes a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), or an antigenic portion thereof.

[0190] Aspects and embodiments relating to transformed poxvirus producer cells

[0191] In one embodiment, a transgenic poxvirus producer cell is provided that is genetically modified to express a transcription repressor protein.

[0192] In one embodiment, the transformed poxvirus producer cell is genetically modified to express a transcription repressor protein permanently or in a regulated manner.

[0193] In one embodiment of a transformed poxvirus producer cell, the transcription repressor protein is implemented as described below in the 'Embodiment relating to the transcription repressor protein'.

[0194] In one embodiment, a transformed poxvirus producer cell was produced by stable transformation of a poxvirus-allowing cell with a plasmid encoding a transcription repressor protein.

[0195] In one embodiment, the transformed poxvirus producer cell is derived from a poxvirus-allowed cell.

[0196] In one embodiment, the transformed poxvirus producer cell or poxvirus-allowing cell is a eukaryotic poxvirus producer cell.

[0197] In one embodiment, the transformed poxvirus producer cell or poxvirus-allowed cell is a cell of a cell line, preferably a continuous or permanent cell line.

[0198] In one embodiment, the cell line is an attached or floating cell line.

[0199] In one embodiment, the poxvirus-allowing cell is an avian cell or is derived from an avian cell.

[0200] In one embodiment, the poxvirus-allowing cells are chicken, duck, or quail cells or are derived from them.

[0201] In one embodiment, the transformed poxvirus producer cell is preferably a transformed vaccinia virus producer cell, most preferably a transformed MVA producer cell.

[0202] In one embodiment, the poxvirus-allowing cell is preferably a vaccinia virus-allowing cell, most preferably an MVA-allowing cell.

[0203] In one embodiment, the poxvirus-allowed cell is a cell of the chicken DF-1 cell line.

[0204] In one embodiment, the transformed poxvirus producer cell is a cell of a cell clone derived from a chicken DF-1 cell line.

[0205] In one embodiment, the transformed poxvirus producer cell is a cell of cell clone DF1-TR59.

[0206] In one embodiment, the poxvirus-accepting cell is a quail CCX cell line, preferably a quail CCX.2C4 or CCX.E10 cell line.

[0207] In one embodiment, the transformed poxvirus producer cell is a cell of a cell clone derived from a quail CCX.2C4 or CCX.E10 cell line, preferably a CCX.2C4 cell line.

[0208] In one embodiment, the transformed poxvirus producer cell is a cell of cell clone CCX.2C4-TR16.

[0209] Other poxviruses such as QOR2 / E11, vaccinia virus, or MVA-accepting cell lines, serial avian cell lines derived from quail embryos (Kraus B, von Fircks S, Feigl S et al. 2011. Avian cell line - Technology for large scale vaccine production. BMC Proc 5: P51.), or AGE.CR1-pIX (commercially available from ProBiogen), or EB66 (duck embryonic stem cell-derived cell line, commercially available from Valneva), or BHK-21 (baby hamster kidney cell line) may also be considered for the production of transformed MVA producer cells.

[0210] In one embodiment, the poxvirus producer cell comprises a recombinant poxvirus as described in 'Aspects and embodiments relating to recombinant poxvirus'.

[0211] Examples relating to transcription repressor proteins

[0212] The following embodiments may relate to any aspect of the present invention.

[0213] In one embodiment, the transcription repressor protein may bind to a binding sequence for the transcription repressor protein included in the recombinant poxvirus.

[0214] In one embodiment, the binding of the transcription repressor protein is a reversible binding.

[0215] In one embodiment, the transcription repressor protein is one of the tetracycline repressor (TetR) or lactose repressor (LacR) series.

[0216] In one embodiment, the transcription repressor protein is one of the TetR family, preferably TetR or a circular TetR, for example, derived from Escherichia coli.

[0217] In one embodiment, the transcription repressor protein may bind to a nucleotide sequence comprising two copies of the Tet operator 2 (TetO2) nucleotide sequence, wherein the TetO2 nucleotide sequence preferably follows SEQ ID NO. 1.

[0218] In one embodiment, the transcription repressor protein may bind to a nucleotide sequence comprising two copies of a Tet operator 2 (TetO2) nucleotide sequence arranged in series and separated by a 2-base spacer, wherein the TetO2 nucleotide sequence preferably follows SEQ ID NO. 1.

[0219] In one embodiment, the transcription repressor protein may include a nucleic acid sequence according to SEQ ID NO. 2 or bind to a nucleotide sequence composed thereof.

[0220] An embodiment regarding the interrelationship between a transcription repressor protein and a corresponding binding sequence

[0221] The following embodiments may relate to any aspect of the present invention.

[0222] In one embodiment, the binding of a transcription repressor protein to a corresponding binding sequence to a transcription repressor protein downregulated the expression of a transgenic gene by a recombinant poxvirus, preferably downregulated compared to a poxvirus that does not contain a binding sequence to a transcription repressor or a recombinant poxvirus.

[0223] In one embodiment, the binding of the transcription repressor protein to the corresponding binding sequence to the transcription repressor protein reduces the expression of the transposable gene by the recombinant poxvirus by at least 40%, or 60%, more preferably 70 to 90%, most preferably at least 90%, and preferably compared to a poxvirus or recombinant poxvirus that does not contain the binding sequence to the transcription repressor.

[0224] In one embodiment, binding of the transcription repressor protein to the corresponding binding sequence for the transcription repressor protein increases the replication of the recombinant poxvirus by at least 2, 3, 5, 10, 20, 30, 40, 50, or 60 times.

[0225] In one embodiment, binding of a transcription repressor protein to a corresponding binding sequence for the transcription repressor protein increases the viral yield of the recombinant poxvirus by at least 2, 3, 5, 10, 20, 30, or 40 times.

[0226] In one embodiment, a binding sequence for a transcription repressor protein contained in the recombinant poxvirus can bind to a transcription repressor protein expressed by a transformed poxvirus producer cell and / or vice versa, that is, a transcription repressor protein expressed by a transformed poxvirus producer cell can bind to a binding sequence for a transcription repressor protein contained in the recombinant poxvirus.

[0227] In one embodiment, the binding sequence to the transcription repressor protein by the recombinant poxvirus (which can bind to the transcription repressor protein expressed by the transformed poxvirus producer cell) preferably comprises two copies of the Tet operator 2 (TetO2) nucleotide sequence arranged in series and separated by a 2-base spacer, and the transcription repressor protein expressed by the transformed poxvirus producer cell (the transcription repressor protein capable of binding to the binding sequence to the transcription repressor protein included in the recombinant poxvirus) is TetR or the original TetR.

[0228] In one embodiment, the binding sequence for a transcription repressor protein included in a recombinant poxvirus (which can bind to a transcription repressor protein expressed by a transformed poxvirus producer cell) preferably comprises two copies of a TetO2 nucleotide sequence following SEQ ID NO. 1, which are arranged in series and separated by a 2-base spacer, and the transcription repressor protein expressed by the transformed poxvirus producer cell (a transcription repressor protein capable of binding to the binding sequence for a transcription repressor protein included by the recombinant poxvirus) is TetR or a circular TetR.

[0229] In one embodiment, the binding sequence for the transcription repressor protein included in the recombinant poxvirus (which can bind to the transcription repressor protein expressed by the transformed poxvirus producer cell) includes or is composed of the nucleotide sequence according to SEQ ID NO. 2, and the transcription repressor protein expressed by the transformed poxvirus producer cell (the transcription repressor protein capable of binding to the binding sequence for the transcription repressor protein included in the recombinant poxvirus) is TetR or the original TetR.

[0230] An embodiment regarding the interrelationship between recombinant poxvirus and transformed poxvirus producer cells

[0231] The following embodiments may relate to any aspect of the present invention.

[0232] In one embodiment, the recombinant poxvirus is a recombinant vaccinia virus, and the transformed poxvirus producer cell is a vaccinia virus producer cell.

[0233] In one embodiment, the recombinant poxvirus is a recombinant MVA, and the transformed poxvirus producer cell is an MVA producer cell.

[0234] Aspects and embodiments regarding medical use

[0235] In one embodiment, the present invention provides a recombinant poxvirus for use in the treatment or prevention of infectious diseases or cancer.

[0236] In one embodiment, the use of a recombinant poxvirus according to the present invention is provided for the manufacture of a medicine or vaccine for use in the treatment or prevention of infectious diseases or cancer.

[0237] In one embodiment, a method for treating or preventing an infectious disease or cancer of a subject is provided, comprising the step of administering a recombinant MVA according to the present invention to the subject.

[0238] In one embodiment, the infectious disease is selected from the group consisting of diseases in which the causative agent is a virus, bacteria, fungus, or parasite.

[0239] In a preferred embodiment, the infectious disease is a viral disease.

[0240] In one embodiment, the causative agent of the viral disease is alphavirus, adenovirus, coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus (CMV), dengue virus, Ebola virus, Epstein-Barr virus (EBV), eastern, western or Venezuelan equine encephalitis virus (EEV), guanaritoh virus, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human herpes simplex virus type 8 (HHV-8), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), human immunodeficiency virus (HIV), influenza virus, Zunin virus, Lassa virus, Machupo virus, Marburg virus, measles virus, human metapneumovirus, mumps virus, Norwalk virus, human papillomavirus (HPV), parainfluenza virus, parvovirus, It originates from a virus selected from the group consisting of poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, rubella virus, saviavirus, severe acute respiratory syndrome virus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), varicella-zoster virus, variola virus, West Nile virus, and yellow fever virus.

[0241] In one embodiment, the viral disease is an Epstein-Barr virus (EBV) infection.

[0242] Aspects and embodiments relating to a transcription repressor protein and a corresponding binding sequence for downregulation of poxvirus transgenic expression

[0243] In one embodiment, a method or use of a transcription repressor protein and a corresponding binding sequence for controlling or downregulating transgenic expression induced by a recombinant poxvirus, primarily or completely late or mid-phase poxvirus promoter-induced transgenic expression is provided.

[0244] In one embodiment, transgenic expression is induced primarily by a fully late promoter, preferably primarily by a late promoter.

[0245] In one embodiment, transgenic expression is induced by the PrS promoter.

[0246] In one embodiment, transgenic expression is induced by the late portion of an early / late promoter, preferably the late portion of a PrS promoter.

[0247] In one embodiment, the transcription repressor protein is one of the tetracycline repressor (TetR) or lactose repressor (LacR) series.

[0248] In one embodiment, the transcription repressor protein is one of the TetR family, preferably derived from TetR or a circular TetR, for example, Escherichia coli.

[0249] In one embodiment, the corresponding binding sequence comprises two copies of the Tet operator 2 (TetO2) nucleotide sequence, wherein the TetO2 nucleotide sequence preferably follows SEQ ID NO. 1.

[0250] In one embodiment, the corresponding binding sequence comprises two copies of a Tet operator 2 (TetO2) nucleotide sequence arranged in series and separated by a 2-base spacer, preferably the TetO2 nucleotide sequence follows SEQ ID NO. 1.

[0251] In one embodiment, the corresponding binding sequence for the transcription repressor protein comprises or consists of the nucleic acid sequence according to SEQ ID NO. 2.

[0252] In one embodiment, the transgenic gene is a virus yield reduction transgenic gene.

[0253] In one embodiment, the recombinant poxvirus is preferably a recombinant vaccinia virus, and most preferably a recombinant MVA.

[0254] Additional explanation

[0255] Variant Vaccinia Virus Ankara (MVA)

[0256] In the past, MVA was generated by 516 consecutive passages of chicken embryonic fibroblasts of the vaccinia virus Ankara strain (CVA) (see literature [Mayr A et al. 1975. Abstammung, Eigenschaften und Verwendung des attenuierten Vaccinia-Stammes MVA. Infection 3:6-14] for review). This virus was renamed from CVA to MVA to describe characteristics substantially altered at 570 passages. MVA was further passaged to a number of passages exceeding 570. As a result of these long passages, the genome of the generated MVA virus was described as having a deletion of approximately 31 kilobases of genomic sequence, and as an advanced host cell with restricted replication into avian cells (19). MVA generated in various animal models was found to be significantly non-toxic compared to the fully replicable starting material (20).

[0257] MVAs useful for the practice of the present invention include MVA-572 (deposited as ECACC V94012707 on January 27, 1994); MVA-575 (deposited as ECACC V00120707 on December 7, 2000), MVA-I721 [see (4)], NIH clone 1 (deposited as ATCC® PTA-5095 on March 27, 2003) and MVA-BN (deposited as European Cell Culture Collection (ECACC) No. V00083008 on August 30, 2000).

[0258] More preferably, the MVA used according to the present invention comprises MVA-BN and MVA-BN derivatives. MVA-BN is described in WO No. 02 / 042480. 'MVA-BN derivatives' refers to any virus that exhibits essentially the same replication characteristics as MVA-BN as described herein, but differs in one or more parts of its genome.

[0259] MVA-BN and its derivatives are incapable of replication, implying a failure of reproductive replication in vivo and in vitro. More specifically, in vitro, MVA-BN or its derivatives may be capable of reproductive replication in chicken embryonic fibroblasts [CEF], but are described as incapable of reproductive replication in human keratinocyte cell line HaCaT (21), human bone osteosarcoma cell line 143B (ECACC deposit number 91112502), human embryonic kidney cell line 293 (ECACC deposit number 85120602), and human cervical adenocarcinoma cell line HeLa (ATCC deposit number CCL-2). Additionally, MVA-BN or its derivatives have a viral amplification rate that is at least 2 times lower than that of MVA-575 in Hela cells and HaCaT cell lines, and more preferably 3 times lower. Tests and verifications regarding these properties of MVA-BN and MVA-BN derivatives are described in WO No. 42480 and WO No. 03 / 048184.

[0260] As previously mentioned, the term "incapable of reproductive replication" in human cell lines in vitro is described, for example, in WO No. 02 / 42480, which also teaches a method for obtaining MVAs having desired characteristics as previously mentioned. This term applies to viruses in which the in vitro viral amplification rate is less than 1 on day 4 after infection using the assay described in WO No. 02 / 42480 or US No. 6,761,893.

[0261] Exemplary generation of recombinant MVA virus

[0262] Several methods may be applicable for the generation of recombinant MVAs as disclosed herein. The DNA sequence to be inserted into the virus may be placed in an E. coli plasmid construct in which DNA homologous to the poxvirus DNA section is inserted. Separately, the DNA sequence to be inserted may be linked to a promoter. The promoter gene linkage may be positioned in the plasmid construct such that DNA homologous to the DNA sequence on the side of the poxvirus DNA region containing non-essential loci is located on both ends. The generated plasmid construct may be amplified by proliferation within E. coli bacteria and isolated. The isolated plasmid containing the DNA gene sequence to be inserted may be transfected into, for example, a cell culture of chicken embryonic fibroblasts [CEF], and simultaneously the culture may be infected with MVA. Recombination between the homologous MVA viral DNA within the plasmid and the viral genome may generate MVAs modified by the presence of foreign (heterogeneous) DNA sequences.

[0263] For example, as CEF cells, cells of a suitable cell culture may be infected with the MVA virus. The infected cells may then be transfected by a first plasmid vector containing foreign or heterogeneous genes or genes, such as one or more nucleic acids provided herein, preferably under the transcriptional control of a poxvirus expression control factor. As described above, the plasmid vector also contains a sequence that may direct the insertion of an exogenous sequence into a selected portion of the MVA virus genome. Optionally, the plasmid vector also contains a cassette containing a marker and / or a selection gene linked in an operable state to a poxvirus promoter. The use of the selection or marker cassette simplifies the identification and isolation of the generated recombinant MVA. However, the recombinant poxvirus may also be identified by PCR technology. Subsequently, additional cells may be infected with the recombinant MVA obtained as described above and transfected with a second vector containing a second foreign or heterogeneous gene or genes. If this gene is to be introduced into a different insertion site in the poxvirus genome, the second vector also has a poxvirus-homologous sequence that instructs the incorporation of a second foreign gene or genes into the poxvirus genome. After homologous recombination occurs, a recombinant virus containing two or more foreign or non-homologous genes can be isolated. To introduce additional foreign genes into the recombinant virus, the infection and transfection steps can be repeated by using the recombinant virus isolated in the previous step for infection and by using an additional vector containing additional foreign genes or genes for transfection. There are many other techniques known to generate recombinant MVAs.

[0264] Unless otherwise specified, the practice of the present invention will utilize the ordinary techniques of immunology, molecular biology, microbiology, cell biology, and recombinant technology, all of which are within the scope of the art. For example, see the literature [Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et al., eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988].

[0265] Examples

[0266] The following examples serve to further illustrate the content of the disclosure. They should not be understood as limiting the invention, and the scope thereof is determined by the appended claims.

[0267] Example 1: Materials and Method

[0268] 1.1 Viruses and Cells

[0269] The recombinant MVA described herein is MVA-BN ®They were derived from (Bavarian Nordic modified vaccinia virus Ankara; also referred to herein as 'MVA-BN'), that is, from MVA-BN seed stock (in the case of MVA-res005) or from bacterial artificial chromosome (BAC) clones constructed from MVA-BN (all other recombinant MVA). MVA-BN wild-type (MVA-wt) and MVA-BN recombinants were proliferated on primary chicken embryonic fibroblast [CEF] cells, DF-1 cells (a continuous cell line of chicken embryonic fibroblasts), or transformed DF1-TR59 cells. The recombinants were TCID 50 The method was used to titrate on CEF cells or DF1-TR59 cells. Shope fibroma virus (SFV) for MVA-BN-BAC reactivation was obtained from ATCC (VR-364), proliferated in rabbit corneal SIRC cells, and titrated.

[0270] Primary CEF cells were prepared from 11-day-old hatched eggs. CEF cells were cultured in VP-SFM medium (Thermo Fisher Scientific) supplemented with 1% gentamicin and 4 mM L-glutamine for transfection and viral stock production, or in DMEM supplemented with 10% fetal calf serum (FCS) for replication analysis and viral titration. Chicken DF-1 cells were obtained from ATCC.

[0271] As well as the chicken DF-1 cell line, the quail cell line, namely CCX.2C4, was used to generate transgenic cell lines expressing the tetracycline inhibitor (TetR). These cell lines can also be used as producer cells for recombinant MVA. The inventors recently developed the CCX.2C4 cell, a permanently adherent cell line derived from quail embryos, and the CCX.E10 cell line, a floating quail cell line, both of which enable the efficient proliferation of MVA-wt and recombinant MVA. Both cell lines are available from Nuvonis. The CCX.2C4 cell line enables satisfactory levels of replication and yield of MVA-wt and forms a sufficiently homogeneous monolayer, allowing for the focus or plaque-based generation and selection of recombinant or other mutant MVA clones.

[0272] 1.2 BAC Recombination and Reactivation of Infectious Recombinant MVA

[0273] The construction of MVA-BN-BAC has been described above (22). Briefly, the inserted BAC cassette contains the miniF plasmid sequence derived from the plasmid pMBO131 (23) for maintenance in E. coli. The BAC cassette was inserted between the MVA orthologues (MVA064L / MVA065L) of the vaccinia virus (VACV)-Copenhagen gene I3L and I4L. All recombinant MVA constructs described herein were produced using variations of the BAC technique, with the exception of MVA-res005, which was produced using conventional homologous recombination between MVA-BN and a transfer plasmid.

[0274] For the generation of MVA-mBNbc440 (see Fig. 1), the neomycin phosphotransferase npt II-IRES-EGFP marker cassette originally included in the BAC cassette was replaced with a bacterial tetracycline expression cassette to remove the enhanced green fluorescence protein (EGFP) gene from the BAC backbone. This enabled the insertion and analysis of the EGFP transgene at other insertion sites.

[0275] MVA-mBNbc440 was generated by linear exchange mutagenesis of MVA-BN-BAC in E. coli as described above, using a BAC cassette with npt II-IRES-EGFP deleted as the starting construct (22). An EGFP ORF under the control of the synthetic poxvirus early / late promoter PrS (24), which lacks the 2xTetO2 operon sequence, was inserted along with a gpt-mRFP fusion gene that leads to PrS in the intergene region [IGR] 044 / 045. For the generation of MVA-res005, an EGFP gene containing the 2xTetO2 operon sequence between the PrS promoter of MVA-res005 and the EGFP ORF was inserted along with the PrS-gpt-mRFP cassette of the IGR between the genes MVA044L / MVA045L (VACV F14L and F15L) (see FIG. 1). Here, MVA-BN was used as the parent, and a co-transfected transport plasmid and classical homologous recombination were used to generate MVA-res005. For the generation of MVA-res034, MVA-res035, MVA-res057, and MVA-res058 (see Fig. 1), MVA-BN-BAC was modified by deletion of an essential gene in MVA-BN-BAC, and recombinant MVA was subsequently generated by homologous recombination and co-reactivation of the modified MVA-BN-BAC using the essential gene as a positive selection marker and SFV as a helper virus.

[0276] To co-recombin and reactivate infectious recombinant MVA in MVA-BN-BAC, FuGENE ® Using HD transfection reagent (Promega) 10 6 Can DF1-TR59 cells (see Example 4.1) were transfected with 3 μg of BAC DNA and infected with SFV after 60 minutes to provide the necessary helper function. Recombinant MVA-res034, MVA-res035, MVA-res057, and MVA-res058 contained the BAC cassette in IGR 64 / 65 cells that still carried the PrS-induced npt II-IRES-EGFP expression cassette (see Fig. 1) used as a marker for the infected cells. The reactivated virus was isolated by further passage in CEF cells (MVA-mBNbc440) or DF1-TR59 cells (MVA-res034, MVAres035, MVA-res057, MVA-res058), and the helper virus was removed as previously described by passage in newly generated recombinant MVA on avian cells that are not tolerated by SFV (22).

[0277] BAC-derived MVA (MVA-mBNbc166) also possesses a BAC cassette with the same EGFP expression cassette as in MVA-res034, MVA-res035, MVA-res057 and MVA-res058, but without other inserts, and was used as a reference MVA (not shown in FIG. 1). MVA-mBNbc166 was previously shown to function like MVA-wt (22), and is therefore sometimes referred to as 'MVA-wt' in this document.

[0278] Recombinant MVA virus stocks were produced in DF-1 or DF1-TR59 cells, and TCID as described 50 The method was used to titrate CEF or DF1-TR59 cells (3).

[0279] 1.3 Flow cytometry analysis of transgene expression by recombinant MVA

[0280] Cell culture monolayers were washed with phosphate-buffered saline (PBS) and harvested by trypsin treatment to obtain single-cell suspensions. For the analysis of EGFP and monomeric red fluorescent protein (mRFP) expression (MVA-res005, MVA-mBNbc440), cells were resuspended in PBS-FACS (2% FCS, 0.1% NaN3) and analyzed directly by flow cytometry using an LSR II flow cytometer (BD Biosciences) and FlowJo software (Tree Star Inc.). For flow cytometry analysis of LMP1 / EBNA2 expression (MVA-res035, MVA-res058), cells were stained with an antibody against EBNA2 (abcam ab90543, 1:500) and a secondary allophycocyanin (APC) coupled anti-mouse antibody [F(ab')₂ goat anti-mouse IgG (H+L) fragment (H+L)] (Jackson Immuno Research, 115-136-146, 1:500).

[0281] 1.4 Virus Replication Analysis

[0282] For multicycle viral replication analysis, a fusion monolayer in a 6-well cell culture plate was infected with sonicated viral dilutions of the indicated multiplicities of infection (MOI) in 500 μl of FCS-free DMEM. After adsorption for 60 minutes at 37 °C and 5% CO2, the inoculum was aspirated, the cells were washed once with DMEM, and further cultured in DMEM / 2% FCS at 37 °C and 5% CO2. Cells and supernatant were harvested at the indicated time points, frozen and thawed three times, and sonicated prior to titration. MVA yield was TCID as described. 50 It was determined in CEF cells using the titration method (3).

[0283] 1.5 Generation of transgenic chicken DF-1 and quail CCX.2C4 cells expressing TetR

[0284] Transformed DF-1 cells expressing TetR were generated by transfecting wild-type DF-1 cells with a plasmid containing a blasticidin selection cassette for selecting stable insertion of the plasmid in cells that express the TetR gene in a eukaryotic cytomegalovirus (CMV) promoter and are transfected with the antibiotic blasticidin.

[0285] A cell line was cloned by seeding a pool of DF-1 cells transfected with 5 µg / ml blasticidin at 5 cells per well of a 96-well plate for 21 days. TetR expression in transfected DF-1 cells was determined by immunoblotting using a TetR-specific antibody (see Example 1.6). Also see Example 4.1.

[0286] TetR-expressing quail cell lines were generated by transfecting wild-type CCX.2C4 cells with the same TetR-expressing plasmid used to generate transformed DF-1 cells as described above. Cell lines were cloned by seeding a pool of transfected CCX.2C4 cells selected with 5 µg / ml blasticidin at a rate of 5 cells per well of a 96-well plate for 8 weeks. TetR expression in the re-transfected CCX.2C4 cells was determined by immunoblotting using a TetR-specific antibody. See also Example 7.1.

[0287] 1.6 Immunoblot Analysis of TetR Protein Expressed by Transformed Cells

[0288] Cells were seeded into a 12-well tissue culture plate the day before infection. Infection was performed as described above (25). At the indicated time after infection, cells were washed with cold phosphate-buffered saline [PBS] and lysed in 200 μl of 1X Lamley loading buffer (65 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 0.1% bromophenol blue, beta-mercaptoethanol [35 μl / ml]) at room temperature for 5 minutes, then sonicated for 3 minutes, followed by heating to 95 °C for 5 minutes. The lysate was centrifuged at 18,000 xg for 1 minute to remove cell debris.

[0289] Soluble proteins in cell lysates were separated on a pre-cast SDS-polyacrylamide gel (MiniProtean TGX, 10%, Bio-Rad) and transferred to a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Turbo blotting system (Bio-Rad) and a Trans-Blot Turbo delivery pack (Bio-Rad). The membrane was blocked using 5% bovine serum albumin (BSA, Carl Roth) in Tris-buffered saline (TBS; 50 mM Tris, 150 mM NaCl, pH 7.5) containing 0.1% Tween-20 and 0.1% NaN3, and incubated overnight with shaking at 4°C with the primary antibodies listed below (diluted with blocking buffer). The membranes were washed four times (total 20–40 minutes) with TBS containing 0.1% Tween-20 between steps and incubated for 1 hour at room temperature with shaking with a secondary antibody coupled to horseradish peroxidase and induced against mouse IgG. The secondary antibody was diluted in TBS containing 5% skim milk powder (VWR International). Bands were visualized by enhanced chemiluminescence (ECL) using two different substrate reagents for high-sensitivity detection: Amersham ECL Select Western Blotting Protection Reagent (GE Healthcare Life Sciences) diluted 1:10 and SuperSignal West Pico (Thermo Fisher Scientific) as the standard reagent. ChemiDoc Touch System and Image Lab were used for image analysis and quantification. TM The signal was recorded using software (Bio-Rad).

[0290] For immunoblot analysis, primary antibodies against TetR (MoBiTec, TET02, mouse; 1:500; expected to detect a signal at 23 kDa, the calculated molecular weight of the 207-amino acid protein encoded by the TetR gene) and anti-mouse β-tubulin (Sigma-Aldrich, T7816, mouse; 1:40000, expected to detect a signal at a molecular weight of 55 kDa) were used.

[0291] Example 2: Design of a recombinant MVA having a TetR-regulatory transposable gene

[0292] A number of recombinant MVAs were produced by including different transgenes used as examples of prototypes of 'neutral' transgenes (i.e., transgenes that do not affect the replication and yield of each recombinant MVA) or as examples of transgenes that significantly reduce the yield of each recombinant MVA. Examples used in the experiments described herein include (1) EGFP as a prototype 'neutral' transgene and (2) a fusion protein of modified versions of LMP1 and EBNA2 proteins (LMP1 / EBNA2) derived from Epstein-Barr virus (EBV) as a yield-reducing transgene.

[0293] A first set of two recombinant MVAs was produced in which a 2xTetO2 sequence (7, 8) that binds to TetR with high affinity was inserted or omitted between the EGFP gene and the early / late PrS promoter (MVA-res005) to produce a 'neutral' EGFP transgene (MVA-mBNbc440, see Fig. 1). By co-inserting a second transcription unit encoding the mRFP fluorescent marker under the same PrS promoter into these recombinant MVAs (Fig. 1), it became possible to conveniently perform flow cytometry and microscopic monitoring of the infection regardless of the potential downregulation of EGFP.

[0294] For the generation of recombinant MVAs carrying the LMP1 / EBNA2 transgene under the control of a PrH5m promoter (MVA-res034, MVA-res035; see FIG. 1) or a PrS promoter (MVA-res057, MVA-res058; see FIG. 1), refer to the following Examples 4.3 and 6.1, respectively.

[0295] Example 3: The 2xTetO2 sequence does not impair MVA-mediated EGFP transgene expression.

[0296] To evaluate the potential effect of the 2xTetO2 sequence inserted between the poxvirus promoter, in this case between the PrS and the transgenic ORF, on transgenic expression, the EGFP levels (i.e., expression of neutral transgenic genes) generated by MVA-res005 (see Fig. 1) were analyzed. Infection of two MVA-accepted cell lines, DF-1 and CCX.2C4, as well as an accepting primary CEF and an unaccepting HeLa cell line, demonstrated that EGFP levels (determined as geometric mean fluorescence intensity = GMFI) were very similar in cells infected with MVA-mBNbc440 or MVA-res005 (Fig. 2). MVA-res005 contains the 2xTetO2 sequence between the PrS promoter and the EGFP transgenic gene, whereas MVA-mBNbc440 does not (see Fig. 1).

[0297] Therefore, the 2xTetO2 sequence between the promoter and the ORF did not reduce the transcription efficiency of EGFP, at least not under the PrS promoter. Additionally, the data indicated that the levels of stable proteins such as EGFP, whose expression was primarily controlled by the late poxvirus PrS promoter, increased significantly by about 7 to 10 times from pi 5 hours in the early late stage of the MVA infection cycle to pi 20 hours in the progressive late stage (Fig. 2), confirming that the PrS promoter is highly active in the late stage of MVA replication.

[0298] Example 4: Downregulation of EGFP or LMP1 / EBNA2 transgene expression in TetR expressing transformed DF-1 cells

[0299] To enable the downregulation of poxvirus transgenes using the TetR system, a transgenic chicken fibroblast DF-1 cell line (26, 27) permanently expressing TetR was generated. DF-1 cells are highly receptive to MVA. The new transgenic DF-1 cell line (referred to as DF1-TR59, see Example 4.1) permanently expressed TetR. Therefore, MVA-mediated expression of the transgene linked to the 2xTetO2 sequence would be 'inactivated' in these cells without the addition of tetracycline or its analog, doxycycline. In non-TetR expressing cells, which also apply to the somatic cells of vaccinated individuals, the transgene would be readily generated because TetR is absent. For in vitro testing purposes in TetR-expressing cells, transgene expression can be switched and activated by the addition of tetracycline or doxycycline.

[0300] 4.1 Generation of DF1-TR59 Cells

[0301] As described in Example 1.5, wild-type DF-1 cells were transfected with a plasmid containing a blasticidin selection cassette expressing TetR from a eukaryotic CMV promoter.

[0302] A total of 38 stably transfected blasticidin-resistant DF-1 cell clones were obtained. Among the stably transfected DF-1 cells, 15 clones expressed moderate to high levels of TetR, as determined by immunoblotting with TetR-specific antibodies (see Example 1.6). Lysates from DF-1 cells transiently transfected with a TetR expression plasmid for 24 hours were used as a reference for achievable TetR levels. Of the 15 cell clones expressing moderate to high TetR levels, 11 exhibited satisfactory proliferative characteristics. The clone DF1-TR59 (Fig. 3), which had high levels of TetR expression, was selected for further development.

[0303] 4.2 PrS-induced downregulation of EGFP transgene expression

[0304] One day after sowing, TetR-expressing DF1-TR59 cells were infected with MVA-res005 containing the EGFP gene linked to the 2xTetO2 sequence (see Fig. 1) at a high infection multiplicity (MOI=5). From the day of sowing throughout the experiment, cells were treated with or not treated with doxycycline. In the absence of doxycycline, EGFP expression was TetR-mediated downregulated, whereas TetR was inactivated upon the addition of doxycycline. Cells were harvested at the indicated time points after infection by accutase treatment (see Fig. 4), and then fixed and permeated. Cells were analyzed for EGFP fluorescence by flow cytometry, and the geometric mean fluorescence intensity [GMFI] was determined as a measure of EGFP expression levels.

[0305] As shown in Figure 4, the addition of doxycycline (DOX) provided distinct EGFP expression, whereas in the absence of DOX, EGFP expression was very low. Therefore, the highly efficient downregulation of EGFP in MVA-res005-infected DF1-TR59 cells demonstrated that TetR expressed in DF1-TR59 cells can substantially downregulate transgenic expression in functional and highly potent poxvirus promoters (i.e., PrS). Furthermore, these results showed that sufficient TetR was produced in DF1-TR59 cells under MVA infection conditions to downregulate TetR-controlled transgenic expression for 24 hours post-infection, which is the time when MVA-infected cells typically begin to undergo apoptosis.

[0306] 4.3 Recombinant MVA encoding LMP1 / EBNA2 fusion protein

[0307] We generated a recombinant MVA that expresses a fusion protein called LMP1 / EBNA2, encoded by two fused Epstein-Barr virus (EBV)-derived genes LMP1 and EBNA2, under the control of the poxvirus early / late PrH5m promoter.

[0308] Using DF1-TR59 cells expressing TetR, recombinant MVAs containing the LMP1 / EBNA2 transgene inserted into IGR 51 / 52 were generated under the control of a PrH5m promoter into which the 2xTetO2 sequence (MVA-res035; see Fig. 1) was inserted. EGFP expression in the npt II-IRES-EGFP cassette within the BAC cassette of IGR 64 / 65 was used as an internal infection control for these recombinants.

[0309] MVA-res035 was produced in TetR-expressing DF1-TR59 cells and promoted the production of recombinants with TetR-regulated LMP1 / EBNA2 transgenes. MVA-res034 (see Fig. 1), which encodes PrH5m-induced LMP1 / EBNA2 but lacks the TetR binding site between the promoter and the transgene (i.e., the 2xTetO2 sequence), could not be rescued by recombinant virus in DF1-TR59 cells, indicating that LMP1 / EBNA2 is a yield-reducing transgene that may disrupt the production of recombinant MVA through uncontrolled expression of this transgene.

[0310] 4.4 LMP1 / EBNA2 is a transgenic gene that reduces yield in MVA.

[0311] Wild-type DF-1 cells allow MVA and produce MVA yields similar to primary CEF cells. When DF-1 cells were infected with MVA-res035 at a low MOI, a significant 38-fold decrease in yield was observed on day 3 compared to MVA-mBNbc166 ('MVA-wt') (Fig. 5, left). The yields of MVA-mBNbc166 and MVA-res035 were not affected by doxycycline treatment over the 3-day infection course (compare Fig. 5, left and right). These results demonstrate that LMP1 / EBNA2 is a yield-reducing transgene in recombinant MVA and that doxycycline treatment does not affect the MVA infection cycle itself.

[0312] 4.5 Downregulation of PrH5m-induced expression of LMP1 / EBNA2 transposable genes

[0313] For the quantitative analysis of LMP1 / EBNA2 transgene downregulation, DF1-TR59 cells were seeded in 6-well plates the day before infection and treated with doxycycline (DOX) to inactivate TetR, or were not treated throughout the experiment starting from the day of seeding [day (-1)] (DOX untreated). DF1-TR59 cells were infected with recombinant MVA-res035 on day 0 at MOI 5. Cells were harvested at the indicated time points after infection by accutase treatment (see Fig. 6), and then fixed and permeated. MVA-res035-infected cells were stained with an EBNA2-specific antibody for the detection of transgene expression by flow cytometry (see Example 1.3).

[0314] In Fig. 6, LMP1 / EBNA2 and EGFP expression levels are denoted by the geometric mean fluorescence intensity [GMFI] of living cells. Here, the time-course kinetics of LMP1 / EBNA2 expression by MVA-res035 were performed by analyzing LMP1 / EBNA2 protein levels at 2, 4, 6, and 24 hours post-infection [pi]. LMP1 / EBNA2 levels at 2 hours post-infection [pi] were very similar depending on the presence or absence of TetR inactivation by doxycycline (Fig. 6a). At 4 and 6 hours post-infection [pi], LMP1 / EBNA2 expression levels were slightly higher under doxycycline treatment compared to the 'no DOX' condition, but again showed very similar levels at 24 hours post-infection [pi] (Fig. 6a).

[0315] Analysis of the expression of the unregulated reference transgene EGFP co-encoded by MVA-res035 indicated that cells had nearly identical EGFP expression levels regardless of treatment with doxycycline (Fig. 6b), and also confirmed that all cells were infected with very similar efficiency. Therefore, the moderate difference in LMP1 / EBNA2 levels (Fig. 6a) is most likely the result of TetR-mediated downregulation of LMP1 / EBNA2 expression.

[0316] The data shown in Figure 6a indicates a transient and very moderate downregulation of LMP1 / EBNA2 expression by active TetR at about 4–6 hours, which is the late start time of the MVA-res035 infection cycle.

[0317] Early poxvirus transcription is known to occur in the viral nucleus. Although TetR is a small protein, this can impair the access of cytoplasmic TetR to the viral genomic DNA that is still capsidized in the nucleus during early transcription. Therefore, specific modes of early poxvirus transcription may prevent regulation by TetR. After the coating of the incoming poxvirus nucleus is removed in the cytoplasm, viral DNA is released, and intermediate and late transcription occur on free cytoplasmic poxvirus genomic DNA to which cytoplasmic TetR can bind. Indeed, a more thorough evaluation of the expression kinetics of the PrH5m promoter has shown that the nominal early / late PrH5m promoter primarily induces early expression and possesses very moderate late activity. Therefore, the inventors' results, showing moderate downregulation in the initiation of intermediate and late gene expression at approximately 2–4 hours pi, are consistent with TetR effects limited to the late MVA replication phase.

[0318] 4.6 Effects of PrH5m-induced LMP1 / EBNA2 transgene downregulation on recombinant MVA replication

[0319] Viral multi-stage growth curve analysis of MVA-res035 in TetR-expressing DF1-TR59 cells was performed with and without doxycycline (see Figure 7). Viral infection in DF1-TR59 cells was initiated at an MOI of 0.05. Regardless of doxycycline addition, MVA-res035 reached a maximum yield, which was approximately 1.5 log lower than that obtained with MVA-mBNbc166 ('MVA-wt'). When TetR was activated ('DOX untreated'), yield increased only slightly on days 1 and 2. On the final day of the analysis, day 3, the yield of MVA-res035 was again very similar regardless of doxycycline treatment. Therefore, activated TetR exhibited only a mild and transient beneficial effect on the yield of MVA-res035. It can be assumed that mild and transient TetR-mediated downregulation of LMP1 / EBNA2 levels (see Fig. 6a) enables a slightly higher peak titer of MVA-res035 occurring earlier (day pi 2 vs. day pi 3) than undisturbed LMP1 / EBNA2 expression under doxycycline treatment conditions (Fig. 7).

[0320] The results confirm that LMP1 / EBNA2 are yield-reducing transgenes that potently degrade the replication kinetics and maximum yield of recombinant MVA-res035, respectively. Slight downregulation of LMP1 / EBNA2 expression induced by PrH5m by TetR showed only minor beneficial effects on MVA-res035 replication kinetics and yield.

[0321] Example 5: Effect of TetR on Early and Late EGFP Transgene Expression

[0322] To determine whether TetR is suitable for controlling late MVA-induced gene expression but less suitable or unsuitable for reducing early MVA-induced gene expression, we analyzed the expression of EGFP under early / late PrS promoters at both early and late stages.

[0323] The effects of TetR on early versus late expression were distinguished using treatment of MVA-res005-infected DF1-TR59 cells with cytosine arabinoside (araC) and / or doxycycline. Time-course kinetic experiments showed that at 2 hours pi, the late stage of the early phase, no difference in EGFP expression was detected regardless of the presence or absence of doxycycline treatment (Fig. 8a). Adding doxycycline (inactivating TetR) to MVA-res005-infected DF1-TR59 cells most rapidly increased EGFP expression at 4 hours pi compared to the 'DOX untreated' condition (i.e., repressed state) (Fig. 8a). At 24 hours pi, the latter part of the MVA replication cycle (i.e., when the late phase had progressed for 22 hours), PrS-induced EGFP expression was clearly downregulated (i.e., repressed state) by up to 3.6% without doxycycline (Fig. 8a).

[0324] Inhibition of viral replication in the early stages by araC treatment significantly reduced total EGFP expression (Fig. 8b), confirming that most of the EGFP expressed under the PrS promoter originates from late transcription in this promoter. No difference in EGFP expression levels was distinguished under araC regardless of doxycycline treatment (Fig. 8b), indicating that TetR does not significantly affect the very intermediate gene expression induced by the early components of the early / late PrS promoter.

[0325] The expression of the PrS-induced mRFP reference transgene, which is also encoded by MVA-res005 and not linked to the 2xTetO2 sequence (see Fig. 1), remained completely unaffected by the active TetR ('DOX untreated'), as demonstrated by comparison with the inactive TetR under doxycycline (Fig. 8c). AraC treatment significantly reduced the maximum mRFP expression induced by the PrS promoter and maintained it at a level similar to that in the early stages (i.e., pi up to 2 hours) (Fig. 8d), confirming that PrS primarily induces late expression. There was no difference in mRFP expression between doxycycline-treated and doxycycline-untreated cells (Fig. 8d), which was expected due to the absence of the 2xTetO2 sequence linked to the mRFP ORF.

[0326] In conclusion, TetR does not affect transgenic expression in the early portions of early / late poxvirus promoters, such as PrS, but efficiently downregulates expression in the late portions of the promoter. Therefore, the TetR system is most suitable for the downregulation of poxvirus transgenic expression induced primarily or completely late promoters.

[0327] In summary, data on both PrH5m and PrS-induced transposable gene expression demonstrated that while the TetR system does not exhibit downregulatory activity against early transposable gene expression induced by recombinant MVA, it is highly effective in downregulating late expression. Data showing significant inhibition of PrS-induced EGFP expression by TetR also indicate that expression in the very potent late poxvirus promoter can be effectively downregulated. Since the metaphase transcript is also generated from free cytoplasmic viral DNA following the degradation of the viral nucleus, gene expression in the metaphase poxvirus promoter is also expected to be efficiently inhibited by the cellularly expressed TetR.

[0328] Example 6: TetR-mediated regulation of yield-reducing LMP1 / EBNA2 transgenes induced primarily by the late PrS promoter

[0329] 6.1 Downregulation of PrS-induced expression of LMP1 / EBNA2 transgenes

[0330] Recombinant MVA-res058, which has the yield-reducing transgene LMP1 / EBNA2 under a late PrS promoter and a 2xTetO2 sequence between the promoter and the LMP1 / EBN2 ORF, was generated using TetR-expressing DF1-TR59 cells (see Fig. 1) to evaluate whether a large and efficient reduction of transgene expression in the late stage achieves a significantly high yield of recombinant MVA that primarily expresses the yield-reducing transgene in the late stage.

[0331] Additionally, we attempted to generate a reference recombinant MVA containing an LMP1 / EBNA2 ORF under a PrS promoter lacking a TetR binding site (MVA-res057, see Fig. 1). As with MVA-res034, which contains an LMP1 / EBNA2 ORF under a PrH5m promoter and lacks the 2xTetO2 sequence (see Example 4.3), the few small viral foci obtained after MVA-res057 recombination / reactivation did not proliferate sufficiently to produce a viral stock, and each recombinant died at passages 2–3 after recombination / reactivation, confirming the results with MVA-res034. These findings are most likely explained by the adverse effect of unhindered LMP1 / EBNA2 expression on MVA replication. In contrast, MVA-res058 having a 2xTetO2 sequence linked to the PrS-LMP1 / EBNA2 gene sequence can be easily generated using DF1-TR59 cells expressing TetR throughout the generation and proliferation process of MVA-res058.

[0332] PrS-induced LMP1 / EBNA2 transgene expression by MVA-res058 in DF1-TR59 cells with or without TetR activity was analyzed by flow cytometry ('DOX untreated' condition or doxycycline treated). 24 hours after infection with MVA-res058, LMP1 / EBNA2 signaling in doxycycline-free DF1-TR59 cells was downregulated by up to 14% compared to LMP1 / EBNA2 signaling in the presence of doxycycline (Fig. 9a). The downregulation of LMP1 / EBNA2 signaling in doxycycline-free DF1-TR59 cells was significantly more effective at the initial infection time of 6 hours pi (LMP1 / EBNA2 signal remained at 7%; see Fig. 9a).

[0333] MVA-res058 also expressed EGFP under the PrS promoter as a reference marker for infection and gene expression (see Fig. 1). Analysis of expression levels at 6 and 24 hours pi revealed that EGFP expression in cells not treated with doxycycline was not lower than EGFP expression in the presence of doxycycline at both time points (Fig. 9b), confirming that the low LMP1 / EBNA2 signal shown in Fig. 9a was not due to infection or a decrease in gene expression efficiency. Conversely, in DF1-TR59 cells incubated without doxycycline, i.e., under conditions of suppressed LMP1 / EBNA2 expression, reference EGFP expression by MVA-res058 was significantly higher at 24 hours pi than under doxycycline treatment (Fig. 9b). This can be explained by the fact that the negative effect on the expression of other viral genes, including the EGFP marker gene co-expressed by MVA-res058, was suppressed as LMP1 / EBNA2 expression was inhibited under 'DOX untreated' conditions.

[0334] In conclusion, TetR in DF1-TR59 cells was able to downregulate PrS-induced LMP1-EBNA2 expression by approximately 85–90% of the LMP1-EBNA2 expression levels obtained when TetR was inactivated by doxycycline.

[0335] 6.2 Effects of PrS-induced LMP1 / EBNA2 transgene downregulation on recombinant MVA replication

[0336] Growth curve experiments were performed to evaluate the replication of MVA-res058 with and without doxycycline in DF1-TR59 cells. As shown in Fig. 10, MVA-res058 expressing LMP1 / EBNA2 in the PrS promoter under doxycycline treatment produced only 3.2 x 10⁶ wells in DF1-TR59 cells on day 2 pi. 6 TCID 50 The highest yield was reached. This confirmed that the replication of recombinant MVA expressing LMP1 / EBNA2 under a late PrS promoter was also severely degraded, which was very similar to what was observed in MVA-res035 in which the PrH5m promoter induces LMP1 / EBNA2 expression (see Example 4.6).

[0337] MVA-res058 produced a 35-fold higher peak viral titer in DF1-TR59 cells when LMP1 / EBNA2 expression was inhibited by TetR ('DOX untreated' condition) compared to LMP1 / EBNA2 expression induced by MVA-res058 in the presence of doxycycline (Fig. 10). When comparing LMP1 / EBNA2 expression with TetR inhibition ('DOX untreated') and non-inhibited inhibition, the mean difference in MVA-res058 replication over days 1–3 of pi was 57-fold. In particular, MVA-res058 replication (i.e., inhibited LMP1 / EBNA2 expression) in DF1-TR59 cells without doxycycline was almost indistinguishable from MVA-mBNbc166 ('MVA-wt') replication with or without doxycycline (Fig. 10).

[0338] The approximately 85% efficiency of downregulation of LMP1 / EBNA2 expression (see Example 6.1), which clearly indicates incomplete downregulation of transgene expression, was sufficient to enable almost unhindered replication of MVA-res058 in DF1-TR59 cells expressing TetR.

[0339] Example 7: Downregulation of LMP1 / EBNA2 transgene expression in TetR expressing transgenic quail CCX.2C4 cells

[0340] 7.1 Generation of CCX.2C4-TR16 Cells

[0341] As described in Example 1.5, a TetR-expressing CCX.2C4 clone was produced by transfecting CCX.2C4 cells growing by attaching to a plasmid containing a blasticidin selection cassette and expressing TetR in a eukaryotic CMV promoter.

[0342] A total of 37 stably transfected CCX.2C4 cell clones exhibiting blasticidin-resistant toxicity were obtained. TetR expression was determined by immunoblotting using TetR-specific antibodies, and the blots were quantified using a luminescence-based ChemiDoc Touch system (see Example 1.6). Of the 37 clones analyzed, 6 showed TetR-specific signals equivalent to or higher than the respective signals observed in DF1-TR59 cells (Fig. 11a). The clone CCX.2C4-TR16 ('2C4-TR16' in Fig. 11a), which had high levels of TetR expression, was selected for further cell banking and development.

[0343] The effect of MVA-wt (MVA-BN wild-type) infection on TetR expression in CCX.2C4-TR16 cells was evaluated by immunoblot analysis in time-course kinetic experiments (Fig. 11b). The signal intensity of the TetR-specific band was analyzed using ImageQuant software and normalized to the level of the housekeeping protein β-tubulin. TetR expression levels showed some change during the infection process. However, clearly, TetR levels were not decreased in CCX.2C4-TR16 cells by MVA infection over the 24-hour infection process, but rather increased slightly (Fig. 11b). Specifically, cellular transcription of the TetR gene under the CMV promoter integrated into the cell genome was not significantly affected, or the TetR protein was very stable, so the decrease in transcription during infection would not alter TetR protein levels during the 24 hours of MVA infection.

[0344] 7.2 Downregulation of LMP1 / EBNA2 Transposable Gene Expression in CCX.2C4-TR16 Cells

[0345] The expression of the yield-reduced LMP1 / EBNA2 transgene was analyzed using a newly created adherent transformed cell line named CCX.2C4-TR16 expressing TetR (see Example 7.1). LMP1 / EBNA2 expression was evaluated under the early PrH5m promoter (MVA-res035, see Fig. 1) and the late PrS promoter (MVA-res058, see Fig. 1). Expression of neutral EGFP (MVA-res035, MVA-res058, see Fig. 1) or mRFP transgene (MVA-res005, see Fig. 1) under the PrS promoter in CCX.2C4-TR16 cells was used as a reference.

[0346] Without doxycycline treatment (i.e., TetR was active), EGFP expression at the PrS promoter in MVA-res005 was downregulated in quail CCX.22C4 cells at 24 hours pi (Fig. 12a, left), but the expression of the co-encoded PrS-mRFP gene was unaffected (Fig. 12a, right).

[0347] No decrease in LMP1 / EBNA2 transgene expression induced by activating TetR was observed in CCX.2C4-TR16 cells infected with MVA-res035 ('DOX untreated' condition) (Fig. 12b, left), where the LMP1 / EBNA2 transgene was primarily expressed at the early PrH5m promoter. The expression of the co-encoded neutral EGPF gene was unaffected (Fig. 12b, right).

[0348] In the case of MVA-res058, 51% of the remaining LMP1 / EBNA2 expression was observed under inhibition conditions ('DOX untreated') at 24 hours pi compared to LMP1 / EBNA2 expression in the presence of doxycycline (Fig. 12c, left). At 6 hours pi, TetR-mediated downregulation was much more efficient, with 27% of LMP1 / EBNA2 expression remaining (Fig. 12c, left). In MVA-res058, the expression levels of EGFP marker genes not regulated by TetR were not reduced in TetR-expressing CCX.2C4-TR16 cells regardless of doxycycline treatment (Fig. 12c, right). Conversely, EGFP expression under 'DOX untreated' conditions was much higher than that after doxycycline treatment, as previously observed in MVA-res058 grown in DF1-TR59 cells (see Example 6.1) (Fig. 12c, right), which appears to reflect the positive effect of the suppressed expression of the LMP1 / EBNA2 transgene that reduces yield in CCX.2C4-TR16 cells. Experiments evaluating reference MVA-mBNbc166, which also possesses a BAC cassette with the same EGFP expression cassette in CCX.2C4-TR16 cells, showed very similar EGFP expression in doxycycline-treated cells versus untreated cells, supporting the conclusion that, apart from TetR inactivation, the potential direct effect of doxycycline treatment on the CCX.2C4-TR16 cells themselves is not the cause of the observed difference in transgene expression.

[0349] Therefore, TetR expression in transformed CCX.2C4-TR16 cells was able to downregulate the LMP1 / EBNA2 transgene, which reduces yield and is primarily expressed late until the end of the MVA infection cycle.

[0350] 7.3 Effects of LMP1 / EBNA2 Transgene Downregulation on Recombinant MVA Replication in CCX.2C4-TR16 Cells

[0351] The effect of TetR-mediated downregulation of LMP1 / EBNA2 expression on the replication of MVA-res058 in CCX.2C4-TR16 cells was determined in multistep growth curve experiments (Fig. 13). BAC-derived MVA-mBNbc166 was used as a reference. When LMP1 / EBNA2 expression was enabled by adding doxycycline to infected CCX.2C4-TR16 cells, MVA-res058 did not replicate at all, and the titers of viral output during days 1–3 post-infection were much lower than those of the inoculum (Fig. 13). In contrast, when TetR was active ('DOX untreated' condition), MVA-res058 replicated to levels similar to those of the reference MVA ('MVA-wt', Fig. 13). This finding is noteworthy in that TetR-mediated downregulation of LMP1 / EBNA2 expression appears to be somewhat moderate during the infection cycle of MVA-res058 in TetR-expressing CCX.2C4-TR16 cells as previously described (see Example 7.2).

[0352] Doxycycline treatment again did not have a significant effect on the replication of reference MVA-mBNbc166 ('MVA-wt', Fig. 13). Therefore, such doxycycline treatment did not affect MVA replication and was not the cause of the significantly reduced replication of MVA-res058 when LMP1 / EBNA2 transgene expression was not suppressed, which was confirmed with MVA-res058 in TetR-expressing DF1-TR59 cells (see Example 6.2, Fig. 10).

[0353] Therefore, despite the apparent moderate effect of TetR-mediated downregulation on LMP1 / EBNA2 expression, this moderate downregulation was sufficient to nearly completely restore the replication of MVA-res058 to the level observed for the reference MVA-mBNbc166.

[0354] These findings demonstrate that downregulating MVA-induced transgene expression to varying degrees, ranging from strong to even slight, has the potential to have a highly dramatic effect on the final yield of each recombinant MVA. Since the strong expression of transgenes that degrade viral replication is also a potent adverse-selective factor for transgene retention, each recombinant virus is likely to rapidly accumulate mutations that lower the expression of each transgene or inactivate its yield-reducing activity through point mutations or deletions. Therefore, the genetic stability of recombinant MVAs containing yield-reducing transgenes will also be significantly increased by the downregulation of transgenes during passage and / or production.

[0355] Conclusion: Throughout the entirety of this specification, various documents have been cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturers' manuals, instructions, etc.) is taken by reference in its entirety. Where any material taken by reference contradicts or does not agree with this specification, this specification replaces such material. Nothing in this document shall be construed as an acknowledgment that the present invention is not qualified to precede this disclosure by any prior invention.

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[0373] 17. 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 79:1159-1167.

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[0387] order

[0388] Sequence No. 1 nucleic acid sequence of TetO2

[0389] TCCCTATCAGTGATAGAGA

[0390] Sequence No. 2 nucleic acid sequence of 2xTetO2

[0391] TCCCTATCAGTGATAGAGATATCCCTATCAGTGATAGAGA

[0392] Sequence No. 3 Nucleic acid sequence of the TetR gene

[0393] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCGCGTACAGCGGATCCCGGGAATTCAGATCTTATTAA

[0394] Sequence No. 4 TetR 유전자에 의해 암호화되는 아미노산 서열

[0395] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSGSREFRSY

[0396] Sequence No. 5 Nucleic acid sequence encoding the LMP1 / EBNA2 fusion protein

[0397]

[0398] Sequence number 6 LMP1 / EBNA2 융합 단백질의 아미노산 서열

[0399] MEHDLERGPPGPRRPPRGPPLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPLGALCILLLMITLLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWMYYHGQRHSDEHHHPTFYLALHGGQTYHLIVDTDSLGNPSLSVIPSNPYQEQLSDTPLIPLTIFVGENTGVPPPPPPQRRDAWTQEPSPLDRDPLGYDVGHGPLASAMRMLWMANYIVRQSRGDRGLILPQGPQTAPQARLVQPHVPPLRPTAPTILSPLSQPRLTPPQPLMMPPRLELEPTPPTPLPPATLTVPPRPTRPTTLPPTPLLTVLQRPTELQPTPSPPRMHLPVLHVPDQSMHPLTHQSTPNDPDSRARAPEPRSPTVFYNIPPMPLPPSQLPPPAAPAQPPPGVINDQQLHHLPSGPPWWPPICDPPQPSKTQGQSRKSRDKQRKPGGPWRPEPNTSSPSMPELSPVLGLHQGQGAGDSPTPGPSNAAPVCRNSHTATPNVSPIHEPESHNSPEAPILFPDDWYPPS

[0400] Sequence No. 7 EGFP 유전자의 핵산 서열

[0401] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

[0402] Sequence No. 8 EGFP 유전자에 의해 암호화된 아미노산 서열

[0403] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0404] Sequence number 9 Nucleic acid sequence encoding the gpt-mRFP fusion protein

[0405]

[0406] Sequence number 10 npt II를 암호화하는 핵산

[0407] ATGGGATCGGCCATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA

[0408] Sequence number 11 PrS 프로모터

[0409] AAAAATTGAA ATTTTATTTT TTTTTTTTGG AATATAAATA

[0410] Sequence No. 12 PrH5m 프로모터

[0411] TACTTAAAAA TTGAAAATAA ATACAAAGGT TCTTGAGGGT TGTGTTAAAT TGAAAGCGAG AAATAATCAT AAATAATTTC ATTATCGCGA TATCCGTTAA GTTTGTATCG TA

Claims

Claim 1 A recombinant poxvirus comprising a nucleotide sequence including a viral yield reduction transgenic gene linked to a poxvirus promoter in an operable state, wherein the nucleotide sequence further comprises a binding sequence to a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame (ORF) of the transgenic gene. Claim 2 A transcription unit comprising a nucleotide sequence comprising a viral yield reduction transgenic gene linked in an operable state to a poxvirus promoter, wherein the nucleotide sequence further comprises a binding sequence to a transcription repressor protein, and the binding sequence is located between the poxvirus promoter and the open reading frame [ORF] of the transgenic gene. Claim 3 In claim 1 or 2, the poxvirus promoter is a recombinant poxvirus or transcription unit that is primarily or completely late-stage promoter or mid-stage promoter. Claim 4 In paragraph 3, the recombinant poxvirus or transcription unit, wherein the main or fully late promoter is selected from the group consisting of promoters Pr11, Pr7,5, PrSSL, PrATI, and PrS, and preferably the poxvirus promoter is the main late promoter PrS. Claim 5 A recombinant poxvirus or transcription unit according to claim 1 or 2, wherein the poxvirus promoter is primarily an early promoter, preferably selected from the group consisting of the promoter long Pr13.5 / Pr13.5, PrHyb, Pr1328 and PrH5m, and more preferably the poxvirus promoter is primarily an early promoter PrH5m. Claim 6 A recombinant poxvirus or transcription unit according to any one of claims 1 and 3 to 5, or any one of claims 2 to 5, wherein the transcription repressor protein is one of the tetracycline repressor (TetR) or lactose repressor (LacR) series, preferably the tetracycline repressor (TetR) series, and more preferably the transcription repressor protein is the tetracycline repressor (TetR) protein. Claim 7 A recombinant poxvirus or transcription unit according to any one of claims 1 and 3 to 6, or any one of claims 2 to 6, wherein the binding sequence to the transcription repressor protein comprises two copies of a Tet operator 2 (TetO2) nucleotide sequence separated by a 2-base spacer, wherein the TetO2 nucleotide sequence preferably follows SEQ ID NO.

1. Claim 8 Any one of claims 1 and 3 to 7, or any one of claims 2 to 7, wherein the binding sequence to the transcription repressor protein comprises or is composed of a nucleic acid sequence according to SEQ ID NO. 2, a recombinant poxvirus or transcription factor. Claim 9 In any one of claims 1 and 3 to 8, or any one of claims 2 to 8, the yield-reducing transgene is a recombinant poxvirus or transcription factor encoding a disease-related antigen, preferably an antigen related to an infectious disease or cancer. Claim 10 In claim 9, the disease-related antigen is derived from Epstein-Barr virus (EBV) or its antigenic portion, preferably selected from the group consisting of EBV proteins BLLF1a / b (gp350 / 220), BALF4 (gB, gp110), BXLF2 (gH, gp85), BKRF2 (gL, gp25), BZLF2 (gp42), BILF2 (gp78), BDLF3 (gp150), BBBRF3 (gM), BLRF1 (gN), BMRF2, EBNA1, EBNA2, EBNA3, LMP1, LMP2, BRLF1, or BZLF1, a recombinant poxvirus or transcription factor. Claim 11 A recombinant poxvirus according to any one of claims 1 and 3 to 10, which is a recombinant vaccinia virus, preferably a modified vaccinia virus Ankara (MVA). Claim 12 A transformed poxvirus producer cell derived from a serial poxvirus-allowed cell line, wherein the transformed cell is genetically modified to express a transcription repressor protein. Claim 13 In paragraph 12, the transgenic poxvirus producer cell, wherein the continuous poxvirus-allowed cell line is an avian cell line, preferably a chicken, duck, or quail cell line, and more preferably a chicken DF-1 or quail CCX cell line. Claim 14 In paragraph 12 or 13, a transformed poxvirus producer cell that is a vaccinia virus producer cell, preferably an MVA producer cell. Claim 15 A transformed poxvirus producer cell according to any one of claims 12 to 14, wherein the transcription repressor protein is one of the tetracycline repressor (TetR) or lactose repressor (LacR) family, preferably the tetracycline repressor (TetR) family, and more preferably the transcription repressor protein is the tetracycline repressor (TetR) protein. Claim 16 A use of a transformed poxvirus producer cell of any one of claims 12 to 15 for propagating a recombinant poxvirus of any one of claims 1 and 3 to 11, wherein the binding sequence for a transcription repressor protein contained in the recombinant poxvirus can bind to the transcription repressor protein expressed by the transformed poxvirus producer cell, and the transcription repressor protein expressed by the transformed poxvirus producer cell can bind to the binding sequence for a transcription repressor protein contained in the recombinant poxvirus. Claim 17 A recombinant poxvirus proliferated in a transformed poxvirus producer cell according to any one of claims 1 and 3 to 11, wherein the binding sequence for a transcription repressor protein included in the recombinant poxvirus can bind to the transcription repressor protein expressed by the transformed poxvirus producer cell, and the transcription repressor protein expressed by the transformed poxvirus producer cell can bind to the binding sequence for a transcription repressor protein included in the recombinant poxvirus. Claim 18 A method for multiplying a recombinant poxvirus, comprising: (1) providing a recombinant poxvirus of any one of claims 1 and 3 to 11; (2) providing a transformed poxvirus producer cell of any one of claims 12 to 15; (3) infecting the transformed cell provided in step (2) with the recombinant poxvirus provided in step (1); (4) culturing the transformed cell of step (3) to multiply the recombinant poxvirus; and (5) harvesting the recombinant poxvirus multiplied in step (4), wherein the binding sequence for a transcription repressor protein included in the recombinant poxvirus provided in step (1) can bind to the transcription repressor protein expressed by the transformed poxvirus producer cell provided in step (2), and the transcription repressor protein expressed by the transformed poxvirus producer cell provided in step (2) can bind to the binding sequence for a transcription repressor protein included in the recombinant poxvirus provided in step (1). Claim 19 A pharmaceutical composition or vaccine comprising a recombinant poxvirus according to any one of claims 1 and 3 through 11, wherein the pharmaceutical composition or vaccine further comprises optionally a pharmaceutically acceptable carrier or excipient. Claim 20 A recombinant poxvirus of any one of paragraphs 1 and 3 through 11 for use in the treatment or prevention of infectious diseases or cancer. Claim 21 In paragraph 20, the infectious disease is a recombinant poxvirus, which is an Epstein-Barr virus (EBV) infection. Claim 22 Use of a transcription repressor protein and a corresponding binding sequence for controlling the expression of a transgenic gene induced by a mainly or fully late or mid-phase poxvirus promoter with a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant MVA.

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