Bulletin "Veterinary biotechnology"

Veterynarna biotehnologija – Veterinary biotechnology, 2018, 32(1), 212-225 [in Ukrainian]. https://doi.org/10.31073/vet_biotech32(1)-28

ROMANENKO O.A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

State Scientific Control Institute of Biotechnology and strains (DNKIBSHM)


Introduction. The mouse challenge test still remains the reference method for the potency determination of human and animal inactivated rabies vaccines, and it is still widely used throughout the world. This tests offers from many disadvantages − it is expensive and time consuming, uses a large number of mice, causes significant animal distress, and suffers from high variability and can be problematic in terms of reaching all of the validity criteria. Recently, the European Pharmacopoeia has recognized the use of a serological potency assay (SPA) as an alternative method to the challenge test. The SPA significantly reduces the number of animals used for the batch potency testing of inactivated rabies vaccines for veterinary use. In addition, it avoids the pain and distress associated with the intracranial injection and subsequent clinical signs in unprotected animals, thereby complying with Three Rs principles.

The goal of our work was to implement the principles of 3 R in the system of control inactivated vaccines against rabies.

Materials and methods. Vaccines previously tested by NIH and European Pharmacopoeia methods: 1. «RabiStar» manufactured «Ukrvetprompostach» Ltd: three batchs. 2. «Antigen of a rabies virus for immunization of horse producers», manufactured PJSC «PHARMSTANDART-BIOLIK», batch number 015E004 expiry date 08.2017; 3. As reference vaccine was used Rabies vaccine (inactivated) for veterinary use BPR, batch number 4.5 with immunogenic activity – 11 IU. It is a freeze-dried vaccine derived from the Pitman-Moore (PM) strain of rabies virus produced in the Nil-2 cell line and inactivated with betapropiolactone. Animals: mice, females, 18–20 g; 10 specimens for the test vaccines and standard. Before immunization, all animals were quarantined for 3 days. Viruses: The Challenge Virus Standard-11 strain (CVS-11) is a standard, fixed laboratory strain (ATCC® Reference VR-959), produced in BHK-21 cells and stored at –80°C. It was used to infect BHK-21 cells used in the RFFIT. The Challenge Virus Standard-27 strain (CVS-27) is a standard, fixed laboratory strain (ATCC® Reference VR-321), produced in live mice and stored lyophilized. It was used to infect mice in the mouse challenge test. Cell Culture: BHK-21. Reference serum: The second International WHO Standard for Rabies Immunoglobulin with activity of 30 IU. The serological assay used involves the immunization of groups of 10 mice with approximately 1/5th the recommended dose volume of the test vaccine diluted appropriately, or of the reference standard vaccine preparation which is adjusted to the minimum potency allowed in the Ph. Eur. 14 days after immunization blood samples are taken and the sera are tested individually for rabies antibody using the described virus neutralization assay. Briefly, sera are titrated on 96-well microtitre plates and incubated for 1h with rabies virus. After adding BHK cells and incubating for 48h the presence of unneutralized rabies virus is revealed by immunofluorescence. Dilutions of the sera that reduce the number of fluorescent cells by 50 per cent are calculated.

Results of research and discussion. All tested vaccines have satisfactory results in serological potency assay (activity> 1 IU / dose), and given their dilution factor before injection to mice, «RabiStar» manufactured «Ukrvetprompostach» Ltd has an activity greater than the minimum (2.0 IU) for the batch number 020416 expiry date 04.2018 with the probability p = 0.020; batch number 051016 expiry date 10.2018 with p = 0.000; batch number 071116 expiry date 11.2018 with p = 0.017; "Antigen of a rabies virus for the immunization of horse producers", manufactured by PJSC "PHARMSTANDART-BIOLIK", batch number 015E004 expiry date 08.2017, has an activity of not less than 2.5 IU per dose with a probability of p = 0.001. The author expresses his sincere gratitude for the help in carrying out the research to the colleagues from the European reference laboratory for rabies ANSES-NANCY, France and PJSC "PHARMSTANDART-BIOLIK", Ukraine.

Conclusion and prospect for further research. The results of immunogenic activity inactivated vaccines against rabies obtained using the NIH and European Pharmacopoeia methods are not identical. The first time in Ukraine in the State Scientific Control Institute of Biotechnology and Strains of Microorganisms was using the serological potency assay for quality control of inactivated vaccines against rabies. In the future it is planned to implement the principles of 3 R in the control system of other vaccines.

Keywords: rabies, inactivated vaccines, quality control, 3 R principles, NIH method, serological potency assay, European Pharmacopoeia.


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