Non-replicating bovine infectious lymphoma virus (BLV) vaccine

Abstract

The purpose of the present invention is to provide a novel non-replicating bovine infectious lymphoma virus (BLV) vaccine. The present invention provides a bovine infectious lymphoma virus (BLV) vaccine in which at least part of the function of the pol gene is deficient. The present invention also provides a method for producing a BLV vaccine, the method including a step for culturing cells that produce non-replicating bovine infectious lymphoma virus (BLV), wherein the non-replicating-BLV-producing cells contain genes of the bovine infectious lymphoma virus (BLV) in which at least a part of the function of the pol gene is deficient. The present invention is advantageous in making it possible to provide a BLV vaccine having high immunogenicity and high safety such that replication does not occur in an infected subject.

Description

Non-replicating bovine infectious lymphoma virus (BLV) vaccine REFERENCE TO RELATED APPLICATIONS

[0001] This application benefits from the priority of an earlier Japanese application, Patent Application No. 2023-183308 (filing date: October 25, 2023), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a non-replicating bovine infectious lymphoma virus (BLV) vaccine.

[0003] The infectious bovine lymphoma virus (BLV) is the causative agent of endemic infectious bovine lymphoma (EBL), a malignant B lymphoma. It is a retrovirus that integrates into the DNA of host cells as a provirus. Approximately 70% of BLV-infected cattle remain healthy and asymptomatic, while approximately 30% develop persistent lymphocytosis. After a long-term incubation period, approximately a few percent develop infectious bovine lymphoma (Non-Patent Documents 1 and 2).

[0004] Although EBL has been increasing in recent years, a vaccine effective in preventing EBL has not yet been developed. One of the main reasons for this is that the amount of virus produced from BLV-infected cells is extremely low, about 1 / 1000 to 1 / 10,000 of that of the AIDS virus, which belongs to the same Retroviridae family, making it difficult to develop an inactivated vaccine or a cell-derived vaccine. Although attempts have been made to develop expression vectors to increase the amount of BLV produced (Patent Document 1), since BLV is a retrovirus, there is a risk that viral genes will be integrated into the host genome after infection, and therefore problems remain in the practical application of live vaccines.

[0005] Japanese Patent Application Laid-Open No. 2019-24351

[0006] Gillet NA, et al., Retrovirology, 2007, 4: 18.Aida Y, et al., Frontiers in Microbiology, 2013, 4: 328.

[0007] The present invention aims to provide a novel non-replicating bovine infectious lymphoma virus (BLV) vaccine.

[0008] The present inventors have now succeeded in establishing a virus-producing cell line that produces infectious but replication-incompetent bovine infectious lymphoma virus (BLV). Analysis of the genetic sequence of the virus produced by the cell line revealed that a portion of the pol gene was deleted. The inventors also found that inoculation of the virus induced antibody production in mice and cattle, and that the neutralizing antibody titers against BLV in sera collected from the mice and cattle increased over time. The present invention is based on these findings.

[0009] The present invention provides the following: [1] A BLV vaccine comprising an infectious bovine lymphoma virus (BLV) in which at least part of the function of the pol gene is deleted. [2] The BLV vaccine according to [1] above, which is administered two or more times with an interval of one week or more between doses. [3] A method for producing a BLV vaccine, which comprises the step of culturing non-replicating infectious bovine lymphoma virus (BLV)-producing cells, wherein the non-replicating BLV-producing cells comprise a gene for an infectious bovine lymphoma virus (BLV) in which at least part of the function of the pol gene is deleted. [4] A method for preventing or treating endemic infectious bovine lymphoma (EBL) (excluding humans) which comprises the step of inoculating a subject with the vaccine according to [1] or [2] above. [5] Use of an infectious bovine lymphoma virus in which at least part of the function of the pol gene is deleted as a BLV vaccine or a preventive or therapeutic agent for endemic infectious bovine lymphoma (EBL), for the manufacture of a BLV vaccine or a preventive or therapeutic agent for EBL, or in a method for preventing or treating EBL.

[0010] The present invention is advantageous in that it can provide a highly immunogenic and highly safe bovine infectious lymphoma virus (BLV) vaccine that does not replicate in infected subjects.

[0011] Figure 1 shows the genetic organization of wild-type BLV and the deleted portion of the pol gene in the CMVΔU3-pBLV-416ΔRT plasmid (pBLV-416ΔRT) compared to the CMVΔU3-pBLV-416 plasmid (pBLV-416). Figure 2A shows the results of PCR amplification of the pol gene regions of pBLV-416ΔRT and pBLV-416. The negative control was the empty vector pBluescript II KS(-) plasmid, and the positive control was FLK-BLV cells, which are persistently infected with BLV. Figure 2B shows the results of Western blot analysis using BLV-infected bovine serum (left) and BLV-uninfected bovine serum (right) to examine the expression of viral proteins produced by pBLV-416ΔRT-transfected and pBLV-416-transfected cells, respectively. The negative control was cells transfected with the empty vector pBluescript II KS(-) plasmid, and the positive control was FLK-BLV cells, which are persistently infected with BLV. Figure 3 shows the results of immunofluorescence antibody analysis of the intracellular localization of viral proteins in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. The negative control was cells transfected with the empty vector pBluescript II KS(-) plasmid, and the positive control was FLK-BLV cells, which are persistently infected with BLV. Figure 4 shows the results of Western blot analysis of the expression levels of viral proteins in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. The negative control was cells transfected with the empty vector pBluescript II KS(-) plasmid, and the positive control was FLK-BLV cells, which are persistently infected with BLV. The results of the Student's t-test are ＊ p<0.05, ＊＊ indicates p<0.01. Figure 5 shows the results of syncytium formation ability in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Cells transfected with the empty vector pBluescript II KS(-) plasmid were used as a negative control. As a result of Student's t-test, ＊＊ p<0.01, ＊＊＊indicates p<0.001. Figure 6 shows the results of cell-to-cell infection in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Cells transfected with the empty vector pBluescript II KS(-) plasmid were used as a negative control, and FLK-BLV cells, persistently infected with BLV, were used as a positive control. Figure 7A shows the amount of viral protein released by pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Figure 7B shows the reverse transcriptase activity of viral proteins produced by pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Figure 7C shows the results of syncytium formation in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Figure 7D shows the results of Western blot analysis of viral proteins released from pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Cells transfected with the empty vector pBluescript II KS(-) plasmid were used as a negative control, and FLK-BLV cells, which are persistently infected with BLV, were used as a positive control. Student's t-test showed that: ＊＊＊ indicates p<0.001. Figure 8 shows the genetic organization of wild-type BLV and the deleted portion of the pol gene in the CMVΔU3-pBLV-IFΔRT plasmid (pBLV-IFΔRT) compared to the CMVΔU3-pBLV-IF plasmid (pBLV-IF). Figure 9A shows the results of Western blot analysis of viral protein expression levels in pBLV-IFΔRT-transfected cells and pBLV-IF-transfected cells. Figure 9B shows the results of syncytium formation in pBLV-IFΔRT-transfected cells and pBLV-IF-transfected cells. Figure 9C shows the results of Capture ELISA analysis of the viral load in the culture supernatants of pBLV-IFΔRT-transfected cells and pBLV-IF-transfected cells. As a negative control, cells into which the empty vector pBluescript II KS(-) plasmid was introduced were used, and as a positive control, FLK-BLV cells, which are persistently infected with BLV, were used. ＊ p<0.05,＊＊ p<0.01, ＊＊＊ indicates p<0.001, respectively. Figure 10A shows the experimental schedule in which mice were inoculated with viral proteins produced by pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Figure 10B shows the results of nested PCR analysis of BLV genes in blood cells 3 weeks after inoculation. Figure 10C shows the results of co-culturing spleen cells isolated from mice with CC81-GREMG cells. Figure 10D shows the results of quantification of anti-p24 antibodies by ELISA from 0 to 6 weeks after inoculation. PBS was used as a negative control, and FLK-BLV cells, which are persistently infected with BLV, were used as a positive control. As a result of Student's t-test, ＊Indicates p<0.05. Figure 11 shows the genetic organization of wild-type BLV and the deleted portion of the BLV pol gene in the PK15-BLVΔRT cell line compared to the CMVΔU3-pBLV-416 plasmid (pBLV-416). Figure 12 shows the results of Western blot analysis of viral protein expression levels in the PK15-BLVΔRT cell line. FLK-BLV cells, which are persistently infected with BLV, were used as a positive control. Figure 13 shows the results of immunofluorescence analysis of the intracellular localization of viral proteins in the PK15-BLVΔRT cell line. FLK-BLV cells, which are persistently infected with BLV, were used as a positive control. Figure 14A shows the amount of viral protein released by the PK15-BLVΔRT cell line. Figure 14B shows a standard curve prepared using His-p24 antigen as a standard. FIG. 15 shows the time course of anti-p24 antibody production in mice inoculated with a virus produced by the PK15-BLVΔRT cell line (PK15-BLVΔRT) and in mice inoculated with PBS (PBS) as a negative control (n=5 per group). Error bars indicate standard deviation. FIG. 16 shows neutralizing antibody titers in sera (weeks 0, 3, and 6) collected from mice inoculated with a virus produced by the PK15-BLVΔRT cell line. FIG. 17 shows the time course of anti-p24 antibody production in cattle inoculated with a virus produced by the PK15-BLVΔRT cell line. FIG. 18 shows the time course of anti-gp51 antibody production in cattle inoculated with a virus produced by the PK15-BLVΔRT cell line. FIG. 19 shows the time course of proviral load in cattle inoculated with a virus produced by the PK15-BLVΔRT cell line. Figure 20 shows the time course of lymphocyte counts in cattle inoculated with viruses produced by the PK15-BLVΔRT cell line, and Figure 21 shows the neutralizing antibody titers in sera (week 0, week 3, and week 6) collected from cattle inoculated with viruses produced by the PK15-BLVΔRT cell line.

[0012] <<Viruses and their manufacturing methods>>

[0013] The bovine leukemia virus (BLV) is a retrovirus that integrates into the DNA of host cells as a provirus and is the causative agent of endemic bovine lymphoma (EBL), a malignant B lymphoma. The wild-type BLV genome is approximately 8,720 bp long, and its genome is composed of the gag, pro, pol, and env genes, which encode structural proteins, the rex and tax genes, which encode regulatory proteins, and the R3 and G4 genes, which encode accessory proteins, all sandwiched between two identical long terminal repeat (LTR) sequences (see Figure 1). The gag gene encodes proteins involved in viral particle formation, the pol gene encodes reverse transcriptase and integrase, and the env gene encodes the envelope protein involved in adsorption to and entry into host cells. An example of the gene sequence of wild-type BLV is shown in Table 1 (the gene sequence of wild-type BLV shows the sequence from the 5'LTR to the 3'LTR, and the underlined portion shows the sequence encoding the pol gene). Note that in the present invention, wild-type BLV sequences other than the gene sequences in Table 1 can also be used.

[0014]

[0015] The present invention provides a bovine infectious lymphoma virus (BLV) in which at least a portion of the pol gene function is deleted. In the present invention, "deleting at least a portion of the pol gene function" means that at least a portion of the pol gene function is deleted compared to wild-type BLV, specifically meaning that at least a portion of the reverse transcriptase and / or integrase function is deleted. That is, the virus of the present invention does not express reverse transcriptase and / or integrase, or, even if it expresses them, these enzymes do not function normally, and does not replicate in an infected subject. For this reason, the virus of the present invention is sometimes referred to herein as a "non-replicating BLV."

[0016] Whether or not at least a portion of the function of the pol gene is lost can be determined by preparing a BLV in which at least a portion of the pol gene is mutated, and determining whether or not the protein encoded by the pol gene is expressed in the prepared virus or whether or not the protein functions normally based on the presence or absence of the enzymatic activity of the protein. The presence or absence of protein expression can be determined by known methods, such as immunological assays using antibodies against the reverse transcriptase and integrase encoded by the pol gene. The presence or absence of enzymatic activity of the protein can be determined by known methods, such as methods for measuring reverse transcriptase activity using colorimetric analysis or methods for measuring integrase activity using strand transfer ability as an indicator.

[0017] The viruses of the present invention have a mutation in at least a portion of the pol gene. In the present invention, "at least a portion of the pol gene is mutated" refers to a mutation in at least a portion of the nucleotide sequence of the pol gene compared to wild-type BLV, and refers to a mutation that results in at least a partial loss of pol gene function. A mutation in the pol gene refers to a deletion, substitution, insertion, and / or addition in at least a portion of the nucleotide sequence of the pol gene, resulting in at least a partial loss of pol gene function. The pol gene function that is missing in the viruses of the present invention is either or both of the reverse transcriptase function and the integrase function, preferably the reverse transcriptase function. In other words, the viruses of the present invention can be referred to as BLVs in which the reverse transcriptase region (RT) and / or the integrase region (IN) encoded by the pol gene has been mutated, or BLVs having a pol gene in which the reverse transcriptase region (RT) and / or the integrase region (IN) has been mutated. Typical examples of the viruses of the present invention include BLVs having a pol gene in which a portion of the RT (e.g., 1 to 1643 bases, 1 to 1600 bases, 1 to 1500 bases, 1 to 1400 bases, 1 to 1300 bases, 1 to 1200 bases, 1 to 1100 bases, 1 to 1000 bases, 1 to 900 bases, 1 to 850 bases, 1 to 800 bases, 10 to 750 bases, 20 to 700 bases, 40 to 650 bases) and / or a portion of the IN (e.g., 1 to 894 bases, 1 to 850 bases, 1 to 800 bases, 10 to 750 bases, 20 to 700 bases, 40 to 650 bases) is deleted. Here, the deletion of bases in a portion of the RT and / or IN may be a deletion of consecutive bases or a deletion of non-consecutive bases.

[0018] In the virus of the present invention, the partial mutation of the base sequence of the pol gene is not limited as long as at least a part of the function of the pol gene is lost. However, when the pol gene is a wild-type pol gene of SEQ ID NO: 2 (Table 2), the RT mutation may be, for example, at positions 1 to 1643, 1 to 1600, 1 to 1500, 1 to 1400, 1 to 1300, 1 to 1200, 1 to 1100, 1 to 1000, 1 to 1111, 1 to 1211, 1 to 1212, 1 to 1313, 1 to 1314, 1 to 1315, 1 to 1416, 1 to 1417, 1 to 1418, 1 to 1419, 1 to 1514, 1 to 1519, 1 to 1519, 1 to 1619, 1 to 1620, 1 to 1621, 1 to 1630, 1 to 1631, 1 to 1643, 1 to 1600, 1 to 1500, 1 to 1400, 1 to 1300, 1 to 1200, 1 to 1100, 1 to 1000, 1 to 1011, 1 to 1512, 1 to 1513, 1 to 1514, 1 to 1515, 1 to 1516, 1 to 1518, 1 to 1621, 1 to 1631, 1 to 1643, 1 to 1600, 1 to 1500, 1 to 1400, 1 to 130 The mutation may be in a base at positions 1644 to 900, 20 to 800, 30 to 700, or 40 to 650, and an IN mutation may be in, for example, a mutation at positions 1644 to 2537, 1650 to 2500, 1700 to 2400, 1800 to 2300, 1700 to 2200, 1650 to 2100, 1650 to 2000, 1650 to 1900, or 1650 to 1800 from the 5' side of the base sequence of the pol gene shown in SEQ ID NO: 2. When the pol gene consists of a base sequence other than that of the wild-type pol gene of SEQ ID NO: 2 (Table 2), the mutation may be in a base corresponding to the base of SEQ ID NO: 2, and can be identified, for example, by appropriately aligning the base sequence of the pol gene to be mutated with the base sequence of SEQ ID NO: 2. That is, the nucleotide sequence of the pol gene to be mutated can be aligned with the nucleotide sequence of SEQ ID NO: 2 using publicly available homology search software or programs such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183:63-69 (1990)), or Smith-Waterman (Meth. Enzym., 164, 765 (1988)), to identify the mutation site.

[0019] The base sequence of the pol gene of SEQ ID NO: 2 in Table 2 is shown in the 5' to 3' direction and corresponds to the underlined base sequence of SEQ ID NO: 1 in Table 1. The number of RT mutations or IN mutations is not particularly limited as long as at least a portion of the function of the pol gene is lost, but the lower limit of the number of mutations can be, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, or 90, and the upper limit of the number of mutations can be 1,000, 900, 800, 700, 600, or 500. These lower and upper limits can be combined in any manner, and the numerical range can be, for example, 1 to 1,000, 10 to 900, 20 to 800, 30 to 700, or 40 to 600.

[0020] In the virus of the present invention, the mutation in the nucleotide sequence of the pol gene may be a mutation spanning both RT and IN. When the pol gene is a wild-type pol gene of SEQ ID NO: 2 (Table 2), such a mutation can be, for example, a mutation in a base at positions 1 to 2537, 10 to 2500, 20 to 2400, 30 to 2300, 40 to 2200, 50 to 2100, 60 to 2000, 70 to 1900, 80 to 1850, 90 to 1800, 100 to 1750, 200 to 1700, 300 to 1650, 400 to 1650, 500 to 1650, 600 to 1650, 700 to 1650, 800 to 1650, 900 to 1650, or 1000 to 1650 from the 5' side of the base sequence of the pol gene shown in SEQ ID NO: 2 in Table 2. When the pol gene consists of a base sequence other than the wild-type pol gene of SEQ ID NO: 2 (Table 2), the mutation can be determined to be in a base corresponding to the base of SEQ ID NO: 2. As described above, for example, the mutation can be identified by appropriately aligning the base sequence of the pol gene to be mutated with the base sequence of SEQ ID NO: 2.

[0021] The number of mutations spanning the above-mentioned RT and IN is not particularly limited as long as at least a portion of the function of the pol gene is lost, but the lower limit of the number of mutations can be, for example, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500, and the upper limit of the number of mutations can be, for example, 2200, 2000, 1800, 1600, 1400, 1200, 1000, 900, 800, 700, 600, or 500. These lower and upper limits can be combined in any manner, and the numerical ranges can be, for example, 1 to 2200, 10 to 2000, 20 to 1800, 30 to 1600, 40 to 1400, 50 to 1200, 60 to 1000, 70 to 900, 80 to 800, 90 to 700, or 100 to 600.

[0022]

[0023] As long as the virus of the present invention lacks at least a portion of the pol gene function and retains the functions of BLV genes other than the pol gene, it may have the entire sequence between the 5' LTR and 3' LTR of wild-type BLV shown in SEQ ID NO: 1, except for the pol gene, or it may have a sequence that is 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, particularly preferably 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more) identical to the region other than the pol gene (wild-type). Here, "identity" refers to the degree of identity, for example, when the sequences to be compared are appropriately aligned, and refers to the percentage of exact amino acid matches between the sequences. Identity calculations take into account, for example, the presence of gaps in the sequences and the properties of the amino acids (Wilbur, Natl. Acad. Sci. USA 80:726-730 (1983)). The alignment can be performed, for example, using any algorithm. Specifically, publicly available homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183:63-69 (1990)), or Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Furthermore, identity can be calculated, for example, using a publicly available homology search program such as the above. For example, identity can be calculated using the default parameters of the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0024] The virus of the present invention can be produced by cells having a BLV gene in which at least part of the function of the pol gene is deleted. The origin of the cells is not limited, and they may be naturally occurring cells, cells obtained by self-cloning, or cells produced by genetic recombination technology. However, from the perspective of use as a vaccine, cells obtained by natural occurrence or self-cloning are preferably used.

[0025] <<Cells and Method for Producing the Same>> The present invention also provides cells comprising a gene for bovine infectious lymphoma virus (BLV) in which at least a portion of the pol gene function is deleted. Here, "comprising a gene" means that the gene is contained in the cell in an expressible manner, and typically refers to the cell being transformed by being introduced with the gene. The cells of the present invention may be naturally occurring cells, cells obtained by self-cloning, or cells produced by genetic engineering, so long as they contain a polynucleotide encoding BLV in which at least a portion of the pol gene function is deleted. However, from the perspective of use as a vaccine, naturally occurring or self-cloned cells are preferably used.

[0026] Another aspect of the present invention provides a method for producing the cells of the present invention. When producing the cells of the present invention using genetic engineering techniques, they can be produced, for example, by introducing into a host cell an expression vector incorporating a polynucleotide encoding a BLV in which at least a portion of the function of the pol gene has been deleted. The function of the BLV pol gene can be deleted by deleting all or part of the RT and / or IN encoded by the pol gene. Alternatively, the pol gene may be mutated by substituting, deleting, inserting, and / or adding bases to prevent normal expression of reverse transcriptase or integrase. Alternatively, a foreign gene may be inserted into the RT or IN. Gene deletion or mutation and foreign gene insertion can be performed, for example, by known techniques such as homologous recombination and site-directed mutagenesis.

[0027] Host cells for producing the viruses of the present invention are not particularly limited as long as they are capable of transcription and translation of the expression vector, and examples include insect cells (e.g., silkworm cells), amphibian cells, reptile cells, avian cells, fish cells, and mammalian cells (e.g., PK15 cells, HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, and CHO cells), with mammalian cells being preferred.

[0028] The transformation of host cells may be carried out by any method selected appropriately depending on the type of host cells, etc., and can be carried out by, for example, electroporation, lithium acetate, calcium phosphate, lipofection, particle gun, etc.

[0029] The cells of the present invention can be produced in accordance with the description of the viruses of the present invention in addition to the above.

[0030] <<Virus Production Method>> The present invention also provides a method for producing a virus. The virus production method of the present invention includes a step of culturing the cells of the present invention. In the present invention, the cell culture conditions are not particularly limited as long as they enable the production of the virus of the present invention. However, the culture conditions can be arbitrarily set taking into account the type and properties of the cells, etc. The virus production method of the present invention may further include a step of recovering the virus produced by the cells of the present invention. In the present invention, the virus recovery method may be appropriately selected from known methods depending on the virus to be produced. In the case of a virus that accumulates within host cells, the virus may be recovered after disrupting the host cells. In the case of a virus that is released outside the host cells, the virus may be recovered from the culture supernatant of the host cells, for example, using an ultracentrifuge or a separation column. The recovered virus may be further purified as necessary. The recovered virus can also be freeze-dried and stored until it is used as an active ingredient in a vaccine. In addition to the above, the virus production method of the present invention can be carried out according to the descriptions of the virus of the present invention, the cell of the present invention, and the method for producing the same.

[0031] <<BLV Vaccine>> The present invention also provides a BLV vaccine. The vaccine of the present invention is characterized by comprising the virus of the present invention. That is, when administered to a subject, the vaccine of the present invention induces an immune response against BLV (such as the production of specific antibodies and the proliferation of cytotoxic T cells), and promotes the production of neutralizing antibodies against BLV and the production of interferon and the like in the subject. Therefore, the vaccine can be used to prevent BLV infection or treat EBL (or improve symptoms). The vaccine of the present invention can be provided as a live vaccine.

[0032] From the viewpoint of effectively inducing an immune response against BLV in a subject, the virus of the present invention contained in the vaccine of the present invention preferably has a gene encoding at least a structural protein, more preferably has at least p24 of the gag gene or gp51 or gp30 of the env gene, and even more preferably has at least gp51. While not being bound by the following theory, it is believed that during BLV infection, when gp51 binds to a receptor on the surface of the host (subject) cell membrane, the membrane fusion activity of gp30 fuses the virus and host cell membranes, allowing the BLV core to invade the host cell (Bai L, et al., FASEB Journal, 2019, fj201901528R.).

[0033] The vaccine of the present invention may contain a pharmaceutically acceptable carrier, lubricant, preservative, stabilizer, wetting agent, emulsifier, salt for adjusting osmotic pressure, buffer, colorant, antioxidant, viscosity adjuster, excipient (a concept that also includes immunostimulants such as carbonate apatite, sodium hydroxide, alum, incomplete / complete Freund's adjuvant, etc.), nanoparticles, etc. Examples of pharmaceutically acceptable carriers include water, various salt solutions, alcohol, vegetable oil, and mineral oil.

[0034] Adjuvants (components that non-specifically stimulate the immune response of a subject) that can be contained in the vaccine of the present invention include, in addition to the above-mentioned incomplete / complete Freund's adjuvant, Ribi adjuvant, Titermax, Specol, aluminum salts (aluminum hydroxide, aluminum phosphate, etc.), alum, calcium hydroxide phosphate, ammonium sulfate, vitamin E, α-tocopherol, nonionic block polymers and polyamines, dextran sulfate, Carbopol, pyran, saponin, Quil A (trademark), Q-vac (trademark), muramyl dipeptide, dimethylglycine, tuftsin, Bayol (trademark), Drakeol (trademark) 6VR, Klearol (trademark), Marcol (trademark) 52, Montanide (trademark), mineral oil, paraffin oil, squalene, squalane, ethyl oleate, ISCOM (immunostimulatory complex), etc.

[0035] Examples of emulsifiers that can be included in the vaccines of the present invention include sorbitan monooleate (Span™ 80), polyoxyethylene-sorbitan monooleate (polysorbate 80, Tween™ 80), etc., and emulsion stabilizers (e.g., benzyl alcohol, triethanolamine sugar) can also be used.

[0036] The vaccine of the present invention may take the form of, for example, an injection, a liquid, a suspension, an emulsion, a powder, a granule, or a capsule. The vaccine may be administered orally or parenterally (subcutaneously, nasally, intraperitoneally, intracapsularly, intramuscularly, or intravenously).

[0037] In the present invention, the "subject" refers to a non-human animal or non-human mammal, and examples thereof include mammals such as cattle (Bos taurus), zebu (Bos indicus), water buffalo (Bubalus bubalis), sheep, goats, pigs, mice, rats, rabbits, cats, and monkeys. Examples of cattle include dairy breeds, meat breeds, dual-purpose breeds, draft breeds, and dual-purpose breeds. Specific examples include Wagyu breeds such as Japanese Black Cattle and Japanese Shorthorn, Holsteins, Jerseys, and native breeds of each country. From the viewpoint of preventing or treating infectious bovine lymphoma, the subject is preferably cattle, zebu, and water buffalo, more preferably cattle and water buffalo, and even more preferably cattle.

[0038] The inoculation dose of the vaccine of the present invention can be appropriately determined taking into consideration the sex, age, and weight of the subject, the form of the vaccine, the mode of inoculation, etc. The inoculation dose of the vaccine of the present invention per adult cattle is, for example, in terms of the viral load of the virus of the present invention (converted into the amount of p24 protein of BLV), and the lower limit (or more than or exceeding) of the inoculation dose per dose is 6 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470 μg, 480 μg, 490 μg, 500 μg, 510 μg, 520 μg, 530 μg, 540 μg, 550 μg, 560 μg, 570 μg, The upper limit (not more than or less than) can be 700 μg, 690 μg, 680 μg, 670 μg, 660 μg, 650 μg, 640 μg, 630 μg, 620 μg, 610 μg, 600 μg, 590 μg, 580 μg, 570 μg, 560 μg, 550 μg, 540 μg, 530 μg, 520 μg, 510 μg, 500 μg, 490 μg, 480 μg, 470 μg, 460 μg, 450 μg, 440 μg, 430 μg, 420 μg, 410 μg, or 400 μg. These lower and upper limits can be combined in any way, for example, 6 μg or more and 700 μg or less, 50 μg or more and 650 μg or less, 100 μg or more and 600 μg or less, 150 μg or more and 550 μg or less, 200 μg or more and 500 μg or less, or 250 μg or more and 450 μg or less.

[0039] The inoculation dose of the vaccine of the present invention may also be determined using the number of syncytia formed as an index. The number of syncytia formed can be calculated, for example, as described in Sato H, et al., Arch Virol. 2018; 163(6): 1519-1530. and JP 2018-23329 A. The inoculation dose of the vaccine of the present invention per adult cow is determined by the number of syncytia formed by the virus of the present invention contained per dose in CC81-GREMG cells (Sato H, et al., Virol J. 2019; 16(1): 66.), with the lower limit (greater than or equal to) being 1 x 10２ , 2 × 10 ２ , 3 x 10 ２ , 4 x 10 ２ , 5 x 10 ２ , 6 x 10 ２ , 7 x 10 ２ , 8 x 10 ２ , 9 x 10 ２ , 1×10 ３ , 2 × 10 ３ , 3 x 10 ３ , 4 x 10 ３ , 5 x 10 ３ , 6 x 10 ３ , 7 x 10 ３ , 8 x 10 ３ , 9 x 10 ３ , 1×10 ４ , 2 × 10 ４ , 3 x 10 ４ , 4 x 10 ４ , 5 x 10 ４ , 6 x 10 ４ , 7 x 10 ４ , 8 x 10 ４ , 9 x 10 ４ or 1 x 10 ５ The upper limit (less than or equal to) can be set to 1×10 ８ , 9 x 10 ７ , 8 x 10 ７ , 7 x 10 ７ , 6 x 10 ７ , 5 x 10 ７ , 4 x 10 ７ , 3 x 10 ７ , 2 × 10 ７ , 1×10 ７ , 9 x 10 ６ , 8 x 10 ６ , 7 x 10 ６ , 6 x 10 ６ , 5 x 10 ６ , 4 x 10 ６ , 3 x 10 ６ , 2 × 10 ６ or 1 x 10 ６ These lower and upper limits can be arbitrarily combined, for example, 1×10 ２ 1x10 or more ８ Below, 3 x 10 ２ 7 x 10 or more ７ Below, 5 x 10２ 4 x 10 or more ７ Below, 1 x 10 ３ 1x10 or more ７ Below, 3 x 10 ３ 7 x 10 or more ６ Below, 5 x 10 ３ 4 x 10 or more ６ or less or 1 x 10 ４ 1x10 or more ６ It can be as follows:

[0040] The inoculation dose of the vaccine of the present invention per adult cattle can also be determined, for example, by the TCID of the virus of the present invention contained per dose. ５０ The TCID may be determined using the Tissue Culture Infectious Dose 50 as an index. ５０ The lower limit (greater than or equal to) is 1 x 10 ２ , 2 × 10 ２ , 3 x 10 ２ , 4 x 10 ２ , 5 x 10 ２ , 6 x 10 ２ , 7 x 10 ２ , 8 x 10 ２ , 9 x 10 ２ , 1×10 ３ , 2 × 10 ３ , 3 x 10 ３ , 4 x 10 ３ , 5 x 10 ３ , 6 x 10 ３ , 7 x 10 ３ , 8 x 10 ３ , 9 x 10 ３ , 1×10 ４ , 2 × 10 ４ , 3 x 10 ４ , 4 x 10 ４ , 5 x 10 ４ , 6 x 10 ４ , 7 x 10 ４ , 8 x 10 ４ , 9 x 10 ４ or 1 x 10 ５ The upper limit (less than or equal to) can be set to 1×10 ８ , 9 x 10 ７ , 8 x 10 ７ , 7 x 10 ７ , 6 x 10 ７, 5 x 10 ７ , 4 x 10 ７ , 3 x 10 ７ , 2 × 10 ７ , 1 x 10 ７ , 9 x 10 ６ , 8 x 10 ６ , 7 x 10 ６ , 6 x 10 ６ , 5 x 10 ６ , 4 x 10 ６ , 3 x 10 ６ , 2 × 10 ６ or 1 x 10 ６ These lower and upper limits can be arbitrarily combined, for example, 1×10 ２ 1x10 or more ８ Below, 3 x 10 ２ 7 x 10 or more ７ Below, 5 x 10 ２ 4 x 10 or more ７ Below, 1 x 10 ３ 1x10 or more ７ Below, 3 x 10 ３ 7 x 10 or more ６ Below, 5 x 10 ３ 4 x 10 or more ６ or less or 1 x 10 ４ 1x10 or more ６ It can be as follows:

[0041] In the present invention, the above-mentioned dose of the active ingredient can be administered once a day, or it can be administered in two to four divided doses. The vaccine of the present invention can be administered multiple times depending on the subject to enhance and maintain the efficacy of vaccination, and additional doses may be administered as the antibody titer in the subject decreases. In the present invention, the above-mentioned dose of the active ingredient can also be administered multiple times, initially once every one, two, three, or four weeks, and then once every one, two, three, three, or four weeks, or once a year.

[0042] Another aspect of the present invention provides a method for producing a vaccine of the present invention. The method for producing a vaccine of the present invention is characterized by comprising a step of culturing the cells of the present invention. The method for producing a virus of the present invention can further comprise a step of recovering the virus produced by the cells of the present invention. The method for producing a virus of the present invention can further comprise a step of mixing the recovered virus with a solution for a vaccine. The mixed virus solution can be used as a vaccine as is.

[0043] The vaccine of the present invention can be used for preventing, treating, or reducing the risk of developing infectious bovine lymphoma. According to another aspect, the present invention provides a method for preventing, treating, or reducing the risk of developing infectious bovine lymphoma, which method comprises the step of inoculating a subject with the vaccine of the present invention.

[0044] The vaccine of the present invention and its production method, as well as the preventive method, therapeutic method, and onset risk reduction method of the present invention can be carried out in accordance with the description of the virus of the present invention and its production method, and the cell of the present invention and its production method, in addition to the above.

[0045] Another aspect of the present invention provides use of the virus of the present invention as a BLV vaccine or an agent for preventing, treating, or reducing the risk of developing endemic bovine infectious lymphoma (EBL). Another aspect of the present invention also provides use of the virus of the present invention for producing a BLV vaccine or an agent for preventing, treating, or reducing the risk of developing EBL. Another aspect of the present invention also provides use of the virus of the present invention in a method for preventing, treating, or reducing the risk of developing EBL. The use of the present invention can be carried out according to the descriptions of the virus of the present invention and the method for producing it, and the cell of the present invention and the method for producing it.

[0046] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0047] Example 1: Establishment of a non-replicating BLV-producing cell line The present inventors previously constructed the CMVΔU3-pBLV-IF plasmid, which exhibits high BLV viral production, as disclosed in JP 2019-24351 A. The present inventors constructed the CMVΔU3-pBLV-416 plasmid using the pBLV-416 plasmid, which was cloned as a wild-type strain exhibiting higher viral production than the CMVΔU3-pBLV-IF plasmid, as a base. The CMVΔU3-pBLV-416 plasmid was prepared in the same manner as the CMVΔU3-pBLV-IF plasmid described in JP 2019-24351 A. This CMVΔU3-pBLV-416 plasmid was introduced into PK15 cells in an attempt to establish a cell line with high BLV production. After cell passage and seven rounds of cloning, we succeeded in producing a CMVΔU3-pBLV-416 plasmid-introduced mutant stable cell line (sometimes referred to herein as the "PK15-BLVΔRT cell line") in which the produced virus retained infectivity but lost replication and integration capabilities. Analysis of the entire nucleotide sequence of the provirus integrated into this cell line confirmed that a deletion from 46 bp to 696 bp in the RT region of the pol gene was present (see Figure 1).

[0048] Example 2: Study using pBLV-416ΔRT-transfected cells (1) In Example 2, a CMVΔU3-pBLV-416ΔRT plasmid was prepared by introducing a deletion from 46 bp to 696 bp into the RT region of the pol gene, and viral proteins produced from cells transfected with this molecular clone (pBLV-416ΔRT-transfected cells) were analyzed.

[0049] (1) Method A. Plasmid The CMVΔU3-pBLV-416 plasmid was prepared in the same manner as the CMVΔU3-pBLV-IF plasmid described in JP 2019-24351 A. The CMVΔU3-pBLV416ΔRT was prepared by site-directed mutagenesis using the CMVΔU3-pBLV-416 plasmid as a template (Bai L, et al., Retrovirology, 2015; 12(1):106, Inabe K, et al., J. Virol., 1999; 73: 1293-1301., Matsuura R, et al., 2019; 11(12):1140.). The deletion of the RT region of the pol gene in pBLV-416ΔRT was confirmed by PCR.

[0050] African green monkey kidney-derived cells (COS-1) were used to transfect the plasmids. COS-1 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1x Penicillin-Streptomycin-Glutamine (PSG) at 37°C in a CO2 incubator.

[0051] C. Transfection: COS-1 cells (5 x 10 ５ The cells were transfected into the 100 cells (100 cells / 60 mm dish), and the cells and culture supernatant were collected 48 hours after transfection. In this specification, the transfected cells may be referred to as "transfected cells."

[0052]

[0053]

[0054]

[0055] (e) Western Blot Expression of viral proteins was confirmed by Western blotting using serum from BLV-infected and non-BLV-infected bovine serum.

[0056] (2) Results The results are shown in Figure 2. PCR amplification of the pol gene region confirmed that a 650-bp shorter fragment was amplified in the pBLV-416ΔRT plasmid compared with pBLV-416, confirming the deletion of the region corresponding to 46 bp to 696 bp of the RT region of the pol gene (Figure 2A). Western blot analysis of BLV-infected bovine serum confirmed that the deletion of the pol gene in pBLV-416ΔRT-transfected cells was not associated with an effect on the expression levels of structural proteins such as Gag protein (p24) and Env proteins (gp51, gp30) compared with pBLV-416-transfected cells (Figure 2B, left). However, no viral proteins were detected in BLV-uninfected bovine serum (Figure 2B, right).

[0057] Example 3: Study using pBLV-416ΔRT-transfected cells (2) In Example 3, the effects of pol gene deletion on the intracellular localization and expression level of viral proteins were studied using pBLV-416ΔRT-transfected cells.

[0058] (1) Method A. Plasmid The same plasmid as in Example 2(1)A was used.

[0059] A. Cells COS-1 cells were used as in Example 2(1)A.

[0060] C. Western Blot Viral proteins were confirmed by Western blotting using anti-Gag and anti-Env antibodies.

[0061] D. Immunofluorescence antibody technique The intracellular localization of viral proteins was confirmed by immunofluorescence antibody technique using anti-Gag antibody and anti-Env antibody.

[0062] (2) Results The results are shown in Figures 3 and 4. In pBLV-416ΔRT-transfected cells, compared with pBLV-416-transfected cells, deletion of the pol gene did not affect the intracellular localization of Gag protein (p24) and Env protein (gp51) (Figure 3), but the expression levels of both p24 and gp51 were confirmed to increase (Figures 4A and 4B).

[0063] Example 4: Study using pBLV-416ΔRT-transfected cells (3) In Example 4, the syncytium-forming ability and cell-to-cell infectivity of the virus produced from pBLV-416ΔRT-transfected cells were studied.

[0064] (1) Methods A. Syncytium formation ability The pBLV-416ΔRT plasmid or pBLV-416 plasmid was introduced into 293T cells together with the pEGFP-N1 plasmid (an EGFP expression plasmid), and the syncytium formation ability was evaluated. As a negative control, 293T cells were introduced with the empty vector pBluescript II KS(-) plasmid together with the EGFP-N1 plasmid.

[0065] To evaluate cell-to-cell infectivity, pBLV-416ΔRT or pBLV-416 plasmids were introduced into COS-1 cells and cocultured with CC81-GREMG cells (BLV reporter cells). CC81-GREMG cells express EGFP in a BLV Tax-dependent manner, and BLV Env protein fused with CC81-GREMG cells to form a syncytium. This syncytium expressed EGFP, allowing evaluation of cell-to-cell infectivity.

[0066] (2) Results The results are shown in Figures 5 and 6. Both pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells were confirmed to form syncytia (Figure 5A). The number of syncytia was greater and the size of the syncytia was larger in pBLV-416ΔRT-transfected cells than in pBLV-416-transfected cells (Figure 5B). These results indicate that deletion of the pol gene enhances the ability to form syncytia in pBLV-416ΔRT-transfected cells.

[0067] Furthermore, with regard to cell-to-cell infection, we confirmed that the number and size of syncytia formed were greater in cells transfected with pBLV-416ΔRT than in cells transfected with pBLV-416 (Fig. 6A and B). These results suggest that deletion of the pol gene may enhance cell-to-cell infection.

[0068] Example 5: Study using pBLV-416ΔRT-transfected cells (4) In Example 5, the amount of virus produced and reverse transcriptase activity produced from pBLV-416ΔRT-transfected cells were analyzed.

[0069] (1) Method A. Plasmid The same plasmid as in Example 2(1)A was used.

[0070] A. Cells COS-1 cells were used as in Example 2(1)A.

[0071] C. Western Blot Viral proteins were detected by Western blotting using anti-Gag and anti-Env antibodies.

[0072] D. Amount of Virus Release To confirm the amount of virus produced by pBLV-416ΔRT-transfected and pBLV-416-transfected cells, the amount of p24 in the culture supernatant was measured by Capture ELISA. The specific procedure is as follows: BLV-positive serum was diluted in carbonate buffer adjusted to pH 9.0 and immobilized on a 96-well plate. Next, culture supernatant of pBLV-416ΔRT-transfected or pBLV-416-transfected cells and Tween 20 were added to the plate with the immobilized serum to capture p24. The amount of captured p24 was then measured by colorimetry using an anti-BLV p24 antibody (BLV3, VMRD) and an HRP-labeled anti-mouse IgG antibody.

[0073] E. Reverse transcriptase activity The viruses released into the culture supernatant were collected by ultracentrifugation, and the reverse transcriptase activity in the virus particles was measured using a reverse transcriptase assay colorimetric kit (Roche).

[0074] To evaluate the infectivity of the virus, virus-containing culture supernatant was added to CC81-GREMG cells, and syncytia formation was confirmed. Furthermore, p24 and gp51 in the virus were quantified by Western blot analysis using 2 μg of virus.

[0075] (2) Results The results are shown in Figure 7. No difference in virus release due to pol gene deletion was observed in pBLV-416ΔRT-transfected cells compared to pBLV-416-transfected cells (Figure 7A). Furthermore, viruses released from pBLV-416ΔRT-transfected cells did not exhibit reverse transcriptase activity (Figure 7B) or form syncytia (Figure 7C). Furthermore, Gag protein (p24) and Env protein (gp51) were detected in pBLV-416ΔRT-transfected cells, indicating that pol gene deletion does not affect virus maturation (Figure 7D).

[0076] Example 6: Study using pBLV-IFΔRT-transfected cells In Example 6, a CMVΔU3-pBLV-IFΔRT plasmid was prepared by introducing a deletion from 46 bp to 696 bp into the RT region of the pol gene, and viral proteins produced from cells transfected with this molecular clone (pBLV-IFΔRT-transfected cells) were analyzed.

[0077] (1) Method A Plasmid The CMVΔU3-pBLV-IF2 plasmid was prepared according to the description of JP 2019-24351 A. Similarly to Example 2(1)A, the CMVΔU3-pBLV-IF2ΔRT plasmid was prepared by site-directed mutagenesis using the CMVΔU3-pBLV-IF plasmid as a template (Tajima S, et al., J. Virol. 2000;74(23):10939-10949.) ( FIG. 8 ).

[0078] B. Cells COS-1 cells and 293T cells were used to introduce the plasmids.

[0079] C. Western Blot Viral proteins were confirmed by Western blotting using anti-Gag and anti-Env antibodies.

[0080] d) Syncytium-forming ability The procedure was carried out in the same manner as in Example 4(1)A.

[0081] (2) Results The results are shown in Figure 9. It was confirmed that the expression levels of Gag protein (p24) and Env protein (gp51) were increased in pBLV-IF2ΔRT-transfected cells (293T cells) compared with pBLV-IF2-transfected cells (293T cells) (Figure 9A). It was also confirmed that the number and size of syncytia were increased in pBLV-IF2ΔRT-transfected cells compared with pBLV-IF2-transfected cells (Figure 9B).

[0082] In pBLV-IF2ΔRT-transfected cells (COS-1 cells), the same amount of Gag protein (p24) was detected by Capture ELISA as in pBLV-IF2-transfected cells (COS-1 cells), and no effect of pol gene deletion was observed (Fig. 9C).

[0083] Example 7: Examination of BLV vaccine (1) In Example 7, the virus produced from pBLV-416ΔRT-transfected cells was inoculated into mice to examine its effectiveness as a vaccine.

[0084] (1) Method A. Plasmid The same plasmid as in Example 2(1)A was used.

[0085] B. Cells COS-1 cells were used to introduce the plasmid.

[0086] C. Virus: The culture supernatant of pBLV-416ΔRT-transfected cells or pBLV-416-transfected cells was centrifuged at 3,000 rpm for 10 minutes to remove cell debris, and then ultracentrifuged at 141,118 × g, 4°C, for 2 hours. The culture supernatant was removed, and the precipitate was resuspended in PBS to purify the virus.

[0087] D. Inoculation into Mice. According to the schedule shown in Figure 10A, mice were inoculated with 100 μg of virus at weeks 0 and 2, with PBS as a negative control (n = 6 per group). Blood samples were collected from weeks 0 to 6 after inoculation, and the mice were euthanized at week 6 after inoculation, followed by removal of the spleens. To confirm viral proliferation in the mice, the BLV gene was detected in blood cells at week 3 after inoculation using nested PCR. Spleen cells were also co-cultured with CC81-GREMG cells, and syncytium formation ability was assessed. Furthermore, anti-p24 antibodies were quantified by ELISA from weeks 0 to 6 after inoculation.

[0088] (2) Results The results are shown in Figure 10. In mice inoculated with virus produced by pBLV-416ΔRT-transfected cells, no BLV gene was detected (Figure 10B), and no syncytia were observed when spleen cells isolated from the mice were co-cultured with CC81-GREMG (Figure 10C). On the other hand, in mice inoculated with virus produced by pBLV-416ΔRT-transfected cells, anti-p24 antibodies were confirmed to increase over time (Figure 10D). These results suggest that virus produced by pBLV-416ΔRT-transfected cells may be useful as a non-replicating BLV vaccine.

[0089] Example 8: Study using a non-replicating BLV-producing cell line (1) In Example 8, the entire nucleotide sequence of the provirus integrated into the PK15-BLVΔRT cell line successfully generated in Example 1 was analyzed again. As a result, it was confirmed that the RT region of the pol gene was deleted from 43 bp to 696 bp (see Figure 11). Compared to the analysis results in Example 1, there was a difference of three nucleotides in the number of nucleotides deleted in the RT region of the pol gene, which is thought to be due to the addition of additional mutations during passaging after the establishment of the PK15-BLVΔRT cell line. Furthermore, compared with the nucleotide sequence of wild-type BLV, two nucleotide mutations and one nucleotide deletion were confirmed after 696 bp of the pol gene, but these mutations did not affect protein expression.

[0090] Example 9: Study using a non-replicating BLV-producing cell line (2) In Example 9, the expression and localization of viral proteins in cells, as well as the amount of released virus, were studied using the PK15-BLVΔRT cell line.

[0091] (1) Methods A. Western Blot Viral proteins were confirmed by Western blotting using anti-Gag antibody and anti-Env antibody.

[0092] (i) Immunofluorescence antibody technique The intracellular localization of viral proteins was confirmed by immunofluorescence antibody technique using anti-Gag antibody and anti-Env antibody.

[0093] C. Virus Release Amount The virus release amount of the PK15-BLVΔRT cell line was measured by Capture ELISA. The specific procedure is as follows: The cell culture supernatant was centrifuged at 141,118 × g for 2 hours, and the supernatant was removed. The virus was then resuspended in 1 / 100th the volume of PBS to concentrate the virus, and the p24 concentration was measured. The viral p24 concentration was calculated using a standard curve prepared using His-p24 antigen expressed and purified in E. coli as a standard (Figure 14B).

[0094] (2) Results The results are shown in Figures 12 to 14. The results in Figure 12 confirmed that the PK15-BLVΔRT cell line expressed the viral structural proteins p24 and gp51. Furthermore, compared to the positive control, FLK-BLV, persistently BLV-infected cells, no change in the molecular weight of p24 or gp51 due to pol gene deletion was observed. The results in Figure 13 confirmed intracellular punctate accumulation, similar to the positive control, FLK-BLV, persistently BLV-infected cells. These results confirmed that pol gene deletion does not affect the intracellular localization of p24. The results in Figure 14A showed a p24 concentration of 25.1 ± 1.2 μg, confirming that a sufficient amount of virus was released from the PK15-BLVΔRT cell line. These results indicated that the virus produced by the PK15-BLVΔRT cell line may be useful as a non-replicating BLV vaccine.

[0095] Example 10: Examination of BLV vaccine (2) In Example 10, the virus produced from PK15-BLVΔRT cells was inoculated into mice to examine its effectiveness as a vaccine.

[0096] (1) Method A) Virus The culture supernatant of PK15-BLVΔRT cells was centrifuged at 3,000 rpm for 10 minutes to remove cell debris, and then ultracentrifuged at 141,118 × g at 4°C for 2 hours. The culture supernatant was removed, and the precipitate was resuspended in PBS to purify the virus and prepare a virus solution.

[0097] B. Inoculation into mice: 200 μL of the mixture (500 μg total viral protein, 10% Montanide) prepared in step A above and added as an adjuvant to the virus solution prepared in step A above was subcutaneously inoculated into mice (BALB / cCrS1c, 4-week-old, female) at weeks 0 and 2. PBS was also inoculated as a negative control, and blood was collected from 0 to 6 weeks after inoculation (n=5 per group). Anti-p24 antibodies were then quantified by ELISA.

[0098] In addition, the neutralizing antibody titers of serum collected from the mice were measured using the following procedures (i) to (vi) (Sato H, et al., Virol J. 2019; 16(1):66.). (i) 1.0 × 10 ４ CC81-GREMG cells were seeded and cultured overnight (37°C, 5% CO ２ (ii) Mouse serum was serially diluted 2-fold from 25 to 200 times with phosphate buffered saline (PBS). (iii) 50 μl of diluted serum was mixed with 50 μl of 200 pg BLV virus solution (containing 200 pg of p24 protein) and allowed to react at room temperature for 1 hour. (iv) 100 μl of the mixture of serum and virus solution was dropped at 100 μl / well onto the well plate from (i) above and incubated for 1 hour (37°C, 5% CO ２ (v) After removing the serum and virus mixture, medium was added and the cells were cultured for 5 days (37°C, 5% CO ２(vi) Five days after virus infection, the cells were fixed with 3.7% formaldehyde containing 0.01% Hoechst 33342, and the number of infected cells was assessed under a fluorescence microscope (EBOS FL AUTO 2 imaging system, Thermo Fisher Scientific). The neutralizing antibody titer was calculated using the 50% effective concentration (NT50) as an index.

[0099] (2) Results The results are shown in Figures 15 and 16. In mice (PK15-BLVΔRT) inoculated with the virus produced from PK15-BLVΔRT cells, the production of anti-p24 antibodies was induced (Figure 15). Furthermore, the neutralizing antibody titers in the serum collected from the mice were observed to increase 3 and 6 weeks after inoculation (Figure 16). These results demonstrated that the virus produced from PK15-BLVΔRT cells is effective as a non-replicating BLV vaccine.

[0100] Example 11: Examination of BLV vaccine (3) In Example 11, the virus produced from PK15-BLVΔRT cells was inoculated into cattle to examine its effectiveness as a vaccine.

[0101] (1) Method A) Virus The culture supernatant of PK15-BLVΔRT cells was centrifuged at 3,000 rpm for 10 minutes to remove cell debris, and then ultracentrifuged at 141,118 × g at 4°C for 2 hours. The culture supernatant was removed, and the precipitate was resuspended in PBS to purify the virus and prepare a virus solution.

[0102] B. Inoculation into cattle: 3 mL of a mixture (virus solution: 2.7 mL (containing 66 μg of p24 protein), Montanide Gel 01 (SEPPIC)) adjuvant was added to the virus solution prepared in A. above. (Virus solution: 2.7 mL (containing 66 μg of p24 protein), Montanide 0.3 mL) was inoculated subcutaneously (in the buttocks) into one cattle (Japanese Black cattle, 4 months old, male, 100-120 kg) at weeks 0 and 2, respectively. Blood was collected from weeks 0 to 6 after inoculation. Anti-p24 antibodies and anti-gp51 antibodies were quantified by ELISA (Bai L, et al., Arch Virol. 2019 Jan;164(1):201-211., Nippon Gene Co., Ltd.). Furthermore, the proviral load was quantified using BLV-CoCoMo-qPCR-2 as described in Takeshima SN, et al., Arch Virol. 2015 May;160(5):1325-32. The proviral load was quantified using Celltac α (Nihon Kohden Corporation).

[0103] In addition, the neutralizing antibody titer of serum collected from the cattle was measured using the following procedures (i) to (vi) (Sato H, et al., Virol J. 2019; 16(1):66.). (i) 1.0 × 10 ４ CC81-GREMG cells were seeded and cultured overnight (37°C, 5% CO ２ (ii) Bovine serum was serially diluted 2-fold from 25 to 800 times with phosphate buffered saline (PBS). (iii) 50 μl of the diluted serum was mixed with 50 μl of 200 pg BLV virus solution (containing 200 pg of p24 protein) and allowed to react at room temperature for 1 hour. (iv) 100 μl of the mixture of serum and virus solution was dropped at 100 μl / well onto the well plate from (i) above and incubated for 1 hour (37°C, 5% CO ２ (v) After removing the serum and virus mixture, medium was added and the cells were cultured for 5 days (37°C, 5% CO ２(vi) Five days after virus infection, the cells were fixed with 3.7% formaldehyde containing 0.01% Hoechst 33342, and the number of infected cells was assessed under a fluorescence microscope (EBOS FL AUTO 2 imaging system, Thermo Fisher Scientific). The neutralizing antibody titer was calculated using the 50% effective concentration (NT50) as an index.

[0104] (2) Results The results are shown in Figures 17 to 21. In cattle inoculated with the virus produced from PK15-BLVΔRT cells, the production of anti-p24 antibodies and anti-gp51 antibodies was induced (Figures 17 and 18). On the other hand, no provirus was detected (Figure 19), and no significant change in lymphocyte counts was observed before and after inoculation (Figure 20). Furthermore, the neutralizing antibody titers in serum collected from the cattle were observed to increase 3 and 6 weeks after inoculation (Figure 21). These results demonstrated that the virus produced from PK15-BLVΔRT cells is effective as a non-replicating BLV vaccine.

Claims

1. A bovine infectious lymphoma virus (BLV) vaccine comprising a BLV in which at least a portion of the function of the pol gene is deleted.

2. The BLV vaccine of claim 1, wherein the number of vaccinations is two or more and the interval between vaccinations is one week or more.

3. A method for producing a bovine infectious lymphoma virus (BLV) vaccine, comprising a step of culturing non-replicating BLV-producing cells, wherein the non-replicating BLV-producing cells contain a bovine infectious lymphoma virus (BLV) gene lacking at least a portion of the function of the pol gene.

4. A method for preventing or treating endemic bovine lymphoma (EBL), comprising the step of inoculating a subject with the vaccine according to claim 1 or 2 (provided that the subject does not include humans).

5. Use of a bovine infectious lymphoma virus in which at least part of the function of the pol gene is deleted as a BLV vaccine or an agent for preventing or treating endemic bovine lymphoma (EBL), for the manufacture of a BLV vaccine or an agent for preventing or treating EBL, or in a method for preventing or treating EBL.

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