A BLUETONGUE VACCINE AND METHODS OF MANUFACTURE THEREOF

Abstract

Disclosed is a novel vaccine formulation for prevention against Bluetongue virus infection in domestic and wild ruminants, cattle, goats, sheep, pigs, and any other mammals for commercial use. The antigen present in the vaccine is an inactivated antigen comprising 5 different serotypes of Bluetongue virus, thereby making a pentavalent vaccine formulation against Bluetongue virus infections. Also disclosed are method of adaptation of Bluetongue virus in suspension culture upto 2000 liters industrial size bioreactors, and inactivation techniques for preparation of the given vaccine formulation with the presence of suitable adjuvants and preservatives.

Claims

1. A method of industrial cultivation of Bluetongue virus, the said method comprising, virus culture of Bluetongue virus in suspension BHK-21 cell lines in stainless steel vessels industrial bioreactor.

2. A method of adaptation of Bluetongue virus in BHK-21 cells in suspension culture capable of industrial production of Bluetongue vaccine.

3. A method of scale up of virus culture of Bluetongue virus on BHK-21 cells from T-175 flask to an industrial bioreactor of 250 liter batch volume, wherein the virus titer attained of the Bluetongue virus ranges from 10.sup.−5.5TCID.sub.50/ml to 10.sup.−7.0TCID.sub.50/ml within 48 hours to 72 hours of infection.

4. A method of scale up of virus culture of Bluetongue virus on BHK-21 cells from 250 liter batch volume to an industrial bioreactor of 2000 liter batch volume, wherein the virus titer attained of the Bluetongue virus ranges from 10.sup.−5.5TCID.sub.50/ml to 10.sup.−7.0TCID.sub.50/ml within 48 hours to 72 hours of infection.

5. A vaccine composition for the prophylaxis of animals from Bluetongue virus infection, comprising a vaccine antigen, wherein the said vaccine antigen comprises of Bluetongue virus serotypes BTV 1, BTV 2, BTV 10, BTV 16 and BTV 23, the said Bluetongue serotypes being adapted to infect BHK-21 cells in suspension cell line culture to obtain a virus titer from 10.sup.−5.5TCID.sub.50/ml to 10.sup.−7.0TCiD.sub.50/ml within 48 hours to 72 hours of infection thereby capable of conferring immune response against Bluetongue virus infections.

6. The vaccine composition according to claim 1, wherein the animal is a domestic or a wild ruminant, cattle, goat, sheep, pig, or any other mammal involved in animal husbandry useful for commercial benefit.

7. The vaccine formulation according to claim 5, wherein the vaccine antigen is inactivated using Binary Ethylenimine.

8. The vaccine formulation according to claim 5, wherein the vaccine composition further comprises a vaccine adjuvant.

9. The vaccine composition according to claim 5, is stable for period of 1 year at 5±3° C.

Description

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

Preparation of Master and Working Seed Virus in BHK21 Suspension Cell Lines

[0014] The Bluetongue virus serotypes BTV1, BTV2, BTV10, BTV16 and BTV23 were received from Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai, India under a Memorandum of Understanding between Indian Council of Agricultural Research and Biovet Private Limited, Bangalore dated 15.sup.th February 2011. These strains are the most current and infective strains present in India and are used to produce bulk antigen. The source material from TANUVAS is aliquoted and stored in liquid nitrogen. These virus are sequenced, characterized and certified by TANUVAS. The master and working seed virus is prepared from source material in BHK21 suspension cell lines and stored at liquid nitrogen above vapour phase.

TABLE-US-00001 Master Bank Total No. of Virus No. of vials Sl. No. Strain vials used Purpose 1 BTV 1  30 7 Identification, Sterility, Virus titer, Mycoplasma and Working Bank 2 BTV 2  30 7 Identification, Sterility, Virus titer, Mycoplasma and Working Bank 3 BTV 10 30 7 Identification, Sterility, Virus titer, Mycoplasma and Working Bank 4 BTV 16 30 7 Identification, Sterility, Virus titer, Mycoplasma and Working Bank 5 BTV 23 30 7 Identification, Sterility, Virus titer, Mycoplasma and Working Bank

TABLE-US-00002 Working Bank Total No. of Virus No. of vials Sl. No. Strain vials used Purpose 1 BTV 1  80 9 Identification, Sterility, Virus titer, Mycoplasma and Antigen batch preparation 2 BTV 2  80 9 Identification, Sterility, Virus titer, Mycoplasma and Antigen batch preparation 3 BTV 10 80 9 Identification, Sterility, Virus titer, Mycoplasma and Antigen batch preparation 4 BTV 16 80 9 Identification, Sterility, Virus titer, Mycoplasma and Antigen batch preparation 5 BTV 23 80 9 Identification, Sterility, Virus titer, Mycoplasma and Antigen batch preparation

Characterization of Master and Working Seed Bank:

[0015] The following tests were carried out for the master and working seed virus, identification of all Bluetongue virus serotypes has been done. Sterility test is done as per I.P.2010 to check the presence of bacteria and fungi by Fluid Thioglycollate Medium and Soybean Casein Digest Medium at 35° C. and 25° C. respectively.

TABLE-US-00003 Working Sl. No. Master Bank Result Bank Result 1 BTV 1  Sterile BTV 1  Sterile 2 BTV 2  Sterile BTV 2  Sterile 3 BTV 10 Sterile BTV 10 Sterile 4 BTV 16 Sterile BTV 16 Sterile 5 BTV 23 Sterile BTV 23 Sterile

[0016] Virus titer is checked for all the Bluetongue virus serotypes by TCID50 method as per in-house specification in Biovet QC lab.

TABLE-US-00004 Working Sl. No. Master Bank Result Bank Result 1 BTV 1  10.sup.−5.47TCID.sub.50/ml BTV 1  10.sup.−5.77TCID.sub.50/ml 2 BTV 2  10.sup.−5.87TCID.sub.50/ml BTV 2  10.sup.−6.12TCID.sub.50/ml 3 BTV 10 10.sup.−6.12TCID.sub.50/ml BTV 10 10.sup.−6.20TCID.sub.50/ml 4 BTV 16 10.sup.−6.30TCID.sub.50/ml BTV 16 10.sup.−6.30TCID.sub.50/ml 5 BTV 23 10.sup.−5.87TCID.sub.50/ml BTV 23 10.sup.−6.12TCID.sub.50/ml

[0017] The mycoplasma test is done and absence of mycoplasma was ascertained.

EXAMPLE 2

Large Scale Production of BHK21 Suspension Cell Lines and Scale Up for Bluetongue Vaccine

[0018] One vial of BHK-21 C13 cells from the working cell bank is taken and is thawed in a water bath at 36° C.±1° C. for 3 to 5 minutes and serially scaled up in cell growth medium. The cell growth medium comprises of Glassgow Essential Medium and new born calf serum or adult bovine serum. Initially, T-75, T-175 tissue culture flasks are used followed by 500 ml, 1 L, 5 L, 10 L, 15 L glass bottles and later in large scale production level batch sizes of 200 L and 2000 L. BHK-21 suspension cells are aseptically transferred into Virus Bioreactor. The cells are then subjected to chilling and sedimentation for a period of 18-24 hours and later the spent media is removed from cell suspension.

Bluetongue Virus:

[0019] Bluetongue virus seed of intended serotype from working seed bank is thawed with a titer value not less than 10.sup.−5.0 Tissue culture infectious dose (TCID.sub.50) is infected as per Multiplicity of Infection (MOI) calculation and followed by virus maintenance medium without any serum. Thereafter, Bluetongue virus is inoculated into BHK-21 C13 suspension cells and kept for incubation at 36° C. for 48-72 hours with continuous stirring along with cell control. Virus culture is harvested when it attains 90-95% Cytopathic Effect (CPE). The viral harvest from bioreactor is subjected to centrifugation to remove cell debris. After complete infection the virus culture is centrifuged, the supernatant collected into a separate sterile container and used as seed for the large scale production.

[0020] The standard virus titer results in the Suspension cell lines of the Bluetongue virus are mentioned in the below table:

TABLE-US-00005 Sl. No. Serotypes Virus titer result Sterility 1 BTV 1  10.sup.−5.12TCID.sub.50/ml Sterile 2 BTV 2  10.sup.−5.87TCID.sub.50/ml Sterile 3 BTV 10 10.sup.−5.42TCID.sub.50/ml Sterile 4 BTV 16 10.sup.−6.12TCID.sub.50/ml Sterile 5 BTV 23 10.sup.−4.87TCID.sub.50/ml Sterile

[0021] Scale up experimental data from T-175cm.sup.2 tissue culture flask to large scale production of Bluetongue virus equivalent to 2000 liter batch-size of five Indian isolates in BHK-21 suspension cell lines in bioreactor is presented in the below mentioned table:

TABLE-US-00006 TABLE 1 Culture of Bluetongue virus in suspension cell lines and scale up from TC flask to 2000 liter commercial scale batch size. 15 liter batch 250 L 250 L 2000 L 2000 L in batch-I in batch-II in batch-I in batch-II in Suspension Suspension Suspension Suspension Suspension T-175 cm.sup.2 Cells Cells Cells Cells Cells TC Flask (BHK-21) (BHK-21) (BHK-21) (BHK-21) (BHK-21) Media conc. GMEM for GMEM for GMEM for GMEM for GMEM for GMEM for cell growth cell growth cell growth cell growth cell growth cell growth with 8% v/v with 8% v/v with 8% v/v with 8% v/v with 8% v/v with 8% v/v Time NBCS ABS ABS ABS ABS ABS Cell count at 0.4 × 10.sup.5 cells/ml 0.65 × 10.sup.6 cells/ml 0.55 × 10.sup.6 cells/ml 0.6 × 10.sup.6 cells/ml 0.5 × 10.sup.6 cells/ml 0.6 × 10.sup.6 cells/ml 0 Hour Cell count at 1.3 × 10.sup.6 cells/ml 1.50 × 10.sup.6 cells/ml  1.4 × 10.sup.6 cells/ml 1.5 × 10.sup.6 cells/ml 1.5 × 10.sup.6 cells/ml 1.6 × 10.sup.6 cells/ml 24 Hour Dicanting of Addition of Addition of Addition of Addition of Addition of Addition of spent media GMEM GMEM GMEM GMEM GMEM GMEM and seed without serum without serum without serum without serum without serum without serum Virus for virus for virus for virus for virus for virus for virus innoculation multiplication multiplication multiplication multiplication multiplication multiplication 48 Hours 10.sup.−4.90TCID.sub.50 10.sup.−4.87TCID.sub.50 10.sup.−5.24TCID.sub.50 10.sup.−5.12TCID.sub.50 10.sup.−4.50TCID.sub.50 10.sup.−4.87TCID.sub.50 after per ml of per ml of per ml of per ml of per ml of per ml of infection BTV-1 BTV-1 BTV-1 BTV-1 BTV-1 BTV-1 10.sup.−5.24TCID.sub.50 10.sup.−5.42TCID.sub.50 10.sup.−5.87TCID.sub.50 10.sup.−5.47TCID.sub.50 10.sup.−5.12TCID.sub.50 10.sup.−5.29TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-2 BTV-2 BTV-2 BTV-2 BTV-2 BTV-2 10.sup.−4.80TCID.sub.50 10.sup.−5.80TCID.sub.50 10.sup.−5.47TCID.sub.50 10.sup.−5.40TCID.sub.50 10.sup.−5.15TCID.sub.50 10.sup.−5.31TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-10 BTV-10 BTV-10 BTV-10 BTV-10 BTV-10 10.sup.−5.80TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−5.88TCID.sub.50 10.sup.−5.80TCID.sub.50 10.sup.−5.87TCID.sub.50 10.sup.−5.50TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-16 BTV-16 BTV-16 BTV-16 BTV-16 BTV-16 10.sup.−5.87TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−5.87TCID.sub.50 10.sup.−5.40TCID.sub.50 10.sup.−5.47TCID.sub.50 10.sup.−5.86TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-23 BTV-23 BTV-23 BTV-23 BTV-23 BTV-23 72 Hours 10.sup.−5.24TCID.sub.50 10.sup.−5.47TCID.sub.50 10.sup.−5.47TCID.sub.50 10.sup.−5.48TCID.sub.50 10.sup.−5.50TCID.sub.50 10.sup.−5.85TCID.sub.50 after per ml of per ml of per ml of per ml of per ml of per ml of infection BTV-1 BTV-1 BTV-1 BTV-1 BTV-1 BTV-1 10.sup.−5.87TCID.sub.50 10.sup.−5.87TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−6.00TCID.sub.50 10.sup.−5.47TCID.sub.50 10.sup.−5.86TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-2 BTV-2 BTV-2 BTV-2 BTV-2 BTV-2 10.sup.−5.12TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−6.20TCID.sub.50 10.sup.−6.30TCID.sub.50 10.sup.−6.32TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-10 BTV-10 BTV-10 BTV-10 BTV-10 BTV-10 10.sup.−6.12TCID.sub.50 10.sup.−6.30TCID.sub.50 10.sup.−6.30TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−6.39TCID.sub.50 10.sup.−6.30TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-16 BTV-16 BTV-16 BTV-16 BTV-16 BTV-16 10.sup.−6.00TCID.sub.50 10.sup.−6.39TCID.sub.50 10.sup.−6.39TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−6.12TCID.sub.50 10.sup.−6.30TCID.sub.50 per ml of per ml of per ml of per ml of per ml of per ml of BTV-23 BTV-23 BTV-23 BTV-23 BTV-23 BTV-23

EXAMPLE 3

Inactivation of Bluetongue Virus

[0022] The clarified virus from 2000 Liter batch size is inactivated with 0.04% v/v formalin for 1 hour followed by addition of 1.5 mM Binary Ethyleneimine (BEI) to the final culture volume at 0 hour and kept under continuous stirring for 24 hours at 36±1 ° C. The culture was transferred to another sterile vessel and second dose of BEI is added having a final concentration of 3 mM and kept for a period of another 24 hours under stirring at 36±1 ° C. The samples were collected at different intervals of time (0, 1.sup.st, 2.sup.nd, 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th, 24.sup.th , and 48.sup.th hours) for studying RNA degradation by using 1% Agarose gel electrophoresis method. FIGS. 1 and 2 shows that there is no presence of RNA after 24 hours of inactivation and 48 hours of post inactivation on respective lanes.

[0023] Concentration of Bluetongue virus: After inactivation, the virus brew is subjected to final concentration of approx 30× using Tangential Flow Filtration (TFF) system and the concentrated bluetongue antigen is collected in a 100 L pressure vessel. The collected antigen in 100 liter pressure vessel is further dispensed under Biosafety cabinet into sterile Polypropylene (PP) bottles and stored at 2° C.-8° C. for vaccine blending. Samples are collected for quality control testing on various parameters like sterility and Indirect ELISA and it was found to be stable for period of 2 years at 5±3° C.