WO2010137036A2 - Novel japanese encephalitis vaccine and method of manufacturing the same - Google Patents

Novel japanese encephalitis vaccine and method of manufacturing the same Download PDF

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WO2010137036A2
WO2010137036A2 PCT/IN2010/000343 IN2010000343W WO2010137036A2 WO 2010137036 A2 WO2010137036 A2 WO 2010137036A2 IN 2010000343 W IN2010000343 W IN 2010000343W WO 2010137036 A2 WO2010137036 A2 WO 2010137036A2
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vaccine
virus
cells
japanese encephalitis
vero
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WO2010137036A3 (en
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Rajesh Jain
Milind V Galgalkar
Kapil Maithal
Sudhanshu Vrati
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Panacea Biotec Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N7/06—Inactivation or attenuation by chemical treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/24011—Flaviviridae
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    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24161—Methods of inactivation or attenuation
    • C12N2770/24163—Methods of inactivation or attenuation by chemical treatment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Definitions

  • the present invention relates to a novel Japanese encephalitis vaccine and a novel process for making the same.
  • the invention in particular relates to a Japanese encephalitis virus strain P20778 vaccine, wherein the viral adaptation and propagation is carried out in a medium which is free of serum and additives of animal origin.
  • Japanese encephalitis is the main cause of viral encephalitis in many countries of Asia.
  • the infection is mosquito-borne and caused by the Japanese encephalitis virus.
  • Clinical presentations vary but may include headache, fever, a change in mental status and onset of seizures, tremors, paresis hypertonia and loss of coordination (WHO- recommended standards for surveillance of selected vaccine-preventable diseases. Geneva: World Health Organization, 2003).
  • the virus exists in a transmission cycle between mosquitoes and pigs and/or water birds. Humans become infected only incidentally when bitten by an infected mosquito (Culex spp.) and the disease is predominantly found in rural and periurban settings.
  • JEV Japanese encephalitis virus
  • Flaviviridae family of animal viruses is an enveloped, positive-sense, single stranded RNA virus.
  • flavivirus virions are spherical, approximately 40 nm in diameter and are composed of a lipid bilayer surrounding a nucleocapsid containing an 11-kb genome complexed with a capsid (C) protein (Rice, C. M. et al. Science 229:726- 33, 1985).
  • C capsid
  • Surface projections on the membrane are composed of glycosylated envelope (E) and membrane (M) proteins.
  • Important physiological activities are associated with the 53-kD E protein, including virus attachment to and fusion with target cell membranes and hence is the primary target for neutralizing antibodies and a critical component of any candidate vaccine (Koshini, E. et al. Virol. 188:714-20, 1992).
  • the cell culture-derived vaccines are manufactured and widely used in China, where the inactivated vaccine is gradually being replaced by the live attenuated vaccine.
  • the SA 14-14-2 vaccine strain was obtained from its wild-type SA 14 parent by serial passages in cell cultures (primary hamster kidney cells- PHK cells) and in animals (mice, hamsters) with successive plaque purifications (in primary chick embryo cells). Concern about possible adventitious agents in primary hamster cells is likely to limit its use outside China.
  • PHK cells in which Chinese vaccine is prepared is not approved by the World Health Organization (WHO) for viral vaccine production or licensed for human use by the developed countries.
  • WHO World Health Organization
  • the present invention suggests development and propagation of JE virus, preferably in continuous cell line, like Vero cells for vaccine production, overcoming previous problems in JE virus produced in mouse brain or primary cell lines.
  • the present invention also identifies methodology developed to cultivate the JE virus, inactivation of JE virus and a downstream process for vaccine production with cost-effectiveness.
  • the present invention identifies methodology improved upon the previously commercialized JE vaccines in the following ways.
  • the invented viral vaccine will not acquire the virulence through the Vero cell cultivation, reducing the hazards of production and affording an additional level of safety to recipients beyond that furnished by stringent control over the virus-inactivation process.
  • Prior art animal media have the potential to bear infective animal viruses which can pose a safety issue.
  • serum free/ animal component free media will ensure that the resulting vaccine is not contaminated with animal derived pathogens.
  • the JE vaccine of the present invention is produced in the absence of serum and animal derived components, providing high yields and inexpensive and scalable production, which are not achieved in the previously commercialized JE vaccines.
  • the present invention provides a novel method for development and production of a Japanese encephalitis vaccine, more specifically a Vero cell derived inactivated Japanese encephalitis vaccine using P20778 strain, comprising the steps of adapting the Vero cell line to serum and animal component free medium, adapting P20778 in Vero cell line by serial passaging, establishment of master and working seed banks of P20778, cultivating cells permissive for productive replication of the virus in a serum free and animal component free medium, infecting the cells with the virus, cultivating the infected cells in serum free and animal component free medium until progeny virus is produced, isolating the virus from the culture, followed by purification, inactivation and. formulation.
  • the present invention further provides a Japanese encephalitis virus strain P20778 obtained by cultivating Vero cells permissive for productive replication of the virus in a serum free and animal component free medium, infecting the cells with the virus, cultivating the infected cells in serum free and animal component free medium until progeny virus is produced, and isolating the virus from the culture.
  • Indian strain of Japanese encephalitis virus (P20778) has been identified as potential vaccine candidate. This strain was clinically isolated from a patient in Christian Medical College, Vellore, India.
  • the JEV genome is a plus-sense single-stranded RNA of about 11 kb.
  • the Vellore P20778 isolate has 10977 bases. It has an additional base compared to the prototype virus.
  • the additional base is part of the 3- non-coding region (NCR), which, does not affect the length of the viral polyprotein and hence predicted site of all viral proteins is similar to other JEV strains like GP78 (India, 1978) and JaOArS982 (Japan, 1982) strains.
  • the JE vaccine made from Nakayama strain has shown cross neutralization with Vellore P20778 strains more efficiently, proving the relatedness of the Vellore strain to the Nakayama strain from Japan. Another interesting finding is that the Vellore strain is found to be closer to Beijing- 1 strain. Thus, the Indian strain, Vellore P20778 is well characterized and phylogenetically close to the Chinese & Japanese isolates.
  • the said virus may be propagated in Vero tissue culture cells.
  • Vero cells are non-tumorigenic cells derived from monkey kidney.
  • the Vero cell line is more advantageous than any other standard cell line, as the Vero cells are more readily adaptable to large scale cell culture and as a transformed cell has an infinite life time.
  • the Vero cell line used is (WHO- Vero 10- 87). The invention is however not restricted to the above mentioned cell lines and other equivalents contemplated by a person of skill in art are within the scope of the present invention.
  • One preferred embodiment of the invention relates to a Japanese Encephalitis vaccine comprising Japanese Encephalitis virus Vellore strain P20778, wherein the virus is propagated in Vero cell line.
  • the Vero cell line (WHO- Vero 10-87) is used for the propagation of the virus.
  • the culture conditions under which a virus strain is multiplied are also of great significance for the achievement of a commercially acceptable yield.
  • both the host system and the culture conditions must therefore be specifically adapted in order to achieve favorable environmental conditions for the desired virus strain.
  • a system that creates optimal growth conditions is therefore required. Efficient production systems are often based on adaptations of the virus population of corresponding culture systems, often using intermediate stages with other host systems and employing protein additives— mostly serum of animal origin.
  • the present invention achieves these results without any addition of serum or animal components.
  • Serum free medium that may be useful for practicing the present invention may include, but need not be limited to Iscove's medium, Ultra-CHO medium (BioWhittaker) , EX-CELL (JRH Bioscience), SFM4MegaVir media and the like.
  • SFM4MegaVir media (Hyclone) formulated with suitable additives like glutamine and gentamicin sulphate may be used as the medium of choice for the propagation of the Vero cells as well as for the propagation of the virus.
  • SFM4MegaVir media (Hyclone) medium formulated with about 2- 5mM stable glutamine and about 20- 100 ⁇ g/mL Gentamicin Sulphate may be used.
  • SFM4MegaVir media (Hyclone) formulated with about 4mM stable glutamine and about 70 ⁇ g/mL Gentamicin Sulphate may be used.
  • 'about' may be used herein to describe the amount of each of the components used during the preparation of the vaccine of the invention, to mean an amount of the said component that is present in amounts of preferably ⁇ 20%, more preferably ⁇ 10% and most preferably ⁇ 5% of the stated amount for that particular component.
  • a monolayer culture method comprises growing and infecting cells that have been cultured on the inner surface of a vessel with a virus of interest and then subjecting the infected cells to a standing culture or a roll-streak culture, so as to prepare the virus in the culture supernatant.
  • the vessels used for this purpose may be a plate culture vessel or a roll-streak culture flask. Specific examples of the vessel include Petri dish, T flasks, roller bottles or multilayer flasks. Material of the vessels is preferably, a non- glass material like plastic.
  • microcarrier method using microcarrier beads.
  • Such microcarrier method comprises allowing cells to replicate on the surfaces of microcarrier beads in a bioreactor (culture tank) and then infecting the cells replicated on the microcarrier beads with virus, followed by culturing the infected cells, so as to prepare viruses of interest in the culture solution.
  • materials of such microcarrier beads may include, but are not limited to ceramic, dextran, glass, silicon, plastic and polyacrylamide.
  • One preferred embodiment of the invention may use microcarriers made of dextran. According to a most preferred embodiment, Cytodex-1 microcarrier beads may be used for the suspension cultures.
  • the concentration of the microcarriers used needs to be suitable so as to provide sufficient surface area for the growth of the cells.
  • One preferred embodiment of the invention relates to the use of about 2-4 g of microcarriers per ml of the medium. According to the most preferred embodiment, about 3 g of microcarriers per ml of the medium may be used.
  • the Vero cell inoculum preparation may be done using standard techniques known in art and the cells may be grown to confluency. After adequate growth, the cells may be harvested and used to seed a bioreactor.
  • the seeding of the bioreactor may preferably be done at a cell density not less than 0.2x 10 6 cells/ ml of the medium. According to a preferred embodiment of the invention, the seeding of the fermentor may be done at a cell count of 300 xlO 6 cells / 100OmL SFM4MegaVir media / 3g cytodex-1 microcarrier beads. Both perfusion and batch systems are within the scope of the current invention. Culture systems in which the medium is continuously supplied and withdrawn are referred to as perfusion systems. As an alternative to this, the cells can also be cultured in a batch system in which the system is run as a largely closed system without supplying medium from inoculation to harvesting.
  • a process of preparing Japanese Encephalitis virus strain P20778 for vaccine comprising the steps of: a) seeding a bioreactor with Vero cells
  • the preferred medium used for the cultivation of the Vero cells in the bioreactor is SFM4MegaVir media (Hyclone) formulated suitable additives like with glutamine and gentamicin sulphate.
  • the cell culture conditions to be used for the current application are variable over a very wide range and can be adapted to the requirements of the current application.
  • the set parameters used are preferably as under: RPM: 40-60
  • pH maintenance in bioreactor may be carried out by using a suitable buffer system. Preferably pH maintenance may be done using 7.5% sterile sodium bicarbonate solution.
  • the cells may be infected by the Japanese encephalitis virus by employing standard techniques known in the art.
  • the cell density of the confluent cells at which the Vero cells in the bioreactor may be infected with the virus may be preferably between about 0.5 x 10 6 to about 2.0 x 10 6 cells/ ml of the medium.
  • the MOI (multiplicity of infection) used for the infection of the cells may be between about 0.01 - 0.5 and preferably about 0.1.
  • the growth parameters used for the production of the virus from the cell biomass is as follows:
  • the present invention also provides suitable methods for harvesting and isolation of the virus.
  • the cells are separated from the culture medium by standard methods like separation, filtration or ultrafiltration.
  • the viruses or the proteins are then concentrated according to methods sufficiently known to those skilled in the art, like gradient centrifugation, filtration, precipitation, chromatography, etc., and then purified.
  • the culture supernatent from the bioreactor may be harvested from the bioreactor at appropriate intervals.
  • the harvesting may be done at every 24 or 48 hours, up to about 168 hours post infections.
  • One preferred embodiment of the invention provides that the harvesting may be done at 72, 96, 120 and 144 hours, post infection.
  • the harvested virus may be clarified by methods known in the art, such as centrifugation, membrane filtration, cartridge filtration or hollow fiber filtration or a combination of these methods.
  • the clarified harvests may be stored at appropriate temperatures so as not to lose its viability.
  • the temperature used for the storage is preferably below 1O 0 C.
  • the harvest may be subjected to purification using high speed zonal centrifugation.
  • the high speed zonal centrifugation may be carried out using sucrose or CsC12 continuous or step gradient of concentrations between 0-60% (w/v), with or without Mg ions, a) feed rate of harvest ranging between 20 ml/ minute to 200 ml/ minute, b) the maximum speed of centrifugation being 97,000 x g, c) the feed being re-circulated once or twice, d) phosphate buffer saline being used to wash the impurities and e) the fractions containing virus being collected by gravity or by applying positive pressure up to 0.5 bars
  • the viral fractions may be further subjected to tangential flow filtration step.
  • virus inactivation can occur, for example, by ⁇ -propiolactone or formaldehyde at any point within the purification process.
  • concentrations of the inactivating agents may be optimized by known methods.
  • Preferred ⁇ -propiolactone concentrations may be in the range of 1 :3000 to 1 :6000 (v/v). In general, this can be achieved by any known chemical or physical means.
  • Inactivated viruses that are produced using the methods of the invention can be formulated for use as vaccines using methods that are known in the art.
  • Numerous pharmaceutically acceptable solutions for use in vaccine preparation are well known in the art and can readily be adapted for use in the present invention by those of skill in this art. (See, e.g., Remington's Pharmaceutical Sciences (18th edition)).
  • the vaccines are prepared as injectables, either as liquid solution or suspension. It is possible to add a stabilizing agent such as carbohydrates (sorbitol, mannitol, starch, sucrose, dextran, glucose, etc), buffers (such as alkali metal phosphate), other excipients, diluents, carriers and adjuvants.
  • the preparation can be lyophilized after adding a stabilizer and it can be vacuum or nitrogen stored.
  • one or more compounds with an adjuvant action can be added. Suitable compounds for this purpose are, for example, aluminum hydroxide, phosphate or oxide, mineral oil, emulsions and saponins.
  • one or more emulsifiers such as Tween and span, are also added to the virus materials.
  • the vaccine of the invention comprises inactivated Japanese Encephalitis virus strain P20778 adsorbed on to an aluminum adjuvant.
  • the vaccines produced using the methods of the invention may be administered to a subject, in amounts and by using methods, which can readily be determined by those of ordinary skill in this art, for the treatment of an infection caused by Japanese encephalitis virus.
  • the immunogenicity and/ or efficacy of the corresponding vaccines can be determined by methods known to one skilled in the art, like protective experiments with loading infection or determination of the antibody titer necessary for neutralization.
  • Determination of the virus amount or amount of antibodies produced can be done by determination of the titer or amount of antigen according to standard methods sufficiently known to one skilled in the art, like virus titration, hemagglutination test, antigen determination or protein determination of different types.
  • Vero cell line (WHO-Vero 10-87) was propagated from the vial of working cell bank.
  • the expansion of the working cell bank may be done in any suitable vessel.
  • the medium used for the propagation of the Vero cells was a serum free and animal component free medium like SFM4MegaVir media (Hyclone) formulated suitable additives like with 4mM stable glutamine and 70 ⁇ g/mL Gentamicin Sulphate.
  • the cells were grown to a density such that the seed used for the seeding of the bioreactor has a cell count not less than 0.2 x 10 6 cells/ ml of the medium.
  • Vero cells may be propagated in a medium like SFM4MegaVir media (Hyclone) formulated with suitable additives like 4mM stable glutamine and 70 ⁇ g/mL Gentamicin Sulphate or any other suitable growth medium, either as monolayer or as a suspension culture using supports like microcarriers, which may be made of dextran or any other suitable material.
  • the cell dissociation may be done using the animal component free HyQTase (Hyclone) solution or any other suitable cell dissociation agent.
  • the seeding of the bioreactor may be done at a cell count of 30OxIO 6 cells / 100OmL SFM4MegaVir media / 3g cytodex-1 microcarrier beads.
  • the preferred bioreactor parameters for cell growth are as under:
  • pH maintenance in bioreactor was done using 7.5% sterile sodium bicarbonate solution
  • the required volume of Japanese encephalitis virus P20778 from seed lot was diluted in a suitable growth medium like SFM4MegaVir media (Hyclone) formulated with suitable additives like 4mM stable glutamine and 70 ⁇ g/mL Gentamicin Sulphate.
  • suitable additives like 4mM stable glutamine and 70 ⁇ g/mL Gentamicin Sulphate.
  • the Vero cells on reaching confluency were infected with the viral suspension. The incubation was done for a required period of time.
  • the virus obtained was used for the infection of the Vero cells during the growth in the bioreactor. Infection of the Vero cells may be done when the cell density is between 0.5x 10 6 and 2x 10 6 cells/ ml.
  • the infection of the cells may be done at a cell count of 0.5 - 1.5 xlO 6 cells/mL with a cell confluency of 70-90% on Cytodex-1 microcarrier beads.
  • Virus MOI multiplicity of infection
  • Bioreactor set parameters for virus production were
  • the harvest collection is ideally done at 72, 96, 120 and 144 hours post infection.
  • the harvest from the upstream processing can be clarified using known methods such as centrifugation, membrane filtration and cartridge filtration or hollow fiber filtration and stored at a temperature below 10 0 C.
  • the clarified harvests (single or pooled harvests- live or inactivated) may be subjected to purification using High speed Zonal centrifugation using sucrose or CsCl 2 continuous or step gradient of concentrations anywhere between 0-60 (w/v) with or without additives like magnesium ions at concentration of 2 to 50 mM.
  • the feed rate for harvest ranged between 20 ml/minute to 200 ml/min.
  • the maximum speed during centrifugation should be ideally 97,000 x g.
  • the banding time provided to virus for getting concentrated in particular zone is ideally 20 mins to 240 mins.
  • the flow through may be re-circulated once or twice with the same set of conditions mentioned above.
  • Phosphate buffer saline may be passed through to wash out any impurities with the same conditioned mentioned above.
  • the rotor speed can be allowed to slow down and once the rotor was stationary, the virus can be collected in fractions either by gravity or by applying positive pressure maximum up to 0.5 bars.
  • Collected fractions after analysis can be pooled and double diluted with phosphate buffer saline before subjecting to further purification and buffer exchange using MWCO of not less than 100 KDa for a TFF or hollow fiber technology.
  • Diafiltration may be done for not more than 4 equal volumes of phosphate buffer saline (pH 7.0 to 7.8).
  • Diafiltered virus sample may then be filtered through 0.45 to 0.1 micron filter.
  • Inactivation of the virus can be done using beta-propiolactone at concentration between 1 :3000 to 1 :6000 (v/v) and at temperature of 2 to 8° C for not more than 120 hrs with continuous stirring.
  • the sample under inactivation may be subjected and maintained to temperatures not more than 40 ° C for duration of not less than 30 mins with continuous stirring.
  • the inactivated virus can then be immediately filtered through 0.45 to 0.1 micron filters before adsorbing it onto an aluminium based adjuvant.
  • the adsorbed inactivated virus can then stored be at temperature between 4° C to 30 ° C.
  • the formulation of the inactivated virus can be carried out by standard techniques known in the art.
  • This example gives a comparison between immunogenicity and cross- reactivity for the antibody response elicited using the vaccine of the invention and a reference, against the challenge by various Japanese encephalitis viral strains.
  • the serum was incubated at 56 0 C for 30 min to inactivate the complement.
  • the serum sample and NIBSC reference serum standard was two-fold diluted starting from 1 :160 to 1 :5120 and 1 :40 to 1 : 1280 respectively in MEM containing 2% FBS.
  • the serum sample 200 ⁇ l was then mixed with an equal volume of JEV culture supernatant of different strains, viz. Beijing, GP-78, Nakayama, JaOAr and Vellore respectively containing 50-150 pfu of the virus.
  • the virus-antibody mixture was incubated at 36.5 + 0.5 0 C for 90 minutes with intermittent shaking every 15-30 minutes, before 200 ⁇ l of it was added to a 6-well culture plate containing around 70% confluent monolayer of PS cells.
  • the plates were incubated at 36.5 0 C + 0.5 0 C for 90 minutes in a CO 2 incubator (5% CO 2 ).
  • the inoculum was removed from the wells and 3 ml overlaying agar medium, containing 2% agarose, was added per well.
  • the plate was kept at 5 0 C ⁇ 3 0 C for 15 minutes to solidify the overlaying agar medium.
  • the plates were then incubated at 36.5 0 C + 0.5 0 C in 5% CO 2 for four to five days for the plaques to develop. Plaques in each well were counted after fixing and staining to calculate the neutralization titer 50% (NT 50 ) values
  • LD 50 Studies A group of 6 male and 6 female Swiss albino mice (4-6 week-old) were injected intracerebrally with different 10-fold dilutions of different virus strains viz. Beijing, GP-78, Nakayama, JaOAr and Vellore respectively. The injected mice were monitored for mortality for 14 days. The LD 50 values were calculated by Read and Muench method (Quality Control of Vaccine and Sera, Manual 1993, CRI Kasauli).

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Abstract

The present invention relates to a novel Japanese encephalitis vaccine and a novel process for making the same. The invention in particular relates to a Japanese encephalitis virus strain P20778 vaccine, wherein the viral adaptation and propagation is carried out in a medium which is free of serum and additives of animal origin. The virus may be propagated particularly in Vero cell line.

Description

NOVEL JAPANESE ENCEPHALITIS VACCINE AND METHOD OF MANUFACTURING THE SAME
FIELD OF INVENTION The present invention relates to a novel Japanese encephalitis vaccine and a novel process for making the same. The invention in particular relates to a Japanese encephalitis virus strain P20778 vaccine, wherein the viral adaptation and propagation is carried out in a medium which is free of serum and additives of animal origin.
BACKGROUND OF THE INVENTION
Japanese encephalitis (JE) is the main cause of viral encephalitis in many countries of Asia. The infection is mosquito-borne and caused by the Japanese encephalitis virus. Clinical presentations vary but may include headache, fever, a change in mental status and onset of seizures, tremors, paresis hypertonia and loss of coordination (WHO- recommended standards for surveillance of selected vaccine-preventable diseases. Geneva: World Health Organization, 2003). The virus exists in a transmission cycle between mosquitoes and pigs and/or water birds. Humans become infected only incidentally when bitten by an infected mosquito (Culex spp.) and the disease is predominantly found in rural and periurban settings.
The disease is endemic with seasonal distribution in parts of China, parts of the Russian Federation and South-East Asia. All year transmission is observed in tropical climatic zones. Currently, Japanese encephalitis is considered hyper endemic in northern India and southern Nepal as well as in parts of Central and Southern India and authorities have responded with immunization campaigns. The spread of JE in new areas has been correlated with agricultural development and intensive rice cultivation supported by irrigation programmes. Japanese encephalitis vaccines have been available since decades and have proven their potential to control the disease. Other control measures such as mosquito control or amplifying pig control have shown to be less reliable. WHO recommends Japanese encephalitis immunization in all regions where the disease is a recognized public health problem (WHO- WER 2006; 81 :325).
Surveillance of the disease is mostly syndromic, while case confirmation and distinction from other causes of encephalitis requires a laboratory diagnosis, which is often conducted in sentinel sites. Case-based surveillance is established in countries that effectively control Japanese encephalitis through vaccination. Overall, the disease is considered under-reported and annual mortality is estimated to range from 10,000- 15,000 deaths, while the total number of clinical cases may be around 50,000. Of these cases, up to 50% result in permanent neuropsychiatric sequel.
Japanese encephalitis virus (JEV) is a member of the Flaviviridae family of animal viruses. Japanese encephalitis virus is an enveloped, positive-sense, single stranded RNA virus. Morphologically, flavivirus virions are spherical, approximately 40 nm in diameter and are composed of a lipid bilayer surrounding a nucleocapsid containing an 11-kb genome complexed with a capsid (C) protein (Rice, C. M. et al. Science 229:726- 33, 1985). Surface projections on the membrane are composed of glycosylated envelope (E) and membrane (M) proteins. A premembrane (prM) glycoprotein, present in intracellular nascent virions, is cleaved by a furin like protease to its mature M protein. Important physiological activities are associated with the 53-kD E protein, including virus attachment to and fusion with target cell membranes and hence is the primary target for neutralizing antibodies and a critical component of any candidate vaccine (Koshini, E. et al. Virol. 188:714-20, 1992).
Currently, there are three major vaccines for JE being used commercially viz. (i) the mouse brain-derived, purified and inactivated vaccine, which is based on either the Nakayama or Beijing strains of the JE virus and produced in several Asian countries; (ii) the cell culture-derived, inactivated JE vaccine based on the Beijing P-3 strain, and (iii) the cell culture-derived, live attenuated vaccine based on the SA 14-14-2 strain of the JE virus, manufactured in China, which has now become the most widely used vaccine in endemic countries. Currently, the only Japanese encephalitis vaccine licensed by the US FDA for human use is JE-VAX, manufactured by the BIKEN Institute in Japan and distributed commonly in the United States by Aventis-Pasteur. It is made from a virulent strain of JE virus, propagated in brains of suckling mouse, purified and inactivated with formalin.
Drawbacks of the mouse-brain derived vaccine are reactogenicity, production in neural tissue, limited duration of induced protection, need for multiple doses due to poor immunogenicity and in most countries not having their indigenous manufacturing, the relatively high price per dose. Further, since this vaccine is produced by intra cerebral injection of infant mice, it is laborious and expensive to manufacture and concerns about the possibility of vaccine related neurological side effects are raised. Though successive refinement in manufacturing process have increased its purity and potency, a moderate frequency of local and systemic reactions have been reported until recently. Furthermore, the requirement of large number of neonatal mice for production of the vaccine has raised issue of cruelty to animals. In addition, theoretical risk from adventitious, infectious agents and trace amounts of mouse brain derived proteins may lead to adverse neurological effect, like acute disseminated encephalomyelitis (ADEM). The principal Japanese manufacturer of mouse brain derived JE vaccine has recently discontinued its production and the quantity of this vaccine produced by other manufacturers has been limited.
The cell culture-derived vaccines are manufactured and widely used in China, where the inactivated vaccine is gradually being replaced by the live attenuated vaccine. The SA 14-14-2 vaccine strain was obtained from its wild-type SA 14 parent by serial passages in cell cultures (primary hamster kidney cells- PHK cells) and in animals (mice, hamsters) with successive plaque purifications (in primary chick embryo cells). Concern about possible adventitious agents in primary hamster cells is likely to limit its use outside China. PHK cells in which Chinese vaccine is prepared is not approved by the World Health Organization (WHO) for viral vaccine production or licensed for human use by the developed countries. The principal disadvantage in using primary hamster cells for the production of vaccines is the uncertainty with regards to the quality of vaccine, as the hamster may become infected with some pathogens and may go undetected which can raise considerable safety issues. With these criticisms, further controlled studies on the safety of the vaccine are required to allow confidence regarding its widespread use. Another disadvantage of the vaccine production from primary cells is the low rate of harvest of the virus and high cost without allowing mass production.
Specific concerns in vaccine products using ingredients or materials primarily originating from serum, animal components, are raised due to high risk of having the presence of animal derived infectious agents, which may be harmful for human use. As a countermeasure against such problems, legal measures for safety have been enhanced, such as the new framework for "bio-originated products" by the amendment of Japanese Pharmaceutical Law in 2003, which desires the development of medical products, free from animal-origin components.
The present invention suggests development and propagation of JE virus, preferably in continuous cell line, like Vero cells for vaccine production, overcoming previous problems in JE virus produced in mouse brain or primary cell lines. The present invention also identifies methodology developed to cultivate the JE virus, inactivation of JE virus and a downstream process for vaccine production with cost-effectiveness.
In summary the present invention identifies methodology improved upon the previously commercialized JE vaccines in the following ways.
1. Safety: The invented viral vaccine will not acquire the virulence through the Vero cell cultivation, reducing the hazards of production and affording an additional level of safety to recipients beyond that furnished by stringent control over the virus-inactivation process. Prior art animal media have the potential to bear infective animal viruses which can pose a safety issue. Thus having serum free/ animal component free media will ensure that the resulting vaccine is not contaminated with animal derived pathogens.
2. Increased supply in safer production substrate: The JE vaccine of the present invention is produced in the absence of serum and animal derived components, providing high yields and inexpensive and scalable production, which are not achieved in the previously commercialized JE vaccines.
3. Less reactogenicity due to elimination of undesirable serum, animal derived components.
4. Possibility of enhanced potency.
5. Cost effectiveness due to the use of standard and easily available media and other raw material used in upstream and downstream processing.
6. Increased acceptability in many countries due to elimination of serum and animal derived components. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1:
Immunogenicity and cross-reactivity of the rabbit sera raised against inactivated JE Virus Vellore strain (P20778). The NT50 values of functional antibodies obtained using Plaque Reduction Neutralization Test (PRNT) are plotted against respective Challenge strains showing the quantitative ability of the sera to neutralize the plaque forming units of different JE virus strains.
DESCRIPTION OF THE INVENTION
In accordance with these and other objects, the present invention provides a novel method for development and production of a Japanese encephalitis vaccine, more specifically a Vero cell derived inactivated Japanese encephalitis vaccine using P20778 strain, comprising the steps of adapting the Vero cell line to serum and animal component free medium, adapting P20778 in Vero cell line by serial passaging, establishment of master and working seed banks of P20778, cultivating cells permissive for productive replication of the virus in a serum free and animal component free medium, infecting the cells with the virus, cultivating the infected cells in serum free and animal component free medium until progeny virus is produced, isolating the virus from the culture, followed by purification, inactivation and. formulation.
The present invention further provides a Japanese encephalitis virus strain P20778 obtained by cultivating Vero cells permissive for productive replication of the virus in a serum free and animal component free medium, infecting the cells with the virus, cultivating the infected cells in serum free and animal component free medium until progeny virus is produced, and isolating the virus from the culture.
In the most preferred aspect of the invention, Indian strain of Japanese encephalitis virus (P20778) has been identified as potential vaccine candidate. This strain was clinically isolated from a patient in Christian Medical College, Vellore, India. The JEV genome is a plus-sense single-stranded RNA of about 11 kb. It has a single open reading frame that codes for a 3432 amino acids long polypeptide protein which is subsequently co- and post-translationally cleaved by host cell proteases and the viral NS2B-NS3 protease to yield the individual viral proteins into three structural proteins namely, capsid (C), glycoslyated envelope (E) and membrane (M) proteins and seven non structural proteins NSl, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The glycoslyated envelope protein E has been found to be immunogenic and responsible for the neutralizing antibody response. Since the genome of JEV is an RNA molecule, it has high potential for evolution as RNA replication machinery that lacks proof reading capability. The Vellore P20778 isolate has 10977 bases. It has an additional base compared to the prototype virus. The additional base is part of the 3- non-coding region (NCR), which, does not affect the length of the viral polyprotein and hence predicted site of all viral proteins is similar to other JEV strains like GP78 (India, 1978) and JaOArS982 (Japan, 1982) strains.
The JE vaccine made from Nakayama strain has shown cross neutralization with Vellore P20778 strains more efficiently, proving the relatedness of the Vellore strain to the Nakayama strain from Japan. Another interesting finding is that the Vellore strain is found to be closer to Beijing- 1 strain. Thus, the Indian strain, Vellore P20778 is well characterized and phylogenetically close to the Chinese & Japanese isolates.
In another preferred embodiment, the said virus may be propagated in Vero tissue culture cells. Vero cells are non-tumorigenic cells derived from monkey kidney. The Vero cell line is more advantageous than any other standard cell line, as the Vero cells are more readily adaptable to large scale cell culture and as a transformed cell has an infinite life time. In a specific embodiment the Vero cell line used is (WHO- Vero 10- 87). The invention is however not restricted to the above mentioned cell lines and other equivalents contemplated by a person of skill in art are within the scope of the present invention.
One preferred embodiment of the invention relates to a Japanese Encephalitis vaccine comprising Japanese Encephalitis virus Vellore strain P20778, wherein the virus is propagated in Vero cell line. According to a more preferred embodiment, the Vero cell line (WHO- Vero 10-87) is used for the propagation of the virus.
In addition to selection of an appropriate cell or host system, the culture conditions under which a virus strain is multiplied are also of great significance for the achievement of a commercially acceptable yield. To maximize the yield of desired virus strains, both the host system and the culture conditions must therefore be specifically adapted in order to achieve favorable environmental conditions for the desired virus strain. In order to achieve a high yield of virus strain, a system that creates optimal growth conditions is therefore required. Efficient production systems are often based on adaptations of the virus population of corresponding culture systems, often using intermediate stages with other host systems and employing protein additives— mostly serum of animal origin. However, the present invention achieves these results without any addition of serum or animal components.
Serum free medium that may be useful for practicing the present invention may include, but need not be limited to Iscove's medium, Ultra-CHO medium (BioWhittaker) , EX-CELL (JRH Bioscience), SFM4MegaVir media and the like. According to a preferred embodiment, SFM4MegaVir media (Hyclone) formulated with suitable additives like glutamine and gentamicin sulphate may be used as the medium of choice for the propagation of the Vero cells as well as for the propagation of the virus. According to a more preferred embodiment, SFM4MegaVir media (Hyclone) medium formulated with about 2- 5mM stable glutamine and about 20- 100 μg/mL Gentamicin Sulphate may be used. In the most preferred embodiment SFM4MegaVir media (Hyclone) formulated with about 4mM stable glutamine and about 70 μg/mL Gentamicin Sulphate may be used.
In accordance with the embodiments of the inventions, 'about' may be used herein to describe the amount of each of the components used during the preparation of the vaccine of the invention, to mean an amount of the said component that is present in amounts of preferably ±20%, more preferably ±10% and most preferably ±5% of the stated amount for that particular component.
Known methods of cell culturing like monolayer culture method or a suspension culture method may be employed for practicing the present invention
A monolayer culture method comprises growing and infecting cells that have been cultured on the inner surface of a vessel with a virus of interest and then subjecting the infected cells to a standing culture or a roll-streak culture, so as to prepare the virus in the culture supernatant. The vessels used for this purpose may be a plate culture vessel or a roll-streak culture flask. Specific examples of the vessel include Petri dish, T flasks, roller bottles or multilayer flasks. Material of the vessels is preferably, a non- glass material like plastic.
An example of suspension culture method is microcarrier method using microcarrier beads. Such microcarrier method comprises allowing cells to replicate on the surfaces of microcarrier beads in a bioreactor (culture tank) and then infecting the cells replicated on the microcarrier beads with virus, followed by culturing the infected cells, so as to prepare viruses of interest in the culture solution. Examples of materials of such microcarrier beads may include, but are not limited to ceramic, dextran, glass, silicon, plastic and polyacrylamide. One preferred embodiment of the invention may use microcarriers made of dextran. According to a most preferred embodiment, Cytodex-1 microcarrier beads may be used for the suspension cultures.
The concentration of the microcarriers used needs to be suitable so as to provide sufficient surface area for the growth of the cells. One preferred embodiment of the invention relates to the use of about 2-4 g of microcarriers per ml of the medium. According to the most preferred embodiment, about 3 g of microcarriers per ml of the medium may be used.
The Vero cell inoculum preparation may be done using standard techniques known in art and the cells may be grown to confluency. After adequate growth, the cells may be harvested and used to seed a bioreactor. The seeding of the bioreactor may preferably be done at a cell density not less than 0.2x 106 cells/ ml of the medium. According to a preferred embodiment of the invention, the seeding of the fermentor may be done at a cell count of 300 xlO6 cells / 100OmL SFM4MegaVir media / 3g cytodex-1 microcarrier beads. Both perfusion and batch systems are within the scope of the current invention. Culture systems in which the medium is continuously supplied and withdrawn are referred to as perfusion systems. As an alternative to this, the cells can also be cultured in a batch system in which the system is run as a largely closed system without supplying medium from inoculation to harvesting.
According to a preferred embodiment of the invention, there is provided a process of preparing Japanese Encephalitis virus strain P20778 for vaccine comprising the steps of: a) seeding a bioreactor with Vero cells
b) growing the cells to confluency at bioreactor set parameters
c) infecting the Vero cells with Japanese Encephalitis virus strain P20778 diluted in a viral growth medium,
d) incubating the infected cells at bioreactor set parameters
e) harvesting the virus, followed by clarification and purification.
As already stated earlier, the preferred medium used for the cultivation of the Vero cells in the bioreactor is SFM4MegaVir media (Hyclone) formulated suitable additives like with glutamine and gentamicin sulphate.
The cell culture conditions to be used for the current application (temperature, cell density, pH, etc.) are variable over a very wide range and can be adapted to the requirements of the current application.
In a preferred embodiment the bioreactor cell culture may be propagated with the following bioreactor set parameters-
RPM: 20-60
Temperature: 35-37°C
% pθ2 (partial pressure of dissolved Oxygen): 30-60% pH: 7.1-7.4
In a most preferred embodiment the set parameters used are preferably as under: RPM: 40-60
Temperature: 370C % pθ2: 40-60% pH: 7.2
pH maintenance in bioreactor may be carried out by using a suitable buffer system. Preferably pH maintenance may be done using 7.5% sterile sodium bicarbonate solution. Once the desired cell count in the bioreactor is reached, the cells may be infected by the Japanese encephalitis virus by employing standard techniques known in the art. The cell density of the confluent cells at which the Vero cells in the bioreactor may be infected with the virus may be preferably between about 0.5 x 106 to about 2.0 x 106 cells/ ml of the medium. The MOI (multiplicity of infection) used for the infection of the cells may be between about 0.01 - 0.5 and preferably about 0.1.
In one of the embodiments the growth parameters for the production of the virus from the cell biomass may be adjusted as follows:
RPM: 20-60 Temperature: 34-370C
% pθ2: 20-60% pH: 7.1-7.7
The growth parameters used for the production of the virus from the cell biomass, according to a preferred embodiment of the invention is as follows:
RPM: 40-60
Temperature: 34°C
% pθ2: 30-60% pH: 7.4-7.6
The present invention also provides suitable methods for harvesting and isolation of the virus. During isolation of viruses, the cells are separated from the culture medium by standard methods like separation, filtration or ultrafiltration. The viruses or the proteins are then concentrated according to methods sufficiently known to those skilled in the art, like gradient centrifugation, filtration, precipitation, chromatography, etc., and then purified.
The culture supernatent from the bioreactor may be harvested from the bioreactor at appropriate intervals. The harvesting may be done at every 24 or 48 hours, up to about 168 hours post infections. One preferred embodiment of the invention provides that the harvesting may be done at 72, 96, 120 and 144 hours, post infection. The harvested virus may be clarified by methods known in the art, such as centrifugation, membrane filtration, cartridge filtration or hollow fiber filtration or a combination of these methods. The clarified harvests may be stored at appropriate temperatures so as not to lose its viability. The temperature used for the storage is preferably below 1O0C.
In a preferred embodiment the harvest may be subjected to purification using high speed zonal centrifugation.
According to a most preferred embodiment, the high speed zonal centrifugation may be carried out using sucrose or CsC12 continuous or step gradient of concentrations between 0-60% (w/v), with or without Mg ions, a) feed rate of harvest ranging between 20 ml/ minute to 200 ml/ minute, b) the maximum speed of centrifugation being 97,000 x g, c) the feed being re-circulated once or twice, d) phosphate buffer saline being used to wash the impurities and e) the fractions containing virus being collected by gravity or by applying positive pressure up to 0.5 bars
According to another preferred embodiment of the invention, the viral fractions may be further subjected to tangential flow filtration step.
In yet another preferred aspect of the invention the virus is inactivated during or after purification. Virus inactivation can occur, for example, by β-propiolactone or formaldehyde at any point within the purification process. The concentrations of the inactivating agents may be optimized by known methods. Preferred β-propiolactone concentrations may be in the range of 1 :3000 to 1 :6000 (v/v). In general, this can be achieved by any known chemical or physical means.
Inactivated viruses that are produced using the methods of the invention can be formulated for use as vaccines using methods that are known in the art. Numerous pharmaceutically acceptable solutions for use in vaccine preparation are well known in the art and can readily be adapted for use in the present invention by those of skill in this art. (See, e.g., Remington's Pharmaceutical Sciences (18th edition)). Preferably the vaccines are prepared as injectables, either as liquid solution or suspension. It is possible to add a stabilizing agent such as carbohydrates (sorbitol, mannitol, starch, sucrose, dextran, glucose, etc), buffers (such as alkali metal phosphate), other excipients, diluents, carriers and adjuvants. The preparation can be lyophilized after adding a stabilizer and it can be vacuum or nitrogen stored. If desired, one or more compounds with an adjuvant action can be added. Suitable compounds for this purpose are, for example, aluminum hydroxide, phosphate or oxide, mineral oil, emulsions and saponins. In addition, if desired, one or more emulsifiers, such as Tween and span, are also added to the virus materials.
In a preferred embodiment of the invention, the vaccine of the invention comprises inactivated Japanese Encephalitis virus strain P20778 adsorbed on to an aluminum adjuvant.
The vaccines produced using the methods of the invention may be administered to a subject, in amounts and by using methods, which can readily be determined by those of ordinary skill in this art, for the treatment of an infection caused by Japanese encephalitis virus.
The immunogenicity and/ or efficacy of the corresponding vaccines can be determined by methods known to one skilled in the art, like protective experiments with loading infection or determination of the antibody titer necessary for neutralization.
Determination of the virus amount or amount of antibodies produced can be done by determination of the titer or amount of antigen according to standard methods sufficiently known to one skilled in the art, like virus titration, hemagglutination test, antigen determination or protein determination of different types.
The following example is used to further illustrate the present invention and advantages thereof. The following specific examples are given with the understanding that it is intended to be illustration without serving as a limitation on the scope of present invention. Example I Upstream processing
Vero cell line (WHO-Vero 10-87) was propagated from the vial of working cell bank. The expansion of the working cell bank may be done in any suitable vessel. The medium used for the propagation of the Vero cells was a serum free and animal component free medium like SFM4MegaVir media (Hyclone) formulated suitable additives like with 4mM stable glutamine and 70μg/mL Gentamicin Sulphate. The cells were grown to a density such that the seed used for the seeding of the bioreactor has a cell count not less than 0.2 x 106 cells/ ml of the medium. In the bioreactor Vero cells may be propagated in a medium like SFM4MegaVir media (Hyclone) formulated with suitable additives like 4mM stable glutamine and 70μg/mL Gentamicin Sulphate or any other suitable growth medium, either as monolayer or as a suspension culture using supports like microcarriers, which may be made of dextran or any other suitable material. The cell dissociation may be done using the animal component free HyQTase (Hyclone) solution or any other suitable cell dissociation agent. In case of suspension cultures, the seeding of the bioreactor may be done at a cell count of 30OxIO6 cells / 100OmL SFM4MegaVir media / 3g cytodex-1 microcarrier beads. The preferred bioreactor parameters for cell growth are as under:
RPM: 40-60 Temperature: 370C
% pθ2: 40-60% pH: 7.2
pH maintenance in bioreactor was done using 7.5% sterile sodium bicarbonate solution
The required volume of Japanese encephalitis virus P20778 from seed lot was diluted in a suitable growth medium like SFM4MegaVir media (Hyclone) formulated with suitable additives like 4mM stable glutamine and 70μg/mL Gentamicin Sulphate. The Vero cells on reaching confluency were infected with the viral suspension. The incubation was done for a required period of time. The virus obtained was used for the infection of the Vero cells during the growth in the bioreactor. Infection of the Vero cells may be done when the cell density is between 0.5x 106 and 2x 106 cells/ ml. Ideally the infection of the cells may be done at a cell count of 0.5 - 1.5 xlO6 cells/mL with a cell confluency of 70-90% on Cytodex-1 microcarrier beads. Virus MOI ( multiplicity of infection) of 0.1 is ideal for the Vero cell Infection. Bioreactor set parameters for virus production were
RPM: 40-60
Temperature: 340C % pθ2: 30-60% pH: 7.4-7.6
The harvest collection is ideally done at 72, 96, 120 and 144 hours post infection.
Downstream processing
The harvest from the upstream processing can be clarified using known methods such as centrifugation, membrane filtration and cartridge filtration or hollow fiber filtration and stored at a temperature below 100C. The clarified harvests (single or pooled harvests- live or inactivated) may be subjected to purification using High speed Zonal centrifugation using sucrose or CsCl2 continuous or step gradient of concentrations anywhere between 0-60 (w/v) with or without additives like magnesium ions at concentration of 2 to 50 mM. The feed rate for harvest ranged between 20 ml/minute to 200 ml/min. The maximum speed during centrifugation should be ideally 97,000 x g. The banding time provided to virus for getting concentrated in particular zone is ideally 20 mins to 240 mins. The flow through may be re-circulated once or twice with the same set of conditions mentioned above. Phosphate buffer saline may be passed through to wash out any impurities with the same conditioned mentioned above. The rotor speed can be allowed to slow down and once the rotor was stationary, the virus can be collected in fractions either by gravity or by applying positive pressure maximum up to 0.5 bars.
Collected fractions after analysis can be pooled and double diluted with phosphate buffer saline before subjecting to further purification and buffer exchange using MWCO of not less than 100 KDa for a TFF or hollow fiber technology. Diafiltration may be done for not more than 4 equal volumes of phosphate buffer saline (pH 7.0 to 7.8). Diafiltered virus sample may then be filtered through 0.45 to 0.1 micron filter. Inactivation of the virus can be done using beta-propiolactone at concentration between 1 :3000 to 1 :6000 (v/v) and at temperature of 2 to 8° C for not more than 120 hrs with continuous stirring. For hydrolysis of remaining beta-propiolactone the sample under inactivation may be subjected and maintained to temperatures not more than 40 ° C for duration of not less than 30 mins with continuous stirring. The inactivated virus can then be immediately filtered through 0.45 to 0.1 micron filters before adsorbing it onto an aluminium based adjuvant. The adsorbed inactivated virus can then stored be at temperature between 4° C to 30 ° C.
Formulation of inactivated Japanese encephalitis virus
The formulation of the inactivated virus can be carried out by standard techniques known in the art.
Example II
This example gives a comparison between immunogenicity and cross- reactivity for the antibody response elicited using the vaccine of the invention and a reference, against the challenge by various Japanese encephalitis viral strains.
Immunogenicity and cross-reactivity testing
Immunization of the Rabbits:
Group of six male and six female New Zealand white rabbits (8-12 weeks) were immunized subcutaneously with inactivated JEV vaccine mixed with aluminium phosphate. The immunized rabbits received booster doses, which was same as the primary dose, at 7 and 21 days after the primary injection. The animals were bled on 42 day post primary injection and the sera was separated and stored at -2O0C.
In-vitro plaque reduction neutralization test (PRNT):
The serum was incubated at 560C for 30 min to inactivate the complement. The serum sample and NIBSC reference serum standard was two-fold diluted starting from 1 :160 to 1 :5120 and 1 :40 to 1 : 1280 respectively in MEM containing 2% FBS. The serum sample (200 μl) was then mixed with an equal volume of JEV culture supernatant of different strains, viz. Beijing, GP-78, Nakayama, JaOAr and Vellore respectively containing 50-150 pfu of the virus. The virus-antibody mixture was incubated at 36.5 + 0.50C for 90 minutes with intermittent shaking every 15-30 minutes, before 200 μl of it was added to a 6-well culture plate containing around 70% confluent monolayer of PS cells. The plates were incubated at 36.50C + 0.50C for 90 minutes in a CO2 incubator (5% CO2).
Subsequently, the inoculum was removed from the wells and 3 ml overlaying agar medium, containing 2% agarose, was added per well. The plate was kept at 50C ± 30C for 15 minutes to solidify the overlaying agar medium. The plates were then incubated at 36.50C + 0.50C in 5% CO2 for four to five days for the plaques to develop. Plaques in each well were counted after fixing and staining to calculate the neutralization titer 50% (NT50) values
Results of Immunogenicity and cross-reactivity study:
Table 1
Figure imgf000017_0001
It is evident from the results that the vaccine prepared using purified and inactivated JE virus Vellore (P20778) strain above is able to elicit immunogenic response in rabbits in terms of functional antibodies, which are capable of neutralizing homologous as well as heterologous JE virus strains, thus providing evidence of immunogenicity as well as cross-reactivity.
LD50 Studies A group of 6 male and 6 female Swiss albino mice (4-6 week-old) were injected intracerebrally with different 10-fold dilutions of different virus strains viz. Beijing, GP-78, Nakayama, JaOAr and Vellore respectively. The injected mice were monitored for mortality for 14 days. The LD50 values were calculated by Read and Muench method (Quality Control of Vaccine and Sera, Manual 1993, CRI Kasauli).
LD5O values obtained Table 2
Figure imgf000018_0001
The above results show level of virulence for different JE virus strains in an in-vivo mice model. Beijing strain is found to be most virulent, whereas strain JaOAr is found to be least virulent amongst the tested strains.
Also included within the scope of the invention, is the propagation of virus previously in the presence of animal sera and subsequent re-derivitization through several rounds of plaque purification by limited dilution in serum free and animal component free medium to reduce the risk of serum contamination.

Claims

1. A Japanese Encephalitis vaccine comprising Japanese Encephalitis virus Vellore strain P20778, wherein the virus is propagated in Vero cell line.
2. A vaccine as claimed in claim 1, wherein the Vero cell line is WHO- Vero 10-87.
3. A vaccine as claimed in claim 1, wherein the virus is propagated in a serum and animal component free medium.
4. A vaccine as claimed in claim 3, wherein the medium used for propagation of the virus is SFM4MegaVir media (Hyclone) which may be formulated with suitable additive(s).
5. A vaccine as claimed in claim 4, wherein the medium comprises about 2-5mM glutamine.
6. A vaccine as claimed in claim 5, wherein the medium comprises about 4mM glutamine.
7. A vaccine as claimed in claim 2, wherein the Vero cell line (WHO- Vero 10-87) is propagated as suspension culture using microcarrier as support for growth of cells.
8. A vaccine as claimed in claim 7, wherein the microcarrier used is made up of material selected from the group consisting of ceramic, dextran, glass, silicon, plastic and polyacrylamide.
9. A vaccine as claimed in claim 8, wherein the microcarrier used is made up of dextran.
10. A vaccine as claimed in claim 9, wherein the microcarrier used is Cytodex-1 microcarrier beads.
11. A process for preparing Japanese Encephalitis virus strain P20778 for vaccine, comprising the steps of: a) seeding a bioreactor with Vero cells b) growing the cells to confluency at bioreactor set parameters c) infecting the Vero cells with Japanese Encephalitis virus strain
P20778 diluted in a viral growth medium, d) incubating the infected cells at bioreactor set parameters, e) harvesting the virus, followed by clarification and purification.
12. The process as claimed in claim 11, wherein the medium used for the cultivation of Vero cells is a serum and animal component free medium
13. The process as claimed in claim 12, wherein the medium is SFM4MegaVir media (Hyclone) formulated with suitable additive(s).
14. The process as claimed in claim 11, wherein the bioreactor set parameters for the growth of cells are: rpm- 20-60; temperature- 35-37°C; % pθ2- 30-60% and pH- 7.1- 7.4.
15. The process as claimed in claim 11, wherein the cell density of the confluent cells is between about 0.5x 106 to about 2.0 x 106 cells /ml.
16. The process as claimed in claim 11, wherein the viral growth medium is a serum and animal component free medium.
17. The process as claimed in claim 16, wherein the viral growth medium is SFM4MegaVir media (Hyclone) formulated with suitable additive(s).
18. The process as claimed in claim 1 1, wherein the infection of the Vero cells using the virus is done at MOI between about 0.01- 0.5.
19. The process as claimed in claim 18, wherein the MOI of 0.1 is used for the infection of the Vero cells.
20. The process as claimed in claim 11, wherein the post infection incubation of the culture is done at the following bioreactor set parameters: rpm 20- 60; temperature- 34- 370C; %pθ2- 20-60% and pH- 7.1-7.7.
21. The process as claimed in claim 11 , wherein the harvest is clarified using any one or a combination of techniques selected from centrifugation, membrane filtration, cartridge filtration and hollow fibre filtration.
22. The process as claimed in claim 11, wherein the harvest is subjected to purification using High Speed Zonal centrifugation.
23. The process as claimed in claim 22, wherein the High speed Zonal centrifugation is carried out using a) Sucrose or CsC12 continuous or step gradient of concentrations between 0- 60% (w/v), with or without Mg+2 ions, b) feed rate of harvest ranging between 20 ml/ minute to 200 ml/ minute, c) the maximum speed of centrifugation being 97,000 x g, d) the feed being re-circulated once or twice, e) phosphate buffer saline being used to wash the impurities and f) the fractions containing virus being collected by gravity or by applying positive pressure up to 0.5 bars
24. The process as claimed in claim 23, wherein the viral fractions are subjected to a further tangential flow filtration step.
25. A process of inactivating Japanese Encephalitis virus strain P20778 using beta- propiolactone.
26. The process as claimed in claim 25, wherein the inactivation is done at a beta- propiolactone concentration between 1:3000 to 1 : 6000 (v/v), at a temperature of 2- 80C for not more than 120 hours.
27. The process as claimed in claim 25, further comprising the step of hydrolyzing the beta- propiolactone remaining in the inactivation sample at a temperature not more than 4O0C for a time period not less than 30 minutes
28. A vaccine as claimed in claim 1 comprising inactivated Japanese Encephalitis virus strain P20778 optionally with other excipients, diluents, stabilizers, carriers and adjuvants.
29. A vaccine as claimed in claim 1 comprising inactivated Japanese Encephalitis virus strain P20778 adsorbed to an aluminium adjuvant.
30. A method of treatment of an infection caused by Japanese Encephalitis virus, by administration of the vaccine as claimed in any of the preceding claims to a subject.
PCT/IN2010/000343 2009-05-25 2010-05-21 Novel japanese encephalitis vaccine and method of manufacturing the same WO2010137036A2 (en)

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CN102925414A (en) * 2012-10-19 2013-02-13 江苏省农业科学院 Swine Japanese encephalitis virus strain and application thereof
CN102925414B (en) * 2012-10-19 2014-09-24 江苏省农业科学院 Porcine Japanese Encephalitis Virus Strain and Its Application
RU2706693C2 (en) * 2013-09-14 2019-11-20 Бхарат Байотек Интернэшнл Лимитед Virus vaccine and methods for production thereof
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