RU2154496C2 - Vaccine against plague, infectious hepatitis and parvoviral enteritis in carnivores - Google Patents

Abstract

FIELD: veterinary science. SUBSTANCE: invention relates to agents of specific prophylaxis of plague, infectious hepatitis and parvoviral enteritis in dogs. Vaccine has the novel attenuated strain "VNIIVViM-88" of carnivore plague virus with infectious activity 3.5-4.5 lg TPD 50/cm³. Strain is cultured in subline CV-1 of African green monkey kidney cells. Vaccine has also vaccine strain "Kornell-2" of canine infectious hepatitis virus with infectious activity 5.5-6.5 lg TPD 50/cm³. Strain is cultured in piglet testicle cells transplanted culture. Also, vaccine has vaccine strain "A" of feline panleukopenia virus with infectious activity 3.5-4.5 lg TPD 50/cm³ and hemagglutinating activity in RGA = (1:32)-(1:512). Strain is cultured in kitten kidney cells transplanted culture (subline Fk). Vaccine has also drying medium based on lactose, sucrose, sorbitol and gelatin. Vaccine shows harmlessness, areactivity, high antigenic and immune activity, stability in storage, standard traits and high technological effectiveness of preparing. EFFECT: improved quality and properties of vaccine. 2 tbl, 3 ex

Description

 The invention relates to veterinary virology, in particular to means for the specific prevention of plague, infectious hepatitis and parvovirus enteritis in dogs.

 For the prevention of these diseases, live and inactivated mono- and associated vaccines are used, obtained both on the basis of primary and transplanted cell cultures.

 For vaccination of carnivores, including dogs, against plague, culture vaccines are used from the strains EPM, VNIIVViM-88, 668-KF, Rockborn, modified Snyder Hill and others (Author, St. USSR Cl. C 12 N 7/08 N 527072, 1977; CL A 61 K 39/12 N 1287592, 1984. U.S. Patents CL 424-89 N 4224412, 1980; N 4351827, 1982; N 5000951, 1991).

 To protect against infectious hepatitis, mainly drugs from the Cornell strain are used, as well as dog type 1 and 2 adenoviruses (US Pat. CL 195-1.3 N 3432391, 1969; CL 195-1.8 N 3616203, 1971; CL 424/89 N 3950512 , 1976. RF patent CL A 61 K 39/295, N 2030917, 1995).

 Vaccines against parvovirus enteritis contain live attenuated and inactivated virulent strains of dog parvoviruses, feline panleukopenia, and mink enteritis (US Pat. Cl. 424-89, N 4193990, 1980; N 4303645, 1981; N 4810494, 1989. Author. v. Czechoslovakia, class A 61 K 39/23 N 262736, 1988; N 252746, 1988; N 252747, 1988. RF patent class A 61 K 39/23 N 871371, 1994. TU 46-21-705-80 on culture vaccine against mink viral enteritis).

 Associated vaccines have been successfully used to form immunity against these infections in the shortest possible time with less labor and risk of complications arising from stressful events when vaccinating against each disease (England Patent CL A 61 K 3/62 N 1016586, 1966. U.S. patent CL 424-89 N 5000951, 1991. Author of St.V. Czechoslovakia CL A 61 K 39/295 N 267937, 1989. RF patent CL A 61 K 39/295, N 2030917, 1995. TU 08064-019-004-94 for the inactivated liquid vaccine against parvovirus enteritis and carnivorous hepatitis TU 10-09-98-91 for the dry culture vaccine otiv distemper, hepatitis and parvovirus, distemper).

 The closest in composition (the prototype of the proposed drug) is a dry culture vaccine against plague, hepatitis and parvovirus carnivores, containing strains: "668-CF" of the virus of carnivores, "Cornell-2" of the hepatitis virus of dogs and "A" of the cat panleukopenia virus, developed by the staff of the Pokrovsky plant of biological products (TU 10-09-98-91). Its main disadvantage is that for the production of virus-containing materials, expensive, non-standard, low-tech and non-contaminant-using primary trypsinized cell cultures are used: from chicken embryo tissues ("668-KF"), piglet kidneys ("Cornell-2") and the kidney of a kitten ("A"). In addition, the strain "668-KF" has a relatively low immunogenicity. The need for further development of this vaccine was indicated in the order for the Main Veterinary Administration of the Ministry of Agriculture and PRF (1993).

 The aim of this invention is an associated dry vaccine against plague, infectious hepatitis and parvovirus enteritis carnivores, with high immunogenic activity, safety, stability during storage and high manufacturability.

The goal is achieved in that the vaccine contains a new attenuated strain "VNIIVViM-88" of the carnivore plague virus (VChP), with an infectious activity of 3.5-4.5 lg TCD 50 / cm ³ , grown in sublimate CV-1 transplanted African kidney cells green monkey, the Cornell-2 strain of the dog infectious hepatitis virus (VIGS) with an infectious activity of 5.5-6.5 lg TCD 50 / cm ³ grown in a transplanted cell culture of piglet testicles (PTP), and the virus strain A panleukopenia of cats (MIC) with an infectious activity of 3.5-4.5 lg ID 50 / cm ³ and hemagglutinating activity in RGA 1: 32-1: 512, grown in a transplanted culture of kitten's kidney cells (subline Fk), as well as a drying medium including lactose, sucrose, sorbitol and gelatin, in the following ratio of components, vol.%:
Viral suspension of the strain "VNIIVViM-88" with a titer of infectious activity of 3.5-4.5 lg TCD 50 / cm ³ - 32-33
Viral suspension of the strain "Cornell-2" with a titer of infectious activity 5.5-6.5 lg TCD 50 / cm ³ - 9-11
Viral suspension of strain "A" with a titer of infectious activity of 3.5-4.5 lg ID 50 / cm ³ and hemagglutinating activity in the RSA 1: 32-1: 512 - 32-33
20% lactose solution - 4.8-5.2
60% sucrose solution - 4.8-5.2
40% sorbitol solution - 4.8-5.2
20% Gelatin Solution - Rest
The cultivation of the above three strains is carried out by a high-tech roller method using highly reproductive, standard, non-tumorigenic transplanted cell lines, which are used at the institute for growing various viruses.

 For the cultivation of the plague of carnivores, the subline CV-1 of a monolayer cell culture obtained in VNIIVViM from the initial culture of transplantable CV-1 cells (USA, ATCC, CCL 70) of the African green monkey kidney (number 43.2 in the catalog of the VNIIVViM cell collection) is used. The manufacturer’s working cell bank (DBC) of the CV-1 subline contains passage 17 cells in an amount of 165 million cells. A monolayer cell culture of the CV-1 subline obtained by serial passaging with a reseeding coefficient of 1: 3-1: 5 was used within 50 consecutive passages.

 For the cultivation of the infectious hepatitis virus of dogs, a monolayer culture of transplantable anti-TB drugs is used, obtained by the Institute from the Ukrainian Scientific Research Veterinary Institute in 1989. (Culture number 10.5 in the catalog of the VNIIVViM cell collection. BRK contains cells of 7-15 passages in the amount of 120 million cells. A monolayer culture of PTP cells is used to cultivate the virus within 50 consecutive passages.

 To grow feline panleukopenia virus, a monolayer cell culture of FK subline cells obtained in VNIIVViM from the original transfected kidney kidney line CRFK (USA, ATCC, CCL 94) is used. The subline is registered under number 34.1 in the catalog of the cell collection VNIIVViM. BRK subline FK contains cells 22-47 passages in the amount of 100 million cells. A monolayer culture of FK subline cells obtained by serial passaging with a reseeding coefficient of 1: 2-1: 4 was used for 50 consecutive passages.

The vaccine strain "VNIIVViM-88" (registration number "VNIIVViM-88 Dep" in the VGNKI collection) was obtained from the EPM strain by adapting it to the CV-1 subline through a series of intermittent passages and subsequent cloning using the limiting dilution method. The new strain is characterized by the following properties:
- possesses morphological properties characteristic of the plague virus of the carnivorous genus Morbillivirus of the family Paramyxoviridae;
- propagated in a transplanted cell culture of the CV-1 subline with the development of a pronounced CPD in the form of symplasts and subsequent cell destruction, the virus titer in this culture reaches 3.5-4.5 lg TCD 50 / cm ³ . Without adaptation, the virus multiplies in a culture of cells from tissues of chicken and quail embryos, causing a specific CPD and accumulating in the titers up to 3.0-4.0 lg TCD 50 / cm ³ ;
- cultured in a monolayer of transplantable cells of the CV-1 subline under stationary and roller conditions, under optimal conditions, the maximum biological activity is reached after 44-52 hours;
- harmless to laboratory and susceptible animals, non-contagious, non-reversible, non-pathogenic to humans;
- induces the formation of intense and prolonged immunity against plague in vaccinated fur animals (minks, thorzofretoks, arctic foxes, foxes, raccoons) and dogs;
- causes the formation of virus-neutralizing antibodies in titers of 1: 8-1: 32 and above; hemagglutinating properties does not possess;
- genetically stable for 10 consecutive passages in cell culture of the subline CV-1.

The vaccine against plague, infectious hepatitis and parvovirus enteritis of carnivores is administered to adult dogs once in a volume of 2 cm ³ , and for puppies - twice with an interval of 14-30 days in a volume of 1 cm ³ .

 Example 1

Virus-containing materials of all three strains are grown in monolayer transplantable cell cultures using a roller plant with a productivity of up to 50 dm ³ per cycle.

 The viral suspension of the vaccine strain VNIIVViM-88 of the plague carnivorous virus is obtained in the subline CV-1 of transplanted cells. As a growth medium, Igla-MEM medium with 10 vol.% Serum of cattle is used. As a supporting medium, Igla-MEM medium with 5 vol.% Serum of cattle is used.

The VNIIVViM-88 production strain of VChP with a titer of not less than 3.5 lg TCD 50 / cm ^{3 is} applied to 20 cm ³ in roller vessels with a 1-2-day monolayer of cells formed, after which the growth medium is drained from them. The contact of the virus with cells is carried out for 30-40 minutes at a temperature of 36.5 - 37.5 ^o C, then 480 cm ³ of support medium, pH 7.2-7.4, are added to each vessel.

The cultivation of the virus is carried out at a temperature of 36.5-37.5 ^o C and the speed of rotation of the vessels 10-12 about. in hour. After 40-48 hours, with the manifestation of a specific CPD in 50% of the cells, the viral suspension is drained, after taking samples from each vessel to control the contamination with bacteria, fungi, mycoplasmas and determine the titer of the virus by CPD in a CV-1 subclan cell culture. After freezing at a temperature of minus 60 ^o C and thawing at 25 ^o C, the resulting virus is poured into a single container.

 The viral suspension of the Cornell-2 vaccine strain VIGS is obtained in a transplanted culture of PTP cells. As a growth medium, Igla-MEM medium with 10 vol.% Serum of cattle is used. As a growth and support medium, Igla-MEM media with 10 and 5 vol.% Serum of cattle, respectively, are used.

The production strain of Cornell-2 of the IHV virus with a titer of at least 5.5 lg TCD 50 / cm ^{3 is} added 6 cm ³ into roller vessels with a 1-2-day monolayer of cells formed, after which the growth medium is drained from them. The virus contact with the cells is carried out for 60 - 90 minutes at a temperature of 36.5 - 37.5 ^o C, then 494 cm ³ of support medium, pH 7.2-7.4, are added to each vessel.

The cultivation of the virus is carried out at a temperature of 36.5-37.5 ^o C and the speed of rotation of the vessels 10-12 about. in hour. After 36-48 hours, with the manifestation of a specific CPD in 50-70% of the cells, the viral suspension is drained, after taking samples from each vessel to control the contamination by bacteria, fungi, mycoplasmas and determine the titer of the virus by the CPD in the culture of PTP cells. After freezing at a temperature of minus 60 ^o C and thawing at 25 ^o C, the resulting virus is poured into a single container.

 The viral suspension of the vaccine strain "A" MIC is obtained in an inoculated Fk cell culture. As a growth and support medium, Igla-MEM media with 10 and 5 vol.% Serum of cattle, respectively, are used.

Production strain "A" VPK with a titer of at least 3.5 lg ID 50 / cm ³ contribute 20 cm ³ in the roll vessels with the formed 1-2-day monolayer of cells, previously draining from them the growth medium. The virus contact with the cells is carried out for 60 - 90 minutes at a temperature of 36.5 - 37.5 ^o C, then 480 cm ³ of support medium, pH 7.2-7.4, are added to each vessel. The cultivation of the virus is carried out at a temperature of 36.5-37.5 ^o C and a speed of rotation of the vessels of 10-12 about. in hour. After 6 days, the viral suspension is drained, after taking samples from each vessel to control for contamination with bacteria, fungi, mycoplasmas, determine the biological activity in the Fk cell culture and hemagglutinating activity in the RGA. After freezing at a temperature of minus 60 ^o C and thawing at 25 ^o C, the resulting virus is poured into a single container.

 In the table. 1 shows the results of three experiments to obtain virus-containing materials used for the production of vaccines.

 There was no contamination with bacteria, fungi and mycoplasmas.

 As can be seen from the table. 1, all series of virus-containing material obtained on the basis of transplanted cell cultures were stable in their indices and had high activity.

Example 2
To 3.2 dm ³ of the carnivore plague virus, add 3.2 dm ³ of the panleukopenia virus of cats and 0.9 dm ³ of the dog infectious hepatitis virus. Drying medium containing 0.48 dm ^{3 of} 20% lactose solution, 60% sucrose solution and 40% sorbitol solution, 1.26 dm ^{3 of} 20% gelatin solution and antibiotics benzylpenicillin is added to the resulting virus mixture with thorough stirring on a shuttle machine; 200 PIECES / cm ³ , monomycin 200 PIECES / cm ³ and polymyxin 100 PIECES / cm ³ . The pH is adjusted to 6.6 with succinic acid at a final concentration of 6%.

In a similar manner, 2 more series of virus-containing mixture are prepared with the following amounts of components (dm ³ ) (see table 2).

The resulting mixture is Packed in 2 cm ³ in sterile ampoules (series 1 and 2), glass vials (series 3) and lyophilized. A total of 15,000 immunizing doses for dogs were made (1 dose per bottle and ampoule).

Example 3
On a commission basis (with the participation of the BAC Institute), 3 vaccine series were studied by the following indicators: biological activity, safety, reactogenicity, and immunogenic activity. Requirements for the vaccine are described in the technical specifications TU 9384-051-00008064-96 "Vaccine against plague, infectious hepatitis and parvovirus enteritis carnivores culture dry", approved in 1996 by the Department of Veterinary Medicine of the Ministry of Agriculture and PRF.

The biological activity of the vaccine is determined by titration of vaccine strains in cell cultures: HPV in the CV-1 subline cell culture, VIGS in the PTP cell culture, VPI in the Fk subline cell culture. The activity of the strains in a dry vaccine should be: "VNIIVViM-88" VChP - 2.75-3.75 lg TCD 50 / cm ³ , "Cornell-2" VIGS - 3.0-4.5 lg TCD 50 / cm ³ , "A" MIC - 3.0-4.5 lg ID 50 / cm ³ .

The safety of the vaccine is determined on dogs 2-4 months of age weighing at least 3 kg, clinically healthy, not vaccinated against the studied diseases that underwent deworming. Four dogs receive 5 cm ^{3 of the} vaccine in their thigh muscles. The animals are clinically monitored for 21 days. All vaccinated animals must remain alive and healthy during the observation period. Allowed for 1-3 days. refusal or incomplete eating of food, as well as liquefaction of feces.

Reactogenicity of the vaccine is determined on dogs 2-4 months. age weighing at least 3 kg, clinically healthy, not vaccinated against the studied diseases that underwent deworming. Four dogs are given a vaccine in the thigh muscles in a volume of 1 vaccination dose (2 cm ³ ). Vaccinated animals are clinically monitored for 21 days. A vaccine is considered to be reactogenic if all vaccinated animals have no deviations from the physiological norm during the observation period. Allowed for 1-2 days. refusal of feed and its incomplete eating, as well as liquefaction of feces.

The immunogenic activity of the vaccine against carnivorous plague is determined on thorzofreths 2–2.5 months old, obtained from farms that are successful in infectious and invasive diseases, clinically healthy, not vaccinated against plague, and not during the rutting season. The vaccine is administered to 4 thorsofretts intramuscularly in 1 cm ³ containing 500-1000 TCD 50 VChP. 21 days after vaccination, the vaccinated and 4 control animals are infected with the virulent Snyder Hill strain at a dose of 1000 LD 50 intramuscularly. The observation period is 25 days. A vaccine is considered immunogenic if it protected against infection and death of all once vaccinated thorzofretc with the death of 4 animals in the control group.

The immunogenic activity of the vaccine against parvovirus enteritis is determined on minks 2-6 months. age obtained from farms that are safe for infectious and invasive diseases, clinically healthy, not vaccinated against the plague, and not during the rutting season. The vaccine is administered to 4 mink intramuscularly at 1 cm ³ containing 1000 ID 50 of the PC virus. 21 days after vaccination, vaccinated and 4 control animals are infected with virulent strain "Rodniki" at a dose of 1000 ID 50 intramuscularly. The observation period is 14 days. A vaccine is considered immunogenic if it protected against infection and death of all once vaccinated minks during the death of 4 animals in the control group. It is allowed the survival of two control minks when their PE is recovered with characteristic signs of the disease.

The immunogenic activity of the vaccine against infectious hepatitis is determined on puppies of dogs 2-4 months of age, out of the experiment to determine reactogenicity. 21 days after vaccination, the vaccinated and 4 control animals are infected with the virulent Karabash strain at a dose of 1000 ID 50 intraperitoneally (5 cm ³ ) and into the conjunctiva of the eye (0.2 cm ³ ). The observation period is 25 days. A vaccine is considered immunogenic if it protects all once vaccinated puppies from the disease. And in control animals, a rise in temperature to 40.3-41 ^o C, an inhibited state, a refusal of food, keratitis of a part of the cornea are noted. Puppies may die for 18-25 days.

 The results of commission tests of 3 vaccine series are summarized in tab. 3.

It was established that in a dry vaccine the infectious activity of the VNIIVViM-88 vaccine strain was 3.0-3.75 lg TCD 50 / cm ³ , of the Cornell-2 strain 3.75-4.5 lg TCD 50 / cm ³ , strain "A" - 3.0-4.0 lg ID 50 / cm and in the RSA 1: 8-1: 256. The vaccine was harmless and areactogenic for dogs, immunogenic for thorzofret, dogs and minks. After twice vaccinating the puppies (the second vaccination after 14-30 days), neutralizing and delaying hemagglutination antibodies were detected in the animals, which reached the maximum titer after 10-14 days. after secondary immunization: against VChP - 1:64 - 1: 128, against VIGS - 1: 16 - 1:64, against VPK - 1: 64-1: 512. Antibodies against three infections were detected in vaccinated dogs for 12 months. after vaccination, while decreasing by 1-2 orders of magnitude.

All immunobiological characteristics of the 3 series of the vaccine meet the requirements of TU-9384-051-00008064-96 and remain for 12 months. storage at a temperature of 4-6 ^o C within the specified standards.

 The use of a new vaccine against plague, infectious hepatitis, and carnivorous parovirus enteritis in veterinary practice will reduce the incidence of dogs with these diseases and increase the effectiveness of anti-epizootic measures.

Claims (1)

Vaccine against plague, infectious hepatitis and parvovirus enteritis carnivores, including a lyophilized mixture of three culture vaccine strains and a drying medium, characterized in that it contains, at effective concentrations, vol.%:
Suspension of a new vaccine strain "VNIIVVmM-88" of carnivore plague grown in the subline SU-1 of transplantable green monkey kidney cells with an infectious activity titer of 3.5 - 4.5 lg TPD 50 / cm ³ - 32 - 33
Suspension of the vaccine strain "Cornell-2" of infectious hepatitis in dogs grown in transplantable cell culture of testicles of the piglet of gnotobiota with a titer of infectious activity 5.5 - 6.5 lg TPD 50 / cm ³ - 9/11
Suspension of vaccine strain A of panleukopenia of cats grown in a transplanted culture of kidney cells of a kitten of a subline Fk with a titer of infectious activity of 3.5 - 4.5 lg ID 50 / cm ³ and hemagglutinating activity of 1: 32 - 1: 512 - 32 - 33
20% lactose solution - 4.8 - 5.2
60% sucrose solution - 4.8 - 5.2
40% sorbitol solution - 4.8 - 5.2
20% Gelatin Solution - Rest

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