MVA-related novel vaccinia virus vectors with broad genome symmetry

文档序号：1785726 发布日期：2019-12-06 浏览：24次中文

阅读说明：本技术 具有广泛基因组对称的mva相关的新型痘苗病毒载体 (MVA-related novel vaccinia virus vectors with broad genome symmetry ) 是由英戈·乔丹福尔克·桑迪希于 2017-02-23 设计创作，主要内容包括：本发明涉及新型改良安卡拉痘苗(MVA)相关病毒。本发明还涉及用于培养所述MVA相关病毒的方法以及用于生产所述MVA相关病毒的方法。此外,本发明涉及药物组合物,所述药物组合物包含所述MVA相关病毒以及一种或多种药学上可接受的赋形剂、稀释剂和/或载体。此外,本发明涉及包含所述MVA相关病毒的疫苗。另外,本发明涉及用于医学用途的所述MVA相关病毒。(The present invention relates to novel improved vaccinia Ankara (MVA) -related viruses. The invention also relates to methods for culturing said MVA-related virus and to methods for producing said MVA-related virus. Furthermore, the present invention relates to a pharmaceutical composition comprising said MVA-related virus together with one or more pharmaceutically acceptable excipients, diluents and/or carriers. Furthermore, the present invention relates to a vaccine comprising said MVA-related virus. In addition, the present invention relates to said MVA-related virus for medical use.)

1. A Modified Vaccinia Ankara (MVA) -associated virus comprising one or more of the following characteristics:

(i) A nucleic acid sequence corresponding to a region comprising a right Inverted Terminal Repeat (ITR) and extending to deletion site III but not deletion site III, but not a nucleic acid sequence comprising a left Inverted Terminal Repeat (ITR) and extending to deletion site V but not deletion site V;

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) No deletion site I;

(v) Two deletion sites IV;

(vi) an open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

(vii) two open reading frames of at least one gene product selected from the group consisting of a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R; and/or

(viii) A nucleic acid sequence encoding an L3L gene product, wherein the nucleic acid sequence comprises at least one mutation that results in a modification of the amino acid sequence of the gene product.

2. The MVA-associated virus of claim 1, wherein,

(i) The region comprising the right ITR and extending to deletion site III but not deletion site III has a nucleic acid sequence according to SEQ ID NO 37 or Genbank accession No. KY633487 (preferably ranging from nucleotide positions 162221 to 190549 or the nucleotide positions corresponding thereto), or is a variant which is at least 95% identical to said nucleic acid sequence;

(ii) Said region comprising the left ITR and extending to but not comprising the deletion site V has a nucleic acid sequence according to SEQ ID NO 37 or Genbank accession No. KY633487 (preferably ranging from nucleotide position 1 to 31261 or the nucleotide position corresponding thereto) or is a variant which is at least 95% identical to said nucleic acid sequence; and/or

(iii) Said nucleic acid sequence comprising deletion site IV and said right ITR has a nucleic acid sequence according to SEQ ID No. 37 or Genbank accession No. KY633487 (preferably ranging from nucleotide positions 179272 to 190549 or the nucleotide positions corresponding thereto), or is a variant which is at least 95% identical to said nucleic acid sequence; and/or

3. the MVA-related virus of claim 1 or 2, wherein the virus further comprises a heterologous nucleic acid sequence.

4. The MVA-related virus of claim 3, wherein the heterologous nucleic acid sequence is selected from the group consisting of sequences encoding:

(i) antigens, in particular antigenic epitopes;

(i) a diagnostic compound; and

(iii) A therapeutic compound.

5. The MVA-associated virus according to any one of claims 1 to 4, wherein the virus is capable of productive replication in avian cells.

6. The MVA-associated virus according to any of claims 1 to 5, wherein the virus is incapable of productive replication in primate cells, more preferably in human cells.

7. The genome of the MVA-related virus of any of claims 1 to 6.

8. A cell comprising the MVA-associated virus according to any of claims 1 to 6 or the genome according to claim 7.

9. the cell of claim 8, wherein the cell is a non-adherent/suspension cell.

10. The cell of claim 8 or 9, wherein the cell is an avian cell.

11. A method for culturing the MVA-associated virus according to any of claims 1 to 6, comprising the steps of:

(a) Providing a cell according to any one of claims 18 to 20;

(b) Culturing the cell; and

12. A method for producing the MVA-associated virus according to any of claims 1 to 6, the method comprising the steps of:

(a) Infecting the cells with MVA virus;

(b) culturing the cell;

(d) Repeating steps (a) to (c) with the MVA virus isolated in step (c) until a MVA-related virus comprising one or more of the following characteristics is detected:

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) No deletion site I;

(v) Two deletion sites IV;

(vi) An open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

(vii) Two open reading frames of at least one gene product selected from the group consisting of a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R; and/or

13. a pharmaceutical composition comprising the MVA-related virus according to any of claims 1 to 6 or the genome according to claim 7, and one or more pharmaceutically acceptable excipients, diluents and/or carriers.

14. A vaccine comprising the MVA-associated virus according to any of claims 1 to 6 or the genome according to claim 7.

15. The MVA-related virus according to any of claims 1 to 6 or the genome according to claim 7 for medical use.

Technical Field

The present invention relates to novel improved vaccinia Ankara (MVA) -related viruses. The invention also relates to methods for culturing said MVA-related virus and to methods for producing said MVA-related virus. Furthermore, the present invention relates to a pharmaceutical composition comprising said MVA-related virus together with one or more pharmaceutically acceptable excipients, diluents and/or carriers. Furthermore, the present invention relates to a vaccine comprising said MVA-related virus. In addition, the present invention relates to said MVA-related virus for medical use.

Background

Vaccinia virus (VACV), belonging to the Orthopoxvirus genus (Orthopoxvirus) of the Poxviridae (Poxviridae), has been used as a live vaccine in the successful eradication of smallpox. However, vaccinia virus can effectively infect humans, and its use as an expression vector in the laboratory has been subject to safety concerns and regulations. The safety concerns of the VACV standard strain have been addressed by the development of vaccinia vectors from highly attenuated virus strains characterized by limited replication capacity in vitro and lack of virulence in vivo.

Modified Vaccinia Ankara (MVA) virus is a strain of vaccinia virus obtained by adaptation of the VACV strain to replication in primary chicken embryo fibroblasts culture (Mayr and Munz, 1964). It belongs to a highly attenuated poxvirus that can be used as a vaccine vector for safe and reactogenic expression of large transgenes to elicit robust T cell-mediated immune responses (Drillien et al, 2004; Liu et al, 2008; Sutter and Moss, 1992; Sutter et al, 1994). It is not inhibited by preexisting immunity and has also been investigated for protective and therapeutic treatment of immunocompromised human patients (ceber et al, 2006; McShane et al, 2002; Milligan et al, 2016; Webster et al, 2005).

Productive replication of MVA is limited to avian cells and to very few mammalian cells, such as cell lines derived from Egyptian Hepterus and BHK (Carroll and Moss, 1997; Drexler et al, 1998; Jordan et al, 2009 a). The molecular basis of the narrow host range of MVA compared to the parental virus appears to be due to six deletions (deletion sites I to VI, the numbers increasing with the size of the deletion rather than the position in the genome) and several more limited mutations that may result from adaptation to avian cells (Blanchard et al, 1998; Meisinger-Henschel et al, 2007; Meyer et al, 1991).

Providing an adequate supply of MVA virus is challenging. Since MVA does not replicate and therefore does not amplify in the recipient, it must be administered at a high dose of 108 infectious units (Wyatt et al, 2004). However, the MVA virus production systems currently available are time consuming and expensive and do not meet the needs of the pharmaceutical industry.

Here, the inventors isolated and characterized for the first time a novel MVA-related virus. The present inventors found that the novel MVA-related viruses are fundamentally different in genomic structure from (wild-type) MVA viruses. The structural differences result in viruses with advantageous properties over (wild-type) MVA viruses. In particular, the novel MVA-related viruses release a higher number of infectious units into the supernatant of infected cultures compared to cultures infected with (wild-type) MVA viruses. Furthermore, the novel MVA-related viruses replicate to very high titers compared to (wild-type) MVA viruses. Furthermore, the novel MVA-related viruses induce less syncytia in adherent culture.

The beneficial properties of the novel MVA-related viruses described above improve their industrial production. In particular, it allows the production of novel MVA-related viruses in high yield and thus also the production of heterologous proteins (e.g. antigens) which may be comprised therein. In addition, the novel MVA-related viruses can be isolated directly from the cell-free supernatant, thereby facilitating purification and thus operation and arrangement of the bioreactor for producing the MVA-related viruses. Which in turn reduces its production costs.

disclosure of Invention

In a first aspect, the present invention relates to a Modified Vaccinia Ankara (MVA) -associated virus comprising one or more of the following characteristics:

(i) A nucleic acid sequence corresponding to a region comprising a right Inverted Terminal Repeat (ITR) and extending to deletion site III but not deletion site III, and not (instead) a nucleic acid sequence comprising a left Inverted Terminal Repeat (ITR) and extending to deletion site V but not deletion site V;

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) No deletion site I;

(v) Two deletion sites IV;

(vi) an open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

In a second aspect, the present invention relates to the genome of a MVA-related virus according to the first aspect.

In a third aspect, the present invention relates to a cell comprising a MVA-related virus according to the first aspect or a genome according to the second aspect.

In a fourth aspect, the present invention relates to a method for culturing a MVA-related virus according to the first aspect, said method comprising the steps of:

(a) providing a cell according to the third aspect;

(b) culturing the cell; and

In a fifth aspect, the present invention relates to a method for producing a MVA-related virus according to the first aspect, the method comprising the steps of:

(a) Infecting the cells with MVA virus;

(b) culturing the cell;

(d) Repeating steps (a) to (c) with the MVA virus isolated in step (c) until a MVA-related virus comprising one or more of the following characteristics is detected:

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) No deletion site I;

(v) Two deletion sites IV;

(vi) An open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

in a sixth aspect, the present invention relates to a pharmaceutical composition comprising a MVA-related virus according to the first aspect or a genome according to the second aspect, and one or more pharmaceutically acceptable excipients, diluents and/or carriers.

In a seventh aspect, the present invention relates to a vaccine comprising a MVA-related virus according to the first aspect or a genome according to the second aspect.

In an eighth aspect, the present invention relates to a MVA-related virus according to the first aspect or a genome according to the second aspect for medical use.

this summary of the invention does not describe all features of the invention.

drawings

FIG. 1: CR-related mutations were inserted into the backbone of the wild-type virus, alone or in various combinations, along with GFP to allow visualization of plaques (spots) without fixation and immunostaining. All infections were performed at an MOI of 0.01 and showed a plaque phenotype at 48h PI. GFP is a GFP-expressing wild-type MVA, wt.a34cr, wt.l3CR and wt.a3A9a34cr represent wt.gfp viruses containing CR-related mutations H639Y, K75E and D86Y in genes A3L, A9L and a34R, and cr19.GFP is a GFP-expressing MVA-CR19.

FIG. 2: deletion site I was lost in MVA-CR19, but not in wild-type MVA (passage 5 in CR cells, MVA-CR5) or MVA obtained by serial passage of adherent cells of egyptian fruit bats (MVA-R18(Jordan et al, 2013 a)).

FIG. 3: predicted (deployed) recombination of MVA-CR19. (a) Schematic representation of the wild type genome with Inverted Terminal Repeats (ITRs) and the expected amplification products spanning 6 deletion sites (roman numerals). The bars starting with "C-" represent contigs (contigs) obtained in the previous (Jordan et al, 2013a) sequence analysis. Recombination regions other than ITRs are shown by bold lines. Left ITR (b) and right ITR (c) in more detail. Open arrows indicate open reading frames. (d) Predicted left ITR of MVA-CR19 after recombination. (e) Sequence chromatogram of recombination sites, reference GenBank U94848 numbering. (a-d) dark gray: sequences derived from the right side of the genome; light gray: sequences derived from the left side of the genome. The closed rectangles represent ITRs and the recombination regions other than ITRs are shown by bold lines. Dark grey and black opposite solid arrows indicate the strand orientation and mark recombination sites.

FIG. 4: confirmation of ITR rearrangement as shown in the above figure. (a) Predicted structure of PCR amplification products derived from left ITRs of GenBank sequence U94848 and MVA-CR19. Genes (open arrows), deletion sites I and IV (filled boxes), ITRs (grey and dark grey pointed rectangles), recombination sites (dark grey and black bold arrows) and target sites for restriction enzymes to confirm the predicted structure of the left ITRs are shown. (b) Agarose gel electrophoresis of the long PCR products shown in (a). For wild type, the expected sizes for cleavage with BclI were 3007bp, 2518bp, 1992bp, 1172bp and 671bp, the expected sizes for cleavage with NruI were 7998bp and 1362bp, and the expected sizes for cleavage with ApaLI were 8464bp and 896 bp; for MVA-CR19, the expected sizes for cleavage with BclI were 13634bp, 1890bp, 1498bp, 1269bp, 984bp, 886bp, 671bp and 480bp, for cleavage with NruI were 11257bp, 8693bp and 1362bp, and for cleavage with ApaLI were 9572bp, 5646bp, 4706bp, 896bp and 492 bp.

FIG. 5: stability of MVA genotype in pix culture. (a) Plaque-purified recombinant wild-type containing GFP expression cassette in deletion site III and CR19 isolate were passaged 20 times in CR. Note the expected size shift of the deletion site III amplification product caused by the GFP cassette and the absence of the deletion site I signal in the CR19 derivative. (b) In the passage interval, the viral DNA purified in (a) also showed a stable pattern of Restriction Fragment Length Polymorphisms (RFLPs) in the A34R gene of both isolates. (c) The presence of the amplification product of RS469 replacing deletion site I in the CR19 derivative was found in the passage of CR19, but not in the passage of the wild type. Applying the sample in the same order as in (b). (d) Stable pattern of RFLP and deletion sites in recombinant viruses carrying a point mutation of the CR genotype in the wild-type backbone or a GFP and mcherry (red) dual expression cassette in the CR19 derivative. (e) The infectious titer obtained with MVA-cr19.gfp. red dual expression vector at the indicated passage level was determined by measurement of PFU with immunostaining for vaccinia virus protein or via measurement of green or red fluorescence signal. The proportion of infectious units is shown in the figure. A decrease in FFU relative to PFU would indicate a loss of the expression cassette. Since some excitation of mCherry also occurs under GFP fluorescence conditions, GFP-based titrations tend to be higher than mCherry-based titrations in dual-expression constructs. The mean values for all ratios were 1.00. + -. 0.09, with no significant difference between the mean values for the ratios of GFP-FFU: PFU and mCherry-FFU: PFU at the 99% confidence level (paired t-test).

FIG. 6: investigation of the L3 mutation. (a) Restriction fragment length polymorphism in MVA-CR19 caused by a mutation in L3L. The 717-bp amplicon was expected to produce HphI fragments of 649bp and 68bp for MVA-CR19 and 380bp, 269bp and 68bp for the wild type. (b-d) two independent MVA-wt.l3lcr.gfp formulations were used for these experiments. (b) Replication kinetics of the designated recombinant viruses in single cell suspension culture. The dashed line shows replication of the designated virus in the conventional method (which induces aggregates). (c, d) infecting adherent CR. pIX cells with GFP expressing rMVA. The mean and standard deviation of three replicates (replicates) of MVA-wt.gfp and MVA-cr19.gfp and the mean and standard deviation of six values (two replicates) of MVA-wt.l3cr.gfp are described. The log10FFU/mL of MVA preparations used for infection in this experiment were 8.5(MVA-wt.gfp), 8.3 and 8.8(MVA-wt.l3cr.gfp) and 9.8 (MVA-cr19.gfp). (. x) represents a significant difference between CR19 and the corresponding 40h time point of the wild-type recombinant virus in the two-tailed independent t-test; the differences were not significant in the comparison between the L3L mutant and the wild type.

Detailed Description

before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A Multilingual gloss of biological technical terms (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B.and H.proceedings (1995), Helvetica Chimica Acta, CH-4010Basel, Switzerland.

Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, GenBank filing sequence accession numbers, etc.) is hereby incorporated by reference, whether supra or infra, in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The elements of the present invention will be described hereinafter. These elements are listed with particular embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and/or preferred elements. Moreover, unless the context indicates otherwise, it is contemplated that any arrangement or combination of all described elements in this application is disclosed by the description of the present application.

throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

The term "attenuated virus" as used herein refers to a virus having attenuated virulence in the intended recipient (e.g., human or animal recipient). Such properties can be achieved by adaptation of the virus to a narrow temperature range or a narrow host range, as well as to other artificial replication environments, including defined chemical composition media. Replication of such viruses is limited in cells derived from the intended recipient (e.g., human or animal recipient) or in cells removed from a tissue environment. The virus can be replicated to high titers outside of the intended recipient, for example in permissive cell cultures or laboratory animals. Examples of attenuated virus strains are the Ender's attenuated measles virus Edmonston strain used to provide protection against severe measles disease, or vaccinia virus strains used in the eradication sport of vaccinia developed by the World Health Organization (WHO) in the 70 s of the 20 th century.

The term "highly attenuated virus" as used herein refers to a virus that has blocked virulence in the intended recipient (e.g., human or animal recipient). Such properties can be achieved by adaptation of the virus to a narrow temperature range or a narrow host range, as well as to other artificial replication environments, including defined chemical composition media. Replication of such viruses is blocked in cells derived from the intended recipient (e.g., human or animal recipient) or in cells removed from a tissue environment. The virus can replicate to high titers outside of the intended recipient (e.g., in permissive cell/cell cultures or laboratory animals). The MVA viruses of the present invention are highly attenuated viruses. The MVA virus does not replicate in human or non-human primate cells.

the term "host-restricted virus" as used herein refers to a virus that replicates (only or predominantly) in a particular host organism, e.g., in a cell (e.g., avian cells) or in an animal (e.g., laboratory animals). The virus does not replicate or only replicates at very low levels in other organisms, for example in cells other than avian cells. Host-restricted viruses can be achieved by (serial) viral passaging of the virus in a host organism, for example in avian cells. The MVA-related viruses of the present invention are restricted to avian cells. The MVA-related virus does not replicate in human cells.

the term "viral passaging" as used herein refers to a process involving infection of a range of host organisms, e.g., cells or animals (e.g., laboratory animals), with a virus. The virus is incubated for some time each time, and then the next host organism is infected with the incubated virus. This process may also be referred to as "serial virus passaging". For example, serial virus passaging allows the production of (highly) attenuated viruses and/or host-restricted viruses. The MVA-related viruses of the present invention are highly attenuated viruses. The MVA-related virus is restricted to avian cells. The MVA-related virus does not replicate in human or non-human primate cells.

When the term "permissive" is used to define a host organism, such as a cell (e.g., avian cell) or an animal (e.g., laboratory animal), it refers to the fact that a virus is able to circumvent the defenses of the organism and is able to invade, replicate in, and escape from the cell. Typically, this occurs when the virus has modulated one or several of the organism's inherent defenses of the cells and/or the organism's immune system.

the term "recipient" as used herein refers to a subject who is acceptable for a virus, e.g., vaccinable with a virus. The subject may be a human or an animal. The animal may be a member of a mammalian species, such as a dog, cat, wolf, ferret, rodent (e.g., mouse, rat, or hamster), horse, cow, sheep, goat, pig, bat (e.g., mega-bat or micro-bat), or non-human primate (e.g., monkey, such as ape). In particular, the MVA-related viruses of the present invention do not replicate in human or non-human primate recipients.

the term "host organism" as used herein refers to an organism that can be used for virus production and/or adaptation. The host organism may be a cell or an animal (e.g., a laboratory animal). The cell may be an avian cell (e.g., a chicken, quail, goose, or duck cell (e.g., a duck retina (CR) cell)). The animal, particularly a laboratory animal, may be a bird (e.g., a chicken, quail, goose, or duck), dog, ferret, rodent (e.g., a mouse, rat, or hamster), sheep, goat, pig, bat (e.g., a giant bat or a miniature bat), or a non-human primate (e.g., a monkey, such as a simian). In particular, the MVA-related viruses of the invention replicate in avian cells (e.g. in chicken, quail, goose or duck cells) or in birds (e.g. in chicken, quail, goose or duck).

The term "infection" as used herein refers to the ability of a virus to replicate in cells and produce viral particles. Infectivity can be assessed by detecting viral load or by observing disease progression in humans or animals.

The term "vaccine" as used herein refers to an agent that can be used to elicit protective immunity in a recipient (e.g., a human or animal recipient). With respect to effectiveness, vaccines are able to elicit immunity in a partially immunized population, and some individuals may not be able to generate a robust or protective immune response, or in some cases, any immune response. This failure may result from the recipient's genetic background or due to an immunodeficiency state (acquired or congenital immunodeficiency) or immune suppression (e.g., due to chemotherapy treatment or use of immune suppressive drugs). Vaccine efficacy can be determined in animal models. The vaccine of the invention comprises a MVA-related virus according to the first aspect or a genome according to the second aspect. In this regard, it is noted that the MVA-related virus may itself be a vaccine. It confers protection against pox (pox). However, the virus may further comprise a heterologous nucleic acid sequence, such as a sequence encoding an antigen, in particular an antigenic epitope, which may elicit additional protective immunity in the recipient against the heterologous nucleic acid sequence. MVA-related viruses comprising heterologous nucleic acid sequences may also be referred to as recombinant MVA-related viruses.

The term "vaccination" as used herein refers to the challenge (challenge) of a recipient (e.g., a human or animal recipient) with an infectious virus (e.g., in an attenuated or inactivated form of the infectious virus) to induce specific immunity. In the present invention, the recipient is challenged with a MVA-related virus according to the first aspect or with a genome according to the second aspect to induce immunity against vaccinia. However, in the context of the present invention, the term "vaccination" also covers the challenge of a recipient with a MVA-related virus further comprising a heterologous nucleic acid sequence. The heterologous sequence is a sequence against which additional protective immunity should be elicited. The heterologous sequence may encode an antigen, in particular an antigenic epitope. MVA-related viruses comprising heterologous nucleic acid sequences may also be referred to as recombinant MVA-related viruses. Examples of such epitopes heterologous to the virus cover, for example, epitopes from proteins of other viruses such as influenza virus, hepatitis virus (e.g., hepatitis c virus), Human Immunodeficiency Virus (HIV), flavivirus, paramyxovirus, hantavirus or filovirus or epitopes derived from proteins associated with the development of tumors and cancer. After administration of the vaccine to a recipient, the epitopes are expressed and presented to the immune system, which induces a specific immune response against these epitopes. The recipient is thereby immunized against the protein containing the epitope.

The term "heterologous nucleic acid sequence" as used herein refers to a nucleic acid sequence which is not normally found in close relation to a virus in nature, in particular to a MVA-related virus according to the present invention. MVA-related viruses comprising heterologous nucleic acid sequences may also be referred to as recombinant MVA-related viruses. The heterologous nucleic acid sequence is preferably selected from the group encoding (i) an antigen (particularly an antigenic epitope); (ii) a diagnostic compound; and (iii) a sequence of a therapeutic compound.

the term "epitope" (also referred to as antigenic determinant) as used herein refers to the part of an antigen that is recognized by the immune system, in particular antibodies, B-cells or T-cells.

The term "protection" as used herein refers to the prevention or treatment (or both) of the development or persistence of a disease (e.g., pox) in a recipient (e.g., a human), as appropriate.

The term "protective immunity" as used herein includes humoral (antibody) immunity or cellular immunity or both, which is effective, for example, to eliminate or reduce the infectious cell or pathogen (e.g., virus, such as poxvirus) load or to produce any other measurable reduction in infection in an immunized (vaccinated) subject.

The term "excipient" as used herein is intended to mean all substances in a pharmaceutical composition that are not active ingredients, such as binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring or coloring agents.

the term "diluent" as used herein relates to a diluent (diluting agent) and/or a viscosity reducing agent (thinning agent). Further, the term "diluent" includes solutions, suspensions (e.g., liquid suspensions or solid suspensions) and/or media.

The term "carrier" as used herein relates to one or more compatible solid or liquid fillers suitable for administration (e.g., to a human). The term "carrier" relates to a natural or synthetic organic or inorganic component combined with an active ingredient to facilitate the use of the active ingredient. Preferably, the carrier component is a sterile liquid, such as water or oil (including mineral oil, animal or vegetable derived oils, such as peanut oil, soybean oil, sesame oil, sunflower oil, and the like). Saline solutions and aqueous dextrose and glycerol solutions can also be used as aqueous carrier compounds.

Pharmaceutically acceptable carriers or diluents for therapeutic use are well known in the Pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R Gennaro edge.1985). Examples of suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Examples of suitable diluents include ethanol, glycerol and water.

the choice of pharmaceutical carrier, diluent and/or excipient may be made according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions of the present invention may comprise any suitable binder, lubricant, suspending agent, coating agent and/or solubilizing agent as (or in addition to) a carrier, excipient or diluent. Examples of suitable binders include starch, gelatin, natural sugars (e.g. glucose), anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums (e.g. gum arabic, tragacanth gum or sodium alginate), carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical compositions. Examples of preservatives include esters of p-hydroxybenzoic acid, sodium benzoate and sorbic acid. Antioxidants and suspending agents may also be used.

The term "primary cell" or "primary cell culture" as used herein refers to a cell or culture that is generally incapable of passaging more than 50 population doublings before suffering from senescence, culture arrest or cell death. The term "secondary cell" or "secondary cell culture" as used herein refers to a cell or culture derived directly from a primary cell or primary cell culture. Population doubling limitations still apply. The term "immortalized cell" or "immortalized cell culture" as used herein refers to a cell or culture and its progeny that are not limited by the number of potential cell doublings. Cell cultures may consist of primary cells, secondary cells or immortalized cells (i.e., cells of a cell line). In a preferred embodiment of the invention, the cells are from the CR or CR. pIX cell lines were derived from immortalized Muscovy duck retina cells designed for Vaccine production (Jordan et al, 2009, Vaccine 27, 748-. The pIX cell line further stably integrates into its genome the gene encoding the adenoviral pIX protein and expresses said gene. In other preferred embodiments of the invention, the cell is a Chicken Embryo Fibroblast (CEF) cell. The cells are primary cells.

the term "isolated cell" as used herein refers to a cell that has been removed from its natural or culture environment. Thus, an isolated cell may be free of some or all of the native components or culture components (i.e., components of the organism in which the cell naturally occurs (e.g., organs, particularly tissues) or components in which the cell is cultured (e.g., culture medium or culture-related impurities, such as culture residues)). The cells may be infected with a MVA-related virus according to the first aspect or transfected with a genome according to the second aspect. Techniques for how to infect or transfect cells are known to those skilled in the art.

the terms "non-adherent cells" and "suspension cells" are used interchangeably herein. In the context of the present invention, the terms "non-adherent cells" and "suspension cells" refer to cells that are capable of surviving suspension culture without adhering to a surface (e.g., a tissue culture plastic carrier or microcarrier). The cells may be cells that are capable of naturally occurring in suspension without adhering to a surface. The cells may also be cells that have been engineered or adapted so that they are able to survive in suspension culture without adhering to a surface (e.g., a tissue culture plastic carrier or microcarrier). Most cells are adherent cells in their original, unmodified or unadapted form. Nonadherent cells can generally be grown to a higher density than allowed by the adherent conditions. Therefore, non-adherent cells are more suitable for culture in industrial levels (e.g. in a bioreactor setting or in stirred culture). Cells must generally be adapted to non-adherent cell culture. Since the original cells will undergo apoptosis in the absence of serum and/or in the absence of suitable surfaces, this adaptation is a long process requiring passage with progressively decreasing serum levels (e.g., dilution lines (rows) from 10% to 0% Fetal Calf Serum (FCS)), thereby selecting a population of irreversibly engineered cells. Adapted non-adherent cells are known in the art. Protocols for transferring cells from an adherent state to a non-adherent state are known to those skilled in the art (see, for example, Appl Microbiol Biotechnol. 2008Mar; 78(3):391-9.Epub 2008Jan 9).

In contrast, the term "adherent cells" as used herein refers to cells that require a surface (e.g., a tissue culture plastic carrier or microcarrier). The surface may be coated with extracellular matrix components to increase the adherent properties and provide other signals required for growth and differentiation. The cells require regular passage but allow easy visual inspection under an inverted microscope. The cells need to be enzymatically dissociated (e.g., with trypsin). Furthermore, the growth of adherent cells is limited by the surface area, which limits the yield.

The term "serum-free conditions" as used herein refers to conditions in which cells are grown in a medium that is completely serum-free from animals. In contrast, cells are grown in a medium that is completely free of any animal-derived components, and preferably in a medium that is free of any complex mixture of biological components (so-called "defined chemical composition medium").

The term "cell proliferation medium" as used herein refers to a medium that supports cell division for at least 10 cell doublings, such that, for example, by passaging through the medium, a seed of 8 × 105 cells can be expanded to about 4 × 108 cells, e.g., sufficient for use in a 200 liter bioreactor. The term "proliferating cells" as used herein refers to dividing cells, i.e., cells capable of at least another 10-fold cell multiplication at a doubling rate of at least one in 48 hours or less.

The term "virus production medium" as used herein refers to a medium that enhances production of a virus in a culture of proliferating cells. By adding virus production medium, cell aggregates are induced and cell proliferation in culture is reduced by at least 2-fold or completely stopped. The virus production medium preferably contains CaCl2, MgSO4 and/or NaCl. The CaCl2 content is preferably in the range between 150mg/l and 250mg/l, more preferably in the range between 180mg/l and 250mg/l, most preferably in the range between 200mg/l and 220mg/l, for example 150mg/l, 155mg/l, 160mg/l, 165mg/l, 170mg/l, 175mg/l, 180mg/l, 185mg/l, 190mg/l, 195mg/l, 200mg/l, 205mg/l, 210mg/l, 215mg/l, 220mg/l, 225mg/l, 230mg/l, 235mg/l, 240mg/l, 245mg/l or 250 mg/l; the MgSO4 content is preferably in the range between 50mg/l and 150mg/l, more preferably in the range between 70mg/l and 150mg/l, most preferably in the range between 90mg/l and 120mg/l, for example 50mg/l, 55mg/l, 60mg/l, 65mg/l, 70mg/l, 75mg/l, 80mg/l, 85mg/l, 90mg/l, 95mg/l, 100mg/l, 105mg/l, 110mg/l, 115mg/l, 120mg/l, 125mg/l, 130mg/l, 135mg/l, 140mg/l, 145mg/l or 150 mg/l; and/or the NaCl content is preferably in the range between 5000mg/l and 7500mg/l, more preferably in the range between 6000mg/l and 7000mg/l, most preferably in the range between 6500mg/l and 6800mg/l, for example 5000mg/l, 5500mg/l, 6000mg/l, 6500mg/l, 7000mg/l or 7500 mg/l. For example, the virus production medium may comprise a salt content of 205mg/l CaCl2, 100mg/l MgSO4, and/or 6500mg/l NaCl.

the term "productive replication" as used herein means that at least one more virus can be recovered from an infected culture than is added to infect the culture. The virus can cause cytopathic effects and replicate to levels that ultimately lead to massive cell death in infected cultures. In contrast to productive replication, reproductive replication can occur at very low levels without concomitant cytopathic effects, and can ultimately lead to the loss of virus in viable culture.

the term "Modified Vaccinia Ankara (MVA) virus" as used herein refers to highly attenuated vaccinia virus strains derived from the ankara strain and developed for use as vaccines and vaccine adjuvants. The original MVA virus was isolated from the wild-type ankara strain by serial passage through chicken embryo cells. So treated, the MVA virus loses about 15% of its wild-type vaccinia virus genome, including its ability to replicate efficiently in primate (including human) cells.

The MVA virus contains a single copy of a double-stranded DNA genome, approximately 178kb in length. The viral genomic DNA comprises a core region flanked by viral telomeres. In particular, the viral telomeres are located at the left and right positions of the viral genomic DNA. The telomere further comprises an Inverted Terminal Repeat (ITR). The virus, which is generally recognized as MVA virus, contains 6 characteristic deletion sites, referred to as deletion sites I, II, III, IV, V and VI. The number increases with the size of the deletion rather than the location in the genome. Deletion site I is located in the left viral telomere and deletion sites II, III, V and VI are located in the core region. Deletion site IV is located in the right telomere. Only viruses containing all six deletion sites are considered true MVAs. Viruses generally recognized as MVA viruses further comprise (an) open reading frame(s) of (functional) gene products selected from the group consisting of C11R, C10L, D7L, a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R. Methods to describe this true/true MVA have been published by Kremer et al (Kremer et al, 2012). The MVA virus may have a sequence according to accession number U94848 (version U94848.1 and GI: 2772662).

The term "Modified Vaccinia Ankara (MVA) -associated virus" as used herein refers to a virus that is structurally different from (the above or wild-type) MVA virus. It can therefore be considered that it no longer constitutes a typical MVA virus. Rather, it is a MVA-related virus. The MVA-related virus may for example no longer comprise a specific deletion site (e.g. deletion site I) and/or comprise a dual specific deletion site (e.g. deletion site IV). Alternatively or additionally, the MVA-related virus may for example no longer comprise an open reading frame for a specific gene product (e.g. gene product C11) and/or comprise a dual open reading frame for a specific gene product (e.g. gene product B1). In a first aspect, MVA-related viruses of the invention are specifically described. The MVA-related virus may have/comprise a sequence according to SEQ ID NO:37 or Genbank accession No. KY633487(MVA-cr19. gfp).

The term "isolated MVA-related virus" as used herein refers to a virus that has been removed from its natural or culture environment. Thus, an isolated MVA-related virus may not contain part or all of the cellular components, i.e. the components of the cells in which the virus is naturally present or the components of the cells in which the virus is cultured (e.g. cytoplasmic components or membrane components). It may also be free of part or all of the culture components (e.g., culture medium or culture-related impurities such as culture residues).

The term "purified MVA-related virus" as used herein refers to a virus that has been isolated under conditions that reduce or eliminate the presence of unrelated materials (i.e., contaminants, including natural materials from which the virus is obtained, such as cell debris, cell residues, cell proteins, cell DNA molecules, and/or cell RNA molecules). The purified MVA-related virus is preferably substantially free of cells and/or culture components. The term "substantially free" as used herein is in the context of operational use in analytical testing of a substance. The purified MVA-related virus substantially free of contaminants is preferably at least 50% pure, more preferably at least 90% pure, and even more preferably at least 99% pure or 100% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, compositional analysis, bioassay, and other methods known in the art.

The term "virus telomere" as used herein refers to the sequence of the left and right sites of the viral genomic DNA (e.g. the genomic DNA of a MVA virus or MVA-related virus). The sequence at the left site of viral genomic DNA is called left viral telomere, and the sequence at the right site of viral genomic DNA is called right viral telomere. Viral telomeres contain Inverted Terminal Repeats (ITRs), which comprise or consist of a region of complementarity between the left and right sides of genomic DNA. Viral telomeres also comprise functional and disrupted (non-functional) genes, which may be repetitive at both ends of the genomic DNA, or may be unique to the left or right side of the genomic DNA. Thus, viral telomeres extend beyond the ITR region itself and may comprise 30000bp (30kbp) or more.

The term "Inverted Terminal Repeat (ITR)" as used herein refers to a region of complementarity between or consisting of the left and right sides of viral genomic DNA (e.g., genomic DNA of MVA virus or MVA-related virus). The ITR on the left side of the genomic DNA is referred to as "left ITR", and the ITR on the right side of the genomic DNA is referred to as "right ITR". The ITRs are part of the virus telomere, in particular the left ITR is contained in the left virus telomere and the right ITR is contained in the right virus telomere. The ITRs can form hairpin structures and appear to provide the origin of viral genomic DNA replication (which may occur through strand displacement of the okazaki fragment and leader strand in the lag strand).

The term "ends" as used herein refers to the left and right ends of the viral genomic DNA (e.g., the genomic DNA of a MVA virus or MVA-related virus).

the term "core region" as used herein refers to a region of the viral genomic DNA (e.g. the genomic DNA of a MVA virus or MVA-related virus) comprising the deletion sites V and III. In particular, the term "core region" as used herein refers to the region extending from deletion site V to deletion site III, including deletion sites V and III.

"amino acid substitutions" may also be referred to herein as "amino acid substitutions". The term "amino acid insertion" as used herein refers to amino acid modifications occurring within the amino acid sequence of the L3L, A3L, a34R and/or A9L gene products, while the term "amino acid addition" as used herein refers to amino acid modifications occurring at the N-terminus or C-terminus of the L3L, A3L, a34R and/or A9L gene products.

In the context of the present invention, amino acid residues in two or more gene products are said to "correspond" to each other if they occupy similar positions in the structure of the gene product. Similar positions in two or more gene products can be determined by aligning the sequences of the gene products based on amino acid sequence or structural similarity, as is well known in the art. Such alignment tools are well known to those skilled in the art and are available, for example, on the world Wide Web (e.g., ClustalW (www.ebi.ac.uk/Clustalw) or Align (http:// www.ebi.ac.u k/EMBOSS/Align/index. html) using standard settings, preferably Align EMBOSS:: needle, Matrix: Blos um62, Gap Open 10.0, Gap extended 0.5). One skilled in the art understands that it may be desirable to introduce gaps in either sequence to produce a satisfactory alignment. Amino acid residues in two or more gene products are said to "correspond" if they are aligned in an optimal sequence alignment. An "optimal sequence alignment" between two gene products is defined as an alignment of identical residues that yields the greatest number of alignments. If the sequence similarity (preferably identity) between two aligned sequences decreases to less than 30%, preferably less than 20%, more preferably less than 10% over a length of 10, 20 or 30 amino acids, the "optimal sequence alignment region" ends and a measure of the length and bounds of the compared sequences is determined therefrom for the purpose of determining a similarity score. The method is also applicable to nucleic acid sequences. This means that in the context of the present invention, nucleic acid sequences are said to "correspond" to each other if the residues occupy similar positions in the nucleic acid structure.

Here, the inventors isolated and characterized for the first time a novel MVA-related virus. The present inventors found that the novel MVA-related viruses differ structurally from (wild-type) MVA viruses. It has a hitherto undescribed genotype. The structural differences result in viruses with advantageous properties over (wild-type) MVA viruses. In particular, the novel MVA-related viruses release a higher number of infectious units into the supernatant of infected cultures compared to cultures infected with (wild-type) MVA viruses. Furthermore, the novel MVA-related viruses replicate to very high titers compared to (wild-type) MVA viruses. Furthermore, the novel MVA-related viruses induce less syncytia in adherent culture.

the beneficial properties of the novel MVA-related viruses described above improve their industrial production. In particular, it allows the production of novel MVA-related viruses in high yield and thus also the production of heterologous proteins (e.g. antigens) which may be comprised therein. In addition, the novel MVA-related viruses can be isolated directly from the cell-free supernatant, thereby facilitating purification and thus operation and arrangement of the bioreactor for producing the MVA-related viruses. Which in turn reduces its production costs.

accordingly, a first aspect of the invention relates to a Modified Vaccinia Ankara (MVA) -associated virus comprising (different from (wild-type) MVA) one or more (e.g. 1, 2, 3, 4, 5, 6, 7 or 8) of the following characteristics:

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) No deletion site I;

(v) two deletion sites IV;

(vi) an open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

(viii) a nucleic acid sequence encoding an L3L gene product, wherein the nucleic acid sequence comprises at least one mutation that results in a modification of the amino acid sequence of the gene product.

The skilled person will understand that the above described nucleic acid sequence is/is comprised in the viral genomic DNA of the MVA-related virus or is/is comprised in the genome of the MVA-related virus.

The MVA-related virus may comprise a characteristic according to (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii). The MVA-related virus may further comprise features according to: (ii) and (iii); (ii) (viii), (iii) and (viii); (ii) and (iv); (ii) (iii), (iv) and (vi); (ii) (viii), (iv) and (viii); (ii) (viii), (iv), (vi), and (viii); (iii) and (v); (iii) (vii), (v) and (vii); (iii) (viii), (v) and (viii); (iii) (viii), (v), (vii), and (viii); (iv) and (v); (iv) (viii), (v) and (viii); (vi) and (vii); or (vi), (vii), and (viii).

In one embodiment, the MVA-related virus comprises a nucleic acid sequence corresponding to a region comprising a right Inverted Terminal Repeat (ITR) and extending to deletion site III but not deletion site III, and not (instead) a nucleic acid sequence comprising a left Inverted Terminal Repeat (ITR) and extending to deletion site V but not deletion site V (feature (i)).

in particular, the region comprising the right ITR and extending to deletion site III but not deletion site III comprises deletion site IV and/or an open reading frame for at least one gene product selected from the group consisting of a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R.

In particular, the region comprising the left ITR and extending to deletion site V but not deletion site V comprises deletion site I and/or at least one open reading frame of at least one gene product selected from the group consisting of C11R, C10L and D7L.

More specifically, the region comprising the right ITR and extending to deletion site III but not deletion site III comprises deletion site IV and/or an open reading frame for at least one gene product selected from the group consisting of a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R; and, the region comprising the left ITR and extending to deletion site V but not deletion site V comprises deletion site I and/or at least one open reading frame of at least one gene product selected from the group consisting of C11R, C10L and D7L.

Thus, in a preferred embodiment, the MVA-related virus does not comprise a deletion site I; comprises two deletion sites IV; an open reading frame that does not contain at least one gene product selected from the group consisting of C11R, C10L, and D7L; and/or two open reading frames comprising at least one gene product selected from the group consisting of a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R.

Preferably, the MVA-related virus comprises the following nucleic acid sequences: the nucleic acid sequence corresponds to a region of the MVA comprising right Inverted Terminal Repeats (ITRs) and extending to deletion site III but not deletion site III, and not (instead) a nucleic acid sequence of the MVA comprising left Inverted Terminal Repeats (ITRs) and extending to deletion site V but not deletion site V.

The MVA-related virus may further comprise the following nucleic acid sequences: the nucleic acid sequence comprises a right Inverted Terminal Repeat (ITR) and extends to a unique (unique) sequence next to the right ITR but does not comprise deletion site III, and not (instead) a nucleic acid sequence comprising a left Inverted Terminal Repeat (ITR) and extends to a unique sequence next to the left ITR but does not comprise deletion site V. In this aspect, the unique sequence is a sequence that does not comprise a complementary region. It may be untranscribed and untranslated, may contain a pseudogene (a previously coding region that has lost its function) or may contain the gene being expressed.

Alternatively, the MVA-related virus may comprise a nucleic acid sequence starting with a right Inverted Terminal Repeat (ITR) and comprising a right ITR and extending to deletion site III but not deletion site III, rather than (instead of) a nucleic acid sequence starting with a left inverted terminal repeat (III) and comprising left III and extending to deletion site V but not deletion site V.

In one embodiment, the MVA-related virus comprises two copies of a nucleic acid sequence comprising deletion site IV and the right ITR (feature (ii)).

In particular, the nucleic acid sequence comprising deletion site IV and the right ITR further comprises an open reading frame for at least one gene product selected from the group consisting of a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R.

Thus, in a preferred embodiment, the MVA-related virus comprises two deletion sites IV; two right ITRs; and two open reading frames of at least one gene product selected from the group consisting of a57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R.

In one embodiment, the MVA-related virus does not comprise the following nucleic acid sequences: the nucleic acid sequence comprises deletion site I and the left ITR (feature (iii)).

In particular, the nucleic acid sequence comprising deletion site I and the left ITR further comprises an open reading frame for at least one gene product selected from the group consisting of C11R, C10L and D7L.

Thus, in a preferred embodiment, the MVA-related virus does not comprise a deletion site I; does not contain a left ITR; and does not contain an open reading frame for at least one gene product selected from the group consisting of C11R, C10L, and D7L.

Preferably:

(i) Said region comprising the right ITR and extending to deletion site III but not comprising deletion site III has a nucleic acid sequence according to SEQ ID NO 37 or Genbank accession No. KY633487 (preferably ranging from nucleotide positions 162221 to 190549 or the nucleotide positions corresponding thereto) or is a variant which is at least 85%, at least 90%, at least 95% or 100% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to said nucleic acid sequence;

(ii) Said region comprising the left ITR and extending to the deletion site V but not comprising the deletion site V has a nucleic acid sequence according to SEQ ID NO 37 or Genbank accession No. KY633487 (preferably ranging from nucleotide position 1 to 31261 or the nucleotide position corresponding thereto) or is a variant which is at least 85%, at least 90%, at least 95% or 100% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to said nucleic acid sequence; and/or

(iii) Said nucleic acid sequence comprising the deletion site IV and said right ITR has a nucleic acid sequence according to SEQ ID No. 37 or Genbank accession No. KY633487 (preferably ranging from nucleotide positions 179272 to 190549 or the nucleotide positions corresponding thereto) or is a variant which is at least 85%, at least 90%, at least 95% or 100% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to said nucleic acid sequence.

the variant is preferably a functionally active variant. This means that the variants do not negatively affect the beneficial properties of the MVA-related viruses according to the invention, such as a higher number of infectious units and/or increased infectious activity in the extracellular space during culture, compared to (wild-type) MVA viruses. Said beneficial properties allow, for example, the production of MVA-related viruses according to the invention in high yield. Experiments to test whether the beneficial properties are still present in the above variants are described in the experimental section.

The variants may also comprise nucleic acid changes due to the degeneracy of the genetic code encoding the same or functionally equivalent amino acids as the nucleic acid sequences described above.

In one embodiment, the MVA-related virus comprises a nucleic acid sequence encoding the L3L gene product, wherein the nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of the gene product (feature (viii)). In an alternative embodiment, the MVA-related virus comprises a nucleic acid sequence encoding an A3L gene product and/or an a34R gene product, wherein said nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of said gene product.

It should be noted that the nucleic acid sequence encoding the gene product described above comprises at least one mutation (e.g., 1, 2, or 3 mutations) resulting in at least one amino acid modification (e.g., 1, 2, or 3 amino acid sequence modifications) of the gene product. The amino acid sequence modification may be an amino acid deletion (e.g., 1, 2, or 3 amino acid deletions), an amino acid insertion (e.g., 1, 2, or 3 amino acid insertions), an amino acid addition (e.g., 1, 2, or 3 amino acid additions), and/or an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions).

Preferably, the nucleic acid sequence further encodes an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2, or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, or 3 amino acid modifications) of the gene product. The amino acid sequence modification may be an amino acid deletion (e.g., 1, 2, or 3 amino acid deletions), an amino acid insertion (e.g., 1, 2, or 3 amino acid insertions), an amino acid addition (e.g., 1, 2, or 3 amino acid additions), and/or an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions). The inventors have surprisingly found that the above mutations further positively influence the virus yield.

more preferably still, the first and second liquid crystal compositions are,

(i) the MVA-related virus comprises a nucleic acid sequence encoding an A3L gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2 or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2 or 3 amino acid modifications) of the gene products (i.e., the A3L gene product and the A9L gene product);

(ii) the MVA-related virus comprises a nucleic acid sequence encoding an a34R gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2 or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2 or 3 amino acid modifications) of the gene products (i.e., the a34R gene product and the A9L gene product);

(iii) The MVA-related virus comprises a nucleic acid sequence encoding an A3L gene product, an a34R gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2 or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2 or 3 amino acid modifications) of the gene products (i.e., the A3L gene product, the a34R gene product and the A9L gene product);

(iv) The MVA-related virus comprises a nucleic acid sequence encoding an L3L gene product and an A3L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2 or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2 or 3 amino acid modifications) of the gene products (i.e., the L3L gene product and the A3L gene product);

(v) the MVA-related virus comprises a nucleic acid sequence encoding an L3L gene product and an a34R gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g. 1, 2 or 3 mutations) resulting in at least one amino acid sequence modification (e.g. 1, 2 or 3 amino acid modifications) of the gene products (i.e. the 3L3 gene product and the a34R gene product); or

(vi) The MVA-related virus comprises a nucleic acid sequence encoding an L3L gene product, an A3L gene product, an a34R gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2, 3 or 4 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, 3 or 4 amino acid modifications) of the gene products (i.e., the L3L gene product, the A3L gene product, the a34R gene product and the A9L gene product).

Preferably:

(i) The amino acid sequence modification is located in a region spanning amino acid positions 634 to 644 or an amino acid position corresponding thereto of the A3L gene product according to SEQ ID No. 1;

(ii) The amino acid sequence modification is located in a region spanning amino acid positions 81 to 91 or the amino acid positions corresponding thereto of the a34R gene product according to SEQ ID No. 2;

(iii) The amino acid sequence modification is located in a region spanning amino acid positions 70 to 80 or the amino acid positions corresponding thereto of the A9L gene product according to SEQ ID No. 3; and/or

(iv) The amino acid sequence modification is located in a region spanning amino acid positions 105 to 115 or the amino acid positions corresponding thereto of the L3L gene product according to SEQ ID NO 13.

Thus, the amino acid sequence modification (e.g., amino acid deletion or amino acid substitution) may be located (i) at amino acid position 634, 635, 636, 637, 638, 639, 640, 641, 642, 643 or 644 of the A3L gene product according to SEQ ID NO:1, or at an amino acid position corresponding thereto; (ii) amino acid positions 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91 of the A34R gene product according to SEQ ID NO 2, or amino acid positions corresponding thereto; (iii) at amino acid position 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 of the A9L gene product according to SEQ ID No. 3, or at an amino acid position corresponding thereto; and/or (iv) at amino acid position 105, 106, 107, 108, 109, 110, 111, 112, 113 or 115 of the L3L gene product according to SEQ ID NO:13, or an amino acid position corresponding thereto.

More preferably:

(i) The amino acid sequence modification is at amino acid position 639 or an amino acid position corresponding thereto of the A3L gene product;

(ii) The amino acid sequence modification is at amino acid position 638 or an amino acid position corresponding thereto of the A3L gene product;

(iii) the amino acid sequence modification is at amino acid position 86 or an amino acid position corresponding thereto of the a34R gene product;

(iv) the amino acid sequence modification is at amino acid position 75 of the A9L gene product or at an amino acid position corresponding thereto;

(v) the amino acid sequence modification is at amino acid position 74 of the A9L gene product or at an amino acid position corresponding thereto; and/or

(vi) The amino acid sequence modification is at amino acid position 110 of the L3L gene product or at an amino acid position corresponding thereto.

More preferably, the amino acid sequence modification is an amino acid deletion or amino acid substitution, wherein:

(i) an H deletion at amino acid position 639 or an amino acid position corresponding thereto of the A3L gene product or a substitution with a hydrophobic amino acid (preferably A, V, I, L, M, F, Y or W), a negatively charged amino acid (preferably D or E), or a polar uncharged amino acid (preferably S, T, N or Q);

(ii) r at amino acid position 638 or the amino acid position corresponding thereto of the A3L gene product is deleted or replaced with a hydrophobic amino acid (preferably A, V, I, L, M, F, Y or W), a negatively charged amino acid (preferably D or E), or a polar uncharged amino acid (preferably S, T, N or Q);

(iii) A D deletion at amino acid position 86 of the a34R gene product or an amino acid position corresponding thereto, or a substitution with a hydrophobic amino acid (preferably A, V, I, L, M, F, Y or W), a positively charged amino acid (preferably R, H or K), or a polar uncharged amino acid (preferably S, T, N or Q);

(iv) A K deletion at or corresponding to amino acid position 75 of the A9L gene product or substitution with a hydrophobic amino acid (preferably A, V, I, L, M, F, Y or W), a negatively charged amino acid (preferably D or E), or a polar uncharged amino acid (preferably S, T, N or Q);

(v) A K deletion at amino acid position 74 of the A9L gene product or an amino acid position corresponding thereto, or a substitution with a hydrophobic amino acid (preferably A, V, I, L, M, F, Y or W), a negatively charged amino acid (preferably D or E), or a polar uncharged amino acid (preferably S, T, N or Q); and/or

(vi) The V deletion at amino acid position 110 of the L3L gene product or at the amino acid position corresponding thereto is replaced by a hydrophobic amino acid (preferably A, V, I, L, M, F, Y or W), a negatively charged amino acid (preferably D or E) or a polar uncharged amino acid (preferably S, T, N or Q).

even more preferably, the amino acid substitutions are the following amino acid substitutions:

(i) replacement of H at amino acid position 639 or an amino acid position corresponding thereto of the A3L gene product with Y (H639Y A3L gene product mutant);

(ii) Replacement of the R at amino acid position 638 or corresponding to amino acid position of the A3L gene product with Y (R638Y A3L gene product mutant);

(iii) substitution of D at amino acid position 86 of the a34R gene product or at an amino acid position corresponding thereto with Y (D86Y a34R gene product mutant);

(iv) Replacement of K at amino acid position 75 or at an amino acid position corresponding thereto of the A9L gene product with E (K75E A9L gene product mutant);

(v) Replacement of K at amino acid position 74 or at an amino acid position corresponding thereto of the A9L gene product with E (K74E A9L gene product mutant); and/or

(vi) Substitution of V with a at amino acid position 110 of the L3L gene product or at the amino acid position corresponding thereto (V110A L3L gene product mutant).

The amino acid substitutions are further even more preferably the following amino acid substitutions:

(i) Substitution of Y for H at amino acid position 639 or an amino acid position corresponding thereto of the A3L gene product and substitution of Y for D at amino acid position 86 or an amino acid position corresponding thereto of the a34R gene product (H639Y A3L/D86Y a34R gene product mutant);

(ii) Substitution of Y for H at amino acid position 639 or an amino acid position corresponding thereto of the A3L gene product and substitution of E for K at amino acid position 75 or an amino acid position corresponding thereto of the A9L gene product (H639Y A3L/K75E A9L gene product mutant);

(iii) substitution of Y for D at amino acid position 86 of the a34R gene product or at an amino acid position corresponding thereto, and substitution of E for K at amino acid position 75 of the A9L gene product or at an amino acid position corresponding thereto (D86Y a34R/K75E A9L gene product mutant);

(iv) substitution of Y for H at amino acid position 639 or an amino acid position corresponding thereto of the A3L gene product, Y for D at amino acid position 86 or an amino acid position corresponding thereto of the a34R gene product, and E for K at amino acid position 75 or an amino acid position corresponding thereto of the A9L gene product (H639Y A3L/D86Y a34R/K75E A9L gene product mutant); and/or

(v) Substitution of Y for H at amino acid position 639 or an amino acid position corresponding thereto of the A3L gene product, substitution of Y for D at amino acid position 86 or an amino acid position corresponding thereto of the a34R gene product, substitution of E for K at amino acid position 75 or an amino acid position corresponding thereto of the A9L gene product, and substitution of a for V at amino acid position 110 or an amino acid position corresponding thereto of the L3L gene product (H639Y A3L/D86Y a34R/K75E A9L/V110A L3L gene product mutant).

most preferably:

(i) The A3L gene product having the H639Y mutation has an amino acid sequence according to SEQ ID No. 4 or a variant which is at least 85%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% or 100% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to said amino acid sequence, wherein said variant (still) comprises amino acid Y at amino acid position 639 or at the amino acid position corresponding thereto;

(ii) The a34R gene product having the D86Y mutation has an amino acid sequence according to SEQ ID No. 5 or is a variant which is at least 85%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% or 100% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to said amino acid sequence, wherein said variant (still) comprises amino acid Y at amino acid position 86 or an amino acid position corresponding thereto;

(iii) the A9L gene product having the K75E mutation has an amino acid sequence according to SEQ ID No. 6 or is a variant which is at least 85%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to said amino acid sequence, wherein said variant (still) comprises the amino acid E at amino acid position 75 or the amino acid position corresponding thereto; and/or

(iv) The L3L gene product having the V110A mutation has an amino acid sequence according to SEQ ID No. 14 or is a variant which is at least 85%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% or 100% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to said amino acid sequence, wherein said variant comprises the amino acid a at amino acid position 110 or an amino acid position corresponding thereto.

Sequence identity is particularly preferably: (i) at least 85%, 90%, 95% or 99% compared to a contiguous sequence stretch of at least 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600 or more amino acids of a corresponding reference amino acid sequence according to SEQ ID No. 4; (ii) at least 85%, 90%, 95% or 99% compared to a contiguous stretch of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 or more amino acids of a corresponding reference amino acid sequence according to SEQ ID NO. 5; (iii) at least 85%, 90%, 95% or 99% compared to a contiguous stretch of at least 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of a corresponding reference amino acid sequence according to SEQ ID NO 6; or (iv) at least 85%, 90%, 95% or 99% compared to a contiguous stretch of at least 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of the corresponding reference amino acid sequence according to SEQ ID NO: 14. The sequence identity is further particularly preferably at least 85% of full length, at least 90% of full length, at least 95% of full length or at least 99% of full length compared to the corresponding reference amino acid sequence according to SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 14.

The variant is preferably a functionally active variant. This means that (further) changes in the amino acid sequence do not have a negative effect on the beneficial properties of the MVA-related viruses according to the invention, such as a higher number of infectious units and/or an increased infectious activity in the extracellular space during culture, compared to known (wild-type) MVA viruses. Said beneficial properties allow, for example, the production of MVA-related viruses according to the invention in high yield. Experiments to test whether the beneficial properties are still present in the above variants are described in the experimental section.

The A3L gene product of MVA described above (also known as P4b protein) is one of the three major core proteins and is processed by the viral protease encoded by I7L to Intracellular Mature Virions (IMVs) during maturation of spherical non-infectious Immature Virions (IV). The A3L gene product of MVA promotes virion morphogenesis in a very early step to allow for proper aggregation and membrane rearrangement in the transition to infectious IMV. Furthermore, the a34R gene product of MVA described above destabilizes the outer membrane of the Extracellular Enveloped Virus (EEV), and is thus of great importance for viral transmission and infectious activity in the extracellular space. EEV has evolved as a vehicle that allows viral spread to distant sites. The additional membrane of EEV is unable to mediate fusion with the target cell and must be disrupted to release the actual viral infectious unit IMV. In addition, the a34R gene product of MVA regulates the rate at which cell-associated enveloped viruses (CEVs) are isolated from producer cells. Furthermore, like the A3L gene product, the A9L gene product of MVA is involved in the early steps of MVA maturation. It is an important factor for the correct agglomeration of the IMV core. In addition, the L3L gene product of MVA is essential for a very early step immediately after entry of the virus into the host cell. L3L is a loosely packed component of the virus particle and appears to be the cause of events that allow nascent mRNA to leave the viral core and appear in the cytoplasm of newly infected host cells.

the MVA-related virus is preferably an isolated MVA-related virus. The isolated MVA virus can be further purified. Thus, the MVA-related virus is more preferably a purified MVA-related virus.

The MVA-related virus preferably further comprises a heterologous nucleic acid sequence. Expression of the heterologous nucleic acid sequence may be under the transcriptional control of a MVA virus promoter. Inserting the heterologous nucleic acid sequence into a nucleic acid sequence of the MVA-associated virus. In a preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted in a non-essential region of the MVA-related viral nucleic acid sequence/genome. In a more preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted into a naturally occurring deletion site (e.g., deletion site III) of the viral genomic DNA/genome. Methods how to insert heterologous nucleic acid sequences into the MVA-related viral genome are known to the person skilled in the art.

more preferably, the heterologous nucleic acid sequence is selected from the group consisting of sequences encoding:

(i) Antigens (particularly antigenic epitopes);

(ii) A diagnostic compound; and

(iii) A therapeutic compound.

The antigen or epitope may be used as a vaccine to induce an immune response against the antigen or epitope. Examples of such antigens heterologous to the virus cover, for example, proteins of other viruses such as influenza virus, hepatitis virus (e.g. hepatitis c virus), Human Immunodeficiency Virus (HIV), flavivirus, paramyxovirus, hantavirus or filovirus, or proteins associated with the development of tumors and cancers such as Her2/neu or MUC-1. Examples of such epitopes heterologous to the virus cover, for example, epitopes from proteins derived from other viruses, such as influenza virus, hepatitis virus (e.g., hepatitis c virus), Human Immunodeficiency Virus (HIV), flavivirus, paramyxovirus, hantavirus or filovirus, or epitopes derived from proteins associated with the development of tumors and cancers (e.g., extracellular peptides of MUC-1 or Her 2/neu).

The antigen may also be a vaccine antigen, for example derived from a virus, fungus, eukaryotic or bacterial pathogen or derived from a tumour.

the therapeutic compound may be any compound having a therapeutic effect. For example, the therapeutic compound may be a compound that affects or participates in tissue growth, cell differentiation; compounds capable of eliciting a biological effect (e.g., an immune response); or a compound that may play any other role in one or more biological processes. In particular, the compound may be an antimicrobial compound, an antiviral compound, an antifungal compound, an immunosuppressive compound, a growth factor, an enzyme, an anti-inflammatory compound, or an antiallergic compound. The therapeutic compound may also be an antisense nucleic acid.

the diagnostic compound may be any compound having a diagnostic effect. For example, the diagnostic compound may be a marker/reporter protein (such as an antibody, GFP, EGFP, β -galactosidase, luciferase) or a protein conferring antibiotic resistance (such as bla (β -lactamase) for ampicillin or npt (neomycin phosphotransferase) for neomycin or G418). The marker/reporter protein may be used to identify or isolate the virus, for example by using hybridization techniques, fluorescence microscopy or ELISA assays. In addition, the protein that confers antibiotic resistance contained in the virus confers resistance to the infected cell against selection for the antibiotic.

as described above, the MVA-related virus is a highly attenuated virus.

In one embodiment of the invention, the MVA-related virus is capable of productive replication in avian cells. The avian cell is preferably a chicken cell, a quail cell, a goose cell or a duck cell (e.g., a duck somite cell or a duck retinal cell). The avian cell (e.g., chicken, quail, goose or duck cell (e.g., duck somite or duck retina cell)) can be a primary cell (or a cell from a primary cell culture), a secondary cell (or a cell from a secondary cell culture), or an immortalized cell (or a cell from a cell line).

In another embodiment of the invention, the MVA-related virus is incapable of productive replication in a mammalian cell, wherein the mammalian cell is not a Baby Hamster Kidney (BHK) cell, a fruit bat R05T cell, a fruit bat R05R cell, or a fruit bat R06E cell. The R05T, R05R, and R06E cells are cells obtained by immortalization of primary cells from Egyptian fruit bat (Egyptian rousette). These are one of the few mammalian cell lines that allow MVA (Jordan et al, 2009, Virus Res 145, 54-62). In a preferred embodiment of the invention, the MVA virus is incapable of productive replication in primate cells (more preferably human cells).

The MVA virus according to the present invention may comprise a nucleic acid sequence encoding the L3L gene product having the amino acid sequence before amino acid modification according to SEQ ID No. 13, the A3L gene product having the amino acid sequence before amino acid modification according to SEQ ID No. 1 and/or the a34R gene product having the amino acid sequence before amino acid modification according to SEQ ID No. 2. The nucleic acid sequence may further encode the A9L gene product having the amino acid sequence before amino acid modification according to SEQ ID NO 3.

The V110A L3L gene product mutant has a sequence according to SEQ ID NO. 14, the H639Y A3L gene product mutant has a sequence according to SEQ ID NO. 4, the D86Y A34R gene product mutant has a sequence according to SEQ ID NO. 5 and/or the K75E A9L gene product mutant has a sequence according to SEQ ID NO. 6.

Furthermore, the corresponding L3L gene may have the nucleic acid sequence before mutation according to SEQ ID NO. 15, the A3L gene may have the nucleic acid sequence before mutation according to SEQ ID NO. 7, the corresponding A34R gene may have the nucleic acid sequence before mutation according to SEQ ID NO. 8 and/or the corresponding A9L gene may have the nucleic acid sequence before mutation according to SEQ ID NO. 9.

furthermore, the mutated L3L gene may have the nucleic acid sequence according to SEQ ID NO. 16, the mutated A3L gene may have the nucleic acid sequence according to SEQ ID NO. 10, the mutated A34R gene may have the nucleic acid sequence according to SEQ ID NO. 11 and/or the mutated A9L gene may have the nucleic acid sequence according to SEQ ID NO. 12.

In addition, MVA viruses (structurally) different from the MVA-related viruses may comprise a nucleic acid sequence (before mutation/rearrangement) according to accession number AY603355 (versions AY603355.1 and GI: 47088326).

in a second aspect, the present invention relates to the genome of a MVA-related virus according to the first aspect.

in a third aspect, the present invention relates to a cell comprising a MVA-related virus according to the first aspect or a genome according to the second aspect. A cell comprising a MVA-related virus according to the first aspect or a genome according to the second aspect may also be referred to as a host cell.

The cells may be for culturing a MVA-related virus according to the first aspect. The cell may be any cell in which the MVA-related virus according to the first aspect is capable of replicating. The cells are preferably not primate cells (in particular human cells). The cell is further preferably an avian cell. The avian cell is preferably a chicken cell, a quail cell, a goose cell or a duck cell (e.g., a duck somite cell or a duck retinal cell). The avian cell (e.g., chicken, quail, goose or duck cell (e.g., duck somite or duck retina cell)) can be a primary cell (or a cell from a primary cell culture), a secondary cell (or a cell from a secondary cell culture), or an immortalized cell (or a cell from a cell line). In a preferred embodiment of the invention, the cells are from the CR or CR. pIX cell lines were derived from immortalized Muscovy duck retina cells designed for Vaccine production (Jordan et al, 2009, Vaccine 27, 748-. The pIX cell line further stably integrates into its genome the gene encoding the adenoviral pIX protein and expresses said gene. In other preferred embodiments of the invention, the cell is a Chicken Embryo Fibroblast (CEF). The cells are primary cells. The cell is preferably an isolated cell.

The cells may be infected with a MVA-related virus according to the first aspect or transfected with a genome according to the second aspect. Techniques for how to infect or transfect cells are known to those skilled in the art.

The cells are further preferably non-adherent/suspension cells. Typically, cells can be grown in suspension or adherent culture. Some cells naturally live in suspension without attaching to surfaces, such as cells present in the bloodstream (e.g., hematopoietic cells). Adherent cells (e.g., primary cells) require a surface (e.g., a tissue culture plastic carrier or microcarrier) that can be coated with extracellular matrix components to increase the adherent properties and provide other signals needed for growth and differentiation.

In a preferred embodiment of the invention, the non-adherent/suspension cells are grown under serum-free conditions.

In a fourth aspect, the present invention relates to a method for culturing a MVA-related virus according to the first aspect, said method comprising the steps of:

(i) Providing a cell according to the third aspect;

(ii) culturing the cell; and

(iii) Isolating the MVA-associated virus.

The cell according to the third aspect comprises a MVA-related virus according to the first aspect or a genome according to the second aspect.

The cells may be cultured in cell proliferation medium and subsequently in virus production medium in step (ii), or the cells may be cultured in cell proliferation medium (only) in step (ii). The cells are preferably cultured in step (ii) (only) in a cell proliferation medium. The use of a single culture medium has the advantage of facilitating the virus culture process, in particular an industrial virus culture process. For example, it facilitates the operation and arrangement of bioreactors for the production of said MVA-related viruses. The cell proliferation medium is preferably serum-free. Serum-free media are in particular completely animal serum-free. In contrast, cells are grown in a medium that is completely free of any animal-derived components, and preferably free of any complex mixture of biological components (so-called defined chemical composition medium). The cell proliferation medium further preferably has a low protein content and/or a low salt content.

the cells are preferably cultured in step (ii) in a stirred culture or bioreactor.

in step (iii), the MVA-associated virus is preferably isolated from cell-free supernatant and/or cell lysate. The isolation of the MVA-related virus in step (iii) may be performed according to standard procedures readily available to the skilled person. Preferably, the MVA-related virus is isolated from the cell-free supernatant. This facilitates virus isolation processes, particularly industrial virus isolation processes. This in turn reduces the cost of virus production. For example, cell lysis for virus isolation is no longer required. In this way contamination of the virus isolate with cellular material, in particular cellular DNA, can be reduced. The DNA limits of the world health organization for virus preparations can thus be more easily obtained.

various virus isolation procedures are known in the art. The separation procedure useful according to the invention does not interfere with the virus to be separated. For example, prolonged exposure to impeller shear forces and other factors that occur during separation should be avoided. The separation in step (iii) is preferably achieved by separating the virus from the cells via centrifugation, sedimentation and/or filtration. The skilled person can easily adapt/adjust suitable separation parameters (e.g. acceleration/G-force and/or time when using centrifugation, filter size when using filtration separation and/or settling time when using sedimentation separation) to separate the virus cultured in the cells.

In a fifth aspect, the present invention relates to a method for producing a MVA-related virus according to the first aspect, the method comprising the steps of:

(a) Infecting the cells with MVA virus;

(b) Culturing the cell;

(d) Repeating steps (a) to (c) with the MVA virus isolated in step (c) until a MVA-related virus comprising one or more of the following characteristics is detected:

(i) a nucleic acid sequence corresponding to a region comprising a right Inverted Terminal Repeat (ITR) and extending to deletion site III but not deletion site III, and not (instead) a nucleic acid sequence comprising a left Inverted Terminal Repeat (ITR) and extending to deletion site V but not deletion site V;

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) No deletion site I;

(v) Two deletion sites IV;

(vi) An open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

Steps (a) to (c) may be repeated until a MVA-associated virus comprising the characteristics according to (i), (ii), (iii), (iv), (v), (vi), (vii) or (viii) is detected. Steps (a) to (c) may also be repeated until MVA-related viruses comprising characteristics according to: (ii) and (iii); (ii) (viii), (iii) and (viii); (ii) and (iv); (ii) (iii), (iv) and (vi); (ii) (viii), (iv) and (viii); (ii) (viii), (iv), (vi), and (viii); (iii) and (v); (iii) (vii), (v) and (vii); (iii) (viii), (v) and (viii); (iii) (viii), (v), (vii), and (viii); (iv) and (v); (iv) (viii), (v) and (viii); (vi) and (vii); or (vi), (vii), and (viii).

In one embodiment, steps (a) to (c) are repeated until the following MVA-associated viruses are detected: the MVA-related virus comprises a nucleic acid sequence corresponding to a region comprising a right Inverted Terminal Repeat (ITR) and extending to deletion site III but not deletion site III, and not (instead) a nucleic acid sequence comprising a left Inverted Terminal Repeat (ITR) and extending to deletion site V but not deletion site V (feature (i)).

Preferably, steps (a) to (c) are repeated until the following MVA-related viruses are detected: the MVA-related virus comprises a nucleic acid sequence corresponding to a region of MVA comprising a right Inverted Terminal Repeat (ITR) and extending to deletion site III but not deletion site III, and not (instead) a nucleic acid sequence of MVA comprising a left Inverted Terminal Repeat (ITR) and extending to deletion site V but not deletion site V.

Steps (a) to (c) may also be repeated until a MVA-associated virus comprising the following nucleic acid sequences is detected: the nucleic acid sequence comprises a right Inverted Terminal Repeat (ITR) and extends to a unique sequence beside the right ITR but not comprises deletion site III, and not (instead) a nucleic acid sequence comprising a left Inverted Terminal Repeat (ITR) and extends to a unique sequence beside the left ITR but not comprises deletion site V. In this aspect, the unique sequence is a sequence that does not comprise a complementary region. The unique sequence may be untranscribed and untranslated, may contain a pseudogene (a previously coding region that has lost its function) or may comprise an expressing gene.

Alternatively, steps (a) to (c) may be repeated until the following MVA-associated viruses are detected: rather than (instead of) a nucleic acid sequence starting with the left inverted terminal repeat (III) and comprising the left III and extending to the deletion site V but not comprising the deletion site V, the MVA-related virus comprises a nucleic acid sequence starting with the right Inverted Terminal Repeat (ITR) and comprising the right ITR and extending to the deletion site III but not comprising the deletion site III.

In one embodiment, steps (a) to (c) are repeated until the following MVA-associated viruses are detected: the MVA-related virus comprises two copies of a nucleic acid sequence comprising deletion site IV and the right ITR (feature (ii)).

in one embodiment, steps (a) to (c) are repeated until a MVA-related virus is detected which does not comprise a nucleic acid sequence of: the nucleic acid sequence comprises deletion site I and the left ITR (feature (iii)).

In one embodiment, steps (a) to (c) are repeated until a MVA-related virus comprising a nucleic acid sequence encoding the L3L gene product is detected, wherein said nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of said gene product. In an alternative embodiment, steps (a) to (c) are repeated until a MVA-related virus comprising a nucleic acid sequence encoding an A3L gene product and/or an a34R gene product is detected, wherein said nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of said gene product.

Preferably, steps (a) to (c) are repeated until a MVA-related virus comprising a nucleic acid sequence further encoding an A9L gene product is detected, wherein said nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of said gene product.

It should be noted that the nucleic acid sequences encoding the above-mentioned gene products comprise at least one mutation (e.g., 1, 2, 3, or 4 mutations) resulting in at least one amino acid modification (e.g., 1, 2, 3, or 4 amino acid sequence modifications) of each of the gene products. The amino acid sequence modification (e.g., 1, 2, 3, or 4 amino acid modification) can be an amino acid deletion (e.g., 1, 2, 3, or 4 amino acid deletions), an amino acid insertion (e.g., 1, 2, 3, or 4 amino acid insertions), an amino acid addition (e.g., 1, 2, 3, or 4 amino acid additions), and/or an amino acid substitution (e.g., 1, 2, 3, or 4 amino acid substitutions).

Further preferred embodiments are described in the context of the first aspect of the invention.

More preferably still, the first and second liquid crystal compositions are,

(i) Detecting a MVA-related virus comprising a nucleic acid sequence encoding an A3L gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2, or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, or 3 amino acid modifications) of the gene products (i.e., the A3L gene product and the A9L gene product);

(ii) Detecting a MVA-related virus comprising a nucleic acid sequence encoding an a34R gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2, or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, or 3 amino acid modifications) of the gene products (i.e., the a34R gene product and the A9L gene product);

(iii) Detecting a MVA-related virus comprising a nucleic acid sequence encoding an A3L gene product, an a34R gene product, and an A9L gene product, wherein said nucleic acid sequence comprises at least one mutation (e.g., 1, 2, or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, or 3 amino acid modifications) of said gene products (i.e., said A3L gene product, said a34R gene product, and said A9L gene product);

(iv) Detecting a MVA-related virus comprising a nucleic acid sequence encoding an L3L gene product and an A3L gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2, or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, or 3 amino acid modifications) of the gene product (i.e., the L3L gene product and the A3L gene product);

(v) Detecting a MVA-related virus comprising a nucleic acid sequence encoding an L3L gene product and an a34R gene product, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2, or 3 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, or 3 amino acid modifications) of the gene product (i.e., the L3L gene product and the a34R gene product); or

(vi) MVA-related viruses comprising a nucleic acid sequence encoding an L3L gene product, an A3L gene product, an a34R gene product, and an A9L gene product are detected, wherein the nucleic acid sequence comprises at least one mutation (e.g., 1, 2, 3, or 4 mutations) resulting in at least one amino acid sequence modification (e.g., 1, 2, 3, or 4 amino acid modifications) of the gene products (i.e., the 3L3 gene product, the A3L gene product, the a34R gene product, and the A9L gene product).

With regard to preferred embodiments of amino acid modifications, reference is made to the first aspect of the invention.

The MVA virus in step (a) may comprise a nucleic acid sequence according to accession number U94848 (version U94848.1 and GI: 2772662).

Preferably, steps (a) to (c) are repeated at least 2 times, preferably at least 7 times, more preferably at least 14 times, most preferably at least 20 times, for example at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20 times.

Further preferably, the cells are cultured in a virus production medium. The virus may be cultured in the virus production medium in step (b) for 1 to 10 days, for example 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

The cells in step (a) may be any cells in which the MVA virus is capable of replicating. The cells are preferably not primate cells (in particular human cells). The cell is further preferably an avian cell. The avian cell is preferably a chicken cell, a quail cell, a goose cell or a duck cell (e.g., a duck somite cell or a duck retinal cell). The avian cell (e.g., chicken, quail, goose or duck cell (e.g., duck somite or duck retina cell)) can be a primary cell (or a cell from a primary cell culture), a secondary cell (or a cell from a secondary cell culture), or an immortalized cell (or a cell from a cell line). In a preferred embodiment of the invention, the cells are from the CR or CR. pIX cell lines were derived from immortalized Muscovy duck retina cells (Jordan et al, 2009, Vaccine 27, 748-756). The pIX cell line further stably integrates into its genome the gene encoding the adenoviral pIX protein and expresses said gene. In other preferred embodiments of the invention, the cell is a Chicken Embryo Fibroblast (CEF). The cells are primary cells.

In a sixth aspect, the present invention relates to a pharmaceutical composition comprising a MVA-related virus according to the first aspect or a genome according to the second aspect, and one or more pharmaceutically acceptable excipients, diluents and/or carriers.

As mentioned above, the MVA-related virus according to the first aspect is highly host-restricted and thus highly attenuated. It is therefore ideal for treatment of a wide range of recipients. The recipient is preferably a primate, more preferably a human.

The pharmaceutical compositions contemplated by the present invention may be formulated and/or administered by a variety of means known to those skilled in the art. The pharmaceutical composition of the invention is preferably in liquid form, for example in the form of a solution (e.g. an injectable solution). The solution may be injected, for example, intramuscularly or parenterally. The mode of administration, the dosage and the number of administrations of the pharmaceutical composition can be optimized by the person skilled in the art in a known manner.

In a seventh aspect, the present invention relates to a vaccine comprising a MVA-related virus according to the first aspect or a genome according to the second aspect.

as mentioned above, the MVA-related virus according to the first aspect is highly host-restricted and thus highly attenuated. It is therefore an ideal vaccine for the treatment of a wide range of recipients.

The recipient is preferably a primate, more preferably a human. In this regard, it is noted that the MVA-related virus may itself be a vaccine. It confers protection against pox. However, the virus or the genome may further comprise a heterologous nucleic acid sequence, such as a sequence encoding an antigen, in particular an antigenic epitope, which may elicit protective immunity (in particular additional protective immunity) in a recipient against the heterologous nucleic acid sequence. Examples of such antigens cover, for example, proteins of other viruses such as influenza virus, hepatitis virus (e.g. hepatitis c virus), Human Immunodeficiency Virus (HIV), flavivirus, paramyxovirus, hantavirus or filovirus, or proteins associated with the development of tumors and cancers such as Her2/neu or MUC-1. Examples of such epitopes cover, for example, epitopes from proteins derived from other viruses, such as influenza virus, hepatitis virus (e.g. hepatitis c virus), Human Immunodeficiency Virus (HIV), flavivirus, paramyxovirus, hantavirus or filovirus, or epitopes derived from proteins associated with tumor and cancer development, such as extracellular peptides of MUC-1 or Her 2/neu. MVA-related viruses comprising heterologous nucleic acid sequences may also be referred to as recombinant MVA-related viruses. Upon administration of the vaccine to a recipient, the antigen (particularly the epitope) is expressed and presented to the immune system, and a specific immune response against the antigen (particularly the epitope) is induced. The recipient is thereby immunized against the antigen, in particular the epitope.

The vaccine comprising the MVA-related virus according to the first aspect or the genome according to the second aspect is preferably a poxvirus vaccine, an influenza virus vaccine, a hepatitis virus (e.g. hepatitis c virus) vaccine, a Human Immunodeficiency Virus (HIV) vaccine, a flavivirus vaccine, a paramyxovirus vaccine, a hantavirus vaccine and/or a filovirus vaccine. It can also be used for vaccination against breast, melanoma, pancreatic or prostate cancer.

Vaccines contemplated by the present invention may be formulated and administered by various means known to those skilled in the art. The vaccine of the invention is preferably in liquid form, e.g. in the form of a solution (such as an injection solution). The solution may be injected, for example, intramuscularly or parenterally. The mode of administration, the dosage and the number of administrations of the vaccine can be optimized by the person skilled in the art in a known manner. For the formulation or preparation of a vaccine, the MVA virus according to the first aspect (in particular a recombinant MVA virus) is converted into a physiologically acceptable form. This can be done based on experience in the preparation of a poxvirus vaccine for vaccination against vaccinia (as described by Stickl et al, 1974, Dtsch Med Wochenschr 99, 2386-. The vaccine is particularly useful for inducing an immune response in an immunocompromised recipient (e.g., a primate including a human). "immunocompromised" describes the state of the recipient's immune system that shows only an incomplete immune response or has reduced efficiency in defense against infectious agents.

in an eighth aspect, the present invention relates to a MVA-related virus according to the first aspect or a genome according to the second aspect for medical use. The MVA-related virus according to the first aspect or the genome according to the second aspect is preferably for use in vaccination and/or therapy. In particular, the recipient is challenged with the MVA-related virus according to the first aspect or with the genome according to the second aspect to induce specific immunity. The recipient is preferably a primate, more preferably a human. The primate (e.g., human) can be immunocompromised. In this regard, it is noted that the MVA-related virus may itself be a vaccine. It confers protection against pox. However, the virus or the genome may further comprise a heterologous nucleic acid sequence, such as a sequence encoding an antigen, in particular an antigenic epitope, which may elicit protective immunity (in particular additional protective immunity) in a recipient against the heterologous nucleic acid sequence. Preferred antigens (in particular epitopes) are described in the first and seventh aspects of the invention. The MVA-related virus or genome is preferably used for vaccination against poxviruses, influenza viruses, hepatitis viruses (e.g. hepatitis c virus), Human Immunodeficiency Virus (HIV), flaviviruses, paramyxoviruses, hantaviruses, filoviruses, tumors and/or cancers, such as breast, melanoma, pancreatic or prostate cancer.

alternatively or additionally, the recipient is challenged with a MVA-related virus according to the first aspect or with a genome according to the second aspect to elicit a therapeutic effect. As described above, the heterologous sequence contained in the virus or genome can encode a therapeutic compound. For example, the therapeutic compound may be a compound that affects or participates in tissue growth, cell differentiation; compounds capable of eliciting a biological effect (e.g., an immune response); or a compound that may play any other role in one or more biological processes. In particular, the compound may be an antimicrobial compound, an antiviral compound, an antifungal compound, an immunosuppressive compound, a growth factor, an enzyme, an anti-inflammatory compound, or an antiallergic compound. The therapeutic compound may also be an antisense nucleic acid.

The person skilled in the art is able to optimize the vaccination mode, the vaccination dose and the number of vaccinations in a known manner. The vaccine may be formulated and administered by various means known to those skilled in the art. The vaccine is preferably administered in liquid form. Preferably, the vaccine is injected (e.g., intramuscularly or parenterally). Preferably the MVA-related virus according to the first aspect or the genome according to the second aspect is administered to said recipient in a pharmaceutically effective amount. The MVA-related virus or genome is effective in a particular route of administration when its amount elicits an immune response in said recipient.

The invention is summarized as follows:

1. A Modified Vaccinia Ankara (MVA) -associated virus comprising one or more of the following characteristics:

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) no deletion site I;

(v) Two deletion sites IV;

(vi) An open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

2. the MVA-associated virus of claim 1, wherein the region comprising the right ITR and extending to deletion site III but not deletion site III comprises deletion site IV.

3. the MVA-related virus of claim 1 or 2, wherein the region comprising the right ITR and extending to deletion site III but not deletion site III comprises an open reading frame for at least one gene product selected from the group consisting of A57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R.

4. The MVA-associated virus of any one of claims 1 to 3, wherein the region comprising the left ITR and extending to deletion site V but not comprising deletion site V comprises deletion site I.

5. The MVA-associated virus of any one of claims 1 to 4, wherein the region comprising the left ITR and extending to deletion site V but not deletion site V comprises at least one open reading frame of at least one gene product selected from the group consisting of C11R, C10L and D7L.

6. The MVA-associated virus of any one of items 1 to 5, wherein the nucleic acid sequence comprising deletion site IV and the right ITR further comprises an open reading frame for at least one gene product selected from the group consisting of A57R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B15R, B16R, B17L, B18R, B19R and B22R.

7. The MVA-associated virus of any one of claims 1 to 6, wherein the nucleic acid sequence comprising deletion site I and the left ITR further comprises an open reading frame for at least one gene product selected from the group consisting of C11R, C10L and D7L.

8. the MVA-associated virus of any one of claims 1 to 7, wherein,

(iii) said nucleic acid sequence comprising the deletion site IV and said right ITR has a nucleic acid sequence according to SEQ ID NO 37 or Genbank accession number KY633487 (preferably ranging from nucleotide positions 179272 to 190549 or the nucleotide positions corresponding thereto) or is a variant which is at least 95% identical to said nucleic acid sequence.

9. the MVA-associated virus of any one of claims 1 to 8, wherein the virus comprises a nucleic acid sequence encoding an A3L gene product and/or an A34R gene product, wherein the nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of the gene product.

10. The MVA-related virus of claim 9, wherein the nucleic acid sequence further encodes the A9L gene product, wherein the nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of the gene product.

11. The MVA-associated virus of claim 10, wherein,

(i) The virus comprises a nucleic acid sequence encoding an A3L gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation that results in an amino acid sequence modification of the gene product; or

(ii) The virus comprises a nucleic acid sequence encoding an a34R gene product and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation resulting in a modification of the amino acid sequence of the gene product.

12. The MVA-related virus of item 10, wherein the virus comprises a nucleic acid sequence encoding an A3L gene product, an a34R gene product, and an A9L gene product, wherein the nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of the gene product.

13. the MVA-related virus of any one of claims 1 to 12, wherein the virus further comprises a heterologous nucleic acid sequence.

14. The MVA-related virus of item 13, wherein the heterologous nucleic acid sequence is selected from the group consisting of sequences encoding:

(i) Antigens (particularly antigenic epitopes);

(i) A diagnostic compound; and

(iii) a therapeutic compound.

15. The MVA-associated virus of any one of claims 1 to 14, wherein the virus is capable of productive replication in avian cells.

16. the MVA-associated virus according to any of claims 1 to 15, wherein the virus is incapable of productive replication in primate cells (more preferably human cells).

17. The genome of the MVA-related virus according to any of claims 1 to 16.

18. A cell comprising the MVA-associated virus according to any of claims 1 to 16 or the genome according to claim 17.

19. the cell of claim 18, wherein the cell is a non-adherent/suspension cell.

20. the cell of claim 18 or 19, wherein the cell is an avian cell.

21. method for culturing a MVA-associated virus according to any of claims 1 to 16, comprising the steps of:

(a) Providing a cell according to any one of claims 18 to 20;

(b) Culturing the cell; and

22. The method of clause 21, wherein the cell is cultured in cell proliferation medium and subsequently in virus production medium, or the cell is cultured in cell proliferation medium only.

23. The method of clause 21 or 22, wherein the MVA-related virus is isolated from cell-free supernatant and/or cell lysate.

24. Method for the production of the MVA-associated virus according to any of claims 1 to 16, comprising the steps of:

(a) Infecting the cells with MVA virus;

(b) culturing the cell;

(d) Repeating steps (a) to (c) with the MVA virus isolated in step (c) until a MVA-related virus comprising one or more of the following characteristics is detected:

(ii) Two copies of a nucleic acid sequence comprising deletion site IV and the right ITR;

(iii) (ii) does not comprise a nucleic acid sequence comprising deletion site I and said left ITR;

(iv) No deletion site I;

(v) Two deletion sites IV;

(vi) An open reading frame free of at least one gene product selected from the group consisting of C11R, C10L, and D7L;

25. The method of clause 24, wherein steps (a) to (c) are repeated until a MVA-related virus comprising a nucleic acid sequence encoding an A3L gene product and/or an a34R gene product is detected, wherein the nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of the gene product.

26. The method of clause 25, wherein steps (a) through (c) are repeated until a MVA-related virus comprising a nucleic acid sequence further encoding an A9L gene product is detected, wherein the nucleic acid sequence comprises at least one mutation resulting in an amino acid sequence modification of the gene product.

27. the method of any one of claims 24 to 26, wherein the cell is cultured in a virus production medium.

28. a pharmaceutical composition comprising a MVA-associated virus according to any of claims 1 to 16 or a genome according to claim 17, and one or more pharmaceutically acceptable excipients, diluents and/or carriers.

29. A vaccine comprising the MVA-associated virus according to any of claims 1 to 16 or the genome according to claim 17.

30. The MVA-associated virus according to any of items 1 to 16 or the genome according to item 17 for medical use.

31. the MVA-related virus or genome according to item 30 for vaccination use.

Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope of this invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, the invention is intended to cover various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields.

The following figures and examples are illustrative of the present invention only and should not be construed as limiting in any way the scope of the invention as indicated by the appended claims.

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