Composition for improving the stability of poxvirus

Abstract

The present invention relates to aqueous compositions that confer improved stability to poxviruses during storage, as well as methods for the use and preparation of such compositions. The compositions comprise recombinant human serum albumin (rHSA), gelatin, or arginine, or a combination thereof.

Description

Technical Field

[0001] The present invention relates to a composition that imparts improved stability to poxvirus during storage. In particular, the present invention relates to an aqueous composition comprising recombinant human serum albumin (rHSA), gelatin or arginine, or a combination thereof. More specifically, the present invention relates to such a composition comprising poxvirus. The present invention further relates to the use of such a composition and a method for preparing the same.

Background Art

[0002] Poxviruses such as vaccinia virus, particularly modified vaccinia Ankara (MVA) virus, have been developed as vectors for vaccines against infectious diseases such as HIV, influenza, malaria and respiratory syncytial virus (RSV), and for immunotherapy and oncolytic therapy against cancer (Choi and Chang Clin Exp Vaccine Res 2013, 2:97-105, Rezaee et al. Curr Opin Virol 2017, 24:70-78, Al Yaghchi et al. Immunotherapy 2015, 7:1249-1258, Verheust et al. Vaccine 2012, 30:2623-2632, Mastrangelo et al. J Clin Invest 2000, 105:1031-1034). They are ideal candidates for vaccine development or gene delivery because of several unique features, namely, (i) large packaging capacity for recombinant DNA, (ii) accurate recombinant DNA expression regulated by strong poxvirus promoters, (iii) no persistence or genomic integration in the host because their replication is cytoplasmic, (iv) high immunogenicity as a vaccine, and (v) easy production of vectors and vaccines (Verheust et al. Vaccine 2012, 30:2623-2632).

[0003] However, for live attenuated vaccines, problems arise in formulation because of the complex macromolecular structure of the virus. This is even more difficult for large enveloped viruses such as poxviruses. For example, vaccinia virus such as MVA is a very large (about 200 - 300 nm) double-stranded DNA enveloped virus with a size of about 192 kbp, consisting of a core region composed of viral DNA covered with a lipoprotein core membrane and various enzymes. The outer layer consists of a lipid bilayer envelope (Al Yaghchi et al. Immunotherapy 2015, 7:1249 - 1258). The virion has two major infectious forms that are morphologically different, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Most of the infectious particles are IMV, which remain in the cytoplasm until cell lysis. EEV is released from the cell and has an additional lipid envelope with at least 10 related proteins that are not present in IMV. The lipid membrane is very fragile, and since the virus is inactivated when the viral envelope is lost, this is an important consideration. Stability can vary even more greatly depending on the preparations and additives used for the preparation and storage of the purified virus.

[0004] One of the difficulties is storage below the freezing point of water to avoid destabilization and / or destruction of the virus during freezing and thawing. To ensure stability, conventionally, stocks of purified infectious virus have generally been stored at less than minus 60 degrees Celsius. One of the problems with storing at such low temperatures is the possibility of thawing and refreezing during transportation or at the point of administration.

[0005] It is well known that there are limitations to the stability of live viruses in aqueous compositions, and most attenuated viruses are lyophilized products such as the vaccinia virus ACAM2000, which is fully replication-competent. ACAM2000 was approved as a lyophilized preparation containing 6 - 8 mM HEPES (pH 6.5 - 7.5), 2% human serum albumin, 0.5 - 0.7% sodium chloride, 5% mannitol, and trace amounts of neomycin and polymyxin B. Lyophilized vaccinia virus is reconstituted with water for injection containing 50% (v / v) glycerol and 0.25% (v / v) phenol (Berhanu et al. Vaccine 2010, 29:289 - 303).

[0006] Hekker et al. reported a lyophilized smallpox vaccine composition containing a combination of peptone - sorbitol with 2% hemacel or 2% polyvinylpyrrolidone (Hekker et al. Journal of Biological Standardization 1973, 1:21 - 32; a summary is in Burke et al. Crit Rev Ther Drug Carrier Syst 1999, 16:1 - 83). Further lyophilized compositions containing MVA or ALVAC are described in WO03 / 053463, WO05 / 066333, WO07 / 056847, WO2011 / 121306, WO2014 / 053571, and Zhang et al. Chemical Research in Chinese Universities 2007, 23:329 - 332. WO2010 / 135495 describes a method for stabilizing viruses in a spray - dried powder composition containing mannitol.

[0007] Just and Finke analyzed freeze-dried MVA compositions containing stabilizers such as albumin, sorbitol, dextran, cysteine or hemaccel, and reported on 5% (w / v) sorbitol and 1% (w / v) human albumin, which are superior for the freeze-dried compositions when stored at +4°C (Just and Finke Zentralbl Bakteriol Orig A 1979, 245:276-282). They also analyzed the stability of non-freeze-dried MVA suspensions containing 1% (w / v) human albumin, but a decrease in virus titer of these formulations was observed when stored at -70°C for more than 8 months.

[0008] Prabhu et al. reported three freeze-dried vaccine formulations of camelpox containing potassium phosphate buffer (pH 6.2) with 3.5% hydrolyzed gelatin and 3.5% sorbitol (Prabhu et al. Biologicals 2014, 42:169-175), which showed a decrease in virus titer even at 4°C after reconstitution.

[0009] Freeze-dried vaccines are typically more thermally stable than non-freeze-dried alternative vaccines, but freeze-drying has several drawbacks such as cost, reconstitution before use, instability after reconstitution, and stress on virus particles due to freezing and drying (Capelle et al. Eur J Pharm Biopharm 2018, 129:215-221). Furthermore, in freeze-dried vaccines, compared to liquid vaccines, administration and dosing errors are more likely to occur due to the need for reconstitution, which can lead to vaccine waste or ineffective vaccine doses (Capelle et al. Eur J Pharm Biopharm 2018, 129:215-221).

[0010] There have also been attempts to use vaccinia virus for oral vaccine application (US6,969,345). The described composition contains mannitol together with other ingredients such as hydroxyethyl starch, fish oil, glycerol, and gelatin.

[0011] Furthermore, the liquid stabilization of live attenuated viral vaccines is the most difficult because the degradation rate and dynamic processes are more favorable (Tlaxca et al. Adv Drug Deliv Rev 2015, 93:56-78).

[0012] WO2010 / 056991 describes a liquid or frozen composition containing MVA virus and mannitol, with mannitol being the sole stabilizer of the composition.

[0013] The beneficial effects on the stability of poxviruses using chelating agents and ethanol when storage is carried out, for example, at +5°C for 12 to 24 months are disclosed in WO2016 / 087457.

[0014] WO2011 / 121301 describes the use of N,N-dimethylglycine or N,N,N-trimethylglycine for the stabilization of MVA in a liquid setting at 37°C for one week.

[0015] Amino acids are used as stabilizers in a wide range of applications, including antibodies, fusion proteins, and vaccine antigens (Maity & Goldstein; Maity & Davagnino; Mistilis et.al.). In particular, histidine or arginine are frequently used as stabilizers for various virus solutions and virus vaccines (Cardoso et.al.). To stabilize virus vaccines in liquid formulations, histidine at a concentration of at least 5 mM, preferably greater than 10 mM, can be used for adenovirus-based vaccines (non-enveloped DNA viruses) (Evans et.al.; WO2014 / 029702), and live attenuated influenza vaccines (enveloped RNA viruses) can be stabilized by arginine (White et.al.), at a minimum concentration of 50 mM (US2007 / 0161085), or in a range of concentrations up to a maximum of 300 mM (WO2014 / 029702). Furthermore, WO2014 / 029702 describes amino acids such as arginine and methionine as efficient stabilizers for four canine viruses (canine parvovirus, canine adenovirus type 2, canine distemper virus, canine parainfluenza virus).

[0016] However, there is still a need for new formulations that enable the stabilization of poxvirus-based materials for large-scale industrial use, provide storage compositions that do not affect the biological activity of the product, and maintain the desired properties of the virus, more specifically, avoid or reduce virus titer reduction. In particular, there is a need for liquid pharmaceutical poxvirus preparations that provide long-term stability and do not require storage below -60°C. It is further desirable to provide high-titer and small-volume compositions suitable for storage at refrigerator temperature and / or at a temperature of about -20°C.

[0017] In particular, there is still a need for the development of a poxvirus liquid composition that is stable at about -20°C for more than about one year and then at +2 to +8°C (preferably for at least 6 or 9 months) and is suitable for subcutaneous, intramuscular and / or intranasal administration. Also, a liquid poxvirus composition that is stable at +2 to +8°C for more than about one year is desirable. Such liquid compositions provide advantages such as reduced product cost, shortened development and / or manufacturing time, and user convenience. The present invention addresses and meets these needs by providing improved poxvirus compositions, particularly MVA compositions, that exhibit enhanced storage stability.

Summary of the Invention

[0018] The present invention provides an aqueous composition with improved stability of poxviruses such as live MVA.

[0019] Accordingly, in one aspect, the present invention provides (a) arginine at a concentration of 50 mM to 150 mM, or (b) albumin at a concentration of 0.25% to 1.0% (w / v) or less than 0.25% to 1.0% (w / v), or (c) gelatin at a concentration of 1.0% to 3% (w / v) or less than 1.0% to 3% (w / v), an aqueous composition having a pH of pH 7.0 to pH 8.5.

[0020] Another aspect provides the above composition further comprising a poxvirus, preferably MVA.

[0021] Another aspect provides the above composition further comprising a poxvirus, preferably MVA, for use as a pharmaceutical or vaccine.

[0022] Another aspect provides the use of the above composition for storing a poxvirus, preferably MVA.

[0023] Another aspect provides a method of preparing a composition further comprising a poxvirus, preferably MVA, comprising the following steps: (i) providing a preparation comprising a poxvirus, preferably MVA, in a pharmaceutically acceptable buffer, and (ii) combining the poxvirus preparation of step (i) with the above composition.

[0024] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate some embodiments of the invention and, together with the description, serve to explain the principles of the invention.

Brief Description of the Drawings

[0025]

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DETAILED DESCRIPTION OF THE INVENTION

[0026] Storage stability, including the preservation of the biological function of MVA-BN-RSV, was previously found by the inventors (PCT / EP2019 / 073825) to be provided by an aqueous composition of 10 mM Tris / 140 mM NaCl containing 10% (w / v) sucrose, 2% (w / v) sorbitol, 1% (w / v) rHSA, and 3% (w / v) gelatin (referred to herein as formulation F23). Excellent stability was observed in aqueous compositions with a pH range of pH 7.0 to pH 8.5.

[0027] However, since rHSA is extremely expensive, it is not the first choice of pharmaceutical additive for bulk manufacturing on an industrial scale. On the other hand, gelatin is an animal-derived product and thus has safety risks, such as the potential for pathogen transmission. Furthermore, there is a trend to avoid animal-derived products (e.g., veganism).

[0028] Therefore, the aim of the study described herein was to investigate whether the concentrations of rHSA and / or gelatin could be reduced while maintaining their stabilizing properties for the poxvirus. Another aim of this study was to investigate whether alternative additives, such as amino acids, had the ability to compensate for the reduction in the concentrations of rHSA and / or gelatin.

[0029] In this study, various formulations containing MVA-BN-RSV were stored at +25 °C / relative humidity (RH) 60%, which simulates "real-time conditions" over a long period, i.e., "accelerated conditions". The real-time tests were started in parallel at +5 °C and -20 °C. Furthermore, freeze / thaw (F / T) experiments were conducted. The stability of MVA-BN-RSV was evaluated based on virus titer reduction and pH changes.

[0030] In particular, the following findings were obtained from the tests: rHSA enhances the stability of the poxvirus. The maximum effect is achieved with 0.5% (w / v) rHSA; there is no difference between 0.5% (w / v) rHSA and 1.0% (w / v) rHSA.

[0031] Gelatin (in the presence of rHSA) enhances the stability of the poxvirus. The maximum effect is achieved with 1.5% (w / v) gelatin; there is no difference between 1.5% (w / v) and 3% (w / v) gelatin.

[0032] Arginine (100 mM) enhances the stability of the poxvirus. Therefore, arginine has the ability to compensate for a decrease in the concentration of rHSA and / or gelatin. Furthermore, arginine can replace rHSA and gelatin.

[0033] Preferred embodiments In one embodiment, a composition containing arginine contains arginine at a concentration of 75 mM to 125 mM, preferably 80 mM to 120 mM, more preferably 90 mM to 110 mM, even more preferably 95 mM to 105 mM, and most preferably 100 mM.

[0034] In one embodiment, a composition containing albumin contains albumin at a concentration of from 0.3% to less than 1.0% (w / v), preferably from 0.4% to less than 1% (w / v), and most preferably from 0.5% to less than 1% (w / v).

[0035] In one embodiment, a composition containing albumin contains albumin at a concentration of 0.25% to 0.9% (w / v), preferably 0.3% to 0.7% (w / v), more preferably 0.4% to 0.6% (w / v), and most preferably 0.5% (w / v).

[0036] In one embodiment, a composition containing gelatin contains gelatin at a concentration of 1.0% to 2% (w / v), more preferably 1.5% to 2% (w / v), and most preferably 1.5 (w / v).

[0037] In one embodiment, a composition containing albumin further contains gelatin. In one embodiment, the composition contains 0.1% (w / v) albumin and 1.5% (w / v) gelatin, or 0.25% (w / v) albumin and 1.5% (w / v) gelatin.

[0038] In one embodiment, the composition further comprises a pharmaceutically acceptable buffer, preferably Tris buffer, more preferably 10 mM Tris buffer.

[0039] In one embodiment, the composition further comprises a pharmaceutically acceptable salt, preferably sodium chloride, most preferably 140 mM sodium chloride.

[0040] In one embodiment, the composition further comprises a disaccharide, preferably sucrose, most preferably 10% (w / v) sucrose.

[0041] In one embodiment, the composition further comprises sorbitol, most preferably 2% (w / v) sorbitol.

[0042] In one embodiment, the composition contains arginine at a concentration of 50 mM to 150 mM, and also contains Tris buffer and sodium chloride, and preferably does not contain a chelating agent and / or glutamic acid. Optionally, the composition further comprises sucrose and sorbitol.

[0043] In one embodiment, the composition is used for storing the poxvirus at -50 °C or -20 °C for at least 2 years.

[0044] In one embodiment, the composition is used for storing the poxvirus at +5 °C or +20 °C for at least 6 months.

[0045] In one embodiment of the composition, use or method, the poxvirus is an orthopoxvirus, more preferably vaccinia virus, even more preferably modified vaccinia virus Ankara (MVA), most preferably MVA-BN®.

[0046] In one embodiment of the composition, use or method, the poxvirus is a recombinant poxvirus encoding at least one respiratory syncytial virus (RSV) protein or a part thereof, most preferably recombinant MVA.

[0047] In one embodiment, the composition (with or without poxvirus) follows any of the formulations F23 and F69 - F80 as defined in Table 1.

[0048] In one embodiment, the composition (with or without poxvirus) contains rHSA and gelatin at concentrations such as any of the formulations defined in Table 1A.

[0049] In one embodiment, the composition (with or without poxvirus) contains rHSA at concentrations such as any of the formulations defined in Table 1B.

[0050] In one embodiment, the composition (with or without poxvirus) contains rHSA and albumin at concentrations of any of the formulations defined in Table 1C.

[0051] In one embodiment, the composition (with or without poxvirus) contains rHSA, gelatin, and arginine at the concentrations defined in Table 1D.

[0052] Poxvirus Poxviruses are generally enveloped viruses and large viruses with double - stranded DNA. Poxviruses belong to the Poxviridae family and include 71 viruses classified into 16 genera (Virus Taxonomy: 2017 release). Two of the best - known orthopoxviruses are the variola virus, the pathogen of smallpox, and the vaccinia virus, which has been converted into a vaccine to enable the eradication of smallpox.

[0053] Poxviruses such as vaccinia virus are known to those skilled in the art and have been used as infectious organisms and more recently to generate recombinant vaccines in the fight against cancer (Mastrangelo et al. J Clin Invest 2000, 105: 1031 - 1034).

[0054] In the context of the present disclosure, poxviruses preferably include orthopoxviruses or avipoxviruses. In a preferred embodiment of the present invention, the poxvirus is an orthopoxvirus.

[0055] Orthopoxviruses include, but are not limited to, variola virus, vaccinia virus, vaccinia virus, and monkeypox virus. Preferably, the orthopoxvirus is vaccinia virus.

[0056] The term "vaccinia virus" can refer to various strains or isolates of replicating vaccinia virus (VACV), such as, for example, Ankara, VACV Western Reserve (WR), VACV Copenhagen (VACV-COP), Temple of Heaven, Paris, Budapest, Dairen, Gam, MRIVP, Per, Tashkent, TBK, Tian Tan, Tom, Bern, Patwadangar, BIEM, B-15, EM-63, IHD-J, IHD-W, Ikeda, DryVax (also known as the VACV Wyeth strain or the New York City Board of Health [NYCBH] strain), NYVAC, ACAM1000, ACAM2000, vaccinia Lister (also known as Elstree), LC16mO or LC16m8.

[0057] In a further embodiment, the poxvirus of the present invention is MVA virus.

[0058] The MVA virus was generated by serial passage of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblasts for 516 passages (for a review, see Mayr et al. Infektion 1975, 3:6-14). As a result of these long-term passages, the genome of the resulting MVA virus has approximately 31 kilobases deleted from its genomic sequence, and thus has been described as having very restricted host cells for replication in avian cells (Meyer et al. J Gen Virol 1991, 72(Pt 5):1031-1038). The resulting MVA has been shown to be highly apathogenic in various animal models compared to its fully replication-competent starting material (Mayr and Danner Dev Biol Stand 1978, 41:225-234).

[0059] Examples of MVA viruses useful in the practice of the present invention include, but are not limited to, MVA-572 (deposited on January 27, 1994 as ECACC V94012707), MVA-575 (deposited on December 7, 2000 as ECACC V00120707), MVA-I721 (referred to in Suter et al. Vaccine 2009, 27:7442-7450), NIH clone 1 (deposited on March 27, 2003 as ATCC® PTA-5095), and MVA-BN (deposited on August 30, 2000 with the European Collection of Cell Cultures (ECACC) under the number V00083008).

[0060] More preferably, MVAs used in accordance with the present invention include MVA-BN and MVA-BN derivatives. MVA-BN is described in international PCT publication WO02 / 042480. "MVA-BN derivatives" refers to any virus that exhibits essentially the same replication characteristics as MVA-BN as described herein, but that shows differences in one or more portions of their genomes.

[0061] MVA-BN and MVA-BN derivatives are unable to replicate, and being unable to replicate means that they cannot reproduce by sexual reproduction in vivo and in vitro. More specifically, in vitro, MVA-BN or MVA-BN derivatives have the ability to reproduce sexually in chicken embryo fibroblasts (CEF), but are described as unable to reproduce sexually in the human keratinocyte cell line HaCat (Boukamp et al (1988), J. Cell Biol. 106:761-771), the human osteosarcoma cell line 143B (ECACC deposit number 91112502), the human fetal kidney cell line 293 (ECACC deposit number 85120602), and the human cervical adenocarcinoma cell line HeLa (ATCC deposit number CCL-2). Furthermore, MVA-BN or MVA-BN derivatives have a virus amplification rate that is at least one-half, more preferably one-third, of MVA-575 in Hela cells and HaCaT cell lines. Tests and assays regarding these properties of MVA-BN and MVA-BN derivatives are described in WO02 / 42480 (US Patent Application Publication No. 2003 / 0206926) and WO03 / 048184 (US Patent Application Publication No. 2006 / 0159699).

[0062] The terms "unable to reproduce sexually" or "lack the ability to reproduce sexually" in human cell lines in vitro as described in the previous paragraphs are described, for example, in WO02 / 42480, which also teaches a method for obtaining MVA with the desired properties as described above. This term applies to viruses with a virus amplification rate of less than 1 in vitro 4 days after infection using the assay described in WO02 / 42480 or US Patent No. 6,761,893.

[0063] The term "unable to reproduce by sexual reproduction" refers to a virus with a virus amplification rate of less than 1 4 days after infection in human cell lines in vitro as described in the previous paragraphs. The assay described in WO02 / 42480 or US Patent No. 6,761,893 is applicable to the determination of the virus amplification rate.

[0064] In vitro amplification or replication of a virus in a human cell line as described in the paragraphs above is typically expressed as the ratio of the virus produced from the infected cells (output) to the original amount (input) initially used to infect the cells, and is referred to as the "amplification rate". An amplification rate of "1" defines an amplification state where the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells, meaning that the infected cells are permissive to virus infection and reproduction. In contrast, an amplification rate less than 1, i.e., a decrease in output compared to the input amount, indicates a lack of productive replication and thus attenuation of the virus.

[0065] In another embodiment, the poxvirus of the present invention is an avipoxvirus such as, but not limited to, fowlpox virus.

[0066] The term "avipoxvirus" refers to any avipoxvirus such as fowlpox virus, canarypox virus, Uncopoxvirus, Mynahpoxvirus, pigeonpox virus, Psittacinepoxvirus, quailpox virus, peacockpox virus, penguinpox virus, sparrowpox virus, starlingpox virus, and budgerigar poxvirus. Preferred avipoxviruses are canarypox virus and fowlpox virus.

[0067] Avipoxvirus is a genus of the family Poxviridae, and the virus can infect and replicate in birds but not in non-avian species (Vanderplasschen and Pastoret Curr Gene Ther 2003, 3:583-595). Avipoxviruses such as fowlpox virus have been shown to be safe and effective non-replicating vectors when used in non-avian species. Ibid.

[0068] An example of canarypox virus is the Rentschler strain. A plaque-purified canarypox strain called ALVAC (U.S. Patent No. 5,766,598) was deposited with the American Type Culture Collection (ATCC) under the accession number VR-2547 based on the requirements of the Budapest Treaty. Another canarypox strain is a commercially available canarypox vaccine strain named LF2 CEP 524 24 10 75, which is available from Institute Merieux, Inc.

[0069] Examples of fowlpox virus are the FP-1 strain, FP-5 strain, TROVAC strain (U.S. Patent No. 5,766,598), POXVAC-TC strain (U.S. Patent No. 7,410,644), and TBC-FPV strain (Therion Biologics-FPV). FP-1 is the Duvette strain modified for use as a vaccine in 1-day-old chickens. This strain is a commercially available fowlpox virus vaccine strain named O DCEP 25 / CEP67 / 2309 October 1980 and is available from Institute Merieux, Inc. FP-5 is a commercially available fowlpox virus vaccine strain derived from chicken pox cups and is available from American Scientific Laboratories (Division of Schering Corp.) (Madison, Wis., United States Veterinary license number 165, serial number 30321).

[0070] In another embodiment, any poxvirus or any of the preferred poxviruses of any of the embodiments of the present invention is a live virus.

[0071] Poxviruses (especially orthopoxviruses, more specifically vaccinia virus or preferably MVA) are preferably at least 10 7 InfU / mL, preferably at least 2×10 7 InfU / mL, at least 3×10 7 InfU / mL, at least 5×107 InfU / mL, at least 6×10 7 InfU / mL, at least 7×10 7 InfU / mL, at least 8×10 7 InfU / mL, at least 9×10 7 InfU / mL, at least 1×10 8 InfU / mL, at least 2×10 8 InfU / mL, at least 3×10 8 InfU / mL, at least 4×10 8 InfU / mL, at least 5×10 8 InfU / mL, at least 6×10 8 InfU / mL, at least 7×10 8 InfU / mL, at least 8×10 8 InfU / mL, at least 9×10 8 InfU / mL, at least 1×10 9 InfU / mL, at least 2×10 9 InfU / mL, at least 3×10 9 InfU / mL, at least 4×10 9 InfU / mL, at least 5×10 9 InfU / mL, at least 6×10 9 InfU / mL, or at least 7×10 9 InfU / mL in titer. For practical reasons, poxvirus (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) is present in the aqueous composition of the present invention at a maximum of 1×10 11 InfU / mL, at a maximum of 5×10 10 InfU / mL, or preferably at a maximum of 1×10 10 InfU / mL in titer.

[0072] In particular, poxvirus (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) is present in the aqueous composition of the present invention at about 1×10 7 InfU / mL to 1×10 11 InfU / mL in titer.

[0073] In particular, poxvirus (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) is present in the aqueous composition of the present invention at a titer of about 1×10 7 InfU / mL to 5×10 10 InfU / mL.

[0074] In particular, poxvirus (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) is present in the aqueous composition of the present invention at a titer of about 1×10 7 InfU / mL to 1×10 10 InfU / mL.

[0075] In particular, poxvirus (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) is present in the aqueous composition of the present invention at a titer of about 1×10 7 InfU / mL to 6×10 9 InfU / mL.

[0076] In certain embodiments, the aqueous composition provided herein is administered to a subject in a single dose, or multiple (i.e., 2, 3, 4, etc.) doses, preferably in a volume of 0.1 - 0.5 ml. In certain embodiments, the aqueous composition provided herein is administered to a subject at a dose of 10 7 to 10 10 InfU, preferably in a volume of 0.1 - 0.5 ml. In other certain embodiments, the aqueous composition is administered in a prime-boost regimen, i.e., a primary (priming) inoculation followed by one or more booster administrations. In certain embodiments, the primary dose contains 10 7 to 10 10 InfU of poxvirus, and the second dose contains an aqueous composition of 10 7 to 10 10 InfU of virus, preferably in a volume of 0.1 - 0.5 ml.

[0077] In certain embodiments, one or more subsequent boosting administrations comprise the same recombinant poxvirus as previously administered, and the method comprises homologous prime-boost vaccination. In certain embodiments, one or more subsequent boosting administrations comprise a different recombinant poxvirus than previously administered, and the method comprises heterologous prime-boost vaccination.

[0078] In certain embodiments, one or more subsequent administrations (i.e., one or more boosting vaccinations) are administered at intervals including days, weeks or months after administration of the initial priming vaccination. In certain embodiments, one or more subsequent administrations (i.e., one or more boosting vaccinations) of the recombinant poxvirus are administered at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more after administration of the initial amount of the recombinant poxvirus (i.e., the priming vaccination). In certain embodiments, one or more subsequent administrations (i.e., one or more boosting vaccinations) of the recombinant poxvirus are administered at intervals of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more after administration of the initial priming vaccination.

[0079] The poxvirus (particularly an orthopoxvirus, more specifically vaccinia virus or preferably MVA) contained in the composition of the present invention can be a wild-type poxvirus, an attenuated poxvirus or a recombinant poxvirus.

[0080] As used herein, the term "recombinant" virus of any of the embodiments described herein refers to a virus that contains, within its genome, an exogenous nucleic acid sequence that is not naturally present in the parental virus, more specifically a poxvirus. Thus, a recombinant virus (e.g., in particular an orthopoxvirus, more specifically vaccinia virus or preferably MVA) refers to a virus produced by the artificial combination of two or more nucleic acid sequence segments of synthetic or semi-synthetic origin that are linked to another nucleic acid in an arrangement that does not occur or is not found in nature. The artificial combination is most often achieved by the artificial manipulation of isolated segments of nucleic acids using well-established genetic engineering techniques. Generally, the "recombinant" poxviruses described herein refer to poxviruses produced by standard genetic engineering methods, and thus, for example, the MVA virus of the present invention is an MVA virus that has been genetically engineered or modified. Thus, the term "recombinant MVA" includes MVA viruses (e.g., MVA-BN) in which at least one recombinant nucleic acid has been incorporated into its genome, preferably in the form of a transcription unit. The transcription unit may include a promoter, enhancer, terminator and / or silencer. The recombinant MVA viruses of the present invention may express heterologous antigenic determinants, polypeptides or proteins (antigens) upon induction of regulatory elements, such as a promoter.

[0081] Method for producing recombinant poxviruses Methods for obtaining recombinant poxviruses (e.g., VACV or MVA) or for inserting exogenous coding sequences into the genome of poxviruses (e.g., VACV or MVA) are well known to those skilled in the art. For example, methods of standard molecular biology techniques such as DNA cloning, DNA and RNA isolation, Western blot analysis, RT-PCR and PCR amplification techniques are described in Molecular Cloning, A laboratory Manual 2 ndIt is described in Ed. (J. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)), and the techniques for handling and manipulating viruses are described in Virology Methods Manual (B.W.J. Mahy et al. (eds.), Academic Press (1996)). Similarly, the techniques and know-how for handling, manipulating and genetically engineering poxviruses are described in Molecular Virology: A Practical Approach (A.J. Davison & R.M. Elliott (Eds.), The Practical Approach Series, IRL Press at Oxford University Press, Oxford, UK (1993), see for example, Chapter 9: Expression of genes by Vaccinia virus vectors), Current Protocols in Molecular Biology (John Wiley & Son, Inc. (1998), see for example, Chapter 16, Section IV: Expression of proteins in mammalian cells using vaccinia viral vector), and Genetic Engineering, Recent Developments in Applications, Apple Academic Press (2011), Dana M. Santos, see for example, Chapter 3: Recombinant-mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA)).The construction and isolation of recombinant MVA are also described in Methods and Protocols, Vaccinia Virus and Poxvirology, ISBN 978-1-58829-229-2 (Staib et al.), Humana Press (2004), see, for example, Chapter 7.

[0082] Methods for producing larger amounts of recombinant poxvirus and for purifying virus-based materials such as virus vectors used according to the invention and / or viruses are known to those skilled in the art. Available methods include the replication of the virus in CEF cells or cell lines, in particular DF-1 (US5,879,924), EBx chicken cell lines (WO2005 / 007840), EB66 duck cells (WO08 / 129058), or Cairina moschata immortalized chicken cells (WO2007 / 077256 or WO2009 / 004016). They can be cultured under conditions well known to those skilled in the art. Serum-free methods for virus culture and virus amplification are preferred. In particular, a serum-free method for virus culture and virus amplification in CEF cells is described, for example, in WO2004 / 022729. The upstream and downstream processes for virus production are well known to those skilled in the art. They can be obtained from WO2012 / 010280 or WO2016 / 087457. Methods useful for purifying the virus of the present application are disclosed in WO03 / 054175, WO07 / 147528, WO2008 / 138533, WO2009 / 100521 and WO2010 / 130753. An exemplary method for the propagation and purification of recombinant poxvirus in duck embryo-derived cells is described in Leon et al. Vaccine 2016, 34:5878-5885.

[0083] Exemplary production of recombinant MVA virus Various methods may be applicable to the production of the recombinant MVA viruses disclosed herein. The DNA sequence to be inserted into the virus may be placed in an E. coli plasmid construct in which DNA homologous to a section of poxvirus DNA has been inserted. Alternatively, the DNA sequence to be inserted can be ligated to a promoter. This promoter-gene conjugate can be placed within a plasmid construct such that the promoter-gene conjugate is flanked at both ends by DNA homologous to the DNA sequence adjacent to the poxvirus DNA region containing the non-essential locus. The resulting plasmid construct can be amplified by growth in E. coli bacteria and isolated. The isolated plasmid containing the DNA gene sequence to be inserted may be transfected, for example, into a cell culture of chicken embryo fibroblasts (CEF) at the same time as the culture is infected with the MVA virus. Recombination between the homologous MVA virus DNA and the virus genome within the plasmid can generate poxviruses modified by the presence of the foreign DNA sequence, respectively.

[0084] According to a preferred embodiment, cells suitable for cell culture, such as CEF cells, can be infected with MVA virus. Subsequently, the infected cells can be transfected with a first plasmid vector containing one or more foreign or heterologous genes(s), such as one of the nucleic acids provided in the present disclosure, preferably under the transcriptional control of a poxvirus expression control element. As described above, the plasmid vector also contains sequences capable of inducing the insertion of foreign sequences into selected MVA virus genome portions. Optionally, the plasmid vector also contains a cassette containing a marker gene and / or a selectable gene operably linked to a poxvirus promoter. The use of a selection cassette or a marker cassette simplifies the identification and isolation of the produced recombinant poxvirus. However, the recombinant poxvirus can also be identified by PCR techniques. Thereafter, additional cells can be infected with the recombinant poxvirus obtained as described above to transfect a second vector containing a second foreign or heterologous gene(s). For safety, this gene should be introduced at a different insertion site in the poxvirus genome, and also in the second vector, the homologous sequences with the poxvirus that induce the integration of the second foreign gene(s) into the poxvirus genome are different. After homologous recombination has occurred, a recombinant virus containing two or more foreign or heterologous genes can be isolated. To introduce additional foreign genes into the recombinant virus, the infection and transfection steps can be repeated by using the recombinant virus isolated in the previous infection step and by using an additional vector containing an additional foreign gene(s) for transfection.

[0085] In other embodiments, any recombinant poxvirus of the embodiments of the present invention (especially orthopoxvirus, more specifically vaccinia virus or preferably MVA) contains an antigen, preferably a nucleic acid encoding at least one antigen.

[0086] For example, suitable antigens according to the present invention may include one or more transgenes having an open reading frame encoding one or more polypeptides for which an immune response is desired in a subject when the virus is used for vaccination purposes. Examples may include, for example, one or more transgenes suitable for generating an immune response against a virus or pathogen, such as, without limitation, RSV, HIV, HPV, HBV, malaria, Ebola, MARV, FMDV, dengue, equine encephalitis virus, or any combination thereof.

[0087] In a preferred embodiment, the antigen is a viral antigen, a co-stimulatory molecule, and / or a tumor-associated antigen (TAA).

[0088] In a preferred embodiment of the present invention, the viral antigen is an immunogenic antigen selected from filoviruses, picornaviruses, papillomaviruses, hepatitis viruses, flaviviruses, retroviruses, orthomyxoviruses, equine encephalitis viruses, paramyxoviruses, and / or combinations thereof.

[0089] In a preferred embodiment of the present invention, the immunogenic antigen is a protein, preferably a full-length protein.

[0090] In a preferred embodiment of the present invention, the paramyxovirus is, for example, a respiratory syncytial virus (RSV) as described in WO2014 / 019718.

[0091] In another preferred embodiment, any recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any of the embodiments of the present invention comprises a nucleic acid encoding an RSV antigen.

[0092] In another preferred embodiment, any recombinant poxvirus (particularly orthopoxvirus, more specifically vaccinia virus or preferably MVA) of any of the embodiments comprises a nucleic acid encoding an RSV protein.

[0093] In another preferred embodiment, any one of the recombinant poxviruses (especially orthopoxviruses, more specifically vaccinia virus or preferably MVA) of the embodiments comprises a nucleic acid encoding the RSV F glycoprotein.

[0094] In another preferred embodiment, any one of the recombinant poxviruses (especially orthopoxviruses, more specifically vaccinia virus or preferably MVA) of the embodiments comprises nucleic acids encoding the RSV F glycoprotein and the RSV G glycoprotein.

[0095] In another preferred embodiment, any one of the recombinant poxviruses (especially orthopoxviruses, more specifically vaccinia virus or preferably MVA) of the embodiments comprises nucleic acids encoding the RSV F glycoprotein and two RSV G glycoproteins.

[0096] In another preferred embodiment, any one of the recombinant poxviruses (especially orthopoxviruses, more specifically vaccinia virus or preferably MVA) of the embodiments comprises nucleic acids encoding the RSV F glycoprotein, two RSV G glycoproteins, and the RSV N protein.

[0097] In another preferred embodiment, any one of the recombinant poxviruses (especially orthopoxviruses, more specifically vaccinia virus or preferably MVA) of the embodiments comprises nucleic acids encoding the RSV F glycoprotein, two RSV G glycoproteins, the RSV N protein, and the RSV matrix protein.

[0098] In another preferred embodiment, any one of the recombinant poxviruses (especially orthopoxviruses, more specifically vaccinia virus or preferably MVA) of the embodiments comprises nucleic acids encoding the RSV F glycoprotein, two RSV G glycoproteins, the RSV N protein, and the RSV M2-1 protein.

[0099] In a preferred embodiment of the present invention, the filovirus is, for example, an Ebola virus and / or a Marburg virus (MARV) as described in WO2016 / 036955, WO2016 / 036971 or WO2016 / 034678.

[0100] In a preferred embodiment of the present invention, the picornavirus is, for example, a foot-and-mouth disease virus (FMDV) as described in WO2016 / 202828.

[0101] In a preferred embodiment of the present invention, the papillomavirus is, for example, a human papillomavirus as described in WO2017 / 192418, WO90 / 10459, WO05 / 09241, WO98 / 04705, WO99 / 03885 or WO2007 / 121894.

[0102] In a preferred embodiment of the present invention, the hepatitis virus is selected from the group consisting of hepatitis A virus, hepatitis B virus, hepatitis C virus and hepatitis E virus as described in WO2004 / 111082.

[0103] In a preferred embodiment of the present invention, the flavivirus is a dengue virus (DENV).

[0104] In a preferred embodiment of the present invention, the retrovirus is HIV-1.

[0105] In a preferred embodiment of the present invention, the orthomyxovirus is an influenza virus.

[0106] In a preferred embodiment of the present invention, the equine encephalitis virus (EEV) is, for example, an eastern equine encephalitis virus (EEEV), a western equine encephalitis virus (WEEV) and / or a Venezuelan equine encephalitis virus (VEEV) as described in WO2017 / 129765.

[0107] In a preferred embodiment of the present invention, the viral antigen is an immunogenic antigen selected from the group consisting of RSV, Ebola virus, MARV, FMDV, HPV, HBV, HIV, influenza virus, DENV, RSV, EEV, and any combination thereof.

[0108] A variety of co-stimulatory molecules are known to those skilled in the art. These include, but are not limited to, ICAM-1, LFA-3, CD72, B7-1, B7-2, CD40, CD40 ligand (CD40L) or other B7-related molecules or combinations thereof, such as TRICOM.

[0109] As the "TRICOM." Triad of Costimlatory Molecules (also known as TRICOM), B7-1 (also known as B7.1 or CD80), intracellular adhesion molecule-1 (ICAM-1, also known as CD54), and lymphocyte function-associated antigen-3 (LFA-3, also known as CD58) are included. Generally, to enhance antigen-specific immune responses, they are included in recombinant viral vectors (e.g., poxvirus vectors) that express specific antigens. The individual components of TRICOM may be under the control of any of the same or different promoters, and may be provided on the same vector as the specific antigen or on separate vectors. Exemplary vectors are disclosed, for example, in Hodge et al. Cancer Res 1999, 59:5800-5807 et al., “A Triad of Costimulatory Molecules Synergize to Amplify T-Cell Activation,” Cancer Res. 59:5800-5807 (1999) and U.S. Patent No. 7,211,432 B2, all of which are incorporated herein by reference.

[0110] TAAs are well-known to those skilled in the art and refer to self-cell antigens that are detected at high frequency or high density in tumor tissues or tumor cells compared to non-tumor tissues or non-tumor cells.

[0111] In a preferred embodiment, the TAA is selected from the group consisting of CEA, MUC-1, TRP-1, NY-ESO-1, TRP-2, p53, PSA, HER-2, PAP, survivin, TYRP1, TYRP2, or Brachyury or any combination thereof.

[0112] In other embodiments, any of the recombinant poxviruses (particularly orthopoxviruses, more specifically vaccinia virus or preferably MVA) of the embodiments of the invention contain a nucleic acid encoding a combination of TAAs. Such exemplary combinations can include HER2 and Brachyury, CEA and MUC-1, or PAP and PSA.

[0113] Sucrose and sorbitol The aqueous composition according to the invention contains the disaccharide sucrose. Alternative disaccharides, such as trehalose or a combination of sucrose and trehalose, may be considered.

[0114] In certain embodiments, the aqueous composition according to the present invention contains a disaccharide (preferably sucrose) at a concentration in the range of 2% (w / v) to 12% (w / v), preferably 4% (w / v) to 12% (w / v). In particular, the aqueous composition according to the present invention contains a disaccharide (preferably sucrose) at a concentration in the range of 2% (w / v) to 11% (w / v), 2% (w / v) to 10% (w / v), 2% (w / v) to 9% (w / v), 2% (w / v) to 8% (w / v), 2% (w / v) to 7% (w / v), 2% (w / v) to 6% (w / v), 2% (w / v) to 5% (w / v), 4% (w / v) to 12% (w / v), 4% (w / v) to 11% (w / v), 4% (w / v) to 10% (w / v), 4% (w / v) to 9% (w / v), 4% (w / v) to 8% (w / v), 4% (w / v) to 7% (w / v), 5% (w / v) to 12% (w / v), 5% (w / v) to 11% (w / v), 5% (w / v) to 10% (w / v), 5% (w / v) to 9% (w / v), 5% (w / v) to 8% (w / v), 6% (w / v) to 12% (w / v), 6% (w / v) to 11% (w / v), 6% (w / v) to 10% (w / v), 6% (w / v) to 9% (w / v), 6% (w / v) to 8% (w / v), 7% (w / v) to 12% (w / v), 7% (w / v) to 11% (w / v), 7% (w / v) to 10% (w / v), 7% (w / v) to 9% (w / v), or 7% (w / v) to 8% (w / v).

[0115] In other embodiments, the aqueous composition according to the present invention contains sucrose at a concentration in the range of 4% (w / v) to 12% (w / v).

[0116] In other embodiments, the aqueous composition according to the present invention contains sucrose at a concentration of 10% (w / v).

[0117] Furthermore, the aqueous composition according to the present invention contains sorbitol.

[0118] In certain embodiments, the aqueous composition according to the present invention contains sorbitol at a concentration in the range of 0.2% (w / v) to 5% (w / v).

[0119] In other embodiments, the aqueous composition according to the present invention contains sorbitol at a concentration in the range of 0.2% (w / v) to 4% (w / v).

[0120] In other embodiments, the aqueous composition according to the present invention contains sorbitol at a concentration in the range of 0.5% (w / v) to 4% (w / v), preferably in the range of 0.5% (w / v) to 3% (w / v).

[0121] In other embodiments, the aqueous composition according to the present invention contains sorbitol at a concentration in the range of 0.2% (w / v) to 2.2% (w / v), preferably in the range of 0.5% (w / v) to 2.2% (w / v).

[0122] In other embodiments, the aqueous composition according to the present invention contains sorbitol at a concentration in the range of 1% (w / v) to 4% (w / v), preferably in the range of 1% (w / v) to 3% (w / v).

[0123] In other embodiments, the aqueous composition according to the present invention contains sorbitol at a concentration of 2% (w / v).

[0124] In other embodiments, the aqueous composition according to the present invention contains sorbitol at a concentration of 2% (w / v).

[0125] Gelatin The gelatin used in the present invention is well known to those skilled in the art. Gelatin is a natural water-soluble protein that can gel or not gel and can be obtained by partial hydrolysis of collagen produced from animal bones, hides and skins, tendons, and sinews such as pigs, cows, fish, and poultry. Type A gelatin is produced by acid treatment of collagen raw materials, while type B is produced by alkali treatment of collagen raw materials. Examples of gelatin for pharmaceutical preparations include purified gelatin described in the European Pharmacopoeia (Ph.Eur.) or the United States Pharmacopoeia (USP).

[0126] The term "gelatin hydrolyzate" according to the present invention is also referred to as hydrolyzed gelatin, hydrolyzed collagen, collagen hydrolyzate, collagen peptide, gelatin hydrolyzate and hydrolyzed gelatin. These terms can be used interchangeably.

[0127] The term "gelatin hydrolyzate" means either a hydrolyzed polypeptide obtained by subjecting gelatin to degradation by hydrolysis cleavage or a polypeptide obtained by polymerizing the above-mentioned hydrolyzed polypeptide. Gelatin hydrolyzate is water-soluble and preferably has a molecular weight of about 35,000 or less. Exemplary gelatin that can be used in the present invention includes Vaccipro® (a trade name for hydrolyzed gelatin or its chemical derivative manufactured and sold by Gelita®, AG, Germany), Gelysate® (a trade name for hydrolyzed gelatin or its chemical derivative manufactured and sold by BBL Co., Ltd., USA), and Rousselot® pharmaceutical gelatin (a trade name for hydrolyzed gelatin or its chemical derivative manufactured and sold by Rousselot B.V, NL), and other commercially available products.

[0128] In other embodiments, the gelatin of the aqueous composition of the present invention is preferably bovine or porcine gelatin, preferably a hydrolyzate of porcine or bovine gelatin. Porcine gelatin is preferably used.

[0129] In other embodiments of the present invention, the gelatin is porcine type A gelatin. In other embodiments of the present invention, the gelatin is porcine type B gelatin.

[0130] In other embodiments, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of about 0.02% (w / v) to 5% (w / v). In a further embodiment, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of about 0.1% (w / v) to 5% (w / v). In other embodiments, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of about 0.2% (w / v) to 5% (w / v). In other embodiments, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of about 0.25% (w / v) to 5% (w / v). In other embodiments, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of about 0.25% (w / v) to 4% (w / v). In other embodiments, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of about 0.2% (w / v) to 3.2% (w / v). In other embodiments, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of about 1% (w / v) to 3.2% (w / v), preferably 2.5% (w / v) to 3% (w / v). In other embodiments, the aqueous composition of the present invention comprises gelatin or hydrolyzed gelatin or any preferred gelatin at a concentration of 0.25% (w / v), 0.5% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), or 3% (w / v).

[0131] In one embodiment, the aqueous composition comprises Tris buffer, sodium chloride, sucrose, sorbitol, and arginine, but does not contain added gelatin.

[0132] Serum albumin In other embodiments, the aqueous composition according to the invention comprises albumin. Albumin is a protein naturally found in mammalian plasma and is the most abundant protein in mammalian plasma. It plays an important role in maintaining the desired osmotic pressure of the blood and in the transport of various substances in the bloodstream. The albumin used in the present invention is preferably human, porcine, murine, rat, rabbit or caprine albumin, but also variants as described in WO2011 / 051489, WO2011 / 051489, WO2010 / 092135, WO2012 / 150319, WO2014 / 072481, WO2011124718, WO2015 / 036579, WO2018 / 065491, WO2017 / 029407, WO2013 / 075066, or Otagiri and Chuang Biol Pharm Bull 2009,32:527-534. Those skilled in the art will also recognize that albumin can be modified by any means known in the art, such as recombinant DNA technology, post-translational modification, proteolytic cleavage and / or chemical means. Substitutions and modifications to albumin that provide a stabilization function essentially equivalent to that of unsubstituted and unmodified albumin are contemplated herein. Preferably, the albumin is human serum albumin. In other embodiments, the albumin is a recombinant albumin, preferably recombinant human serum albumin (rHSA) expressed and purified from Pichia pastoris, Saccharomyces cerevisiae or Oryza sativa. Preferably, the albumin used according to the present invention is Recombumin® expressed in Saccharomyces cerevisiae. However, other recombinant albumins, such as human recombinant albumin expressed in Pichia pastoris (e.g., Albagen™, rHSA, CAS number 70024-90-7, Sigma-Aldrich), etc., are suitable for the present invention.

[0133] In other embodiments, the aqueous composition according to the invention comprises albumin (preferably any of the preferred albumins mentioned herein) at a concentration in the range of 0.02% (w / v) to 3% (w / v), preferably at a concentration in the range of 0.02% (w / v) to 2% (w / v).

[0134] In other embodiments, the aqueous composition according to the invention comprises albumin (preferably any of the preferred albumins mentioned herein) at a concentration in the range of 0.2% (w / v) to 2% (w / v), preferably at a concentration in the range of 0.2% (w / v) to 1.5% (w / v).

[0135] In other embodiments, the aqueous composition according to the invention comprises albumin (preferably any of the preferred albumins mentioned herein) at a concentration in the range of 0.5% (w / v) to 2% (w / v), preferably at a concentration in the range of 0.5% (w / v) to 1.5% (w / v).

[0136] In other embodiments, the aqueous composition according to the invention comprises albumin (preferably any of the preferred albumins mentioned herein) at a concentration in the range of 0.8% (w / v) to 1.2% (w / v).

[0137] In other embodiments, the aqueous composition according to the invention comprises albumin (preferably any of the preferred albumins mentioned herein) at a concentration of 0.1% (w / v), 0.25 (w / v), 0.5% (w / v), or 1% (w / v).

[0138] In one embodiment, the aqueous composition comprises Tris buffer, sodium chloride, sucrose, sorbitol, and arginine, but does not contain added serum albumin.

[0139] Buffer and pH The aqueous composition according to any of the embodiments of the invention preferably has a pH in the range of pH 7.0 to pH 8.5.

[0140] In other embodiments of the present invention, the aqueous composition according to any of the embodiments has a pH in the range of pH 7.3 to pH 8.1.

[0141] In other embodiments of the present invention, the aqueous composition according to any of the embodiments has a pH of 7.7.

[0142] Those skilled in the art proficient in pharmaceutical development are well aware, for example, of the buffers that can be used to achieve a pH in the range of pH 7.0 to pH 8.5. Such buffers are preferably selected from the group consisting of phosphate buffer, Tris (tris(hydroxymethyl)aminomethane), Tris-HCl (tris(hydroxymethyl)aminomethane-HCl), tricine (N-[tris(hydroxymethyl)methyl]-methyl]-glycine), and HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid). The phosphate buffer preferably comprises a mixture of Na2HPO4 and KH2PO4 or a mixture of Na2HPO4 and NaH2PO4. In certain embodiments, the buffer of the aqueous composition is a Tris buffer, preferably a Tris-HCl buffer.

[0143] In certain embodiments, the aqueous composition according to any of the embodiments does not contain citrate or citrate buffer.

[0144] In certain embodiments, the buffer of the aqueous composition according to any of the embodiments is preferably present at a concentration in the range of 1 mM to 50 mM, preferably in the range of 1 mM to 25 mM.

[0145] In certain embodiments, the buffer of the aqueous composition according to any of the embodiments is present at a concentration in the range of 1 mM to 15 mM, preferably in the range of 5 mM to 11 mM, more preferably in the range of 7 mM to 10 mM.

[0146] In certain embodiments, the buffer of the aqueous composition according to any of the embodiments is present at a concentration of 10 mM.

[0147] In certain embodiments, the buffer of the aqueous composition according to any of the embodiments is present at a concentration of 7.5 mM.

[0148] In a preferred embodiment, the buffer is 10 mM Tris-HCl buffer.

[0149] Salt In other embodiments, the aqueous composition according to any of the embodiments contains a monovalent salt. The monovalent salt is preferably sodium chloride (NaCl) or potassium chloride (KCl), preferably NaCl. The NaCl is preferably present at a concentration of 40 mM to 200 mM, preferably 40 mM to 150 mM.

[0150] In another embodiment, NaCl can be present at a concentration of 40 mM to 140 mM, 40 mM to 130 mM, 40 mM to 120 mM, 40 mM to 110 mM, 40 mM to 100 mM, 40 mM to 90 mM, 40 mM to 80 mM, 50 mM to 150 mM, 50 mM to 140 mM, 50 mM to 130 mM, 50 mM to 120 mM, 50 mM to 110 mM, 50 mM to 100 mM, 50 mM to 90 mM, 50 mM to 80 mM, 60 mM to 150 mM, 60 mM to 140 mM, 60 mM to 130 mM, 60 mM to 120 mM, 60 mM to 110 mM, 60 mM to 100 mM, 60 mM to 90 mM, 60 mM to 80 mM, 70 mM to 150 mM, 70 mM to 140 mM, 70 mM to 130 mM, 70 mM to 120 mM, 70 mM to 110 mM, 70 mM to 100 mM, 70 mM to 90 mM, or 70 mM to 80 mM.

[0151] In another embodiment, NaCl can be present at a concentration of 60 mM to 80 mM.

[0152] In another embodiment, NaCl can be present at a concentration of 70 mM.

[0153] In other embodiments, the aqueous composition according to any of the embodiments contains a divalent salt. The divalent salt is preferably magnesium chloride (MgCl2). The MgCl2 is preferably present at a concentration of 1 mM to 300 mM.

[0154] In another embodiment, MgCl2 may be present at a concentration of 2 mM to 300 mM, 5 mM to 300 mM, 10 mM to 300 mM, 20 mM to 300 mM, 30 mM to 300 mM, 40 mM to 300 mM, 50 mM to 300 mM, 60 mM to 300 mM, 70 mM to 300 mM, 80 mM to 300 mM, 90 mM to 300 mM, 100 mM to 300 mM, 150 mM to 300 mM, 180 mM to 300 mM, 200 mM to 300 mM, 2 mM to 260 mM, 5 mM to 260 mM, 10 mM to 260 mM, 20 mM to 260 mM, 30 mM to 260 mM, 40 mM to 260 mM, 50 mM to 260 mM, 60 mM to 260 mM, 70 mM to 260 mM, 80 mM to 260 mM, 90 mM to 260 mM, 100 mM to 260 mM, 150 mM to 260 mM, 180 mM to 260 mM, 200 mM to 260 mM, 75 mM to 300 mM, 75 mM to 260 mM, 100 mM to 260 mM, 120 mM to 260 mM, 150 mM to 260 mM, 200 mM to 260 mM, 2 mM to 200 mM, 5 mM to 200 mM, 10 mM to 200 mM, 20 mM to 200 mM, 30 mM to 200 mM, 40 mM to 200 mM, 50 mM to 200 mM, 60 mM to 200 mM, 70 mM to 200 mM, 80 mM to 200 mM, 90 mM to 200 mM, 2 mM to 150 mM, 5 mM to 150 mM, 10 mM to 150 mM, 20 mM to 150 mM, 30 mM to 150 mM, 40 mM to 150 mM, 50 mM to 150 mM, 60 mM to 150 mM, 70 mM to 150 mM, 80 mM to 150 mM, 90 mM to 150 mM, 2 mM to 100 mM, 5 mM to 100 mM, 10 mM to 100 mM, 20 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 50 mM to 100 mM, 60 mM to 100 mM, 70 mM to 100 mM, 80 mM to 100 mM, 90 mM to 100 mM, 2 mM to 75 mM, 5 mM to 75 mM, 10 mM to 75 mM, 20 mM to 75 mM, 30 mM to 75 mM, 40 mM to 75 mM, 50 mM to 75 mM, 60 mM to 75 mM, 70 mM to 75 mM, 2 mM to 50 mM, 5 mM to 50 mM, 10 mM to 50 mM, 20 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 2 mM to 40 mM, 5 mM to 40 mM, 10 mM to 40 mM, 20 mM to 40 mM, or 30 mM to 40 mM.

[0155] In another embodiment, MgCl2 may be present at a concentration of 75 mM to 300 mM.

[0156] In another embodiment, MgCl2 may be present at a concentration of 100 mM to 300 mM.

[0157] In another embodiment, MgCl2 may be present at a concentration of 200 mM to 300 mM.

[0158] In another embodiment, MgCl2 may be present at a concentration of 220 mM to 260 mM.

[0159] In another embodiment, MgCl2 may be present at a concentration of 240 mM to 260 mM.

[0160] In other embodiments, an aqueous composition according to any of the embodiments contains a monovalent salt and a divalent salt, preferably at any concentration shown above.

[0161] In another embodiment, an aqueous composition according to any of the embodiments contains a monovalent salt at a concentration of 60 mM to 80 mM and a divalent salt at a concentration of 220 to 260 mM.

[0162] In another embodiment, an aqueous composition according to any of the embodiments contains NaCl at a concentration of 40 mM to 150 mM and MgCl2 at a concentration of 220 mM to 260 mM.

[0163] In another embodiment, an aqueous composition according to any of the embodiments contains NaCl at a concentration of 70 mM to 150 mM and MgCl2 at a concentration of 220 mM to 260 mM.

[0164] In another embodiment, an aqueous composition according to any of the embodiments contains NaCl at a concentration of 60 mM to 80 mM and MgCl2 at a concentration of 220 mM to 260 mM.

[0165] In another embodiment, the aqueous composition does not contain MgCl2 and / or CaCl2.

[0166] In a preferred embodiment, the aqueous composition contains 140 mM of NaCl.

[0167] Amino acid The aqueous composition according to any of the embodiments described herein may contain one or more amino acids. Preferred amino acids are histidine, arginine, lysine, glycine and / or glutamic acid or salts thereof, in particular the L-isomers L-histidine, L-arginine, L-lysine, L-glycine and / or L-glutamic acid or salts thereof. The above amino acid(s) is / are not the amino acids encoded by the recombinant or non-recombinant virus of the present invention. Thus, the amino acid is not contained in the composition through the process of purifying the virus (e.g., MVA), but is added during the purification of the composition for manufacturing the vaccine. Mainly preferred is L-arginine, for example, L-arginine HCl.

[0168] The amino acid is preferably present at a concentration of less than 150 mM, less than 130 mM, less than 120 mM, preferably less than 110 mM.

[0169] In a further embodiment, the amino acid is present at a concentration in the range of 10 mM to 110 mM, 20 mM to 110 mM, 30 mM to 110 mM, 40 mM to 110 mM, 50 mM to 110 mM, 60 mM to 110 mM, 70 mM to 110 mM, or 80 mM to 110 mM.

[0170] In a further embodiment, the amino acid is present at a concentration in the range of 40 mM to 110 mM.

[0171] In a further embodiment, the amino acid is present at a concentration in the range of 90 mM to 110 mM.

[0172] In a further embodiment, the amino acid is present at a concentration of 100 mM.

[0173] In a further embodiment, the amino acid is present at a concentration of 50 mM.

[0174] In a further embodiment, the aqueous composition of the present invention may contain histidine (preferably L-histidine) at a concentration of 40 mM to 60 mM.

[0175] In a further embodiment, the aqueous composition of the present invention may contain arginine (preferably L-arginine) at a concentration of 40 mM to 60 mM.

[0176] In a further embodiment, the aqueous composition of the present invention may contain lysine (preferably L-lysine) at a concentration of 40 mM to 60 mM.

[0177] In a further embodiment, the aqueous composition of the present invention may contain glycine (preferably L-glycine) at a concentration of 40 mM to 60 mM.

[0178] In a further embodiment, the aqueous composition of the present invention may contain histidine, arginine, lysine and glycine, preferably at concentrations of 40 mM to 60 mM each.

[0179] In a further embodiment, the aqueous composition of the present invention may contain histidine, arginine, lysine and glycine, preferably at concentrations of 40 mM to 60 mM each.

[0180] The above amino acid concentrations may be used for any of the specific amino acids mentioned in the above section (e.g., histidine, arginine, lysine, and / or glycine).

[0181] Glutamic acid or its salt (e.g., sodium glutamate or sodium glutamate monohydrate) is preferably present at a concentration of 2.5 mM to 7.5 mM.

[0182] In a further embodiment, glutamic acid or a salt thereof (e.g., monosodium glutamate monohydrate or monosodium glutamate monohydrate) is present at a concentration in the range of 3 mM to 6 mM. Preferably, glutamic acid or a salt thereof (e.g., monosodium glutamate monohydrate or monosodium glutamate monohydrate)) is present at a concentration of 5 mM.

[0183] In a preferred embodiment, the aqueous composition of the present invention substantially does not contain or does not contain glutamic acid. Preferably, the aqueous composition substantially does not contain or does not contain added amino acids other than arginine.

[0184] In a preferred embodiment, the aqueous composition of the present invention contains Tris buffer, sodium chloride, sucrose, sorbitol, and arginine, but does not contain other added amino acids.

[0185] Additional additives The aqueous composition according to any of the embodiments described herein may further contain octanoate. Preferably, the aqueous composition according to any of the embodiments contains octanoate ions at a concentration of 5 mM or less, preferably 1 mM or less, more preferably 0.001 to 1 mM.

[0186] The aqueous composition according to any of the embodiments described herein may further contain one or more additional carriers, additives, antibiotics, preservatives, adjuvants, and / or diluents, and preferably, any of the additional carriers, additives, antibiotics, preservatives, adjuvants, and / or diluents is pharmaceutically acceptable.

[0187] In a further embodiment, the aqueous composition of the present invention does not contain mannitol.

[0188] In a further embodiment, the aqueous composition of the present invention substantially does not contain citrate.

[0189] In a further embodiment, the aqueous composition of the present invention does not contain citrate.

[0190] In further embodiments, the aqueous composition of the present invention is substantially pharmaceutically acceptable or pharmaceutically acceptable.

[0191] In other embodiments, the aqueous composition of the present invention is a vaccine or a pharmaceutical composition.

[0192] In further embodiments, the aqueous composition of the present invention is substantially free of mannitol.

[0193] In further embodiments, the aqueous composition is substantially free of chelating agents. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA), ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic acid (DMSA), diethylenetriaminepentaacetic acid (DTPA), and 2,3-dimercapto-1-propanesulfonic acid (DMPS). By "substantially free of chelating agents" according to the present invention is meant less than 50 μM of chelating agent.

[0194] In further embodiments, the aqueous composition of the present invention does not contain a chelating agent.

[0195] In further embodiments, the aqueous composition of the present invention is substantially free of polysorbate. Examples of polysorbate include polysorbate 80.

[0196] In further embodiments, the aqueous composition of the present invention does not contain polysorbate.

[0197] In further embodiments, the aqueous composition of the present invention is substantially free of C2-C3 alcohols, where the C2-C3 alcohols are ethanol and / or isopropanol. By "substantially free of C2-C3 alcohols" according to the present invention is meant less than 0.05 (v / v) of ethanol or isopropanol.

[0198] In a further embodiment, the aqueous composition of the present invention does not contain C2-C3 alcohol, where the C2-C3 alcohol is ethanol and / or isopropanol.

[0199] In a further embodiment, the aqueous composition of the present invention substantially does not contain mannitol, citrate, chelating agent, C2-C3 alcohol (i.e., ethanol and / or isopropanol) and / or polysorbate.

[0200] In a further embodiment, the aqueous composition of the present invention does not contain mannitol, citrate, chelating agent, C2-C3 alcohol (i.e., ethanol and / or isopropanol) and / or polysorbate.

[0201] In a further embodiment, the aqueous composition of the present invention substantially does not contain HPBCD and mannitol.

[0202] In a further embodiment, the aqueous composition of the present invention does not contain HPBCD and mannitol.

[0203] In a further embodiment, the aqueous composition of the present invention substantially does not contain HPBCD, mannitol and citrate.

[0204] In a further embodiment, the aqueous composition of the present invention does not contain HPBCD, mannitol and citrate.

[0205] In a further embodiment, the aqueous composition of the present invention substantially does not contain HPBCD, mannitol, citrate, chelating agent, and C2-C3 alcohol (i.e., ethanol and / or isopropanol).

[0206] In a further embodiment, the aqueous composition of the present invention does not contain HPBCD, mannitol, citrate, chelating agent, and C2-C3 alcohol (i.e., ethanol and / or isopropanol).

[0207] In a further embodiment, the aqueous composition of the present invention is substantially free of or does not contain HPBCD, mannitol, citrate, chelating agent, C2-C3 alcohol (i.e., ethanol and / or isopropanol) and polysorbate.

[0208] In a further embodiment, the aqueous composition of the present invention does not contain HPBCD, mannitol, citrate, chelating agent, C2-C3 alcohol (i.e., ethanol and / or isopropanol) and polysorbate.

[0209] In a further embodiment, the aqueous composition of the present invention is substantially free of or does not contain serum albumin. In particular, an aqueous composition containing arginine may not contain serum albumin.

[0210] In a further embodiment, the aqueous composition of the present invention is substantially free of or does not contain gelatin. In particular, an aqueous composition containing arginine may not contain gelatin.

[0211] In a further embodiment, the aqueous composition of the present invention is substantially free of or does not contain arginine. In particular, an aqueous composition containing serum albumin and / or gelatin may not contain arginine.

[0212] Others In other embodiments, the aqueous composition according to the invention is contained in a vial. The term "vial" refers to any container, vessel, cartridge, device, glass ampoule, or syringe capable of storing active pharmaceutical ingredients such as viruses disclosed herein. Thus, the terms vial, container, vessel, cartridge, device, glass ampoule, or syringe can be used interchangeably. Vials are typically made of inert materials, particularly glass (such as DIN 2R type I borosilicate glass vials) or polymeric materials. In a preferred embodiment, the composition is contained in a DIN 2R type I borosilicate glass vial. In a preferred embodiment, the composition is contained in a syringe.

[0213] The composition of the present invention can be administered to a subject, preferably a human, by any means known in the art. Routes of administration include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous application, intranasal administration, transdermal administration, transcutaneous administration, or percutaneous administration. The method of administration, dosage, and frequency of administration can be optimized by methods known to those skilled in the art. In a preferred embodiment, the aqueous composition of the present invention is suitable for parenteral administration or application. In other preferred embodiments, the aqueous composition of the present invention is suitable for intranasal administration or application. In other preferred embodiments, the aqueous composition of the present invention is suitable for intramuscular or subcutaneous administration or application.

[0214] In certain embodiments, any aqueous composition of an embodiment of the present invention is further defined as having an infectivity of at least 50%, 60%, 79%, 80%, or 90% of the starting infectivity (day 0) when stored at +5°C for 3 months.

[0215] In certain embodiments, any aqueous composition of an embodiment of the present invention is further defined as having an infectivity of at least 70%, 80%, or 90% of the starting infectivity (day 0) when stored at -20°C for 3 months.

[0216] In certain embodiments, any aqueous composition of the embodiments of the present invention is further defined to have an infectivity of at least 70%, 80% or 90% of the starting infectivity (day 0) when stored at +5 °C for 6 months.

[0217] In certain embodiments, any aqueous composition of the embodiments of the present invention is further defined to have an infectivity of at least 70%, 80% or 90% of the starting infectivity (day 0) when stored at -20 °C for 6 months.

[0218] In certain embodiments, any aqueous composition of the embodiments of the present invention is further defined to have an infectivity of at least 70%, 80% or 90% of the starting infectivity (day 0) when stored at +25 °C / 60% relative humidity for 5 months.

[0219] According to certain embodiments, the aqueous composition of the present invention is stable.

[0220] According to certain embodiments, the aqueous composition of the present invention preferably has an overall decrease in virus titer of less than 0.5 log 10 InfU / mL, preferably less than 0.4 log 10 InfU / mL, more preferably less than 0.3 log 10 InfU / mL, most preferably less than 0.2 log 10 InfU / mL when determined by fluorescence-activated cell sorter (FACS) assay and is stable when stored at +5 °C for at least 3 months.

[0221] According to certain embodiments, the aqueous composition of the present invention preferably has an overall decrease in virus titer of less than 0.5 log 10 InfU / mL, preferably less than 0.4 log 10 InfU / mL, more preferably less than 0.3 log 10 InfU / mL, most preferably less than 0.2 log 10If it is less than InfU / mL, it is stable.

[0222] According to a particular embodiment, the aqueous composition of the invention preferably has an overall decrease in virus titer of 0.5 log over at least 9 months at +5°C, as determined by a fluorescence-activated cell sorter (FACS) assay 10 less than InfU / mL, preferably 0.4 log 10 less than InfU / mL, more preferably 0.3 log 10 less than InfU / mL, most preferably 0.2 log 10 If it is less than InfU / mL, it is stable.

[0223] According to a particular embodiment, the aqueous composition of the invention preferably has an overall decrease in virus titer of 0.5 log over at least 12 months at +5°C, as determined by a fluorescence-activated cell sorter (FACS) assay 10 less than InfU / mL, preferably 0.4 log 10 less than InfU / mL, more preferably 0.3 log 10 less than InfU / mL, most preferably 0.2 log 10 If it is less than InfU / mL, it is stable.

[0224] According to a particular embodiment, the aqueous composition of the invention preferably has an overall decrease in virus titer of 0.5 log over at least 3 months at -20°C, as determined by a fluorescence-activated cell sorter (FACS) assay 10 less than InfU / mL, preferably 0.4 log 10 less than InfU / mL, more preferably 0.3 log 10 less than InfU / mL, most preferably 0.2 log 10 If it is less than InfU / mL, it is stable.

[0225] According to a particular embodiment, the aqueous composition of the invention preferably has an overall decrease in virus titer of 0.5 log over at least 6 months at -20°C, as determined by a fluorescence-activated cell sorter (FACS) assay 10Less than InfU / mL, preferably 0.4 log 10 Less than InfU / mL, more preferably 0.3 log 10 Less than InfU / mL, most preferably 0.2 log 10 When it is less than InfU / mL, it is stable.

[0226] According to a particular embodiment, the aqueous composition of the invention preferably has an overall decrease in virus titer of 0.5 log over at least 9 months at -20 °C, as determined by a fluorescence-activated cell sorter (FACS) assay 10 Less than InfU / mL, preferably 0.4 log 10 Less than InfU / mL, more preferably 0.3 log 10 Less than InfU / mL, most preferably 0.2 log 10 When it is less than InfU / mL, it is stable.

[0227] According to a particular embodiment, the aqueous composition of the invention preferably has an overall decrease in virus titer of 0.5 log over at least 12 months at -20 °C, as determined by a fluorescence-activated cell sorter (FACS) assay 10 Less than InfU / mL, preferably 0.4 log 10 Less than InfU / mL, more preferably 0.3 log 10 Less than InfU / mL, most preferably 0.2 log 10 When it is less than InfU / mL, it is stable.

[0228] According to a particular embodiment, the aqueous composition of the invention preferably has an overall decrease in virus titer of 0.5 log over at least 5 months at +25 °C / relative humidity 60%, as determined by a fluorescence-activated cell sorter (FACS) assay 10 Less than InfU / mL, preferably 0.4 log 10 Less than InfU / mL, more preferably 0.3 log 10 Less than InfU / mL, most preferably 0.2 log 10 When it is less than InfU / mL, it is stable.

[0229] According to certain embodiments, the aqueous composition of the present invention is stable upon storage at about -20°C for at least 12 months followed by storage at +2 to +8°C for 3 months, where the overall decrease in virus titer upon storage at -20°C for a specified period of at least 12 months followed by storage at +2 to +8°C for at least 3 months is less than 0.5 log 10 InfU / mL. Preferably, the overall decrease in virus titer determined by fluorescence-activated cell sorter (FACS) assay is preferably less than 0.4 log 10 InfU / mL, more preferably less than 0.3 log 10 InfU / mL, most preferably less than 0.2 log 10 InfU / mL.

[0230] According to certain embodiments, the aqueous composition of the present invention is stable upon storage at about -20°C for at least 12 months followed by storage at +2 to +8°C for 9 months, where the overall decrease in virus titer upon storage at -20°C for a specified period of at least 12 months followed by storage at +2 to +8°C for at least 9 months is less than 0.5 log 10 InfU / mL. Preferably, the overall decrease in virus titer determined by fluorescence-activated cell sorter (FACS) assay is preferably less than 0.4 log 10 InfU / mL, more preferably less than 0.3 log 10 InfU / mL, most preferably less than 0.2 log 10 InfU / mL.

[0231] According to certain embodiments, the aqueous composition of the present invention is stable upon storage at about -20°C for at least 24 months followed by storage at +2 to +8°C for 9 months, where the overall decrease in virus titer upon storage at -20°C for a specified period of at least 24 months followed by storage at +2 to +8°C for at least 9 months is less than 0.5 log 10 InfU / mL. Preferably, the overall decrease in virus titer determined by fluorescence-activated cell sorter (FACS) assay is preferably less than 0.4 log 10 InfU / mL, more preferably less than 0.3 log10 Less than InfU / mL, most preferably 0.2 log 10 Less than InfU / mL.

[0232] The "overall decrease in virus titer" according to the present invention is defined as the cumulative decrease in virus titer expressed in log InfU / mL, measured during storage of the composition at the indicated temperature n (e.g., +5°C) and time t (e.g., 6 months). The overall decrease in virus titer is expressed as x log 10 InfU / mL. For example, 0.5 log as measured at +5°C for 6 months is expressed as 10 such (e.g., 0.5 log as measured at +5°C for 6 months) 10 and is represented as.

[0233] According to certain embodiments, the aqueous composition of the present invention is stable, and the composition shows a decrease in potency of less than 0.3 log InfU / mL when stored at -20°C for a period of 12 months. 10

[0234] According to certain embodiments, the aqueous composition of the present invention is stable, and the composition shows a decrease in potency of less than 0.3 log InfU / mL when stored at +4°C to +8°C for a period of 12 months. 10

[0235] According to certain embodiments, the aqueous composition of the present invention is stable, and the composition shows a decrease in potency of less than 0.2 log InfU / mL when stored at -20°C for a period of 12 months. 10

[0236] According to certain embodiments, the aqueous composition of the present invention is stable, and the composition shows a decrease in potency of less than 0.2 log InfU / mL when stored at +4°C to +8°C for a period of 12 months. 10

[0237] According to certain embodiments, the aqueous composition of the present invention is stable, and the composition shows a decrease in potency of less than 0.1 log InfU / mL when stored at -20°C for a period of 12 months. 10

[0238] According to certain embodiments, the aqueous composition of the present invention is stable, and the composition exhibits a potency decrease of less than 0.1 log 10 InfU / mL when stored at +4°C to +8°C for a period of 12 months.

[0239] According to certain other embodiments, the aqueous composition of the present invention is a liquid composition or a liquid frozen composition.

[0240] According to certain other embodiments, the aqueous composition of the present invention is an aqueous frozen composition.

[0241] According to certain other embodiments, the aqueous composition of the present invention is an aqueous liquid composition.

[0242] According to certain other embodiments, the aqueous composition of the present invention is not a dry composition, and preferably is neither a freeze-dried composition nor a lyophilized composition.

[0243] Another aspect provides the aqueous composition of the present invention for treating or preventing a disease, preferably an infectious disease or cancer.

[0244] Another aspect provides the use of the aqueous composition of the present invention for manufacturing a pharmaceutical or a vaccine for treating or preventing an infectious disease or cancer.

[0245] Another aspect provides a method for treating or preventing an infectious disease by administering a composition of any of the embodiments of the present invention to a subject.

[0246] Definitions and Terms It should be understood that both the foregoing summary and detailed description are for purposes of illustration and description only, and are not intended to limit the claimed invention. Since the specific methods, protocols and reagents described herein are subject to change, the invention is not limited thereto. It should also be understood that the terms used herein are for the purpose of describing only individual embodiments and are not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0247] The terms are defined and explained so that the invention can be more readily understood. Further definitions are set forth throughout the detailed description.

[0248] As used herein, it should be noted that the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes one or more nucleic acid sequences, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that can be modified or substituted for the methods described herein.

[0249] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood as referring to each individual element in that series. One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0250] As used herein, the connective "and / or" between a plurality of recited elements is understood to encompass both individual choices and combined choices. For example, when two elements are connected by "and / or", in the first option, it refers to the first element being applicable without using the second element. The second option refers to the second element being applicable without including the first element. The third option refers to the first element and the second element being applicable together. Any one of these options falls within the scope of its meaning, and thus it is understood to meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options also falls within the scope of its meaning, and thus is understood to meet the requirements of the term "and / or".

[0251] Throughout this specification and the following claims, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to imply the inclusion of the stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps, unless the context requires otherwise. When used in connection with an aspect or embodiment of the present invention, the term "comprising" is amendable and thus can also be replaced with the term "containing" or "including", or when used herein, can also be replaced with "having". Similarly, any of the foregoing terms (comprising, containing, including, having), when used in any case in connection with an aspect or embodiment of the present invention, includes the terms "consisting of" or "consisting essentially of" which represent different specific legal meanings by law.

[0252] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claims. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims.

[0253] As used herein, the term "substantially free of" a component, unless otherwise stated herein, excludes trace amounts of that component that do not substantially affect the stability of the compositions of the invention. For example, the term "free of" mannitol means that the aqueous compositions of the invention do not contain mannitol.

[0254] As used in this application, "about" means ±10% unless otherwise indicated. It should also be noted that, unless otherwise specified, any numerical values such as concentrations or concentration ranges described herein are to be understood as being modified in each case by the term "about". Throughout this specification, the term "about" with respect to any quantity or concentration is intended to include that quantity. For example, "about 5 mM" is intended herein to include 5 mM and values that are understood to be about 5 mM with respect to the entity described. As used herein, the use of a numerical range explicitly includes any possible sub-ranges within that range, any individual numerical values, and integers and fractions of those values within such range, unless the context clearly indicates otherwise. Similarly, the term "about" prefaced to any numerical value or range used herein in the context of the present invention may, although less preferably, be deleted and replaced by that numerical value or range without the term "about".

[0255] The terms "nucleic acid", "nucleotide sequence", "nucleic acid sequence" and "polynucleotide" can be used interchangeably and refer to RNA or DNA, which can be linear or branched, single-stranded or double-stranded, or hybrids thereof. The polynucleotide may be obtained by chemical synthesis or may be derived from a microorganism. The term "foreign" nucleic acid sequence when used in the context of recombinant viruses means a foreign nucleic acid sequence, i.e., a nucleic acid sequence that is not contained in the non-recombinant virus used to produce the recombinant virus, or a nucleic acid sequence that is inserted into the viral genome during the production of the recombinant virus.

[0256] "Pharmaceutically acceptable" means that a carrier, additive, antibiotic, preservative, adjuvant, diluent, stabilizer or additive at the dosage and concentration used does not substantially cause any undesirable or harmful effects in the subject(s) to which they are administered. A "pharmaceutically acceptable" additive is any inert substance that is combined with an active molecule such as a virus to prepare a preferred or convenient dosage form. A "pharmaceutically acceptable" additive is non-toxic to the recipient at the dosage and concentration used and is compatible with the other components of the formulation containing the virus preparation. Examples of additives are cryoprotectants, non-ionic detergents, buffers, salts and inhibitors of free radical oxidation. A "pharmaceutically acceptable carrier" is described, for example, in Remington’s Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Ed., Mack Publishing Company (1990), Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, eds., Taylor & Francis

[2000] , and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000).

[0257] The terms "stable", "stabilized", "stability" or "stabilization", all of which can be used interchangeably, mean that the poxvirus contained in the composition of the present invention essentially retains its physical stability, identity, integrity, and / or chemical stability, identity, integrity, particle morphology and / or biological activity or efficacy during storage, as required for the shelf life of the pharmaceutical composition. As used herein, the term "shelf life" means the time during which a product retains its activity and / or stability in accordance with its product characteristics under specific storage conditions (e.g., storage at +2°C to +8°C) for use as a human drug. The shelf life corresponds to the point at which the lower or upper limit of a given specification is exceeded.

[0258] Stability can be evaluated by determining various characteristics, such as the amount, potency, and / or other quality aspects of the poxvirus (e.g., MVA) in the formulation, over a certain period of time under specific storage conditions. These characteristics of a poxvirus (e.g., MVA) formulation can be measured under high temperature (predicting the actual storage temperature) or other stress conditions. For example, to examine the effects of various formulations to maximize the shelf life, the formulation can be subjected to incubation at +20°C, +25°C, -20°C or +5°C, and also to freeze / thaw cycles and agitation. Methods for determining the stability of a poxvirus (e.g., MVA) are well known to those skilled in the art and can be determined by at least one method selected from the group of visual inspection, pH measurement, turbidity assay, particle morphology and potency (infectivity) assay.

[0259] Turbidity measurement measures the decrease in the intensity of transmitted light (apparent absorbance) due to scattering of particles in a sample, detected at a wavelength at which the molecules in the sample do not absorb light (e.g., 350 nm for a sample where protein is the main chromophore). When molecules aggregate or form supramolecular complexes, the light scattering, which was random when coming from separate particles, becomes coherent here, and thus the measured intensity increases. For this reason, light scattering and turbidity measurement are useful techniques for detecting aggregation and complex formation or dissociation.

[0260] In the turbidity assay, the sample is transferred in triplicate to a UV-transparent flat-bottom microplate. The absorbance spectrum is recorded by a microplate reader from 230 to 500 nm, the absorbance at 975 nm is measured, and the difference in optical path length is determined and, if necessary, corrected. A control sample consisting of a formulation without MVA was included in the assay to correct for scattering or absorption of matrix components, if necessary. The apparent absorbance at 350 nm was used as a quantitative measure of turbidity.

[0261] The turbidity assay is an assessment of the stability of MVA samples. Aggregation of MVA results in an increase in turbidity, while dissociation of the capsid results in a decrease. The assay precision is less than 5% (CV%) for turbidity values above 1 NTU.

[0262] Methods for determining particle morphology are well known to those skilled in the art. For example, particle morphology can be determined using transmission electron microscopy and immune electron microscopy (immune-EM) as described, for example, in Schweneker et al. J Virol 2017, 91: e00343-00317. An alternative method for determining particle morphology is nanoparticle tracking analysis (NTA) as described, for example, in Filipe et al. Pharm Res 2010, 27: 796-810. Nanoparticle tracking analysis (NTA) is a method for directly and real-time visualizing and analyzing the particle size distribution and aggregation in a liquid. Based on laser irradiation microscopy technology, the Brownian motion of nanoparticles is analyzed in real-time by a charge-coupled device (CCD) camera, and each particle is visualized and tracked simultaneously but separately by a dedicated particle tracking image analysis program. The ability of NTA to measure particle size and particle scattering intensity simultaneously enables the separation of heterogeneous particle mixtures and the direct estimation of particle concentration.

[0263] As used herein in connection with a virus, the term "potency" or "infectivity" refers to the ability of the virus to infect cells, and refers to the entry and proliferation of the virus in cells or organisms. Thus, infectivity refers to the activity of a poxvirus (e.g., vaccinia virus or MVA) expressed as infectious units (InfU), usually expressed as InfU / mL. The terms "potency" and "infectivity" can both be used interchangeably in the present invention. The potency of a poxvirus such as MVA can be determined using various methods known to those skilled in the art, for example, after virus infection, the percentage of virus-positive cells such as baby hamster kidney cells 21 (BHK-21) can be determined. A preferred assay is, for example, a fluorescence-activated cell sorter (FACS) assay as described in the examples.

[0264] The terms "subject" and "patient" are used interchangeably. As used herein, a subject is typically a mammal such as a non-primate (e.g., female cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human), and in some preferred embodiments is a human.

[0265] According to the present invention, the term "virus" means virus, virus particle and virus vector. All of these terms can be used interchangeably. This term includes wild-type virus, recombinant and non-recombinant viruses, live virus, as well as attenuated live virus.

[0266] According to the present invention, a concentration expressed as %(w / v) means weight in grams (g) per volume in units of 100 mL, for example, 20% (w / v) means 20 g / 100 mL. A concentration expressed as %(v / v) means volume in mL per volume in units of 100 mL, for example, 20% (v / v) means 20 mL / 100 mL.

[0267] Throughout the specification, unless otherwise stated, values of physical parameters such as pH are measured at +25°C or around +25°C. In particular, pH measurements may be carried out at ambient temperature.

[0268] Several documents are cited throughout the body of this specification. Each of the documents cited in this specification (all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety, whether supra or infra. To the extent that the material incorporated by reference conflicts with or is inconsistent with this specification, this specification shall prevail over any such material. No recitation herein shall be construed as an admission that the present invention has no right to antedate such disclosure on the basis of prior invention.

[0269] In the practice of the present invention, unless otherwise specified, the prior art of immunology, molecular biology, microbiology, cell biology, and recombinant techniques is used, all of which are within the skill of those in the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et al., eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988. Note that although this application relates to the invention described in the claims, the following may also be included as other aspects. 1. (a) Arginine at a concentration of 50 mM to 150 mM, or (b) Albumin at a concentration of 0.25% to 1.0% (w / v) or less than 0.25% to 1.0% (w / v), or (c) Gelatin at a concentration of 1.0% to 3% (w / v) or less than 1.0% to 3% (w / v), an aqueous composition having a pH of pH 7.0 to pH 8.5. 2. The composition according to 1(a) above, containing arginine at a concentration of 75 mM to 125 mM, preferably 80 mM to 120 mM, more preferably 90 mM to 110 mM, even more preferably 95 mM to 105 mM, and most preferably 100 mM. 3. The composition according to 1(b) above, containing albumin at a concentration of less than 0.3% to 1.0% (w / v), preferably less than 0.4% to 1% (w / v), and most preferably less than 0.5% to 1% (w / v). 4. The composition according to 1(b) above, containing albumin at a concentration of 0.25% to 0.9% (w / v), preferably 0.3% to 0.7% (w / v), more preferably 0.4% to 0.6% (w / v), and most preferably 0.5% (w / v). 5. The composition according to 1(c) above, containing gelatin at a concentration of 1.0% to 2% (w / v), more preferably 1.5% to 2% (w / v), and most preferably 1.5 (w / v). 6. The composition according to 1(b) above, further containing gelatin. 7. The composition according to 6 above, containing 0.1% (w / v) albumin and 1.5% (w / v) gelatin, or 0.25% (w / v) albumin and 1.5% (w / v) gelatin. 8. The composition according to any one of 1 to 7 above, further containing a pharmaceutically acceptable buffer, preferably Tris buffer, more preferably 10 mM Tris buffer. 9. The composition according to any one of 1 to 8 above, further containing a pharmaceutically acceptable salt, preferably sodium chloride, and most preferably 140 mM sodium chloride. 10. The composition according to any one of 1 to 9 above, further containing a disaccharide, preferably sucrose, and most preferably 10% (w / v) sucrose. 11. The composition according to any one of items 1 to 10 above, further comprising sorbitol, most preferably 2% (w / v) sorbitol. 12. The composition according to any one of items 1 to 11 above, further comprising a poxvirus, preferably an orthopoxvirus, more preferably vaccinia virus, even more preferably modified vaccinia virus Ankara (MVA), and most preferably MVA-BN®. 13. The composition according to item 12 above, wherein the poxvirus is a recombinant poxvirus encoding at least one respiratory syncytial virus (RSV) protein or a part thereof, and most preferably recombinant MVA. 14. The composition according to any one of items 12 or 13 above, for use as a medicament or a vaccine. 15. Use of the composition according to any one of items 1 to 12 above for storing a poxvirus, preferably an orthopoxvirus, more preferably vaccinia virus, even more preferably modified vaccinia virus Ankara (MVA), and most preferably MVA-BN®. 16. The use according to item 15 above, wherein the poxvirus is a recombinant poxvirus encoding at least one respiratory syncytial virus (RSV) protein or a part thereof, and most preferably recombinant MVA. 17. The use according to item 15 or 16 above for storing the poxvirus at -50°C or -20°C for at least two years. 18. The use according to item 15 or 16 above for storing the poxvirus at +5°C, +20°C or +25°C for at least six months. 19. A method for preparing the composition according to item 12 or 13 above, comprising: (i) providing a preparation containing a poxvirus, preferably an orthopoxvirus, more preferably vaccinia virus, even more preferably modified vaccinia virus Ankara (MVA), and most preferably MVA-BN® in a pharmaceutically acceptable buffer; and (ii) combining the poxvirus preparation of step (i) with the composition according to any one of items 1 to 11 above. 20. The method according to 19 above, wherein the poxvirus is a recombinant poxvirus encoding at least one respiratory syncytial virus (RSV) protein or a part thereof, most preferably recombinant MVA. 21. An aqueous composition having a pH of pH 7.0 to pH 8.5, containing arginine, Tris buffer, sodium chloride, sucrose, and sorbitol at a concentration of 50 mM to 150 mM, preferably not containing a chelating agent and / or glutamic acid.

Example

[0270] The following examples illustrate the present invention, but should not be construed as limiting the scope of the claims in any way. They merely serve to clarify the present invention.

[0271] Example 1: Preparation of Recombinant MVA The recombinant MVA used in the stability study was MVA-BN-RSV (MVA-mBN294B) as described in WO2015 / 136056, which is incorporated herein by reference. MVA-mBN294B encodes the RSV-F (Along) protein, G(A) and G(B) proteins, and N and M2-1 proteins, where the N and M2-1 sequences are encoded by a single open reading frame separated by the 2A self-cleaving protease domain of the FMDV virus. IGR 64 / 65 and IGR 148 / 149 were used as antigen integration sites. As described in WO2015 / 136056, genes were expressed under the poxvirus promoters Pr7.5e / l (Cochran et al. J Virol 1985, 54:30-37), PrS (Chakrabarti et al. Biotechniques 1997, 23:1094-1097), PrLE1 (Baur et al. J Virol 2010, 84:8743-8752) and PrH5m (Wennier et al. PLoS One 2013, 8:e73511).

[0272] MVA-BN-RSV (MVA-mBN294B) was produced in chicken embryo fibroblast (CEF) cells. CEF cells were generated using standard methods well known to those skilled in the art as described in WO2012 / 010280. Several methods are available for amplifying the recombinant MVA virus and purifying the bulk drug substance (BDS), which are also well known to those skilled in the art (e.g., as described in WO2006 / 052826).

[0273] MVA-BN-RSV (MVA-mBN294B) was produced in CEF cells using a bioreactor process. Cells were seeded into a bioreactor containing additives (L-glutamine and poloxamer) and VP-SFM medium. The cells were cultured on microcarriers for 6-7 days until the desired cell density for infection was obtained.

[0274] After the infection and production of MVA-BN-RSV (MVA-mBN294B), the cells were harvested by agitation, and the microcarriers were removed by sieving. The cells were harvested, homogenized by sonication to lyse the cells, and the produced virus particles were released. The purified virus was concentrated by centrifugation to finally obtain the purified bulk drug substance. The BDS was stored at -80°C ± 10°C.

[0275] Example 2: Formulation of Recombinant MVA The individual compositions of Formulations F23 and F69 - F88 regarding recombinant human serum albumin (rHSA), gelatin, and arginine are shown in Table 1 below. The formulation numbers, and thus the order in which they were prepared, were randomized using a random sequence generator. Each formulation contained MVA-BN-RSV (always from the same BDS batch) in an aqueous composition (pH 7.6 - 7.9) of 10 mM Tris buffer, 140 mM NaCl, 10% (w / v) sucrose, and 2% (w / v) sorbitol. The concentrations of rHSA, gelatin, and arginine are shown in Table 1.

[0276] [Table 1]

[0277] [Table 2]

[0278] [Table 3]

[0279] [Table 4]

[0280] Preparation of the formulation: The concentrated BDS of MVA-BN-RSV generated in Example 1 above was mixed with a formulation buffer (10 mM Tris, 140 mM NaCl, 10% (w / v) sucrose, 2% (w / v) sorbitol) containing rHSA, gelatin, arginine, and each of the combinations at 3-fold concentrations (as shown in Table 1 at the final 1-fold concentration). The formulation buffer was filtered using a 0.2 μm syringe before use. The formulated MVA-BN-RSV was filled into DIN 2-R glass vials (0.5 mL for FACS samples and 0.8 mL for pH samples).

[0281] The following raw materials were used for the preparation of the formulation buffer: Sorbitol EMPROVE® exp Ph Eur, BP, NF, JP Parteck® SI 400 LEX, Merck; sucrose, EMPROVE® exp Ph Eur, BP, JP, NF, low endotoxin Merck; recombinant human albumin, Recombumin®, Albumedix; hydrolyzed gelatin, Vaccipro®, Gelita; L-arginine, PharmaGrade, USP, Sigma Aldrich; sodium chloride BioXtra Sigma; Tris hydrochloride, VWR. MVA-mBN294B in 10 mM Tris and 140 mM sodium chloride (pH 7.7 ± 0.4) was used as a control.

[0282] For each formulation, the samples were equally divided using triplicates for storage and analysis at various time points (+25 °C / relative humidity 60%) (0, 3 weeks / 19 days, and 5 weeks / 33 days). For each time point, the vials were filled with 0.5 mL of the corresponding MVA-BN-RSV formulation and then stored at each respective temperature for subsequent stability analysis. The vials used for pH measurement were filled with 0.8 mL.

[0283] At the start of the test, the vials containing the various formulations corresponding to time zero (t = 0) were transferred to -80 °C, and the remaining vials were transferred to +25 °C / relative humidity 60% (accelerated storage conditions), +5 °C (real time), and -20 °C (real time), respectively.

[0284] Example 3: Determination of Virus Titer As a stability parameter, the virus titer of MVA-BN-RSV was determined. A decrease in virus titer indicates instability, which is generally expected during storage.

[0285] The titer (InfU / mL) of MVA-BN-RSV was determined by a potency (infectivity) assay using fluorescence-activated cell sorting (FACS). Baby hamster kidney 21 (BHK-21) cells infected with MVA-BN-RSV were immunostained with a fluorescent dye-conjugated antibody specific for vaccinia virus (VACV), and then quantified using a FACSVerse™ (BD Bioscience) instrument equipped with a BD Flow Sensor for quantitative cell counting.

[0286] More specifically, in a 12-well plate, 2.5x10 5BHK21 cells (source: ATCC) were seeded in GMEM / 9% FBS / 1.8% Ala-Gln. The next day, the cells were infected with a serial dilution of the MVA virus stock of interest. After incubation at 37 °C for 1 hour, rifampicin (100 μg / mL in GMEM / 9% FBS / 1.8% Ala-Gln) was added. Cells were harvested 19 ± 2 hours post-infection and fixed and permeabilized with the BD Perm / Wash™ kit prior to antibody staining. The fixed cells were incubated with anti-vaccinia FITC (Fitzgerald Industries International, catalog number 60-v68) for 60 - 90 minutes. The percentage of virus-positive cells was then determined by flow cytometry using a BD FACSVerse™ cytometer. In parallel, the total cell number was determined using a BD FACSVerse™ flow sensor on unstained fixed cells. The calculation of virus titer (InfU / mL) was based on the percentage of virus-positive cells, the virus dilution used at the time of infection, the infection volume, and the average number of cells per well. To limit the effect of variation in cell number between wells, the cell number was established by averaging the cell numbers from multiple wells. Only dilutions containing 2 - 35% VACV-positive cells were included in the calculation of InfU / mL for the virus samples. The calculation of InfU / mL for each sample dilution was performed according to the following formula:

Number

[0287] Example 4: Effects of rHSA, gelatin, and arginine on virus titer at +25 °C Based on the results obtained from the FACS assay (see Example 4 above), the virus titer gradient was calculated by linear regression analysis for each formulation (see Table 2 below). Generally, the higher the gradient value, the greater the increase in the stability of MVA-BN-RSV in a given formulation.

[0288]

Table 5

[0289] The data of the virus titer gradient shown in Table 2 and their respective standard errors were processed to obtain Figures 2 to 5. The data used in Figure 1 were obtained from separate experiments.

[0290] Figure 1 shows that formulation F23 containing 1% rHSA and 3% gelatin produced a much flatter gradient than formulation F1 containing 10 mM Tris / 140 mM NaCl.

[0291] In Figure 2, F23 represents the reference. As shown, several formulations showed virus titer gradients with values similar to those of F23. Significantly, there was no difference at least between formulations F23 and F72, and the differences between F23 and F74 - F81 were very small. In contrast, the formulations without rHSA (F86) or containing rHSA at concentrations of 0.1% (F71) and 0.25% (F84) respectively did not have the ability to sufficiently maintain the stability of MVA - BN - RSV.

[0292] Figure 3 shows data obtained from formulations containing only rHSA in the basic composition (10 mM Tris / 140 mM NaCl containing 10% sucrose and 2% sorbitol). As shown, the stability of MVA - BN - RSV increased from 0.1% to 0.5% rHSA. No significant difference in the virus titer gradient was observed between 0.5% and 1.0% rHSA. In particular, no further increase in stability occurred between 0.5% and 1% rHSA. Therefore, the concentration of 0.5% rHSA is sufficient to produce acceptable virus stability.

[0293] Figure 4 shows that when gelatin was present at concentrations of 1.5% - 3%, virus titer gradients with similar values were generated at different concentrations of rHSA. Therefore, 1.5% - 3% gelatin has the ability to compensate for a decrease in rHSA concentration. Acceptable stability was already obtained using the formulation containing 1.5% gelatin and 0.1% rHSA, which was comparable to formulation F23 containing 3% gelatin and 1% rHSA.

[0294] As considered in relation to FIG. 3 (see above), the stabilizing ability of rHSA varied depending on its concentration. FIG. 5 shows that by additionally presenting 100 mM of arginine, a similar viral titer gradient was obtained at all rHSA concentrations tested. Notably, in formulations containing 0.5% or 1% rHSA, no significant effect of arginine was observed. Overall, the results shown in FIG. 5 indicate that 100 mM of arginine alone can produce a viral titer gradient similar to that seen in the case of high concentrations of rHSA.

[0295] In summary, rHSA, gelatin, and arginine may have the potential to improve the stability of MVA - BN - RSV.

[0296] In particular, it is known that formulation F23 containing 1% rHSA and 3% gelatin stabilizes MVA - BN - RSV very well (FIGS. 1 and 2). Formulations containing 0.5% rHSA (F73) and 0.1% rHSA and 1.5% gelatin (F87) each show a slightly lower stabilizing ability, but are still similar to the values seen in F23 (FIG. 2). Advantageously, F73 and F87 contain lower concentrations of rHSA and gelatin compared to F23.

[0297] Similarly, a formulation containing 100 mM of arginine instead of rHSA and gelatin (F72) appears to stabilize MVA - BN - RSV slightly less strongly than F23 (FIG. 2). However, advantageously, F72 does not contain the expensive rHSA and gelatin.

[0298] Example 5: Effects of HSA, Gelatin, and Arginine on pH As an indirect parameter of stability, the pH of the formulations was determined. An increase in pH can indicate instability.

[0299] Based on these results, linear regression analysis was used to calculate the pH gradient of each formulation (see Table 3 below).

[0300]

Table 6

[0301] The pH gradient data and standard errors shown in Table 3 were processed to obtain Figures 7 to 10. The data used in Figure 6 were obtained from separate experiments.

[0302] Figure 6 shows the difference in pH stability between formulation F23 containing 1% rHSA and 3% gelatin, and formulation F1 containing 10 mM Tris / 140 mM NaCl.

[0303] As shown in Figures 7 and 8, rHSA had no stabilizing effect on the pH of the formulation. In contrast, gelatin at concentrations of 1.5% and 3% increased the pH stability regardless of the presence or concentration of rHSA (Figure 9). Similarly, 100 mM arginine increased the pH stability of the formulation regardless of rHSA (Figure 10).

[0304] Example 6: Effects of rHSA, gelatin, and arginine on virus titer during freeze-thaw cycles In another study, the formulation was exposed to freeze-thaw (F / T) cycles (-50 °C / ambient temperature).

[0305] Linear regression analysis was performed on the virus titers obtained at the start of the test (time zero, t = 0), after 5 cycles of F / T, and after 10 cycles of F / T (see Table 4 below).

[0306]

Table 7

[0307] The data on the virus titer gradient shown in Table 4 and their respective standard errors were processed to obtain Figure 11.

[0308] Figure 11 shows that formulations F75 (0.25% rHSA, 1.5% gelatin) and F72 (100 mM arginine) have good virus stability in the F / T cycle. Furthermore, the pH is not affected by the F / T cycle (data not shown).

[0309] References Al Yaghchi,C.,Zhang,Z.,Alusi,G.,Lemoine,N.R.,and Wang,Y.(2015).Vaccinia virus,a promising new therapeutic agent for pancreatic cancer.Immunotherapy 7:1249-1258. Baur,K.,Brinkmann,K.,Schweneker,M.,Patzold,J.,Meisinger-Henschel,C.,Hermann,J.,Steigerwald,R.,Chaplin,P.,Suter,M.,and Hausmann,J.(2010).Immediate-early expression of a recombinant antigen by modified vaccinia virus ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses.J Virol 84:8743-8752. Berhanu,A.,King,D.S.,Mosier,S.,Jordan,R.,Jones,K.F.,Hruby,D.E.,and Grosenbach,D.W.(2010).Impact of ST-246(R)on ACAM2000 smallpox vaccine reactogenicity,immunogenicity,and protective efficacy in immunodeficient mice.Vaccine 29:289-303. Burke, C.J., Hsu, T.A., and Volkin, D.B. (1999). Formulation, stability, and delivery of live attenuated vaccines for human use. Crit Rev Ther Drug Carrier Syst 16:1-83. 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(1973). A stable freeze-dried smallpox vaccine made in monolayer cultures of primary rabbit kidney cells. Journal of Biological Standardization 1:21-32. Hodge, J.W., Sabzevari, H., Yafal, A.G., Gritz, L., Lorenz, M.G., and Schlom, J. (1999). A triad of costimulatory molecules synergize to amplify T-cell activation. Cancer Res 59:5800-5807. Just, I., and Finke, H. (1979). [A contribution to the stabilisation of the MVA-vaccine (author’s transl)]. Zentralbl Bakteriol Orig A 245:276-282. Leon, A., David, A.L., Madeline, B., Guianvarc’h, L., Dureau, E., Champion-Arnaud, P., Hebben, M., Huss, T., Chatrenet, B., and Schwamborn, K. (2016). The EB66(R) cell line as a valuable cell substrate for MVA-based vaccines production. Vaccine 34:5878-5885. Mastrangelo, M.J., Eisenlohr, L.C., Gomella, L., and Lattime, E.C. (2000). Poxvirus vectors: orphaned and underappreciated. J Clin Invest 105: 1031 - 1034. Mayr, A., and Danner, K. (1978). 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Claims

1. An aqueous composition for storing modified vaccinia virus Ankara (MVA), comprising: - Arginine at a concentration of 50 mM to 150 mM, - Tris buffer at a concentration of 1 mM to 25 mM, - Sodium chloride at a concentration of 40 mM to 200 mM, - Sucrose at a concentration of 2% (w / v) to 12% (w / v), and - Sorbitol at a concentration of 0.2% (w / v) to 5% (w / v), wherein the composition has a pH of pH 7.0 to pH 8.

5.

2. The composition according to claim 1, comprising arginine at a concentration of 75 mM to 125 mM.

3. The composition according to claim 1 or 2, wherein the Tris buffer is present at a concentration of 1 mM to 15 mM.

4. The composition according to any one of claims 1 to 3, wherein the sodium chloride is present at a concentration of 40 mM to 140 mM.

5. The composition according to any one of claims 1 to 4, comprising sucrose at a concentration of 4% (w / v) to 12% (w / v).

6. The composition according to any one of claims 1 to 5, comprising sorbitol at a concentration of 0.2% (w / v) to 4% (w / v).

7. The composition according to any one of claims 1 to 6, wherein the modified vaccinia virus Ankara (MVA) is MVA-BN®.

8. The composition according to any one of claims 1 to 7, wherein the modified vaccinia virus Ankara (MVA) is a recombinant MVA encoding at least one respiratory syncytial virus (RSV) protein or a portion thereof.

9. The composition according to any one of claims 1 to 8, for storing the modified vaccinia virus Ankara (MVA) at -50 °C or -20 °C for at least 2 years.

10. The composition according to any one of claims 1 to 8, for storing the modified vaccinia virus Ankara (MVA) at +5 °C, +20 °C or +25 °C for at least 6 months.

11. - Arginine at a concentration of 50 mM to 150 mM, - Tris buffer at a concentration of 1 mM to 25 mM, - Sodium chloride at a concentration of 40 mM to 200 mM, - Sucrose at a concentration of 2% (w / v) to 12% (w / v), and - Sorbitol at a concentration of 0.2% (w / v) to 5% (w / v), comprising further modified vaccinia virus Ankara (MVA). An aqueous composition having a pH of pH 7.0 to pH 8.

5.

12. The composition according to claim 11, wherein the modified vaccinia virus Ankara (MVA) is a recombinant MVA encoding at least one respiratory syncytial virus (RSV) protein or a part thereof.

13. The composition according to claim 11 or 12, wherein the modified vaccinia virus Ankara (MVA) is present at a titer of 1×10^7 InfU / mL to 1×10^11 InfU / mL.

14. The composition according to any one of claims 11 to 13 for use as a medicament or a vaccine.

15. - Arginine at a concentration of -50 mM to 150 mM, - Tris buffer at a concentration of 1 mM to 25 mM, - Sodium chloride at a concentration of 40 mM to 200 mM, - Sucrose at a concentration of 2% (w / v) to 12% (w / v), and - Sorbitol at a concentration of 0.2% (w / v) to 5% (w / v), Use for storing modified vaccinia virus Ankara (MVA) of an aqueous composition containing the same and having a pH of pH 7.0 to pH 8.

5.

16. The use according to claim 15, wherein the modified vaccinia virus Ankara (MVA) is a recombinant MVA encoding at least one respiratory syncytial virus (RSV) protein or a part thereof.

17. The use according to claim 15 or 16 for storing the modified vaccinia virus Ankara (MVA) at -50 °C or -20 °C for at least 2 years.

18. The use according to claim 15 or 16 for storing the modified vaccinia virus Ankara (MVA) at +5 °C, +20 °C or +25 °C for at least 6 months.

19. A method for preparing the composition according to any one of claims 11 to 14, comprising: (i) providing a preparation containing modified vaccinia virus Ankara (MVA) in a pharmaceutically acceptable buffer, and (ii)combining the modified vaccinia virus Ankara (MVA) preparation of step (i) with an aqueous composition having a pH of pH 7.0 to pH 8.5 and containing arginine at a concentration of 50 mM to 150 mM, Tris buffer at a concentration of 1 mM to 25 mM, sodium chloride at a concentration of 40 mM to 200 mM, sucrose at a concentration of 2% (w / v) to 12% (w / v), and sorbitol at a concentration of 0.2% (w / v) to 5% (w / v), the method comprising:

20. The method according to claim 19, wherein the modified vaccinia virus Ankara (MVA) is a recombinant MVA encoding at least one respiratory syncytial virus (RSV) protein or a portion thereof.

21. An aqueous composition according to any one of claims 1 to 14, which does not contain a chelating agent and / or glutamic acid.

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