RU2187333C2 - Vaccine for protecting swine against reproductive and respiratory syndromes and method for protection - Google Patents

Abstract

FIELD: veterinary science. SUBSTANCE: the suggested vaccine is obtained out of viral agent NEB-1-P94 kept in the American collection of type cultures under registration number of VR-2525. The method for protecting deals with introducing the present vaccine for swine at efficient quantity. The innovation enables to considerably decrease the death rate in piglets and infection of vaccinated sows. EFFECT: higher efficiency. 7 cl, 5 ex, 4 tbl

Description

 The present invention relates to a vaccine for the treatment of reproductive and respiratory syndrome in pigs (PRRS).

 In 1987, the pig industry in the United States faced an unknown infectious disease that caused him a serious economic blow. There have been reports of the disease syndrome in Europe, including Germany, Belgium, the Netherlands, Spain and England.

 The disease was characterized by impaired reproductive function, respiratory diseases, and various clinical symptoms, including loss of appetite, fever, shortness of breath, and mild neurological symptoms. The main component of the syndrome is reproductive damage, which manifests itself as premature birth, miscarriage in the later stages, weakness of newborn piglets, the birth of dead piglets, mummification of the fetus in the womb, slowing down the farrowing process and delaying the return of estrus. Clinical symptoms of respiratory disease are most evident in dairy pigs under 3 weeks of age, but there are reports of their occurrence at all stages of raising pigs. The affected piglets grow slowly, their hair coarsens, they have difficulty breathing ("dull bumps") and mortality increases.

 The disease syndrome has been described using a variety of different terms, including such as “mysterious swine disease” (MSD), “swine miscarriage and respiratory distress syndrome” (PEARS), “pig infertility and respiratory distress syndrome” (SIRS). The currently commonly used name is pig reproductive and respiratory syndrome (PRRS); this term will be used throughout the text of this patent application.

 The object of the invention is to provide a vaccine that protects pigs from clinical diseases caused by PRRS. Another object is the creation of a vaccine, which, given to pigs when they are raised in a herd, reduces the presence of PRRS in the population.

SUMMARY OF THE INVENTION
The object of the present invention is to provide a new vaccine that protects pigs from a clinical disease caused by the Reproductive and Respiratory Syndrome Virus (PRRS).

 A further object of the present invention is to provide a vaccine protecting pigs against the PRRS virus NEB-1 strain.

 Another object of the invention is to provide a method for protecting pigs from a clinical disease caused by the virus of reproductive and respiratory disorders in pigs.

 The next object of the present invention is to obtain a virus in a vaccine, the phenotypic characteristics of which can distinguish it from wild-type PRRS virus.

DETAILED DESCRIPTION OF THE INVENTION
The invention relates to the creation of a composition containing a weakened virus of reproductive and respiratory syndrome in pigs (PRRS), which is modified by laboratory treatment before use in vaccination.

 In addition, the composition has phenotypic properties that allow it to be used for diagnostic purposes in order to distinguish a pig that is naturally infected with PRRS virus from animals that detect only the vaccine strain. The PRRS virus isolate NEB-1-P94 is stored in the American Type Culture Collection, registration VB-2525 ("American Type Culture Collection, Accession VP-2525).

 Virulent PRRS virus isolate was obtained from dead pig tissue samples from the University of Nebraska Diagnostic Laboratory. Homogenate tissue from a dead pig was inoculated onto primary porcine alveolar macrophages, and the presence of the virus was determined by the cytopathic effects on inoculated cultures and their absence on control cultures. An isolated virus (designated NEB-1) was sequentially characterized as a PRRS virus based on its physical properties (sensitivity to chloroform and ether, floating density and lack of hemagglutinating activity), reactivity to specific antibodies, and genetic analysis data. Inoculation of suckling piglets virus resulted in a respiratory illness accompanied by intense fever, altered respiration and lung pathology, consistent with viral interstitial pneumonia. In addition, inoculation of pregnant sows with the virus resulted in reproductive damage, characterized by mummification of the fetus in the womb, the birth of dead piglets and weak piglets, which subsequently died. The respiratory and reproductive damage caused by this virus was typical of the syndrome transmitted by the PRRS virus.

 NEB-1 virus was attenuated by serial reseeding in tissue culture. The virus was first seeded by inoculation of primary cultures of porcine alveolar macrophage (SAM) (for the first two transfers), and then by serial transfer on MA104 cells (provided by Microbiological associates, Inc., Rockville, MD) to a total number of transfers of 94. During In this process, virus clones were isolated by purification to individual plaques and examined for phenotypic properties. The vaccine clone designated NEB-1-P94 was selected taking into account the weakened development on pig alveolar macrophages, the lack of reactivity to the PRRS-specific monoclonal antibody SDOW17 (ATCC HB 10997) and the absence of the disease in piglets and pregnant sows. The amount of NEB-1-P94 virus was increased in volume on MA104 cells and frozen as a model virus for plating, also designated PRRS-MSV-94-1, for further use in vaccine development.

The vaccine was prepared using MA104 cells as a substrate (although alternative cell lines that support the development of the PRRS virus can be used, for example, MARC 145 cells (provided by Dr. Wang, Agriculture Research Station, Clay Center NE)). MA104 cells were grown to a monolayer in suitable cell culture vessels, for example, 850 cm ² roller bottles, using the minimum required Needle medium (EMEM) containing 5-10% bovine serum, 30 mM HEPES (N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid, 2 mM L-glutamine and antibiotics, e.g. 30 μm / ml gentamicin). Alternative tissue culture media that can support MA104 cell growth, such as Dulbecco's Modified Essential Medium (DMEM), Medium 199, or others, can also be used. Monolayers of MA104 cells were inoculated with NEB-1-P94 virus at a multiplicity of infection (MOI) in a ratio of 1: 5 to 1: 1000 and preferably in a ratio of 1: 10. After incubation for 3-5 days at 37 ^° C, culture supernatant fluids were collected by decantation.

Viral fluids were titrated by serial dilutions in EMEM with the aforementioned additives and inoculation of at least 4 replicate wells with a monolayer of MA104 or MARS 145 cells in a 96-well tissue culture plate in an amount of 0.2 ml per well. The cultures were incubated for 5 days at 37 ^° C and a CO ₂ content _of 3-5% in a humidified chamber and studied to detect cytopathic effects. Titers (50% of endpoints) were counted according to the methods of Spearman and Karber (Methods in Virology, Volume IV, K. Maramorosch and H. Koprowski (Eds). Academic Press, New York, 1977). Cells can be fixed with 80% acetone and tested for lack of reactivity with monoclonal antibodies VO17 or EP147 (provided by Dr. E. Nelson, South Dakota State University, Brookings, SD) (expected positive result) in order to confirm phenotypic identity the virus.

To prepare a killed vaccine, viral fluids were incubated with an inactivating chemical agent. Examples of inactivating agents are formaldehyde, glutaraldehyde, binary ethyleneimine or beta-propiolactone. Then the viral fluids were stored at 4 ^o C until then, until they started to receive the vaccine. The vaccine was prepared by mixing viral fluids (containing 10 ⁶ to 10 ⁹ TCD _{50 of the} virus; based on pre-inactivation titers) with a physiologically acceptable diluent (such as EMEM, Hank's balanced saline, phosphate buffered saline) and immunostimulating adjuvant (e.g. mineral oil, vegetable oil, aluminum hydroxide, saponin, non-ionic detergents, squalene, block copolymers or other compounds known to the skilled person and used individually or in combination). The dose of the vaccine was usually in the range between 1 and 5 ml.

To obtain a live vaccine, viral fluids were stored frozen at a temperature of -50 ^o C or lower until use. Viral fluids with a TC1D ₅₀ content in the range of 10 ^4.0 - 10 ^7.0 and preferably with a content of 10 ^{6.0 were} diluted with a physiologically acceptable diluent (such as EMEM, Hank's balanced salt solution, phosphate-buffered saline) and physiologically acceptable a mixture of compounds designed to stabilize the virus. Compounds that are known to one skilled in the art can be used to stabilize viruses individually or in combination, include sucrose, lactose, NZ amine, glutathione, neopeptone, gelatin, dextran and tryptone. The vaccine was stored frozen until use (at a temperature of -50 ^° C or lower) or lyophilized (at a temperature of 4 ^° C). Typically, the vaccine dose was in the range of 1-5 ml, preferably 2 ml.

 To prevent disease caused by PRRS, the pig vaccine was administered orally, intranasally or parenterally. Examples of parenteral administration are intradermal, intramuscular, intravenous, intraperitoneal and subcutaneous administration.

 When administered as a solution, the proposed vaccine can be prepared in the form of an aqueous solution, syrup, elixir or tincture. Such formulations are known to the skilled person and are prepared by dissolving the antigen and other appropriate additives in suitable solvent systems. These solvents include water, saline, ethanol, ethylene glycol, glycerin, Al-liquid, etc. Suitable additives known to the person skilled in the art include certified colorants, flavors, sweeteners, and antimicrobial preservatives such as thimerosal (sodium ethyl mercurithiosalicylate). Such solutions can be stabilized, for example, by the addition of partially hydrolyzed gelatin, sorbitol or a cell culture medium, and can be buffered by methods known to those skilled in the art using known reagents such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate and / or potassium dihydrogen phosphate.

 Liquid preparations may also be suspensions and emulsions. The preparation of suspensions, for example in a colloid mill, and emulsions, for example in a homogenizer, is known in the art.

 Parenteral dosage forms intended for injection into body fluids require compliance with certain isotonicity and pH values corresponding to these values in the pig's body fluids. Parenteral drugs must also be sterilized before use.

 Isotonicity can be brought to the desired level with sodium chloride and other salts. Other solvents such as ethanol or propylene glycol can be used to increase the solubility of the ingredients of the composition and the stability of the solution. Additional types of additives used in the form provided may include dextrose, conventional antioxidants, and chelating agents, for example ethylenediaminetetraacetic acid (EDTA).

 Revaccination can be carried out two to four weeks after the initial immunization. To prevent damage to the reproductive function, vaccination is usually carried out from 6 weeks before to 1 week after mating. To prevent diseases of the respiratory tract in piglets, vaccination can be carried out at an early age such as 3 weeks. The body's response to vaccination can be monitored by measuring the titer of the antibody directed against the PRRS virus using an enzyme-linked immunosorbent assay (ELISA), serum neutralization assay, indirect immunofluorescence or Western blotting.

 The vaccine strain has phenotypic properties that allow it to be used to diagnose wild-type PRRS infection in pigs, in contrast to cases when pigs are exposed to the vaccine strain only.

 Animals exposed to different strains of the PRRS virus can be distinguished from animals exposed only to the vaccine strain NEB-1-P94 by evaluating the antibody response to the epitope recognized by the SDOW17 monoclonal antibody (Mab). The presence of antibodies reactive with the SDOW17 epitope is an indicator of exposure to the wild-type virus.

 To complete the assessment of antibody responses to the SDOW17 epitope, a competitive ELISA can be used. Plates (96-well) were coated with NEB-1 PRRS virus (or other PRRS viruses expressing the SDOW17 epitope). The plates were then incubated with pork serum from test animals and an SDOW17 monoclonal antibody labeled with an enzyme (e.g., conjugated to horseradish peroxidase). The ability of pork sera to recognize the SDOW17 epitope was assessed by inhibition of binding to the enzyme-labeled Mab SDOW17 plate, the inhibition being determined by the absence of a color change in the enzyme substrate. Alternatively, direct ELISA analysis can be used. The amino acid sequence containing the SDOW17 epitope can be obtained as a synthetic peptide or by using recombinant DNA expression methods in an appropriate vector system such as E. coli. SDOW17 antigen coated plates were incubated with pork serum. The binding of pig antibodies to the SDOW17 antigen was determined by incubation with antigens immunoglobulin antisera conjugated to the enzyme, followed by incubation with the enzyme substrate and evaluation of color change.

 The invention is described in detail in the following examples. It will be clear to the skilled person that in practice modifications of materials and methods are possible without deviating from the purpose and essence of this description.

Example 1
Establishment of the phenotypic characteristic of NEB-1-P94 for development on alveolar macrophages.

The vaccine strain of the NEB-1-P94 virus after five passages from the harvested seed was analyzed by the development of MA104, MARC 145 and porcine alveolar macrophages. Pig alveolar macrophages (SAM) were obtained by broncho-alveolar washing with saline and subsequent centrifugation to obtain cells in the form of balls. Macrophages were resuspended in EMEM supplemented with 10% bovine fetal serum and 50 μg / ml gentamicin and placed on 96-well tissue culture plates in an amount of about 7 • 10 ⁴ cells per well. MA104 and MARC 145 cells were plated on 96-well tissue culture plates in medium (EMEM containing 10% bovine fetal serum, 30 mM HEPES, 2 mM L-glutamine and 50 μg / ml gentamicin). NEB-1-P94 virus or parental NEB-1 virus were sequentially diluted in medium and replicate wells of 96-well plates were inoculated with 0.2 ml of each solution, the wells containing SAM, MA104 or MARC 145. The cultures were incubated for 5 days at 37 ^o C in a humidified chamber containing 3-5% CO ₂ , and examined for the presence of cytopathic effects typical of the PRRS virus. Titers (50% of the endpoints) were counted using the Spearman and Karber method. NEB-1-P94 showed reduced or non-measurable titers on three separate SAM cultures compared to those obtained on MA104 and MARC 145 cells (Table 1). This is a phenotypic change compared to the parent strain, which shows the same titers on all tested cultures. Therefore, the weakened development on pig alveolar macrophages is a selective phenotypic marker for the vaccine strain NEB-1-P94.

Example 2
Establishing the phenotypic characterization of NEB-1-P94 for the absence of virulence in pigs
Four gnotobiont piglets (aged 7 to 10 days) from sows seronegative for PRRS virus were inoculated with harvested inoculum of NEB-1-P94 virus (10 ^5.3 TCID ₅₀ / ml) intranasally (3 ml / nostril) . Piglets were observed to detect clinical signs of respiratory tract disease, and the vaccine strain of the virus was again isolated from serum five days after inoculation. Serum from the first group of pigs was used for intranasal inoculation of the gnotobiont pigs of the second group, which were controlled in the same way. This process was repeated for all five serial piggyback passages to determine if the vaccine virus could return to a virulent state. The vaccine virus was removed from each successive passage of the animal, however, respiratory disease was not observed in gnotobiont pigs. Additionally, the virus isolated from the fifth pig reverse passage was intranasally inoculated in ordinary piglets at the age of 1 week and three weeks (about 10 ^5.3 TCID ₅₀ / ml was introduced to each piglet). The animals were monitored for 42 days after the inoculation of the virus and no clinical signs of the disease (for example, intense, prolonged fever, respiratory disorders, lung injuries) associated with infection with virulent PRRS were found. Therefore, it was concluded that the NEB-1-P94 virus is not virulent for the initiation of respiratory disease in piglets.

The NEB-1-P94 virus was then tested for its ability to cause reproductive dysfunction. PRRS seronegative sows with a gestational age of 85 days were inoculated intranasally (3 ml / nostril) with a vaccine strain from harvested seed (10 ^4.5 TCID ₅₀ / ml). All sows were farrowed at the appropriate time, and 96% of the piglets were born alive and healthy. For comparison, control, non-inoculated sows gave birth, where 87% of piglets were alive and well. Thus, the vaccine strain did not cause reproductive damage typical of the virulent PRRS virus (see example 4). These data confirmed the avirulent phenotype of the vaccine strain NEB-1-P94.

Example 3
Establishing the phenotypic characterization of NEB-1-P94 for reactivity with monoclonal antibodies specific for PRRS virus
MA104 cells infected with the parental NEB-1 strain or the vaccine strain of the NEB-1-P94 virus were tested for reactivity to monoclonal antibodies specific for the PRRS virus using indirect immunofluorescence. In short, 96-well plates with a monolayer of MA104 cells were fixed with 80% acetone for 10 minutes two days after infection with each virus. Then the monolayers were incubated with monoclonal antibodies SDOW17, VO17 or EP147. After washing, the reactivity of the monoclonal antibody to each virus was determined by incubation with anti-mouse IgG conjugated with fluorescein isothiocyanate, followed by washing and fluorescence assessment using microscopy. For the parent strain NEB-1, positive fluorescence was observed with all three monoclonal antibodies (see table 2). However, the vaccine strain NEB-1-P94 has lost reactivity with the SDOW17 monoclonal antibody, but has shown a positive result with other monoclonal antibodies. These data indicate that strain NEB-1-P94 does not detect expression of an epitope recognized by the SDOW17 monoclonal antibody. The lack of reactivity with this monoclonal antibody most likely means a genetic mutation in the RNA sequence of NEB-1-P94, which led to a change in the amino acid sequence in the portion of the nucleocapsid protein recognized by SDOW17.

Example 4
Prevention of reproductive damage by vaccination of sows with strain NEB-1-P94
The vaccine was prepared by inoculating a monolayer of MA104 cells in roller bottles of strain NEB-1-P94 (4 passages from harvested seed) with a multiplicity of lesion of approximately 1:10 in EMEM containing 10% bovine fetal serum, 2 mM L-glutamine and 30 μg / ml gentamicin. The cultures were incubated for three days at 37 ^{° C.} and then supernatant fluids were collected by decantation. Viral fluids were diluted in an amount of 50% by volume with a stabilizer (75 g / l tryptone, 30 g / l dextran, 2 g / l gelatin, 100 g / l lactose, 2 g / l sodium glutamate, 1.05 g / l KH ₂ PO ₄ , 2.5 g / l K ₂ HPO ₄ , 10 g / l albumin fraction V), were frozen and lyophilized. The vaccine was again moistened with sterile deionized water and 2 ml of it (10 ^5.1 TCID ₅₀ / ml) was administered intramuscularly to gilts 4-6 weeks before mating.

On the 85th day of pregnancy, vaccinated and control non-vaccinated gilts were intranasally infected with NEB-1 virus (about 10 ^6.3 TCID ₅₀ / ml). Animals were observed for seven weeks after farrowing in order to establish the death of the fetus or newborn associated with the PRRS virus. PRRS virus-induced viremia developed in 11 of 12 control sows (92%) and in 100% of their live piglets. Infection with PRRS virus during pregnancy resulted in the loss of 16% of piglets born dead (large mummification of the fetus and premature birth) in the control group (see table 4), while only 6% of piglets died in vaccinated sows. In addition, the vaccination resulted in a 50% decrease in the birth of weak and trembling piglets and a 94% decrease in the number of low birth weight piglets (weighing less than 2 pounds at birth) compared to the control. Vaccination prevented congenital infection with the PRRS virus, as evidenced by the absence of PRRS in the blood and tissues of all piglets from immunized sows and approximately 55% prophylaxis for the death of piglets under 7 weeks of age (see table 3) compared to the control. The statistically significant prevention of death of piglets and viral infection in vaccinated sows and their offspring has clearly demonstrated the effectiveness of this vaccine for the prevention of those forms of reproductive damage caused by the PRRS virus.

Example 5
Prevention of airway disease by vaccinating sows with NEB-1-P94 virus
The vaccine was prepared by inoculating a monolayer of MA104 cells in roller bottles with the NEB-1-P94 virus (at 4 passages from harvested seed) with a multiplicity of infection of approximately 1:10 in EMEM containing 10% bovine fetal serum, 2 mM L-glutamine and 30 mcg / ml gentamicin. The cultures were incubated for five days at 37 ^{° C.} and then supernatant fluids were collected by decantation. Viral fluids were diluted in an amount of 50% by volume with a stabilizer (75 g / l tryptone, 30 g / l dextran, 2 g / l gelatin, 100 g / l lactose, 2 g / l sodium glutamate, 1.05 g / l KH ₂ PO ₄ , 2.5 g / l K ₂ HPO ₄ , 10 g / l albumin fraction), were frozen and lyophilized. The vaccine was again moistened with sterile deionized water and 1 ml (10 ^4,9 TCID ₅₀ / ml) was injected intramuscularly to piglets, seronegative against PRRS, at the age of three weeks.

 Four weeks after vaccination, the piglets were infected with the virulent PRRS virus NEB-1 intranasally, as described for gilts (Example 4). Piglets were observed to detect symptoms of respiratory disease within 14 days after infection. All unvaccinated control piglets developed clinical symptoms of respiratory disease, and a comparison showed that in vaccinated animals this happened in 3/40 (8%) cases. Vaccination caused a statistically significant decrease in fever, respiratory symptoms and clinical diseases in vaccinated animals compared with the control (see table 4). This study clearly showed the effectiveness of the vaccine in preventing PRRS virus infections in young pigs.

Claims (7)

 1. A vaccine to protect pigs from reproductive and respiratory syndrome, containing the PRRS virus isolate NEB-1-P94, stored in the American Type Culture Collection under registration number VR-2525.  2. The vaccine according to claim 1, available in the form of a live, killed or modified live (attenuated) vaccine.  3. The vaccine according to claim 2, in which the viral agent is modified live (attenuated) and is available in liquid, frozen or dried form. 4. The vaccine according to claim 1, containing a virus in an amount of 10 ^4.0 - 10 ^9.0 TCID ₅₀ of PRRS virus per ml.  5. The vaccine according to claim 1, in which the attenuated virus can be distinguished from wild forms of the virus by the lack of reactivity with the SDOW17 monoclonal antibody and the absence of the effective development of porcine alveolar macrophages on the cells.  6. A method of protecting pigs from a clinical disease caused by the virus of the reproductive and respiratory syndrome, comprising administering to the pig an effective amount of the vaccine according to claim 1 in the case when it is necessary to take precautions against the disease caused by the aforementioned virus.  7. The method according to p. 6, according to which the vaccine is administered orally, intranasally, intramuscularly, intradermally, intravenously or subcutaneously.

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