Porcine circovirus infection-preventing vaccine composition effective against PCV2b and PCV2d

Abstract

The present invention relates to a porcine circovirus infection-preventing vaccine composition in which the amount of an antigen is adjusted. The vaccine composition according to the present invention has been verified to be safe, and, unlike existing patents, has also been identified to effectively induce protective immunity against PCV2d challenge vaccination in pigs (SPF minipigs) which are target animals. In addition, it has been identified that animals inoculated with the vaccine composition of the present invention have significantly reduced PCV2-related lesions. As such, effective protection against currently prevalent porcine circovirus is indicated, and thus the present invention can be widely utilized in the fields of porcine circovirus infection prevention and pig farming.

Description

Vaccine composition for preventing porcine circovirus infection effective against PCV2b and PCV2d

[0001] The present invention relates to a vaccine composition for preventing porcine circovirus infection with controlled antigen content.

[0002] Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). Pigs infected with PCV2 are characterized by chronic wasting, pale skin, and enlarged lymph nodes. They also exhibit respiratory symptoms such as dyspnea and chronic pneumonia, as well as digestive symptoms such as diarrhea and gastric ulcers. Furthermore, PCV2 infection results in a high mortality rate (~50%) in weaned piglets, resulting in significant economic losses worldwide. PCV2 is also associated with numerous other infections, but it is highly resistant to the environment and organic compounds, making it resistant to common disinfectants. Therefore, vaccination is known as the only alternative.

[0003] Accordingly, the inventors of the present invention developed a vaccine composition for preventing porcine circovirus infection and confirmed that antibodies against PCV2 were produced excellently in target animals vaccinated with the vaccine composition, thereby completing the present invention.

[0004] Accordingly, the purpose of the present invention is to provide a vaccine composition for preventing porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2 (PCV2); and an excipient; wherein the mixing ratio of the recombinant protein derived from porcine circovirus type 2 and the excipient is 0.1-10:1 by volume.

[0005] Another object of the present invention is to provide a method for preventing porcine circovirus infection, comprising the step of inoculating a vaccine composition into a pig.

[0006] Another object of the present invention is to provide a pharmaceutical composition for preventing diseases caused by porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2; and an excipient; wherein the mixing ratio of the recombinant protein derived from porcine circovirus type 2 and the excipient is 0.1-10:1 by volume.

[0007] Another object of the present invention is to provide a method for preventing or treating a disease caused by porcine circovirus infection, comprising the step of administering to a subject in need thereof a composition comprising a porcine circovirus type 2-derived recombinant protein and an excipient, wherein the mixing ratio of the porcine circovirus type 2-derived recombinant protein and the excipient is 0.1-10:1 by volume.

[0008] To achieve the above purpose, the present invention provides a vaccine composition for preventing porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2; and an excipient; wherein the mixing ratio of the recombinant protein derived from porcine circovirus type 2 and the excipient is 0.1-10:1 by volume.

[0009] The present invention also provides a method for preventing porcine circovirus infection, comprising the step of inoculating a vaccine composition into a pig.

[0010] In addition, the present invention provides a pharmaceutical composition for preventing a disease caused by porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2; and an excipient; wherein the mixing ratio of the recombinant protein derived from porcine circovirus type 2 and the excipient is 0.1-10:1 by volume.

[0011] In addition, the present invention provides a method for preventing or treating a disease caused by porcine circovirus infection, comprising the step of administering to a subject in need thereof a composition comprising a porcine circovirus type 2-derived recombinant protein and an excipient, wherein the mixing ratio of the porcine circovirus type 2-derived recombinant protein and the excipient is 0.1-10:1 by volume.

[0012] The vaccine composition according to the present invention not only demonstrated safety but also effectively induced protective immunity against PCV2 in pigs, the target animal. Furthermore, individuals vaccinated with the vaccine composition of the present invention demonstrated a significant reduction in PCV2-related lesions. This indicates that the composition effectively protects against the recently prevalent porcine circovirus, and thus has diverse applications in the prevention of porcine circovirus infection and in pig farming.

[0013] Figure 1 is a diagram showing a vaccination schedule and evaluation schedule for evaluating the efficacy and PCV2d challenge vaccination protection ability of a vaccine composition according to the present invention in pigs.

[0014] Figure 2 is a diagram showing the results of measuring body weight by group according to vaccination with the vaccine composition of the present invention and PCV2d challenge vaccination.

[0015] Figure 3 is a diagram showing the results of calculating the average daily weight gain (ADWG) according to the vaccination with the vaccine composition of the present invention and the PCV2d challenge vaccination.

[0016] Figure 4 is a diagram showing the results of measuring PCV2b-specific IgG antibody titers following vaccination with the vaccine composition of the present invention and PCV2d challenge vaccination.

[0017] Figure 5 is a diagram showing the results of measuring PCV2d-specific IgG antibody titers following vaccination with the vaccine composition of the present invention and PCV2d challenge vaccination.

[0018] Figure 6 is a diagram showing the results of measuring PCV2b neutralizing antibodies following vaccination with the vaccine composition of the present invention and PCV2d challenge vaccination.

[0019] Figure 7 is a diagram showing the results of measuring PCV2d neutralizing antibodies following vaccination with the vaccine composition of the present invention and PCV2d challenge vaccination.

[0020] Figure 8 is a diagram showing the results of measuring the amount of IFN-γ secreted following vaccination with the vaccine composition of the present invention and PCV2d challenge vaccination.

[0021] Figure 9 is a diagram showing the results of detecting PCV2 viral DNA in serum samples following inoculation with the vaccine composition of the present invention and challenge inoculation with PCV2d.

[0022] Figure 10 is a diagram showing the results of detecting PCV2 viral DNA in a nasal sample following inoculation with the vaccine composition of the present invention and challenge inoculation with PCV2d.

[0023] Figure 11 is a diagram showing the results of detecting PCV2 viral DNA in a workplace sample following inoculation with the vaccine composition of the present invention and PCV2d challenge inoculation.

[0024] Figure 12 is a diagram showing the results of detecting PCV2 viral DNA in lung samples following inoculation with the vaccine composition of the present invention and PCV2d challenge inoculation.

[0025] Figure 13 is a diagram showing the results of detecting PCV2 viral DNA in a lymph node sample following inoculation with the vaccine composition of the present invention and challenge inoculation with PCV2d.

[0026] Hereinafter, the present invention will be described in detail.

[0027] According to an aspect of the present invention, the present invention provides a vaccine composition for preventing porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2 (PCV2); and an excipient; wherein the mixing ratio of the recombinant protein derived from porcine circovirus type 2 and the excipient is 0.1-10:1 by volume.

[0028] In the present invention, the 'vaccine' is a veterinary vaccine containing an antigenic substance, and is administered for the purpose of inducing specific active or passive immunity against porcine circovirus.

[0029] In a specific example of the present invention, the mixing ratio of the porcine circovirus type 2-derived recombinant protein and the excipient is more preferably a volume ratio of 0.5 to 5:1, and most preferably a volume ratio of 1:1.

[0030] The recombinant protein derived from the porcine circovirus type 2 can be produced as follows. The ORF2 gene of PCV2d DNA isolated from porcine serum is inserted into the pFastBac1 vector (Gibco, USA) to produce recombinant Bacmid DNA, which is then transfected into ExpiSf9 cells (Gibco) to produce a recombinant baculovirus.

[0031] In a specific embodiment of the present invention, the recombinant protein derived from porcine circovirus type 2 preferably includes an amino acid sequence represented by SEQ ID NO: 1, and more preferably may be composed of an amino acid sequence represented by SEQ ID NO: 1.

[0032] The PCV2-derived recombinant protein of the present invention may include any polypeptide having at least 70%, 80%, 90%, 95%, 98%, and preferably at least 99% homology with the amino acid sequence of SEQ ID NO: 1. "Homology" refers to a measure of similarity between protein or polynucleotide sequences. These polypeptides may have deletions, additions, or substitutions of at least one amino acid compared to the amino acid sequence of SEQ ID NO: 1. The degree of homology between two sequences, as scored, is based on the percentage of identities and / or preservation of sequence substitutions.

[0033] When the recombinant proteins of the present invention are used in the production of a vaccine, they may be added to the vaccine in a purified protein form or may be included in a lysate form that is not purified after being expressed in E. coli, animal cells, or insect cells.

[0034] In a specific embodiment of the present invention, the recombinant protein derived from porcine circovirus type 2 may be included at a concentration of 0.1 to 1000 μg / dose, preferably at a concentration of 0.5 to 100 μg / dose, more preferably at a concentration of 1 to 50 μg / dose, still more preferably at a concentration of 10 to 30 μg / dose, and most preferably at a concentration of 20 μg / dose.

[0035] The vaccine composition of the present invention may include an adjuvant mixture and one or more pharmaceutically or veterinarily acceptable carriers, excipients or diluents as an immunopotentiator that enhances the immunogenicity of the vaccine and can induce protective immunity even with minimal administration. The term "pharmaceutically or veterinarily acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions when administered to an animal. Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives may be included. Suitable carriers for use include, but are not limited to, aqueous media including saline, phosphate buffered saline, minimal essential medium (MEM), or MEM in HEPES buffer.

[0036] In a specific embodiment of the present invention, the excipient is preferably IMS1313, an IMS series excipient, considering safety and immune persistence. In addition, the IMS1313 may have a concentration of 10 to 90% (v / v), preferably 30 to 70% (v / v), and most preferably 50% (v / v).

[0037]

[0038] The vaccine composition of the present invention may include one or more immunostimulants suitable for forming the vaccine composition.

[0039] An adjuvant that may be included in the composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art. The adjuvants include, but are not limited to, Freund's complete and incomplete adjuvants, vitamin E, non-ionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and mineral-free oil, Carbopol, water-in-oil emulsion adjuvants, and the like.

[0040] Additionally, the vaccine composition of the present invention can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. The formulation can be in the form of powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders, etc. The vaccine composition of the present invention can be administered via the intramuscular, subcutaneous, intradermal, transdermal, intravenous, intranasal, intraperitoneal, or oral routes, and is preferably administered via the intramuscular or subcutaneous route. The dosage of the vaccine can be appropriately selected depending on various factors such as the route of administration, the age, sex, weight, and severity of the animal.

[0041] Additionally, the vaccine composition of the present invention can be prepared by standard methods in the art, except for methods specific to the present invention, such as the preparation of a PCV2-derived recombinant protein. For example, the organism can be grown in a culture medium, such as complete medium, and the growth of the organism can be monitored using standard techniques, such as measuring color change units (CCU), and harvested when a sufficiently high titer is achieved. The stock can be further concentrated or lyophilized by conventional methods before being incorporated into a vaccine for formulation.

[0042]

[0043] According to another aspect of the present invention, the present invention provides a method for preventing porcine circovirus infection, comprising the step of inoculating a vaccine composition into a pig.

[0044] The above pigs may include, without limitation, individuals susceptible to porcine circovirus infection, and such infection prevention methods may be utilized in conjunction with other treatment or prevention methods known in the art. The term "vaccination" as used herein refers to administering a predetermined composition of the present invention to an individual by any suitable method.

[0045] In a specific embodiment of the present invention, the vaccine composition can be administered by one or more methods selected from the group consisting of intramuscular, subcutaneous, intradermal, transdermal, intravenous, intranasal, intraperitoneal, and oral.

[0046] The vaccine composition according to the present invention can be administered once at 1 to 5 weeks of age, and more preferably, once at 3 weeks of age.

[0047] The purpose of the preventive method of the present invention is to prevent the outbreak of an infectious disease that can be caused by the porcine circovirus in pigs.

[0048]

[0049] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing a disease caused by porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2 (PCV2); and an excipient; wherein the mixing ratio of the recombinant protein derived from porcine circovirus type 2 and the excipient is 0.1-10:1 by volume.

[0050] In a specific example of the present invention, the diseases caused by the porcine circovirus infection include Porcine Respiratory Disease Complex (PRDC), enzootic pneumonia (EP), postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), sow abortion and mortality syndrome (SAMS), Porcine Reproductive and Respiratory Syndrome (PRRS), pseudorabies, Glasser's disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietary hepatosis, and suppurative bronchopneumonia. Any one or more selected from the group consisting of bronchopneumonia, Eustachian tube inflammation, polyserositis, mycoplasmal pneumonia, and pleuropneumonia may be applied without limitation as long as it is a disease or illness caused by porcine circovirus infection.

[0051] The pharmaceutical composition of the present invention is preferably administered orally or parenterally.

[0052] In the case of oral administration, including oral administration, the pharmaceutical composition of the present invention can be administered orally in any orally acceptable form, including but not limited to pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions.

[0053] For oral tablets, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When an aqueous suspension is administered orally, the active ingredient is combined with emulsifying and suspending agents. Sweetening and / or flavoring and / or coloring agents may be added, if desired.

[0054] Pharmaceutical compositions for oral administration can be prepared by mixing the active ingredient with solid excipients, and can be prepared in the form of granules for preparation in the form of tablets or dragees. Suitable excipients include sugars such as lactose, sucrose, mannitol, and sorbitol; carbohydrates such as starch, methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose from corn, wheat flour, rice, potatoes, or other plants; gums including gum arabic and tagacanth; or protein fillers such as gelatin or collagen. If necessary, disintegrating or solubilizing agents in the form of cross-linked polyvinylpyrrolidone, agar, and alginic acid or sodium alginate, respectively, can be added.

[0055] In the present invention, 'parenteral' includes subcutaneous, intradermal, intranasal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0056] The pharmaceutical composition of the present invention may be in the form of a sterile injectable aqueous or oily suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Acceptable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, a sterile fixed oil is typically used as a solvent or suspending medium. For this purpose, any fixed oil with minimal irritation, including synthetic mono- or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in injectable formulations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), particularly their polyoxyethylated forms. Furthermore, for parenteral administration, the pharmaceutical compositions of the present invention may be formulated as aqueous solutions. Preferably, physically suitable buffers such as Hank's solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions may incorporate substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active ingredient may be formulated as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Polycationic amino polymers can also be used as carriers.Optionally, the suspension may contain suitable stabilizers or agents to increase the solubility of the compound and to prepare a highly concentrated solution.

[0057] The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration. These compositions can be prepared by mixing the recombinant protein derived from porcine circovirus type 2 of the present invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0058] When the pharmaceutical composition of the present invention is applied topically to the skin, the pharmaceutical composition should be formulated as a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the porcine circovirus type 2-derived recombinant protein of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes, and water. The pharmaceutical composition of the present invention may also be formulated as a suitable lotion or cream containing the active ingredient suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The pharmaceutical composition of the present invention may also be applied topically to the lower intestinal tract by rectal suppositories or as suitable enemas. Topically applied transdermal patches are also encompassed by the present invention.

[0059] The pharmaceutical composition of the present invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the pharmaceutical field and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption promoters to increase bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0060] The specific effective dose for a particular pig may vary depending on several factors including the activity of the porcine circovirus type 2 derived recombinant protein used, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, and severity of the specific disease being prevented or treated.

[0061]

[0062] According to another aspect of the present invention, the present invention provides a method for preventing or treating a disease caused by porcine circovirus infection, comprising the step of administering to a subject in need thereof a composition comprising a porcine circovirus type 2-derived recombinant protein and an excipient, wherein the mixing ratio of the porcine circovirus type 2-derived recombinant protein and the excipient is 0.1-10:1 by volume.

[0063] In a specific embodiment of the present invention, the subject may be, but is not limited to, a subject expected to develop a disease caused by porcine circovirus infection; a subject that has developed the disease; or a subject that has been judged to have been cured.

[0064] The method for preventing or treating a disease caused by porcine circovirus infection of the present invention preferably increases an in vivo immune response to porcine circovirus type 2 through intradermal administration of a composition according to the present invention, and more specifically, it is preferable to increase the level of neutralizing antibodies.

[0065]

[0066] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0067] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0068]

[0069] [Experimental Example]

[0070] Experimental Example 1. Preparation of vaccine composition

[0071] To prepare a vaccine composition, a purified recombinant PCV2d protein antigen was prepared. The purified recombinant PCV2d protein antigen was produced using a baculovirus system and is represented by SEQ ID NO: 1.

[0072] The purified recombinant PCV2d protein antigen (20 μg / dose) was mixed with 2X PBS (up to 0.5 mL / dose) and stirred for at least 20 minutes. Then, an excipient (IMS1313, Seppic) was added in the same volume and stirred for at least 20 minutes to prepare a vaccine composition. The specific composition of the vaccine composition is as shown in Table 1.

[0073] Raw material drug name Quantity Antigen Recombinant PCV2d protein purified antigen ≥ 20 μg AdjuvantIMS131350% (v / v)

[0074]

[0075] Experimental Example 2. Animal Testing Design, Schedule, and Methods

[0076] 2-1. Experimental design in pigs as the target animal

[0077] Three-week-old pigs (SPF minipigs) were used as test subjects, and the groups were divided as shown in Table 2.

[0078] County name County description Number of individuals Vaccination dose Vaccination Challenge Vaccination Negative control group Non-vaccinated group Unperformed group 2-Challenge Vaccination Control group Non-vaccinated group Performed group 3-Vaccinated group Vaccinated group 51 dose (1 mL)

[0079] The challenge inoculation strain used in the experiment was isolated directly by Innovac Co., Ltd. from pig lung and lymph node tissue samples (from a farm in Pocheon, Gyeonggi Province, in November 2020) obtained from Optipharm Co., Ltd. Specific challenge inoculation conditions are shown in Table 3.

[0080] Substance name, titer, inoculation dose, PCV2d isolate (HID9071) 10 5.5 TCID 50 / mL2 mL

[0081] The test items and timing for evaluating the protective efficacy using pigs were as shown in Figure 1. One dose of the test vaccine is 1 mL, and the inoculation volume and route were established with reference to the national release approval testing standards. Before challenge vaccination, the negative control group was raised in an SPF area to prevent virus transmission, and all test groups except the negative control group were raised in a shielded facility.

[0082] 2-2. Vaccination and blood collection

[0083] The vaccine was administered once (1 mL / dose of vaccine) into the right neck muscle of the pigs. For the challenge vaccination, 2 mL of the PCV2d isolate was injected into each nostril of the pigs, 1 mL each.

[0084] Blood samples were collected via the jugular vein (5 mL each) at 0 and 2 WPV (Weeks postvaccination) to isolate serum. In addition, 8 mL each were collected via the jugular vein at 4, 6, 7, and 8 WPV to isolate PBMC and serum.

[0085]

[0086] 2-3. Clinical evaluation

[0087] - Observation of side effects after vaccination

[0088] Body temperature was measured for two days after vaccination, and the injection site was observed for seven days to observe and record any swelling or inflammation. Clinical adverse effects, such as loss of appetite, decreased energy, coughing, dyspnea, choking, shock, vomiting, and diarrhea, were also observed and recorded. During the trial, the number and duration of respiratory symptoms in each group were observed, and the type and duration of medication used for each group were recorded.

[0089]

[0090] - Measured body tempreture

[0091] Body temperature was measured and recorded rectally at 0, 4, 24, and 48 hours after vaccination.

[0092]

[0093] - Weight measurement (daily weight gain)

[0094] Body weight was measured by placing each pig in a cage equipped with a scale. The measured body weight was used to analyze the average daily weight gain (ADWG) for each pig.

[0095]

[0096] 2-4. Immunological evaluation

[0097] - Antibody titer measurement

[0098] The recombinant PCV2d protein purified antigen, which is the coating antigen, was diluted in carbonate buffer and dispensed into a 96-well plate at 100 ng / 100 μL / well. The antigen was coated for 16 hours at 4°C. The coating solution was removed, and 300 μL / well of 0.05% phosphate-buffered saline-tween 20 (PBS-T) was dispensed and washed three times by removing the solution. 200 μL / well of blocking solution [1% bovine serum albumin (BSA) in PBS-T] was dispensed and blocked for 2 hours at 37°C. The blocking solution was removed, and 300 μL / well of 0.05% PBS-T was dispensed and washed three times by removing the solution. The primary antibody was diluted 1:2,000 in 0.1% BSA solution (in PBS-T) and dispensed at 90 μL / well, and reacted with the coating antigen for 2 hours at 37°C. The primary antibody was removed, and the plate was washed three times by dispensing 300 μL / well of 0.05% PBS-T and removing the plate. The secondary antibody was diluted 1:10,000 in 0.1% BSA solution and dispensed at 100 μL / well of HRP-conjugated goat anti-pig IgG fragment antibody [Bethyl] and reacted for 1 hour at 37°C. The secondary antibody was removed, and the plate was washed three times by dispensing 300 μL / well of 0.05% PBS-T and removing the plate. The TMB solution [Surmodics] was dispensed at 100 μL / well and reacted in the dark at room temperature for 5 minutes. The reaction was stopped by dispensing 100 μL / well of stop solution, and the absorbance was measured at 450 nm.

[0099]

[0100] - Neutralizing antibodies measured

[0101] Test serum was inactivated at 56°C for 30 minutes. 50 μL of serum and 150 μL of DMEM were dispensed into the first well of a 96-well plate for dilution, and then 100 μL of DMEM per well was dispensed into the remaining wells for binary dilution. PCV2b [HID9029] or PCV2d [HID9071] virus was incubated at 400 TCID 50 After diluting to 100 μL / mL, the same amount (100 μL / well) was dispensed into the well containing the diluted serum and incubated at 37°C for 1 hour. PCV1-free Porcine kidney (PK-15) cells were seeded at 2 x 10 4 Cells were dispensed at 100 μL / well. After culturing for 24 h in a 37°C, 5% CO2 incubator, 300 mM glucosamine was treated at 200 μL / well for 30 min and then removed. After washing three times by dispensing and then removing PBS, 200 μL / well of DMEM containing 1% antibiotic-antimycotic was added and cultured for 2 days.

[0102] After culture, immunofluorescence assay was performed as follows. Specifically, the culture medium was discarded, PBS was dispensed, and then washed once by removing it. Then, the PBS was completely removed and the plate was placed in a 37°C incubator and dried for 30 minutes. 100 μL / well of 80% cold acetone (prepared in advance and stored at -20°C) was dispensed onto the dried plate and fixed at 4°C for 1 hour (or -20°C for 15 minutes). The plate was washed once by discarding the fixative, dispensing PBS, and then removing it. Pig anti-PCV2 antiserum diluted 1:500 in PBS was dispensed at 100 μL / well and reacted at 37°C for 1 hour. The plate was washed three times by dispensing and then removing PBS.

[0103] Pig IgG-heavy and light chain antibodies, goat polyclonal, FITC conjugate [Bethyl] were diluted 1:200 in PBS, dispensed at 100 μL / well, and incubated at 37°C for 30 minutes. The plates were washed once by dispensing and removing PBS.

[0104] The neutralizing antibody titer was determined as the reciprocal of the highest serum dilution that inhibited virus proliferation (90% inhibition in 1 well) by observation under a fluorescence microscope.

[0105]

[0106] - Measurement of IFN-γ secretion

[0107] Blood samples were collected from all individuals in each group at the time of challenge vaccination and 2 and 4 weeks after challenge vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated from the collected blood. The isolated PBMCs were stimulated with PCV2d antigen to induce IFN-γ secretion. Analysis was performed using the IFN-γ Porcine ELISA kit [Invitrogen, #KSC4021].

[0108]

[0109] Experimental Example 3. Microbiological Evaluation

[0110] 3-1. RT-qPCR (serum, nasal swab, rectal swab, lung, lymph node)

[0111] Serum was isolated from the blood of all individuals in each group, and nasal and rectal specimens were collected using cotton swabs. After autopsy, lung and lymph node tissue specimens were collected. The specimens were placed in a 2 mL E-tube, mixed with 1 mL of PBS, and vortexed thoroughly to mix the specimen with the PBS. The tissues were mixed with PBS at a concentration of 1 g / mL in a 2 mL round-bottom E-tube, and disrupted with beads using a TissueLyser II. The supernatant was then separated by centrifugation at 10,000 × g for 5 minutes. Viral DNA was isolated using the Gene-spin Viral DNA / RNA Extraction Kit.

[0112] RT-qPCR primer information for quantitative analysis is shown in Table 4.

[0113] GenePrimer sequencesPCV2Forward5'-AACCACATACTGGAAACCAC-3'(SEQ ID NO: 2)Reverse5'-TTGAGGAGTACCATTCCAAC-3'(SEQ ID NO: 3)

[0114] The reagent composition for RT-qPCR is as shown in Table 5.

[0115] Reagent volume (μL)TOPreal TM qPCR 2X PreMIX10Viral DNA template510 pM Forward primer110 pM Reverse primer1Sterile DDW3

[0116] RT-qPCR conditions are as shown in Table 6.

[0117] PCR conditions Temperature (℃) Time Number (cycle) Initial denaturation9510 min1Denaturation9510 sec35Annealing5315 secElongation7215 secMelt Curve stage9515 sec1601 min160 ~ 950.3℃ / 1 step15 sec / 1 step1

[0118] When performing RT-qPCR, always use a negative control and a standard sample for PCV2d (10 3 ~ 10 9 DNA copy) was performed together. In addition, the number of DNA copies in the sample was measured by substituting it into the standard quantitative curve obtained through the standard sample after RT-qPCR, and no amplification product should be observed in the negative control group.

[0119] The results of the test samples were summarized and recorded as PCV2d positive or negative.

[0120]

[0121] Experimental Example 4. Pathological Evaluation

[0122] 4-1. Autopsy and gross lesion evaluation

[0123] To examine the internal organ findings of SPF minipigs, autopsies and gross lesion assessments were performed. After observing the gross findings, major internal organs were removed and some were fixed in a 10% neutral-buffered formalin solution. After fixation, the tissues were trimmed to allow cross-section observation of each organ, embedded in paraffin according to standard tissue processing methods, and sectioned to approximately 3–4 μm thickness to produce tissue slides. The slides were stained with hematoxylin & eosin (H&E) and examined using an optical microscope (Olympus BX53, Japan).

[0124]

[0125]

[0126] [Example]

[0127] Example 1. Clinical evaluation

[0128] 1-1. Observation of clinical symptoms or side effects after vaccination

[0129] For 7 days after vaccination, clinical symptoms such as swelling at the injection site, inflammation, loss of appetite, decreased energy, cough, dyspnea, suffocation, shock, vomiting, and diarrhea were observed. The results of observation of clinical symptoms and adverse effects after vaccination are shown in Table 7.

[0130] Clinical symptom assessment according to the number of days since vaccination 2) (Day) Military name 1) Individual number 1234567 Negative control group 4-100000004-20000000 Challenge vaccinated control group 3-100000003-200000003-30000000 Vaccinated group 2-100000002-200000002-300000002-400000002-500000001) The negative control group and the challenge vaccinated control group are the unvaccinated groups. 2) Clinical symptom score notation: Normal 0, Mild 1, Moderate 2, Severe 3.

[0131] As shown in Table 7, no clinical symptoms or side effects related to vaccination were observed in the vaccinated group.

[0132] 1-2. Temperature measurement after vaccination

[0133] Body temperature was measured before vaccination (0 hours) and after vaccination (4, 24, 48 hours), and the results are shown in Table 8.

[0134] Mean body temperature by group according to the time elapsed since vaccination (℃, AVE ± SE) 0 H4 H24 H48 H Negative control group 39.0 ± 0.4 39.5 ± 0.1 39.2 ± 0.1 39.0 ± 0.1 Challenge vaccination control group 39.2 ± 0.1 39.6 ± 0.1 39.0 ± 0.1 39.4 ± 0.2 Vaccination group 39.0 ± 0.2 39.7 ± 0.2 39.1 ± 0.1 38.9 ± 0.1

[0135] As shown in Table 8, body temperature rose slightly 4 hours after vaccination, but dropped to the pre-vaccination level after 24 hours.

[0136] 1-3. Weight change and average daily weight gain (ADWG) by group

[0137] The body weight of all groups was measured according to the vaccination course, and the results are shown in Figure 2.

[0138] As shown in Fig. 2, all test groups had an average body weight of 3.1-3.3 kg before vaccination (0 WPV). The 4 WPV vaccinated group had an average body weight of 5.8 kg, which was slightly lower than the challenge control group (5.9 kg) and the negative control group (6.1 kg). However, the body weight of the 8 WPV challenge control group was the lowest at an average of 9.1 kg, while the vaccinated group had an average of 9.9 kg, which was similar to or higher than the negative control group (9.5 kg).

[0139]

[0140] Based on the above weight measurement results, the daily weight gain was calculated, and the results are shown in Figure 3.

[0141] As shown in Fig. 3, from vaccination to challenge vaccination (0-4 WPV), the daily weight gain of the vaccinated group was 96.4 g, which did not show a significant difference compared to the challenge control group (90.5 g) and the negative control group (108.9 g). However, from challenge vaccination to the end of the test (5-8 WPV), the daily weight gain of the challenge control group was the lowest at an average of 113.7 g, while the daily gain of the vaccinated group was higher at an average of 147.1 g compared to the negative control group (119.6 g).

[0142] The above results indicate that vaccination did not result in weight loss, indicating that there were no adverse effects, such as decreased appetite, following vaccination. Furthermore, this suggests that vaccination provided a protective effect against the PCV2d challenge, resulting in increased weight gain compared to the challenge group.

[0143]

[0144] Example 2. Immunological evaluation

[0145] 2-1. Measurement of PCV2 antibody levels in pigs (SPF minipigs) by ELISA

[0146] - PCV2b-specific IgG antibody test results

[0147] PCV2b-specific IgG antibody titers were measured in pigs, the target animals. The results of PCV2b-specific IgG antibody titer measurement are shown in Figure 4.

[0148] As shown in Figure 4, before vaccination (0 WPV), all test groups had an average absorbance of 0.2 (OD 450 ) or lower, the level was low. The vaccinated group began to produce IgG antibody titers specific to PCV2b from 2 WPV and showed a high absorbance of 1.0 or higher at 4 WPV. In particular, the IgG antibody titers in the vaccinated group were significantly higher than those in the challenge control group and the negative control group from 6 WPV. The challenge control group produced antibody titers from 2 weeks after the challenge vaccination (6 WPV) and showed antibody titers similar to those in the vaccinated group at 8 WPV.

[0149] Meanwhile, the negative control group showed low levels of antibody titers throughout the test period.

[0150]

[0151] - PCV2d-specific IgG antibody test results

[0152] PCV2d-specific IgG antibody titers were measured in pigs, the target animals. The results of PCV2d-specific IgG antibody titer measurement are shown in Figure 5.

[0153] As shown in Figure 5, before vaccination (0 WPV), all test groups had an average absorbance of 0.2 (OD 450 ) was lower than that. The vaccinated group began to produce IgG antibody titers specific to PCV2d from 2 WPV and showed a high absorbance of 3.0 or higher at 4 WPV, which was significantly higher than that of the challenge control group and the negative control group. In particular, the vaccinated group maintained a significantly high level of IgG antibody titers from the challenge vaccination to 8 WPV.

[0154] The challenge vaccination control group produced antibody titers starting two weeks after the challenge vaccination (6 WPV) and showed antibody titers similar to those of the vaccinated group at 8 WPV.

[0155] Meanwhile, the negative control group showed low levels of antibody titers throughout the test period.

[0156]

[0157] 2-2. Measurement of PCV2 neutralizing antibodies in pigs (SPF minipigs)

[0158] - PCV2b neutralizing antibody measurement results

[0159] PCV2b neutralizing antibody titers were measured in pigs, the target animals, and the results are shown in Figure 6.

[0160] As shown in Figure 6, all test groups showed low levels of PCV2b neutralizing antibody titers before vaccination (0 WPV). The vaccinated group began to form neutralizing antibodies against PCV2b from 2 WPV, and showed a significant difference compared to the negative control group and the challenge control group at 6 WPV. In particular, the neutralizing antibody titer in the vaccinated group continuously increased from 4 WPV after the challenge vaccination, and showed a significantly higher value than the negative control group and the challenge group at 8 WPV.

[0161] The challenge vaccination control group developed neutralizing antibodies after the challenge vaccination, but at a lower level than the vaccinated group.

[0162] Meanwhile, neutralizing antibodies in the negative control group were observed at very low levels throughout the test.

[0163]

[0164] - PCV2d neutralizing antibody measurement results

[0165] PCV2d neutralizing antibody titers were measured in pigs, the target animals, and the results are shown in Figure 7.

[0166] As shown in Figure 7, all test groups showed low levels of neutralizing antibody titers before vaccination (0 WPV). In the vaccinated group, neutralizing antibody titers against PCV2d began to form from 2 WPV, and a significant difference was observed compared to the negative control group at 6 WPV. In particular, the neutralizing antibody titers in the vaccinated group continuously increased from 4 WPV after the challenge vaccination, and significantly higher levels were observed at 8 WPV.

[0167] The challenge control group showed neutralizing antibodies after challenge vaccination, but at a lower level than the vaccinated group.

[0168] Meanwhile, the negative control group showed very low levels of neutralizing antibodies throughout the test period.

[0169]

[0170] 2-3. Measurement of IFN-γ secretion in pigs (SPF minipigs)

[0171] After vaccination, PBMCs were isolated from the blood of all groups at 4 WPV and then sensitized with PCV2d to induce IFN-γ secretion. The results of the subsequent measurement of IFN-γ secretion are shown in Figure 8.

[0172] As shown in Figure 8, it was confirmed that IFN-γ specific to the PCV2d antigen was secreted in the vaccinated group. In contrast, IFN-γ secretion was minimal in the challenge control group and the negative control group.

[0173]

[0174] Example 3. Microbiological evaluation

[0175] 3-1. Results of quantitative evaluation of the challenge inoculation virus DNA

[0176] Samples (serum, nasal, and rectal swabs) were collected from all animals from the time of vaccination until the challenge vaccination (4 WPV), and lung and lymph node tissue specimens were collected after autopsy. Viral DNA was isolated from these samples and specimens, and PCV2 DNA was detected by RT-qPCR.

[0177]

[0178] - Serum

[0179] The results of detecting PCV2 DNA in serum samples are shown in Figure 9.

[0180] As shown in Figure 9, no PCV2 DNA (i.e., viremia) was detected in the blood of any group from the time of vaccination until the challenge vaccination (4 WPV). Furthermore, PCV2 DNA was not observed in the vaccinated group after the challenge vaccination, as was the case in the negative control group. In contrast, viremia was measured in the challenge control group two weeks after the challenge vaccination (6 WPV), showing a significant difference compared to the other test groups.

[0181] Additionally, no viremia was observed in any group at 7-8 WPV.

[0182]

[0183] - Nasal and rectal swabs

[0184] The results of detecting PCV2 DNA in nasal swab samples are shown in Fig. 10, and the results of detecting PCV2 DNA in rectal swab samples are shown in Fig. 11.

[0185] As shown in Figures 10 and 11, PCV2 DNA was not detected in nasal and rectal swabs in any group from post-vaccination until challenge (4 WPV). However, PCV2 DNA was detected only in the challenge control group from 6 WPV, and significantly higher levels of PCV2 DNA were detected in nasal and rectal swabs, showing a significant difference compared to the other groups. In addition, the challenge control group showed a gradual decrease in PCV2 DNA up to 8 WPV.

[0186]

[0187] - Lungs and lymph nodes

[0188] The results of detecting PCV2 DNA in lung samples are shown in Figure 12, and the results of detecting PCV2 DNA in lymph node samples are shown in Figure 13.

[0189] As shown in Figures 12-13, small amounts of PCV2 DNA were detected in the lungs of the vaccinated group, but the PCV2 DNA copy number was significantly lower than that of the challenge control group. Furthermore, PCV2 DNA was not detected in the lymph nodes of the vaccinated group, similar to the negative control group. In contrast, very high levels of PCV2 DNA copy number were detected in the lungs and lymph nodes of the challenge control group.

[0190] Meanwhile, PCV2 DNA was not detected in the lungs and lymph nodes of the negative control group.

[0191]

[0192] Example 4. Pathological evaluation (observation of lesion sites following challenge vaccination)

[0193] 4-1. Visual lesion evaluation

[0194] To examine the internal organ findings of the pigs, necropsy was performed. Inguinal lymph nodes, lungs, and hearts were isolated from the necropsied pigs and examined macroscopically. No lesions were observed macroscopically in the vaccinated and negative control groups. In contrast, lesions were observed in the inguinal lymph nodes, lungs, and heart of the challenged control group. Macroscopic evaluation of lesions revealed enlarged lymph nodes in the challenged control group. Additionally, lymph node size measurements revealed that both lymph nodes in the challenged control group were enlarged compared to the other experimental groups.

[0195] In the lungs, purplish-red pulmonary consolidation was formed centered on the anterolateral lobe, and pulmonary consolidation was confirmed only in two individuals of the challenged control group.

[0196] Additionally, one animal in the challenge control group had pericardial adhesions on the heart surface, with inflammatory exudate presumed to be milky-white fibrin. No significant macroscopic changes were observed in other internal organs.

[0197]

[0198] In summary, the present inventors have developed a vaccine composition for preventing porcine circovirus infection. The vaccine is characterized by (i) increased antigen content and reduced excipient content compared to previously developed vaccine compositions; and (ii) a specific ratio of antigen and excipients. The safety of the vaccine of the present invention was verified through clinical evaluation, and body weight and daily weight gain were improved compared to the challenge control group. Immunological evaluations showed that the vaccine effectively induced humoral immunity (IgG antibody, neutralizing antibody) and cellular immunity (IFN-γ), thereby activating a protective immune response against PCV2. This resulted in a significant reduction in viremia and PCV2 antigen content in the nasal cavity, rectum, lungs, and lymph nodes. Pathological evaluations also showed a significant reduction in PCV2-related lesions in the lungs, lymph nodes, and kidneys. This indicates that the vaccine effectively protects against the recently prevalent porcine circovirus, and thus can be utilized in various fields such as porcine circovirus infection prevention and pig farming.

[0199]

[0200] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Contains a recombinant protein derived from porcine circovirus type 2 (PCV2); and an excipient; A vaccine composition for preventing porcine circovirus infection, wherein the mixing ratio of the porcine circovirus type 2-derived recombinant protein and excipients is 0.1-10:1 by volume.

2. A vaccine composition according to claim 1, wherein the recombinant protein derived from porcine circovirus type 2 is included at a concentration of 0.1 to 1000 μg / dose.

3. A vaccine composition according to claim 1, wherein the recombinant protein derived from porcine circovirus type 2 comprises an amino acid sequence represented by sequence number 1.

4. A vaccine composition according to claim 1, wherein the excipient is IMS1313.

5. A vaccine composition according to claim 4, wherein the IMS1313 is contained at a concentration of 10 to 90% (v / v).

6. A method for preventing porcine circovirus infection, comprising the step of inoculating a pig with a vaccine composition according to paragraph 1.

7. A method according to claim 6, wherein the vaccine composition is administered by one or more methods selected from the group consisting of intramuscular, subcutaneous, intradermal, transdermal, intravenous, intranasal, intraperitoneal, and oral.

8. Containing a recombinant protein derived from porcine circovirus type 2 (PCV2); and excipients; A pharmaceutical composition for preventing diseases caused by porcine circovirus infection, wherein the mixing ratio of the recombinant protein derived from porcine circovirus type 2 and the excipient is 0.1-10:1 by volume.

9. In paragraph 8, the disease caused by the porcine circovirus infection is Porcine Respiratory Disease Complex (PRDC), enzootic pneumonia (EP), postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), sow abortion and mortality syndrome (SAMS), Porcine Reproductive and Respiratory Syndrome (PRRS), pseudorabies, Glasser's disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietary hepatosis, suppurative bronchopneumonia. A pharmaceutical composition characterized by at least one selected from the group consisting of bronchopneumonia, Eustachian tube inflammation, polyserositis, mycoplasmal pneumonia, and pleural pneumonia.

10. A method for preventing or treating a disease caused by porcine circovirus infection, comprising the step of administering to a subject in need thereof a composition comprising a porcine circovirus type 2-derived recombinant protein and an excipient, wherein the mixing ratio of the porcine circovirus type 2-derived recombinant protein and the excipient is 0.1-10:1 by volume.

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