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

Abstract

The 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, and provides virus-like particles or nanoparticles produced by co-expressing VP4, VP1, VP2 and VP3 in FMDV structural protein or expressing VP1, VP2 and VP3 except for VP4, and a preparation method of the virus-like particles or nanoparticles. The virus-like particles or nanoparticles prepared according to the method can be effectively used for preventing 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 Art

[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. When infected, the virus causes various symptoms such as fever, lameness, and the formation of blisters, and can also be transmitted through contact or by humans, vehicles, and various objects passing through contaminated areas. In addition, it has a high degree of infectivity and can be transmitted through the air up to a distance of more than 250 km. This has a serious impact on the livestock industry and causes economic losses such as pregnancy problems and reduced milk production. Foot-and-mouth disease is a disease designated as a Class 1 livestock infectious disease in the "Livestock Infectious Disease Prevention and Control Law" of the World Organization for Animal Health (WOAH).

[0003] FMDV is a single-stranded positive-sense RNA virus belonging to the genus Aphtovirus of the family Picornaviridae. A total of 7 serotypes, namely O, A, Asia1, C, SAT1, SAT2, and SAT3, are known so far, and various topotypes are known to exist for each serotype. Type O mainly persists globally, and in East Asian regions such as China and North Korea, type O occurs and types Asia1 and A occur intermittently. FMDV exhibits significant genetic or antigenic differences between viruses of different serotypes, such that there is no serological neutralization and no cross-protection by vaccines.

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

[0005] To prevent foot-and-mouth disease, inactivated virus vaccines are commercialized and sold. Usually, the virus is cultured in cells and inactivated with ethyleneimine and mixed with an adjuvant to be used as a vaccine. The disadvantages of foot-and-mouth disease vaccines pointed out so far include a short duration of antibodies and low immunogenicity in pigs. Therefore, more effective and stable vaccines need to be developed. In particular, when intramuscularly inoculating pigs, there are local side effects such as fibrosis and granuloma at the inoculation site and low stability. The commercial foot-and-mouth disease vaccine is inoculated by intramuscular injection, but it still exists in the form of granuloma or suppuration until shipment, resulting in abnormal meat quality. This has become the main reason for farms suffering economic losses due to abnormal meat quality to resist vaccination. An ideal vaccine design is needed to overcome these limitations of current commercial vaccines.

[0006] Therefore, the present inventors produced virus-like particles or nanoparticles by co-expressing VP4, VP1, VP2, VP3 in the FMDV structural protein or expressing VP1, VP2, VP3 except VP4, and confirmed that the vaccine composition using the same can induce an effective immune response, thus completing the present invention. Summary of the Invention

[0007] Technical Problem

[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 preparing the same.

[0009] Another object of the present invention is to provide a vaccine composition against foot-and-mouth disease virus and a method for preventing diseases caused by foot-and-mouth disease virus infection.

[0010] Another object of the present invention is to provide a use of the virus-like particles or nanoparticles for preventing foot-and-mouth disease virus diseases.

[0011] Technical Solution

[0012] To achieve the above object, the present invention provides a recombinant expression vector, which contains a first promoter and genes encoding foot-and-mouth disease virus (FMDV) proteins VP2, VP3, VP1, and 3C operably linked to the first promoter.

[0013] Furthermore, the present invention provides a recombinant expression vector comprising: a first promoter and genes encoding FMDV proteins VP2, VP3, VP1 and 3C operably linked to the first promoter; and a second promoter and a gene encoding FMDV protein VP4 operably linked to the second promoter.

[0014] Furthermore, the present invention provides a transformant transformed with the recombinant expression vector, virus-like particles or nanoparticles produced by the transformant, and a method for preparing the same.

[0015] Furthermore, the present invention provides a vaccine composition against foot-and-mouth disease virus, which comprises the virus-like particles or nanoparticles as an active ingredient.

[0016] Furthermore, the present invention provides a method for preventing diseases caused by foot-and-mouth disease virus infection, the method comprising the step of administering the vaccine composition.

[0017] Furthermore, the present invention provides a use of the virus-like particles or nanoparticles for preventing foot-and-mouth disease virus diseases.

[0018] Effects of the Invention

[0019] 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 co-expressing VP4, VP1, VP2, VP3 among FMDV structural proteins or expressing VP1, VP2, VP3 except VP4, and a method for preparing the same. The virus-like particles or nanoparticles prepared according to the method of the present invention can be effectively used for preventing diseases caused by foot-and-mouth disease virus infection. Brief Description of the Drawings

[0020] Figure 1 It is a schematic diagram showing the co-expression form of VP4 of the present invention.

[0021] Figure 2 It is a schematic diagram showing the expression form of Delta-VP4 of the present invention.

[0022] Figure 3 It is a schematic diagram showing the full form.

[0023] Figure 4 It is a figure showing the result of confirming whether the recombinant virus of the present invention expresses the target protein by Coomassie Brilliant Blue staining.

[0024] Figure 5 It is a figure showing the result of confirming whether the recombinant virus of the present invention expresses VP1 protein by Western blotting.

[0025] Figure 6 A figure showing the result of confirming the expression of VP1 protein in a culture after culturing with a recombinant virus transformed into the Delta-VP4 form.

[0026] Figure 7 A figure showing the result of confirming whether a recombinant virus transformed into the full form expresses the target protein by Coomassie Brilliant Blue staining.

[0027] Figure 8 A figure showing the result of confirming the expression of VP1 protein in a culture cultured with a recombinant virus transformed into the full form.

[0028] Figure 9 A figure showing the result of comparing the amount of antigen by ELISA using an antibody specific to the FMDV VP1 protein.

[0029] Figure 10 A figure showing the result of confirming the expression of type O FMDV VP1 protein by Western blotting.

[0030] Figure 11 A figure showing the result of confirming the expression of type O FMDV VP2 protein by Western blotting.

[0031] Figure 12 A figure showing the result of confirming the expression of type O FMDV VP3 protein by Western blotting.

[0032] Figure 13 A figure showing the result of confirming the expression of type O FMDV VP4 protein by Western blotting.

[0033] Figure 14 A figure showing the result of confirming the expression of type A FMDV VP1 protein by Western blotting.

[0034] Figure 15 A figure showing the result of confirming the expression of type A FMDV VP2 protein by Western blotting.

[0035] Figure 16 A figure showing the result of confirming the expression of type A FMDV VP3 protein by Western blotting.

[0036] Figure 17 A figure showing the result of confirming the expression of type A FMDV VP0 protein by Western blotting.

[0037] Figure 18 A figure showing the result of confirming the expression of type A FMDV VP4 protein by Western blotting.

[0038] Figure 19 A figure for observing nanoparticles of an FMDV antigen candidate substance by TEM.

[0039] Figure 20 A figure for observing VLP particles of an FMDV antigen candidate substance by TEM.

[0040] Figure 21 A figure showing the administration method of the vaccine composition of the present invention.

[0041] Figure 22 A chart showing the results of confirming the immunogenicity of a vaccine candidate substance.

[0042] Figure 23 A chart showing the results of measuring antibody titers according to the concentration of vaccine candidates against serotype O and A.

[0043] Figure 24 A chart showing the results of measuring neutralizing antibody titers according to the concentration of vaccine candidates against serotype O and A.

[0044] Figure 25 A figure showing the results of histopathological observation of tissues of pigs administered with a vaccine composition produced by a recombinant virus transformed into a co-expression form of VP4.

[0045] Figure 26 A figure showing the results of histopathological observation of tissues of pigs administered with a control vaccine.

[0046] Best Mode

[0047] Hereinafter, the present invention will be described in detail.

[0048] 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 first promoter.

[0049] Furthermore, the present invention provides a recombinant expression vector comprising: a first promoter and genes encoding FMDV proteins VP2, VP3, VP1, and 3C operably linked to the first promoter; and a second promoter and a gene encoding FMDV protein VP4 operably linked to the second promoter.

[0050] In the present invention, "foot-and-mouth disease virus (hereinafter referred to as FMDV)" belongs to the genus Aphtovirus of the family Picornaviridae. The virus is composed of 60 copies of 4 capsid proteins (VP1, VP2, VP3, and VP4) and a single-stranded RNA genome (about 8.5 kb). It is a highly contagious disease that infects cloven-hoofed animals, especially cattle, pigs, and sheep.

[0051] The 4 capsid proteins of FMDV are VP1, VP2, VP3, and VP4. Among them, VP1, VP2, and VP3 are proteins exposed on the surface of the capsid. VP1 has the greatest relationship with the infectivity of the virus, and VP4 is a small protein located inside. There are multiple serotypes according to different regions and are divided into 7 serotypes, namely A, O, C, SAT1, SAT2, SAT3, and Asia1, according to the antigenic structure. There are more than 80 serum subtypes of these serotypes.

[0052] In the present invention, "Virus-Like Particle (VLP) and Nanoparticle" are antigens with shapes almost identical to those of viruses containing non-structural proteins responsible for virus replication and intracellular penetration. They do not contain genetic material and have a form almost identical to that of viruses by binding the structural surface (capsid) proteins representing the antigenicity of the virus using genetic recombination technology.

[0053] The capsid part of the foot-and-mouth disease virus is the antigenic part that is most clearly detected by the immune system after in vivo infection. A safe and effective new vaccine can be prepared using the proteins that form the capsid.

[0054] The recombinant expression vector contains a polynucleotide in which the base sequences of VP2, VP3, VP1, and 3C of the foot-and-mouth disease virus are arranged in sequence. When expressed as a protein, it can self-assemble to prepare foot-and-mouth disease virus-like particles. And the base sequence of VP4 can be expressed in the direction opposite to that of the polynucleotide.

[0055] In the present invention, the foot-and-mouth disease virus can be selected from serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3.

[0056] Furthermore, the foot-and-mouth disease virus serotype O can 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, serotype O can utilize the gene sequence of O-Andong (GenBank: KF112887.1). Also, in some embodiments, in order to increase the protein expression level, in the present invention, the nucleic acid sequence is optimized using insect cell-preferred codons. As an example, the base sequences encoding the VP1, VP2, VP3, VP4, and 3C proteins can be the base sequences shown in 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. Also, the O-type VP1, VP2, VP3, or VP4 protein can be composed of or contain variants of the amino acid sequences selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively.

[0057] Furthermore, the foot-and-mouth disease virus serotype A can 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, serotype A can utilize the gene sequence of A-Pocheon (GenBank: KC588943.1) or A-Yeoncheon (GenBank: KY766148.1). Also, in some embodiments, in order to increase the protein expression level, in the present invention, the nucleic acid sequence is optimized using insect cell-preferred codons. As an example, the base sequences encoding the VP1, VP2, VP3, VP4, and 3C proteins can be the base sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, or variants thereof. Also, the A-type VP1, VP2, VP3, or VP4 protein can be composed of or contain variants of the amino acid sequences selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively.

[0058] The 3C can be a base sequence encoding an amino acid of the base sequence shown in SEQ ID NO: 9 or a variant thereof, and can be composed of the amino acid sequence shown in SEQ ID NO: 18 or include a variant thereof. The FMDV 3C is a protease, and can achieve the self-assembly of FMDV VLP by cleaving FMDV VP4, VP2, VP3, and VP1 proteins.

[0059] The sequence of the 3C does not vary with the FMDV serotype and has a common conserved sequence. However, the sequences of FMDV VP4-VP2-VP3-VP1 are different among the O serotype, A serotype, Asia1 serotype, SAT serotype, and C serotype, and even among different serum subtypes within the same serotype.

[0060] In the present invention, the recombinant expression vector can also contain any sequence, preferably a promoter and / or a sequence for promoting expression (such as, 2A sequence and / or Burst sequence, etc.), as long as it can increase the expression level or efficiency of the target protein.

[0061] In the present invention, the "promoter" can be applied without limitation as long as it is a sequence sufficient to induce transcription. As an example, the promoter of the polyhedrin-encoding gene (Polyhedrin promoter) is located in the front part of the target protein of VP2-VP3-VP1-3C, and can improve the expression efficiency of the target protein. The promoter of the polyhedrin-encoding gene can be the base sequence shown in SEQ ID NO: 19 or a variant thereof. And, the promoter of the polyhedrin-encoding gene can be located in the front part of the vp39 promoter (SEQ ID NO: 20). On the other hand, different promoters can be used to express the target protein of VP4. As an example, the p10 promoter is located in the front part of the target protein of VP4, and can increase the expression efficiency of the target protein. The p10 promoter can be the base sequence shown in SEQ ID NO: 21 or a variant thereof.

[0062] The recombinant expression vector may further comprise a 2A sequence. The 2A gene sequence encodes 18 to 22 amino acids, among which the 4 amino acids at the end, namely asparagine (N), proline (P), glycine (G), and proline (P), are important conserved amino acids among species. When synthesized into a peptide, this sequence shows a tendency to undergo self-cleavage. Due to this property, when ribosomes perform protein transcription, if they reach the genetic code encoding N, P, and G at the end of the 2A sequence, they sequentially recognize NPG to form a peptide bond and bring in a releasing factor (RF), rather than the prolyl-tRNA that binds to the proline-encoding codon as the next amino acid. After the binding of the RF factor, the previously formed peptide no longer continues the peptide bond connection and is released from the ribosome. Moreover, the codons encoded after the 2A sequence work normally and proceed with the next protein transcription. Eventually, by inserting the 2A sequence, multiple genes can be expressed using a single promoter. The recombinant expression vector of the present invention can insert such a 2A sequence between each gene, thereby enabling the simultaneous expression of these genes. As an example, when the base sequences encoding the VP2-VP3-VP1-3C proteins are arranged in sequence, a 2A sequence may also be included between the VP1 and 3C genes. As an example, the 2A sequence may be a base sequence as shown in SEQ ID NO:22 or SEQ ID NO:23 or a variant thereof.

[0063] The recombinant expression vector may further comprise a Burst sequence. The Burst sequence is a part of the polyhedrin promoter and is the sequence between the translation initiation site and TAAG. It is known that at the end stage of baculovirus infection, Vlf-1 specifically binds to the Burst sequence and promotes transcription. As an example, the Burst sequence may be a base sequence as shown in SEQ ID NO:24 or a variant thereof.

[0064] In the present invention, a "vector" refers to any medium used for cloning and / or transferring bases to a host cell. A vector can be a replicon that binds to other DNA fragments and brings about the replication of the bound fragments. A "replicon" refers to any genetic unit (e.g., plasmid, phage, cosmids, chromosome, virus) that functions as an autonomous unit of DNA replication in an organism, i.e., can replicate through self-regulation. The term "vector" includes viral and non-viral media for introducing bases into a host cell in vitro, ex vivo, or in vivo.

[0065] In the present invention, the term "recombinant expression vector" refers to a vector prepared to be capable of expressing a target protein in a suitable host cell, and is a gene construct containing essential regulatory factors operably linked to express a gene insert. The recombinant expression vector of the present invention may contain genes encoding FMDV proteins VP2, VP3, VP1, and 3C or 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 can be respectively linked to two different promoters in a single vector, and by making the directions of the two promoters in the vector opposite, the influence between the two promoters can be minimized.

[0066] In the present invention, "variant" as used for nucleic acids may refer to: (i) a part or fragment of the nucleotide sequence of reference; (ii) a complementary sequence of the nucleotide sequence of reference or a part thereof; (iii) a nucleic acid substantially identical to the nucleic acid of reference or its complementary sequence; or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleic acid of reference, its complementary sequence, or a sequence substantially identical thereto.

[0067] In the present invention, a "variant" of a peptide or polypeptide has a different amino acid sequence due to amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. A variant may also refer to a protein having an amino acid sequence substantially identical to the protein of reference, and the protein of reference has amino acids that retain at least one biological activity. Conservative substitution of amino acids, i.e., amino acid substitution using different amino acids with similar properties (e.g., degree and distribution of hydrophilicity, charged regions) is generally considered to include minor changes in the art.

[0068] Furthermore, the present invention provides a transformant transformed with the recombinant expression vector.

[0069] In the present invention, the description of "recombinant expression vector" is as described above.

[0070] In the present invention, "transformation" refers to the phenomenon of artificially inducing gene alteration by introducing external DNA into a cell, where DNA becomes replicable as a chromosomal factor or through chromosomal integration by introducing DNA into a host.

[0071] The term "transformant" as used in this specification refers to a transgenic plant or transgenic animal produced by transformation, including a gene recombinant produced by inducing modification or variation of a specific gene using genetic recombination technology.

[0072] In the present invention, the host cell for preparing the "transformant" is preferably a host cell with high DNA introduction efficiency and high expression efficiency of the introduced DNA, and all microorganisms including prokaryotic microorganisms and eukaryotic microorganisms can be used. The host cell can be selected from the group consisting of bacteria of the genus Escherichia; bacteria of the genus Bacillus; bacteria of the genus Pseudomonas; lactic acid bacteria; yeasts; animal cells; and insect cells. Preferably, the host cell can be an insect cell.

[0073] The transformant is used for preparing foot-and-mouth disease virus-like particles or nanoparticles.

[0074] Furthermore, the present invention provides a virus-like particle or nanoparticle produced by a transformant.

[0075] In the present invention, the descriptions of "transformation", "virus-like particle or nanoparticle" are as described above.

[0076] Furthermore, the present invention provides a method for preparing a virus-like particle or nanoparticle for preventing a disease caused by foot-and-mouth disease virus infection, the method comprising the following steps: step (1), constructing the recombinant expression vector of the present invention; step (2), preparing a transformant using the recombinant expression vector; step (3), transfecting the transformant into a host cell; step (4), culturing the transfected host cell and obtaining its culture; and step (5), obtaining the virus-like particle or nanoparticle from the culture.

[0077] In the present invention, the descriptions of "recombinant expression vector", "host cell", "transformant", "foot-and-mouth disease", "virus-like particle or nanoparticle" are as described above.

[0078] Furthermore, the present invention provides a vaccine composition against foot-and-mouth disease virus, which contains the virus-like particle or nanoparticle as an active ingredient.

[0079] In the present invention, the term "vaccine" refers to a biological preparation containing an antigen that provides immunity to an organism, and is an immunogen or antigenic substance that is administered to a human or animal by injection or orally to cause the organism to develop immunity for preventing infectious diseases.

[0080] Relative to the total weight of the vaccine composition, the content of the antigen can be from 1 wt% to 15 wt%.

[0081] The vaccine composition of the present invention may further comprise one or more adjuvants.

[0082] In the present invention, an "adjuvant" generally refers to any substance that enhances the humoral and / or cellular immune response against an antigen. Traditional vaccines consist of unprocessed preparations of inactivated pathogenic microorganisms, and impurities associated with the culture broth of the pathogenic microorganisms can be used as adjuvants to enhance the immune response. However, when a homogeneous preparation of a purified protein subunit is used as the antigen for vaccination, it is necessary to add some foreign substances as adjuvants due to insufficient immunity induced by the antigen as described above. By using an adjuvant, a smaller dose of antigen may be required to stimulate the immune response, thereby reducing the production cost of the vaccine. In some embodiments, the adjuvant includes EMULSIGEN, aluminum hydroxide, Carbigen, saponin, and CpG or a combination thereof. In another embodiment, the adjuvant may be a known oil emulsion, preferably a single oil emulsion.

[0083] The vaccine composition of the present invention may further comprise one or more second adjuvants selected from the group consisting of stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH regulators, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, bacterial cell walls, derivatives of bacterial cell walls, bacterial vaccines, animal poxvirus proteins, subviral particle adjuvants, cholera toxin, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propanediamine, monophosphoryl lipid A, dioctadecyldimethylammonium bromide, and mixtures thereof.

[0084] Moreover, the vaccine composition of the present invention may comprise a veterinarily acceptable carrier. In the present invention, the term "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coating agents, immunoadjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc. The carriers, excipients, and diluents that can be included in the composition for the vaccine may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0085] Moreover, the vaccine composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods. When formulating, common diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. can be used for preparation. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing one or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in the lecithin emulsifier. Moreover, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration can use suspensions, internal liquids, emulsions, syrups, etc., and in addition to common simple diluents such as water or liquid paraffin, various excipients can be included, such as wetting agents, sweeteners, flavoring agents, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used as non-aqueous solvents and suspensions.

[0086] VLP vaccines express one or more structural proteins of a virus through molecular biology techniques. These structural proteins have the natural ability to self-assemble, so they can form a spatial structure and antigenic determinants similar to natural virus particles, but without viral nucleic acid, and not only have strong immunogenicity but also no infectivity. Moreover, due to the presence of a high density of viral antigens on the surface, they can be delivered to immune cells in the same way as the virus infects an organism, thus effectively inducing humoral immunity and cellular immunity of the organism's immune system and shortening the incubation period.

[0087] Moreover, the present invention provides a method for preventing diseases caused by foot-and-mouth disease infection, and the method includes the step of administering the vaccine composition to mammals other than humans.

[0088] In the present invention, the description of the "vaccine composition" is as described above.

[0089] In the present invention, "prevention" refers to all behaviors of inhibiting or delaying the infection of the foot-and-mouth disease virus by administering a composition containing the foot-and-mouth disease virus-like particle or nanoparticle protein self-assembled from the recombinant expression vector of the present invention as an active ingredient.

[0090] In the present invention, the mammal can be unrestrictedly applied to animals such as chickens, pigs, monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, goats, etc. Preferably, the mammal is an even-toed ungulate such as a pig or a cow.

[0091] In the present invention, the administration can be carried out by any administration method known in the art. For example, the administration can be directly administered to an individual intravenously, intramuscularly, orally, transdermally, mucosally, intranasally, intratracheally, or subcutaneously. The administration can be systemic administration or local administration.

[0092] In the present invention, the composition of the present invention can be administered in a therapeutically or prophylactically effective amount. A person of ordinary skill in the art can appropriately select the "therapeutically or prophylactically effective amount" considering the severity of the symptoms, the gender, age, and weight of the individual, etc. For example, the therapeutically or prophylactically effective amount can be 1 pg to 5 g per 1 kg of the individual of the foot-and-mouth disease virus-like particles or nanoparticles self-assembled using the polynucleotide, the protein extract of the transformant, or the recombinant protein isolated from the transformant.

[0093] Furthermore, the present invention provides the use of the virus-like particles or nanoparticles for preventing foot-and-mouth disease virus diseases.

[0094] In the present invention, the description of "foot-and-mouth disease virus" or "virus-like particles or nanoparticles" is as described above. Detailed Description of the Invention

[0095] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that a person of ordinary skill in the art to which the present invention pertains can easily implement them. However, the present invention can be implemented in various different forms and is not limited to the embodiments described in this specification.

[0096] Example 1. Construction of a recombinant expression vector and virus expressing FMDV antigen candidates

[0097] A transfer vector capable of expressing the structural proteins and main antigen candidates of FMDV was constructed to utilize the high-expression baculovirus expression system.

[0098] Specifically, using the high-expression vector pPol-6, the target genes arranged in the order of VP2, VP3, VP1, and 3C were cloned behind the polyhedrin promoter to construct a Delta-VP4 expression form ( Figure 1)。 Then, clone the p10 promoter and VP4 in the direction opposite to the Polyhedrin promoter in the Delta-VP4 expression form to construct the VP4 co-expression form ( Figure 2 )。 On the other hand, clone the target genes arranged in the order of VP4, VP2, VP3, VP1, and 3C behind the Polyhedrin promoter to construct the full form as a control group ( Figure 3 )。 To improve the cloning efficiency, insert the codon-optimized gene sequence into the vector. Construct a recombinant transfer vector containing the antigenic determinant site of serotype O corresponding to the codon-optimized base sequence and amino acid sequence. And construct a recombinant transfer vector containing the antigenic determinant site of serotype A corresponding to the codon-optimized base sequence and amino acid sequence.

[0099] Insert each of the constructed transfer vectors into a bacmid of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) from which the viral chitinase and cathepsin have been removed to construct a recombinant virus, and proliferate it in Sf9 cells, which are an insect cell line, to mass-produce the recombinant virus.

[0100] Example 2. Confirmation of the proteins expressed by the recombinant virus expressing FMDV structural proteins

[0101] 2-1. Results of electrophoretic analysis of the proteins expressed by the recombinant virus expressing FMDV structural proteins

[0102] Conduct an experiment to confirm whether the recombinant virus expressing FMDV structural proteins constructed in Example 1 expresses the target protein.

[0103] Specifically, inoculate the recombinant virus into insect cells Hi5. After several days, centrifuge at 4000 rpm and 4 °C for 5 minutes to recover the cells and the culture medium. Resuspend the recovered cells by treating them with the same volume of PBS as the culture medium. Perform SDS-PAGE and Western blotting on the recovered cell and culture medium samples respectively.

[0104] First, analyze the proteins expressed by the recombinant virus transformed into the Delta-VP4 form and the VP4 co-expression form.

[0105] As a result, as Figure 4 shown, distinct bands were confirmed at the positions of the target proteins. Among them, a size of 23.1 kDa corresponded to the VP2 protein, a size of 24.4 kDa corresponded to the VP2 protein, a size of 23.9 kDa corresponded to the VP3 protein, a size of 8.9 kDa corresponded to the VP4 protein, and a high yield of protein secreted into the culture medium was confirmed by the naked eye.

[0106] Moreover, as Figure 5 shown, from the results of Western blotting using an antibody specific to the VP1 protein, it was clearly confirmed that the protein identified in SDS-PAGE was the VP1 protein. Also, the cleavage of VP1 from P1 was verified by the normal expression of 3C. In particular, a high secretion efficiency was also confirmed in the Delta-VP4 form, but the highest expression level of the structural proteins could be confirmed in the VP4 co-expression form.

[0107] Furthermore, the proteins expressed by the cultures (cells and supernatants) of the recombinant virus transformed into the Delta-VP4 form were analyzed.

[0108] As a result, as Figure 6 shown, similar to the VP4 co-expression form, a high efficiency of secretion into the culture medium after expression was also confirmed in the Delta-VP4 form.

[0109] From the above results, it was found that when VP4 was removed from the FMDV structural protein P1 (VP4-VP2-VP3-VP1) and expressed, such as in the VP4 co-expression form and the Delta-VP4 form, the protein was secreted into the culture medium after expression, and this secretion contributed greatly to the productivity of the antigen candidate.

[0110] On the other hand, the proteins expressed by the cultures (cells and supernatants) of the recombinant virus transformed into the full form were analyzed.

[0111] As a result, as Figure 7 and Figure 8 shown, in the full form, it was found that a large amount of protein was present in the cells after expression, but no secretion into the supernatant was observed even as the number of days post-inoculation (dpi) increased.

[0112] The above results show that vectors obtained by removing VP4 from the FMDV structural protein P1 (VP4-VP2-VP3-VP1) and expressing them (Delta-VP4 form, VP4 co-expression form) play an important role in the production of antigen candidates.

[0113] 2-2. Comparing antigen amounts by ELISA using an antibody specific for FMDV VP1

[0114] ELISA was performed to compare the amounts of FMDV antigen produced by the recombinant viruses of the present invention.

[0115] Specifically, the recombinant virus was inoculated into insect cells Hi5. After several days, the culture solution was centrifuged at 4000 rpm and 4°C for 5 minutes and recovered. ELISA was performed on the recovered culture solution sample using an antibody specific for FMDV VP1. BIOAFTOGEN FMD vaccine was used as a commercial vaccine for the control group.

[0116] As a result, as Figure 9 shown, higher result values were confirmed in the VP4 co-expression form, and thus the VP4 co-expression form was selected as the optimal expression form of the FMDV structural protein.

[0117] Example 3. Analyzing expressed proteins using antibodies specific for VP1, VP2, VP3, and VP4 structural proteins

[0118] An experiment was conducted to confirm whether all FMDV structural proteins are expressed by the recombinant virus.

[0119] Specifically, in the culture solution obtained by culturing the recombinant virus in large quantities, Western blotting was performed using specific antibodies corresponding to VP0, VP1, VP2, VP3, and VP4, respectively. BIOAFTOGEN FMD vaccine was used as a commercial vaccine for the control group.

[0120] As a result, as Figures 10 to 13 shown, each band of the FMDV structural proteins VP1, VP2, VP3, and VP4 could be confirmed by the recombinant virus against serotype O. From the above results, it was confirmed that all proteins existed separately after expression, which means that the 3C protease was normally expressed and functioned.

[0121] Moreover, as Figures 14 to 18As shown, it was confirmed that the FMDV structural proteins VP1, VP2, VP3, and VP4 were simultaneously expressed by the recombinant virus against type A. In particular, in the VP4 co-expression form, VP2 was expressed alone, and a band with a size of approximately 24.4 kDa was confirmed. In the full form, it was confirmed that VP2 and VP4 were expressed in the form of binding as VP0 ( Figure 15 ). Also, in the VP4 co-expression form, since VP0 was not confirmed, it was known that VP0 was separated into VP2 and VP4, but in the full form, it was expressed in the form of VP0, which is a form in which VP2 and VP4 are bound ( Figure 17 ).

[0122] The above results indicate that, different from the full form, in the VP4 co-expression form, each protein VP1, VP2, VP3, and VP4 was expressed alone and existed in a form secreted extracellularly, which means that the 3C protease was normally expressed and functioned.

[0123] Example 4. Morphological observation of the FMDV antigen candidate using TEM An experiment was conducted to analyze the characteristics of the proteins expressed in the above example.

[0124] Specifically, the culture solution of the recombinant virus using the VP4 co-expression form was concentrated and purified. The culture solution was passed through a 100 kDa hollow fiber filter using a tangential flow filtration (TFF) system and concentrated 20-fold, and then exchanged with 20 mM Tris-HCl, pH 6.5 buffer. Ion exchange chromatography (IEX) was performed on the concentrated solution of the obtained culture solution using Captocore Q impress. The purified FMDV antigen candidate was confirmed by SDS-PAGE and Western blotting.

[0125] The purified FMDV antigen candidate was photographed with a transmission electron microscope (TEM) for morphological observation.

[0126] As a result, as Figure 19 shown, it was confirmed that the size of the nanoparticles was approximately 10 - 15 nm. Also, asFigure 20 As shown, it was confirmed that all VLP particles had a suitable size of approximately 25 to 30 nm.

[0127] Example 5. Verification of the immunogenicity of the vaccine candidate

[0128] A vaccine for inoculation into the target animal was prepared to confirm the immunological efficacy of the vaccine candidate.

[0129] Specifically, the vaccine candidate used was composed of the structural proteins against serotype O and was produced by a recombinant virus constructed in a co-expression form of VP4, and was prepared by emulsification with an oil emulsion. The vaccine was co-administered twice. The first dose was administered at week 0 at the start of the experiment, and the second dose was administered at week 4. Blood was collected weekly until week 15 and porcine serum ( Figure 21 ) was separated. The antibody titer was measured using the separated serum by an ELISA kit (PrioCHECK FMDV Type O Antibody SP ELISA kit). The BIOAFTOGEN FMD vaccine was used as a commercial vaccine for the control group.

[0130] As a result, as Figure 22 shown, a 100% inhibition rate was confirmed in all individuals, and it was confirmed that the immunogenicity was superior to that of the control group.

[0131] Example 6. Examination of the immunogenicity of the vaccine candidate concentration

[0132] The antibody titer and the neutralizing antibody titer (VNT) were evaluated according to the concentration of the vaccine candidate.

[0133] Specifically, the vaccine was co-administered twice. The first dose was administered at week 0 at the start of the experiment, and the second dose was administered at week 4. Vaccines against serotype O and serotype A were prepared separately and applied. Serum was collected from pigs inoculated with each serotype, and the weekly antibody titer was measured using the recovered serum by an ELISA kit (PrioCHECK FMDV Type O Antibody SP ELISA kit, PrioCHECK FMDV Type A Antibody SP ELISA kit). The BIOAFTOGEN FMD vaccine was used as a commercial vaccine for the control group.

[0134] As a result, as Figure 23As shown, in the O type, the antibody titer equivalent to that of the control group was exhibited at all antigen amounts. In the A type, the antibody titer at low dose reached 50% 2 weeks later than that of the control group, and increased similarly at other doses.

[0135] And, as Figure 24 shown, as a result of measuring the neutralizing antibody titer (VNT), in the O type, it reached 32-fold at all doses from the 2nd week and then remained above 100-fold. In the A type, the neutralizing antibody was slowly formed at low dose, increased and gradually increased from the 2nd week at intermediate dose, and reached 32-fold from the 2nd week at high dose.

[0136] Example 7. Confirming the safety of the candidate vaccine-inoculated group

[0137] Histopathological observation was performed on the skin of pigs inoculated with the vaccine candidate substance.

[0138] Specifically, the vaccine candidate substance used was composed of the structural protein against the O serotype and was produced by a recombinant virus constructed in the co-expression form of VP4. The vaccine was inoculated 2 times. The first dose was inoculated at the 0th week of the start of the test, and the second dose was inoculated at the 4th week and the inoculation site was observed. The BIOAFTOGEN FMD vaccine was used as the commercial vaccine for the control group.

[0139] Table 1

[0140]

[0141]

[0142] NM * : No remarkable gross lesions

[0143] As a result, as shown in Table 1, Figure 25 and Figure 26 shown, it was confirmed that the overall degree of lesion formation of the candidate vaccine using the co-expression form of VP4 was low. On the other hand, obvious gross lesions were observed in the control group, and severe chronic granulomatous inflammation was confirmed, and extensive myocyte degeneration and necrosis were confirmed to have progressed.

[0144] The above results mean that the vaccine candidate substance of the present invention has a low side effect of abnormal meat quality.

Claims

1. A recombinant expression vector, characterized in that, Comprising a first promoter and genes encoding foot-and-mouth disease virus proteins VP2, VP3, VP1 and 3C operably linked to the first promoter.

2. The recombinant expression vector according to claim 1, wherein The recombinant expression vector further comprises a second promoter and a gene encoding FMDV protein VP4 operably linked to the second promoter.

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

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

5. The recombinant expression vector according to 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, characterized in that, The transformant is transformed from the recombinant expression vector according to claim 1 or 2.

7. A virus-like particle or nanoparticle, characterized in that, The virus-like particles or nanoparticles are produced from the transformant according to claim 6.

8. A method for preparing virus-like particles or nanoparticles for preventing diseases caused by foot-and-mouth disease virus infection, characterized in that, Comprising the following steps: Step (1), constructing the recombinant expression vector according to claim 1 or 2; Step (2), preparing a transformant using the recombinant expression vector; Step (3), transfecting the transformant into a host cell; Step (4), culturing the transfected host cell and obtaining its culture; And Step (5), obtaining virus-like particles or nanoparticles from the culture.

9. A vaccine composition against foot-and-mouth disease virus, characterized in that, Comprising the virus-like particles or nanoparticles according to claim 7 as an active ingredient.

10. The vaccine composition against foot-and-mouth disease virus according to claim 9, characterized in that, The composition further comprises an adjuvant.

11. The vaccine composition against foot-and-mouth disease virus according to claim 10, characterized in that, The adjuvant is an oil emulsion.

12. A method for preventing diseases caused by foot-and-mouth disease infection, characterized in that, Comprising the step of administering the vaccine composition according to claim 9.

13. Use of the virus-like particle or nanoparticle according to claim 7, characterized in that, For preventing foot-and-mouth disease virus diseases.

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