CS265945B1 - Infectious rhinotrachelid vaccine Inactivated and its production method - Google Patents

Abstract

The solution concerns a vaccine against infectious bovine rhinotracheitis. The vaccination dose contains 1θ7,SStkid^q of inactivated virus of the CAMP V-317 strain. Primary calf kidney cultures are used for virus cultivation, which are cultivated in Earl's salt mixture with 10% serum free of antibodies against the IBR virus, at a temperature of 34 to 38 °C for 2 to 5 days, then freed from cell detritus, chemically inactivated and prepared into an application form. The antigen is concentrated, for example, by ultrafiltration or ultracentrifugation. Chemical inactivation is carried out with glutaraldehyde in a final concentration of 0.02 to 0.05 wt. at temperatures of 4 to 40 °C. For use, the inactivated virus is mixed with a lipoid adjuvant to form a liquid vaccine or lyophilized with a lyophilization medium based on sucrose and gelatin and dissolved in a lipoid adjuvant before use.

Description

The invention relates to a vaccine against infectious bovine rhinotracheitis (IBR) and a method of its production. In a broader sense, the invention concerns the prophylaxis of infectious bovine rhinotracheitis, a disease that plays a significant role in the complex of infectious diseases of cattle. For this reason, great attention is paid to the study and prevention. Diagnostic investigations based on the use of immunoenzymatic tests have shown that IBR is widely spread in our farms <1 has a significant impact on the economy, the breeding of cattle in individual agricultural enterprises. First of all, the process of reproduction, the rearing of healthy calves and the infection-free bulls from potential mothers.

The disease is characterized by its clinical manifestation in several forms and threatens animals of all ages, and can cause direct and indirect losses. The most serious and most frequently detected form in our country is respiratory disease in calves and disruption of pregnancy (fetal death, abortions) in cows.

The causative agent, the IBR virus, tends to persist in the animal organism without obvious symptoms and to be excreted only at certain moments provoked by external environmental factors (corticosteroid treatment, animal transport, simultaneous invasion of Dictyocaulus viniparus, superinfection with the PI-3 virus, parturition in cows, etc.). However, these infected animals play a significant role in the spread of the disease. Latently infected mothers can transmit the infectious virus to their calves, which can be a source of infection for other animals, due to the exacerbation of the infection provoked by parturition.

Current epizootological findings derived from extensive serological examinations show that antibody titers in blood serum of individual animals exposed to natural contact infection are different and fluctuate in a wide range - from negative to highly positive values. The concentration of antibodies in blood serum and in colostrum and milk of cows reflects the degree of overall immunity of the given animal and determines both the possibility of exacerbation of latent infectious processes (growth of the virus into the pregnant uterus) and the susceptibility of newborn calves to infection.

From the perspective of comprehensive IBR prophylaxis, it is therefore important to induce and permanently maintain a high level of immunity in all animals housed in the farm and exposed to infection.

The goal of the author team was therefore to develop a suitable harmless and highly effective immunopreparation - an inactivated vaccine with a sufficiently long expiration period and to define an indication for its use in the prevention of IBR i' within the framework of comprehensive prophylactic programs aimed at completely recovering herds from this disease.

Currently, in countries with developed livestock production and advanced veterinary services, both live vaccines containing attenuated IBR virus and inactivated vaccines are used for specific immunoprophylaxis of IBR.

The clear advantage of attenuated vaccines, i.e. low production costs, is significantly reduced by the fact that IBR virus strains induce a latent infection state and can be excreted by the host together with virulent strains and spread to susceptible unvaccinated animals /PASTORET et al., Infect. Immun., 29, 1980, 483 to 488). Attenuated vaccines are therefore only useful for reducing losses and limiting the incidence of clinically manifest infections. In no case can their use be expected for the overall suppression of the infection and recovery (NETTLETON et al., Vet. Rec., 107, 1980, 379 to 382).

For this reason, the use of inactivated vaccines is currently preferred, in which the emergence of latent infection is excluded, which increase the overall level of the herd, limit the reactivation of latent infections, reliably protect the fetus and prevent infection. They allow for the gradual displacement of the virus from the cattle population. They are therefore also applicable within immunoprophylactic programs oriented towards the gradual recovery of herds from IBR.

The basic parameter of the effectiveness of inactivated vaccines are the antibody titers determined in the blood sera of vaccinated animals 28 to 42 days after immunization. The antibody titers determined after the application of the vaccine prepared according to this PV (1:8 to 1:128) correspond to the titers achieved with foreign preparations prepared from other strains and by other working procedures. (SWEAT, R. T,., J. Am. Vet. Med. Ass . , 182, 1983 , 809 to 811).

The advantage of the vaccine prepared according to the procedure described in this application is that it induces not only humoral immunity of the same level as the BORINAK vaccine (Bioveta Nitra), but also demonstrably specific cell-mediated immunity. The fact that the vaccine according to this procedure is prepared in a lyophilized state extends its expiration period to at least 12 months.

The essence of the invention is an inactivated vaccine against infectious bovine rhinotracheitis

8 and a method of its production, characterized in that it contains at least 10 ' ΤΚΙϋ^θ of inactivated virus of strain CAMP V-317 in one vaccination dose. The virus is cultivated in a primary calf kidney cell culture, for the cultivation of which serum free of specific antibodies against infectious bovine rhinotracheitis virus was used, at a temperature of 34 to 38 ° C for 2 to 5 days. The obtained virus is freed from cell detritus, chemically inactivated and prepared into an application form. The antigen is concentrated, for example, by ultrafiltration and ultracentrifugation. Chemical inactivation is carried out with glutaraldehyde in a final concentration of 0.02 to 0.05% by weight, at temperatures of 4 to 40 ° C. For use, the inactivated virus is mixed with a lipoid adjuvant into the form of a liquid vaccine, or lyophilized with a lyophilization medium based on sucrose and gelatin and dissolved in a lipoid adjuvant before use.

The advantage of the proposed vaccine is a good antibody response after vaccination and revaccination of animals with a dose of 2 cm ¹ , both in animals without the presence of humoral antibodies and in their presence.

Achieved geometric means of antibody titers detected in different groups of animals, or in their colostrum

Number of ___________Antibody levels

animals Before the vacc. 21 days after vaccination. In 21 days after the revak. In colostrum in calves after drinking colostrum Animals infected without vaccination. 9 8.00 5.65 5.65 6.34 14.25 Animals infected with vacc. 30 7.55 57.01 120.81 170.85 80.63 Animals not infected with vaccine 23 0 2.20 47.55 - -

Cell-mediated specific immune response of calves after administration of an inactivated vaccine against IBR and after challenge i. n. infection with a virulent IBR strain.

Tele-1 Tele-2 Tele-3 Tele-4 Before BTL vaccination SI = 0 SI = 0 SI = 0 SI = 0 (infection) LMI 0% 0% 0% 0% LAI 0% 0% 0% 0% First BTL reaction 6d., SI = 4 From, SI = 0 - after LMI revaccination 3d., 20% 17d., 15% — — LAI 3d., 45% 3d., 20% - — Highest BTL 21d., SI = 9 5 Od. , SI = 0 18d., SI = 70 - reaction after LMI 3d., 20% 17d., 15% 31d., 40% — LAI revaccination 3d., 45% 21d., 40% 3d., 63% —

r

265945 Tele-1 4 Tele-2 Tele-3 Tele-4 First reactions after i. sat. infection with IBR virus BTL LMI LAI 3d. 7d. 3d. , SI = 5 , 40% , 35% 3d., SI = 2.9 7d., 45% 14 days, 20% - 14d., SI = 2 3d, 29% 3d, 60% No jνγΓιΠ í t o· •Ί k < ·υ 11< > 1 . sat. infection with IBR virus ΠΤΓ, LMI LAI people /<1. 14d. , SI - 1 1, , 40% , 45% 1 1(,.1., SI - 75 7(1., 45% 14d., 20% people, 7d. , 40% 14d., 30% 7 / 1 4<1. , lil -2 3d., 29% 3d., 60% IBR virus shedding from the nasal mucosa after i^. sat. inf. neg 3 days 10 ³ - 10 ⁰ ' ⁵ TK!D ₅₀ neg. 7 days 10 ⁵ ' ⁷⁵ - 10 ^{1 TKID} 50 Serum antibody titer at the time of i. sat. inf. (ELISA) 1:6 300 1:1 350 1:7 100 neg. Clinical symptoms of illness after i. sat. inf. - - - -

Legend: Tele-1 - vaccinated 2x Tele-2 - vaccinated 2x Tele-3 - vaccinated 3x Tele-4 - control, unvaccinated BTL = blast transformation test of lymphocytes LMI = leukocyte immigration inhibition test LAI = leukocyte adhesion inhibition test SI = stimulation test d = days after vaccination or infection

He gave an example.

The given example does not limit or exhaust the subject matter of the invention in any way.

Viral antigen

The vaccine is produced using the infectious bovine rhinotracheitis virus strain CAMP V-317, which was isolated from calves suffering from acute respiratory disease in primary calf kidney cell cultures cultured in Earle's salt mixture supplemented with 0.3% lactalbumin6 8 3

-hydrolysate. For the production of the vaccine, a virus with a titer of 10 ' ΤΚΙΟ^θ in 1 cm and higher can be used.

Cell culture for virus propagation

For infection, a 5-day-old culture of primary calf kidney cells propagated in Earle's salt mixture with 0.3% by weight of lactalbumin hydrolysate and 10% by weight of bovine serum albumin is used.

inactivated serum free of antibodies against bovine rhinotracheitis virus. The absence of specific antibodies against IBR virus is checked by ELISA test and virus neutralization test.

The grown culture is stripped of the growth medium with serum and infected with the IBR virus by absorption at a dose of 0.05 TKID ₅₀ per cell. After the virus has been absorbed into the cell culture, maintenance medium is added - Earl's salt mixture with 0.3% by weight of lactalbumin hydrolysate. The infected cell cultures are incubated for 4 days at 37 °C. During this time, a cytopathic effect develops.

The amplified virus is freed from cell debris by centrifugation at 3,000 g for 15 minutes. The pure virus is then concentrated through an ultrafilter that captures particles with a molecular weight greater than 20,000. The virus is concentrated 10x. The concentrated virus is inactivated with glutaraldehyde at a concentration of 0.125% by weight at 37°C for 2 hours. The pH of the inactivated virus is adjusted to 7.0; a protective lyophilization medium containing gelatin and sucrose is added and lyophilization is performed. The lyophilized antigen is diluted in a lipoid adjuvant before use and administered to animals. The advantage of the vaccine prepared in this way is a long expiration period.

A lipoid adjuvant of the following composition can be used to dilute the vaccine:

Distilled water

Paraffin oil

Lanolin

Tween 80 (polyoxyethylated sorbitol monooleate)

000 ^cm3

600 ctn ³

300g

100g

Example 2

The concentrated and inactivated virus prepared according to Example 1 is mixed with a lipoid adjuvant and the vaccine thus formed is stored in a liquid state until use.

Composition of the liquid vaccine:

Concentrated antigen IBR 30cm

Paraffin oil 62cm

Montanide 103 8cm

Claims (6)

An inactivated infectious bovine rhinotracheitis vaccine, characterized in that 6 8 contains at least 10 'ΤΚΙϋ ₅ θ of inactivated infectious bovine rhinotracheitis virus strain CAMP V-317 in one vaccine dose. 2. A method for producing a vaccine, wherein the virus is cultured in a primary calf kidney cell culture using a serum free of specific antibodies against infectious bovine rhinotrocheitis virus at a temperature of 34 to 38 ° C for 2 to 5 days, followed by depletion. cell detritus, is chemically inactivated and adjusted to the application form. 3. A process for the manufacture of a vaccine according to claim 2, characterized in that the viral antigen is concentrated, for example by ultrafiltration or ultracentrifugation. 4. A process for the production of a vaccine according to claim 2, characterized in that the chemical inactivation is carried out with glutaraldehyde in a final concentration of 0.02 to 0.5% by weight, at temperatures of 4 to 40 ° C. 5. The method of manufacturing a vaccine according to item 2, characterized in that the inactivated virus is mixed with the lipoid adjuvant into a liquid vaccine before use. 6. The method of manufacturing a vaccine according to item 2, characterized in that the inactivated virus is lyophilized with a lyophilization medium based on sucrose and gelatin and dissolved in a lipoid adjuvant before use.