Process for preparing an attenuated tetravalent dengue vaccine
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Publication Number
US-10201600-B2
Publication Date
2019-02-12
Expiration Date
Abstract
The present invention refers to a process for preparing an attenuated tetravalent dengue vaccine and its product. The present invention also refers to a process for preparing a tetravalent dengue vaccine for administration to a subject, to a method for inducing an immune response to virus dengue serotype 1, 2, 3 and 4 in a patient and to a tetravalent dengue vaccine kit.
Core Innovation
The invention relates to an attenuated tetravalent dengue vaccine produced by a process that uses Vero cells adapted for growth in serum-free medium. The Vero cells are grown in serum-free medium, are sub-cultured with trypsin of non-animal origin, and are propagated from Vero cell Master and Working banks in 225 cm2 TC-flasks and later in a multi-layered cell culture system and/or Cell Factory System.
Dengue virus amplification is performed for dengue virus serotypes 1, 2, 3 and 4 using separate serum-free cultures. The process infects Vero cells independently with dengue virus serotypes 1, 2, 3 and 4 from a Seed or Working bank of each virus, incubates the infected cultures, and harvests viral supernatants.
Each dengue virus suspension is filtered through a membrane with 0.2 µm of porosity, and the filtered dengue virus is stored at -80°C (±15°C). Dengue virus bulks are prepared for the serotypes, monovalent vaccines are formulated for subsequent tetravalent formulation, and the tetravalent vaccine is filled into vials, lyophilized in the vials, sealed, and stored at 2–8°C. A vaccine kit and a specific phosphate-based reconstitution diluent are disclosed, and the lyophilized vaccine is reported to be stable for up to about 1 year at 2–8°C.
Claims Coverage
The patent includes one independent claim. It covers a production process with serum-free adaptation of Vero cells, separate infection with dengue virus serotypes 1–4, harvesting and 0.2 µm filtration, formulation into monovalent and then tetravalent vaccines, and lyophilization, packaging, and storage at 2–8°C. Dependent claims add administration to induce an immune response and a vaccine kit with a specified phosphate reconstitution composition.
Serum-free adapted Vero cells with non-animal trypsin in Master and Working banks
Vero cells are adapted for growth in serum-free medium, are grown in serum-free medium, and are sub-cultured with trypsin of non-animal origin, propagated from Vero cell Master and Working banks in 225 cm2 TC-flasks and later in a multi-layered cell culture system and/or Cell Factory System.
Separate serum-free infection with dengue serotypes 1-4 from Seed or Working banks
Vero cells are independently infected with dengue virus serotypes 1, 2, 3 and 4 from a Seed or Working bank of each virus in separate serum-free cultures.
Harvesting and 0.2 µm filtration of each dengue virus suspension
Supernatants of each culture are harvested, each dengue virus suspension is filtered through a membrane with 0.2 µm of porosity, and the filtered dengue virus is stored at -80°C (±15°C).
Monovalent formulation and mixing into tetravalent vaccine followed by lyophilization, sealing, and 2-8°C storage
Dengue virus bulks of serotypes 1, 2, 3 and 4 are used to prepare monovalent vaccines, which are formulated into a tetravalent vaccine by mixing; vials are filled, lyophilized in the vials, sealed, and stored at 2–8°C.
Immune response induction in a subject by administering the vaccine
The vaccine is administered to a subject to induce an immune response to dengue virus serotypes 1, 2, 3 and 4.
Vaccine kit with specified phosphate reconstitution composition
A tetravalent dengue vaccine kit comprises the vaccine and a reconstitution composition comprising 0.2 M sodium phosphate monobasic dihydrate, 0.2 M sodium phosphate dibasic heptahydrate, and water.
The inventive coverage centers on producing an attenuated tetravalent dengue vaccine using serum-free adapted Vero cells with non-animal-origin trypsin, independently infecting serum-free cultures with dengue serotypes 1–4, harvesting and filtering viral supernatants through a 0.2 µm membrane, formulating monovalent vaccines and mixing them into a tetravalent lyophilized product stored at 2–8°C, with dependent claims adding administration to induce immune responses and a kit including a specific phosphate reconstitution composition.
Stated Advantages
Higher yield and reproducibility.
Increased productivity via Cell Factory System and multiple harvests.
Avoidance of animal-origin contaminants by using no serum and trypsin of non-animal origin.
Stability of the lyophilized vaccine for up to about 1 year at 2–8°C.
Reported safety and immunogenicity in human phase II testing in Brazil.
Documented Applications
Human phase II testing in Brazil reporting safety and immunogenicity.
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