JPH11506122A - Swine reproductive and respiratory syndrome vaccine - Google Patents

Abstract

  (57) [Summary] The present invention discloses vaccines and methods for the treatment of porcine reproductive and respiratory syndrome. This vaccine is derived from the viral factor NEB-1-P94, deposited with the American Type Culture Collection under accession number VR-2525. Further, the present invention discloses vaccine viruses having phenotypic characteristics that can be distinguished from wild-type PRRS virus.

Description

DETAILED DESCRIPTION OF THE INVENTION Swine Reproductive and Respiratory Syndrome Vaccine Background of the Invention The present invention relates to vaccines for the treatment of swine reproductive and respiratory syndrome (PRRS). In 1987, the US pig manufacturing industry experienced an unknown infectious disease that had serious economic impact on the pig industry. This disease syndrome has been reported in Europe, including Germany, Belgium, the Netherlands, Spain, and the United Kingdom. The disease is characterized by reproductive failure, respiratory disorders, and various clinical signs, including loss of appetite, fever, dyspnea, and mild neurological signs. A major component of the syndrome is reproductive failure, manifested as preterm birth, late abortion, weakly born pigs, stillbirth, mummified fetuses, reduced birth rates, and delayed recovery of estrus. Clinical signs of respiratory disease are most pronounced in pigs below 3 weeks of age, but have been reported to occur in pigs at all stages of production. Affected piglets grow slowly, have a rough hair coat, respiratory distress ("marvel"), and increased mortality. This disease syndrome has been referred to by a number of different terms including Mystery Swine Disease (MSD), Swine Epidemic Miscarriage and Respiratory Syndrome (PEARS), Swine Infertility and Respiratory Syndrome (SIRS). The name currently commonly used is Swine Reproductive and Respiratory Syndrome (PRRS); this term is used throughout this patent application. It is an object of the present invention to provide a vaccine that protects pigs against clinical disease caused by PRRS. Another object is to provide a vaccine that, when administered to a herd of pigs in breeding, reduces the presence of PRRS in those populations. SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel vaccine that protects pigs against clinical disease caused by the porcine reproductive and respiratory syndrome (PRRS) virus. It is a further object of the present invention to provide a vaccine that protects pigs against the PRB virus NEB-1 strain. It is a further object of the present invention to provide a method of protecting pigs against clinical disease caused by porcine reproductive and respiratory syndrome virus. It is a further object of the present invention to provide vaccine viruses with phenotypic characteristics that can be distinguished from wild-type PRRS virus. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a composition of matter comprising an attenuated porcine respiratory and reproductive syndrome (PRRS) virus, modified by laboratory operation for use in vaccination. In addition, the composition has a phenotypic trait that allows its use for diagnostic purposes to distinguish pigs that are naturally infected with the PRRS virus against animals that are only exposed to the vaccine strain. Have. The PRRS virus isolate NEB-1-P94 has been deposited with the American Type Culture Collection (accession number VR-2525). A virulent isolate of the PRRS virus was obtained from a tissue sample from a dead pig provided to the University of Nebraska Diagnostics Laboratory. Tissue homogenates from dead pigs were inoculated into primary porcine alveolar macrophages and the presence of virus was detected by cytotoxic effects on the inoculated but not control cultures. The isolated virus (designated NEB-1) was later purified based on physical properties (ether and chloroform sensitivity, buoyant density, and lack of hemagglutinating activity), reactivity with specific antibodies, and PRRS based on genetic analysis. Characterized as a virus. Inoculation of the virus into lactating piglets resulted in respiratory disorders characterized by high fever, altered respiration, and pulmonary pathology consistent with viral interstitial pneumonia. In addition, inoculation of pregnant sows with the virus resulted in reproductive disorders characterized by fetal mummification, stillborn piglets, and weakly born piglets, which subsequently died. The respiratory and reproductive disorders caused by this virus were typical of the syndrome reported for the PRRS virus. The NEB-1 virus was attenuated by successive passages in tissue culture. Virus was obtained from MA104 cells (Microbiological Associates, Inc., Rockville, MD), first by inoculation of primary porcine alveolar macrophage (SAM) culture (first two passages) and then for a total of 94 passages. (Possible). During this process, virus clones were isolated by plaque purification and characterized for phenotypic characteristics. A vaccine clone, designated NEB-1-P94, was used to prevent impaired growth in porcine alveolar macrophages, lack of reactivity with the PRRS-specific monoclonal antibody SDOW17 (ATCC HB10997), and in piglets and pregnant sows. Selected for lack of disease induction. NEB-1-P94 was expanded on MA104 cells and frozen as a master seed virus (also referred to as PRRS-MSV-94-1) for use in vaccine development studies. Vaccines are prepared using MA104 cells as a substrate (but other cell lines that support the growth of PRRS virus, such as MARC 145 [available from Dr. Wang, Agriculture Research Station, Clay Center NE], are also available. Can also be used). MA104 cells were incubated with 5-10% bovine serum, 30 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) in a suitable tissue culture vessel (eg, 850 cm ² roller bottle). )) Grow to confluence using Eagle's Minimum Essential Medium (EMEM) containing 2 mM L-glutamine and an antibiotic (eg, 30 μg / ml gentamicin). Other tissue culture media that can support the growth of MA104 cells, such as Dulbecco's Modified Essential Medium [DMEM], Medium 199, or others, may also be used. Confluent monolayers of MA104 cells are inoculated with NEB-1-P94 virus at a multiplicity of infection (MOI) in the range of 1: 5 to 1: 1000, and preferably in the range of 1:10. After incubation at 37 ° C. for 3-5 days, the culture supernatant is recovered by decanting. Viral fluid was made up in serial dilutions in EMEM supplemented as described above, and 0.2 ml / well into at least four replicate wells of confluent MA104 or MARC 145 cells in a 96-well tissue culture plate. It is titrated by inoculation. Cultures are incubated for 5 days at 37 ° C. in a humidified chamber with 3-5% CO ₂ and observed for cytotoxic effects. Titers (50% endpoint) are calculated according to the method of Spearm an and Karber (Methods in Virology, Volume IV, K. Maramorosch and H. Kopowski (eds.), Academic Press, New York, 1977). Cells can be fixed with 80% acetone and lack of reactivity with SDOW17 and VO17 or EP147 (Dr. E. Nelson, South Dakota State University, Brookings) to confirm phenotypic identity of the virus. (Available from SD) can be tested for positive reactivity with monoclonal antibodies (expected positive results). For preparation of a killed vaccine, the virus fluid is incubated with a chemical inactivator. Examples of inactivating agents include formaldehyde, glutaraldehyde, binary ethyleneimine, or β-propiolactone. The virus fluid is then stored at 4 ° C. until formulated into a vaccine. The vaccine contains a virus solution (containing 10 ⁶ to 10 ⁹ TCID ₅₀ of virus; based on pre-inactivation titers) in a physiologically acceptable diluent (eg, EMEM, Hanks balanced salt solution, phosphate buffered saline). Saline) and immunostimulatory adjuvants (eg, mineral oil, vegetable oil, aluminum hydroxide, saponins, nonionic surfactants, squalene, block copolymers or other compounds known in the art, alone or in combination) ) Is prepared by mixing Vaccine doses are typically between 1 ml and 5 ml. For live vaccine formulations, the virus solution is stored frozen at -50 ° C or lower until use. 10 ^4.0 ~10 ^7.0 TCID ₅₀ / dose in the range, as well as viral solution preferably contains 10 ^6.0 TC ID ₅₀ / dose, physiologically suitable diluent (e.g., EMEM, Hank's balanced salt solution, phosphate-buffered Saline) and a physiologically relevant mixture of compounds designed to stabilize the virus. Compounds known in the art that can be used alone or in combination to stabilize the virus include sucrose, lactose, NZ amine, glutathione, neopeptone, gelatin, dextran, and tryptone. The vaccine is stored frozen (below -50 ° C) or lyophilized and stored at 4 ° C until use. Vaccines typically have a dose size range of 1-5 ml, and preferably 2 ml. The vaccine is administered to pigs orally, intranasally or parenterally for prevention against PRRS-induced disease. Examples of parenteral routes of administration include intradermal, intramuscular, intravenous, intraperitoneal, and subcutaneous routes of administration. When administered as a solution, the vaccines of the invention may be prepared in the form of aqueous solutions, syrups, elixirs, or tinctures. Such formulations are known in the art, and are prepared by dissolving the antigen and other suitable additives in a suitable solvent system. Such solvents include water, saline, ethanol, ethylene glycol, glycerol, Al solution and the like. Suitable additives known in the art include approved dyes, flavors, sweeteners, and antimicrobial preservatives such as thimerosal (sodium ethylmercury thiosalicylate). Such solutions may be stabilized, for example, by the addition of partially hydrolyzed gelatin, sorbitol, or cell culture media, and prepared using reagents known in the art (eg, sodium hydrogen phosphate, dihydrogen dihydrogen phosphate). Sodium, potassium hydrogen phosphate, and / or potassium dihydrogen phosphate) can be buffered by methods known in the art. Liquid formulations may also include suspensions and emulsions. The preparation of suspensions, for example using a colloid mill, and emulsions using, for example, a homogenizer, is known in the art. Parenteral dosage forms designed for infusion into bodily fluid systems require appropriate isotonicity and pH buffering of porcine bodily fluids to the corresponding levels. Parenteral formulations must also be sterilized before use. Isotonicity may be adjusted as necessary with sodium chloride and other salts. Other solvents, such as ethanol or propylene glycol, can be used to increase the solubility of the components of the composition and the stability of the solution. Additional additives that can be used in the formulations of the present invention include dextrose, conventional antioxidants, and conventional chelators such as ethylenediaminetetraacetic acid (EDTA). Booster vaccinations can be given 2-4 weeks after the first immunization. To prevent reproductive disease, vaccination regimens are typically performed up to six weeks before mating and up to one week after mating. For prevention of respiratory disease in piglets, vaccination can be given as early as 3 weeks of age. Response to vaccination can be monitored by measuring antibody titers against the PRRS virus using an enzyme-linked immunosorbent assay (ELISA), serum neutralization assay, indirect immunofluorescence, or Western blot. The vaccine strain has phenotypic characteristics that can be used for the diagnosis of pigs with wild-type PRRS infection versus exposure to the vaccine strain alone. Animals exposed to the PRRS virus wild type can be distinguished from animals exposed only to the NEB-1-P94 vaccine strain by measuring the antibody response to the epitope recognized by the monoclonal antibody (MAb) SDOW17. The presence of antibodies reactive with the SDOW17 epitope is indicative of exposure to wild-type virus. Measurement of antibodies to the SDOW17 epitope can be achieved using a competitive ELISA. Plates (96 wells) are coated with the NEB-1 PRRS virus (or other PRRS virus expressing the SDOW17 epitope). The plate is then incubated with porcine serum from test animals and an enzyme-labeled (eg, horseradish peroxidase conjugated) SDOW17 monoclonal antibody. The ability of porcine serum to recognize the SDOW17 epitope is measured by the inhibition of binding of the enzyme-bound SDOW17 MAb to the plate, as detected by the absence of a color change in the substrate of the enzyme. Alternatively, a direct ELISA can be used. The amino acid sequence containing the SDOW17 epitope can be prepared as a synthetic peptide or by recombinant DNA expression in a suitable vector system (eg, E. coli). Plates coated with SDOW17 antigen are incubated with porcine serum. Binding of the porcine antibody to the SDOW17 antigen is detected by incubation with anti-swine immunoglobulin antiserum conjugated to the enzyme, followed by incubation with the enzyme substrate and detection of a color change. The following examples describe the invention in detail. It will be apparent to one skilled in the art that changes in materials and methods may be made without departing from the purpose and spirit of the disclosure. Example 1 Phenotypic Characterization of NEB-1-P94 for Proliferation on Alveolar Macrophages NEB-1-P94 vaccine strain virus, passaged 5 times from the original strain, was isolated from MA104, MARC 145, and porcine alveolar macrophages. Was characterized for growth. Porcine alveolar macrophages (SAM) were obtained by broncho-alveolar lavage with saline followed by pelleting the cells by centrifugation. Macrophages were resuspended in EMEM with 10% fetal calf serum and 50 μg / ml gentamicin and plated at approximately 7 × 10 ⁴ cells per well of a 96-well tissue culture plate. MA104 and MARC 145 cells were plated in 96-well tissue culture plates in medium (EMEM containing 10% fetal calf serum, 30 mM HEPES, 2 mM L-glutamine, and 50 μg / ml gentamicin). NEB-1-P94 or parental NEB-1 virus is serially diluted in culture medium and 0.2 ml of each dilution is inoculated into duplicate wells of a 96-well plate containing SAM, MA104, or MARC 145 cells did. Cultures were incubated for 5 days in a humidified chamber at 37 ° C. with 3% to 5% CO ₂ and monitored for cytotoxic effects specific to the PRRS virus. Titers (50% endpoint) were calculated according to the method of Spearman and Karber. NEB-1-P94 showed reduced or unmeasurable titers in three separate SAM cultures as compared to those obtained with MA104 and MARC 145 cells (Table 1). This is a phenotypic change compared to the parent strain that showed similar titers in all of the cultures tested. Thus, reduced proliferation on porcine alveolar macrophages was the phenotypic marker of choice for vaccine strain NEB-1-P94. Example 2 Phenotypic Characterization of NEB-1-P94 for Lack of Pathogenicity in Pigs Four purely isolated piglets (7-10 days old) from PRRS seronegative sows were treated with NEB-1 -1-P94 progenitor virus (10 ^5.3 TCID ₅₀ / ml) were inoculated intranasally (3 ml / nostril). Piglets were observed for clinical signs of respiratory disease, and the vaccine strain virus was reisolated from serum 5 days after inoculation. Serum from the first group of pigs was used to intranasally inoculate a second group of purely isolated pigs, which were also monitored. This process was repeated for a total of five consecutive backpassages in piglets to determine whether the vaccine strain could reverse the pathogenic state. Vaccine virus was recovered from each successive animal passage. However, respiratory disease was not observed in the purely isolated pigs. Further, the fifth return viruses isolated from passage pigs, (was administered about 10 ^5.3 TCID _{5 0} / ml per piglet) for 1 week and 3-week-old normal were inoculated intranasally piglets reared . Animals were monitored for 42 days after virus inoculation and no signs of clinical disease consistent with pathogenic PRRS infection (ie, prolonged high fever, respiratory signs, lung lesions) were found. Therefore, it was concluded that the NEB-1-P94 virus was non-pathogenic for inducing respiratory disease in piglets. Next, the NEB-1-P94 virus was tested for its ability to produce reproductive disease. The sows PRRS seronegative pregnant 8 day 5, ancestors vaccine strain (10 ^4.5 TCID ₅₀ / ml) were inoculated intranasally (3 ml / nostril). All sows delivered at their predicted time points and 96% of the piglets survived and were born healthy. By comparison, unvaccinated control sows produced 87% of surviving and healthy litters. Thus, the vaccine strain NEB-1-P94 failed to induce reproductive diseases specific to the pathogenic PRRS virus (see Example 4). These data confirmed the non-pathogenic phenotype of the vaccine strain NEB-1-P94. Example 3 Phenotypic Characterization of NEB-1-P94 for Reactivity with PRRS Virus-Specific Monoclonal Antibodies MA104 cells infected with the parental strain NEB-1 or vaccine strain NEB-1-P94 virus were subjected to PRRS Reactivity with virus-specific monoclonal antibodies was tested by indirect immunofluorescence. Briefly, confluent MA104 cells in 96-well plates were fixed with 80% acetone for 10 minutes two days after infection with each virus. The monolayer was then incubated with a SDOW17, VO17, or EP147 monoclonal antibody. After washing, the reactivity of the monoclonal antibody with each virus was detected by incubation with anti-mouse IgG conjugated with fluorescein isothiocyanate, followed by washing and microscopic fluorescence inspection. Positive fluorescence was expressed with all three monoclonal antibodies for the NEB-1 parent strain (Table 2). However, although vaccine strain NEB-1-P94 lost its reactivity with the SDOW17 monoclonal antibody, it tested positive with the other two monoclonal antibodies. These data indicate that NEB-1-P94 strain lost expression of the epitope recognized by the SDOW17 antibody. This loss of reactivity with the monoclonal antibody is most likely to indicate a genetic variation in the RNA sequence of NEB-1-P94 that results in an altered amino acid sequence in the nucleocapsid protein region recognized by SDOW17. Example 4 Prevention of Reproductive Disease by Vaccination of Sow with NEB-1-P94 Vaccine was applied to roller bottles of confluent MA104 cells at approximately 1:10 with NEB-1-P94 (4 passages from the original). Prepared by inoculation in EMEM containing 10% fetal calf serum, 2 mM L-glutamine, and 30 μg / ml gentamicin at a multiplicity of infection of The culture was incubated at 37 ° C. for 3 days, and the supernatant was then decanted. The virus solution was treated 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 / LK ₂ HPO ₄ , 10 g / L albumin fraction V), diluted to 50% (v / v), frozen and lyophilized. Vaccine rehydrated with sterile deionized water, and administered intramuscularly to gilts 2ml of (10 ^5.1 TCID ₅₀ / ml) in four to six weeks prior to mating. On day 85 of gestation, vaccinated and unvaccinated control young sows were challenged by intranasal administration of NEB-1 virus (about ^106.3 TCID ₅₀ ). Animals were monitored throughout the 7 weeks postpartum for signs of fetal or neonatal death due to the PRRS virus. PRRS viremia was expressed in 11/12 (92%) of control sows and 100% of their live litters. PRRS infection during pregnancy resulted in 16% mortality loss at birth (large mummy and stillborn pigs) in the control group compared to only 6% mortality loss in vaccinated sows (Table 3). ). In addition, vaccination results in a 50% reduction in the incidence of frail and diseased piglets and a 94% reduction in piglets with low birth weight (less than 2 pounds at birth) compared to controls Was. Vaccination is congenital as demonstrated by the absence of PRRS virus in the blood or tissue of any piglet from the immunized sow and a 55% prevention of mortality loss through 7 weeks of age when compared to controls. Sex PRRS was prevented (Table 3). The statistically significant prevention of mortality loss and viral infection in vaccinated sows and their offspring clearly demonstrated the efficacy of this vaccine in preventing reproductive forms of PRRS virus-induced disease. Example 5 Prevention of Respiratory Disease by Vaccination of Sow with NEB-1-P94 Vaccine was injected approximately 1:10 with NEB-1-P94 (4 passages from the original) of MA104 cells in confluent roller bottles. Prepared by inoculation in EMEM containing 10% fetal calf serum, 2 mM L-glutamine, and 30 μg / ml gentamicin at a multiplicity of infection of The culture was incubated at 37 ° C. for 5 days, and the supernatant was then collected by decantation. The virus solution was used as 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 / LK ₂ HPO ₄ , Diluted to 50% (v / v) with 10 g / L albumin fraction V), frozen and lyophilized. The vaccine was rehydrated with sterile deionized water and 1 ml ( ^104.9 TCID ₅₀ / ml) was administered intramuscularly to 3 week old PRRS seronegative piglets. Four weeks after vaccination, piglets were challenged with the pathogenic NEB-1 PRRS virus by the intranasal route, as described for young sows (Example 4). Piglets were monitored for signs of respiratory disease up to 14 days after challenge. All unvaccinated control piglets developed clinical signs of respiratory disease as compared to 3/40 (8%) of the vaccinated animals. Vaccination resulted in a statistically significant reduction in fever, respiratory signs, and clinical illness in vaccinated animals compared to controls (Table 4). This study clearly demonstrated the efficacy of the vaccine in preventing respiratory disease caused by the PRRS virus in young piglets.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG), UA (AM, AZ, BY, KG, KZ , MD, RU, TJ, TM), AL, AM, AU, AZ , BA, BB, BG, BR, BY, CA, CN, CZ, EE, GE, HU, IL, IS, JP, KG, KR, K Z, LC, LK, LR, LT, LV, MD, MG, MK , MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, TJ, TM, TR, TT, UA, UZ, V N, YU

Claims (1)

[Claims] 1. Suitable for use in porcine protection against porcine reproductive and respiratory syndrome, comprising a PRRS virus essentially corresponding to the NEB-1-P94 isolate deposited with the American Type Culture Collection under accession number VR-2525 vaccine. 2. 2. The vaccine of claim 1, which is in a live, killed, or modified live (attenuated) form. 3. 3. The vaccine according to claim 2, wherein said viral agent is modified live (attenuated) and is in liquid, frozen or dried form. Containing 10 ^4.0 to 10 of ^9.0 TCID ₅₀ amount of PRRS virus per 4.1 ml, vaccine according to claim 1. 5. A method of protecting pigs from a clinical disease caused by swine reproductive and respiratory syndrome virus, said method comprising administering an effective amount of the vaccine of claim 1 to a pig in need of prophylaxis against a disease caused by said virus. Administering to the subject. 6. 6. The method of claim 5, wherein the vaccine is administered orally, intranasally, intramuscularly, intradermally, intravenously, or subcutaneously.