RU2306949C2 - Protective medium for virus vaccine production in aviculture - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to using of lipopherol-liposomal preparation of antioxidant action for intravenous administration as protective medium. Said lipopherol contains soy psospholipids, DL-alpha-tocopherol, sorbitol-phosphate buffer. Protective medium of present invention prevents infection of bovine spongiform encephalopathy.

EFFECT: bioactive vaccines of high quality and immunity.

2 ex, 1 tbl

Description

The present invention relates to the field of virology and relates to the manufacture of drugs used in veterinary medicine, in particular, it can be used in the manufacture of specific protection against infectious laryngotracheitis of birds and Newcastle disease, etc.

It is known to use the protective environment SPLF - 20% in the manufacture of a combined vaccine against Newcastle disease (NB) and infectious laryngotracheitis of birds (ILT) by mixing virus-containing materials of the NB virus from the La Sota strain and the ILT virus from the VNIIBP strain [1 ].

It is also known to use a protective medium containing peptone, edible gelatin and sucrose, taken in a ratio of 2: 5: 5, or peptone with sucrose, or lactose with peptone and nonfat cow's milk when receiving means for the prevention of ILT based on the strain "VNIIBP" [2 ].

It is known the use of liposomes as a protective drying environment in the manufacture of a dry virus vaccine from the strain "VNIIBP" against ILT [3].

Known liposomal protective environment includes soya lecithin (SL), soybean acid phospholipids (CF), cholesterol (cholesterol) and α-tocopherol in a ratio of 67.4: 9.5: 22.0: 1.1.

However, the use of protective media in the production of virus vaccines that contain peptone or cholesterol-containing components is currently undesirable due to the possible contamination of cattle with spongiform encephalopathy by their pathogen, which is hazardous to human health [4].

The objective of the invention is the creation of a protective environment used in the production of virus vaccines, eliminating the possibility of infection with spongiform encephalopathy in cattle and at the same time contributing to the production of high-quality, highly immune and biologically active vaccines.

The problem is solved in that as a protective environment for the virus vaccine, lipoferol is used - a liposome preparation of antioxidant action for intravenous administration.

Lipoferol is a liposome preparation with DL-alpha-tocopherol for intravenous administration.

The main lipid components of lipoferol are isolated from domestic raw materials - soybean phospholipids. The preparation was obtained by the film-dispersion method, followed by homogenization of the components under high pressure. The composition of the drug includes soy phospholipids, DL-alpha-tocopherol, sorbitol-phosphate buffer. Lipoferol has an antioxidant and membrane stabilizing effect, it is harmless, non-toxic, does not cause allergic reactions, does not accumulate in the body due to complete metabolism.

It is used independently and in complex therapy regimens for burn shock, shock injury, toxemia of various origins, toxic and infectious hepatitis.

The developer and manufacturer of the drug is the Russian Research Institute of Hematology and Transfusiology [5].

To determine the possibility of using lipoferol as a protective environment in the production of virus vaccines in poultry, pilot batches of the liposomal virus vaccine against infectious bird laryngotracheitis (ILT) and the associated dry liposomal virus vaccine against bird infectious laryngotracheitis (ILT) and Newcastle disease (NB) were developed )

To produce the experimental series of dry liposomal virus vaccines against ILT, the VNIIBP strain was used, and for the associated dry liposomal virus vaccine, the La Sota dry strain against NB and the VNIIBP strain against ILT were used. To obtain vaccines, a viral suspension of these strains was inoculated into the allantoic cavity of 9-day-old chicken embryos (CE). On the sixth day, chorioallantoic membranes (CAO) were collected from embryos infected with the VNIIBP strain, and extraembryonic fluid was collected from embryos infected with the La Sota strain.

Virus-containing material of the strain "VNIIBP" and the liposomal emulsion were mixed in a ratio of 2: 1, and the same material of the strains "VNIIBP", "La Sota" and the liposomal emulsion in the ratio of 1: 1: 1.

The mass of liposomal vaccines after freeze drying was homogeneous, porous, whitish-pink in color, when dissolved in distilled water for 2-3 minutes formed a homogeneous suspension. The vaccines were sterile, not contaminated with foreign viruses, biologically active and immunogenic. Ten-fold immunizing doses of vaccines with ocular administration were found to be harmless.

Example 1

In order to comprehensively evaluate the dry liposomal vaccine virus for specific prophylaxis of bird ILT, a commission check was carried out under production conditions at ZAO Sinyavinskaya Poultry Farm, where Haysex White egg-bird is grown.

The dry liposomal vaccine against ILT and the commercial vaccine against ILT were used twice as an aerosol method: for the first time, at the age of 34-35 days, at a dose of 2.5 EID _{50 / goal} , and the second time, at the age of 52-53 days at a dose of 7, 5 EID _{50 / goal} , and at the same time she was prescribed a vaccine from the La Sota strain against Newcastle disease at a dose of 1100 EID _{50 / goal} .

On the same day, the method of "spray" was immunized with a vaccine from strain "N-120" against IB.

For the tests, two batches of chickens were formed: the experimental one for testing the liposomal vaccine (series 1), in the amount of 37,300 heads, and the control, where the commercial vaccine was tested (series No. 58), in the amount of 38,000 heads.

For vaccination, a dry liposomal virus vaccine was used against avian ILT from the VNIIBP strain series No. 1 with biological activity of 5.88 lg EID _{50 / cm} ³ and a dry virus vaccine from the same strain was dry series No. 58-5.7 lg EID _{50 / cm} ³ .

The effectiveness of aerosol immunization of these vaccines was established by determining the titer of antibodies in the blood serum of chickens by ELISA using kits of the company "Biocek".

The average antibody titer in all blood serum samples prior to vaccine administration belonged to the zero titro group. After the first and second immunization with the virus vaccine, dry liposomal and commercial average titer increased by 3-5 times. There was no significant difference in antibody titers after the second vaccination between biologics. Tests of the dry liposomal ILT vaccine virus vaccine showed that it provides effective protection against the control of pathogenic ILT virus infection (strain 24a) and is not inferior in terms of this indicator (94.4%) to the commercial vaccine (88.8%), in which Peptone is used as a protective medium (TU 9384-001-00495674-96).

Example 2

The biological activity of the associated dry liposomal virus vaccine against NB (La Sota strain) and ILT (VNIIBP strain) was determined immediately after manufacture and after 10 months of storage. The titration results were compared with a control combination vaccine prepared with standard protective medium (VNIVIP medium containing cattle peptone).

The experimental data on titration of vaccines prepared on various protective media are presented in the table.

Table Comparative indicators of the biological activity of ILT and NB viruses when using various protective environments. Protective environment Biological titer lg EID _{50 / cm} ³ After manufacturing 10 months later ILT NB ILT NB Liposomal emulsion 6.03 10.5 5.58 9.92 Environment "VNIVIP" based on peptone 6.03 10.5 5.98 9.50

As can be seen from this table, the titer of ILT and NB virus in the vaccine prepared on the basis of various protective environments was equivalent. After 10 months of storage, the titer of ILT virus in a liposomal suspension decreased by 0.4 lg EID _{50 / cm} ³ , and the titer of NB virus on a medium with peptone decreased by 0.42 lg EID _{50 / cm} ³ , which is permissible within the method error.

Thus, the composition of the medium practically did not affect the titer of viruses, i.e. both protective media can be used in the production of the associated dry liposomal virus vaccine against NB and ILT.

The resulting associated dry liposomal virus vaccine against ILT and NB was sterile. The introduction of a 10-fold intranasal dose of this vaccine to the chickens did not cause clinical signs of pain or depression within 10 days of observation, i.e. the vaccine was harmless. When determining the immunogenicity of the vaccine, chickens were vaccinated twice at the age of 20 and 40 days by enteral and nasal methods.

During the challenge with virulent ILT virus infection in the unvaccinated control group, 19 out of 19 chickens (100%) got sick with the ILT clinic (oppression, refusal to feed, shortness of breath, wheezing). While, in the group of chickens immunized with the associated dry liposomal and commercial virus vaccine by the enteral method, 2 heads (11.7%) fell ill. With the nasal method of vaccination, there were no diseased chickens, and in the group vaccinated with a commercial vaccine, 1 chicken fell ill. The correlation coefficient (r) for immunized chickens was 0.89-1.0, which indicates a close correlation with the infected unvaccinated control. The immunization efficiency for the associated dry liposomal virus vaccine was 88.3-100%, and for the commercial 88.39-94.1%.

When determining the immunogenic properties of the associated dry liposomal virus vaccine against NB in RHCA, the titer was 7.0-7.5 log ₂ , and commercial - 6.0 log ₂ . After the second vaccination, the titer of antihemagglutinins in all groups of chickens was relatively the same and ranged from 5.8 to 6.5 log ₂ , i.e. a reliable level of protection was created.

Thus, the proposed protective environment can be used in the production of the associated dry liposomal virus vaccine against NB and ILT, however, when using a liposome emulsion - lipoferol as a protective medium - there are advantages, since it does not contain a potential source of contamination of bovine spongiform encephalopathy, dangerous to humans.

Information sources

1. RF patent No. 2106152, A61K 39/17, 39/245, 1998

2. Malushko VV, Sokolov VD, Bykov VF, Chabanova LP, Shornikov VV "Poultry", No. 12, 1983, S. 21-23.

3. Novosadyuk T.V. "Abstract of dissertation for the degree of candidate of veterinary sciences" Liposomal form of vaccine against bird ILT ", St. Petersburg, 1998, p.10.

4. Kostenko Yu.G., Lisitsyn A.B., Shagova T.S., Timoshenko N.V., Kobzev A.N. “The problem of spongiform encephalopathy and animal feed production”, “BIO”, July 2002, pp. 26-28.

5. RF patent No. 2071765, A61K 9/127, 1997.

Claims (1)

The use of lipoferol, a liposomal antioxidant preparation, as a protective medium for the manufacture of virus vaccines in poultry farming.