KR100297537B1 - Pig reproductive and respiratory syndrome vaccine - Google Patents

Abstract

Vaccines and methods for treating swine reproduction and respiratory syndromes are described herein. The vaccine of the present invention is derived from the viral agent NEB-1-P94 deposited with accession number VR-2525 in the American Type Culture Collection. Also described herein are vaccine viruses with phenotypic characteristics that can be distinguished from wild-type PRRS viruses.

Description

[Name of invention]

Porcine Reproductive and Respiratory Syndrome Vaccine

[Background]

The present invention relates to a vaccine for the treatment of Porcine Reproductive and Respiratory Syndrome (PRRS).

In 1987, the pig-producing industry in the United States experienced an unknown infectious disease that had a serious and economic impact on the pig industry. This disease syndrome has also been reported in Europe, including Germany, Belgium, the Netherlands, Spain and the United Kingdom.

This disease is characterized by various clinical signs including infertility, respiratory disease and loss of appetite, high fever, dyspnea and mild neuropathy. The main components of this syndrome are obvious reproductive insufficiency, such as premature birth, late abortion, weak pig delivery, stillbirth, mummified fetuses, reduced delivery rate and delayed estrous recovery. Clinical signs of respiratory disease are most common in pigs at 3 weeks of age, but have been reported to occur in all reproductive stages of pigs. Affected piglets are slow to grow, rough in the shell, have difficulty breathing (“daughter”), and increase mortality.

This disease syndrome has been referred to in several different terms, including mysterious swine disease (MSD), swine epidemic abortion and respiratory syndrome (PEARS), swine infertility and respiratory syndrome (SIRS). Recently, the term porcine reproductive and respiratory syndrome (PRRS) is commonly used and will be used throughout this patent application.

It is an object of the present invention to provide a vaccine that protects swine against clinical disease caused by PRRS. Another object is to provide a vaccine which, when administered to a breeding pig herd, may reduce the presence of PRRS in these populations.

[Summary of invention]

It is an object of the present invention to provide a novel vaccine that protects pigs against clinical diseases caused by porcine reproductive and respiratory syndrome (PRRS) viruses.

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

Another object of the present invention is to provide a method of protecting swine against clinical diseases caused by swine reproductive and respiratory disease viruses.

Another object of the present invention is to provide a vaccine virus having phenotypic characteristics that can be distinguished from wild type PRRS virus.

Detailed description of the invention

The present invention provides compositions comprising attenuated swine respiratory and reproductive syndrome (PRRS) viruses modified by laboratory manipulation for use in vaccination. In addition, these compositions have phenotypic properties used for diagnostic purposes only to distinguish pigs naturally infected with the PRRS virus from animals exposed to the vaccine species. PRRS virus isolate NEB-1-P94 was deposited in the American Type Culture Collection (Accession No. VR-2525; Deposit Date: March 1, 1996).

Toxic isolates of the PRRS virus are obtained from tissue samples of dead pigs provided by the University of Nebraska Diagnostic Laboratory. Tissue homogenates from dead pigs are inoculated into major swine alveolar macrophages and the presence of the virus is detected by cytopathic effect on the inoculated culture and not the control culture. Subsequently, the isolated virus (named NEV-1) was characterized as a PRRS virus based on physical properties (ether and chloroform sensitivity, lack of suspended density and hemagglutination activity), reactivity with specific antibodies and genetic analysis It became. Inoculation of the virus into lactating piglets results in respiratory disease characterized by lung disease accompanied by high fever, respiratory changes and viral interstitial pneumonia. In addition, when the virus is inoculated with a sow that conceived the virus, reproductive diseases are characterized by the death of the baby following the mummification of the fetus, the stillbirth of the swine pig, and the birth of a weak piglet. Respiratory and reproductive diseases caused by these viruses are typical syndromes reported for the PRRS virus.

NEB-1 virus is attenuated by serial passage of tissue culture. These viruses were first inoculated in primary swine alveolar macrophage (SAM) culture (for the first two passages) and passaged, followed by a total of 94 on MA104 cells (available from Microbiological Associates, Inc., Rockville, MD). Passage consecutive times. During this process, virus clones are isolated by plaque purification and characterized for phenotypic properties. A vaccine clone named NEB-1-P94 was used for growth impairment on porcine alveolar macrophages, lack of reactivity with the PRRS-specific monoclonal antibody SDOW17 (ATCC HB10997), and lack of disease induction in piglets and pregnant sows. Screening. NEB-1-P94 is stretched and frozen on MA104 cells and used in vaccine development studies as the master seed virus designated PRRS-MSV-94-1.

Vaccines are prepared using MA104 cells as substrate (although another cell line may be used that supports the growth of PRRS virus, such as MARC 145 (available from Dr. Wang, Agriculture Research Station, Clay Center NE) cells). . Eagle containing 5-10% bovine serum, 30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 2 mM L-glutamine and antibiotics (e.g. 30 μg / ml gentamicin) Minimal essential medium (EMEM) is used to grow MA104 cells in a suitable tissue culture vessel (eg 850 cm 2 roller bottle). Another tissue culture medium may be used that can support the growth of MA104 cells, such as Dulbecco's Modified Essential Medium (DMEM), Medium 199, and the like. Frontal monolayers of MA104 cells are inoculated with NEB-1-P94 virus in the multiplicity of infection (MOI) 1: 5 to 1: 1000 and preferably 1:10. After incubation at 37 ° C. for 3-5 days, the culture supernatant is collected by decantation.

Viral fluid is titrated by serial dilution in EMEM supplemented as described above and inoculating 0.2 ml per well into 4 or more replicated wells of frontal MA104 or MARC 145 cells in 96-well tissue culture plates. Cultures are incubated in a wetting chamber under 3-5% CO ₂ at 37 ° C. for 5 days and the cytopathic effect is observed. The titer (50% endpoint is described in Spearman and Karber's method (Methods in Virology, Volume IV, K. Maramorosch and H. Koprowski (Eds). Academic Press, New York, 1977). The cells are fixed in 80% acetone and lack reactivity with SDOW17 and positive reactivity with VO17 or EP147 (available from Dr. E. Nelson, South Dakota State University, Brookings, SD) (positive results expected). Test to determine the phenotype homology of the virus.

To prepare a dead viral vaccine, the viral fluid is incubated with a chemical inactivating agent. Examples of inactivating agents include formaldehyde, glutaraldehyde, binary ethylenimine, or beta-propiolactone. The viral fluid is then stored at 4 ° C. until formulated into a vaccine. Vaccine virus fluid (based on pre-inactivation titer 10 ⁶ to 10 ⁹ containing from virus TCID ₅₀₎ of diluent to be physiologically acceptable (for example: EMEM, Hank's balanced salt solution, phosphate buffered saline) and immune - It is prepared by mixing with stimulating aids such as mineral oils, vegetable oils, aluminum hydroxide, saponins, nonionic detergents, squalene, block copolymers or other compounds known in the art used alone or in combination. The vaccine dose is typically 1-5 ml.

For live viral vaccine formulations, freeze storage at −50 ° C. or lower until viral fluid is used. Viral fluids containing 10 ^4.0 to 10 ^7.0 TCID ₅₀ / dose and preferably 10 ^6.0 TCID ₅₀ / dose for physiologically suitable diluents (e.g., EMEM, Hank's balanced salt solution, phosphate buffered saline) and virus Dilute with a physiologically suitable mixture of compounds. Compounds known in the art that can be used alone or in combination to stabilize viruses include sucrose, lactose, NZ amines, glutathione, neopeptone, gelatin, dextran and tryptone. Vaccines are stored at 4 ° C. by freeze storage (-50 ° C. or below) or lyophilized until use. Typical dosages of the vaccine are 1-5 ml and preferably 2 ml.

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

When administered in solution, the vaccines of the present 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 or other suitable additive in a suitable solvent system. Such solvents include water, saline, ethanol, ethylene glycol, glycerol, Al fluid and the like. Suitable additives known in the art include recognized dyes, flavors, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethyl mercurythiosalicylate). Such solutions may be sterilized, for example, by addition of partially hydrolyzed gelatin, sorbitol, or cell culture medium, or reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, hydrogen phosphate Potassium and / or potassium dihydrogen phosphate can be used to buffer by methods known in the art.

Liquid formulations may also include suspending agents and emulsions. For example, methods for preparing suspensions using colloid mills and methods for preparing emulsions, for example using homogenizers, are known in the art.

Parenteral dosage forms designed for injection in a bodily fluid system should have adequate isotonicity and pH buffers to correspond to pig body fluid levels. Parenteral formulations should also be sterilized prior to use.

Isotonicity can be adjusted with sodium chloride and other salts as needed. Other solvents such as ethanol or propylene glycol can be used to increase the solubility of the composition components and the stability of the solution. Still other additives that may be used in the formulations of the present invention include dextrose, conventional antioxidants and conventional chelating agents, such as ethylenediamine tetraacetic acid (EDTA).

Booster vaccine treatment can be administered 2 to 4 weeks after the initial immunization. For the prevention of reproductive diseases, vaccination regimens can typically be carried out 6 weeks before breeding to 1 week after breeding. To prevent respiratory disease in piglets, vaccination can be given as early as three weeks of age. Response to vaccination can be monitored by measuring antibody titers induced against PRRS virus using enzyme-linked immunosorbent assay (ELISA), serum neutralization assay, indirect immunofluorescence, or western blotting.

Vaccine species have phenotypic characteristics that can be used to diagnose wild-type PRRS infected pigs from pigs exposed only to the vaccine species. Animals exposed to wild-type PRRS virus species can be distinguished from animals exposed only to the NEB-1-P94 vaccine species by measuring antibody response to epitopes recognized by the monoclonal antibody (MAb) SDOW17. The presence of antibodies reactive with the SDOW17 epitope indicates exposure to wild type virus.

Measurement of antibodies against SDOW17 epitopes can be performed using a competitive ELISA. Plates (96-wells) are coated with NEB-1 PRRS virus (or other PRRS virus expressing SDOW17 epitope). Plates are then incubated with porcine serum and enzyme-labeled SDOW17 monoclonal antibodies (eg conjugated with horseradish peroxidase) from test animals. The ability of sera in pigs to recognize SDOW17 epitopes is measured by inhibition of enzyme-bound SDOW17 MAb binding to plates by detecting a lack of enzyme substrate color conversion. ELISA can also be used directly. Amino acid sequences comprising the SDOW17 epitope can be prepared from synthetic peptides or by recombinant DNA expression methods in a suitable vector system such as Escherichia coli. Plates coated with SDOW17 antigen are incubated with pig serum. Binding of porcine antibodies to the SDOW17 antigen is detected by incubation with enzyme-conjugated anti-pig immunoglobulin antiserum followed by incubation with enzyme substrate to detect color change.

The following examples describe the invention in detail. It will be apparent to those skilled in the art that modifications can be made in the materials and methods without departing from the object and spirit of the invention.

EXAMPLE

Example 1

Phenotypic Characterization of NEB-1-P94 on Growth on Alveolar Macrophages

The NEB-1-P94 vaccine species virus passaged five times from the base seed virus is characterized for growth on MA104, MARC 145 and porcine alveolar macrophages. Porcine alveolar macrophages (SAM) are obtained by washing the bronchial-alveoli with saline and then pelleting the cells by centrifugation. Macrophages are resuspended in EMEM containing 10% fetal bovine serum and 50 μg / ml gentamycin and plated at about 7 × 10 ⁴ cells per well of 96-well tissue culture plates. MA104 cells and MARC 145 cells are plated in 96-well tissue culture plates in medium (EMEM containing 10% fetal bovine 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 medium and 0.2 ml of each dilution is inoculated into replica wells of 96-well plates containing SAM, MA104, or MARC 145 cells. Cultures are incubated in a wet chamber under 3-5% CO ₂ at 37 ° C. for 5 days and the typical cytopathic effect of PRRS virus is monitored. Titer (50% endpoint) is calculated according to Spearman and Carver's method. NEB-1-P94 showed reduced or no measurable titers obtained for three isolated SAM cultures compared to titers obtained for MA104 and MARC 145 cells (Table 1). This is a phenotypic change compared to the parent strain showing similar titers for all cultures tested. Thus, growth impairment on porcine alveolar macrophages is a selected phenotypic marker for vaccine species NEB-1-P94.

Table 1 Growth comparison of NEB-1-P94 virus against various cell types

Example 2

Phenotypic Characterization of NEB-1-P94 for Toxicity Deficiency in Pigs

Four sterile piglets (7-10 days old) from PRRS seronegative sows are intranasally inoculated with NEB-1-P94 basal seed virus (10 ^5.3 TCID ₅₀ / ml) (3 ml / nostrils). Piglets are observed for clinical signs of respiratory disease and vaccine virus species are re-separated from serum 5 days after inoculation. Serum from group 1 pigs is used to intranasally inoculate group 2 sterile pigs and monitored in the same manner. This process is repeated in piglets for a total of five consecutive reverse passages to determine whether the vaccine species has been converted to a toxic state. Vaccine virus was recovered from each serial passage, but no respiratory disease was observed in sterile pigs. In addition, the virus isolated from the fifth passage pig was intranasally inoculated into 1 and 3 week old piglets (approximately 10 ^5.3 TCID ₅₀ / ml per pig). Animals were monitored for 45 days post vaccination, but no clinical signs of disease accompanying toxic PRRS infection (ie, persistent fever, signs of breathing, lung lesions) were observed. Thus, it was concluded that NEB-1-P94 virus was not toxic to induction of respiratory disease in piglets.

The NEB-1-P94 virus is then tested for the ability to induce reproductive disease. PRRS seronegative sows 85 days old are intranasally inoculated with base seed vaccine species (10 ^4.5 TCID ₅₀ / ml) (3 ml / nostrils). All sows gave birth to scheduled time, and 96% of piglets were born alive and well. In comparison, uninoculated control sows were delivered, with 87% of piglets born healthy. Thus, the vaccine species NEB-1-P94 did not induce a typical reproductive disease of toxic PRRS virus (see Example 4). These data confirm that the vaccine species NEB-1-P94 is a nontoxic phenotype.

Example 3

Phenotypic Characterization of NEB-1-P94 for Reactivity with PRRS Virus-Specific Monoclonal Antibodies

MA104 cells infected with the parent strain NEB-1 or vaccine species NEB-1-P94 virus are tested for reactivity with monoclonal antibodies specific for the PRRS virus by indirect immunofluorescence. In summary, 96-well plates of front MA104 cells were fixed in 80% acetone for 10 minutes two days after infection with each virus. Monolayers are then incubated with SDOW17, VO17, or EP147 monoclonal antibodies. After washing, the monoclonal antibody reactivity with each virus is detected by incubation with fluorescent dye isothiocyanate conjugated anti-mouse IgG, washing and fluorescence under a microscope. Positive fluorescence was observed in all three monoclonal antibodies against the NEB-1 parent strain (Table 2). However, the vaccine species NEB-1-P94 was not reactive with the SDOW17 monoclonal antibody but was tested positive with the other two monoclonal antibodies. These data indicate that NEB-1-P94 species lost expression of the epitope recognized by the SDOW17 antibody. The loss of reactivity with this monoclonal antibody is expected to result from a modification of the amino acid sequence in the nucleocapsis protein region recognized by SDOW17 due to the presence of a genetic mutation in the RNA sequence of NEB-1-P94.

TABLE 2 Reactivity of the parental and vaccine species PRRS to specific monoclonal antibodies

Example 4

Prevention of reproductive diseases by vaccination of sows with NEB-1-P94

The vaccine was treated with NEB-1-P94 (4 times from primary seed) in a front roller bottle of MA104 cells at approximately 1:10 multiplicity of infection in EMEM containing 10% fetal bovine serum, 2 mM L-glutamine and 30 μg / ml gentamicin Prepared by inoculation). Cultures are incubated at 37 ° C. for 3 days and the supernatant is harvested by decantation. Stabilizing viral fluids (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 aliquots V) are diluted 50% (v / v), frozen and lyophilized. The vaccine is rehydrated with sterile deionized water and intramuscularly administered to sows 4-6 weeks prior to breeding 2 ml (10 ^5.1 TCID ₅₀ / ml).

85 days after conception, NEB-1 virus (about 10 ^6.3 TCID ₅₀ ) is administered intranasally to infect vaccinated sows and untreated control sows. Animals are monitored for signs of fetal or neonatal death due to PRRS virus over 7 weeks postpartum. PRRS virus infections occurred in 11 of 12 control sows (92%) and 100% of piglets that had normal delivery. PRRS infections in conception died 16% after delivery in the control group (mostly mummified and stillbirth pigs) compared to only 6% after delivery in vaccinated sows (Table 3). Vaccination also reduced the incidence of fragile and staggering piglets by 50% compared to controls and 94% reduction in low birth weight (less than 2 pounds at birth). Vaccination prevented congenital PRRS as evidenced by the absence of PRRS virus in the blood or tissue of all piglets from immunized sows and prevented 55% mortality loss over 7 weeks compared to controls (Table 3). . Statistically significant prevention of death and viral infection in vaccinated sows and their offspring clearly demonstrates the vaccine efficacy of the present invention in preventing reproductive types of PRRS virus-induced diseases.

Table 3 Summary of reproductive diseases observed after PRRS virus infection of vaccinated sows against control sows

Example 5

Prevention of Respiratory Diseases by Vaccination of Sows Using NEB-1-P94

The vaccine passaged the front roller bottle of MA104 cells at NEB-1-P94 (4 times from seed) with multiple infections at approximately 1:10 in EMEM containing 10% fetal bovine serum, 2 mM L-glutamine and 30 μg / ml gentamicin. Inoculated). Cultures are incubated at 37 ° C. for 3 days and the supernatant is harvested by decantation. Stabilizing viral fluids (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 aliquots V) are diluted 50% (v / v), frozen and lyophilized. The vaccine is rehydrated with sterile deionized water and 1 ml (10 ^4.9 TCID ₅₀ / ml) is administered intramuscularly to 3 week old PRRS seronegative piglets.

Four weeks after vaccination, piglets are infected with toxic NEB-1 PRRS virus by nasal route as described in Example 4 for sows. Piglets are monitored for signs of respiratory disease for 14 days after infection. Clinical signs of respiratory disease were seen in all control piglets not ringworm compared to 3 of 40 vaccinated animals (8%). Compared to the control group, vaccination showed a statistically significant decrease in high fever, respiratory signs and clinical disease in vaccinated animals (Table 4). This study demonstrates the efficacy of the vaccine according to the invention in the prevention of respiratory diseases caused by the PRRS virus in young pigs.

TABLE 4 Summary of clinical disease in vaccinated and control animals after PRRS virus infection

Claims (6)

Immunizing swine against swine reproductive and respiratory syndrome, including swine reproductive and respiratory syndrome (PRRS) virus having all identified characteristics of isolate NEB-1-P94 deposited as deposit number VR-2525 in the American Type Culture Collection Suitable vaccines. The vaccine of claim 1, wherein the vaccine is in the form of live virus, dead virus or modified (attenuated) live virus. 3. The vaccine of claim 2, wherein the virus is in modified, attenuated live virus in liquid, frozen or dried form. The vaccine of claim 1, wherein the vaccine contains PRRS virus in an amount of 10 ^4.0 to 10 ^9.0 TCID ₅₀ per ml. A method for protecting a pig against clinical diseases caused by said virus, comprising administering an effective amount of the vaccine according to claim 1 to a pig in need of preventing a disease caused by the pig reproductive and respiratory syndrome virus. The method of claim 5, wherein the vaccine is administered orally, nasal, intramuscular, intradermal, intravenous or subcutaneous.