Edition: BP 2025 (Ph. Eur. 11.6 update)
1 DEFINITION
Clostridium novyi (type B) vaccine for veterinary use is prepared from a liquid culture of a suitable strain of Clostridium novyi (type B).
The whole culture or its filtrate (alpha toxin) or a mixture of the two is inactivated to eliminate its toxicity while maintaining adequate immunogenic properties. This monograph applies to vaccines intended for active immunisation of animals and/or to protect passively their progeny against disease caused by C. novyi (type B).
2 PRODUCTION
2-1 PREPARATION OF THE VACCINE
C. novyi (type B) used for production is grown in an appropriate liquid medium. Toxoids and/or inactivated cultures may be treated with a suitable adjuvant, after concentration if necessary.
2-2 CHOICE OF VACCINE COMPOSITION
The vaccine is shown to be satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) for the animals for which it is intended. For the latter, it shall be demonstrated that for each target species the vaccine, when administered according to the schedule to be recommended, stimulates an immune response (for example, induction of antibodies) consistent with the claims made for the product.
The following test for safety (section 2-2-1) may be used during the demonstration of safety.
2-2-1 Safety
Carry out the tests for each route and method of administration to be recommended for vaccination and where applicable, in animals of each category for which the vaccine is intended, using in each case animals not older than the minimum age to be recommended for vaccination. Use a batch of vaccine containing not less than the maximum potency that may be expected in a batch of vaccine.
For each test, use not fewer than 8 animals that do not have antibodies against C. novyi (type B). Administer to each animal 1 dose of the vaccine. If the schedule to be recommended requires a 2nd dose, administer another dose after an interval of at least 14 days. Observe the animals at least daily until 14 days after the last administration.
The vaccine complies with the test if no animal shows abnormal local or systemic reactions or dies from causes attributable to the vaccine. If the test is carried out in pregnant animals, no adverse effects on gestation or the offspring are noted.
2-3 MANUFACTURER’S TESTS
2-3-1 Residual toxicity
Residual toxicity is assessed immediately after detoxification by a suitable method (preferably in vitro e.g. in cell cultures). The result complies with the value specified for the product.
2-3-2 Antigen content
The antigen content is determined by a suitable method preferably in vitro, such as total combining power (TCP) using appropriate cell cultures as indicators of toxicity, an enzyme-linked immunosorbent assay (ELISA) or any other validated method.
2-3-3 Batch potency test
The potency test (section 3-3) is not carried out for each batch of vaccine if it has been carried out using a batch of vaccine with a minimum potency.
An alternative validated method is used, the criteria for acceptance being set with reference to a batch of vaccine that has given satisfactory results in the test described under Potency and that has been shown to be satisfactory with respect to immunogenicity in the target species. The following test may be used after a satisfactory correlation with the test under Potency (section 3-3) has been established.
Vaccinate rabbits as described under Potency and prepare sera. Determine the level of antibodies against the alpha toxin of C. novyi in the individual sera by a suitable method such as an immunochemical method (2.7.1) or neutralisation in cell cultures. Use a homologous reference serum against C. novyi alpha antitoxin. Clostridia (multicomponent) rabbit antiserum BRP is suitable for use as a reference serum.
The vaccine complies with the test if the level of antibodies is not less than that found for a batch of vaccine that has given satisfactory results in the test described under Potency and that has been shown to be satisfactory with respect to immunogenicity in the target species.
3 BATCH TESTS
3-1 Identification
The vaccine contains the antigenic components of C. novyi (type B) stated under Definition.
3-2 Bacteria and fungi
The vaccine, including where applicable the diluent supplied for reconstitution, complies with the test for sterility prescribed in the monograph Vaccines for veterinary use (0062).
3-3 Potency
Use for the test not fewer than 10 healthy rabbits, 3-6 months old. Administer to each rabbit by the subcutaneous route a quantity of vaccine not greater than the minimum dose stated on the label as the 1st dose. After 21-28 days, administer to the same animals a quantity of the vaccine not greater than the minimum dose stated on the label as the 2nd dose. 10- 14 days after the 2nd injection, bleed the rabbits and pool the sera.
The vaccine complies with the test if the potency of the pooled sera is not less than 3.5 IU/mL.
The International Unit is the specific neutralising activity for C. novyi alpha toxin contained in a stated amount of the International Standard, which consists of a quantity of dried immune horse serum. The equivalence in International Units of the International Standard is stated by the World Health Organization.
The potency of the pooled sera obtained from the rabbits is determined by comparing the quantity necessary to protect mice or other suitable animals against the toxic effects of a fixed dose of C. novyi alpha toxin with the quantity of a reference preparation of Clostridium novyi alpha antitoxin, calibrated in International Units, necessary to give the same protection. For this comparison, a suitable preparation of C. novyi alpha toxin for use as a test toxin is required. The dose of the test toxin is determined in relation to the reference preparation; the potency of the serum to be examined is determined in relation to the reference preparation using the test toxin.
Clostridia (multicomponent) rabbit antiserum BRP is suitable for use as a reference serum.
3-3-1 Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 5-day culture in liquid medium of C. novyi type B and dry by a suitable method. Select the test toxin by determining for mice the L+/10 dose and the LD50, the observation period being 72 h.
A suitable alpha toxin contains not less than 1 L+/10 dose in 0.05 mg and not less than 10 LD50 in each L+/10 dose.
3-3-2 Determination of test dose of toxin. Prepare a solution of the reference preparation in a suitable liquid so that it contains 1 IU/mL. Prepare a solution of the test toxin in a suitable liquid so that 1 mL contains a precisely known amount such as 1 mg. Prepare mixtures of the solution of the reference preparation and the solution of the test toxin such that each mixture contains 1.0 mL of the solution of the reference preparation (1 IU), one of a series of graded volumes of the solution of the test toxin and sufficient of a suitable liquid to bring the total volume to 2.0 mL. Allow the mixtures to stand at room temperature for 60 min. Using not fewer than 2 mice, each weighing 17-22 g, for each mixture, inject a dose of
0.2 mL by the intramuscular or the subcutaneous route into each mouse. Observe the mice for 72 h. If all the mice die, the amount of toxin present in 0.2 mL of the mixture is in excess of the test dose. If none of the mice die, the amount of toxin present in 0.2 mL of the mixture is less than the test dose. Prepare fresh mixtures such that 2.0 mL of each mixture contains 1.0 mL of the solution of the reference preparation (1 IU) and one of a series of graded volumes of the solution of the test toxin separated from each other by steps of not more than 20 per cent and covering the expected end-point. Allow the mixtures to stand at room temperature for 60 min. Using not fewer than 2 mice for each mixture, inject a dose of
0.2 mL by the intramuscular or the subcutaneous route into each mouse. Observe the mice for 72 h. Repeat the determination at least once and combine the results of the separate tests that have been made with mixtures of the same composition so that a series of totals is obtained, each total representing the mortality due to a mixture of a given composition.
The test dose of toxin is the amount present in 0.2 mL of that mixture which causes the death of one half of the total number of mice injected with it.
3-3-3 Determination of the potency of the serum obtained from rabbits
Preliminary test Dissolve a quantity of the test toxin in a suitable liquid so that 1.0 mL contains 10 times the test dose (solution of the test toxin). Prepare a series of mixtures of the solution of the test toxin and of the serum to be examined such that each mixture contains 1.0 mL of the solution of the test toxin, one of a series of graded volumes of the serum to be examined and sufficient of a suitable liquid to bring the final volume to 2.0 mL. Allow the mixtures to stand at room temperature for 60 min. Using not fewer than 2 mice for each mixture, inject a dose of 0.2 mL by the intramuscular or the subcutaneous route into each mouse. Observe the mice for 72 h. If none of the mice die, 0.2 mL of the mixture contains more than 0.1 IU. If all the mice die, 0.2 mL of the mixture contains less than 0.1 IU.
Final test Prepare a series of mixtures of the solution of the test toxin and the serum to be examined such that 2.0 mL of each mixture contains 1.0 mL of the solution of the test toxin and one of a series of graded volumes of the serum to be examined, separated from each other by steps of not more than 20 per cent and covering the expected end-point as determined by the preliminary test. Prepare further mixtures of the solution of the test toxin and of the solution of the reference preparation such that 2.0 mL of each mixture contains 1.0 mL of the solution of the test toxin and one of a series of graded volumes of the solution of the reference preparation, in order to confirm the test dose of the toxin. Allow the mixtures to stand at room temperature for 60 min. Using not fewer than 2 mice for each mixture, proceed as described in the preliminary test.
The test mixture that contains 0.1 IU in 0.2 mL is that mixture which kills the same or almost the same number of mice as the reference mixture containing 0.1 IU in 0.2 mL. Repeat the determination at least once and calculate the average of all valid estimates. The test is valid only if the reference preparation gives a result within 20 per cent of the expected value.
The confidence limits (P = 0.95) have been estimated to be:
— 85 per cent and 114 per cent when 2 animals per dose are used;
— 91.5 per cent and 109 per cent when 4 animals per dose are used;
— 93 per cent and 108 per cent when 6 animals per dose are used.
4 LABELLING
The label states:
— whether the product is a toxoid, a vaccine prepared from a whole inactivated culture or a mixture of the two;
— for each target species, the immunising effect produced (for example, antibody production, protection against signs of disease or infection).
