Modified african swine fever virus and vaccine using 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

Modified African swine fever virus and vaccine using the same

[0001] The present invention relates to a modified African swine fever virus and a vaccine using the same.

[0002] African swine fever (ASF) is an infectious disease of Suidae animals (pigs, wild boars, etc.) caused by the African swine fever virus (ASFV) and has a high mortality rate. Originally, ASFV is found only on the African continent, but it has accidentally spread from the continent through the movement of people and goods between continents. Currently, ASF is prevalent outside of Africa, mainly in Eastern Europe and Asia.

[0003] A vaccine against ASF is considered one way to bring the disease to an end. Recently, a vaccine using a genetically modified attenuated ASFV strain was approved in Vietnam and is being used in the country as the world's first ASF vaccine. While pigs immunized with this vaccine strain have high vaccine efficacy in preventing the onset of virulent strains, the long-term persistence of the vaccine strain in pigs has raised safety concerns. Therefore, there is a strong demand for the development of a virus that does not persist in the bodies of vaccinated animals and can confer protection against ASF.

[0004] Regarding such viruses that do not persist in pigs, Sanford et al. have reported that they created ASFV-G / VP-ΔTK, in which the thymidine kinase (TK) gene was deleted, and that inoculation of this virus with the virus did not result in a state in which the virus persisted in the blood (viremia) (the virus was below the detection limit) (Non-Patent Document 1). However, pigs immunized with this strain were completely unable to protect against challenge with a highly virulent strain. Thus, there was a problem in that a virus that did not cause any viremia lost its protective effect as a vaccine.

[0005] B Sanford et al., Virus Res. , February 2, 2016, 213:165-171. Huisheng Liu et al., J Virol. , August 25, 2021, 95(18): e0082421. Edward Spinard et al., Microbiol Resource Announcement. , December 15, 2022, 11(12): e0088122.

[0006] The present invention has been made in consideration of the problems associated with the prior art, and aims to provide an African swine fever virus that does not remain in the body of an animal after inoculation and that has a vaccine effect against African swine fever.

[0007] As a result of extensive research aimed at achieving the above-mentioned objective, the present inventors have succeeded in producing an African swine fever virus (AQSΔE120R) in which the nucleotide sequence encoding the full-length E120R protein has been deleted. They have also demonstrated that when pigs are inoculated with this modified ASFV, the virus concentration in the blood is extremely low, below the detection limit, unlike when the parent strain is inoculated. Furthermore, they have found that inoculation with AQSΔE120R does not cause fever or death. On the other hand, they have also demonstrated that when pigs inoculated with AQSΔE120R are challenged with the parent strain, a vaccine effect is achieved.

[0008] The present inventors also succeeded in producing ASFVs (AQS E120RΔN, AQS E120RΔC, and AQS E120RΔNC) that retain the E120R protein with deletions of the N-terminal and / or C-terminal regions, and analyzed their pathogenicity and their concentrations in the bodies of 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. Furthermore, pigs inoculated with AQS E120RΔN did not develop fever or other symptoms, but tended to remain in the spleen and gastrohepatic lymph nodes. On the other hand, in pigs inoculated with AQS E120RΔNC, no fever or other symptoms were observed, and it was also revealed that the presence of the modified ASFV in the 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 in which the nucleotide sequence encoding the amino-terminal region (N-terminal region) and the nucleotide sequence encoding the carboxy-terminal region (C-terminal region) of the E120R protein are deleted in the genome.

[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 of the 10th to 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 of the 10th to 30th amino acids from the C-terminus of the E120R protein.

[0012] [3] The modified African swine fever virus according to [1], wherein the nucleotide sequence encoding the full-length E120R protein is deleted in the genome.

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

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

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

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

[0017] [8] The method according to any one of [5] to [7], wherein the cells are immortalized macrophages of a boar.

[0018] [9] A method for producing a vaccine composition against African swine fever, comprising the steps of 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 pharmacologically acceptable carrier or medium.

[0019]

[10] A method for producing a modified African swine fever virus, comprising the steps of infecting a cell with the modified African swine fever virus according to any one of [1] to [3] and allowing the virus to grow.

[0020]

[11] The production method according to

[10] , wherein the cells are immortalized macrophages of a boar.

[0021]

[12] A method for producing a vaccine composition against African swine fever, comprising the steps of producing a modified African swine fever virus by the production method according to

[10] or

[11] , and mixing the modified African swine fever virus with a pharmacologically acceptable carrier or medium.

[0022] The E120R protein of ASFV encodes a gene involved in viral budding and immune control. More specifically, the E120R protein suppresses the host's antiviral response by inhibiting interferon-β production, and it has been shown that the 72nd and 73rd amino acids are essential for this suppressive function. Furthermore, it has been shown that inoculation of a modified ASFV lacking these two amino acids results in improved interferon-β production compared to the parent strain, thereby activating the host's antiviral response (Non-Patent Document 2).

[0023] However, as shown in the Examples below, when the 72nd and 73rd amino acids of the E120R protein were deleted, the pathogenicity of ASFV could not be reduced, and pigs inoculated with the modified ASFV became lethal.

[0024] On the other hand, in the modified ASFV of the present invention, the deletions in the E120R protein are in the N-terminal region and the C-terminal region, which differs from the deletion targets in the E120R protein in Non-Patent Document 2. Furthermore, as will be shown in the Examples below, no pigs were found to be lethal when inoculated with the modified ASFV of the present invention, demonstrating that pathogenicity is suppressed.

[0025] Furthermore, several research groups have attempted to create a strain in which the E120R gene is completely deleted, but have not been successful (Non-Patent Documents 2 and 3). However, as shown in the Examples below, the present inventors have succeeded in creating a strain in which the E120R gene is completely deleted, and have also demonstrated its effectiveness.

[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 that has a vaccine effect against African swine fever. In particular, according to the present invention, even if inoculated, the virus does not cause pathogenicity such as fever, and furthermore, the virus does not remain in the body, and therefore it is possible to confer immunity against African swine fever in animals with high safety.

[0027] This figure shows an outline of the E120R gene in African swine fever virus (ASFV) AQS-C-1-2, and an outline of the E120R gene modified in AQSΔE120R, AQS E120RΔN, AQS E120RΔC, and AQS E120RΔNC, which are derived from AQS-C-1-2 as a parent strain. This figure shows an outline of the genome of modified ASFV (AQSΔE120R) and the results of observing the modified ASFV under a fluorescence microscope. In the figure, "a" shows that the E120R gene was replaced with the copGFP gene by homologous recombination in the genome of the parent strain (ASFV AQS-C-1-2), resulting in the deletion of the former. "b" shows that AQSΔE120R was produced, as GFP-derived fluorescence indicating expression of the copGFP gene was detected. 1 is a graph showing the results of measuring the body temperature over time of pigs inoculated with a modified ASFV (AQSΔE120R) in which the nucleotide sequence encoding the full-length E120R protein has been deleted. １ , 10 ３ or 10５ TCID ５０ "Inoculated group" shows the results of a group of pigs inoculated (infected) with each immunization dose (virus titer) shown in each item. "AQS-C-1-2 inoculated group" shows the results (control) of a group of pigs inoculated (infected) with ASFV AQS-C-1-2, the parent strain of AQSΔE120R. Each broken line shows the measurement results for each individual pig. However, the solid line at 40°C indicates the pig's normal body temperature (upper limit). This graph shows the results of measuring the viral gene quantity in the blood of pigs inoculated with AQSΔE120R over time. In the figure, 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, multiplied by 1000, and then converted to a common logarithm (log10), which represents the gene quantity per 1 mL of each sample. Other item notations and broken lines are the same as in Figure 3A. The broken lines with daggers indicate the results of individuals that died or were euthanized, as in Figure 3A. However, the broken line of 3.0, which indicates the blood viral gene load, indicates the measurement detection limit. 28 days after inoculation with AQSΔE120R, 10 ２ TCID ５０ 3A is a graph showing the results of measuring the body temperature of pigs inoculated with (challenged with) the parent strain of AQSΔE120R over time. The item notations, broken lines, and daggers in the figure are the same as those in FIG. 3A. However, the solid line at 40°C indicates the normal body temperature (upper limit) of the pigs. 28 days after inoculation with AQSΔE120R, the body temperature of 10 pigs was ２ TCID ５０3B. The dashed line at 3.0 indicates the detection limit for the measurement. This graph shows the results of measuring the body temperature over time of pigs inoculated (infected) with ASFV harboring a partial deletion mutant of E120R (AQS E120RΔN, AQS E120RΔC, or AQS E120RΔNC). The lines and daggers in the graph are the same as those in FIG. 3A. The solid line at 40°C indicates the pig's normal body temperature (upper limit). This graph shows the results of measuring the viral gene quantity in blood, gastrohepatic lymph nodes, or spleen collected from pigs 7 days after inoculation with AQS E120RΔN, AQS E120RΔC, or AQS E120RΔNC. 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, expressed as the gene quantity per μL of each sample. "Pigs 1 to 7" show the measurement results for each individual pig. The solid line representing a viral gene quantity of 1.0 indicates the detection limit for viral gene measurement in the gastrohepatic lymph nodes and spleen, and the solid line representing a viral gene quantity of 0 indicates the detection limit for viral gene measurement in blood. This graph shows the results of measuring the body temperature over time of pigs inoculated (infected) with AQS E120RΔNC. The broken lines in the figure are the same as those in Figure 3A. This graph shows the results of measuring the viral gene quantity over time in the blood of pigs inoculated (infected) with AQS E120RΔNC. In the figure, "1-4" indicate the measurement results for each individual pig. The broken lines are the same as in Figure 3B. However, the dashed line at 3.0, which indicates the blood viral gene quantity, indicates the detection limit. This graph shows the results of measuring the viral gene quantity in the blood, stomach, liver, lymph nodes, or spleen of pigs inoculated with ASFV (AQS E120RΔ72-73) harboring a mutant in which the 72nd and 73rd amino acids of the E120R protein are deleted, and then collected at necropsy. In the figure, "pig1-2" indicate the measurement results for each individual pig. The vertical axis indicates the absolute quantitative value obtained by real-time PCR using purified nucleic acid from 1 μL of sample as a template, expressed as the gene quantity per μL of each sample.The solid line representing a viral gene amount of 1.0 indicates the detection limit for viral gene measurement in the stomach, liver, lymph nodes and spleen, and the solid line representing a viral gene amount of 0 indicates the detection limit for viral gene measurement in blood.

[0028] As shown in the Examples below, the present inventors have demonstrated that an African swine fever virus carrying the E120R protein in which the N-terminal and C-terminal regions have been deleted confers a vaccine effect against African swine fever on inoculated animals, while not being pathogenic, and further that the presence of this modified ASFV in the bodies (blood, spleen, and gastrohepatic lymph nodes) of inoculated pigs is extremely low, below the detection limit.

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

[0030] (African Swine Fever Virus) In the present invention, "African Swine Fever Virus (Asfarviridae Asfivirus, ASFV)" refers to a virus of the genus Asfivirus in the family Asfarviridae, which has double-stranded DNA in its genome. In the present invention, the ASFV to be subjected to the E120R gene modification described below is not particularly limited, and may be, for example, any of the 24 reported genotypes (type II, type I, type X, etc.). Furthermore, the pathogenicity (for example, highly virulent or attenuated, or peracute, acute, subacute, chronic, or subapparent) does not matter, and the virus may be of either a field type or an adapted type.

[0031] (Modifications in E120R) In the present invention, the "E120R" to be modified is a structural protein expressed (transcribed) in ASFV in the late stage of its replication cycle, and is also referred to as p14.5. E120R is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (a protein consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1). However, nucleotide sequences can naturally mutate. Accordingly, 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 above-mentioned typical sequence, but also includes natural variants (such as homologs). Examples of such natural variants include, but are not limited to, proteins comprising an amino acid sequence having 80% or more homology or identity to the amino acid sequence set forth in SEQ ID NO: 1. More specifically, proteins identified by UniProtKB P0C9Y5·P14_ASFK5 or Q65201·P14_ASFB can be mentioned. Furthermore, the homology or identity here 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" refers to the proportion of sites where the amino acid types are identical between the amino acid sequences being compared, and "homology" refers to the proportion of sites where similar amino acids are identical, plus the proportion of sites where similar amino acids are identical. 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 an amino acid sequence by BLASTP, the parameters are set, for example, as score=50 and wordlength=3.Furthermore, when analyzing amino acid sequences using the Gapped BLAST program, the method can be performed as described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

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

[0033] On the other hand, examples of the "carboxy-terminal region (C-terminal region)" to be deleted include a region consisting of the amino acid sequence from the 5th to 50th amino acid from the C-terminus, a region consisting of the amino acid sequence from the 6th to 40th amino acid from the C-terminus, a region consisting of the amino acid sequence from the 8th to 30th amino acid from the C-terminus, and a region consisting of the amino acid sequence from the 10th to 20th amino acid from the C-terminus. More specifically, examples include a region consisting of the amino acid sequence from the C-terminus to the 50th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 49th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 45th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 40th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 35th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 30th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 25th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 20th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 18th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 15th amino acid, a region consisting of the amino acid sequence from the C-terminus to the 10th amino acid, and a region consisting of the amino acid sequence from the C-terminus to the 5th amino acid. The position (number) of an amino acid in the C-terminal region is determined by the amino acid number assigned in ascending order toward the N-terminus, starting with the C-terminus, i.e., the final amino acid encoded by the codon immediately preceding the termination codon (for example, lysine at position 122 in the amino acid sequence of SEQ ID NO: 1).

[0034] Furthermore, the positions (numbers) of amino acids in the N-terminal region and C-terminal region are the amino acid numbers based on the amino acid sequence set forth in SEQ ID NO: 1. In the case of the naturally occurring mutant of E120R, these should be read as the amino acids corresponding to each site set forth in SEQ ID NO: 1 (for example, "a region consisting of the amino acid sequence from the N-terminus to the 10th to 30th amino acids" in SEQ ID NO: 1 should be read as "a region consisting of the amino acid sequence from the N-terminus to the amino acids corresponding to the 10th to 30th amino acids set forth in SEQ ID NO: 1" in the case of the naturally occurring mutant of E120R). Note that "corresponding" means that when the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence of the naturally occurring mutant of E120R are aligned (subjected to alignment) using nucleotide and amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), they are aligned in the same order.

[0035] Furthermore, as will be shown in the Examples below, in the present invention, the deletion of the N-terminal region and C-terminal region of E120R may be the deletion of the entire length of E120R. In this case, the amino acids to be deleted may also include those encoded by the initiation codon.

[0036] Furthermore, as shown in the examples below, a modified ASFV having an E120R protein lacking such an N-terminal region and a C-terminal region does not exhibit pathogenicity such as fever when inoculated, and furthermore, does not remain in the body, making it highly safe and capable of conferring immunity to animals against African swine fever.

[0037] In the present invention, "pathogenic" refers to the property of causing symptoms due to ASFV infection, and "non-pathogenic" refers to, for example, a state in which fever, one of the symptoms due to ASFV infection, is not observed for one week after inoculation with the modified ASFV of the present invention. Here, "fever" refers to a state in which an animal's body temperature exceeds 1°C above its normal body temperature; for example, as shown in the Examples below, a body temperature exceeding 41°C in pigs (whose normal body temperature is less than 40°C) is considered to be feverish.

[0038] Furthermore, "does not remain in the body" means that one week or more after inoculation with the modified ASFV of the present invention, the gene derived from the ASFV is below the detection limit in at least one tissue in the body. Among such tissues in the body, the tissue in which the gene derived from the modified ASFV of the present invention is below the detection limit is preferably at least the blood, and more preferably at least the blood, spleen, and gastrohepatic lymph nodes. The gene derived from the modified ASFV of the present invention is not particularly limited, but is preferably the p72 gene, and more preferably below the detection limit in real-time PCR targeting the gene. More specifically, for example, the real-time PCR can be performed using a concentration series of a standard (e.g., plasmid DNA obtained by cloning the amplification product in the real-time PCR) as a template, and the lowest detectable concentration can be set as the detection limit.

[0039] "Providing immunity against African swine fever" means that an animal inoculated with the modified ASFV of the present invention is provided with humoral immunity and / or cellular immunity against African swine fever, thereby enabling at least one prevention selected from prevention of ASFV infection, prevention of onset due to ASFV infection, and prevention of aggravation 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 ASFV in a cell infected with the ASFV.

[0041] For example, knockout techniques utilizing homologous recombination can be used for such "nucleotide sequence deletion," as shown in the Examples below. In knockout techniques, a targeting DNA construct having a sequence homologous to at least a portion of a target gene region (in the present invention, the E120R gene region) is introduced into a cell, as described below, so that homologous recombination occurs between the target gene region and the target gene region. The targeting DNA construct typically has a structure in which DNA consisting of a sequence homologous to the sequence of the target DNA site 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 in which the sequences are completely (i.e., 100%) identical, but also cases in which the sequences differ partially, as long as homologous recombination occurs. Typically, the sequences are at least 95% identical, preferably 97% identical, and more preferably 99% identical. The length of these arms may be any number sufficient to induce homologous recombination, for example, 200 to 3,000 nucleotides, preferably 300 to 2,000 nucleotides, and more preferably 500 to 1,000 nucleotides. When a target gene region on the genome interacts with a homologous sequence on the targeting DNA construct, a specific sequence in the target gene region is exchanged with DNA on the targeting DNA construct, thereby deleting a portion or the entire target gene. The genome sequence of ASFV is known (e.g., the nucleotide sequence listed in GenBank: OR660089.1). In this sequence, the nucleotide sequence encoding the E120R protein consists of nucleotides 167,839 to 168,207. Therefore, a person skilled in the art can appropriately design the homologous sequence based on this sequence information and induce homologous recombination. Furthermore, in preparing a targeting DNA construct, for example, as shown in the Examples below, DNA in which a marker gene (a fluorescent protein gene such as the GFP gene or the mCherry gene) has been inserted into a cloned target gene can be used.Furthermore, the targeting DNA construct may be a linear DNA or a circular DNA.

[0042] In the present invention, to induce homologous recombination, for example, the targeting DNA construct is first introduced into the cells described below. There are no particular limitations on the method of introduction, and any known gene transfer method can be used as appropriate. Known gene transfer methods include, for example, lipofection, electroporation, calcium phosphate, DEAE-dextran, microinjection, and particle gun methods.

[0043] Next, the cells into which the targeting DNA construct has been introduced are infected with the above-mentioned ASFV. Infection of cells with ASFV can be achieved, for example, by adding ASFV to a cell culture system (e.g., medium) and contacting the cells with the ASFV. The timing of infection is not particularly limited, and examples include 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or the next day after the introduction of the targeting DNA construct. The culture period after infection (e.g., contact) of cells with ASFV is not particularly limited, as long as it is a period during which homologous recombination can occur, and examples include 3 days to 3 weeks, or 1 to 2 weeks. Furthermore, the "culture medium" for ASFV infection is not particularly limited, as long as it is a medium that can support the cells described below, and can be prepared by appropriately adding well-known and commonly used medium additives to a known basal medium. Examples of "basal media" include Dulbecco's modified Eagle's medium (DMEM medium), DMEM medium (high glucose), DMEM medium (low glucose), RPMI 160 medium, RPMI 1640 medium, Ham's F12 medium, KSOM medium, Eagle's MEM medium, Glasgow's MEM medium, αMEM medium, Ham's medium, Fisher's medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc Option Improved Medium, IMDM medium, Medium 199 medium, and any mixed medium thereof.Examples of "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, rhodopsin, etc.), and the like. Examples of suitable organic compounds include, but are not limited to, ferric nitrate, iron(II) 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 (selenious acid), organic compounds (para-aminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine, pyruvic acid, 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 to 42°C, preferably 37°C. The carbon dioxide concentration in the gas in contact with the medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume.

[0044] Next, ASFV resulting from homologous recombination is isolated from the culture of cells infected with ASFV. Such isolation can be achieved, for example, by subjecting the cell culture (e.g., culture supernatant) to limiting dilution to purify ASFV, as shown in the Examples below. Furthermore, when a targeting DNA construct containing a marker gene within the target gene is used, ASFV can also be isolated using expression of the marker gene as an indicator.

[0045] Furthermore, the modified ASFV isolated in this manner can be propagated by contacting it again with the cells described below and culturing the infected cells. Culture conditions (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 (e.g., 18 to 24 days, 19 to 23 days, 20 to 22 days, or 21 days).

[0046] Those skilled in the art can determine whether the modified ASFV has proliferated using known methods. Such methods include, for example, methods for detecting genes derived from ASFV or their expression. Here, gene expression may be at the transcription level (mRNA level) or translation 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, gene or mRNA levels can be quantitatively detected by counting the number of reads in so-called next-generation sequencing. Furthermore, methods for detecting proteins include, for example, antibody-based detection methods (immunological techniques) 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 a cytopathic effect (CPE) test using CPE as an indicator, and a hemadsorption (HAD) test using the HAD reaction, which is specifically observed in ASFV-infected cells, as an indicator.

[0047] In the present invention, the "cells" used to produce and further propagate the modified ASFV are not particularly limited as long as they are cells that can be infected with ASFV, and examples thereof include macrophages. The origin of the macrophages is not particularly limited, but they are preferably derived from animals of the Suidae family. Furthermore, the macrophages may be primary cultured cells, or immortalized cells can be used. In the present invention, immortalized macrophages from animals of the Suidae family are preferably used.

[0048] (Cells derived from immortalized macrophages of boars) In the present invention, the term "boars" refers to animals belonging to the order Cetacea, suborder Suidae, family Suidae, class Mammalia, including animals belonging to the genus Sus (such as wild boars and their domesticated species, pigs) and animals belonging to the genus Potamochoerus (such as red river boars).

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

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

[0051] 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 thereof include viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors. Examples of viral vectors include retroviral vectors such as lentivirus, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors. Examples of plasmid vectors include plasmid vectors for expression in animal cells, such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Among these vectors, retroviral vectors are preferred, and lentiviruses are more preferred, from the viewpoint of increasing the efficiency of gene transfer into macrophages.

[0052] In addition to the immortalization gene, the vector may contain expression control sequences such as a promoter, enhancer, poly(A) addition signal, and terminator; a replication origin or a nucleotide sequence encoding a protein that binds to the replication origin and controls replication; a 5' untranslated region including a 5' cap structure, a Shine-Dalgarno sequence, a Kozak sequence, and the like; a 3' untranslated region including a polyadenylation signal, an AU-rich element, a GU-rich element, and the like; nucleotides encoding other proteins; and the like. The immortalization gene can be operably positioned downstream of the promoter to efficiently transcribe each polynucleotide. Examples of such "promoters" include the EF1α promoter, CMV promoter, SRα promoter, SV40 early 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] When multiple immortalization genes are introduced, 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. When incorporating them into a single vector, for example, by inserting an IRES, a 2A peptide sequence, or the like into the vector, it becomes possible to express multiple immortalization genes in a polycistronic manner.

[0054] Methods for introducing the vector into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun methods. Furthermore, when 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 contained in 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. Furthermore, 293 cells and 293T cells, which have high transfection efficiency, can also be used as packaging cells. Furthermore, viral particles prepared in this manner can be introduced into cells using the Polybrene method, the Protamine method, the RetroNectin method, and the like.

[0055] Immortalized macrophages established by introducing an immortalizing gene in this manner exhibit a proliferation rate of at least one month, preferably two months or more, more preferably three months or more, even more preferably four months or more, and even more preferably 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 the immortalized macrophages maintain macrophage characteristics. For example, at least one gene selected from 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. Furthermore, the immortalized macrophages may express at least one gene selected from CD163 and CD169, which are marker genes for specific macrophage subpopulations, and MHC-II, which is an antigen-presenting cell marker gene. Furthermore, the immortalized macrophages according to the present invention retain at least one of the following macrophage properties: the ability to produce inflammatory cytokines in response to stimulation by bacterial cell wall components (such as LPS), phagocytic activity, and the ability to mature IL-18 associated with inflammasome activity, and preferably retain two or more of these functions. Unlike primary culture macrophages, immortalized macrophages proliferate in a densely spread (sheet-like) state.

[0056] In addition, immortalized macrophages can be produced by those skilled in the art with appropriate reference to previous reports. Examples of such previous reports 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., JP 2021-61772 A, and WO 2022 / 107793.

[0057] (Vaccine composition and method for producing same) As shown in the examples below, the modified ASFV of the present invention does not exhibit pathogenicity such as fever when administered, does not remain in the body, and is highly safe, and can confer immunity to animals against ASF. Therefore, it is suitable for use in vaccine compositions against ASF. In other words, the present invention can also provide a vaccine composition against ASF that contains 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 above-mentioned method from cells and mixing it with a pharmacologically acceptable carrier or medium.

[0059] "Isolation" of the modified ASFV refers to separation, purification, and / or concentration from the cells and / or the cell culture system (e.g., culture supernatant). Methods for isolating the virus include, for example, filtration of the culture medium, cell disruption (sonication, hypotonic solution treatment, freeze-thawing, 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 (i.e., a live vaccine) as is, or in a live-attenuated form (i.e., a live attenuated virus). A live attenuated virus refers to 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 growth in the presence of a mutagen, adaptation to cultured cells by continuous (long-term) in vitro passage, or growth under conditions that deviate from the natural growth environment (e.g., high temperature conditions). Live attenuated viruses can also be obtained by deleting or recombining specific viral genes using genome editing, gene modification techniques, etc.

[0061] Examples of the "pharmacologically acceptable carrier" to be mixed with the isolated modified ASFV include stabilizers, excipients, preservatives, surfactants, chelating agents, and binders. Examples of the "pharmacologically acceptable medium" include water, physiological saline, phosphate buffer, and Tris-HCl buffer. Those skilled in the art can select and use these carriers and media appropriately or in combination from known carriers and media used in the art, depending on the vaccine formulation and method of use. Furthermore, the form of the vaccine is not particularly limited, and may be, for example, a suspension or a lyophilized form.

[0062] To enhance the vaccine effect, an adjuvant may be further mixed in. Examples of adjuvants include inorganic substances such as aluminum gel adjuvant, microorganisms or substances derived from microorganisms (e.g., BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, cholera toxin), surfactants (e.g., saponin, deoxycholic acid), emulsions of oily substances (e.g., mineral oil, vegetable oil, animal oil), and alum.

[0063] Furthermore, the "animal" to which the vaccine composition of the present invention produced in this manner is administered is not particularly limited as long as it is an animal that can be infected with ASFV, and examples thereof include animals of the Suidae family, more specifically, pigs, wild boars, red river boars, etc. Furthermore, the animal may be a livestock animal, a companion animal (pet), an experimental animal, or an animal for other purposes.

[0064] The vaccine composition of the present invention can be administered to animals via known routes of administration, including intramuscular, subcutaneous, intradermal, intravenous, oral, and intranasal, to confer immunity against ASF. The dose may be any amount sufficient to confer the immunity, and may be adjusted appropriately depending on the animal species, age, and body weight, the type of ASFV (e.g., differences in the degree of pathogenicity), and the method and route of administration. For example, when administered intramuscularly to pigs, the titer of the modified virus of the present invention (median tissue culture infectious dose: TCID ) is 1000 mg / kg. ５０ ) is, for example, 10 ３ or more, preferably 10 ４ More preferably, 10 ５ The administration may be carried out multiple times, in which case the interval between administrations is, for example, 1 to 2 weeks.

[0065] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples. The 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 An African swine fever virus lacking the nucleotide sequence encoding the full-length E120R protein was created as follows. The African swine fever virus (ASFV) used was the isolate ASFV AQS-C-1-22. For details of this strain, see Ken-Ichiro Kameyama, Tomoya Kitamura et al., Viruses. 2022 Aug 16; 14(8):1794. An immortalized porcine kidney macrophage cell line (IPKM cells) was used as a host cell for amplifying ASFV. For details of this cell line, see Takato Takenouchi et al. , Front Vet Sci. 2017 Aug 21:4:132. Please refer to.

[0067] Specifically, to delete the E120R gene of the ASFV strain by homologous recombination, the ASFV p72 promoter and copGFP gene were ligated, and the resulting ligation sequence was inserted approximately 1,000 bases before and after the E120R gene. This sequence was then integrated into pGEM T easy vector (Promega). (The plasmid prepared in this manner is also referred to as "pTEΔE120R / GFP.") This plasmid was lipofected into IPKM cells, and the following day, the cells were infected with AQS-C-1-22 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-deleted strain (AQSΔE120R).

[0068] IPKM cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 μg / mL bovine insulin, 25 μM monothioglycerol, and penicillin-streptomycin solution (100x). The following incubators (all manufactured by Sumitomo Bakelite Co., Ltd.) were used for culturing IPKM cells: 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 applies below to IPKM cell culture.)

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

[0070] Each of the modified viruses produced was cultured in a flask or the like, and after proliferation, it was dispensed into tubes and stored at -80°C. One of them was thawed and the above TCID ５０ Based on this value, the stored virus was adjusted to the amount of virus actually inoculated into pigs, and used in the following experiment.

[0071] 3. Analysis of the pathogenicity of AQSΔE120R in pigs and vaccine efficacy 10 １ , 10 ３ or 10 ５ TCID ５０AQSΔE120R was inoculated into the gluteal muscles of 6-8 week old pigs (LWD, mixed sex) (n=6 for each), and clinical symptoms were observed for 28 days after infection. ２ TCID ５０ The mice were challenged with the parent strain of the above strain, and clinical symptoms were observed for 30 days thereafter.

[0072] In addition, 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). A Roche High Pure Viral Nucleic Acid Kit was used for nucleic acid extraction. Real-time PCR was performed using a series of concentrations of a plasmid (standard) into which the amplification product of real-time PCR had been cloned 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 μL, which was converted to 1 mL (multiplied by 1000) to set the detection limit at 10. ３ (Common logarithm (log10): 3.0).

[0073] 4. Creation of ASFV (AQS E120RΔN, ΔC, or ΔNC) Carrying a Partial Deletion Mutant of E120R In ASFV AQS-C-1-22 and other strains, E120R is a protein consisting of 122 amino acids. An attempt was made to create an ASFV carrying a partial deletion mutant of this protein. Specifically, an attempt was made to create an ASFV (AQS E120RΔN, ΔC, or ΔNC) carrying a gene encoding a mutant in which 15 amino acids were deleted from the N-terminus of E120R (E120RΔN), a mutant in which 18 amino acids were deleted from the C-terminus (E120RΔC), or a mutant in which both of these were deleted (E120RΔNC). To this end, the nucleotide sequence encoding E120RΔN, ΔC, or ΔNC was sequentially linked to the ASFV p72 promoter and the mCherry gene. The binding sequence was then inserted approximately 1,000 bases before and after the E120R gene, and the resulting sequence was incorporated into pGEM T easy vector (each of the plasmids prepared in this manner is also referred to as pTE E120RΔN, ΔC, or ΔNC). These plasmids were each lipofected into IPKM cells, which were then infected the following day 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.

[0074] FIG. 1 shows an outline of the E120R gene modified in AQS E120RΔN, ΔC, and ΔNC, and the above-mentioned AQSΔE120R, as well as an outline of 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 was inoculated into the gluteal muscle of 3-5 day old pigs (LWD, mixed sex) (n=2 for each), and clinical symptoms were observed for 7 days after infection.

[0076] Blood, gastrohepatic lymph nodes, and spleen were also collected on day 7 after infection. The gastrohepatic lymph nodes and spleen were each mashed and a solvent (PBS) was added to prepare a 10% (mass percent) emulsion. 200 μL of each was then subjected to nucleic acid extraction, and quantitative detection of the viral gene (p72 gene) by real-time PCR was attempted using the purified nucleic acid obtained as a template (see Donald P. King et al., 2003, supra). A Roche High Pure Viral Nucleic Acid Kit was used for nucleic acid extraction. Real-time PCR was also performed using a concentration series of plasmids (standards) into which the amplification products of real-time PCR had been cloned as templates, and the minimum detectable gene amount was set as the detection limit. As a result, the minimum detectable gene amount was 1 copy per μL, so the detection limit for blood was set at 1 (common logarithm (log10): 0), and for the stomach, liver, lymph nodes, and spleen, the detection limit was converted to 100% emulsion (10 times) and set at 10 (common logarithm (log10): 1.0).

[0077] 6. Analysis of the pathogenicity of AQS E120RΔNC 10 ２ TCID ５０ AQS E120RΔNC was inoculated into the gluteal muscles of 6-8 week-old pigs (LWD, mixed sex) to infect them (n=4). Clinical symptoms were observed for 21 days after infection, and blood samples were taken periodically. Using the same method as above, viral genome detection was attempted for each sample.

[0078] 7. Creation of ASFV (AQS E120RΔ72-73) Carrying a Two-Amino Acid Deletion Mutant of E120R To create ASFV (AQS E120RΔ72-73) carrying a mutant (E120RΔ72-73) in which the 72nd and 73rd amino acids of the E120R protein were deleted, the nucleotide sequence encoding E120RΔ72-73 was ligated to the ASFV p72 promoter and the mCherry gene in that order. The ligated sequence was then inserted approximately 1,000 bases before and after the E120R gene, and the resulting sequence was integrated into 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] The sequence numbers of the various sequences used in the production of the above-described modified ASFVs are shown in Table 1. Note that for some proteins in this table, the amino acid sequence and the nucleotide sequence encoding it (cDNA sequence) are shown.

[0080]

[0081] 8. Analysis of the pathogenicity of AQS E120RΔ72-73 10 ２ TCID ５０ AQS E120RΔ72-73 was inoculated into the gluteal muscle of 3- to 5-day-old pigs (LWD, mixed sex) (n=2) and infected. Clinical symptoms were observed for 7 days after infection.

[0082] On day 7 after infection, blood, gastrohepatic lymph nodes, and spleen were collected. The gastrohepatic lymph nodes and spleen were each mashed and a solvent (PBS) was added to prepare a 10% (mass percent) emulsion. 200 μL of each was then subjected to nucleic acid extraction, and quantitative detection of the viral gene (p72 gene) was attempted by real-time PCR using the purified nucleic acid obtained as a template (see Donald P. King et al., 2003, supra). Roche's High Pure Viral Nucleic Acid Kit was used for nucleic acid extraction. Real-time PCR was also performed using a concentration series of plasmids (standards) into which the amplification products of real-time PCR had been cloned as templates, and the minimum detectable gene amount was set as the detection limit. As a result, the minimum detectable gene amount was 1 copy per μL, so the detection limit for blood was set at 1 (common logarithm (log10): 0), and for the stomach, liver, lymph nodes, and spleen, the detection limit was converted to 100% emulsion (10 times) and set at 10 (common logarithm (log10): 1.0).

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

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

[0085] Next, 10 １ , 10 ３ or 10 ５ TCID ５０AQSΔE120R or its parent strain (ASFV AQS-C-1-22 strain) was inoculated into pigs. As a result, as shown in Figure 3A, pigs inoculated with the parent strain (control) developed fever, and all of the pigs died or were euthanized. On the other hand, pigs inoculated with AQSΔE120R did not die at any of the immunization doses (virus titers), and no pigs showed a clear fever.

[0086] In addition, blood samples were collected periodically during the observation period and analyzed for the presence or absence of viral genes. As a result, as shown in Figure 3B, unlike the control, the above genes were not detected at all in the pigs inoculated with AQSΔE120R, regardless of the immunization dose. In other words, it was revealed that AQSΔE120R does not cause viremia.

[0087] Furthermore, 28 days after inoculation with AQSΔE120R, ２ TCID ５０ As a result, as shown in Figure 4A, １ In the AQSΔE120R inoculated group, all six pigs died, but 10 ３ One animal survived in the AQSΔE120R-vaccinated group, and 10 ５ Four pigs survived in the AQSΔE120R-vaccinated group. In other words, the vaccine effect was confirmed according to the immunization dose of AQSΔE120R. Furthermore, as shown in Figure 4B, fever and viremia were observed in all immunized pigs after challenge, but the severity was reduced with increasing immunization dose.

[0088] (Example 2, Comparative Examples 1 and 2) Creation and Analysis of ASFV Carrying Partial Deletion Mutants of E120R IPKM cells transfected with pTE E120RΔN, ΔC, or ΔNC were infected with AQSΔE120R to induce homologous recombination. The culture supernatant approximately one week after induction of homologous recombination was subjected to limiting dilution. After approximately seven days of culture, AQS E120RΔN, ΔC, and ΔNC were successfully isolated using mCerry fluorescence as an indicator.

[0089] Next, these are 10 ２ TCID ５０AQS E120RΔN was inoculated into pigs at 100°C. As a result of inoculating pigs with AQS E120RΔN, no clear fever was observed in any of the pigs, as shown in Figure 5A. However, on the final day of the observation period, blood, stomach, liver, lymph nodes, and spleen were collected and analyzed for the presence or absence of viral genes. As shown in Figure 5B, viral genes were detected in one of two pigs.

[0090] Furthermore, all pigs inoculated with AQS E120RΔC developed a clear fever and died within the observation period, as shown in Figure 5A. When the stomach, liver, lymph nodes, and spleens were collected at the time of death and analyzed for the presence of viral genes, viral genes were detected in all samples.

[0091] On the other hand, none of the pigs inoculated with AQS E120RΔNC showed any clear fever, as shown in Figures 5A and 6A. Furthermore, on the final day of the observation period, blood, gastrohepatic lymph nodes, and spleens were collected and analyzed for the presence or absence of viral genes. As a result, no viral genes were detected, as shown in Figure 5B. Furthermore, blood was collected periodically during the observation period and analyzed for the presence or absence of viral genes. As shown in Figure 6B, no viral genes were detected.

[0092] Thus, when either the N-terminal or C-terminal region of the E120R protein was deleted, the modified ASFV persisted 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, including viremia, occurred in the pigs. Furthermore, no pigs died at any immunization dose.

[0093] (Comparative Example 3) Creation and analysis of ASFV carrying a two-amino acid deletion mutant of E120R As described above, ASFV carrying a two-amino acid deletion mutant of E120R was created. ２ TCID ５０Two pigs were inoculated with the virus at 1000 kJ / kg / day, and although not shown in the figure, one of them died on the fourth day after inoculation. The remaining pig was euthanized from the perspective of animal welfare because it was found to have lost energy, had a decreased appetite, and had diarrhea on the fifth day. Furthermore, the spleen and gastrohepatic lymph nodes were collected at autopsy (blood was also collected from the euthanized pigs only) and analyzed for the presence or absence of viral genes. As shown in Figure 7, viral genes were detected in all samples.

[0094] According to Non-Patent Document 2, when a modified ASFV in which the 72nd and 73rd amino acids of E120R have been deleted is inoculated, interferon β production is improved compared to the parent strain, i.e., the host's antiviral response is activated.

[0095] However, when these two amino acids of the E120R protein were deleted in this way, the pathogenicity of ASFV could not be reduced, and pigs inoculated with the modified ASFV were lethal. On the other hand, as described above, no pigs were lethal when inoculated with the modified ASFV of the present invention (AQS E120RΔNC, AQSΔE120R, etc.), and pathogenicity was suppressed.

[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 that has a vaccine effect against African swine fever. In particular, the present invention can confer immunity to animals against African swine fever safely without causing pathogenicity such as fever upon inoculation and without remaining in the body. Therefore, the present invention is useful as a vaccine against African swine fever.

Claims

A modified African swine fever virus in which the nucleotide sequence encoding the amino-terminal region (N-terminal region) and the nucleotide sequence encoding the carboxy-terminal region (C-terminal region) of the E120R protein are deleted in the genome. 　 The modified African swine fever virus according to claim 1, wherein the N-terminal region is a region consisting of the amino acid sequence from the N-terminus to the 10th to 30th amino acids of the E120R protein, and the C-terminal region is a region consisting of the amino acid sequence from the C-terminus to the 10th to 30th amino acids of the E120R protein. 　 The modified African swine fever virus according to claim 1, wherein the nucleotide sequence encoding the full-length E120R protein is deleted in the genome. 　 A vaccine composition against African swine fever containing the modified African swine fever virus according to any one of claims 1 to 3 as an active ingredient. 　 A method for producing a modified African swine fever virus, comprising: A method for producing the modified African swine fever virus of any one of claims 1 to 3, comprising the step of deleting, by homologous recombination in a cell infected with the African swine fever virus, nucleotide sequences encoding the N-terminal and C-terminal regions of the E120R protein of the virus. 　 The method according to claim 5 , wherein the cells are immortalized macrophages of a boar. 　 A method for producing a vaccine composition against African swine fever, comprising: A step of producing a modified African swine fever virus by the production method according to claim 5; and a step of mixing the modified African swine fever virus with a pharmacologically acceptable carrier or vehicle; A method comprising: 　 A method for producing a modified African swine fever virus, comprising: A production method comprising the steps of infecting a cell with the modified African swine fever virus according to any one of claims 1 to 3 and propagating the virus. 　 The method according to claim 8, wherein the cells are immortalized macrophages of a boar. 　 A method for producing a vaccine composition against African swine fever, comprising: A step of producing a modified African swine fever virus by the production method according to claim 8; and a step of mixing the modified African swine fever virus with a pharmacologically acceptable carrier or vehicle; A method comprising:

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