PROCESS FOR PREPARING AN ATTENUATED TETRAVALENT DENGUE VACCINE

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.

Claims

1-18. (canceled)

19: An attenuated tetravalent dengue vaccine produced by a process comprising: (i) amplifying Vero cells in culture to produce Master and Working banks of Vero cells, wherein the 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 of this cell in 225 cm.sup.2 Tissue Culture (TC)-flasks and later in a multi-layered cell culture system; (ii) infecting Vero cells from the Master or Working bank with dengue virus serotypes 1, 2, 3 and 4 from a Seed or Working bank of each virus, wherein the Vero cells are independently infected with dengue virus serotypes 1, 2, 3, and 4 in separate cultures with serum free medium; (iii) incubating the 225 cm.sup.2 TC-flasks or multi-layered cell culture system containing the Vero cells infected with each dengue virus at 36.5 C. (1 C.) for 10 to 20 days; (iv) harvesting the supernatants of each culture; (v) filtering each dengue virus suspension from step (iv) through a membrane with 0.2 m of porosity and storing the filtered dengue virus at 80 C. (5 C.); (vi) preparing dengue virus bulks of the serotypes 1, 2, 3 and 4; (vii) formulating monovalent vaccines; (viii) formulating tetravalent vaccine by mixing the monovalent vaccines; (ix) filling vials with the tetravalent vaccine; (x) lyophilizing the tetravalent vaccine in the vials; (xi) sealing the lyophilized tetravalent vaccine in the vials; and (xii) storing the lyophilized and sealed product at 2-8 C., thereby preparing an attenuated tetravalent dengue vaccine.

20: A method for inducing an immune response to virus dengue serotypes 1, 2, 3 and 4 in a subject that comprises administering the vaccine of claim 19 to the subject.

21: A tetravalent dengue vaccine kit that comprises the vaccine of claim 19, a reconstitution composition comprising 0.2M sodium phosphate monobasic dihydrate, 0.2M sodium phosphate dibasic heptahydrate and water.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The purpose of the disclosure, together with further advantages thereof, can be better understood by reference to the accompanying drawing and the following descriptions:

[0028] FIG. 1 is a summary of the disclosure, describing all the steps of the process for preparing an attenuated tetravalent dengue vaccine.

DESCRIPTION

[0029] Although the present invention may be susceptible to different embodiments, certain embodiments are shown in the drawings and following detailed discussion, with the understanding that the present disclosure can be considered an exemplification of the principles of the invention and is not intended to limit the scope of invention to that which is illustrated and disclosed in this description.

A Process for Preparing an Attenuated Tetravalent Dengue Vaccine

[0030] In a first embodiment, the present invention refers to a process for preparing an attenuated tetravalent dengue vaccine comprising any subset or all of the following steps: adapting Vero cells to growth in serum-free medium and trypsin of non-animal origin; amplifying Vero cells in culture of this cell in 225 cm.sup.2 TC-flasks and later in Cell Factory System (CFS); producing Master and Working banks of Vero cells and Seed and Working banks of dengue's virus serotypes 1, 2, 3 and 4; infecting Vero cells contained in 225 cm.sup.2 TC-flasks or CFS with dengue's virus serotypes 1, 2, 3 and 4 from banks; incubating the 225 cm.sup.2 TC-flasks or CFS containing the Vero cells/virus suspension infected with dengue virus at 36.5 C. (1 C.) for 10 to 20 days; harvesting the supernatants of these cultures, filtering these dengue virus suspension in membrane with 0.2 m of porosity and storing at 80 C. (5 C.); preparing dengue virus bulks of serotypes 1, 2, 3 and 4; formulating monovalent vaccines with these bulks; formulating tetravalent vaccine mixing the monovalent vaccines; filling, lyophilizing; sealing and storing the product at 2-8 C. In a further embodiment the Vero cell line used is ATCC CCL-81.4 (cGMPVero, Kidney African Green MonkeyCercopithecus aeothiops; available from the ATCC, Manassas, Va., USA). In a further embodiment the dengue virus strains used are rDEN130-1545 (SEQ ID NO:1) or variants thereof; rDEN2/430(ME)-1495,7163 (SEQ ID NO:2) or variants thereof; rDEN330/31-7164 (SEQ ID NO:3) or variants thereof; and rDEN430-7132,7163,8308 (SEQ ID NO:4) or variants thereof. Variants of the aforementioned dengue virus strains that can be used include but are not limited to: (1) variants of rDEN130-1545 (SEQ ID NO:1) having a genome with at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity across the entire length of SEQ ID NO:1 and variants with the aforementioned percent sequence identities that encode a viral polyprotein with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the viral polyprotein encoded by SEQ ID NO:1; (2) variants of rDEN2/430(ME)-1495,7163 (SEQ ID NO:2) having a genome with at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity across the entire length of SEQ ID NO:2 and variants with the aforementioned percent sequence identities that encode a viral polyprotein with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the viral polyprotein encoded by SEQ ID NO:2; (3) variants of rDEN330/31-7164 (SEQ ID NO:3) having a genome with at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity across the entire length of SEQ ID NO:3 and variants with the aforementioned percent sequence identities that encode a viral polyprotein with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the viral polyprotein encoded by SEQ ID NO:3; and (4) variants of rDEN430-7132,7163,8308 (SEQ ID NO:4) having a genome with at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity across the entire length of SEQ ID NO:3 and variants with the aforementioned percent sequence identities that encode a viral polyprotein with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the viral polyprotein encoded by SEQ ID NO:4.

[0031] rDEN130 (GenBank access number: AY145123) is a live attenuated virus derived from the DEN1 Western Pacific (WP) wild-type strain by means of a deletion of 30 nucleotides (30) in the 3 untranslated region (3UTR). The rDEN130-1545 strain (SEQ ID NO: 1) used herein encodes a single Lys.fwdarw.Arg mutation at amino acid residue number 484 (A1545G mutation) in the viral polyprotein.

[0032] For the development of the DEN2 virus, the ME region of DEN2 was substituted for the corresponding genes of rDEN430 to create the vaccine candidate rDEN2/4L30(ME). The rDEN2/430(ME)-1495,7163 strain (SEQ ID NO: 2) used herein encodes a Ser.fwdarw.Phe mutation at amino acid residue number 186 (C1495T mutation) and a Leu.fwdarw.Phe mutation at amino acid residue number 112 (A7163C mutation) in the viral polyprotein.

[0033] rDEN330/31 is a live attenuated virus derived from rDEN330 strain. Initially it was constructed a complete cDNA copy of the strain DEN3 Sleman/78, creating a deletion of 30 nucleotides (30) in the 3UTR. As from the resulting rDEN330 virus, an additional deletion of about 31 nucleotides was carried out in the 3UTR [2]. Therefore, rDEN330/31 includes the original 30 deletion and a non-contiguous 31 nt deletion that removes both the original TL-2 and TL-3 structures. The resultant rDEN330/31-7164 strain (SEQ ID NO: 3) used herein encodes a Val.fwdarw.Ala mutation at amino acid residue number 115 (T7164C mutation) in the viral polyprotein.

[0034] rDEN430 is a live attenuated virus derived from the wild-type DEN4 Dominica/81 using recombinant DNA technology. One stem-loop structure, identified as TL2 in the secondary structure of the 3 UTR, was previously removed by deletion of 30 nucleotides from the DEN4 genome (3d 172-143) and has subsequently been designated as 30 mutation. The rDEN4L30-7132, 7163, 8308 strain (SEQ ID NO: 4) used herein encodes a Thr.fwdarw.Ile mutation at amino acid residue number 102 (C7132T mutation), a Leu.fwdarw.Phe mutation at amino acid residue number 112 (A7163C mutation) and a Lys.fwdarw.Arg mutation at amino acid residue number 249 (A8308G mutation) in the viral polyprotein.

[0035] In a further embodiment the MOI of dengue virus strains varies for each dengue serotype: 0.01 to 0.03 for DENV 1 and 4, 0.02 to 0.04 for DENV 2 and 0.05 to 0.08 for DENV3. In a further embodiment the monovalent vaccines are mixed in the same ratio of volume to obtain the tetravalent dengue vaccine serotypes 1, 2, 3, 4 (attenuated). In a further embodiment the parameters used in the freeze drying process are: freezing (30 to 50 C.), vacuum (20 to 100 bar), drying from 30 to 50 C. (36 to 40 h), from 5 to 10 C. (18 to 24 h) and 25 to 29 C. (8 to 15 h). In certain embodiments, the adaptation of Vero cell to serum-free medium was carried out with passage 123; the working cell bank was carried out with passage 134; and, the process for production of dengue virus used Vero cells with passage 138 to 149.

[0036] In certain embodiments, a stabilizer is used before step (vii) of formulation of monovalent vaccines. Suitable stabilizers for certain embodiments of the present invention include, but are not limited to, trehalose, sucrose, maltose, lactose, galactose, ASO4 (an stabilizer system including a mixture of stable aluminum hydroxide and monophosphoryl lipid A), human serum albumin (HSA), Pluronic block copolymers F127, F68 (BASF), P85 (BASF) and P123 (BASF), polysaccharide chitosan, and recombinant HSA (rHSA) [8, 9].

An Attenuated Tetravalent Dengue Vaccine

[0037] In another embodiment the present invention refers to an attenuated tetravalent dengue vaccine produced by the process as described above.

Use of a Composition for Reconstituting the Dried Vaccine

[0038] In another embodiment a composition comprising sodium phosphate monobasic dihydrate 0.2 M, sodium phosphate dibasic heptahydrate, 0.2 M and WFI water is used to reconstitute the vaccine as described above. In a further embodiment 5 mL of the composition is used to reconstitute the dried vaccine.

A Method for Inducing an Immune Response to Virus Dengue Serotypes 1, 2, 3 and 4 in a Patient

[0039] In another embodiment the present invention refers to a method for inducing an immune response to virus dengue serotypes 1, 2, 3 and 4 in a subject by administering the vaccine as described above to the subject.

[0040] For prophylactic treatment against Dengue infection, it is intended that the vaccine of the present invention can be administered prior to exposure of an individual to Dengue virus serotypes 1-4 and that the resulting immune response can inhibit or reduce the severity of the Dengue infection.

A Tetravalent Dengue Vaccine Kit

[0041] In another embodiment the present invention refers to a tetravalent dengue vaccine kit comprising the vaccine as described above, a reconstitution composition comprising sodium phosphate monobasic dihydrate 0.2 M, sodium phosphate dibasic heptahydrate, 0.2 M and WFI water.

EXAMPLES

Example 1. Description of Production Process

[0042] The process of production of dengue vaccine 1, 2, 3, 4 (attenuated) comprises the following steps:

[0043] Step 1. Preparation of Culture Media and Solutions Used in the Process of Vaccine's Production

[0044] The serum-free culture media for maintenance of Vero cells, preparation of Bulks and formulation of vaccine are prepared as follows:

[0045] VP-SFM AGT or AGT OptiPRO (GIBCO) serum-free media: flask of powdered culture medium is diluted in WFI water, and thereto is added L-Glutamine so that, at the end, the culture medium present 200 mM of this reagent. The medium is sterilized by filtration in membrane of 0.2 m and samples are taken for measurement of pH and Sterility test.

[0046] Leibovitz (L-15) culture media without phenol red: flasks containing the powdered culture medium are diluted in WFI water. Then, the medium is filtered in membrane of 0.2 m. Samples are taken for sterility, bacterial endotoxin, pH and appearance testing.

[0047] The culture media filtered are packed in polycarbonate flasks and stored at 2-8 C.

[0048] Buffered saline solution with 0.02M Phosphate is composed of sodium chloride, dibasic sodium phosphate, monobasic potassium phosphate, and WFI water. This solution is used for washing the cultures during the amplification cell process and in the dengue virus suspensions concentration.

[0049] Step 2. Preparation of Banks of Master and Working Vero Cells

[0050] The Vero cell banks were obtained from adaptation of Vero cell line ATCC CCL-81.4 (e.g. cGMPVero, Kidney African Green MonkeyCercopithecus aeothiops p. 123 Batch 7388125) to the culture in serum-free medium and non-animal origin trypsin. This adaptation was carried out by successive subculture of this cell in culture cell 225 cm.sup.2 T-flasks for cell culture using the serum-free medium (VP-SFM AGTGIBCO) and recombinant trypsin (TrypLE SelectGibco). After adaptation of the cells that only grow in medium with serum for growth in serum-free medium, cultures grown in serum-free medium are used to prepare the cell banks.

[0051] In the preparation of Master and Working cell banks, adapted Vero cells contained in culture flasks with a confluence of 90 to 100% are detached with trypsin, suspended in medium OptiPRO AGT (Gibco), centrifuged and the pelleted is resuspended in the same medium containing 5% DMSO. The cell suspension is homogenized and distributed into cryotubes containing 4 to 1010.sup.6 cells/ml. The cryotubes are placed in a freezer at 80 C. (5 C.), for 48 hours and then stored in liquid nitrogen. Samples are taken for bank certification through the following quality control tests: Sterility, Karyotyping, Cell Identity, Adventitious Agents in Cells and Animals, Hemadsorbents Virus and Mycoplasmas.

[0052] Step 3. Amplification of Vero Cells Used as Cellular Substrate in the Production of Dengue Virus

[0053] The cell amplification process includes thawing of a cryotube containing Vero cells from an origin cell (ATCC-CCL81.4) or from master or working cell banks in a water bath at 37 C. (1 C.). After thawing, the suspension of Vero cell is placed in T-flask with serum-free medium and incubated at 36.5 C. (1 C.) until the coverage of the cell monolayer is 90 to 100% of the T-flask cultivation area. The flasks are removed from the incubator and the cells are submitted to a new subculture. In this process, the cell monolayer is washed with saline solution buffered with phosphate 0.02M and detached with recombinant trypsin (Tryple SelectGIBCO). The cells are suspended in serum-free medium and split into TC-flasks containing the same medium. The TC-flasks are incubated again at 36.5 C. (1 C.) until reaching a coverage of 90 to 100% and then further subcultured. Amplification of the cells is initially, made in 225 cm.sup.2 TC-flasks and later in a Cell Factory System (CFS) with 10 tray layers.

[0054] Step 4. Preparation of Working Dengue Virus Banks DEN1, DEN2, DEN3 and DEN4

[0055] TC-flasks with 225 cm.sup.2 of culture area containing amplified Vero cells are infected with the dengue virus strains rDEN130-1545 (SEQ ID NO:1); rDEN2/430(ME)-1495,7163 (SEQ ID NO:2); rDEN330/31-7164 (SEQ ID NO:3); and rDEN430-7132,7163,8308 (SEQ ID NO:4), separately. The MOI (Multiplicity of Infection) used for virus infection is different for each serotype: 0.01 to 0.03 for DENV 1 and DENV 4, 0.02 to 0.04 for DENV 2 and 0.05 to 0.08 for DENV3. The infected cultures are incubated at 36.5 C. (1 C.). After 8 days of incubation, supernatants of the cultures infected with DEN130, DEN2/4830(ME)-1495,7163, DEN330/31-7164 and DEN430-7132,7163,8308 are separately harvested, filtered through a sterilizing membrane and stored in a freezer at 80 C. (5 C.). The culture medium of flasks is replaced, and the flasks are again incubated at 36.5 C. (1 C.). This procedure is repeated for three consecutive days to produce at the end four samples of the supernatants. For cultures infected with DEN3, this procedure begins on the 10th day of incubation. Samples of each harvest of the cultures' supernatant are taken for sterility and virus titration tests.

[0056] In the preparation of working banks, harvests approved in sterility tests, with titers higher than 10.sup.5.0 PFU/ml are mixed, distributed into cryotubes with 2 to 4 mL and maintained in liquid nitrogen. The bank is used after being approved in the following tests: Viral Identity, Sterility, Titration, Adventitious Agents in Cells and Animals, Hemadsorbents Virus and Mycoplasmas.

[0057] Step 5. Production of Dengue Virus Serotypes 1, 2, 3 and 4 for Dengue Vaccine Formulation

[0058] After amplification, Vero cells contained in TC-flasks or Cell Factory System obtained from the amplification process, as described in step 3, are trypsinized and suspended in serum-free medium (OptiPRO AGTGIBCO). The Vero cell suspension obtained is inoculated with dengue virus strains rDEN130-1545, rDEN2/430(ME)-1,495.7163, rDEN330/31-7164 and rDEN430-7132,7163,8308, from the banks of dengue virus prepared in step 4 of the process for production of dengue vaccine. For inoculation the different MOIs (Multiplicity of Infection) for each serotype are used: 0.01 to 0.03 for DENV 1 and DENV 4, 0.02 to 0.04 for DENV 2 and 0.05 to 0.08 for DENV3. After inoculation, the virus/cell suspension is stirred at 32 C. (1 C.) for 30 to 60 minutes and then distributed in 225 cm.sup.2 TC-flasks or CFS with 10 tray layers. Serum-free culture media is added to cultures until it reaches the volume of 100 to 150 mL in the TC-flask and 1,200 to 1,800 ml for CFS. For CFS with different numbers of tray layers, it is calculated that the volume of culture medium to be added by making a rule of three. The cultures are incubated at 36.5 C. (1 C.). On the 8th or 10th day of incubation, 50% to 60% of the medium is removed, and the same volume of serum-free medium is added in the cultures. The cultures are incubated again at 36.5 C. (1 C.). The harvest of the supernatants of infected Vero cell cultures occur from the 10th to the 20th day after inoculation of dengue virus.

[0059] The harvest process includes the removal of the supernatants of the TC-flasks or CFS cultures infected, mixture of the supernatants harvested, a sterilizing filtration of this mixture, distribution of the dengue virus suspension filtered in polypropylene/polycarbonate flasks and storage in a freezer at 80 C. (5 C.). Samples are taken for Sterility and Viral Titration tests. After approbation in the tests, the flasks with the virus dengue suspension are removed from the freezer and forwarded to the concentration process.

[0060] The virus dengue suspension harvested are thawed and concentrated by tangential filtration process using a Pellicon System (Millipore) with a membrane of 30 to 50 kDa of porosity. Samples are taken for control quality tests: Viral titration and Sterility. The viral concentrate (C1) of rDEN130-1545, rDEN2/430(ME)-1495,7163, rDEN330/31-7164, or rDEN430-7132,7163,8308 is denominated and stored at 80 C. (5 C.).

[0061] Step 6. Preparation of Dengue Virus Bulks

[0062] Dengue virus concentrate C1 (rDEN130-1545, rDEN2/430(ME)-1495,7163, rDEN330/31-7164, or rDEN430-7132,7163,8308) are removed from the freezer at 80 C. (+5 C.), thawed and subjected to the following process: the virus concentrate is diluted with Leibovitz medium without phenol red and dilution factor used is 5 to 10 times its initial volume. The concentrate diluted is concentrated again, by tangential filtration (Pellicon system) to a volume 2.5 to 3 times its initial volume. This concentrate is called C2.

[0063] The concentrate C2 is filtered in membrane with 0.2 m of porosity, distributed in tubes/flasks and stored in a freezer at 80 C. (5 C.). Samples are taken for quality control tests (Sterility and Bacterial Endotoxin, Mycoplasmas, Adventitious Agents in cells, Hemadsorbents Virus, Identity and Viral titration). After approbation in quality control tests, the Bulk is released to the formulation of monovalent vaccine.

[0064] The dengue virus Bulks lots produced in 2013 and 2014 are in table 1.

TABLE-US-00001 TABLE 1 Bulks of rDEN130-1545, rDEN2/430(ME)-1495, 7163, rDEN330/31-7164, and rDEN430-7132, 7163, 8308 dengue virus produced in 2013 and 2014. Viral Quality Control Tests Number titer Bacterial of Log.sub.10 Endotoxin Other Bulks Lots flasks PFU/ml (UE/mL) tests IB-DEN130/ 01/13 18 6.8 <1.25 Approved Vero/M 02/13 40 5.8 <1.25 Approved 01/14 43 6.4 <0.50 Approved 02/14 16 6.9 <0.73 Approved 03/14 24 6.1 <0.51 Approved 04/14 26 6.8 <0.50 Approved IB-DEN2/430/ 01/13 19 5.7 <1.25 Approved Vero/M 02/13 32 5.9 <1.25 Approved 01/14 39 6.4 <0.50 Approved 02/14 21 7.0 4.43 Approved 03/14 21 7.0 <0.50 Approved 04/14 40 6.5 <0.50 Approved IB-DEN330/ 01/13 17 6.1 <1.25 Approved 31Vero/M 02/13 45 6.1 <1.25 Approved 02/14 22 6.8 <0.50 Approved 03/14 23 6.6 <0.50 Approved IB-DEN430/ 01/13 50 5.9 <1.25 Approved Vero/M 02/13 35 6.3 <1.25 Approved 01/14 23 6.9 0.68 Approved 02/14 19 6.0 <0.50 Approved 03/14 30 6.8 0.68 Approved PS. The endotoxin value until 50 UE/mL is considered satisfactory, since the final product must be smaller or equal to 10 UE/mL.

[0065] Step 7. Formulations of Monovalent Vaccines rDEN130-1545, rDEN2/430(ME)-1495,7163, rDEN3830/31-7164, and rDEN430-7132,7163,8308 and Dengue Vaccine Serotypes 1, 2, 3, 4 (Attenuated)

[0066] Four dengue monovalent vaccines are formulated, one for each type of dengue virus (rDEN130-1545, rDEN2/430(ME)-1495,7163, rDEN330/31-7164, and rDEN4830-7132,7163,8308). The calculations for formulation consist in determining a dilution factor so that the monovalent vaccines according with each serotype are provided in the following amounts: 5.70.2, 5.60.2, 6.10.2 and 5.80.2 Log.sub.10 PFU/ml for DENV1, DENV2, DENV3, and DENV4, respectively.

[0067] The formula to determine the dilution factor is: antilog of the bulk titer (Log.sub.10 PFU/ml) divided by the antilog of viral titer (Log.sub.10 PFU/ml) desired for each type of monovalent. The formulations of rDEN130-1545, rDEN2/430(ME)-1495,7163, rDEN330/31-7164, and rDEN430-7132,7163,8308 monovalent are made with Leibovitz (L-15) medium without phenol red concentrate twice, i.e., the medium remains with its original components twice concentrated.

[0068] To make the dengue vaccine serotypes 1, 2, 3, 4 (attenuated) formulation, the monovalent 1, 2, 3 and 4 vaccines are mixed in the same ratio of volume. After homogenization of the formulated tetravalent vaccine, the product is subjected to a filtration (membrane with 0.2 m of porosity) and samples are taken to the flowing quality control tests: Sterility, Bacterial Endotoxin, Viral Titration, pH, and Appearance of the product.

[0069] Step 8. Filling, Lyophilization and Sealing of the Tetravalent Dengue Vaccine.

[0070] After the tetravalent dengue vaccine formulation, the product is used to fill vials with 3 ml of vaccine. Samples of filled vials are taken for quality control tests (Sterility, Endotoxin Bacterial, Viral Titration, Appearance and pH).

[0071] After the filling of the vials they are transported to the lyophilizer and start the freeze-drying process. In this process, the following parameters are established: freezing (30 to 50 C.), vacuum (20 to 100 bar), drying from 30 to 50 C. (36 to 40 h), from 5 to 10 C. (18 to 24 h) and 25 to 29 C. (8 to 15 h).

[0072] At the end of freeze-drying process, the vials with the lyophilized vaccine are subjected to a sealing process. The final product, dengue vaccine 1, 2, 3, 4 (attenuated), is stored at 2-8 C. Samples of the vaccine lot are tested for Sterility, Bacterial Endotoxin, Viral Titration, Product Appearance before and after reconstitution with the diluent, pH, Residual DNA and Residual Moisture tests.

[0073] The product can be denominated dengue vaccine serotypes 1, 2, 3, 4 (attenuated), when following the Brazilian regulations for designation of vaccines.

[0074] The product can be reconstituted with 5.0 mL of the specific diluent to this vaccine (mixture of sodium phosphates), which corresponds to 10 doses/0.5 mL/vial. Each dose contains 10.sup.2.7 to 10.sup.3.7 PFU/dose of each of dengue virus used in the dengue vaccine 1, 2, 3, 4 (attenuated) formulation. The results of quality control tests obtained from six batches produced in 2014, are shown in table 2.

TABLE-US-00002 TABLE 2 Dengue vaccine serotypes 1, 2, 3, 4 (attenuated) lots produced in 2014. Results of quality control tests Viral titer Bacterial Residual Residual Sterility Log.sub.10/PFU/dose Endotoxin DNA moisture Product Lots DEN1 DEN2 DEN3 DEN4 (UE/mL) PH (pg/dose) % appearance 01/14 3.0 3.4 3.0 3.2 <0.500 6.9 32.6 1.33 Approved 02/14 2.7 3.1 2.8 3.2 <0.500 6.9 27.2 2.01 Approved 03/14 3.1 3.2 2.9 3.2 <0.500 6.9 52.6 1.99 Approved 04/14 3.2 3.6 3.1 3.6 0.956 6.9 40.8 0.89 Approved 05/14 3.5 3.6 3.0 3.5 1.030 6.9 31.5 0.52 Approved 06/14 3.4 3.5 3.5 3.4 <0.500 6.9 35.1 0.35 Approved Values for lot approval: Bacterial endotoxin = 10 UE/ml; Viral titration = 10.sup.2.7 to 10.sup.3.7 PFU/dose; pH = 6.8 to 7.2; Residual Cellular DNA 100 pg/dose; Residual moisture 3%; Product appearance before the reconstitution: slightly yellowish (homogeneous cake (SYHC) and Product appearance after reconstitution: slightly yellowish clear liquid (SYCL).

III Diluent for the Reconstitution of Dengue Vaccine 1, 2, 3, 4 (Attenuated)

Composition:

[0075] For the preparation of 1,000 mL
Solution 1 (sodium phosphate monobasic dihydrate 0.2 M) . . . 195 mL
Solution 2 (sodium phosphate dibasic heptahydrate, 0.2 M) . . . 05 mL
WFI water qsp . . . 1,000 mL

[0076] Presentation: vials or ampoules with 5.0 mL

IV Stability Studies of Dengue Vaccine 1, 2, 3, 4 (Attenuated) Stability Studies at 2-8 C.

[0077] The results of the tests carried out in the samples of three batches of dengue vaccine 1, 2, 3, 4 (attenuated) stored at 2-8 C. are shown in tables 3 and 4.

TABLE-US-00003 TABLE 3 Results of sterility and physical-chemical tests found in the lots of dengue vaccine 1, 2, 3, 4 (attenuated) stored at 2-8 Results of Samples Months of Appearance Residual Vaccine Storage before and after moisture Lots 2-8 C. Sterility pH reconstitution (%) 01/10 12 Approved 7.1 SYHC and SYCL 2.91 02/10 12 Approved 7.1 SYHC and SYCL 2.79 01/11 12 Approved 7.1 SYHC and SYCL 2.48 SYHC: slightly yellowish homogeneous dried cake. SYCL: slightly yellowish clear liquid.

III Diluent for the Reconstitution of Dengue Vaccine 1, 2, 3, 4 (Attenuated)

Composition:

[0078] The analysis of the results of Table 3 indicates that for up to at least one year of storage the titers of dengue virus serotypes 1, 2, 3 and 4 remained satisfactory. After 18 months of storage at 2-8 C., titers of DENV3 and DENV4 fell below the minimum required (10.sup.2.7 PFU/dose of vaccine).

TABLE-US-00004 TABLE 4 Results of dengue virus titers components of dengue vaccine 1, 2, 3, 4 (attenuated) stored at 2-8 C. Dengue virus titers (Log.sub.10 PFU/dose) Vaccine Months of Storage at 2-8 C. Lots Serotypes 0 3 6 9 12 18 01/10 DEN1 3.1 3.2 3.3 3.3 3.1 3.0 DEN2 3.1 3.2 3.3 3.3 3.1 3.0 DEN3 3.2 3.3 3.1 3.1 3.2 2.0 DEN4 3.3 3.4 3.4 3.4 3.3 2.8 02/10 DEN1 3.1 3.1 3.6 3.3 3.1 3.2 DEN2 3.2 3.2 3.3 3.3 3.2 3.0 DEN3 3.2 3.2 3.1 3.1 3.1 2.2 DEN4 3.2 3.2 3.4 3.4 3.2 2.2 01/11 DEN1 3.1 3.4 3.1 3.1 3.1 3.0 DEN2 3.2 3.3 3.1 3.0 3.0 3.0 DEN3 3.2 3.1 3.1 3.1 3.0 2.4 DEN4 3.1 3.1 3.1 3.0 3.0 1.7

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[0088] Having described certain embodiments of the invention, one skilled in the art will appreciate in the appended claims that many modifications and variations of the present invention are possible in light of the above teachings. It is therefore, to be understood that, within the scope of the appended claims and disclosure provided herein, the invention may be practiced otherwise than as specifically described in certain embodiments.