Modified African swine fever virus and vaccine using the same

Abstract

By deleting a nucleotide sequence encoding the amino terminal region and a nucleotide sequence encoding the carboxy terminal region of the E120R protein in the genome, the modified African swine fever virus does not remain in the body of the inoculated animal and exhibits a vaccine effect against African swine fever.

Description

[Technical Field]

[0001] This invention relates to a modified African swine fever virus and a vaccine using the same. [Background technology]

[0002] African swine fever (ASF) is a highly fatal infectious disease of the Suidae family (pigs, wild boars, etc.) caused by the African swine fever virus (ASFV). Originally, ASFV is found only on the African continent, but it can accidentally escape Africa through the movement of people and goods between continents. Currently, ASF is prevalent outside of Africa, mainly in Eastern Europe and Asia.

[0003] Vaccines against African Swine Fever (ASF) are considered one way to contain the outbreak. Recently, Vietnam approved a vaccine using a genetically modified attenuated ASFV strain, and it is being used domestically as the world's first ASF vaccine. While pigs immunized with this vaccine strain exhibit high efficacy in preventing the onset of disease from highly virulent strains, the long-term persistence of the vaccine strain in the pigs' bodies has raised safety concerns. Therefore, there is a strong need to create a virus that does not persist in the bodies of vaccinated animals and yet provides protection against ASF.

[0004] Regarding viruses that do not persist in the pig body, Sanford et al. created ASFV-G / VP-ΔTK, which lacks the thymidine kinase (TK) gene, and reported that pigs inoculated with this virus did not develop a condition where the virus persists in the blood (viremia) (the levels were below the detection limit) (Non-Patent Literature 1). However, pigs immunized with this strain were not protected at all from attacks by highly virulent strains. Thus, viruses that do not cause viremia at all have the problem of losing their protective effect as a vaccine. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] B. Sanford et al., Virus Res., February 2, 2016, 213:165-171. [Non-Patent Document 2] Huisheng Liu et al., J Virol., 25 August 2021, 95(18):e0082421. [Non-Patent Document 3] Edward Spinard et al., Microbiol Resour Announc., December 15, 2022, 11(12):e0088122. [Overview of the project] [Problems that the invention aims to solve]

[0006] This invention has been made in view of the problems of the prior art described above, and aims to provide an African swine fever virus that does not remain in the body of an inoculated animal and that exhibits a vaccine effect against African swine fever. [Means for solving the problem]

[0007] The inventors, through diligent research to achieve the above objective, succeeded in creating an African swine fever virus (AQSΔE120R) in which the nucleotide sequence encoding the full-length E120R protein was deleted. They then revealed that when this modified ASFV was inoculated into pigs, the blood virus concentration was extremely low, below the detection limit, unlike when the parent strain was inoculated. Furthermore, they found that inoculation with AQSΔE120R did not cause fever or lead to death. On the other hand, they also revealed that when pigs inoculated with AQSΔE120R were attacked with the parent strain, a vaccine effect was achieved.

[0008] The inventors also succeeded in creating ASFV (AQS E120RΔN, AQS E120RΔC, AQS E120RΔNC) that retains the E120R protein lacking the N-terminal region and / or C-terminal region, and analyzed their pathogenicity and their in vivo concentrations in inoculated pigs. As a result, pigs inoculated with AQS E120RΔC developed fever and died. Furthermore, it was revealed that the modified ASFV was present in the spleen and gastrohepatic lymph nodes. In addition, although no fever or the like was observed in pigs inoculated with AQS E120RΔN, a tendency to remain in the spleen and gastrohepatic lymph nodes was observed. On the other hand, in pigs inoculated with AQS E120RΔNC, no fever or the like was observed, and furthermore, it was clarified that the presence of the modified ASFV in blood, spleen and gastrohepatic lymph nodes was extremely low, below the detection limit, leading to the completion of the present invention.

[0009] That is, the present invention provides the following aspects.

[0010] [1] A modified African swine fever virus lacking in its genome the nucleotide sequence encoding the amino-terminal region (N-terminal region) of the E120R protein and the nucleotide sequence encoding the carboxy-terminal region (C-terminal region).

[0011] [2] The modified African swine fever virus according to [1], wherein the N-terminal region is a region consisting of the amino acid sequence from the 10th to the 30th amino acid from the N-terminus of the E120R protein, and the C-terminal region is a region consisting of the amino acid sequence from the 10th to the 30th amino acid from the C-terminus of the E120R protein.

[0012] [3] The modified African swine fever virus according to [1], lacking in its genome the nucleotide sequence encoding the full length of the E120R protein.

[0013] [4] A vaccine composition against African swine fever, containing as an active ingredient the modified African swine fever virus according to any one of [1] to [3].

[0014] [5] A method for producing a modified African swine fever virus, comprising: In cells infected with African swine fever virus, by homologous recombination, deleting the nucleotide sequences encoding the N-terminal region and the C-terminal region of the E120R protein of the virus, and obtaining the modified African swine fever virus according to any one of [1] to [3].

[0015] [6] The N-terminal region is a region consisting of the amino acid sequence from the 10th to the 30th amino acids from the N-terminus of the E120R protein, and the C-terminal region is a region consisting of the amino acid sequence from the 10th to the 30th amino acids from the C-terminus of the E120R protein. The production method according to [5].

[0016] [7] The nucleotide sequence to be deleted is the nucleotide sequence encoding the full length of the E120R protein. The production method according to [5].

[0017] [8] The cell is a cell immortalized from macrophages of an inoshishi family animal. The production method according to any one of [5] to [7].

[0018] [9] A method for producing a vaccine composition against African swine fever, comprising: Producing a modified African swine fever virus by the production method according to any one of [5] to [8], and Mixing the modified African swine fever virus with a pharmaceutically acceptable carrier or vehicle The method comprising.

[0019]

[10] A method for producing a modified African swine fever virus, comprising: Infecting a cell with the modified African swine fever virus according to any one of [1] to [3] and proliferating it.

[0020]

[11] The cell is a cell immortalized from macrophages of an inoshishi family animal. The production method according to

[10] .

[0021]

[12] A method for producing a vaccine composition against African swine fever, A step of producing a modified African swine fever virus by the manufacturing method described in

[10] or

[11] , and The process of mixing the modified African swine fever virus with a pharmacologically acceptable carrier or medium. Methods that include...

[0022] Furthermore, the E120R protein of ASFV is a protein that encodes genes related to viral budding and immune regulation. More specifically, the E120R protein is a protein that suppresses the host's antiviral response by inhibiting the production of interferon-β, and it has been shown that amino acids 72 and 73 are essential for this suppressive function to be exerted. In addition, it has been shown that when a modified ASFV lacking these two amino acids is inoculated, interferon-β production is improved compared to the parent strain, that is, the host's antiviral response can be activated (Non-Patent Literature 2).

[0023] However, as shown in the examples described later, deleting the 72nd and 73rd amino acids of the E120R protein did not reduce the pathogenicity of ASFV, and pigs inoculated with this modified ASFV were lethal.

[0024] On the other hand, in the modified ASFV of the present invention, the deletion in the E120R protein is in the N-terminal and C-terminal regions, which differs from the deletion target in Non-Patent Literature 2. Furthermore, as shown in the examples described later, no lethal pigs were observed when inoculated with the modified ASFV of the present invention, clearly demonstrating that pathogenicity is suppressed.

[0025] Furthermore, several research groups have attempted to create strains completely lacking the E120R gene, but have not been successful (Non-Patent Documents 2 and 3). However, as shown in the examples described below, the present inventors have succeeded in creating strains completely lacking the E120R gene, and have also demonstrated their effectiveness. [Effects of the Invention]

[0026] According to the present invention, it is possible to provide an African swine fever virus that does not remain in the body of an inoculated animal and yet exhibits a vaccine effect against African swine fever. In particular, according to the present invention, immunity against African swine fever can be conferred to animals with high safety, without causing pathogenicity such as fever after inoculation, and without remaining in the body. [Brief explanation of the drawing]

[0027] [Figure 1] This figure shows an overview of the E120R gene in African swine fever virus (ASFV) AQS-C-1-2, and an overview of the modified E120R gene in AQSΔE120R, AQS E120RΔN, AQS E120RΔC, and AQS E120RΔNC, which are derived from this parent strain. [Figure 2] This figure shows an overview of the genome in the modified ASFV (AQSΔE120R) and the results of observing the modified ASFV using a fluorescence microscope. In the figure, "a" shows that in the genome of the parent strain (ASFV AQS-C-1-2), the E120R gene was replaced with the copGFP gene by homologous recombination, resulting in the deletion of the former. In "b," AQSΔE120R was created as a result of detecting fluorescence derived from GFP, indicating the expression of the copGFP gene. [Figure 3A]This graph shows the results of measuring the body temperature over time in pigs inoculated with a modified ASFV (AQSΔE120R) in which the nucleotide sequence encoding the full length of the E120R protein was deleted. In the figure, "101, 103, or 105 TCID50 inoculation group" shows the results of pig groups inoculated (infected) with each immunization dose (viral titer) indicated in each item. "AQS-C-1-2 inoculation group" shows the results (control) of pig groups inoculated (infected) with ASFV AQS-C-1-2, the parent strain of AQSΔE120R. Each line shows the measurement results for each individual pig. However, the solid line at 40°C indicates the normal body temperature (upper limit) of the pig. [Figure 3B] This graph shows the results of measuring the amount of viral gene in the blood of pigs inoculated with AQSΔE120R over time. In the figure, the vertical axis shows the absolute quantitative value obtained by real-time PCR using purified nucleic acid from 1 μL of sample as a template, multiplied by 1000, and then taken as the common logarithm (log10), representing the amount of gene per 1 mL of each sample. Other item notations and each line are the same as in Figure 3A. Lines marked with a dagger indicate the results of individuals that died or were euthanized, as in Figure 3A. However, the dashed line for a blood viral gene amount of 3.0 indicates the detection limit. [Figure 4A] This graph shows the results of measuring the body temperature over time in pigs that were inoculated with the parent strain of 102 TCID50 (attacked with the parent strain) 28 days after inoculation with AQSΔE120R. The item notations, line graphs, and daggers in the graph are the same as in Figure 3A. However, the solid line at 40°C represents the normal body temperature (upper limit) of the pigs. [Figure 4B] This graph shows the results of measuring the blood viral gene levels over time in pigs that were inoculated with the parent strain of 102 TCID50 (attacked with the parent strain) 28 days after inoculation with AQSΔE120R. The item notations, line graphs, and daggers in the figure are the same as in Figure 3B. However, the dashed line at blood viral gene level 3.0 indicates the detection limit. [Figure 5A]This graph shows the results of measuring the body temperature over time in pigs inoculated (infected) with ASFV (AQS E120RΔN, AQS E120RΔC, or AQS E120RΔNC) carrying a partial deletion mutant of E120R. In the figure, each line and dagger is the same as in Figure 3A. However, the solid line at 40°C represents the normal body temperature (upper limit) of the pigs. [Figure 5B] This graph shows the results of measuring the viral gene load in blood, gastric-liver lymph nodes, or spleen collected from pigs 7 days after inoculation with AQS E120RΔN, AQS E120RΔC, or AQS E120RΔNC. In the graph, the vertical axis shows the absolute quantitative value obtained by performing real-time PCR using purified nucleic acid from 1 μL of sample as a template, as the gene load per 1 μL of each sample. "pig1~7" shows the measurement results for each individual pig. The solid line for viral gene load 1.0 indicates the detection limit for viral gene measurement in gastric-liver lymph nodes and spleen, and the solid line for viral gene load 0 indicates the detection limit for viral gene measurement in blood. [Figure 6A] This graph shows the results of measuring the body temperature over time in pigs that were inoculated (infected) with AQS E120RΔNC. Each line in the graph is the same as in Figure 3A. [Figure 6B] This graph shows the results of measuring the amount of viral gene in the blood of pigs that were inoculated (infected) with AQS E120RΔNC over time. In the figure, "1-4" shows the measurement results for each individual pig. Each line is the same as in Figure 3B. However, the dashed line at 3.0 for the amount of viral gene in the blood indicates the detection limit. [Figure 7]This graph shows the results of measuring the amount of viral gene in blood, gastric-liver lymph nodes, or spleen collected from pigs at necropsy after inoculation with ASFV (AQS E120RΔ72-73), which carries a mutant lacking the 72nd and 73rd amino acids of the E120R protein. In the figure, "pig1~2" shows the measurement results for each individual pig. On the vertical axis, the absolute quantitative value obtained by performing real-time PCR using purified nucleic acid from 1 μL of sample as a template is shown as the amount of gene per 1 μL of each sample. The solid line at a viral gene amount of 1.0 indicates the detection limit for viral gene measurement in gastric-liver lymph nodes and spleen, and the solid line at a viral gene amount of 0 indicates the detection limit for viral gene measurement in blood. [Modes for carrying out the invention]

[0028] As shown in the examples described below, the inventors have demonstrated that African swine fever virus (ASFV) possessing the E120R protein with deleted N-terminal and C-terminal regions confers a vaccine effect against African swine fever to inoculated animals, while remaining non-pathogenic. Furthermore, the presence of this modified ASFV in the bodies of inoculated pigs (blood, spleen, and gastrohepatic lymph nodes) is extremely low, below the detection limit.

[0029] Therefore, the present invention provides a modified African swine fever virus in which the genome lacks the nucleotide sequence encoding the amino-terminal region and the nucleotide sequence encoding the carboxy-terminal region of the E120R protein.

[0030] (African swine fever virus) In this invention, "African swine fever virus (Asfarviridae Asfivirus, ASFV)" is a virus of the Asfarviridae family, genus Asfivirus, that has double-stranded DNA in its genome. In this invention, there are no particular restrictions on the ASFV that is the target of the E120R gene modification described later; for example, any of the 24 reported genotypes (type II, type I, type X, etc.) may be used. Furthermore, the level of pathogenicity (e.g., highly virulent or weakly virulent, or peracute, acute, subacute, chronic or asymptomatic) is not a factor, and it may be a field type or a habituated type.

[0031] (Modifications in E120R) In the present invention, "E120R" to be modified is a type of structural protein expressed (transcribed) in the late state of the replication cycle in ASFV, and is also referred to as p14.5. E120R is typically a protein consisting of the amino acid sequence described in SEQ ID NO: 2 (a protein consisting of the amino acid sequence encoded by the nucleotide sequence described in SEQ ID NO: 1). However, nucleotide sequences can mutate in nature. Consequently, the amino acid sequence of the protein encoded by the nucleotide sequence can also change. Therefore, E120R according to the present invention is not limited to the typical sequence described above, but also includes natural variants (homogenizers, etc.). There are no particular restrictions on such natural variants, however, Sequence ID: 2Examples include proteins containing amino acid sequences that have 80% or more homology or identity with the amino acid sequences described above. More specifically, examples include proteins identified by UniProtKB P0C9Y5·P14_ASFK5 or Q65201·P14_ASFB. Here, homology or identity is preferably 85% or more, more preferably 87% or more, even more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more), and more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Note that "identity" means the percentage of sites where the types of amino acids are the same between the amino acid sequences being compared, and "homology" means the percentage including the percentage of sites where similar amino acids are the same. Sequence homology or identity can be determined using the BLASTP (amino acid level) program (Altschul et al. J.Mol.Biol., 215:403-410, 1990). This program is based on the BLAST algorithm by Karlin and Altschul (Proc.Natl.Acad.Sci.USA, 87:2264-2268, 1990, Proc.Natl.Acad.Sci.USA, 90:5873-5877, 1993). When analyzing amino acid sequences using BLASTP, the parameters should be, for example, score=50 and wordlength=3. Alternatively, when analyzing amino acid sequences using the Gapped BLAST program, it can be done as described by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using the BLAST and Gapped BLAST programs, use the default parameters for each program. The specific methods for these analysis techniques are publicly known.

[0032] In the present invention, ASFV is modified to delete the amino-terminal region (N-terminal region) and carboxy-terminal region (C-terminal region) of E120R described above. Examples of the "amino-terminal region (N-terminal region)" to be deleted include the region consisting of amino acid sequences from the 5th to the 80th amino acid from the N-terminus, the region consisting of amino acid sequences from the 7th to the 70th amino acid from the N-terminus, the region consisting of amino acid sequences from the 8th to the 50th amino acid from the N-terminus, the region consisting of amino acid sequences from the 9th to the 40th amino acid from the N-terminus, the region consisting of amino acid sequences from the 10th to the 30th amino acid from the N-terminus, and the region consisting of amino acid sequences from the 10th to the 20th amino acid from the N-terminus. More specifically, examples include the amino acid sequence from the N-terminus to the 80th amino acid, the amino acid sequence from the N-terminus to the 75th amino acid, the amino acid sequence from the N-terminus to the 71st amino acid, the amino acid sequence from the N-terminus to the 70th amino acid, the amino acid sequence from the N-terminus to the 60th amino acid, the amino acid sequence from the N-terminus to the 50th amino acid, the amino acid sequence from the N-terminus to the 40th amino acid, the amino acid sequence from the N-terminus to the 30th amino acid, the amino acid sequence from the N-terminus to the 25th amino acid, the amino acid sequence from the N-terminus to the 20th amino acid, the amino acid sequence from the N-terminus to the 16th amino acid, the amino acid sequence from the N-terminus to the 15th amino acid, the amino acid sequence from the N-terminus to the 10th amino acid, or the amino acid sequence from the N-terminus to the 5th amino acid. Note that the position (number) of the amino acid in the N-terminal region is determined by the number of amino acids assigned in ascending order toward the C-terminus, with the first amino acid encoded by the N-terminus, i.e., the start codon (e.g., methionine), being number 1. Furthermore, the start codon is the starting point for protein synthesis. Therefore, the N-terminal region targeted for deletion does not contain (is excluded from) the amino acid encoded by the start codon.For example, in AQS E120RΔN and AQS E120RΔC, as shown in the examples described later, the N-terminal region to be deleted is the region consisting of the amino acid sequence from the N-terminus to the 16th amino acid, but the number of amino acids deleted is 15 amino acids.

[0033] On the other hand, examples of "carboxy-terminal regions (C-terminal regions)" that are subject to deletion include the amino acid sequence from the 5th to the 50th amino acid from the C-terminus, the amino acid sequence from the 6th to the 40th amino acid from the C-terminus, the amino acid sequence from the 8th to the 30th amino acid from the C-terminus, and the amino acid sequence from the 10th to the 20th amino acid from the C-terminus. More specifically, examples include the amino acid sequence from the 50th amino acid from the C-terminus, the amino acid sequence from the 49th amino acid from the C-terminus, the amino acid sequence from the 45th amino acid from the C-terminus, the amino acid sequence from the 40th amino acid from the C-terminus, the amino acid sequence from the 35th amino acid from the C-terminus, the amino acid sequence from the 30th amino acid from the C-terminus, the amino acid sequence from the 25th amino acid from the C-terminus, the amino acid sequence from the 20th amino acid from the C-terminus, the amino acid sequence from the 18th amino acid from the C-terminus, the amino acid sequence from the 15th amino acid from the C-terminus, the amino acid sequence from the 10th amino acid from the C-terminus, or the amino acid sequence from the 5th amino acid from the C-terminus. Note that the position (number) of an amino acid in the C-terminal region refers to the last amino acid encoded by the codon immediately preceding the C-terminus, i.e., the stop codon (for example, Sequence ID: 2 In the amino acid sequence described, the 122nd amino acid (lysine) is considered the first amino acid, and the amino acids are numbered in ascending order towards the N-terminus.

[0034] Furthermore, the positions (positions) of the amino acids in the above N-terminal and C-terminal regions are as follows: Sequence ID: 2 The amino acid numbers are based on the amino acid sequence described above. In the natural mutant of E120R mentioned above, Sequence ID: 2Each of the parts described shall be interpreted as the amino acid corresponding to that part (for example, Sequence ID: 2 In E120R, the "region consisting of amino acid sequences from the N-terminus to the 10th to 30th amino acid" is defined as "from the N-terminus, Sequence ID: 2 (This should be read as "the region consisting of amino acid sequences from the 10th to the 30th amino acid corresponding to the amino acid described in [the relevant section]"). Note that "corresponding" means using nucleotide and amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Sequence ID: 2 This refers to the relationship where the amino acid sequence described in [the document] and the amino acid sequence of a natural variant of E120R are aligned (applied to alignment) and are in the same position.

[0035] Furthermore, as shown in the examples described later, in the present invention, the deletion of the N-terminal and C-terminal regions of E120R may be a deletion of the entire length of E120R. In this case, the deletion target may also include the amino acid encoded by the start codon.

[0036] Furthermore, the modified ASFV having the E120R protein lacking the N-terminal and C-terminal regions, as shown in the examples described below, does not exhibit pathogenicity such as fever upon inoculation, and does not remain in the body, thus safely conferring immunity to African swine fever to animals.

[0037] In the present invention, "pathogenicity" means the property of causing symptoms due to ASFV infection, and "not pathogenic" means, for example, that fever, which is one of the symptoms of ASFV infection, is not observed for one week after inoculation with the modified ASFV of the present invention. Here, "fever" means a state in which the body temperature is more than 1°C above the normal body temperature of the animal. For example, as shown in the examples described later, in pigs (normal body temperature less than 40°C), a state in which the body temperature exceeds 41°C is considered a fever.

[0038] Furthermore, "not remaining in the body" means that, one week or more after inoculation with the modified ASFV of the present invention, the ASFV-derived gene is below the detection limit in at least one tissue in the body. In such tissues, the tissue in which the modified ASFV-derived gene is below the detection limit is preferably blood, and preferably at least blood, spleen, and gastrohepatic lymph nodes. There are no particular restrictions on the modified ASFV-derived gene to be targeted, but it is preferably the p72 gene, and it is even more preferable that it is below the detection limit in a real-time PCR method targeting this gene. More specifically, for example, the real-time PCR method can be performed using a concentration series of a standard (for example, plasmid DNA cloned from the amplification product in the real-time PCR method) as a template, and the lowest detectable concentration can be set as the detection limit.

[0039] "Conferring immunity to African swine fever" means conferring humoral and / or cellular immunity to African swine fever to animals inoculated with the modified ASFV of the present invention, thereby enabling at least one of the following forms of prevention: prevention of ASFV infection, prevention of disease onset due to ASFV infection, and prevention of severe illness due to ASFV infection.

[0040] (Method for producing modified African swine fever virus) The modified ASFV of the present invention described above can be produced, for example, by deleting the nucleotide sequences encoding the N-terminal and C-terminal regions of the E120R protein in the genome of the ASFV within an ASFV-infected cell.

[0041] Such "deletions of nucleotide sequences" can be addressed using, for example, a knockout technique utilizing homologous recombination, as shown in the examples described later. In the knockout technique, a targeting DNA construct having a sequence homologous to at least a portion of the sequence of the target gene region (in this invention, the E120R gene region) is introduced into the cells described later so that homologous recombination occurs between the target gene region and the target DNA construct. Typically, the targeting DNA construct has a structure in which DNA consisting of a sequence homologous to the sequence of the target DNA region is adjacent to each end of the DNA for disrupting the target gene. That is, the homologous DNA is located on the left and right arms of the targeting DNA construct, and the DNA for disrupting the target gene is located between these two arms. Here, "homologous" includes not only cases where the sequences are completely (i.e., 100%) identical, but also cases where some sequences are different, as long as homologous recombination occurs. Usually, at least 95%, preferably 97%, and more preferably 99% or more of the sequences are identical. Furthermore, the chain length of these arms can be any number that allows homologous recombination to occur, for example, 200 to 3000 nucleotides, preferably 300 to 2000 nucleotides, and more preferably 500 to 1000 nucleotides. Through interaction between the target gene region on the genome and homologous sequences of the targeting DNA construct, a specific sequence in the target gene region is exchanged with DNA on the targeting DNA construct, thereby causing deletion of part or the entire target gene. The genome sequence of ASFV is publicly known (for example, the nucleotide sequence described in GenBank:OR660089.1). In this sequence, the nucleotide sequence encoding the E120R protein consists of nucleotides 167839 to 168207. Therefore, those skilled in the art can appropriately set the homologous sequences, etc., based on this sequence information and induce homologous recombination. In addition, in the preparation of the targeting DNA construct, for example, as shown in the examples below, DNA in which a marker gene (fluorescent protein gene such as GFP gene or mCherry gene) is inserted inside the cloned target gene can be used.Furthermore, the targeting DNA construct may be linear DNA or circular DNA.

[0042] In the present invention, in order to induce homologous recombination, for example, the targeting DNA construct is first introduced into the cells described later. There are no particular limitations on the method of such introduction, and it can be carried out using any known gene transfer method. Examples of known gene transfer methods include lipofection, electroporation, calcium phosphate, DEAE-dextran, microinjection, and particle gun.

[0043] Next, the cells into which the targeting DNA construct has been introduced are infected with the aforementioned ASFV. Infection of cells with ASFV can be carried out, for example, by adding ASFV to the cell culture system (e.g., culture medium) and bringing it into contact with the cells. There are no particular restrictions on the timing of infection, but examples include 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or the following day after the introduction of the targeting DNA construct. There are no particular restrictions on the culture period after infecting (e.g., contacting) the cells with ASFV, as long as homologous recombination can occur, but examples include 3 days to 3 weeks or 1 to 2 weeks. Furthermore, there are no particular restrictions on the "culture medium" used to infect the cells with ASFV, as long as it can maintain the cells described later, and it can be prepared by appropriately adding well-known and conventional culture medium additives to a known basic medium. Examples of "basic media" include Dulbecco's modified Eagle medium (DMEM medium), DMEM medium (high glucose), DMEM medium (low glucose), RPMI 160 medium, RPMI 1640 medium, Ham F12 medium, KSOM medium, Eagle MEM medium, Glasgow MEM medium, αMEM medium, Ham medium, Fishers medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc option improved medium, IMDM medium, Medium 199 medium, and any mixture of these.Examples of "culture medium additives" include serum (e.g., fetal bovine serum), reducing agents (monothioglycerol, 2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc.), antibiotics (penicillin, streptomycin, gentamicin, etc.), antifungal agents, functional proteins (insulin, transferrin, lactoferrin, etc.), lipids other than fatty acids (cholesterol, etc.), amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides, etc. (nucleosides, cytidine, adenosine 5'-monophosphate, etc.). Examples of suitable media include, but are not limited to, oxyxanthine, thymidine, etc., metal salts (ferrous nitrate, ferrous sulfate, copper sulfate, zinc sulfate, etc.), inorganic salts (sodium, potassium, calcium, magnesium, phosphorus, chlorine, etc.), carbon sources (glucose, galactose, fructose, sucrose, etc.), vitamins, inorganic compounds (selenic acid), organic compounds (para-aminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine, pyruvate, lactic acid, triiodothyronine, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), and pH indicators (phenol red, etc.). The culture temperature is not particularly limited, but is usually 30-42°C, preferably 37°C. The concentration of carbon dioxide in the gas in contact with the culture medium is not particularly limited, but is usually 1-10% by volume, preferably 2-5% by volume.

[0044] Next, homologous recombination-induced ASFV is isolated from the cell culture system infected with ASFV. This isolation can be performed, for example, by subjecting the cell culture system (e.g., culture supernatant) to limiting dilution to purify the ASFV, as shown in the examples described later. Alternatively, if a targeting DNA construct containing a marker gene within the target gene is used, isolation can also be performed using the expression of the marker gene as an indicator.

[0045] Furthermore, the modified ASFV isolated in this manner can be grown by contacting the cells described below again and culturing the infected cells. The culture conditions (culture medium, culture temperature, etc.) are as described above. The culture period after contact with the modified ASFV is not particularly limited, but is usually 1 to 5 weeks, preferably 2 to 4 weeks, and more preferably about 3 weeks (for example, 18 to 24 days, 19 to 23 days, 20 to 22 days, or 21 days).

[0046] Whether modified ASFV has proliferated can be determined by methods known to those skilled in the art. Such methods include, for example, detecting genes derived from ASFV or their expression. Here, gene expression may be at the transcriptional level (mRNA level) or the translational level (protein level). Methods for detecting genes (genomic DNA) or mRNA include, for example, PCR (RT-PCR, real-time PCR, quantitative PCR), DNA microarray analysis, Northern blotting or Southern blotting, in situ hybridization, dot blotting, RNase protection assay, and mass spectrometry. Furthermore, genes or mRNA levels can be quantitatively detected by counting the number of reads in so-called next-generation sequencing. Methods for detecting proteins include, for example, antibody-based detection methods (immunological methods) such as ELISA, antibody arrays, immunoblotting, imaging cytometry, flow cytometry, radioimmunoassay, immunoprecipitation, and immunohistochemical staining, as well as mass spectrometry. Furthermore, the proliferation of modified ASFV can also be detected by the CPE test, which uses cytopathic effects (CPE) as an indicator, and the HAD test, which uses the hematopoietic adsorption (HAD) reaction specifically observed in ASFV-infected cells as an indicator.

[0047] In this invention, the "cells" used to produce and further proliferate modified ASFV are not particularly limited as long as they are cells that ASFV can infect, for example, macrophages. There are no particular restrictions on the origin of the macrophages, but they are preferably derived from animals of the Suidae family. The macrophages may be primary cultured cells or immortalized cells may be used. In this invention, immortalized macrophages from animals of the Suidae family are preferably used.

[0048] (Immortalized macrophages from wild boars) In this invention, "Suidae animals" refers to animals belonging to the Suidae family, suborder Suoidei, order Artiodactyla, class Mammalia. Examples include animals belonging to the genus Sus (wild boar, its domesticated species the pig, etc.) and animals belonging to the genus Potamochoerus (red river hog, etc.).

[0049] In the present invention, the "macrophages" to be immortalized may be active macrophages (inflammatory M1-type macrophages, anti-inflammatory M2-type macrophages) or resting macrophages. Furthermore, there are no particular restrictions on the tissues in which the macrophages exist. Examples of macrophages according to the present invention (so-called tissue macrophages) include renal macrophages, alveolar macrophages, blood macrophages, intestinal macrophages, liver macrophages, brain macrophages, and osteoclasts.

[0050] There are no particular restrictions on the method for immortalizing primary cultured macrophages derived from various tissues, but it can be done by introducing at least one immortalization gene. Examples of immortalization genes include SV40 large T antigen (SV40T antigen), telomerase reverse transcriptase (TERT), Myc, and Ras. It is preferable to introduce SV40T antigen and TERT, and from the viewpoint of increasing the efficiency of macrophage immortalization, it is even more preferable to introduce SV40T antigen and porcine-derived TERT.

[0051] The introduction of an immortalization gene can be carried out by using a vector encoding the gene. The vector may be linear or circular, and examples include viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors. Examples of viral vectors include retroviral vectors such as lentiviruses, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, sylvisvirus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors. Examples of plasmid vectors include animal cell expression plasmid vectors such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Of these vectors, retroviral vectors are preferred, and lentiviruses are more preferred, from the viewpoint of increasing the efficiency of gene introduction into macrophages.

[0052] The vector may contain, in addition to the immortalization gene, expression control sequences such as promoters, enhancers, poly-A addition signals, and terminators; nucleotide sequences encoding replication origins and proteins that bind to replication origins and regulate replication; 5' untranslated regions including 5' cap structures, Shine-Dalgano sequences, and Kozak sequences; 3' untranslated regions including polyadenylation signals, AU-rich elements, and GU-rich elements; and nucleotides encoding other proteins. By operably positioning the immortalization gene downstream of the promoter, each polynucleotide can be efficiently transcribed. Examples of such "promoters" include the EF1α promoter, CMV promoter, SRα promoter, SV40 initial promoter, LTR promoter, RSV promoter, HSV-TK promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, metallothionein promoter, and heat shock promoter. Examples of "nucleotides encoding other proteins" include marker genes such as reporter genes and drug resistance genes.

[0053] Furthermore, when introducing multiple types of immortalization genes, these genes may be incorporated into a single vector or into separate vectors, but from the viewpoint of increasing expression efficiency, it is preferable to incorporate them into separate vectors. Also, when incorporating into a single vector, for example, by inserting IRES, 2A peptide sequences, etc. into the vector, it becomes possible to express multiple types of immortalization genes polycistronically.

[0054] Methods for introducing the vector into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun. Furthermore, if the vector of the present invention is a retroviral vector, appropriate packaging cells may be selected based on the LTR sequence and packaging signal sequence of the vector, and retroviral particles may be prepared using these cells. Examples of packaging cells include PG13, PA317, GP+E-86, GP+envAm-12, and Psi-Crip. Additionally, 293 cells or 293T cells, which have high transfection efficiency, can be used as packaging cells. The viral particles thus prepared can then be introduced into cells by methods such as the Polybrene method, Protamine method, and RetroNectin method.

[0055] Immortalized macrophages established by introducing immortalization genes in this manner exhibit proliferative activity for at least one month, preferably for two months or more, more preferably for three months or more, even more preferably for four months or more, and even more preferably for five months or more. The doubling time of immortalized macrophages is at least four days, preferably three days, more preferably two days, and even more preferably one day. Furthermore, it is preferable that immortalized macrophages maintain the characteristics of macrophages. For example, at least one of the macrophage-specific genes Iba1, CD172a, CD203a, CD16, and CD204 (MSR-A) is expressed, preferably two or more genes are expressed, more preferably three or more genes are expressed, even more preferably four or more genes are expressed, and particularly preferably all of these genes are expressed. In addition, immortalized macrophages may express at least one of the marker genes CD163 and CD169, which are specific macrophage subpopulations, and the antigen-presenting cell marker gene MHC-II. Furthermore, the immortalized macrophages according to the present invention retain at least one of the following macrophage characteristics: the ability to produce inflammatory cytokines in response to stimulation by bacterial cell wall components (such as LPS), phagocytic ability, and IL-18 maturation ability associated with inflammasome activity, and preferably two or more functions. Unlike primary cultured macrophages, immortalized macrophages proliferate in a dense, sheet-like manner.

[0056] Furthermore, immortalized macrophages can be prepared by those skilled in the art, with due reference to previously published information. Examples of such previously published information include Takato Takenouchi et al., Front Vet Sci. 2017 Aug 21:4:132., Takato Takenouchi et al., Front Vet Sci. 2022 Jul 18:9:919077., Takato Takenouchi et al., Front Vet Sci. 2022 Nov 18:9:1058124., Japanese Patent Publication No. 2021-61772, and International Publication No. 2022 / 107793.

[0057] (Vaccine composition and method for producing the same) As shown in the examples described below, the modified ASFV of the present invention does not cause pathogenicity such as fever when administered, and does not remain in the body, thus safely conferring immunity to ASF to animals. Therefore, it is suitably used in vaccine compositions against ASF. In other words, the present invention can also provide a vaccine composition against ASF containing the modified ASFV as an active ingredient.

[0058] In the present invention, such a vaccine composition can be prepared by isolating the modified ASFV produced by the method described above from cells and mixing it with a pharmacologically acceptable carrier or medium.

[0059] "Isolation" of modified ASFV means separation, purification, and / or concentration from the above-mentioned cells and / or the culture system of those cells (e.g., culture supernatant). Methods for isolating the virus include, for example, filtration of the culture medium, cell disruption (sonication, hypotonic treatment, freeze-thaw cycle, etc.), centrifugation (ultracentrifugation, density gradient centrifugation, etc.), and concentration (ammonium sulfate, resin column, polyethylene glycol salting-out, etc.).

[0060] The modified ASFV isolated in this manner may be used as a vaccine (a so-called live vaccine) or as a live attenuated form (a so-called live attenuated virus). A live attenuated virus is a virus that has a reduced level of toxicity (pathogenicity, etc.) compared to a virus isolated from the field. Attenuated viruses can be obtained by known methods, such as proliferation in the presence of mutagenic agents, acclimatization to cultured cells by continuous (long-term) passage in vitro, or proliferation under conditions deviating from the natural growth environment (e.g., high-temperature conditions). Live attenuated viruses can also be obtained by deleting or recombining specific genes of the virus using genome editing, gene modification technology, etc.

[0061] Examples of "pharmacologically acceptable carriers" to be mixed with isolated modified ASFV include stabilizers, excipients, preservatives, surfactants, chelating agents, and binders. Examples of "pharmacologically acceptable media" include water, physiological saline, phosphate buffer, and Tris-HCl buffer. Those skilled in the art can appropriately select or combine known carriers and media used in the art, depending on the vaccine dosage form and method of use. Furthermore, there are no particular restrictions on the form of the vaccine; for example, it may be in the form of a suspension or in a lyophilized form.

[0062] From the perspective of enhancing the vaccine's effectiveness, adjuvants may be added. Examples of adjuvants include inorganic substances such as aluminum gel adjuvants, microorganisms or substances derived from microorganisms (BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, cholera toxin, etc.), surfactants (saponins, deoxycholic acid, etc.), emulsions of oily substances (mineral oil, vegetable oil, animal oil, etc.), and alum.

[0063] Furthermore, there are no particular restrictions on the "animals" to which the vaccine composition of the present invention, manufactured in this manner, is administered, as long as they are animals that can be infected with ASFV. Examples include animals of the Suidae family, and more specifically, pigs, wild boars, red river hogs, etc. It may also be livestock, pets, laboratory animals, or animals used for other purposes.

[0064] The vaccine composition of the present invention can confer immunity against ASF to animals when administered via known routes of administration, including intramuscular, subcutaneous, intradermal, intravenous, oral, and nasal. The dosage can be adjusted as appropriate depending on the animal species, age, weight, type of ASFV (e.g., differences in pathogenicity), and method and route of administration. For example, when administered intramuscularly to pigs, the titer of the modified virus of the present invention (50% tissue infectious dose, median tissue culture infectious dose: TCID) may be used. 50 ) is, for example, 10 3 Preferably 10 4 The above is more comfortable 10 5 That concludes the explanation. Furthermore, the drug may be administered multiple times, with an interval of, for example, 1 to 2 weeks between doses. [Examples]

[0065] The present invention will be described more specifically below based on examples and comparative examples, but the present invention is not limited to the following examples. Furthermore, these examples were carried out using the materials and methods shown below.

[0066] <Materials and Methods> 1. Creation of an African swine fever virus strain (AQSΔE120R) lacking the E120R gene. African swine fever virus (ASFV) lacking the nucleotide sequence encoding the full-length E120R protein was created as follows. The isolate ASFV AQS-C-1-22 was used as the ASFV strain. For details on this strain, please refer to Ken-Ichiro Kameyama, Tomoya Kitamura et al., Viruses. 2022 Aug 16;14(8):1794. Immortalized porcine kidney macrophage cell line (IPKM cells) was used as the host cell for amplifying ASFV. For details on this cell line, please refer to Takato Takenouchi et al., Front Vet Sci. 2017 Aug 21:4:132.

[0067] Specifically, to delete the E120R gene of the ASFV strain by replacing it with the copGFP gene using homologous recombination, the p72 promoter of ASFV and the copGFP gene were linked, and the linking sequence was inserted approximately 1000 base pairs before and after the E120R gene. This sequence was then incorporated into a pGEM T easy vector (Promega). (The plasmid prepared in this way is also referred to as "pTEΔE120R / GFP"). This plasmid was lipofected into IPKM cells, and the following day, AQS-C-1-22 was used to induce homologous recombination. One week after viral infection, the culture supernatant was collected, and limiting dilution was performed using GFP fluorescence as an indicator to isolate the E120R gene deletion strain (AQSΔE120R).

[0068] For culturing IPKM cells, Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 10 μg / mL bovine insulin, 25 μM monothioglycerol, and penicillin-streptomycin solution (×100) was used. For culturing IPKM cells, the following incubators (all manufactured by Sumitomo Bakelite Co., Ltd.) were used as appropriate: suspension cell culture plate 6F (MS-8006R), suspension cell culture plate 96F (MS-8096R), suspension cell culture flask 75 (MS-2125R), and suspension cell culture flask 225 (MS-2180R) (the same procedure was followed for IPKM cell culture).

[0069] 2. Virus titer In this example, the titer of viruses such as AQSΔE120R was determined as the 50% tissue culture infectious dose (median tissue culture infectious dose: TCID 50 ). Specifically, first, IPKM cells were seeded in each well of a 96-well plate, and the serially diluted virus solution was inoculated (4 to 8 wells were used for each dilution ratio). One week after inoculation, the wells with a dilution ratio at which virus infection (cytopathic effect: CPE) could be detected were counted, and according to the Behrens-Karber method, TCID 50 was calculated using the following formula. logTCID 50 = D + (h3 + h4 + h5 + ·····) × d + 0.5 × d D: Common logarithm of the highest dilution ratio at which all wells were CPE positive (positive rate = 1) h: CPE positive rate of each well (wells with less than 1) d: Common logarithm of the dilution ratio per one dilution of the sample = 1.

[0070] Each modified virus produced was cultured and propagated in a flask or the like, then dispensed into tubes and stored at -80°C. One of them was thawed, and the above TCID 50 value was calculated. Then, based on this value, the amount of the stored virus was adjusted to the amount of virus actually inoculated into pigs, and used in the following experiments.

[0071] 3. Analysis of the pathogenicity and vaccine effect of AQSΔE120R on pigs 10 1 、10 3 or 10 5 TCID 50 of AQSΔE120R was inoculated intramuscularly into the gluteal muscles of pigs (LWD, mixed sexes) at 6 to 8 weeks of age (n = 6 for each group), and clinical symptoms were observed for 28 days after infection. Furthermore, on the 28th day after infection, the pigs were challenged with 10 2 TCID 50 of the parental strain, and clinical symptoms were observed for 30 days thereafter.

[0072] Furthermore, blood samples were collected periodically, and quantitative detection of the viral gene (p72 gene) by real-time PCR was attempted (see Donald P King et al., J Virol Methods. 2003 Jan;107(1):53-61). Roche's High Pure Viral Nucleic Acid Kit was used for nucleic acid extraction. Additionally, real-time PCR was performed using a concentration series of plasmids (standards) cloned from the amplification products of real-time PCR as a template, and the minimum detectable gene amount was set as the detection limit. As a result, the minimum detectable gene amount was 1 copy per 1 μL, so this was converted to 1 mL (multiplied by 1000), and the detection limit was set to 10 3 (Common logarithm (log10): 3.0) was used.

[0073] 4. Creation of ASFV (AQS E120RΔN, ΔC, or ΔNC) that retain a partial deletion mutant of E120R. In ASFV AQS-C-1-22 and other strains, E120R is a protein consisting of 122 amino acids. We attempted to create ASFVs that carry partial deletion mutants of this protein. Specifically, we attempted to create ASFVs (AQS E120RΔN, ΔC, or ΔNC) that carry genes encoding mutants with 15 amino acids deleted from the N-terminus of E120R (E120RΔN), 18 amino acids deleted from the C-terminus (E120RΔC), or both deleted (E120RΔNC). To do this, we sequentially ligated the nucleotide sequences encoding E120RΔN, ΔC, or ΔNC with the p72 promoter of ASFV and the mCherry gene. Then, the sequence in which the binding sequence was inserted approximately 1000 base pairs before and after the E120R gene was incorporated into a pGEM T easy vector (each plasmid prepared in this way is also referred to as pTE E120RΔN, ΔC, or ΔNC). These plasmids were lipofected into IPKM cells, and the following day, AQSΔE120R was used to induce homologous recombination. One week after viral infection, the culture supernatant was collected and isolated by limiting dilution using mCherry fluorescence as an indicator.

[0074] Figure 1 shows an overview of the modified E120R gene in AQS E120RΔN, ΔC, and ΔNC, as well as the E120R gene in their parent strain, ASFV AQS-C-1-2.

[0075] 5. Analysis of the pathogenicity of AQS E120RΔN, ΔC, or ΔNC 10 2 TCID 50 AQS E120RΔN, ΔC, or ΔNC were inoculated intramuscularly into the rump muscles of 3-5 day old pigs (LWD, mixed sexes) (n=2 for each), and clinical symptoms were observed for 7 days from infection.

[0076] Furthermore, blood, gastrohepatic lymph nodes, and spleen samples were collected on day 7 of infection. The gastrohepatic lymph nodes and spleen were ground and mixed with a solvent (PBS) to create 10% (mass percentage) emulsions. 200 μL of each emulsion was then used for nucleic acid extraction, and the resulting purified nucleic acids were used as templates for quantitative detection of the viral gene (p72 gene) by real-time PCR (see Donald P King et al., 2003). The Roche High Pure Viral Nucleic Acid Kit was used for nucleic acid extraction. Additionally, real-time PCR was performed using a concentration series of plasmids (standards) cloned from the amplification products of real-time PCR as templates, and the minimum detectable gene amount was set as the detection limit. As a result, the minimum detectable gene amount was determined to be 1 copy per 1 μL. Therefore, for blood, the detection limit was set as 1 (common logarithm (log10): 0), while for gastric, hepatic, and lymph nodes and the spleen, the detection limit was set to 10 (common logarithm (log10): 1.0) by converting to a 100% emulsion (multiplying by 10).

[0077] 6. Analysis of the pathogenicity of AQS E120RΔNC 10 2 TCID 50AQS E120RΔNC was inoculated intramuscularly into the rump muscles of 6-8 week old pigs (LWD, mixed sexes) to infect them (n=4). Clinical symptoms were observed for 21 days after infection, and blood samples were collected regularly. The viral gene was then detected in all samples using the same method as described above.

[0078] 7. Creation of ASFV (AQS E120RΔ72-73) that retains a two-amino acid deletion mutant of E120R. To create an ASFV (AQS E120RΔ72-73) carrying a mutant (E120RΔ72-73) lacking amino acids 72 and 73 of the E120R protein, the nucleotide sequence encoding E120RΔ72-73, the ASFV p72 promoter, and the mCherry gene were sequentially ligated. This ligation sequence was then inserted approximately 1000 base pairs before and after the E120R gene, and the resulting sequence was incorporated into a pGEM T easy vector (the plasmid thus prepared is also referred to as "pTE E120RΔ72-73"). IPKM cells lipofected with this plasmid were infected with AQSΔE120R to induce homologous recombination. One week after viral infection, the culture supernatant was collected and isolated by limiting dilution using mCherry fluorescence as an indicator.

[0079] Table 1 shows the sequence numbers of the various sequences used in the creation of the modified ASFV described above. Note that in this table, for proteins, both the amino acid sequence and the nucleotide sequence (cDNA sequence) encoding it are also shown.

[0080] [Table 1]

[0081] 8. Analysis of the pathogenicity of AQS E120RΔ72-73 10 2 TCID 50AQS E120RΔ72-73 was inoculated into the hind muscles of 3-5 day old pigs (LWD, mixed sexes) to infect them (n=2). Clinical symptoms were observed for 7 days from the time of infection.

[0082] Furthermore, blood, gastrohepatic lymph nodes, and spleen were collected on day 7 of infection. The gastrohepatic lymph nodes and spleen were each ground up, and a solvent (PBS) was added to prepare a 10% (mass percentage) emulsion. 200 μL of each emulsion was then used for nucleic acid extraction, and the resulting purified nucleic acid was used as a template to attempt quantitative detection of the viral gene (p72 gene) by real-time PCR (see Donald P King et al., 2003). Roche's High Pure Viral Nucleic Acid Kit was used for nucleic acid extraction. Additionally, real-time PCR was performed using a concentration series of plasmids (standards) cloned from the amplification products of real-time PCR as a template, and the minimum detectable gene amount was set as the detection limit. As a result, the minimum detectable gene amount was determined to be 1 copy per 1 μL. Therefore, for blood, the detection limit was set as 1 (common logarithm (log10): 0), while for gastric, hepatic, and lymph nodes and the spleen, the detection limit was set to 10 (common logarithm (log10): 1.0) by converting to a 100% emulsion (multiplying by 10).

[0083] The results obtained using the above materials and methods are shown below.

[0084] (Example 1) Creation and analysis of ASFV lacking the E120R gene As shown in Figure 2a, IPKM cells introduced with pTEΔE120R / GFP were infected with the ASFV AQS-C-1-22 strain to induce homologous recombination. Approximately one week after the induction of homologous recombination, the culture supernatant was subjected to the limiting dilution method. The cells isolated by limiting dilution were then cultured for about 14 days. As a result, as shown in Figure 2b, AQSΔE120R was successfully produced.

[0085] Next, 10 1 , 10 3 Or 105 TCID 50 Pigs were inoculated with AQSΔE120R or its parent strain (ASFV AQS-C-1-22 strain). As shown in Figure 3A, the pigs inoculated with the parent strain (control) developed fever, and all individuals died or were euthanized. On the other hand, in pigs inoculated with AQSΔE120R, no deaths occurred at any immunosuppression level (viral titer), and no individuals showed clear fever.

[0086] Furthermore, blood samples were collected regularly during the observation period and analyzed for the presence or absence of viral genes. As shown in Figure 3B, unlike the control group, the aforementioned gene was not detected at any immunization level in pigs inoculated with AQSΔE120R. In other words, it was clear that AQSΔE120R does not cause viremia.

[0087] Furthermore, 28 days after receiving AQSΔE120R, 10 2 TCID 50 The parent plant was used for the attack. As a result, as shown in Figure 4A, 10 1 In the AQSΔE120R vaccinated group, all 6 pigs died, but 10 3 One animal survived in the AQSΔE120R vaccinated group, and 10 5 Four pigs survived in the AQSΔE120R vaccinated group. This indicates that the vaccine effect was proportional to the amount of immunity received by the AQSΔE120R. Furthermore, as shown in Figure 4B, fever and viremia were observed in all vaccinated pigs after the attack, but the severity of these symptoms decreased with higher immunity levels.

[0088] (Example 2, Comparative Examples 1 and 2) Creation and analysis of ASFVs carrying a partial deletion mutant of E120R IPKM cells introduced with pTE E120RΔN, ΔC, or ΔNC were infected with AQSΔE120R to induce homologous recombination. Approximately one week after induction of homologous recombination, the culture supernatant was subjected to limiting dilution. After culturing for approximately 7 days, AQS E120RΔN, ΔC, and ΔNC were successfully isolated using mCerry fluorescence as an indicator.

[0089] Next, these are 10 2 TCID 50 The vaccine was administered to pigs. As a result of inoculating the pigs with AQS E120RΔN, no clear fever was observed in any of the individuals, as shown in Figure 5A. However, on the last day of the observation period, blood, gastric and hepatic lymph nodes, and spleen were collected and analyzed for the presence or absence of viral genes, and as shown in Figure 5B, viral genes were detected in one out of two pigs.

[0090] Furthermore, as shown in Figure 5A, all pigs inoculated with AQS E120RΔC exhibited clear fever and died within the observation period. Gastric, hepatic, and spleen samples were collected at the time of death, and analysis for the presence of viral genes revealed the presence of viral genes in all samples.

[0091] On the other hand, the pigs inoculated with AQS E120RΔNC did not show any clear fever, as shown in Figures 5A and 6A. Furthermore, blood, gastric and hepatic lymph nodes, and spleen were collected on the last day of the observation period and analyzed for the presence or absence of viral genes. As shown in Figure 5B, no viral genes were detected at all. In addition, blood was collected regularly throughout the observation period and analyzed for the presence or absence of viral genes. As shown in Figure 6B, no viral genes were detected at all.

[0092] Thus, when either the N-terminal or C-terminal region of the E120R protein was deleted, the modified ASFV remained in the inoculated pigs. On the other hand, as shown by the analysis results of AQS E120RΔNC and AQSΔE120R, when both the N-terminal and C-terminal regions were deleted, no persistence occurred in the pigs, including viremia. Furthermore, no pigs died at any of the immunization levels.

[0093] (Comparative Example 3) Creation and analysis of ASFV possessing a two-amino acid deletion mutant of E120R As described above, we created an ASFV that possesses a two-amino acid deletion mutant of E120R. 2 TCID50 Two pigs were inoculated, and although not shown in the figure, one of them died on the fourth day after inoculation. The remaining pig showed lethargy, decreased appetite, and diarrhea on the fifth day, and was euthanized from an animal welfare perspective. Furthermore, the spleen and gastric and hepatic lymph nodes were collected during necropsy (blood was also collected only from the euthanized pigs), and the presence or absence of viral genes was analyzed. As shown in Figure 7, viral genes were detected in all of the materials.

[0094] Furthermore, according to Non-Patent Literature 2, it has been revealed that when a modified ASFV in which the 72nd and 73rd amino acids of E120R are deleted is inoculated, interferon-β production is improved compared to the parent strain, meaning that the host's antiviral response can be activated.

[0095] However, deleting these two amino acids from the E120R protein did not reduce the pathogenicity of ASFV, and pigs inoculated with this modified ASFV were lethal. On the other hand, as described above, no lethal pigs were observed when inoculated with the modified ASFV of the present invention (AQS E120RΔNC and AQSΔE120R, etc.), indicating that pathogenicity was suppressed. [Industrial applicability]

[0096] As described above, the present invention makes it possible to provide an African swine fever virus that does not remain in the body of an inoculated animal and exhibits a vaccine effect against African swine fever. In particular, the present invention allows for the safe administration of immunity to African swine fever to animals without causing pathogenicity such as fever, and without the virus remaining in the body. Therefore, the present invention is useful as a vaccine against African swine fever.

Claims

1. In the genome of African swine fever virus, (1) In the nucleotide sequence encoding the E120R protein, only the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 2 to 16 from the amino terminus as described in SEQ ID NO: 2, and the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 1 to 18 from the carboxy terminus as described in SEQ ID NO: 2 are deleted. The aforementioned African swine fever virus is strain ASFV AQS-C-1-22. The E120R protein is a protein consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence described in SEQ ID NO:

2. Modified African swine fever virus.

2. A modified African swine fever virus is contained as an active ingredient, The modified African swine fever virus, in the genome of the African swine fever virus, (1) In the nucleotide sequence encoding the E120R protein, only the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 2 to 16 from the amino terminus as described in SEQ ID NO: 2 and the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 1 to 18 from the carboxy terminus as described in SEQ ID NO: 2 are deleted, or (2) Only the nucleotide sequence encoding the full length of the E120R protein is deleted. The aforementioned African swine fever virus is strain ASFV AQS-C-1-22. The E120R protein is a protein consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence described in SEQ ID NO:

2. A vaccine composition for African swine fever.

3. A method for producing a modified African swine fever virus, In cells infected with African swine fever virus, homologous recombination occurs in the genome of the virus, (1) In the nucleotide sequences encoding the E120R protein, only the nucleotide sequences encoding the region consisting of amino acids corresponding to positions 2 to 16 from the amino terminus as described in SEQ ID NO: 2 and the nucleotide sequences encoding the region consisting of amino acids corresponding to positions 1 to 18 from the carboxy terminus as described in SEQ ID NO: 2 The process includes deleting a component to obtain the modified African swine fever virus described in claim 1, The aforementioned African swine fever virus is strain ASFV AQS-C-1-22. The E120R protein is a protein consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence described in Sequence ID No.

2. The aforementioned cells are immortalized macrophage cells derived from pig kidney. method.

4. A method for producing a vaccine composition against African swine fever, In cells infected with African swine fever virus, homologous recombination occurs from the genome of the virus, (1) In the nucleotide sequence encoding the E120R protein, delete only the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 2 to 16 from the amino terminus as described in SEQ ID NO: 2, and the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 1 to 18 from the carboxy terminus as described in SEQ ID NO: 2, or (2) Delete only the nucleotide sequence that codes for the full length of the E120R protein, The process for producing modified African swine fever virus, and, The process of mixing the modified African swine fever virus with a pharmacologically acceptable carrier or medium. Includes, The aforementioned African swine fever virus is strain ASFV AQS-C-1-22. The E120R protein is a protein consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence described in Sequence ID No.

2. A method wherein the cells are immortalized macrophage cells derived from porcine kidney.

5. A method for producing a modified African swine fever virus, The modified African swine fever virus described in claim 1 is used to infect cells and propagate, A method for producing the aforementioned cells, wherein the cells are immortalized macrophage cells derived from porcine kidney.

6. A method for producing a vaccine composition against African swine fever, The process of infecting cells with a modified African swine fever virus and propagating it, and The process of mixing the replicated modified African swine fever virus with a pharmacologically acceptable carrier or medium. Includes, The modified African swine fever virus, in the genome of the African swine fever virus, (1) In the nucleotide sequence encoding the E120R protein, only the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 2 to 16 from the amino terminus as described in SEQ ID NO: 2 and the nucleotide sequence encoding the region consisting of amino acids corresponding to positions 1 to 18 from the carboxy terminus as described in SEQ ID NO: 2 are deleted, or (2) Only the nucleotide sequence encoding the full length of the E120R protein is deleted. The aforementioned African swine fever virus is strain ASFV AQS-C-1-22. The E120R protein is a protein consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence described in Sequence ID No.

2. A method wherein the cells are immortalized macrophage cells derived from porcine kidney.

Images