Purification of Japanese encephalitis virus

Details Purification of Japanese encephalitis virus Purification of Japanese encephalitis virus

1997-03-25

2001-03-27

Weber, Jon P. (Department: 1633)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

Recovery or purification

C435S235100, C435S236000, C424S218100

Reexamination Certificate

active

06207439

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a process for purification of Japanese encephalitis virus (JEV), in particular a large-scale process suitable for use in vaccine preparation.
2. Description of the Related Art Japanese encephalitis virus (JEV), a spherical RNA virus belonging to genus Flavivirus of virus family Flaviviridae, which is an important, severe, human pathogen which is spread through the vector mosquito, causing serious public health problems during summer time in the West Pacific and Southeast Asian Regions. The infection may result in permanent neurological or psychiatric injury or even death. The spread of the disease has been effectively controlled through vaccination. The vaccine employed is traditionally manufactured from isolated, purified and formalin-inactivated virus harvested from infected mouse brain tissue. To date, because of its relatively low cost and high efficacy, vaccinating children with inactivated JE vaccine remains the most effective means for the disease control of JE in many countries in the above-mentioned areas.
Unfortunately, the mouse brain-derived JE vaccine has the drawback of provoking allergic reaction easily in the human body upon repeated vaccination due to the presence of residual mouse tissue proteins which are difficult to remove away during purification procedures. For decades, efforts have been made and thereby several methods, including protamine sulfate precipitation (Ada, G. L., Anderson, S. G., and Abbot, A. J. (1961) , Purification of Murray Valley encephalitis virus,
Gen. Microbiol.,
24: 177-186; and Cheng, P.-Y. (1961), Purification, size and morphology of mosquito borne animal virus Semliki Forest encephalitis viruses,
Virology,
14: 124-131), active carbon treatment (Steven, T. M., and Schlesinger, R. W. (1965), Studies on the nature of Dengue viruses 1. Correlation of particle density, infectivity and RNA content of type 2 virus,
Virology,
27: 103-112), alcohol precipitation (Nakamura, J. (1969), Studies on the purified Japanese encephalitis vaccines,
NIBS Bull. Biol. Res.,
8: 78-99), polyethylene glycol precipitation (Aizawa, C., Hasegawa, S., Cheng, C.-Y., and Yoshioka, I. (1980), Large-scale purification of Japanese encephalitis virus from infected mouse brain for preparation of vaccine,
APPl. Env. Microbiol.,
39: 54-57) , separation by hydroxyapatite column (Pfefferkorn, E. R., and Hunter, H. S. (1963), Purification and partial chemical analysis of Sindbis virus,
Virology,
20: 433-445), sucrose density gradient sedimentation (Okuda, K., Ito, K., Miyake, K., Morita, M., Ogonuki, M. and Matsui, S. (1975), Purification of Japanese encephalitis vaccine by zonal centrifugation,
J. Clin. Microbiol.,
1: 96-101), as well as various combinations of the above, were found to obtain vaccines with reasonable purities.
Although some successes have been seen in the above-discussed methods, the achievements employing these purification procedures are limited due to the remaining trace amounts of impurities originating from mouse brain myelin proteins, or the complexity of the procedures to be practically utilized. In addition, in order to meet the regulation promulgated by the WHO that the protein content in each dosage of JE vaccine should be less than 80 &mgr;g/ml, dilution of the JE vaccine has to be made occasionally and this will undesirably cause the virus titer in the vaccine to be lower than the reference vaccine. To solve these problems, certain JEV-susceptible established cell lines, such as BHK21, MK, Singh's
Aedes albopictus
(SA), Vero and C6/36 cells, have been employed in the culture of JE virus. It has been reported that high purity of JEV particles can be obtained from the above infected cells by the combined use of polyethylene glycol precipitation and sucrose gradient sedimentation (Akira, I., Takahisa, F., Fuyoko, S., Suranga, S., and Konosuke, F. (1973), Purification of Japanese encephalitis virus grown in BHK21 and Singh's
Aedes albopictus
cells by polyethylene glycol precipitation,
Biken J.,
16: 67-73).
As liquid chromatography has been widely applied in the purification of biomolecules, cell organelles, and viral particles, Wezel et al. disclosed a sequential purification process comprising a combination of filter clarification, ultrafiltration, gel filtration, and anion-exchange liquid chromatography, in order to remove residual serum proteins in inactivated poliovirus and rabiesvirus suspensions harvested from infected cells (
Dev. Biol. Stand.,
42: 65-69, 1978). However, it is unknown as to whether a similar method is applicable to the purification of other different viral particles, including JEV, without impairing the native structures and biological activities thereof.
Therefore, there is still a need to develop a less complicated and more efficient purification process under a physiological condition for the mass production of the JE virus for use in vaccine preparation.
SUMMARY OF THE INVENTION
One object of the invention is to provide a process for large-scale purification of living Japanese encephalitis virus (JEV) suitable for use in vaccine preparation from a JEV source, including JEV-infected cell cultures and JEV-infected mouse brains. The process comprises the following steps:
(a) obtaining a sample from JEV-infected mouse brains or JEV-infected cell cultures,
(b) subjecting said sample to a preliminary separation to remove cell and cell debris from the sample of step (a),
(c) concentrating said sample from step (b) by ultrafiltration to remove substances having a molecular weight below 100 kDa, and
(d) subjecting the sample concentrate to gel filtration to obtain a substantially pure fraction of JEV.
With the present process, at least 95%, preferably at least 99%, of the contaminating proteinaceous substances are removed from the virus sample, whilst the viral particle still remains substantially intact without losing its infectivity. In addition, the present process can achieve a virus recovery yield of at least 80%, and more preferably 90%.
Another object of the present invention is to provide a process for purification of live JEV in high recovery yield and high purity, which comprises the three steps of microfiltration, ultrafiltration and gel filtration.
This invention also provides a JEV vaccine, which is prepared by inactivating the JEV obtained from the present process with an inactivation agent, e.g. formalin and binary ethyleneimide (BEI). Since the JE vaccine according to the present invention is of higher purity than the currently widely used mouse-brain vaccine and has no significant loss in antigenicity, it is suitable for use in the preparation of multi-valent vaccine, such as DPT-JE, DPT-HBV-JE and so forth.


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van Wezel, et al.; Large-S

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