KR20120026436A - Novel porcine reproductive respiratory syndrome virus and uses thereof - Google Patents

Abstract

PURPOSE: An immunogenic composition containing novel PRRSV and inactive PRRSV for preventing or treating PRRSV is provided. CONSTITUTION: A novel PRRSV(porcine reproductive respiratory syndrome virus) is PRRS-GC-6262(deposit number KCTC 11852BP), PRRS-GC-4019(deposit number KCTC 11850BP), PRRS-GC-DM(deposit number KCTC 11853BP), PRRS-GC-4172(deposit number KCTC 11851BP), or PRRS-GC-EU0907(deposit number KCTC 11854BP). An immunogenic composition for preventing or treating PRRS contains inactive PRRS-GC-6262(deposit number KCTC 11852BP), PRRS-GC-4019(deposit number KCTC 11850BP), PRRS-GC-DM(deposit number KCTC 11853BP), PRRS-GC-4172(deposit number KCTC 11851BP), PRRS-GC-EU0907(deposit number KCTC 11854BP), or mixture thereof.

Description

Novel porcine reproductive respiratory syndrome virus and uses

The present invention provides a novel immunogenic composition for the prevention or treatment of porcine respiratory syndrome (PRRSV), including the novel reproductive respiratory syndrome virus (PRRSV), inactivated porcine respiratory syndrome virus (PRRSV) and mammalian porcine respiratory syndrome using the same It relates to a method of preventing or treating.

Porcine reproductive respiratory syndrome virus (PRRSV) is an enveloped, single-strand-positive sense RNA virus that includes the genus Nidovirales, Arteriviridae, and Arterivirus genus. Belong. PRRSV is a recently developed pathogen, which causes sows miscarriage, stillbirth, and pneumonia and dysplasia in fetuses, low birth rate, low growth rate, other secondary bacterial infections, and respiratory disease in pigs of all ages, both domestically and internationally. Is a contagious disease that has seriously affected pig industry. PRRSV, which was first isolated and reported in 1993 in Korea, preferentially proliferates in macrophages. Its onset forms are genital, respiratory, and mixed, primarily transmitted from pigs to pigs, and can be transmitted to piglets without colostrum antibodies through feces, urine, milk, It can also be transmitted by needles, insects and air.

PRRSV has a wide variety of genetically diverse strains, depending on the geopolitical location at the same time. The two main genotypes are European (genotype 1) and North American (genotype 2), which show 40% nucleotide differences in terms of genome level. The strain that occurs mainly in Korea has been found to be VR2332. According to the study, North American PRRSV has been evolving since it spread to Korean piglets, and it is predicted that genomic alteration of PRRSV gene is caused by geopolitical influence.

The overall genome size of PRRSV is 15 kb and has 9 open reading frames (ORFs) (ORFs 1a, 1b, 2a, 2b, 3-7) with the lower genome mRNA overlapping each other. ORF 1a and 1b are replication related nonstructural proteins and ORF 2-7 constitute structural proteins as GP2a, GP2b, GP3, GP4, GP5, membrane / matrix (M) protein and nucleocapsid (N) protein. The major structural proteins are N protein, M protein and GP5, the primary envelope glycoprotein. Of these, GP5 (ORF 5) protein is the most abundant protein known to be capable of inducing neutralizing antibodies in the host upon infection.

As a method for preventing or treating PRRS, a method of administering a live poison vaccine using attenuated VR2332 strain to pigs has been proposed. However, this method is somewhat less effective, so that the infection of PRRSV may not be successfully controlled and may cause infection by the vaccine strain VR2332.

Globally, farm damage caused by PRRSV infection is not easily reduced despite many efforts. According to the data released by the Korean Association of Veterinary Veterinarians, the total domestic loss due to PRRSV infection as of 2008 is estimated to be 53.3 billion won. The problem is that this enormous damage is occurring despite the enormous use of PRRS imported vaccines. In fact, only two types of vaccines used in Korea are sold, such as live vaccines from multinational companies and killed vaccines from domestic companies. In the case of live poison vaccines, there are many reports on their effects, but due to reports of virus release, there have been questions about safety, and their application in piglets was limited due to interference effects by maternal transfer antibodies. In addition, only one North American virus was used as a vaccine strain for the poisoned vaccine, and the safety of the poisoned vaccine was higher than that of the live vaccine, but the frequent genetic variation of PRRSV had a high effect on the protective ability. The use of a kind of virus as a vaccine has limitations in providing effective immunity. Moreover, despite the increased use of these vaccines, the proportion of monthly PRRS-positive farms from 2008 to 2009 is seldom reduced and remains high.

PRRSV is divided into two subtypes, North America and Europe. Genetic differences of about 40% are observed between North America and Europe, and cross-immunity is not achieved. Individual strategies for strains are needed. Until 2006, only North American states have been known to have been infected in domestic pig farms, and vaccines using North American viruses have been used. Since 2006, however, domestic infections in Europe have been identified. Moreover, European infections, a sporadic form of small infections in 2006, were reported in 2010 joint research data from Green Cross Veterinary Medicine and Seoul National University Veterinary Medicine (Lee et al., Virus genes, 2010, 40). The proportion of infected farmers rose sharply to half.

According to the prior art as described above, there is still a need for a vaccine capable of defending against European-type PRRSV and / or to protect against various North American-type PRRSVs found in Korea.

One embodiment provides novel swine respiratory syndrome virus (PRRSV).

Another embodiment provides an immunogenic composition for the prevention or treatment of high safety and high immunogenicity, including the inactivated swine genital respiratory syndrome virus (PRRSV).

Another embodiment provides a method of effectively preventing or treating a PRRS of a mammal using the composition.

In one embodiment, the novel porcine respiratory syndrome selected from the group consisting of PRRS-GC-6262, PRRS-GC-4019, PRRS-GC-DM, PRRS-GC-4172, PRRS-GC-EU0907, and combinations thereof. Virus (PRRSV) is provided.

These new PRRSVs are all isolated in Korea, and four states, PRRS-GC-6262, PRRS-GC-4019, PRRS-GC-DM and PRRS-GC-4172, have a North American genotype. On the other hand, PRRS-GC-EU0907 has a European genotype.

The coding nucleotide sequences of GP5 of these novel PRRSVs are shown in SEQ ID NOs: 1-5 [PRRS-GC-6262 GP5 coding sequence (VR2332 GP5 standard 1-603): SEQ ID NO: 1, PRRS-GC-4019 GP5 coding sequence (VR2332 GP5 standard) 1-603): SEQ ID NO: 2, PRRS-GC-DM GP5 coding sequence (44-540 based on VR2332 GP5): SEQ ID NO: 3, PRRS-GC-4172 GP5 coding sequence (VR2332 GP5 based 44-540): SEQ ID NO: 4 And PRRS-GC-EU0907 GP5 coding sequence (Lelystad GP5 standard 1-605): SEQ ID NO: 5]. In addition, the results of comparing the nucleotide sequence of the GP5 coding sequence of these novel PRRSV and the GP5 coding sequence of the North American standard strains and European standard strains VR2332 and Lelystad are shown in Table 1.

VR2332 PRRSV-GC-6262 PRRSV-GC-4019 PRRSV-GC-DM PRRSV-GC-4172 PRRSV-GC-EU0907 Lelystad VR2332 - 0.844 0.996 0.877 0.883 0.64 0.637 PRRSV-GC-6262 0.844 - 0.844 0.851 0.85 0.616 0.612 PRRSV-GC-4019 0.996 0.844 - 0.874 0.879 0.64 0.637 PRRSV-GC-DM 0.877 0.851 0.874 - 0.931 0.638 0.635 PRRSV-GC-4172 0.883 0.85 0.879 0.931 - 0.627 0.624 PRRSV-GC-EU0907 0.64 0.616 0.64 0.638 0.627 - 0.988 Lelystad 0.637 0.612 0.637 0.635 0.624 0.988 -

Table 1 shows a comparison of the PRRSV of the invention with the GP5 coding sequences of North American and European standard strains. In Table 1, the figures represent the homology obtained by sequence alignment using CLustal W using Bioedit (North Carolina Univ. NC. USA) software. Since the GP5 moiety plays an important role in neutralizing antibody formation, the sequence homology of GP5 is indicative of whether it is within the range of cross-defense and how the genetic variation has progressed.

As shown in Table 1, all four other states, namely PRRS-GC-6262, PRRS-GC-, except that PRRS-GC-4019 showed 99.6% homology compared to the North American standard state VR2332. DM, PRRS-GC-4172 and PRRS-GC-EU0907 showed homology between 64.0% and 88.3%, confirming that they had significant genetic variation with North American standard strains. These correspond to the week in which the antigenic variation has progressed from the standard strain, and the use of these antigenic mutations as inactivating vaccines can broaden the defense against PRRSV.

In addition, the PRRS-GC-EU0907 showed 98.8% homology compared to the European standard week Lelystad, except that all four other states, PRRS-GC-6262, PRRS-GC-4019, and PRRS-GC-DM And PRRS-GC-4172 showed a homology of 61.2% to 63.7%, confirming that it had a significant genetic variation with European standard strains.

In addition, compared to the five states, PRRS-GC-DM and PRRS-GC-4172 show a homogeneity of 61.6% to 88.3%, except that they have 93.1% homology. It was confirmed to have.

Therefore, these five PRRSV strains correspond to the strains in which the antigenic mutations were advanced between each other and the standard strains, and the range of defense against PRRSV can be broadened by using the strains in which the antigenic mutations were used as inactivating vaccines.

The new PRRSV notes, PRRS-GC-6262, PRRS-GC-4019, PRRS-GC-DM, PRRS-GC-4172, and PRRS-GC-EU0907, as of January 21, 2011, are subject to the provisions of Article 7 of the Budapest Treaty. It was deposited with KCTC (Korean Collection for Type Cultures) under the Korea Institute of Bioscience and Biotechnology, an international depository institution. Access numbers are KCTC 11852BP, KCTC 11850BP, KCTC 11853BP, KCTC 11851BP and KCTC 11854BP.

1 shows a phylogenetic tree based on the homology of the novel PRRSV strain and the GP5 coding sequence of North American and European PRRSV. As shown in FIG. 1, it can be seen that the five new PRRSVs have different groups.

Other embodiments include inactivated PRRS-GC-6262 (accession number KCTC 11852BP), inactivated PRRS-GC-4019 (accession number KCTC 11850BP), inactivated PRRS-GC-DM (accession number KCTC 11853BP), For the prevention or treatment of porcine respiratory syndrome (PRRS) selected from the group consisting of activated PRRS-GC-4172 (Accession No. KCTC 11851BP), inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP) and combinations thereof It provides an immunogenic composition.

The composition can be one or all of the five inactivated PRRSV strains. That is, it can be a combination vaccine containing five PRRSVs. In this specification, the term "immunogenic composition" is used interchangeably with the term "vaccine".

The composition may further comprise a pharmaceutically acceptable carrier or adjuvant. The carrier may comprise one or more components selected from the group consisting of diluents, buffers, preservatives, excipients, disintegrants, binders and lubricants. The adjuvant is a substance used to enhance the immunogenicity of antigens, including mineral salts such as alum, aluminum hydroxide, aluminum phosphate and calcium phosphate; Surfactants and particulates such as nonionic block polymer surfactants (such as cholesterol), virosomes, saponins (such as Quil A, QS-21 and GPI-0100), proteosomes, immune stimulation Complexes, coclate, quaternary amines (dimethyl dioctadecyl ammonium bromide (DDA)), abridine, vitamin A, vitamin E; Bacterial products such as RIBI adjuvant system (Ribi Inc.), Mycobacterum phlei cell wall skeleton (Detox®), muramyl dipeptide (MDP) and tripeptide (MTP), monophosphoryl lipid A, Bacillus Calmete-Guerin (BCG), heat-labile E. coli enterotoxin, cholera toxin, trehalose dimicholate, CpG oligodeoxynucleotides; cytokines and hormones such as interleukin (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte colony stimulating factor, dehydroepiandrosterone, 1,25-dihydroxy vitamin D3; polyanions such as dextran; poly Acrylics (eg, polymethylmethacrylate, Carbopol 934P); carriers such as tetanus and diphtheria toxides, cholera toxin B subunit, mutant heat-labile enterotoxin (rmLT) of endotoxin producing E. coli, heat shock protein ; Oil-in-water oil painting Such as AMPHIGEN® (Hydronics, USA); IMS1312N or Montanide Gel, RehydraGel, and water-in-oil emulsions such as Freund's complete and incomplete adjuvant in the IMS series from Seppic (France). The amount of adjuvant depends on the subject's weight, sex, age, disease severity, virus titer, etc., but may be, for example, 10-15% (w / w). For example, thimerosal, formaldehyde and / or gentamicin.

Inactivation of the novel PRRSV can be accomplished by methods known in the art. The inactivation may be achieved, for example, by passage in a host cell and by treatment with chemicals such as formaldehyde (or formalin) or bromoethylamine hydrobromide. The inactivation may be, for example, by immersing the bulk of the virus in a solution of bromoethylamine hydrobromide (BEA: BrCH ₂ CH ₂ NH ₂ HBr: molecular weight: 204.9 Da). The virus bulk here refers to a state before vaccine production obtained by centrifugation from a virus culture medium prepared for vaccine production. The BEA solution may be 0.0001 to 0.1M, or 0.0005 to 0.01M BEA solution. The inactivation may be to immerse the virus in a BEA solution and leave at 30-45 ° C., for example 37 ° C., with or without stirring. The neglect may be for a suitable time, for example, 10 hours to 24 hours, for example 18 hours. Inactivation with formalin can be achieved, for example, by adding formalin at a concentration of 0.2-0.5%. After inactivation, sodium thiosulfate may be added to neutralize the inactivating agent used. For example, 1M sodium thiosulfate may be added, and it may be neutralized by stirring gradually at 37 degreeC for 1 hour and overnight at 4 degreeC.

If the composition is a mixed vaccine comprising two or more inactivated viruses, the method includes mixing a solution of the inactivated virus. The ratio of mixing the cultures is not limited, but inactivated PRRS-GC-6262 (Accession No. KCTC 11852BP), Inactivated PRRS-GC-4019 (Accession No. KCTC 11850BP), Inactivated PRRS-GC-DM ( Accession No. KCTC 11853BP), Inactivated PRRS-GC-4172 (Accession No. KCTC 11851BP) and Inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP), for example, in a volume ratio of 1: 1.5-3.5: 1.5 -3.5: 1.5-3.5: 1.5-3.5: 1.5-3.5, for example, 1: 2: 2: 2: 2. In addition, immunopotentiators such as IMS1312N (Seppic, France) or RehydraGel can be added to enhance the individual's immunity.

Swine Respiratory Syndrome (PRRS) can include any of a variety of symptoms associated with reproductive and respiratory diseases in pigs caused by PRRSV, including, for example, similar acid in sows and pulmonary cyanosis in fetuses, lower parity, and other secondary Symptoms are caused by bacterial infection.

The immunogenic compositions of the invention can be in any form known in the art, for example in the form of solutions and injections. In the case of solutions or injections, it may contain 10-40% propylene glycol, if necessary, and an amount sufficient to prevent hemolysis, such as about 1% sodium chloride. The solutions or injections may include any diluent or buffer known in the art. In addition, the immunogenic composition of the present invention can be prepared immediately before use by lyophilizing and preserving in a container such as a vial and adding a carrier or adjuvant, saline, etc. required for injection before use.

The immunogenic compositions of the invention can be administered by any means of administration known in the art. For example, the administration can be administered directly intravenously, intramuscularly, orally, transdermal, mucosal, intranasal, intratracheal or subcutaneous. The immunogenic composition can be administered systemically or locally.

In the immunogenic composition of the present invention, the mammal may be human, cow, pig, goat, dog, sheep and the like.

The present invention provides a method for preventing or treating a PRRS of a mammal, comprising administering the immunogenic composition to a mammal other than a human.

In the method of the present invention, the mammal may be cattle, pigs, goats, dogs, sheep and the like.

In the methods of the invention, the immunogenic compositions of the invention can be administered by any means of administration known in the art. For example, the administration can be administered directly intravenously, intramuscularly, oral, transdermal, mucosal, nasal, intratracheal or subcutaneous. The strain or immunogenic composition may be administered systemically or locally.

In the immunogenic compositions or methods of the invention, the immunogenic compositions of the invention may be administered in an effective amount. "Effective amount" means the amount required to alleviate or eliminate the condition to be treated or prevented and may be appropriately selected by those skilled in the art. For example, a therapeutically or prophylactically effective amount may comprise from 1 × 10 ^4.5 to 1 × 10 ^6.5 TCID ₅₀ / ml, for example 1 × 10 ^5.0 to 1 × 10 ^6.0 TCID ₅₀ / ml of the inactivated PRRSV per unit dose. For example, the composition may be inactivated PRRS-GC-6262 (Accession No. KCTC 11854BP), inactivated PRRS-GC-4019 (Accession No. KCTC 11850BP), with a viral titer of 1 × 10 ^6.0 TCID ₅₀ / ml, respectively. 10 vol%: 20 volumes of activated PRRS-GC-DM (accession number KCTC 11853BP), inactivated PRRS-GC-4172 (accession number KCTC 11851BP) and inactivated PRRS-GC-EU0907 (accession number KCTC 11854BP) %: 20% by volume: 20% by volume: 20% by volume, 10% by volume montanide gel ^TM (SEPPIC, Paris, France). montanide gel ^TM (SEPPIC, Paris, France) is a composition consisting of a synthetic polymer and an aqueous gel.

According to the PRRSV strain according to the present invention, since the GP5 coding sequence is different from that of the conventionally known North American strain and European strain, it can be used to prepare an immunogenic composition.

According to the immunogenic composition for the prevention or treatment of porcine respiratory syndrome (PRRS) according to the present invention, it is possible to prevent or treat porcine respiratory syndrome (PRRS) in an individual. In particular, since a strain having antigenic variability was used as compared to the standard strain, it can protect against a wide range of PRRSV.

According to the method for preventing or treating a PRRS of a mammal according to the present invention, it is possible to prevent or treat pig respiratory syndrome (PRRS) in an individual.

1 shows a phylogenetic tree based on the homology of the novel PRRSV strain and the GP5 coding sequence of North American and European PRRSV.
2 is a view showing the results of measuring the neutralizing antibody titers in the vaccine group and the control group.

Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited by the following examples.

Example : Preparation of Mixed Vaccine and Confirmation of Effect

In this embodiment, PRRS-GC-6262 (accession number KCTC 11852BP), PRRS-GC-4019 (accession number KCTC 11850BP), PRRS-GC-DM (accession number KCTC 11853BP), PRRS-GC-4172 (accession number KCTC 11851BP) ) And PRRS-GC-EU0907 (Accession No. KCTC 11854BP) were cultured, propagated and inactivated, respectively, to prepare an immunogenic composition comprising five inactivated viruses. Next, the effect on the PRRS of the composition was confirmed.

(1) used viruses

Viruses used in the combined vaccine were PRRS-GC-6262 (Accession No. KCTC 11852BP), PRRS-GC-4019 (Accession No. KCTC 11850BP), PRRS-GC-DM (Accession No. KCTC 11853BP), PRRS- isolated from domestic farms. GC-4172 (Accession No. KCTC 11851BP) and PRRS-GC-EU0907 (Accession No. KCTC 11854BP) were used. These strains were isolated from veterinary laboratories of Green Cross Pharmaceutical Co., Ltd. by inoculating an emulsion of tissues confirmed to be infected with cells susceptible to PRRSV.

(2) Of master seed virus  Passage method

(2.1) Cultivation and Maintenance of Host Cells

Culture and maintenance of MARC-15, the host cell of these PRRSV, was performed as follows. The dispersed MARC-145 cells were digested with trypsin solution (Trypsin EDTA, Gibco, Cat. No. 15400-054), suspended in proliferation medium, subdivided into culture vessels, and then cultured at 37 ° C. for 2-3 days. It was used for subculture after 3 days of culture. The medium for MARC-145 cell proliferation is 900 ml α-MEM, 100 ml FBS (fetal serum albumin), 1% antibiotic-antimycotics (PAA, Cat.NO.p11-002), non-essential amino acids (NEAA) Consisting of 1%, 1% L-glutamine and 1% 7% sodium bicarbonate solution, adjusted to pH 7.0-7.4, MARC-145 cells have high tolerance to PRRSV of MA-104 cells, monkey kidney cells. It is derived from the PRRSV high permissive subpopulation.

(2.2) Virus Culture and Passage

Protozoa was inoculated with a virus inoculated to cells formed with a monolayer of about 80% or more of the virus isolated from the Veterinary Research Institute of Green Cross Pharmaceuticals Co., Ltd. after sensitization at 37 ° C. for 1 to 2 hours, and cultured for 72 hours. When more than 80% of the cytopathic effect (CPE) appeared, the culture was aseptically lyophilized by freezing or stored at -80 ℃. Seed virus was inoculated, cultured and harvested in the same manner as the original poison and lyophilized or frozen at -80 ° C. The maintenance medium was 1000 ml α-MEM, 20 ml FBS, 1% antibiotic-antimycotics (PAA, Cat. No. p11-002), 1% non-essential amino acids (NEAA), 1% L-glutamine and It consists of 1% of 7% sodium bicarbonate solution.

Viruses produced in bulk were not passaged more than five times in the original venom. Inoculation, incubation, and harvesting were carried out in the same manner as the original poison and stored at −80 ° C. and used for vaccine production.

Culture of the virus was performed as follows. After removing the medium from the cell culture vessel in which the monolayer was formed, each virus was inoculated at 10 ³ TCID ₅₀ / ml and adsorbed for 1 hour. After 1 hour, the cell monolayers were washed with PBS, and then, maintenance medium was added thereto, and the cells were left incubated for 3 to 4 days at 37 ° C.

Harvesting of the virus was carried out as follows. Virus-inoculated cells were cultured for 3 to 4 days to harvest infected cells, centrifuged at 2,500 rpm for 15 minutes, and the precipitated cells were resuspended in PBS at 1/10 of the culture solution, followed by freezing and thawing three times. . After complete destruction of these cells, the supernatant was harvested by centrifugation at 2,500 rpm for 15 minutes. Virus titers were PRRS-GC-4019 10 ^6.0 TCID ₅₀ / ml, PRRS-GC-6262 10 ^6.0 TCID ₅₀ / ml, PRRS-GC-4172 10 ^6.0 TCID ₅₀ / ml, PRRS-GC-DM 10 ^6.0 TCID ₅₀ / ml , PRRS-GC-EU0907 10 ^6.0 TCID ₅₀ / ml or more was confirmed.

(3) vaccine manufacturing

Inactivation of the virus bulk was performed as follows. First, BEA (bromoethylamine hydrobromide: BrCH ₂ CH ₂ NH ₂ HBr, molecular weight 204.9 Da) (Sigma B 9258) was used as an inactivating material. BEA was dissolved in a 0.2 N NaOH solution (8 g / l) heated to 37 ° C. to a concentration of 0.1 M (20.49 g of BEA was added to 1 L of 0.2 N NaOH solution). Shake well for 1 hour in a 37 ℃ constant temperature water bath to complete the BEI (Binary Ethylenimine) solution. 0.1 M BEI solution was added to the virus bulk at 1% (100-fold dilution) to a final concentration of 0.001 M (1 mM). Inactivation was performed at 37 ° C. for about 18 hours with stirring of the magnetic bar. After cooling to 4 ° C., sodium thiosulfate was added to a final concentration of 2 mM to neutralize the action of BEI (2M of sodium thiosulfate, 31.62 g of sodium thiosulfate, in 100 ml of distilled water. The solution dissolved in sterilized and then diluted 1000-fold was used). 1% thimerosal solution was added to a final concentration of 0.01%. In this way, each of the five PRRSVs described above was inactivated.

Next, each inactivated PRRSV and adjuvant were mixed to prepare a mixed vaccine. First, the inactivated PRRSV antigen was centrifuged at 3,000 rpm for 30 minutes to extract the supernatant. Virus titers in the obtained supernatant were all 10 ^6.0 TCID ₅₀ / ml or more. Inactivated PRRS-GC-4019, Inactivated PRRS-GC-6262, Inactivated PRRS-GC-DM, Inactivated PRRS-GC-4172, Inactivated, all with titers of 10 ^6.0 TCID ₅₀ / ml or higher Montanide gel ^TM , a 10% volume synthetic polymer gel for 20 minutes while mixing PRRS-GC-EU0907 at a volume ratio of 10: 20: 20: 20 and stirring with a magnetic bar Was added. The mixed vaccine obtained by mixing for about 1 hour was dispensed into an appropriate container and sealed.

(4) Safety test of vaccine

Eight healthy 15-20 g mice, four guinea pigs weighing 300-350 g, and two healthy weaning pigs with negative antibody to porcine respiratory syndrome virus were disclosed. Eight mice were subcutaneously vaccinated with 0.5 ml of vaccine, and four guinea pigs were subcutaneously and intraperitoneally inoculated at 2 ml, respectively, and observed for 7 days to determine that they survived abnormally. Two heads (2ml) of two weaning pigs were observed for 21 days after inoculation and determined to be safe when surviving without abnormalities such as purulent injection, necrosis, fever, and respiratory symptoms. Table 2 shows the safety test results for experimental animals.

Table 3 shows the safety test results for pigs.

In Table 3, * indicates changes in body temperature, dyspnea, depression, vomiting, swelling of inoculation sites, purulent inoculation sites, and anorexia.

(5) of the vaccine Activity test

The potency test was carried out according to the following Test 1 or Test 2.

Exam 1 :

Ten guinea pigs weighing 300-350 g were used. Eight guinea pigs were intramuscularly injected with 1.0 ml of the vaccine and inoculated again by 1.0 ml two weeks later, and two weeks later, blood samples were collected with two control groups to measure neutralizing antibody titers. The test method is as follows. First, serum was isolated and serum was inactivated at 56 ° C. for 30 minutes in a medium (α-MEM 1000ml, FBS 20ml, antibiotic-antifungal 1% (Antibiotic / Antimycotic, PAA, Cat. No. p11-002), non-essential amino acids. (NEAA) consisting of 1%, 1% L-glutamine, and 1% 7% sodium bicarbonate solution), followed by homogenous mixing with swine Respiratory Syndrome Virus (300 TCID ₅₀ /0.1ml) at 48 ° C. After neutralizing for over an hour, 50 μl of the medium containing fresh guinea pig serum (3 to 5%) was added, followed by reaction at 37 ° C. for 60 minutes. After 50 days of culture medium containing 3% FBS containing MARC-145 cells, MA-104 cells, or cells susceptible to Swine Respiratory Syndrome Syndrome virus, the cells were cultured at 37 ° C for 5 days, or the cells were denatured. Was fixed in 80% acetone and the antibody titer was measured by an indirect immunoflorescence assay (IFA).

Neutralizing antibody titers to Swine Respiratory Syndrome Virus should be determined to be positive if at least 80% of the inoculated animals are at least 8 fold, and the control should be negative.

Table 4 shows the results of potency tests in test animals conducted in accordance with Test 1.

The five species used as controls in Table 4 represent phosphate buffered saline (PBS) solutions.

(6) defense against immunization and challenge vaccination

Pig groups used in the challenge test were assigned as follows. Three week old pigs who did not eat colostrum or were negative for the PRRS virus were assigned to the vaccine-vaccinated group, the vaccine control group and the inoculation control group, three heads each.

Vaccination was performed as follows. Vaccine containing 1 ml of 5 inactivated PRRSV of the present invention was intramuscularly injected into the 3 week old vaccine group and subjected to the second inoculation at 5 week old. At this time, the inoculation control was inoculated in the same manner with a medium containing no virus.

Field viral inoculation was performed as follows. Four weeks after the last vaccination, when the pigs were 9 weeks old, they were challenged with the field virus isolates. The challenge inoculation virus was PRRSV-GC-4019, which was not inactivated as the field isolate, and 2 ml of the nasal cavity was inoculated with a titer of 10 ⁴ TCID ₅₀ / ml. The vaccine group and the inoculation control group were challenged, respectively, and the vaccine control group was inoculated intranasally with the same amount of medium.

Blood antibody concentrations of challenge inoculated pigs were measured. First, blood was secured through blood collection at intervals of two weeks after the vaccination date. Blood samples were collected from 3, 5, 7, 9, and 11 weeks old heads and neutralized antibodies were measured using the Serum Neutralization test (SN test).

2 is a view showing the results of measuring the neutralizing antibody titers in the vaccine group and the control group. As shown in Figure 2, it was confirmed that the neutralizing antibody titer in the vaccine group.

In addition, the clinical symptoms were observed in pigs after challenge inoculation and measured whether the virus was released. Specifically, the body temperature and clinical symptoms were observed at 2 days interval after challenge inoculation. Observations were made for each group of individuals, and if there were any specificities, they were immediately recorded. Viral excretion detected antigens of the virus in nasal secretions using RT-PCR for 14 days after challenge. RT-PCR uses a total RNA isolated from nasal secretions as a template, performs a reverse transcription reaction using a random hexamer as a reverse transcription primer, uses the obtained cDNA as a template, a forward primer (SEQ ID NO: 6) and a reverse primer (SEQ ID NO: 6). PCR was performed using No. 7) as a primer.

Table 5 and Table 6 show the results of observing clinical symptoms for the vaccine group and the control group, respectively.

From the above results, the immunogenic composition of the present invention had the effect of defending the individual against a wider range of PRRSV than the known vaccine.

Korea Research Institute of Bioscience and Biotechnology KCTC11852BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11850BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11853BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11851BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11854BP 20110121

<110> Green Cross Veterinary Products Co., Ltd. <120> Novel porcine reproductive respiratory syndrome virus and uses          the <130> PN092741 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 603 <212> DNA <213> PRRS-GC-6262 <400> 1 atgttgggga aatgcttgac cgcgggttgt tgctcgcaat tgcctttttt gtggtgtatc 60 gtgccgttct gttttgctgc gctcgtcaac gccagcagca acagcagctc ccatttacag 120 ttgatatata acttgacgat atgcgagctg aatggcacag agtggttaaa tgaaaagttt 180 ggctgggcag tggaaacgtt cgtcatcttt cctgtgttga ctcatattgt ttcctacggc 240 gccctcacca ccagccatct ccttgacaca gttggtctga tcactgtgtc caccgccgga 300 tattaccatg ggcggtatgt cttgagtagt gtctatgctg tttgcgcctt ggctgcgtta 360 acttgcttcg tcattagatt gacaaaaaac tgcatggctt ggcgctactc gtgtaccaga 420 tacactaact ttcttttgga caccaagggc agactctatc gctggcgatc acccgtcatc 480 atagagaaag ggggtaaggt tgaggttggg ggtcacctaa tcgacctcaa gagagtcgtg 540 cttgatggtt ccgcggcaac ccctataacc aaagtttcgg cggaacaatg gggtcgtccc 600 tag 603 <210> 2 <211> 603 <212> DNA <213> PRRS-GC-4019 <400> 2 atgttggaga aatgcttgac cgcgggctgt tgctcgcaat tgctttcttt gtggtgtatc 60 gtgccgttct gttttgctgt gctcgccaac gccagcaacg acagcagctc ccatctacag 120 ctgatttaca acttgacgct atgtgagctg aatggcacag attggctagc taacaaattt 180 gattgggcag tggagagttt tgtcatcttt cccgttttga ctcacattgt ctcctatggt 240 gccctcacta ccagccattt ccttgacaca gtcgctttag tcactgtgtc taccgccggg 300 tttgttcacg ggcggtatgt cctaagtagc atctacgcgg tctgtgccct ggctgcgttg 360 acttgcttcg tcattaggtt tgcaaagaat tgcatgtcct ggcgctacgc gtgtaccaga 420 tataccaact ttcttctgga cactaagggc agactctatc gttggcggtc gcctgtcatc 480 atagagaaaa ggggcaaagt tgaggtcgaa ggtcatctga tcgacctcaa aagagttgtg 540 cttgatggtt ccgtggcaac ccctataacc agagtttcag cggaacaatg gggtcgtcct 600 tag 603 <210> 3 <211> 540 <212> DNA <213> PRRSV-GC-DM <400> 3 tttttttgtg gtatatcgtg ccgtcctgtt ttgttgcgct cgtcagcgcc agcaacaaca 60 gcagctccaa atttcagttg atttataact tgacactatg cgagctgaac ggcacagact 120 ggctagccag taagtttgat tgggcagttg agaccttcgt catttttccc gtgttgactc 180 acattgtctc ctatggtgcc cttactacta gccatttcct tgacacagct ggtctggcca 240 ctgtgtctac cgccggttat tatcatgggc ggtatgtttt gagtagcatc tacgcggtct 300 gtgccctggc tgcgttgatt tgtttcatca tcaggttcgt gaaaaattgc atgtcttggc 360 gctactcatg taccaggtat accaactttc ttctggacac caagggcaga ctctatcgtt 420 ggcgttcacc tgtcatcata gagaaagggg gtaaagttga ggtcgaaggt catctaatcg 480 atctcaagag agttgtgctt gacggttccg cggcaacccc tgtaaccaaa gtttcagcgg 540                                                                          540 <210> 4 <211> 540 <212> DNA <213> PRRSV-GC-4172 <400> 4 tttttttgtg gtgtatcgtg ccgtcctgtt ttgttgcgct cgtcagcgcc aacaacagca 60 gcagctccca tttgcagttg atttataatt tgacgctatg cgagctgaac ggcacagatt 120 ggctagccga caaattttcc tgggcagttg agacttttgt catctttccc gtgttgaccc 180 acatcgtctc ttatggtgcc ctcactacta gccatttcct tgacacagtt ggtctggtca 240 ctgtgtctgc cgccggttat tatcacaggc ggtatgtttt gagtagcatt tacgcggtct 300 gtgccctggc tgcgttaatt tgtttcatca tcaggttcgt gaaaaactgc atgtcctggc 360 gctactcatg taccaggtat accaactttc ttctggacac caagggcaga ctctatcgtt 420 ggcgctcacc tgtcatcata gagagagggg gtaaagttga ggtcgaaggt catctgatcg 480 acctcaagag agttgtgctt gatggttccg cggcaacccc tgtaaccaaa gtttcagcgg 540                                                                          540 <210> 5 <211> 605 <212> DNA <213> PRRSV-GC-EU0907 <400> 5 atgagatgtt ctcacaaatt ggggcgtttc ttgactccgc actcttgctt ctggtggttt 60 tttttgctgt gtaccggctt gtcctggtcc tttgccgatg gcaacggcaa cagctcgaca 120 taccaataca tatataactt gacgatatgc gagctgaatg ggaccgactg gttgtccagc 180 cattttggtt gggcagtcga gacctttgtg ttttacccgg ttgccactca tatcctctca 240 ctgggttttc tcacaacaag ccattttttt gacgcgctcg gtctcggcgc tgtatccact 300 gcaggatttg ttggcgggcg gtatgtactc tgcagcgtct acggcgcttg tgctttcgca 360 gcgttcgtat gttttgtcat ccgtgctgct aaaaattgca tggcctgccg ctatgcccgt 420 acccggttta ccaacttcat tgtagacaac cgggggagag ttcatcgatg gaagtctcca 480 atagtggtag aaaaattggg caaagccgaa gtcgacggca acctcgtcac catcaaacat 540 gtcgtcctcg aaggggttaa agctcaaccc ttgacgagga cttcggctga gcaatgggag 600 gccta 605 <210> 6 <211> 20 <212> DNA <213> PRRSV-GC-4019 <400> 6 gtacattctg gcccctgccc 20 <210> 7 <211> 20 <212> DNA <213> PRRSV-GC-4019 <400> 7 gccctaattg aataggtgac 20

Claims (6)

PRRS-GC-6262 (accession number KCTC 11852BP), PRRS-GC-4019 (accession number KCTC 11850BP), PRRS-GC-DM (accession number KCTC 11853BP), PRRS-GC-4172 (accession number KCTC 11851BP) and PRRS- Novel swine respiratory syndrome virus (PRRSV) selected from the group consisting of GC-EU0907 (Accession No. KCTC 11854BP) and combinations thereof. Inactivated PRRS-GC-6262 (Accession No. KCTC 11852BP), Inactivated PRRS-GC-4019 (Accession No. KCTC 11850BP), Inactivated PRRS-GC-DM (Accession No. KCTC 11853BP), Inactivated PRRS- Immunogenic composition for the prevention or treatment of porcine respiratory syndrome (PRRS) selected from the group consisting of GC-4172 (Accession No. KCTC 11851BP), Inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP) and combinations thereof. The inactivated PRRS-GC-6262 (accession number KCTC 11852BP), the inactivated PRRS-GC-4019 (accession number KCTC 11850BP), the inactivated PRRS-GC-DM (accession number KCTC 11853BP). , Inactivated PRRS-GC-4172 (Accession No. KCTC 11851BP) and inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP). The inactivated PRRS-GC-6262 (accession number KCTC 11852BP), the inactivated PRRS-GC-4019 (accession number KCTC 11850BP), the inactivated PRRS-GC-DM (accession number KCTC 11853BP). Inactivated PRRS-GC-4172 (Accession No. KCTC 11851BP) and Inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP) are contained in a volume ratio of 10: 20: 20: 20: 20, each with a titer of 1 × 10 ^4.5 to 1 × 10 ^6.5 TCID ₅₀ / ml. The inactivated PRRS-GC-6262 (Accession Number KCTC 11852BP), Inactivated PRRS-GC-4019 (Accession Number KCTC 11850BP), Inactivated PRRS-GC. -DM (Accession No. KCTC 11853BP), Inactivated PRRS-GC-4172 (Accession No. KCTC 11851BP) and Inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP) were inactivated by bromoethylamine hydrobromide Composition. A method for preventing or treating a PRRS of a mammal, comprising administering the immunogenic composition according to any one of claims 1 to 5 to a mammal other than a human.

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