JP3135069B2 - Swine reproductive and respiratory syndrome vaccine - Google Patents

Description

BACKGROUND OF THE INVENTION The present invention relates to a vaccine for the treatment of porcine 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 occurred in Germany, Belgium, the Netherlands,
Reported in Europe, including 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 this syndrome is reproductive failure, which is premature birth,
It manifests as 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 ("hiccup"), 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 purpose is when administered to a herd of breeding pigs,
It is to provide a vaccine that reduces the presence of PRRS in those populations.

SUMMARY OF THE INVENTION An object of the present invention is to provide pig reproductive and respiratory syndrome (PRRS).
It is to provide a novel vaccine that protects pigs against clinical disease caused by the virus.

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

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 a vaccine virus having 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 that has been 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. 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 is available at the University
Obtained from tissue samples from dead pigs provided to the Nebraska Diagnostic 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 gene analysis.
Characterized as a virus. Inoculation of lactating piglets with the virus 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 first inoculated by inoculation of primary porcine alveolar macrophage (SAM) culture (first 2
MA104 cells (Microbi) for a total of 94 passages
(available from Biological Associates, Inc., Rockville, MD). During this process, virus clones were isolated by plaque purification and characterized for phenotypic characteristics. A vaccine clone, designated NEB-1-P94, was cloned from a swine alveolar macrophage with impaired growth, PRRS-specific monoclonal antibody SDOW.
17 (ATCC HB10997) and lack of disease induction in piglets and pregnant sows. NEB-1-P94 was expanded on MA104 cells and purified for use in vaccine development studies.
ster seed) virus, which is also PRRS-MSV-94-1
).

Vaccines are prepared using MA104 cells as a substrate (but MARC 145 [Dr. Wang, Agriculture Resear
ch Station, Clay Center NE]. Other cell lines that support the growth of PRRS virus, such as cells, may also be used.) MA104 cells were prepared in a suitable tissue culture vessel (eg, 850 cm ² roller bottle) with 5-10% bovine serum, 30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 2 mM. Grow to confluence using Eagle's Minimum Essential Medium (EMEM) containing L-glutamine and an antibiotic (eg, 3 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. In a confluent monolayer of MA104 cells, the NEB-1-P94 virus has a multiplicity of infection (MOI) in the range of 1: 5 to 1: 1000, and preferably in the range of 1:10.
Inoculated in. 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 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. Titer (50% end point)
Are Spearman and Karber (Methods in Virology, IV
Vol., K. Maramorosch and H. Koprowski (eds.), Academic P
ress, New York, 1977). Cells can be fixed using 80% acetone, and lack of reactivity with SDOW17 and V017 or EP147 (Dr. E, Nelson, South Da) to confirm phenotypic identity of the virus.
(available from kota State University, Brookings, SD)
It can be tested for positive reactivity with the monoclonal antibody (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 the virus solution (10 ⁶
Containing ~ 10 ⁹ TCID ₅₀ of virus; based on pre-inactivation titers) with a physiologically acceptable diluent (eg, EME
M, Hanks balanced salt solution, phosphate buffered saline) and immunostimulatory adjuvants (eg, mineral oil, vegetable oil, aluminum hydroxide, saponin, non-ionic surfactant,
Squalene, block copolymers or other compounds known in the art, used alone or in combination). Vaccine doses are typically between 1 ml and 5 ml.

For live vaccine formulations, the virus solution is stored frozen at -50 ° C or below until use. 10 ^4.0 〜 10 ^7.0 TC
ID ₅₀ / dose range, as well as preferably 10 ^6.0 TCID ₅₀ /
The viral fluid containing the dose may be a physiologically relevant diluent (e.g., EMEM, Hanks Balanced Salt Solution, phosphate buffered saline) and a physiologically relevant diluent of the compound designed to stabilize the virus. Diluted with a suitable mixture. 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. Vaccines are stored frozen (below -50 ° C) or lyophilized at 4 ° C until use.
Stored in. Vaccines typically have a dose size range of 1-5 ml, and preferably 2 ml.

To prevent PRRS-induced disease,
Administered to pigs orally, intranasally or parenterally. 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 a solution
For example, it can be stabilized by the addition of partially hydrolyzed gelatin, sorbitol, or cell culture media and known in the art (eg, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate). ,
And / or potassium dihydrogen phosphate) using methods known in the art.

Liquid formulations may also include suspensions and emulsions.
For example, a suspension using a colloid mill, and, for example,
The preparation of an emulsion using a homogenizer is known in the art.

Parenteral dosage forms designed for infusion into bodily fluid systems should be adequately isotonic to the corresponding level of porcine bodily fluids and
Requires pH buffering. 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. Further additives that can be used in the formulations of the present invention include:
Contains dextrose, conventional antioxidants, and conventional chelating agents (eg, 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 diagnosis of pigs with wild-type PRRS infection versus exposure to the vaccine strain alone. Animals exposed to the wild-type PRRS virus 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. SDOW
The presence of antibodies reactive with the 17 epitope is indicative of exposure to wild-type virus.

Measurement of antibodies against the SDOW17 epitope was performed using a competitive ELIS
A can be achieved with A. Plate (96 well) is NEB
-1 Coated with PRRS virus (or other PRRS virus expressing 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 lack 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, as a synthetic peptide,
Alternatively, it can be prepared by recombinant DNA expression in a suitable vector system (eg, E. coli). Plates coated with SDOW17 antigen are incubated with porcine serum. Binding of porcine antibody to the SDOW17 antigen was performed by incubation with anti-porcine immunoglobulin antiserum conjugated to the enzyme,
It is detected by subsequent 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 intent of the present disclosure.

Example 1 Phenotypic Characterization of NEB-1-P94 for Proliferation of Alveolar Macrophages The NEB-1-P94 vaccine strain virus, passaged 5 times from the original strain, was isolated from MA104, MARC 145, and porcine alveolar macrophages. 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 in a 96-well tissue culture plate. MA104 cells and MARC 145 cells were added to a 96-well tissue culture plate in medium (10% fetal bovine serum, 30 mM HEPE
S, 2 mM L-glutamine, and EMEM containing 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 compared to the titers 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. Therefore,
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-P94 prototypic virus (10
^5.3 TCID ₅₀ / ml) was 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 if 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. In addition, the virus isolated from the fifth back-passaged pig was
Week-old and three-week-old normally reared piglets were inoculated intranasally (approximately ^105.3 TCID ₅₀ / ml was administered per piglet). 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 for 85 days gestation, 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. Therefore, the vaccine strain NEB-1-P94 failed to induce reproductive diseases specific to the pathogenic PRRS virus (Example 4).
checking). These data are based on the vaccine strain NEB-
The non-pathogenic phenotype of 1-P94 was confirmed.

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 the vaccine strain NEB-1-P94 virus were purified from PRRS virus Were tested by indirect immunofluorescence for reactivity with a monoclonal antibody specific for. Briefly, confluent MA104 cells in 96-well plates were fixed with 80% acetone for 10 minutes two days after infection with each virus. Then, the monolayer,
Incubated with SDOW17, V017, 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 reactivity with the SDOW17 monoclonal antibody, it tested positive with the other two monoclonal antibodies. These data are
4 shows that the B-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 bottle confluent MA10
About 1:10 in 4 cells with NEB-1-P94 (4 passages from the original)
Were 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. Add the virus solution to 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 ,
50 g using 2.5 g / LK ₂ HPO ₄ and 10 g / L albumin fraction V)
% (V / v), frozen and lyophilized. The vaccine is rehydrated with sterile deionized water and 2 ml (10
^5.1 TCID ₅₀ / ml) intramuscularly administered to young sows 4-6 weeks prior to mating On day 85 of gestation, vaccinated and unvaccinated control young sows were treated with NEB-1 virus. (About 10 ^6.3 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 11/1 in control sows
2 (92%) 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 is associated with a 50% reduction in the incidence of frail and sick piglets, as compared to controls, and in piglets with low birth weight (less than 2 pounds at birth).
Resulted in a 94% reduction. Vaccination was absent of PRRS virus in the blood or tissue of any piglet from the immunized sow and 7 when compared to controls.
Congenital PRRS was prevented as evidenced by a 55% prevention of mortality loss throughout the week (Table 3). Statistically significant prevention of mortality loss and viral infection in vaccinated sows and their offspring is due to the reproductive form of PR
The efficacy of this vaccine in preventing RS virus-induced disease was clearly demonstrated.

Example 5 Prevention of Respiratory Disease by Vaccination of Sow with NEB-1-P94 Vaccine was applied to MA10 in confluent roller bottles
About 1:10 in 4 cells with NEB-1-P94 (4 passages from the original)
Were 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. Add the virus solution to 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 ,
50 g using 2.5 g / LK ₂ HPO ₄ and 10 g / L albumin fraction V)
% (V / v), frozen and lyophilized. The vaccine is rehydrated with sterile deionized water and 1 ml (10
^4.9 TCID ₅₀ / ml) was administered intramuscularly to 3 week old PRRS seronegative piglets.

Four weeks after vaccination, piglets are given pathogenic NEB-1
PRRS virus was challenged 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 reduces fever, respiratory signs, and clinical illness in vaccinated animals compared to controls.
Produced a statistically significant decrease (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 the front page (56) References JP-A-7-289250 (JP, A) JP-A-7-138186 (JP, A) JP 11-505248 (JP, A) JP 11-509087 (JP) JP-A-8-506486 (JP, A) JP-T-7-505049 (JP, A) JP-T-6-5055692 (JP, A) British Patent 2282811 (GB, A) (58) (Int.Cl. ⁷ , DB name) A61K 39/12 A61P 15/00 A61P 31/12 BIOTECHABS (STN) CA (STN) EMBASE (STN) MEDLINE (STN)

Claims (6)

(57) [Claims] An isolated NEB-1 deposited with the American Type Culture Collection under accession number VR-2525.
A vaccine suitable for use in the protection of pigs against porcine reproductive and respiratory syndrome, comprising a PRRS virus essentially corresponding to P94. 2. The vaccine according to claim 1, which is in a live, killed, or modified live (attenuated) form. 3. The vaccine according to claim 2, wherein said viral factor is modified live (attenuated) and is in liquid, frozen or dried form. 4. A PRRS in an amount of 10 ^4.0 to 10 ^9.0 TCID ₅₀ per ml.
2. The vaccine according to claim 1, comprising a virus. 5. A method of protecting pigs from a clinical disease caused by the porcine reproductive and respiratory syndrome virus, said method comprising the step of preventing an effective amount of the vaccine according to claim 1 against the disease caused by the virus. Administering to a pig in need thereof. 6. The method of claim 5, wherein said vaccine is administered orally, intranasally, intramuscularly, intradermally, intravenously, or subcutaneously.