Recombinant expression vector for producing foot-and-mouth disease virus-like particles or nanoparticles and vaccine compositions using the same

Abstract

The present invention relates to a recombinant expression vector for producing foot-and-mouth disease virus-like particles or nanoparticles and a vaccine composition using the same. The present invention provides virus-like particles or nanoparticles produced by simultaneously expressing VP4 with VP1, VP2, and VP3 among FMDV structural proteins, or by expressing VP1, VP2, and VP3 excluding VP4, and a method for producing the same. The virus-like particles or nanoparticles produced according to the method of the present invention can be effectively used to prevent diseases caused by foot-and-mouth disease virus infection.

Description

[Technical Field]

[0001] The present invention relates to a recombinant expression vector for producing foot-and-mouth disease virus-like particles or nanoparticles and a vaccine composition using the same. [Background technology]

[0002] Foot and Mouth Disease Virus (FMDV) is a virus that causes a fatal, acute, infectious disease in cloven-hoofed animals, including cattle and pigs. The virus induces a variety of symptoms, including fever, lameness, and blister formation, and can be transmitted through direct contact or by people, vehicles, and various objects passing through contaminated areas. It is also highly contagious, capable of spreading through the air up to 250 km or more. This has a serious impact on the livestock industry, resulting in economic losses such as pregnancy problems and reduced milk production. Foot and mouth disease is designated as a Type 1 livestock infectious disease by the World Organization for Animal Health (WOAH) under the Act on the Prevention of Infectious Diseases of Livestock.

[0003] FMDV is a single-stranded, positive-sense RNA virus belonging to the Aphthovirus genus of the Picornaviridae family. Seven serotypes are known: O, A, Asia1, C, SAT1, SAT2, and SAT3. Each serotype is known to have diverse regional topotypes. Globally, O type has been predominantly prevalent, while in East Asian regions such as China and North Korea, O, Asia1, and A types tend to occur intermittently. FMD virus serotypes are not serologically neutralized, and significant genetic and antigenic differences exist, making cross-protection by vaccines ineffective.

[0004] FMDV has a naked capsid with an icosahedral structure. The P1 region of the FMDV polyprotein encodes structural proteins, while the P2 and P3 regions encode nonstructural proteins. The structural protein precursor P1 is cleaved by viral protease 2A, and the P1 precursor is processed into capsid proteins VPO, VP3, and VP1. 3C is the viral protease responsible for processing the P1 precursor into capsid proteins. In the virion, the protein VP0 is cleaved into two proteins, VP4 and VP2.

[0005] Inactivated virus vaccines for preventing FMD are commercially available and are typically inactivated with ethyleneimine after cell culture and mixed with an adjuvant. Current FMD vaccines have been criticized for their short antibody persistence and low immunogenicity in pigs, necessitating the development of more effective and stable vaccines. In particular, intramuscular administration of commercial FMD vaccines to pigs can cause local side effects, such as the formation of muscle lesions at the injection site, such as fibrosis and granulomas, and can also result in poor safety. Commercial FMD vaccines administered intramuscularly can cause granulomas and suppuration that persist until the time of shipment, leading to problems with abnormal meat. This has become a major reason for farms to avoid vaccination due to the economic losses caused by abnormal meat.

[0006] An ideal vaccine design is needed to overcome these limitations of current commercial vaccines.

[0007] Therefore, the present inventors produced virus-like particles or nanoparticles by simultaneously expressing VP4 and VP1, VP2, and VP3 among the FMDV structural proteins, or by expressing VP1, VP2, and VP3 excluding VP4, and confirmed that a vaccine composition using these can induce an effective immune response, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a recombinant expression vector containing an FMDV antigen, a transformant transformed with the recombinant expression vector, virus-like particles or nanoparticles produced by the transformant, and a method for producing the same. It is another object of the present invention to provide a vaccine composition for FMD virus and a method for preventing disease caused by FMD virus infection.

[0009] Yet another object of the present invention is to provide a use of said virus-like particles or nanoparticles for the prevention of foot-and-mouth disease virus disease. [Means for solving the problem]

[0010] To achieve the above object, the present invention provides a recombinant expression vector comprising a first promoter and genes encoding Foot and Mouth Disease Virus (FMDV) proteins VP2, VP3, VP1, and 3C operably linked to the promoter.

[0011] The present invention also provides a recombinant vector comprising a first promoter and genes encoding FMDV VP2, VP3, VP1 and 3C operably linked to the promoter; and a second promoter and a gene encoding FMDV protein VP4 operably linked to the promoter.

[0012] The present invention also provides a transformant transformed with the recombinant expression vector, a virus-like particle or nanoparticle produced by the transformant, and a method for producing the same.

[0013] The present invention also provides a vaccine composition for foot-and-mouth disease virus, which contains the virus-like particles or nanoparticles as an active ingredient.

[0014] The present invention also provides a method for preventing a disease caused by foot-and-mouth disease virus infection, comprising administering the vaccine composition.

[0015] The present invention also provides a use of the virus-like particles or nanoparticles for the prevention of foot-and-mouth disease virus disease. [Effects of the Invention]

[0016] The present invention relates to a recombinant expression vector for producing foot-and-mouth disease virus-like particles or nanoparticles and a vaccine composition using the same. The present invention provides virus-like particles or nanoparticles produced by simultaneously expressing VP4 with VP1, VP2, and VP3 among FMDV structural proteins, or by expressing VP1, VP2, and VP3 excluding VP4, as well as a method for producing the same. The virus-like particles or nanoparticles produced according to the method of the present invention can be effectively used to prevent diseases caused by foot-and-mouth disease virus infection. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram showing the VP4 co-expression form of the present invention. [Figure 2] FIG. 1 is a schematic diagram showing the expression form of Delta-VP4 of the present invention. [Figure 3] FIG. 1 is a schematic diagram showing the full form. [Figure 4] FIG. 1 shows the results of Coomassie staining to confirm whether the recombinant virus of the present invention expresses a target protein. [Figure 5] FIG. 1 shows the results of Western blotting to confirm whether the recombinant virus of the present invention expresses VP1 protein. [Figure 6] This figure shows the results of confirming the expression of VP1 protein using a culture obtained by culturing a recombinant virus transformed with Delta-VP4 form. [Figure 7]FIG. 1 shows the results of Coomassie staining to confirm whether the recombinant virus transformed in full form expresses the target protein. [Figure 8] FIG. 1 shows the results of confirming the expression of VP1 protein using a culture cultured with a recombinant virus transformed with the full form. [Figure 9] FIG. 1 shows the results of comparing the antigen amounts obtained by ELISA using an antibody specific to the FMDV VP1 protein. [Figure 10] FIG. 1 shows the results of Western blotting to confirm the expression of O type FMDV VP1 protein. [Figure 11] FIG. 1 shows the results of Western blotting to confirm the expression of O type FMDV VP2 protein. [Figure 12] FIG. 1 shows the results of Western blotting to confirm the expression of O type FMDV VP3 protein. [Figure 13] FIG. 1 shows the results of Western blotting to confirm the expression of O type FMDV VP4 protein. [Figure 14] FIG. 1 shows the results of Western blotting to confirm the expression of type A FMDV VP1 protein. [Figure 15] FIG. 1 shows the results of Western blotting to confirm the expression of type A FMDV VP2 protein. [Figure 16] FIG. 1 shows the results of Western blotting to confirm the expression of type A FMDV VP3 protein. [Figure 17] FIG. 1 shows the results of Western blotting to confirm the expression of type A FMDV VP0 protein. [Figure 18] FIG. 1 shows the results of Western blotting to confirm the expression of type A FMDV VP4 protein. [Figure 19] FIG. 1 shows nanoparticles of candidate FMDV antigen substances observed by TEM. [Figure 20]FIG. 1 shows TEM observation of VLP particles of FMDV antigen candidate substances. [Figure 21] FIG. 1 shows a method for administering the vaccine composition of the present invention. [Figure 22] 1 is a graph showing the results of confirming the immunogenicity of vaccine candidate substances. [Figure 23] 1 is a graph showing the results of measuring antibody titers against O and A types as a function of the concentration of a vaccine candidate substance. [Figure 24] 1 is a graph showing the results of measuring neutralizing antibody titers against O and A types as a function of the concentration of a vaccine candidate substance. [Figure 25] FIG. 1 shows the histopathological observation results of pigs administered with a vaccine composition produced from a recombinant virus transformed with the VP4 Co expression form. [Figure 26] FIG. 1 shows histopathological observations of pigs administered the control vaccine. BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The present invention will be described in detail below.

[0019] The present invention provides a recombinant expression vector comprising a first promoter and genes encoding Foot and Mouth Disease Virus (FMDV) proteins VP2, VP3, VP1, and 3C operably linked to the promoter.

[0020] The present invention also provides a recombinant vector comprising a first promoter and genes encoding FMDV VP2, VP3, VP1 and 3C operably linked to the promoter; and a second promoter and a gene encoding FMDV protein VP4 operably linked to the promoter.

[0021] In the present invention, the "foot-and-mouth disease virus (hereinafter referred to as FMDV)" belongs to the genus Aphtovirus in the family Picornaviridae. The virus is composed of 60 copies of four capsid proteins (VP1, VP2, VP3, and VP4) and a single-stranded RNA genome (approximately 8.5 kb), and is a highly contagious disease that infects cloven-hoofed animals, particularly cattle, pigs, and sheep.

[0022] FMDV has four capsid proteins: VP1, VP2, VP3, and VP4. Of these, VP1, VP2, and VP3 are exposed on the capsid surface, with VP1 being most closely related to viral infectivity, and VP4 being a small protein located internally. There are many serotypes depending on the region, and seven serotypes, including A, O, C, SAT1, SAT2, SAT3, and Asia1, are classified according to antigenic structure, with these serotypes having over 80 serosubtypes.

[0023] In the present invention, "virus-like particles (VLPs) and nanoparticles" refer to antigens that have a pattern that is almost identical to that of a virus, including nonstructural proteins that are responsible for viral replication and intracellular penetration. They do not contain genetic material, but have a shape that is almost identical to that of a virus due to the combination of structural surface (coat) proteins that represent the antigenicity of the virus, using genetic recombination technology.

[0024] The envelope of the FMD virus is the antigenic site most reliably detected by the immune system after internal infection, and it is possible to produce a safe and effective new vaccine using the proteins that form this envelope.

[0025] The recombinant expression vector contains a polynucleotide in which the base sequences of FMD virus VP2, VP3, VP1, and 3C are sequentially arranged, and when expressed as a protein, it can self-assemble to produce FMD virus-like particles. It can also be engineered to express the VP4 base sequence in the opposite direction to the polynucleotide.

[0026] The foot-and-mouth disease virus in the present invention can be selected from O, A, Asia1, C, SAT1, SAT2, and SAT3 types.

[0027] The FMD virus O type may be selected from the group consisting of O-Andong, O-PanAsia2 (O-PA2), O-manisa, O-Taiwan97 (O-Twn97), O-Campos, O-Boeun (O-BE), O-Jincheon (O-JC), O-Anseong (O-AS), and O-Gimje (O-GJ). In some embodiments of the present invention, the O type may utilize the gene sequence of O-Andong (GenBank: KF112887.1). In some embodiments, the nucleic acid sequence is codon-optimized using insect cell-preferred codons to improve protein expression. In one example, the nucleotide sequences encoding the VP1, VP2, VP3, VP4, and 3C proteins may be the nucleotide sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 9, respectively, or variants thereof. Additionally, the O type VP1, VP2, VP3, or VP4 protein may each consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, or may comprise a variant thereof. The FMD virus type A may be selected from the group consisting of A-Pocheon (A-PC), A-Yeoncheon (A-YC), A-Bangladesh (A-Ban), A-Malaysia97 (A-May97), A-Gimpo (A-GP), and A22-Iraq. In some embodiments of the present invention, the A type may be the gene sequence of A-Pocheon (GenBank: KC588943.1) or A-Yeoncheon (GenBank: KY766148.1). In some embodiments, the nucleic acid sequence is codon-optimized using insect cell-preferred codons to improve protein expression. For example, the nucleotide sequences encoding the VP1, VP2, VP3, VP4, and VP3C proteins may be the nucleotide sequences represented by SEQ ID NOs: 5, 6, 7, 8, and 9, respectively, or variants thereof. Furthermore, the A type VP1, VP2, VP3, or VP4 protein can each consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or a variant thereof.

[0028] The 3C may be a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 or a variant thereof, and may consist of the amino acid sequence of SEQ ID NO: 18 or a variant thereof. The FMDV 3C is a protease that cleaves FMDV VP4, VP2, VP3, and VP1 proteins, enabling self-assembly of FMDV VLPs.

[0029] The 3C sequence does not differ among FMDV serotypes and has a commonly conserved sequence. However, the VP4-VP2-VP3-VP1 sequence of FMDV differs among serotypes O, A, Asia1, SAT, and C, and even within the same serotype, the sequence differs among serosubtypes.

[0030] In the present invention, the recombinant expression vector can further include any sequence, preferably a promoter and / or a sequence for promoting expression (e.g., a 2A sequence and / or a Burst sequence, etc.), as long as it can increase the expression level or efficiency of the protein of interest.

[0031] The term "promoter" as used herein is not limited to any sequence sufficient to induce transcription. For example, a polyhedrin promoter may be located in front of the VP2-VP3-VP1-3C target protein to increase the expression efficiency of the target protein. The polyhedrin promoter may have the nucleotide sequence of SEQ ID NO: 19 or a variant thereof. Alternatively, the polyhedrin promoter may be located in front of the vp39 promoter (SEQ ID NO: 20). A different promoter may be used for the expression of the VP4 target protein. For example, a p10 promoter may be located in front of the VP4 target protein to increase the expression efficiency of the target protein. The p10 promoter may have the nucleotide sequence of SEQ ID NO: 21 or a variant thereof.

[0032] The recombinant expression vector may further include a 2A sequence. The 2A gene sequence encodes 18 to 22 amino acids, of which the four terminal amino acids, asparagine (N), proline (P), glycine (G), and proline (P), are highly conserved across species. This sequence tends to undergo self-cleavage during peptide synthesis. Due to this property, when the ribosome transcribes a protein and reaches the genetic code encoding the N, P, and G amino acids at the end of the 2A sequence, it recognizes NPG in order to form a peptide bond. Next, it recruits a prolyl-tRNA bound to the proline amino acid encoding code, instead of a translation release factor (RF). After the RF factor binds, the previously formed peptide is released from the ribosome without further peptide bond formation. The code encoded after the 2A sequence then functions normally, allowing protein transcription to proceed. In conclusion, by inserting a 2A sequence, several genes can be expressed using a single promoter. The recombinant expression vector of the present invention can simultaneously express the genes by inserting such a 2A sequence between each gene. In one embodiment, when the nucleotide sequence encoding the VP2-VP3-VP1-3C protein is sequentially arranged, a 2A sequence can be further included between the VP1 and 3C genes. In one embodiment, the 2A sequence can be the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 23, or a variant thereof.

[0033] The recombinant expression vector may further comprise a Burst sequence. The Burst sequence is a portion of the polyhedrin promoter, located between the translation initiation site and TAAG, and it is known that Vlf-1 specifically binds to the Burst sequence at the late stage of baculovirus infection to promote transcription. In one embodiment, the Burst sequence may be the nucleotide sequence represented by SEQ ID NO: 24 or a variant thereof.

[0034] In the present invention, the term "vector" refers to any vehicle for cloning and / or transferring a base into a host cell. A vector may also be a replication unit to which another DNA fragment is attached, allowing replication of the attached fragment. The term "replication unit" refers to any genetic unit (e.g., a plasmid, a phage, a cosmid, a chromosome, a virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replicating under its own control. The term "vector" includes viral and non-viral vehicles for introducing a base into a host cell in vitro, ex vivo, or in vivo.

[0035] In the present invention, the term "recombinant expression vector" refers to a vector constructed to express a protein of interest in an appropriate host cell, comprising essential regulatory elements operably linked to allow expression of a gene insert. The recombinant expression vector of the present invention may contain genes encoding FMDV proteins VP2, VP3, VP1, and 3C, or may contain a gene encoding FMDV protein VP4. In this case, the genes encoding FMDV proteins VP2, VP3, VP1, and 3C and the gene encoding FMDV protein VP4 are each linked to two different promoters within a single vector, and the promoters can be positioned in opposite directions within the vector to minimize interference between the two promoters.

[0036] In the present invention, the term "variant" as used herein with respect to a nucleic acid can mean (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement of a referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0037] In the present invention, a "variant" of a peptide or polypeptide differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. A variant can also refer to a protein having substantially the same amino acid sequence as a reference protein, with amino acids that retain at least one biological activity. Conservative substitutions of amino acids, i.e., the replacement of an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes.

[0038] The present invention also provides a transformant transformed with the recombinant expression vector.

[0039] In the present invention, the "recombinant expression vector" is as described above.

[0040] In the present invention, "transformation" refers to the process by which DNA is introduced into a host and becomes replicable as a chromosomal integrant or by complete chromosomal integration, and refers to the phenomenon of introducing external DNA into a cell to artificially cause a genetic change.

[0041] The term "transformant" as used herein means a transformed plant or animal produced by transformation, and includes genetically modified organisms produced by inducing modification or mutation of a specific gene using genetic engineering techniques.

[0042] In the present invention, host cells for producing a "transformant" are preferably host cells that have high DNA introduction efficiency and high expression efficiency of the introduced DNA, and any microorganism, including prokaryotic and eukaryotic, can be used. The host cells can be selected from the group consisting of Escherichia bacteria, Bacillus bacteria, Pseudomonas bacteria, lactic acid bacteria, yeast, animal cells, and insect cells, and preferably, the host cells can be insect cells.

[0043] The transformant is for producing foot-and-mouth disease virus-like particles or nanoparticles.

[0044] The present invention also provides virus-like particles or nanoparticles produced by the transformants.

[0045] In the present invention, the explanations for "transformation" and "virus-like particles or nanoparticles" are as described above.

[0046] The present invention also provides a method for producing virus-like particles or nanoparticles for preventing diseases caused by foot-and-mouth disease virus infection, comprising the steps of: (1) constructing a recombinant expression vector of the present invention; (2) preparing a transformant using the recombinant expression vector; (3) transfecting host cells with the transformant; (4) culturing the transfected host cells and harvesting the culture; and (5) harvesting the virus-like particles or nanoparticles from the culture.

[0047] In the present invention, the "recombinant expression vector," "host cell," "transformant," "foot-and-mouth disease," and "virus-like particle or nanoparticle" are as described above.

[0048] The present invention also provides a vaccine composition for foot-and-mouth disease virus, which contains the virus-like particles or nanoparticles as an active ingredient.

[0049] In the present invention, the term "vaccine" refers to a biological preparation containing an antigen that induces immunity in a living body, and is an immunogen or antigenic substance that induces immunity in a living body when injected or orally administered to humans or animals for the prevention of infectious diseases.

[0050] The content of the antigen may be 1 to 15% by weight based on the total weight of the vaccine composition.

[0051] The vaccine compositions of the present invention may further comprise one or more adjuvants.

[0052] In the present invention, the term "adjuvant" generally refers to any substance that enhances humoral and / or cellular immune responses to an antigen. Traditional vaccines are composed of unprocessed preparations of killed pathogenic microorganisms, and impurities associated with the culture medium of the pathogenic microorganisms can act as adjuvants to enhance immune responses. However, when a homogenous preparation of purified protein subunits is used as an antigen for vaccination, the immunity elicited by such antigens is insufficient, necessitating the addition of some exogenous substance as an adjuvant. The use of an adjuvant may allow for a smaller dose of antigen to stimulate an immune response, thereby reducing vaccine production costs. In some embodiments, the adjuvant includes EMULSIGEN, aluminum hydroxide, Carbigen, saponin, and CpG, or a combination thereof. In another embodiment, the adjuvant is preferably a commonly known oil emulsion, and may be a single oil emulsion.

[0053] The vaccine composition according to the present invention may further contain one or more second adjuvants selected from the group consisting of stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, immunostimulating complexes (iscoms), synthetic glycopeptides, bulking agents, carboxypolymethylene, bacterial cell walls, bacterial cell wall derivatives, bacterial vaccines, animal poxvirus proteins, subviral particle adjuvants, cholera toxin, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide, and mixtures thereof.

[0054] The vaccine composition of the present invention may also contain a veterinarily acceptable carrier. In the present invention, the term "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, adsorption delaying agents, etc. Examples of carriers, excipients, and diluents that can be contained in vaccine compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0055] The vaccine composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as sterile injectable solutions, according to conventional methods. When formulated, these compositions can be prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. These solid formulations can be prepared by mixing the lecithin-like emulsifier with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, water-insoluble preparations, suspensions, emulsions, and lyophilized preparations. Examples of water-insoluble preparations and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0056] VLP vaccines express one or more structural proteins of a virus through molecular biology technology. These structural proteins have the natural ability to self-assemble and can form a three-dimensional structure and antigenic determinants similar to those of natural virus particles, but lack viral nucleic acid, making them highly immunogenic and non-infectious. In addition, because they contain a high density of viral antigens on their surface, they can be delivered to immune cells in a manner similar to that of a virus infecting a living body, effectively inducing humoral and cellular immunity in the body's immune system and shortening the incubation period.

[0057] The present invention also provides a method for preventing diseases caused by foot-and-mouth disease virus infection, which comprises administering the vaccine composition to a mammal other than a human.

[0058] In the present invention, the "vaccine composition" is as described above.

[0059] In the present invention, "prevention" refers to any action of suppressing or delaying infection with the foot-and-mouth disease virus by administering a composition containing as an active ingredient a foot-and-mouth disease virus-like particle or nanoparticle protein that is self-assembled using the recombinant expression vector of the present invention.

[0060] In the present invention, the mammal may be, without limitation, a chicken, pig, monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, goat, etc. Preferably, the mammal is an artiodactyl, such as a pig or a cow.

[0061] In the present invention, the administration can be by any administration means known in the art. For example, the administration can be directly administered to an individual via intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous routes. The administration can be systemic or local.

[0062] In the present invention, the composition of the present invention may be administered in a therapeutically or prophylactically effective amount. The "therapeutically or prophylactically effective amount" can be appropriately selected by those skilled in the art, taking into consideration the severity of symptoms, the sex, age, and weight of the individual, etc. The therapeutically or prophylactically effective amount may be, for example, 1 pg to 5 g per 1 kg of the individual to be administered of FMD virus-like particles or nanoparticles self-assembled using the polynucleotide, a protein extract of the transformant, or a recombinant protein isolated from the transformant.

[0063] The present invention also provides a use of the virus-like particles or nanoparticles for preventing foot-and-mouth disease virus disease.

[0064] The "foot-and-mouth disease virus" or "virus-like particles or nanoparticles" in the present invention are as described above.

[0065] MODE FOR CARRYING OUT THE INVENTION DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings so that those skilled in the art can easily understand the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments set forth herein.

[0066] Example 1: Construction of recombinant vectors and viruses expressing FMDV antigen candidate substances To use the high-expression baculovirus expression system, transfer vectors capable of expressing FMDV structural proteins and candidate major antigens were constructed.

[0067] Specifically, we used the high-expression vector pPol-6 to clone the target gene (VP2, VP3, VP1, and 3C) downstream of the polyhedrin promoter to create the Delta-VP4 expression form (Fig. 1). We then cloned the p10 promoter and VP4 in the opposite direction to the polyhedrin promoter of the Delta-VP4 expression vector to create the VP4 co-expression form (Fig. 2). As a control, we also created a full-expression form in which the target gene (VP4, VP2, VP3, VP1, and 3C) was cloned downstream of the polyhedrin promoter (Fig. 3). To improve cloning efficiency, we inserted a codon-optimized gene sequence into the vector. We constructed a recombinant transfer vector containing the codon-optimized nucleotide sequence and the corresponding O-type antigenic determinant. We also constructed a recombinant transfer vector containing the codon-optimized nucleotide sequence and the corresponding A-type antigenic determinant.

[0068] Each of the transfer vectors constructed was inserted into a chitinase- and chathepsin-depleted Bacmid vector based on AcMNPV (Autographa californica multiple nucleopolyhedrovirus), to generate recombinant viruses, which were then propagated in Sf9 cells, an insect cell line, for mass production.

[0069] <Example 2> Confirmation of proteins expressed from recombinant viruses expressing FMDV structural proteins <2-1> Electrophoretic analysis of proteins expressed from recombinant viruses expressing FMDV structural proteins An experiment was carried out to confirm whether the recombinant virus expressing the FMDV structural proteins prepared in Example 1 expresses the target protein.

[0070] Specifically, the recombinant virus was inoculated into Hi5 insect cells. After a few days, the cells were centrifuged at 4000 rpm for 5 minutes at 4°C, and the cells and culture medium were then collected. The collected cells were then resuspended in an equal volume of PBS as the culture medium. The collected cells and culture medium were then subjected to SDS-PAGE and Western blotting, respectively.

[0071] First, we analyzed the proteins expressed from the recombinant viruses transformed with the Delta-VP4 form and the VP4 co-expression form.

[0072] As a result, as shown in Figure 4, clear bands were observed at the target protein positions of 23.1 kDa, 24.4 kDa, 23.9 kDa, and 8.9 kDa, corresponding to the VP2 protein, VP3 protein, and VP4 protein, respectively, confirming with the naked eye that a high yield of protein was secreted into the culture medium.

[0073] Furthermore, as shown in Figure 5, Western blot analysis using a VP1-specific antibody clearly confirmed that the protein identified by SDS-PAGE was VP1. Furthermore, normal expression of 3C confirmed that VP1 was cleaved from P1. In particular, while high secretion efficiency was confirmed in the Delta-VP4 form, the VP4 co-expression form demonstrated the highest expression of structural proteins.

[0074] In addition, proteins expressed from cultures (cells and supernatants) of recombinant viruses transformed with Delta-VP4 formm were analyzed.

[0075] As a result, as shown in Figure 6, it was confirmed that the Delta-VP4 form was secreted into the culture medium with high efficiency after expression, similar to the VP4 co-expression form.

[0076] These results suggest that when VP4 is removed from the FMDV structural protein P1 (VP4-VP2-VP3-VP1) and expressed, as in the VP4 co-expression form and Delta-VP4 form, the protein is secreted into the culture medium after expression, and that this secretion significantly contributes to the productivity of the antigen candidate substance.

[0077] In addition, proteins expressed from cultures (cells and supernatants) of the recombinant viruses transformed in full form were analyzed.

[0078] As a result, as shown in Figures 7 and 8, after expression of the full form, a large amount of protein was observed in the cells, but secretion by the supernatant was not observed even with increasing dpi (days post inoculation).

[0079] These results suggest that the vector expressing the FMDV structural protein P1 (VP4-VP2-VP3-VP1) after removing VP4 (Delta-VP4 form, VP4 co-expression form) plays an important role in the production of antigen candidate substances.

[0080] <2-2> ELISA was performed using antibodies specific to FMDV VP1 to compare the amount of antigen. ELISA was performed to compare the amount of FMDV antigen produced by the recombinant viruses of the present invention.

[0081] Specifically, the recombinant virus was inoculated into Hi5 insect cells. After several days, the cells were centrifuged at 4000 rpm for 5 minutes at 4°C, and the culture medium was collected. ELISA was performed on the collected culture medium samples using an antibody specific to FMDV VP1. The commercial vaccine used as a control was the BIOAFTOGEN FMD vaccine.

[0082] As a result, as shown in Figure 9, higher results were confirmed in the VP4 co-expression form, and the VP4 co-expression form was selected as the optimal expression form for FMDV structural proteins.

[0083] Example 3: Analysis of expressed proteins using antibodies specific to VP1, VP2, VP3, and VP4 structural proteins An experiment was carried out to confirm whether all FMDV structural proteins were expressed by the recombinant virus.

[0084] Specifically, Western blot analysis was performed on the culture medium of large-scale culture of the recombinant virus using specific antibodies corresponding to VP0, VP1, VP2, VP3, and VP4. As a control, a commercial vaccine, BIOAFTOGEN FMD vaccine, was used.

[0085] As a result, as shown in Figures 10 to 13, bands corresponding to the FMDV structural proteins VP1, VP2, VP3, and VP4 were confirmed using the recombinant virus against type O. These results confirmed that all proteins were expressed and existed individually, which means that the 3C protease was normally expressed and functioning.

[0086] Furthermore, as shown in Figures 14 to 18, we were able to confirm that all of the FMDV structural proteins VP1, VP2, VP3, and VP4 were expressed by the recombinant virus against type A. In particular, the VP4 co-expression form showed the expression of VP2 alone, with a band of approximately 24.4 kDa, while the full form was expressed in the form of VP0, with VP2 and VP4 bound together (Figure 15). Furthermore, since VP0 was not detected in the VP4 co-expression form, it was suggested that VP0 was separated into VP2 and VP4, while the full form was expressed in the form of VP0, with VP2 and VP4 bound together (Figure 17).

[0087] The above results indicate that, unlike the full form, in the VP4 co-expression form, each protein, VP1, VP2, VP3, and VP4, was expressed singly and secreted outside the cell, which means that the 3C protease was normally expressed and activated.

[0088] Example 4: Morphological observation of FMDV antigen candidate substances using TEM Experiments were carried out to analyze the properties of the proteins expressed in the above examples.

[0089] Specifically, the culture medium of the recombinant virus using the VP4 co-expression form was concentrated and purified. Using a tangential flow filtration (TFF) system, the culture medium was passed through a 100 kDa hollow fiber filter to concentrate 20-fold, and then the medium was exchanged with 20 mM Tris-HCl, pH 6.5 buffer. The concentrated culture medium was subjected to ion exchange chromatography (IEX) using a Captocore Q impress column. The purified FMDV antigen candidate was confirmed by SDS-PAGE and Western blot.

[0090] The purified FMDV antigen candidate material was photographed using a transmission electron microscope (TEM) for morphological observation.

[0091] As a result, it was confirmed that the nanoparticles were approximately 10 to 15 nm in size, as shown in Figure 19. Furthermore, it was confirmed that all VLP particles were approximately 25 to 30 nm in size, which was an appropriate size, as shown in Figure 20.

[0092] Example 5: Immunogenicity assay of vaccine candidate substances To confirm the immunological efficacy of the vaccine candidate, a vaccine was prepared for inoculation into target animals.

[0093] Specifically, the vaccine candidate used consisted of structural proteins against serotype O, produced from a recombinant virus constructed using VP4 co-expression, and emulsified in oil emulsion. The vaccine was administered twice, with the primary vaccination administered at week 0 and the secondary vaccination administered at week 4. Blood samples were collected weekly until week 15, and pig serum was isolated (Figure 21). Antibody titers were measured using an ELISA kit (PrioCHECK FMDV Type O Andibody SP ELISA kit) using the isolated serum. The commercial control vaccine, BIOAFTOGEN FMD vaccine, was used.

[0094] As a result, as shown in FIG. 22, 100% percentage inhibition was confirmed in all individuals, demonstrating superior immunogenicity compared to the control group.

[0095] Example 6: Immunogenicity assay depending on the concentration of vaccine candidate substance Antibody titers and neutralizing antibody titers (VNT) were evaluated according to the concentration of the vaccine candidate substance.

[0096] Specifically, the vaccine was administered twice, with the first vaccination administered at week 0 and the second at week 4 of the study. Vaccines against type O and type A were prepared and administered separately. Serum was collected weekly from pigs inoculated with each serotype, and antibody titers were measured weekly using ELISA kits (PrioCHECK FMDV Type O Andibody SP ELISA kit, PrioCHECK FMDV Type A Andibody SP ELISA kit). The commercial vaccine used in the control group was the BIOAFTOGEN FMD vaccine.

[0097] As a result, as shown in Figure 23, the antibody titers of the O type at all antigen doses were comparable to those of the control group. The antibody titer of the A type reached 50% at the low dose, two weeks later than the control group, and increased similarly at all other doses.

[0098] Furthermore, as shown in Figure 24, neutralizing antibody titers (VNT) were measured for the O type, and the VNT reached 32-fold from the second week at all doses and remained above 100-fold thereafter. For the A type, neutralizing antibodies were formed slowly at low doses, but began to increase at the intermediate dose from the second week and gradually increased, reaching 32-fold at the high dose from the second week.

[0099] <Example 7> Confirmation of safety in candidate vaccine recipient groups The skin of pigs inoculated with the vaccine candidate was examined histopathologically.

[0100] Specifically, the vaccine candidate used consisted of structural proteins against serotype O and was produced from a recombinant virus engineered in the VP4 co-expression form. The vaccine was administered twice, with the first vaccination administered at week 0 and the second at week 4, and the injection sites were observed. The commercial vaccine used in the control group was the BIOAFTOGEN FMD vaccine.

[0101] [Table 1]

[0102] As shown in Table 1, Figures 25 and 26, the candidate vaccine using the VP4 co-expression form demonstrated a low overall lesion formation level. In contrast, the control group showed significant macroscopic lesions, evidence of severe chronic granulomatous inflammation, and extensive muscle cell degeneration and necrosis.

[0103] The above results indicate that the vaccine candidate substance of the present invention has a low side effect of causing abnormal meat.

Claims

1. A recombinant expression vector comprising a first promoter and genes encoding Foot and Mouth Disease Virus (FMDV) proteins VP2, VP3, VP1 and 3C operably linked to said promoter.

2. 2. The recombinant vector of claim 1, further comprising a second promoter and a gene encoding the FMDV protein VP4 operably linked to the promoter.

3. The recombinant vector according to claim 1 , wherein the first promoter and the second promoter are arranged in opposite directions within the vector.

4. The recombinant expression vector according to claim 1, wherein the recombinant vector is expressed by a baculovirus vector.

5. 2. The recombinant expression vector of claim 1, wherein the foot-and-mouth disease virus is selected from the group consisting of serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3.

6. A transformant transformed with the recombinant expression vector according to claim 1 or 2.

7. A virus-like particle (VLP) or nanoparticle (NP) produced by the transformant according to claim 6.

8. (1) preparing a recombinant expression vector according to claim 1 or 2; (2) preparing a transformant using the recombinant expression vector; (3) transfecting the transformant into a host cell; (4) culturing the transfected host cells and harvesting the culture; and (5) harvesting virus-like particles or nanoparticles from the culture; A method for producing virus-like particles or nanoparticles for preventing disease caused by foot-and-mouth disease virus infection, comprising:

9. A vaccine composition for foot-and-mouth disease virus, comprising the virus-like particle or nanoparticle according to claim 7 as an active ingredient.

10. The vaccine composition of claim 9, further comprising an adjuvant.

11. The vaccine composition according to claim 10, wherein the adjuvant is an oil emulsion.

12. A method for preventing a disease caused by foot-and-mouth disease virus infection, comprising administering the vaccine composition of claim 9.

13. 8. Use of the virus-like particles or nanoparticles according to claim 7 for preventing foot and mouth disease virus disease.

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