Porcine circovirus type 2 infection-preventing vaccine composition for intradermal injection

Abstract

The present invention relates to a porcine circovirus type 2 infection-preventing vaccine composition for intradermal injection. The porcine circovirus type 2 infection-preventing vaccine composition for intradermal injection, according to the present invention, has been verified to be safe through clinical evaluation, and has been confirmed to induce a higher level of a neutralizing antibody titer compared to an intramuscular injection group. This means that the porcine circovirus type 2 infection-preventing vaccine composition for intradermal injection, according to the present invention, effectively protects against porcine circovirus which is recently prevalent, and thus may be utilized in various ways in the fields of porcine circovirus infection prevention and pig farming.

Description

Vaccine composition for preventing porcine circovirus type 2 infection for intradermal inoculation

[0001] The present invention relates to a vaccine composition for preventing porcine circovirus type 2 infection for intradermal inoculation.

[0002] Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus associated disease (PCVAD), which causes growth retardation, respiratory disease, abortion, skin disease, and some cases even death, causing significant economic damage to the pig industry worldwide. PCV2 genotypes are classified as PCV2a to PCV2h based on the ORF2 (open reading frame 2) gene sequence encoding the capsid protein. Since genotype shifts to PCV2d were reported in China and the United States in 2009 and 2012, respectively, the PCV2d genotype has been prevalent worldwide. The PCV2 capsid protein encoded by ORF2 is a major immunogenic protein that plays an important role in inducing protective immunity against PCV2 infection and binding to heparin sulfate receptors. The ORF2 gene of PCV2a has a high similarity to that of PCV2b, but it is significantly different from the gene of PCV2d, which is rapidly spreading worldwide. According to related studies, commercial PCV2a-based vaccines showed neutralizing activity against PCV2b isolates, but showed low neutralizing efficiency against PCV2d isolates. Therefore, existing commercial vaccines manufactured based on PCV2a or PCV2b genotypes have limitations in completely preventing the currently prevalent new PCV2d variants. Therefore, a vaccine with a high protective rate manufactured based on a new PCV2d is required for effective prevention against a new PCV2d variant.

[0003] Currently, most commercially available PCV2 vaccines are administered intramuscularly. While this approach offers the advantages of rapid absorption and action and the ability to administer high doses, it also carries the risk of nerve and blood vessel damage, pain, swelling, and redness at the injection site, and the risk of needle-borne infection. For these reasons, intradermal, intranasal, or oral vaccinations have the potential to replace intramuscular injections in the future. Intradermal vaccination, in particular, is rich in dendritic cells as well as lymphatic vessels that facilitate rapid antigen processing, enabling high immunity with a small antigen dose. Furthermore, intradermal vaccination uses a needle-free syringe, reducing stress and pain in pregnant sows and minimizing adverse reactions at the injection site and animal suffering. Therefore, research is actively underway on various swine vaccines utilizing intradermal vaccination techniques, including PEDV, FMD, PCV2, Mhp, PRRSV-2, and ASF. Currently, MSD's Porcilis is a commercially available PCV2 ID vaccine. ® MHYOSPHERE of PCV ID (Inactivated vaccine) and HIPRA ® There is a PCV ID (Inactivated Recombinant Mycoplasma hyopneumoniae, cp PCV2) vaccine. However, the existing ID vaccine is manufactured using the PCV2a genotype, which limits its ability to provide complete protection. Therefore, the development of an ID vaccine based on the currently prevalent PCV2d genotype is urgently needed.

[0004] Accordingly, the present inventors developed a porcine circovirus type 2 vaccine composition for intradermal inoculation based on the PCV2d genotype, and confirmed that the vaccine composition induces a high level of neutralizing antibodies against PCV2d in the target animal, thereby completing the present invention.

[0005] 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 2d (PCV2d); and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) based on the volume of the vaccine composition.

[0006] Another object of the present invention is to provide a method for preventing porcine circovirus infection, comprising the step of intradermally inoculating the vaccine composition into a pig.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2d; and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) based on the composition's dosage.

[0008] 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 intradermally administering to a pig a composition comprising a recombinant protein derived from porcine circovirus type 2d; and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) of the composition dose.

[0009] 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 2d; and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) based on the vaccine composition volume.

[0010] The present invention also provides a method for preventing porcine circovirus infection, comprising the step of intradermally inoculating the vaccine composition into a pig.

[0011] The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2d; and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) based on the composition volume.

[0012] In addition, the present invention provides a method for preventing or treating a disease caused by porcine circovirus infection, comprising the step of intradermally administering to a pig a composition comprising a recombinant protein derived from porcine circovirus type 2d; and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) of the composition dosage.

[0013] The vaccine composition for preventing porcine circovirus type 2 infection for intradermal inoculation according to the present invention has been clinically evaluated for safety and was confirmed to induce higher levels of neutralizing antibodies compared to the intramuscular inoculation group. This indicates that the vaccine composition for preventing porcine circovirus type 2 infection for intradermal inoculation according to the present invention 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.

[0014] Figure 1 is a diagram showing the results of measuring antibody titers against PCV2d according to the type of immune enhancer when inoculated with the vaccine composition of the present invention in pigs (**: P<0.01, ***: P<0.001 compared with the negative control group).

[0015] Figure 2 is a diagram showing the results of measuring neutralizing antibodies against PCV2d according to the type of immune enhancer when inoculated with the vaccine composition of the present invention in pigs (***: P<0.001 compared with negative control group).

[0016] Figure 3 is a diagram showing the results of measuring antibody titers against PCV2d according to the content of an immune enhancer when inoculated with the vaccine composition of the present invention in pigs (*: P<0.05, **: P<0.01, ***: P<0.001 compared with the negative control group).

[0017] Figure 4 is a diagram showing the results of measuring neutralizing antibodies against PCV2d according to the content of an immune enhancer when inoculated with the vaccine composition of the present invention in pigs (**: P<0.01, ***: P<0.001 compared with the negative control group).

[0018] Figure 5 is a diagram showing the results of measuring antibody titers against PCV2d according to the amount of antigen when inoculated with the vaccine composition of the present invention in pigs (**: P<0.01, ***: P<0.001 compared with the negative control group).

[0019] Figure 6 is a diagram showing the results of measuring neutralizing antibodies against PCV2d according to the antigen amount when inoculated with the vaccine composition of the present invention in pigs (***: P<0.001 compared with negative control group, #: P<0.05 compared with test group-1).

[0020] Hereinafter, the present invention will be described in detail.

[0021] 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 2d (PCV2d); and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) based on the dose of the vaccine composition.

[0022] 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.

[0023] The recombinant protein derived from the porcine circovirus type 2d 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.

[0024] In a specific embodiment of the present invention, the recombinant protein derived from porcine circovirus type 2d may be represented by the amino acid sequence of SEQ ID NO: 1. The recombinant protein derived from porcine circovirus type 2d of the present invention may include all polypeptides 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, which is scored, is based on the percentage of identities and / or preservation of sequence substitutions.

[0025] 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 included in a lysate form that is expressed in E. coli and is not purified.

[0026] The excipients are preferably present in an amount of 10 to 50% (v / v) of the vaccine composition, more preferably 25 to 45% (v / v), even more preferably 30 to 40% (v / v), and most preferably 40% (v / v).

[0027] In a specific embodiment of the present invention, the recombinant protein derived from the porcine circovirus type 2d may be included at a concentration of 1 to 50 μg / dose, preferably 5 to 40 μg / dose, more preferably 10 to 30 μg / dose, and most preferably 20 μg / dose.

[0028] 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 immune enhancers that enhance the immunogenicity of the vaccine and 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.

[0029] An immunostimulant 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 immunostimulants are known to those skilled in the art. Such immunostimulants include, but are not limited to, Freund's complete and incomplete immunostimulants, vitamin E, non-ionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and mineral-free oil, Carbopol, water-in-oil emulsion immunostimulants, and the like.

[0030] In a specific embodiment of the present invention, the excipient may be at least one selected from the group consisting of ISA15A, GEL02, IMS1313, and carbopol 974p in consideration of safety and immune persistence, and preferably, IMS1313, which is an IMS series excipient, is preferable. As mentioned above, the IMS1313 may be 10 to 50% (v / v) of the vaccine composition dose, preferably 25 to 45% (v / v), more preferably 30 to 40% (v / v), and most preferably 40% (v / v).

[0031] 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 a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, sterile powder, or the like.

[0032] In specific embodiments of the present invention, the vaccine composition of the present invention may be administered via intramuscular, subcutaneous, intradermal, transdermal, intravenous, intranasal, intraperitoneal, or oral routes, and is preferably administered via the intradermal route. The dosage of the vaccine may be appropriately selected depending on various factors, such as the route of administration, the age, sex, weight, and severity of the animal.

[0033] Additionally, the vaccine composition of the present invention can be prepared using standard methods known in the art. 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 using conventional methods before being incorporated into a vaccine for formulation.

[0034] In a specific embodiment of the present invention, the inoculation volume of the composition is preferably 0.1 to 1 ml, more preferably 0.5 ml, but the scope of the present invention is not limited thereto.

[0035]

[0036] According to another aspect of the present invention, a method for preventing porcine circovirus infection is provided, comprising the step of intradermally inoculating the vaccine composition into a pig.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] In a specific embodiment of the present invention, the inoculation is preferably intradermal using a needle-free syringe. Furthermore, the pressure of the needle-free syringe may be 10 to 100 psi, preferably 30 to 80 psi, more preferably 50 to 70 psi, and most preferably 60 psi.

[0041] In a specific embodiment of the present invention, the vaccination may be administered intradermally to one or more sites selected from the group consisting of the back of the ear, the back of the neck, the hind legs, and the buttocks.

[0042] As with the preventive method of the present invention, intradermal inoculation of the vaccine composition of the present invention allows for high immunity induction with a small antigen dose due to the abundance of dendritic cells as well as lymphatic vessels that promote rapid antigen processing. Furthermore, intradermal inoculation has the advantage of reducing stress and pain responses in pregnant sows, minimizing adverse reactions at the injection site, and minimizing animal suffering.

[0043]

[0044] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by porcine circovirus infection, comprising a recombinant protein derived from porcine circovirus type 2d; and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) based on the composition's dosage.

[0045] 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. The present invention may be at least one selected from the group consisting of bronchopneumonia, Eustachian tube inflammation, polyserositis, mycoplasmal pneumonia, and pleural pneumonia, but the scope of the present invention is not limited thereto.

[0046] The pharmaceutical composition of the present invention may be in the form of an aqueous or oily suspension for injection. 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 injectable preparation may also be an 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. Sterile, fixed oils are also commonly 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 preparations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), particularly their polyoxyethylated forms. Furthermore, for parenteral administration, the pharmaceutical composition of the present invention may be prepared as an aqueous solution. Preferably, a physically appropriate buffer solution, such as Hank's solution, Ringer's solution, or physically buffered saline, may be used. Aqueous injection suspensions may contain a substrate that increases the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active ingredient may be prepared as 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 non-lipid amino polymers may 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.

[0047] The specific effective dose for a particular pig may vary depending on several factors including the activity of the recombinant protein derived from porcine circovirus type 2d 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.

[0048]

[0049] 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 intradermally administering to a pig a composition comprising a recombinant protein derived from porcine circovirus type 2d; and an excipient; wherein the excipient is present in an amount of 10 to 50% (v / v) based on the dosage of the composition.

[0050] 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, particularly type 2d, through intradermal administration of a composition according to the present invention, and more specifically, it is preferable to increase the level of neutralizing antibodies.

[0051]

[0052] 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.

[0053] 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.

[0054]

[0055] [Experimental Example]

[0056] Experimental Example 1. Experimental Animals

[0057] The experimental animals used in this example were 3-week-old pigs, and various test groups were formed depending on the purpose of the test.

[0058] The vaccine was administered intradermally or intramuscularly, with a 0.5 ml dose per test group, depending on the purpose of the study. Each test group received one dose of the vaccine, administered once into the right cervical region, using a needle-free syringe (Trian Pulse 50). However, the positive control group received one dose into the right temporal region.

[0059] Blood samples were collected through the jugular vein at 0, 2, 4, 6, and 8 WPV, and serum was separated from more than 5 mL of blood.

[0060]

[0061] Experimental Example 2. Clinical Evaluation

[0062] 2-1. Observation of side effects and adverse reactions

[0063] Adverse reactions, such as suppuration, necrosis, fever, and skin lesions at the injection site, were observed and recorded for 7 days after vaccination. Additionally, during the post-vaccination study period, any abnormal symptoms, such as shock, vomiting, and diarrhea, or death were observed daily. The individual number, date of symptom onset, and symptoms were recorded.

[0064]

[0065] 2-2. Temperature measurement

[0066] Body temperature was measured and recorded at the workplace before and 4 hours after vaccination.

[0067]

[0068] Experimental Example 3. Immunological Evaluation

[0069] 3-1. Antibody measurement

[0070] 2 X 10 4 PCV1-free Porcine kidney (PK-15) cells and PCV2d [HID9071] virus (600 TCID) at 100 μL / well 50 / mL and dispensed into 96-well plates at 100 μL / well. After culturing for 24 hours in an incubator (37°C, 5% CO2), 300 mM glucosamine was added at 200 μL / well, reacted for 30 minutes, and then removed. To remove the PBS after dispensing, the cells were washed, and then 200 μL / well of DMEM containing 1% antibiotic-antimycotic was dispensed and cultured for 4 days. The culture medium in the 96-well plate was removed and washed once with PBS. 100 μL / well of 80% cold acetone (prepared in advance and stored at -20°C) was dispensed and fixed at 4°C for 1 hour (or -20°C for 15 minutes). After removing the fixative, the plate was washed once with PBS and stored at -20°C.

[0071] A fixed plate was used for antibody titer testing. Pig serum samples were prepared by binary dilution with DMEM, dispensed at 100 μL / well onto the fixed plate, and incubated at 37°C for 1 hour. The pig serum samples were removed and washed three times with PBS. Antibodies (Goat anti-pig IgG-heavy and light chain FITC-conjugated polyclonal antibody [Bethyl]) were diluted 1 / 200 in PBS, dispensed at 100 μL / well, and incubated at 37°C for 30 minutes. After incubation, the plates were washed with PBS and observed under a fluorescence microscope. The reciprocal of the highest dilution of the serum sample was determined as the antibody titer as observed under a fluorescence microscope.

[0072]

[0073] 3-2. Measurement of neutralizing antibodies

[0074] The 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. 400 TCID of PCV2d[HID9071] virus was added. 50After diluting to 100 μL / mL, the same volume (100 μL / well) was dispensed into wells containing binary diluted serum and reacted at 37°C for 1 hour. PCV1-free Porcine kidney (PK-15) cells were cultured at 2 x 10 4 Cells were dispensed at a concentration of 100 μL / well. Cells were cultured in a 5% CO2 incubator at 37°C for 24 hours, and 300 mM glucosamine was treated with 200 μL / well for 30 minutes and then removed. Cells were washed three times with PBS, and 200 μL / well of DMEM containing 1% antibiotic-antimycotic was added and cultured for 2 days. After culture, an immunofluorescence assay was performed. Specifically, cells were washed once with PBS, PBS was completely removed, and the plates were placed in a 37°C incubator and dried for 30 minutes. 100 μL / well of cold 80% acetone (prepared in advance and stored at -20°C) was dispensed to the dried plates, and the plates were fixed at 4°C for 1 hour (or at -20°C for 15 minutes). After removing the fixative, the plates were washed once with PBS. Pig anti-PCV2 antiserum diluted 1 / 500 in PBS was dispensed at 100 μL / well and incubated at 37°C for 1 hour. The reaction solution was removed and washed three times with PBS. Antibody (Goat anti-pig IgG-heavy and light chain FITC conjugated polyclonal antibody [Bethyl]) diluted 1 / 200 in PBS was dispensed at 100 μL / well and incubated at 37°C for 30 minutes. The reaction solution was removed and washed once with PBS. The neutralizing antibody titer was determined as the reciprocal of the highest serum dilution that inhibited virus replication (90% inhibition in one well) observed under a fluorescence microscope.

[0075]

[0076] [Example]

[0077] Example 1. Preparation of PCV2 vaccine composition containing purified recombinant PCV2d protein antigen

[0078] An intradermal vaccine (hereinafter referred to as “InnoSeco ID vaccine”) was manufactured using a needle-free syringe developed by InnoVac Co., Ltd. Specifically, the InnoSeco ID vaccine is as follows.

[0079]

[0080] [InnoSeco ID Vaccine]

[0081] - Inoculation volume (1 dose): 0.5 ml

[0082] - Antigen type and amount: 20 μg of purified recombinant PCV2d protein antigen

[0083] - Type and content of immune booster: IMS1313 40% (v / v)

[0084]

[0085] The above recombinant PCV2d protein purified antigen was cultured in an Erlenmeyer flask and purified by Q Sepharose ® This antigen was produced by purifying using Fast Flow and then quantifying it using BCA assay, and is represented by the amino acid sequence of sequence number 1.

[0086]

[0087] Example 2. Screening test for immune enhancers in PCV2 vaccine compositions

[0088] In this example, an immune enhancer that induces immunogenicity equivalent to or greater than that of the intramuscular innocirco vaccine when administered intradermally (i.e., the innocirco ID vaccine) was selected.

[0089] The experimental group for screening immune enhancers was organized as shown in Table 1.

[0090] Military Army NameImmunity Booster Antigen Dose (1 dose)Vaccination RouteVaccination Dose (1 dose)Number of individualsProduct NameContent (1 dose)G1 Test Group-1 1) --Recombinant PCV2d protein purified antigen 3), 20 μg intradermally 0.5 mL 4G2 test group-2 ISA 15A 15% (v / v) recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 4G3 test group-3 Montanide TM GEL 0210 %(v / v) Recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 4G4 Test group - 4IMS131350 %(v / v) Recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 4G5 Test group - 5Carbopol 974p1 mg Recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 4G6 Positive control groupIMS131350 %(v / v) Recombinant PCV2d protein purified antigen, 20 μg muscle 1.0 mL 4G7 Negative control group 2) - -Intradermal 0.5 mL4 1) Test group-1 is the Antigen only group with no added immune booster. 2) The negative control group is the group that received physiological saline solution (PBS). 3) The recombinant PCV2d protein purified antigen is an antigen produced by culturing in an Erlenmeyer flask, purifying using Q Sepharose® Fast Flow, and then quantifying using a BCA assay.

[0091]

[0092] 2-1. Evaluation of side effects and adverse reactions

[0093] For 7 days after vaccination, clinical symptoms such as swelling, inflammation, loss of appetite, decreased vitality, cough, dyspnea, suffocation, shock, vomiting, and diarrhea were observed at the vaccination site for each individual.

[0094] As a result of observing side effects and adverse reactions, no unusual findings were found in any group.

[0095]

[0096] 2-2. Temperature measurement

[0097] The results of rectal temperature measurements before and after vaccination are shown in Table 2.

[0098] Mean body temperature by group (time after vaccination, AVE±SE) 0 h 4 h Test group-1 (Antigen only) 39.5±0.2 39.4±0.1 Test group-2 (ISA15A) 38.9±0.2 39.1±0.1 Test group-3 (GEL02) 39.7±0.4 39.6±0.3 Test group-4 (IMS1313) 39.4±0.3 39.5±0.3 Test group-5 (Carbopol 974p) 39.5±0.3 39.5±0.2 Positive control group 39.5±0.2 39.7±0.1 Negative control group 39.1±0.4 39.2±0.3

[0099] As shown in Table 2, all vaccinated groups had similar body temperatures compared to the negative control group, and there was no statistical significance compared to the negative control group, and no unusual findings were found.

[0100]

[0101] 2-3. Antibodies to PCV2d

[0102] To compare the immunogenicity of vaccines according to the immune booster, antibody titers against PCV2d were measured. The results of the PCV2d antibody titer measurements are presented in Table 3 and Figure 1.

[0103] PCV2d-specific IgG antibody titer average (dilution factor, AVE±SE) 0 WPV2 WPV4 WPV6 WPV8 WPV Test group-1 (Antigen only) 272±139 396±239 128±0 213±43 256±0 Test group-2 (ISA15A) 1,100±1,000 448±212 320±641,024±36 21,664±820 Test group-3 (GEL02) 169±117 256±91 576±16 1640±128 853±171 Test group-4 (IMS1313) 105±57 416±96 512±0 768±148 1,152±322 Test group-5 (Carbopol) 974p)26±14272±92192±37512±0853±171 Positive control group272±92368±222512±1811024±01,536±296 Negative control group352±224128±4556±820±413±3

[0104] As shown in Table 3 and Fig. 1, relatively high levels of PCV2d-specific IgG antibody titers were observed in all groups before vaccination due to maternal antibodies. As time elapsed after vaccination, the maternal antibodies in the negative control group gradually decreased, but the vaccinated test group maintained high levels of PCV2d-specific IgG antibody titers until the end of the test (8 WPV). In particular, test groups 2, 3, 4, and 5 containing the immune booster and the positive control group showed significantly higher antibody titers than the negative control group from 4 WPV (P<0.001), and showed high antibody titers that were significantly different from the negative control group up to 8 WPV.

[0105]

[0106] 2-4. Neutralizing antibodies against PCV2d

[0107] To compare the immunogenicity of vaccines according to the immune booster, neutralizing antibody titers against PCV2d were measured. The results of the neutralizing antibody titers against PCV2d are presented in Table 4 and Figure 2.

[0108] PCV2dAverage neutralizing antibody titer (dilution factor, AVE±SE)0 WPV2 WPV4 WPV6 WPV8 WPV Test group-1 (Antigen only) 11±720±437±1443±1164±0 Test group-2 (ISA15A) 6±236±1048±980±16144±40 Test group-3 (GEL02) 3±132±1180±1696±19107±21 Test group-4 (IMS1313) 3±148±988±24192±37176±48 Test group-5 (Carbopol) 974p)6±320±432±064±0107±21 Positive control group6±336±1080±16176±48144±40 Negative control group4±12±02±02±03±1

[0109] As shown in Table 4 and Fig. 2, all vaccinated test groups showed significant differences compared to the negative control group from 2 WPV (P<0.001), and it was confirmed that the neutralizing antibody titer against PCV2d continuously increased up to 8 WPV. In particular, test group-4 (IMS1313) showed a significantly high level of neutralizing antibody titer of approximately 192 to 6 WPV, and it was confirmed that a higher level of neutralizing antibody was produced than the positive control group, intramuscular innocirco vaccine, from 2 WPV to 8 WPV.

[0110] Based on the above results, IMS1313 was selected as an immune enhancer for the InnoSec ID vaccine.

[0111]

[0112] Example 3. Test to determine the content of immune enhancer in PCV2 vaccine composition

[0113] In Example 2 above, it was confirmed that the neutralizing antibody titer of the InnoSerco ID vaccine was higher than that of the InnoSerco vaccine for intramuscular injection when the immune enhancer IMS1313 was included. Accordingly, in this Example, the content of IMS1313 that exhibited the most effective immune-inducing ability in pigs, the target animal, was investigated.

[0114] The experimental group for determining the content of the immune enhancer was composed as shown in Table 5.

[0115] Military Military Adjuvant Antigen Dose (1 dose) Vaccination Route 2) Inoculation dose (1 dose) Number of individuals Product name Content (1 dose) G1 Test group - 1 IMS 131 3 50% (v / v) Recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 5 G2 Test group - 2 IMS 131 3 40% (v / v) Recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 5 G3 Test group - 3 IMS 131 3 30% (v / v) Recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 5 G4 Test group - 4 IMS 131 3 20% (v / v) Recombinant PCV2d protein purified antigen, 20 μg intradermally 0.5 mL 5 G5 Negative control group 1)---Intradermal 0.5 mL5 1) The negative control group is the group that received physiological saline solution (PBS). 2) The inoculation pressure is 60 psi.

[0116]

[0117] 3-1. Evaluation of side effects and adverse reactions

[0118] For 7 days after vaccination, clinical symptoms such as swelling, inflammation, loss of appetite, decreased vitality, cough, dyspnea, suffocation, shock, vomiting, and diarrhea were observed at the vaccination site for each individual.

[0119] As a result of observation, no side effects or adverse reactions were observed in any group.

[0120]

[0121] 3-2. Temperature measurement

[0122] Rectal temperature was measured before and 4 hours after vaccination. The results are shown in Table 6.

[0123] Mean body temperature by group (time after vaccination, AVE±SE) 0 h 4 h Test group - 1 (IMS1313 50%) 39.5±0.1 39.7±0.1 Test group - 2 (IMS1313 40%) 40.0±0.2 40.0±0.2 Test group - 3 (IMS1313 30%) 39.9±0.2 40.0±0.3 Test group - 4 (IMS1313 20%) 39.9±0.1 40.0±0.2 Negative control group 40.0±0.1 39.8±0.1

[0124] As shown in Table 6, all vaccinated groups had similar body temperatures compared to the negative control group, and no unusual findings were found.

[0125]

[0126] 3-3. Antibodies to PCV2d

[0127] To compare the immunogenicity of vaccines with different levels of immune booster, antibody titers against PCV2d were measured. The antibody titer measurement results are presented in Table 7 and Figure 3.

[0128] Group name PCV2d-specific IgG antibody average (dilution factor, AVE±SE) 0 WPV2 WPV4 WPV6 WPV Test group-1 (IMS1313 50%) 474±228 333±77 704±1922,048±0 Test group-2 (IMS1313 40%) 84±44 282±63 512±1402,253±502 Test group-3 (IMS1313 30%) 418±187 192±40 358±1692,662±614 Test group-4 (IMS1313 20%) 232±117 141±47 154±267 17±125 Negative control group 646±398 250±19 472±47 36±24

[0129] As shown in Table 7 and Fig. 3, relatively high levels of PCV2d-specific IgG antibody titers were confirmed in all groups before vaccination due to maternal antibodies. As time elapsed after vaccination, the maternal antibodies in the negative control group gradually decreased, but the vaccinated test group maintained high levels of PCV2d-specific IgG antibody titers until the end of the test (6 WPV). In particular, test group-1 (IMS1313 50%), test group-2 (IMS1313 40%), and test group-3 (IMS1313 30%) showed high antibody titers of approximately 2048 (P<0.05), 2253 (P<0.01), and 2662 (P<0.001) at 6 WPV, respectively, showing significant differences compared to the negative control group.

[0130]

[0131] 3-4. Neutralizing antibodies against PCV2d

[0132] To compare the immunogenicity of vaccines with different levels of immune enhancers, neutralizing antibody titers against PCV2d were measured. The neutralizing antibody titer results are presented in Table 8 and Figure 4.

[0133] PCV2d neutralizing antibody average (dilution factor, AVE±SE) 0 WPV2 WPV4 WPV6 WPV Test group-1 (IMS1313 50%) 3±0 22±480±16 224±32 Test group-2 (IMS1313 40%) 4±129±990±16 410±63 Test group-3 (IMS1313 30%) 4±135±877±13 358±94 Test group-4 (IMS1313 20%) 8±6 26±477±13 154±26 Negative control group 8±22±03±06±1

[0134] As shown in Table 8 and Fig. 4, the vaccinated test groups showed a continuous increase in neutralizing antibody titers against PCV2d from WPV 2 to WPV 6. In particular, test groups 2 (IMS1313 40%) and 3 (IMS1313 30%) showed significantly high neutralizing antibody titers of approximately 410 (P<0.001) and 358 (P<0.01), respectively, against WPV 6, showing a significant difference compared to the negative control group.

[0135] Based on the above results, it was confirmed that the optimal content of IMS1313 in the InnoSeco ID vaccine was 40% (v / v).

[0136]

[0137] Example 4. Antigen content determination test of PCV2 vaccine composition

[0138] In the above Examples 2 and 3, it was confirmed that the neutralizing antibody titer of the InnoSeco ID vaccine was high when the immune enhancer IMS1313 was added at 40% (v / v). Accordingly, in this Example, the content of the purified recombinant PCV2d protein antigen that exhibited the most effective immune-inducing ability in the target animal, pigs, was investigated.

[0139] The experimental group for determining the antigen amount was organized as shown in Table 9.

[0140] Military Military Military Description Vaccination Dose (1 dose) 2)Antigen content per dose: Adjuvant G1, Test group - 1, Recombinant PCV2d protein purified antigen, 20 μg, IMS131340 % (v / v) 0.5 mL, 5G2, Test group - 2, Recombinant PCV2d protein purified antigen, 10 μg, 0.5 mL, 5G3, Test group - 3, Recombinant PCV2d protein purified antigen, 5 μg, 0.5 mL, 5G4, Test group - 4, Recombinant PCV2d protein purified antigen, 2 μg, 0.5 mL, 5G5, Negative control group, Phosphate Buffered Saline (hereinafter PBS) 1) 0.5 mL5 1) The negative control group is the group vaccinated with physiological saline solution (PBS). 2) It was administered intradermally and the injection pressure was 60 psi.

[0141]

[0142]

[0143]

[0144] 4-1. Evaluation of side effects and adverse reactions

[0145] For 14 days after vaccination, clinical symptoms such as swelling, inflammation, loss of appetite, decreased energy, cough, dyspnea, suffocation, shock, vomiting, and diarrhea were observed at the vaccination site for each individual.

[0146] As a result of observation, atrophy occurred in one subject (G2-5) of the test group-2 and one subject (G5-4) of the negative control group in the second week after vaccination, but G5-4 showed a recovery pattern from 4 WPV and G2-5 showed a recovery pattern from 6 WPV.

[0147]

[0148] 4-2. Temperature measurement

[0149] Rectal temperature was measured before vaccination and 4 and 24 hours after vaccination. The results are shown in Table 10.

[0150] Mean body temperature by group (time after vaccination, AVE±SE) 0 h 4 h 2 4 h Test group - 1 (20 μg) 39.7 ± 0.1 39.8 ± 0.1 39.5 ± 0.1 Test group - 2 (10 μg) 40.2 ± 0.2 40.3 ± 0.3 39.8 ± 0.1 Test group - 3 (5 μg) 40.1 ± 0.0 40.1 ± 0.2 39.6 ± 0.1 Test group - 4 (2 μg) 39.8 ± 0.2 39.8 ± 0.2 39.7 ± 0.2 Negative control group 39.8 ± 0.2 40.0 ± 0.2 39.6 ± 0.1

[0151] As shown in Table 10, all vaccinated groups had similar body temperatures compared to the negative control group, and no unusual findings were found.

[0152]

[0153] 4-3. Antibodies to PCV2d

[0154] To compare the immunogenicity of vaccines according to antigen dose, antibody titers against PCV2d were measured. The antibody titer measurement results are shown in Table 11 and Figure 5.

[0155] Group name PCV2d-specific IgG antibody average (dilution factor, AVE±SE) 0 WPV2 WPV4 WPV6 WPV Test group-1 (20 μg) 4,198±1,654 320±179 973±307 1,331±307 Test group-2 (10 μg) 3,686±1,842 525±382 563±125 1,843±597 Test group-3 (5 μg) 2,662±1,424 998±433 870±320 922±102 Test group-4 (2 μg)2,278±8021,690±3581,024±280614±174 Negative control group2,611±1,445550±208442±238120±56

[0156] As shown in Table 11 and Figure 5, relatively high levels of PCV2d-specific IgG antibody titers were observed in all groups before vaccination due to maternal antibodies. While maternal antibodies in the negative control group gradually decreased over time after vaccination, the vaccinated test group maintained high levels of PCV2d-specific IgG antibody titers until the end of the study (6 WPV), showing a significant difference compared to the negative control group. In particular, antibody titers in test groups 1, 2, 3, and 4 were statistically similar.

[0157]

[0158] 4-4. Neutralizing antibodies against PCV2d

[0159] To compare the immunogenicity of vaccines according to antigen dose, neutralizing antibody titers against PCV2d were measured. The neutralizing antibody titer measurement results are shown in Table 12 and Figure 6.

[0160] PCV2d neutralizing antibody average (dilution factor, AVE±SE) 0 WPV2 WPV4 WPV6 WPV Test group-1 (20 μg) 5±132±0 192±40 205±77 Test group-2 (10 μg) 5±158±6 179±31 154±43 Test group-3 (5 μg) 9±219±370±16 102±16 Test group-4 (2 μg) 9±238±683±19 58±19 Negative control group 8±28±04±02±0

[0161] As shown in Table 12 and Figure 6, it was confirmed that the neutralizing antibody titer against PCV2d continuously increased from WPV 2 to WPV 6 in all vaccinated test groups after vaccination, and there was a significant difference compared to the negative control group. In particular, the average neutralizing antibody titer of test group-1 (20 μg) at WPV 6 was the highest at approximately 206, and showed a significant difference from test group-4 (2 μg) (P<0.05). In addition, a correlation was confirmed in which the higher the antigen dose, the more the neutralizing antibody against PCV2d increased.

[0162]

[0163] In summary, the present inventors have developed a vaccine composition for preventing PCV2d infection for intradermal administration (i.e., an innocirco ID vaccine). The innocirco ID vaccine is characterized by (i) having an inoculation volume (1 dose) of 0.5 ml, (ii) containing 20 μg of purified recombinant PCV2d protein antigen, and (iii) containing 40% (v / v) of the immune enhancer IMS1313. The innocirco ID vaccine of the present invention has been verified for safety through clinical evaluation, and was confirmed to induce a higher level of neutralizing antibody titer than the intramuscular vaccination group. This means that the innocirco vaccine effectively protects against the recently prevalent porcine circovirus, and thus can be utilized in various fields such as the prevention of porcine circovirus infection and pig farming.

[0164]

[0165] 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 2d (PCV2d); and an excipient; A vaccine composition for preventing porcine circovirus infection, wherein the above excipient is present in an amount of 10 to 50% (v / v) of the vaccine composition volume.

2. A composition according to claim 1, wherein the recombinant protein derived from porcine circovirus type 2d is represented by the amino acid sequence of sequence number 1.

3. A composition according to claim 1, wherein the recombinant protein derived from porcine circovirus type 2d is included at a concentration of 1 to 50 μg / dose.

4. A composition according to claim 1, wherein the excipient is at least one selected from the group consisting of ISA15A, GEL02, IMS1313, and carbopol 974p.

5. A composition according to claim 1, wherein the composition is for intradermal injection.

6. A composition according to claim 1, wherein the inoculation volume of the composition is 0.1 to 1 ml.

7. A method for preventing porcine circovirus infection, comprising the step of intradermally inoculating a vaccine composition according to Article 1 into a pig.

8. A method according to paragraph 7, wherein the inoculation is intradermal inoculation using a needle-free syringe.

9. In the 8th paragraph, the pressure of the needle-free syringe is 10 to 100 psi.

10. A method according to claim 7, wherein the inoculation is performed intradermally at one or more sites selected from the group consisting of the back of the ear, the back of the neck, the hind legs, and the buttocks.

11. Containing a recombinant protein derived from porcine circovirus type 2d; and excipients; A pharmaceutical composition for preventing or treating a disease caused by porcine circovirus infection, wherein the above excipient is present in an amount of 10 to 50% (v / v) based on the composition volume.

12. In paragraph 11, 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 comprising at least one selected from the group consisting of bronchopneumonia, Eustachian tube inflammation, polyserositis, mycoplasmal pneumonia, and pleural pneumonia.

13. A step of intradermally administering to a pig a composition comprising a recombinant protein derived from porcine circovirus type 2d; and an excipient; The above excipients are 10 to 50% (v / v) of the composition capacity. Method for preventing or treating diseases caused by porcine circovirus infection.

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