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Category Active immunizing agent.

      Inactivated Japanese Encephalitis Vaccine is a sterile liquid or freeze-dried preparation of a suitable strain of Japanese encephalitis virus, either Nakayama or Beijing, grown in mouse brain or in cell cultures and inactivated by a suitable method.

      The vaccine, reconstituted if necessary as stated on the label, complies with the requirements stated under Vaccines, with the following modifications.

Description Liquid vaccine is clear or slightly whitish turbid and colourless liquid.

      Freeze-dried vaccine is a white amorphous pellet; when reconstituted, it becomes clear or slightly whitish turbid and colourless liquid. 

Strengths available As specified by the manufacturers.

Dose According to the strength specified.

Warning

1. It may cause tenderness, redness and swelling at the injection site.

2. Headache, rash, edema and generalized urticaria or angioedema may occur shortly after vaccination or up to 17 days following vaccination.

3. It should not be administered to anyone who developed a reaction to a previous dose.

4. It is not recommended for infants and neonates.

Expiration date When stored under the prescribed conditions, the expiration date of liquid vaccine is not later than 1 year or shall be based on the stability data of the vaccine, and of the freeze-dried vaccine is not later than 5 years, from the date of the last satisfactory test for potency.

Labelling Complies with the “General Information for Biological Products”, p. 177. In addition the label on the container states (1) whether the vaccine was prepared by an in vivo or an in vitro method; (2) the strain included in the vaccine; (3) the method used to inactivate the virus; (4) if the vaccine is in dried form, a statement that, after its reconstitution, it shall be used as soon as possible or stored at 2º to 8º and discarded at the end of the day; (5) the name and the concentration of the preservative.

Identification The test for potency may serve as an identification test.

pH 6.8 to 7.4 (Appendix 4.11).

Protein content Not more than 80 μg per ml when neural tissue is used, and not more than 200 μg per ml when human albumin is added in cell culture. Carry out the test as described in the “Determination of Nitrogen” (Method III, Appendix 6.7).

Inactivation test Inject intracerebrally at a dose of 0.03 ml of vaccine into at least 10 mice of about 4 weeks of age. Observe the animals for 14 days. No animal shows any abnormal sign during the observation period.

Pyrogens Complies with the “Pyrogen Test” (Appendix 8.2), using 1.0 ml of the vaccine per kg of the rabbit’s weight.

Assay

      The assay of Japanese Encephalitis Vaccine is determined by assessing the ability of the vaccine to stimulate the production of antibody to Japanese encephalitis virus in mice to which the appropriate vaccine has been administered. The sera of the mice are titrated for antibody by comparing their ability to neutralize 50 per cent of a fixed dose of Japanese encephalitis virus with the ability of that of the National or International Reference Preparation of Japanese Encephalitis Vaccine to give the same effects.

      Preparation of challenge virus suspension The challenge virus is prepared by inoculating intracerebrally the virus strain for challenge into suckling mice of about 2 days of age. The brains of mice showing typical signs of infection are harvested and triturated in a suitable diluent containing calf serum to make a 10 per cent w/v suspension. Centrifuge the brain suspension at about 2000 × g for 30 minutes. The supernatant is diluted to contain about 100 PFU of virus per 0.2 ml to serve as virus suspension for challenge.

      Determination of the potency of the vaccine Dilute the test vaccine and the reference preparation with saline TS. Each dilution is injected intraperitoneally in two doses of 0.5 ml each at 7-day intervals into at least 10 mice of 4 weeks of age. Bleed the animals after 7 days of the second injection. The separated serum is pooled at each dilution of vaccine and then inactivated at 56º for 30 minutes and may be stored at –20º or below.

      The serum is appropriately diluted and mixed with an equal volume of the challenge virus. The mixtures are kept at 37º for 90 minutes for neutralization and inoculated at a dose which contains challenge virus about 100 PFU, onto at least three wells of chick embryo cell or BHK-21 or other suitable cell.

      The challenge virus suspension is diluted and inoculated onto chick embryo cell or BHK-21 cell to serve as the virus control. All the inoculated cell cultures are kept standing at 35º to 37º for 90 minutes in a CO₂-adjusted incubator, and then overlayed with agar or methylcellulose.

      After incubation for 5 to 7 days, the inoculated cell cultures are stained and further incubated for one day. The number of plaques on cell cultures are counted to obtain the plaque reduction rates for the test vaccine and the Reference preparation. The neutralizing antibody titres are calculated for each group. The mean number of plaques of the virus control shall be 50 to 150 per well.

      Validity conditions The test is not valid unless:

      – for both the test vaccine and the reference preparation, the slopes of the plaque neutralizing reduction rates of the highest and the lowest serum dilutions are significant,

      – the statistical analysis shows no significant deviation from linearity or parallelism. The test may be repeated but when more than one test is performed, the results of all valid tests must be combined in the estimated potency and its confidence limit.