African swine fever vaccine composition

Abstract

The present invention relates to polypeptides, polynucleotides, and plasmids involved in generating an immune response against African swine fever, and vaccine compositions containing them. The present invention also relates to a method for generating an immune response against African swine fever in an individual. The present invention also relates to polypeptides, polynucleotides, and plasmids involved in generating an immune response against African swine fever, and pharmaceutical compositions containing them for treating or preventing African swine fever. The present invention also relates to a method for preventing or treating African swine fever in an individual.

Description

Technical Field

[0001] The present invention relates to polypeptides, polynucleotides, plasmids involved in generating an immune response against African swine fever, and vaccine compositions containing them. The present invention also relates to a method for generating an immune response against African swine fever in an individual. The present invention also relates to a pharmaceutical composition for treating or preventing African swine fever containing polypeptides, polynucleotides, plasmids involved in generating an immune response against African swine fever, and them. The present invention also relates to a method for preventing or treating African swine fever in an individual.

Background Art

[0002] African swine fever (ASF) is a fatal viral hemorrhagic swine infectious disease. Once it breaks out, it is highly infectious, there is no treatment method, and the fatality rate of infected pigs is very high, mostly dying, so it is a disease that causes great damage to the pig farming industry. Therefore, when African swine fever occurs, the World Organization for Animal Health (OIE) must be immediately informed of the fact, and international trade related to pigs is also immediately interrupted. African swine fever is classified as a very important disease by the World Organization for Animal Health (OIE), and in Korea, it is designated and managed as a Class 1 notifiable infectious disease under the Livestock Infectious Diseases Prevention Act.

[0003] African swine fever virus (ASFV) is a DNA virus of about 200 nm belonging to the genus Asfivirus in the family Asfarviridae. African swine fever virus is reported to infect only animals belonging to the family Suidae and does not infect humans or other animals.

[0004] African swine fever virus (AFF) is present in large quantities in the saliva, respiratory secretions, urine, and feces of infected animals, and can be transmitted to healthy animals that come into contact with these substances. The virus can also survive in the blood and tissues of dead pigs, so feeding pigs unheated leftovers containing the tissues of infected animals can lead to rapid transmission. Alternatively, the virus can be transmitted through blood shed during fights between pigs or through diarrhea mixed with blood. In addition, blood-sucking insects such as soft ticks (belonging to Ornithodoros spp.), mosquitoes, and biting flies can act as vectors, carrying the AFF virus and transmitting the disease when they bite or feed on pigs.

[0005] African swine fever can infect pigs of all ages. Morphology varies depending on the infected virus and exposure route, and in cases of natural infection, the incubation period varies from 4 to 19 days. The mortality rate is almost 100% if infected with a highly pathogenic virus.

[0006] African swine fever viruses can be classified into highly pathogenic, moderately pathogenic, and lowly pathogenic types based on their pathogenicity. Highly pathogenic African swine fever viruses typically cause peracute (pigs die 1-4 days after infection) and acute (pigs die 3-8 days after infection) disease, while moderately pathogenic African swine fever viruses cause acute (pigs die 11-15 days after infection) and subacute (pigs die 20 days after infection) disease. Lowly pathogenic African swine fever viruses are reported only in endemic areas and cause semi-clinical or chronic disease.

[0007] African swine fever (ASF) is difficult to diagnose in its early stages, especially when the number of infected pigs is small. This is because the clinical symptoms of ASF can be confused with other hemorrhagic swine diseases such as septicaemic salmonellosis and porcine dermatitis nephropathy syndrome (PDNS). Accurate differentiation is only possible through laboratory diagnostics, such as detecting the virus in blood and internal organs, or detecting antibodies in the serum of infected pigs. By the time ASF is diagnosed, the disease is often already widespread. For example, in China, the infectivity of ASF is extremely high; in less than nine months since its first confirmed case in 2018, ASF had spread to all administrative regions of China. Furthermore, in China, the world's largest pig-producing country, approximately 130 million pigs died or were culled due to African swine fever in 2019. This represents a reduction of about one-third from the 430 million pigs that existed before the outbreak.

[0008] Research into African swine fever vaccines has been steadily progressing since the 1960s. While various attempts have been made to date, including attenuated live vaccines, inactivated vaccines, recombinant protein vaccines, and viral vector vaccines, no vaccine with sufficient efficacy for commercial application has yet been developed. Recently, research has shown that even when neutralizing antibodies are formed after inactivated vaccine administration, their protective effect during actual attack vaccination (challenge) is minimal or nonexistent. This suggests that inducing a cellular immune response, in addition to generating neutralizing antibodies, is an important mechanism for enhancing the vaccine's ability to protect against the virus (Takamatsu et al., Virus Res. 2013 Apr, 173(1):110-21).

[0009] Another reason why developing an effective vaccine for African swine fever, a disease deadly to pigs, is difficult is the large and complex nature of the African swine fever virus. While typical viruses have 10 to 12 proteins, the African swine fever virus has more than 150 proteins, and there is also a wide variety of viral types (24 genotypes). Therefore, experts considered it difficult to commercialize a vaccine within the next 10 years at the very least.

[0010] Therefore, there is a pressing need for a vaccine or treatment for African swine fever. In particular, it is crucial to prevent African swine fever reliably and effectively, and there is a very large unmet demand for new vaccines for this purpose. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Takamatsu et al., Virus Res.2013 Apr, 173(1):110-21 [Overview of the project] [Problems that the invention aims to solve]

[0012] One object of the present invention is to provide polypeptides and polynucleotides that can generate an immune response against African swine fever.

[0013] Another object of the present invention is to provide a plasmid capable of generating an immune response against African swine fever.

[0014] Another object of the present invention is to provide a vaccine composition against African swine fever.

[0015] Another object of the present invention is to provide a method for generating an immune response against African swine fever from an individual.

[0016] Another object of the present invention is to provide pharmaceutical compositions for the treatment or prevention of African swine fever from individual organisms.

[0017] Another object of the present invention is to provide a method for treating or preventing African swine fever from an individual. [Means for solving the problem]

[0018] To achieve the above objective, one aspect of the present invention relates to the African swine fever virus. protein The present invention provides polypeptides containing one or more amino acid sequences from among p30, p54, C-type lectin, CD2v, p49, pp62, EP364R, F317L, A104R, and K205R.

[0019] Another embodiment of the present invention provides a polypeptide comprising one or more amino acid sequences of sequence numbers 1 to 10, or amino acid sequences having 90% or more homology thereto.

[0020] As used herein, the term “homology” means the degree to which a given polypeptide sequence or polynucleotide sequence matches, and may be expressed as a percentage. Hereinafter, homologous sequences having identical or similar activity to a given polypeptide sequence are expressed as “% homology”. Such homology can be confirmed, for example, by using standard software that calculates parameters such as score, identity, and similarity, such as BLAST 2.0, or by comparing sequences in hybridization experiments performed under justified and rigorous conditions, the appropriate justified hybridization conditions of which can be determined by methods well known to those skilled in the art.

[0021] The amino acid sequences of SEQ ID NO: 1 to 10 are, in order, consensus sequences derived by comparing the sequence homology of genes p30 (CP204L), p54 (E183L), C-type lectin (EP153R), CD2v (EP402R), p49 (B438L), pp62 (CP530R), EP364R, F317L, A104R, K205R from various strains of African swine fever virus.

[0022] One or more polypeptides containing the amino acid sequences of SEQ ID NO: 1 to 10, or amino acid sequences having 90% or more homology thereto, can effectively act as antigens capable of generating an immune response against African swine fever.

[0023] The polypeptide may contain one of the amino acid sequences, or may contain two or more thereof. Further, the polypeptide 、I may further contain a gE leader (IgE leader) sequence, a ubiquitin sequence, and Cut a cleavage site sequence One or more amino acid sequences selected from the following For example, the polypeptide 、I can be composed of a gE leader sequence - the amino acid sequence of SEQ ID NO: 1. As another example, the polypeptide can be composed of an IgE leader sequence - a ubiquitin sequence - the amino acid sequence of SEQ ID NO: 1. As another example, the polypeptide 、I can be composed of a gE leader sequence - a ubiquitin sequence - the amino acid sequence of SEQ ID NO: 1 - a cleavage site sequence - the amino acid sequence of SEQ ID NO: 2. The ubiquitin sequence may preferably be SEQ ID NO: 41, and the cleavage site sequence may preferably be SEQ ID NO: 43.

[0024] One aspect of the present invention provides a polynucleotide containing one or more base sequences from the African swine fever virus genes p30, p54, C-type lectin, CD2v, p49, pp62, EP364R, F317L, A104R, and K205R.

[0025] Another embodiment of the present invention provides a polynucleotide comprising one or more of the base sequences of SEQ ID NOs: 11 to 20, or base sequences having 90% or more homology thereto.

[0026] Alternatively, yet another aspect of the present invention provides a polynucleotide coding one or more amino acid sequences of sequence numbers 1 to 10, or amino acid sequences having 90% or more homology thereto. The polynucleotide coding the amino acid sequences of sequence numbers 1 to 10 may be the base sequences of sequence numbers 11 to 20, respectively.

[0027] The nucleotide sequences of sequence numbers 11 through 20 are, in order, consensus sequences derived by comparing the sequence homology of the genes p30 (CP204L), p54 (E183L), C-type lectin (EP153R), CD2v (EP402R), p49 (B438L), pp62 (CP530R), EP364R, F317L, A104R, and K205R from various strains of African swine fever virus.

[0028] Alternatively, the polynucleotide may be a modified version in which the amino acid sequence of SEQ ID NOs. 1 to 10, or the base sequence of SEQ ID NOs. 11 to 20, is modified with codons optimized for the target organism that is to generate an immune response. The target organism is, for example, an animal. The animal may be a mammal, such as a pig, cattle, horse, sheep, goat, deer, or human. Preferably, the animal is a pig.

[0029] The polynucleotide may contain one of the base sequences, or two or more. However, this is also acceptable. Furthermore, the polynucleotide may be a Kozak sequence, an IgE leader sequence. , ubiquitin sequence and Cut Cleavage site arrangement Select one or more nucleotide sequences from the following: Further elements may be included. For example, the polynucleotide may consist of the nucleotide sequence Kozak sequence-IgE leader sequence-SEQ ID NO: 11. Another example is that the polynucleotide may consist of the nucleotide sequence Kozak sequence-IgE leader sequence-ubiquitin sequence-SEQ ID NO: 11. Yet another example is that the polynucleotide may consist of the nucleotide sequence Kozak sequence-IgE leader sequence-ubiquitin sequence-SEQ ID NO: 11-cleavage site sequence-SEQ ID NO: 12. The ubiquitin sequence may preferably be SEQ ID NO: 42, and the cleavage site sequence may preferably be SEQ ID NO: 44.

[0030] One embodiment of the present invention provides a plasmid containing a polynucleotide comprising one or more base sequences from among the African swine fever virus genes p30, p54, C-type lectin, CD2v, p49, pp62, EP364R, F317L, A104R, and K205R.

[0031] Another embodiment of the present invention provides a plasmid containing a polynucleotide comprising one or more nucleotide sequences of sequence numbers 11 to 20, or sequences having 90% or more homology thereto.

[0032] Another embodiment of the present invention provides a plasmid comprising a polynucleotide coding one or more amino acid sequences that have 90% homology to the amino acid sequence of SEQ ID NO: 1, or to the amino acid sequence of SEQ ID NO: 1.

[0033] As used herein, the term “plasmid” means a DNA product containing a DNA sequence operably ligated to a suitable regulatory sequence capable of expressing DNA in a suitable host or subject. The plasmid may be a vector, a grasping particle, or, simply put, a potential genomic insert. In this specification, plasmids may be used interchangeably with vectors (or viral vectors).

[0034] The plasmid can be manufactured by conventional methods well known in the art. For example, the plasmid according to the present invention can be manufactured by inserting the polynucleotide into a plasmid vector through gene cloning and used as a DNA vaccine.

[0035] The plasmid may contain one or more of the polynucleotides. The polynucleotide may further contain a Kozak sequence, an IgE leader sequence, a ubiquitin sequence, and / or a cleavage site sequence.

[0036] For example, the plasmid may contain the sequence of Sequence ID No. 31, where Sequence ID No. 31 is a sequence of Sequence ID Nos. 11 to 20 linked together. Another example is the plasmid which contains the sequence of Sequence ID No. 36, where Sequence ID No. 36 is a sequence of ubiquitin and Sequence ID Nos. 11 to 20 linked together. Yet another example is the plasmid which contains the sequence of Sequence ID No. 50, where Sequence ID No. 50 is a sequence of ubiquitin and Sequence ID Nos. 11 to 20 linked together, with each of Sequence ID Nos. 11 to 20 linked together by cleavage site sequences.

[0037] The ubiquitin sequence may preferably be the nucleotide sequence of SEQ ID NO: 42. CD8 +T cells recognize MHC class I-associated peptides derived from endogenous antigens, such as oncogene products or viral antigens, located in the cytosol. Before such MHC class I molecules can present their antigens, the antigens undergo ubiquition and are processed with antigenic peptides by the proteasome. Therefore, ubiquitin-fused proteins can enter the proteasome-dependent degradation pathway, enhancing their MHC class I peptide presentation ability and increasing the induction of cytotoxic T lymphocyte responses (CTLs).

[0038] The cleavage site sequence may preferably be the nucleotide sequence of Sequence ID No. 44. The cleavage site refers to a peptide that is recognized and cleaved by a protease. In the present invention, polypeptides expressed from a plasmid containing the polynucleotide can be cleaved by a degrading enzyme, thereby enabling each cleaved polypeptide to effectively act as an antigen capable of generating an immune response against African swine fever. The cleavage site may be cleaved by an endogenous enzyme present in the cell, specifically by a furine protease, but is not limited thereto.

[0039] As another example, the plasmid may contain the nucleotide sequence of SEQ ID NO: 32, where SEQ ID NO: 32 is a sequence of nucleotide sequences of SEQ ID NOs: 11 to 14. Alternatively, the plasmid may contain the nucleotide sequence of SEQ ID NO: 37, where SEQ ID NO: 37 is a sequence of ubiquitin and nucleotide sequences of SEQ ID NOs: 11 to 14.

[0040] As another example, the plasmid may contain the nucleotide sequence of SEQ ID NO: 33, where SEQ ID NO: 33 is a sequence of nucleotide sequences of SEQ ID NOs: 15 to 20. Alternatively, the plasmid may contain the nucleotide sequence of SEQ ID NO: 38, where SEQ ID NO: 38 is a sequence of ubiquitin and nucleotide sequences of SEQ ID NOs: 15 to 20.

[0041] As another example, the plasmid may contain the nucleotide sequence of SEQ ID NO: 34, where SEQ ID NO: 34 is a sequence of nucleotide sequences 11, 12, 16, 13, and 14. Alternatively, the plasmid may contain the nucleotide sequence of SEQ ID NO: 39, where SEQ ID NO: 39 is a sequence of ubiquitin and nucleotide sequences 11, 12, 16, 13, and 14.

[0042] As another example, the plasmid may contain the nucleotide sequence of SEQ ID NO: 35, where SEQ ID NO: 35 is a sequence of nucleotide sequences 15, 17, 18, 19, and 20. Alternatively, the plasmid may contain the nucleotide sequence of SEQ ID NO: 40, where SEQ ID NO: 40 is a sequence of ubiquitin and nucleotide sequences 15, 17, 18, 19, and 20.

[0043] As another example, the plasmid may also contain the nucleotide sequence of SEQ ID NO: 50, where SEQ ID NO: 50 is a sequence of nucleotide sequences of SEQ ID NOs: 11, 44, 12, 44, 13, 44, 14, 44, 15, 44, 16, 44, 17, 44, 18, 44, 19, 44, and 20, which include the ubiquitin sequence of SEQ ID NO: 42 and the cleavage site sequence of SEQ ID NO: 44.

[0044] As another example, the plasmid may also contain the sequence of Sequence ID No. 51, where Sequence ID No. 51 is a sequential concatenation of the sequences of Sequence ID Nos. 11, 44, 12, 44, 13, 44, and 14, which include the ubiquitin sequence of Sequence ID No. 42 and the cleavage site sequence of Sequence ID No. 44.

[0045] As another example, the plasmid may also contain the nucleotide sequence of SEQ ID NO: 52, where SEQ ID NO: 52 is a sequence of nucleotide sequences of SEQ ID NOs: 15, 44, 16, 44, 17, 44, 18, 44, 19, 44, and 20, which include the ubiquitin sequence of SEQ ID NO: 42 and the cleavage site sequence of SEQ ID NO: 44.

[0046] As another example, the plasmid may also contain the sequence of Sequence ID No. 53, where Sequence ID No. 53 is a sequence of sequences of Sequence ID Nos. 11, 44, 12, 44, 16, 44, 13, 44, and 14, which include the ubiquitin sequence of Sequence ID No. 42 and the cleavage site sequence of Sequence ID No. 44.

[0047] As another example, the plasmid may also contain the sequence of Sequence ID No. 54, where Sequence ID No. 54 is a sequence of sequences of Sequence ID Nos. 15, 44, 17, 44, 18, 44, 19, 44, and 20, which include the ubiquitin sequence of Sequence ID No. 42 and the cleavage site sequence of Sequence ID No. 44.

[0048] Another embodiment of the present invention provides an African swine fever vaccine composition comprising the polypeptide, polynucleotide, or plasmid of the present invention.

[0049] In the African swine fever vaccine composition, the polypeptide, polynucleotide, and plasmid are as described above unless otherwise specified.

[0050] The term "vaccine" refers to a biological preparation containing an antigen that gives immunity to an individual, and is an immunogenic or antigenic substance administered to humans or animals, for example by injection or oral administration, to induce immunity for disease prevention.

[0051] The aforementioned vaccine may be a DNA vaccine. The term "DNA vaccine" refers to a vaccine that induces an immune response by artificially replicating a portion of the genes of pathogenic bacteria or viruses and then administering it. Such DNA vaccines have several advantages over existing protein vaccines: i) They can be synthesized using only the genetic information of the pure target pathogen antigen, eliminating the need to directly handle dangerous pathogens; ii) They use only a portion of the genes necessary for inducing toxicity, so there is no risk of them exhibiting toxicity when administered to a recipient; and iii) Their simplicity, consisting only of plasmid DNA, makes it possible to quickly develop vaccines in response to a variety of rapidly emerging infectious diseases.

[0052] The African swine fever vaccine composition may preferably have protective immunity against the African swine fever virus.

[0053] The vaccine compositions of the present invention may contain veterinary-acceptable carriers. The term "veterinary-acceptable carriers" includes any and all solvents, dispersion media, coatings, antigen reinforcers, stabilizers, diluents, preservatives, antimicrobial and antifungal agents, isotonic agents, adsorption retarders, and the like. Examples of carriers, excipients, and diluents that may be included in the vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, the vaccine compositions of the present invention may be used in the form of oral dosage forms such as acids, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as sterile injection solutions, by conventional methods. When formulating, diluting agents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used may be used. Preferably, poly-L-Glutamic acid can be used to aid in stabilization.

[0054] The vaccine composition can be administered to an individual in various forms. "Administration" can be performed by any one method selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, and oral administration.

[0055] The vaccine composition may contain one or more adjuvants to improve or enhance the immune response. Suitable adjuvants include peptides, aluminum hydroxides, aluminum phosphates, aluminum oxides, and compositions consisting of mineral oils or vegetable oils such as Marcol 52 and one or more emulsifiers, or surface-active substances such as lysolecithin, polyvalent cations, and polyvalent anions.

[0056] One aspect of the present invention provides a method for generating an immune response from an individual to African swine fever virus, comprising the step of administering the vaccine composition of the present invention to the individual.

[0057] In the method for generating the immune response described above, the vaccine composition is as described above unless otherwise specified.

[0058] As used herein, the term “immune response” means the activation of the host’s immune system in response to the introduction of an antigen. An immune response may take the form of a cellular response, a humoral response, or both.

[0059] As used herein, the term “individual” means an animal, including humans, and may be, for example, a mammal, more specifically, a pig, a cattle, a horse, a sheep, a goat, a deer, or a human. Preferably, a pig. The term “individual” can be used interchangeably with “subject” or “host.”

[0060] In the method for generating the immune response described above, the vaccine composition of the present invention may contain an effect-size active ingredient, i.e., a totally recombinant polypeptide, a recombinant polynucleotide, or a plasmid containing the same, together with a pharmaceutically acceptable carrier and adjutants. The term "effect-size" means an amount of the vaccine component sufficient to induce a specific immune response against African swine fever virus in the vaccinated animal. The effect-size can be readily determined by an expert in the art, for example, through conventional experiments in animals.

[0061] The administration can be carried out via any route suitable for the administration of the DNA vaccine, for example, by subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0062] Another embodiment of the present invention provides a method for treating or preventing African swine fever from an individual, comprising the step of administering a vaccine composition, polypeptide, polynucleotide, or plasmid of the present invention to the individual.

[0063] Another embodiment of the present invention provides a pharmaceutical composition for the treatment or prevention of African swine fever comprising a polypeptide, polynucleotide, or plasmid according to the present invention.

[0064] In the method for treating or preventing African swine fever and the pharmaceutical composition for treating or preventing African swine fever according to the present invention, each term has the same meaning as described above unless otherwise specified.

[0065] As used in this invention, the term "prevention" means all actions by which African swine fever is suppressed or delayed by the administration of the composition according to the present invention. Furthermore, as used in this invention, the term "treatment" means all actions by which the symptoms of African swine fever are improved, cured, alleviated, or partially treated by the administration of the composition according to the present invention. [Effects of the Invention]

[0066] The vaccine composition according to the present invention is a DNA vaccine that has excellent protective immunity against African swine fever virus. In particular, the vaccine composition according to the present invention is extremely effective in inducing a cellular immune response against African swine fever.

[0067] When the vaccine composition of the present invention is administered, antibodies against African swine fever are produced at a remarkably high level from the vaccinated individual, thereby effectively inducing an immune response and providing excellent preventive effects against African swine fever. [Brief explanation of the drawing]

[0068] [Figure 1]This invention relates to a vector cleavage map containing African Swine Fever (ASF) virus genes, which includes Kozak sequences, IgE leader sequences, ubiquitin sequences, and Furin cleavage site sequences, and to a vector containing ASF virus genes such as p30, p54, C-type lectin, CD2v, p49, pp62, EP364R, F317L, A104R, and K205R. [Figure 2] This is a cleavage map of a vector containing ASF virus genes, including Kozak sequences, IgE leader sequences, ubiquitin sequences, and furin cleavage site sequences, and relates to a vector containing p30, p54, C-type lectin, and CD2v as ASF virus genes. [Figure 3] This invention relates to a vector containing ASF virus genes, comprising a cleavage map including a Kozak sequence, an IgE leader sequence, a ubiquitin sequence, and a furin cleavage site sequence, and containing ASF virus genes p49, pp62, EP364R, F317L, A104R, and K205R. [Figure 4] This is a cleavage map of a vector containing ASF virus genes, including Kozak sequences, IgE leader sequences, ubiquitin sequences, and furin cleavage site sequences, and relates to a vector containing ASF virus genes such as p30, p54, pp62, C-type lectin, and CD2v. [Figure 5] This is a cleavage map of a vector containing ASF virus genes, comprising Kozak sequences, IgE leader sequences, ubiquitin sequences, and furin cleavage site sequences, and relating to a vector containing ASF virus genes p49, EP364R, F317L, A104R, and K205R. [Figure 6]This graph shows the number of cytotoxic T cells (CD8+ T cells) generated by ASF virus antigens (p30, p54, or CD2v) after DNA vaccination containing ASF virus genes, indicating the level of cellular immune response induced by the DNA vaccine. 'Mock' represents the control group administered a DNA vaccine that does not contain ASF virus genes, 'ASF_4G' represents experimental group 1 administered a DNA vaccine containing ASF virus genes (p30, p54, C type lectin, and CD2v) but without ubiquitin, and 'ASF_Ubi_4G' represents experimental group 2 administered a DNA vaccine containing ASF virus genes (p30, p54, C type lectin, and CD2v) but with ubiquitin. [Figure 7] This graph shows the survival rate of pigs infected with the ASF virus after DNA vaccination containing ASF virus genes. 'Mock' represents the control group that received a DNA vaccine that did not contain ASF virus genes, while 'ASF_Ubi_10G' represents the experimental group that received a DNA vaccine containing ASF virus genes (p30, p54, C-type lectin, CD2v, p49, pp62, EP364R, F317L, A104R, and K205R) and ubiquitin. [Modes for carrying out the invention]

[0069] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

[0070] Example 1. African swine fever virus gene The amino acid sequence of Sequence ID No. 1 and the nucleotide sequence of Sequence ID No. 11 are consensus sequences derived by comparing the sequence homology of the p30(CP204L) gene from various strains of African swine fever virus. Specifically, the amino acid sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 11 are from the African swine fever virus strains ANG / 70 (GenBank accession no. EU874271), Malawi / 1978 (GenBank accession no. JQ744998), NAM / 1 / 80 (GenBank accession no. JQ745005), Dedza (GenBank accession no. JQ745028), BUR / 90 / 1 (GenBank accession no. EU874299), MOZ / 94 / 1 (GenBank accession no. EU874263), DED 91 / 1 (GenBank accession no. KC867513), GUL 88 / 1 (GenBank accession no. KC867514), KAC 91 / 2 (GenBank accession no. KF736439), KANA 89 / 1 (GenBank accession no.KF736440), Killean I(GenBank accession no.JQ764860), Killean II(GenBank accession no.KC867521), Killean III(GenBank accession no.JQ764861), Kimakia I(GenBank accession no.JQ764954), Kimakia II(GenBank accession no. no.KC867515), KIRT 89 / 2(GenBank accession no.JQ764858), KIRT 89 / 3(GenBank accession no.JQ764856), KIRT 89 / 4(GenBank accession no.JQ764857), KLI 88 / 2(GenBank accession no.KC867516), LIL 89 / 1(GenBank accession no.KC867517), LIL 90 / 1 (GenBank accession no.KF736437)、LIV 5 / 40(GenBank accession no.KC867518)、LIV 9 / 31(GenBank accession no.JQ764966)、LIV 9 / 35(GenBank accession no.JQ764965)、LIV 10 / 11(GenBank accession no.KC867519)、LIV 12 / 17(GenBank accession no.JQ764967)、Mchinji 075(GenBank accession no.JQ764880)、MOZ 2001 / 1(GenBank accession no.KC867524)、MPO 89 / 1(GenBank accession no.KC867520)、NYA1 / 2(GenBank accession no.EU874302)、SAL 92 / 1(GenBank accession no.KF736441)、TEN 89 / 1(GenBank accession no.KF736442)、THY 90 / 1(GenBank accession no.KF736438)、Trench(GenBank accession no.JQ764859)、MOZ / 1960(GenBank accession no.EU874309)、Lillie(GenBank accession no.EU874306)、24823(GenBank accession no.KC867500)、MOZ / 1979(GenBank accession no.EU874310)、SPEC / 154(GenBank accession no.EU874291)、SPEC / 205(GenBank accession no.EU874305)、SPEC / 209(GenBank accession no.EU874290)、SPEC / 257(GenBank accession no.EU874265)、MOZ / 94 / 8(GenBank accession no.EU874276)、E70(GenBank accession no.AF462272)、ZAM / 2017 / Mbala / 1(GenBank accession no.LC322014)、CN201801(GenBank accession no.MH735141)、DB / HLJ / 2018(GenBank accession no.MK333184)、M-78(GenBank accession no.MK211505)、BA71(GenBank accession no.KP055815)、BA71V(GenBank accession no.U18466)、E75(GenBank accession no.FN557520)、OURT 88 / 3(GenBank accession no.AM712240)、Georgia 2007(GenBank accession no.FR682468)、Ken06.Bus(GenBank accession no.KM111295)、Estonia 2014(GenBank accession no.LS478113)、Benin 97 / 1(GenBank accession no.AM712239)、Italy / 26544 / OG10(GenBank accession no.KM102979)、Portugal / NHV / 1968(GenBank accession no.KM262845)、Italy / 47 / SS / 2008(GenBank accession no.KX354450)、Uganda / R35 / 2015(GenBank accession no.MH025920)、Uganda / R25 / 2015(GenBank accession no.MH025918)、Uganda / R8 / 2015(GenBank accession no.MH025916)、Pol16_20186_o7(GenBank accession no.MG939583)、Pol17_03029_C201(GenBank accession no.MG939587)、Belgium 2018 / 1(GenBank accession no.LR536725)、China / 2018 / AnhuiXCGQ(GenBank accession no.MK128995)、DB / LN / 2018(GenBank accession no.MK333181)、Pig / HLJ / 2018(GenBank accession no.MK333180)、ASFV-SY18(GenBank accession no.The p30 (CP204L) gene was constructed by comparing sequence homology from MH766894), Ken05 / Tk1 (GenBank accession no. KM111294), Ken05 / Tk6 (GenBank accession no. HM745363), Ken05.DPk2 (GenBank accession no. HM745368), Ken08WH / 4 (GenBank accession no. HM745390), Ken08Tk.2 / 1 (GenBank accession no. HM745380), Ken09Tk.13 / 1 (GenBank accession no. HM745382), Ken09Tk.19 / 2 (GenBank accession no. HM745386), and Ken09Tk.19 / 11 (GenBank accession no. HM745388).

[0071] The amino acid sequence of Sequence ID No. 2 and the nucleotide sequence of Sequence ID No. 12 are consensus sequences derived by comparing the sequence homology of the p54(E183L) gene from various strains of African swine fever virus. Specifically, the amino acid sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 12 are found in the following African swine fever virus strains: Tengani / 60 (GenBank accession no. KF015886), ANG / 70 (GenBank accession no. EU874327), BA71 (GenBank accession no. KP055815), Malawi / 1978 (GenBank accession no. KC662380), Brazil / 79 (GenBank accession no. KC535549), DomRep / 79 (GenBank accession no. FJ238534), KAV / 89 / 1 (GenBank accession no. KF015902), BUR / 90 / 1 (GenBank accession no. EU874363), MOZ / 94 / 1 (GenBank accession no. EU874342), CHK 89 / 2 (GenBank accession no.KF015921), KAC 91 / 2(GenBank accession no.KF736421), KANA 89 / 1(GenBank accession no.KF736422), Killean III(GenBank accession no.KF736423), Kimakia I(GenBank accession no.KF015924), KIRT 89 / 4(GenBank accession no.KF736414), LIL 90 / 1(GenBank accession no.KF736416), LIV 9 / 31(GenBank accession no.KF015928), MAN 89 / 2(GenBank accession no.KF015940), MOZ 2001 / 1(GenBank accession no.KF736428), MPO 89 / 1(GenBank accession no.KF736418), Trench(GenBank accession no.KF736420)、MOZ / 1960(GenBank accession no.EU874371)、Lillie(GenBank accession no.EU874341)、Madagascar(GenBank accession no.KC662387)、ZIM / 92 / 1(GenBank accession no.EU874345)、SPEC / 205(GenBank accession no.EU874329)、Co62(GenBank accession no.FJ174387)、E70(GenBank accession no.FJ174389)、Ba71V(GenBank accession no.U18466)、E75(GenBank accession no.FN557520)、Hu90(GenBank accession no.FJ174399)、SS81(GenBank accession no.FJ174403)、Ori90(GenBank accession no.FJ174407)、Nu98.8B(GenBank accession no.FJ174418)、OURT 88 / 3(GenBank accession no.AM712240)、Almodovar 99(GenBank accession no.DQ028315)、Almodovar 99 / NE1(GenBank accession no.DQ028317)、Georgia 2007(GenBank accession no.FR682468)、Angola(GenBank accession no.FJ174424)、Nig01(GenBank accession no.FJ174426)、Ug03H.1(GenBank accession no.FJ174431)、Ug64(GenBank accession no.FJ174430)、Ken05.DPU1(GenBank accession no.HM745354)、Ken08WH / 4(GenBank accession no.HM745333)、ken09Tk.20 / 5(GenBank accession no.HM745344)、Con09 / PN003(GenBank accession no.HQ645949)、TAN / 08 / Mazimbu*(GenBank accession no.GQ410767)、TAN / 08 / Mabibo*(GenBank accession no.GQ410768)、Arm07(GenBank accession no.JX857494)、Az08D(GenBank accession no.JX857501)、Oren08(GenBank accession no.JX857498)、Rostov09(GenBank accession no.JX857504)、Tver0312 / Novo(GenBank accession no.KJ627190)、Bel13 / Grodno(GenBank accession no.KJ627192)、LT14 / 1490(GenBank accession no.KJ627193)、ETH / 1(GenBank accession no.KT795366)、ET13 / 1504(GenBank accession no.KU291452)、ETH / 017(GenBank accession no.KT795369)、Ken06.Bus(GenBank accession no.KM111295)、Ken07.Kia(GenBank accession no.FJ174437)、CON09 / Bzz020(GenBank accession no.HQ645950)、Ken10 / KakFA1(GenBank accession no.KC112568)、Ug10.Kumi(GenBank accession no.KC990876)、BUR / 90 / 2(GenBank accession no.KF015897)、Ug12.Wakiso(GenBank accession no.KC990885)、Benin 97 / 1(GenBank accession no.AM712239)、Italy / 26544 / OG10(GenBank accession no.KM102979)、Portugal / NHV / 1968(GenBank accession no.KM262845)、Italy / 47 / SS / 2008(GenBank accession no.KX354450), Uganda / R35 / 2015(GenBank accession no.MH025920), Uganda / R7 / 2015(GenBank accession no.MH025917), Pol17_04461_C210(GenBank accession no.MG939588), Belgium 2018 / 1(GenBank accession no.LR536725), China / 2018 / AnhuiXCGQ(GenBank accession no.MK128995), DB / LN / 2018(GenBank accession no.MK333181), Pig / HLJ / 2018(GenBank accession no.MK333180), ASFV-SY18(GenBank accession no. no.MH766894), CN201801(GenBank accession The p54(E183L) gene was constructed by comparing sequence homology from no.MH735140, Estonia 2014 (GenBank accession no.LS478113), and Nig6_JS10 (GenBank accession no.KT961344).

[0072] The amino acid sequence of Sequence ID No. 3 and the nucleotide sequence of Sequence ID No. 13 are consensus sequences derived by comparing the sequence homology of the C-type lectin (EP153R) gene from various strains of African swine fever virus. Specifically, the amino acid sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 13 are found in the following African swine fever virus strains: Katanga / 63 (GenBank accession no. KM609340), Uganda (GenBank accession no. KM609361), BA71 (GenBank accession no. KP055815), Davis (GenBank accession no. KM609336), Ba71V (GenBank accession no. U18466), E75 (GenBank accession no. FN557520), NH / P68 (GenBank accession no. AF481875), Mafra 86 (GenBank accession no. DQ026269), Coimbra 87 (GenBank accession no. DQ026268), OURT 88 / 3 (GenBank accession no. AM712240), Portalegre 90 (GenBank accession no.DQ026270), Barrancos 93(GenBank accession no.DQ026267), Almodovar 99(GenBank accession no.DQ026265), Almodovar 99 / NE1(GenBank accession no.DQ026266), Georgia 2007(GenBank accession no.FR682468), Ken05 / Tk1(GenBank accession no.KM111294), Ken06.Bus(GenBank accession no.KM111295), Estonia 2014(GenBank accession no.LS478113), Benin 97 / 1(GenBank accession no.AM712239), Italy / 26544 / OG10(GenBank accession no.KM102979)、Portugal / NHV / 1968(GenBank accession no.KM262845)、Italy / 47 / SS / 2008(GenBank accession no.KX354450)、Uganda / R35 / 2015(GenBank accession no.MH025920)、Uganda / R25 / 2015(GenBank accession no.MH025918)、Uganda / R8 / 2015(GenBank accession no.MH025916)、Pol16_20186_o7(GenBank accession no.MG939583)、Pol17_03029_C201(GenBank accession no.MG939587)、Pol17_04461_C210(GenBank accession no.MG939588)、Belgium 2018 / 1(GenBank accession no.LR536725)、China / 2018 / AnhuiXCGQ(GenBank accession no.MK128995)、DB / LN / 2018(GenBank accession no.MK333181)、Pig / HLJ / 2018(GenBank accession no.MK333180)、ASFV-SY18(GenBank accession no.MH766894)、UGA(GenBank accession no.AF017039)、PR5(GenBank accession no.AF017037)、M1(GenBank accession no.AF017034)、K1(GenBank accession no.AF017033)、CR3(GenBank accession no.AF017029)、CR1(GenBank accession no.AF017028)、LC-PP(GenBank accession no.KM609345)、Magadi(GenBank accession no.kM609348)、Bartlett(GenBank accession no.KM609335)、Volgograd_2012 / wb(GenBank accession no.KM609363), Volgograd_2012 / dom(GenBank accession no.KM609362), Tver_2012 / wb(GenBank accession no.KM609360), Rhodesia(GenBank accession no.KM609354), TSP80(GenBank accession no.KM609359), STP-1(GenBank accession no. no. KM609355), O-77 (GenBank accession no. KM609350), F-32 (GenBank accession no. KM609337), MK-200 (GenBank accession no. KM609347), Spencer (GenBank accession no. KM609357), Silva-1 (GenBank accession no. no.kM609356), Ndjassi-77(GenBank The C-type lectin (EP153R) gene was constructed by comparing sequence homology from accession no. KM609349, KK-262 (GenBank accession no. KM609341), K-49 (GenBank accession no. KM609339), L-57 (GenBank accession no. KM609344), and L-50 (GenBank accession no. KM609343).

[0073] The amino acid sequence of Sequence ID No. 4 and the nucleotide sequence of Sequence ID No. 14 are consensus sequences derived by comparing the sequence homology of the CD2v(EP402R) gene from various strains of African swine fever virus. Specifically, the amino acid sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 14 are from the following African swine fever virus strains: China / 2018 / AnhuiXCGQ (GenBank accession no.MK128995), DB / LN / 2018 (GenBank accession no.MK333181), Pig / HLJ / 2018 (GenBank accession no.MK333180), ASFV-SY18 (GenBank accession no.MH766894), Belgium 2018 / 1 (GenBank accession no.LR536725), Pol17_03029_C201 (GenBank accession no.MG939587), Pol16_20186_o7 (GenBank accession no.MG939583), Uganda / R8 / 2015 (GenBank accession no. no.MH025916), Uganda / N10 / 2015(GenBank accession no.MH025919), Estonia 2014(GenBank accession no.LS478113), Davis(1959_Kenya)(GenBank accession no.KM609336), Killean II(1959_Kenya)(GenBank accession no.KM609372), Kimakia II(1961_Kenya)(GenBank accession no.KM609374), Ba71V(1971_Spain)(GenBank accession no.U18466), E75(1975_Spain)(GenBank accession no.FN557520), Ca78(Italy_1978)(GenBank accession no.KT718663), Ori85(Italy_1985)(GenBank accession no.KT718668), Nu91.5(Italy_1991)(GenBank accession no.KT718673)、NH / P68(Portugal_1968)(GenBank accession no.AF481875)、Mafra 86(Portugal_1986)(GenBank accession no.DQ026269)、Coimbra 87(Portugal_1987)(GenBank accession no.DQ026268)、Portalegre 90(GenBank accession no.DQ026270)、Barrancos 93(GenBank accession no.DQ026267)、Almodovar 99 / NE1(GenBank accession no.DQ026266)、OURT 88 / 3(GenBank accession no.AM712240)、FR682468(GenBank accession no.Georgia 2007)、Ken05 / Tk1(GenBank accession no.KM111294)、Ken06.Bus(GenBank accession no.KM111295)、Italy / 47 / SS / 2008(GenBank accession no.KX354450)、Portugal / NHV / 1968(GenBank accession no.KM262845)、Italy / 26544 / OG10(GenBank accession no.KM102979)、Benin 97 / 1(GenBank accession no.AM712239)、Bartlett(GenBank accession no.KM609335)、Zavidovo-2012(GenBank accession no.KM609392)、Tver_2012 / wb(GenBank accession no.KM609360)、Rhodesia(GenBank accession no.KM609354)、Nanyuki(GenBank accession no.KM609382)、Krasnodar_2012 / dom(GenBank accession no.KM609342)、TS-7 / 27-230(GenBank accession no.KM609388)、TSP80(GenBank accession no.KM609359)、K-49(GenBank accession no.KM609339)、KK-262(GenBank accession no.KM609341)、Spencer(GenBank accession no.KM609357)、MK-200(GenBank accession no.KM609347)、691 / 88(GenBank accession no.KM609334)、F-32(GenBank accession no.KM609337)、O-77(GenBank accession no.KM609350)、P-60(GenBank accession no.KM609351)、PPA(GenBank accession no.KM609352)、STP-1(GenBank accession no.KM609355)、BA71(GenBank accession no.KP055815)、STP-1-79(GenBank accession no.KM609384)、Malta(GenBank accession no.KM609380)、Yamba-74(GenBank accession no.KM609391)、TKF(GenBank accession no.KM609387)、Kimuele-6(GenBank accession no.KM609375)、E-70(GenBank accession no.KM609369)、Cuba-71(GenBank accession no.KM609366)、CU-80(GenBank accession no.KM609365)、Brazil-80(GenBank accession no.KM609364)、MNI-82(GenBank accession no.KM609378)、K-73(Le Bray)(GenBank accession no.KM609370)、Madeira(GenBank accession no.KM609379)、Diamang(GenBank accession no.KM609367)、L-50(GenBank accession no.KM609343)、L-57(GenBank accession no.The CD2v (EP402R) gene was constructed by comparing its sequence homology with that of KM609344, Kikasa-77 (GenBank accession no. KM609371), and VL (GenBank accession no. KM609390).

[0074] The amino acid sequence of Sequence ID No. 5 and the nucleotide sequence of Sequence ID No. 15 are consensus sequences derived by comparing the sequence homology of the p49(B438L) gene from various strains of African swine fever virus.Specifically, the amino acid sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 15 are from the following African swine fever virus strains: BA71 (GenBank accession no. KP055815), Ba71V (GenBank accession no. U18466), E75 (GenBank accession no. FN557520), OURT 88 / 3 (GenBank accession no. AM712240), Georgia 2007 (GenBank accession no. FR682468), Ken05 / Tk1 (GenBank accession no. KM111294), Ken06.BUS (GenBank accession no. KM111295), Estonia 2014 (GenBank accession no. LS478113), Benin 97 / 1 (GenBank accession no. AM712239), Italy / 26544 / OG10 (GenBank accession no.KM102979), Portugal / NHV / 1968(GenBank accession no.KM262845), Italy / 47 / SS / 2008(GenBank accession no.KX354450), Uganda / R35 / 2015(GenBank accession no.MH025920), Uganda / N10 / 2015(GenBank accession no.MH025919), Pol16_20186_o7(GenBank accession no.MG939583), Pol17_04461_C210(GenBank accession no.MG939588), Belgium 2018 / 1(GenBank accession no.LR536725), China / 2018 / AnhuiXCGQ(GenBank accession The p49(B438L) gene was constructed by comparing sequence homology from no.MK128995), DB / LN / 2018 (GenBank accession no.MK333181), Pig / HLJ / 2018 (GenBank accession no.MK333180), and ASFV-SY18 (GenBank accession no.MH766894).

[0075] The amino acid sequence of Sequence ID No. 6 and the nucleotide sequence of Sequence ID No. 16 are consensus sequences derived by comparing the sequence homology of the pp62(CP530R) gene from various strains of African swine fever virus. Specifically, the amino acid sequence of SEQ ID NO: 6 and the nucleotide sequence of SEQ ID NO: 16 are from the following African swine fever virus strains: BA71 (GenBank accession no. KP055815), Ba71V (GenBank accession no. U18466), E75 (GenBank accession no. FN557520), OURT 88 / 3 (GenBank accession no. AM712240), Georgia 2007 (GenBank accession no. FR682468), Ken05 / Tk1 (GenBank accession no. KM111294), Ken06.BUS (GenBank accession no. KM111295), Estonia 2014 (GenBank accession no. LS478113), Benin 97 / 1 (GenBank accession no. AM712239), Italy / 26544 / OG10 (GenBank accession no.KM102979), Portugal / NHV / 1968(GenBank accession no.KM262845), Italy / 47 / SS / 2008(GenBank accession no.KX354450), Uganda / R35 / 2015(GenBank accession no.MH025920), Uganda / N10 / 2015(GenBank accession no.MH025919), Pol16_20186_o7(GenBank accession no.MG939583), Pol17_04461_C210(GenBank accession no.MG939588), Belgium 2018 / 1(GenBank accession no.LR536725), China / 2018 / AnhuiXCGQ(GenBank accession no.MK128995), DB / LN / 2018(GenBank accession no.The pp62 (CP530R) gene was constructed by comparing sequence homology from MK333181), Pig / HLJ / 2018 (GenBank accession no. MK333180), ASFV-SY18 (GenBank accession no. MH766894), and Krasnodar (2012) (GenBank accession no. KJ380911).

[0076] The amino acid sequence of Sequence ID No. 7 and the nucleotide sequence of Sequence ID No. 17 are consensus sequences derived by comparing the sequence homology of the EP364R gene from various strains of African swine fever virus.Specifically, the amino acid sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 17 are from the following African swine fever virus strains: BA71 (GenBank accession no. KP055815), Ba71V (GenBank accession no. U18466), E75 (GenBank accession no. FN557520), OURT 88 / 3 (GenBank accession no. AM712240), Georgia 2007 (GenBank accession no. FR682468), Ken05 / Tk1 (GenBank accession no. KM111294), Ken06.BUS (GenBank accession no. KM111295), Estonia 2014 (GenBank accession no. LS478113), Benin 97 / 1 (GenBank accession no. AM712239), Italy / 26544 / OG10 (GenBank accession no.KM102979), Portugal / NHV / 1968(GenBank accession no.KM262845), Italy / 47 / SS / 2008(GenBank accession no.KX354450), Uganda / R35 / 2015(GenBank accession no.MH025920), Uganda / N10 / 2015(GenBank accession no.MH025919), Pol16_20186_o7(GenBank accession no.MG939583), Pol17_04461_C210(GenBank accession no.MG939588), Belgium 2018 / 1(GenBank accession no.LR536725), China / 2018 / AnhuiXCGQ(GenBank accession The EP364R gene was constructed by comparing its sequence homology with that of no.MK128995, DB / LN / 2018 (GenBank accession no.MK333181), Pig / HLJ / 2018 (GenBank accession no.MK333180), and ASFV-SY18 (GenBank accession no.MH766894).

[0077] The amino acid sequence of Sequence ID No. 8 and the nucleotide sequence of Sequence ID No. 18 are consensus sequences derived by comparing the sequence homology of the F317L gene from various strains of African swine fever virus. Specifically, the amino acid sequence of SEQ ID NO: 8 and the nucleotide sequence of SEQ ID NO: 18 are found in the following African swine fever virus strains: BA71 (GenBank accession no. KP055815), Ba71V (GenBank accession no. U18466), E75 (GenBank accession no. FN557520), OURT 88 / 3 (GenBank accession no. AM712240), Georgia 2007 (GenBank accession no. FR682468), Ken05 / Tk1 (GenBank accession no. KM111294), Ken06.BUS (GenBank accession no. KM111295), Estonia 2014 F317L (GenBank accession no. LS478113), Benin 97 / 1 F317L (GenBank accession no.AM712239), Italy / 26544 / OG10 F317L(GenBank accession no.KM102979), Portugal / NHV / 1968 F317L(GenBank accession no.KM262845), Italy / 47 / SS / 2008 F317L(GenBank accession no.KX354450), Uganda / R35 / 2015 F317L(GenBank accession no.MH025920), Uganda / N10 / 2015(GenBank accession no.MH025919), Pol16_20186_o7(GenBank accession no. MG939583), Pol17_04461_C210 (GenBank accession no. MG939588), Belgium 2018 / 1 (GenBank accession no. LR536725), China / 2018 / AnhuiXCGQ (GenBank accession no. MK128995), DB / LN / 2018 (GenBank accession no.The F317L gene was constructed by comparing sequence homology from MK333181, Pig / HLJ / 2018 (GenBank accession no. MK333180), and ASFV-SY18 (GenBank accession no. MH766894).

[0078] The amino acid sequence of Sequence ID No. 9 and the nucleotide sequence of Sequence ID No. 19 are consensus sequences derived by comparing the sequence homology of the A104R gene from various strains of African swine fever virus.Specifically, the amino acid sequence of SEQ ID NO: 9 and the nucleotide sequence of SEQ ID NO: 19 are from the following African swine fever virus strains: BA71 (GenBank accession no. KP055815), Ba71V (GenBank accession no. U18466), E75 (GenBank accession no. FN557520), OURT 88 / 3 (GenBank accession no. AM712240), Georgia 2007 (GenBank accession no. FR682468), Ken05 / Tk1 (GenBank accession no. KM111294), Ken06.BUS (GenBank accession no. KM111295), Estonia 2014 (GenBank accession no. LS478113), Benin 97 / 1 (GenBank accession no. AM712239), Italy / 26544 / OG10 (GenBank accession no.KM102979), Portugal / NHV / 1968(GenBank accession no.KM262845), Italy / 47 / SS / 2008(GenBank accession no.KX354450), Uganda / R35 / 2015(GenBank accession no.MH025920), Uganda / N10 / 2015(GenBank accession no.MH025919), Pol16_20186_o7(GenBank accession no.MG939583), Pol17_04461_C210(GenBank accession no.MG939588), Belgium 2018 / 1(GenBank accession no.LR536725), China / 2018 / AnhuiXCGQ(GenBank accession The A104R gene was constructed by comparing sequence homology from no.MK128995, DB / LN / 2018 (GenBank accession no.MK333181), Pig / HLJ / 2018 (GenBank accession no.MK333180), and ASFV-SY18 (GenBank accession no.MH766894).

[0079] The amino acid sequence of Sequence ID No. 10 and the nucleotide sequence of Sequence ID No. 20 are consensus sequences derived by comparing the sequence homology of the K205R gene from various strains of African swine fever virus.Specifically, the amino acid sequence of SEQ ID NO: 10 and the nucleotide sequence of SEQ ID NO: 20 are from the following African swine fever virus strains: BA71 (GenBank accession no. KP055815), Ba71V (GenBank accession no. U18466), E75 (GenBank accession no. FN557520), OURT 88 / 3 (GenBank accession no. AM712240), Georgia 2007 (GenBank accession no. FR682468), Ken05 / Tk1 (GenBank accession no. KM111294), Ken06.BUS (GenBank accession no. KM111295), Estonia 2014 (GenBank accession no. LS478113), Benin 97 / 1 (GenBank accession no. AM712239), Italy / 26544 / OG10 (GenBank accession no.KM102979), Portugal / NHV / 1968(GenBank accession no.KM262845), Italy / 47 / SS / 2008(GenBank accession no.KX354450), Uganda / R35 / 2015(GenBank accession no.MH025920), Uganda / N10 / 2015(GenBank accession no.MH025919), Pol16_20186_o7(GenBank accession no.MG939583), Pol17_04461_C210(GenBank accession no.MG939588), Belgium 2018 / 1(GenBank accession no.LR536725), China / 2018 / AnhuiXCGQ(GenBank accession The K205R gene was constructed by comparing sequence homology from no.MK128995, DB / LN / 2018 (GenBank accession no.MK333181), Pig / HLJ / 2018 (GenBank accession no.MK333180), and ASFV-SY18 (GenBank accession no.MH766894).

[0080] Example 2. Preparation of African swine fever vaccine A vaccine was manufactured using the African swine fever virus gene from Example 1 described above.

[0081] Specifically, a DNA plasmid containing the aforementioned gene, Kozak sequence, IgE leader sequence, ubiquitin sequence, and / or Furin cleavage site sequence was manufactured using a method commonly known in the industry. Subsequently, E. coli containing the DNA plasmid was inoculated into 2.5 L of LB medium and cultured at 37°C in oil-in-water (O / W) culture. Plasmid was extracted from the cultured E. coli using the EndoFree Plasmid Giga kit (QIAGEN, Cat#12391), and the extracted plasmid was subsequently used for inoculation as a vaccine.

[0082] The cleavage maps of the DNA plasmids produced are shown in Figures 1 to 5.

[0083] Example 3. Effect of African swine fever vaccine on inducing cellular immune response. In Example 2, the DNA vaccine produced was found to have a preventive effect against African swine fever by inducing a cellular immune response (CTL, Cytotoxic T Lymphocyte response).

[0084] Fifteen C57BL / 6 mice (8 weeks old, female) were divided into three groups of five mice each, and then vaccinated three times at two-week intervals. The administered DNA vaccine concentration was 30 ug / mouse, and administration and vaccination were performed using an electroporation device (cellectra 2000). The types of DNA vaccines administered to each control group and experimental group were set as follows.

[0085] - Control group: Mock Plasmid DNA vaccine (a DNA vaccine that does not contain African swine fever virus genes) - Experimental group 1: DNA vaccine containing African swine fever virus genes but without ubiquitin (ASF_4G DNA vaccine; p30, p54, C type lectin and CD2v) - Experimental group 2: DNA vaccine containing African swine fever virus gene and ubiquitin (ASF_Ubi_4G DNA vaccine; ubiquitin, p30, p54, C type lectin, and CD2v)

[0086] After administering the aforementioned DNA vaccine three times, the spleen was removed from the mice one week later, and splenocytes were isolated. Mouse IFN-gamma ELISpot analysis was then performed on the isolated splenocytes using the IFN-gamma ELISpot KIT (Cellular Technology Limited / USA). Specifically, the splenocytes administered with each vaccine were treated with polypeptides of each antigen gene, and then IFN-γ-secreting cytotoxic T cells (CD8) were analyzed. + By analyzing the number of T cells, the extent of the cellular immune response induced by viral infection after vaccination was confirmed.

[0087] As a result, as can be seen in Figure 6 and Table 1 below, in experimental groups 1 and 2, which were administered a DNA vaccine containing the African swine fever virus gene, when the polypeptide of each antigen gene was treated, cytotoxic T cells (CD8) secreted IFN-γ. + We confirmed a significant increase in the number of T cells.

[0088] [Table 1]

[0089] In particular, compared to when a DNA vaccine without ubiquitin was administered (experimental group 1), when a DNA vaccine containing ubiquitin was administered (experimental group 2), cytotoxic T cells (CD8) that secrete IFN-γ were more likely to be stimulated. +We confirmed that the number of T cells increased by approximately 1.6 to 2.2 times. In addition, among the diverse antigen genes included in the DNA vaccine, cytotoxic T cells (CD8) that secrete IFN-γ due to p54 or CD2v were also identified. + The number of T cells was particularly increased, and depending on p30, cytotoxic T cells (CD8) that secreted IFN-γ were only administered when a ubiquitin-containing vaccine was given (experimental group 2). + We confirmed that the number of T cells increased.

[0090] The results described above show that when a DNA vaccine containing African swine fever virus genes and ubiquitin is administered, it is possible to efficiently induce a cellular immune response, such as a significant increase in the number of cytotoxic T cells that secrete IFN-γ, even when infected with African swine fever virus. Therefore, it was found that the DNA vaccine can be usefully utilized as a vaccine composition for the prevention of African swine fever.

[0091] Example 4. Survival rate increasing effect of African swine fever vaccine. In Example 2, the vaccine produced was found to have a preventive effect against African swine fever, specifically by increasing the survival rate after infection with the African swine fever virus.

[0092] Specifically, the aforementioned DNA vaccine was administered to piglets a total of three times (at week 0, week 2, and week 4). Subsequently, at week 7, an attack inoculation (challenge) with African swine fever virus was performed, and the survival rate for the following 28 days was investigated. At this time, the types of DNA vaccine administered to each control group and experimental group were set as follows.

[0093] - Control group: DNA vaccine (Mock) that does not contain the African swine fever virus gene. - Experimental group: DNA vaccine containing African swine fever virus gene and ubiquitin (ASF_Ubi_10G DNA vaccine; ubiquitin, p30, p54, C-type lectin, CD2v, p49, pp62, EP364R, F317L, A104R and K205R)

[0094] As shown in Figure 7, in the control group, only one out of three pigs survived on day 28 after vaccination, indicating a low survival rate of approximately 33%. In contrast, in the experimental group administered the DNA vaccine containing the African swine fever virus gene, four out of six pigs survived on day 28 after vaccination, showing a high survival rate of approximately 66%, which was more than double that of the control group.

[0095] Based on the results described above, it was found that when a DNA vaccine containing African swine fever virus genes and ubiquitin is administered, the subjects exhibit excellent survival ability even after infection with African swine fever virus. Therefore, the DNA vaccine can be usefully utilized as a vaccine composition for the prevention of African swine fever.

Claims

1. (i) Sequence IDs 1 to 4, (ii) Sequence numbers 5-10, or (iii) Sequence numbers 1-10, A polypeptide containing an amino acid sequence that includes [specific amino acid sequence].

2. The polypeptide according to claim 1, further comprising one or more amino acid sequences selected from an IgE leader sequence, a ubiquitin sequence, and a furin cleavage site sequence.

3. The polypeptide according to claim 1, further comprising the amino acid sequence of SEQ ID NO:

41.

4. The polypeptide according to claim 1, further comprising the amino acid sequence of SEQ ID NO:

43.

5. (i) Sequence IDs 11-14, (ii) Sequence numbers 15-20, or (iii) Sequence IDs 11-20, A polynucleotide containing a base sequence that includes [specific bases].

6. The polynucleotide according to claim 5, further comprising one or more nucleotide sequences selected from a Kozak sequence, an IgE leader sequence, a ubiquitin sequence, and a cleavage site sequence.

7. The polynucleotide according to claim 5, further comprising the base sequence of sequence number 42.

8. The polynucleotide according to claim 5, further comprising the base sequence of sequence number 44.

9. A plasmid comprising the polynucleotide described in claim 5.

10. The plasmid according to claim 9, comprising any one of the nucleotide sequences of sequence numbers 31 to 40 and sequence numbers 50 to 54.

11. African swine fever vaccine composition comprising a polypeptide according to any one of claims 1 to 4, a polynucleotide according to any one of claims 5 to 8, or a plasmid according to any one of claims 9 and 10.

12. A method for generating an immune response to African swine fever virus from an animal other than a human, comprising the step of administering the vaccine composition described in claim 11 to the animal.

13. The method according to claim 12, characterized in that the administration is performed by electroporation.

14. A method for treating or preventing African swine fever, comprising the step of administering the vaccine composition according to claim 11 to an animal other than a human.

15. The method according to claim 14, characterized in that the administration is performed by electroporation.

16. A pharmaceutical composition for the treatment or prevention of African swine fever, comprising a polypeptide according to any one of claims 1 to 4, a polynucleotide according to any one of claims 5 to 8, or a plasmid according to any one of claims 9 and 10.

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