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Category Active immunizing agent.

      Inactivated Rabies Vaccine for human use is a sterile liquid or freeze-dried preparation of a suitable strain of fixed rabies virus grown in cell cultures or embryonated duck eggs, purified 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 slightly whitish turbid that may be coloured owing to the presence of a pH indicator.

      Freeze-dried vaccine, when reconstituted, becomes clear liquid that may be coloured owing to the presence of a pH indicator.

Strengths available Liquid or reconstituted preparation, not less than 2.5 IU rabies virus antigen per 0.5 or 1.0 ml. 

Dose Intramuscular, one human dose, at deltoid region (for small children and infants injection at anterolateral thigh is more preferable).

Warning

1. It may cause transient pain, erythema, swelling or itching at the injection site.

          2. Nausea, vomiting, abdominal pain, diarrhea, headache, fatigue, sore throat, low-grade fever (up to 38.3º), chills, muscle aches, arthralgia, myalgia, fainting, dizziness, and malaise may occur.

3. Patients should be closely observed during the period of immunization.

4. It should not be injected into the gluteal region or near blood vessels or nerve.

Additional information

1. Pregnancy is not a contra-indication to postexposure therapy.

          2. For postexposure therapy in previously unvaccinated individuals, passive immunization with a single dose of rabies immunoglobulin should always be administered in conjunction with the first dose of rabies vaccine.

Expiration date When stored under the prescribed conditions, the expiration date is not later than 2 years for the liquid vaccine and not later than 3 years for the freeze-dried vaccine, 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) that it contains rabies virus antigen equivalent to not less than 2.5 IU per dose; (2) the cell culture in which the vaccine was prepared or embryonated duck eggs; (3) the method used for inactivating the virus; (4) that after reconstitution, it shall be used immediately unless data are provided to show that it may be stored for a limited time without loss of potency; (5) the name and amount of adjuvant(s) and preservative(s).

Identification The vaccine is shown to contain rabies virus antigen by a suitable immunochemical method (Appendix 14.5) using specific antibodies, preferably monoclonal; alternatively, the assay serves also to identify the vaccine.

Residual infectious virus Inoculate a quantity equivalent to not less than 25 human doses of vaccine into cell cultures of the same type as those used for production of the vaccine. A passage may be made after 7 days. Maintain the cultures for a total of 21 days and then examine the cell cultures for rabies virus using an immunofluorescence test. No rabies virus is detected.

Bovine serum albumin Not more than 50 ng per single human dose. Carry out the “Immunochemical Methods” (Appendix 14.5).

Ovalbumin If the vaccine is produced in a duck embryonated egg, it contains not more than 1 μg of ovalbumin per human single dose, determined by a suitable immunochemical method (Appendix 14.5).

Bacterial endotoxins When tested as described in the “Test for Bacterial Endotoxins” (Appendix 8.5), it contains less than 25 Endotoxin Units per single human dose.

Pyrogens Complies with the “Pyrogen Test” (Appendix 8.2), injecting into each rabbit a single human dose of the vaccine diluted to 10 times its volume.

Assay The potency of rabies vaccine is determined by comparing the dose necessary to protect mice against the effects of a lethal dose of rabies virus, administered intracerebrally, with the quantity of a reference preparation of rabies vaccine necessary to provide the same protection. For this comparison a reference preparation of rabies vaccine, calibrated in International Units, and a suitable preparation of rabies virus for use as the challenge preparation are necessary.

      The International Unit is the activity contained in a stated quantity of the International Standard.

      The test described below uses a parallel-line model with at least three points for the vaccine to be examined and the reference preparation.

      Selection and distribution of the test animals Use healthy mice about 4 weeks old, each weighing 11 to 15 g, from the same stock. Distribute the mice into six groups of 16 for potency test and four groups of 10 for titrating the challenge virus. The mice must all be of the same sex, or, if of different sexes, the sexes must be distributed equally among the groups.

      Preparation of the challenge virus suspension Inoculate mice intracerebrally with the Challenge Virus Standard (CVS) strain of rabies virus and when the mice show signs of rabies, but before they die, euthanize them, remove the brains and prepare a homogenate of the brain tissue in a suitable diluent. Centrifuge and use the supernatant liquid as the challenge suspension. Distribute the suspension in small volumes in ampoules, seal and store at a temperature below –60º. Determine the LD₅₀ of challenge virus suspension by thawing one ampoule of the suspension and make serial dilutions in a suitable diluent. Allocate each dilution to a group of five mice and inject intracerebrally into each mouse 0.03 ml of the dilution allocated to its group. Observe the mice for 14 days. Calculate the LD₅₀ of the undiluted suspension using the number in each group that, between the fifth and fourteenth days, die or develop signs of rabies.

      Determination of potency of the vaccine to be examined Prepare at least three fivefold serial dilutions of the vaccine to be examined and at least three fivefold serial dilutions of the reference preparation of rabies vaccine. Prepare the dilutions such that the most concentrated suspensions may be expected to protect more than 50 per cent of the animals to which they are administered and the least concentrated suspensions may be expected to protect less than 50 per cent of the animals to which they are administered. Allocate the six dilutions, one to each of the six groups of 16 mice, and inject intraperitoneally into each mouse 0.5 ml of the dilution allocated to its group. After 7 days, prepare three identical dilutions of the vaccine to be examined and of the reference preparation and repeat the injections. Seven days after the second injection, prepare a suspension of the challenge virus such that, on the basis of the preliminary titration, 0.03 ml contains 12 to 50 LD₅₀. Inject intracerebrally into each vaccinated mouse 0.03 ml of this suspension.

      Prepare three suitable serial dilutions of the challenge suspension. Allocate the challenge suspension and the three dilutions one to each of the four groups of 10 control mice and inject intracerebrally into each mouse 0.03 ml of the suspension or one of the dilutions allocated to its group. Observe the animals in each group for 14 days and record the number in each group that die or show signs of rabies for a period of 5 to 14 days after challenge.

      Validity conditions The test is not valid unless: 

      – for both the vaccine to be examined and the reference preparation the 50 per cent protective dose lies between the largest and smallest doses given to the mice;

      – the titration of the challenge suspension shows that 0.03 ml of the suspension contained 12 to 50 LD₅₀;

      – the statistical analysis shows a significant slope and no significant deviations from linearity or parallelism of the dose-response curves;

      – the confidence limits (P = 0.95) are not less than 25 per cent and not more than 400 per cent of the estimated potency.

      The vaccine complies with the test if the estimated potency is not less than 2.5 IU per human dose.