Generation of poxvirus from quail cell cultures

Abstract

The present invention relates to a method for producing a poxvirus viral vector-based vaccine product from a avian cell line. In some embodiments, the avian cell line is a suspended quail cell line. Pharmaceutical compositions, such as vaccines, produced by the method of the present invention are also provided. In some embodiments, the poxvirus viral vector is modified vaccinia virus ankara ("MVA") or recombinant MVA. In some embodiments, the recombinant MVA encodes a heterologous antigen and can be used to produce a vaccine against that antigen. In some embodiments, the recombinant MVA encodes an antigen of respiratory syncytial virus (RSV), and the avian cell line is used to produce a vaccine against RSV containing recombinant MVA and / or the encoded antigen.

Description

[Technical Field]

[0001] The present invention relates to a method for optimizing the production of poxvirus viral vector-based vaccine products from avian cell cultures. The poxvirus viral vector may be modified vaccinia virus ankara ("MVA") or recombinant MVA encoding a heterologous antigen. Viruses from cell cultures can be used in the production of vaccines. [Background technology]

[0002] Poxviruses have a long history of providing vaccines for immune defense against infection and disease. One example is modified vaccinia virus Ankara ("MVA"), a highly attenuated strain of vaccinia virus (genus Orthopoxvirus). The MVA-BN® virus, developed by Bavarian Nordic® A / S, is used as a vaccine against smallpox and monkeypox and is marketed under the trade names IMVAMUNE®, IMVANEX®, and JYNNEOS®. Furthermore, recombinant MVA-BN® viruses encoding various heterologous antigens are also used as vaccines. For example, recombinant MVA-BN® viruses encoding antigens from four different filoviruses (Ebola virus, Sudan virus, Thai forest virus, and Marburg virus) provide an improved vaccine against Ebola virus and are disclosed, for example, in WO2016 / 034678. Used in combination with the Zabdeno® vaccine as part of a two-component vaccine regimen, the MVABEA® vaccine is approved for use in the prevention of Ebola virus disease.

[0003] The cells approved for generating the MVA-BN® virus were primary chicken embryo fibroblasts ("CEF" cells). CEF cells are widely used in studies of cell-virus interactions and in vaccine production. However, primary CEF cells in this context have several drawbacks, for example, they require time, cost, and effort to prepare (see, e.g., Farzaneh et al. (2017) British Poultry Science 58:681-686), there is variability in cell substrates in each batch, and the preparation procedure is prone to contamination.

[0004] One alternative to CEF cells is the EB66® cell line derived from duck embryos (see, e.g., Leon et al. (2016) Vaccine 34:5878-85). Serial chicken cell lines have also been developed from Taiwanese ducks (Jordan et al. (2016) Avian Pathology 45:137-155) and peacocks (Wang et al. (2022) Poultry Sci. 101:102147). Another alternative to CEF cells is the serial quail cell line, which has been generated by various means (see, e.g., Kraus et al. (2011) BMC Proceedings 5(Suppl.8):P52; Lee et al. (2008) J. Virol. Meth. 153:22-8). Quail cells lack most of the endogenous retrovirus (ERV) sequences detectable in chicken cells. For example, sequences from a subgroup of the endogenous avian retrovirus family called EAV-HP were found in chickens but completely absent in quail (see, e.g., Smith et al. ((1999) J.Gen.Virol.80:261-268)). Comparative mapping of quail and chicken genomes revealed that only 393 intact ERVs were identified in quail, compared to 1212 in chickens (Morris et al. ((2020) BMC Biol.18:14)).

[0005] However, the cost of vaccine production remains high, partly due to cell lines often failing to meet the desired yield (see, e.g., Hoeksema et al. (2018) Vaccine 36:2093-2103), or the resulting product having unacceptably high levels of impurities, such as total protein and host cell DNA. Regulatory authorities require that the level of host cell DNA in the final vaccine product be very low, e.g., less than 100 nanograms per vaccine dose. Therefore, there remains a need for safe, efficient, and commercially viable methods for processing virus-infected cell lines to produce vaccines, including those containing recombinant poxviruses. [Overview of the project]

[0006] The present invention relates to a method for producing a viral vector-based vaccine product by culturing and processing poxvirus-infected chicken cells. In some embodiments, the viral vector is a modified vaccinia virus Ankara ("MVA"), e.g., MVA-BN® virus. In some embodiments, the viral vector is recombinant MVA encoding one or more heterologous antigens, and the chicken cells are used to produce a vaccine containing recombinant MVA and / or encoded antigens. In some embodiments, the recombinant MVA encodes one or more antigens of respiratory syncytial virus (RSV), and the chicken cells are used to produce a vaccine that protects against diseases caused by RSV. In some embodiments, the recombinant MVA encodes tumor-associated antigens, and the chicken cells are used to produce a vaccine that stimulates an immune response to said antigens.

[0007] In some embodiments, methods for culturing bird cells to increase viral titer are also provided. In some embodiments, the bird cells are quail cells. A higher viral titer in the infected cell line may be useful in producing a superior vaccine product with reduced levels of contaminants such as host cell DNA. Therefore, in some embodiments, the method of the present invention includes an “upstream process” designed to maximize the viral titer produced by cultured cells, and also includes a “downstream process” for isolating and purifying the virus for use in a vaccine. Vaccines containing MVA and recombinant MVA are produced by the method of the present invention and are therefore also provided by the present invention. [Brief explanation of the drawing]

[0008] [Figure 1] The X-axis shows the host cell DNA ("HCD") present in the collected material (i.e., material collected from poxvirus-infected cell cultures), and the effect of adding Denerase® enzyme on reducing HCD levels at various steps in the downstream process (see Example 1). The Y-axis shows the HCD content per vaccine dose. Note the logarithmic scale. Each line connects data points from specific tests in the downstream process. The abbreviations on the horizontal axis represent the following downstream process stages: PI = post-poxvirus infection (day 3); PT = after treatment with trypsin; PN = after NaCl addition; PL = after cell lysis; PF = after deep filtration (5 μM); TF1 = sample after TFF filtration; PD = sample after second treatment with Denerase® enzyme; BDS = final product sample ("API"). The data demonstrate that Denerase® enzyme acts to reduce HCD even in the presence of high salt (1 M NaCl). [Figure 2]The effect of NaCl addition on the reduction of host cell DNA ("HCD") during the subsequent downstream purification process is shown (see Example 1; note the logarithmic scale). The abbreviations on the horizontal axis represent the following downstream process stages: PI = after poxvirus infection; PT = after treatment with trypsin; PN = after NaCl addition; PL = after cell lysis; PF = after deep filtration (5 μM); TF = sample after TFF filtration; PD = after treatment with Denarase® enzyme; TF2 UF = sample after the second TFF ultrafiltration step; TF2 DF = sample after the second TFF diafiltration step; BDS = final product sample ("API"). These data show the surprising result that, despite an initial increase in HCD levels in the earlier steps of the process, the addition of NaCl results in lower HCD levels in the later steps. [Figure 3]The virus yield from quail suspension cells cultured in various media is shown (see Example 3). Quail cells (CCX.E10 cells (Nuvonis, Vienna, Austria)) were seeded in SCGM medium ("SCGM-ac") without the addition of anti-clamping agents ("ac") in a stirrer-equipped flask. After 3 days of culture, the cells were collected and reseeded at a cell density of 8 × 10⁵ to 1.1 × 10⁶ cells / mL in one of the following media: SCGM-ac with CaCl₂, OptiPRO® medium (with Pluronic F68 additive and GlutaMAX® supplement at a final concentration of 4 mM), or a 50:50 mixture of SCGM-ac and OptiPRO® medium (with Pluronic F68 additive). Cells were transferred to either 30°C or 32°C for 1 hour, then infected with MVA-BN-RSV at an MOI (Multiple Infection Intake) of 0.1 (= Day 0). To analyze viral yield, cell culture samples were collected 1–4 days post-infection, sonicated, frozen at -20°C, and then titrated by a standard TCID50 assay (vortexed for 1 minute or more). Figure 3 shows viral yield as the geometric mean (GM) of TCID50 / mL along with its geometric standard deviation (geoSD). Data points represent results from nine independent experiments. The magnification differences shown below the graphs are comparisons with yields using SCGM-ac + CaCl2 at the corresponding time points (Day 1 ("d1") to Day 4 ("d4"). [Figure 4A]The virus yield from quail suspension cells cultured in media containing different proportions (%) of conditioned SCGM and OptiPRO® medium is shown (see Example 4). Quail cells (CCX.E10) were seeded on day 3 in SCGM medium without anti-agglutinating agent ("SCGM-ac medium") in a stirrer-equipped flask to obtain conditioned medium ("cSCGM-ac medium"). On day 0 (day of virus infection), a fixed amount of cell culture from this conditioned medium was transferred to a shaking flask and mixed with: equal volumes of OptiPRO® medium (resulting in a 50:50 mixture); cSCGM-ac pre-mixed with OptiPRO® medium (resulting in a ratio of cSCGM-ac to OptiPRO® medium ranging from 60:40 to 90:10); or cSCGM with CaCl2 (without OptiPRO® medium). All culture media were supplemented with GlutaMAX® supplement and Pluronic F68 additive at a final concentration of 2 mM. After transferring cells to 30°C for 1 hour, they were infected with MVA-BN-RSV, which also encodes mRFP (monomer red fluorescent protein), to allow analysis of viral infection and spread (see Figure 4B). Infection was performed at an MOI (multiple infections) of 0.1, and cells were then cultured at 30°C. To analyze viral yield, duplicate samples were collected on post-infection days 1–4, sonicated, and frozen at -20°C. Samples from day 1 / 2 (d1, d2) and day 3 / 4 (d3, d4) were titrated in parallel using a standard TCID50 assay (vortexed for 1 minute or more). Figure 4A shows viral yield as the geometric mean (GM) of TCID50 / mL along with its geometric standard deviation (geoSD). To analyze viral infection and spread, mRFP expression was analyzed by flow cytometry 2–4 days post-infection (Figure 4B) (GMFI, geometric mean fluorescence intensity).In both Figure 4A and Figure 4B, for each number of days represented by the bar graph, the data is shown from left to right for the following media: 50% cSCGM-ac / 50% OptiPRO(trademark) medium ("50:50"); 60% cSCGM-ac / 40% OptiPRO(trademark) medium ("60:40"); 70% cSCGM-ac / 30% OptiPRO(trademark) medium ("70:30"); 80% cSCGM-ac / 20% OptiPRO(trademark) medium ("80:20"); 90% cSCGM-ac / 10% OptiPRO(trademark) medium ("90:10"); cSCGM+CaCl2. [Figure 4B]The virus yield from quail suspension cells cultured in media containing different proportions (%) of conditioned SCGM and OptiPRO® medium is shown (see Example 4). Quail cells (CCX.E10) were seeded on day 3 in SCGM medium without anti-agglutinating agent ("SCGM-ac medium") in a stirrer-equipped flask to obtain conditioned medium ("cSCGM-ac medium"). On day 0 (day of virus infection), a fixed amount of cell culture from this conditioned medium was transferred to a shaking flask and mixed with: equal volumes of OptiPRO® medium (resulting in a 50:50 mixture); cSCGM-ac pre-mixed with OptiPRO® medium (resulting in a ratio of cSCGM-ac to OptiPRO® medium ranging from 60:40 to 90:10); or cSCGM with CaCl2 (without OptiPRO® medium). All culture media were supplemented with GlutaMAX® supplement and Pluronic F68 additive at a final concentration of 2 mM. After transferring cells to 30°C for 1 hour, they were infected with MVA-BN-RSV, which also encodes mRFP (monomer red fluorescent protein), to allow analysis of viral infection and spread (see Figure 4B). Infection was performed at an MOI (multiple infections) of 0.1, and cells were then cultured at 30°C. To analyze viral yield, duplicate samples were collected on post-infection days 1–4, sonicated, and frozen at -20°C. Samples from day 1 / 2 (d1, d2) and day 3 / 4 (d3, d4) were titrated in parallel using a standard TCID50 assay (vortexed for 1 minute or more). Figure 4A shows viral yield as the geometric mean (GM) of TCID50 / mL along with its geometric standard deviation (geoSD). To analyze viral infection and spread, mRFP expression was analyzed by flow cytometry 2–4 days post-infection (Figure 4B) (GMFI, geometric mean fluorescence intensity).In both Figure 4A and Figure 4B, for each number of days represented by the bar graph, the data is shown from left to right for the following media: 50% cSCGM-ac / 50% OptiPRO(trademark) medium ("50:50"); 60% cSCGM-ac / 40% OptiPRO(trademark) medium ("60:40"); 70% cSCGM-ac / 30% OptiPRO(trademark) medium ("70:30"); 80% cSCGM-ac / 20% OptiPRO(trademark) medium ("80:20"); 90% cSCGM-ac / 10% OptiPRO(trademark) medium ("90:10"); cSCGM+CaCl2. [Figure 5] The virus yield from quail suspension cells cultured in various media is shown (see Example 5). Quail suspension cells (CCX.E10) were initially grown in SCGM medium (SCGM-ac) without the addition of anti-clamping (ac) medium. On day 0, a fixed volume of cell culture was transferred to different shaking flasks and mixed with OptiPRO® medium, DMEM-F12 medium, and / or cSCGM medium to obtain OptiPRO® medium or DMEM-F12 medium ("F12") at a final concentration of 50% or 30% in a 40 mL culture volume having 1.1 × 10⁶ cells / mL. All media were supplemented with Pluronic F68 additive and a final 2 mM GlutaMAX® supplement. After transferring cells to 30°C for 1 hour, they were infected with MVA-BN-RSV (together encoding mRFP) at an MOI (Moment of Infection Multiplicity) of 0.1 and further cultured at 30°C. Samples were collected 1–4 days post-infection and titrated using a standard TCID50 assay. Viral yields are shown in Figure 5 as the geometric mean of TCID50 / mL, along with the geometric standard deviation (geoSD). [Figure 6]Data comparing the MVA-BN® virus yield obtained from cultures of chicken embryonic fibroblasts (CEF cells) with the MVA-BN® virus yield obtained from quail suspension cells (CCX.E10 cells (Nuvonis, Vienna, Austria); see Example 6) are presented. CCX.E10 cells were seeded on day -3 in SCGM medium (SCGM-ac) without the addition of an anti-agglutinating agent in an S-125 shaking flask. On day 0, cells were counted one hour before infection and mixed with an equal volume of OptiPRO® medium supplemented with Pluronic F68 additive and a final 4 mM GlutaMAX® supplement to obtain a 50:50 mixture of SCGM-ac and OptiPRO® medium. CEF cells were seeded on day -1 in VP-SFM medium (Thermo Fisher Scientific) in a 6-well plate and counted on the day of infection. CCX.E10 and CEF cells were infected in parallel with MVA-BN® virus (IMVAMUNE® vaccine) at an MOI (multiple infections) of 0.1 and then cultured at 32°C. To analyze viral yield, triple samples of CEF and CCX.E10 cells were collected daily from 1 to 4 days post-infection, sonicated, frozen at -20°C, and titrated in parallel using a standard TCID50 assay (vortexed for more than 1 minute). The data shown in Figure 6 show viral yield as the geometric mean (GM) of TCID50 / mL, along with its geometric standard deviation (geoSD). The number above the bar on day 4 (d4) indicates the cell-specific peak viral yield (TCID50 / cell). [Figure 7]A schematic diagram shows the hypothetical effects of the first enzymatic treatment step with trypsin enzyme activity and nucleases (e.g., TrypLE and Denarase® enzymes) on the isolated cells, amplified virus, and impurities resulting from cell lysis during the virus purification process (MVA-BN-RSV is shown as an exemplary virus). Although the present invention is not bound by any particular mechanism of action, it is thought that adding these enzymatic activities before cell disruption reduces cell clamping, allowing the enzymes to access their substrates more effectively, thereby facilitating the removal of impurities and resulting in an improvement in the virus yield observed in this process. This anti-clumping effect can also be seen in the transmission electron micrographs in Figure 7, showing cellular material without trypsin (TrypLE) (left micrograph) and cellular material with trypsin (TrypLE) (right micrograph). These micrographs demonstrate that the addition of trypsin reduces the formation of large clumps of cells, which are likely to be difficult for the enzymes to permeate. Figure 7 also demonstrates that a protease with trypsin activity functions in this process to deaggregate these cells grown in suspension. This is quite different from the more common use of trypsin to detach cells grown as adherent cell cultures (e.g., cells grown as a monolayer in a cell culture dish). [Modes for carrying out the invention]

[0009] The present invention relates to a method for producing a viral vector-based vaccine product from a avian cell culture. In some embodiments, the viral vector is a modified vaccinia virus Ankara ("MVA"), e.g., MVA-BN® virus. In some embodiments, the viral vector is recombinant MVA encoding one or more heterologous antigens, and the method is used to produce a vaccine containing recombinant MVA. In some embodiments, the recombinant MVA encodes one or more antigens of, for example, respiratory syncytial virus (RSV), and the method is used to produce a vaccine containing recombinant MVA that protects against RSV-induced lower respiratory tract disease. In some embodiments, the recombinant MVA encodes a tumor-associated antigen that can stimulate an immune response to said antigen. In some embodiments, the avian cells are quail cells from a suspension cell line, which are processed according to the method of the present invention to provide a vaccine containing MVA or recombinant MVA. In some embodiments, the quail cells are CCX.E10 cells (Nuvonis (Vienna, Austria)).

[0010] The present invention provides a method for producing a vaccine by processing bird cell lines infected with MVA and recombinant MVA. In some embodiments, the method includes an “upstream process” designed to maximize the viral titer produced by cultured cells, and also includes a “downstream process” for isolating and purifying the virus for use in a vaccine. Vaccines containing MVA (including the MVA-BN® virus) and recombinant MVA are produced using the method of the present invention and are therefore also provided by the present invention.

[0011] The use of vaccinia viruses to protect humans from smallpox has a long history, including the long-term use of vasocallic vaccinia virus Ankara (CVA), which was maintained at the Vaccine Institute in Ankara, Turkey. However, due to frequent severe post-vaccination complications associated with these viruses, efforts have been made to produce more attenuated and safer smallpox vaccines. Modified vaccinia virus Ankara ("MVA"), an attenuated CVA-derived virus, was obtained by serially breeding CVA for more than 570 passages in primary chicken embryo fibroblasts ("CEF" cells; see Mayr et al. (1975) Infection 3:6-14 for an overview). As a result of the passages used to attenuate MVA, several different MVA strains or isolates exist, which are sometimes named according to the passage number in CEF cells. For example, MVA-572 was used in Germany during the smallpox eradication program, and MVA-575 was widely used as an animal vaccine. MVA-575 was deposited with the European Collection of Animal Cell Cultures (ECACC) on December 7, 2000, under accession number V00120707.

[0012] MVA and Recombinant MVA Strains of MVA with enhanced safety profiles for the development of safer products such as vaccines or pharmaceuticals are being developed by Bavarian Nordic® A / S. For example, MVA has been further passaged by Bavarian Nordic® A / S and named the MVA-BN® virus. MVA and MVA-BN® viruses lack approximately 15% of their genome compared to the ancestral CVA virus, specifically 31kb from six regions. These deletions affect several pathogenicity and host-range genes, as well as the genes of type A inclusion bodies. A sample of the MVA-BN® virus corresponding to passage 583 was deposited with the European Collection of Cell Cultures (ECACC) on August 30, 2000, under accession number V00083008.

[0013] The MVA-BN® virus can attach to and enter human cells, where the genes encoded by the virus are expressed very efficiently. However, progeny virus construction and release do not occur, and no infectious viruses are produced. The MVA-BN® virus does not replicate in human cells, but it is strongly compatible with primary chicken embryo fibroblast (CEF) cells. According to the U.S. Centers for Disease Control and Prevention, the MVA-BN® virus is classified as a biosafety level 1 organism. Preparations of the MVA-BN® virus and its derivatives have been administered to many types of animals and to more than 2,000 humans, including immunocompromised individuals. All vaccinations have generally been proven safe and well-tolerated. Despite its high attenuation and reduced pathogenicity, preclinical studies have shown that the MVA-BN® virus can induce both humoral and cellular immune responses to heterologous gene products encoded by genes cloned into the vaccinia and MVA genomes (see Harrer et al. (2005) Antivir.Ther. 10(2):285-300; Cosma et al. (2003) Vaccine 22(1):21-9; Di Nicola et al. (2003) Hum.Gene Ther. 14(14):1347-1360; and Di Nicola et al. (2004) Clin.Cancer Res. 10(16):5381-5390).

[0014] The MVA-BN® virus is approved in the European Union (EU) under the trade name IMVANEX® for the prevention of smallpox infection, and in Canada under the trade name IMVAMUNE® for the prevention of smallpox, monkeypox, and other orthopoxvirus infections. In the United States, the MVA-BN® virus is approved under the trade name JYNNEOS® for active immunization against smallpox and monkeypox in adults considered to be at high risk of these diseases. Therefore, the MVA-BN® virus itself (i.e., without expressing any additional heterologous antigens) is also a vaccine that can be manufactured using the method of the present invention.

[0015] A “derivative” or “variant” of MVA is a virus that exhibits essentially the same replication characteristics as MVA as described herein, but has differences in one or more parts of its genome. MVA-BN® virus and its derivatives or variants cannot replicate proliferatively in vivo in humans and mice, even in severely immunosuppressed mice. More specifically, MVA-BN® virus or its derivatives or variants still have proliferative replication ability in chicken embryo fibroblasts (CEF), preferably, but do not have proliferative replication ability in human keratinocyte cell line HaCat (Boukamp et al. (1988) J. Cell Biol. 106:761-771), human bone osteosarcoma cell line 143B (ECACC number 91112502), human embryonic kidney cell line 293 (ECACC number 85120602), and human cervical adenocarcinoma cell line HeLa (ATCC number CCL-2). In addition, derivatives or variants of the MVA-BN® virus have at least twice, more preferably three times, lower viral amplification rates than MVA-575 in HeLa cells and HaCaT cell lines. Tests and assays relating to these properties of MVA variants are described in WO2002 / 042480 (US2003 / 0206926) and WO2003 / 048184 (US2006 / 0159699). Viral amplification or replication is usually expressed as the ratio (called the “amplification rate”) of the virus produced from an infected cell (output) to the amount initially used to infect the cell (input). An amplification rate of “1” defines an amplification state in which the amount of virus produced from an infected cell is the same as the amount initially used to infect the cell, i.e., the infected cell is allowed to infect and replicate with the virus. In contrast, an amplification rate of less than 1 (i.e., a decrease in output compared to the input level) indicates a lack of proliferative replication and therefore attenuation of the virus.

[0016] The advantages of MVA-based vaccines include their safety profile and the availability of large-scale vaccine production. Preclinical studies have shown that the MVA-BN® virus demonstrates superior attenuation and efficacy compared to other MVA strains (WO2002 / 042480). A further characteristic of the MVA-BN® virus strain is its ability to induce substantially the same level of immunity in vaccinia virus prime and vaccinia virus boost prime / boost regimens compared to regimens utilizing DNA prime and vaccinia virus boost. Recombinant MVA-BN® viruses, the most preferred embodiment described herein, are considered safe due to their clear replication defects in mammalian cells and their well-established nonpathogenicity. Furthermore, the feasibility of industrial-scale production of MVA-BN® viruses may be beneficial. Moreover, MVA-based vaccines can deliver multiple heterologous antigens, enabling the simultaneous induction of humoral and cellular immunity.

[0017] In addition to recombinant MVA expressing the RSV antigen (collectively referred to herein as "MVA-RSV"), other recombinant MVAs can be produced using the methods of the present invention, which include culturing in quail cells, as will be discussed in detail below. Any recombinant MVA that can be grown in quail cells can be produced using the methods of the present invention. Accordingly, the present invention provides a method for preparing a vaccine comprising any recombinant MVA that can be grown in quail cells. Examples of such recombinant MVAs include those expressing heterologous genes such as EBV antigen ("MVA-EBV"), equine encephalitis virus antigen, foot-and-mouth disease virus antigen, filovirus antigen, and other disease, virus, or cancer-related antigens, or tumor-related antigens, or monomeric red fluorescent protein (mRFP, see, e.g., Campbel et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:7877-82). Thus, it will be understood that recombinant MVAs for use in the methods and compositions of the present invention contain heterologous nucleotide sequences encoding one or more heterologous antigens.

[0018] The MVA and recombinant MVA viruses described herein are highly attenuated and therefore highly restricted in replication, making them ideal candidates for the treatment of a wide range of mammals, including humans and even immunocompromised humans. The methods of the present invention, the MVA and recombinant MVA produced in cells, cell cultures, and populations of cells, can be isolated and / or purified, and used to provide compositions for further use, including pharmaceutical compositions such as vaccines. Techniques and formulations suitable for these purposes are known in the art.

[0019] In another embodiment, suitable MVA virus strains for generating recombinant viruses may be MVA-572, MVA-575, or any similarly attenuated MVA strain. Also suitable may be mutant MVA, e.g., deleted vasicinia virus ankara (dCVA). dCVA contains the del I, del II, del III, del IV, del V, and del VI deletion sites of the MVA genome. These deletion sites are particularly useful for inserting multiple heterologous sequences. dCVA can be proliferated in human cell lines (such as human 293, 143B, and MRC-5 cell lines) (with amplification rates greater than 10), which then allows for further optimization through mutation and may be useful in virus-based vaccination strategies (see WO2011 / 092029).

[0020] MVA-RSV RSV is a major respiratory pathogen and the most clinically significant cause of acute lower respiratory tract (LRT) infections. It causes significant morbidity and mortality in infants and children under 5 years of age worldwide (see, e.g., Aliyu et al. (2010), Bayero J. Pure Appl. Sci. 3(1):147-155). Primary RSV infection does not induce complete immunity to RSV, leading to frequent reinfections throughout life, with the most severe infections occurring in very young, very old, and immunocompromised individuals of all ages (see, e.g., Murata (2009) Clin. Lab. Med. 29(4):725-39).

[0021] RSV is an enveloped RNA virus belonging to the family Paramyxoviridae. Each RSV virion contains an unsegmented minus-stranded single-stranded RNA molecule containing 10 genes encoding 11 distinct proteins, including 8 structural proteins (G, F, SH, M1, N, P, M2-1, and L) and 3 non-structural proteins (NS1, NS2, and M2.2). M2 contains two open reading frames (Murata (2009) Clin. Lab. Med. 29(4):725-39). Much is known about the roles and interactions of RSV proteins.

[0022] Recombinant MVA expressing at least one RSV antigen is generally referred to herein as MVA-RSV. Some embodiments of MVA-RSV include the MVA-BN® virus, which is generally referred to herein as MVA-BN-RSV.

[0023] Vaccination with recombinant vaccinia virus ankara (MVA) expressing at least one antigen of the RSV membrane glycoprotein and at least one antigen of the RSV nucleocapsid protein (e.g., MVA-mBN201B) has been shown to induce better immunoprotection than RSV vaccines containing only the RSV-F and / or RSV-G antigens (see WO2014 / 019718). In addition, such constructs induced near-complete sterile immunity when applied via the intranasal route, and enhanced protection was obtained by administering candidate RSV vaccines intranasally compared to intramuscular or subcutaneous administration (see, for example, Wyatt et al. (2000) Vaccin 18:392-97). Unfortunately, in a Phase 3 clinical trial conducted by Bavarian Nordic A / S, this recombinant MVA failed to meet all of the primary endpoints for preventing lower respiratory tract disease from RSV, and commercial development of this vaccine candidate was discontinued (see Bavarian Nordic A / S press release dated July 22, 2023).

[0024] RSV nucleotide sequence and protein Exemplary RSV vaccines that can be produced using the methods of the present invention are known in the art and are described, for example, in WO2014019718 (the entirety of which is specifically incorporated herein by reference). When used in the methods of the present invention, recombinant MVA encoding the RSV gene referred to herein (hereinafter, “MVA-RSV”) means the gene encoding the corresponding protein in any RSV strain or isolate, or a homolog or variant of that gene (even if the exact sequence and / or genomic location of that gene may differ between strains or isolates). Similarly, RSV protein referred to herein means the protein encoded and expressed by the corresponding gene as defined above, or a homolog or variant of that protein. Generally, MVA-RSV encodes the RSV protein, which is the antigen. In some embodiments, MVA-RSV is “MVA-BN-RSV”, which contains the MVA-BN® virus, for example, MVA-mBN294B.

[0025] When referring to the RSV F protein gene, other terms such as “F protein gene,” “F glycoprotein gene,” “RSV F glycoprotein gene,” or “F gene” may also be used, all of which refer to the gene encoding a transmembrane fusion glycoprotein in any RSV strain or isolate, or its homolog or variant (even if the exact sequence and / or genomic location of that F protein gene may differ between strains or isolates). For example, in the RSV A2 strain, the F(A2) protein gene contains nucleotides 5601–7499 (including endpoints) numbered with GenBank acceptance number M11486. ​​The F(A2) protein gene further contains a protein encoding an open reading frame (ORF) extending to nucleotides 5614–7338 (including endpoints) numbered with GenBank acceptance number M11486. ​​The nucleotide sequence of the F protein gene from RSV A2 (SEQ ID NO: 1) is publicly known in the art (see WO2014019718).

[0026] The terms “F protein,” “F glycoprotein,” “RSV F protein,” “RSV F glycoprotein,” or “F” are also used interchangeably herein to refer to a heavily glycosylated transmembrane fusion glycoprotein, or a homolog or variant of that protein, encoded and expressed by the RSV F protein gene as defined above. The amino acid sequence of the F protein from RSV A2 (SEQ ID NO: 2) is publicly known in the art (see WO2014019718). The RSV(A2)F protein comprises a signal peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic domain (see, for example, UniProtKB / Swiss-Prot acceptance number P03420). Other proteins and fragments are publicly known in the art and can be used to provide MVA-RSV, and by extension, can be produced using the methods of the present invention to provide a vaccine.

[0027] When referring to the RSV G protein G, other terms such as “G protein gene,” “G glycoprotein gene,” “RSV G protein gene,” “RSV G glycoprotein gene,” and “G gene” are used interchangeably herein. For example, in the RSV A2 strain, the G(A2) protein gene contains nucleotides 4626–5543 (including endpoints) numbered with GenBank acceptance number M11486. ​​The G(A2) protein gene further contains a protein encoding an open reading frame (ORF) extending to nucleotides 4641–5537 (including endpoints) numbered with GenBank acceptance number M11486. ​​The nucleotide sequence of the G protein gene from RSV A2 (SEQ ID NO: 3) is publicly known in the art (see WO2014019718).

[0028] The terms “G protein,” “G glycoprotein,” “RSV G protein,” “RSV G glycoprotein,” or “G” refer to a heavily glycosylated transmembrane glycoprotein, or a homolog or variant of that protein. The amino acid sequence of the G protein from RSV A2 (SEQ ID NO: 4) is publicly known in the art (see WO2014019718). The RSV A2 G protein includes an extracellular domain, a transmembrane domain, and a cytoplasmic domain (see, for example, UniProtKB / Swiss-Prot acceptance number P03423). These domains are also publicly known in the art and are taught in WO2014019718. For example, the extracellular domain of the RSV A2 G protein consists of amino acids 67-298 of sequence number 4 of WO2014019718, the transmembrane domain of the RSV A2 G protein consists of amino acids 38-66 of sequence number 4, and the cytoplasmic domain of the RSV A2 G protein consists of amino acids 1-37 of sequence number 4.

[0029] The terms also used interchangeably herein are “M2-1 protein gene,” “M2.1 protein gene,” “M2-1 transcription elongation factor,” “RSV M2-1 protein gene,” “RSV M2.1 protein gene,” “RSV M2-1 transcription elongation factor gene,” or “M2 gene.” For example, in the RSV A2 strain, the M2(A2) protein gene contains nucleotides 7550–8506 (including endpoints) numbered with GenBank acceptance number M11486. ​​The M2(A2) protein gene further contains a protein encoding an open reading frame (ORF) extending to nucleotides 7559–8143 (including endpoints) numbered with GenBank acceptance number M11486. The nucleotide sequence of the M2 protein gene from RSV A2 (SEQ ID NO: 5) and the amino acid sequence of the M2 protein from RSV A2 (SEQ ID NO: 6) are publicly known in the art (see WO2014019718; see, for example, UniProtKB / Swiss-Prot acceptance number P04545).

[0030] When referring to the RSV N protein gene, the terms “N protein gene,” “N nucleocapsid protein gene,” “RSV N nucleocapsid protein gene,” or “N gene” may be used interchangeably in this specification. For example, in the RSV A2 strain, the N(A2) protein gene contains nucleotides 1081–2277 (including endpoints) numbered with GenBank acceptance number M11486. ​​The N(A2) protein gene further contains a protein encoding an open reading frame (ORF) extending from nucleotides 1096–2271 (including endpoints) numbered with GenBank acceptance number M11486. ​​The nucleotide sequence of the N protein gene from RSV A2 (Sequence ID 7) is publicly known in the art (see WO2014019718). The amino acid sequence (SEQ ID NO: 8) of the RSV N protein (also known as "N protein," "N nucleocapsid protein," "RSV N nucleocapsid protein," or "N") from RSV strain A2 is publicly known in the art (see WO2014019718; UniProtKB / Swiss-Prot acceptance number P03418).

[0031] A specific recombinant MVA that codes for the RSV antigen The recombinant MVA used in the methods and compositions of the present invention can encode one or more RSV antigens suitable for use in RSV vaccines (collectively referred to herein as "MVA-RSV"). For example, recombinant MVA for use in an RSV vaccine may comprise at least one nucleotide sequence encoding an antigen of the RSV membrane glycoprotein and at least one nucleotide sequence encoding an antigen of the RSV nucleocapsid protein.

[0032] In some embodiments, MVA-RSV encodes an antigen of surface fusion protein (F), two glycoproteins (G) from RSV subtype A and RSV subtype B, and an internal protein which is nucleoprotein N and transcription elongation factor M2-1. In some embodiments, protein F is based on the native surface protein F from RSV subtype A.

[0033] In certain embodiments, MVA-RSV encodes at least one antigen of the RSV membrane glycoprotein. In certain embodiments, MVA-RSV encodes the RSV F antigen and / or the RSV G antigen. In certain embodiments, recombinant MVA encodes the RSV F antigen and / or the RSV G antigen derived from RSV strain A2. In certain embodiments, MVA-RSV encodes at least two antigens of the RSV membrane glycoprotein, which are the RSV F antigen and the RSV G antigen. In certain embodiments, the RSV F antigen and / or the RSV G antigen are derived from RSV strain A2.

[0034] In certain embodiments, MVA-RSV comprises at least one heterologous nucleotide sequence encoding an antigen of the RSV membrane glycoprotein and an antigen of the RSV nucleocapsid protein. In certain embodiments, recombinant MVA-RSV encodes at least one antigen of the RSV F membrane glycoprotein and at least one antigen of the RSV M2 nucleocapsid protein or the RSV N nucleocapsid protein. In certain embodiments, MVA-RSV encodes at least one antigen of the RSV G membrane glycoprotein and an antigen of the RSV M2 nucleocapsid protein and / or an antigen of the RSV N nucleocapsid protein.

[0035] In certain embodiments, MVA-RSV encodes at least one antigen of each of the following: RSV F membrane glycoprotein; RSV G membrane glycoprotein; and RSV M2 or N nucleocapsid protein. In certain embodiments, both the RSV F antigen and the RSV G antigen are derived from RSV strain A2. In certain embodiments, MVA-RSV encodes at least one antigen of each of the following: RSV F membrane glycoprotein, RSV G membrane glycoprotein, RSV M2 nucleocapsid protein, and RSV N nucleocapsid protein. In certain embodiments, both the RSV F antigen and the RSV G antigen are derived from RSV strain A2.

[0036] In certain embodiments, MVA-RSV encoding the antigen of RSV F membrane glycoprotein encodes the full-length RSV F membrane glycoprotein, or alternatively, a cleaved or partial RSV F membrane glycoprotein, and / or a variant of wild-type RSV F membrane glycoprotein. In certain embodiments, MVA-RSV encodes the full-length RSV F membrane glycoprotein from strain A2, or RSV strain A Long It encodes the full-length, cleaved, or variant RSV F antigen derived from [the original RSV]. In some embodiments, MVA-RSV is a cleaved RSV (A) lacking the cytoplasmic and transmembrane domains of the native RSV F protein. Long It codes for the )F antigen and / or RSV(A2)F antigen.

[0037] In certain embodiments, MVA-RSV encodes an RSV G membrane glycoprotein antigen from RSV strain A2 or B, which may be full-length, cleaved, or a variant of the wild-type RSV G protein. In certain embodiments, MVA-RSV encodes a cleaved RSV G antigen lacking the cytoplasmic and transmembrane domains of the full-length RSV G protein.

[0038] In certain embodiments, MVA-RSV encodes the antigen of the RSV M2 nucleocapsid protein, which is a variant of the full-length, cleaved, or wild-type RSV M2 protein, and in some embodiments, derived from RSV strain A2. In certain embodiments, MVA-RSV encodes the antigen of the RSV N nucleocapsid protein, which is a variant of the full-length, cleaved, or wild-type RSV N protein, and in some embodiments, derived from RSV strain A2. In certain embodiments, MVA-RSV encodes the antigens of RSV N and RSV M2, which are encoded by a single open reading frame and isolated by an autocleaved protease domain, such as an autocleaved protease 2A fragment from foot-and-mouth disease virus. In certain embodiments, MVA-RSV comprises heterologous nucleotide sequences encoding the RSV N and RSV M2 antigens, which comprises the nucleotide sequence described in SEQ ID NO: 9 and / or the amino acid sequence of SEQ ID NO: 10.

[0039] In a particular embodiment (referred to herein for convenience as “Embodiment A”), the recombinant modified vaccinia virus ankara (MVA) comprises: (a) a nucleotide sequence encoding at least one nucleotide sequence of respiratory syncytial virus (RSV) membrane glycoprotein antigen, which encodes the full-length RSV F membrane glycoprotein; (b) a nucleotide sequence of at least one nucleotide sequence encoding the RSV nucleocapsid antigen, which encodes both the full-length RSV N nucleocapsid protein and the full-length RSV M2-1 transcription elongation factor protein, which are encoded by a single open reading frame, the single open reading frame comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12 or the nucleotide sequence of SEQ ID NO: 11; and (c) a nucleotide sequence of at least one nucleotide sequence encoding the RSV G membrane glycoprotein antigen, which further comprises a nucleotide sequence encoding the full-length RSV G membrane glycoprotein.

[0040] Embodiment (B) is a recombinant MVA of Embodiment (A), wherein the nucleotide sequence encoding the RSV F membrane glycoprotein is preferably A2 and / or A2 from RSV strain A. Long Embodiment (C) is a recombinant MVA of Embodiment (A) or Embodiment (B), wherein the nucleotide sequence encoding the RSV F membrane glycoprotein includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or includes the nucleotide sequence of SEQ ID NO: 1. Embodiment (D) is a recombinant MVA of any of Embodiments (A), (B), or (C), wherein the nucleotide sequence encoding the RSV G membrane glycoprotein is from RSV strain A, preferably strain A2 and / or strain B. Embodiment (E) is a recombinant MVA of Embodiments (A), (B), (C), or (D), wherein the nucleotide sequence encoding the RSV G membrane glycoprotein includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 or includes the nucleotide sequence of SEQ ID NO: 3.

[0041] Embodiment (F) is a recombinant MVA of Embodiment (A), (B), (C), (D), or (E), wherein the MVA used to produce the recombinant MVA is the MVA-BN® virus deposited in the European Collection of Cell Culture (ECACC) under number V00083008. Embodiment (G) is a recombinant MVA of Embodiment (A), (B), (C), (D), (E), or (F) for use in preventing at least one symptom of RSV infection or in preventing disease caused by RSV. Embodiment (H) is a recombinant MVA of Embodiment (G) for use in preventing at least one symptom of RSV infection or in preventing disease caused by RSV, wherein the recombinant MVA is administered intranasally and / or subcutaneously, preferably intranasally.

[0042] In some embodiments, the recombinant MVA (MVA-RSV) for use in the methods and compositions of the present invention is MVA-BN-RSV, e.g., MVA-mBN294B (see, for example, WO2014019718, which is specifically incorporated herein by reference). Several clinical trials of MVA-mBN294B have been conducted, and the safety and immunogenicity of this vaccine have been successfully demonstrated. These trials include Phase 1 trial RSV-MVA-001 and Phase 2 trial RSV-MVA-002, which yielded promising safety and immunogenicity results. Unfortunately, Phase 3 clinical trials of this RSV vaccine candidate in adults aged 60 years and older failed to meet all of the primary endpoints for the prevention of lower respiratory tract disease (LRTD) caused by RSV, and commercial development of this vaccine candidate has been discontinued (see Bavarian Nordic A / S press release dated July 22, 2023).

[0043] Immunogenicity is assessed by evaluating antibody and T cell responses, and the vaccine dose is 1 × 10⁶ per 0.5 mL. 8 5 × 10⁶ per infectious unit (IU) or 0.5 mL 8 The vaccine was administered as an IU. Results from completed Phase 1 and Phase 2 trials showed that a single dose of MVA-BN-RSV induced a broad humoral, cellular, and mucosal immune response that lasted for at least 6 months. A single dose of the MVA-BN-RSV vaccine induced increases in neutralizing antibodies against all five inserts encoded by the vaccine (identified using PRNT for RSV-A and B subtypes) and total antibodies (IgG and IgA ELISA), as well as a broad Th1-biased cellular immune response (IFN-γ / IL-4 ELISPOT), which were also confirmed by non-clinical results in various animal models. Overall, the vaccine-induced antibody response (geometric mean titer (GMT)) remained above baseline for 30 weeks post-vaccination.

[0044] Vaccine and pharmaceutical composition The MVA and recombinant MVA viruses described herein have highly restricted replication and are thus highly attenuated, making them ideal candidates for the treatment of a wide range of mammals, including humans and immunocompromised humans. Accordingly, the present disclosure provides methods for generating these recombinant MVAs and compositions containing them for use as pharmaceutical compositions and vaccines, all of which are intended to induce an immune response in a living animal, including a human.

[0045] For this purpose, MVA or recombinant MVA, vaccine, or pharmaceutical composition can be formulated in solution within a concentration range of about 10 4 ~10 9 IU / mL, 10 5 ~5×10 8 IU / mL, 10 6 ~10 8 IU / mL, or 10 7 ~10 8 IU / mL. Preferred dosages for humans include 10 6 ~10 9 IU / mL and include at least about 10 6 IU / mL, 10 7 IU / mL, 10 8 IU / mL, or 5×10 8 IU / mL dosages. In some embodiments, a pharmaceutical composition that is a vaccine containing MVA or MVA-RSV contains 1×10 8 IU / 0.5 mL or 5×10 8 IU / 0.5 mL (i.e., 1×10 8 IU in a volume of 0.5 mL, or 5×10 8 IU in a volume of 0.5 mL), or contains about 1.58×10 9 InfU / mL in a final (vaccine) volume of 0.5 mL.

[0046] The pharmaceutical compositions produced by the method of the present invention may generally contain one or more pharmaceutically acceptable and / or approved buffers, carriers, additives, antibiotics, preservatives, adjuvants, diluents, and / or stabilizers. Such auxiliary substances may include water, physiological saline, glycerol, ethanol, wetting agents, or emulsifiers, pH buffers, etc. Suitable carriers are typically large molecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and lipid aggregates.

[0047] For vaccine preparation, the MVA or recombinant MVA produced by the method of the present invention can be converted into a physiologically acceptable form. This can be done based on the experience of preparing poxvirus vaccines used for smallpox vaccination, for example, as described by Stickl et al. ((1974) Dtsch.med.Wschr.99:2386-2392). For example, 5 × 10¹⁶ MVA formulated in about 10 mM Tris and 140 mM NaCl (pH 7.7). 8 Purified virus with a titer of IU / mL can be stored at -80°C. To prepare vaccine doses, for example, 10 2 ~10 8 or 10 2 ~10 9The virus particles can be lyophilized in 100 ml of phosphate-buffered saline (PBS) in an ampoule, preferably a glass ampoule, in the presence of 2% peptone and 1% human albumin. Alternatively, vaccine doses can be generated by stepwise lyophilization of the virus in the formulation. This formulation may contain additional additives, such as mannitol, dextran, sugar, glycine, lactose, or polyvinylpyrrolidone, or other adjuvants suitable for in vivo administration, such as antioxidants, or inert gases, stabilizers, or recombinant proteins (e.g., human serum albumin). In some embodiments, the glass ampoule can then be sealed and stored for several months at 4°C to room temperature. However, the ampoule can also be stored at temperatures below -20°C, for example.

[0048] When administered to a subject, the lyophilized product may be dissolved in an aqueous solution, preferably physiological saline or Tris buffer, and administered systemically or topically, i.e., by parenteral, subcutaneous, intravenous, intramuscular, intranasal, or any other suitable route of administration. The mode of administration, dosage, and number of doses can be optimized in ways known to those skilled in the art. However, most commonly, subjects are vaccinated with a second injection approximately 1 month to 6 weeks after the first vaccination injection.

[0049] In some embodiments, vaccination with MVA or recombinant MVA of the present invention (i.e., recombinant MVA of the present invention or recombinant MVA produced by the method of the present invention) induces sterile immunity in the treated subject. However, sterile immunity is not required of the vaccine to benefit the vaccinated subject. The usefulness of recombinant MVA as a vaccine is illustrated, for example, by MVA-BN-RSV (i.e., MVA-mBN294B; see data provided in the examples). Vaccination of a subject with MVA-BN-RSV provides optimal protection from RSV-induced disease by stimulating various aspects of the adaptive immune system, such as the production of RSV-specific antibodies and / or T cells. Efficacy can be evaluated using animal models, such as the RSV challenge model in BALB / c mice (see, e.g., Waris et al. (1996) J. Virol. 70:2852-60). The effectiveness of stimulating the immune response can be evaluated, for example, by a reduction in the viral load of the vaccinated subject if they are exposed to RSV and potentially subsequently infected. Effectiveness can also be evaluated as a statistical reduction in the incidence or severity of infections across the entire vaccinated population, as is well known in the art.

[0050] In this way, the methods and compositions of the present invention provide vaccines useful for protecting subjects from diseases, such as lower respiratory tract diseases caused by RSV, or smallpox or monkeypox. Other recombinant MVA or MVA (such as MVA-BN® virus) produced using the methods of the present invention can be similarly evaluated in corresponding assays known in the art. In some embodiments, MVA or recombinant MVA produced using the methods of the present invention stimulate an immune response in vaccinated subjects to increase the production of specific antibodies and / or T cells above the background levels observed before vaccination.

[0051] definition As used herein, the term "antigen" refers to any molecule that stimulates the host immune system to produce an antigen-specific immune response, whether cellular or humoral. Antigens may include proteins, polypeptides, protein fragments, and epitopes that induce an immune response in the host. Thus, antigens, which are proteins, polypeptides, protein fragments, and epitopes, are not limited to specific native amino acid sequences but also include modifications to native sequences, such as deletions, additions, insertions, and substitutions, that result in variant amino acid sequences. Antigen-coding sequences may be from another virus or pathogen, or they may be associated with a disease such as cancer or tumor, or they may be other sequences.

[0052] As used herein, “day” means approximately 24 hours, or at least 12 hours but less than 36 hours. “Overnight” means approximately 6 or 8 hours, or approximately 8 to 12 hours, or approximately 12 to 16 hours.

[0053] Preferably, “sequence variant” or “variant” has at least about 80% or 85%, or at least about 90%, 91%, 92%, 93%, or 94%, or at least about 95%, 96%, 97%, 98%, or 99% identity with the referenced nucleic acid or amino acid sequence, as used herein. The term “variant” also includes cleaved, deleted, or otherwise modified nucleic acid or protein sequences, such as soluble RSV-F or RSV-G proteins lacking a signal peptide, as well as the transmembrane and / or cytoplasmic domains and encoding nucleic acids of full-length RSV-F or RSV-G proteins, as well as deleted, cleaved, or otherwise mutated forms of full-length RSV-M2 or RSV-N proteins and encoding nucleic acids. Deletion or truncation of a protein or nucleic acid may differ from its full-length form due to the deletion or truncation of one or more elements, and / or the deletion or truncation of specific amino acids or nucleic acids, for example, fewer than 30, 20, or 10 amino acids or nucleic acids.

[0054] Techniques for determining sequence identity between different nucleic acids and between different amino acids are known in the art. Two or more sequences can be compared by determining their “percentage of identity (%)”. The percentage of identity (%) of two sequences is obtained by dividing the number of exact matches between the two aligned sequences, whether nucleic acid or amino acid sequences, by the length of the shorter sequence and multiplying by 100. Alternatively, if it is necessary to introduce gaps in the sequence alignment to achieve the maximum percentage of sequence identity (%), then, as used herein, the “percentage of sequence identity (%)” is the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to a nucleotide or amino acid residue in a reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage of sequence identity (%). Alignment for the purpose of determining the percentage of amino acid sequence identity (%) can be achieved in various ways within the scope of the art using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for generating sequence alignments and calculating sequence identity, including any algorithms necessary to achieve the greatest possible alignment over the full length of the sequences being compared. Details of these programs are publicly known in the art and can be found, for example, on the website of the National Center for Biotechnology Information.

[0055] As used herein, “heterogenetic” genes, nucleic acids, or proteins are understood to have nucleic acids or amino acid sequences that are not present in the wild-type poxvirus genome (e.g., sequences encoding antigens of another virus or pathogen, or sequences associated with diseases such as cancer or tumors, or other heterogenetic sequences). If a nucleic acid that is a “heterogene” is present in a recombinant poxvirus such as MVA, it will be understood by those skilled in the art that this nucleic acid should be incorporated into the poxvirus genome so that it is expressed as a corresponding heterogenetic product, i.e., a “heterogenetic antigen” and / or “heterogenetic protein,” after administration of the recombinant poxvirus to host cells. Expression is typically achieved by operably linking the heterogene to a regulatory element that enables expression in poxvirus-infected cells. In some embodiments, the regulatory element includes a natural or synthetic poxvirus promoter.

[0056] "Aseptic immunity," as used herein, means protective immunity provided by a vaccine in the absence of a detectable pathogen in the subject (e.g., absence of the RSV genome when a highly sensitive detection method such as RT-qPCR is applied). "Subject" is intended to be any animal being treated (e.g., by administration of vaccine), and as used herein, a subject may be a mammal including livestock or companion animals, or a human subject or patient.

[0057] "Increased inflammatory response" or "enhanced inflammatory response," as used herein, is characterized by one or more of the following: increased production of IL-12p70, M-CSF, and / or IL-33; increased antigen-specific CD8+ T cells, increased percentage of CD8+ T cells expressing IFN-gamma and TNF-alpha; decreased tumor size and / or growth rate; and improved survival rate of the treated subject, etc. These can be detected by assays known in the art. As used herein, "increased inflammatory response" generally refers to an increase in the production of specific cytokines or cell types associated with inflammation compared to baseline levels before treatment (e.g., treatment with the composition of the present invention). In "increased inflammatory response," the amount of cytokine or cell type increases by at least 10%, 20%, 30%, 50%, 70%, or 100% or more compared to baseline levels in the subject before treatment.

[0058] Viral titer can be measured in several ways. Those skilled in the art are familiar with techniques for determining viral titer and comparing measurements of different titers. 50 TCID is an abbreviation for "Tissue Culture Infectious Dose," which is the amount of pathogen that causes pathological changes in 50% of seeded cell cultures. 50 It is expressed as / ml. TCID 50 Methods for determining this are well known to those skilled in the art, as described, for example, in Example 2 of WO03 / 053463. When used herein, "IU" represents "infectious units" and is also a measure of viral titer, typically expressed as IU / mL.

[0059] Tori cells and methods The method of the present invention involves processing poxvirus-infected chicken cells to produce a viral vector-based vaccine product, and can be carried out using chicken cells. Therefore, the method of the present invention can be carried out using chicken cells, such as chicken embryonic fibroblasts ("CEF" cells), duck cells, and quail cells.

[0060] Sequential quail cell lines, including CCX.E10 (Nuvonis, Vienna, Austria), were obtained from embryonic quail cell starting materials without introducing exogenous genes, viral sequences, or chemical treatments. The most likely mechanism of immortalization was the occurrence of spontaneous mutations in the embryonic quail cells used as starting materials. Briefly, cell lines were generated from quail cells by subculturing adherent cells and subsequently adapting them to growth in suspension. A single clone was then selected, amplified, and adapted to serum-free cell suspension growth medium. In the case of CCX.E10, the final selected clone was amplified in a spinner flask and frozen as a cell bank.

[0061] In some embodiments, a “conditioning medium” is present during the infection of cells. In some embodiments, this refers to a medium in which the cells have been expanded for a certain period of time prior to infection (e.g., about 12 hours, about 24 hours, about 36 hours, or about 48 hours or more) and can be used to continue infecting those cells by optionally adding the same additional fresh medium as the original medium and / or by adding a different additional fresh medium than the original medium.

[0062] Cells from a frozen cell bank can be thawed and used to produce MVA or recombinant MVA for use in vaccines, as further described below. In this way, the present invention provides quail cells, quail cell lines, and bird cells, including populations of bird cells (e.g., quail cells), for use in the manufacture of vaccines containing MVA and recombinant MVA.

[0063] Method for culturing bird cells In some embodiments, cells are grown in different media at different developmental stages, which can be characterized as the cell growth phase, the virus infection phase, and the virus production phase. That is, in some embodiments, cells are seeded in a container containing an initial medium and grown or cultured during an initial stage (also referred to herein as the “cell growth phase”). This cell growth phase may continue for a certain period, or until a certain cell density is reached or thereafter, or until a certain cellular characteristic (e.g., increased aggregation of cells in culture) is expressed or observed or thereafter. Generally, bird cells, such as quail cells, tend to grow faster when suspended as single cells rather than as a group or aggregated cells. Therefore, in some embodiments, the composition, temperature, and cell density of the medium are adjusted so that cells grow mostly as single cells during the cell growth phase, or substantially as single cells during the growth phase. In some embodiments, quail cells have a cell density of 1 × 10⁶ during the cell growth phase. 6 cells / mL~5×10 6 cells / mL, or preferably 2 × 10⁶ 6 cells / mL~4×10 6 Cells / mL, or more preferably about 3 × 10⁶ 6 The cells are cultured until the cell count reaches [number] cells / mL. The cells can be evaluated by microscopy, FACS analysis, or other techniques known in the art.

[0064] In some embodiments, the cell proliferation phase is carried out in a bioreactor capable of perfusion (i.e., stepwise removal of old medium and introduction of new medium while cell culture continues). Alternatively, the cell proliferation phase can be carried out in cell bags (also called "wave bags"), shaking flasks, or spinner flasks. In some embodiments, the cell proliferation phase is carried out in roller bottles, and the cells are suspended in the medium before the viral infection phase.

[0065] Following the completion of this cell proliferation phase is the viral infection phase, in which the MVA to be produced or recombinant MVA is added to the cell culture, attached to the cells for growth, and introduced. Generally, viral infection is enhanced by the aggregation or clamping of cells in the culture. In preparation for viral cell infection, the medium in which the cells are cultured may have the same composition as the previous cell proliferation phase, or it may be modified or replaced to include a medium with a different composition. In some embodiments, the cells are transferred from the initial growth medium to a second medium. In some embodiments, the cells are cultured in a continuous cell culture in the initial medium for the cell proliferation phase, and then the composition of the medium is modified in preparation for the viral infection phase by gradually or in fixed amounts adding different or subsequent media to the cell culture. In some embodiments, fresh medium with the same composition as the original medium may be added. In some embodiments, the aforementioned different or subsequent media are added to a continuous cell culture (e.g., in a bioreactor or perfusion vessel), while the old medium is removed, thereby essentially replacing the aforementioned initial or previous medium with the aforementioned different or subsequent medium. In some embodiments, the aforementioned different or subsequent media are added to the continuous cell culture until the culture vessel contains a mixture of the aforementioned previous medium and the aforementioned different or subsequent medium in a ratio of approximately 1:1 (volume-to-volume, or v / v). In some embodiments, the different or subsequent medium constitutes approximately 1 / 5, 1 / 3, 1 / 2, 2 / 3, or 3 / 4 of the total volume of the medium mixture in the vessel after it is added, or constitutes an approximately 80:20 (v / v) mixture. In some embodiments, the total volume of the cell culture (cells plus medium) remains approximately the same before and after the addition of the different or subsequent medium, and in some embodiments, the total volume increases with the addition of the different or subsequent medium. In some embodiments, additional media are added to the culture later, and these additional media may include the initial media and / or any of the different media or subsequent media described above.

[0066] In some embodiments, quail cells or other bird cells are seeded in a flask for the cell proliferation phase and cultured in FS / YIE or SCGM-ac medium for several days (e.g., about 2, 3, 4, or 5 days). The culture medium may be SCGM (Suspension Cell Growth Medium), which, as used herein, comprises FreeSize 293® Expression Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 g / L of Difco yeast extract (Difco, Becton Dickinson & Co., Franklin Lakes, NJ, USA); 10 μg / L of human recombinant EGF (Gibco, Thermo Fisher Scientific, Waltham, MA, USA); 50 μg / L of Long R3 human (recombinant) IGF (Sigma-Aldrich, St. Louis, MO, USA); 20 mg / L of L-ornithine monohydrochloride; and 20 mg / L of putrescine dihydrochloride (both from Sigma-Aldrich, St. Louis, MO, USA).

[0067] In some embodiments, an anticlamping agent (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) is added in a ratio of 1:100 or in the range of 1:50 to 1:150. Since commercially available media often contain anticlamping agents, if an anticlamping agent is not added, the medium is referred to herein as "SCGM-ac". Therefore, preferably, SCGM does not contain an anticlamping agent and is therefore called SCGM-ac. All media used to grow cells according to the method of the present invention are serum-free. In some embodiments, L-ornithine and putrescine are excluded from SCGM, and this medium is generally referred to herein as "FS / YIE". FS / YIE is a serum-free FreeStyle 293™ expression medium supplemented with yeast extract, EGF, and IGF (e.g., 1 g / L Difco yeast extract; 10 μg / L EGF; and 50 μg / L Long R3 IGF human (recombinant)).

[0068] Those skilled in the art will understand that some variation in the amount of additives to the cell culture medium is permissible and yields the same results. Therefore, for each additive, an amount of + / - 50% of the above amount is considered to be included in this description. In general, SCGM comprises FreeStyle 293® expression medium supplemented with 0.5–1.5 g / L of Difco yeast extract; 5–15 μg / L of human recombinant EGF; 25–75 μg / L of Long R3 human recombinant IGF; 10–30 mg / L of L-ornithine monohydrochloride; and 10–30 mg / L of putrescine dihydrochloride. As used herein, “SCGM” does not contain any animal serum; that is, it is “serum-free.” In “FS / YIE” medium, FreeStyle 293® expression medium can be supplemented, for example, with 0.5–1.5 g / L of Difco yeast extract; 5–15 μg / L of EGF; and 25–75 μg / L of Long R3 IGF Human (recombinant). OptiPRO® medium, when used herein, is generally supplemented with 1–5 mM of GlutaMAX® supplement and 0.01%–0.5% of Poloxamer 188 (also referred herein as Pluronic F68 or simply “F68”). Optionally, in some embodiments, OptiPRO® medium is used without additional glutamine, L-glutamine, or GlutaMAX® supplement, and / or without Poloxamer 188 or similar additives. The terms “Poloxamer 188” or “Poloxamer” are used herein interchangeably with the related trade name “Pluronic F68” (sometimes also referred to as “F68”). In general, the components of cell culture media can be replaced with similar compounds. For example, in some cases, glutamine or L-glutamine can be replaced with GlutaMAX® supplement to obtain similar results. FS / YIE medium does not contain any animal serum; that is, it is "serum-free". In some embodiments of the method of the present invention, the medium used does not contain any animal serum; that is, it is "serum-free".

[0069] For the preparation of the viral infection phase, cells are cultured in a mixture of conditioned SCGM-ac or FS / YIE medium and OptiPRO® medium (approximately 1:1 by volume). In some embodiments, this is accomplished by adding an equal volume of OptiPRO® medium to an existing cell culture. In some embodiments, quail cells are cultured in FS / YIE or SCGM-ac medium for a certain period of time, and then the medium is modified by adding OptiPRO® medium before infection. In some embodiments, the cells are in a continuous culture state such that the composition of the medium is modified by adding OptiPRO® medium and removing the previous medium until the desired medium mixture is achieved. In some embodiments, cells are initially cultured in FS / YIE or SCGM-ac medium, the composition of which is modified by adding OptiPRO® medium to include a 40:60 (v / v) or 50:50 (v / v) mixture of FS / YIE or SCGM-ac to OptiPRO® medium.

[0070] In some embodiments, this pre-infection cell proliferation phase is continued until the cells in the culture have almost aggregated into a group of cells, or have grown into a “clump.” Generally, cells are considered aggregated when, when a sample of a cell culture is viewed microscopically, the cells appear as clumps rather than individual cells. “Almost aggregated” means that at least 50% of the cells in the culture appear to be clumping together in the suspension culture. In some embodiments, quail cells are cultured during this pre-infection cell proliferation phase until the cells have almost aggregated, or until about 50%–70%, or about 60–80%, or about 90% of the cells have aggregated. The cells can be evaluated by microscopy or other techniques known in the art. In some embodiments, this pre-infection cell proliferation phase is continued for about 1 hour, or about 2–4 hours, or about 8–12 hours, or overnight. In some embodiments, MVA or recombinant MVA can be added to the cell culture before cell aggregation so that cell infection can occur during aggregation.

[0071] For viral infection, MVA or recombinant MVA (e.g., MVA-BN-RSV) is added to the culture at an MOI (Multiplicity of Infection) of approximately 0.1, or approximately 0.05–0.15, to allow the MVA or recombinant MVA to attach to and enter the cells in the culture. MOI (Multiplicity of Infection) refers to the ratio of the number of infectious viral particles, such as MVA or recombinant MVA, to the estimated number of cells in the culture. In some embodiments, MVA or recombinant MVA is added to the culture at an MOI of approximately 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or approximately 0.5 or higher. As those skilled in the art will understand, due to the limitations of the art, each of these values ​​(infectious virus particles of MVA or recombinant MVA and the number of cells in the culture) is susceptible to measurement errors, and therefore the actual MOI may vary from the calculated MOI, but generally it will be within about 100%, 50%, 25%, 20%, or 10% of the calculated MOI. In some embodiments, before adding MVA or recombinant MVA to the cells, the cells are transferred to a lower temperature (e.g., about 30°C or 32°C, or about 30°C–34°C) and incubated until the culture reaches that temperature.

[0072] Cells and / or cell cultures containing MVA or recombinant MVA are then continuously cultured in the same medium or, for example, in a perfusion bioreactor where, as the culture continues, preferably some of the medium is removed and replaced with fresh medium, during the virogenesis phase. After about 2 days, or after about 24 to 36 hours, or after about 36 to 48 hours, or after about 48 to 72 hours, or after about 72 to 96 hours, or after about 2 to 4 days, preferably after about 3 days, the culture can be harvested and products such as the virus can be purified. "Harvested" or "harvesting," as used herein, means that the cell culture is prepared for further processing to isolate the virus from the cell culture. In some embodiments, "harvesting" involves centrifugation to separate the cells from the cell culture medium, and in some embodiments, the cell culture, including both cells and medium, is processed to isolate the virus by adding reagents such as salts, nucleases, proteases, etc. In some embodiments, the cell culture is placed in a tank or container suitable for the treatment, which results in "collection".

[0073] For the viral infection phase and / or viral generation phase, in some embodiments a mixture of culture media is used, which consists of OptiPRO® medium (2 mM GlutaMAX® supplement and 0.025% Pluronic F68 (Poloxamer 188; all manufactured by Gibco, Thermo Fisher Scientific, Waltham, MA, USA)), FS / YIE (Yeast Extract (Difco, Becton Dickinson & Co., Franklin Lakes, NJ, USA), IGF-1 Long R3 (Sigma-Aldrich, St. Louis, MO, USA), and EGF (Gibco, Thermo Fisher Scientific)). The medium comprises FreeStyle® 293 expression medium (Scientific, Waltham, MA, USA) and the two media are mixed in a ratio of approximately 1:1, or the final mixture contains approximately 1 part OptiPRO® medium to 9 parts FS / YIE medium, or approximately 20:80 (v / v) mixtures of OptiPRO® medium to FS / YIE medium, or 30:70, 40:60, preferably 50:50, 60:40, or 70:30 (v / v) mixtures. In some embodiments, the final mixture preferably contains approximately 1:1 mixtures of OptiPRO® medium and FS / YIE medium.

[0074] In some embodiments, the cell growth phase, virus infection phase, and virus production phase are carried out as follows: Quail cells from a suspension cell line are seeded in a shaking or spinner flask containing a medium such as a suspension cell growth medium (SCGM-ac) or FS / YIE medium (see above) that does not contain anticlamping agents. In some embodiments, the quail suspension cell line is CCX.E10 cells (Nuvonis (Vienna, Austria)). In some embodiments, L-ornithine and putrescine are excluded from SCGM-ac. Cells are approximately 1.5 × 10⁻⁶ 5Seeds are seeded in this medium at a cell density of / mL and incubated at a temperature of 35°C–39°C (+ / -2°C) or 37°C (+ / -2°C). Cells are then propagated and subcultured into larger containers and volumes of medium as needed (e.g., first a shaking flask, then a 50 L bioreactor, then a 200 L bioreactor). Before infecting cells with MVA or recombinant MVA, the medium is changed or adjusted to an approximately 1:1 mixture of OptiPRO® medium (containing GlutaMAX® supplement and Poloxamer 188) and FS / YIE or SCGM-ac, and the temperature is adjusted to approximately 30°C–34°C, or approximately 31°C + / -0.5°C. MVA or recombinant MVA is added to the cell culture at an MOI of 0.05 (+ / -0.010) IU / cell, and incubation is continued at a temperature of, for example, approximately 30°C to 34°C, or approximately 31°C + / -0.5°C. In some embodiments, the cell concentration at the time of infection is approximately 1 × 10⁻⁶ 6 ~5×10 6 Cells / mL, or approximately 2 × 10⁶ 6 ~4×10 6 Cells / mL, or approximately 3 × 10⁶ 6 ~3.5×10 6 The concentration is cells / mL. In some embodiments, the cells are incubated at the same temperature or a higher temperature for approximately 24–36 hours, or approximately 36–48 hours, or approximately 60–84 hours to allow the virus to grow.

[0075] These methods of culturing quail cells in a mixture of culture media during the viral infection phase and / or viral generation phase were superior to other methods, such as culturing in only one of the media (see Example 3) or culturing CEF cells on microcarriers. These methods yielded viral titers of approximately 40–49 IU / cell, compared to 121–147 IU / cell using quail cells. This dramatic improvement in specific yield increases the amount of product available for recovery and reduces the amount of impurities and residues that must be removed for product purification in downstream processes. Thus, the present invention provides an improved method for culturing poxvirus in quail cells for the production of viral vector-based vaccine products, for example, MVA or recombinant MVA.

[0076] Downstream processes for purifying viruses from cell cultures In short, the downstream process involves collecting the product (e.g., MVA or recombinant MVA) from the culture and removing impurities. In an exemplary embodiment, this process includes the steps of: collecting a cell culture containing cells infected with MVA or recombinant MVA; treating the collected cell culture or cells ("Material") with a protease (e.g., trypsin); inactivating the protease with an inhibitor; treating the Material with a nuclease (e.g., Denarase® endonuclease or Bensonase® endonuclease) and incubating it; lysing the cells in the Material (e.g., using high-pressure homogenization or sonication); removing cell debris and remaining intact cells in a first filtration step (e.g., using deep filtration to remove components larger than 5 μm); continuing incubation under conditions suitable for nuclease activity; and concentrating the product (e.g., using tangential flow filtration to remove smaller impurities while retaining components larger than 100 nm). The solution or buffer containing the product can then be replaced, if desired, using diafiltration.

[0077] In some embodiments of this process, the pH and salt concentration of the material can be adjusted before, after, or concurrently with the addition of a nuclease (e.g., Denarase® endonuclease or Bensonase® endonuclease). In some embodiments, the salt concentration of the material is adjusted to 0.5 M NaCl or higher, or about 1 M NaCl, after the nuclease has been added to the material. In some embodiments, the process further includes a second enzymatic treatment with the nuclease; a second concentration of the product (e.g., using tangential flow filtration); and, optionally, exchange of the buffer by diafiltration. In some embodiments, the product is MVA or recombinant MVA provided in a pharmaceutically acceptable buffer or other solution, and is therefore a pharmaceutical composition. This product can then be stored, for example, at a very low temperature (below -20°C). The nuclease step assists in the removal of host cell DNA. In some embodiments, the nuclease is Denarase® nuclease, and in other embodiments, any suitable nuclease such as Bensonase® endonuclease (Sigma Aldrich, St. Louis, MO, USA) or TurboNuclease® nuclease (VITA Scientific, Beltsille, MD, USA) may be used. In some embodiments, the product, which is MVA or recombinant MVA, can be separated from impurities and other residues using the sucrose cushion technique and / or ultracentrifugation.

[0078] Where used herein, “material” refers to the residue from a cell culture being processed to recover products such as viruses, and at various stages, “material” may include cells, lysed cells, host cell DNA, residual proteins, etc. Where used herein, the term “product” refers to MVA or recombinant MVA used to infect a cell culture and which can be purified by the methods described herein. In some embodiments, the product is suitable for use as a vaccine when sufficiently purified. Where used herein, “purified” means that most of the residue and other contaminants such as host cell DNA have been removed from the cell culture material, and a product is provided that essentially contains MVA or recombinant MVA with little to no additional contamination, or without significant additional contamination that would interfere with the therapeutic function of the product when used as a vaccine, for example. In some embodiments, the product is sufficiently purified for use as a pharmaceutical composition such as a vaccine. For example, as intended herein, the product has a host cell DNA level of less than 100 ng per vaccine dose (e.g., 1 × 10⁻⁶). 8 IU / 0.5mL or 5 x 10 8 A product is considered sufficiently purified for use as a vaccine if it meets the following criteria (per IU / 0.5 mL dose, or equivalent per dose and volume): that is, the host cell DNA level of the product must be 1 × 10⁻⁶. 8 IU or 5x10 8 If the amount of IU virus in a vaccine preparation is less than 100 ng per 0.5 mL of volume, it can be considered sufficiently purified.

[0079] When used in the context of parameter values ​​for the methods and compositions of the present invention, "approximately" means that the actual value of the parameter(s) may vary somewhat from the stated value, but will still possess the same characteristics and yield the benefits and results of the methods and compositions. Therefore, as used herein, a parameter described with "approximately" will have an actual value within 10% of the stated value unless the context clearly indicates otherwise (for example, a solution with a temperature of "approximately 30°C" will have a temperature of 27°C to 33°C). Thus, "approximately" encompasses the stated value of the parameter (for example, a solution with a temperature of "approximately 30°C" may have a temperature of 30°C).

[0080] In exemplary embodiments, the details of the downstream process are as follows: The material is collected in a mixer tank to harvest the cell culture and collect the product. Then, a first series of enzymatic steps is performed (sometimes referred to as “Enzymatic Treatment 1”). The material is treated with a protease. In some embodiments, the protease has trypsin activity (e.g., trypsin or recombinant trypsin, e.g., TrypLE (Gibco, Fisher Scientific, Waltham, MA, USA)). In some embodiments, the material is treated with recombinant trypsin (e.g., in a 1:5 ratio) and incubated at ambient temperature for, for, for, 30 minutes to 1 hour, to allow time for digestion. Before the addition of the nuclease, the material is treated to inactivate the protease (e.g., with a trypsin inhibitor (1:5) for 15 minutes at ambient temperature) and also treated to adjust the pH, salt concentration, and volume of the material as needed. These adjustments can be performed in any order and may be performed simultaneously or separately. For example, the pH of the material is adjusted to approximately 7.5, 8.0, 8.5, 8.7, or any suitable pH, for example, any pH within the range of 8.0 to 8.6, for example, approximately 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, or 8.7. In some embodiments, MgCl2 is added to a concentration of approximately 2 mM or in the range of 2 mM to 25 mM, and a nuclease is added (for example, Denarase® endonuclease (c-LEcta (Leipzig, Germany)), for example, at a concentration of 20 U / mL). NaCl or another salt or chaotropic agent is added to a concentration of approximately 1 M, or at least approximately 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, or at least approximately 1.4 M. Optionally, the material may be cooled to approximately 4°C or 2°C-8°C for approximately 2 hours, 4 hours, 12 hours, 2-24 hours, or 14-24 hours while being stirred or agitated, or the material may proceed to the next step without incubation (for example, the material may be held overnight at 4°C, which can be called "holding 1").Optionally, if a higher biomass load is present (i.e., more cells are being treated), further impurities and other residues can be removed by increasing the amount of nuclease, raising the incubation temperature, and / or extending the incubation time between these steps. In some embodiments, the material is also diluted as a result of or in addition to the above-mentioned adjustment of pH and salt, for example, by increasing the volume of the material by about 40%, 50%, 60%, or about 100% or 200%, thereby aiding in the removal of impurities.

[0081] The cells are then lysed to collect intracellular viruses or other products. This step may be called “cell lysis” and is carried out by any preferred technique such as high-pressure homogenization (HPH) or sonication, which releases cellular debris containing host cell DNA and host cell proteins. The impurities, including cellular debris and residual cells, are then removed, for example, by deep filtration (e.g., through a 5 μm or 3 μm depth filter), and this step may be called “clarification.” The material is then stored at ambient temperature or heated to about 30°C, or within the range of 25–37°C, and incubated overnight, or about 1 hour, or about 2–6 hours, or about 8–12 hours, or about 22–30 hours, to allow a nuclease (e.g., Denarase® endonuclease) to degrade the host cell DNA (a step which may be called “holding 2”). In some embodiments, the material is continuously agitated or stirred during this step. Optionally, the material is held at ≤10°C. This step typically removes a large portion of the host cell DNA.

[0082] Next, further filtration is performed, for example, using tangential flow filtration (TFF), and this step may be referred to as "TFF1". This step can be performed using any suitable filter, for example, using a 0.1 μm PESU E screen to obtain a certain concentration (e.g., 5-6 times) by ultrafiltration while removing small impurities, and then a further concentration (e.g., 1.3 times) can be obtained using diafiltration (in some embodiments, this is done for a total concentration of at least about 2 times, about 4 times, about 6 times, or about 8 times). In some embodiments, in this step, filtration is performed using hollow fiber (HF) based filtration with a polysulfone (PS) based filter having a diameter of 0.5 mm and a cutoff of 0.05 μm. In some embodiments, filtration is performed using HF based filtration with a mixed cellulose ester ("ME") based filter having a diameter of 0.63 mm and a cutoff of 0.1 μm, and in some embodiments, the filter contains modified polyethersulfone (mPES) having a diameter of 0.5 mm and a cutoff of 750 kDa. As described above, the product is concentrated in this TFF, and then diafiltration is performed with diafiltration buffer DF1 (10 mM Tris, 2 mM MgCl2, pH 8.2) to remove impurities and Denarase® endonuclease. Optionally, the system is flushed at the end of this step, and this flush solution is pooled with the filtered material in a suitable buffer.

[0083] In preliminary tests, these methods removed large aggregates present in the material after cell lysis, resulting in excellent outcomes in subsequent steps and the final product, including a reduction in the amount of host cell DNA and total protein. These excellent results were unexpected for several reasons. This includes the fact that the Denarase® endonuclease manufacturer (c-LEcta GmbH, Leipzig, Germany) recommends using this endonuclease at low salt concentrations to avoid a decrease in enzyme efficiency. Also, it was surprising that adding a relatively large amount of NaCl early in the process improved the removal of host cell DNA from the product in the subsequent filtration step (see Figure 2), an effect that could not be obtained by adding NaCl later in the process. Furthermore, it was unexpected that adding an enzyme with trypsin-like activity early in the process also reduced the level of aggregates and improved overall recovery and host cell DNA removal, because trypsin is typically used in upstream processes and, when used as described herein, can be considered an unwanted contaminant. This downstream process worked well for virus purification from various types of cultures, including bioreactor cultures, perfusion bioreactor cultures, and wave bag cultures.

[0084] In some embodiments of the downstream process, a second enzymatic treatment with a nuclease (e.g., Denarase® endonuclease) is performed to further digest any remaining host cell DNA. For example, Denarase® endonuclease is added to a concentration of 75–150 U / mL and incubated overnight (e.g., for about 4–8 hours, 8–12 hours, or 14–22 hours) at ambient temperature (approximately 22°C) or in the range of 25–37°C. This step, if performed, may be called "enzymatic treatment 2". In some cases, MgCl2 is added (e.g., up to 2 mM) to adjust the pH to increase nuclease activity. These adjustments may be performed before, after, or concurrently with the addition of the nuclease to the material. Optionally, the material is kept at a low temperature (e.g., below approximately 10°C or below approximately 4°C). This may be called "keeping 3". In some embodiments, the material is continuously stirred or agitated during this step to avoid sedimentation and / or aggregation.

[0085] In some embodiments, a second filtration step is then performed using tangential flow filtration (TFF), which is carried out, for example, using a PESU E screen with a cutoff of 0.1 μm (e.g., producing a 14-15 times concentration increase by ultrafiltration (including at least 2 to about 10 diafiltration buffer changes)). Another filter suitable for this step is, for example, an mPES cassette with a cutoff of 0.01 μm. This filtration can be carried out using diafiltration buffer (DF2) (10 mM Tris, 140 mM NaCl, pH 7.7), and a final concentration of about 4 times can be obtained by flushing the system at the end of this step and pooling the flush solution with the product. Alternatively, this second filtration step may be carried out using an HF (hollow fiber)-based TFF filtration step with any suitable filter, e.g., a PS, a hollow fiber with a diameter of 0.5 mm and a cutoff of 0.05 μm, a mixed cellulose ester ("ME")-based filter having a diameter of 0.63 mm and a cutoff of 0.1 μm, or a modified polyethersulfone (mPES) filter having a diameter of 0.5 mm and a cutoff of 750 kDa. First, the material can be concentrated and then diafiltration to replace the buffer. Optionally, the system can be flushed at the end of this step, and the flush liquid can be pooled with the product to obtain a product concentrated about 10-fold. The product can then be stored, for example, at about -20°C or about -80°C.

[0086] In some embodiments, it is important to avoid sedimentation or precipitation of the material during the process, particularly during the overnight and / or incubation step. This is because adverse effects on yield may be observed, possibly due to aggregation of viral products with residual debris from host cells or other viral particles. Therefore, in some embodiments, it is necessary to agitate or otherwise stir the material intermittently or continuously between each process step to prevent sedimentation and / or precipitation. In some embodiments, the material is agitated or stirred during steps requiring incubation, such as during overnight holding, while other steps may be carried out without agitation or stirring. Agitation or stirring may be carried out by any preferred means, such as using a magnetic stirrer bar, a shaking platform, or circulating gas or liquid in the material.

[0087] In some embodiments, the process can be paused or “held” at any suitable step, or the duration of a step can be extended, as long as the effects of the step and / or the effects of the entire process are achieved. Thus, although some steps are indicated herein as “held” steps, these incubations can be shortened or lengthened, or “held” can be performed at other steps, for the convenience or ease of manufacturing.

[0088] The present invention provides a method for purifying active viruses without using chromatographic methods (e.g., hydrophobic interaction chromatography "HIC"), and thus has the advantage of not requiring additional materials such as HIC matrices for virus purification. Therefore, the present invention provides a purification method that does not use hydrophobic interaction chromatography or other chromatographic techniques.

[0089] In preliminary trials, the results of this process favorably achieved the goals of a high overall virus (product) recovery rate of at least 50% and a host cell DNA level of less than 100 ng per vaccine dose. Furthermore, the remaining host cell DNA was fragmented, thereby reducing the likelihood of any viable coding nucleic acids in the vaccine. These results were superior to those obtained in previous trials using EB66 duck cells and CEF cells.

[0090] In summary, these methods for producing vaccines containing MVA or recombinant MVA from bird cells result in higher yields and lower impurity levels. These methods can also be used to prepare vaccines containing MVA or recombinant MVA from infected quail cell lines, thereby enabling the use of cell banks, reducing the possibility of exogenous contamination, and allowing the production of vaccine products without the use of antibiotics. Thus, using the methods of the present invention in the production of MVA and recombinant MVA can increase the quality and safety of vaccines containing these formulation materials.

[0091] Specific Embodiments Specific embodiments of the present invention also include the following:

[0092] Item 1 is a method for culturing quail cells to produce a poxvirus (or alternatively, a method for producing a poxvirus in quail cells), comprising the following steps: (a) culturing quail cells to a cell density of approximately 1 × 10 6 cells / mL~5×10 6 Cells / mL, preferably about 2 × 10⁶ 6 cells / mL~4×10 6 Cells / mL, more preferably about 3 × 10⁶ 6 cells / mL or at least 3 × 10⁶ 6The method comprises the steps of: (b) culturing the cells in suspension in a culture medium until the cell volume reaches a concentration of cells / mL ("cell proliferation phase"); (b) modifying the culture medium to induce cell aggregation; (c) adding MVA or recombinant MVA to the cell culture to infect the cells with the MVA or recombinant MVA ("viral infection phase"); (d) continuing the cell culture for about 1 day or about 1 to 4 days to increase the amount of MVA or recombinant MVA ("viral production phase"); and (e) collecting the cell culture. In some embodiments, the method further comprises purifying the MVA or recombinant MVA described herein.

[0093] Item 2 is the method of Item 1, wherein step (a) comprises culturing the quail cells in a medium containing FS / YIE or SCGM-ac medium, and step (b) comprises modifying the medium to contain a mixture of FS / YIE or SCGM-ac and fresh OptiPRO® medium in a ratio of approximately 60:40. Item 3 is the method of Item 2, wherein a certain amount of OptiPRO® medium is added to the cell culture so that the medium contains a mixture of FS / YIE or SCGM-ac and OptiPRO® medium in a ratio of approximately 1:1, and each medium is either fresh or conditioned. Item 4 is the method of Item 1, wherein step (a) comprises culturing the quail cells in FS / YIE or SCGM-ac medium, and step (b) comprises modifying the medium to contain a mixture of FS / YIE or SCGM-ac medium and OptiPRO® in a ratio of about 35:65 to 55:45 (v / v), preferably about 1:1, wherein the FS / YIE or SCGM-ac medium is partially or completely fresh or acclimatized medium. Item 5 is the method of Item 2, wherein step (b) comprises adding a certain amount of OptiPRO® medium to the cell culture so that the medium contains a mixture of fresh OptiPRO® medium and the previous medium in a ratio of about 40:60 (v / v) or a mixture of the previous medium and fresh OptiPRO® medium in a ratio of about 1:1. Item 6 is the method of Item 4 or Item 5, wherein step (b) further comprises culturing the cells until about 50% to 70% of the cells have aggregated. Item 7 is the method of Item 1, wherein the cells are cultured for approximately 2 days, or approximately 24 to 36 hours, approximately 40 to 48 hours, approximately 60 to 72 hours, approximately 3 days, or approximately 4 days during the virus generation phase of step (d). Item 8 is the method of any of Items 1 to 7, wherein the cells are cultured for approximately 12 to 24 hours, approximately 24 to 36 hours, approximately 36 to 48 hours, approximately 48 to 72 hours, or approximately 72 to 96 hours, preferably 2 to 4 days, more preferably approximately 3 days during the virus generation phase of step (d). Item 9 is the method of any of Items 1 to 8, wherein step (c) comprises adding the MVA-BN® virus to the cell culture.Item 10 is the method according to any one of items 1 to 9, wherein step (c) comprises adding MVA-BN-RSV to the cell culture. Item 11 is the method according to any one of items 1 to 10, wherein the quail cells are CCX.E10 quail cells (Nuvonis (Vienna, Austria)).

[0094] Item 12 is a method for producing MVA or recombinant MVA in quail cells, comprising: (a) culturing a continuous quail cell suspension to increase the number of cells; (b) modifying the culture medium to induce aggregation of the cells during culture; (c) adding the MVA or recombinant MVA to the culture to infect the cells; (d) further culturing the infected cells to increase the amount of MVA or recombinant MVA in the cell culture; and (e) taking the cell culture for further processing. Item 13 is the method of Item 12, further comprising step (a) culturing the cells in a medium which is FS / YIE or SCGM-ac, and step (b) modifying the medium to contain a mixture of FS / YIE or SCGM-ac medium and OptiPRO® medium in a ratio of approximately 1:1 (v / v).

[0095] Item 14 is the method according to any one of items 1-8 or 10-13, wherein the recombinant MVA comprises (i) at least one nucleotide sequence encoding the antigen of respiratory syncytial virus (RSV) membrane glycoprotein, the nucleotide sequence encoding the full-length RSV F membrane glycoprotein; (ii) at least one nucleotide sequence encoding the RSV nucleocapsid antigen, the nucleotide sequence encoding both the full-length RSV N nucleocapsid protein and the full-length RSV M2 transcription elongation factor protein; and (c) at least one nucleotide sequence encoding the antigen of RSV G membrane glycoprotein, the nucleotide sequence encoding the full-length RSV G membrane glycoprotein. Item 15 is the method according to item 14, wherein the nucleotide sequence encoding the RSV G membrane glycoprotein is from RSV strain A.

[0096] Item 16 is a cell culture which is a population of quail cells infected with MVA or recombinant MVA or a suspended quail cell culture, wherein the population or cell culture contains approximately 1 × 10⁶ cells per 1 mL. 6 ~1 × 10 8 1 quail cell, and 1 × 10 per 1 mL 3 The aforementioned MVA of the virus or recombinant MVA, or preferably 1 × 10 per 1 mL. 6 Individual MVA or recombinant MVA ~ 1 × 10 per 1 mL 8 A population or cell culture containing MVA or recombinant MVA. Item 17 is a cell culture or population of quail cells which is a suspended quail cell culture containing MVA or recombinant MVA, wherein the cell culture or population contains about 1 × 10⁶ cells. 6 cells / mL~5×10 8 Cells / mL and approximately 1 × 10⁶ cells per mL. 7 Virus ~ 1 x 10 per 1 mL 9 The cell culture or population that has been cultured to contain the virus. Item 18 is a cell culture that is a suspended quail cell culture as described in Item 16 or 17, containing CCX.E10 quail cells, or a population of quail cells as described in Item 16 or 17, which is CCX.E10 quail cells. Item 19 is a cell culture as described in Item 16, 17, or 18, containing MVA-BN® or MVA-BN-RSV.

[0097] Item 20 is a method for providing a pharmaceutical composition comprising MVA or recombinant MVA from a cell culture or population of cells (for example, items 16, 17, 18, or 19), or a method for producing a vaccine comprising the same, the method comprising the following steps: (a) collecting material or taking a cell culture or population of cells containing cells and / or viruses to produce collected material; (b) treating the collected material with a protease having trypsin activity; (c) optionally adjusting the pH of the material to 8.0 to 8.6; (d) treating the material with a nuclease; (e) lysing the cells in the material; (f) filtering the material to remove cell debris and remaining cells; (g) continuing incubation with the nuclease for a period of time; and (h) concentrating the product using tangential flow filtration. Item 21 is the method of Item 20, further comprising the steps: (j) treating the material with a nuclease; (k) concentrating the MVA or recombinant MVA product using tangential flow filtration. Optionally, with respect to each of Items 20 and 21, there is an additional step of exchanging the buffer by diafiltration, and optionally, there is a final step of suspending the product in a suitable buffer to yield a pharmaceutical composition.

[0098] Item 22 is the method according to Item 20 or 21, wherein the nuclease in step (d) and / or step (j) is Denarase® nuclease. Item 23 is the method according to Item 22, further comprising adding NaCl to a final concentration of about 1 M after step (d), during step (d), and / or before step (e). Item 24 is the method according to Item 22, further comprising adding NaCl to a final concentration of about 1 M after the addition of the nuclease in step (d); optionally, after the addition of NaCl, the material is incubated with the nuclease at 22°C to 37°C for at least 2 hours. In some embodiments of Items 3 and 4, NaCl is added to a final concentration of at least 0.5 M.

[0099] Item 25 is a pharmaceutical composition containing MVA-RSV or MVA-BN-RSV prepared by any of the methods described in Items 20-24 for the prevention of lower respiratory tract disease caused by RSV. Item 26 is a pharmaceutical composition containing MVA or recombinant MVA prepared by any of the methods described in Items 20-24, and optionally containing 100 ng or less of host cell DNA per vaccine dose.

[0100] Item 27 is a recombinant modified vaccinia virus ankara (MVA) prepared by any of the methods described in items 20-24, comprising a nucleotide sequence encoding an antigen of at least one respiratory syncytial virus (RSV) membrane glycoprotein, for treating or preventing diseases caused by RSV infection, for example, by intranasal administration.

[0101] Item 28 is a recombinant modified vaccinia virus ankara (MVA) prepared by any of the methods described in items 20-24, comprising at least one nucleotide sequence encoding a respiratory syncytial virus (RSV) membrane glycoprotein antigen and at least one nucleotide sequence encoding an RSV nucleocapsid antigen.

[0102] Item 29 is a recombinant MVA according to item 27 or 28, wherein the nucleotide sequence encoding the antigen of the RSV membrane glycoprotein encodes the RSV F antigen. Item 30 is a recombinant MVA according to item 27 or 28, wherein the nucleotide sequence encoding the antigen of the RSV membrane glycoprotein encodes the full-length RSV F membrane glycoprotein. Item 31 is a recombinant MVA according to item 31, wherein the nucleotide sequence encoding the antigen of the RSV F membrane glycoprotein encodes RSV strain A, preferably A2 and / or A Long It is a recombinant MVA derived from item 29 or 30.

[0103] Item 32 is a recombinant modified vaccinia virus ankara (MVA) prepared by the method described in any of Items 20-24, comprising: (a) at least one nucleotide sequence encoding an antigen of respiratory syncytial virus (RSV) membrane glycoprotein, wherein the nucleotide sequence encodes the full-length RSV F membrane glycoprotein; (b) at least one nucleotide sequence encoding an RSV nucleocapsid antigen, wherein the nucleotide sequence encodes both the full-length RSV N nucleocapsid protein and the full-length RSV M2 matrix protein, which are encoded by a single open reading frame, wherein the single open reading frame comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10 or a nucleotide sequence of SEQ ID NO: 9; and (c) at least one nucleotide sequence encoding the full-length RSV G membrane glycoprotein.

[0104] Item 33 states that the nucleotide sequence encoding the RSV F membrane glycoprotein is preferably A2 and / or A from RSV strain A. LongItem 34 is a recombinant MVA according to item 32, which is derived from the RSV F membrane glycoprotein. Item 34 is a recombinant MVA according to item 32 or 33, wherein the nucleotide sequence encoding the RSV F membrane glycoprotein includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or includes the nucleotide sequence of SEQ ID NO: 1. Item 35 is a recombinant MVA according to any of items 27 to 32, wherein the nucleotide sequence encoding the RSV G membrane glycoprotein is derived from RSV strain A, preferably strain A2 and / or strain B. Item 36 is a recombinant MVA according to any of items 27 to 35, which includes a nucleotide sequence encoding the RSV G membrane glycoprotein, which includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 or includes the nucleotide sequence of SEQ ID NO: 3. Item 37 is a recombinant MVA according to any of items 27 to 36, wherein the MVA used to produce the recombinant MVA is the MVA-BN® virus deposited with the European Collection of Cell Culture (ECACC) under number V00083008.

[0105] Item 38 is a vial containing the pharmaceutical composition described in Item 25 or 26, wherein the pharmaceutical composition contains at least 1 × 10 8 IU / mL, at least 3 × 10⁻⁶ 8 IU / mL, at least 5 × 10 8 IU / mL, or at least 7 × 10 8 The vial contains IU / mL of MVA or recombinant MVA.

[0106] Item 39 is a pharmaceutical composition containing recombinant MVA as described in any of items 25-37. Item 40 is at least 1 × 10 8 IU / mL, at least 3 × 10⁻⁶ 8 IU / mL, at least 5 × 10 8 IU / mL, or at least 7 × 10 8A pharmaceutical composition comprising recombinant MVA as described in any of items 25-37 in IU / mL. Item 41 is a vial containing a vaccine comprising the pharmaceutical composition described in item 39 or 40, optionally containing 100 ng or less of host cell DNA per vaccine dose.

[0107] Item 42 is a method that includes the steps described in any of items 1 through 15, followed by the steps described in any of items 20 through 24.

[0108] Item 43 is a method for producing a poxvirus by culturing and processing quail cells (or alternatively, a method for producing a poxvirus comprising culturing and processing quail cells), the following steps: (a) culturing quail cells to a cell density of about 1 × 10 6 cells / mL~5×10 6 Culture in suspension in culture medium until the cell count reaches 1 × 10⁻¹⁶ cells / mL. 6 cells / mL~5×10 6(b) a step of producing a cell culture containing cells / mL; (c) a step of modifying the medium to contain FS / YIE or SCGM-ac medium and OptiPRO® medium in a ratio of 40:60 to 60:40 (v / v) and continuing to culture the quail cells; (d) a step of adding MVA or recombinant MVA to the cell culture to infect the cells with MVA or recombinant MVA; (e) a step of continuing to culture the cells for about 1 to 4 days so that the amount of MVA or recombinant MVA increases; (f) a step of extracting material containing cells and / or viruses from the cell culture. The method comprises the steps of: (f) collecting and generating the collected material; (g) treating the collected material with a protease having trypsin activity; (h) adjusting the pH of the material to 8.0-8.6; (i) treating the material with a nuclease; (j) mechanically lysing the cells in the material; (g) filtering the material to remove cell debris and residual cells; (k) continuing incubation with the nuclease for a period of time; and (l) concentrating the product using tangential flow filtration. Item 44 is the method of item 43, wherein the nuclease in step (h) is Denarase® nuclease. Item 45 is the method of item 43 or 44, further comprising adding NaCl to a final concentration of about 1 M after step (h), during step (h), and / or before step (h); optionally, after the addition of NaCl, the material is incubated at 22°C to 37°C for at least 2 hours. Item 46 is the method of item 43, 44, or 45, further comprising the following steps: (m) after step (l), the material is treated with a nuclease; and (n) the MVA or recombinant MVA product is concentrated using tangential flow filtration. Optionally, for each of items 43 to 46, there is an additional step of exchanging the buffer with a diafiltration and then performing tangential flow filtration to obtain the product (MVA or recombinant MVA) in a suitable solution such as buffer. [Examples]

[0109] Example 1: Purification of recombinant MVA from chicken cell cultures Each of the MVA-BN-RSV and MVA-BN® viruses was amplified (separately) in a suspended quail cell culture as described above, and then purified as follows: The material (cell culture) was collected in a mixer tank and treated with TrypLE (Gibco, Fisher Scientific, Waltham, MA, USA) at ambient temperature for 1 hour, followed by inactivation with a trypsin inhibitor for 15 minutes. The pH of the material was adjusted to 8.5, MgCl2 was added to 2 mM, and 20 U / mL of Denarase® endonuclease was added. NaCl aqueous solution was added to a final concentration of 1 M, and the material was incubated overnight at 5-8°C with stirring. Cells were lysed using high-pressure homogenization (HPH), and cell debris and remaining cells were removed by deep filtration using a 5 μm depth filter. The material was then incubated overnight at 30°C with stirring and filtered using a PESU filter with a cutoff of 0.1 μm or a cassette-based tangential flow filtration (TFF) with PS hollow fibers having a cutoff of 0.05 μm and an inner diameter of 0.5 mm. Ultrafiltration was performed in this TFF, followed by diafiltration using DF1 buffer (10 mM Tris, 2 mM MgCl2, pH 8.2) (including flushing the system at the end and pooling it with the retained material). A second enzymatic treatment was performed with 100 U / mL Denarase® endonuclease in 2 mM MgCl2 and incubated overnight at room temperature with stirring. A second TFF step was then performed using a PESU filter with a cutoff of 0.1 μm or a cassette-based TFF with PS hollow fibers having a cutoff of 0.05 μm and an inner diameter of 0.5 mm. In this filtration process, diafiltration was performed using DF2 buffer (10 mM Tris, 140 mM NaCl, pH 7.7). A second buffer change was performed as an option.

[0110] In four exemplary trials, this process yielded a recovery rate of approximately 65%, with host cell DNA at 85 ng / dose and total protein at 1 mg / dose. These results closely meet the regulatory recommended standard of less than 10 ng / dose for the final concentration of host cell DNA in the final product. Furthermore, the process achieved an overall recovery rate of over 50%, making it economically viable as a vaccine manufacturing process. In addition, the remaining host cell DNA was fragmented, thereby reducing the likelihood of any viable coding nucleic acids being present in the vaccine.

[0111] A particularly surprising aspect of these experiments is that trypsin enzyme activity had a positive effect on later process steps and the final product, even though trypsin activity was only present early in the process, even before the cells were lysed. Figure 1 shows the effect of Denerase® endonuclease on the amount of host cell DNA ("HCD") per dose of vaccine at various stages of an exemplary downstream process. Figure 2 shows that the addition of NaCl during the early steps of the downstream process results in a remarkable decrease in the amount of host cell DNA (HCD) per dose of vaccine at later stages of the exemplary downstream process, despite an increase in the amount of host cell DNA (HCD) per dose of vaccine at early stages of the process. Figure 7 includes a schematic diagram showing the hypothetical effects of the first enzymatic treatment step with trypsin enzyme activity (e.g., TrypLE) and nuclease (e.g., Denarase® enzyme) on the harvested cells, amplified virus, and impurities resulting from cell lysis during the virus purification process. Figure 7 also shows transmission electron microscope images of cells treated with and without trypsin enzyme therapy. In these images, cells that have not been treated with trypsin enzyme therapy form dense clusters (left image), while cells treated with trypsin enzyme therapy appear to be much more loosely associated in smaller groups (right image), with each cell appearing to be in contact with the surrounding environment.

[0112] Another surprising aspect of the present invention is that Denarase® endonuclease was able to digest a large portion of host cell DNA (HCD) at a high NaCl concentration of approximately 1 M (far exceeding the manufacturer's recommended level of up to 150 mM NaCl, and the level at which the manufacturer indicates the enzyme should be active).

[0113] The downstream purification process was compared in parallel with and without pH adjustment in the first enzymatic treatment step. The results were very similar. Specifically, without pH adjustment, the recovery rate as solubility (%) was 52.9%, and the HCD was 3.1 ng / dose with a resulting dose of 5341 doses per liter of bioreactor, compared to the standard process including pH adjustment, with a recovery rate of 55.0%, and the HCD was 1.3 ng / dose with a resulting dose of 5572 doses per liter of bioreactor. Therefore, in some embodiments, pH adjustment in the first enzymatic treatment can be omitted without adversely affecting the results.

[0114] Example 2: Suspended quail cell line for generating poxvirus Serial suspension quail cell lines were generated from quail cells by passage of adherent cells and subsequent adaptation for growth in a suspension state. A single clone was then selected, amplified, and adapted to serum-free cultured cell suspension medium (SCGM). One serial suspension quail cell line was selected for further use (referred herein as "CCX.E10"), and an adherent quail cell line (CCX.2C4) was also characterized (both cell lines are commercially available from Nuvonis (Vienna, Austria)).

[0115] Initial experiments were conducted to evaluate the suitability of quail cell suspension and adherent strains for virus generation. Schoepp's fibroma virus (SFV), fowlpox virus (FPV), and vaccinia virus (VACV) were tested. As expected, SFV did not replicate in CEF cells or quail cells. FPV and VACV produced peak titers in quail cells within the same range as those produced in CEF cells under several conditions.

[0116] Example 3: Virus yield from suspended quail cells cultured in various media. Cells from the serial suspension quail cell line CCX.E10 (Nuvonis (Vienna, Austria)) were seeded in a stirrer-equipped flask in a suspension cell growth medium (SCGM-ac medium) without anticlamping agents during the cell growth phase. Viral infection was excluded from this study because previous experiments had shown that it is inhibited by anticlamping agents (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) commonly added to conventional suspension cultures.

[0117] Three days later (Day 0), the cells were divided into 8x10 cells for the viral infection phase. 5 ~1.1 × 10 6 Cells were transferred to different culture media at different cell densities (cells / mL). The media tested were SCGM-ac + CaCl2, OptiPRO® medium, and a 50:50 mixture of SCGM-ac + OptiPRO® medium. Previous experiments have shown that adding CaCl2 to SCGM-ac medium increases viral yield because CaCl2 induces cell aggregation, thereby facilitating viral spread. However, OptiPRO® also promotes cell aggregation. GlutaMAX® and Pluronic F68 were supplemented to the media to reduce shear force and foaming.

[0118] Cells were incubated at 30°C or 32°C for 1 hour, then infected with MVA-BN-RSV (i.e., MVA-mBN294B) at an MOI (Multiplicity of Infection) of 0.1. To analyze viral yield, cell samples were collected 1–4 days post-infection, sonicated, frozen at -20°C, and then subjected to standard TCID testing. 50 Titer was measured by assay (vortexing for more than 1 minute). Figure 3 shows the viral yield in TCID. 50 The geometric mean (GM) of the volume / mL is shown along with its geometric standard deviation (geoSD). The values ​​are shown in Table 1 below. The data points represent results from nine separate experiments. The multiple difference shown below the graph is compared to the yield obtained with SCGM-ac + CaCl2.

[0119] [Table 1]

[0120] The data in Figure 3 shows that when the cell culture medium for the viral infection phase was completely changed to OptiPRO® medium (with Pluronic F68 additive), the MVA-BN-RSV yield was higher than that obtained using SCGM-ac + CaCl2 cell culture medium. Cells incubated in OptiPRO® medium produced a peak viral titer (d3) that was approximately three times higher than that of cells incubated in SCGM-ac + CaCl2 (d2; Table 1 and Figure 3; note the logarithmic scale). The data shown in Figure 3 also demonstrates that the viral yield from cells incubated in a 50:50 mixture of cSCGM-ac + OptiPRO® medium was consistently higher than the viral yield obtained using either SCGM-ac + CaCl2 or OptiPRO® medium. In cells cultured in a 50:50 mixture of cSCGM-ac + OptiPRO® medium, the peak yield of MVA-BN-RSV at post-infection day 2 ("d2") was 180 TCID, which is the cell-specific yield. 50These corresponded to cells, and their peak yields were approximately 4.4 times and 13.8 times higher, respectively, than those of OptiPRO(trademark)(d3) and SCGM-ac+CaCl2(d2) alone.

[0121] Example 4: Virus yield from quail cells cultured in a culture medium mixture Cells from the serial suspension quail cell line CCX.E10 (Nuvonis (Vienna, Austria)) were seeded in SCGM-ac medium (SCGM without anti-clamping agent) in a stirrer-equipped flask on day 3. Cell culture in the medium converts the SCGM-ac medium to conditioned SCGM-ac medium (cSCGM-ac). On day 0, 20 mL of this cell culture (including the medium) was transferred to each of six different shaking flasks and mixed with equal volumes of various media. The culture medium may include: (i) OptiPRO® medium (a 50:50 mixture of cSCGM-ac and OptiPRO® medium); (ii) OptiPRO® medium premixed with varying amounts of cSCGM-ac (the ratio of cSCGM-ac to OptiPRO® medium will range from 60:40 to 90:10); or (iii) cSCGM + CaCl2 (without OptiPRO® medium). All conditions require a culture volume of 40 mL and 1 × 10⁻⁶ 6 The tests were performed using cells / mL. The culture medium was supplemented with GlutaMAX® supplement (final concentration 2 mM) and Pluronic F68 (final volume 0.125%) to reduce shear force and foaming.

[0122] After transferring the cells to 30°C for 1 hour, they were infected with MVA-BN-RSV (which co-encodes the mRFP fluorescent protein) at an MOI (Multiple Infection Infection) of 0.1, and then cultured at 30°C. To analyze viral yield, duplicate samples were collected from the CCX.E10 cultures on days 1-4 post-infection, sonicated, and frozen at -20°C. Samples from days 1 and 2 (d1, d2) and days 3 and 4 (d3, d4) were analyzed using standard TCID. 50Titers were measured in parallel by assay (vortexed for 1 minute or more). Figure 4A shows the virus yield as the geometric mean with geometric standard deviation as TCID 50 / mL. To analyze virus infection and spread, mRFP expression was analyzed by flow cytometry on days 2 - 4 post-infection (Figure 4B) (GMFI, geometric mean fluorescence intensity).

[0123] As shown in Figure 4A, the virus yield of MVA - BN - RSV was affected by the ratio of SCGM:OptiPRO™ medium used during the infection of quail cells with recombinant MVA (i.e., the virus infection stage). The highest virus yield (1.8×10 8 TCID 50 / mL) was reached on day 3 post - infection (d3) with a 50:50 mixture of cSCGM:OptiPRO™ medium, although the yield was already very close to this maximum yield at this medium composition on day 2 post - infection (d2).

[0124] Compared to the 50:50 mixture (also called 1:1 herein), the virus yield decreased slightly in media containing less OptiPRO™ medium. In the 60:40 mixture, the peak virus yield was 1.5×10 8 TCID 50 / mL on day 3, but still within the same range as the 50:50 mixture. In the 70:30 mixture, the peak virus yield of 7.5×10 5 TCID 50 / ml on both d3 and d4 was 2 - fold lower than that obtained with the 50:50 mixture. In media containing less than 20% OptiPRO™ medium, the virus yield on day 2 post - infection was clearly much lower. The TCID 50 / cell values were 208 on day 3 with a 50:50 mixture of media, 168 on day 3 with a 60:40 mixture, 78 on days 3 and 4 with a 70:30 mixture, 75 on day 4 with an 80:20 mixture, 66 on day 4 with a 90:10 mixture, and 42 on day 4 with cSCGM + CaCl2 medium.

[0125] These viral yield results were reflected in data obtained by evaluating mRFP expression (which can be used as a marker for infected cells) induced by MVA-BN-RSV used here. Viral spread was monitored by mRFP analysis on post-infection days 2–4 ("d2"–"d4"). Flow cytometry results are shown in Figure 4B. Viral spread was most efficient in media containing 50% or 40% OptiPRO® medium, with over 80% of cells already infected by post-infection day 2 (i.e., after introduction of MVA-BN-RSV into cell cultures). Levels of mRFP expression in infected cells (Figure 4B, GMFI mRFP) were higher in cell cultures infected in the presence of higher levels of OptiPRO® medium, and this difference further increased over time. This increased mRFP expression suggests that viral protein expression may also be increased, which could explain the facilitation of viral replication and the resulting higher viral yield per cell.

[0126] In media containing lower levels of OptiPRO®, viral spread was significantly slower. The percentage of infected cells in media with lower levels of OptiPRO® increased over time, but remained lower than in media with higher levels of OptiPRO®.

[0127] Previous experiments have shown that quail cells cultured in OptiPRO® medium do not grow efficiently during the cell proliferation phase. This is likely because quail cells cultured in OptiPRO® medium tend to aggregate, which aided in viral infection and yield but negatively affected quail cell proliferation. Therefore, increased viral production can be achieved by expanding quail cells in SCGM culture medium during the cell proliferation phase and switching the medium to OptiPRO® medium or SCGM-ac + OptiPRO® medium during the viral infection phase.

[0128] The above experiment demonstrates that viral production can be increased by a process that includes, for example, seeding and growing quail cells in SCGM-ac medium, then adding approximately equal volumes of OptiPRO® medium to this cell culture, and subsequently infecting these cells with MVA or recombinant MVA. Subsequent experiments showed that FS / YIE medium can be substituted for SCGM-ac medium without any reduction in viral titer or other process parameters or results.

[0129] Example 5: Yield of MVA and recombinant MVA from quail cell cultures Quail cells were further investigated for their ability to increase the titers of MVA and recombinant MVA. Additional experiments were conducted to test various media to maximize virus production from quail suspension cells (Figure 5). Quail suspension cells (CCX.E10) were seeded in SCGM-ac medium in a stirrer-equipped flask on day 3. On day 0, to test all conditions, 20 ml of cell culture in cSCGM-ac was transferred to different S-125 shaking flasks and mixed with OptiPRO® medium, or DMEM-F12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for comparison, at a concentration of 1.1 × 10⁶. 6 OptiPRO® medium or DMEM-F12 medium at a final concentration of 50% or 30% was obtained in a 40 mL culture volume containing cells / mL. Medium additives included GlutaMAX® supplement (final concentration 2 mM) and Pluronic F68 (final volume 0.125%), to reduce shear force and foaming as needed. Cells were transferred to 30°C for 1 hour and then infected with MVA-BN-RSV (together encoding mRFP) at an MOI (Moment of Infection) of 0.1. Cells were then cultured at 30°C. Duplicate samples were collected on post-infection days 1–4, sonicated, and frozen at -20°C. Samples from day 1 / 2 (d1, d2) and day 3 / 4 (d3, d4) were subjected to standard TCID testing. 50 Titer measurements were performed in parallel using an assay (vortexing for more than 1 minute). Virus yield is shown in Figure 5, TCID.50 It is shown together with the geometric standard deviation as the geometric mean per mL. To analyze the infection and spread of the virus, mRFP expression was analyzed by flow cytometry on days 1 to 4 after infection. The numbers in Table 2 below are the peak virus yields = TCID 50 / mL in these experiments.

[0130]

Table 2

[0131] Further experiments were carried out to investigate the effect of cell density at the time of infection on virus yield. Suspended CCX.E10 quail cells (Nuvonis (Vienna, Austria)) were grown in SCGM+ac medium, harvested, and 1×10 6 , 2×10 6 , or 3×10 6 cells / mL were reseeded in OptiPRO™ medium at a cell density. After equilibrating the cells at 32°C for 1 hour, they were infected with MVA-BN encoding mRFP (monomeric red fluorescent protein; see, for example, Campbell et al. (2002) Proc. Nat’l. Acad. Sci. USA 99:7877-82) at an MOI of 0.1. Thereafter, the cells were cultured at 32°C in a flask with a stirrer. Samples of the cells were taken on days 1 to 4 after infection for analysis of the cells. Analysis of the cells includes using mRFP as a marker for virus spread (evaluated by microscopy and flow cytometry). Titration of duplicate samples (vortexed for more than 1 minute) was evaluated using a standard TCID 50 assay. From the results (see Table 3 below), it was shown that as the cell density at the time of infection increased up to 3×10 6 cells / mL, virus spread became easier and the virus yield was high early after infection. The total virus yield showed that the virus output per cell was disproportionately high compared to that expected from the multiple by which the starting cell number increased. At an infected cell density of 1×10 6 cells / mL, the virus titer was approximately 56 TCID 50 / At the level of becoming cells, it peaked on day 4, whereas 3×10 6 At an infected cell density of cells / mL, the viral titer is approximately 217 TCID. 50 At the cellular level, it reached its peak earlier, on the second day.

[0132] [Table 3]

[0133] Example 6: Viral yield of MVA-BN® virus from suspended quail cell cultures Methods for increasing poxvirus titer in suspended quail cells were further investigated for the MVA-BN® virus and compared with the virus produced in chicken embryonic fibroblast (CEF) cells. To evaluate the viral yield of MVA-BN® virus from suspended quail cells (CCX.E10 cells (Nuvonis, Vienna, Austria)), cells were seeded in SCGM-ac medium in a shaking flask on day -3. On day 0, cells were counted 1 hour before infection and mixed with an equal volume of OptiPRO® medium (resulting in a 50:50 mixture of SCGM-ac medium and OptiPRO® medium; 1:1), supplemented with Pluronic F68 and GlutaMAX®. CEF cells were seeded in VP-SFM medium in a 6-well plate on day -1. CCX.E10 and CEF cells were infected in parallel at a MOI of 0.1 and then cultured at 32°C. To analyze viral yield, triple samples of CEF and CCX.E10 cells were collected 1–4 days post-infection, sonicated, frozen at -20°C, and subjected to standard TCID testing. 50 Titer was measured in parallel by assay (vortexing for more than 1 minute). The data shown in Figure 6 represent the viral yield as measured by TCID. 50 This shows the geometric mean of / mL along with its geometric standard deviation. TCID 50 The values ​​in / mL are also shown in Table 4 below. The peak titer was reached on day 4. At this time, the cell specific yield (TCID) was 50The number of cells was approximately 200 in CEF cells and approximately 900 in CCX.E10 cells.

[0134] [Table 4]

[0135] To evaluate the stability of MVA and recombinant MVA in quail cell lines, the stability was compared after infection and culture in chicken embryo fibroblast (CEF) cells and suspended quail cell lines. Briefly, the following recombinant MVA-cell line combinations underwent 10 cell passages at an initial MOI of approximately 0.05: MVA-BN-RSV (MVA-mBN294B) in CCX.E10 cells in OptiPRO® medium; MVA-BN-RSV (MVA-mBN294B) in CCX.B11 cells in OptiPRO® medium; and MVA-BN-RSV (MVA-mBN294B) in CCX.E10 cells in SCGM medium. The stability of MVA-BN (-mRFP) in CCX.E10 cells cultured in OptiPRO® medium was also investigated. The spread of the virus in cultured cell populations was investigated using the marker protein red fluorescent protein (RFP). Stability was assessed by sequencing the MVA vector backbone, and inserts within recombinant MVA were evaluated using PCR and sequencing of the inserted gene. The results showed that the stability of the inserts in both quail cell lines was not inferior to the stability shown in CEF cells, and that passage did not introduce mutations into the MVA vector "backbone" (i.e., the non-recombinant portion of recombinant MVA).

[0136] Example 7: Purification of recombinant MVA from chicken cell cultures The MVA-BN® virus was amplified in a suspended quail cell culture, and the active pharmaceutical ingredient (BDS) was then purified as follows. This method is similar to the method described in Example 1, but includes only one tangential flow filtration (TFF) step (sometimes referred to as the “short process”).

[0137] The material (cell culture) was collected in a mixer tank and treated with TrypLE® enzyme (Gibco®, Fisher Scientific®, Waltham, MA, USA) at ambient temperature for 1 hour, followed by inactivation with a trypsin inhibitor for 15 minutes. The pH of the material was adjusted to 8.5, MgCl2 was added to 2 mM, and 20 U / mL of Denarase® endonuclease was added. NaCl was added to a final concentration of 1 M, and the material was incubated at 5-8°C for approximately 15 hours with agitation. Cells were lysed using high-pressure homogenization (HPH) at 500 bar, and cell debris and remaining cells were removed by deep filtration using a 5 μm depth filter (sometimes called "clarification"). The material was then incubated overnight at room temperature with agitation. The material was then filtered using tangential flow filtration (TFF) with PS hollow fibers with a cutoff of 0.05 μm. Diafiltration was performed using DF2 buffer (10 mM Tris, 140 mM NaCl, pH 7.7) (including flushing the system at the end and pooling it with the retained material). A 4.5-fold overall concentration was achieved. Similar results were obtained when BB2 buffer was used for diafiltration in this process.

[0138] Different tests of this process were performed on materials (cell cultures) collected from bioreactors containing approximately 3 liters and approximately 50 liters of culture, and the results were compared. In 11 tests from 3 liters of culture, the average HCD (unit ng / dose) was 1.3, while in tests from 50 liters of culture, the HCD (unit ng / dose) was 5.3. Similar HCD (ng / dose) results were obtained from the "short process" compared to the "long process." In these experiments, the "long process" further included an additional enzymatic treatment using Denarase® enzyme (100 U / mL) with 2 mM MgCl2, incubation at room temperature for approximately 15 hours, and a second tangential flow filtration (TFF). This second TFF used PESU with a 0.1 micrometer cutoff, and involved a 10-fold overall enrichment and a total of 10-fold DF in a two-step DF process, first 5-fold in DF2 buffer and then 5-fold in BB2 buffer, plus flushing to produce the final Built Drug Substance (BDS). This "long process" produced almost the same HCD (unit ng / dose) as the "short process," and the "short process" was used for further production due to reduced reagent costs and shorter time required for BDS production. Thus, the present invention provides several processes for producing MVA-BN® APIs having less than 10 ng of host cell DNA per dose.

[0139] Example 8: Purification of MVA-BN-RSV from chicken cell cultures A representative MVA-BN-RSV is MVA-mBN294, which is a recombinant MVA that encodes at least the antigenic determinants of the RSV nucleocapsid N protein, RSV M2 matrix protein, RSV G membrane glycoprotein, and RSV F membrane glycoprotein (see, for example, WO2014019718, which is incorporated herein by reference in its entirety).

[0140] MVA-mBN294 was amplified in a suspended quail cell culture and then purified as follows: The material (cell culture) was collected in a mixer tank and treated with TrypLE® enzyme (Gibco®, Fisher Scientific®, Waltham, MA, USA) at ambient temperature for 1 hour, followed by inactivation with a trypsin inhibitor for 15 minutes. The pH of the material was adjusted to 8.5, MgCl2 was added to 2 mM, and 20 U / mL of Denarase® endonuclease was added. NaCl aqueous solution was added to a final concentration of 1 M, and the material was incubated overnight at 5-8°C with stirring. Cells were lysed using high-pressure homogenization (HPH) at 500 bar, and cell debris and remaining cells were removed by deep filtration using a 5 μm depth filter ("clarification"). Next, the material was incubated overnight at room temperature with stirring and filtered using tangential flow filtration (TFF) with PS hollow fibers with a cutoff of 0.05 μm. In this TFF, ultrafiltration was performed to obtain a 5.75-fold concentration, followed by a second ultrafiltration after one diafiltration to obtain a 7.65-fold concentration, and then seven diafiltrations were performed (including flushing the system at the end and pooling it with the retained material). All of these steps were performed using DF1 buffer (10 mM Tris, 2 mM MgCl2, pH 8.2).

[0141] The second enzymatic treatment was performed using 100 U / mL Denarase® nuclease with 2 mM MgCl2, and incubated at room temperature for approximately 15 hours with stirring. Subsequently, a second TFF step was performed using PS hollow fibers with a cutoff of 0.05 μm and an inner diameter of 0.5 mm. In this filtration, diafiltration was performed five times with DF2 buffer (10 mM Tris, 140 mM NaCl, pH 7.7), then five times with BB2 buffer (10 mM Tris, 140 mM NaCl, pH 7.7, 10% w / w sucrose, 2% sorbitol, 100 mM L-arginine), followed by flushing. This was then combined with the retained material to produce the final active pharmaceutical ingredient ("BDS"), resulting in a tenfold increase in overall concentration.

[0142] This process was carried out using 3L, 50L, and 200L bioreactors. In five trials using the 3L bioreactor, the average HCD (ng / dose) was 61.6. In three trials using the 40L bioreactor, the average HCD (ng / dose) was 72.3, and in two trials using the 200L bioreactor, the average HCD (ng / dose) was 78.0. These results indicate that this process is scalable and that BDS can be produced with an HCD of less than 100 ng / dose.

[0143] Example 9: Purification of MVA-BN-WEV from chicken cell culture MVA-BN-WEV is a recombinant MVA containing nucleic acids encoding antigens for Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, and Venezuelan Equine Encephalitis Virus, and is being developed for use as a vaccine against these viruses (see, for example, MVA-mBN396B described in WO2017129765). MVA-BN-WEV was amplified in a suspension of quail cell cultures and then purified as follows: The material (cell culture) was collected in a mixer tank and treated with TrypLE® enzyme (Gibco®, Fisher Scientific®, Waltham, MA, USA) at ambient temperature for 1 hour, followed by inactivation with a trypsin inhibitor for 15 minutes. The pH of the material was adjusted to 8.5, MgCl2 was added to 2 mM, and 20 U / mL of Denarase® nuclease was added. NaCl was added to a final concentration of 1 M, and the material was incubated at 5-8°C for approximately 15 hours with stirring. Cells were lysed using high-pressure homogenization (HPH) at approximately 500 bar, and cell debris and remaining cells were removed by deep filtration using a 5 μm depth filter ("clarification"). The material was then incubated overnight at room temperature with agitation and filtered using tangential flow filtration (TFF) with a filter containing PS hollow fibers with a cutoff of 0.05 μm and an inner diameter of 0.5 mm. In this TFF, ultrafiltration was performed to increase the concentration sixfold, followed by one diafiltration and then another ultrafiltration to increase it eightfold, and finally nine diafiltrations (including flushing the system and pooling it with the retained material). All of these steps were performed using DF1 buffer (10 mM Tris, 2 mM MgCl2, pH 8.2).

[0144] The second enzymatic treatment was performed using 100 U / mL Denarase® nuclease with 2 mM MgCl2, incubated at room temperature for approximately 15 hours with stirring. Subsequently, a second diafiltration (TFF) was performed using PS hollow fibers with a cutoff of 0.05 μm and an inner diameter of 0.5 mm. All were diafiltrations performed 10 times using BB2 buffer, followed by flushing to obtain a 10.5-fold increase in overall concentration and the final active pharmaceutical ingredient (BDS).

[0145] This process was performed on materials produced in 3L bioreactors ("3L"), 50L bioreactors ("50L"), and 200L bioreactors ("200L"). In seven trials using the 3L bioreactor, the average HCD (unit ng / dose) was 16.3. In one trial using the 50L bioreactor, the HCD was 40.8 ng / dose, and in one trial using the 200L bioreactor, the HCD was 12.9 ng / dose. These data demonstrate that this process is scalable and produces BDS suitable for commercial vaccines with an HCD within the range of 10 ng / dose.

[0146] These tests also showed that, in the case of MVA-BN-WEV, a “short process” ending after a single tangential flow filtration can yield similar results to a “longer process” that further includes a second Denarase® nuclease treatment and a second tangential flow filtration. The “short process” offers further advantages for commercial production because it uses fewer reagents and requires less time.

[0147] Example 10: Comparison of quail cell process and CEF cell process Several experiments were conducted to compare the downstream purification of viruses from quail cells with the results obtained from CEF cells. MVA-BN® virus and recombinant MVA were amplified in chicken embryo fibroblast (CEF) cultures or quail cell cultures and then purified using the downstream process of the present invention. Cells were amplified, infected with the virus, and cultured in wave bag cultures (e.g., 300 liters (300 L) total volume) or bioreactors (e.g., 250 liter (250 L) bioreactors) for viral amplification. The virus was then purified essentially as follows:

[0148] For wavebag cultures, cell material was collected, then sonicated, and clarified using a Viafuge® centrifuge (CARR Biosystems®, Clearwater, FL, US). Tangential flow filtration with ultrafiltration followed by 3x diafiltration was then performed using a cartridge containing 0.1 μm PES. The material was then treated with Denarase® nuclease, followed by a second tangential flow filtration with ultrafiltration followed by 15x diafiltration.

[0149] For bioreactor cultures, CEF cells were grown in VP-SFM medium (Thermo Fisher Scientific®, Waltham, MA, US) containing Cytodex® microcarriers (Sigma Aldrich®, St. Louis, MO, USA). Following viral infection and replication, the bioreactor cultures were mixed at maximum speed (approximately 145 rpm) for about 5 hours to release the virus from the cells, and the microcarriers were removed by passing the virus-containing supernatant through a Harvestainer® system (Thermo Fisher Scientific®, Waltham, MA, US). The remaining bioreactor cell debris was mixed with culture medium to release further virus, and the collection process was repeated. The collected and pooled material was then sonicated and clarified by centrifugation using a Viafuge® centrifuge (CARR Biosystems®, Clearwater, FL, US). Finally, tangential flow filtration was performed using a cartridge containing 0.1 μm PES. The first filtration involved ultrafiltration and 3x diafiltration. The material was then treated with 100 U / mL Denarase for approximately 3 hours, agitated at ambient temperature. The second tangential flow filtration involved ultrafiltration and 15x diafiltration. The material was then batch-centrifuged at 10,800 rcf for 45 minutes at 4°C.

[0150] The results of these processes using CEF cells were compared with the results from the processes using quail cells described above (see Table 5). In these calculations, the dose was 1.58 × 10⁶ in a final (vaccine) volume of 0.5 mL. 9 It was considered to be InfU / mL.

[0151] [Table 5]

[0152] Therefore, the results of the downstream process in quail cell cultures were unexpectedly better than those in chicken embryo fibroblast (CEF) cells, with much higher dose yields per unit volume of cell culture and much lower levels of host cell DNA (HCD) per unit dose.

[0153] [Table 6]

[0154] As shown in Table 6 for MVA-BN®, the downstream process results in quail cell cultures were unexpectedly superior to those in chicken embryo fibroblast (CEF) cells, with much higher dose yields per unit volume of cell culture and much lower levels of host cell DNA (HCD) per dose.

[0155] Table 6 shows that some of the recovery rates (%) relative to the collected sample volume exceeded 100%. This likely reflects that much of the virus remains inside the cells at the time of sample collection and is subsequently released during the downstream purification process. Therefore, the viral titer after lysis may be more useful for comparison, as it has been shown to represent the peak viral titer during the process.

[0156] [Table 7]

[0157] As shown in Table 7 for MVA-BN-WEV, the downstream process results for MVA-BN® in quail cell cultures were unexpectedly better than those in chicken embryo fibroblast (CEF) cells, with much higher dose yields per unit volume of cell culture and much lower levels of host cell DNA (HCD) per dose.

[0158] It should be understood that the general and detailed descriptions above are illustrative and descriptive only and do not limit or restrict the claimed invention. The accompanying drawings incorporated herein and constituting part thereof illustrate various embodiments of the invention and, together with their description, serve to illustrate the principles of the invention.

Claims

1. A method for producing a pharmaceutical composition from a chicken cell culture infected with MVA or recombinant MVA, comprising the following steps: (a) A step of collecting a cell culture containing MVA or recombinant MVA-infected chicken cells to produce collected material; (b) The step of treating the collected material with a protease having trypsin activity; (c) A step of treating the material with a nuclease; (d) A step of adjusting the pH and / or salt content of the material; (e) A step of dissolving the cells in the material; (f) A step of filtering the material to remove cell debris and remaining cells; (g) A step of continuing incubation with a nuclease for a certain period of time; (h) a step of concentrating the product using tangential flow filtration; and (i) The step of suspending the product in a suitable buffer to obtain a pharmaceutical composition. The method, including the method described above.

2. After step (h) and before step (i), the following steps are taken: (A) The step of treating the material with a nuclease; and (B) A step of concentrating the product using tangential flow filtration. The method according to claim 1, further comprising:

3. The method according to claim 1 or 2, wherein the nuclease in step (c) and / or step (A) is Denarase® endonuclease.

4. The method according to claim 3, further comprising adding NaCl to a final concentration of at least 0.5 M or about 1 M during step (d).

5. The method according to claim 1, wherein step (i) includes using diafiltration to replace the buffer in which the product is suspended.

6. The method according to claim 1, wherein the bird cells are quail cells.

7. The method according to claim 6, wherein the quail cells are CCX.E10 cells.

8. The method according to claim 1, wherein the MVA is the MVA-BN® virus.

9. The method according to claim 1, wherein the recombinant MVA is MVA-BN-RSV.

10. The chicken cell cultures infected with MVA-BN® virus or recombinant MVA are subjected to the following steps: (i) Quail cells in a culture medium containing FS / YIE or SCGM-ac medium, with the cell density being 1 × 10 ６ cells / mL~5×10 ６ Culture in suspension until the cell count reaches 1 × 10⁶ cells / mL. ６ cells / mL~5×10 ６ A step of generating a cell culture containing cells / mL; (ii) Modify the culture medium to include FS / YIE or SCGM-ac medium and OptiPRO® medium in a ratio of 40:60 to 60:40 (v / v), and continue culturing the quail cells; (iii) Adding MVA or recombinant MVA to the cell culture to infect the cells with the MVA or recombinant MVA; (iv) A step of continuing the culture of the cells for about 1 to 4 days so that the amount of MVA or recombinant MVA increases. The method according to claim 1, which is prepared by a method comprising:

11. The method according to claim 10, wherein step (ii) comprises modifying the culture medium to contain FS / YIE or SCGM-ac medium and OptiPRO® medium in a 50:50 (v / v) ratio.

12. The method according to claim 10, wherein step (ii) is continued until approximately 50% to 70% of the cells have aggregated.

13. A pharmaceutical composition comprising MVA-BN® virus or MVA-BN-RSV prepared by the method described in any of claims 1 to 7 or 10 to 12.

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