Dengue vaccine formulation

Abstract

The present invention relates to a dengue vaccine formulation comprising a tetravalent dengue virus composition comprising live attenuated dengue virus serotype 1, live attenuated dengue virus serotype 2, live attenuated dengue virus serotype 3, and live attenuated dengue virus serotype 4, at least one non-reducing disaccharide, at least one poloxamer, urea, at least one amino acid having a positively charged side chain at neutral pH, tromethamine, and human serum albumin.

Description

[Technical Field]

[0001] The present invention relates to dengue vaccine formulations, unit doses, vials, kits of dengue vaccine formulations, and the use of dengue vaccine formulations in methods for preventing dengue disease, methods of preparing dengue vaccine formulations, and methods of administration. [Background technology]

[0002] Dengue fever, caused by infection with the dengue virus, is the fastest-spreading mosquito-borne viral disease, with an estimated 390 million cases and 500,000 hospitalizations worldwide annually. Because preventive options are limited, dengue virus is estimated to cause approximately 20,000 deaths worldwide annually. Dengue virus infection can lead to debilitating and painful symptoms, including sudden high fever, headache, joint and muscle pain, nausea, vomiting, and skin rash. To date, four serotypes of dengue virus have been identified: dengue 1 (DENV-1), dengue 2 (DENV-2), dengue 3 (DENV-3), and dengue 4 (DENV-4). Dengue virus serotypes 1 to 4 can also cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the most severe cases, DHF and DSS can be life-threatening. All four serotypes of dengue virus are endemic throughout tropical regions of the world and represent the most significant mosquito-borne viral threat to humans in these regions. Dengue virus is transmitted to humans primarily by the Aedes aegypti mosquito, but also by the Aedes albopictus mosquito. Infection with one dengue virus serotype confers lifelong protection against reinfection with that serotype, but does not prevent secondary infection with one of the other three dengue virus serotypes. In fact, previous infection with one dengue virus serotype can increase the risk of severe disease (DHF / DSS) during secondary infection with a different serotype.

[0003] Vaccines for protection against viral infections have been used effectively to reduce the incidence of human disease. One of the most successful technologies for viral vaccines involves immunizing animals or humans with weakened or attenuated virus strains ("live attenuated viruses"). Due to limited replication after immunization, attenuated virus strains do not cause disease. However, limited viral replication is sufficient to express the full repertoire of viral antigens and generate a strong, long-lasting immune response against the virus. Thus, upon subsequent exposure to a pathogenic virus strain, immunized individuals are protected from disease. These live attenuated virus vaccines are among the most successful vaccines in public health.

[0004] For live attenuated vaccines to be effective, they must be replicable after immunization, and the virus itself must be protected from degradation during preparation and transportation prior to administration to a subject, for example, to ensure that the correct dosage concentration is delivered to the subject. Some vaccines are sensitive to extreme temperatures. Either excessive heat or accidental freezing can inactivate the vaccine. Maintaining this "cold chain" throughout distribution is particularly difficult in developing countries. Therefore, there remains a need to improve the stability of live attenuated viruses in vaccine formulations, such as dengue vaccine formulations, to improve vaccine manufacturing, as well as transportation and delivery of the formulations.

[0005] A stable dengue vaccine formulation is described in WO2020 / 051334A1. A major driver for the development of new excipient compositions is to further improve virus stability, which is also expected to reduce the amount of virus required to ensure the required end-of-shelf-life (ESL) potency of the vaccine formulation. Summary of the Invention

[0006] An object of the present invention is to provide an improved dengue vaccine formulation for reducing degradation of live attenuated dengue viruses (e.g., serotypes 1 to 4) during manufacture, storage, and transportation of the dengue vaccine formulation.

[0007] An object of the present invention is to provide an improved dengue vaccine formulation for reducing the consumption of live attenuated dengue virus (eg, serotypes 1 to 4) during the production of the dengue vaccine formulation.

[0008] It is an object of the present invention to provide an improved dengue vaccine formulation to reduce degradation of live attenuated dengue virus (eg, serotypes 1-4) during freeze-drying.

[0009] It is an object of the present invention to provide an improved dengue vaccine formulation to reduce drying time.

[0010] An object of the present invention is to provide an improved dengue vaccine formulation for reducing degradation of live attenuated dengue virus (eg, serotypes 1 to 4) after lyophilization.

[0011] It is an object of the present invention to provide an improved dengue vaccine formulation to improve the local physiological compatibility of the dengue vaccine formulation after reconstitution.

[0012] It is an object of the present invention to provide an improved dengue vaccine formulation that maintains low foaming properties during reconstitution of the formulation.

[0013] It is an object of the present invention to provide a freeze-dried dengue vaccine formulation that retains its immunogenic potential during preparation and for the required pharmaceutically acceptable period and that can be dissolved in a diluent with minimal foaming.

[0014] It is a further object of the present invention to provide improved methods for preparing dengue vaccine formulations to reduce the consumption of live attenuated dengue virus.

[0015] Another object of the present invention is to provide an improved method for preparing a dengue vaccine formulation to reduce the length of the freeze-drying cycle.

[0016] Another object of the present invention is to provide an improved method for preparing a dengue vaccine formulation to improve production capacity.

[0017] A further object of the present invention is to provide a safe and effective vaccine against all serotypes of dengue virus for dengue endemic and non-dengue endemic populations and for a wide age range, independent of previous exposure to dengue virus and the corresponding seropositive or seronegative status before vaccination.

[0018] Accordingly, the present invention provides, in part, a dengue vaccine formulation comprising: a tetravalent dengue virus composition (e.g., "TAK-003") comprising live attenuated dengue virus serotype 1, live attenuated dengue virus serotype 2, live attenuated dengue virus serotype 3, and live attenuated dengue virus serotype 4; at least one non-reducing disaccharide (e.g., trehalose and / or sucrose); at least one poloxamer (e.g., poloxamer 407); Urea and at least one amino acid having a positively charged side chain at neutral pH (e.g., arginine, lysine, and / or histidine); Tromethamine and Human serum albumin, The present invention relates to a dengue vaccine formulation, including

[0019] The present invention relates, inter alia, in part, to a dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain); (ii) a live attenuated dengue virus serotype 2 (e.g., a dengue serotype 2 strain); (iii) a live attenuated dengue virus serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain); and (iv) a live attenuated dengue virus serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain); a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; an excipient composition comprising: The present invention relates to a dengue vaccine formulation, including

[0020] In particular, a combination of trehalose, poloxamer, urea, arginine, tromethamine, and human serum albumin has been found to provide superior stability of tetravalent dengue virus compositions (e.g., "TAK-003"). In particular, the excipient compositions disclosed herein are useful for improving the stability of dengue vaccine formulations after drying, e.g., during storage and transportation of the lyophilized dengue vaccine formulations described herein. The excipient compositions disclosed herein are also useful for improving the stability of dengue vaccine formulations during manufacturing, e.g., during freezing when the dengue vaccine formulations described herein are subjected to a lyophilization process. In certain such embodiments, the excipient compositions are useful for avoiding pH changes in the dengue vaccine formulations during freezing, thereby reducing degradation of the live-attenuated dengue virus. In certain embodiments, the excipient compositions are useful for reducing the time to lyophilize the dengue vaccine formulations described herein. In certain embodiments, the excipient compositions disclosed herein are useful for maintaining the low-foaming properties of the dengue vaccine formulations described herein during reconstitution and withdrawal.

[0021] The present invention relates in particular to such a dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain); (ii) a live attenuated dengue virus serotype 2 (e.g., a dengue serotype 2 strain); (iii) a live attenuated dengue virus serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain); and (iv) a live attenuated dengue virus serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain); a tetravalent dengue virus composition comprising: B. An excipient composition, based on the dry weight of the vaccine formulation, comprising: a. about 65-95% w / w, for example, about 87.0% w / w, of trehalose; b. about 0.05-5.0% w / w, e.g., about 2.9% w / w, of a poloxamer; c. about 0.1-6.0% w / w, for example, about 2.9% w / w, of urea; d. about 0.1-5.0% w / w, for example, about 2.4% w / w, of arginine hydrochloride; e. about 0.05 to 5.0% w / w, for example, about 1.2% w / w, of tromethamine; f. about 0.05 to 3.0% w / w, for example, about 0.96% w / w, of human serum albumin; an excipient composition comprising: The present invention relates to a dengue vaccine formulation, including

[0022] The present invention relates in particular to such a dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain); (ii) a live attenuated dengue virus serotype 2 (e.g., a dengue serotype 2 strain); (iii) a live attenuated dengue virus serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain); and (iv) a live attenuated dengue virus serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain); a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and an excipient composition comprising: The ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 4:1; the chloride salt is present in the dengue vaccine formulation in an amount of less than 4.0% w / w, based on the dry weight of the vaccine formulation; phosphate is included in the dengue vaccine formulation in an amount of less than 1.0% w / w, based on the dry weight of the vaccine formulation; This applies to dengue vaccine formulations.

[0023] The present invention relates in particular to such a dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and an excipient composition comprising: dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, dengue serotype 2 is a non-chimeric dengue serotype 2 strain, dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; The dengue serotype 2 strain is derived from the live attenuated DEN-2 PDK-53 virus strain, and the three chimeric dengue strains are derived from the dengue serotype 2 strain by replacing the structural proteins prM and E from the dengue serotype 2 strain with the corresponding structural proteins of other dengue serotypes, resulting in four live attenuated dengue virus strains, TDV-1, TDV-2, TDV-3, and TDV-4 (e.g., "TAK-003"); This applies to dengue vaccine formulations.

[0024] The present invention relates in particular to such a dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) TDV-1; and (ii) TDV-2; and (iii) TDV-3; and (iv) TDV-4; and a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. Human serum albumin. an excipient composition comprising: The present invention relates to a dengue vaccine formulation, including

[0025] The present invention relates in particular to such a dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and an excipient composition comprising: dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, dengue serotype 2 is a non-chimeric dengue serotype 2 strain, dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; Dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 and differ from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); Three chimeric dengue strains were derived from serotype 2 strains by replacing the structural proteins prM and E from serotype 2 strains with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, The present invention relates to a dengue vaccine formulation that provides

[0026] The present invention relates in particular to such a dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising four live-attenuated dengue virus serotypes: (i) a chimeric dengue serotype 2 / 1 strain; (ii) dengue serotype 2 strains; (iii) chimeric dengue serotype 2 / 3 strains; (iv) chimeric dengue serotype 2 / 4 strains; Including, Dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 and differ from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); Three chimeric dengue strains were derived from serotype 2 strains by replacing the structural proteins prM and E from serotype 2 strains with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, a tetravalent dengue virus composition providing B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and an excipient composition comprising: the human serum albumin is contained in the dengue vaccine formulation in an amount of about 0.1 to 1.0% w / w based on the dry weight of the vaccine formulation, and the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 4:1; This applies to dengue vaccine formulations.

[0027] The present invention particularly relates to such a dengue vaccine formulation (e.g., a lyophilized dengue vaccine formulation), comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and a tetravalent dengue virus composition comprising: B. An excipient composition, based on the dry weight of the vaccine formulation, comprising: a. approximately 65-95% w / w trehalose; b. about 0.05-5.0% w / w of poloxamer; c. about 0.1-6.0% w / w urea; d. about 0.1-5.0% w / w arginine hydrochloride; e. about 0.05-5.0% w / w tromethamine; f. about 0.05-3.0% w / w human serum albumin; g. about 0.05 to 3.0% w / w of chloride salt; h. about 0.05-0.5% w / w of phosphate; and an excipient composition comprising: dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, dengue serotype 2 is a non-chimeric dengue serotype 2 strain, dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; Dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 and differ from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); Three chimeric dengue strains were derived from serotype 2 strains by replacing the structural proteins prM and E from serotype 2 strains with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, The present invention relates to a dengue vaccine formulation that provides

[0028] The present invention particularly relates to such a dry form dengue vaccine formulation (e.g., a freeze-dried dengue vaccine formulation), comprising: A. A tetravalent dengue virus composition comprising four live-attenuated dengue virus serotypes: (i) a chimeric dengue serotype 2 / 1 strain; (ii) dengue serotype 2 strains; (iii) chimeric dengue serotype 2 / 3 strains; (iv) chimeric dengue serotype 2 / 4 strains; Including, Dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 and differ from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); Three chimeric dengue strains were derived from serotype 2 strains by replacing the structural proteins prM and E from serotype 2 strains with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, a tetravalent dengue virus composition providing B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and an excipient composition comprising: A unit dose of dengue vaccine formulation, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, gives: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL; a. Trehalose at a concentration of 40 to 80 g / L, for example, about 55 g / L; b. poloxamer at a concentration of about 0.1 to 5.0 g / L, for example, about 1.65 g / L; c. urea at a concentration of about 0.1 to 6.0 g / L, for example, about 1.65 g / L; d. arginine hydrochloride at a concentration of about 0.5-5.0 g / L, for example, about 1.4 g / L; e. Tromethamine at a concentration of about 0.1 to 1.5 g / L, for example, about 0.7 g / L; f. human serum albumin at a concentration of 0.1 to less than 1 g / L, for example, about 0.55 g / L; The present invention relates to a dengue vaccine formulation, including

[0029] The present invention particularly relates to such a dry form dengue vaccine formulation (e.g., a freeze-dried dengue vaccine formulation), comprising: A. A tetravalent dengue virus composition comprising four live-attenuated dengue virus serotypes: (i) a chimeric dengue serotype 2 / 1 strain; (ii) dengue serotype 2 strains; (iii) chimeric dengue serotype 2 / 3 strains; (iv) chimeric dengue serotype 2 / 4 strains; Including, Dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 and differ from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); Three chimeric dengue strains were derived from serotype 2 strains by replacing the structural proteins prM and E from serotype 2 strains with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, a tetravalent dengue virus composition providing B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; Including, A unit dose of dengue vaccine formulation, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, contains: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 mL or at least 3.1 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL; a. Trehalose at a concentration of 40 to 80 g / L, for example, about 43 g / L (equivalent to about 47 g / L of trehalose dihydrate); b. poloxamer at a concentration of about 0.1 to 5.0 g / L, for example, about 1.42 g / L; c. urea at a concentration of about 0.1 to 6.0 g / L, for example, about 1.42 g / L; d. arginine hydrochloride at a concentration of about 0.5-5.0 g / L, for example, about 1.18 g / L; e. tromethamine at a concentration of about 0.1 to 1.5 g / L, for example, about 0.6 g / L; f. human serum albumin at a concentration of 0.1 to less than 1 g / L, for example, about 0.48 g / L; The present invention relates to a dengue vaccine formulation, including

[0030] The present invention is further directed, in part, to a container, eg, a vial, that contains a dengue vaccine formulation described herein, preferably in a dried form, eg, a lyophilized form.

[0031] The present invention is particularly directed to such containers, e.g., vials, containing one unit dose or multiple (e.g., 5) unit doses, such as 1 to 10 unit doses, of the dengue vaccine formulation described herein, preferably in dry form, e.g., lyophilized form.

[0032] The present invention particularly relates to such containers, e.g., vials, containing a dengue vaccine formulation as described herein, preferably in dry form, e.g., lyophilized form, a. Trehalose is contained in an amount of about 100 to 170 mg, for example, about 149 mg; b. Poloxamer is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; c. urea is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; d. arginine hydrochloride is contained in an amount of about 1.5 to 7.5 mg, for example, about 4.1 mg; e. Tromethamine is contained in an amount of about 0.5 to 4.0 mg, for example, about 2.0 mg; f. Human serum albumin is contained in an amount of about 0.1 to 2.5 mg, for example, about 1.65 mg; Containers, for example vials, are of interest.

[0033] The present invention further relates to such containers, e.g., vials, containing, inter alia, 5 unit doses of a dengue vaccine formulation as described herein, a. Trehalose is contained in an amount of about 100 to 170 mg, for example, about 149 mg; b. Poloxamer is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; c. urea is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; d. arginine hydrochloride is contained in an amount of about 1.5 to 7.5 mg, for example, about 4.1 mg; e. Tromethamine is contained in an amount of about 0.5 to 4.0 mg, for example, about 2.0 mg; f. Human serum albumin is contained in an amount of about 0.1 to 2.5 mg, for example, about 1.65 mg; Containers, for example vials, are of interest.

[0034] The invention is further directed, in part, to a kit comprising one or more, e.g., ten, containers described in the preceding paragraph, and optionally comprising one or more, e.g., ten, containers containing a pharmaceutically acceptable aqueous diluent for reconstitution.

[0035] The present invention is further directed, in part, to a kit for preparing a dengue vaccine formulation in a reconstituted form, comprising: a) a container, e.g., a vial, containing a dengue vaccine formulation described herein in dry form, e.g., a lyophilized dengue vaccine formulation described herein; and b) a container, e.g., a vial, containing a pharmaceutically acceptable aqueous diluent for reconstitution.

[0036] The present invention is particularly directed to kits for preparing 1 unit dose or a multiple (e.g., 5) unit doses, such as 1 to 10 unit doses, of a dengue vaccine formulation in reconstituted form, the kit comprising: a) a container, e.g., a vial, containing 1 unit dose or a multiple (e.g., 5) unit doses, such as 1 to 10 unit doses, of a dengue vaccine formulation described herein in dry form, e.g., a lyophilized dengue vaccine formulation described herein; and b) a container, e.g., a vial, containing 1 to 10 aliquots, e.g., 1 aliquot (e.g., 0.5 mL / unit dose) or multiple (e.g., 5) aliquots (e.g., 2.5 mL) of a pharmaceutically acceptable aqueous diluent for reconstitution (e.g., 37 mM aqueous sodium chloride solution), wherein the number of aliquots corresponds to the number of unit doses. The aliquot(s) are preferably provided in a single container, e.g., a single vial.

[0037] The present invention is further directed, in part, to five unit doses of a dengue vaccine formulation in aqueous form obtained by reconstituting five unit doses of the dengue vaccine formulation in dry form described herein with five aliquots of a pharmaceutically acceptable aqueous diluent.

[0038] The present invention is further directed, in part, to a unit dose of a dengue vaccine formulation in aqueous form obtained by reconstituting a unit dose of a dengue vaccine formulation in dry form described herein with an aliquot (e.g., 0.5 mL / unit dose) of a pharmaceutically acceptable aqueous diluent (e.g., 37 mM aqueous sodium chloride solution).

[0039] The present invention is further directed, in part, to a unit dose of a dengue vaccine formulation in aqueous form obtained by reconstituting multiple unit doses, e.g., 5 unit doses, of a dengue vaccine formulation in dry form described herein with multiple aliquots, e.g., 5 aliquots, of a pharmaceutically acceptable aqueous diluent (e.g., 37 mM aqueous sodium chloride solution), the number of aliquots corresponding to the number of unit doses, and removing a unit dose (e.g., 0.5 mL) volume of the reconstituted dengue vaccine formulation.

[0040] The present invention further relates, in part, to a method for preparing a dengue vaccine formulation in dry form, comprising the steps of: The present invention is further directed to a method comprising: (a) providing a dengue vaccine formulation in aqueous form; (b) freezing the dengue vaccine formulation in aqueous form to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature ranging from above the glass transition temperature Tg of the formulation to a temperature not exceeding 25°C or not exceeding 20°C above the glass transition temperature Tg of the formulation to form a primary dried product; and (d) subjecting the primary dried product to a secondary drying step at a pressure of less than 1 mbar in an apparatus having a storage temperature higher than the storage temperature of the primary drying step to form a secondary dried product.

[0041] The present invention further relates, in part, to a method for preparing a dengue vaccine formulation in dry form, comprising the steps of: The present invention is further directed to a method comprising: (a) providing a dengue vaccine formulation in aqueous form; (b) freezing the dengue vaccine formulation in aqueous form to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature ranging from above the glass transition temperature Tg of the formulation to a temperature not exceeding 15°C above the glass transition temperature Tg of the formulation to form a primary dried product; and (d) subjecting the primary dried product to a secondary drying step at a pressure of less than 1 mbar in an apparatus having a storage temperature higher than the storage temperature of the primary drying step to form a secondary dried product.

[0042] The present invention relates, in part, to a method for preparing a dengue vaccine formulation described herein in dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form, the dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: a tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; B. providing an excipient composition comprising: a. trehalose; b. poloxamer; c. urea; d. arginine hydrochloride; e. tromethamine; and f. human serum albumin; (b) freezing the aqueous form of the dengue vaccine formulation in a device having a storage temperature of less than -35°C to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step in an apparatus having a storage temperature of about −31 to −19° C. at a pressure of less than 0.1 mbar to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of at least 15°C at a pressure of less than 1 mbar to form a secondary dried product; The present invention is further directed to a method, comprising:

[0043] The present invention relates, in part, to a method for preparing a dengue vaccine formulation described herein in dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form, the dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: a tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; B. providing an excipient composition comprising: a. trehalose; b. poloxamer; c. urea; d. arginine hydrochloride; e. tromethamine; and f. human serum albumin; (b) freezing the aqueous form of the dengue vaccine formulation in a device having a storage temperature of less than -35°C to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step in an apparatus having a storage temperature of about −31 to −21° C. at a pressure of less than 0.1 mbar to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of at least 20°C at a pressure of less than 1 mbar to form a secondary dried product; The present invention is further directed to a method, comprising:

[0044] The advantageous configuration of the method for preparing the dengue vaccine formulation in dry form described herein helps to reduce the time for drying the dengue vaccine formulation described herein and helps to improve the appearance of the dengue vaccine formulation in dry form.

[0045] The present invention relates, in part, to a method for preparing five unit doses of a dengue vaccine formulation described herein in dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form, the dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1, e.g., a chimeric dengue serotype 2 / 1 strain, (ii) a live attenuated dengue virus serotype 2, e.g., a dengue serotype 2 strain; (iii) a live attenuated dengue virus serotype 3, e.g., a chimeric dengue serotype 2 / 3 strain; (iv) a live attenuated dengue virus serotype 4, e.g., a chimeric dengue serotype 2 / 4 strain; a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. about 9-11% w / v, e.g., about 10% w / v, trehalose; b. about 0.2-0.4% w / v, for example, about 0.3% w / v, of poloxamer; c. about 0.2-0.4% w / v, for example, about 0.3% w / v, of urea; d. about 0.2-0.3% w / v, for example, about 0.25% w / v, of arginine hydrochloride; e. about 0.05 to 0.15% w / v, for example, about 0.1% w / v, of tromethamine; f. about 0.05 to 0.15% w / v, for example, about 0.1% w / v, human serum albumin; and an excipient composition comprising: (b) freezing the aqueous dengue vaccine formulation in a device having a storage temperature of about −40 to −50° C. to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step in an apparatus having a storage temperature of about −31 to −19° C., or about −31 to −21° C., at a pressure of less than 0.1 mbar for about 30 to 100 hours to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of about 15-40°C at a pressure of less than 1 mbar for about 3-10 hours to form a secondary dried product; Including, The dengue vaccine formulation in aqueous form is subjected to the freezing step b) in a volume approximately half the volume used to reconstitute 5 unit doses of the dengue vaccine formulation in dry form; The method is further directed to:

[0046] The present invention relates, in part, to a method for preparing five unit doses of a dengue vaccine formulation described herein in dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form, the dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1, e.g., a chimeric dengue serotype 2 / 1 strain, (ii) a live attenuated dengue virus serotype 2, e.g., a dengue serotype 2 strain; (iii) a live attenuated dengue virus serotype 3, e.g., a chimeric dengue serotype 2 / 3 strain; (iv) a live attenuated dengue virus serotype 4, e.g., a chimeric dengue serotype 2 / 4 strain; a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. about 9-11% w / v, e.g., about 10% w / v, trehalose; b. about 0.2-0.4% w / v, for example, about 0.3% w / v, of poloxamer; c. about 0.2-0.4% w / v, for example, about 0.3% w / v, of urea; d. about 0.2-0.3% w / v, for example, about 0.25% w / v, of arginine hydrochloride; e. about 0.05 to 0.15% w / v, for example, about 0.1% w / v, of tromethamine; f. about 0.05 to 0.15% w / v, for example, about 0.1% w / v, human serum albumin; and an excipient composition comprising: (b) freezing the aqueous dengue vaccine formulation in a device having a storage temperature of about −40 to −50° C. to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step in an apparatus having a storage temperature of about −31 to −19° C., or about −31 to −21° C., at a pressure of less than 0.1 mbar for about 30 to 100 hours to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of about 20-40°C at a pressure of less than 1 mbar for about 3-10 hours to form a secondary dried product; Including, The dengue vaccine formulation in aqueous form is subjected to the freezing step b) in a volume approximately half the volume used to reconstitute 5 unit doses of the dengue vaccine formulation in dry form; The method is further directed to:

[0047] The advantageous dengue vaccine formulations described herein, in combination with the advantageous methods described herein for preparing them in dry form, can help reduce waste of dengue virus (e.g., TDV-3) by using reduced fill volumes for drying multiple unit doses (e.g., 5 unit doses) of the dengue vaccine formulation, and can reduce fill time and time in solution of the dengue vaccine formulation during manufacture, as well as the length of the lyophilization cycle, resulting in increased formulation volume.

[0048] The present invention is further directed to a dengue vaccine formulation as described herein for use in a method for preventing dengue disease in a subject, the method comprising administering a primary vaccination, which comprises only two administrations of unit doses, and which comprises the following steps: administering a first unit dose of the dengue vaccine formulation to the subject; and administering a second unit dose of the dengue vaccine formulation to the subject, e.g., administered within three months of administration of the first unit dose; and optionally administering a booster dose at least 12 months after administration of said second unit dose of the dengue vaccine.

[0049] The present invention is further directed to a method for preventing dengue disease in a subject, comprising administering a primary vaccination of a dengue vaccine formulation described herein, which method comprises only two administrations of unit doses, and comprises the following steps: administering a first unit dose of the dengue vaccine formulation to the subject; administering a second unit dose of the dengue vaccine formulation to the subject within three months of administration of the first unit dose; and optionally administering a booster dose at least twelve months after administration of said second unit dose of the dengue vaccine. [Brief explanation of the drawings]

[0050] [Figure 1]

[0023] Figure 1 shows data from an experiment using a dengue vaccine formulation prepared according to Example 1 disclosed herein and a dengue vaccine control formulation ("Control") prepared according to a comparative example disclosed herein to measure the log loss of TDV-1 and TDV-2 titers after exposure of the lyophilized formulations to approximately 5°C for 12 months. [Figure 2] 1 shows data from an experiment using a dengue vaccine formulation prepared according to Example 1 disclosed herein and a dengue vaccine control formulation ("Control") prepared according to a comparative example disclosed herein to measure the log loss of TDV-3 and TDV-4 titers after exposure of the lyophilized formulations to approximately 5°C for 12 months. [Figure 3] The genetic structure of the four dengue strains contained in TDV, as published in WO2020 / 051334A1, is shown. The triangles indicate three attenuating mutations present in the 5'NCR, NS1, and NS3 proteins. TDV-1, TDV-3, and TDV-4 strains are chimeric viruses in which the prM and E genes from dengue serotypes 1, 3, and 4, respectively, have been inserted into the TDV-2 backbone. [Figure 4] 1 shows data from an experiment using a dengue vaccine formulation prepared according to Example 3 disclosed herein and a dengue vaccine control formulation ("Control") prepared according to a comparative example disclosed herein to measure the log loss of TDV-1 and TDV-2 titers after exposure of the lyophilized formulations to approximately 5°C for 6, 9, 12, 18, and 24 months. [Figure 5] 1 shows data from an experiment using a dengue vaccine formulation prepared according to Example 3 disclosed herein and a dengue vaccine control formulation ("Control") prepared according to a comparative example disclosed herein to measure the log loss of TDV-3 and TDV-4 titers after exposure of the lyophilized formulations to approximately 5°C for 6, 9, 12, 18, and 24 months. [Figure 6]1 shows data from an experiment using a dengue vaccine formulation prepared according to Example 4 disclosed herein and dengue comparative formulation 20 disclosed herein, in which titers were measured on days 0 and 30 after exposing the liquid formulations to temperatures between 2 and 8°C.

[0051] All references and publications cited herein are hereby incorporated by reference in their entirety for all purposes. DETAILED DESCRIPTION OF THE INVENTION

[0052] definition In describing the present invention, the following terminology will be used as indicated below: As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0053] As used herein, the terms "dose," "unit dose," "unit dose of dengue vaccine composition," and "unit dose of the invention described herein" refer to an amount of dengue vaccine administered to a subject in a single dose. In one embodiment, a single unit dose is present in a container, e.g., a vial, which is administered to a subject, optionally after reconstitution. In one embodiment, multiple unit doses of a dengue vaccine composition may be present in a vial such that multiple subjects can be vaccinated. In a particular embodiment, multiple (e.g., 5) unit doses of a dengue vaccine formulation are present in a vial. When multiple (e.g., 5) unit doses are present in a vial in dry form, a unit dose of the dengue vaccine formulation can be obtained by reconstituting the multiple (e.g., 5) unit doses with an equal number (e.g., 5) aliquots of a pharmaceutically acceptable aqueous diluent and removing a unit dose (e.g., 0.5 mL) volume of the reconstituted dengue vaccine formulation.

[0054] As used herein, the term "aliquot" refers to the volume of pharmaceutically acceptable aqueous diluent per unit dose, expressed in mL / unit dose, that is provided to a unit dose of a dengue vaccine formulation in dry form for reconstitution. Thus, if one unit dose is present in a container, e.g., a vial, one aliquot (e.g., 0.5 mL / unit dose) of a pharmaceutically acceptable aqueous diluent is added for reconstitution. If multiple (e.g., 5) unit doses are present in a container, e.g., a vial, an equal number of aliquots (e.g., 5 aliquots) of a pharmaceutically acceptable aqueous diluent are added for reconstitution. In certain such embodiments, multiple (e.g., 5) aliquots for reconstitution are provided in a single container, such as a vial or syringe, and the volume of multiple (e.g., 5) aliquots (e.g., 2.5 mL) is preferably added at one time to the dengue vaccine formulation in dry form. The volume of the reconstituted vaccine formulation is greater than the aliquot(s) used for reconstitution because the dissolved components of the vaccine formulation are added to the final volume.

[0055] As used herein, the term "container, e.g., vial, containing one or more unit doses (e.g., 1 unit dose, 1 to 10 unit doses, multiple unit doses, or 5 unit doses) of a dengue vaccine formulation" allows for overfilling of the dengue vaccine formulation by a factor of approximately 1.3 per unit dose. This allows for accurate removal of unit doses from the container, e.g., vial. For example, a container, e.g., vial, containing a unit dose of a dengue vaccine formulation in dry form contains 1.3 times the amount of dengue vaccine administered to a subject in a single dose. The volume of the pharmaceutically acceptable aqueous diluent added for reconstitution is therefore increased by the same factor (e.g., 1.3 x 0.5 mL / unit dose = 0.65 mL). From the total amount of reconstituted dengue vaccine formulation, a unit dose volume (e.g., 0.5 mL) is removed, e.g., with a syringe, and administered to a subject. Alternatively, the total amount (e.g., 0.7 mL) of the reconstituted dengue vaccine formulation is withdrawn, for example, with a syringe, and a unit dose volume (e.g., 0.5 mL) is administered to the subject. In a preferred embodiment, the dengue vaccine formulation in a vial is reconstituted with the entire contents of the vial or pre-filled syringe of diluent (e.g., 37 mM aqueous sodium chloride solution), thereby allowing a 0.5 mL dose of vaccine to be withdrawn and administered.

[0056] As used herein, the term "upon reconstitution in 0.5 mL" refers to the amount of virus and excipients present in a unit dose of a dengue vaccine formulation when 0.5 mL of diluent is used for reconstitution, although it is not intended to limit reconstitution to using 0.5 mL of diluent. Using a different volume (e.g., 0.8 mL) for reconstitution will result in a different concentration of virus in the reconstituted unit dose, whereas administering a total unit dose volume (e.g., 0.8 mL) will result in the same total amount of administered virus. The same applies to the amount of excipients contained in a reconstituted unit dose, unless the pharmaceutically acceptable aqueous diluent used for reconstitution contains additional excipients. If the diluent contains additional excipients (e.g., sodium chloride), the total amount of excipients contained in the reconstituted unit dose is increased by the amount of excipients added by the diluent. In a preferred embodiment of the present invention, the diluent does not add any excipients other than sodium chloride to the unit dose. In certain such embodiments, the diluent used for reconstitution adds less than 1.5 mg / unit dose (eg, about 1.1 mg / unit dose) of sodium chloride to the dengue vaccine formulation.

[0057] A "reconstituted unit dose," or a "unit dose in reconstituted form," results from reconstituting a unit dose of a dengue vaccine formulation in dry form with a pharmaceutically acceptable aqueous diluent. The diluent does not contain dengue virus. The reconstituted unit dose is a liquid that can be administered to a subject by injection, e.g., subcutaneous injection.

[0058] As used herein, the term "dengue vaccine formulation in dry form" refers to a formulation obtained and / or obtainable by subjecting a given volume of a liquid dengue vaccine formulation to a drying procedure, such as lyophilization, to remove the solvent. A lyophilized dengue vaccine formulation is obtained and / or obtainable by subjecting a given volume of a liquid dengue vaccine formulation to lyophilization, i.e., a drying procedure in which the formulation is first frozen and then the ice, or freezing solvent, is removed by sublimation under vacuum. As used herein, a lyophilized formulation is a formulation in dry form. The dengue vaccine formulation in dry form described herein preferably has a residual moisture content of less than 5% w / w.

[0059] As used herein, "% w / w," or "% (w / w)," refers to % mg / mg. As used herein, the % w / w of an ingredient "based on the dry weight of the vaccine formulation" can be calculated by dividing the amount of said ingredient in mg by the amount of the dengue vaccine formulation in mg in dry form, and multiplying by 100. Alternatively, as used herein, the % w / w of an ingredient "based on the dry weight of the vaccine formulation" can be calculated by dividing the amount of said ingredient in mg by the total amount of excipients contained in the dengue vaccine formulation in dry form, and multiplying by 100, since the weight of the tetravalent dengue virus composition contained in the dengue vaccine formulation in dry form is negligible and the difference in the results is insignificant.

[0060] As used herein, the term "about" when used in numerical ranges refers to a variation of ±10%.

[0061] As used herein, the term "aqueous dengue vaccine formulation" refers to a water-based liquid formulation. Aqueous dengue vaccine formulations may be subjected to a drying procedure, such as lyophilization, to obtain a dry dengue vaccine formulation. Aqueous dengue vaccine formulations may also be formed from a dry dengue vaccine formulation by reconstitution with a pharmaceutically acceptable diluent. Thus, the terms "reconstituted dengue vaccine formulation" and "reconstituted dengue vaccine formulation" refer to a formulation obtained from a dry dengue vaccine formulation, such as a lyophilized dengue vaccine formulation, by reconstitution with a pharmaceutically acceptable aqueous diluent, such as 37 mM sodium chloride aqueous solution or water for injection. The diluent does not contain dengue virus. The reconstituted dengue vaccine formulation may be administered to a subject by injection, such as subcutaneous injection.

[0062] As used herein, "% w / v," or "% (w / v)," refers to % mg / mL; for example, 100 mg / mL is 10% w / v.

[0063] As used herein, "a concentration of at least X log 10 pfu / 0.5 mL" refers to the concentration of a dengue serotype in 0.5 mL, but does not limit the unit dose to 0.5 mL. If the unit dose has a volume other than 0.5 mL, or is lyophilized from a volume other than 0.5 mL, or is reconstituted in a volume other than 0.5 mL, the concentration will be different from "a concentration of at least X log 10 pfu / 0.5 mL." However, if the volume of the unit dose is 0.5 mL, or is lyophilized from a volume of 0.5 mL, or is reconstituted in a volume of 0.5 mL, the concentration will be "a concentration of at least X log 10 pfu / 0.5 mL." Thus, while the concentration may vary, the total amount of virus in the unit dose remains the same.

[0064] The "total concentration in pfu / 0.5 ml," which serves as the reference value for the calculation of the concentration percentage for each individual component of the tetravalent dengue vaccine, is shown for one exemplary tetravalent vaccine composition containing dengue serotype 1 at a concentration of 3.60 log pfu / 0.5 ml, dengue serotype 2 at a concentration of 4.00 log pfu / 0.5 ml, dengue serotype 3 at a concentration of 4.60 log pfu / 0.5 ml, and dengue serotype 4 at a concentration of 5.11 log pfu / 0.5 ml. Typically, the logarithmic values ​​of the concentrations are converted to numerical values. This conversion results in a 4 x 10 3 pfu / 0.5 mL, for serotype 2, 1 × 10 4 pfu / 0.5 mL, for serotype 3, 4 × 10 4 pfu / 0.5 mL, and for serotype 4, 1.3 × 10 5 pfu / 0.5 mL. The total concentration in pfu / 0.5 mL is the sum of the preceding values, 1.84 x 10 5 yielding pfu / 0.5mL.

[0065] The "percentage concentration" for each of serotypes 1, 2, 3, and 4 is obtained by dividing the concentration value of the individual serotype (expressed as pfu / 0.5 mL) by the total concentration (expressed as pfu / 0.5 mL) and multiplying the result by 100, i.e.:

[0066] Serotype 1 concentration percentage = (4 x 10 3 pfu / 0.5mL ÷ 1.84 × 10 5 pfu / 0.5mL) x 100 = 2%.

[0067] Serotype 2 concentration percentage = (1 x 10 4 pfu / 0.5mL ÷ 1.84 × 10 5 pfu / 0.5mL) x 100 = 5%.

[0068] Serotype 3 concentration percentage = (4 x 10 4 pfu / 0.5mL ÷ 1.84 × 10 5 pfu / 0.5mL) x 100 = 22%.

[0069] Serotype 4 concentration percentage = (1.3 x 10 5 pfu / 0.5mL ÷ 1.84 × 10 5 pfu / 0.5mL) x 100 = 71%.

[0070] Concentration percentages are rounded to the nearest whole number.

[0071] As used herein, the terms "dengue serotype" and "dengue virus serotype" refer to species of dengue virus that are defined by their cell surface antigens and therefore can be distinguished by serological methods known in the art. Currently, four serotypes of dengue virus are known: dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3), and dengue serotype 4 (DENV-4).

[0072] As used herein, the term "tetravalent dengue virus composition" refers to a dengue virus composition that comprises four different immunogenic components, preferably four different live attenuated dengue viruses (each representing one dengue serotype), derived from four different dengue serotypes, DENV-1, DENV-2, DENV-3, and DENV-4, and that aims to stimulate an immune response against all four dengue serotypes.

[0073] As used herein, the term "live attenuated dengue virus" refers to a viable dengue virus that has been mutated to reduce its pathogenicity. A live attenuated dengue virus can be a dengue virus in which all components are derived from the same dengue serotype, or it can be a chimeric dengue virus having portions from more than one dengue serotype.

[0074] A "virus strain," and in particular a "dengue virus strain," is a genetic subtype of virus, particularly a dengue virus, characterized by a particular nucleic acid sequence. Dengue serotypes can include different strains with different nucleic acid sequences that have the same cell surface antigen. A dengue virus strain can be a dengue virus in which all components are derived from the same dengue serotype, or it can be a chimeric dengue virus with portions from more than one dengue serotype.

[0075] As used herein, "TDV-2" refers to a molecularly characterized and cloned dengue serotype 2 strain derived from the live-attenuated DEN-2 PDK-53 virus strain. The PDK-53 strain is described, for example, in Bhamarapravati et al. (1987) Bulletin of the World Health Organization 65(2):189-195. In one embodiment, the TDV-2 strain served as the backbone for chimeric TDV-1, TDV-3, and TDV-4 strains incorporating portions from the TDV-1, TDV-3, and TDV-4 strains. TDV-1, TDV-2, TDV-3, and TDV-4 combined form a tetravalent dengue virus composition, "TAK-003," which is Takeda's recent regulatory submission for a dengue vaccine candidate.

[0076] A "non-chimeric dengue virus," or "non-chimeric dengue serotype strain," or "non-chimeric dengue strain" contains only portions derived from one dengue serotype. In particular, a non-chimeric dengue virus does not contain portions derived from a different flavivirus, such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, or tick-borne encephalitis virus. TDV-2 is an example of a non-chimeric dengue virus.

[0077] A "chimeric dengue virus," or "chimeric dengue serotype strain," or "chimeric dengue strain" contains portions derived from at least two different dengue serotypes. Preferably, the chimeric dengue virus does not contain portions derived from different flaviviruses, such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, or tick-borne encephalitis virus. More preferably, the chimeric dengue viruses described herein do not contain portions derived from yellow fever virus. As used herein, a "chimeric dengue serotype 2 / 1 strain," or "DENV-2 / 1 chimera," or "TDV-1" refers to a dengue virus chimeric construct containing portions derived from both DENV-2 and DENV-1. Preferably, in a chimeric dengue serotype 2 / 1 strain, the prM and E proteins from DENV-1 replace the prM and E proteins from DENV-2, as described in more detail below. As used herein, a "chimeric dengue serotype 2 / 3 strain," or a "DENV-2 / 3 chimera," or a "TDV-3" refers to a dengue virus chimeric construct containing portions derived from both DENV-2 and DENV-3. Preferably, in a chimeric dengue serotype 2 / 3 strain, the prM and E proteins from DENV-3 replace the prM and E proteins from DENV-2, as described in more detail below. As used herein, a "chimeric dengue serotype 2 / 4 strain," or a "DENV-2 / 4 chimera," or a "TDV-4" refers to a dengue virus chimeric construct containing portions derived from both DENV-2 and DENV-4. Preferably, in a chimeric dengue serotype 2 / 4 strain, the prM and E proteins from DENV-4 replace the prM and E proteins from DENV-2, as described in more detail below.

[0078] As used herein, "TDV" refers to a tetravalent live-attenuated dengue vaccine comprising a mixture of four live-attenuated dengue virus strains, TDV-1, TDV-2, TDV-3, and TDV-4, which express surface antigens from four dengue serotypes, DENV-1, DENV-2, DENV-3, and DENV-4, respectively. In one embodiment (e.g., also in the Examples), TDV-1 is characterized by a nucleotide sequence according to SEQ ID NO: 1 and / or an amino acid sequence according to SEQ ID NO: 2. In one embodiment, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and / or an amino acid sequence according to SEQ ID NO: 4. In one embodiment, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and / or an amino acid sequence according to SEQ ID NO: 6. In one embodiment, TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and / or an amino acid sequence according to SEQ ID NO: 8.

[0079] As used herein, the term "lyophilization loss" refers to the log10 loss in titer of a live attenuated dengue virus (e.g., TDV-3) due to lyophilization, and is calculated by subtracting the amount of live attenuated dengue virus in log10 pfu / unit dose of the lyophilized dengue vaccine formulation from the amount of live attenuated dengue virus in log10 pfu / unit dose of the dengue vaccine formulation in liquid form before lyophilization. For analysis, the dengue vaccine formulation in liquid form is frozen and stored at -80°C and thawed before testing. The lyophilized dengue vaccine formulation is reconstituted before testing.

[0080] As used herein, the term "stability loss" refers to the log loss in titer of a live, attenuated dengue virus (e.g., TDV-3) upon storage at a particular temperature (e.g., 2-8°C), calculated by subtracting the amount of live, attenuated dengue virus in log pfu / unit dose of a lyophilized dengue vaccine formulation stored at a particular temperature, e.g., 2-8°C, for a particular period, e.g., 3 months, 6 months, 8 months, or 12 months, from the amount of live, attenuated dengue virus in log pfu / unit dose of the lyophilized dengue vaccine formulation before storage. The lyophilized dengue vaccine formulation(s) are reconstituted prior to testing.

[0081] As used herein, the term "accelerated stability loss" refers to the log 10 loss in titer of a live attenuated dengue virus (e.g., TDV-3) due to storage at accelerated conditions, and is calculated by subtracting the amount of live attenuated dengue virus in log 10 pfu / unit dose of a lyophilized dengue vaccine formulation stored at 25° C. for a specified period, e.g., 2 weeks, 5 weeks, or 12 weeks, from the amount of live attenuated dengue virus in log 10 pfu / unit dose of the lyophilized dengue vaccine formulation before storage. The lyophilized dengue vaccine formulation(s) are reconstituted prior to testing.

[0082] As used herein, the term "accelerated conditions" refers to a temperature of 25°C and 65% relative humidity RH, or a temperature of 25°C and about 60% relative humidity RH in a stability chamber. Accelerated conditions can be used to screen and select superior dengue vaccine formulations.

[0083] As used herein, the term "Tg" refers to the glass transition temperature as determined by differential scanning calorimetry, such as low temperature differential scanning calorimetry (LT-DSC). LT-DSC analysis is performed according to the United States Pharmacopeial Convention's USP <891> This can be done using the methods described in

[0084] As used herein, the term "dengue fever" refers to a disease caused by infection with the dengue virus. Symptoms of dengue fever include, for example, sudden high fever, headache, joint and muscle pain, nausea, vomiting, and skin rash. The term also includes dengue hemorrhagic fever (DHF) and more severe forms of dengue shock syndrome (DSS). Symptoms of DHF include increased vascular permeability, hypovolemia, and abnormal blood clotting mechanisms. Subjects with DHF may exhibit severe signs of plasma leakage and bleeding. If a subject with DHF experiences shock, they are classified as having DSS. Symptoms of DSS include bleeding, which can appear as small dots of blood on the skin and large patches of blood under the skin. Sustained shock is a major factor associated with complications, including massive gastrointestinal bleeding, which can lead to death. As used herein, a DHF case is defined as a case of VCD that meets the WHO 1997 criteria for DHF. In the context of preventing dengue disease in elderly subjects, the term "preventing dengue disease" preferably includes preventing DHF and / or DSS. In the context of preventing dengue disease in elderly subjects, the term "preventing dengue disease" preferably includes preventing severe end-organ manifestations of dengue, such as hepatomegaly and acute renal failure.

[0085] As used herein, "preventing dengue disease" refers to preventing a subject from developing one or more symptoms of dengue disease due to infection with a dengue virus. In particular, preventing dengue disease is achieved by vaccinating or inoculating a subject with a dengue vaccine composition, such as a reconstituted unit dose described herein. As used herein, the term "prophylactically treating dengue disease" is equivalent to "preventing dengue disease." In certain embodiments, preventing dengue disease includes preventing DHS and / or DSS.

[0086] As used herein, "vaccination" or "inoculation" refers to the administration of a vaccine to a subject with the aim of preventing the subject from developing one or more symptoms of the disease. As used herein, "vaccination against dengue disease" or "inoculation against dengue disease" refers to the administration of a dengue vaccine composition to a subject with the aim of preventing the subject from developing one or more symptoms of dengue disease. In principle, the method includes a primary vaccination and, optionally, one or more booster vaccinations. Primary vaccination is defined as a primary administration schedule for administering a composition or unit dose disclosed herein to establish a protective immune response, consisting of, for example, two administrations, for example, within three months. Whenever administration is mentioned within the scope of the present disclosure, such administration refers to a primary vaccination unless specified as a booster vaccination. Booster vaccination refers to an administration or administration schedule that occurs at least one year, or even five or ten years, after the primary vaccination, for example, the last administration, e.g., the second administration, of the primary vaccination schedule. A booster administration is intended, for example, to enhance the immune response of a primary vaccination.

[0087] As used herein, the term "subject(s)" is limited to a human subject (e.g., an infant, a child, or an adult). The term "elderly subject(s)" refers to a subject over 60 years of age, e.g., 61-100 years of age, 61-90 years of age, 61-80 years of age, 61-75 years of age, or 61-70 years of age.

[0088] As used herein, a "subject population" refers to a group of subjects. A subject population can refer to at least 40 subjects, at least 50 subjects, at least 60 subjects, at least 100 subjects, or at least 1000 subjects, and is defined by certain parameters. Parameters that can be used to define a subject population include, but are not limited to, the age of the subjects, whether the subjects are from a dengue-endemic or non-dengue-endemic area, and the serostatus of the subjects.

[0089] As used herein, "endemic area" refers to an area where a disease or infectious agent is consistently present and / or typically prevalent in a population within that area. As used herein, "non-endemic area" refers to an area where a disease is not present or typically prevalent. Thus, a "dengue-endemic area" refers to a geographic area where dengue virus infections are consistently maintained at baseline levels. A "non-dengue-endemic area" is a geographic area where dengue virus infections are not consistently maintained at baseline levels. Thus, a subject population or subject "from a dengue-endemic area" or "from a dengue-non-endemic area" refers to a subject population or subject residing in a geographic area as defined above. Whether a geographic region or a target population is dengue endemic or not can be determined by different calculation methods, such as those described in Bhatt et al. (2013) Nature 496(7446):504-507 and supplementary materials, and Stanaway et al. (2016) Lancet Infect Dis. 16(6):712-723 and supplementary materials. Dengue-endemic regions and summaries of dengue epidemiology are regularly published, for example, by the World Health Organization (WHO) or the Centers for Disease Control and Prevention (CDC). Typical dengue-endemic regions are found in Central and South America, Southeast Asia, and the Pacific Islands. Dengue-endemic countries include, but are not limited to, Australia, Brazil, Bangladesh, Colombia, China, the Dominican Republic, Indonesia, India, Mexico, Malaysia, Nicaragua, Nigeria, Pakistan, Panama, the Philippines, Puerto Rico, Singapore, Sri Lanka, Thailand, and Vietnam. The infectiousness of a region is measured by seroprevalence surveys, which provide a seroprevalence rate. Areas with very high infectivity are considered to have a seroprevalence of greater than 80%. As used herein, the term "area," when referring to seroprevalence, refers to a geographic area where seroprevalence can be determined or is known, e.g., a village, town, city, region, county, state, district, or part of any of the above, or an entire country.

[0090] As used herein, "serostatus" refers to the amount of antibodies a subject has against a particular infectious agent, particularly dengue virus. As used herein, "seronegative" or "seronaive" means that a subject does not have neutralizing antibodies in their serum against any one of the dengue serotypes DENV-1, DENV-2, DENV-3, and DENV-4. A seronegative or seronaive subject or subject population is defined by a neutralizing antibody titer of less than 10 for each of the four dengue serotypes. A subject or subject population with a neutralizing antibody titer of 10 or greater for at least one dengue serotype is defined as "seropositive" for that dengue serotype. Baseline serostatus refers to the serostatus prior to administration of the dengue vaccine composition described herein.

[0091] As used herein, "neutralizing antibody titer" refers to the amount of antibodies in a subject's serum that neutralize each dengue serotype. Neutralizing antibody titers against DENV-1, DENV-2, DENV-3, and DENV-4 are determined in a subject's serum sample using known methods, such as the plaque reduction neutralization test (PRNT) described in WHO guidelines (World Health Organization Department of Immunization Vaccines Biologicals (2007) Guidelines for plaque reduction neutralization testing of human antibodies to dengue viruses, WHO / IVB / 07.07) or the microneutralization (MNT50) assay described herein. As used herein, "a ratio of the neutralizing antibody titer for dengue serotype 2 to the neutralizing antibody titer for dengue serotype 4 of 20 or less" means that the neutralizing antibody titer for dengue serotype 2 is divided by the neutralizing antibody titer for dengue serotype 4, and the resulting ratio is 20 or less. That is, the dengue serotype 2 neutralizing antibody titer in the subject is no more than 20 times as high as the dengue serotype 4 neutralizing antibody titer.

[0092] As used herein, the terms "geometric mean neutralizing antibody titer" and "GMT" refer to the geometric mean of the titers of neutralizing antibodies against the corresponding dengue serotype in the serum of subjects in a subject population. The geometric mean is calculated by well-known formulas. As used herein, "a ratio of GMT for dengue serotype 2 to GMT for dengue serotype 4 of 20 or less" means that the geometric mean neutralizing antibody titer for dengue serotype 2 (GMT DENV-2) is divided by the geometric mean neutralizing antibody titer for dengue serotype 4 (GMT DENV-4), and the resulting ratio is 20 or less. That is, the geometric mean neutralizing antibody titer for dengue serotype 2 in a subject population is 20-fold or less higher than the geometric mean neutralizing antibody titer for dengue serotype 4.

[0093] As used herein, "immune response" refers to a subject's response to administration of a dengue vaccine. In particular, the immune response includes the formation of neutralizing antibodies against one or more dengue serotypes. The immune response may also include the stimulation of a cell-mediated response or the formation of antibodies against nonstructural proteins, e.g., NS1. An immune response is stimulated by administration of a unit dose of the invention described herein if, after said administration of the unit dose, there is an increase in the titer of neutralizing antibodies against at least one dengue virus serotype, preferably all four dengue virus serotypes. An immune response is stimulated by administration of a unit dose of the invention described herein if, after said administration of the unit dose, there is an increase in the secretion of interferon gamma by peripheral blood mononuclear cells stimulated with peptides derived from dengue virus proteins. An immune response is stimulated by administration of a unit dose of the invention described herein if, after said administration of the unit dose, there is an increase in the titer of antibodies against nonstructural proteins, e.g., NS1. In certain embodiments, administration of a reconstituted unit dose of the invention described herein stimulates the formation of neutralizing antibodies against one or more dengue serotypes, cell-mediated responses, and the formation of antibodies against nonstructural proteins, e.g., NS1.

[0094] As used herein, a "balanced immune response" means that the immune response to the four dengue serotypes is sufficient to provide protection against infection by all four dengue serotypes, and preferably the immune responses to the four dengue serotypes are of similar strength. Specifically, the neutralizing antibody titers to the four dengue serotypes at 180 days or 365 days after administration of the first reconstituted unit dose of the invention described herein are similar, i.e., they differ by less than 30-fold, less than 25-fold, or less than 20-fold.

[0095] RNA viruses can be characterized by their RNA sequence.Alternatively or additionally, it is also common practice to characterize the genome of RNA viruses by corresponding DNA sequence, and corresponding DNA sequence is easily understood to reflect the RNA genome in the virus.Therefore, when referring to genome RNA virus sequence, references to "t" or "thymine" throughout this disclosure should be understood as references to "u" or "uracil", respectively.

[0096] Dengue vaccine formulation The present invention relates to a dengue vaccine formulation, the dengue vaccine formulation comprising a tetravalent dengue virus composition comprising live attenuated dengue virus serotype 1, live attenuated dengue virus serotype 2, live attenuated dengue virus serotype 3, and live attenuated dengue virus serotype 4 (e.g., TDV-1, TDV-2, TDV-3, and TDV-4, i.e., "TAK-003"), at least one non-reducing disaccharide (e.g., trehalose and / or sucrose), at least one poloxamer (e.g., poloxamer 407), urea, at least one amino acid having a positively charged side chain at neutral pH (e.g., arginine, lysine, and / or histidine), tromethamine (e.g., the free base form of tromethamine and tromethamine hydrochloride), and human serum albumin.

[0097] In certain embodiments, the dengue vaccine formulation comprises sucrose. In certain other embodiments, the dengue vaccine formulation does not comprise sucrose.

[0098] In certain embodiments, the dengue vaccine formulation does not include mannitol and / or sorbitol.

[0099] In a preferred embodiment, the dengue vaccine formulation of the invention comprises a tetravalent dengue virus composition (e.g., TDV-1, TDV-2, TDV-3, and TDV-4, i.e., "TAK-003") and an excipient composition comprising trehalose, poloxamer, urea, arginine hydrochloride, tromethamine, and human serum albumin.

[0100] In one embodiment, the dengue vaccine formulation described herein comprises, in addition to the excipient composition disclosed in the previous paragraph, a phosphate salt, such as potassium dihydrogen phosphate or disodium hydrogen phosphate.

[0101] In one embodiment, the dengue vaccine formulation described herein comprises, in addition to the excipient composition disclosed in the previous two paragraphs, a chloride salt, e.g., sodium chloride or potassium chloride. "Acid addition salts," e.g., arginine hydrochloride or tromethamine hydrochloride, are not chloride salts within the meaning of the present invention. In one embodiment, the dengue vaccine formulation described herein further comprises hydrochloric acid.

[0102] Dry preparation In one aspect of the present invention, the dengue vaccine formulation described herein is in the form of a dry formulation, i.e., a dry form of the dengue vaccine formulation. The dry form of the dengue vaccine formulation can be obtained by lyophilization of an aqueous form of the dengue vaccine formulation. The dry formulation can be stored and reconstituted with a pharmaceutically acceptable aqueous diluent, such as water for injection or 37 mM aqueous sodium chloride solution, before administration. The lyophilized dengue vaccine formulation is the final product after manufacturing and is preferably the stored form of the dengue vaccine formulation.

[0103] In a preferred embodiment, the dengue vaccine formulation in dry form has a residual moisture content of 5.0% w / w or less, preferably 3.0% w / w or less, as determined by Karl Fischer measurement. In certain embodiments, the residual moisture content is in the range of 0.1% w / w to 5% w / w, or 0.1% w / w to 3% w / w, or 0.1% w / w to less than 2% w / w, or 0.1% w / w to 1% w / w. In certain preferred embodiments, the dengue vaccine formulation in dry form has a residual moisture content of less than 1% w / w, more preferably less than 0.9% w / w or less than 0.8% w / w.

[0104] In one embodiment, a lyophilized dengue vaccine formulation is obtained by subjecting an aqueous dengue vaccine formulation described herein to lyophilization. In a preferred embodiment, the lyophilized dengue vaccine formulation has a residual moisture content of 5.0% w / w or less, preferably 3% w / w or less, as determined by Karl Fischer measurement. In certain embodiments, the residual moisture content is in the range of 0.1% w / w to 5% w / w, or 0.1% w / w to 3% w / w, or 0.1% w / w to 2% w / w, or 0.1% w / w to 1% w / w. In certain preferred embodiments, the lyophilized dengue vaccine formulation has a residual moisture content of less than 1% w / w, more preferably less than 0.9% w / w or less than 0.8% w / w.

[0105] In one embodiment, the lyophilized unit dose is obtained by subjecting a volume of 0.5 mL of the aqueous dengue vaccine formulation described herein to lyophilization. In a preferred embodiment, the lyophilized unit dose is obtained by subjecting a volume of about 0.25 mL of the aqueous dengue vaccine formulation described herein to lyophilization.

[0106] In one embodiment, the lyophilized unit dose is present in a container, preferably a vial, and said unit dose is administered to a subject after reconstitution. In one embodiment, multiple unit doses (e.g., 5 unit doses) of the dengue vaccine composition are present in a single container, preferably a vial, and the contents of a single container, preferably a vial, can be used to vaccinate multiple subjects (e.g., 5 subjects). In one embodiment, a container, preferably a vial, containing multiple unit doses (e.g., 5 unit doses) of a dengue vaccine formulation described herein is used to provide reconstituted unit doses for use in the methods of the invention described herein.

[0107] In certain embodiments, a container containing one or more unit doses, e.g., 1-10 unit doses, of a dengue vaccine formulation of the invention is part of a kit. Accordingly, the invention is directed, in part, to a kit for preparing a reconstituted dengue vaccine formulation, comprising a dengue vaccine formulation described herein in dry form (e.g., a lyophilized dengue vaccine formulation) and a pharmaceutically acceptable reconstitution diluent.

[0108] In certain embodiments, the reconstitution diluent is provided in a container, preferably a vial or a pre-filled syringe. In some embodiments, the reconstitution diluent is selected from water for injection, phosphate buffered saline, or aqueous sodium chloride solution. In a preferred embodiment, the reconstitution diluent is 30-40 mM sodium chloride, for example, 37 mM sodium chloride solution.

[0109] In one embodiment of the invention, when a unit dose of a dengue vaccine formulation in dry form described herein is part of a kit for preparing a unit dose of a dengue vaccine formulation in reconstituted form, an aliquot (e.g., 0.5 mL / unit dose) of a pharmaceutically acceptable aqueous diluent (e.g., aqueous sodium chloride solution) for reconstitution is provided in a container, preferably a vial. In one embodiment of the invention, when five unit doses of a dengue vaccine formulation in dry form are part of the kit, five aliquots (e.g., 2.5 mL) of a pharmaceutically acceptable aqueous diluent (e.g., aqueous sodium chloride solution) are provided in a container, preferably a vial. Thus, in a specific embodiment, a kit for preparing multiple unit doses of a dengue vaccine formulation in reconstituted form comprises a) a container, e.g., a vial, containing multiple unit doses of a dengue vaccine formulation in dry form described herein, and b) a container, e.g., a vial, containing multiple aliquots of a pharmaceutically acceptable aqueous diluent for reconstitution, wherein the number of aliquots corresponds to the number of unit doses.

[0110] In one such embodiment, the aliquot has a volume of about 0.4-0.8 mL / unit dose, preferably about 0.4-0.7 mL / unit dose, more preferably about 0.45-0.65 mL / unit dose, for example 0.5 mL / unit dose.

[0111] Also contemplated within the scope of the present invention are kits of parts comprising one or more dengue vaccine formulation containers, e.g., vials, as described in the preceding paragraph. In certain such embodiments, the kit comprises a plurality, e.g., 10, dengue vaccine formulation vials, each containing 5 unit doses of a dengue vaccine formulation described herein. In one embodiment, the kit comprises one or more diluent container(s) and one or more dengue vaccine formulation container(s), e.g., vials. In certain such embodiments, the kit comprises 10 dengue vaccine formulation vials, each containing 5 unit doses of a dengue vaccine formulation described herein, and a plurality of diluent containers for preparing 50 unit doses of the dengue vaccine formulation in reconstituted form.

[0112] aqueous preparations In one aspect, the dengue vaccine formulation described herein is a liquid formulation, i.e., an aqueous form of the dengue vaccine formulation, which may be subjected to a drying procedure, such as lyophilization, to obtain a dry form of the dengue vaccine formulation.

[0113] In certain such embodiments, the dengue vaccine formulation in aqueous form, which is subjected to a drying procedure such as lyophilization, comprises about 5-15% w / v, preferably about 6-12% w / v, more preferably about 8-11% w / v, of trehalose (e.g., about 9% w / v of α,α-trehalose based on 10% w / v of α,α-trehalose dihydrate) and about 0.05-1.0% w / v, preferably about 0.1-0.5% w / v, more preferably about 0.2-0.4% w / v of poloxamer (e.g., about 0.3% w / v of Kolliphor P407) and about 0.05 to 1.0% w / v, preferably about 0.1 to 0.5% w / v, more preferably about 0.2 to 0.4% w / v urea (e.g., about 0.3% w / v urea) and about 0.05 to 1.0% w / v, preferably 0.1 to 0.5% w / v, more preferably about 0.2 to 0.3% w / v arginine hydrochloride (e.g., about 0.25% w / v L-arginine hydrochloride) and about 0.01 to 0.5% w / v, preferably about 0.02 to 0.5% w / v, more preferably about , about 0.05-0.15% w / v tromethamine (e.g., about 0.12% w / v tromethamine in the form of the free base, or about 0.02% w / v tromethamine combined with about 0.13% w / v tromethamine HCl, i.e., about 10 mM tromethamine), and about 0.01-0.5% w / v, preferably about 0.02-0.5% w / v, more preferably about 0.05-0.15% w / v human serum albumin (e.g., about 0.1% w / v human serum albumin).

[0114] In certain such embodiments, small amounts of phosphate and chloride salts are included in the dengue vaccine formulations described herein. For example, an aqueous dengue vaccine formulation that has been subjected to a drying procedure, such as lyophilization, contains less than 0.1% w / v, preferably about 0.01-0.06% w / v, and more preferably about 0.01-0.04% w / v, of phosphate salts (e.g., about 0.028% w / v disodium hydrogen phosphate dihydrate and 0.004% w / v potassium dihydrogen phosphate) and less than 0.5% w / v, preferably about 0.01-0.3% w / v, and more preferably about 0.01-0.25% w / v, of chloride salts (e.g., about 0.22% w / v sodium chloride and 0.003% w / v potassium chloride).

[0115] Reducing the amount of chloride salt helps reduce the time to lyophilize the dengue vaccine formulations described herein.

[0116] In certain such embodiments, the dengue vaccine formulation in aqueous form, which is subjected to a drying procedure such as lyophilization, comprises about 9-11% w / v, e.g., about 10% w / v, trehalose, about 0.2-0.4% w / v, e.g., about 0.3% w / v, poloxamer, about 0.2-0.4% w / v, e.g., about 0.3% w / v, urea, about 0.2-0.3% w / v, e.g., about 0.25% w / v, arginine hydrochloride, about 0.05-0.15% w / v, e.g., about 0.1% w / v, tromethamine, and about 0.05-0.15% w / v, e.g., about 0.1% w / v, human serum albumin.

[0117] In certain such embodiments, the pH of the aqueous form of the dengue vaccine formulation is determined according to the United States Pharmacopeial Convention's USP for pH Measurement. <791> According to the test method, it is about 7.0 to about 8.5, preferably about 7.3 to 8.0, more preferably about 7.4 to 7.6, for example, about 7.4, or about 7.5, or about 7.6.

[0118] Hydrochloric acid can be used for pH adjustment. Thus, in one embodiment, hydrochloric acid is included in the dengue vaccine formulation described herein. In other embodiments, preferably in embodiments in which tromethamine is provided by a combination of tromethamine and tromethamine HCl, hydrochloric acid is not added to the dengue vaccine formulation described herein. Tromethamine HCl can be used for pH adjustment without the need for hydrochloric acid. Thus, in a preferred embodiment, tromethamine HCl is used to adjust the pH.

[0119] The aqueous form of the dengue vaccine formulation may also be formed from the dry form of the dengue vaccine formulation as a reconstituted form of the dengue vaccine formulation. In one embodiment, the reconstituted unit dose is preferably obtained by subjecting a lyophilized unit dose to reconstitution with a pharmaceutically acceptable diluent prior to administration of the dengue vaccine.

[0120] In certain such embodiments, reconstitution is achieved by adding a pharmaceutically acceptable diluent, such as water for injection, phosphate buffered saline, or aqueous sodium chloride solution, to the lyophilized unit dose. In one embodiment, aqueous sodium chloride solution, for example, 37 mM aqueous sodium chloride solution, is added to the lyophilized unit dose for reconstitution.

[0121] Thus, in one embodiment, the dengue vaccine formulation in aqueous form is obtained by reconstituting the dengue vaccine formulation described herein in dry form using a pharmaceutically acceptable aqueous diluent.

[0122] In one embodiment, a unit dose of a dengue vaccine formulation in aqueous form is obtained by reconstituting a unit dose of a dengue vaccine formulation described herein in dry form with an aliquot of a pharmaceutically acceptable aqueous diluent.

[0123] In one embodiment, a unit dose of a dengue vaccine formulation in aqueous form is obtained by reconstituting a plurality of unit doses (e.g., 5 unit doses) of a dengue vaccine formulation described herein in dry form using a plurality of aliquots (e.g., 5 aliquots) of a pharmaceutically acceptable aqueous diluent, where the number of aliquots corresponds to the number of unit doses, and removing 0.5 mL of the reconstituted dengue vaccine formulation.

[0124] In one embodiment, five unit doses of a dengue vaccine formulation in aqueous form are obtained by reconstituting five unit doses of a dengue vaccine formulation described herein in dry form using five aliquots of a pharmaceutically acceptable aqueous diluent.

[0125] In one embodiment, a container, preferably a vial, containing a dengue vaccine formulation described herein is used to provide a reconstituted dengue vaccine formulation, hi certain such embodiments, the container contains one or more unit doses of a dengue vaccine formulation described herein.

[0126] In a preferred embodiment, a container, preferably a vial, containing a plurality of unit doses (e.g., at least 5 unit doses) of a dengue vaccine formulation described herein is used to provide the reconstituted dengue vaccine formulation for use in the methods of the invention described herein.

[0127] In one such embodiment, the lyophilized dengue vaccine formulation is reconstituted with 0.4-0.8 mL / unit dose, or 0.4-0.7 mL / unit dose, or 0.45-0.65 mL / unit dose, e.g., 0.5 mL / unit dose, of diluent. In a preferred embodiment, the lyophilized dengue vaccine formulation is reconstituted with 0.5 mL / unit dose of 37 mM aqueous sodium chloride solution. In another preferred embodiment, the lyophilized dengue vaccine formulation is reconstituted with 37 mM aqueous sodium chloride solution to a volume of 0.5 mL / unit dose. In one embodiment, the reconstituted unit dose administered subcutaneously or intramuscularly has a volume of 0.5 mL. The reconstituted dengue vaccine formulation observes physiological pH, osmolality, and tonicity considerations.

[0128] In certain such embodiments, the pH of the reconstituted formulation is determined according to the United States Pharmacopeial Convention's USP for the Determination of pH. <791> According to the test method, it is about 7.0 to about 8.5, for example, about 7.4, or about 7.5, or about 7.6.

[0129] In one embodiment, a unit dose of the dengue vaccine formulation in dry form, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, has a pH at 25°C of about 7.0 to 8.5, preferably about 7.3 to 8.0, more preferably about 7.4 to 7.8, or about 7.6.

[0130] In one embodiment, a unit dose of the dengue vaccine formulation in dry form, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, has a pH at 25°C of about 7.0 to 8.5, preferably about 7.0 to 8.0, more preferably about 7.3 to 7.8, or about 7.4.

[0131] In certain such embodiments, the United States Pharmacopeial Convention's USP for the determination of osmolality is <785> According to the test method, the osmolality of the reconstituted formulation is about 100 to about 700 mOsmol / kg, for example, about 300 mOsmol / kg.

[0132] In one embodiment, a unit dose of the dengue vaccine formulation in dry form, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, has an osmolality of about 100-700 mOsmol / kg, preferably less than about 100-700 mOsmol / kg, more preferably about 100-400 mOsmol / kg, or about 300 mOsmol / kg.

[0133] In one embodiment, a unit dose of the dengue vaccine formulation in dry form, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, has an osmolality of about 100-700 mOsmol / kg, preferably less than about 100-700 mOsmol / kg, more preferably about 100-500 mOsmol / kg.

[0134] Excipient Composition The dengue vaccine formulation of the present invention preferably comprises a combination of trehalose, at least one poloxamer, urea, arginine hydrochloride, tromethamine, and human serum albumin.

[0135] Tromethamine can be used in the form of its free base, or, for example, in the form of an acid addition salt (e.g., tromethamine hydrochloride, also known as Tris HCl), or a combination of the two forms. In a preferred embodiment, a mixture of tromethamine (base) and tromethamine hydrochloride is used in the preparation. Unless otherwise indicated, the tromethamine contained in the dengue vaccine formulation is calculated based on tromethamine in the free base form. For example, if a) 0.01 mmol (equivalent to 1.21 mg) of tromethamine or b) 0.01 mmol (equivalent to 1.58 mg) of tromethamine hydrochloride are used in the preparation, the amount of tromethamine in the dengue vaccine formulation within the meaning of the present invention is in both cases 1.21 mg, i.e., 0.01 mmol.

[0136] In particular, the presence of tromethamine, urea, and arginine hydrochloride helps improve the stability of the human serum albumin-containing dengue vaccine formulations described herein. The presence of tromethamine, in particular, helps to avoid pH changes during freezing, which contribute to loss of viral potency due to conformational changes in the viral structure.

[0137] In one embodiment, tromethamine is contained in the dengue vaccine formulation in an amount of about 0.05 to 1.0 mg / unit dose, preferably about 0.1 to 0.6 mg / unit dose, and more preferably about 0.2 to 0.5 mg / unit dose. That is, when a unit dose is administered in a volume of 0.5 mL, the amount of tromethamine contained in the dengue vaccine formulation is about 0.05 to 1.0 mg / 0.5 mL, preferably about 0.1 to 0.6 mg / 0.5 mL, and more preferably about 0.2 to 0.5 mg / 0.5 mL.

[0138] In one embodiment, tromethamine is contained in the dengue vaccine formulation in an amount of about 0.1 to 10.0 μmol / unit dose, preferably 0.1 to 6.0 μmol / unit dose, and more preferably 1.0 to 3.0 μmol / unit dose. That is, when a unit dose is administered in a volume of 0.5 mL, the amount of tromethamine contained in the dengue vaccine formulation is about 0.1 to 10.0 μmol / 0.5 mL, preferably 0.1 to 6.0 μmol / 0.5 mL, and more preferably 1.0 to 3.0 μmol / 0.5 mL.

[0139] In one embodiment, tromethamine is contained in the dengue vaccine formulation in an amount of about 0.05 to 5.0% w / w, preferably about 0.1 to 3.0% w / w, and more preferably about 0.5 to 2.0% w / w.

[0140] The trehalose may be α,α-trehalose, the poloxamer may be poloxamer 407, the arginine hydrochloride may be L-arginine hydrochloride, and the human serum albumin may be native human serum albumin or may include recombinant human serum albumin (rHSA).

[0141] In a preferred embodiment, the poloxamer is poloxamer 407. Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of poly(propylene oxide) flanked by two hydrophilic chains of poly(ethylene oxide). The length of the polymer blocks can be customized, resulting in different poloxamers with slightly different properties. As used herein, "poloxamer 407" is a hydrophilic nonionic surfactant consisting of a triblock copolymer consisting of a central propylene glycol block with approximately 56 repeating units and two adjacent hydrophilic polyethylene glycol blocks, each containing approximately 101 repeating units. "Poloxamer 407" is also known by its trade names Pluronic F127 and Synperonic PE / F127.

[0142] In one embodiment, the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 4:1, preferably about 2:1 to 3:1.

[0143] In one embodiment, the ratio of the amount of arginine hydrochloride to the amount of tromethamine contained in the dengue vaccine formulation is about 1:1 to 3:1, preferably about 1.5:1 to 2.5:1.

[0144] In one embodiment, the ratio of the amount of urea to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 5:1, preferably about 2:1 to 4:1.

[0145] In one embodiment, the ratio of the amount of urea to the amount of arginine hydrochloride contained in the dengue vaccine formulation is about 1:1 to 2:1.

[0146] In one embodiment, the ratio of the amount of urea to the amount of poloxamer contained in the dengue vaccine formulation is about 1:1 to 2:1.

[0147] In one embodiment, the ratio of the amount of trehalose to the amount of urea contained in the dengue vaccine formulation is about 20:1 to 40:1, preferably about 25:1 to 35:1.

[0148] In one embodiment, the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 4:1, and the human serum albumin is contained in the dengue vaccine formulation described herein in an amount of about 0.1 to 1.0% w / w, based on the dry weight of the vaccine formulation.

[0149] In one embodiment, the dengue vaccine formulation comprises trehalose, poloxamer, urea, arginine hydrochloride, tromethamine, human serum albumin, chloride salts, and phosphate salts. Preferably, the chloride salts comprise or consist of sodium chloride and potassium chloride. Preferably, the phosphate salts comprise or consist of potassium dihydrogen phosphate and disodium hydrogen phosphate. Most preferably, the excipient is a combination consisting of α,α-trehalose dihydrate, poloxamer 407 (Kolliphor® P407 (F-127)), urea, L-arginine hydrochloride, tromethamine, Tris-HCl, human serum albumin, potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, potassium chloride, and sodium chloride.

[0150] In certain such embodiments, the chloride salt is present in the dengue vaccine formulation in an amount of less than 4.0% w / w based on the dry weight of the vaccine formulation, the phosphate salt is present in the dengue vaccine formulation in an amount of less than 1.0% w / w based on the dry weight of the vaccine formulation, and the ratio of the amount of arginine hydrochloride to the amount of human serum albumin present in the dengue vaccine formulation is between about 1:1 and 4:1.

[0151] In certain embodiments, phosphate is present in the dengue vaccine formulation in an amount of less than 1.0% w / w, or about 0.05-0.5% w / w, preferably about 0.2-0.4% w / w, and more preferably 0.3% w / w or less, based on the dry weight of the vaccine formulation.

[0152] In certain embodiments, the chloride salt is present in the dengue vaccine formulation in an amount less than 4.0% w / w, or between about 0.05 and 3.0% w / w, based on the dry weight of the vaccine formulation.

[0153] In one embodiment, hydrochloric acid is contained in the dengue vaccine formulation in an amount of less than 1.0% w / w, or about 0.05 to 0.5% w / w, preferably about 0.2 to 0.4% w / w, and more preferably 0.3% w / w or less, based on the dry weight of the vaccine formulation.

[0154] In one embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 0.1 to 5.0% w / w arginine hydrochloride, urea in an amount of about 0.1 to 6.0% w / w urea, human serum albumin in an amount of about 0.05 to 3.0% w / w, trehalose in an amount of about 65 to 95% w / w, poloxamer in an amount of about 0.05 to 5.0% w / w, and tromethamine in an amount of about 0.05 to 5.0% w / w, based on the dry weight of the vaccine formulation. In certain such embodiments, phosphate salts may be included in the dengue vaccine formulation in an amount of less than 1.0% w / w, or about 0.05-0.5% w / w, and / or chloride salts may be included in the dengue vaccine formulation in an amount of less than 4.0% w / w, or about 0.05-3.0% w / w, based on the dry weight of the vaccine formulation.

[0155] In a particularly preferred embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 0.5 to 5.0% w / w, urea in an amount of about 0.5 to 5.0% w / w, human serum albumin in an amount of about 0.1 to 2.0% w / w, trehalose in an amount of about 70 to 95% w / w, poloxamer in an amount of about 0.1 to 5.0% w / w, and tromethamine in an amount of about 0.1 to 3.0% w / w, based on the dry weight of the vaccine formulation. In certain such embodiments, phosphate salts may be included in the dengue vaccine formulation in an amount of about 0.2-0.4% w / w, and / or chloride salts may be included in the dengue vaccine formulation in an amount of less than 4.0% w / w, or about 0.05-3.0% w / w, based on the dry weight of the vaccine formulation.

[0156] In an even more preferred embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 1.0 to 4.0% w / w, urea in an amount of about 1.0 to 4.0% w / w, human serum albumin in an amount of about 0.1 to 1.5% w / w, trehalose in an amount of about 75 to 95% w / w, poloxamer in an amount of about 0.5 to 4.0% w / w, and tromethamine in an amount of about 0.5 to 2.0% w / w, based on the dry weight of the vaccine formulation. In certain such embodiments, phosphate salts may be included in the dengue vaccine formulation in an amount of 0.3% w / w or less, and / or chloride salts may be included in the dengue vaccine formulation in an amount of less than 4.0% w / w, or between about 0.05 and 3.0% w / w, based on the dry weight of the vaccine formulation.

[0157] In a particularly preferred embodiment, arginine hydrochloride is contained in the dengue vaccine formulation in an amount of about 2.0 to 3.0% w / w, for example, about 2.4% w / w, urea is contained in the dengue vaccine formulation in an amount of about 2.0 to 4.0% w / w, for example, about 2.9% w / w, and human serum albumin is contained in the dengue vaccine formulation in an amount of about 0.1 to 1.0% w / w, or 1.0% w / w or less, based on the dry weight of the vaccine formulation; The dengue vaccine formulation may contain trehalose in an amount of at least 80% w / w, e.g., 80-95% w / w, or 80-90% w / w, or 85-95% w / w, or 85-90% w / w, or about 87% w / w, the poloxamer in an amount of about 2.9% w / w or less, and the tromethamine in an amount of less than 2.0% w / w, e.g., about 1.2% w / w, based on the dry weight of the vaccine formulation. In certain such embodiments, the dengue vaccine formulation may contain phosphate salts in an amount of 0.3% w / w or less and / or chloride salts in an amount of less than 4.0% w / w, or about 0.05-3.0% w / w, based on the dry weight of the vaccine formulation.

[0158] In a particularly preferred embodiment, arginine hydrochloride is contained in the dengue vaccine formulation in an amount of about 2.0 to 3.0% w / w, for example, about 2.4% w / w, urea is contained in the dengue vaccine formulation in an amount of about 2.0 to 4.0% w / w, for example, about 2.9% w / w, and human serum albumin is contained in the dengue vaccine formulation in an amount of about 0.5 to 1.5% w / w, for example, about 1.0% w / w, based on the dry weight of the vaccine formulation. In certain such embodiments, the dengue vaccine formulation may contain trehalose in an amount of about 85-90% w / w, e.g., about 87% w / w, poloxamer in an amount of about 2.0-4.0% w / w, e.g., about 2.9% w / w, and tromethamine in an amount of about 0.5-2.0% w / w, e.g., about 1.2% w / w, based on the dry weight of the vaccine formulation. In certain such embodiments, the dengue vaccine formulation may contain a phosphate salt in an amount of about 0.2-0.4% w / w, e.g., about 0.3% w / w, and / or a chloride salt in an amount of about 1.0-3.0% w / w, e.g., about 1.9% w / w, based on the dry weight of the vaccine formulation.

[0159] In one embodiment, sodium chloride is contained in the dengue vaccine formulation in an amount of about 1.0 to 3.0% w / w, preferably 2.1% w / w or less, for example, 1.9% w / w, based on the dry weight of the vaccine formulation. In one embodiment, potassium chloride is contained in the dengue vaccine formulation in an amount of about 0.01 to 0.05% w / w, preferably 0.03% w / w or less, based on the dry weight of the vaccine formulation.

[0160] In one embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 0.1 to 2.0 mg / unit dose, urea in an amount of about 0.1 to 3.0 mg / unit dose, human serum albumin in an amount of about 0.05 to 0.5 mg / unit dose, trehalose in an amount of about 14 to 34 mg / unit dose or about 20 to 34 mg / unit dose, poloxamer in an amount of about 0.1 to 3.0 mg / unit dose, and tromethamine in an amount of about 0.05 to 1.0 mg / unit dose. In certain such embodiments, phosphate salts may be included in the dengue vaccine formulation in an amount of less than 0.3 mg / unit dose, or about 0.01-0.2 mg / unit dose, and / or chloride salts may be included in the dengue vaccine formulation in an amount of less than 2.0 mg / unit dose.

[0161] In a particularly preferred embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 0.2 to 1.5 mg / unit dose or about 0.5 to 1.2 mg / unit dose, urea in an amount of about 0.1 to 2.0 mg / unit dose or about 0.3 to 1.5 mg / unit dose, human serum albumin in an amount of about 0.1 to 0.4 mg / unit dose, trehalose in an amount of about 16 to 32 mg / unit dose or about 22 to 32 mg / unit dose, poloxamer in an amount of about 0.3 to 1.5 mg / unit dose, and tromethamine in an amount of about 0.1 to 0.6 mg / unit dose. In certain such embodiments, the phosphate salt may be included in the dengue vaccine formulation in an amount of about 0.08-0.1 mg / unit dose, and / or the chloride salt may be included in the dengue vaccine formulation in an amount of about 0.1-1.0 mg / unit dose.

[0162] In a more preferred embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 0.5 to 1.0 mg / unit dose, urea in an amount of about 0.6 to 1.2 mg / unit dose, human serum albumin in an amount of less than 0.4 mg / unit dose, trehalose in an amount of about 18 to 30 mg / unit dose or about 23 to 30 mg / unit dose, poloxamer in an amount of about 0.6 to 1.2 mg / unit dose, and tromethamine in an amount of about 0.2 to 0.5 mg / unit dose. In certain such embodiments, the phosphate salt may be included in the dengue vaccine formulation in an amount of 0.1 mg / unit dose or less, and / or the chloride salt may be included in the dengue vaccine formulation in an amount of about 0.55-0.75 mg / unit dose.

[0163] In a particularly preferred embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 0.6 to 0.8 mg / unit dose, urea in an amount of about 0.75 to 1.0 mg / unit dose, human serum albumin in an amount of about 0.25 to 0.33 mg / unit dose, or 0.3 mg / unit dose or less, trehalose in an amount of about 23 to 27 mg / unit dose, or 27 mg / unit dose or less, poloxamer in an amount of about 0.75 to 1.0 mg / unit dose, or 1.0 mg / unit dose or less, and tromethamine in an amount of about 0.3 to 0.4 mg / unit dose, or 0.4 mg / unit dose or less. In certain such embodiments, the phosphate salt may be included in the dengue vaccine formulation in an amount of 0.1 mg / unit dose or less, and / or the chloride salt may be included in the dengue vaccine formulation in an amount of about 0.55-0.75 mg / unit dose.

[0164] In a most preferred embodiment, the dengue vaccine formulation contains arginine hydrochloride in an amount of about 0.5 to 0.7 mg / dose, urea in an amount of about 0.6 to 0.8 mg / dose, human serum albumin in an amount of about 0.2 to 0.3 mg / dose, trehalose in an amount of about 19 to 24 mg / dose (equivalent to about 21 to 27 mg / dose of α,α-trehalose dihydrate), poloxamer in an amount of about 0.6 to 0.8 mg / dose, and tromethamine in an amount of about 0.2 to 0.4 mg / dose (e.g., about 0.2 to 0.35 mg / dose of tromethamine-HCl and about 0.02 to 0.08 mg / dose of tromethamine in the form of free base). In certain such embodiments, the phosphate salt may be included in the dengue vaccine formulation in an amount of 0.1 mg / dose or less, e.g., about 0.06-0.1 mg / dose, and / or the chloride salt may be included in the dengue vaccine formulation in an amount of about 0.4-0.6 mg / dose. In a preferred embodiment, the dose is administered in a volume of 0.5 mL.

[0165] In one embodiment, sodium chloride is included in the dengue vaccine formulation in an amount of 0.72 mg / unit dose or less. In one embodiment, potassium chloride is included in the dengue vaccine formulation in an amount of 0.01 mg / unit dose or less.

[0166] In one embodiment, hydrochloric acid is contained in the dengue vaccine formulation in an amount of about 0.01 to 0.5 mg / unit dose, preferably less than 0.2 mg / unit dose, or about 0.07 to 0.1 mg / unit dose, more preferably 0.1 mg / unit dose.

[0167] In one embodiment, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises arginine hydrochloride at a concentration of about 0.5-5.0 g / L, urea at a concentration of about 0.1-6.0 g / L, human serum albumin at a concentration of about 0.1-less than 1 g / L, trehalose at a concentration of about 40-80 g / L, poloxamer at a concentration of about 0.1-5.0 g / L, and tromethamine at a concentration of about 0.1-1.5 g / L. In certain such embodiments, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form may further comprise phosphate at a concentration of less than 0.3 g / L, or about 0.16-0.22 g / L, and / or chloride salt at a concentration of less than 5 g / L.

[0168] In one embodiment, when reconstituted with a pharmaceutically acceptable aqueous diluent to a volume of 0.5 mL, a unit dose of the dengue vaccine formulation in dry form comprises arginine hydrochloride at a concentration of about 0.5-5.0 g / L, urea at a concentration of about 0.1-6.0 g / L, human serum albumin at a concentration of about 0.1-less than 1 g / L, trehalose at a concentration of about 40-80 g / L, poloxamer at a concentration of about 0.1-5.0 g / L, and tromethamine at a concentration of about 0.1-1.5 g / L. In certain such embodiments, when reconstituted with a pharmaceutically acceptable aqueous diluent to a volume of 0.5 mL, a unit dose of the dengue vaccine formulation in dry form may further comprise phosphate at a concentration of less than 0.3 g / L, or about 0.13-0.22 g / L, and / or chloride salt at a concentration of less than 5 g / L.

[0169] In a preferred embodiment, a unit dose of the dengue vaccine formulation in dry form, upon reconstitution with 0.5 mL of a pharmaceutically acceptable aqueous diluent, comprises arginine hydrochloride at a concentration of about 0.5 to less than 2.5 g / L, urea at a concentration of about 0.5 to less than 3.0 g / L, human serum albumin at a concentration of about 0.3 to 0.9 g / L, trehalose at a concentration of about 40 to 70 g / L, poloxamer at a concentration of about 0.5 to less than 3.0 g / L, and tromethamine at a concentration of about 0.5 to less than 1.2 g / L. In certain such embodiments, a unit dose of the dengue vaccine formulation in dry form, upon reconstitution with 0.5 mL of a pharmaceutically acceptable aqueous diluent, may further comprise phosphate at a concentration of less than 0.22 g / L, e.g., about 0.18 g / L, and / or chloride salt at a concentration of about 3.3 to 3.6 g / L.

[0170] In one embodiment, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises arginine hydrochloride at a concentration of about 0.5 to less than 2.0 g / L, urea at a concentration of about 0.5 to less than 3.0 g / L, human serum albumin at a concentration of about 0.3 to 0.9 g / L, trehalose at a concentration of about 40 to 70 g / L, poloxamer at a concentration of about 0.5 to less than 3.0 g / L, and tromethamine at a concentration of about 0.5 to less than 1.2 g / L. In certain such embodiments, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form may further comprise phosphate salt at a concentration of 0.22 g / L or less, e.g., about 0.13 g / L, and / or chloride salt at a concentration of about 3.1 to 3.6 g / L.

[0171] In a more preferred embodiment, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises arginine hydrochloride at a concentration of about 1.3-1.65 g / L, urea at a concentration of about 1.5-2.0 g / L, human serum albumin at a concentration of about 0.5-0.7 g / L, trehalose at a concentration of about 45-60 g / L, poloxamer at a concentration of about 1.5-2.0 g / L, and tromethamine at a concentration of about 0.5-1.0 g / L. In certain such embodiments, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form may further comprise phosphate salt at a concentration of 0.22 g / L or less, e.g., about 0.18 g / L, and / or chloride salt at a concentration of 3.6 g / L or less, e.g., about 3.4 g / L.

[0172] In an even more preferred embodiment, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises arginine hydrochloride at a concentration of about 1.0-1.65 g / L, urea at a concentration of about 1.0-2.0 g / L, human serum albumin at a concentration of about 0.4-0.7 g / L, trehalose at a concentration of about 40-48 g / L (corresponding to about 44-53 g / L of α,α-trehalose dihydrate), poloxamer at a concentration of about 1.0-2.0 g / L, and tromethamine at a concentration of about 0.5-1.0 g / L (e.g., about 0.5-0.7 g / L of tromethamine-HCl and about 0.05-0.2 g / L of free form tromethamine). In certain such embodiments, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, e.g., 37 mM aqueous sodium chloride solution, a unit dose of the dengue vaccine formulation in dry form may further comprise phosphate salt at a concentration of 0.22 g / L or less, e.g., about 0.16 g / L, and / or chloride salt at a concentration of 3.6 g / L or less, e.g., about 3.1 g / L.

[0173] In a particularly preferred embodiment, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises arginine hydrochloride at a concentration of 1.65 g / L or less, e.g., about 1.4 g / L; urea at a concentration of about 2.0 g / L or less, e.g., about 1.65 g / L; human serum albumin at a concentration of about 0.7 g / L or less, e.g., about 0.6 g / L; trehalose at a concentration of 60 g / L or less, e.g., about 55 g / L; poloxamer at a concentration of about 2.0 g / L or less, e.g., about 1.65 g / L; and tromethamine at a concentration of 0.8 g / L or less, e.g., about 0.7 g / L. In certain such embodiments, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form may further comprise phosphate salt at a concentration of 0.22 g / L or less, e.g., about 0.18 g / L, and / or chloride salt at a concentration of 3.6 g / L or less, e.g., about 3.4 g / L.

[0174] In a most preferred embodiment, when reconstituted with a pharmaceutically acceptable aqueous diluent to a volume of 0.5 mL, a unit dose of the dengue vaccine formulation in dry form comprises arginine hydrochloride at a concentration of not more than 1.65 g / L, e.g., about 1.2 g / L; urea at a concentration of not more than about 2.0 g / L, e.g., about 1.4 g / L; human serum albumin at a concentration of not more than about 0.7 g / L, e.g., about 0.5 g / L; trehalose at a concentration of not more than 60 g / L, e.g., about 43 g / L; poloxamer at not more than about 2.0 g / L, e.g., about 1.4 g / L; and tromethamine at not more than 0.8 g / L, e.g., about 0.6 g / L. In certain such embodiments, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, e.g., 37 mM aqueous sodium chloride solution, a unit dose of the dengue vaccine formulation in dry form may further comprise phosphate salt at a concentration of 0.22 g / L or less, e.g., about 0.13 g / L, and / or chloride salt at a concentration of 3.6 g / L or less, e.g., about 3.1 g / L.

[0175] In one embodiment, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of a dengue vaccine formulation described herein in dry form contains sodium chloride at a concentration of less than 5 g / L, preferably about 3.3 to 3.6 g / L, more preferably 3.6 g / L or less, for example, about 3.4 g / L.

[0176] In one embodiment, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of a dengue vaccine formulation described herein in dry form contains sodium chloride at a concentration of less than 5 g / L, preferably about 3.1 to 3.6 g / L, more preferably 3.6 g / L or less, e.g., about 3.1 g / L.

[0177] In one embodiment, when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, a unit dose of a dengue vaccine formulation described herein in dry form contains hydrochloric acid at a concentration of about 0.05 to 0.3 g / L, preferably about 0.15 to 0.19 g / L, more preferably 0.19 g / L or less, e.g., about 0.16 g / L.

[0178] In one embodiment, when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of a dengue vaccine formulation described herein in dry form contains hydrochloric acid at a concentration of about 0.05 to 0.3 g / L, preferably about 0.15 to 0.19 g / L, more preferably 0.19 g / L or less, e.g., about 0.14 g / L.

[0179] The excipient concentrations after reconstitution can also be expressed in terms of amount per dose. In a preferred embodiment, a dose administered in 0.5 mL comprises or consists of the following excipients: α,α-trehalose dihydrate in an amount of 18-30 mg / dose, preferably 20-28 mg / dose, more preferably 22-26 mg / dose (e.g., 23.46 mg / dose); poloxamer (e.g., Kolliphor® P407 (F127)) in an amount of 0.1-1.5 mg / dose, preferably 0.5-1.0 mg / dose, more preferably 0.6-0.8 mg / dose (e.g., 0.71 mg / dose); and 0.5-1.0 mg / dose of 0.6-0.8 mg / dose (e.g., 0.71 mg / dose). Human serum albumin in an amount of 0.05 to 0.5 mg / dose, preferably 0.05 to 0.4 mg / dose, more preferably 0.1 to 0.3 mg / dose (e.g., 0.24 mg / dose), tromethamine in an amount of 0.1 to 0.7 μmol / dose, preferably 0.2 to 0.6 μmol / dose, more preferably 0.3 to 0.5 μmol / dose (e.g., 0.39 μmol / dose), and tetrahydrofuran in an amount of 0.5 to 6.0 μmol / dose, preferably 1.0 to 5.0 μmol / dose, more preferably 1.0 to 3.0 μmol / dose (e.g., 1.97 μmol / dose). Tris HCl and L-arginine HCl in an amount of 0.5 to 8.0 μmol / dose, preferably 1.0 to 6.0 μmol / dose, more preferably 2.0 to 4.0 μmol / dose (e.g., 2.81 μmol / dose), and urea in an amount of 5.0 to 20.0 μmol / dose, preferably 8.0 to 16.0 μmol / dose, more preferably 10.0 to 14.0 μmol / dose (e.g., 11.81 μmol / dose), and urea in an amount of 0.001 to 1.0 μmol / dose, preferably 0.01 to 0.1 μmol / dose, more preferably 0.05 to 0.09 μmol / dose (e.g., 11.81 μmol / dose). potassium dihydrogen phosphate in an amount of 0.01 to 1.0 μmol / dose, preferably 0.1 to 0.6 μmol / dose, more preferably 0.3 to 0.5 μmol / dose (e.g., 0.38 μmol / dose); disodium hydrogen phosphate dihydrate in an amount of 0.001 to 1.0 μmol / dose, preferably 0.01 to 0.5 μmol / dose, more preferably 0.05 to 0.2 μmol / dose (e.g., 0.10 μmol / dose); and potassium chloride in an amount of 15.0 to 40.0 μmol / dose, preferably 15.0 to 35.0 μmol / dose.Sodium chloride in an amount of 0 μmol / dose, more preferably 25.0 to 30.0 μmol / dose (e.g., 26.66 μmol / dose).

[0180] In one embodiment, the dengue vaccine formulation described herein is present in a container, e.g., a vial. In certain such embodiments, the container contains arginine hydrochloride in an amount of about 1.5-7.5 mg, urea in an amount of about 2.0-8.0 mg, human serum albumin in an amount of about 0.1-2.5 mg, trehalose in an amount of about 100-170 mg, poloxamer in an amount of about 2.0-8.0 mg, and tromethamine in an amount of about 0.5-4.0 mg. In a preferred embodiment, the container contains about 2.0 to 6.0 mg of arginine hydrochloride, about 3.0 to 7.0 mg of urea, about 0.5 to 2.0 mg of human serum albumin, about 120 to 170 mg of trehalose, about 4.0 to 6.0 mg of poloxamer, and about 1.0 to 3.0 mg of tromethamine. In a preferred embodiment, the container contains about 3.0 to 5.0 mg of arginine hydrochloride, about 4.0 to 6.0 mg of urea, about 1.0 to 2.0 mg of human serum albumin, about 140 to 160 mg of trehalose, about 4.0 to 6.0 mg of poloxamer, and about 1.0 to 3.0 mg of tromethamine. In a particularly preferred embodiment, the container contains arginine hydrochloride in an amount of about 3.5 to 4.5 mg, for example, about 4.1 mg; urea in an amount of about 4.5 to 5.5 mg, for example, about 4.95 mg; serum albumin in an amount of about 1.5 to 1.8 mg, for example, about 1.65 mg; trehalose in an amount of about 145 to 155 mg, for example, about 149 mg; poloxamer in an amount of about 4.5 to 5.5 mg, for example, about 4.95 mg; and tromethamine in an amount of about 1.5 to 2.5 mg, for example, about 2.0 mg.

[0181] In a preferred embodiment, the excipients in the amounts described in the previous paragraph are contained in a container, e.g., a vial, comprising 5 unit doses of the dengue vaccine formulation to provide 5 unit doses of the dengue vaccine formulation in reconstituted form.

[0182] Tetravalent Dengue Virus Compositions Dengue virus strains Dengue virus is a single-stranded positive-sense RNA virus in the Flaviviridae family. The taxonomy is summarized in Table 1. The Flaviviridae family includes three genera: Flavivirus, Hepacivirus, and Pestivirus. The Flavivirus genus contains highly pathogenic and potentially hemorrhagic fever viruses, such as yellow fever virus and dengue virus, encephalitis viruses, such as Japanese encephalitis virus, Murray Valley encephalitis virus, and West Nile virus, as well as many less pathogenic viruses.

[0183] [Table 1]

[0184] The flavivirus genome consists of the following in the 5' to 3' direction: - 5'-non-coding region (5'-NCR), - the capsid protein (C) coding region, - the precursor membrane protein (prM) coding region, - the envelope protein (E) coding region, - the region encoding the nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5), and - 3' non-coding region (3'-NCR), (See Figure 3).

[0185] The viral structural proteins are C, prM, and E, and the nonstructural proteins are NS1 to NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by intracellular and viral proteases.

[0186] The dengue vaccine formulations of the invention described herein include tetravalent dengue virus compositions comprising four live-attenuated dengue virus strains, representing dengue serotypes 1, 2, 3, and 4. Preferably, the composition comprises a chimeric dengue virus and, optionally, at least one non-chimeric dengue virus. For example, a tetravalent dengue virus composition may comprise four live-attenuated dengue serotypes, DENV-1, DENV-2, DENV-3, and DENV-4, at least one of which is a dengue-dengue chimera, and the other serotypes are each a dengue-dengue chimera or a non-chimeric dengue serotype. In particular, the dengue virus compositions include molecularly characterized and cloned dengue serotype 2 strains derived from the live attenuated DEN-2 PDK-53 virus strain (TDV-2), and the structural proteins prM and E from the TDV-2 strains are replaced with the corresponding structural proteins from other dengue serotypes to produce the following chimeric dengue strains: - DENV-2 / 1 chimera (TDV-1), - DENV-2 / 3 chimera (TDV-3), and - DENV-2 / 4 chimera (TDV-4), and three chimeric dengue strains derived from TDV-2 strains by resulting in Non-limiting examples of four such suitable dengue strains are the four drug substances of TAK-003, which are marketed under the product "Qdenga."

[0187] The genetically modified tetravalent dengue vaccine, TDV, is based on the molecularly characterized and cloned dengue-2 virus strain (TDV-2). This attenuated TDV-2 strain was generated by cDNA cloning of the laboratory-derived attenuated DEN-2 PDK-53 virus strain, originally isolated at Mahidol University, Bangkok, Thailand (Kinney et al. (1997) Virology 230(2):300-308). DEN-2 PDK-53 was generated by 53 serial passages at 32°C in primary canine kidney (PDK) cells (Bhamarapravati et al. (1987) Bull. World Health Organ. 65(2):189-195).

[0188] The attenuated DEN-2 PDK-53 strain (the precursor of TDV-2) is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in nine nucleotides, as characterized below (Kinney et al. (1997) Virology 230(2):300-308): (i) 5′-noncoding region (NCR)-57 (nt-57 C to T): major attenuation locus; (ii) prM-29 Asp to Val (nt-524 A to T); (iii) nt-2055 C to T (E gene) silent mutation; (iv) NS1-53 Gly to Asp (nt-2579 G to A): major attenuation locus; (v) NS2A-181 Leu to Phe (nt-4018 C to T); (vi) NS3-250 Glu to Val (nt-5270 A to T): major attenuation locus; (vii) nt-5547 (NS3 gene) T to C silent mutation; (viii) NS4A-75 Gly to Ala (nt-6599 G to C); *nt-8571 C to T (NS5 gene) silent mutation.

[0189] Three nucleotide changes located in the 5' non-coding region (NCR) (nucleotide 57) (mutation (i)), the NS-1 (amino acid 828 of SEQ ID NO:4) (mutation (iv)), and the NS-3 gene (amino acid 1725 of SEQ ID NO:4) (mutation (vi)) form the basis for the attenuated phenotype of the DEN-2 PDK-53 strain (Butrapet et al. (2000) J. Virol. 74(7):3111-3119) (Table 2). These three mutations are referred to herein as "attenuating mutations" and are contained in TDV-1, TDV-2, TDV-3, and TDV-4.

[0190] [Table 2]

[0191] In one embodiment, the TDV-2 contains, in addition to the three attenuating mutations: a) an adenine to thymine mutation at nucleotide 524 in the prM gene, resulting in an amino acid change from aspartic acid to valine at position 143; and / or b) a silent mutation from cytosine to thymine at nucleotide 2055 in the E gene, and / or c) a cytosine to thymine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymine to cytosine mutation at nucleotide 5547 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2168; and / or f) a silent mutation from thymine to cytosine at nucleotide 900 in the prM gene; The present invention includes one or more mutations selected from the following: The silent cytosine-to-thymine mutation at nucleotide 8571 in the NS5 gene of DEN-2 PDK-53 is absent in TDV-2 strains.

[0192] In another embodiment, the TDV-2 contains, in addition to the three attenuating mutations: g) an adenine to guanine mutation at nucleotide 592 in the prM gene, resulting in an amino acid change from lysine to glutamic acid at position 166; and / or h) an adenine to guanine mutation at nucleotide 8803 in the NS5 gene, resulting in an amino acid change from isoleucine to valine at position 2903; The present invention includes one or more mutations selected from the following:

[0193] In another embodiment, the TDV-2 comprises, in addition to the three attenuating mutations, mutations a) and g), preferably mutations a), g), c), e), and h), more preferably mutations a), g), c), e), h), and b), even more preferably mutations a), g), c), e), h), b), and d), and most preferably mutations a) through h). The nucleotide and amino acid positions of TDV-2 refer to the nucleotide sequence set forth in SEQ ID NO:3 and the amino acid sequence set forth in SEQ ID NO:4.

[0194] The structural envelope (E) and precursor membrane (prM) proteins of dengue virus have been identified as primary antigens that elicit neutralizing and protective antibody responses (Plotkin 2001). To generate a tetravalent dengue vaccine (TDV), standard molecular genetic engineering techniques (Huang et al. (2003) J. Virol. 77(21):11436-11447) were used to modify DENV-2 by replacing the nucleic acid sequences encoding the prM and E glycoproteins of DENV-2 with those encoding the corresponding wild-type prM and E glycoproteins from wild-type strains of DENV-1, DENV-3, and DENV-4, DENV-1 16007, DENV-3 16562, or DENV-4 1036 viruses, respectively (see Table 3).

[0195] [Table 3]

[0196] A diagram of the four TDV strains included in the dengue vaccine formulation is shown in Figure 3 .

[0197] The chimeric dengue strains TDV-1, TDV-3, and TDV-4 express the surface antigens prM and E of DENV-1, DENV-3, or DENV-4 viruses, respectively, and retain the genetic changes responsible for the attenuation of TDV-2, as shown in Table 3. Thus, each of the TDV-1, TDV-3, and TDV-4 strains contains the attenuating mutations listed in Table 2.

[0198] In one embodiment, the TDV-1 contains, in addition to the three attenuating mutations: c) a cytosine to thymine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymine to cytosine mutation at nucleotide 5547 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2168; and / or i) a silent mutation from thymine to cytosine at nucleotide 1575 in the E gene, and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymine-guanine to cytosine-cytosine mutation at nucleotides 2381 / 2382 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 762; The present invention includes one or more mutations selected from the following:

[0199] In another embodiment, the TDV-1 comprises, in addition to the three attenuating mutations: l) an adenine to cytosine mutation at nucleotide 3823 in the NS2A gene, resulting in an amino acid change from isoleucine to leucine at position 1243; and / or m) an adenine to thymine mutation at nucleotide 4407 in the NS2B gene, resulting in an amino acid change from glutamic acid to aspartic acid at position 1437; and / or n) a silent adenine to guanine mutation at nucleotide 7311 in the NS4B gene; The present invention includes one or more mutations selected from the following:

[0200] In another embodiment, the TDV-1 strain comprises, in addition to the three attenuating mutations, mutations l) and m), preferably mutations l), m), c), and e), even more preferably mutations l), m), c), e), d), and n), and most preferably mutations l), m), c), e), d), n), i), j), and k). The TDV-1 nucleotide and amino acid positions refer to the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2.

[0201] In one embodiment, the TDV-3 contains, in addition to the three attenuating mutations: c) a cytosine to thymine mutation at nucleotide 4012 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1306; and / or d) a silent thymine to cytosine mutation at nucleotide 5541 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6593 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2166; and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymine-guanine to cytosine-cytosine mutation at nucleotides 2375 / 2376 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 760; and / or o) a silent mutation from cytosine to thymine at nucleotide 552 in the prM gene, and / or p) an adenine to thymine mutation at nucleotide 1970 in the E gene, resulting in an amino acid change from histidine to leucine at position 625; The present invention includes one or more mutations selected from the following:

[0202] In another embodiment, the TDV-3 contains, in addition to the three attenuating mutations: q) an adenine to thymine mutation at nucleotide 1603 in the E gene, resulting in an amino acid change from threonine to serine at position 503; and / or r) a silent adenine to guanine mutation at nucleotide 7620 in the NS5 gene; The present invention includes one or more mutations selected from the following:

[0203] In another embodiment, the TDV-3 comprises, in addition to the three attenuating mutations, mutations p) and q), preferably mutations p), q), c), and e), even more preferably mutations p), q), c), e), d), and r), and most preferably mutations p), q), c), e), d), r), j), k), and o). The nucleotide and amino acid positions of TDV-3 refer to the nucleotide sequence set forth in SEQ ID NO:5 and the amino acid sequence set forth in SEQ ID NO:6.

[0204] In one embodiment, the TDV-4 contains, in addition to the three attenuating mutations: c) a cytosine to thymine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymine to cytosine mutation at nucleotide 5547 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2168; and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymine-guanine to cytosine-cytosine mutation at nucleotides 2381 / 2382 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 762; and / or s) an adenine to cytosine mutation at nucleotide 396 in the C gene, resulting in an amino acid change from arginine to serine at position 100; and / or t) a silent adenine to guanine mutation at nucleotide 1401 in the E gene, and / or u) a cytosine to thymine mutation at nucleotide 2027 in the E gene, resulting in an amino acid change from alanine to valine at position 644; and / or v) an adenine to cytosine mutation at nucleotide 2275 in the E gene, resulting in an amino acid change from methionine to leucine at position 727; The present invention includes one or more mutations selected from the following:

[0205] In another embodiment, the TDV-4 contains, in addition to the three attenuating mutations: w) a silent adenine to thymine mutation at nucleotide 225 in the C gene, and / or x) an adenine to guanine mutation at nucleotide 3674 in the NS2A gene, resulting in an amino acid change from aspartic acid to glycine at position 1193; and / or y) a mutation in the NS2A gene at nucleotide 3773 from adenine to adenine / guanine mix, resulting in an amino acid change at position 1226 from lysine to lysine / arginine mix, and / or z) a silent mutation from cytosine to thymine at nucleotide 5391 in the NS3 gene, and / or aa) a cytosine to thymine mutation at nucleotide 6437 in the NS4A gene, resulting in an amino acid change from alanine to valine at position 2114; and / or bb) a silent mutation in the NS4B gene at nucleotide 7026 from thymine to a thymine / cytosine mix or cytosine; and / or cc) a silent adenine to cytosine mutation at nucleotide 9750 in the NS5 gene; The present invention includes one or more mutations selected from the following:

[0206] In another embodiment, the TDV-4 comprises, in addition to the three attenuating mutations, mutations s), u), and v), preferably mutations s), u), v), c), e), x), y), and aa), even more preferably mutations s), u), v), c), e), x), y), aa), and w), even more preferably mutations s), u), v), c), e), x), y), aa), w), d), z), bb), and cc), and most preferably mutations s), u), v), c), e), x), y), aa), w), d), z), bb), cc), j), k), and t). The nucleotide and amino acid positions of TDV-4 refer to the nucleotide sequence set forth in SEQ ID NO:7 and the amino acid sequence set forth in SEQ ID NO:8.

[0207] In a preferred embodiment, TDV-1 is characterized by the nucleotide sequence of SEQ ID NO: 1, TDV-2 is characterized by the nucleotide sequence of SEQ ID NO: 3, TDV-3 is characterized by the nucleotide sequence of SEQ ID NO: 5, and / or TDV-4 is characterized by the nucleotide sequence of SEQ ID NO: 7. In a further preferred embodiment, TDV-1 is characterized by the amino acid sequence of SEQ ID NO: 2, TDV-2 is characterized by the amino acid sequence of SEQ ID NO: 4, TDV-3 is characterized by the amino acid sequence of SEQ ID NO: 6, and TDV-4 is characterized by the amino acid sequence of SEQ ID NO: 8. In a further preferred embodiment, TDV-1 is characterized by a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6, and TDV-4 is characterized by a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8.

[0208] [Table 4]

[0209] Thus, in certain preferred embodiments, the dengue vaccine formulations of the invention described herein comprise live attenuated dengue virus strains TDV-1, TDV-2, TDV-3, and TDV-4, which are based on TDV-2 and contain the prM and E regions of DENV-1, -3, and -4, respectively. In another particularly preferred embodiment, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO:1 and the amino acid sequence set forth in SEQ ID NO:2; TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO:3 and the amino acid sequence set forth in SEQ ID NO:4; TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO:5 and the amino acid sequence set forth in SEQ ID NO:6; and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO:7 and the amino acid sequence set forth in SEQ ID NO:8.

[0210] The DENV-3 E protein has two fewer amino acids than the DENV-2 E protein, so the TDV-2 nucleotide and encoded amino acid backbone, which begin after the DENV-3 E region at nucleotide 2374 of SEQ ID NO:5 and amino acid 760 of SEQ ID NO:6, are 6 nucleotides and 2 amino acids fewer than the original TDV-2 nucleotide and amino acid positions, respectively.

[0211] In a particularly preferred embodiment, the dengue vaccine formulation described herein comprises the tetravalent dengue virus composition "TAK-003."

[0212] Virus concentration The present invention relates, in part, to a dengue vaccine composition, a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) at a concentration of at least 2.7 log pfu / 0.5 mL, or at least 3.1 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) at a concentration of at least 4.5 log pfu / 0.5 mL; and a tetravalent dengue virus composition comprising: Dengue vaccine compositions are directed.

[0213] In one embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain, e.g., TDV-1), preferably at a concentration of at least 3.3 log pfu / 0.5 mL to 3.8 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., a dengue serotype 2 strain, e.g., TDV-2), preferably at a concentration of at least 2.7 log pfu / 0.5 mL, or at least 3.1 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain, e.g., TDV-3), preferably at a concentration of at least 4.0 log pfu / 0.5 mL; and (iv) a dengue serotype 4 strain (e.g., a chimeric dengue serotype 2 / 4 strain, e.g., TDV-4), preferably at a concentration of at least 4.5 log pfu / 0.5 ml, or 4.6 log pfu / 0.5 mL, optionally up to 6.2 log pfu / 0.5 ml; The present invention includes a tetravalent dengue virus composition comprising:

[0214] The invention further relates, in part, to a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 mL, or at least 3.1 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL; and a tetravalent dengue virus composition comprising: A unit dose of a dengue vaccine composition is provided.

[0215] In one embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL to 3.8 log pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 mL, or at least 3.1 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL, or at least 4.6 log pfu / 0.5 mL, optionally up to 6.2 log pfu / 0.5 mL; The present invention includes a tetravalent dengue virus composition comprising:

[0216] Preferably, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4.

[0217] In one embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / 0.5 mL to 5.3 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / 0.5 mL to 5.0 log pfu / 0.5 mL, or 3.1 log pfu / 0.5 mL to 5.0 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / 0.5 mL to 6.0 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 log pfu / 0.5 mL to 6.5 log pfu / 0.5 mL; Includes tetravalent dengue virus compositions.

[0218] In one such embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / 0.5 mL to 5.0 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / 0.5 mL to 4.9 log pfu / 0.5 mL, or 3.1 log pfu / 0.5 mL to 4.9 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / 0.5 mL to 5.7 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / 0.5 mL to 6.2 log pfu / 0.5 mL; Includes tetravalent dengue virus compositions.

[0219] In further such embodiments, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / dose to 5.0 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 4.9 log pfu / dose, or 3.1 log pfu / dose to 4.9 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 5.7 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / dose to 5.5 log pfu / dose; Includes tetravalent dengue virus compositions.

[0220] In further such embodiments, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / dose to 4.1 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 3.6 log pfu / dose, or 3.1 log pfu / dose to 3.6 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 4.7 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / dose to 5.3 log pfu / dose; Includes tetravalent dengue virus compositions.

[0221] In further such embodiments, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / 0.5 mL to 3.6 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / 0.5 mL to 4.0 log pfu / 0.5 mL, or 3.1 log pfu / 0.5 mL to 4.0 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / 0.5 mL to 4.6 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / 0.5 mL, or 4.6 log pfu / 0.5 mL to 5.1 log pfu / 0.5 mL; Includes tetravalent dengue virus compositions.

[0222] In another embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 4.3 log pfu / 0.5 mL to 4.4 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.7 log pfu / 0.5 mL to 3.8 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.5 log pfu / 0.5 mL to 5.0 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.5 log pfu / 0.5 mL to 5.6 log pfu / 0.5 mL; Includes tetravalent dengue virus compositions.

[0223] In another particularly preferred embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.6 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 4.0 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strain) has a concentration of 4.6 log pfu / 0.5 mL; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 log pfu / 0.5 mL; Includes tetravalent dengue virus compositions.

[0224] In another preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is less than 6.7 log pfu / 0.5 mL, preferably less than 5.5 log pfu / 0.5 mL. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 log pfu / 0.5 mL. In a preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is within the range of 4.6 log pfu / 0.5 mL to 6.7 log pfu / 0.5 mL, preferably within the range of 4.6 log pfu / 0.5 mL to 5.5 log pfu / 0.5 mL.

[0225] Preferably, in said embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2, TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4, TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6, and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 8.

[0226] The invention relates, in part, to a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) at a concentration of at least 3.3 log pfu / dose; (ii) dengue serotype 2 (e.g., a dengue serotype 2 strain) at a concentration of at least 2.7 log pfu / dose, or at least 3.1 log pfu / dose; (iii) dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of at least 4.0 log pfu / dose; (iv) dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) at a concentration of at least 4.5 log pfu / dose; and a tetravalent dengue virus composition comprising: A unit dose of a dengue vaccine composition is provided.

[0227] In one embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / dose to 5.3 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 5.0 log pfu / dose, or 3.1 log pfu / dose to 5.0 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 6.0 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / dose to 6.5 log pfu / dose; Includes tetravalent dengue virus compositions.

[0228] In one such embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / dose to 5.0 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 4.9 log pfu / dose, or 3.1 log pfu / dose to 4.9 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 5.7 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / dose to 6.2 log pfu / dose; Includes tetravalent dengue virus compositions.

[0229] In further such embodiments, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / dose to 5.0 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 4.9 log pfu / dose, or 3.1 log pfu / dose to 4.9 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 5.7 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / dose to 5.5 log pfu / dose; Includes tetravalent dengue virus compositions.

[0230] In further such embodiments, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / dose to 4.1 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 3.6 log pfu / dose, or 3.1 log pfu / dose to 3.6 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 4.7 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / dose to 5.3 log pfu / dose; Includes tetravalent dengue virus compositions.

[0231] In further such embodiments, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log pfu / dose to 3.6 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 4.0 log pfu / dose, or 3.1 log pfu / dose to 4.0 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 4.6 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / dose, 4.6 log pfu / dose, to 5.1 log pfu / dose; Includes tetravalent dengue virus compositions.

[0232] In another embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 4.3 log pfu / dose to 4.4 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.7 log pfu / dose to 3.8 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.5 log pfu / dose to 5.0 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 5.5 log pfu / dose to 5.6 log pfu / dose; Includes tetravalent dengue virus compositions.

[0233] In a particularly preferred embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 4.4 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.8 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strain) has a concentration of 4.5 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.6 log pfu / dose; Includes tetravalent dengue virus compositions.

[0234] In another particularly preferred embodiment, the dengue vaccine composition is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.6 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 4.0 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strain) has a concentration of 4.6 log pfu / dose; (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 log pfu / dose; Includes tetravalent dengue virus compositions.

[0235] In another preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is less than 6.7 log pfu / dose, preferably less than 5.5 log pfu / dose. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 log pfu / dose. In a preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is in the range of 4.6 log pfu / dose to 6.7 log pfu / dose, preferably in the range of 4.6 log pfu / dose to 5.5 log pfu / dose.

[0236] In one embodiment, in the composition, (i), (ii), (iii), and (iv) provide a total concentration in pfu / 0.5 mL, based on said concentrations, the concentration of (iii) is at least 10% of the total concentration in pfu / 0.5 mL.

[0237] In one embodiment, in the composition, (i), (ii), (iii), and (iv) provide a total concentration in pfu / 0.5 mL, and based on said total concentration, the concentration of (ii) in pfu / 0.5 mL is less than 10%, the concentration of (iv) in pfu / 0.5 mL is at least 50%, the concentration of (i) in pfu / 0.5 mL is at least 1%, and the concentration of (iii) in pfu / 0.5 mL is at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0238] (iii) The concentration in pfu / 0.5 mL in the reconstituted unit dose is preferably at least 10%.

[0239] In one embodiment, in the composition, (i), (ii), (iii), and (iv) provide a total concentration in pfu / 0.5 mL, and based on said total concentration, the concentration of (ii) in pfu / 0.5 mL is less than 2%, the concentration of (iv) in pfu / 0.5 mL is at least 50%, the concentration of (i) in pfu / 0.5 mL is at least 1%, and the concentration of (iii) in pfu / 0.5 mL is at least 6%.

[0240] Preferably, in said embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2, TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4, TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6, and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 8.

[0241] The concentrations of different dengue viruses are preferably determined by immunofocus assays known in the art. For example, concentrations can be determined by immunofocus assays in which serial dilutions of dengue virus are applied to a monolayer of adherent cells, such as Vero cells. After a period allowing infectious virus to bind and be internalized by the cells, an overlay containing a thickening agent, such as agarose or carboxymethylcellulose, is added to prevent viral spread, so that progeny virus can only infect cells adjacent to the originally infected cell. After an incubation period to allow viral replication, the cells are fixed and stained using a serotype-specific anti-dengue monoclonal antibody and a secondary antibody, such as an antibody labeled with alkaline phosphatase. Bright spots are stained by adding a suitable substrate for the enzyme conjugated to the secondary antibody, such as 5-bromo-4-chloro-3-indolylphosphate / nitroblue tetrazolium phosphatase substrate. The number of plaques on the plate corresponds to the plaque-forming units of virus in the solution applied to the cells. For example, a concentration of 1,000 pfu / μl indicates that 1 μl of solution applied to cells contains enough virus to produce 1,000 plaques in the cell monolayer.

[0242] The dengue vaccine composition includes a tetravalent dengue virus composition containing four live-attenuated dengue virus strains, including a chimeric dengue serotype 2 / 1 strain, a dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain, providing a total concentration of pfu / 0.5 mL. The term "total concentration in pfu / 0.5 mL" or "total concentration in pfu / dose" is defined as the sum of the concentrations of dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain), dengue serotype 2 (e.g., a dengue serotype 2 strain), dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain), and dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain), preferably the sum of the concentrations of TDV-1, TDV-2, TDV-3, and TDV-4, and is defined as 100% dengue virus concentration, determined by pfu (plaque-forming units) in 0.5 mL or in a dose.

[0243] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live-attenuated dengue virus strains, wherein dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain), dengue serotype 2 (e.g., a dengue serotype 2 strain), dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain), and dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) provide a total concentration in pfu / 0.5 mL, and based on said total concentration, the concentration of dengue serotype 2 (e.g., a dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 10% of the total concentration, or less than 8%, or less than 6% of the total concentration, and the concentration of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is at least 50%, or at least 60%, or at least 65% of the total concentration. In one embodiment, based on the total concentration, the concentration of dengue serotype 2 (e.g., dengue serotype 2 strains) measured in pfu / 0.5 mL is 0.3 to 10%, or 0.5 to 8%, of the total concentration, and the concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) measured in pfu / 0.5 mL is 50% to 90%, or 60% to 88% of the total concentration. This means that the concentration of dengue serotype 2 (e.g., dengue serotype 2 strains) is lower than the concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains).

[0244] In one such embodiment, the concentration of dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is at least 1% of the total concentration, and / or the concentration of dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 6% of the total concentration, or at least 7%, or 8%, 10%, 12%, 14%, 16%, or 18% of the total concentration. In one such embodiment, the concentration of dengue serotype 2 (e.g., chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1% to 7%, or 2% to 6%, or 2.0% to 5.0% of the total concentration, and / or the concentration of dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is 6% to 25%, or 7% to 25%, or 10% to 25%, or 18% to 25% of the total concentration. This means that the concentration of dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) is lower than the concentration of dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strain).

[0245] In a preferred embodiment, the concentration of dengue serotype 2 strains, such as TDV-2, measured in pfu / 0.5 mL is less than 10%, preferably less than 6%, or less than 2% of the total concentration; the concentration of dengue serotype 4, such as TDV-4 (e.g., chimeric dengue serotype 2 / 4 strains), measured in pfu / 0.5 mL, is at least 50%, preferably at least 65%, of the total concentration; the concentration of dengue serotype 1, such as TDV-1 (e.g., chimeric dengue serotype 2 / 1 strains), measured in pfu / 0.5 mL, is at least 1%, preferably between 1% and 7%, or 2.0% and 5.0%, of the total concentration; and the concentration of dengue serotype 3, such as TDV-3 (e.g., chimeric dengue serotype 2 / 3 strains), measured in pfu / 0.5 mL, is at least 6%, preferably between 6% and 25%, or 10% and 25%, or 18% and 25% of the total concentration.

[0246] In a further preferred embodiment, a dengue virus composition is provided that includes dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain), dengue serotype 2 (e.g., a dengue serotype 2 strain), dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 3 strain), and dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain), such as TDV-1, TDV-2, TDV-3, and TDV-4, wherein the concentration of dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain), measured in pfu / 0.5 mL, is The concentration of dengue serotype 2 (e.g., dengue serotype 2 strains), measured in pfu / 0.5 mL, is at least 1%, preferably 1%-7%, or 2.0%-5.0% of the total concentration; the concentration of dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains), measured in pfu / 0.5 mL, is at least 6%, preferably between 6%-25%, or 10%-25%, or 18%-25% of the total concentration. Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains), is particularly preferred, as it has the highest concentration of all four dengue serotypes.

[0247] In a further preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the concentration of dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1% to 7% of the total concentration, the concentration of dengue serotype 2 (e.g., a dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 8% of the total concentration, for example, in the range of 1% to 8% of the total concentration, the concentration of dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 10% of the total concentration, and the concentration of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is at least 65% of the total concentration, for example, in the range of 65% to 80%. In certain such embodiments, the arithmetic sum of all four serotypes is in the range of 4.6 log10 pfu / 0.5 mL to 6.7 log10 pfu / 0.5 mL, preferably in the range of 4.6 log10 pfu / 0.5 mL to 5.5 log10 pfu / 0.5 mL.

[0248] In further preferred embodiments, dengue serotype 1, such as TDV-1 (e.g., a chimeric dengue serotype 2 / 1 strain), and dengue serotype 2, such as TDV-2 (e.g., a dengue serotype 2 strain), are each present at a concentration within 5 percentage points of each other, based on total concentration in pfu / 0.5 mL, and / or together account for less than about 10% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), is preferably present at at least about 10% of the total concentration in pfu / 0.5 mL, and more preferably, dengue serotype 4, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), is present at at least about 70% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 4, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), represents the highest concentration in the composition of all four serotypes, preferably at least about 70% of the total concentration in pfu / 0.5 mL; dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), represents the second highest concentration in the composition of all four serotypes, preferably at least about 10% of the total concentration in pfu / 0.5 mL; and dengue serotype 1, such as TDV-1 (e.g., a chimeric dengue serotype 2 / 1 strain), and dengue serotype 2, such as TDV-2 (e.g., a dengue serotype 2 strain), each represent a concentration lower than that of serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), and optionally together represent less than about 10% of the total concentration in pfu / 0.5 mL.

[0249] Preferably, in said embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2, TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4, TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6, and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 8.

[0250] According to a further embodiment, the chimeric dengue serotype 2 / 4 strain, preferably TDV-4, has the highest concentration in the dengue vaccine composition, followed by the chimeric dengue serotype 2 / 3 strain, preferably TDV-3, then the chimeric dengue serotype 2 / 1 strain, preferably TDV-1, then the dengue serotype 2 strain, preferably TDV-2. It is particularly preferred that the dengue serotype 2 strain has the lowest concentration of the four strains present in the dengue vaccine composition.

[0251] Whenever a concentration / 0.5 mL is mentioned, this does not limit the volume of the unit dose described herein to 0.5 mL. 0.5 mL is the reference volume for determining the concentration of the virus strain in the composition in pfu / ml. The volume and / or amount per unit dose is described in the respective chapters.

[0252] Virus Stability The dengue vaccine formulations described herein provide excellent stability, e.g., during lyophilization and storage, of a tetravalent dengue virus composition (e.g., "TAK-003") comprising live attenuated dengue virus serotype 1, live attenuated dengue virus serotype 2, live attenuated dengue virus serotype 3, and live attenuated dengue virus serotype 4.

[0253] In one embodiment, the amount of dengue virus in a dengue vaccine formulation described herein before lyophilization differs from the amount of dengue virus immediately after lyophilization by less than 0.46 log pfu / unit dose, preferably less than 0.4 log pfu / unit dose, more preferably less than 0.3 log pfu / unit dose, or less than 0.25 log pfu / unit dose for each of serotypes 1 to 4. Thus, in a preferred embodiment, the lyophilization loss for each of serotypes 1 to 4 in a dengue vaccine formulation described herein is less than 0.4 log pfu / unit dose.

[0254] In one embodiment, the amount of dengue virus in a dengue vaccine formulation described herein immediately after lyophilization and after lyophilization and storage for 12 weeks at about 25° C. and 65% relative humidity differs by less than 1.6 log pfu / unit dose, preferably less than 1.4 log pfu / unit dose, more preferably less than 1.2 log pfu / unit dose, or less than 1.0 log pfu / unit dose for each of serotypes 1 to 4. Thus, in a preferred embodiment, the loss of stability upon storage at accelerated conditions for 12 weeks is less than 1.4 log pfu / unit dose for each of serotypes 1 to 4.

[0255] In one embodiment, the amount of dengue virus in a dengue vaccine formulation described herein immediately after lyophilization and after lyophilization and storage for 12 months at 2-8° C. differs by less than 0.44 log pfu / unit dose, preferably less than 0.4 log pfu / unit dose, and more preferably less than 0.35 log pfu / unit dose for each of serotypes 1 to 4. Thus, in a preferred embodiment, the loss of stability after 12 months of storage at 2-8° C. is less than 0.4 log pfu / unit dose for each of serotypes 1 to 4 in a dengue vaccine formulation described herein.

[0256] In one embodiment, the amount of dengue virus in a dengue vaccine formulation described herein immediately after lyophilization and after lyophilization and storage for 18 months at 2-8° C. differs by less than 0.7 log pfu / unit dose, preferably less than 0.6 log pfu / unit dose, and more preferably less than 0.5 log pfu / unit dose for each of serotypes 1 to 4. Thus, in a preferred embodiment, the loss of stability after 18 months of storage at 2-8° C. is less than 0.6 log pfu / unit dose for each of serotypes 1 to 4 in a dengue vaccine formulation described herein.

[0257] In one embodiment, the amount of dengue virus in a dengue vaccine formulation described herein immediately after lyophilization and after lyophilization and 24 months of storage at 2-8° C. differs by less than 0.7 log pfu / unit dose, preferably less than 0.6 log pfu / unit dose, and more preferably less than 0.5 log pfu / unit dose for each of serotypes 1 to 4. Thus, in a preferred embodiment, the loss of stability after 24 months of storage at 2-8° C. is less than 0.6 log pfu / unit dose for each of serotypes 1 to 4 in a dengue vaccine formulation described herein.

[0258] In one embodiment, the amount of dengue virus in a dengue vaccine formulation described herein immediately after lyophilization and after lyophilization and storage for 24 months at -20°C differs by less than 0.2 log pfu / unit dose, preferably less than 0.15 log pfu / unit dose, and more preferably less than 0.1 log pfu / unit dose for each of serotypes 1 to 4. Thus, in a preferred embodiment, the loss of stability after 24 months of storage at 2-8°C is less than 0.15 log pfu / unit dose for each of serotypes 1 to 4 in a dengue vaccine formulation described herein.

[0259] In one embodiment, the lyophilized dengue vaccine formulation described herein has a shelf life of at least 2 years at 2-8° C., preferably greater than 2 years at 2-8° C. In certain such embodiments, the end of shelf life (ESL) specifications for dengue serotypes 1-4 are at least 3.3 log PFU / dose for serotype 1, at least 2.7 log PFU / dose for serotype 2, at least 4.0 log PFU / dose for serotype 3, and at least 4.5 log PFU / dose for serotype 4. In certain other embodiments, the end of shelf life (ESL) specifications for dengue serotypes 1-4 are at least 3.3 log PFU / dose for serotype 1, at least 3.1 log PFU / dose for serotype 2, at least 4.0 log PFU / dose for serotype 3, and at least 4.5 log PFU / dose for serotype 4.

[0260] In one embodiment, the lyophilized dengue vaccine formulation described herein provides stability that qualifies for Vaccine Vial Monitor (VVM) Type 7. VVM Type 7 conditions are: 7 days at 37°C, 45 days at 25°C, and greater than 2 years at 2°C-8°C. The VVM is a small indicator attached to the vaccine vial that changes color as the vaccine is exposed to cumulative heat, informing healthcare professionals whether the vaccine has exceeded a pre-set limit at which it should not be used for immunization. Depending on the stability of the vaccine when exposed to heat, the vaccine is assigned a VVM category.

[0261] In one embodiment, the lyophilized dengue vaccine formulation meets end of shelf life (ESL) specifications for dengue serotypes when exposed to 37° C. for 7 days, 25° C. for 45 days, or 2° C.-8° C. for more than 2 years. In such an embodiment, the end of shelf life (ESL) specifications for dengue serotypes 1-4 are at least 3.3 log PFU / dose for serotype 1, at least 2.7 log PFU / dose or at least 3.1 log PFU / dose for serotype 2, at least 4.0 log PFU / dose for serotype 3, and at least 4.5 log PFU / dose for serotype 4.

[0262] In one embodiment, the liquid dengue vaccine formulation described herein loses less than 1.0 log pfu / unit dose of dengue virus stability upon 30 days of storage at 2-8°C for each of serotypes 1-4.

[0263] Manufacturing method The present invention is directed, in part, to a method for preparing a dengue vaccine formulation in a dried form, the method comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form; (b) freezing the dengue vaccine formulation in aqueous form to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature ranging from above the glass transition temperature Tg of the formulation to not more than 25°C above, or not more than 20°C above, or not more than 15°C above, the glass transition temperature Tg of the formulation, to form a primary dried product; and (d) subjecting the primary dried product to a secondary drying step at a pressure of less than 1 mbar in an apparatus having a storage temperature above the storage temperature of the primary drying step to form a secondary dried product.

[0264] The present invention is particularly directed, in part, to a method for preparing a dengue vaccine formulation as described herein in dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form, the dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4. a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose; b. poloxamer; c. urea; d. arginine hydrochloride; e. tromethamine; and f. human serum albumin. and an excipient composition comprising: (b) freezing the aqueous form of the dengue vaccine formulation in a device having a storage temperature of less than -35°C to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature of about -31 to -19°C, or about -31 to -21°C, to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of at least 15°C at a pressure of less than 1 mbar, preferably less than 0.1 mbar, to form a secondary dried product; The present invention is directed to a method, including:

[0265] The present invention is particularly directed, in part, to a method for preparing a dengue vaccine formulation as described herein in dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form, the dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4. a tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose; b. poloxamer; c. urea; d. arginine hydrochloride; e. tromethamine; and f. human serum albumin. and an excipient composition comprising: (b) freezing the aqueous form of the dengue vaccine formulation in a device having a storage temperature of less than -35°C to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature of about -31 to -19°C, or about -31 to -21°C, to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of at least 20°C at a pressure of less than 1 mbar to form a secondary dried product; The present invention is directed to a method, including:

[0266] In one embodiment, the freezing step (b) in the method described in the preceding paragraph is carried out in an apparatus having a storage temperature below -35°C, or below -40°C, preferably between about -40 and -50°C, for example about -45°C.

[0267] In certain embodiments, the dengue vaccine formulation in aqueous form is subjected to freezing step b) of the method described in the preceding paragraph in containers, e.g., vials, each containing a plurality (e.g., 5) of unit doses of the dengue vaccine formulation. In certain such embodiments, the dengue vaccine formulation in aqueous form is subjected to freezing step b) in a volume that is less than the volume that would be used to reconstitute 5 unit doses of the lyophilized dengue vaccine formulation. In certain preferred embodiments, the volume of the dengue vaccine formulation in aqueous form that is subjected to freezing step b) is about half the volume that would be used to reconstitute the lyophilized dengue vaccine formulation.

[0268] In certain other embodiments, the dengue vaccine formulation in aqueous form is subjected to freezing step b) of the method described in the preceding paragraph in containers, e.g., vials, each containing a unit dose of the dengue vaccine formulation. In certain such embodiments, the dengue vaccine formulation in aqueous form is subjected to freezing step b) in a volume that is less than the volume used to reconstitute a unit dose of the lyophilized dengue vaccine formulation. In certain preferred embodiments, the volume of the dengue vaccine formulation in aqueous form that is subjected to freezing step b) is about half the volume used to reconstitute the lyophilized dengue vaccine formulation.

[0269] In one embodiment, the dengue vaccine formulation in aqueous form used in the drying method described in the preceding paragraph comprises: about 9-11% w / v, for example, about 10% w / v, of trehalose; about 0.2-0.4% w / v, for example, about 0.3% w / v, of poloxamer; about 0.2 to 0.4% w / v, for example, about 0.3% w / v, of urea; About 0.2 to 0.3% w / v, for example, about 0.25% w / v, of arginine hydrochloride; about 0.05 to 0.15% w / v, for example, about 0.1% w / v, of tromethamine; about 0.05 to 0.15% w / v, for example, about 0.1% w / v, of human serum albumin; Includes:

[0270] In one embodiment, the primary drying step (c) of the method described in the preceding paragraph is carried out in an apparatus having a storage temperature of about −31° C. to −19° C., or about −31 to −21° C., preferably about −29 to −25° C., e.g., about −27° C., optionally at a pressure of less than 0.05 mbar, preferably about 0.01 to 0.05 mbar, or about 0.04 mbar. In certain such embodiments, the primary drying step (c) is carried out for a period of about 20 to 120 hours, preferably about 30 to 100 hours, more preferably about 40 to 70 hours, or about 50 to 70 hours.

[0271] In a preferred embodiment, the primary drying step (c) of the method described in the preceding paragraph is carried out in an apparatus having a storage temperature of about −31° C. to −21° C., such as about −27° C. or about −24° C., at a pressure of less than 0.05 mbar, preferably about 0.01 to less than 0.05 mbar, such as about 0.03 mbar.

[0272] In one embodiment, secondary drying step (d) is carried out in an apparatus having a storage temperature of at least 15° C., or at least 20° C., or about 15-40° C., or about 20-40° C., or about 25° C., optionally at a pressure of less than 0.5 mbar, preferably less than about 0.1-0.4 mbar, or about 0.3 mbar. In certain such embodiments, secondary drying step (d) is carried out for a period of about 1-15 hours, preferably about 3-10 hours, more preferably about 6-10 hours, or about 7-9 hours.

[0273] In a preferred embodiment, the secondary drying step (d) is carried out in an apparatus having a storage temperature of at least 15°C, e.g., 25°C, at a pressure of less than 0.05 mbar, preferably less than about 0.01-0.04 mbar, e.g., about 0.03 mbar.

[0274] In preferred embodiments, the same pressure is used in the primary drying step and the secondary drying step, and in certain such embodiments, the pressure in the primary drying step and the secondary drying step is about 0.025 mbar to 0.035 mbar, for example, about 0.030 mbar.

[0275] In one embodiment, the primary and secondary drying steps in the method described in the preceding paragraph are carried out for a period of less than 160 hours, preferably about 50-120 hours, more preferably less than about 50-100 hours, or about 60-90 hours.

[0276] In a preferred embodiment, steps b), c), and d) of the method for preparing a lyophilized dengue vaccine formulation described in the preceding paragraph are carried out for a period of less than 120 hours, preferably less than about 50-100 hours, more preferably less than about 40-90 hours.

[0277] In one embodiment, the primary dried product of the method described in the preceding paragraph has a residual moisture content of about 5-10% w / w, or less than 10% w / w, as determined by the Karl Fischer method. In one embodiment, the secondary dried product has a residual moisture content of 3.0% w / w or less, or about 0.01-2% w / w, preferably less than 2% w / w, and more preferably less than 1% w / w, as determined by the Karl Fischer method.

[0278] Prevention methods and uses The present invention is directed, in part, to methods for preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject. Accordingly, in certain embodiments, the present invention is directed to methods for preventing dengue disease in a subject, comprising administering to the subject a unit dose, particularly a reconstituted unit dose, of a dengue vaccine formulation described herein, e.g., by subcutaneous injection or intramuscular administration.

[0279] The present invention relates, inter alia, to a method for preventing dengue disease in a subject, comprising administering a primary vaccination with only two administrations of a unit dose of a dengue vaccine formulation described herein, comprising the following steps: administering to the subject a first unit dose of the dengue vaccine formulation; administering to the subject a second unit dose of the dengue vaccine formulation within three months of administration of the first unit dose; and optionally administering a booster dose at least twelve months after administration of said second unit dose of the dengue vaccine.

[0280] The present invention is further directed, in part, to a dengue vaccine formulation described herein for use in a method for preventing dengue disease (particularly virologically confirmable dengue, VCD) in a subject. Accordingly, in certain embodiments, the present invention is directed to a dengue vaccine formulation described herein for use in a method for preventing dengue disease in a subject, the method comprising administering to the subject a unit dose, particularly a reconstituted unit dose, of the dengue vaccine formulation described herein, e.g., by subcutaneous injection or intramuscular administration.

[0281] The present invention is particularly directed, in part, to a dengue vaccine formulation as described herein for use in a method for preventing dengue disease in a subject, the method comprising administering a primary vaccination in only two administrations of unit doses, the method comprising the following steps: administering to the subject a first unit dose of the dengue vaccine formulation, e.g., within three months of administration of the first unit dose, administering to the subject a second unit dose of the dengue vaccine formulation, and optionally administering a booster dose at least twelve months after administration of the second unit dose of the dengue vaccine.

[0282] The present invention is further directed, in part, to the use of a dengue vaccine formulation described herein for the manufacture of a medicament for preventing dengue disease (particularly virologically confirmable dengue, VCD) in a subject. Accordingly, in certain embodiments, the present invention is directed to the use of a dengue vaccine formulation described herein for the manufacture of a medicament for preventing dengue disease in a subject by administering to the subject a unit dose of the dengue vaccine formulation, particularly a reconstituted unit dose, for example, by subcutaneous injection or intramuscular administration.

[0283] The present invention is particularly directed, in part, to the use of a dengue vaccine formulation described herein for the manufacture of a medicament for preventing dengue disease in a subject by administering a primary vaccination in only two administrations of unit doses, comprising: administering to the subject a first unit dose of the dengue vaccine formulation; and administering to the subject a second unit dose of the dengue vaccine formulation, e.g., within three months of administration of the first unit dose; and optionally, administering a booster dose at least twelve months after administration of the second unit dose of the dengue vaccine. [Example]

[0284] The following examples are included to illustrate certain aspects and embodiments of the claimed invention. However, it should be understood by those skilled in the art that the following descriptions are merely illustrative and should not be construed as limiting the invention in any way. The concentrations and amounts shown in the tables below have been rounded to, for example, three significant digits or less.

[0285] Preparation of Dengue Virus Strains and Tetravalent Dengue Virus Compositions

[0286] The methods used to generate the chimeric dengue strains TDV-1, -3, and -4 were standard molecular cloning and DNA engineering methods, as described in Huang et al. (2003) J. Virology 77(21):11436-11447. The following well-known methods were used to construct and introduce the prM-E genes of dengue serotypes 1, 3, and 4 into the TDV-2 backbone: reverse transcriptase PCR (RT-PCR), PCR, restriction enzyme digestion, DNA fragment ligation, bacterial transformation by electroporation, plasmid DNA preparation, in vitro transcription with T7 RNA polymerase, and transfection of Vero cells by electroporation. Growth and purification of the different dengue serotypes were performed as described in Huang et al. (2013) PLOS Neglected Dis, 7(5):e2243.

[0287] Chimeric dengue strains TDV-1, -3, and -4 were combined with TDV-2 to form tetravalent dengue virus compositions at the concentrations described herein.

[0288] Example 1 Preparation of an aqueous dengue vaccine formulation

[0289] The tetravalent dengue virus composition was combined with pharmaceutically acceptable excipients to obtain an aqueous dengue vaccine formulation containing the excipient concentrations shown in Table 5.

[0290] [Table 5]

[0291] Preparation of a dry form of dengue vaccine formulation

[0292] To provide 5 unit doses of the dengue vaccine formulation, 1.65 mL of the aqueous dengue vaccine formulation was lyophilized in vials according to the process shown in Table 6.

[0293] [Table 6]

[0294] The excipient composition of the lyophilized dengue vaccine formulation is shown in Table 7. The residual moisture content determined by Karl Fischer method was 0.58% w / w.

[0295] [Table 7]

[0296] The cake characteristics of the lyophilized dengue vaccine formulation are summarized in Table 8.

[0297] [Table 8]

[0298] The cake, i.e., the lyophilized dengue vaccine formulation, was reconstituted with 3.25 mL of 37 mM aqueous sodium chloride and appeared clear and colorless after 70 seconds with a ring of air bubbles on the surface. The observed pH was measured according to the United States Pharmacopeial Convention's USP for pH measurement. <791> According to the test method, the observed osmolality was 7.6. <785> According to the test method, it was about 300 mOsmol / kg. The excipient concentrations in the reconstituted dengue vaccine formulations are shown in Table 9.

[0299] [Table 9]

[0300] Example 2 A dengue vaccine formulation containing 10 mM tromethamine (0.12% w / v) prior to lyophilization was prepared using a combination of tromethamine and tromethamine hydrochloride (Tris-HCl) using the process described in Example 1. The excipient composition of the lyophilized dengue vaccine formulation is shown in Table 10.

[0301] [Table 10]

[0302] One vial contains five unit doses of the dengue vaccine formulation, as described in Example 1. At the time of administration, the vial is reconstituted with the entire contents of the vial or a pre-filled syringe of diluent (e.g., 37 mM aqueous sodium chloride), from which five 0.5 mL doses of vaccine can be withdrawn and administered.

[0303] After reconstitution of the vial with 3.38 mL of 37 mM aqueous sodium chloride to a volume of approximately 3.49 mL, each 0.5 mL dose contains, for example, approximately 0.47 g / L of human serum albumin (1.65 mg / vial: 3.49 mL), or approximately 0.24 mg of human serum albumin (0.47 g / L x 0.5 mL).

[0304] Example 3 Preparation of an aqueous dengue vaccine formulation

[0305] The tetravalent dengue virus composition was combined with pharmaceutically acceptable excipients to obtain an aqueous dengue vaccine formulation containing the excipient concentrations shown in Table 11.

[0306] [Table 11]

[0307] Preparation of a dry form of dengue vaccine formulation

[0308] To provide 5 unit doses of the dengue vaccine formulation, 1.65 mL of the aqueous dengue vaccine formulation was lyophilized in vials according to the process shown in Table 12. Upon successful completion of the pressure rise test, the chamber pressure is increased to 700 mbar using dry nitrogen (N2) for stoppering. In preparation for stoppering, the storage temperature is reduced to 5°C. The total lyophilization time from 5°C equilibration to the stoppering step is approximately 72 hours.

[0309] [Table 12]

[0310] The excipient composition of the lyophilized dengue vaccine formulation is shown in Table 13.

[0311] [Table 13]

[0312] The excipient concentrations in the reconstituted dengue vaccine formulation after reconstitution with 3.38 mL of 37 mM aqueous sodium chloride to a volume of approximately 3.49 mL are shown in Table 14.

[0313] [Table 14]

[0314] The physiochemical properties of the dengue vaccine formulations are summarized in Table 15.

[0315] [Table 15]

[0316] Example 4 A dengue vaccine formulation containing 10 mM tromethamine (0.12% w / v) before lyophilization was prepared using a combination of tromethamine and tromethamine hydrochloride (Tris-HCl) using the process described in Example 3. The excipient concentrations in the dengue vaccine formulation before lyophilization are shown in Table 16.

[0317] [Table 16]

[0318] To provide 5 unit doses of the dengue vaccine formulation, 1.65 mL of the aqueous dengue vaccine formulation was lyophilized in vials. The total lyophilization cycle time was approximately 77 hours. The excipient composition of the lyophilized dengue vaccine formulation is shown in Table 17.

[0319] [Table 17]

[0320] The excipient concentrations in the reconstituted dengue vaccine formulation after reconstitution with 3.38 mL of 37 mM aqueous sodium chloride to a volume of approximately 3.49 mL are shown in Table 18.

[0321] [Table 18]

[0322] To provide one unit dose of the dengue vaccine formulation, 0.33 mL of the aqueous form of the dengue vaccine formulation was lyophilized in a vial. The total lyophilization cycle time was approximately 49 hours. The excipient composition of the lyophilized dengue vaccine formulation is shown in Table 19.

[0323] [Table 19]

[0324] The excipient concentrations in the reconstituted dengue vaccine formulation after reconstitution with 0.68 mL of 37 mM aqueous sodium chloride to a volume of approximately 0.70 mL are shown in Table 20.

[0325] [Table 20]

[0326] The composition of the dengue vaccine formulation prepared according to Example 4 is shown in Table 21. The physicochemical properties of the dengue vaccine formulation correspond to those described in Table 15 above.

[0327] [Table 21]

[0328] Comparative Example Preparation of an aqueous dengue virus control formulation

[0329] The tetravalent dengue virus composition was combined with pharmaceutically acceptable excipients to provide an aqueous dengue vaccine control formulation ("Control") containing the excipient concentrations shown in Table 22.

[0330] [Table 22]

[0331] Preparation of a dry form dengue virus control formulation

[0332] To provide 5 unit doses of the dengue vaccine control formulation, 3.25 mL of the aqueous form of the dengue vaccine control formulation was lyophilized in vials according to the process shown in Table 23.

[0333] [Table 23]

[0334] The excipient composition of the lyophilized dengue vaccine control formulation is shown in Table 24. The residual moisture content determined by Karl Fischer method was 0.77% w / w.

[0335] [Table 24]

[0336] The cake characteristics of the lyophilized dengue vaccine control formulation are summarized in Table 25.

[0337] [Table 25]

[0338] The cake, i.e., the lyophilized dengue vaccine control formulation, was reconstituted with 3.25 mL of 37 mM aqueous sodium chloride and appeared clear and colorless after 20 seconds with a ring of air bubbles on the surface. The observed pH was measured according to the United States Pharmacopeial Convention's USP for pH Measurement. <791> According to the test method, the observed osmolality was 7.6. <785> According to the test method, it was approximately 800 mOsmol / kg. The excipient concentrations in the reconstituted control formulation are shown in Table 26.

[0339] [Table 26]

[0340] [Table 27]

[0341] [Table 28]

[0342] Stability Measurements The virus concentration is determined by immune focus assay, which yields plaque-forming units (pfu). In one example, immune focus assay is performed as described in detail in section 2.5 of Brewoo et al. (Vaccine. 2012 February 14; 30(8): 1513-1520. doi:10.1016 / j.vaccine.2011.11.072.). Each sample was measured in triplicate, and the results were reported as the average of the values.

[0343] Freeze drying loss

[0344] The log10 loss in viral potency due to lyophilization was determined for the dengue vaccine formulation prepared according to Example 1 and the dengue vaccine control formulation prepared according to the Comparative Example. The results are shown in Table 29.

[0345] [Table 29]

[0346] stability loss

[0347] The log loss in viral titer was determined for the dengue vaccine formulation prepared according to Example 1 and the dengue vaccine control formulation prepared according to the Comparative Example at 1, 2, 4, 6, 8, and 12 weeks of storage at a temperature of 25° C. and a relative humidity RH of 65%. The results at 12 weeks are shown in Table 30.

[0348] [Table 30]

[0349] The log loss in viral titer was determined for the dengue vaccine formulation prepared according to Example 1 and the dengue vaccine control formulation prepared according to the Comparative Example upon storage for 3, 6, 9, and 12 months at temperatures between 2 and 8° C. The results are shown in Table 31 and Figures 1 and 2.

[0350] [Table 31]

[0351] The log loss in viral titer was determined for the dengue vaccine formulation prepared according to Example 3 and the dengue vaccine control formulation prepared according to the Comparative Example after storage for 6, 9, 12, 18, and 24 months at temperatures between 2 and 8° C. The results are shown in Table 32 and Figures 4 and 5.

[0352] [Table 32]

[0353] The log loss in viral titer was determined for the dengue vaccine formulation prepared according to Example 3 and the dengue vaccine control formulation prepared according to the Comparative Example at a temperature of -20°C over 24 months of storage. The results are shown in Table 33.

[0354] [Table 33]

[0355] The dengue vaccine formulations prepared according to Example 4 and the dengue vaccine control formulations prepared according to the Comparative Example were tested for time in solution (TIS) duration up to 36 hours. The TIS after formulation begins at the completion of the formulation addition and ends at the start of the lyophilization process. The results are shown in Table 34.

[0356] [Table 34]

[0357] The post-formulation TIS degradation rate for all TDV serotypes is improved for the formulations according to the invention compared to the control formulation. TDV3 shows the greatest improvement, with a loss rate 3.1 times lower than the post-formulation TIS for the control formulation. TDV2, the second best improvement, showed a loss rate 1.84 times lower than the post-formulation TIS for the control formulation. TDV1 and TDV4 showed slight improvements in loss rate.

[0358] comparative study The purpose of this study was to evaluate the performance and property differences between a formulation according to the present invention and formulation 20 proposed in US2020 / 0390877, Example 11. The excipient concentrations of formulation 20 are shown in Table 35.

[0359] [Table 35]

[0360] To compare the stabilizing effect of formulation 20 and a formulation of the present invention against dengue viruses TDV1, TDV2, TDV3, and TDV4, a drug substance was formulated in formulation 20 (hereinafter "Comparative Formulation 20") and tested against a formulation of the present invention according to Example 4 disclosed herein. As shown in Table 36, the final concentration of the drug substance was the same in both formulations.

[0361] [Table 36]

[0362] Preliminary observations

[0363] The excipient composition of Formulation 20 was more than twice as viscous as the excipient composition of Example 4. While 1 L of the excipient composition of Example 4 could be filtered through a 0.22 μm filter for sterilization, the excipient composition of Formulation 20 clogged the 0.22 μm cup filter after filtering 300 mL, and the entire volume was filtered using two 1 L 0.22 μm filter cups and one 500 mL 0.22 μm filter cup. The filter clogging may be due to the high viscosity of the excipient composition of Formulation 20 and / or the presence of excipients such as sodium carboxymethylcellulose.

[0364] Each excipient composition was combined with a tetravalent dengue virus composition to produce an aqueous dengue vaccine formulation. The biophysical properties of the aqueous dengue vaccine formulations are shown in Table 37.

[0365] [Table 37]

[0366] Stability Measurements

[0367] The liquid stability of Comparative Formulation 20 and the formulation according to Example 4 was evaluated for efficacy using an immunofocus assay (IFA) at 2-8°C for a total of 30 days. Samples were taken at the following time points: Day 0 and Day 30. The Day 0 sample was frozen at -80°C until testing. The results are shown in Table 38 and Figure 6.

[0368] [Table 38]

[0369] Comparative formulation 20 showed significantly higher potency loss after 30 days compared to the formulation according to Example 4.

[0370] Further provisions of the present invention

[0371] The present invention relates in particular to the following further provisions:

[0372] 1. A dengue vaccine formulation comprising: a tetravalent dengue virus composition comprising a live attenuated dengue virus serotype 1, a live attenuated dengue virus serotype 2, a live attenuated dengue virus serotype 3, and a live attenuated dengue virus serotype 4; at least one non-reducing disaccharide; at least one poloxamer; Urea and at least one amino acid having a positively charged side chain at neutral pH; Tromethamine and Human serum albumin, The dengue vaccine formulation comprising:

[0373] 2. The dengue vaccine formulation of clause 1, wherein the at least one non-reducing disaccharide is trehalose and the at least one amino acid having a positively charged side chain at neutral pH is arginine hydrochloride.

[0374] 3. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1, e.g., a chimeric dengue serotype 2 / 1 strain, (ii) a live attenuated dengue virus serotype 2, e.g., a dengue serotype 2 strain; (iii) a live attenuated dengue virus serotype 3, e.g., a chimeric dengue serotype 2 / 3 strain; (iv) a live attenuated dengue virus serotype 4, e.g., a chimeric dengue serotype 2 / 4 strain; the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; the excipient composition comprising: The dengue vaccine formulation comprising:

[0375] 4. The dengue vaccine formulation according to any one of clauses 1 to 3, wherein the arginine hydrochloride is contained in the dengue vaccine formulation in an amount of about 0.1 to 5.0% w / w, preferably about 0.5 to 5.0% w / w, more preferably about 1.0 to 4.0% w / w, particularly preferably about 2.0 to 3.0% w / w, for example, about 2.4% w / w, based on the dry weight of the vaccine formulation.

[0376] 5. The dengue vaccine formulation according to any one of clauses 1 to 4, wherein the arginine hydrochloride is contained in the dengue vaccine formulation in an amount of about 0.1 to 2.0 mg / unit dose, preferably about 0.2 to 1.5 mg / unit dose, or about 0.5 to 1.2 mg / unit dose, more preferably about 0.5 to 1.0 mg / unit dose, and particularly preferably about 0.6 to 0.8 mg / unit dose.

[0377] 6. The dengue vaccine formulation according to any one of clauses 1 to 5, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form comprises said arginine hydrochloride at a concentration of about 0.5 to 5.0 g / L, preferably 0.5 to less than 2.5 g / L, more preferably about 1.3 to 1.65 g / L, particularly preferably 1.65 g / L or less, for example about 1.4 g / L.

[0378] 7. The dengue vaccine formulation of any one of clauses 2 to 6, wherein said arginine hydrochloride is L-arginine hydrochloride.

[0379] 8. The dengue vaccine formulation according to any one of clauses 1 to 7, wherein the urea is contained in the dengue vaccine formulation in an amount of about 0.1 to 6.0% w / w of urea, preferably about 0.5 to 5.0% w / w, more preferably about 1.0 to 4.0% w / w, particularly preferably about 2.0 to 4.0% w / w, for example, about 2.9% w / w, based on the dry weight of the vaccine formulation.

[0380] 9. The dengue vaccine formulation according to any one of clauses 1 to 8, wherein the urea is contained in the dengue vaccine formulation in an amount of about 0.1 to 3.0 mg / unit dose, preferably about 0.1 to 2.0 mg / unit dose, or about 0.3 to 1.5 mg / unit dose, more preferably about 0.6 to 1.2 mg / unit dose, and particularly preferably about 0.75 to 1.0 mg / unit dose.

[0381] 10. The dengue vaccine formulation according to any one of clauses 1 to 9, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form contains the urea at a concentration of about 0.1 to 6.0 g / L, preferably less than 0.5 to 3.0 g / L, more preferably about 1.5 to 2.0 g / L, and particularly preferably about 2.0 g / L or less, for example about 1.65 g / L.

[0382] 11. The dengue vaccine formulation according to any one of clauses 1 to 10, wherein the human serum albumin is contained in the dengue vaccine formulation in an amount of about 0.05 to 3.0% w / w, preferably about 0.1 to 2.0% w / w, more preferably about 0.1 to 1.5% w / w, and particularly preferably about 0.1 to 1.0% w / w, or 1.0% w / w or less, based on the dry weight of the vaccine formulation.

[0383] 12. The dengue vaccine formulation according to any one of clauses 1 to 11, wherein the human serum albumin is contained in the dengue vaccine formulation in an amount of about 0.05 to 0.5 mg / unit dose, preferably about 0.1 to 0.4 mg / unit dose, more preferably less than 0.4 mg / unit dose, and particularly preferably about 0.25 to 0.33 mg / unit dose, or 0.3 mg / unit dose or less.

[0384] 13. The dengue vaccine formulation of any one of clauses 1 to 12, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises the human serum albumin at a concentration of less than 0.1 to 1 g / L, preferably about 0.3 to 0.9 g / L, more preferably about 0.5 to 0.7 g / L, and particularly preferably 0.7 g / L or less, for example about 0.6 g / L.

[0385] 14. The dengue vaccine formulation of any one of clauses 1 to 13, wherein said human serum albumin is native human serum albumin.

[0386] 15. The dengue vaccine formulation of any one of clauses 1 to 14, wherein said human serum albumin comprises recombinant human serum albumin.

[0387] 16. The dengue vaccine formulation according to any one of clauses 1 to 15, wherein the trehalose is contained in the dengue vaccine formulation in an amount of about 65 to 95% w / w, preferably about 70 to 95% w / w, more preferably about 75 to 95% w / w, and particularly preferably at least 80% w / w, for example, 80 to 95% w / w, or 80 to 90% w / w, or 85 to 95% w / w, or 85 to 90% w / w, or about 87% w / w, based on the dry weight of the vaccine formulation.

[0388] 17. The dengue vaccine formulation according to any one of clauses 1 to 16, wherein the trehalose is contained in the dengue vaccine formulation in an amount of about 20 to 34 mg / unit dose, preferably about 22 to 32 mg / unit dose, more preferably about 23 mg to 30 mg / unit dose, and particularly preferably about 23 to 27 mg / unit dose, or 27 mg / unit dose or less.

[0389] 18. The dengue vaccine formulation of any one of clauses 1 to 17, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises the trehalose at a concentration of 40 to 80 g / L, preferably about 40 to 70 g / L, more preferably about 45 to 60 g / L, and particularly preferably 60 g / L or less, for example about 55 g / L.

[0390] 19. The dengue vaccine formulation of any one of clauses 2 to 18, wherein said trehalose is α,α-trehalose.

[0391] 20. The dengue vaccine formulation according to any one of clauses 1 to 19, wherein the poloxamer is contained in the dengue vaccine formulation in an amount of about 0.05 to 5.0% w / w, preferably about 0.1 to 5.0% w / w, more preferably about 0.5 to 4.0% w / w, and particularly preferably less than 4.0% w / w or about 2.9% w / w or less, based on the dry weight of the vaccine formulation.

[0392] 21. The dengue vaccine formulation according to any one of clauses 1 to 20, wherein the poloxamer is contained in the dengue vaccine formulation in an amount of about 0.1 to 3.0 mg / unit dose, preferably about 0.3 to 1.5 mg / unit dose, more preferably about 0.6 to 1.2 mg / unit dose, and particularly preferably about 0.75 to 1.0 mg / unit dose, or 1.0 mg / unit dose or less.

[0393] 22. The dengue vaccine formulation of any one of clauses 1 to 21, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form comprises the poloxamer at a concentration of about 0.1 to 5.0 g / L, preferably 0.5 to less than 3.0 g / L, more preferably about 1.5 to 2.0 g / L, and particularly preferably about 2.0 g / L or less, for example about 1.65 g / L.

[0394] 23. The dengue vaccine formulation of any one of clauses 1 to 22, wherein said poloxamer is poloxamer 407.

[0395] 24. A dengue vaccine formulation according to any one of clauses 1 to 23, wherein the tromethamine is contained in the dengue vaccine formulation in an amount of about 0.05 to 5.0% w / w, preferably about 0.1 to 3.0% w / w, more preferably about 0.5 to 2.0% w / w, and particularly preferably less than 2.0% w / w, for example about 1.2% w / w, based on the dry weight of the vaccine formulation.

[0396] 25. A dengue vaccine formulation according to any one of clauses 1 to 24, wherein the tromethamine is contained in the dengue vaccine formulation in an amount of about 0.05 to 1.0 mg / unit dose, preferably about 0.1 to 0.6 mg / unit dose, more preferably about 0.2 to 0.5 mg / unit dose, and particularly preferably about 0.3 to 0.4 mg / unit dose, or 0.4 mg / unit dose or less.

[0397] 26. The dengue vaccine formulation of any one of clauses 1 to 25, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of the dengue vaccine formulation in dry form contains the tromethamine at a concentration of about 0.1 to 1.5 g / L, preferably less than 0.5 to 1.2 g / L, more preferably about 0.5 to 1.0 g / L, and particularly preferably 0.8 g / L or less, for example about 0.7 g / L.

[0398] 27. The dengue vaccine formulation comprises, based on the dry weight of the vaccine formulation: a. approximately 65-95% w / w trehalose; b. about 0.05-5.0% w / w of poloxamer; c. about 0.1-6.0% w / w urea; d. about 0.1-5.0% w / w arginine hydrochloride; e. about 0.05-5.0% w / w tromethamine; f. about 0.05-3.0% w / w human serum albumin; 27. The dengue vaccine formulation of any one of clauses 1 to 26, comprising:

[0399] 28. A dengue vaccine formulation according to any one of clauses 1 to 27, a. trehalose in an amount of about 20-34 mg / unit dose; b. poloxamer in an amount of about 0.1-3.0 mg / unit dose; c. urea in an amount of about 0.1-3.0 mg / unit dose; d. arginine hydrochloride in an amount of about 0.1-2.0 mg / unit dose; e. tromethamine in an amount of about 0.05 to 1.0 mg per unit dose; f. human serum albumin in an amount of about 0.05-0.5 mg / unit dose; The dengue vaccine formulation comprising:

[0400] 29. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: a. Trehalose at a concentration of 40-80 g / L; b. Poloxamer at a concentration of approximately 0.1 to 5.0 g / L; c. urea at a concentration of approximately 0.1 to 6.0 g / L; d. arginine hydrochloride at a concentration of about 0.5 to 5.0 g / L; e. Tromethamine at a concentration of about 0.1 to 1.5 g / L; f. Human serum albumin at a concentration of 0.1 to less than 1 g / L; 29. The dengue vaccine formulation of any one of clauses 1 to 28, comprising:

[0401] 30. The dengue vaccine formulation of any one of clauses 1 to 29, further comprising a phosphate salt, for example potassium dihydrogen phosphate or disodium hydrogen phosphate.

[0402] 31. The dengue vaccine formulation according to any one of clauses 1 to 30, wherein the phosphate is contained in the dengue vaccine formulation in an amount of less than 1.0% w / w, or about 0.05 to 0.5% w / w, preferably about 0.2 to 0.4% w / w, more preferably 0.3% w / w or less, based on the dry weight of the vaccine formulation.

[0403] 32. The dengue vaccine formulation according to any one of clauses 1 to 31, wherein the phosphate is contained in the dengue vaccine formulation in an amount of less than 0.3 mg / unit dose, or about 0.01 to 0.2 mg / unit dose, preferably about 0.08 to 0.1 mg / unit dose, more preferably 0.1 mg / unit dose or less.

[0404] 33. The dengue vaccine formulation of any one of clauses 1 to 32, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form comprises said phosphate at a concentration of less than 0.3 g / L, or about 0.16 to 0.22 g / L, preferably 0.22 g / L or less, for example about 0.18 g / L.

[0405] 34. The dengue vaccine formulation of any one of clauses 30 to 33, wherein the phosphate salts are potassium dihydrogen phosphate and disodium hydrogen phosphate.

[0406] 35. The dengue vaccine formulation of any one of clauses 1 to 34, further comprising a chloride salt, for example sodium chloride, or potassium chloride.

[0407] 36. The dengue vaccine formulation of any one of clauses 1 to 35, wherein the chloride salt is present in the dengue vaccine formulation in an amount of less than 4.0% w / w, or about 0.05 to 3.0% w / w, based on the dry weight of the vaccine formulation.

[0408] 37. The dengue vaccine formulation of any one of clauses 1 to 36, wherein the sodium chloride is contained in the dengue vaccine formulation in an amount of about 1.0 to 3.0% w / w, preferably 2.1% w / w or less, based on the dry weight of the vaccine formulation.

[0409] 38. The dengue vaccine formulation of any one of clauses 1 to 37, wherein the potassium chloride is contained in the dengue vaccine formulation in an amount of about 0.01 to 0.05% w / w, preferably 0.03% w / w or less, based on the dry weight of the vaccine formulation.

[0410] 39. The dengue vaccine formulation of any one of clauses 1 to 38, wherein the chloride salt is contained in the dengue vaccine formulation in an amount of less than 2.0 mg / unit dose, preferably about 0.1 to 1.0 mg / unit dose, more preferably about 0.55 to 0.75 mg / unit dose.

[0411] 40. The dengue vaccine formulation of any one of clauses 1 to 39, wherein the sodium chloride is contained in the dengue vaccine formulation in an amount of 0.72 mg / unit dose or less.

[0412] 41. The dengue vaccine formulation of any one of clauses 1 to 40, wherein the potassium chloride is contained in the dengue vaccine formulation in an amount of 0.01 mg / unit dose or less.

[0413] 42. The dengue vaccine formulation of any one of clauses 1 to 41, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form comprises said chloride salt at a concentration of less than 5 g / L, preferably about 3.3 to 3.6 g / L, more preferably 3.6 g / L or less, for example about 3.4 g / L.

[0414] 43. The dengue vaccine formulation of any one of clauses 1 to 42, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form comprises said sodium chloride at a concentration of less than 5 g / L, preferably about 3.3 to 3.6 g / L, more preferably 3.6 g / L or less, for example about 3.4 g / L.

[0415] 44. The dengue vaccine formulation of any one of clauses 1 to 43, further comprising hydrochloric acid.

[0416] 45. The dengue vaccine formulation of any one of clauses 1 to 44, wherein the hydrochloric acid is contained in the dengue vaccine formulation in an amount of less than 1.0% w / w, or about 0.05 to 0.5% w / w, preferably about 0.2 to 0.4% w / w, more preferably about 0.3% w / w or less, based on the dry weight of the vaccine formulation.

[0417] 46. ​​The dengue vaccine formulation of any one of clauses 1 to 45, wherein the hydrochloric acid is contained in the dengue vaccine formulation in an amount of about 0.01 to 0.5 mg per unit dose, preferably less than 0.2 mg per unit dose, or about 0.07 to 0.1 mg per unit dose, more preferably 0.1 mg per unit dose or less.

[0418] 47. The dengue vaccine formulation of any one of clauses 1 to 46, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form comprises said hydrochloric acid at a concentration of about 0.05 to 0.3 g / L, preferably about 0.15 to 0.19 g / L, more preferably about 0.19 g / L or less, for example about 0.16 g / L.

[0419] 48. The dengue vaccine formulation, a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; g. Chloride salts; h. phosphate; 48. The dengue vaccine formulation of any one of clauses 1 to 47, comprising:

[0420] 49. The dengue vaccine formulation comprises, based on the dry weight of the vaccine formulation: a. approximately 65-95% w / w trehalose; b. about 0.05-5.0% w / w of poloxamer; c. about 0.1-6.0% w / w urea; d. about 0.1-5.0% w / w arginine hydrochloride; e. about 0.05-5.0% w / w tromethamine; f. about 0.05-3.0% w / w human serum albumin; g. about 0.05 to 3.0% w / w of chloride salt; h. about 0.05-0.5% w / w of phosphate; 49. The dengue vaccine formulation of any one of clauses 1 to 48, comprising:

[0421] 50. The dengue vaccine formulation comprises, based on the dry weight of the vaccine formulation: a. approximately 65-95% w / w trehalose; b. about 0.05-5.0% w / w of poloxamer; c. about 0.1-6.0% w / w urea; d. about 0.1-5.0% w / w arginine hydrochloride; e. about 0.05-5.0% w / w tromethamine; f. about 0.05-3.0% w / w human serum albumin; g. about 0.05 to 3.0% w / w of chloride salt; h. about 0.05-0.5% w / w of phosphate; i. about 0.05-0.5% w / w hydrochloric acid; 50. The dengue vaccine formulation of any one of clauses 1 to 49, comprising:

[0422] 51. A dengue vaccine formulation according to any one of clauses 1 to 50, a. trehalose in an amount of about 20-34 mg / unit dose; b. poloxamer in an amount of about 0.1-3.0 mg / unit dose; c. urea in an amount of about 0.1-3.0 mg / unit dose; d. arginine hydrochloride in an amount of about 0.1-2.0 mg / unit dose; e. tromethamine in an amount of about 0.05 to 1.0 mg per unit dose; f. human serum albumin in an amount of about 0.05-0.5 mg / unit dose; g. Chloride salt in an amount of less than 2.0 mg / unit dose; h. phosphate in an amount of less than 0.3 mg / unit dose; The dengue vaccine formulation comprising:

[0423] 52. A dengue vaccine formulation according to any one of clauses 1 to 51, a. trehalose in an amount of about 20-34 mg / unit dose; b. poloxamer in an amount of about 0.1-3.0 mg / unit dose; c. urea in an amount of about 0.1-3.0 mg / unit dose; d. arginine hydrochloride in an amount of about 0.1-2.0 mg / unit dose; e. tromethamine in an amount of about 0.05 to 1.0 mg per unit dose; f. human serum albumin in an amount of about 0.05-0.5 mg / unit dose; g. Chloride salt in an amount of less than 2.0 mg / unit dose; h. phosphate in an amount of less than 0.3 mg / unit dose; i. hydrochloric acid in an amount of about 0.01-0.5 mg / unit dose; The dengue vaccine formulation comprising:

[0424] 53. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: a. Trehalose at a concentration of 40-80 g / L; b. poloxamer at a concentration of about 0.1 to 5.0 g / L; c. urea at a concentration of approximately 0.1 to 6.0 g / L; d. arginine hydrochloride at a concentration of about 0.5 to 5.0 g / L; e. Tromethamine at a concentration of about 0.1 to 1.5 g / L; f. Human serum albumin at a concentration of 0.1 to less than 1 g / L; g. chloride salts at a concentration of approximately 3.3 to 3.6 g / L; h. Phosphate at a concentration of about 0.16 to 0.22 g / L; 53. The dengue vaccine formulation of any one of clauses 1 to 52, comprising:

[0425] 54. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: a. Trehalose at a concentration of 40-80 g / L; b. poloxamer at a concentration of about 0.1 to 5.0 g / L; c. urea at a concentration of approximately 0.1 to 6.0 g / L; d. arginine hydrochloride at a concentration of about 0.5 to 5.0 g / L; e. Tromethamine at a concentration of about 0.1 to 1.5 g / L; f. Human serum albumin at a concentration of 0.1 to less than 1 g / L; g. chloride salts at a concentration of approximately 3.3 to 3.6 g / L; h. Phosphate at a concentration of about 0.16 to 0.22 g / L; i. hydrochloric acid at a concentration of about 0.05 to 0.3 g / L; 54. The dengue vaccine formulation of any one of clauses 1 to 53, comprising:

[0426] 55. A dengue vaccine formulation according to any one of clauses 2 to 54, wherein the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is between about 1:1 and 4:1.

[0427] 56. A dengue vaccine formulation according to any one of clauses 2 to 55, wherein the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is between about 2:1 and 3:1.

[0428] 57. A dengue vaccine formulation according to any one of clauses 2 to 56, wherein the ratio of the amount of arginine hydrochloride to the amount of tromethamine contained in the dengue vaccine formulation is between about 1:1 and 3:1.

[0429] 58. A dengue vaccine formulation according to any one of clauses 2 to 57, wherein the ratio of the amount of arginine hydrochloride to the amount of tromethamine contained in the dengue vaccine formulation is between about 1.5:1 and 2.5:1.

[0430] 59. The dengue vaccine formulation of any one of clauses 1 to 58, wherein the ratio of the amount of urea to the amount of human serum albumin contained in the dengue vaccine formulation is between about 1:1 and 5:1.

[0431] 60. The dengue vaccine formulation of any one of clauses 1 to 59, wherein the ratio of the amount of urea to the amount of human serum albumin contained in the dengue vaccine formulation is between about 2:1 and 4:1.

[0432] 61. The dengue vaccine formulation of any one of clauses 2 to 60, wherein the ratio of the amount of urea to the amount of arginine hydrochloride contained in the dengue vaccine formulation is about 1:1 to 2:1.

[0433] 62. The dengue vaccine formulation of any one of clauses 1 to 61, wherein the ratio of the amount of urea to the amount of poloxamer contained in the dengue vaccine formulation is about 1:1 to 2:1.

[0434] 63. The dengue vaccine formulation of any one of clauses 2 to 62, wherein the ratio of the amount of trehalose to the amount of urea contained in the dengue vaccine formulation is between about 20:1 and 40:1.

[0435] 64. The dengue vaccine formulation of any one of clauses 2 to 63, wherein the ratio of the amount of trehalose to the amount of urea contained in the dengue vaccine formulation is between about 25:1 and 35:1.

[0436] 65. The dengue vaccine formulation of any one of clauses 1 to 64, wherein said dengue vaccine formulation does not contain mannitol.

[0437] 66. The dengue vaccine formulation of any one of clauses 1 to 65, wherein said dengue vaccine formulation does not contain sorbitol.

[0438] 67. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: (i) dengue virus serotype 1 at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) dengue virus serotype 2 at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) dengue virus serotype 3 at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) dengue virus serotype 4 at a concentration of at least 4.5 log pfu / 0.5 mL; 67. The dengue vaccine formulation of any one of clauses 1 to 66, comprising:

[0439] 68. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: (i) Dengue virus serotype 1 at a concentration of at least 3.3 log pfu / 0.5 mL to 3.8 log pfu / 0.5 mL; (ii) dengue virus serotype 2 at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) dengue virus serotype 3 at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) a dengue virus serotype 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL, or at least 4.6 log pfu / 0.5 mL to optionally 6.2 log pfu / 0.5 mL; 68. The dengue vaccine formulation of any one of clauses 1 to 67, comprising:

[0440] 69. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: (i) Dengue virus serotype 1 at a concentration of 3.3 log10 pfu / 0.5 mL to 3.6 log10 pfu / 0.5 mL; (ii) Dengue virus serotype 2 at a concentration of 2.7 log10 pfu / 0.5 mL to 4.0 log10 pfu / 0.5 mL; (iii) Dengue virus serotype 3 at a concentration of 4.0 log10 pfu / 0.5 mL to 4.6 log10 pfu / 0.5 mL; (iv) Dengue virus serotype 4 at a concentration of 4.5 log10 pfu / 0.5 mL, or 4.6 log10 pfu / 0.5 mL to 5.1 log10 pfu / 0.5 mL; 69. The dengue vaccine formulation of any one of clauses 1 to 68, comprising:

[0441] 70. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, in a unit dose of said dengue vaccine formulation in dry form, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, and based on said total concentration of pfu / 0.5 mL: the concentration of (i) in pfu / 0.5 mL is at least 1%; The concentration of (ii) in pfu / 0.5 mL is less than 10%; the concentration of (iii) in pfu / 0.5 mL is at least 10%; The concentration of (iv) in pfu / 0.5 mL is at least 50%; 70. The dengue vaccine formulation of any one of clauses 1 to 69.

[0442] 71. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, in a unit dose of said dengue vaccine formulation in dry form, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, and based on said total concentration of pfu / 0.5 mL: The concentration of (i) in pfu / 0.5 mL is 1% to 7%; The concentration of (ii) in pfu / 0.5 mL is 8% or less, for example, in the range of 1% to 8%; the concentration of (iii) in pfu / 0.5 mL is at least 10%; The concentration of (iv) in pfu / 0.5 mL is at least 65%, for example, in the range of 65% to 80%; 71. The dengue vaccine formulation of any one of clauses 1 to 70.

[0443] 72. The dengue vaccine formulation of any one of clauses 1 to 71, wherein, upon reconstitution with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or upon reconstitution to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, in a unit dose of said dengue vaccine formulation in dry form, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, and based on said total concentration of pfu / 0.5 mL, the concentration of (iii) in pfu / 0.5 mL is at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0444] 73. The dengue vaccine formulation of any one of clauses 1 to 72, wherein said tetravalent dengue virus composition comprises at least one non-chimeric dengue virus and at least one chimeric dengue virus.

[0445] 74. The dengue vaccine formulation of any one of clauses 1 to 73, wherein said dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, said dengue serotype 2 is a non-chimeric dengue serotype 2 strain, said dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and said dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain.

[0446] 75. The dengue vaccine formulation of any one of clauses 1 to 74, wherein the dengue serotype 2 strain is derived from the live attenuated DEN-2 PDK-53 virus strain, and the three chimeric dengue strains are derived from the dengue serotype 2 strain by replacing the structural proteins prM and E from the dengue serotype 2 strain with the corresponding structural proteins of other dengue serotypes.

[0447] 76. The dengue vaccine formulation of any one of clauses 1 to 75, wherein said dengue serotype 1 is TDV-1, said dengue serotype 2 is TDV-2, said dengue serotype 3 is TDV-3, and said dengue serotype 4 is TDV-4.

[0448] 77. The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); The three chimeric dengue strains are derived from the serotype 2 strain by replacing the structural proteins prM and E from the serotype 2 strain with the corresponding structural proteins from other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, 77. The dengue vaccine formulation of any one of clauses 74 to 76, which results in

[0449] 78. The dengue vaccine formulation of any one of clauses 1 to 77, wherein the dengue serotype 1 is characterized by the amino acid sequence of SEQ ID NO: 2, the dengue serotype 2 is characterized by the amino acid sequence of SEQ ID NO: 4, the dengue serotype 3 is characterized by the amino acid sequence of SEQ ID NO: 6, and the dengue serotype 4 is characterized by the amino acid sequence of SEQ ID NO: 8.

[0450] 79. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL; Including, Upon reconstitution, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, based on the total concentration of pfu / 0.5 mL: the concentration of (i) in pfu / 0.5 mL is at least 1%; The concentration of (ii) in pfu / 0.5 mL is less than 10%; the concentration of (iii) in pfu / 0.5 mL is at least 10%; the concentration of (iv) in pfu / 0.5 mL is at least 50%; The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57) b) NS1-53 Gly to Asp (nt-2579) c) NS3-250 Glu to Val (nt-5270); The three chimeric dengue strains are derived from the serotype 2 strain by replacing the structural proteins prM and E from the serotype 2 strain with the corresponding structural proteins from other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, 79. The dengue vaccine formulation of any one of clauses 1 to 78, which results in

[0451] 80. When reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form is: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL; Including, Upon reconstitution, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, based on the total concentration of pfu / 0.5 mL: the concentration of (i) in pfu / 0.5 mL is at least 1%; The concentration of (ii) in pfu / 0.5 mL is less than 10%; the concentration of (iii) in pfu / 0.5 mL is at least 10%; the concentration of (iv) in pfu / 0.5 mL is at least 50%; The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); The three chimeric dengue strains are derived from the serotype 2 strain by replacing the structural proteins prM and E from the serotype 2 strain with the corresponding structural proteins from other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, brings about Upon reconstitution, the dengue vaccine formulation comprises: a. The trehalose at a concentration of 40 to 80 g / L, for example, about 55 g / L, or about 43 g / L; b. the poloxamer at a concentration of about 0.1 to 5.0 g / L, e.g., about 1.65 g / L, or about 1.42 g / L; c. the urea at a concentration of about 0.1 to 6.0 g / L, for example, about 1.65 g / L, or about 1.42 g / L; d. the arginine hydrochloride at a concentration of about 0.5 to 5.0 g / L, for example, about 1.4 g / L, or about 1.18 g / L; e. the tromethamine at a concentration of about 0.1 to 1.5 g / L, for example, about 0.7 g / L, or about 0.6 g / L; f. the human serum albumin at a concentration of 0.1 to less than 1 g / L, for example, about 0.55 g / L, or about 0.48 g / L; 80. The dengue vaccine formulation of any one of clauses 1 to 79, comprising:

[0452] 81. The dengue vaccine formulation of any one of clauses 1 to 80, wherein said tetravalent dengue virus composition is "TAK-003."

[0453] 82. A dengue vaccine formulation according to any one of clauses 1 to 81, in a dried form, having a residual moisture content of less than 2% w / w, preferably less than 1% w / w, more preferably less than 0.9% w / w or less than 0.8% w / w in the dried form.

[0454] 83. The dengue vaccine formulation of any one of clauses 1 to 82, which is a freeze-dried dengue vaccine formulation.

[0455] 84. A dengue vaccine formulation according to any one of clauses 1 to 83, wherein the amount of dengue virus before lyophilization and the amount of dengue virus immediately after lyophilization differ by less than 0.46 log10 pfu / unit dose, preferably less than 0.4 log10 pfu / unit dose, more preferably less than 0.3 log10 pfu / unit dose, or less than 0.25 log10 pfu / unit dose, for each of serotypes 1 to 4.

[0456] 85. A dengue vaccine formulation according to any one of clauses 1 to 84, wherein the amount of dengue virus immediately after lyophilization and after lyophilization and storage for 12 weeks at about 25°C and 65% relative humidity differs by less than 1.6 log10 pfu / unit dose, preferably less than 1.4 log10 pfu / unit dose, more preferably less than 1.2 log10 pfu / unit dose, or less than 1.0 log10 pfu / unit dose, for each of serotypes 1 to 4.

[0457] 86. A dengue vaccine formulation according to any one of clauses 1 to 85, wherein the amount of dengue virus immediately after lyophilization and after lyophilization and storage at about 2 to 8°C for 12 months differs by less than 0.44 log10 pfu / unit dose, preferably less than 0.4 log10 pfu / unit dose, more preferably less than 0.35 log10 pfu / unit dose, for each of serotypes 1 to 4.

[0458] 87. A dengue vaccine formulation according to any one of clauses 1 to 86, wherein the amount of dengue virus immediately after freeze-drying and after freeze-drying and storage at about 2-8°C for 18 months differs by less than 0.7 log10 pfu / unit dose, preferably less than 0.6 log10 pfu / unit dose, more preferably less than 0.5 log10 pfu / unit dose, or less than 0.4 log10 pfu / unit dose, for each of serotypes 1 to 4.

[0459] 88. A dengue vaccine formulation according to any one of clauses 1 to 87, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form has a pH at 25°C of about 7.0 to 8.5, preferably about 7.3 to 8.0, more preferably about 7.4 to 7.8, or about 7.6.

[0460] 89. The dengue vaccine formulation of any one of clauses 1 to 88, wherein when reconstituted with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or when reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, a unit dose of said dengue vaccine formulation in dry form has an osmolality of about 100 to 700 mOsmol / kg, preferably less than about 100 to 700 mOsmol / kg, more preferably about 100 to 400 mOsmol / kg, or about 300 mOsmol / kg.

[0461] 90. A dengue vaccine formulation in aqueous form obtainable by reconstituting a dengue vaccine formulation according to any one of clauses 1 to 89 in dry form with a pharmaceutically acceptable aqueous diluent.

[0462] 91. The dengue vaccine formulation of any one of clauses 6 to 90, wherein the pharmaceutically acceptable aqueous diluent is selected from the group consisting of water for injection and aqueous sodium chloride solution.

[0463] 92. The dengue vaccine formulation of any one of clauses 6 to 91, wherein the pharmaceutically acceptable aqueous diluent is a 37 mM aqueous sodium chloride solution.

[0464] 93. A container, for example a vial, containing a dengue vaccine formulation according to any one of clauses 1 to 92.

[0465] 94. A container, for example a vial, containing 1 to 10 unit doses of a dengue vaccine formulation according to any one of clauses 1 to 92.

[0466] 95. A container, for example a vial, containing a unit dose of a dengue vaccine formulation according to any one of clauses 1 to 92.

[0467] 96. A container, for example a vial, containing multiple unit doses of the dengue vaccine formulation of any one of clauses 1 to 92.

[0468] 97. A container, for example a vial, containing 5 unit doses of a dengue vaccine formulation according to any one of clauses 1 to 92.

[0469] 98. A container, e.g., a vial, according to clause 97, containing the arginine hydrochloride in an amount of about 1.5 to 7.5 mg, preferably about 2.0 to 6.0 mg, more preferably about 3.0 to 5.0 mg, particularly preferably about 3.5 to 4.5 mg, e.g., about 4.1 mg.

[0470] 99. A container, e.g., a vial, according to clause 97 or 98, containing urea in an amount of about 2.0 to 8.0 mg, preferably about 3.0 to 7.0 mg, more preferably about 4.0 to 6.0 mg, particularly preferably about 4.5 to 5.5 mg, for example, about 4.95 mg.

[0471] 100. A container, e.g., a vial, according to any one of clauses 97 to 99, wherein the human serum albumin is contained in an amount of about 0.1 to 2.5 mg, preferably about 0.5 to 2.0 mg, more preferably about 1.0 to 2.0 mg, particularly preferably about 1.5 to 1.8 mg, for example, about 1.65 mg.

[0472] 101. A container, e.g., a vial, according to any one of clauses 97 to 100, wherein the trehalose is contained in an amount of about 100 to 170 mg, preferably about 120 to 170 mg, more preferably about 140 to 160 mg, particularly preferably about 145 to 155 mg, e.g., about 149 mg.

[0473] 102. A container, e.g., a vial, according to any one of clauses 97 to 101, wherein the poloxamer is contained in an amount of about 2.0 to 8.0 mg, preferably about 3.0 to 7.0 mg, more preferably about 4.0 to 6.0 mg, particularly preferably about 4.5 to 5.5 mg, for example, about 4.95 mg.

[0474] 103. A container, e.g., a vial, according to any one of clauses 97 to 102, wherein the tromethamine is contained in an amount of about 0.5 to 4.0 mg, preferably about 0.5 to 3.5 mg, more preferably about 1.0 to 3.0 mg, particularly preferably about 1.5 to 2.5 mg, e.g., about 2.0 mg.

[0475] 104. A container, for example a vial, containing a dengue vaccine formulation according to any one of clauses 1 to 92, a. The trehalose is contained in an amount of about 100 to 170 mg, for example, about 149 mg; b. The poloxamer is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; c. The urea is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; d. The arginine hydrochloride is contained in an amount of about 1.5 to 7.5 mg, for example, about 4.1 mg; e. The tromethamine is contained in an amount of about 0.5 to 4.0 mg, for example, about 2.0 mg; f. The human serum albumin is contained in an amount of about 0.1 to 2.5 mg, for example, about 1.65 mg. The container, for example, a vial.

[0476] 105. A container, e.g., a vial, containing 5 unit doses of a dengue vaccine formulation according to any one of clauses 1 to 92, a. The trehalose is contained in an amount of about 100 to 170 mg, for example, about 149 mg; b. The poloxamer is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; c. The urea is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; d. The arginine hydrochloride is contained in an amount of about 1.5 to 7.5 mg, for example, about 4.1 mg; e. The tromethamine is contained in an amount of about 0.5 to 4.0 mg, for example, about 2.0 mg; f. The human serum albumin is contained in an amount of about 0.1 to 2.5 mg, for example, about 1.65 mg. The container, for example, a vial.

[0477] 106. A container, e.g., a vial, according to any one of clauses 97 to 105, wherein the phosphate is contained in an amount of about 0.05 to 2.0 mg, preferably about 0.1 to 1.0 mg, more preferably about 0.3 to 0.7 mg, particularly preferably about 0.4 to 0.6 mg, e.g., about 0.5 mg.

[0478] 107. A container, e.g., a vial, according to any one of clauses 97 to 106, wherein the chloride salt is contained in an amount of less than 5.0 mg, or about 0.5 to 4.0 mg, preferably about 1.0 to 4.0 mg, e.g., about 3.6 mg.

[0479] 108. A container, e.g., a vial, according to any one of clauses 97 to 107, wherein the hydrochloric acid is contained in an amount of about 0.05 to 2.0 mg, preferably about 0.1 to 1.0 mg, more preferably about 0.3 to 0.7 mg, particularly preferably about 0.4 to 0.6 mg, for example, about 0.5 mg.

[0480] 109. A container, for example a vial, containing 5 unit doses of a dengue vaccine formulation according to any one of clauses 1 to 92, a. The trehalose is contained in an amount of about 100 to 170 mg, for example, about 149 mg; b. The poloxamer is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; c. The urea is contained in an amount of about 2.0 to 8.0 mg, for example, about 4.95 mg; d. The arginine hydrochloride is contained in an amount of about 1.5 to 7.5 mg, for example, about 4.1 mg; e. The tromethamine is contained in an amount of about 0.5 to 4.0 mg, for example, about 2.0 mg; f. The human serum albumin is contained in an amount of about 0.1 to 2.5 mg, for example, about 1.65 mg; g. the chloride salt is present in an amount of less than 5.0 mg; h. The phosphate is contained in an amount of about 0.05 to 2.0 mg; i. The hydrochloric acid is contained in an amount of about 0.05 to 2.0 mg; The container, for example, a vial.

[0481] 110. A container, eg a vial, according to any one of clauses 93 to 109, wherein the dengue vaccine formulation is contained in a dried form, preferably in a freeze-dried form.

[0482] 111. A container, e.g., a vial, according to any one of clauses 96 to 110, for providing 5 unit doses of the dengue vaccine formulation in reconstituted form.

[0483] 112. A kit comprising a plurality, preferably ten, of containers according to any one of clauses 93 to 111 and, optionally, one or more, preferably ten, of containers containing a pharmaceutically acceptable aqueous diluent for reconstitution.

[0484] 113. A kit according to clause 112 for preparing 50 unit doses of a dengue vaccine formulation in reconstituted form.

[0485] 114. A kit for preparing a dengue vaccine formulation in reconstituted form, comprising: a) a dengue vaccine formulation according to any one of clauses 1 to 92 in a dried form, such as a lyophilized form; b) a pharmaceutically acceptable aqueous diluent for reconstitution; and The kit comprises:

[0486] 115. A kit for preparing 1 to 10 doses of a dengue vaccine formulation in reconstituted form, comprising: a) a container, e.g., a vial, containing 1 to 10 unit doses of the dengue vaccine formulation of any one of clauses 1 to 92 in a dried form, such as a freeze-dried form; b) a container, e.g., a vial, containing 1 to 10 aliquots of a pharmaceutically acceptable aqueous diluent for reconstitution; Including, the number of aliquots corresponds to the number of unit doses; The kit.

[0487] 116. A kit for preparing a unit dose of a dengue vaccine formulation in reconstituted form, comprising: a) a container, e.g., a vial, containing a unit dose of the dengue vaccine formulation of any one of clauses 1 to 92 in a dry form, such as a lyophilized form; b) a container, e.g., a vial, containing an aliquot of a pharmaceutically acceptable aqueous diluent for reconstitution; The kit comprises:

[0488] 117. A kit for preparing a multiple unit dose dengue vaccine formulation in reconstituted form, comprising: a) a container, e.g., a vial, containing multiple unit doses of the dengue vaccine formulation of any one of clauses 1 to 92 in a dried form, such as a lyophilized form; b) a container, e.g., a vial, containing multiple aliquots of a pharmaceutically acceptable aqueous diluent for reconstitution; Including, the number of aliquots corresponds to the number of unit doses; The kit.

[0489] 118. A kit for preparing a dengue vaccine formulation in a reconstituted form in 5 unit doses, comprising: a) a container, e.g., a vial, containing 5 unit doses of the dengue vaccine formulation of any one of clauses 1 to 92 in a dry form, such as a lyophilized form; b) a container, e.g., a vial, containing 5 aliquots of a pharmaceutically acceptable aqueous diluent for reconstitution; The kit comprises:

[0490] 119. A kit according to any one of clauses 112 to 118, wherein the aliquots have a volume of about 0.4 to 0.8 mL per unit dose, preferably about 0.4 to 0.7 mL per unit dose, more preferably about 0.45 to 0.65 mL per unit dose.

[0491] 120. A kit according to any one of clauses 112 to 119, wherein the pharmaceutically acceptable aqueous diluent is selected from the group consisting of water for injection and aqueous sodium chloride solution.

[0492] 121. A kit according to any one of clauses 112 to 120, wherein the pharmaceutically acceptable aqueous diluent is a 37 mM aqueous sodium chloride solution.

[0493] 122. Five unit doses of a dengue vaccine formulation in aqueous form obtained by reconstituting five unit doses of a dengue vaccine formulation according to any one of clauses 1 to 92, in dry form, such as freeze-dried form, with five aliquots of a pharmaceutically acceptable aqueous diluent.

[0494] 123. A unit dose of a dengue vaccine formulation in aqueous form obtained by reconstituting a unit dose of a dengue vaccine formulation according to any one of clauses 1 to 92, in dry form, with one aliquot of a pharmaceutically acceptable aqueous diluent.

[0495] 124. A unit dose of a dengue vaccine formulation in aqueous form obtained by reconstituting a plurality of unit doses of a dengue vaccine formulation according to any one of clauses 1 to 92, in dry form, with a plurality of aliquots of a pharmaceutically acceptable aqueous diluent, the number of said aliquots corresponding to the number of said unit doses, and removing a unit dose volume of the reconstituted dengue vaccine formulation.

[0496] 125. A unit dose of a dengue vaccine formulation in aqueous form obtained by reconstituting five unit doses of a dengue vaccine formulation according to any one of clauses 1 to 92, in dry form, with five aliquots of a pharmaceutically acceptable aqueous diluent, and removing a unit dose volume of the reconstituted dengue vaccine formulation.

[0497] 126. The unit dose(s) according to any one of clauses 122 to 125, wherein the aliquots have a volume of about 0.4 to 0.8 mL per unit dose, preferably about 0.4 to 0.7 mL per unit dose, more preferably about 0.45 to 0.65 mL per unit dose, for example 0.5 mL per unit dose.

[0498] 127. The unit dose(s) according to any one of clauses 122 to 126, wherein the pharmaceutically acceptable aqueous diluent is selected from the group consisting of water for injection and aqueous sodium chloride solution.

[0499] 128. The unit dose(s) according to any one of clauses 122 to 127, wherein said pharmaceutically acceptable aqueous diluent is a 37 mM aqueous sodium chloride solution.

[0500] 129. A dengue vaccine formulation according to any one of clauses 1 to 128 for use in a method for preventing dengue disease in a subject, said method comprising administering a primary vaccination in only two administrations of a unit dose, said administration comprising: administering a first unit dose of the dengue vaccine formulation to the subject; administering a second unit dose of the dengue vaccine formulation to the subject; The dengue vaccine formulation comprising:

[0501] 130. A dengue vaccine formulation for use in the method of clause 129, wherein said second dose is administered within three months of administration of said first unit dose.

[0502] 131. A dengue vaccine formulation for use in the method of clause 129 or 130, wherein a booster dose is administered at least 12 months after administration of said second unit dose of said dengue vaccine.

[0503] 132. A method for preventing dengue disease in a subject, comprising administering a primary vaccination with only two administrations of a unit dose of a dengue vaccine formulation according to any one of clauses 1 to 128, wherein said administrations administering a first unit dose of the dengue vaccine formulation to the subject; administering a second unit dose of the dengue vaccine formulation to the subject; The method comprising:

[0504] 133. The method for preventing dengue disease in a subject according to clause 132, wherein said second dose is administered within three months of administration of said first unit dose.

[0505] 134. The method for preventing dengue disease in a subject according to clause 132 or 133, wherein a booster dose is administered at least 12 months after administration of said second unit dose of said dengue vaccine.

[0506] 135. A method for preparing a dengue vaccine formulation in a dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form; (b) freezing the aqueous dengue vaccine formulation to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature ranging from above the glass transition temperature Tg of the formulation to no more than 25°C above, or no more than 20°C above, or no more than 15°C above, the glass transition temperature Tg of the formulation to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature above the storage temperature of the primary drying step at a pressure of less than 1 mbar to form a secondary dried product; The method comprising:

[0507] 136. The method of clause 135, wherein said freezing step (b) is carried out in a device having a storage temperature of less than -35°C.

[0508] 137. The method of clause 135 or 137, wherein the primary drying step (c) is carried out in an apparatus having a storage temperature of about -31 to -19°C, or about -31 to -21°C.

[0509] 138. A method for preparing a dengue vaccine formulation according to any one of clauses 1 to 131 in a dried form, comprising the following steps: (a) providing a dengue vaccine formulation in aqueous form, said dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine f. human serum albumin; and the excipient composition comprising: (b) freezing the aqueous form of the dengue vaccine formulation in a device having a storage temperature of less than -35°C to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature of about -31 to -19°C, or about -31 to -21°C, to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of at least 15°C, e.g., at least 20°C, at a pressure of less than 1 mbar to form a secondary dried product; The method comprising:

[0510] 139. The method according to clause 138, wherein the freezing step (b) is carried out in an apparatus having a storage temperature below -40°C, preferably between about -40 and -50°C, for example about -45°C.

[0511] 140. The method of any one of clauses 135 to 139, wherein the primary drying step (c) is carried out in an apparatus having a storage temperature of about -31 to -21°C, preferably about -29 to -25°C, for example about -27°C.

[0512] 141. The method of any one of clauses 135 to 140, wherein the primary drying step (c) is carried out at a pressure of less than 0.05 mbar, preferably less than about 0.01 to 0.05 mbar, or about 0.04 mbar.

[0513] 142. A method according to any one of clauses 135 to 141, wherein the primary dried product has a residual moisture content of about 5 to 10% w / w, or less than 10% w / w.

[0514] 143. The method of any one of clauses 135-142, wherein the secondary drying step (d) is carried out in an apparatus having a storage temperature of at least 15°C, or at least 20°C, or about 15-40°C, or about 20-40°C, or about 25°C.

[0515] 144. The method of any one of clauses 135 to 143, wherein the secondary drying step (d) is carried out at a pressure of less than 0.5 mbar, preferably less than about 0.1 to 0.4 mbar, or about 0.3 mbar, more preferably less than 0.05 mbar.

[0516] 145. A method according to any one of clauses 135 to 144, wherein the secondary dried product has a residual moisture content of about 0.01 to 2% w / w, preferably less than 2% w / w, more preferably less than 1% w / w.

[0517] 146. The method according to any one of clauses 135 to 145, wherein the dengue vaccine formulation in aqueous form is subjected to the freezing step b) in containers such as vials each containing a unit dose of the dengue vaccine formulation.

[0518] 147. The method of any one of clauses 135 to 146, wherein the dengue vaccine formulation in aqueous form is subjected to the freezing step b) in containers such as vials each containing multiple unit doses of the dengue vaccine formulation, for example 5 unit doses.

[0519] 148. The aqueous dengue vaccine formulation is about 9-11% w / v, for example, about 10% w / v, of trehalose; about 0.2-0.4% w / v, for example, about 0.3% w / v, of poloxamer; about 0.2 to 0.4% w / v, for example, about 0.3% w / v, of urea; About 0.2 to 0.3% w / v, for example, about 0.25% w / v, of arginine hydrochloride; about 0.05 to 0.15% w / v, for example, about 0.1% w / v, of tromethamine; about 0.05 to 0.15% w / v, for example, about 0.1% w / v, of human serum albumin; 148. The method of any one of clauses 135 to 147, comprising:

[0520] 149. The method of any one of clauses 135 to 148, wherein the aqueous form of the dengue vaccine formulation is subjected to the freezing step b) in a volume less than the volume used to reconstitute the lyophilized dengue vaccine formulation, preferably about half the volume.

[0521] 150. The method of any one of clauses 135 to 149, wherein the aqueous form of the dengue vaccine formulation is subjected to the freezing step b) in containers, e.g. vials, each containing a unit dose of the dengue vaccine formulation, in a volume approximately half the volume used to reconstitute a unit dose of the freeze-dried dengue vaccine formulation.

[0522] 151. The method of any one of clauses 135 to 149, wherein the aqueous form of the dengue vaccine formulation is subjected to the freezing step b) in containers, e.g. vials, each containing 5 unit doses of the dengue vaccine formulation, in a volume approximately half the volume used to reconstitute 5 unit doses of the freeze-dried dengue vaccine formulation.

[0523] 152. The method of any one of clauses 135-151, wherein the primary drying step (c) is carried out for a period of about 20-120 hours, preferably about 30-100 hours, more preferably about 40-70 hours, or about 50-70 hours.

[0524] 153. The method of any one of clauses 135-152, wherein the secondary drying step (d) is carried out for a period of about 1 to 15 hours, preferably about 3 to 10 hours, more preferably about 6 to 10 hours, or about 7 to 9 hours.

[0525] 154. The method of any one of clauses 135 to 153, wherein the primary and secondary drying steps are carried out for a period of less than 160 hours, preferably about 50 to 120 hours, more preferably less than about 50 to 100 hours, or about 60 to 90 hours.

[0526] 155. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising: the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 4:1; the chloride salt is present in the dengue vaccine formulation in an amount of less than 4.0% w / w, based on the dry weight of the vaccine formulation; the phosphate is present in the dengue vaccine formulation in an amount of less than 1.0% w / w, based on the dry weight of the vaccine formulation; The dengue vaccine formulation.

[0527] 156. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising: the dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, the dengue serotype 2 is a non-chimeric dengue serotype 2 strain, the dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and the dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; The dengue serotype 2 strain is derived from the live attenuated DEN-2 PDK-53 virus strain, and the three chimeric dengue strains are derived from the dengue serotype 2 strain by replacing the structural proteins prM and E from the dengue serotype 2 strain with the corresponding structural proteins of other dengue serotypes, resulting in four live attenuated dengue virus strains, TDV-1, TDV-2, TDV-3, and TDV-4. The dengue vaccine formulation.

[0528] 157. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) TDV-1; and (ii) TDV-2; and (iii) TDV-3; and (iv) TDV-4; and the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising: The dengue vaccine formulation.

[0529] 158. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1 (e.g., TDV-1); (ii) a live attenuated dengue virus serotype 2 (e.g., TDV-2); and (iii) a live attenuated dengue virus serotype 3 (e.g., TDV-3); and (iv) a live attenuated dengue virus serotype 4 (e.g., TDV-4); and the tetravalent dengue virus composition comprising: B. An excipient composition, based on the dry weight of the vaccine formulation, comprising: a. about 65-95% w / w, for example, about 87.0% w / w, of trehalose; b. about 0.05-5.0% w / w, for example, about 2.9% w / w, of a poloxamer; c. about 0.1-6.0% w / w, for example, about 2.9% w / w, of urea; d. about 0.1-5.0% w / w, for example, about 2.4% w / w, of arginine hydrochloride; e. about 0.05 to 5.0% w / w, for example, about 1.2% w / w, of tromethamine; f. about 0.05 to 3.0% w / w, for example, about 0.96% w / w, of human serum albumin; and the excipient composition comprising:

[0530] 159. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising: the dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, the dengue serotype 2 is a non-chimeric dengue serotype 2 strain, the dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and the dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); The three chimeric dengue strains were derived from the serotype 2 strain by replacing the structural proteins prM and E from the serotype 2 strain with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, The dengue vaccine formulation.

[0531] 160. A freeze-dried dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising four live-attenuated dengue virus serotypes: (i) a chimeric dengue serotype 2 / 1 strain; (ii) dengue serotype 2 strains; (iii) chimeric dengue serotype 2 / 3 strains; (iv) chimeric dengue serotype 2 / 4 strains; Including, The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); The three chimeric dengue strains were derived from the serotype 2 strain by replacing the structural proteins prM and E from the serotype 2 strain with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, the tetravalent dengue virus composition providing B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising: the human serum albumin is contained in the dengue vaccine formulation in an amount of about 0.1 to 1.0% w / w based on the dry weight of the vaccine formulation, and the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 4:1; The freeze-dried dengue vaccine formulation.

[0532] 161. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and the tetravalent dengue virus composition comprising: B. An excipient composition, based on the dry weight of the vaccine formulation, comprising: a. approximately 65-95% w / w trehalose; b. about 0.05-5.0% w / w of poloxamer; c. about 0.1-6.0% w / w urea; d. about 0.1-5.0% w / w arginine hydrochloride; e. about 0.05-5.0% w / w tromethamine; f. about 0.05-3.0% w / w human serum albumin; g. about 0.05 to 3.0% w / w of chloride salt; h. about 0.05-0.5% w / w of phosphate; and the excipient composition comprising: the dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, the dengue serotype 2 is a non-chimeric dengue serotype 2 strain, the dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and the dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); The three chimeric dengue strains are derived from the serotype 2 strain by replacing the structural proteins prM and E from the serotype 2 strain with the corresponding structural proteins from other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, The dengue vaccine formulation.

[0533] 162. A freeze-dried dengue vaccine preparation, comprising: A. A tetravalent dengue virus composition comprising four live-attenuated dengue virus serotypes: (i) a chimeric dengue serotype 2 / 1 strain; (ii) dengue serotype 2 strains; (iii) chimeric dengue serotype 2 / 3 strains; (iv) chimeric dengue serotype 2 / 4 strains; Including, The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57); b) NS1-53 Gly to Asp (nt-2579); c) NS3-250 Glu to Val (nt-5270); The three chimeric dengue strains were derived from the serotype 2 strain by replacing the structural proteins prM and E from the serotype 2 strain with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera, - DENV-2 / 3 chimeras, and - DENV-2 / 4 chimera, the tetravalent dengue virus composition providing B. An excipient composition comprising: a. trehalose, b. poloxamer, C. urea, d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising: Upon reconstitution with 0.5 mL of a pharmaceutically acceptable aqueous diluent, or upon reconstitution to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, the unit dose of said dengue vaccine formulation (i) the chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) the dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) the chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) the chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL; a. The trehalose at a concentration of 40 to 80 g / L, for example, about 55 g / L, or about 43 g / L; b. the poloxamer at a concentration of about 0.1 to 5.0 g / L, e.g., about 1.65 g / L, or about 1.42 g / L; c. the urea at a concentration of about 0.1 to 6.0 g / L, for example, about 1.65 g / L, or about 1.42 g / L; d. the arginine hydrochloride at a concentration of about 0.5 to 5.0 g / L, for example, about 1.4 g / L, or about 1.18 g / L; e. the tromethamine at a concentration of about 0.1 to 1.5 g / L, for example, about 0.7 g / L, or about 0.6 g / L; f. the human serum albumin at a concentration of 0.1 to less than 1 g / L, for example, about 0.55 g / L, or about 0.48 g / L; The freeze-dried dengue vaccine formulation comprising:

[0534] 163. A method for preparing 5 unit doses of a dengue vaccine formulation in dry form, comprising the following steps: (a) providing a dengue vaccine formulation in aqueous form, said dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1, e.g., a chimeric dengue serotype 2 / 1 strain, (ii) a live attenuated dengue virus serotype 2, e.g., a dengue serotype 2 strain; (iii) a live attenuated dengue virus serotype 3, e.g., a chimeric dengue serotype 2 / 3 strain; (iv) a live attenuated dengue virus serotype 4, e.g., a chimeric dengue serotype 2 / 4 strain; the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. about 9-11% w / v, e.g., about 10% w / v, trehalose; b. about 0.2-0.4% w / v, for example, about 0.3% w / v, of poloxamer; c. about 0.2-0.4% w / v, for example, about 0.3% w / v, of urea; d. about 0.2-0.3% w / v, for example, about 0.25% w / v, of arginine hydrochloride; e. about 0.05 to 0.15% w / v, for example, about 0.1% w / v, of tromethamine; f. providing the aqueous dengue vaccine formulation, comprising the excipient composition, and about 0.05-0.15% w / v, for example, about 0.1% w / v, of human serum albumin; (b) freezing the aqueous dengue vaccine formulation in a device having a storage temperature of about −40 to −50° C. to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step in an apparatus having a storage temperature of about −31 to −19° C., or about −31 to −21° C., at a pressure of less than 0.1 mbar for about 30 to 100 hours to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of about 15-40°C, or about 20-40°C, at a pressure of less than 1 mbar for about 3-10 hours to form a secondary dried product; Including, the dengue vaccine formulation in aqueous form is subjected to the freezing step b) in a volume that is approximately half the volume used to reconstitute 5 unit doses of the dengue vaccine formulation in dry form; The method.

[0535] 164. When reconstituted to a volume of 0.5 mL with a pharmaceutically acceptable aqueous diluent, the unit dose of the dengue vaccine formulation in dry form has a pH of about 7.0 to 8.5; (i) the dengue virus serotype 1 at a concentration of at least 3.3 log pfu / 0.5 mL; (ii) the dengue virus serotype 2 at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) the dengue virus serotype 3 at a concentration of at least 4.0 log pfu / 0.5 mL; (iv) the dengue virus serotype 4 at a concentration of at least 4.5 log pfu / 0.5 mL; a. Trehalose in an amount of approximately 20-34 mg / 0.5 mL; b. poloxamer in an amount of about 0.1-3.0 mg / 0.5 mL; c. urea in an amount of approximately 0.1 to 3.0 mg / 0.5 mL; d. arginine hydrochloride in an amount of about 0.1-2.0 mg / 0.5 mL; e. Tromethamine in an amount of approximately 0.05 to 1.0 mg / 0.5 mL; f. human serum albumin in an amount of about 0.05 to 0.5 mg / 0.5 mL; Chloride salts in an amount less than 2.0 mg / 0.5 mL; h. Phosphate in an amount less than 0.3 mg / 0.5 mL; 93. The dengue vaccine formulation of any one of clauses 1 to 92, comprising:

Claims

1. 1. A dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. a poloxamer; c. urea; d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising:

2. 2. The dengue vaccine formulation of claim 1, wherein the ratio of the amount of arginine hydrochloride to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 4:

1.

3. 3. The dengue vaccine formulation of claim 1, wherein the ratio of the amount of urea to the amount of human serum albumin contained in the dengue vaccine formulation is about 1:1 to 5:1, and / or the ratio of the amount of urea to the amount of poloxamer contained in the dengue vaccine formulation is about 1:1 to 2:

1.

4. The dengue vaccine formulation of any one of claims 1 to 3, wherein phosphate is contained in the dengue vaccine formulation in an amount of less than 0.3 mg per unit dose.

5. The dengue vaccine formulation of any one of claims 1 to 4, wherein the chloride salt is contained in the dengue vaccine formulation in an amount of less than 2.0 mg per unit dose.

6. The dengue vaccine formulation a. the trehalose in an amount of about 20-34 mg / unit dose; b. the poloxamer in an amount of about 0.1-3.0 mg / unit dose; c. the urea in an amount of about 0.1-3.0 mg / unit dose; d. the arginine hydrochloride in an amount of about 0.1-2.0 mg / unit dose; e. the tromethamine in an amount of about 0.05 to 1.0 mg per unit dose; f. the human serum albumin in an amount of about 0.05 to 0.5 mg per unit dose; The dengue vaccine formulation according to any one of claims 1 to 5, comprising:

7. The dengue vaccine formulation of any one of claims 1 to 6, wherein hydrochloric acid is contained in the dengue vaccine formulation in an amount of about 0.01 to 0.5 mg per unit dose.

8. The dengue vaccine formulation comprises, based on the dry weight of the vaccine formulation: a. about 65-95% w / w of said trehalose; b. about 0.05-5.0% w / w of said poloxamer; c. about 0.1-6.0% w / w of said urea; d. about 0.1-5.0% w / w of said arginine hydrochloride; e. about 0.05-5.0% w / w of said tromethamine; f. about 0.05-3.0% w / w of said human serum albumin; A dengue vaccine formulation according to any one of claims 1 to 7, preferably in dry form, comprising:

9. A unit dose of said dengue vaccine formulation in dry form, when reconstituted with a pharmaceutically acceptable aqueous diluent to a volume of 0.5 mL, a. the trehalose at a concentration of 40 to 80 g / L; b. the poloxamer at a concentration of about 0.1-5.0 g / L; c. urea at a concentration of about 0.1 to 6.0 g / L; d. the arginine hydrochloride at a concentration of about 0.5-5.0 g / L; e. the tromethamine at a concentration of about 0.1 to 1.5 g / L; f. the human serum albumin at a concentration of 0.1 to less than 1 g / L; The dengue vaccine formulation according to any one of claims 1 to 8, comprising:

10. 10. The dengue vaccine formulation of any one of claims 1 to 9, wherein a unit dose of the dengue vaccine formulation in dry form, when reconstituted with a pharmaceutically acceptable aqueous diluent to a volume of 0.5 mL, comprises a concentration of hydrochloride salt of less than 5 g / L.

11. 11. The dengue vaccine formulation of any one of claims 1 to 10, wherein a unit dose of the dengue vaccine formulation in dry form, when reconstituted with a pharmaceutically acceptable aqueous diluent to a volume of 0.5 mL, has an osmolality of about 100 to 700 mOsmol / kg.

12. A unit dose of said dengue vaccine formulation in dry form, when reconstituted with a pharmaceutically acceptable aqueous diluent to a volume of 0.5 mL, (i) the dengue virus serotype 1 in an amount of at least 3.3 log pfu / 0.5 mL; (ii) the dengue virus serotype 2 in an amount of at least 2.7 log pfu / 0.5 mL; (iii) the dengue virus serotype 3 in an amount of at least 4.0 log pfu / 0.5 mL; (iv) the dengue virus serotype 4 in an amount of at least 4.5 log pfu / 0.5 mL; The dengue vaccine formulation of any one of claims 1 to 11, comprising:

13. The dengue vaccine formulation of any one of claims 1 to 12, wherein the tetravalent dengue virus composition comprises at least one non-chimeric dengue virus and at least one chimeric dengue virus.

14. The dengue vaccine formulation according to any one of claims 1 to 13, wherein the dengue virus serotype 1 is a chimeric dengue serotype 2 / 1 strain, the dengue virus serotype 2 is a non-chimeric dengue serotype 2 strain, the dengue virus serotype 3 is a chimeric dengue serotype 2 / 3 strain, and the dengue virus serotype 4 is a chimeric dengue serotype 2 / 4 strain, preferably, the dengue serotype 1 is TDV-1, the dengue serotype 2 is TDV-2, the dengue serotype 3 is TDV-3, and the dengue serotype 4 is TDV-4.

15. The dengue vaccine formulation according to any one of claims 1 to 14, wherein the tetravalent dengue virus composition is "TAK-003".

16. The dengue vaccine formulation according to any one of claims 1 to 15, which is a freeze-dried dengue vaccine formulation.

17. 17. The dengue vaccine formulation of any one of claims 1 to 16, wherein for each of serotypes 1 to 4, the amount of dengue virus before lyophilization and the amount of dengue virus immediately after lyophilization differ by less than 0.46 loglO pfu / unit dose.

18. 18. The dengue vaccine formulation of any one of claims 1 to 17, wherein for each of serotypes 1 to 4, the amount of dengue virus immediately after lyophilization and the amount of dengue virus after lyophilization and storage for 12 weeks at about 25°C and 65% relative humidity differ by less than 1.6 loglO pfu / unit dose.

19. 19. The dengue vaccine formulation of any one of claims 1 to 18, wherein for each of serotypes 1 to 4, the amount of dengue virus immediately after lyophilization and the amount of dengue virus after lyophilization and storage at 2-8°C for 12 months differ by less than 0.44 loglO pfu / unit dose.

20. A container, preferably a vial, containing one or more unit doses of the dengue vaccine formulation of any one of claims 1 to 19.

21. A container, preferably a vial, containing 5 unit doses of a dengue vaccine formulation according to any one of claims 1 to 19.

22. 22. A container, preferably a vial, according to claim 21, a. the trehalose is present in an amount of about 100 to 170 mg; b. the poloxamer is present in an amount of about 2.0 to 8.0 mg; c. the urea is present in an amount of about 2.0 to 8.0 mg; d. the arginine hydrochloride is present in an amount of about 1.5 to 7.5 mg; e. the tromethamine is present in an amount of about 0.5 to 4.0 mg; f. The human serum albumin is contained in an amount of about 0.1 to 2.5 mg. The container is preferably a vial.

23. 23. A kit comprising more than one, preferably ten, containers according to any one of claims 20 to 22, and optionally one or more, preferably ten, containers containing a pharmaceutically acceptable aqueous diluent for reconstitution.

24. 1. A kit for preparing a dengue vaccine formulation in a reconstituted form, comprising: a) a container, preferably a vial, containing a dengue vaccine formulation according to any one of claims 1 to 19 in dry form; b) a container, preferably a vial, containing a pharmaceutically acceptable aqueous diluent for reconstitution; The kit comprises:

25. 25. The kit of claim 23 or 24, wherein the pharmaceutically acceptable aqueous diluent is selected from the group consisting of water for injection and aqueous sodium chloride solution, preferably, the pharmaceutically acceptable aqueous diluent is 37 mM aqueous sodium chloride solution.

26. 20. A unit dose of a dengue vaccine formulation in aqueous form obtained by reconstituting 1 to 10, preferably 5, unit doses of a dengue vaccine formulation according to claims 1 to 19 in dry form with an equal number of aliquots of a pharmaceutically acceptable aqueous diluent and removing a unit dose volume of said reconstituted dengue vaccine formulation.

27. 27. The unit dose according to claim 26, wherein the aliquot has a volume of about 0.4 to 0.8 mL per unit dose, preferably about 0.4 to 0.7 mL per unit dose, more preferably about 0.45 to 0.65 mL per unit dose.

28. 1. A method for preparing a dengue vaccine formulation in dry form, comprising the steps of: (a) providing a dengue vaccine formulation in aqueous form; (b) freezing the aqueous dengue vaccine formulation to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step at a pressure of less than 0.1 mbar in an apparatus having a storage temperature ranging from above the glass transition temperature Tg of the formulation to no more than 25°C above the glass transition temperature Tg, e.g., no more than 15°C above the glass transition temperature Tg, to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature above the storage temperature of the primary drying step at a pressure of less than 1 mbar to form a secondary dried product; The method comprising:

29. 29. The method of claim 28, wherein the primary drying step (c) is carried out in an apparatus having a storage temperature of about -31 to -19°C, for example about -31 to -21°C.

30. 30. The method of claim 28 or 29, wherein the secondary drying step (d) is carried out in an apparatus having a storage temperature of at least 15°C, such as at least 20°C.

31. A method for preparing a dengue vaccine formulation according to any one of claims 1 to 19 in a dry form, comprising the following steps: (a) providing a dengue vaccine formulation in aqueous form, said dengue vaccine formulation comprising: A. A tetravalent dengue virus composition comprising: (i) a live attenuated dengue virus serotype 1; (ii) a live attenuated dengue virus serotype 2; and (iii) a live attenuated dengue virus serotype 3; and (iv) a live attenuated dengue virus serotype 4; and the tetravalent dengue virus composition comprising: B. An excipient composition comprising: a. trehalose, b. a poloxamer; c. urea; d. arginine hydrochloride; e. Tromethamine, f. human serum albumin; and the excipient composition comprising: (b) freezing the aqueous form of the dengue vaccine formulation in a device having a storage temperature of less than −35° C. to form a frozen dengue vaccine formulation; (c) subjecting the frozen dengue vaccine formulation to a primary drying step in an apparatus having a storage temperature of about −31 to −19° C., e.g., about −31 to −21° C., at a pressure of less than 0.1 mbar to form a primary dried product; (d) subjecting the primary dried product to a secondary drying step in an apparatus having a storage temperature of at least 15°C, e.g., at least 20°C, at a pressure of less than 1 mbar to form a secondary dried product; The method comprising:

32. 32. The method of any one of claims 28 to 31, wherein the dengue vaccine formulation in aqueous form is subjected to the freezing step b) in a volume that is smaller than the volume used to reconstitute the lyophilized dengue vaccine formulation, preferably the dengue vaccine formulation in aqueous form is subjected to the freezing step b) in containers, e.g. vials, each containing one or more unit doses of the dengue vaccine formulation in a volume that is about half the volume used to reconstitute one or more unit doses of the lyophilized dengue vaccine formulation.

33. 33. A dengue vaccine formulation according to any one of claims 1 to 32 for use in a method for preventing dengue disease in a subject, said method comprising administering a primary vaccination in only two administrations of a unit dose, comprising the steps of: administering a first unit dose of the dengue vaccine formulation to the subject; administering to said subject a second unit dose of said dengue vaccine formulation, preferably within three months of administration of said first unit dose; Optionally, administering a booster dose at least 12 months after administration of the second unit dose of the dengue vaccine formulation; and The dengue vaccine formulation comprising:

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