BG64693B1 - Vaccine against the reproductive and respiratory syndrome in pigs - Google Patents

Abstract

The invention relates to a vaccine and methods for the treatment of the reproductive and respiratory syndrome in pigs. The vaccine is produced from the NEB-1-P94 viral agent deposited in the American collection of types of cultures under No. VR-2525. The invention also relates to vaccinal virus of phenotype characteristics by which it can be distinguished from the PRRS wild type virus.

Description

(54) Vaccine against swine reproductive and respiratory syndrome

BACKGROUND OF THE INVENTION

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

In 1987, the pig industry in the United States was confronted with an unknown infectious disease that had serious economic consequences for pig production. This syndrome has been reported in Europe, including in Germany, Belgium, the Netherlands, Spain and England.

The disease is characterized by inability to reproduce, respiratory disease and various clinical signs, including loss of appetite, fever, dyspnoea, and mild neurological signs. A major component of the syndrome is the inability to reproduce, which manifests as premature births, late term abortions, weak pigs at birth, stillbirths, mummified fetuses, decreased frequencies of dusting, and delayed subsequent dispersal. The clinical signs of respiratory disease are most pronounced in pigs at 3 weeks of age, but it is also reported in pigs at all stages of pig production. The affected piglets grow slowly, have bristle, respiratory distress (“thumping”) and increased mortality.

The disease syndrome is referred to in various terms, including mysterious swine disease (MSD), respiratory syndrome for epidemic swine abortions (PEARS), swine infertility and respiratory syndrome (SIRS). The common name now is porcine reproductive and respiratory syndrome (PRRS); this term will be used throughout this patent application.

It is an object of the invention to provide a vaccine that protects pigs from the clinical disease caused by PRRS. Another task is to provide a vaccine whose use in breeding pig herds leads to a decrease in their PRRS population.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a new vaccine that protects pigs from a clinical disease caused by porcine reproductive and respiratory syndrome (PRRS) virus.

Another object of the present invention is to provide a vaccine that protects pigs from strain NEB-1 of the PRRS virus.

Another object of the present invention is to provide a method of protecting pigs from a clinical disease that is caused by the swine reproductive and respiratory disease virus.

Another object of this invention is to provide a vaccine virus with phenotypic features that may distinguish it from wild-type PRRS virus.

The invention relates to a vaccine for administration for the protection of pigs from reproductive and respiratory syndrome in pigs, comprising a PRRS virus corresponding exactly to the isolate NEB-1-P94, deposited in the American Type Culture Collection under code number VR-2525; wherein NEB-1-P94 is characterized by being distinct from the wild-type forms of the virus based on a lack of response to monoclonal antibody SDOW17 and impaired growth on porcine alveolar macrophage cells.

The invention further relates to a preparation comprising an attenuated swine reproductive and respiratory syndrome (PRRS) virus, which has been modified by laboratory manipulation for use as a vaccine. The preparation also has phenotypic properties that allow its use for diagnostic purposes where a distinction is to be made between pigs that have been naturally infected with the PRRS virus and animals that are infected with the vaccine strain alone. The PRRS virus isolate NEB-1-P94 has been deposited with the American Type Culture Collection (code number VR-2525).

The virulent isolate of the PRRS virus was obtained from dead swine tissue specimens submitted to the University of Nebraska diagnostic laboratory. Dead pig tissue tissue homogenate was inoculated into primary porcine alveolar macrophages, the presence of the virus being determined by its cytopathic effects on inoculated but not control cultures. Subsequently, the isolated virus (designated NEB-1) was characterized as a PRRS virus based on physical features (sensitivity to ether and chloroform, density and absence of haemagglutinating activity), specific antibody response and genetic analysis. The inoculation of the virus in non-weaned piglets led to a respiratory disease characterized by high fever, altered breathing and pulmonary pathology such as viral interstitial pneumonia. Moreover, inoculation of the virus in pregnant pigs has led to a reproductive disease characterized by mummification of fetuses, stillbirths and weak pigs at birth that later die.

The respiratory and reproductive disease caused by this virus was typical of the syndrome described for the PRRS virus.

The NEB-1 virus was attenuated (serially) by serial tissue culture replication. The virus was first replicated by inoculating primary porcine alveolar macrophage (SAM) cultures (for the first two strains), and then by serial transplantation on MA104 cells (obtained from Microbiological Associates, Inc., Rockville, MD) for a total of 94 strains. During this process, virus clones were isolated by plaque purification and characterized for phenotypic features. The vaccine clone, designated NEB-1-P94, was selected for impaired growth on porcine alveolar macrophages, lack of response to the monoclonal antibody SDOW17 (ATCC HB10997), specific for PRRS, and non-pathogenicity in piglets and pregnant pigs. NEB-1-P94 was propagated in MA 104 cells and frozen as a virus sample, also designated PRRS -MSV94-1, for use in vaccine development studies.

The vaccine is obtained by using MA104 cells as a substrate (although other cell lines that grow the PRRS virus, such as MARC, 145 [which may be obtained from Dr. Wang, Agriculture Research Station, may be used. Clay Center NO]). MA 104 c cells are cultured to form a dense monolayer (confluence) in tissue culture vessels, e.g. 850 cm ² oval mattresses using Eagle's minimum essental media (EMEM) medium containing 5 to 10% bovine serum, 30 mM HEEP8 (No. 2-hydroxyethylpiperazine-No.-2-ethanesulfonic acid, 2 mm MM L-glutamine and antibiotics (such as 30 pkg / ml gentamicin) Other tissue culture media may be used in which MA104 cells can grow, such as Dulbecco's modified essential media [DMEM], Medium 199, or other. Confluent monolayers of MA cells 104 are inoculated with the NEB-1-P94 virus at multiplicity of infection (MO1) in the range of 1: 5 to 1: 1000, and preferably in the range 1:10. After incubation for three to five days at 37 ° C, the culture fluids above the cells were collected by pipette.

Virus fluids were titrated by serial dilutions in EMEM supplemented as above. In 96-well tissue culture plates, 0.2 ml of dilutions per well were inoculated with at least four replicates of wells with confluent cells MA104 or MARC 145. The cultures were incubated for five days at 37 ° C, 3-5%. CO ₂ in the chamber a humidified chamber and observed for cytopathic effects. Tigers (50% endpoints) were calculated according to the methods of Spearman and Karber (Methods in Virology, Volume IV, K. Maramorosch and H. Koprowski (Eds). Academic Press, New York, 1977). To confirm the phenotypic identity of the virus, cells can be fixed with 80% acetone and tested for absence of reaction with the monoclonal antibody SDOW / 17 and for positive reaction with the monoclonal antibodies V017 or EP147 (which can be obtained from Dr E. Nelson Dakota State University, Brookings, SD,) (expected positive result).

To obtain the "killed" vaccine, the viral fluids are incubated with a chemically inactivating substance. Examples of inactivating agents include formaldehyde, glutaraldehyde, secondary ethyleneimine or β-propiolac3 ton. The viral fluids are then stored at 4 ° C until included in the vaccine. The vaccine is prepared by mixing viral fluids (containing 10 ⁶ to 10 'TCID ^ of the virus; tiger-based pre-inactivation) with a physiologically acceptable diluent (such as EMEM, Hank's balanced saline, phosphate-buffered saline) and an immunostimulating adjuvant (such as mineral oil, vegetable oil, aluminum hydroxide, saponin, non-ionic detergents, squalene, block copolymers, or other known compounds, used singly or in combination). The dose of the vaccine is usually between 1 and 5 ml.

For the preparation of a live vaccine, virus fluids are stored frozen at -50 ° C or colder until used. Viral fluids in the range of 10 ^{4 0} and 10 ^{7 0} TCID / dose and preferably vessels containing 10 ^{6 0} TCID dose are diluted with a physiologically suitable diluent (such as EMEM, Hank balanced saline, phosphate buffered saline) and physiologically a suitable mixture of compounds intended to stabilize the virus. Compounds known in the art that can be used alone or in combination to stabilize viruses include sucrose, lactose, NZ-amine, glutathione, neopeptone, gelatin, dextran and tryptone. The vaccine is stored frozen (-50 ° C or colder) or lyophilized at 4 ° C until used. The vaccine usually has a dose range of 1 to 5 ml, preferably 2 ml.

For the prevention of PRRS-induced disease, the vaccine is administered to the pig orally, intranasally or parenterally. Examples of parenteral administration include intradermal, intramuscular, intravenous, intraperitoneal and subcutaneous routes of administration.

When administered as a solution, the present vaccine may be formulated as an aqueous solution, syrup, elixir or tincture. Such dosage forms are known in the art and are prepared by dissolving the antigen and other suitable additives in the respective solvent systems. Such solvents include water, saline, ethanol, ethylene glycol, glycrol, A1-liquid, etc. Suitable additives known in the art include approved dyes, colorants, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethylmercury) . Such solutions may be stabilized, for example, by the addition of partially hydrolyzed gelatin, sorbitol or cell culture medium, and may be buffered by methods known in the art using known reagents such as sodium hydrogen phosphate, sodium dihydrogen phosphate , potassium hydrogen phosphate and / or potassium dihydrogen phosphate.

Suspensions and emulsions may also be assigned to liquid dosage forms. The preparation of suspensions, for example by the use of a colloidal mill, as well as emulsions by the use of a homogenizer, are known in the art.

Dosage forms for parenteral administration for injection into body fluid systems require accurate isotonicity and pH buffering to the appropriate levels of porcine body fluids. Parenteral dosage forms must also be sterilized before use.

Isotonicity can be achieved with sodium chloride or, if necessary, with other salts. Other solvents, such as ethanol or propylene glycol, may also be used to increase the solubility of the constituents of the preparation and the stability of the solution. Other additives that may be used in the present dosage forms include dextrose, traditional antioxidants and traditional chelating agents such as ethylenediaminetetraacetic acid (EDTA).

Two to four weeks after initial immunization, booster vaccination can be done. To prevent reproductive disease, the vaccination regimen is usually performed up to 6 weeks before and 1 week after fertilization. To protect piglets from respiratory disease, the vaccine can be given at the early age of 3 weeks. The response to the vaccine can be monitored by measuring the tiger of antibodies against the PRRS virus, using an enzyme-linked immunosorbent assay (ELISA), serum neutralization test, indirect immunofluorescence, or protein blot (Western blot).

The vaccine strain has phenotypic features that can be used to diagnose swine wild-type PRRS virus infection that may be different from infection with the vaccine strain alone. Animals infected with natural strains of PRRS virus can be distinguished from animals infected only with the vaccine strain NEB-1-P94 by measuring the response by producing antibodies against an epitope recognized by the monoclonal antibody (MAb) SDOW17 . The presence of antibodies that respond to the SDOW17 epitope is indicative of wild-type virus infection.

Measurement of antibodies against the epitope of SDOW17 can be performed by competitive ELISA. Plates (96-well) are coated with the PRRS NEB-1 virus (or other PRRS viruses that express the SDOW / 17 epitope). The plates are then incubated with porcine serum from the test animals and labeled with enzyme (eg horseradish peroxidase conjugated) monoclonal antibody SDOW17. The ability of porcine sera to recognize the epitope of SDOW 17 was measured by inhibiting the binding of the enzyme-labeled MAb SDOW 17 to the plaque, which was established by the lack of discoloration of the enzyme substrate. Alternatively, a direct ELISA can be used. The amino acid sequence comprising the epitope of SDOW 17 can be obtained as a synthetic peptide or by methods of expressing recombinant DNA in a suitable vector system, such as E. coli. Plates coated with the SDOW 17 antigen were incubated with porcine serum. The binding of porcine antibodies to the antigen of SDOW 17 was established by incubation with sera containing enzyme-conjugated antibodies against porcine immunoglobulins, incubation with the enzyme substrate, and reading of color change.

The following examples describe the invention in detail. It will be appreciated by those skilled in the art that materials and methods can be modified without departing from the purposes and intentions of this specification.

Examples of carrying out the invention

Example 1

Phenotype characteristic of NEB-1P-94 for growth on alveolar macrophages

After five strands of the original sample, the vaccine strain of the NEB-1-P94 virus was characterized for growth on MA 104, MARC 145 and on porcine alveolar macrophages. Porcine alveolar macrophages (SAM) are obtained by broncho-alveolar lavage in saline followed by centrifugation to precipitate cells. Macrophages were resuspended in EMEM with 10% fetal bovine serum and 50 pkg / ml gentamicin and seeded approximately 7 x 10 ⁴ cells per well in 96-well tissue culture plates. MA 104 and MARC 145 cells were seeded in 96-well tissue culture plates in medium (EMEM containing 10% fetal bovine serum, 30 mM HEPES, 2 mM L-glutamine and 50 pkg / ml gentamicin). The NEB-1-P96 or parental NEB-1 virus was serially diluted in medium and 0.2 ml of each dilution was inoculated into two wells of 96-well plates containing SAM, MA 104 or MAEC cells 145. The cultures were incubated 5 days at 37 ° C, 3% to 5% CO ₂ in the chamber a humidified and monitored for cytopathic effects typical of the virus PRRS. Tigers (50% endpoints) were calculated according to the Spearman & Karber method. NEB-1-P94 showed reduced or immeasurable tigers on three separate cultures or SAM compared to tigers obtained on MA104 and MARC 145 cells (Table 1). This is a phenotypic change compared to the original strain, showing similar tigers on all cultures tested. Thus, impaired growth on porcine alveolar macrophages is a selective phenotypic marker for the NEB-1-P94 vaccine strain.

Table 1. Comparison of NEB-1-P94 virus growth on different cell types

The virus Titer on MA 104 Caption 'on MARC 145 Caption 'on SAM 7 Caption on SAM 8 Caption 'on SAM 22 NEB-1-P94 5,3 6,1 1.2 2.5 <1.2 NEN-1 5.2 6.5 5.2 6.5 6.5

Titer = 1 of ₁₀ TLC50 / ml (sensitivity of this test is 1.2)

Example 2.

Phenotype characteristic of NEB-1-P94 for lack of virulence in pigs

Four gnotobiotic piglets (seven to 10 days of age) from seronegative PRRS pigs were inoculated intranasally (3 ml / nostril) with the original virus model NEB-1-P94 (10 ⁵ .TClD5 (l / ml). The piglets are monitored for clinical signs of respiratory disease and the vaccine strain of the virus is isolated again from the serum five days after inoculation Serum from the first group of pigs is used to intranasally inoculate a second group of gnotobiotic pigs, which are monitored in the same way. of re-inoculation in the piglets repeated a total of five times to determine whether the vaccine strain could return to a virulent condition.The vaccine virus was isolated after each subsequent transplantation in animals, but no respiratory disease was observed in the gnotobiotic pigs. one and three weeks, the virus isolated after the fifth piglet was inoculated intranasally (approximately 10 ⁵ · ³ TCID w / ml per pig). Animals were monitored 42 days after inoculation of the virus, with no clinical signs of disease (ie, prolonged fever, respiratory signs, pulmonary lesions) typical of infection with the virulent PRRS virus. Therefore, it is concluded that the NEB-1-P94 virus is not virulent and does not induce respiratory disease in piglets.

The NEB-1-94 virus is then tested for its ability to cause reproductive disease. Seronegative PRRS pigs were inoculated intranasally (3 ml / nostril) on the 85th day of pregnancy with the NEB-1-P94 virus sample sample (10 ^{4 5} TCID / ml). All pigs are grazed at the expected time and 96% of the piglets are born alive and well. Thus, the vaccine strain NEB-1-P94 did not induce a reproductive disease typical of the virulent PRRS virus (see Example 4). These data confirm the non-virulent phenotype of the vaccine strain, NEB-1-P94.

Example 3.

Phenotype characteristic of NEB-1-P94 for reaction with monoclonal antibodies specific for PRRS virus

MA 104 cells infected with the parent strain NEB-1 or with the vaccine strain strain NEB-1-P94 were tested for reaction with monoclonal antibodies specific for the PRRS virus by indirect immunofluorescence. Briefly, 96-well MA104 cell monolayer plates were fixed with 80% acetone for 10 min 2 days after infection with each virus. The monolayers were then incubated with the monoclonal antibodies SDOW17, V017 or EP147. After washing, the reaction of the monoclonal antibodies to each of the viruses was detected by incubation with fluorescinisothiocyanate conjugated with anti-mouse IgG followed by washing and fluorescence examination with a microscope. For the parent strain NEB-1, positive fluorescence was observed with all three monoclonal antibodies (Table 2). However, the vaccine strain, NEB-1 P94, has lost its ability to react with the monoclonal antibody SDOW17, but the test with the other two monoclonal antibodies is positive. These data indicate that strain NEB-1-P94 does not express the epitope recognized by the SDOW17 antibody. The loss of reactivity with this monoclonal antibody is most likely a genetic mutation in the RNA sequence of NEB-1-P94, which results in a changed amino acid sequence in the region of the nucleocapsid protein recognized by SDOW17.

Table 2. Reactivity of the parental and vaccine strain of PRRS with specific monoclonal antibodies.

reaction with SDOW17 reaction cV017 reaction with EP147 NEB-1-P94 - + + NEB-1 + + +

Example 4

Prevention of reproductive disease by vaccination of pigs with NEB-1-P94

The vaccine is prepared by inoculating oval mattresses with solid monolayers of MA104 cells with NEB-1-P94 (4 primers) at an infection multiplicity of approximately 1:10 in EMEM containing 10% fetal bovine serum, 2 mM L-glutamine and 30 pkg / ml gentamicin. The cultures were incubated for three days at 37 ° C and then the supernatant liquids were separated. Viral fluids were diluted 50% (v / v) with 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). 1 KN ₂ PO ₄ , 2.5 g / g K ₂ HPO ₄ , 10 g / l albumin fraction V), frozen and lyophilized. The vaccine was rehydrated with sterile deionized water and the pigs were injected intramuscularly with 2 ml (10 ^s · ¹ TCID _M / ml) four to six weeks before fertilization.

On day 85 of pregnancy, vaccinated and unvaccinated control pigs were infected by intranasal administration of the NEB-1 virus (approximately 10 ^{6 3} TCID ^). For seven weeks after the pollination, the animals are examined for signs of fetal

2f or neonatal death caused by the PRRS virus. PRRS viremia developed in 11/12 (92%) of control pigs and in 100% of their live births. Infection with PRRS during pregnancy resulted in 16% mortality at birth (large mummies and stillbirths) in the control group (Table 3) compared to only 6% mortality in vaccinated pigs. In addition, vaccination results in a 50% reduction in cases of lean and cadaveric piglets and a 94% reduction in low birthweight piglets (weighing less than 2 pounds at birth) when compared with controls. Vaccination prevented congenital PRRS, as evidenced by the absence of PRRS virus in the blood or tissues of any pig pigs of immunized pigs and 55% prevention of mortality within 7 weeks (Table 3) when compared with controls. The statistically significant prevention of mortality and viral infection in vaccinated pigs and their offspring clearly indicates the efficacy of this vaccine in preventing the reproductive form of the disease induced by the PRRS virus.

Table 3. Summary of reproductive disease observed after infection with vaccinated against control pigs with PRRS virus.

group number of sains (average pig / pigs % large mummies and stillbirths % born alive, numb % weak and cacky Crapaoan weighing <2 liters mortality within our 7 weeks vaccinated 21 (10.8) 6 « 2% 3% 1% 17% controls 12 (11.1) 16% 3% 6% 17% 38%

Example 5.

Prevention of respiratory disease by vaccination of pigs with NEB-1-P94

The vaccine is prepared by inoculating oval mattresses with solid monolayers of MA 104 cells with NEB-1-P94 (4 primers) at an infection multiplicity of approximately 1:10 in EMEM containing 10% fetal bovine serum, 2 mM L- glutamine and 30 µg / ml of gentamicin. The cultures were incubated for three days at 37 ° C and then the supernatant liquids were separated. Viral fluids were diluted 50% (v / v) with 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). l KHjP0 ₄ 2,5 g / l K ₂ HPO _4, 10 g / l albumin fraction V), frozen, and lyophilized. The vaccine is rehydrated with sterile deionized water and seronegative piglets at three weeks of age are injected intramuscularly with 1 ml (10 ⁴⁹ TCID _5O / ml).

Four weeks after vaccination, the piglets were infected intranasally with the virulent virus of PRRS NEB-1 as described for sows (Example 4). Pigs are screened for signs of respiratory disease within 14 days of infection. All non-vaccinated control pigs developed clinical signs of respiratory disease compared with 3/40 (8%) of the vaccinated animals. Vaccination resulted in a statistically significant reduction in the signs of respiratory inflammation and clinical disease in the vaccinated animals compared to controls (Table 4). This study clearly demonstrates the efficacy of the vaccine to prevent respiratory disease caused by the PRRS virus in young pigs.

Table 4. Summary of clinical disease in vaccinated and control animals after infection with PRRS virus.

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HE = missing data controls n = 30 vaccinated η = 40

Claims (4)

Claims A vaccine for administration to protect pigs from reproductive and respiratory syndrome in pigs, comprising a PRRS virus corresponding exactly to the isolate NEB-1-P94 deposited with the American Type Culture Collection under code number VR-2525; wherein NEB1-P94 is characterized by being distinct from the wild-type forms of the virus based on a lack of response to the monoclonal antibody SDOW17 and impaired growth on porcine alveolar macrophage cells. A vaccine according to claim 1, characterized in that the PRRS virus is in the killed or modified (attenuated) form of life. A vaccine according to claim 2, wherein the PRRS is in a liquid, frozen- 5 in or lyophilized form. The vaccine of claim 1, wherein the amount of PRRS virus is in the range of 10 ⁴ · ⁰ to 10 ⁹⁰ TCID ₅₀ per 1 ml. Use of the vaccine according to claims 1-4 for the preparation of a pharmaceutical product intended to prevent pigs from a clinical disease caused by the swine reproductive and respiratory syndrome 15 virus.