Using microRNAs to down-regulate cytotoxic transgene expression by Modified Vaccinia Virus Ankara (MVA)

Abstract

The present invention relates to a recombinant modified vaccinia virus Ankara (MVA) comprising a series of miRNA target sequences arranged in a miRblock linked to a transgene, each miRNA target sequence corresponding to a miRNA in a eukaryotic MVA producing cell. The present invention also relates to medical uses of the recombinant MVA.

Description

[Technical field]

[0001] The present invention relates to the field of viral vectors, in particular to viral vector-based vaccines. More specifically, the present invention relates to a recombinant modified vaccinia virus Ankara (MVA) that contains a set of microRNA (miRNA) target sequences arranged in a so-called miRblock linked to a transgene, each miRNA target sequence corresponding to a miRNA expressed in eukaryotic MVA-producing cells. The present invention also relates to the medical use of the recombinant MVA. [Background technology]

[0002] A common problem with recombinant viral vectors is the adverse effect of transgene products expressed by such vectors on cellular processes in vector-producing cells. This ultimately leads to impaired yield of a given recombinant viral vector (1). Cytotoxic effects can be the result of expression of single or multiple transgenes, or of combinations of transgene products that do not show cytotoxic effects when expressed separately. The problem of cytotoxic transgene products reducing viral vector yield is also relevant for recombinant MVA vectors derived from vaccinia virus, the prototype species of the genus Orthopoxvirus in the family Poxviridae. For example, replication defects have been observed in HIV-env-expressing MVA (2).

[0003] MVA-BN® is a well-characterized viral vector isolated from the MVA (modified vaccinia virus Ankara) virus stock. MVA originates from the replicating vaccinia virus, cutaneous vaccinia virus Ankara strain (Chorioallantois vaccinia virus Ankara (CVA)) (3). The attenuated CVA-derived virus MVA was obtained by continuously propagating CVA in primary chicken embryo fibroblasts (CEF or CEF cells) for over 570 passages. Further passage of this MVA by Bavarian Nordic resulted in a further attenuated MVA strain, MVA-BN® (4). MVA-BN® lacks approximately 15% of the genome compared to the ancestral CVA virus (31 kb missing, resulting in six major deletion sites). These deletions affect multiple virulence and host range genes as well as genes in the type A inclusion body. MVA-BN® is able to attach to, enter, and express virus-encoded genes very efficiently in human cells, without the assembly and release of progeny viruses in human cells. As such, MVA-BN® is an important and versatile vaccine vector that can efficiently express antigen-encoding transgenes for use in vaccination approaches targeting diseases with previously unmet medical needs, such as Ebola virus disease (5). Preparations of MVA-BN® and its derivatives have been administered to many types of animals and to over 10,500 human subjects, including immunocompromised patients, in clinical trials without any serious adverse events.

[0004] The reduction in viral yield of some MVA recombinants expressing cytotoxic transgenes can vary over a wide range, ranging from a marked reduction in viral yield to severe replication defects, and even the inability to generate recombinant MVA expressing a particular transgene. Recombinant MVA replication defects can be caused by a single highly cytotoxic transgene alone, but can also be caused by a combination of multiple transgenes. Although these transgenes may not appear to be cytotoxic individually upon MVA-mediated expression, their minimal cytotoxic effects appear to add up or even synergize to significantly reduce MVA yield. The inability to generate recombinant MVA containing a particular transgene may be due to a selective disadvantage that the deleterious transgene confers on the replication process of the MVA recombinant, to the extent that it cannot be successfully selected and isolated from the parental MVA background due to inefficient replication. Furthermore, the genetic stability of the transgenic insert or genome of the viral vector expressing this transgene can be compromised by the expression of the deleterious transgene during viral vector production (1, 2).

[0005] Therefore, there is a need for recombinant MVA capable of expressing multiple potentially cytotoxic transgenes while minimizing the loss of viral replicative capacity.

[0006] We aimed to assess whether the microRNA machinery of MVA vector-producing cells could be exploited to downregulate transgene expression during recombinant MVA production.

[0007] MicroRNAs (miRNAs) are small non-coding RNAs, typically 21–23 nucleotides (nt) long, that are produced by all eukaryotic cells for post-transcriptional control of gene expression. These RNAs are encoded in the cellular genome as long non-coding RNAs of 100–1000 nucleotides, called primary miRNAs (pri-miRNAs), which are trimmed in the nucleus by the RNase Drosha to approximately 80 nt hairpin structures, called precursor miRNAs (pre-miRNAs). These hairpin RNAs are further trimmed in the cytoplasm by the RNase Dicer to yield mature 21–23 nt miRNAs. Mature miRNAs are loaded into the so-called RISC multiprotein complex, which mediates miRNA action. Binding of miRNAs to their cognate target sequences in the coding sequence or 5'- or 3'-untranslated regions (UTRs) of cellular mRNAs leads to reduced translation in the case of imperfect matches, or even mRNA degradation in the case of perfect matches. In the latter process, miRNAs that mediate mRNA degradation are not themselves degraded but are internalized by the microRNA effector machinery. Thus, they can initiate a new cycle of mRNA degradation by recognizing their target sequences. Notably, downregulation of cellular protein levels by miRNAs is usually less than two-fold (6, 7), but these effects can be enhanced, for example, by perfect target matching and by tandem arrangement of target sequences (8).

[0008] Many miRNA genes are highly conserved throughout the animal kingdom, while lineage-, species-, and even tissue-specific miRNAs exist. There are several examples where viruses have been engineered to be targeted by cellular miRNAs with the general goal of achieving specific tissue tropism or preventing replication in non-tumor tissues. Examples of successful replication restriction include poliovirus, which was modified by the insertion of two separate single miRNA target sequences, and vesicular stomatitis virus (VSV), which was modified with three target sequences. These studies demonstrate that in vivo viral replication can be controlled by miRNAs (9, 10). Similarly, attenuation of influenza virus or oncolytic picornaviruses was achieved by inserting species- or tissue-specific microRNA target sequences into the viral genome (11, 12). It has also been shown that species-specific attenuation in mice (but not in chicken eggs) of influenza A virus was achieved by inserting miRNA-93 target sequences into the influenza ORF (13).

[0009] In addition to deliberate attenuation of viral replication by miRNA targeting, the microRNA machinery has also been used to suppress transgene expression in viral gene therapy vectors. Packaging of adeno-associated virus (AAV) vector genomes into vector particles can be severely compromised by expression of the encoded transgene under the control of a eukaryotic promoter if the transgene product is cytotoxic. miRNA-mediated downregulation of cytotoxic transgene expression increased the packaging efficiency and yield of the respective AAV (14). So far, this approach has been suggested to be effective only by overexpressing artificial miRNAs (amiRNAs) in pri-miRNA-like scaffolds (14) or as short hairpin RNAs (shRNAs) (15) to enhance AAV yield. Typically, AAV vectors are generated by transfecting a set of plasmids encoding the vector genome and helper functions, so it was technically feasible to co-transfect amiRNA- or shRNA-expressing plasmids. However, miRNA transfection approaches are not feasible for MVA or poxvirus-based vectors in general, which grow by infection of sensitive producer cells. For CEF-adapted MVA, the common producer cells are primary CEF cells or some avian cell lines, such as the chicken continuous fibroblast cell line DF-1. Primary CEFs exhibit very limited transfection efficiency, so the transfection procedure is generally not ideal for industrial-scale production processes.

[0010] Therefore, we aimed to utilize intracellular miRNAs in primary CEFs to downregulate the expression of transgenes driven by MVA vectors during vector production, thereby achieving improved yields of recombinant MVA for vaccination purposes.

[0011] Publicly available knowledge regarding the interplay between the miRNA machinery and vaccinia virus infection is limited. Vaccinia virus appears to downregulate the cellular miRNA machinery at various levels, including induction of miRNA degradation (16, 17) and downregulation of the intracellular RNase Dicer, which is required for miRNA biogenesis (17, 18). This suggested that miRNAs are not suitable for controlling vaccinia virus-driven transgene expression. There is an example of targeted miRNA-mediated downregulation of expression of a vaccinia virus protein named B5, where the goal was to impair B5-dependent vaccinia virus morphogenesis and thus progeny virus production (19, 20). However, the early / late poxvirus B5 promoter, which drives expression of the B5R gene, is not a very strong promoter (21, 22). In contrast, the problem of controlling poxvirus-mediated transgene expression in producer cells is compounded by the fact that the poxvirus promoters used to drive transgene expression are purposefully designed and selected to be as strong as possible in order to direct the synthesis of the maximum amount of recombinant protein that will serve as an immunogen (e.g., the widely used synthetic early / late PrS promoter (23) and the strong immediate-early promoter Pr13.5long (24)). Summary of the Invention

[0012] It is an object of the present invention to provide means and methods for producing recombinant MVA in high yields.

[0013] The problem underlying the present invention is solved by providing a recombinant MVA modified such that expression of a transgene is downregulated during propagation of the MVA in susceptible producer cells. In particular, the present invention is defined by the appended claims as well as the following aspects and embodiments thereof.

[0014] In a first aspect, the present invention provides a recombinant modified vaccinia virus Ankara (MVA) comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a miRNA target sequence linked to the transgene, the miRNA target sequence corresponding to a miRNA in a eukaryotic MVA-producing cell.

[0015] In a first aspect, the present invention provides a recombinant modified vaccinia virus Ankara (MVA) comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a series of miRNA target sequences arranged within a miRblock linked to the transgene, each miRNA target sequence within the miRblock corresponding to a miRNA in a eukaryotic MVA-producing cell.

[0016] In a further aspect, the present invention provides a recombinant MVA comprising a transcription unit comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a miRNA target sequence linked to the transgene or a series of miRNA target sequences located within a miRblock linked to the transgene, wherein each miRNA target sequence or each miRNA target sequence within a miRblock corresponds to a miRNA in a eukaryotic MVA producing cell.

[0017] In yet a further aspect, the present invention provides a recombinant MVA comprising a first and a second transcription unit, or more transcription units, each transcription unit comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising an miRNA target sequence linked to the transgene, or a series of miRNA target sequences located within a miRblock linked to the transgene, wherein the miRNA target sequence, or each miRNA target sequence within the miRblock, corresponds to a miRNA in a eukaryotic MVA producing cell.

[0018] In another aspect, the present invention provides a transcription unit comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, preferably suitable for use in recombinant MVA, the nucleotide sequence further comprising a miRNA target sequence linked to the transgene or a series of miRNA target sequences located within a miRblock linked to the transgene, wherein each miRNA target sequence or each miRNA target sequence within a miRblock corresponds to a miRNA in a eukaryotic MVA producing cell.

[0019] In yet another aspect, the present invention provides a series of miRNA target sequences arranged within a miRblock, preferably suitable for use in recombinant MVA, each miRNA target sequence corresponding to a miRNA in a eukaryotic MVA-producing cell.

[0020] In yet another aspect, the present invention provides a plasmid comprising a nucleotide sequence comprising a transgene operably linked to a promoter active in a eukaryotic production cell, the nucleotide sequence further comprising a miRNA target sequence linked to the transgene or a series of miRNA target sequences located within a miRblock linked to the transgene, wherein each miRNA target sequence or each miRNA target sequence within a miRblock corresponds to a miRNA in the eukaryotic production cell.

[0021] In yet another aspect, the present invention provides a method for producing a recombinant MVA according to the present invention, comprising the steps of: (1) providing a miRNA target sequence or a series of miRNA target sequences located within a miRblock according to the present invention; (2) preparing a transcription unit according to the present invention using the miRNA target sequence or miRblock prepared in step (1); (3) inserting the transcription unit prepared in step (2) into MVA; (4) infecting and propagating eukaryotic MVA-producing cells with the MVA obtained in step (3); (5) harvesting the recombinant MVA propagated in step (4).

[0022] In yet another aspect, the present invention provides a recombinant MVA produced by a process according to the present invention.

[0023] In yet another aspect, the present invention provides the use of a recombinant MVA according to the invention for the industrial-scale production of a vaccine.

[0024] In yet another aspect, the present invention provides the use of a miRNA target sequence according to the present invention for downregulating expression of an MVA-encoding transgene in a eukaryotic MVA-producing cell, in particular for industrial-scale production of a vaccine.

[0025] In yet another aspect, the present invention provides the use of a set of miRNA target sequences located within a miRblock according to the present invention for downregulating expression of an MVA-encoding transgene in a eukaryotic MVA producing cell, in particular for large-scale production of a vaccine.

[0026] In yet another aspect, the present invention provides a pharmaceutical composition or vaccine comprising a recombinant MVA according to the invention, and optionally further comprising a pharma- ceutically acceptable carrier or excipient.

[0027] In yet another aspect the present invention provides a recombinant MVA according to the invention for use as a medicament or vaccine, preferably for use in the treatment or prevention of a disease.

[0028] In yet another aspect, the present invention provides a recombinant MVA according to the present invention for use in the treatment or prevention of an infectious disease or cancer.

[0029] In yet another aspect, the present invention provides the use of a recombinant MVA according to the invention for the manufacture of a medicament or vaccine for use in the treatment or prevention of an infectious disease or cancer.

[0030] In yet another aspect, the present invention provides a method for treating or preventing an infectious disease or cancer in a subject, the method comprising administering to the subject a recombinant MVA according to the present invention.

[0031] These aspects and embodiments thereof are described in further detail below in conjunction with the description of the invention. [Brief description of the drawings]

[0032] [Figure 1] Figure 1 shows the design of plasmid inserts expressing EGFP and containing miRNA target sequences placed within a hetero-oligomeric miRblock in the EGFP 3'-UTR. EGFP plasmids without miRNA target sequences ("EGFP without miRb") (A), with hetero-oligomeric miRblock-1 (B) and -2 (C), and with a control miRblock containing four scrambled miRNA target sequences ("EGFP-scrbl2") (D). pCMV = human cytomegalovirus immediate early promoter / enhancer; EGFP = enhanced green fluorescent protein; SV40 polyA = polyadenylation signal from simian virus-40; nt = nucleotides; ORF = open reading frame. [Diagram 2]Figure 1 shows a comparison of the effect of miRblock on plasmid-driven EGFP expression in primary CEF cells. CEF cells in VP-SFM medium were seeded in 96-well plates at 37 °C on day 0 (4 × 104 cells / well). Cells were co-transfected with EGFP- and blue fluorescent protein (BFP)-encoding plasmids in triplicate on day 1. EGFP-encoding plasmids carrying 10 different hetero-oligomeric miRblocks in the 3'-UTR of the EGFP gene were named miRb-1 to miRb-10 (see Table 4 for miRNA target sequences). Transfection with EGFP-encoding plasmid without miRblock served as the reference for EGFP expression ("no miRb"). Cells were analyzed for EGFP and BFP expression by flow cytometry on day 2. Geometric mean fluorescence intensity (GMFI) of EGFP (top) and BFP (bottom) of BFP-positive cells is shown (error bars indicate geometric mean (GM) and geometric standard deviation (geoSD)). The percentages indicate the % EGFP expression level relative to the EGFP expression standard without miRblock. [Diagram 3] Analysis of the effect of miRblock on EGFP expression at 30°C and 37°C after plasmid transfection in CEF and DF-1 cells is shown. CEF cells in VP-SFM medium (left) and DF-1 cells in DMEM / 10% FCS (right) were seeded in 96-well plates (4x104 cells / well) on day 0. Cells were co-transfected with plasmids encoding EGFP ("miRb-1", "miRb-2", miRblock control "scrbl") and BFP in triplicate on day 1. Transfection with plasmid encoding EGFP without miRblock served as EGFP expression baseline ("no miRb"). Cells were incubated at 30°C or 37°C and EGFP and BFP expression was analyzed by flow cytometry 23 hours after transfection. GMFI of EGFP (top) and BFP (bottom) in BFP positive cells is shown (GM and geoSD). % EGFP expression level relative to EGFP expression baseline. [Figure 4]1 shows the design of a recombinant MVA insert encoding EGFP and containing a miRNA target sequence located within a hetero-oligomeric miRblock in the EGFP 3'-UTR. Recombinant MVA encoding EGFP without a miRNA target sequence ("EGFP"), with hetero-oligomeric miRblock-1 and -2 ("EGFP-miRb-1", "EGFP-miRb-2"), and with a control miRblock ("EGFP-scrbl2"). PrS = synthetic poxvirus early / late promoter; RFP = red fluorescent protein; gpt = guanine phosphoribosyltransferase; nt = nucleotides; TTS = early transcription termination signal; IGR = intergenic region within the MVA genome. [Diagram 5] Analysis of the effect of miRblock on EGFP expression levels with recombinant MVA is shown. CEF cells in VP-SFM medium (left) and DF-1 cells in DMEM / 10% FCS (right) were seeded in 96-well plates (4x104 cells / well) on day 0. On day 1, in triplicate, cells were infected with EGFP- and RFP-expressing MVA-BN recombinants containing no miRblock ("no miRb", EGFP expression reference), miRblock-1 or -2 ("miRb-1", "miRb-2"), or miRblock-scrbl2 control ("scrbl2") at a multiplicity of infection (MOI)=5. During infection, cells were incubated at 30°C or 37°C. 19 hours after infection, cells were trypsinized, washed, and resuspended in PBS-FACS-FORM (1% FCS, 0.1% NaN3, 1% paraformaldehyde (PFA)). EGFP and RFP expression was analyzed by flow cytometry. GMFI of EGFP (top) and RFP (bottom) in RFP positive cells is shown (GM and geoSD). EGFP expression level % relative to EGFP expression baseline. [Figure 6]Analysis of the effect of miRblock on EGFP expression after infection with recombinant MVA at different MOIs is shown. CEF cells (left) in VP-SFM medium (left) and DF-1 cells (right) in DMEM / 10% FCS (right) were seeded in triplicate in 96-well plates (4x104 cells / well) on day 0. On day 1, cells were infected with EGFP- and RFP-expressing MVA-BN recombinants containing no miRblock ("no miRb", EGFP expression reference), miRblock-1 or -2 ("miRb-1", "miRb-2"), or miRblock-scrbl2 control ("scrbl2") at a multiplicity of infection (MOI) of 5, 1, or 0.2 as indicated. Six hours after infection, cells were trypsinized, washed, and resuspended in PBS-FACS-FORM (1% FCS, 0.1% NaN3, 1% PFA). EGFP expression was analyzed by flow cytometry. GMFI of EGFP (top; GM and geoSD) and GMFI% of EGFP expression level relative to EGFP expression baseline (bottom; mean and standard error of the mean (SEM)) are shown. RFP expression was not recorded. [Figure 7] Figure 1 shows the analysis of the time-course effect of miRblock on EGFP expression (multiple cycles of replication) after infection with recombinant MVA at a low MOI. CEF cells in VP-SFM medium were seeded in triplicate in 96-well plates (4x104 cells / well) on day 0. On day 1, cells were infected with EGFP- and RFP-expressing MVA-BN recombinants containing no miRblock ("no miRb"), miRblock-1 or -2 ("miRb-1", "miRb-2"), or miRblock-scrbl2 ("scrbl2", control) at an MOI of 0.1. At the indicated times post-infection, cells were trypsinized, washed, and resuspended in PBS-FACS-FORM (1% FCS, 0.1% NaN3, 1% PFA). EGFP and RFP expression was analyzed by flow cytometry. The SGMFI (GM and geoSD) of EGFP (top left) and RFP (top right) of RFP positive cells, as well as GMFI EGFP% and GMFI RFP% relative to the EGFP expression standard are shown (bottom; mean and SEM). [Figure 8] Figure 2 shows the design of recombinant MVA inserts containing miRNA target sequences arranged within a hetero-oligomeric miRblock and expressing EGFP under different promoters. PrS = synthetic poxvirus early / late promoter; Pr13.5long = immediate early promoter. [Figure 9] Comparison of the effect of miRblock on EGFP expression under different poxvirus promoters is shown. CEF cells in VP-SFM medium (left) and DF-1 cells in DMEM / 10% FCS (right) were seeded in 96-well plates on day 0 (4x104 or 3x104 cells / well). On day 1, in triplicate, cells were infected with EGFP- and RFP-expressing MVA-BN recombinants containing miRblock-2 ("miRb-2") or miRblock-scrbl2 ("scrbl2", control) and a miRNA-targeted EGFP gene under the control of either the PrS promoter or the Pr13.5long promoter (MOI=10). At 6 and 18 hours post-infection, cells were trypsinized, washed, and resuspended in PBS-FACS-FORM (1% FCS, 0.1% NaN3, 1% PFA). EGFP and RFP expression was analyzed by flow cytometry. Shown are the GMFI% of EGFP relative to the EGFP expression level in RFP-positive cells infected with the "scrbl2" control (mean and SEM). PrS or Pr13.5long promoters and infection times as indicated. % of EGFP expression level relative to the "scrbl2" control. [Figure 10]A comparison of the effects of hetero- and related homo-oligomeric miRblocks on plasmid-driven EGFP expression is shown. CEF cells (4x104 cells / well) in VP-SFM medium were seeded in 96-well plates on day 0. Cells were co-transfected with EGFP- and BFP-encoding plasmids in triplicate on day 1. EGFP expression was analyzed from plasmids containing hetero-oligomeric miRblock-1 or -2 ("miRb-1", "miRb-2") or homo-oligomeric miRblock-13-20. miRblock-13-16 contained three repeats of the miRNA target sequence contained in miRblock-1, and miRblock-17-20 contained four repeats of the miRNA target sequence contained in miRblock-2. A plasmid containing no miRblock ("no miRb") served as a reference for EGFP expression, and a plasmid containing miRblock-scrbl2 ("scrbl2") served as a control. Shown are GMFI (GM and geoSD) of EGFP (top) and BFP (bottom) in BFP-positive cells quantified by flow cytometry 1 day after transfection. Percentage of EGFP expression relative to baseline EGFP expression. A set of plasmids containing miRblock was analyzed in two separate experiments. [Figure 11] Figure 2 shows the design of recombinant MVA inserts expressing EGFP and containing selected miRNA target sequences from miRblock-2 positioned within homo-oligomeric miRblocks. Recombinant MVA without miRNA target sequence ("EGFP"), with hetero-oligomeric miRblock-2 ("EGFP-miRb-2"), with homo-oligomeric miRblock-17 or -18 ("EGFP-miRb-17", "EGFP-miRb-18"), and with control miRblock ("EGFP-scrbl2"). [Figure 12]Comparison of the effect of hetero- and related homo-oligomeric miRblocks on EGFP expression by recombinant MVA is shown. CEF cells in VP-SFM were seeded in 96-well plates (4x104 cells / well) on day 0. On day 1, cells were infected with MVA-BN recombinants (MOI=5) containing hetero-oligomeric miRblock-2 ("miRb-2") or homo-oligomeric miRblock-17 or -18 ("miRb-17", "miRb-18"). miRblock-17 and -18 contained 4 repeats of the miRNA target sequence contained in miRblock-2. 18 hours after infection, cells were trypsinized, washed, and resuspended in PBS-FACS-FORM (1% FCS, 0.1% NaN3, 1% PFA). EGFP and RFP expression was analyzed by flow cytometry. GMFI of EGFP (left) and RFP (right) expression in RFP positive cells is shown (GM and geoSD). % EGFP expression levels relative to EGFP expression baseline ("no miRb"). [Figure 13]Comparison of plasmid-driven EGFP expression controlled by homo-oligomeric miRblocks designed based on selected miRNAs is shown. CEF cells (4x104 cells / well) in VP-SFM medium (left) and DF-1 cells (3x104 cells / well) in DMEM / 10% FCS (right) were seeded in 96-well plates on day 0. Cells were transfected with EGFP and BFP expression plasmids in triplicate on day 1. EGFP expression by plasmids containing homo-oligomeric miRblock-25 to -36, composed of four repeats of the miRNA target sequence, was analyzed. Plasmids containing hetero-oligomeric miRblock-1 or -2 were included for comparison. Plasmids without miRblock ("no miRb") served as the EGFP expression reference, and plasmids containing miRblock-scrbl2 ("scrbl2") served as the control. Shown are GMFI (GM and geoSD) of EGFP (top) and BFP (bottom) of BFP positive cells quantified by flow cytometry 1 day after transfection. % EGFP expression relative to EGFP expression baseline ("no miRb"). Datasets including miRblock-25 and 26 data are from independent experiments. [Figure 14]Analysis of EGFP downregulation in cells infected with recombinant MVA containing hetero-oligomeric miRblocks composed of the most effective miRNA target sequences is shown. CEF cells (4x104 cells / well) in VP-SFM medium (left) and DF-1 cells (3x104 cells / well) in DMEM / 10% FCS (right) were seeded in 96-well plates on day 0. Cells were transfected with EGFP and BFP expressing plasmids containing hetero-oligomeric miRblocks in triplicate on day 1. miRblock-37 to -47 were composed of miRNA target sequences from homo-oligomeric miRblock-13, -17-, -18, -20 (see FIG. 10) and miRblock-25, -26, -31 to -33 (see FIG. 13). Plasmid containing hetero-oligomeric miRblock-2 was included for comparison. Plasmid without miRblock ("No miRb") served as EGFP expression baseline, and plasmid with miRblock-scrbl2 ("scrbl2") served as control. Cells were analyzed for EGFP and BFP expression by flow cytometry 24 hours after transfection. GMFI (GM and geoSD) of EGFP (top) and BFP (bottom) in BFP positive cells are shown. % EGFP expression relative to EGFP expression baseline ("No miRb"). [Figure 15]Figure 1 shows the design of recombinant MVA-BN-RSV and its modified versions containing a miRblock within the 3'-UTR of an RSV-derived transgene. MVA-BN recombinants encoding RSV-derived transgenes under the control of different promoters ("MVA-BN-RSV", "MVA-BN-RSV-miRb1 / 2", "MVA-BN-RSV-miRb39 / 41") are illustrated as indicated. In MVA-BN-RSV, the encoded RSV-derived transgene is not linked to the miRblock. In MVA-BN-RSV-miRb1 / 2 and MVA-BN-RSV-miRb39 / 41, all three or four transgenes, respectively, are linked to a hetero-oligomeric miRblock. G(A), G(B) = ORFs of the RSV G protein in the A and B serotypes; N = RSV nucleoprotein ORF; 2A = picornavirus-derived self-cleaving 2A peptide; M2-1 = M2-1 ORF of the RSV M2 gene; F(A-long BN) = ORF of the modified A-long serotype of the RSV-F protein; poxvirus promoters as indicated. [Figure 16] Analysis of miRNA-mediated downregulation of MVA-encoded RSV G, F, and N / M2-1 transgenes is shown. On day 0, 1x106 CEF cells were seeded in VP-SFM. The following day, cells were mock infected or infected with MVA-BN, or recombinant MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, or MVA-BN-RSV-miRb39 / 41 at an MOI of 1. Cell lysates ("CL") were prepared 12 hours (left) and 18 hours (right) post-infection, and proteins were separated according to size by SDS-PAGE and analyzed by immunoblotting with anti-RSV G, F, or N antibodies, or anti-D8 VACV antibody (MVA vector control). [Figure 17]Replication capacity of MVA-BN-RSV-miRb1 / 2 and MVA-BN-RSV-miRb39 / 41 compared to MVA-BN-RSV is shown. CEF cells were seeded in VP-SFM medium in 6-well plates one day prior to infection. Confluent monolayers were infected in triplicate with MVA recombinants: MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, or MVA-BN-RSV-miRb39 / 41 in 1 ml VP-SFM at an MOI of 0.1 (left) or 0.01 (right). MVA-BN wild type (i.e., non-recombinant) was included for comparison. Infected cells were cultured at 30°C and directly frozen on days 3 and 4 post-infection for subsequent TCID50 titration. Viral yields are shown as geometric mean and SD of TCID50 / 2 ml. The table shows the fold difference in viral titer calculated from the data shown in the figure (above). [Figure 18] Figure 1 shows the analysis of RSV-specific CD8+ T cell responses in peripheral blood cells of mice analyzed by dextramer staining. Groups of 10 female BALB / c mice were immunized intramuscularly with 1x108 TCID50 per mouse of MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, or MVA-BN-RSV-miRb39 / 41 on days 0 and 21. TBS-treated mice (n=5) were included as controls. PBMCs were collected on days 7, 28 (=7 days post-boost), and 34 (=13 days post-boost) after priming and stained with MHC class I dextramers specific for immunodominant epitopes within RSV M2-1 and MVA E3 proteins (vector control), as well as expression of surface markers CD4, CD8, and CD44. The percentages of CD8+ T cells that were activated (CD44+) and specific for RSV M2-1 (left) or MVA-derived E3 (right) are shown (mean and SEM). [Figure 19]Figure 1 shows the analysis of RSV-specific T cell responses in mouse splenocytes analyzed by intracellular cytokine staining (ICCS) and ELISpot. Female BALB / c mice were treated as described in Figure 18. 13 days after boost, single cell splenocyte suspensions were prepared for ICCS and ELISpot analysis of T cells. (A) For ICCS, splenocytes were restimulated for 6 hours with immunodominant peptides from the indicated RSV proteins or immunodominant MVA epitopes from MVA E3 (vector control). The frequency of CD44+IFN-γ+ cells in CD8+ T cells after peptide stimulation is shown (mean and SEM). (B) For ELISpot analysis, splenocytes were restimulated with immunodominant RSV G, F, and M2-1 derived peptides or immunodominant MVA-E3 peptide. The IFN-γ+ spot-forming colonies (SFC) per 1x106 splenocytes are shown (mean and SEM). [Figure 20] Analysis of RSV-specific IgG antibody titers and RSV plaque reduction neutralization titers in mouse serum is shown. Female BALB / c mice were treated as described in FIG. 18. Sera were collected 1 day before immunization (day -1), 20 days after priming, and 34 days (= day 13 after boost). (A) RSV- and MVA-specific IgG antibodies (left and right, respectively) were detected by ELISA and results are shown as geometric mean titers (GMT) and geoSD. (B) RSV A2 strain-specific neutralizing antibody titers in serum samples were quantified (GMT and geoSD) by plaque reduction neutralization test (PRNT). Percentage of seroconversion (defined as appearance of plaque reduction neutralization titers ≧15 in initially seronegative mice) is shown in the table.

[0033] A brief description of the sequence [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2] [Table 4] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The aim was to improve the growth of recombinant MVA in its producer cells to increase virus yields, especially in large-scale vaccine production.

[0035] The challenge was to reduce cytotoxic transgene expression by the recombinant MVA during propagation while retaining the potential of the recombinant MVA to induce a transgene-specific immune response in the vaccine recipient.

[0036] To address this issue, we exploited miRNAs endogenously expressed in avian MVA-producing cells to downregulate transgene expression by recombinant MVA. For this purpose, miRNA target sequences corresponding to the miRNAs were inserted into the 3′-UTR region of the transgene.

[0037] The inventors followed a strategy of first using a transfected model gene (EGFP) to screen for miRNAs that are most efficient at downregulating transgene expression by recombinant MVA, and then evaluating the selected miRNA target sequences to optimize their use in recombinant MVA.

[0038] Here, the concept of linking miRNA target sequences to recombinant MVA-expressed transgenes is shown to be feasible for downregulation of transgene expression in MVA-producing cells, a finding that was unexpected not only because vaccinia virus is assumed to mediate impairment of the cellular microRNA machinery, but also because extensive activation of poxvirus promoters is used to achieve large amounts of transgene product.

[0039] Two sets of heterologous miRNA target sequences, each of which is located within a so-called miRblock, namely miRblock-39 and -42, were selected for the preparation of modified MVA-BN-RSV. The criteria for miRblock selection were (i) high activity in mediating downregulation of transgene expression in MVA-producing cells, (ii) low sequence similarity between miRNA target sequences within the miRblock, and (iii) low expression of related miRNAs in the target tissues of vaccination, namely blood and skeletal muscle.

[0040] Finally, we have shown that MVA-BN-RSV recombinants modified by the insertion of miRNA target sequences (e.g., miRblock-39 and -42) produce higher virus yields during growth in MVA-producing cells than unmodified MVA-BN-RSV recombinants. Furthermore, we have shown in mouse immunization experiments that the miRNA target sequences linked to the transgenes in the recombinant MVA-BN-RSV do not impair the immunogenicity of RSV-derived antigens in vivo.

[0041] definition It should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a nucleic acid" includes one or more nucleic acid sequences.

[0042] As used herein, the conjunction "and / or" between multiple described elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meet the requirements of the term "and / or" as used herein. Two or more of the options being simultaneously applicable is also understood to be within the meaning and thus meet the requirements of the term "and / or".

[0043] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising" are understood to imply the inclusion of a stated integer or step or group of integers or steps, and not the exclusion of any other integer or step or group of integers or steps. When used in the context of a described aspect or embodiment of the invention, the term "comprising" is modifiable and thus may be replaced with the term "containing" or "including" or, as used herein, with the term "having". Similarly, all of the foregoing terms (comprising, containing, including, having), whenever used in the context of a described aspect or embodiment of the invention, include the terms "consisting of" or "consisting essentially of", each of which carries a specific legal meaning depending on the jurisdiction.

[0044] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0045] The term "recombinant MVA" as described herein refers to an MVA that contains a foreign nucleic acid sequence inserted into its genome that does not naturally occur in the wild-type virus. Thus, a recombinant MVA refers to an MVA that is created by artificially combining two or more segments of nucleic acid sequence of synthetic or semi-synthetic origin that do not occur in nature or are linked to another nucleic acid in an arrangement not found in nature. Artificial combination is most commonly achieved by artificially manipulating isolated nucleic acid segments using established genetic engineering techniques. Generally, the term "recombinant MVA" as described herein refers to an MVA that has been produced by standard genetic engineering methods, and thus, for example, a recombinant MVA is a genetically engineered or genetically modified MVA. Thus, the term "recombinant MVA" includes an MVA (e.g., MVA-BN®) that has incorporated at least one recombinant nucleic acid into its genome, preferably in the form of a transcription unit. A recombinant MVA may express a heterologous antigenic determinant, polypeptide, or protein (antigen) by induction of a regulatory element (e.g., a promoter).

[0046] As used herein, the term "heterologous" refers to a gene or transgene or DNA sequence that is not native (or foreign) to the MVA, but that has been artificially inserted into the MVA using recombinant techniques. Similarly, the term "heterologous miRblock" refers to a miRblock that is heterologous to the MVA that contains it.

[0047] The term "miRNA" (short for "microRNA") refers to small, single-stranded, non-coding RNA molecules, typically 21-23 nucleotides (nt) in length. miRNAs function in RNA silencing and post-transcriptional regulation of gene expression by binding to mRNA.

[0048] The name of miRNA is mainly based on the simple sequential number of the identified miRNA, which is preceded by the abbreviation of the organism in which the miRNA is identified.All miRNAs mentioned herein are identified in CEF cells or chicken tissues, so each miRNA name must be preceded by gga (for gallus gallus (chicken)), for example, the official name of "miR-17-5p" (used herein) is gga-miR-17-5p.Since only chicken miRNAs were tested in the study presented herein, gga was omitted from all miRNA names for ease of reading.

[0049] As used herein, the term "miRNA sequence" refers to the nucleotide sequence of a mature miRNA.

[0050] The term "seed sequence" refers to a section within the nucleotide sequence of a miRNA that is essential for the binding between the miRNA and an mRNA, which is 7-8 nucleotides in length and is perfectly complementary to the relevant section within the mRNA sequence.

[0051] As used herein, "miRNA target sequence" refers to a nucleic acid sequence that corresponds to the nucleotide sequence of a miRNA. Matching (i.e., nucleotide complementarity) between the sequence of a miRNA and its corresponding target sequence can be 100% match or less.

[0052] As used herein, the term "corresponding" or "corresponding to" refers to a nucleotide sequence (e.g., of an miRNA target sequence) relative to a related nucleotide sequence (e.g., of an miRNA). More precisely, an miRNA target sequence corresponding to an miRNA refers to the counterpart or complementary sequence of the miRNA sequence.

[0053] The term "complementary" refers to two nucleotide sequences that match each other so that the nucleotides form a double-stranded structure.

[0054] As used herein, the term "miRblock" refers to a set of miRNA target sequences. A set in this context means two, three, or more consecutive or concatenated miRNA target sequences.

[0055] A "homo-oligomeric" miRblock is composed of a series of identical miRNA target sequences.

[0056] A hetero-oligomer "miRblock" is composed of a set of miRNA target sequences that differ in nucleotide sequence. Usually, all miRNA target sequences within a miRblock are different from each other. Alternatively, two or more miRNA target sequences are different from each other, and other sequences within the miRblock are identical.

[0057] The term "downregulation" or "down-regulation" in the context of transgene expression relates to a decrease or reduction in the amount of the transgene product, which may result from a decrease in the amount of transgene mRNA or a decrease in translation of the transgene mRNA.

[0058] As used herein, a "transcription unit" comprises a transgene and, operably linked thereto, a promoter, a terminator, and optionally a series of miRNA target sequences.

[0059] As used herein, the term "operably linked" means that a first nucleic acid sequence (e.g., a transgene) is in a functional relationship with a second nucleic acid sequence (e.g., a promoter). For example, a promoter is operably linked to a coding sequence of a transgene if the promoter is positioned such that it is capable of directing transcription of the coding sequence.

[0060] Abbreviation BFP: Blue fluorescent protein bp: base pair(s) CEF: chicken embryo fibroblasts CMV: Cytomegalovirus DF-1: chicken continuous fibroblast cell line EGFP: Enhanced Green Fluorescent Protein miRb: miRblock, a set of miRNA target sequences miRNA: MicroRNA MOI: Multiplicity of infection MVA: Modified vaccinia virus Ankara MVA-BN: MVA-BN (registered trademark) (Bavarian Nordic) nt: nucleotide ORF: Open reading frame pi: after infection RFP: Red fluorescent protein RSV: Respiratory syncytial virus TCID 50 :50% tissue culture infectious dose TTS: transcription termination signal

[0061] Embodiment In one embodiment, at least one of the miRNA target sequences within the miRblock is capable of mediating downregulation of expression levels of a transgene in a eukaryotic MVA producing cell.

[0062] In one embodiment, at least one of the miRNA target sequences in the miRblock mediates down-regulation of expression levels of a transgene in a eukaryotic MVA producing cell upon binding of the miRNA target sequence to its corresponding miRNA.

[0063] In one embodiment, downregulation of the expression level of a transgene means that there is less amount of transgene product (e.g., per cell) compared to a transgene that is not bound to a miRNA target sequence.

[0064] In one embodiment, downregulation of transgene expression levels refers to a reduction in the levels of transgene mRNA or a reduction in the translation of transgene mRNA.

[0065] In one embodiment, the reduction or decrease in the expression level of the transgene compared to the expression level of a transgene not bound to any miRNA target sequence is about 20, 40, 60, 80, 90, or 99%.

[0066] In one embodiment, the set of miRNA target sequences in the miRblock is a set of 2, 3, 4, 5, 6, 7, 8 or more miRNA target sequences, preferably a set of 3, 4, 5, 6, or 7 miRNA target sequences, more preferably a set of 3 or 4 target sequences, and most preferably a set of 4 target sequences.

[0067] In one embodiment, the series of miRNA target sequences in the miRblock is a series of 2 to 10, preferably 2 to 8, more preferably 3 to 7, even more preferably 2 to 5, and most preferably 3 to 5, miRNA target sequences.

[0068] In one embodiment, the miRblock is inserted into the 3'-UTR region of the open reading frame (ORF) of the transgene.

[0069] In one embodiment, the miRblock is attached to the transgene such that the first 5' nucleotide of the first 5' miRNA target sequence is linked to the stop codon of the transgene ORF via a spacer nucleotide sequence of about 1-500 nucleotides, or about 2-100 nucleotides, or about 3-50 nucleotides, preferably 5-25 nucleotides, more preferably about 10-20 nucleotides, even more preferably about 13-17 nucleotides, and most preferably 15 nucleotides. Alternatively, but less preferably, the miRblock is attached to the transgene such that the first 5' nucleotide of the first 5' miRNA target sequence is linked directly, i.e., without a spacer nucleotide sequence, to the stop codon of the transgene ORF.

[0070] In one embodiment, a transgene operably linked to a poxvirus promoter is inserted into an intergenic region (IGR) of recombinant MVA together with a miRblock linked to the transgene, preferably an IGR selected from the group consisting of IGR44 / 45, 64 / 65, 88 / 89, and 148 / 149.

[0071] In one embodiment, the transcription unit is inserted into an intergenic region (IGR) of the recombinant MVA, preferably an IGR selected from the group consisting of IGR44 / 45, 64 / 65, 88 / 89, and 148 / 149.

[0072] miRNA Target Sequence Embodiments In one embodiment, the miRNA target sequence corresponds to a miRNA such that the miRNA target sequence is capable of binding or partially binding to the miRNA.

[0073] In one embodiment, the miRNA target sequence corresponding to a miRNA is partially or completely complementary to the nucleotide sequence of the miRNA.

[0074] In one embodiment, at least one miRNA target sequence in a miRblock corresponds to a miRNA sequence with about 80-100%, preferably about 90-100%, more preferably about 95-100%, and most preferably about 100% sequence similarity, most preferably 100% sequence identity.

[0075] In one embodiment, at least one miRNA target sequence in a miRblock comprises a nucleotide sequence outside the seed sequence that corresponds to a miRNA sequence with about 80-100%, preferably about 90-100%, more preferably about 95-100%, and most preferably about 100% sequence similarity, most preferably 100% sequence identity.

[0076] In one embodiment, at least one miRNA target sequence within a miRblock is complementary to a miRNA sequence, preferably with about 100% sequence similarity, most preferably 100% sequence identity.

[0077] In one embodiment, at least one miRNA target sequence within a miRblock is selected from the group consisting of nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p), SEQ ID NO:2 (miR-20a-5p), SEQ ID NO:3 (miR-21-5p), SEQ ID NO:4 (miR-221a-3p), SEQ ID NO:5 (miR-18a-5p), SEQ ID NO:6 (miR-19a-3p), SEQ ID NO:7 (miR-199-3p), SEQ ID NO:8 (miR-33-5p), SEQ ID NO:9 (miR-218b-5p).

[0078] In one embodiment, at least one miRNA target sequence within the miRblock is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p), SEQ ID NO:2 (miR-20a-5p), SEQ ID NO:3 (miR-21-5p), SEQ ID NO:4 (miR-221a-3p), SEQ ID NO:6 (miR-19a-3p), and SEQ ID NO:7 (miR-199-3p).

[0079] miRblock embodiments In one embodiment, the set of miRNA target sequences within a miRblock comprises or consists of less than about 200 bp, preferably less than about 150 bp, and more preferably about 90-100 bp.

[0080] In one embodiment, the miRNA target sequence within a miRblock is arranged within a hetero- or homo-oligomeric miRblock, preferably a hetero-oligomeric miRblock.

[0081] In one embodiment, all miRNA target sequences in the hetero-oligomeric miRblock are different from each other. In another embodiment, at least two or most miRNA target sequences in the hetero-oligomeric miRblock are different from each other. In particular, the miRNA target sequences are different in their nucleotide sequences.

[0082] In one embodiment, 3 or 4, preferably 4 miRNA target sequences are arranged in hetero-oligomeric miRblock.Preferably, 3 or 4, preferably 4 miRNA target sequences in hetero-oligomeric miRblock are all different from each other.In particular, miRNA target sequences are different in their nucleotide sequence.

[0083] In one embodiment, the two miRNA target sequences from each miRNA target sequence in the miRblock are separated by a spacer nucleotide sequence of about 1-10 nucleotides, preferably about 2-8 nucleotides, more preferably about 3-6 nucleotides, and most preferably about 4 nucleotides.Alternatively, but less preferably, the miRNA target sequences in the miRblock are not separated by a spacer nucleotide sequence.

[0084] In one embodiment, the miRblock is followed by a poxvirus transcription termination signal (TTS).

[0085] In one embodiment, the miRNA target sequence within the hetero-oligomeric miRblock is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p), SEQ ID NO:2 (miR-20a-5p), SEQ ID NO:3 (miR-21-5p), SEQ ID NO:4 (miR-221a-3p), SEQ ID NO:5 (miR-18a-5p), SEQ ID NO:6 (miR-19a-3p), SEQ ID NO:7 (miR-199-3p), SEQ ID NO:8 (miR-33-5p), SEQ ID NO:9 (miR-218b-5p).

[0086] In one embodiment, the miRNA target sequence within the hetero-oligomeric miRblock is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p), SEQ ID NO:2 (miR-20a-5p), SEQ ID NO:3 (miR-21-5p), SEQ ID NO:4 (miR-221a-3p), SEQ ID NO:6 (miR-19a-3p), and SEQ ID NO:7 (miR-199-3p).

[0087] In one embodiment, the miRblock comprises the nucleotide sequence set forth in SEQ ID NO:1 (corresponding to miR-17-5p of miRblock-1).

[0088] In one embodiment, the miRblock comprises the nucleotide sequences set forth in SEQ ID NO: 2 (corresponding to miR-20a-5p of miRblock-2), SEQ ID NO: 3 (miR-21-5p of miRblock-2), and SEQ ID NO: 4 (miR-221a-3p of miRblock-2).

[0089] In one embodiment, the miRblock comprises the nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p of miRblock-37), SEQ ID NO:2 (miR-20a-5p of miRblock-37), SEQ ID NO:3 (miR-21-5p of miRblock-37), and SEQ ID NO:6 (miR-19a-3p of miRblock-37).

[0090] In one embodiment, the miRblock comprises the nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p of miRblock-38), SEQ ID NO:3 (miR-21-5p of miRblock-38), SEQ ID NO:5 (miR-18a-5p of miRblock-38), and SEQ ID NO:6 (miR-19a-3p of miRblock-38).

[0091] In one embodiment, the miRblock comprises the nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p of miRblock-39), SEQ ID NO:5 (miR-18a-5p of miRblock-39), SEQ ID NO:6 (miR-19a-3p of miRblock-39), and SEQ ID NO:7 (miR-199-3p of miRblock-39).

[0092] In one embodiment, the miRblock comprises the nucleotide sequences set forth in SEQ ID NO:1 (corresponding to miR-17-5p of miRblock-41), SEQ ID NO:4 (miR-221a-5p of miRblock-41), SEQ ID NO:8 (miR-33-5p of miRblock-41), and SEQ ID NO:9 (miR-218b-5p of miRblock-41).

[0093] In one embodiment, the miRblock comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO:48 (corresponding to miRblock-1), SEQ ID NO:49 (miRblock-2), SEQ ID NO:55 (miRblock-scrbl-2), SEQ ID NO:51 (miRblock-37), SEQ ID NO:52 (miRblock-38), SEQ ID NO:53 (miRblock-39), and SEQ ID NO:54 (miRblock-41).

[0094] In one embodiment, the miRblock comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 48 (corresponding to miRblock-1), SEQ ID NO: 49 (miRblock-2), SEQ ID NO: 53 (miRblock-39), and SEQ ID NO: 54 (miRblock-41).

[0095] In one embodiment, the miRblock comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 53 (corresponding to miRblock-39) and SEQ ID NO: 54 (miRblock-41).

[0096] In one embodiment, the miRblock comprises or consists of the nucleotide sequence set forth in SEQ ID NO:54 (corresponding to miRblock-41).

[0097] Promoter Embodiments In one embodiment, the promoter is an early / late promoter or an early promoter, preferably an immediate early promoter.

[0098] In one embodiment, the early / late promoter is selected from the group consisting of PrS, Pr7.5, and PrH5m promoters.

[0099] In one embodiment, the immediate early promoter is selected from the group consisting of Pr13.5long, Pr1328, PrLE1 (pHyb) promoters.

[0100] In a preferred embodiment, the promoter is the Pr13.5long promoter.

[0101] Transgene Embodiments In one embodiment, the transgene encodes a protein or peptide, preferably a protein or peptide comprising one or more antigenic determinants, more preferably a proteinaceous or peptidic antigen.

[0102] In one embodiment, the transgene encodes an antigen, or an antigenic portion thereof, selected from the group consisting of viral, bacterial, fungal, plant, parasitic, non-human animal, and human antigens.

[0103] In one embodiment, the transgene encodes a viral antigen or a portion thereof.

[0104] In one embodiment, the viral antigen is selected from the group consisting of 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), Guanarito virus, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human herpes 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), The virus is derived from a virus selected from the group consisting of influenza virus, Junin 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, Sabia virus, severe acute respiratory syndrome virus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), varicella zoster virus, smallpox virus, West Nile virus, and yellow fever virus.

[0105] In one embodiment, the viral antigens are derived from RSV.

[0106] In one embodiment, the transgene encodes a protein, or antigenic portion thereof, derived from RSV, preferably selected from the group consisting of the RSV G(A), G(B), F, N, and M2-1 proteins, and the N / M2-1 fusion protein.

[0107] In one embodiment, viral antigens are derived from Eastern, Western, or Venezuelan EEV.

[0108] In one embodiment, the transgene encodes a protein derived from Eastern, Western, or Venezuelan EEV, or an antigenic portion thereof, preferably a protein selected from the group consisting of envelope polyproteins E3, E2, 6k, and E1.

[0109] In one embodiment, the viral antigens are derived from Epstein-Barr virus.

[0110] In one embodiment, the transgene encodes a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), or an antigenic portion thereof.

[0111] miRNA Embodiments In one embodiment, the miRNA in the eukaryotic MVA-producing cell is present, detectable, or expressed in the eukaryotic MVA-producing cell.

[0112] In one embodiment, the miRNA is endogenous to the eukaryotic MVA producing cell.

[0113] In one embodiment, the miRNA is encoded by, and preferably expressed in, a heterologous nucleotide sequence in a transgenic cell line.

[0114] In a preferred embodiment, the miRNA is not expressed, or is only lowly or moderately expressed, in skeletal muscle cells and blood cells (eg, white blood cells).

[0115] Eukaryotic MVA-producing cell embodiments In one embodiment, the eukaryotic MVA producing cells are avian (eg, chicken) cells, preferably primary avian cells or cells of a permanent avian cell line.

[0116] In one embodiment, the eukaryotic MVA producing cells, preferably avian primary cells, are chicken embryo fibroblasts (CEF).

[0117] In one embodiment, the eukaryotic MVA producing cells, preferably cells of a permanent avian cell line, are DF-1 or quail cells.

[0118] MVA embodiment In one embodiment, the recombinant MVA is derived from MVA or an MVA derivative that is capable of reproductive replication in vitro in chicken embryo fibroblasts (CEF) but not in the human keratinocyte cell line HaCaT, the human bone osteosarcoma cell line 143B, the human embryonic kidney cell line 293, and the human cervical adenocarcinoma cell line HeLa.

[0119] In one embodiment, the recombinant MVA is derived from MVA-BN®, deposited at the European Collection of Animal Cell cultures (ECACC) on Aug. 30, 2000 under accession number V00083008.

[0120] Embodiments relating to recombinant MVA with several transgenes In one embodiment, the recombinant MVA comprises multiple, for example, two, three, four, five or even more transgenes or transcription units.

[0121] In one embodiment, the recombinant MVA comprises a first, a second and a third, or a first to a fourth, or a first to a fifth, or a first to a sixth, or more transcription units.

[0122] In a preferred embodiment, the recombinant MVA comprises four or a first to a fourth transcription unit.

[0123] In one embodiment of a recombinant MVA comprising more than two transgenes, each transgene is different, preferably each transcription unit comprises a different transgene.

[0124] In one embodiment, the recombinant MVA further comprises one or more transcription units that do not contain a miRNA target sequence, preferably comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter and that does not contain a miRNA target sequence linked to the transgene.

[0125] Recombinant MVA embodiments carrying transgenes encoding RSV-derived proteins In one embodiment, the recombinant MVA comprises a transcription unit comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a series of miRNA target sequences arranged within a miRblock linked to the transgene, each miRNA target sequence corresponding to or complementary to a miRNA expressed in the eukaryotic MVA producing cell; (a) the transgene encodes a RSV G(A) protein, and preferably the poxvirus promoter is the Pr7.5 promoter; (b) the transgene encodes the RSV G(B) protein, and preferably the poxvirus promoter is the PrS promoter; (c) the transgene encodes a RSV F protein, and preferably the poxvirus promoter is a PrH5m promoter; and / or (d) the transgene encodes a RSV N / M2-1 fusion protein, and preferably the poxvirus promoter is the PrLE1 promoter.

[0126] In one embodiment, the recombinant MVA comprises a transcription unit comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a series of miRNA target sequences arranged within a miRblock linked to the transgene, each miRNA target sequence corresponding to or complementary to a miRNA expressed in a eukaryotic MVA producing cell, the transgene encoding a RSV N / M2-1 fusion protein, and the poxvirus promoter is the PrLE1 promoter.

[0127] In one embodiment, the recombinant MVA comprises a transgene encoding an RSV-derived protein in a first to third transcription unit, each transcription unit comprising a nucleotide sequence comprising the transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a series of miRNA target sequences located within a miRblock linked to the transgene, each miRNA target sequence corresponding to or complementary to a miRNA sequence in a eukaryotic MVA-producing cell; (aa) the transgene in the first transcription unit encodes the RSV G(A) protein; (bb) the transgene in the second transcription unit encodes the RSV G(B) protein; (cc) The transgene in the third transcription unit encodes the RSV F protein.

[0128] In one embodiment, the recombinant MVA comprises a transgene encoding an RSV-derived protein within the first to third transcription units, (aa') the miRblock linked to the transgene encoding the RSV G(A) protein comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 49 (corresponding to miRblock-2); (bb') the miRblock linked to the transgene encoding the RSV G(B) protein comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 48 (miRblock-1); (cc') The miRblock linked to the transgene encoding the RSV F protein comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 48 (miRblock-1).

[0129] In one preferred embodiment, the recombinant MVA comprises a transgene encoding an RSV-derived protein in first through fourth transcription units, each transcription unit comprising a nucleotide sequence comprising the transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a set of miRNA target sequences located within a miRblock linked to the transgene, each miRNA target sequence corresponding to or complementary to a miRNA sequence in a eukaryotic MVA-producing cell; (aaa) the transgene in the first transcription unit encodes the RSV G(A) protein; (bbb) the transgene in the second transcription unit encodes the RSV G(B) protein; (ccc) the transgene in the third transcription unit encodes the RSV F protein; (ddd) The transgene in the fourth transcription unit encodes the RSV-N / M2-1 proteins.

[0130] In one embodiment, the recombinant MVA comprises a transgene encoding a protein derived from RSV within the first to fourth transcription units, (aaa') the miRblock linked to the transgene encoding the RSV G(A) protein comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 54 (corresponding to miRblock-41); (bbb') the miRblock linked to the transgene encoding the RSV G(B) protein comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 53 (miRblock-39); (ccc') the miRblock linked to the transgene encoding the RSV F protein comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 53 (miRblock-39); (ddd') The miRblock linked to the transgene encoding the RSV-N / M2-1 protein comprises or consists of the nucleotide sequence shown in SEQ ID NO: 54 (miRblock-41).

[0131] In one embodiment, (w) the transgene encodes the RSV G(A) protein and the poxvirus promoter is the Pr7.5 promoter; (x) the transgene encodes the RSV G(B) protein and the poxvirus promoter is the PrS promoter; (y) the transgene encodes a RSV F protein and the poxvirus promoter is a PrH5m promoter; and / or (z) The transgene encodes a RSV N / M2-1 fusion protein and the poxvirus promoter is the PrLE1 promoter.

[0132] In one embodiment, the recombinant MVA comprises a transcription unit comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a series of miRNA target sequences arranged within a miRblock linked to the transgene, each miRNA target sequence corresponding to or complementary to a miRNA sequence in the eukaryotic MVA producing cell, the transgene encodes a RSV N / M2-1 fusion protein, the poxvirus promoter is a PrLE1 promoter, and the miRblock comprises or consists of the nucleotide sequence set forth in SEQ ID NO:54 (miRblock-41).

[0133] Medical Use or Treatment Embodiments In one embodiment, the recombinant MVA for use as a medicament or vaccine, preferably for use in the treatment or prevention of a disease, more preferably for use in the treatment or prevention of an infectious disease or cancer, is for use in a subject.

[0134] In one embodiment, the subject is not avian, and is preferably a human or non-human mammal.

[0135] In one embodiment, the infectious disease is an RSV infection, or an Eastern, Western, or Venezuelan Equine Encephalitis Virus (EEV) infection, or an Epstein-Barr virus infection, preferably an RSV infection.

[0136] In one embodiment, the recombinant MVA is administered intramuscularly or subcutaneously, preferably intramuscularly.

[0137] Further embodiments In one embodiment, the recombinant MVA is grown in eukaryotic production cells at a temperature of about 30°C to 37°C, preferably at a temperature selected from the group consisting of about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, and 37°C, most preferably at a temperature of about 30°C or 37°C.

[0138] In one embodiment, recombinant MVA is propagated in eukaryotic producer cells at temperatures of about 30°C or 37°C in DF-1 cells.

[0139] In one embodiment, the recombinant MVA is propagated in a eukaryotic production cell culture after infection with the recombinant MVA at a multiplicity of infection (MOI) of 0.001 to 5, preferably at an MOI of 0.001, 0.05, 0.01, 0.05, 0.1, 0.2, 1, 2, or 5.

[0140] In one embodiment, the recombinant MVA is propagated in eukaryotic production cell cultures in a multi-cycle replication setup.

[0141] Further details Modified vaccinia virus Ankara (MVA) In the past, MVA was generated by serial passage of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblast cells for 516 generations (for review, see Mayr et al. 1975). The virus was renamed from CVA to MVA at passage 570, in view of its substantially altered properties. MVA underwent further passages up to passage 570. As a result of these long passages, the genome of the resulting MVA virus was deleted of approximately 31 kilobases of genomic sequence and was therefore described as being highly host cell restricted with respect to replication in avian cells (Meyer et al. 1991). The resulting MVA was shown in various animal models to be largely non-pathogenic compared to the fully replication-competent starting material (Mayr and Danner 1978).

[0142] MVAs useful in the practice of the invention include MVA-572 (deposited January 27, 1994 under ECACC V94012707), MVA-575 (deposited December 7, 2000 under ECACC V00120707), MVA-I721 (reviewed in Suter et al. 2009), NIH Clone 1 (deposited March 27, 2003 under ATCC® PTA-5095), and MVA-BN (deposited August 30, 2000 with the European Collection of Cell Cultures (ECACC) under number V00083008).

[0143] More preferably, the MVA used according to the present invention includes MVA-BN and MVA-BN derivatives. MVA-BN is described in WO02 / 042480. "MVA-BN derivatives" refers to any virus that exhibits essentially the same replication characteristics as MVA-BN as described herein, but exhibits differences in one or more parts of their genome.

[0144] MVA-BN and MVA-BN derivatives are replication-incompetent, i.e., unable to reproduce reproductively in vivo and in vitro. More specifically, in vitro MVA-BN or MVA-BN derivatives have been described as capable of reproductive replication in chicken embryo fibroblasts (CEF), but incapable of reproductive replication in human keratinocyte cell line HaCaT (Boukamp et al 1988), 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 (ATACC deposit number CCL-2). Furthermore, MVA-BN or MVA-BN derivatives have a viral amplification ratio that is at least two-fold, more preferably three-fold, lower than MVA-575 in HeLa and HaCaT cell lines. Tests and assays for these properties of MVA-BN and MVA-BN derivatives are described in WO02 / 42480 and WO03 / 048184.

[0145] The term "incapable of reproductive replication" in human cell lines in vitro as described above is explained for example in WO02 / 42480, which also teaches how to obtain MVA with the desired properties as described above. The term applies to viruses that have an in vitro viral amplification factor of less than 1 4 days after infection using the assays described in WO02 / 42480 or US 6,761,893.

[0146] Exemplary Generation of Recombinant MVA Viruses Different methods may be applied to generate the recombinant MVA disclosed herein. The DNA sequence to be inserted into the virus can be placed in an E. coli plasmid construct in which DNA homologous to a section of poxvirus DNA is inserted. Separately, the DNA sequence to be inserted may be ligated to a promoter. The promoter-gene linkage can be placed in a plasmid construct such that it is flanked on both sides by DNA sequences homologous to DNA sequences flanking a region of poxvirus DNA containing a non-essential locus. The resulting plasmid construct can be amplified by growth in E. coli and isolated. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into a cell culture, for example a cell culture of chicken embryo fibroblasts (CEF), and the culture is simultaneously infected with MVA. Recombination between the MVA viral DNA homologous in the plasmid and in the viral genome, respectively, can generate an MVA modified by the presence of a foreign (heterologous) DNA sequence.

[0147] The cells of a suitable cell culture, such as CEF cells, can be infected with the MVA virus. The infected cells can then be transfected with a first plasmid vector containing a foreign or heterologous gene(s) (e.g., one or more of the nucleic acids provided herein), preferably under the transcriptional control of a poxvirus expression control element. As explained above, the plasmid vector also contains a sequence capable of directing the insertion of the foreign sequence into a selected part of the MVA virus genome. Optionally, the plasmid vector also contains a cassette containing a marker gene and / or a selection gene operably linked to a poxvirus promoter. The use of a selection or marker cassette simplifies the identification and isolation of the recombinant MVA produced. However, recombinant poxviruses can also be identified by PCR techniques. Further cells can then be infected with the recombinant MVA obtained as described above and transfected with a second vector containing a second foreign or heterologous gene(s). To be sure, this gene should be introduced into a different insertion site of the poxvirus genome, and the second vector also has a different sequence homologous to the poxvirus, which induces the integration of the second foreign gene(s) into the genome of the poxvirus. After homologous recombination has occurred, a recombinant virus containing two or more foreign or heterologous genes can be isolated. If additional foreign genes are to be introduced into the recombinant virus, the infection and transfection steps can be repeated by using the recombinant virus isolated in the previous infection step and by using an additional vector containing the additional foreign gene(s) for transfection. There are many other techniques known for producing recombinant MVA.

[0148] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant technology, all of which are within the skill of one of ordinary skill in the art (see, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et al., eds, 1987; Methods in Enzymology (Academic Press, Inc.) series; PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988). EXAMPLES

[0149] The following examples serve to further illustrate the present disclosure and should not be understood as limiting the invention, the scope of which is defined by the appended claims.

[0150] Example 1: Materials and Methods 1.1 Cells and viruses Chicken fibroblast cell line DF-1 was obtained from ATCC. Primary CEF cells were prepared from 11-day-old embryonated chicken eggs. CEF cells were cultured in VP-SFM medium (ThermoFisher Scientific) (supplemented with 1% gentamicin and 4 mM L-glutamine) for transfection and virus stock production, and in DMEM (supplemented with 10% FCS) for replication analysis and virus titration. MVA used in this study was derived from a bacterial artificial chromosome (BAC) clone constructed from MVA-BN® (Bavarian Nordic; referred to herein as “MVA-BN”) and has been previously described (35) (WO02 / 42480). MVA-BN wild type and MVA-BN recombinants were grown in CEF or DF-1 cells and TCID 50 The Shope Fibroma virus for MVA-BAC reactivation was obtained from ATCC (VR-364) and propagated and titrated in rabbit corneal SIRC cells.

[0151] 1.2 BAC recombination engineering and reactivation of infectious recombinant MVA The construction of MVA-BAC has been described previously (35). Briefly, the inserted BAC cassette contains the miniF plasmid sequence derived from the plasmid pMBO131 (36) for maintenance in E. coli. The BAC cassette was inserted between the MVA orthologues of VACV-Copenhagen genes I3L and I4L (MVA064L / MVA065L). The originally included neomycin-phosphotransferase (npt)II-IRES-EGFP marker cassette was replaced with a bacterial tetracycline expression cassette to remove the enhanced green fluorescent protein (EGFP) gene from the BAC backbone, allowing the insertion and analysis of EGFP transgenes linked to miRNA target sequences. The MVA-BAC was modified by allelic exchange mutagenesis with a counterselectable rpsL / neo cassette as described (35). The EGFP gene was inserted together with the PrS-gpt-RFP cassette (see Fig. 4) into the intergenic region (IGR) between genes MVA044L / MVA045L (F14L and F15L) in the VACV Copenhagen nomenclature under the control of the synthetic poxvirus early / late promoter PrS (23). The target sequences of the respective miRNAs (listed in Tables 1 and 2) or miRblock-scrbl2 (a control miRblock with a scrambled version of miRblock-2) (see Table 3) were inserted directly following the stop codon of the 3'-UTR of the EGFP gene. Starting from miRblock-13 (see Tables 5 and 6 for miRblocks), a 15-nucleotide spacer was inserted between the stop codon of the EGFP ORF and the first nucleotide of the 5'-proximal miRNA target sequence. The poxvirus transcription termination sequences of the early genes were inserted downstream of the miRNA target sequence. In the 3'-UTR of the EGFP gene of the reference recombinant MVA-EGFP without miRblock, only the early transcription termination sequence (TTS) was inserted ("EGFP expression reference"). 6BHK-21 cells were transfected with 3 μg of BAC DNA using FuGENE® HD Transfection Reagent (Promega) and infected 60 min later with Shope Fibroma virus to provide the necessary helper functions. Reactivated virus was isolated by further passaging in CEF cells, and helper virus was removed as previously described (35).

[0152] Recombinant MVAs containing miRblock-17 and -18 (under the control of the PrS promoter), as well as EGFP-miRblock-2 and EGFP-scrbl2 under the control of the Pr13.5long immediate-early promoter, were generated similarly to the BAC-derived recombinant MVAs described above, using transfer plasmids with flanking homology regions targeting the transgenes to IGR MVA044L / MVA045L by standard homologous recombination in CEF cells. The resulting recombinants were purified by three rounds of plaque purification in CEF cells.

[0153] All recombinant MVA virus stocks were generated in DF-1 cells and administered at TCID 50 Recombinant MVA for mouse experiments was grown on CEF cells and purified by two successive rounds of sucrose cushion centrifugation and titrated in CEF cells using the TCID 50 The titration was performed on CEF cells using the titration method.

[0154] 1.3 Plasmid design and cloning for miRNA-targeted EGFP expression analysis The various miRNA target sequences or miRblocks to be inserted into the 3'-UTR of EGFP were sequenced as oligonucleotide primers that, together with the forward primer, served as reverse PCR primers for the amplification of the EGFP gene. A DNA fragment carrying the complete EGFP ORF, including the miRNA target sequence at the 3' end of the ORF and restriction sites for cloning at both ends, was amplified by PCR and cloned into the mammalian expression vector pEGFP-C1. From this vector, the various EGFP genes were expressed under the control of the active human CMV promoter in all mammalian cells as well as avian cells. The miRNA target sequences tested with the various miRblocks are listed in Tables 1 and 2.

[0155] 1.4 Transfection assay On day 0, freshly prepared CEF cells (4 × 10 4 cells / well) and DF-1 cells (3 × 10 4 Cells (1000 x 1000 cells / well) were seeded in 96-well plates. Cells were transfected in triplicate on day 1 with FuGENE® HD Transfection Reagent (Promega) according to the manufacturer's instructions, using 5 μl of transfection mix containing 0.3 μl of transfection reagent and 0.1 μg of plasmid DNA (containing a 1:2 ratio of plasmids expressing EGFP or BFP driven by the human CMV promoter) per 96-well. Transfection with a plasmid encoding EGFP without miRblock served as the EGFP expression reference, and a plasmid encoding EGFP with scrambled miRblock-2 served as the control. Cells were then incubated at 37°C for the indicated time periods. One day after transfection, cells were trypsinized, washed, and resuspended in PBS-FACS-FORM (1% FCS, 0.1% NaN3, 1% PFA) and then analyzed for EGFP and BFP expression by flow cytometry.

[0156] 1.5 Flow cytometry for fluorescent marker expression analysis Cell culture monolayers were washed with PBS and cells were harvested by trypsinization to prepare single cell suspensions. For EGFP, red fluorescent protein (RFP), and blue fluorescent protein (BFP) expression analysis, cells were resuspended in PBS-FACS (2% FCS, 0.1% NaN3) and directly analyzed by flow cytometry using an LSR II flow cytometer (BD Biosciences) and FlowJo software (Tree Star Inc.).

[0157] 1.6 Viral replication analysis For analysis of multicycle virus replication, confluent monolayers of 6-well cell culture plates were infected with sonicated virus dilutions at the indicated multiplicity of infection (MOI) in 500 μl of DMEM without FCS. After 60 min of adsorption at 37°C and 5% CO2, the inoculum was aspirated, cells were washed once with DMEM and further incubated in DMEM / 2% FCS at 37°C and 5% CO2. Cells and supernatants were harvested at the indicated time points, freeze-thawed three times, sonicated and titrated. MVA yields in CEF cells were determined as TCID 50 This was measured using a titration method (3).

[0158] 1.7 Immunoblot analysis of RSV protein expression by recombinant MVA Cells were seeded into 12-well tissue culture plates the day before infection. Infections were performed as previously described (37). At the indicated times postinfection, cells were washed with cold phosphate-buffered saline (PBS) and lysed in 200 μl of 1X Laemmli 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 min, then sonicated for 3 min, followed by heating at 95°C for 5 min. Lysates were centrifuged at 18,000 × g for 1 min to remove cell debris. Soluble proteins in cell lysates were separated on precast SDS-polyacrylamide gels (MiniProtean TGX, 10%; Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo blotting system (Bio-Rad) and Trans-Blot Turbo transfer pack (Bio-Rad). The membranes were blocked with 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 at 4°C with shaking with the following primary antibodies (diluted in blocking buffer): Membranes were washed four times (total 20–40 min) with TBS containing 0.1% Tween-20 between steps and incubated with secondary antibodies conjugated to horseradish peroxidase and directed against mouse or rabbit IgG for 1 h at room temperature with shaking. Secondary antibodies were diluted in TBS containing 5% skim milk powder (VWR International). Bands were visualized by enhanced chemiluminescence (ECL) using two different substrate reagents, SuperSignal West Pico (Thermo Fisher Scientific) as a standard reagent and Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences) (1:10 dilution) for highly sensitive detection.Signals were recorded using a ChemiDoc Touch System and Image Lab™ Software (Bio-Rad) and used for image analysis and quantification.

[0159] The following primary antibodies were used for immunoblot analysis: anti-RSV G (acris BM1268), anti-RSF F (abcam ab43812), RSV N (abcam ab94806), or anti-D8 VACV (clone AB12IT-012-001M1) (all mouse, 1:1000).

[0160] 1.8 Mouse immunization Groups of 10 female BALB / c mice (Janvier SAS, Saint-Berthevin Cedex, France) were immunized with 10 8 TCID 50 Mice were immunized by the intramuscular (im) route with a total of 100 μl inoculum (50 μl in each hind leg) containing the respective MVA recombinants of . TBS-treated mice (n=5) were included as controls. For analysis, blood was collected from the tail vein at the indicated time points into PBS containing 2% FCS, 0.1% sodium azide, and 2.5 U / ml heparin and processed as described below. Mice were euthanized 34 days after immunization and splenocytes were prepared for intracellular cytokine staining (ICCS) of T cells. All animal experiments were approved by the Government of Upper Bavaria (Regierung von Oberbayern).

[0161] 1.9 T cell assay: Dextramer staining for RSV-M2-1 and MVA-E3 specific CD8 T cells For analysis of RSV- and MVA-specific CD8+ T cell responses, blood was collected from mice on days 7, 28, and 34, and peripheral blood mononuclear cells (PBMCs) were prepared by lysing red blood cells using red blood cell lysis buffer (Sigma-Aldrich) according to the manufacturer's instructions. PBMCs were stained with MHC class I dextramers (Immudex) specific for immunodominant epitopes of RSV M2-1 or MVA E3 (control) in a BALB / c background, as well as expression of surface markers CD4, CD8, and CD44.

[0162] 1.10 Intracellular cytokine staining and ELISpot analysis Mice were sacrificed 13 days after the boost (day 34), and spleens were harvested and single-cell suspensions were prepared by collagenase / DNase digestion with mechanical disruption of the tissue through a 70 μm cell strainer followed by red blood cell lysis (Sigma-Aldrich). For intracellular cytokine staining (ICCS), splenocytes were restimulated with the indicated peptides or controls for 6 h, then fixed using IC Fixation & Permeabilization Staining kit (eBioscience) and stained for expression of cell surface markers and intracellular cytokines IFN-γ, TNF-α, and IL-2. For ELISpot analysis, 5 × 10 5 Splenocytes / well were restimulated in duplicate with the indicated RSV proteins in the H-2d haplotype and immunodominant peptides against MVA E3, and responses were assayed according to the manufacturer's protocol (BD™ ELISPOT).

[0163] 1.11 ELISA for RSV and MVA specific antibodies For analysis of RSV- and MVA-specific IgG antibody titers, sera were collected 1 day before immunization (day -1), 20 days after the priming inoculation, and 34 days (= 13 days after the boosting inoculation). RSV- and MVA-specific IgG levels in serum were measured by direct ELISA. 96-well ELISA plates were coated overnight with RSV antigen (Meridian) or crude extracts from MVA-BN infected cells. Samples were titrated using serial dilutions starting at 1:100 against serum. Sheep anti-mouse IgG-HRP (AbD Serotec) or goat anti-mouse IgG-HRP (Abcam) were used as detection antibodies. Antibody titers were calculated by a 4-parameter fit (Magellan Software) and defined as the serum dilution resulting in an optical density of 0.24. Geometric mean titers (GMT) and standard error of the mean (SEM) were calculated using Excel (Microsoft Office 365). Antibody titers below the assay cutoff (OD<0.24) were assigned an arbitrary value of "1" for calculation purposes.

[0164] 1.12 PRNT of RSV neutralizing antibodies For analysis of RSV plaque reduction neutralization titers, sera were collected 1 day before immunization (day -1), 20 days after priming inoculation, and 34 days (=13 days after boosting inoculation). RSV-specific neutralization titers were measured by plaque reduction neutralization test (PRNT). Virus neutralization was performed by incubating two-fold serial dilutions of serum samples with a defined number of RSV-A2 plaque forming units (pfu) for 30 min. The mixture was then adsorbed onto Vero cells for 70 min. Overlay medium was added and the plates were incubated for 5 days. After staining with Crystal Violet, PRNT titers were quantified and calculated based on plaque counts using the neural network plaque counting package. Neutralization titers are expressed as the serum dilution capable of neutralizing 50% of mature virus.

[0165] Example 2: Design of miRNA target sequences and miRblocks First, we extracted the miRNAs that have been reported to be most abundant in CEF cells from the scientific literature (28-34), and then quantified the miRNAs in uninfected CEF cells and in CEF cells infected with non-recombinant MVA by RNA sequencing.

[0166] The miRNA target sequences used were nucleotide sequences that perfectly matched the nucleotide sequences of the respective miRNAs. Usually, four miRNA target sequences were arranged consecutively in a so-called "miRblock" (sometimes abbreviated as "miRb" herein). In some cases, three (in one exceptional case, eight) miRNA target sequences were combined in a miRblock instead of four. The composition of the miRblock with respect to the individual miRNA target sequences was hetero- or homo-oligomeric.

[0167] The selected miRNAs, their corresponding target sequences, and the miRblocks constructed respectively are listed in Tables 5 and 6 below. miRblock-13–20 were constructed from miRNA target sequences based on chicken miRNA abundance and specificity data from the literature (25–31). In a few cases (miR-9999, miR-10000), miRNAs were selected from the miRviewer database. miRblock-25–36 were constructed from miRNA target sequences identified based on our own miRNA sequence data, and miRblock-37–47 were constructed from miRNA target sequences previously used in miRblock-13–36. [Table 5-1] [Table 5-2] [Table 5-3] [Table 6]

[0168] Example 3: miRNA target sequences in plasmids 3.1 Construction of plasmids containing miRNA target sequences To assess the potential of miRNA target sequences to mediate downregulation of transgene expression in chicken cells, plasmids were constructed and the insert of such a plasmid construct is shown in FIG.

[0169] As a model transgene, we used the enhanced green fluorescent protein (EGFP) gene. Expression of the EGFP reporter gene was driven by the canonical cytomegalovirus IE promoter (pCMV) (Figure 1).

[0170] The miRNA target sequences in miRblock-1 and -2 ("EGFP-miRb-1" and "EGFP-miRb-2" in Figure 1B and Figure 1C, respectively; see also Table 3) were selected because they corresponded to the most abundant miRNAs in CEF cells (25, 27, 30). The four miRNA target sequences in miRblock-1 and -2 were separated from each other by a 4-nucleotide (nt) spacer (Figure 1B and Figure 1C, respectively). The first nucleotide of the miRNA target sequence at the 5' end of the miRblock was directly linked to the stop codon of the EGFP open reading frame (ORF). This concept was also applied to miRblock-3 to -10. In all other cases, a 15-nt spacer was inserted between the EGFP stop codon and the first miRNA target sequence.

[0171] As a control, an EGFP expression plasmid ("EGFP expression standard") containing an EGFP ORF without any additional insertion sequence in the 3'-UTR was used ("EGFP without miRb" in FIG. 1A). A plasmid containing an EGFP ORF with a miRblock called "scrambled-2 (or scrbl2)" in the 3'-UTR served as an additional control ("scrbl2" in FIG. 1D). miRblock-scrbl2 was designed by scrambling each nucleotide of the four miRNA target sequences of miRblock-2 and religating the scrambled miRNA target sequences with a 4-nt spacer (Table 3). For scrambling, a tool provided by GeneScript® was used.

[0172] 3.2 Downregulation of EGFP expression The EGFP down-regulation activity of miRblock-1-10 was tested.

[0173] For this purpose, CEF cells were co-transfected with a plasmid containing each miRblock and a second plasmid expressing blue fluorescent protein (BFP) under the CMV promoter. This BFP-expressing plasmid did not contain any additional inserted sequence within the 3'-UTR coding region and served as a transfection control and internal standard for quantifying EGFP expression levels in BFP-positive cells.

[0174] As shown in Figure 2 (top), miRblock-1 mediated downregulation of EGFP expression to approximately 25% of the control ("no miRb"), and miRblock-2 further mediated EGFP downregulation to approximately 9% of the control, an effect not observed with BFP expression by co-transfection of a control plasmid (Figure 2 bottom).

[0175] Compared with miRblock-1 and -2, miRblock-3 mediated moderate EGFP downregulation, whereas miRblock-5 to -10 showed no significant effect on EGFP expression (Figure 2, top).

[0176] 3.3 Effect of temperature on EGFP downregulation Based on the results described in Example 3.2, miRblock-1 and -2 were selected for further characterization.

[0177] Downregulation of EGFP expression mediated by miRblock-1 and -2 was tested at 30° C. or 37° C. EGFP downregulation in CEF cells was also compared to downregulation in the chicken fibroblast cell line DF-1 using the same constructs and temperatures.

[0178] As shown in Figure 3 (top), the EGFP expression standard ("no miRb") and the miRblock control ("scrbl2") yielded similar levels of EGFP expression at a given temperature in both cell types (24 hours after transfection). This finding indicated that inserting a miRblock (i.e., a stretch of sequence of approximately 90-100 nucleotides) into the 3'-UTR coding region of EGFP does not so alter the expression level of the transgene, and that miRblock-scrbl2 is a suitable control.

[0179] As further shown, miRblock-1 and -2 mediated downregulation of EGFP expression in CEF and DF-1 cells (Figure 3, top). In both cell types and at both temperatures, the effect of miRblock-2 was more pronounced than that of miRblock-1.

[0180] Furthermore, both miRblock-1 and -2 mediated downregulation of EGFP expression more effectively at 37° C. than at 30° C. This temperature effect was more pronounced in DF-1 cells (FIG. 3, top).

[0181] BFP expression by the co-infected control plasmid was again independent of EGFP downregulation (FIG. 3, bottom).

[0182] Example 4: miRNA target sequences in recombinant MVA 4.1 Construction of recombinant MVA containing miRNA target sequence We next investigated whether EGFP downregulation could be achieved in the context of MVA infection using miRNA targeting sequences.

[0183] For this purpose, two MVA recombinants were constructed with the insertion of the EGFP gene linked to miRblock-1 or miRblock-2 in the intergenic region (IGR) 44 / 45 ("EGFP-miRb-1", "EGFP-miRb-2" in Figure 4). Each miRblock is followed by a poxvirus early transcription termination signal (TTS), which functions to limit the length of the initial transcript to approximately 50 nucleotides. The TTS is a standard element in gene expression cassettes in poxvirus vectors. A monomeric red fluorescent protein (RFP) was coexpressed by all MVA recombinants (Figure 4). The RFP gene was not modified by the insertion of a miRNA target sequence and therefore should not be subject to regulation by the miRNA machinery of infected cells. It was inserted as a marker to monitor and compare the infection levels provided by the different MVA constructs. Both EGFP and RFP were under the control of the early / late PrS promoter in the recombinant MVA constructs (Figure 4).

[0184] As controls, we constructed a recombinant MVA containing the EGFP ORF without the miRNA target sequence ("EGFP" in Figure 4), i.e., an EGFP expression standard, and a recombinant MVA carrying miRblock-scrbl2 within the 3'-UTR of EGFP ("EGFP-scrbl2" in Figure 4).

[0185] 4.2 Downregulation of EGFP expression and effect of temperature Next, the EGFP down-regulation mediating activity of miRblock-1 and -2 was analyzed in cells infected with the respective MVA recombinants and cultured at 30°C or 37°C.

[0186] As shown in Figure 5 (top), recombinant MVA-induced EGFP expression was downregulated by miRblock-1 and -2 in both CEF and DF-1 cells. Similar to the observations with the plasmid, miRblock-2 was more effective, especially in DF-1 cells.

[0187] Furthermore, EGFP downregulation was detectable at both temperatures but was more pronounced at 37°C than at 30°C in both cell types, with this temperature effect being more pronounced in DF-1 cells (Fig. 5, top).

[0188] The simultaneous expression of RFP was independent of EGFP downregulation (Figure 5, bottom), demonstrating that the expression of an MVA transgene (here, the EGFP gene) can be selectively controlled by a miRNA target sequence without simultaneously affecting other transgenes (here, the RFP gene) present in the same recombinant MVA.

[0189] 4.3 Effect of MOI on EGFP downregulation To test whether the multiplicity of infection (MOI) affects EGFP downregulation by miRblock-1 or -2, cells were infected with the respective MVA recombinants at an MOI of 5, 1, or 0.2.

[0190] As shown in Figure 6 (top), downregulation of EGFP expression by miRblock-1 and -2 was detectable at 6 hours post-infection at all MOIs tested. Notably, when EGFP expression is shown as a % of control (i.e., "no miRb" EGFP expression baseline) (Figure 6 bottom), it becomes clear that the downregulation effect of miRblock-1 and -2 is independent of the MOI used. Thus, the high EGFP expression levels in infected cell cultures due to high MOI did not affect the downregulation of EGFP expression.

[0191] 4.4 Effect of multicycle MVA replication on EGFP downregulation Transgene downregulation was further investigated in a similar setting as is usually applied during virus propagation to produce virus stocks or vaccine lots. For this purpose, CEF cells were infected with recombinant MVA containing miRblock-1 or -2 at a low MOI (0.1) and cultured for a continuous incubation period of 3 days.

[0192] As shown in Figure 7 (top, left), EGFP expression levels increased over time (at least from 6 to 48 hours postinfection), but the increase in EGFP expression after infection with recombinant MVA containing miRblock-1 or -2 was significantly reduced compared to that observed with the EGFP expression baseline ("no miRb") and the miRblock control ("scrbl2").

[0193] When EGFP expression was expressed as a percentage of the control ("no miRb") (Figure 7, bottom left), it was found that the downregulation of EGFP by miRblock-1 and -2 remained nearly constant over the entire observation period of 72 h postinfection. miRblock-2 mediated the downregulation of EGFP expression more effectively than miRblock-1. Again, The levels of RFP co-expressed by the recombinant MVA also increased over time (up to 72 h postinfection) (Fig. 7, top left), but without the downregulation observed with EGFP (Fig. 7, top right and bottom right), indicating similar infection efficiency and virus spread across cell cultures.

[0194] Thus, the efficiency of EGFP downregulation mediated by miRblock-1 and -2 remained stable over the course of multi-cycle MVA replication.

[0195] 4.5 Effect of poxvirus promoters Poxvirus promoters used to drive transgene expression are often selected from a class of composite promoters that initiate expression at early and late times in viral infection, with the synthetic PrS promoter (23), designed to drive strong transgene expression, being a classic example and widely used.

[0196] Despite the presence of optimized early promoter motifs within the PrS promoter, transgene expression under the control of this promoter occurs predominantly late, however, early or even immediate early transgene expression is preferred to induce the best possible T cell response to the transgene product.

[0197] Therefore, we performed an experiment in which the previously used PrS promoter (see, for example, Figure 4) was replaced with the immediate-early Pr13.5long promoter (24) (WO2014 / 063832). EGFP expression under the control of the PrS or Pr13.5long promoter was compared in recombinant MVA containing miRblock-2 within the 3'-UTR coding region of EGFP (Figure 8). Recombinant MVA carrying miRblock-scrbl2 and the PrS or Pr13.5long promoter was used as a control (Figure 8).

[0198] As shown in Figure 9, EGFP expression directed by both PrS and Pr13.5 promoters was downregulated by miRblock-2 in CEF and DF-1 cells at 6 and 18 hours postinfection. Notably, however, downregulation of Pr13.5long-driven EGFP expression was significantly more effective than PrS-driven EGFP expression in both cell types and at both time points (Figure 9).

[0199] Example 5: Evaluation of miRNA target sequences using homo- and hetero-oligomeric miRblocks 5.1 EGFP downregulation by individual miRNA target sequences in plasmids To quantify the contribution of individual miRNA target sequences to EGFP downregulation mediated by hetero-oligomeric miRblocks, we generated plasmid expression constructs carrying homo-oligomeric repeats of individual miRNA target sequences previously placed within the hetero-oligomeric miRblocks.

[0200] First, eight different target sequences of miRblock-1 and -2 were examined. Each miRNA target sequence contained in miRblock-1 was arranged in three identical copies (three repeats) in the homo-oligomeric miRblock (see miRblock-13 to -16 in Table 6). Each miRNA target sequence contained in miRblock-2 was arranged in four copies (four repeats) (see miRblock-17 to -20 in Table 6). Using the target sequence in miR-125b-5p (contained in miRblock-1) as an example, it was observed that there was no significant difference in EGFP downregulation when three or four copies were arranged in the homo-oligomeric miRblock. Individual miRNA target sequences in the homo-oligomeric miRblock were separated by a 4-nt spacer as described above for the hetero-oligomeric miRblock (see Example 3.1).

[0201] As shown in Figure 10 (top), miRblock-13, -17, and -18 (composed of target sequences in miR-17-5p, miR-20a-5p, and miR-21-5p, respectively) were the most efficient among the homo-oligomers miRblock-13 to -20 in downregulating EGFP expression. The extent of EGFP downregulation mediated by miRblock-13, -17, and -18 was within the range of the effect mediated by the hetero-oligomers miRblock-1 and -2, in % relative to the control ("no miRb") (Figure 10 top).

[0202] miRblock-15, -16, and -20 (target sequences in miR-125b-5p, miR-222a, and miR-221-3p, respectively) mediated moderate levels of EGFP downregulation, whereas mRblock-14 and -19 (target sequences in miRNA103-3p and 148a-3p, respectively) had only a minor effect (Fig. 10, top).

[0203] BFP expression was very similar in all transfected CEF cultures, confirming the similar transfection efficiency of the plasmid constructs (FIG. 10, bottom).

[0204] The results obtained were interesting because miR-222a and miR-221a-3p (miRblock-16 and -20, respectively) have been described as the most abundant miRNAs in CEF cells (28). However, in our experiments, their corresponding target sequences mediated only moderate downregulation of EGFP expression (Figure 10, top). On the other hand, the abundance of miR-17-5p and miR-20a-5p (miRblock-13 and -17, respectively) has been reported to be only about 10% of that of miR-222a and miR-221-3p (28), but in our experiments, their corresponding target sequences were found to be even more effective (Figure 10, top).

[0205] 5.2 EGFP downregulation by selected miRNA target sequences in recombinant MVA As described above in Example 5.1 and shown in FIG. 10, the homo-oligomers miRblock-13, -17, and -18 were particularly effective in mediated down-regulation of EGFP expression by plasmid transfection.

[0206] Next, the efficiency of EGFP downregulation by miRblock-17 and -18 was quantified in the context of MVA infection. Two recombinant MVAs expressing EGFP under the PrS promoter and containing either miRblock-17 or miRblock-18 in the 3'-UTR coding region of EGFP ("EGFP-miRb-17" and "EGFP-miRb-18", respectively, in Figure 11) were constructed. For comparison, a recombinant MVA containing miRblock-2 was used ("EGFP-miRb-2" in Figure 11). The miRNA target sequences constituting miRblock-17 and -18 were also included in the hetero-oligomeric miRblock-2.

[0207] As shown in Figure 12 (left), EGFP expression in CEF cells was downregulated by miRblock-2 to about 29% of the control ("no miRb"), whereas in MVA recombinants containing miRblock-17 or -18, EGFP expression was only reduced to about 62% and 66%, respectively (Figure 12 (left)).

[0208] The expression of RFP by the different MVA recombinants was very similar (Fig. 12, right), indicating similar levels of MVA infection.

[0209] It is noteworthy that miRblock-17 and -18 achieved only about half the degree of downregulation of EGFP expression as miRblock-2 did (Figure 12, left). This result was different from that obtained with the plasmid. As shown above (see also Example 5.1, Figure 10), the downregulation of EGFP by miRblock-17 and -18 when expressed from a plasmid was quite similar to that achieved by miRblock-2. Thus, the arrangement of miRNA target sequences within hetero-oligomeric miRblocks may be particularly advantageous when applied to MVA recombinants.

[0210] 5.3 Screening of further potential miRNA target sequences in plasmids We performed our own RNA sequencing analysis of small RNAs in CEF cells and examined EGFP downregulation by miRNAs obtained from this analysis.

[0211] The target sequences in the selected miRNAs were arranged in four repeats separated by 4-nt spacers within homo-oligomeric miRblocks (see miRblock-25 to -36 in Table 6). The miRblocks were placed within a sequence stretch of the EGFP ORF, and EGFP was expressed by plasmid transfection.

[0212] As shown in Figure 13 (top), miRblock-26 and -31 (target sequences in miR-19a-3p and miR-199-3p, respectively) were the most efficient among miRblock-25 to -36 in down-regulating EGFP expression in CEF and DF-1 cells.

[0213] miRblock-25 and -32 (target sequences in miR-18a-5p and miR-33-5p, respectively) mediated moderate levels of EGFP downregulation. The same was true for miRblock-33 (target sequence in miR-218b-5p), at least in CEF cells. In contrast, the remaining miRblock-27 to -30 and miRblock-33 to -36 showed little or no downregulation activity or even an enhancement effect on EGFP expression (Figure 13, top).

[0214] Similar levels of RFP expression in CEF and DF-1 cells confirmed equal transfection efficiency (FIG. 13, bottom).

[0215] 5.4 Defining the most suitable miRNAs for down-regulating EGFP expression by recombinant MVA The findings obtained about the EGFP down-regulation activity of different miRNA target sequences (see Examples 5.1, 5.2, 5.3) served as the basis for optimizing the design of hetero-oligomeric miRblocks for use in recombinant MVA. The miRNAs selected for this effect are listed in Table 7 below. [Table 7]

[0216] Generally, repeats of nucleotide sequences have the risk of homologous recombination during MVA replication.Therefore, homo-oligomeric repeats of part or all of the miRNA target sequence in miRblock may lead to undesired deletion or rearrangement in newly created recombinant MVA genome.It is assumed that the probability of such events is highest when identical or highly similar sequence stretches are tandemly arranged, and therefore close or directly adjacent to each other.

[0217] Therefore, the inventors aimed to design hetero-oligomeric miRblocks with maximum differences in the nucleotide sequences of miRNAs. As a result, hetero-oligomers of miRblock-37 to -47 were created (Table 5). miRblock-43 and -44 contained the same miRNA target sequences as miRblock-39 and -41, respectively, but in different orders.

[0218] In a plasmid containing one of miRblock-37 to -47, a sequence encoding EGFP was linked as described above, and EGFP downregulation was analyzed.

[0219] As shown in Figure 14 (top left), miRblock-37, -38, and -39 mediated EGFP downregulation in CEF cells most effectively among miRblock-37 to -47. The degree of downregulation of EGFP expression (% relative to the control "no miRb") was even better than that induced by miRblock-2. In DF-1 cells (Figure 14 top right), EGFP downregulation by at least miRblock-37 and -38 was within the range induced by miRblock-2. The remaining miRblock-40 to -47 were less effective at mediating EGFP downregulation compared to these (Figure 14 top).

[0220] The expression of RFP by the different MVA recombinants was again very similar (FIG. 14, bottom).

[0221] A recombinant MVA containing a hetero-oligomeric miRblock with eight different miRNA target sequences (miRblock-45 in Table 4) was also tested but showed only marginal activity in EGFP down-regulation.

[0222] From the miRblocks showing the best EGFP down-regulation activity, lead candidate miRblocks were selected based on the following considerations: First, miRNA target sequences with high or very high expression in human blood, leukocytes, or skeletal muscle were avoided. The reason behind was that intramuscular or subcutaneous application was considered as the most common route of vaccination. Data on miRNA expression in human blood, leukocytes, and skeletal muscle were obtained from the miRgeneDB database (http: / / mirgenedb.org / ). Since miRNA-21-5p is highly expressed in human leukocytes, miRblocks containing the corresponding target sequence, i.e., miRblock-37 and -38, were excluded from further consideration. Second, miRblocks containing target sequences in both miR-17-5p and miR-20a-5p, i.e., miRblock-37, -46, and -47, were not further considered due to the high sequence similarity of these two target sequences.

[0223] Finally, miRblock-39 and -41, which met the exclusion criteria of miRNA target sequences and had the closest EGFP down-regulation activity to miRblock-37 and -38, were identified as the most suitable for application in recombinant MVA.

[0224] Example 6: miRNA target sequences in recombinant MVA-BN-RSV 6.1 Construction of MVA-BN-RSV modified with miRNA target sequence The effect of miRNA target sequences on the expression of transgenes present in MVA-BN®-RSV (referred to as "MVA-BN-RSV") (WO2014 / 019718) was investigated.

[0225] MVA-BN-RSV encodes five proteins of human respiratory syncytial virus (RSV) (Figure 15). Two ORFs of glycoproteins (G) from both circulating RSV antigenic subtypes A and B are expressed under the control of the early / late promoters Pr7.5 (G(A)) and PrS (G(B)), respectively. The fusion glycoprotein (F) gene is expressed under the control of the PrH5m early / late promoter. The nucleoprotein (N) and M2-1 proteins, which primarily serve as targets for antiviral CD8 T cell responses, are encoded as fusion proteins N / M2-1, and the respective ORFs are expressed under the control of the immediate early promoter PrLE1 (Figure 15) ((35), in which PrLE1 is referred to as pHyb).

[0226] A first version of modified MVA-BN-RSV containing miRblock-1 and -2 ("MVA-BN-RSV-miRb1 / 2" in FIG. 15) was generated to downregulate the expression of RSV glycoproteins G(A), G(B), and F. miRblock-1 was inserted into the 3'-UTR of the G(B) and F genes, while miRblock-2 was inserted into the 3'-UTR of the G(A) gene (FIG. 15).

[0227] In the second version of the modified MVA-BN-RSV ("MVA-BN-RSV-miRb39 / 41" in Figure 15), all RSV-derived transgenes were linked to miRNA target sequences. MVA-BN-RSV-miRb39 / 41 was designed and engineered using the miRblocks identified as most suitable for transgene downregulation in recombinant MVA, namely miRblock-39 and -41 (see Example 5.4).

[0228] 6.2 Downregulation of transgenes in modified MVA-BN-RSV Expression of RSV G, F, and N / M2-1 proteins in CEF cells infected with MVA-BN-RSV-miRb1 / 2 or MVA-BN-RSV-miRb39 / 41 was analyzed by immunoblot and compared with the expression of the respective proteins in MVA-BN-RSV.

[0229] In the lysates of CEF cells infected with MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, or MVA-BN-RSV-miRb39 / 41 for 12 or 18 hours, the fully glycosylated mature form of RSV G with a molecular weight of approximately 90 kDa was mainly detected (FIG. 16). In this regard, it should be noted that the antibody used for RSV G detection did not distinguish between the two antigenic subtypes A and B of the RSV G protein. Therefore, the RSV G-specific signal seen in the immunoblot in FIG. 16 is considered to be composed of overlapping signals of RSV G(A) and G(B). Compared to cells infected with the MVA-BN-RSV control, the expression levels of both mature RSV G and immature RSV G were lower in cells infected with MVA-BN-RSV-miRb1 / 2 or MVA-BN-RSV-miRb39 / 41 (FIG. 16). Thus, miRblock-1 and -2 (binding to RSV G(B) and (G)A, respectively, in MVA-BN-RSV-miRb1 / 2) and miRblock-39 and -41 (binding to RSV G(B) and G(A), respectively, in MVA-BN-RSV-miRb39 / 41) mediated downregulation of RSV G(A) / (B) protein expression.

[0230] The RSV F protein was detectable as a precursor protein F0 and a large F1 subunit (generated by proteolytic cleavage of F0 by the cellular furin protease) (FIG. 16). Expression of RSV F0 / F1 by MVA-BN-RSV-miRb1 / 2 was only moderately reduced compared to the MVA-BN-RSV control, but was clearly reduced by MVA-BN-RSV-miRb39 / 41 (FIG. 16). Thus, miRblock-39 downregulated the expression of RSV F0 / F1 more effectively than miRblock-1.

[0231] Expression of the RSV N / M2-1 fusion protein (approximately 62 kDa) in MVA-BN-RSV-miRb39 / 41-infected cells was significantly reduced compared to cells infected with MVA-BN-RSV control or MVA-BN-RSV-miRb1 / 2 (FIG. 16). In this regard, it should be noted that MVA-BN-RSV-miRb1 / 2 did not contain any miRNA target sequence within the RSV N / M2-1 ORF (see FIG. 15).

[0232] The expression of vaccinia virus D8 protein, used as an endogenous expression control, was comparable in cells infected with MVA-BN-RSV-miRb1 / 2 or MVA-BN-RSV-miRb39 / 41 (Fig. 16). Interestingly, the D8 signal was slightly stronger in lysates from cells infected with wild-type, i.e., non-recombinant MVA-BN, compared to the three recombinants (Fig. 16). This result may indicate that the cytotoxic effect of the co-expressed RSV-derived transgene without any miRNA-mediated downregulation affected the expression of spontaneous MVA genes, such as D8. On the other hand, miRNA targeting of the RSV transgene did not completely restore normal D8 expression.

[0233] In summary, the expression of RSV G(A) / (B) and RSV F0 / F1 is downregulated in MVA-BN-RSV-miRb1 / 2 and MVA-BN-RSV-miRb39 / 41. However, the downregulation of RSV F0 / F1 expression was more pronounced in MVA-BN-RSV-miRb39 / 41. The expression of N / M2-1 is particularly well downregulated, as expected, since its expression is driven by the early promoter.

[0234] 6.3 Yield of modified MVA-BN-RSV recombinants The yield of recombinant MVA from CEF cells infected with MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, and MVA-BN-RSV-miRb39 / 41 was quantified at two different MOIs, i.e., 0.1 and 0.01, on days 3 and 4 postinfection.

[0235] As shown in Figure 17, MVA-BN-RSV had a significantly reduced yield compared to wild-type MVA-BN at both MOI = 0.1 and 0.01 on days 3 and 4 after infection (approximately 4.2- to 21.5-fold reduction; see "MVA-BN vs.-RSV" in the table of Figure 17). However, compared to MVA-BN-RSV, the yield of MVA-BN-RSV-miRb1 / 2 was increased by approximately 1.2- to 4.2-fold ("MVA-RSV-miRb1 / 2 vs. MVA-RSV"). The yield of MVA-BN-RSV-miRb39 / 41 was further increased (at least at MOI=0.1 on days 3 and 4, and at MOI=0.01 on day 4) over MVA-BN-RSV-miRb1 / 2, ranging from 1.2-fold to 2.6-fold ("MVA-RSV-miRb39 / 41 vs.-miRb1 / 2"). The greatest increase over MVA-BN-RSV was observed with MVA-BN-RSV-miRb39 / 41 at MOI=0.1 on days 3 and 4 post-infection (approximately 3.2-fold and 6.8-fold, respectively) ("MVA-RSV-miRb39 / 41 vs. MVA-RSV").

[0236] Nevertheless, MVA-BN-RSV-miRb39 / 41 did not fully restore the replication behavior of MVA-BN (see "MVA-BN vs. RSV-miRb39 / 41" in the table of Figure 17), and the yield of MVA-BN-RSV-miRb39 / 41 was still reduced by approximately 3.2-fold (MOI = 0.1) and 2.6-fold (MOI = 0.01) compared to MVA-BN.

[0237] In conclusion, the best effect of miRNA target sequences on production yield was obtained with MVA-BN-RSV-miRb39 / 41 at MOI=0.1 and day 4 post-infection.

[0238] 6.4 Immunogenicity of RSV Proteins from Modified MVA-BN-RSV 6.4.1 T cell assays To examine the immunogenicity of the RSV transduction products from MVA-BN-RSV-miRb39 / 41 and MVA-BN-RSV-miRb1 / 2, mouse immunization experiments were performed.

[0239] When produced in BALB / c mice, the M2-1 protein contains a potent immunodominant CD8 T cell epitope, and analysis of this epitope has thus provided a highly sensitive assay with a wide dynamic range for quantifying RSV M2-1-specific CD8 T cell responses.

[0240] CD8 T cell responses of BALB / 1 mice specific for RSV M2-1 or MVA E3 (used as vector control) were analyzed by Dextramer staining. Mouse PBMCs were collected and stained 7 days after priming, and 7 and 13 days after the 21 day boost (i.e., 28 and 34 days after the first immunization).

[0241] As shown in FIG. 18 (left panel), MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, and MVA-BN-RSV-miRb39 / 41 suppressed RSV M2-1-specific CD8 +T cells were induced at similar frequencies at all time points analyzed, therefore, the immunogenicity of RSV N / M2-1 was not affected in vivo by the miRNA target sequences present in the modified MVA-BN-RSV recombinant.

[0242] Furthermore, MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, and MVA-BN-RSV-miRb39 / 41 upregulated the E3-specific CD8 + T cells were induced at similar frequencies (Figure 18 right panel). This indicates that the T cell responses to the MVA vector backbone are comparable and that immunization with the different recombinants is generally similarly effective. Furthermore, analysis of the frequency of memory T cell phenotypic subsets based on the expression patterns of certain cell surface markers (CD4, CD8, CD44, CD62L, CD127, CD29, CX3CR1) showed no differences among the three MVA-BN-RSV recombinants.

[0243] 6.4.2 Intracellular cytokine staining and ELISpot analysis CD8 in terms of cytokine production + To further evaluate the functional response of T cells and quantitatively compare CD8 T cell responses to RSV F and G proteins, mouse splenocytes were harvested 13 days after boost and immediately stimulated with peptides derived from RSV G, F, or M2-1, or peptides derived from MVA E3 (vector control). Responses were analyzed by intracellular cytokine staining (ICCS) and ELISpot analysis.

[0244] In mice immunized with MVA-BN-RSV, MVA-BN-RSV-miRb1 / 2, or MVA-BN-RSV-miRb39 / 41, CD44 specific for RSV G, F, and N / M2-1 proteins or MVA E3, respectively, was expressed. + IFN-γ + CD8 +The overall frequency of T cells was very similar among groups of mice immunized with one of the three MVA-BN-RSV recombinants (Fig. 19A). After stimulation with RSV F-derived peptides, IFN-γ + CD8 + Although the overall frequency of T cells was the lowest among the antigens tested (<2%), the highest frequency was obtained with the RSV N / M2-1-derived peptide (Figure 19A). In addition, the patterns of surface markers CD62L, CD127, and CX3CR1 were very similar among all mouse groups.

[0245] T cell responses in immunized mice were also analyzed using an ELISPOT assay, including analysis of CD4 and CD8 T cells, and the results are shown in Figure 19B.

[0246] ELISpot analysis after stimulation of splenocytes with RSV M2-1-derived peptides revealed that IFN-γ+ spot-forming colonies were too numerous to count (and therefore not shown in Fig. 19B).The results confirmed that RSV N / M2-1 proteins induced strong CD8 T cell responses, consistent with other reports that RSV M2-1 contains immunodominant epitopes in H-2d mice (36, 37).

[0247] ELISpot analysis of splenocytes after stimulation with peptides derived from RSV G and F, and MVA E3 confirmed that the response to the RSV G peptide was stronger than the response to the RSV F peptide (Figure 19B). Similar to ICCS (Figure 19A and above), no differences were observed in the frequency of T cells specific for each of the RSV G and F proteins and MVA E3 between groups of mice immunized with one of the three MVA-BN-RSV recombinants (Figure 19B).

[0248] 6.4.3 ELISA and PRNT Analysis Humoral responses to the encoded RSV proteins were analyzed by quantitating antibody titers that neutralized infectious RSV in vitro using an ELISA assay in which immunoglobulin G (IgG) antibodies bind to whole RSV as an antigen. RSV- and MVA-specific IgG titers were analyzed 1 day before priming (day -1), 20 days after priming, and 34 days after priming (i.e., 13 days after the boost).

[0249] After priming or boosting, no differences in total RSV-specific IgG titers were observed in the sera of mice immunized with one of the three MVA-BN-RSV recombinants (Fig. 20A, left). Similar results were observed for MVA-specific IgG titers (Fig. 20A, right).

[0250] RSV-specific neutralizing antibody titers in the serum of immunized mice 34 days after priming (ie, 13 days after boosting) were quantified by plaque reduction neutralization test (PRNT).

[0251] There was no detectable difference in the amount of neutralizing antibodies induced after immunization of mice with one of the three different MVA-BN-RSV recombinants (Figure 20B). The seroconversion rate to RSV neutralizing antibodies was 100% in all groups of immunized mice (see table in Figure 20B).

[0252] 6.4.4 Conclusions from immunogenicity studies In summary, T cell responses were driven by RSV-specific CD8 +The frequency and functionality of T cells, as well as the induction of RSV-specific IgG and neutralizing antibodies, were very similar between the groups of mice immunized with one of the three different MVA-BN-RSV recombinants. Thus, the addition of miRNA target sequences within the 3'-UTR of the transgene did not adversely affect the immunogenicity of the respective transgene products in mice compared to the transgene products from the unmodified MVA-BN-RSV construct. Both results demonstrated that downregulation of cytotoxic transgene expression mediated by miRNA target sequences favorably affected MVA yields from CEF cells without detectably affecting the immunogenicity of the respective transgene products in vivo.

[0253] Conclusion: Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is incorporated herein by reference in its entirety. In the event that the material incorporated by reference conflicts or is inconsistent with this specification, this specification takes precedence over any such material. Nothing in this specification shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

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Claims

1. A recombinant modified vaccinia virus Ankara (MVA) comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, wherein the nucleotide sequence further comprises a series of miRNA target sequences arranged within a heterologous miRblock linked to the transgene, each miRNA target sequence corresponding to a miRNA in a eukaryotic MVA-producing cell, and at least one of the miRNA target sequences within the miRblock is capable of mediating down-regulation of expression of the transgene in the eukaryotic MVA-producing cell.

2. A transcription unit comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a series of miRNA target sequences arranged within heterologous miRblocks linked to the transgene, each miRNA target sequence corresponding to a miRNA sequence in a eukaryotic MVA-producing cell, and at least one of the miRNA target sequences within the miRblocks being capable of mediating down-regulation of expression of the transgene in the eukaryotic MVA-producing cell.

3. A series of miRNA target sequences arranged within a heterologous miRblock, each miRNA target sequence corresponding to a miRNA in a eukaryotic MVA-producing cell, and at least one of the miRNA target sequences within the miRblock is capable of mediating down-regulation of expression of an introduced gene bound to the miRblock in the eukaryotic MVA-producing cell.

4. The recombinant MVA of claim 1, wherein at least one miRNA target sequence corresponds to the sequence of said miRNA with about 80-100% nucleotide sequence similarity.

5. A transcription unit as described in claim 2, wherein at least one miRNA target sequence corresponds to the sequence of the miRNA with approximately 80 to 100% nucleotide sequence similarity.

6. The miRblock described in claim 3, wherein at least one miRNA target sequence corresponds to the sequence of the miRNA with approximately 80 to 100% nucleotide sequence similarity.

7. The recombinant MVA of claim 1 or 4, wherein at least one miRNA target sequence is selected from the group consisting of nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

8. A transcription unit described in claim 2 or 5, wherein at least one miRNA target sequence is selected from the group consisting of nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

9. A miRblock described in claim 3 or 6, wherein at least one miRNA target sequence is selected from the group consisting of nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

10. The recombinant MVA of claim 1 or 4, wherein the miRblock comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO:

55.

11. A transcription unit described in claim 2 or 5, wherein the miRblock comprises or consists of a nucleotide sequence selected from the group consisting of nucleotide sequences shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO:

55.

12. The miRblock described in claim 3 or 6, wherein the miRblock comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO:

55.

13. The recombinant MVA of claim 1 or 4, wherein the transgene encodes a protein from respiratory syncytial virus (RSV), or an antigenic portion thereof.

14. The recombinant MVA described in claim 1 or 4, wherein the introduced gene encodes a protein selected from the group consisting of RSV G(A), G(B), F, N, and M2-1 proteins, and N / M2-1 fusion proteins, or an antigenic portion thereof.

15. A transcription unit described in claim 2 or 5, wherein the introduced gene encodes a protein derived from respiratory syncytial virus (RSV), or an antigenic portion thereof.

16. The transcription unit of claim 2 or 5, wherein the transgene encodes a protein, or an antigenic portion thereof, selected from the group consisting of RSV G(A), G(B), F, N, and M2-1 proteins, and N / M2-1 fusion proteins.

17. The miRblock described in claim 3 or 6, wherein the introduced gene encodes a protein derived from respiratory syncytial virus (RSV), or an antigenic portion thereof.

18. The miRblock of claim 3 or 6, wherein the transgene encodes a protein selected from the group consisting of RSV G(A), G(B), F, N, and M2-1 proteins, and N / M2-1 fusion proteins, or an antigenic portion thereof.

19. The recombinant MVA according to claim 1 or 4, wherein the eukaryotic MVA-producing cells are avian primary cells or permanent avian cell lines.

20. A transcription unit described in claim 2 or 5, wherein the eukaryotic MVA-producing cell is an avian primary cell or an avian permanent cell line.

21. The miRblock described in claim 3 or 6, wherein the eukaryotic MVA-producing cells are avian primary cells or avian permanent cell lines.

22. The recombinant MVA described in claim 19, wherein the avian permanent cell line is a quail cell.

23. The transcription unit described in claim 20, wherein the avian permanent cell line is a quail cell.

24. The miRblock described in claim 21, wherein the avian permanent cell line is a quail cell.

25. The recombinant MVA of claim 1 or 4, wherein the promoter is an immediate early promoter selected from the group consisting of Pr13.5long, Pr1328, and PrLE1 (pHyb) promoters.

26. A transcription unit described in claim 2 or 5, wherein the promoter is an immediate early promoter selected from the group consisting of Pr13.5long, Pr1328, and PrLE1 (pHyb) promoters.

27. 10. A method for producing a recombinant MVA according to claim 1 or 4, comprising: (1) preparing a series of miRNA target sequences arranged within the miRblock according to claim 3 or 6; (2) preparing the transcription unit according to claim 2 or 5 using the miRblock prepared in step (1); (3) inserting the transcription unit prepared in step (2) into MVA; (4) infecting and propagating eukaryotic MVA-producing cells with the MVA obtained in step (3); (5) harvesting the recombinant MVA propagated in step (4); The method comprising:

28. Use of a miRNA target sequence for down-regulating the expression of an MVA-encoding transgene in a eukaryotic MVA-producing cell in vitro, wherein the miRNA target sequence is selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

29. Use of a series of miRNA target sequences located within a miRblock for down-regulating expression of an MVA-encoding transgene in eukaryotic MVA-producing cells in vitro, wherein the miRblock comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO:

55.

30. A pharmaceutical composition or vaccine comprising the recombinant MVA of claim 1 or 4.

31. A pharmaceutical composition or vaccine as described in claim 30 for treating or preventing an infectious disease or cancer.

32. A recombinant MVA according to claim 1 or 4 for use as a medicine or vaccine.

33. A recombinant MVA according to claim 1 or 4 for use in the treatment or prevention of an infectious disease or cancer.

34. A recombinant MVA comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, wherein the nucleotide sequence further comprises an miRNA target sequence linked to the transgene, the miRNA target sequence corresponding to an miRNA in a eukaryotic MVA-producing cell, and the miRNA target sequence within the miRblock is capable of mediating down-regulation of expression of the transgene in the eukaryotic MVA-producing cell.

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