Non-replicating bovine infectious lymphoma virus (BLV) vaccine

Abstract

To provide a novel non-replicating bovine infectious lymphoma virus (BLV) vaccine.SOLUTION: 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 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.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a non-replicating bovine infectious lymphoma virus (BLV) vaccine. [Background technology]

[0002] The infectious bovine lymphoma virus (BLV) is the causative agent of endemic infectious bovine lymphoma (EBL), a malignant B lymphoma, and is a retrovirus that is integrated as a provirus into the DNA of host cells. Approximately 70% of BLV-infected cattle are healthy and unaffected, while approximately 30% have persistent lymphocytosis, and after a long period of incubation, approximately a few percent develop infectious bovine lymphoma (Non-Patent Documents 1 and 2).

[0003] In recent years, the incidence of EBL has been increasing, but a vaccine with preventive effects against 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 small, about 1 / 1000 to 1 / 10000 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 incorporated into the genome of the host after infection, and therefore problems remain in the practical application of live vaccines. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-24351 A [Non-patent literature]

[0005] [Non-Patent Document 1] Gillet NA, et al., Retrovirology, 2007, 4: 18. [Non-Patent Document 2] Aida Y, et al., Frontiers in Microbiology, 2013, 4: 328. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a novel non-replicating bovine infectious lymphoma virus (BLV) vaccine. [Means for solving the problem]

[0007] The present inventors have now succeeded in establishing a virus-producing cell line that produces an infectious but replication-incompetent bovine infectious lymphoma virus (BLV), and have analyzed the genetic sequence of the virus produced by the cell line, finding that a portion of the pol gene is deleted. The present inventors have also found that inoculation of the virus induces antibody production in mice and cattle, and that the neutralizing antibody titers against BLV in the sera collected from the mice and cattle increase over time. The present invention is based on these findings.

[0008] According to the present invention, the following inventions are provided. [1] A bovine infectious lymphoma virus (BLV) vaccine containing a BLV in which at least part of the function of the pol gene is deleted. [2] The BLV vaccine described in [1] above, in which the number of vaccinations is two or more and the interval between vaccinations is one week or more. [3] A method for producing a BLV vaccine, comprising a step of culturing non-replicating bovine lymphoma virus (BLV)-producing cells, wherein the non-replicating BLV-producing cells contain a bovine lymphoma virus (BLV) gene in which at least part of the function of the pol gene is deleted. [4] A method for preventing or treating BLV, comprising the step of administering the vaccine described in [1] or [2] above to a subject (provided that the subject does not include humans).

[0009] The present invention is advantageous in that it can provide a bovine infectious lymphoma virus (BLV) vaccine that is highly immunogenic and highly safe since it does not replicate in infected subjects. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the genetic organization of wild-type BLV and also shows the deleted portion of the pol gene of the CMVΔU3-pBLV-416ΔRT plasmid (pBLV-416ΔRT) in comparison with the CMVΔU3-pBLV-416 plasmid (pBLV-416). [Diagram 2] Figure 2A shows the results of PCR amplification of the pol gene region 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 of the expression of viral proteins produced by pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells using BLV-infected bovine serum (left) and non-BLV-infected bovine serum (right). 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. [Diagram 3] The results of immunofluorescence antibody analysis of the intracellular localization of viral proteins in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells are shown in Figure 3. 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]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. As a result of the Student's t-test, * indicates p<0.05 and ** indicates p<0.01. [Diagram 5] Figure 5 shows the results of syncytium formation 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 the Student's t-test, ** indicates p<0.01 and *** indicates p<0.001. [Figure 6] Figure 6 shows the results of cell-to-cell infection in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. As a negative control, cells transfected with the empty vector pBluescript II KS(-) plasmid were used, and as a positive control, FLK-BLV cells, which are persistently infected with BLV, were used. [Figure 7] FIG. 7A shows the amount of viral protein released by pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. FIG. 7B shows the reverse transcriptase activity of viral protein produced by pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. FIG. 7C shows the results of syncytium formation ability in pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. FIG. 7D shows the results of Western blot for viral protein released from pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells. Note that 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. Results of Student's t-test: *** indicates p<0.001. [Figure 8]FIG. 8 shows the genetic organization of wild-type BLV and also shows the deleted portion of the pol gene of CMVΔU3-pBLV-IFΔRT plasmid (pBLV-IFΔRT) in comparison with CMVΔU3-pBLV-IF plasmid (pBLV-IF). [Figure 9] Figure 9A shows the results of Western blot analysis of the expression levels of viral proteins 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 measuring the amount of virus in the culture supernatant of pBLV-IFΔRT-transfected cells and pBLV-IF-transfected cells by Capture ELISA. 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, **, and ***, respectively. [Figure 10] FIG. 10A shows the schedule of an experiment in which viral proteins produced by pBLV-416ΔRT-transfected cells and pBLV-416-transfected cells were inoculated into mice. FIG. 10B shows the results of nested PCR for the BLV gene in blood cells 3 weeks after inoculation. FIG. 10C shows the results of co-culturing spleen cells extracted from mice with CC81-GREMG cells. FIG. 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. The results of the Student's t-test are as follows: * indicates p<0.05. [Figure 11] FIG. 11 shows the genetic organization of wild-type BLV and the deleted portion of the BLV pol gene in the PK15-BLVΔRT cell line in comparison with the CMVΔU3-pBLV-416 plasmid (pBLV-416). [Figure 12]12 shows the results of Western blot analysis of the expression levels of viral proteins in the PK15-BLVΔRT cell line, with FLK-BLV cells, which are persistently infected with BLV, used as a positive control. [Figure 13] 13 shows the results of immunofluorescence antibody analysis of the intracellular localization of viral proteins in the PK15-BLVΔRT cell line, with FLK-BLV cells, which are persistently infected with BLV, used as a positive control. [Figure 14] Figure 14A shows the amount of viral protein released by the PK15-BLVΔRT cell line, and Figure 14B shows a standard curve prepared using His-p24 antigen as a standard. [Figure 15] Figure 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 as a negative control (PBS) (n=5 per group). Error bars indicate standard deviation. [Figure 16] FIG. 16 shows neutralizing antibody titers of sera (0 weeks, 3 weeks, 6 weeks) collected from mice inoculated with virus produced by the PK15-BLVΔRT cell line. [Figure 17] FIG. 17 shows the time course of anti-p24 antibody production in cattle inoculated with viruses produced by the PK15-BLVΔRT cell line. [Figure 18] FIG. 18 shows the time course of anti-gp51 antibody production in cattle inoculated with viruses produced by the PK15-BLVΔRT cell line. [Figure 19] FIG. 19 shows the time course of proviral load in cattle inoculated with virus produced by the PK15-BLVΔRT cell line. [Figure 20] FIG. 20 shows the time course of lymphocyte counts in cows inoculated with viruses produced by the PK15-BLVΔRT cell line. [Figure 21] FIG. 21 shows neutralizing antibody titers of sera (0 weeks, 3 weeks, 6 weeks) collected from cows inoculated with virus produced by the PK15-BLVΔRT cell line. Description of the Invention

[0011] <<Viruses and their manufacturing methods>>

[0012] Bovine leukemia virus (BLV) is the causative virus of endemic bovine lymphoma (EBL), a malignant B lymphoma, and is a retrovirus that is integrated into the DNA of host cells as a provirus. The full length of the wild-type BLV genome is approximately 8720 bp, and the genes are sandwiched between two identical long terminal repeat (LTR) sequences, and consist of the gag, pro, pol, and env genes that code for structural proteins, the rex and tax genes that code for regulatory proteins, and the R3 and G4 genes that code for accessory proteins (see Figure 1). Here, the gag gene codes for proteins involved in the formation of virus particles, the pol gene codes for reverse transcriptase and integrase, and the env gene codes for the envelope protein involved in adsorption to and penetration 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 5'LTR to 3'LTR, and the underlined part shows the sequence that codes for the pol gene). In the present invention, wild-type BLV sequences other than the gene sequences in Table 1 can be used.

[0013] [Table 1] TIFF2025072877000003.tif235153TIFF2025072877000004.tif235153TIFF2025072877000005.tif234152TIFF2025072877000006.tif66153

[0014] According to the present invention, there is provided a bovine infectious lymphoma virus (BLV) in which at least a portion of the function of the pol gene is deleted. In the present invention, "deleting at least a portion of the function of the pol gene" means that at least a portion of the function of the pol gene is deleted in comparison with wild-type BLV, and specifically means that at least a portion of the function of reverse transcriptase and / or integrase is deleted. In other words, the virus of the present invention does not express reverse transcriptase and / or integrase, or, even if expressed, these enzymes do not function normally, and does not replicate in an infected subject. For this reason, in the present specification, the virus of the present invention is sometimes referred to as "non-replicating BLV".

[0015] Whether or not at least a part of the function of the pol gene is deleted can be determined by preparing a BLV in which at least a part of the pol gene is mutated, and determining whether or not the protein encoded by the pol gene in the prepared virus is expressed or not, or by determining 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 the protein expression can be determined by known methods, for example, immunological assays using antibodies against the reverse transcriptase and integrase encoded by the pol gene. The presence or absence of the enzymatic activity of the protein can be determined by known methods, for example, a method for measuring reverse transcriptase activity using colorimetric analysis or a method for measuring integrase activity using strand transfer ability as an index.

[0016] The virus of the present invention has at least a part of the pol gene mutated. In the present invention, "at least a part of the pol gene mutated" refers to at least a part of the nucleotide sequence of the pol gene mutated in comparison with the wild-type BLV, and means a mutation that causes at least a part of the function of the pol gene to be deleted. The mutation of the pol gene means a mutation in which at least a part of the nucleotide sequence of the pol gene is deleted, substituted, inserted and / or added, causing at least a part of the function of the pol gene to be deleted. The function of the pol gene that is deleted in the virus of the present invention is either or both of the function of reverse transcriptase and the function of integrase, and preferably the function of reverse transcriptase. In other words, the virus of the present invention can be said to be a BLV in which the reverse transcriptase region (RT) and / or the integrase region (IN) encoded by the pol gene is mutated, or a BLV having a pol gene in which the reverse transcriptase region (RT) and / or the integrase region (IN) is mutated. Typical examples of the virus of the present invention include BLV having a pol gene in which a portion of 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 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 RT and / or IN may be a deletion of consecutive bases or a deletion of non-consecutive bases.

[0017] 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 deleted. 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 1100, 1 to 1200, 1 to 1300, 1 to 1400, 1 to 1500, 1 to 1600, 1 to 1700, 1 to 1800, 1 to 1900, 1 to 2000, 2 to 2100, 2 to 2200, 2 to 2300, 2 to 2400, 2 to 2500, 2 to 2600, 2 to 2700, 2 to 2800, 2 to 3000, 3 to 3100, 3 to 3200, 3 to 3300, 3 to 3400, 3 to 3500, 3 to 3600, 3 to 3700, 3 to 3800, 3 to 3900, 4 to 4000, 4 to 4100, 4 to 4200, 4 to 4300, 4 to 4400, 4 to 4500, 4 to 4600, 4 to 4700, 4 to 4800, 4 to 5000, 5 to 5000, 6 to 510 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 a base 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 may be specified, 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 base sequence of the pol gene to be mutated can be aligned with the base 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.

[0018] The base sequence of the pol gene in SEQ ID NO: 2 in Table 2 is shown in the direction from the 5' to the 3' side, and corresponds to the underlined base sequence of SEQ ID NO: 1 in Table 1. The number of RT mutations or the number of IN mutations is not particularly limited as long as at least a part 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 1000, 900, 800, 700, 600, or 500. These lower and upper limits can be arbitrarily combined, and the numerical range can be, for example, 1 to 1000, 10 to 900, 20 to 800, 30 to 700, or 40 to 600.

[0019] 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 base 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 in a base corresponding to the base of SEQ ID NO: 2, and 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.

[0020] The number of mutations spanning the above 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 combination, and the numerical range 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.

[0021] [Table 2] TIFF2025072877000008.tif151155

[0022] The virus of the present invention may have the region other than the pol gene in the entire sequence between 5'LTR and 3'LTR of wild-type BLV shown in SEQ ID NO: 1 as it is, or may have a sequence having an identity of 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) to the region other than the pol gene (wild type), so long as at least a part of the function of the pol gene is deleted and the functions of the genes of BLV other than the pol gene are maintained. Here, "identity" refers to the degree of identity when the sequences to be compared are appropriately aligned, for example, and means the occurrence rate (%) of exact amino acid matches between the sequences. When calculating the identity, for example, the presence of gaps in the sequence and the properties of the amino acids are taken into consideration (Wilbur, Natl. Acad. Sci. USA 80:726-730(1983)). The alignment can be performed, for example, by 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)), and Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. In addition, the identity can be calculated, for example, by using a publicly available homology search program such as the above, for example, by using the default parameters in the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0023] The virus of the present invention can be produced by cells having a BLV gene lacking at least a part of the function of the pol gene. The origin of the cells is not limited, and they may be natural occurrence cells, cells obtained by self-cloning, or cells produced by genetic recombination technology. From the viewpoint of use as a vaccine, it is preferable to use cells obtained by natural occurrence or self-cloning.

[0024] <<Cells and their manufacturing method>> The present invention also provides a cell comprising a gene of bovine infectious lymphoma virus (BLV) in which at least a part of the function of the pol gene is deleted. Here, "comprising a gene" means that the gene is contained in the cell in an expressible manner, and typically means that the gene has been introduced into the cell and is in a transformed state. As long as the cell contains a polynucleotide encoding a BLV in which at least a part of the function of the pol gene is deleted, the cell of the present invention may be a natural occurrence cell, a cell obtained by self-cloning, or a cell produced by genetic recombination technology. From the viewpoint of use as a vaccine, however, it is preferable to use a cell obtained by natural occurrence or self-cloning.

[0025] According to another aspect of the present invention, a method for producing the cell of the present invention is provided. When the cell of the present invention is produced by genetic recombination technology, for example, it can be produced by introducing an expression vector incorporating a polynucleotide encoding a BLV in which at least a part of the function of the pol gene is deleted into a host cell. In order to delete the function of the pol gene of BLV, all or a part of the RT and / or IN encoded by the pol gene may be deleted. In addition, the gene may be mutated by substituting, deleting, inserting and / or adding bases of the pol gene so that normal reverse transcriptase or integrase is not expressed. Alternatively, a foreign gene may be inserted into the RT or IN. The deletion or mutation of the gene and the insertion of the foreign gene can be performed by known methods such as homologous recombination and site-directed mutagenesis.

[0026] Host cells for producing the virus of the present invention are not particularly limited as long as they are capable of transcription and translation of the expression vector, and examples thereof include insect cells (e.g., silkworm cells), amphibian cells, reptile cells, avian cells, fish cells, mammalian cells (e.g., PK15 cells, HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells and CHO cells), etc., with mammalian cells being preferred.

[0027] Transformation of host cells can 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.

[0028] The cells of the invention can be carried out in accordance with the description of the viruses of the invention in addition to the above.

[0029] <<How the virus is produced>> The present invention also provides a method for producing a virus. The method for producing a virus of the present invention includes a step of culturing the cells of the present invention. In the present invention, the culture of the cells is not particularly limited as long as the culture conditions are such that the virus of the present invention can be produced, but the culture conditions can be arbitrarily set in consideration of the type and properties of the cells. The method for producing a virus 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 method for recovering the virus may be appropriately selected from known methods according to the virus to be produced, but in the case of a virus that accumulates in a host cell, the virus may be recovered after disrupting the host cell, and in the case of a virus that is released outside the host cell, the virus may be recovered, for example, from the culture supernatant of the host cell using an ultracentrifuge or a separation column. The recovered virus may be further purified as necessary. The recovered virus may be freeze-dried and stored until it is used as an active ingredient of a vaccine. In addition to the above, the method for producing a virus of the present invention may be carried out according to the description of the virus of the present invention and the cell of the present invention and the method for producing the same.

[0030] <<BLVワクチン> > The present invention also provides a BLV vaccine. The vaccine of the present invention is characterized by containing 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 (production of specific antibodies, proliferation of cytotoxic T cells, etc.), and promotes the production of neutralizing antibodies against BLV and the production of interferon, etc. in the subject, and can therefore 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.

[0031] 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 at least a gene encoding 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. Here, without being bound by the following theory, it is believed that in BLV infection, when gp51 binds to a receptor on the cell membrane surface of the host (subject), the membrane fusion activity of gp30 fuses the virus and the host cell membrane, thereby allowing the core of BLV to invade the host cell (Bai L, et al., FASEB Journal, 2019, fj201901528R.).

[0032] The vaccine of the present invention may contain a pharma- ceutically acceptable carrier, a lubricant, a preservative, a stabilizer, a wetting agent, an emulsifier, a salt for adjusting osmotic pressure, a buffer, a colorant, an antioxidant, a viscosity modifier, an activator (a concept that also includes carbonate apatite, sodium hydroxide, alum, incomplete / complete Freund's adjuvant, etc., as immunoactivators), or nanoparticles, etc. Examples of pharma- ceutically acceptable carriers include water, various salt solutions, alcohol, vegetable oil, and mineral oil.

[0033] Adjuvants (components that non-specifically stimulate a subject's immune response) that can be contained in the vaccine of the present invention include, in addition to the incomplete / complete Freund's adjuvants described above, 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), and the like.

[0034] Emulsifiers that can be included in the vaccines of the present invention include, for example, sorbitan monooleate (Span™ 80), polyoxyethylene-sorbitan monooleate (polysorbate 80, Tween™ 80), and emulsion stabilizers (e.g., benzyl alcohol, triethanolamine sugar) can also be used.

[0035] 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, a capsule, etc. The vaccine may be administered orally or parenterally (subcutaneously, nasally, intraperitoneally, intracapsularly, intramuscularly, intravenously, etc.).

[0036] In the present invention, a "subject" is a non-human animal or a non-human mammal, for example, a bovine ( Bos Taurus ), Zebu cow ( Bos Indicus ), water buffalo ( Bubalus bubalis), sheep, goats, pigs, mice, rats, rabbits, cats, monkeys, and other mammals. Cattle include dairy breeds, meat breeds, dual-purpose dairy and meat breeds, draft breeds, and dual-purpose draft breeds. Specific examples include Wagyu breeds such as Japanese Black Cattle and Japanese Shorthorn, Holstein, Jersey, and native breeds of each country. From the viewpoint of preventing or treating infectious bovine lymphoma, the subject is preferably cattle, zebu cattle, and water buffalo, more preferably cattle and water buffalo, and even more preferably cattle.

[0037] The inoculation amount of the vaccine of the present invention can be appropriately determined taking into consideration the sex, age, and body weight of the subject, the form of the vaccine, the mode of inoculation, etc. The inoculation amount of the vaccine of the present invention per adult cow is, for example, 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, 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, 580 μg, 590 μg, 600 μg, 610 μg, 620 μg, 630 μg, 640 μg, 650 μg, The upper limit can be 0 μg, 280 μg, 290 μg or 300 μg, and 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 manner, 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.

[0038] The inoculation amount 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 amount of the vaccine of the present invention per adult cow is the number of syncytia formed in CC81-GREMG cells (Sato H, et al., Virol J. 2019; 16(1): 66.) of the virus of the present invention contained per dose, with the lower limit (above or exceeding) being 1×10 2 , 2×10 2 , 3×10 2 , 4×10 2 , 5×10 2 , 6×10 2 , 7×10 2 , 8×10 2 , 9×10 2 , 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 or 1×10 5 The upper limit (less than or equal to) is 1×10 8 , 9×10 7 , 8×10 7 , 7×10 7 , 6×10 7 , 5×10 7 , 4×10 7 , 3×10 7 , 2×10 7 , 1×10 7 , 9×106 , 8×10 6 , 7×10 6 , 6×10 6 , 5×10 6 , 4×10 6 , 3×10 6 , 2×10 6 or 1×10 6 These lower and upper limits can be arbitrarily combined, for example, 1×10 2 More than 1×10 8 Below, 3 x 10 2 More than 7×10 7 Below, 5 x 10 2 More than 4×10 7 Below, 1×10 3 More than 1×10 7 Below, 3 x 10 3 More than 7×10 6 Below, 5 x 10 3 More than 4×10 6 or less than 1×10 4 More than 1×10 6 It can be as follows:

[0039] The inoculum dose of the vaccine of the invention per adult cattle can also be, for example, the TCID 50 The above TCID (Tissue Culture Infectious Dose 50) may be used as an index. 50 The lower limit (greater than or equal to) is 1×10 2 , 2×10 2 , 3×10 2 , 4×10 2 , 5×10 2 , 6×10 2 , 7×10 2 , 8×10 2 , 9×10 2 , 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×104 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 or 1×10 5 The upper limit (less than or equal to) is 1×10 8 , 9×10 7 , 8×10 7 , 7×10 7 , 6×10 7 , 5×10 7 , 4×10 7 , 3×10 7 , 2×10 7 , 1×10 7 , 9×10 6 , 8×10 6 , 7×10 6 , 6×10 6 , 5×10 6 , 4×10 6 , 3×10 6 , 2×10 6 or 1×10 6 These lower and upper limits can be arbitrarily combined, for example, 1×10 2 More than 1×10 8 Below, 3 x 10 2 More than 7×10 7 Below, 5 x 10 2 More than 4×10 7 Below, 1×10 3 More than 1×10 7 Below, 3 x 10 3 More than 7×10 6 Below, 5 x 10 3 More than 4×10 6 or less than 1×10 4 More than 1×10 6 It can be as follows:

[0040] In the present invention, the above-mentioned dose of the active ingredient can be administered once a day, or can be administered in two to four separate doses. The vaccine of the present invention can be administered multiple times depending on the subject in order to enhance and maintain the effect of vaccination, and the number of vaccinations may be increased each time the antibody titer in the subject decreases. In the present invention, the above-mentioned dose of the active ingredient can be administered multiple times, initially once every one, two, three or four weeks, and then once every one, two, three, four or four months.

[0041] According to another aspect of the present invention, there is provided a method for producing the vaccine of the present invention. The method for producing the vaccine of the present invention is characterized by comprising a step of culturing the cell of the present invention. The method for producing the virus of the present invention may further comprise a step of recovering the virus produced by the cell of the present invention. The method for producing the virus of the present invention may 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 it is.

[0042] The vaccine of the present invention can be used for the prevention or treatment of bovine infectious lymphoma. According to another aspect of the present invention, there is provided a method for preventing or treating bovine infectious lymphoma, comprising the step of inoculating a subject with the vaccine of the present invention.

[0043] The vaccine of the present invention and the method for producing the same, as well as the preventive and therapeutic methods of the present invention, can be carried out in accordance with the descriptions of the virus of the present invention and the method for producing the same, as well as the cells of the present invention and the method for producing the same, in addition to the above. EXAMPLES

[0044] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0045] Example 1: Establishment of a non-replicative BLV-producing cell line The present inventors previously constructed the CMVΔU3-pBLV-IF plasmid, which has a high BLV virus production amount as disclosed in JP 2019-24351 A, and constructed the CMVΔU3-pBLV-416 plasmid using the pBLV-416 plasmid cloned as a wild-type strain with a higher virus production amount 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 to attempt to establish a cell line with high BLV production. After cell passage and seven repeated clonings, we succeeded in producing a CMVΔU3-pBLV-416 plasmid-introduced mutant stable cell line (sometimes referred to as the "PK15-BLVΔRT cell line" in this specification) in which the virus produced retained infectivity but lost its replication and integration capabilities. We then analyzed the entire base sequence of the provirus integrated into this cell line, and confirmed that the RT region of the pol gene from 46 bp to 696 bp was deleted (see Figure 1).

[0046] Example 2: Study using pBLV-416ΔRT-transfected cells (1) In Example 2, a CMVΔU3-pBLV-416ΔRT plasmid was constructed by introducing a deletion from 46 bp to 696 bp into the RT region of the pol gene, and the viral proteins produced from cells transfected with this molecular clone (pBLV-416ΔRT-transfected cells) were analyzed.

[0047] (1) Method A Plasmid The CMVΔU3-pBLV-416 plasmid was prepared in the same manner as the preparation of 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.

[0048] B. Cells African green monkey kidney-derived cells (COS-1) were used as the cells to introduce the plasmid. COS-1 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1× Penicillin-Streptomycin-Glutamine (PSG) at 37°C in a CO2 incubator.

[0049] C. Transfection The reagents and plasmids shown in Tables 3 to 5 were used to treat COS-1 cells (5 × 10 5 The cells were transfected into the cells (60 mm dish) and the cells and culture supernatant were collected 48 hours after transfection. In the present specification, the transfected cells may be referred to as "transfected cells."

[0050] [Table 3]

[0051] [Table 4]

[0052] [Table 5]

[0053] D. Western blot The expression of viral proteins was confirmed by Western blot analysis using serum from BLV-infected and non-BLV-infected cattle.

[0054] (2) Results The results are shown in Figure 2. As a result of amplifying the pol gene region by PCR, a 650 bp shorter fragment was amplified in the pBLV-416ΔRT plasmid compared with pBLV-416, and it was confirmed that the region corresponding to 46 bp to 696 bp of the RT region of the pol gene was deleted (Figure 2A). In addition, Western blotting confirmed that the expression levels of structural proteins such as Gag protein (p24) and Env protein (gp51, gp30) due to the deletion of the pol gene were not affected in the BLV-infected bovine serum in the pBLV-416ΔRT-transfected cells compared with the pBLV-416-transfected cells (Figure 2B, left), whereas no viral proteins were detected in the BLV-uninfected bovine serum (Figure 2B, right).

[0055] Example 3: Study using pBLV-416ΔRT-transfected cells (2) In Example 3, the effects of deletion of the pol gene on the intracellular localization and expression level of viral proteins were examined using pBLV-416ΔRT-transfected cells.

[0056] (1) Method A Plasmid The same plasmid as in Example 2(1)A was used.

[0057] B. Cells COS-1 cells were used as in Example 2(1)(i).

[0058] C Western blot Viral proteins were identified by Western blotting using anti-Gag and anti-Env antibodies.

[0059] D. Immunofluorescence antibody technique The intracellular localization of viral proteins was confirmed by immunofluorescence using anti-Gag and anti-Env antibodies.

[0060] (2) Results The results are shown in Figures 3 and 4. In pBLV-416ΔRT-transfected cells, the deletion of the pol gene had no effect on the intracellular localization of Gag protein (p24) and Env protein (gp51) compared with pBLV-416-transfected cells (Figure 3), but the expression levels of both p24 and gp51 were confirmed to be increased (Figures 4A and B).

[0061] 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 examined.

[0062] (1) Method A. Syncytium formation ability The pBLV-416ΔRT or pBLV-416 plasmid was co-transfected with pEGFP-N1 plasmid (EGFP expression plasmid) into 293T cells, and the syncytium formation ability was evaluated. As a negative control, 293T cells were transfected with the empty vector pBluescript II KS(-) plasmid co-transfected with the EGFP-N1 plasmid.

[0063] B. Evaluation of cell-to-cell infectivity To evaluate the cell-to-cell infectivity, pBLV-416ΔRT or pBLV-416 plasmid was introduced into COS-1 cells and co-cultured with CC81-GREMG cells (BLV reporter cells). CC81-GREMG cells express EGFP in a BLV Tax-dependent manner, and BLV-producing cells and CC81-GREMG cells fuse with BLV Env protein to form syncytia, which express EGFP. This allows the cell-to-cell infectivity to be evaluated.

[0064] (2) Results The results are shown in Figures 5 and 6. Both pBLV-416ΔRT-introduced cells and pBLV-416-introduced cells were confirmed to form syncytia (Figure 5A). The number of syncytia was greater and the size of the syncytia was larger in the pBLV-416ΔRT-introduced cells than in the pBLV-416-introduced cells (Figure 5B). These results indicate that the deletion of the pol gene enhances the syncytium-forming ability in the pBLV-416ΔRT-introduced cells.

[0065] In addition, the number of syncytia formed was greater and the size of the syncytia was larger in pBLV-416ΔRT-transfected cells than in pBLV-416-transfected cells (Fig. 6A and B). These results suggest that the deletion of the pol gene may enhance the ability of cell-to-cell infection.

[0066] 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.

[0067] (1) Method A Plasmid The same plasmid as in Example 2(1)A was used.

[0068] B. Cells COS-1 cells were used as in Example 2(1)(i).

[0069] C Western blot Viral proteins were detected by Western blot using anti-Gag and anti-Env antibodies.

[0070] D. Amount of virus released To confirm the amount of virus produced by pBLV-416ΔRT-transfected cells 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 cells or pBLV-416-transfected cells and Tween 20 were added to the plate on which the serum was immobilized, and p24 was captured. The amount of captured p24 was then measured by colorimetry using anti-BLV p24 antibody (BLV3, VMRD) and HRP-labeled anti-mouse IgG antibody.

[0071] 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).

[0072] Infectiousness of mosquito viruses To evaluate the infectivity of the virus, we added virus-containing culture supernatant to CC81-GREMG cells and confirmed the formation of syncytia, and quantified viral p24 and gp51 by Western blot analysis using 2 μg of virus.

[0073] (2) Results The results are shown in Figure 7. In pBLV-416ΔRT-transfected cells, no difference in the amount of virus released due to deletion of the pol gene was observed compared to pBLV-416-transfected cells (Figure 7A). In addition, viruses released from pBLV-416ΔRT-transfected cells did not show reverse transcriptase activity (Figure 7B) and did not form syncytia (Figure 7C). Furthermore, Gag protein (p24) and Env protein (gp51) were detected in pBLV-416ΔRT-transfected cells, indicating that deletion of the pol gene does not affect virus maturation (Figure 7D).

[0074] Example 6: Study using pBLV-IFΔRT-transfected cells In Example 6, a CMVΔU3-pBLV-IFΔRT plasmid was constructed by introducing a deletion from 46 bp to 696 bp into the RT region of the pol gene, and the viral proteins produced from cells transfected with this molecular clone (pBLV-IFΔRT transfected cells) were analyzed.

[0075] (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).

[0076] B. Cells COS-1 cells and 293T cells were used as cells into which the plasmids were introduced.

[0077] C Western blot Viral proteins were identified by Western blotting using anti-Gag and anti-Env antibodies.

[0078] D. Syncytium formation ability The procedure was carried out in the same manner as in Example 4(1)A.

[0079] (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 to pBLV-IF2-transfected cells (293T cells) (Figure 9A). It was also confirmed that the number of syncytia was increased and the size of syncytia was increased in pBLV-IF2ΔRT-transfected cells compared to pBLV-IF2-transfected cells (Figure 9B).

[0080] 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 the deletion of the pol gene was observed (Fig. 9C).

[0081] Example 7: Study of infectious bovine lymphoma vaccine (1) In Example 7, the virus produced from the pBLV-416ΔRT-introduced cells was inoculated into mice to examine its effectiveness as a vaccine.

[0082] (1) Method A Plasmid The same plasmid as in Example 2(1)A was used.

[0083] B. Cells COS-1 cells were used as the cells into which the plasmid was introduced.

[0084] C Virus The culture supernatant of pBLV-416ΔRT- 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 and 4°C for 2 hours. The culture supernatant was removed and the precipitate was resuspended in PBS to purify the virus.

[0085] D. Inoculation into mice According to the schedule shown in Figure 10A, 100 μg of virus was inoculated into mice at weeks 0 and 2, and PBS was inoculated as a negative control (n=6 per group). Blood was collected from weeks 0 to 6 after inoculation, and the mice were euthanized at week 6 after inoculation, after which the spleens were removed. To confirm the proliferation of the virus in the mice, the BLV gene in blood cells at week 3 after inoculation was detected by nested PCR. In addition, spleen cells were co-cultured with CC81-GREMG cells, and the syncytium formation ability was evaluated. Furthermore, anti-p24 antibodies were quantified by ELISA from week 0 to week 6 after inoculation.

[0086] (2) Results The results are shown in Figure 10. In mice inoculated with the virus produced by pBLV-416ΔRT-transfected cells, the BLV gene was not detected (Figure 10B), and no syncytia were observed when spleen cells extracted from the mice were co-cultured with CC81-GREMG (Figure 10C). On the other hand, in mice inoculated with the virus produced by pBLV-416ΔRT-transfected cells, it was confirmed that anti-p24 antibodies increased over time (Figure 10D). These results suggest that the virus produced by pBLV-416ΔRT-transfected cells may be useful as a non-replicating BLV vaccine.

[0087] Example 8: Study using non-replicative BLV-producing cell lines (1) In Example 8, the entire base sequence of the provirus integrated into the PK15-BLVΔRT cell line successfully produced 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 FIG. 11). Compared to the analysis results in Example 1, the number of deleted bases in the RT region of the pol gene differed by 3 bases, but it is considered that further mutations were added during subculture after the establishment of the PK15-BLVΔRT cell line. In addition, compared to the base sequence of the wild-type BLV, 2 base mutations and 1 base deletion were confirmed after 696 bp of the pol gene, but these were not mutations that affected protein expression.

[0088] Example 9: Study using non-replicative BLV-producing cell lines (2) In Example 9, the expression and localization of viral proteins in cells, as well as the amount of released virus, were examined using the PK15-BLVΔRT cell line.

[0089] (1) Method A Western Blot Viral proteins were identified by Western blotting using anti-Gag and anti-Env antibodies.

[0090] B. Immunofluorescence antibody technique The intracellular localization of viral proteins was confirmed by immunofluorescence using anti-Gag and anti-Env antibodies.

[0091] C. Amount of virus released The amount of virus released from the PK15-BLVΔRT cell line was measured by the Capture ELISA method. 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 concentration of p24 was measured. Here, the concentration of viral p24 was calculated from a standard curve created using His-p24 antigen expressed and purified in E. coli as a standard (Figure 14B).

[0092] (2) Results The results were as shown in Figures 12 to 14. From the results in Figure 12, it was confirmed that the PK15-BLVΔRT cell line expressed p24 and gp51, which are structural proteins of the virus. In addition, compared to the positive control BLV persistently infected cells FLK-BLV, no change in molecular weight of p24 and gp51 due to deletion of the pol gene was observed. From the results in Figure 13, as with the positive control BLV persistently infected cells FLK-BLV, spotted accumulation was confirmed in the cells. From this result, it was confirmed that the deletion of the pol gene does not affect the intracellular localization of p24. From the results in Figure 14A, the concentration of p24 was 25.1 ± 1.2 μg, confirming that a sufficient amount of virus was released from the PK15-BLVΔRT cell line. From these results, it was shown that the virus produced from the PK15-BLVΔRT cell line may be useful as a non-replicative BLV vaccine.

[0093] Example 10: Study of bovine infectious lymphoma vaccine (2) In Example 10, the virus produced from PK15-BLVΔRT cells was inoculated into mice to examine its effectiveness as a vaccine.

[0094] (1) Method A virus The culture supernatant of PK15-BLVΔRT cells was centrifuged at 3000 rpm for 10 minutes to remove cell debris, and then ultracentrifuged at 141,118 × g and 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.

[0095] B. Inoculation into mice The virus solution prepared in the above (A) was mixed with Montanide (Montanide Gel 01, SEPPIC) as an adjuvant to give a mixture of 200μL (500μg total virus protein, Montanide 10%) and subcutaneously inoculated into mice (BALB / cCrSlc, 4-week-old, female) at 0 and 2 weeks. PBS was 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.

[0096] 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 two-fold from 25-fold to 200-fold with phosphate buffered saline (PBS). (iii) 50 μl of the diluted serum was mixed with 50 μl of a 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 onto the well plate in (i) above at 100 μl / well 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).

[0097] (2) Results The results are shown in Figures 15 and 16. In mice inoculated with the virus produced from PK15-BLVΔRT cells (PK15-BLVΔRT), the production of anti-p24 antibodies was induced (Figure 15). In addition, it was observed that the neutralizing antibody titers of the serum collected from the mice increased 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.

[0098] Example 11: Study of bovine infectious lymphoma vaccine (3) In Example 11, the virus produced from PK15-BLVΔRT cells was inoculated into cattle to examine its effectiveness as a vaccine.

[0099] (1) Method A virus The culture supernatant of PK15-BLVΔRT cells was centrifuged at 3000 rpm for 10 minutes to remove cell debris, and then ultracentrifuged at 141,118 × g and 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.

[0100] B. Inoculation of cattle The virus solution prepared in the above step (A) was mixed with Montanide (Montanide Gel 01, SEPPIC) as an adjuvant to give a mixture of 3 mL (virus solution: 2.7 mL (containing 66 μg of p24 protein), Montanide 0.3 mL) and inoculated subcutaneously (buttocks) into a cow (Japanese black cattle, 4 months old, male, 100-120 kg) at weeks 0 and 2, respectively, and blood was collected from 0 to 6 weeks 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.). In addition, 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., and lymphocytes were quantified using Celltac α (Nihon Kohden Corporation).

[0101] In addition, the neutralizing antibody titer of serum collected from the cows 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 two-fold from 25 to 800 times with phosphate buffered saline (PBS). (iii) 50 μl of the diluted serum was mixed with 50 μl of a 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 onto the well plate in (i) above at 100 μl / well 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).

[0102] (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 count was observed before and after inoculation (Figure 20). In addition, it was observed that the neutralizing antibody titer of serum collected from the cattle increased 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 in which at least a portion of the function of the pol gene is deleted.

4. A method for preventing or treating BLV, comprising a step of administering the vaccine described in claim 1 or 2 to a subject (provided that the subject does not include humans).

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