JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1975, p. 96-101 Copyright 0 1975 American Society for Microbiology

Vol. 1, No. 1 Printed in U.S.A.

Purification of Japanese Encephalitis Virus Vaccine by Zonal Centrifugation KOREMASA OKUDA, KOZO ITOH, KOICHI MIYAKE, MICHIO MORITA, MASAO OGONUKI, AND SEIJI MATSUI Chiba Serum Institute, Ichikawa, Chiba, Japan

Received for publication 1 November 1974

A large-scale procedure for purification and concentration of Japanese encephalitis virus vaccine by a continuous-flow isopycnic banding technique in a sucrose density gradient solution, using a K-III zonal centrifuge rotor, is presented. The quality of zonal-purified vaccine was compared with commercial and Japanese National Institutes of Health reference vaccines for antigenicity, immunodiffusion, and allergic encephalitogenicity tests to show its high purity. Both live and inactivated Japanese encephalitis (JE) virus vaccines have been studied in the United States and Japan. In spite of many studies, a suitable strain of live vaccine that is acceptable for human use has not been found (8, 9). Inactivated vaccine has been produced by several methods including tissue cultures (3, 15, 16) of mouse or chicken embryo origin. JE virus propagates well in mouse brain and this type of vaccine has a high antigenic potential. In Japan, this type of vaccine has been produced since 1954. Since 1954, the number of people vaccinated has increased yearly, and in recent years as many as 24 x 106 doses have been produced and used every year. As people are injected many times in their lifespan, a vaccine that contains a minimum of impurities is required. The purity of the early vaccine was not high; however, it has been increased by chemical and centrifugation purification techniques (12, 17). It is known that experimental allergic encephalitis is caused by injection of nervous system tissue of various animal species with Freund adjuvant clinically or histologically (4, 5). The type of vaccine prepared from brains or spinal cords of infected animals is thus capable of causing allergic encephalitis. Although Okinaka and his associates have recorded very few cases of demyelination after repeated injection of the vaccine to many thousands of people (13), the Japanese National Institutes of Health (JNIH) restricts the protein-nitrogen (P-N) content of JE vaccine to under 10 gg/ml (11) to avoid possible hazardous side reactions. Although current JE vaccine is highly purified, small amounts of impurities originating from tissue protein are still permitted. It is vital that a purification procedure minimize the amount of hazardous allergic tissue protein present. 96

At present, vaccine production requires large quantities of crude viral material. In addition, the purification procedures are both laborious and very complicated. The development of large capacity continuous-flow zonal centrifugation by Anderson et al. has made possible the concentration and purification of large volumes of crude viral materials (1, 7). This paper describes the application of continuous-flow zonal centrifugation for mass purification of JE virus vaccine. MATERIALS AND METHODS Virus strain. Nakayama NIH virus strain supplied by the JNIH was used. After one passage in suckling mouse brain in our laboratory, the strain was frozen and stored at -80 C. At the time of thawing, the virus titered 10-80 mean lethal dose per 0.03 ml by intracerebral injection of adult mice. This virus was used both for production of the vaccine and as a challenge virus in the antigenicity test. Vaccine preparation and purification. A stock virus preparation was injected intracerebrally into adult, 21- to 28-day-old mice. After full development of paralysis, moribund mice were killed, and their brains were harvested aseptically. A 20% suspension of brain tissue was prepared by grinding in a homogenizer with sterile phosphate-buffered saline (PBS) at pH 8.0. This suspension was then continuously centrifuged at 14,000 x g with a flow rate of 2 liters/h in a Kokusan centrifuge (Tokyo, Japan). The supernatant fluid collected from this centrifugation was then treated with protamine sulfate (Salmine, Nutritional Biochemical Corp.) at a final concentration of 1.5 mg/ml, permitted to stand for 2 h in a cold room to form floccules, and then centrifuged. The supernatant was removed after a second continuous centrifugation at 2,500 x g with a flow rate of 10 liters per h. This supernatant was then introduced into the zonal centrifuge as the crude viral material for further purification. Isopycnic separation by zonal centrifugation. The zonal centrifuge consisted of a K-Ill rotor driven

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PURIFICATION BY ZONAL CENTRIFUGATION

in a K-IIC zonal ultracentrifuge manufactured by Electro-Nucleonics, Inc., Fairfield, N.J. The sucrose gradient solution was formed by the reorienting, self-forming method by a static loading of 1,600 ml of PBS and 1,600 ml of 60% (wt/wt) buffered sucrose. Then, the rotor was accelerated to an operational speed of 35,000 rpm. When operational speed was attained, the sample of crude viral material was introduced into the rotor from the top for continuous flow isopycnic separation to purify the viral material. To increase the recovery of the viral material, the effluent was recycled through the centrifuge once. Flow rate of the sample was kept at 6 liters per h. After recycling the effluent, the centrifuge was decelerated to rest and the gradient solution was unloaded from the bottom by air pressure and distributed in 32 fractions of 100 ml. The procedure for vaccine purification is schematically shown in Fig. 1. Each fraction was immediately analyzed for sucrose concentration, ultraviolet absorbancy, and hemagglutination (HA) titer. The sucrose concentration was determined by using a Bausch and Lomb refractometer and the ultraviolet absorbancy of each fraction was determined by a Hitachi 139 spectrophotometer at 280-nm wave length. Inactivation of vaccine. The fractions with high HA titer, usually 10 or 11 fractions, were pooled and inactivated with 1:2,500 formalin for about 30 days in a cold room. After inactivation, sucrose was removed with Sephadex G-75 (Pharmacia Fine Chemicals, Sweden). Thimerosal was added as a preservative (0.01% wt/vol). T'he final vaccine was prepared by dilution of the virus concentrate to the desired level of antigenicity for use. P-N determination. The nitrogen content originating from the protein of the vaccine was measured as follows. Trichloroacetic acid was added to the vaccine in a final concentration of 5% (wt/vol), and then the mixture was heated to 100 C and kept at this temperature for 15 min. The resulting insoluble material was

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then subjected to nitrogen content measurement by the micro-Kjeldahl method (lOa). HA assays. A HA assay was performed according to the method of Clark and Casals (2). Goose erythrocytes were used and the reaction was allowed to proceed in a plastic tray at 37 C, pH 6.4. Then, virus preparations were diluted in twofold steps, starting with a dilution of 1:10. Titers were expressed as the reciprocal of the highest dilution with positive HA after 1 h of incubation.

RESULTS

Table 1 shows the results of five typical runs. The effluent contained about 90% of the original P-N content and the remaining 10% was retained in the sucrose gradient. The final gradient characteristics of a typical run are shown in Fig. 2. Most of the HA activity was banded in fractions 4 to 22 between 25 and 52% (wt/wt) sucrose concentration. Maximal activity of more than 1:216 x 10/0.4 ml was obtained between 42 and 47% (wt/wt) sucrose concentration (1.19 and 1.22 g/ml in density). As shown in Fig. 2 by the optical density absorbancy (280 nm), the protein component was banded in a region centering around 20% (wt/wt) sucrose concentration (1.08 g/ml in density). It is shown that by zonal centrifugation it is possible to separate most of the nonspecific protein from the HA activity, which is essential in vaccine production. About 10 fractions with high HA activity were collected as the purified-concentrated vaccine. Electron microscopy. Electron microscopy was performed with Hitachi model HU-11B. Test specimens were dialyzed through cellulose tubing (Visking Co., N.Y.) and sedimented by ultracentrifugation (Hitachi, model 55P-2, To20% brain homogenate kyo, Japan) at 106,000 x g for 5 h. The pellets were immediately resuspended in a small Centrifuge amount of PBS. Specimens were negatively (14,000 x g, continuous) stained with 1% phosphotungstic acid, pH 7.5. An electron micrograph of the purified-concentrated vaccine is shown in Fig. 3a. A similar Sup Add protamine sulfate Ppt electron micrograph of a commercial vaccine is (1.5 mg/ml, final) shown in Fig. 3b for comparison. In Fig. 3a, 2hat4 C spheres, about 41 nm in diameter with the CentrIifuge (2,500 x g, continuous) dense central 26-nm core, are presumed to be JE virus from their size and shape (6, 10). The Sup. Ppt zonal-purified vaccine contained a small proportion of doughnut-shaped particles in addiZonal centrifugation tion to predominant virus particles, whereas the commercial vaccine contained many amorDistribute to 32 fractions phous particles. Thus, it appears that most of Pool high HA titer fractions the impurities, contained in the viral material and inactivate with 1:2,500 were removed by the zonal isopycnic separation. formalin Comparative effectiveness with comFIG. 1. Procedure for purification of JE vaccine. mercial and reference vaccine. The effectiveAbbreviations: (ppt) precipitate; (sup.) supernatant. ness of the zonal purified-concentrated vaccine

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OKUDA ET AL.

J. CLIN. MICROBIOL.

TABLE 1. Recovery of HA and P-N after zonal centrifugation LHA' ExptIAmtoffltid| Vol of Amt of fluid no. processed (ml) fluid (ml) Original fraction Effluent

Volloected_______

Expt

1 2 3 4 5 a '

21,400 25,500 27,500 23,500 21,300

1,000 1,000 1,100 1,000 900

10 10 10 9 8

16 < 16 < 16

Purification of Japanese encephalitis virus vaccine by zonal centrifugation.

A large-scale procedure for purification and concentration of Japanese encephalitis virus vaccine by a continuous-flow isopycnic banding technique in ...
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