The virus strain pestis suum used to control immunogenic and antigenic activity of vaccines and biologics manufacturing for specific prophylaxis and diagnosis of classical swine fever
The invention relates to the field of veterinary biotechnology, Virology and Microbiology. Proposed new strain of the virus of classical swine fever (CSF) obtained by direct passage of the pigs virulent CSF virus. The strain has a stable infection, antigenic and immunogenic activity. The strain used for the manufacture of highly immunogenic vaccines against CSF, receiving hyperimmune sera for the prevention of CSF and allows the use of antigen and received him in serum serological reactions for the diagnosis of CSF. The new strain may also be used as a test object for quality analysis of Biopharmaceuticals.
The present invention relates to the field of veterinary Virology, in particular to a new Pestis strain suum used to control immunogenic and antigenic activity of vaccines, production of biological products for specific prophylaxis and diagnosis of classical swine fever (CSF).
CSF is a severe, often fatal, contagious viral disease of swine characterized by a variety of clinical forms and manifestations, namely fever, lesions of blood and chromatosphere intestine. The causative agent is a virus belonging to the genus Pestivirus of the family Flaviviridae.
Currently CoES on the relevance is one of the first places in infectious diseases of pigs and considered for countries with intensive system hog one of the economically important diseases, veterinary and sanitary plan - one of the most difficult liquidated epizootics. Over the past years, there had been several periods of activation of epizootic process of this disease with subsequent reduction of its intensity. CoES is registered in almost all economic regions of Russia (from Kaliningrad to the Far East and from the Arkhangelsk region to Krasnodar Krai). Although the reservoirs and transmission routes and infection is well known, practical experience shows that to implement complex security of the country and farms from the introduction of the pathogen CoES, and especially to eliminate the epidemic is extremely difficult.
For the specific prevention of pigs used live lepidosirenidae or culture vaccine derived from an attenuated virus strains CoES.
To control immunogenic vaccines and for the manufacture of inactivated vaccines against-Mine" in Russia, "Washington" and "Ames" in the United States, "Alfort" in France, "ALD" in Japan, "Shimen" in China (1-7).
The disadvantages of these strains are: 1) the relative volatility with long-term (3-10 years) storage of biomaterials even at low temperatures, 2) the variability in the level of infectious activity in biomaterials (105-107LD50/cm3), 3) different duration of infection in infected pigs, not immune to the virus CoES (7-21 days).
The purpose of the invention to provide a new strain of the virus Pestis suum "Shi-Ming" stable biological and antigenic activity during long-term storage, with the possibility of its use for control immunogenic and antigenic activity of vaccines and biologics manufacturing for specific prophylaxis and diagnosis of classical swine fever.
New Pestis strain suum "Shi-Ming" obtained from a dedicated in 1992 isolate virus Pestis suum, from patient gilt 3 months of age by serial passages on the pigs. In 2001, the strain deposited in the Collection of microorganisms of the Federal state institution “all-Russian state research Institute for control, standardisation and certificaion room "Shi-Mini-47"-DEPT.
Strain Pestis suum "Shi-Mini-47" VGNKI characterized by the following features and properties.
When electron-microscopic study of the strain Shi-Mini-47" presents typical pestiviruses separate virions are spherical or round shape with a diameter of 40 to 44 nm. In ultrathin sections, the magnitude of the nucleocapsid defined in 34-38 nm, and the thickness of the lipoprotein membrane 3 nm. When biophysical research the virus to their sedimentation coefficient in Svedberg equal 10418 S, floating density is 1.16-1.20 g/cm3.
Genetic engineering and biochemical methods established that the viral genome is a single-stranded plus-RNA with a molecular weight of 410 Yes, comprising 12284 nucleotides with a single long open reading frame corresponding to polyprotein long 3898 amino acid residues. In the genome of the virus CSF genes are from T-end-to-end in the following sequence: gp44/48-gp33-gp55. Purified in the gradient virions (electrophoresis in SDS page) contain 3 main types virousspecificakih proteins with a molecular mass 54-55 kDa, 45-47 kDa and 35-37 kDa. The two most large polypeptide are glycoproteins located(gp46) and (gp36). Polypeptide gp55 contains major immunoreactive dominants involved in the neutralization of the infectivity of the virus in CSF.
The virus reproduces well in transplantable cell cultures kidney pig (RC-15) and primary cultures of cells: leukocytes pigs (LS), and bone marrow pigs (CCM), and other cells of porcine origin. It is expressed cytopathic effect (JRC) in infected cell cultures with the exception of a sharp reduction in phagocytic activity of macrophages in the LAN. Viral infection of cell cultures detected by using the method fluoresceine antibodies. Under cultivation in cell cultures of RK-15 and (LS) the titer of the virus is 5-7 lg CCID/cm3.
The optimal conditions for the cultivation of strain Shi-Ming are odnostoechnye transplantable cell culture RK-15 making in a supportive environment 5-10% fetal cattle serum and 5-day primary cultures of cells of drugs and CCMS making in a supportive environment 5-10% serum of pigs that do not contain pestivirus antibodies, autologous blood serum of pigs.
Strain Shi-Mini-47" of the CSF virus in injecting adult swingersi of microblades). Virulent properties.
After injection of strain Shi-Ming virus CoES pigs at a dose of 10-1000000 LD50/cm3in 100% of vaccinated animals after 2-6 days after infection note the increase of body temperature to 40.1-42,0, which is held for 7-10 days in the presence of syndromic CoES (hyperthermia, diarrhea, vomiting, cyanosis, depression, loss of appetite) and destruction of all infected animals at 7-21 day.
Strain Shi-Mini-47 virus CSF maintains stable infectious and antigenic activity for 20 serial passages in pigs (observation period on the 67th passage).
Strain Shi-Mini-47 virus CoES active in the neutralization with specific serum.
Strain Shi-Mini-47 virus CoES not agglutinate erythrocytes of animals.
Emissions laboratory animals.
Strain Shi-Mini-47 virus CoES harmless for white mice, Guinea pigs and rabbits by intramuscular, subcutaneous and nasal administration.
The ability of the strain to spread in vivo.
Infection of pigs in the environment is alimentary, airborne, TGNA.
Main storage conditions.
Strain Shi-Mini-47 virus CoES well stored at minus 50-70C in dried form in vials under hermetically sealed tubes and rolled metal caps or in ampoules sealed under vacuum. The titer of lyophilized virus for 10 years of storage in ampoules at virtually the same level, and biological activity is not changed. However, the native storage of the virus within 5 years at minus 10-40S reduces its contagious.
For more information about the strain.
Free from viral, bacterial, Mycoplasma and fungal contaminants.
The invention is illustrated by the following examples:
Prepare dried vaccinated material from strain Shi-Mini-47” VGNKI. For this pigs (not immune to pestiviruses and having a mass of 20-60 kg) intramuscularly injected a suspension of strain Shi-Mini-47" in a dose of 10 000 LD50in volume 1 cm3. In animals daily measure body temperature. Pigs reacting through 42-52 h temperature rise to 40.5-41,8 With, totally Deplete on day 5-7. From each animal blood is taken in a separate sterile vial containing 1000 IU of heparin. The blood is then poured into one of the s 4-8C and observing sterile conditions, centrifuged at 3000 rpm for 20 min to release from membranes destructively erythrocytes and possible clots. The supernatant is collected in a bottle and add antibiotics in an effective amount. Received vaccinated material combine with medium drying in the ratio required for obtaining vaccinated biomaterial with a given infectious activity. Components of a protective environment using commonly used for these purposes, ingredients: sucrose, gelatin and hydrolyzed lactalbumin. The mixture of antigen and protective environment is thoroughly mixed, if necessary, bring the pH of the mixture to 7.3 to 7.4, poured into sterile vials (ampoules) 1-2 cm3and subjected to freeze-drying. After drying, the vials closed with rubber stoppers and running aluminum caps, ensuring the integrity of the bottles.
Ampoules sealed under vacuum. Ready biomaterial is a dry amorphous mass of red-brown color, easily soluble adding saline solution. Vaccinated material from strain Shi-Mini-47” retains its biological activity when Hurley dry VirusWall LK-Vniivvim against classical swine fever.
To check immunogenic activity VirusWall LK-Vniivvim used 11 not immune to the virus CoES pigs (weight 20-65 kg). The contents of five bottles or 10 vials dissolved in physiological solution (pH 7.0 to 7.4) to its original status. Prepare 10-fold dilution of VirusWall (10-1-10-6) physiological solution (pH 7.0 to 7.4). Form 4 groups of animals, of which the first 3 are subjected to immunization intramuscularly culture dry venusvalley LK-Vniivvim against CSF at a dose of 1 cm3and dilution 10-43 other - in a dose of 1 cm3and dilution 10-5; 3 pigs of the third group at a dose of 1 cm3and dilution 10-6two control pigs not vaccinated. In 15 days after inoculation experimental and two control pigs intramuscularly infect suspension of strain Shi-Mini-47” VGNKI virus CoES obtained in example 1, at a dose of 4.0 lg LD50/cm3. For infected pigs see within 14 days.
Sick after the introduction of the suspension of the strain Shi-Mini-47” VGNKI virus CoES and died 7-12 day with the characteristic features of the CoES (hyperthermia to 42S, depression, loss of appetite, cyanosis of skin and mucous membranes, vomiting, diarrhea) all control gilts and two of the three animals t is STI VirusWall was 10of 5.83IPD50/cm3. Thus, the vaccine is immunogenic and is suitable for practical use.
Check immunogenicity lepidosirenidae dry VirusWall of strain SENLAC” against classical swine fever.
Conduct test VirusWall on 6 pigs (2 control) weighing 25-35 kg at the age of 60-80 days without blood pestivirus antibodies. The contents of five bottles or 10 vials of VirusWall, pre-titrated in rabbits with title 104ID50/cm3, dissolved in saline solution (pH 7.0 to 7.4) to the initial condition (2 cm3) and then diluted 1000 times.
Each of the 4 Guinea pigs intramuscularly behind the ear or on the inner side of the thigh 2 cm3the reconstituted vaccine containing 10% pravilnoy dose, i.e. 10 ID50/cm3(rabbit infectious doses). The period of observation of vaccinated animals is 14 days.
At the end of the observation period all animals infect intramuscular injection of the suspension of strain “Shi-Mini-47” VGNKI virus CoES similarly as in example 2. The period of observation of infected animals 2 weeks. Within 2 weeks, all vaccinated animals octanol for practical application.
Assess the antigenic activity of the production strain LK-Vniivvim virus classical swine fever.
For this purpose, three pigs 2-3 months of age are subjected to immunization specified in volumetric strain dose of 2 cm3and in a dilution of 10-3(1000 IPD50/cm3). On 22 days in all vaccinated pigs take blood samples and get the blood serum. To determine the titer of neutralizing antibodies to classical swine fever virus obtained serum examined in the neutralization with 100-1000 KID50/cm3strain Shi-Mini-47" VGNKI obtained in example 1. The estimation of the reaction spend 2-3 days in reaction immunofluorescence assay for the presence of transplantable monolayer transplantable cell cultures kidney pig (RC-15) - "MicroBlaze", i.e. groups of cells infected with CSF virus and fluoresceine emerald-green glow. Control is infected and uninfected transplantable cell culture kidney pig (RC-15). The titer of neutralizing antibody in the serum is 1:64-1:1024. Thus, the production strain LK-Vniivvim” of classical swine fever virus is immunogenic.
Prepare inactivity in example 1, vaccinated material dissolved in physiological solution with a pH of 7.2 to 7.4, titrated in the culture of drugs, and reduce the titer of the virus to 7 lg CCID50/cm3(fabric option). Vaccinated material for cultural variant vaccines receive the standard methods of monolayer and roller 5-7-day-old primary cultures of cells leukocytes and bone marrow cells of pigs (cultural variant of the vaccine). Title vaccinated material in the culture of drugs is 7,0-8,5 lg CCID50/cm3.
Then for inactivation in vaccinated material make a 10-fold concentrated solution of the drug AND24(5 mg/cm3) in volumetric ratio of 10:1 and spend incubation with S in 6-12 hours. The calculation of the amount of time required for inactivation, is determined by the formula
where T is time in hours, x - titer vaccinated material, 1,54 and 4.51 - constant coefficients.
In an inactivated preparation add 50% aluminium hydroxide (GOA) and incubated for 30 minutes. The finished vaccine sterile Packed in vials of 100-200 cm3, closed with rubber stoppers and running aluminum caps, ensuring the integrity of the bottles, check its activity in compliance which retains immunogenic activity for 1 year when stored under the conditions temperature 2-8C.
Obtained according to example 7 of inactivated vaccine from strain Shi-Mini-47” VGNKI used to immunize five pigs 3 months of age. To this end, the vaccine is administered twice with an interval of 14 days subcutaneously in the ear at a dose of 4 cm3. Animals at 21 days after the 2nd vaccination take blood samples, get the serum and in the neutralization determine the antibody titers in accordance with example 4. All immunized animals set neutralizing antibody titers ranging from 1:16 to 1:128. Thus, the resulting vaccine is immunogenic and is suitable for practical use.
Check immunogenic activity of inactivated vaccine against classical swine prepared according to example 5.
To check immunogenic vaccines use 4 not immune to the virus CoES pigs (weight 20-65 kg). Contents 3 bottles mixed and subjected to immunization 3 pigs intramuscularly at a dose of 4 cm3twice with an interval of 14 days. Control gilt not vaccinated. After the second immunization (21 days) experimental and control pigs intramuscularly infect pre-cooked suspe nymi the pigs see within 14 days.
Reference gilt fell ill after the introduction of vaccinated suspension and died on 12 days with the characteristic symptoms of the disease CSF. The rest of the animals showed resistance to infection control. Thus, inactivated vaccine is immunogenic and is suitable for practical use.
Receive hyperimmune serum from strain Shi-Mini-47” VGNKI for diagnostic and preventive purposes. For this two, is not immune to the virus CoES pigs weighing 50-60 kg subjected to immunization subcutaneously inactivated vaccine against CSF from strain Shi-Mini-47” VGNKI obtained in accordance with example 5, at a dose of 4 cm3. After 10 days in immunized pigs infect intramuscularly with the suspension of the strain Shi-Mini-47” VGNKI obtained in example 1, at a dose of 6.0 lg LD50/cm3. Hyperimmune serum get from sick pigs through 45-70 days. Serum get the conventional method. Set the titer obtained serum neutralization reaction using transplantable cell culture kidney pig (RC-15) and a method of direct immunofluorescence assay, which is 1:2569-1:10240.
Ready hyperimmune serum is the way ecrivait rubber plugs and running aluminum caps, ensuring the integrity of the vials, and stored in the native state at a temperature of 4C for 12 months.
Obtained according to example 8 hyperimmune serum used 10 piglets 1.5 to 3 months of age. The serum is injected once subcutaneously behind the ear, at a dose of 5-10 cm3. On 1-15 day the animals infect previously prepared suspension of strain Shi-Mini-47” VGNKI virus CoES obtained in example 1, at a dose of 4.0 lg LD50/cm3. Animals observed for 14 days after infection. All animals do not indicate deviations from normal physiological state, which confirms the high efficiency of the obtained biological product.
Prepare specific FITZ-immunoglobulin (fluorescent antibodies to classical swine fever virus) for diagnostic purposes.
Receive hyperimmune serum against CSF from strain Shi-Mini-47” VGNKI in accordance with example 8. Selection globulin fraction from native hyperimmune sera of pigs spend the conventional method at 40% saturation of ammonium sulfate. Of the total globulin fraction on the column with DEAE-cellulose allocate a fraction of immunoglobulins. Label immun is m, fluoreszierendem green.
To do this within 24 hours at the temperature of 2-4C carry out conjugation of antibodies with FITZ in the ratio of 1:50 (fluorochrome/protein). Purification of the conjugate from unbound fluorochrome spend gelfiltration through a column of Sephadex G-50, the elution to 0.14 M phosphate buffer solution with pH 7.5, collecting fractions 10-15 cm3. If gelfiltration solution FITZ-immunoglobulin diluted to 0.5% protein content. The protein concentration determined using a spectrophotometer according to a formula based on measurement of light absorption of the conjugate at a wavelength of 280 and 495 nm. The degree of purification of the conjugate from unbound fluorochrome carried out by the method of ascending chromatography on paper. To eliminate nonspecific fluorescence conjugates treated powders of the liver is not immune to CSFV pigs (50 to 100 mg per 1 mg of protein) and uninfected transplantable cell culture kidney pig (RC-15) - 10-20 mg per 1 mg of protein.
Fluorescent antibodies screened for specificity and activity in direct and indirect immunofluorescence assay reactions. Painting title FITZ-immunoglobulin was 1:16. Nonspecific glow in the control preparations were absent. Protection the capsules stored in a dark place at a temperature of 4C for 24 months and is used for the given reaction is a direct immunofluorescence assay.
Use in veterinary practice Pestis strain suum "Shi-Mini-47" VGNKI will allow sufficient reliability to judge the quality of the produced and used biologics against classical swine fever, manufactured on its basis, highly immunogenic vaccine against CSF, to obtain hyperimmune serum on the pigs for the prevention of classical swine fever. To use the antigen and received him in serum serological reactions for the diagnosis of classical swine fever.
The virus strain Pestis suum VGNKI "Shi-Mini-47 DEPT", used to control immunogenic and antigenic activity of vaccines and biologics manufacturing for specific prophylaxis and diagnosis of classical swine fever.