MXPA98007083A - Vaccine against the reproductive and respiratory syndrome porc - Google Patents

Abstract

The present invention relates to a vaccine and method for the treatment of porcine reproductive and respiratory syndrome. The vaccine is obtained from the viral agent NEB-1-P94. In addition, the invention describes a vaccine virus with phenotypic characteristics that can be distinguished from the wild type PRRS virus. Deposit in the American Type Crop Association No. VR-25

Description

VACCINE AGAINST PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME BACKGROUND OF THE INVENTION The present invention relates to a vaccine for the treatment of porcine reproductive and respiratory syndrome (PRRS). In 1987, the pig breeding industry in the United States suffered from an unknown infectious disease that had a severe economic impact on the pig farming industry. The pathological syndrome was reported in Europe, including Germany, Belgium, the Netherlands, Spain and England. The disease is characterized by lack of reproduction, respiratory disorders and various clinical signs including loss of appetite, fever, dyspnea and neurological signs ieves. A fundamental component of this syndrome is the reproductive deficiency manifested in the form of premature births, near-term abortions, weak-born pigs, stillbirths, mummified fetuses, reduced birth rates and return of delayed estrus. Respiratory diseases are more pronounced in pigs less than 3 weeks old, although their appearance in pigs has been reported in all stages of production. The affected piglets are developed with lentituo have their harshest hairs sutren ae respiratory msuticiencia ("golpeteo") and increased mortality The pathological syndrome has been referred to in many different ways, including mysterious porcine disease (MSD), porcine epidemic respiratory and abortion syndrome (PEARS), porcine respiratory infertility syndrome (SIRS). The name commonly used today is Porcine Reproductive and Respiratory Syndrome (PRRS). This term is used throughout this patent application.

It is an object of the invention to produce a vaccine that protects a vaccine against clinical disease caused by PRRS. Another objective is to produce a vaccine that, when administered to a herd of pigs for reproduction, reactivates the presence of PRRS in its SUMMARY OF THE INVENTION An objective of the present invention is to present a novel vaccine that protects a pig against the clinical disease caused by the porcine reproductive and respiratory syndrome virus (PRRS). Another objective of the present invention is to present a vaccine that protect a cerao against the NEB-1 strain of PRRS virus An objective of the present invention is to present a method to protect a cerao against the causative disease caused by a porcine reproductive and respiratory disease virus. Other objectives the present invention is to present a virus or vaccination with phenotypic characteristics that can differentiate get rid of the aei virus PRRS or bighorn or wild type DETAILED DESCRIPTION OF THE INVENTION The invention presents a composition of matter comprising a respiratory and respiratory Reproductive Porcine Reproductive Virus (PRRS) that has been modified by manipulation of the oooratope for use in vaccination In addition, the composition has phenotypic properties that allow its use for diagnostic purposes to distinguish between pigs that are naturally infected with the virus or the PRRS of animals that have only been exposed to the strain of the vaccine. The isolated strain oei PRRS virus NEB -1-P94 has been deposited in the American Collection of Type Crops (Registry Number VR - A virulent isolate of the PRRS virus was obtained from samples of tissues from a dead fence submitted to the Diagnostic Laboratory of the University of Neoras? tissue homogenate from the dead fence was inoculated into primary alveolar pig macrophages and the presence of the virus was detected by cytopathic effects in the inoculated cultures, unlike the control cultures The isolated virus (designated with the name NEB-1) was then characterized as a PRRS virus based on physical properties (sensitivity to ether and chloroform, strong density), and lack of hemoagiutiary activity), reactivation with specific antibodies, and genetic analysis. The inoculation of the virus in mamonas resulted in a respiratory disease characterized by high temperature, altered respiration, and lung disease compatible with viral interstitial pneumonia. oei virus in pregnant sows gave rise to the reproductive disease that is characterized by the mummification of the fetuses, the birth of dead chickens, and lechonins that were born oogues and then died The respiratory and reproductive disease caused by this virus turned out to be typical of the reported syndrome Regarding virus or PRRS, NEB-1 virus was attenuated by serial passage in tissue cultures. The virus was transferred by inoculation of cultures of primary porcine alveolar macrophages (SAM (in the first steps) and then by transference in senes. oe MA104 cells (which can be oothener in Micro biological Associates, me, Rockviile, MD), for a total of 94 transfers During this process, cei virus clones were isolated by plaque purification and these were characterized by their phenotypic properties. The clone of the vaccine, designated NEB-1 -P94, was selected for insufficient development in porcine alveolar macrophages, lack of reactivity with specific monoclonal antibody or PRRS SDOW17 (ATCC HB10997), and lack of induction to disease in pregnant spines and sows. The NEB-1 -P94 was expanded in MA104 cells and frozen as master seed virus also designated PRRS-MSV-91 -1, for use in the vaccine development studies. The vaccine is prepared using MA104 cells as a substrate (although alternative cell lines supporting the development can also be used). of PRRS virus, such as MARC 145 [obtained from Dr Wang Agricultural Research Station, Ciay Center EN]) MA10 plants are cultivated 4 up to confluence in suitable containers for tissue cultures, for example 850 cnr rolling bottles using Eagie minimum essential means (EMEM) containing 5 to 10% bovine serum, 30 mM HEPES (N-2-hydroxyl acid) ? etiip? peraz? na-N '-2-etansuifón? co, 2 mM L-giutamma, and antibiotics (such as, 30 μg / ml gentamicma) You can also start other alternative tissue culture media that can sustain ei growth of MA104 cells such as modified essential oils oe Dulbecco (DMEM), Medium 199, or others The confluent single strata of MA104 cells are inoculated with the NEB-1 -P94 virus at a multiplicity of infection (MI) in the range of 1: 5 to 1 1000, and preferably, in the range a 1 to 10. After incubation for three to five days at 37 ° C, the supernatant fluids are harvested by decanting. The viral fluids were titrated by dilutions in semen in EMEM supernatant in the ante form. Aescppta and inoculations are 0.2 ml per receptacle in at least four replicate receptacles of MA104 or MARC 145 cells in a 96-well tissue culture dish. The cultures are incubated for five days at 37 ° C 3-4% C02 in a humid chamber and its cuopathic effects were observed Titles (end points ai 50%) are calculated with the methods of Spearman and Karber (Methods in Virology, Volume IV, K Maramorosch and H Koprowski (Edits and Acaoemic Press, New York, 1977). The cells can be fixed with 80% acetone and verify their lack of reactivity with SDOW17 and their positive reactivity with the monovalent antibodies V017 or EP147 (obtainable from Dr E Nelson, South Dakota State University , Brookmgs, SD) (estimated positive result) to confirm the phenotypic identity of the virus. For the preparation of a killed vaccine, the viral fluids are incubated with a chemical deactivating agent. Examples of deactivating agents include formaldehyde, glutaraldehyde, Diuria, or beta-propiolactopa. Next, viral fluids are stored at 4 ° C until the vaccine is prepared. The vaccine is prepared by mixing viral fluids (containing 10d to 10s TCID50). virus based on pre-deactivation values) with a physiologically acceptable diluent (such as EMEM, HanK's Sanna Balanced Solution, Saima with Phosphate Buffer), and a immunostimulant adjuvant (such as mineral oil, vegetable oil, hydroxy aluminum or saponin, detergents nonionics, squalene, block copolymers or other compounds known in the art, used alone or in combination) A vaccination oosis is typically between 1 and 5 ml. For a live vaccine formulation, viral fluids are stored, frozen at -50 ° C or at a lower temperature nasta its use The viral fluids, in the range of 10"° to 10 'or TCID5o / two? S, and preferably co A content of 106 or TCID / doses are diluted with a physiologically acceptable diluent (such as EMEM, HanK Balanced Saline Solution, Saline with Buffer or Phosphate) and a physiologically acceptable mixture of compounds intended to stain the virus. The technique that can be used alone or in preparation to stannate viruses, are sucrose, lactose amine NZ, glutathione, neopeptone, gelatin, or extracell and tnptone. The vaccine is stored frozen (-50 ° C or less) or iiofihzaaa with storage at 4 ° C until use). The vaccine typically has a dose size in the range of 1 to 5 ml, and preferably 2 ml. For prophylaxis against PRRS-induced disease, the vaccine is administered to the pig orally, mtranasai or parenteral. parenteral administration include the intradermal intramuscular, intravenous mtraperitoneai, and subcutaneous routes of administration. When administered in the form of a solution, the present vaccine can be prepared in the form of an aqueous solution, syrup, elixir, or tincture. Such formulations are known in the art, and they prepare by dissolving the antigen and other suitable additives in solvent systems aaecuaaos Such solvents include water, saima, ethanol, ethylene col, glycerol, fluid A1, etc. The additives known in the art include dyes, flavors, sweeteners and antimicrobial preservatives allowed, such as thimerosai (ethyl citrate sodium). These solutions can be to be stabilized, for example, by the addition of partially hydrolyzed geythm, sorbitol or cell culture medium, and can be regulated by known methods in the environment, using reagents known in the art, such as sodium hydrophosphate, sodium dihydrophosphate, potassium hydrophosphate and / or potassium dihydrophosphate. In addition, suspensions and emulsions can be prepared. The preparation of the suspensions is carried out, for example, by a coloiaai mill. and emuisic, using for example a homogenizer, is known in the art. The forms of parenteral aosification, intended for injection into systems of body fluids, require isotonicity and regulation of pH appropriate to the corresponding levels of porcine body fluids. parenteraies can also be sterilized prior to use. Isotonicidae can be regulated with sodium chloride and other salts, as needed. Other solvents, such as ethanol or propylene glycol, can be used to increase the solubility of composition ingredients and stability. The other additives that may be used in the present formulation include dextrose, conventional antioxidants, and conventional chelating agents, such as ethylenediaminetetraacetic acid (EDTA). A booster vaccination may be applied two to four weeks after administration. initial immunization For prevention of reproductive disease, the vaccination regimen is typically carried out up to 6 weeks before and 1 week after procreation. For the prevention of respiratory disease in the young, vaccination can be administered after 3 weeks of vaccination. eoad The response to vaccination can be monitored by measuring the antibody titre against the PRRS virus using the enzyme-linked immunosorbent assay (ELISA), the serum neutralization assay, the indirect immunofluorescence assay, or the Western blot) The strain oe Vaccination has phenotypic properties that can be used to diagnose PRRS infection of the bighorn type in pigs, unlike exposure to only the strain of vaccination. Animals exposed to field strains to the PRRS virus can be distinguished from the exposed animals. to the NEB-1 -P94 vaccination strain by measuring the antibody response to the epitope recognized The presence of antibodies that react with the epitope of SDOW17 indicates the exposure to the virus or the bipolar type. The measurement of the antibodies to the epitope and SDOW17 can be carried out using a competitive ELISA. receptacles) with the NEB-1 -PRRS virus (or other aei PRRS viruses) expressing the SDOW17 epitope) The plates are then incubated with porcine serum obtained from the animals to the assay and enzymatically labeled monoclonal antibody SDOW17 (for example conjugated with peroxidase). aeo raoano) The ability of porcine sera to recognize the SOW17 epitope is mediated by the inhibition of SDOW 17 MAb binding enzymatically linked to the plate as detected by the lack of chromatic conversion of the enzymatic substrate. Direct ELISA The amino acid sequence comprising the SDOW17 epitope can be prepared in synthetic peptide form or by ex-methods. Recombinant DNA pressure in a suitable vector system, such as E co Coated plates with the SDOW17 antigen are incubated with the porcine serum The attachment of the porcine antibodies to the SDOW17 antigen is detected by? ncuoac? on with antisera or anti-porcine immunoglobulin. enzymatically conjugated followed by the mcuDacion with substrate enzm, attic and the aeteccion e a change in color The following examples descpoen the invention in detail The people trained in the art will recognize that you can practice the moaificaaon materials and metoaos without departing for the purpose and the intention of this description EXAMPLE 1 Phenotype characterization or NEB-1-p94 for the Development in Alveolar Macrophages The virus of the NEB-1-P94 vaccination strain in five passages from the master sieve was characterized with respect to its aesarroiy. in cells MA104 MARC 145 and porcine alveolar macrophages Porcine alveolar macrophages (SAM) were ooteniaos by Dronquio-alveolar lavage with saline, followed by centrifugation to tablet the cells Macrophages were resuspended in EMEM with fetal bovine serum ai 10% and 50 μg / ml gentamicin and applied at a rate of approximately 7 x 10"cells per receptacle of tissue culture plates to 96 receptacles MA104 cells and MARC 145 cells were applied to tissue culture plates of 96 receptacles in meatos (EMEM with a serum content). fetal covinoid at 10% 30 mM HEPES, 2 mM and L-glutamine and 40 μg / ml gentamicma) NEB-1- p94 or parental NEB-1 virus were serially diluted in media and 0.2 ml Each dilution was inoculated into receptacles to replicate plates to 96 receptacles containing SAM, MA104, or MARC 145 cells (Table 1). These represent a phenotypic camoium compared to the progenitor strain that exhibited t Similar titers in all the analyzed crops Therefore, the reduced development in pig alveolar macrophages constituted a phenotypic marker selected for vaccine strain NEB-1 -P94 Tada 1 Comparison of Development of Virus NEB-1 -P94 in various types of Cells * Title =? Og? 0 i ClD5o / ml (imitate aete-tion in this essay = 1, 2) EXAMPLE 2 Phenotypic characterization of NEB-1 -P94 regarding the lack of virulence in pigs Four gnoto-otic leonons (from seven to 10 years after the serosgative PRRS sows were inoculated via mtranasai (3 ml / opf) with master seed NEB-1-P94 (105 3 TCIDso / ml). Piglets were observed for clinical signs of respiratory disease. and the virus from the vaccination strain was isolated again from the serum at five years after the inoculation. Serum was used in the first group of ceraos to innocuously feed a second group of gnoto-otic pigs that were monitored in the same way. This process was repeated during a total of five back-transfers in senes in piglets to determine if the vaccination strain could return to a virulence stage. The vaccination virus was recovered from each successive animal transfer, without However, no respiratory disease was observed in the gnotooiotic ceramics. Aaemas, the virus isolated from the fifth retrotransfer was inoculated intranasally in conventional one and three-week-old lechrons (approximately 105 j TCID5o ml was administered per piglet). animals were monitored for 42 days after virus inoculation and no clinical signs of the disease were found (ie, prolonged fever, respiratory symptoms, lung lesions) compatible with PRRS infection. Therefore, it was concluded that the NEB virus -1- P94 lacked virulence for inactivation of respiratory disease in groups The NEB-1-P94 virus was then examined to aemate its ability to cause reproauctive disease. Seronegative PRRS sows were inoculated with 85 ae gestation by mtranasai (3 ml / opf) with the master seed vaccination strain (1014 3 TCID50 / ml) All the sows gave birth in the pl Azo estimate and 96% of the leonones were born alive and healthy. By comparison, the non-inoculatory control pigs gave birth to leonates, and 87% of the lemons were born alive and healthy. Therefore, the vaccination strain NEB-1 -P94 did not inactivate the disease and the typical reproduction of a virulent PRRS virus (See Example 4). These data confirmed the virulent phenotype of the NEB- "P94 vaccination strain. EXAMPLE 3 Phenotype Characterization of NEB-1-P94 Regarding Reactivia with Specific PRRSV Monoantibodies MA104 cells infected with NEB-1 parental strain or NEB-1-p94 vaccination strain were analyzed for reactivity with monoclonal antibodies specific to PRRS virus by indirect immunofiuorescence A large traits, plates of 96 receptacles or confluent MA104 cells were fixed with 80% acetone for 10 minutes within two days after infection with each virus. Then the single layers were incubated with monoclonal antibodies SDOW17, V017, OR EP147. detected the reactivity of the monoclonal antibody with caaa virus incubation with anti-mouse IgG conjugated with fluorescein isothiocyanate conjugate followed by iavaase and fluorescence analysis by microscopy Positive fluorescence was observed with the three monoclonal antibodies for the parental strain NEB-1 (Table 2 ) However, the vaccination strain, NEB-1-P94, had lost reactivity with the monoclonal antibody SOW17 but had positive results with the other monoclonal antibodies. These ates inactivated that the NEB-1 -P94 strain haoia peraiao the expression of the epitope Recognized by the SDOW17 antibody The lack of reactivation with this monoclonal antibody represents very the genetic mutation in the RNA sequence of NEB-1-P94 that synthesized a modified amino acid sequence in the region of the nucleocapsis of proteins recognized by SDOW17 Taoia 2 Reactivial Virus and Parental Virus and PRRS Vaccine with Specific Monoclonal Antibodies EXAMPLE 4 Prevention of Reproductive Disease by Vaccination of Pigs with NEB-1-P94 The vaccine was prepared by inoculating confluent rolling bottles of cells MA104 with NEB-1 -P94 (4 passes from the master seed) to a multiplicity of infection of approximately 1 10 in EMEM containing 10% fetal bovine serum, 2 mM L-glutamma, and 30 μg / ml gentamicin. The cultures were incubated for three days at 37 ° C and then the supernatants were harvested by decantation. Viral fluids were diluted at 50% or (v / v) with stabilizer (75 g / L of tpptona, 30 g / l of dextran, 2 g / l of gelatin, 100 g / l of lactose, 2 g / l of sodium glutamate, 1.05 g / i of KH3PO4, 2.5 g of K3HP04, 10 g / l and fraction V of a umma), frozen, and lyophilized The vaccine was rehiarataa with sterile deionized water and 2 ml were added ( 10 'TCID50 / ml) intramuscularly to young sows four to six weeks before procreation At 85 days of gestation, vaccinated fences and unvaccinated control fences were challenged by intranasal administration of NEB-1 virus. (approximately 10 or 3 TCID50) The animals were monitored until seven weeks after calving for signs of fetal or neonatal death attributed to the PRRSV virus. The PRRS viraemia was increased by 1 1/12 (92%) to the sows. control and 100% of their babies born alive. The infection of PRRS during pregnancy resulted in a loss of life. by death of 16% at calving (large mummies and dead pigs born) in the control group (Taola 3) compared to only one pear per diem and 6% in the vaccinated sows. In addition, vaccination resulted in 50% of reduction in the incidence of deoiies and shimmings and a 94% reduction in piglets with low birth weight (weighing less than 2 pounds at birth) compared to controls. Vaccination prevented the congenital PRRS as evidenced by the absence The PRRS virus in the blood or tissues of all the children born to immunized pigs and 55% of prevention or loss by death up to seven weeks of age (Tabia 3) in comparison with the controls. The prevention of loss due to death and Virus infection statistically significant in vaccinated sows and their offspring clearly demonstrated the efficacy of this vaccine in preventing the reproauctive form of maucia disease caused by the virus d PRRS Tabia 3 Summary of Reproductive Disease Ooservaaa after challenge with PRRS virus from vaccinated sows versus unvaccinated controls EXAMPLE 5 Prevention of Respiratory Disease by Vaccination of Pigs with NEB-1 -P94 The vaccine was prepared by inoculation of confluent rodent ootheliae of MA104 plants with NEB-1 -P94 (4 passes from the master seed) to a multip ... infection was approximately 1 10 in EMEM containing fetal serum Dovino ai 10% 2 ml of L-giutamine, and 30 μg / ml of gentamicin The cultures were incubated for three days at 37 ° C and then the supernatants were harvested by decantation . The viral fluids were diluted at 50% (v / v) with stabilizer (75 g / L of tnptone, 30 g / l of dextran, 2 g / l of gelatin, 100 g / l of lactose, 2 g / l of glutamate sodium, 1.05 g / l of KH3P04, 2.5 g of r < 3HPO4, 10 g / l of fraction V of albumin) frozen and ofilized. The vaccine was rehydrated with sterile deionized water and 2 ml were administered (105'1 TCID50 / ml) intramuscularly to PRRS seronegative piglets at three weeks of age Four weeks after vaccination, the piglets were challenged with virulent PRRS virus NEB-1 via mtranasai in the form descppta for young sows (Example 4) Piglets were monitored for signs of respiratory disease for 14 days after challenge All unvaccinated control piglets developed clinical signs of respiratory disease compared to 3/40 (8%) of vaccinated animals Vaccination It led to a reduction in fever, respiratory signs, and statistically significant clinical disease in vaccinated animals compared to controls (Table 4). This study strongly demonstrated the efficacy of the vaccine in the prevention of respiratory disease caused by the virus. PRRS in young pigs Table 4. Summary of Clinical Disease in Vaccinated Animals and Controls After the Challenge with the Virus of the PRRS Nd = Data not available. Controls n = 30 Vaccinated n = 40.

Claims (1)

16 CLAIMS 5 1 A vaccine used to treat reproauctive and respiratory syndrome that corresponds essentially to the isolate of the American Type of Culture and the number of the record VR-2525 2 The vaccine to the outside with the type 1 that is alive, dead or modified (attenuated) 3 Vaccination with reivination 2 in which the viral agent is alive modified (atenuaa) or is in liquid, frozen or dried form. 10 * to 10s or TCID50 of the PRRS virus per my vaccine according to claim 1 in which the attenuated virus can be distinguished from the bighorn-type forms of the virus in Dase in the absence of reactivation with the monoclonal antibody SDOVV17 and lack of efficient development in alveolar porcine macrophage cells 20 6 A method to protect a wax from clinical disease is caused by the porcine reproductive and respiratory syndrome virus that is 8 A method for diagnosing infection with the vaccine virus of claim 1 only versus infection with the bighorn type virus in Dase to the isolation of the virus from the serum or tissue to an infectious wax and the phenotypic characterization in accordance with IO. Specified in the reivipation 5 9 A method is used with claim 8 in which the diagnosis of the virus infection of the vaccine or with the bighorn type virus includes the meation of the antibodies in the infected pigs that react with the epitope. Protein aei virus to PRRS recognized by the monoclonal antibody SDOW17