(Ph. Eur. monograph 0746)
1 DEFINITION
Rabies vaccine (live, oral) for foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) is a preparation of a suitable immunogenic strain of an attenuated rabies virus. The virus strain has one or more stable genetic markers that discriminate the vaccine strain from other rabies virus strains. The vaccine is incorporated in bait in such a manner as to enable the tests prescribed below to be performed aseptically. The bait casing, attractive to the target species, may contain a biomarker (e.g. tetracycline). This monograph applies to vaccines intended for the active immunisation of foxes, or foxes and raccoon dogs against rabies.
2 PRODUCTION
2-1 PREPARATION OF THE VACCINE
The vaccine virus is grown in cell cultures. The virus suspension is harvested on one or more occasions within 14 days of inoculation. Multiple harvests from a single cell lot may be pooled and considered as a single harvest. It may be mixed with
a suitable stabiliser.
2-2 SUBSTRATE FOR VIRUS PROPAGATION
2-2-1 Cell cultures
The cell cultures comply with the requirements for cell cultures for the production of vaccines for veterinary use (5.2.4).
2-3 CHOICE OF VACCINE VIRUS
The vaccine virus is shown to be satisfactory with respect to safety (5.2.6) for the target species and the non-target species, and efficacy (5.2.7) for the species for which it is intended. The vaccine strain is genetically characterised by gene
sequencing.
The following tests for safety of the virus strain (section 2-3-1), stability of the genetic marker (section 2-3-2) and immunogenicity (2-3-3) may be used during the demonstration of safety and efficacy.
In natural and experimental conditions, the virus strain does not spread from one animal to another in wild rodents.
2-3-1 Safety of the virus strain
Administer the virus strain by the oral route. Use vaccine virus at the least attenuated passage level that will be present in a batch of the vaccine.
For each test performed in the target species (foxes, or foxes and raccoon dogs), use not fewer than 20 animals that do not have antibodies against rabies virus. Administer orally to each animal a quantity of the vaccine virus equivalent to not less than 10 times the maximum virus titre likely to be contained in 1 vaccine bait. Observe the animals at least daily for 180 days.
For each test performed in the non-target species (dogs, cats, and if appropriate, raccoon dogs), use not fewer than 10 animals that do not have antibodies against rabies virus. Administer orally to each animal a quantity of the vaccine virus equivalent to not less than 10 times the maximum virus titre likely to be contained in 1 vaccine bait. Observe the animals at least daily for 180 days.
The vaccine virus complies with the test if no animal shows signs of disease and if the presence of the vaccine virus is not demonstrated in the brain of any animal. The presence of rabies virus in the brain is tested using reference diagnostic
tests (immunofluorescence test and cell-culture test).
2-3-2 Stability of the genetic marker
Carry out the test using suckling mice that have not been vaccinated against rabies. Passage the vaccine virus sequentially through 5 groups via the intracerebral route.
Inoculate each of the 5 mice of the 1st group with a quantity of the vaccine virus that will allow recovery of virus for the passages described below (e.g. not more than 0.02 mL). When the mice show signs of rabies, but not later than 14 days after inoculation, euthanise the mice and remove the brain of each mouse. Prepare a suspension from the brain of each mouse and pool the samples. Administer not more than 0.02 mL of the pooled samples to each mouse of the next group. Carry out this passage operation not fewer than 4 times; verify the presence of the virus at each passage. If the virus is not found at a passage level, repeat the passage by administration to a group of 10 animals.
Verify the genetic marker in the vaccine virus recovered from the last passage.
The vaccine virus complies with the test if the genetic marker remains stable.
2-3-3 Immunogenicity
A test is carried out for the oral route of administration and with the bait to be stated on the label using animals of the target species (foxes, or foxes and raccoon dogs) at least 3 months old. The quantity of vaccine virus to be administered to each fox, or fox and raccoon dog, is not greater than the minimum virus titre to be stated on the label and the virus is at the most attenuated passage level that will be present in a batch of vaccine.
Use for the test not fewer than 35 animals of each target species, that do not have antibodies against rabies virus. In each target species, apply the following protocol, validity criteria and acceptance limits.
Vaccinate not fewer than 25 animals, according to the schedule to be recommended. Maintain not fewer than 10 animals as controls. Observe the animals for 180 days after vaccination. The test is not valid if fewer than 25 vaccinated animals survive after this observation period. Challenge all the animals at least 180 days after vaccination by intramuscular injection of a sufficient quantity of a virulent rabies virus strain approved by the competent authority. Observe the animals at least daily for 90 days after challenge. Animals that die from causes not attributable to rabies are eliminated.
The test is not valid if the number of such deaths reduces the number of vaccinated animals in the test to fewer than 25 and the test is invalid unless at least 9 control animals (or a statistically equivalent number if more than 10 control animals are challenged) show signs of rabies and the presence of rabies virus in their brain is demonstrated by the immunofluorescence test or some other reliable method.
The vaccine virus complies with the test if not more than 2 of 25 vaccinated animals (or a statistically equivalent number if more than 25 vaccinated animals are challenged) show signs of rabies.
2-4 BAIT STABILITY
Incubate the bait at 25 °C for 5 days. Titrate the vaccine. The virus titre must be at least the minimum virus titre stated on the label. Heat the bait at 40 °C for 1 h. The bait casing complies with the test if it remains in its original shape and adheres to the vaccine container.
3 BATCH TESTS
3-1 Identification
3-1-1 The vaccine virus is identified using a suitable method. For example, when mixed with a monospecific rabies antiserum, it is no longer able to infect susceptible cell cultures into which it is inoculated.
3-1-2 A test is carried out to demonstrate the presence of the genetic marker.
3-2 Bacteria and fungi
The vaccine complies with the test for sterility prescribed in the general monograph Vaccines for veterinary use (0062).
3-3 Mycoplasmas (2.6.7)
The vaccine complies with the test for mycoplasmas.
3-4 Extraneous agents
3-4-1 Extraneous agents (5.2.5)
The vaccine is free from extraneous agents.
3-4-2 Test for contaminating rabies virus
A test to demonstrate the absence of extraneous rabies virus strains is performed. The following test may be used. Inoculate 1 in 10 and 1 in 1000 dilutions of the vaccine into susceptible cell cultures. Incubate at 37 °C. After 2, 4 and 6 days, stain the cells with a panel of monoclonal antibodies that do not react with the vaccine strain but that react with other strains of rabies vaccine (for example, street virus, Pasteur strain). The vaccine complies with the test if it shows no evidence of contaminating rabies virus.
3-5 Virus titre
Titrate the vaccine virus in suitable cell cultures. The vaccine complies with the test if 1 dose contains not less than the minimum virus titre stated on the label.
3-6 Potency
The vaccine complies with the requirements of the test prescribed under Immunogenicity (section 2-3-3) when administered by a recommended route and method. It is not necessary to carry out the potency test for each batch of the vaccine if it has been carried out on a representative batch using a vaccinating dose containing not more than the minimum virus titre stated on the label.
3-7 Biomarker
If the bait contains a biomarker, the stability of the biomarker is verified by a suitable method. When tetracycline is used, the vaccine complies with the test if chemical analysis of the bait casing shows less than 30 per cent conversion of the total amount of tetracycline into the epitetracycline isomer.
4 LABELLING
The label states:
— the nature of the genetic marker of the virus strain;
— where applicable, the nature of the biomarker of the bait.
