Canine Coronavirus Vaccine, Inactivated 

Canine Coronavirus Vaccine, Inactivated is a preparation containing canine coronavirus, inactivated in such a manner that its immunogenic activity is retained. It may be issued as a liquid or as a freeze-dried preparation to be reconstituted with a suitable liquid immediately before use. The liquid vaccine may contain a suitable adjuvant. 

Production 

The virus is grown in suitable cell culture systems. The cell culture complies with the requirements for cell culture for production of veterinary vaccines (2.7.13). The test for residual live virus is carried out using a quantity of inactivated virus harvest equivalent to at least 10 doses of the vaccine. Two passages at an interval of 6 to 8 days in the cell cultures of the same type used for the production are carried out. The inactivated virus harvest complies with the test if no live virus is detected using suitable tests. The vaccine may contain a suitable adjuvant. 

Choice of vaccine strain 

A reference strain obtained from an authentic source shall be used for the vaccine production. The master seed which has been established as pure, safe and immunogenic shall be used for vaccine production 

Identification 

When inoculated into dogs, the vaccine stimulates the production of specific neutralizing antibodies against canine coronavirus as determined by suitable serological tests. 

Tests 

Safety. Carry out the test for each route and method of administration to be recommended for the vaccination. Double dose of the batch prepared from the master seed shall be injected to ten healthy dogs in the age group and by the route stated on the label. Observe the animals for 14 days and no abnormal systemic or local reaction occurs. Such animals used in the test shall be preferably free of canine coronavirus antibodies. At least three representative batches from the master seed shall be tested for safety. 

If vaccine is intended to be use or may  be used in the pregnant bitches, use not less than 10 bitches at the stage of pregnancy to be recommended or at a range of pregnancy according to the schedule to be recommended. Administer to each bitch a double dose of the vaccine. If the schedule to be recommended requires a second dose, administer double dose after the interval to be recommended. Observe the bitches daily until one day after whelping. The vaccine complies with the test if no bitch shows local or systemic reactions, sign of diseases or dies from causes attributed to the vaccine virus and if no adverse effects on the pregnancy or the offspring are noted. 

Potency. Inject each of 6 healthy susceptible dogs between 8 and 14 weeks having antibody titer less than 6 SN₅₀ per 50 µl of serum with a representative batch with the dose recommended on the label. Use 2 dogs of the same age as control. If a second dose is recommended, the second dose shall be administered at the time specified on the label. Not less than 14 days booster or not less than 21 days after single vaccination, challenge all the animals through appropriate route with a virulent virus strain of canine coronaovirus. Observe the animals for 14 days. The vaccine complies with the test if the 5 vaccinated dogs remain healthy and show no sign of disease. The test is not valid unless the controls die or show clinical signs of canine coronavirus infection. 

Manufacturer’s tests 

Identification 

Vaccine complies the requirements of the test mentioned under production. 

Residual live virus. Vaccine complies the requirements of the test mentioned under production. 

Potency. If is not necessary to carry out the potency test for each batch of the vaccine if it has been carried out using a batch of vaccine with a minimum potency. Where the test is not carried out, an alternative validated methods is used, the criteria for acceptance being set with reference to a batch of vaccine that has been given satisfactory results in the test described under potency. 

Batch tests 

Identification 

Vaccine complies the requirements of the test mentioned under production. Alternatively, identification on the final lot by molecular techniques is acceptable and can be used in the routine batch release tests after proper validation of antigen extraction protocol from adjuvanted vaccine and test applied (2.8.1). 

Residual live virus. Vaccine complies the requirements of the test mentioned under production. If the vaccine contains an adjuvant, separate the adjuvant from the liquid phase by a method that does not inactivate or otherwise interfere with the detection of live virus before test for inactivation. 

Water (2.3.43). Not more than 3.0 per cent (for freeze dried vaccine only). 

Safety. Inject each of two healthy susceptible dogs in the recommended age group free from canine coronavirus antibodies with a quantity equivalent to 2 doses by the route stated on the label. Observe the animals for 14 days. No abnormal systemic or local reaction occurs. 

Sterility (2.2.11). Complies with the test for sterility. 

Potency. Either of the test A or B may be carried out. 

1. Inject each of five healthy susceptible guinea pigs, each weighing between 350 and 400 g, with half the minimum dose and by the route stated on the label. Repeat the injection after14 days. Fourteen days after the second injection collect blood samples and obtain the serum from each guinea pig separately. Inactivate each serum by heating at 56° for 30 minutes. Examine the serum samples for antibodies by the following method.

Prepare 2-fold serial dilutions of serum in a medium suitable for canine cells. Add to each dilution an equal volume of a virus suspension containing approximately 10²TCID50 and incubate the mixtures at 37° for 1 hour. Inoculate a suitable volume of canine cells into at least 4 replicates of serum virus mixtures and incubate at 37° for 4 days. Examine for evidence of specific cytopathic effects and calculate the antibody titre. The vaccine complies with the test if the mean antibody titre is not less than 45 SN₅₀per 50 µl of serum. 

1. Inject each of 2 healthy susceptible dogs between 8 and 14 weeks having antibody titer less than 6 SN₅₀per 50 µl of serum with a representative batch with the dose recommended on the label. If a second dose is recommended, the second dose shall be administered at the time specified on the label. For single dose schedule, collect blood after 21 days or for two dose schedule, collect blood 14 days after booster from each dog. Inactivate each serum sample by heating at 56^°for 30 minutes. Examine the serum sample individually for the neutralizing antibodies. Prepare 2 fold serial dilutions of the serum in a suitable for canine cells. Add to each dilution an equal volume of a virus suspension containing approximately 10² TCID 50 and incubate the mixture at 37° for 1 hour. Inoculate a suitable volume of canine cells into at least 4 replicates of serum virus mixture and incubate at 37° for 4 days. Examine for evidence of specific cytopathic effects and calculate the mean antibody titer. Vaccine complies with the test, if the mean antibody titer in vaccinated dogs is not less than 45 SN₅₀ per 50 µl of serum. 

Labelling. The label states (1) the recommended routes of administration; (2) that the preparation should be shaken well before use; (3) that the liquid preparation should not be allowed to freeze; (4) that the vaccine should be used immediately after reconstitution for freeze dried vaccine (5) storage temperature (6) expiry date.