Non-replicating bovine lymphoma virus (BLV) and its producing cells

Abstract

The purpose of the present invention is to provide: a novel non-replicating bovine infectious lymphoma virus (BLV); and a cell for producing the same. According to the present invention, provided is a bovine infectious lymphoma virus (BLV) in which at least part of the function of pol genes is lost. Moreover, according to the present invention, provided is a non-replicating BLV-producing cell which contains genes of the BLV in which at least part of the function of pol genes is lost. The present invention is advantageous in that a BLV vaccine having high immunogenicity and high safety in which replication does not occur in an infected subject can be provided.

Description

Cross-reference to related applications

[0001] This application claims the benefit of priority of Japanese Patent Application No. 2022-69710 (filing date: April 20, 2022), the entire disclosure of which is incorporated herein by reference.

Technical Field

[0002] The present invention relates to non-replicating bovine leukemia virus (BLV) and its producing cells.

Background Art

[0003] Bovine leukemia virus (BLV) is the causative virus of enzootic bovine leukosis (EBL), a malignant B-lymphoma, and is a retrovirus that integrates as a provirus into the DNA of host cells. Approximately 70% of BLV-infected cattle are asymptomatic and healthy, approximately 30% have persistent lymphocytosis, and after a long latency period, approximately a few percent develop enzootic bovine leukosis (Non-Patent Documents 1 and 2).

[0004] In recent years, EBL has continued to increase, but no vaccine with a preventive effect against EBL has been developed. One of the main factors is that the virus production amount from BLV-infected cells is extremely low, about 1 / 1000 to 1 / 10000 of the amount of the AIDS virus belonging to the same Retroviridae family, making it difficult to develop inactivated vaccines and cell-derived vaccines. So far, attempts have been made to develop expression vectors for increasing the production amount of BLV (Patent Document 1), but since BLV, a retrovirus, has a risk of integrating viral genes into the host genome after infection, problems in the practical application of live vaccines still remain.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006] [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. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a novel non-replicating bovine lymphoma virus (BLV) and its producing cells. [Means for solving the problem]

[0008] The inventors have now succeeded in establishing a virus-producing cell line that produces bovine lymphoma virus (BLV) that is infectious but has lost its replication ability. The inventors also analyzed the gene sequence of the virus produced by this cell line and found that a portion of the pol gene was deleted. Furthermore, the inventors found that cells into which a BLV gene with a portion of the pol gene was introduced (pBLV-416ΔRT-introduced cells) showed increased expression and release of the virus produced, as well as enhanced syncytium formation ability, compared to control cells (pBLV-416-introduced cells). The inventors also found that when this virus was inoculated into mice, anti-p24 antibodies increased over time, while the BLV gene was not detected. The inventors also investigated using a non-replicating BLV-producing cell line (PK15-BLVΔRT cell line) and found that the virus produced from this cell line may be useful as a non-replicating BLV vaccine. The present invention is based on these findings.

[0009] The present invention provides the following inventions. [1] Bovine lymphoma virus (BLV) in which at least part of the function of the pol gene is lost. [2] The virus described in [1] above, wherein the function of the pol gene is the function of a reverse transcriptase and / or integrase. [3] The viruses described in [1] or [2] above, wherein at least a portion of the pol gene is mutated. [4] The virus as described in [3] above, wherein the mutation is a deletion of 1 to 1643 bases in the reverse transcriptase region of the pol gene and / or 1 to 894 bases in the integrase region of the pol gene. [5] The virus as described in [3] above, wherein the mutation is a mutation in the bases corresponding to the 1st to 1643rd bases from the 5' end and / or the 1644th to 2537th bases in the base sequence of the pol gene shown in Sequence ID No. 2. [6] Any of the viruses described in [1] to [5] above that do not replicate in the infected subject. [7] Non-replicating BLV-producing cells comprising a gene for bovine lymphoma virus (BLV) in which at least part of the function of the pol gene is lost. [8] A method for producing non-replicating BLV, comprising the step of culturing the cells described in [7] above. [9] A BLV vaccine containing any of the viruses described in [1] to [6] above.

[10] A method for preventing or treating BLV, comprising the step of administering an inoculation targeting any of the viruses described in [1] to [6] above or the vaccine described in [9] above (excluding humans from the target).

[11] A method for producing non-replicating BLV-producing cells, comprising the step of introducing a gene of bovine lymphoma virus (BLV) in which at least part of the function of the pol gene is deficient into host cells.

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

[0011] [Figure 1]Figure 1 shows the genetic makeup of wild-type BLV, and also illustrates the deletion of the pol gene in the CMVΔU3-pBLV-416ΔRT plasmid (pBLV-416ΔRT) in comparison with the CMVΔU3-pBLV-416 plasmid (pBLV-416). [Figure 2] Figure 2A shows the results of PCR amplification of the pol gene region of pBLV-416ΔRT and pBLV-416, respectively. The pBluescript II KS(-) plasmid, an empty vector, was used as the negative control, and FLK-BLV cells, which are persistently infected with BLV, were used as the positive control. Figure 2B shows the results of Western blotting on the expression of viral proteins produced by pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, using BLV-infected cattle serum (left) and BLV-uninfected cattle serum (right), respectively. The pBluescript II KS(-) plasmid-transformed cells, an empty vector, were used as the negative control, and FLK-BLV cells, which are persistently infected with BLV, were used as the positive control. [Figure 3] Figure 3 shows the results of immunofluorescence assay for the intracellular localization of viral proteins in pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Cells transduced with the empty vector pBluescript II KS(-) plasmid were used as the negative control, and FLK-BLV cells, which are persistently infected BLV cells, were used as the positive control. [Figure 4] Figure 4 shows the results of Western blots on viral protein expression levels in pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Cells transduced with the empty vector pBluescript II KS(-) plasmid were used as the negative control, and FLK-BLV cells, which are persistently infected with BLV, were used as the positive control. Student's t-test results show * for p<0.05 and ** for p<0.01. [Figure 5]Figure 5 shows the results of syncytium formation ability in pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Cells transformed with the empty vector pBluescript II KS(-) plasmid were used as a negative control. Student's t-test results show that ** indicates p<0.01 and *** indicates p<0.001. [Figure 6] Figure 6 shows the results of intercellular infectivity in pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Cells transformed with the empty vector pBluescript II KS(-) plasmid were used as negative controls, and FLK-BLV cells, which are persistently infected with BLV, were used as positive controls. [Figure 7] Figure 7A shows the release levels of viral proteins produced by pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Figure 7B shows the reverse transcriptase activity of viral proteins produced by pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Figure 7C shows the results of syncytium formation ability in pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Figure 7D shows the results of Western blotting for viral proteins released from pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, respectively. Cells transformed with the empty vector pBluescript II KS(-) plasmid were used as the negative control, and FLK-BLV cells, which are persistently infected BLV cells, were used as the positive control. Student's t-test results indicate p<0.001 where ***. [Figure 8] Figure 8 shows the genetic makeup of wild-type BLV, and also illustrates the deletion of the pol gene in the CMVΔU3-pBLV-IFΔRT plasmid (pBLV-IFΔRT) in comparison with the CMVΔU3-pBLV-IF plasmid (pBLV-IF). [Figure 9]Figure 9A shows the results of Western blotting for the expression levels of viral proteins in pBLV-IFΔRT-introduced cells and pBLV-IF-introduced cells, respectively. Figure 9B shows the results of syncytium formation ability in pBLV-IFΔRT-introduced cells and pBLV-IF-introduced cells, respectively. Figure 9C shows the results of measuring the amount of virus in the culture supernatants of pBLV-IFΔRT-introduced cells and pBLV-IF-introduced cells by Capture ELISA, respectively. Note that cells transfected with the empty vector pBluescript II KS(-) plasmid were used as negative controls, and FLK-BLV cells, which are BLV persistently infected cells, were used as positive controls. As a result of Student's t-test, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001, respectively. [Figure 10] Figure 10A shows the schedule of an experiment in which mice were inoculated with viral proteins produced by pBLV-416ΔRT-introduced cells and pBLV-416-introduced cells. Figure 10B shows the results of the Nested PCR method for the BLV gene in blood cells at 3 weeks after inoculation. Figure 10C shows the results of co-culturing spleen cells excised from mice with CC81-GREMG cells. Figure 10D shows the results of quantifying 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 BLV persistently infected cells, were used as a positive control. As a result of Student's t-test, * indicates p < 0.05. [Figure 11] Figure 11 shows the gene constitution of wild-type BLV and shows the deleted part of the pol gene of BLV in the PK15-BLVΔRT cell line in comparison with the CMVΔU3-pBLV-416 plasmid (pBLV-416). [Figure 12] Figure 12 shows the results of Western blotting for the expression level of viral proteins in the PK15-BLVΔRT cell line. Note that FLK-BLV cells, which are BLV persistently infected cells, were used as a positive control. [Figure 13]Figure 13 shows the results of an immunofluorescence antibody method for the intracellular localization of viral proteins in the PK15-BLVΔRT cell line. For the positive control, FLK-BLV cells, which are BLV persistently infected cells, were used. [Figure 14] Figure 14A shows the release amount of viral proteins produced by the PK15-BLVΔRT cell line. Figure 14B shows a standard curve prepared using His-p24 antigen as a standard. Detailed description of the invention

[0012] <<Virus and method for producing the same>> "Bovine leukemia virus (BLV)" is the causative virus of enzootic bovine leukosis (EBL), a malignant B lymphoma, and is a retrovirus that integrates as a provirus into the DNA of host cells. The full length of the genome of wild-type BLV is approximately 8720 bp, and the genes are in a form sandwiched between two identical long terminal repeat (LTR) sequences, and consist of the gag, pro, pol, and env genes that encode structural proteins, the rex and tax genes that encode regulatory proteins, and the R3 and G4 genes that encode accessory proteins (see Figure  1). Here, the gag gene encodes a protein involved in virus particle formation, the pol gene encodes reverse transcriptase and integrase, and the env gene encodes an envelope protein involved in adsorption and invasion of 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 part shows the sequence encoding the pol gene). In the present invention, wild-type BLV sequences other than the gene sequence of Table 1 can be used.

[0013]

Table 1

[0014] The present invention provides a bovine lymphoma virus (BLV) in which at least a portion of the function of the pol gene is deficient. In the present invention, "at least a portion of the function of the pol gene is deficient" means that at least a portion of the function of the pol gene is deficient compared to wild-type BLV, and specifically means that at least a portion of the function of reverse transcriptase and / or integrase is deficient. That is, the virus of the present invention does not express reverse transcriptase and / or integrase, or if expressed, these enzymes do not function normally and do not replicate in infected subjects. For this reason, the virus of the present invention may be referred to as "non-replicating BLV" in this specification.

[0015] Whether or not at least part of the function of the pol gene is lost can be determined by preparing a BLV in which at least part of the pol gene is mutated, and then determining whether the protein encoded by the pol gene is functioning normally by checking whether the prepared virus expresses the protein or whether the protein has enzymatic activity. 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, respectively. 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 transferability as an indicator.

[0016] The virus of the present invention has a mutation in at least a portion of the pol gene. In the present invention, "a mutation in at least a portion of the pol gene" means that, compared to wild-type BLV, at least a portion of the nucleotide sequence of the pol gene has mutated, and that the mutation results in a state in which at least a portion of the function of the pol gene is lost. A mutation in the pol gene means a mutation in which deletion, substitution, insertion and / or addition occurs in at least a portion of the nucleotide sequence of the pol gene, resulting in a state in which at least a portion of the function of the pol gene is lost. In the virus of the present invention, the function of the pol gene that is lost is either the function of reverse transcriptase and / or the function of integrase, or both, preferably the function of reverse transcriptase. That is, the virus of the present invention can be said to be a BLV in which the reverse transcriptase region (RT) and / or integrase region (IN) encoded by the pol gene has mutated, or a BLV having a pol gene in which the reverse transcriptase region (RT) and / or integrase region (IN) has mutated. A typical example of the virus of the present invention is a BLV having a pol gene in which a portion of the RT (e.g., 1-1643 bases, 1-1600 bases, 1-1500 bases, 1-1400 bases, 1-1300 bases, 1-1200 bases, 1-1100 bases, 1-1000 bases, 1-900 bases, 1-850 bases, 1-800 bases, 10-750 bases, 20-700 bases, 40-650 bases) and / or a portion of the IN (e.g., 1-894 bases, 1-850 bases, 1-800 bases, 10-750 bases, 20-700 bases, 40-650 bases) is deleted. Here, the base deletions in the RT and / or IN may be consecutive base deletions or non-consecutive base deletions.

[0017] In the virus of the present invention, mutations in a portion of the pol gene sequence are not limited to those that result in a loss of at least a portion of the pol gene's function. However, if the pol gene is the wild-type pol gene shown in SEQ ID NO: 2 (Table 2), then the RT mutations may occur from the 5' end of the pol gene sequence shown in SEQ ID NO: 2 in Table 2, for example, at positions 1-1643, 1-1600, 1-1500, 1-1400, 1-1300, 1-1200, 1-1100, 1-1000, and 10 The mutations can be at bases ~900, 20~800, 30~700, or 40~650. As IN mutations, starting from the 5' end of the pol gene sequence shown in Sequence ID No. 2, the mutations can be at bases such as 1644~2537, 1650~2500, 1700~2400, 1800~2300, 1700~2200, 1650~2100, 1650~2000, 1650~1900, or 1650~1800. If the pol gene consists of a nucleotide sequence other than the wild-type pol gene in Sequence ID No. 2 (Table 2), the mutations can be at bases corresponding to the bases in Sequence ID No. 2. For example, this can be identified by appropriately aligning the pol gene sequence to be mutated with the nucleotide sequence of Sequence ID No. 2. In other words, the mutation site can be identified by aligning the base sequence of the pol gene targeted for mutation with the base sequence of Sequence 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)).

[0018] Note that the nucleotide sequence of the pol gene in SEQ ID NO: 2 in Table 2 is shown in the direction from the 5' end to the 3' end, and corresponds to the nucleotide sequence of the underlined part of SEQ ID NO: 1 in Table 1. Furthermore, the number of RT mutations or 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 1,000, 900, 800, 700, 600, or 500. These lower and upper limits can be combined arbitrarily, 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.

[0019] In the virus of the present invention, the mutation in the pol gene sequence may also be a mutation spanning RT and IN. If the pol gene is the wild-type pol gene shown in Sequence ID No. 2 (Table 2), such mutations can occur in the bases at positions 1-2537, 10-2500, 20-2400, 30-2300, 40-2200, 50-2100, 60-2000, 70-1900, 80-1850, 90-1800, 100-1750, 200-1700, 300-1650, 400-1650, 500-1650, 600-1650, 700-1650, 800-1650, 900-1650, or 1000-1650, starting from the 5' end of the pol gene sequence shown in Sequence ID No. 2 in Table 2. If the pol gene consists of a base sequence other than that of the wild-type pol gene in Sequence ID No. 2 (Table 2), the mutation can be attributed to a base corresponding to the base in Sequence ID No. 2. As mentioned 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 Sequence ID No. 2.

[0020] The number of mutations spanning RT and IN described above 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 such 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 such 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 way, and the resulting 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.

[0021] [Table 2] TIFF0007853724000007.tif151155

[0022] The virus of the present invention may have the entire sequence of the wild-type BLV sequence from 5'LTR to 3'LTR shown in Sequence ID No. 1, excluding the pol gene, insofar as at least a portion of the function of the pol gene is missing and the functions of BLV genes other than the pol gene are preserved, 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 when the sequences to be compared are appropriately aligned, and means the percentage of exact amino acid matches between the sequences. In calculating identity, for example, the presence of gaps in the sequence and the properties of amino acids are taken into consideration (Wilbur, Natl. Acad. Sci. USA 80:726-730 (1983)). The aforementioned 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)), and Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Furthermore, identity can be calculated, for example, using a publicly available homology search program as described above. For example, it can be calculated using the BLAST homology algorithm (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) from the National Center for Biotechnology Information (NCBI) using default parameters.

[0023] The virus of the present invention can be obtained by producing it in cells having a BLV gene in which at least a portion of the function of the pol gene is deficient. The origin of these cells is not limited, but they may be cells from natural occurrences, cells obtained by self-cloning, or cells produced by genetic engineering technology. However, from the viewpoint of use as a vaccine, cells obtained from natural occurrences or by self-cloning are preferably used.

[0024] <<Cells and methods for producing them>> The present invention also provides cells comprising a gene for bovine lymphoma virus (BLV) in which at least a portion of the function of the pol gene is deficient. Here, "comprising the gene" means that the gene is expressibly contained in the cell, and typically refers to a state in which the gene has been introduced into the cell and transformed. The cells of the present invention may be cells of natural occurrence, cells obtained by self-cloning, or cells produced by genetic engineering technology, as long as they contain a polynucleotide encoding BLV in which at least a portion of the function of the pol gene is deficient. From the viewpoint of use as a vaccine, cells obtained by natural occurrence or self-cloning are preferably used.

[0025] 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 by genetic engineering technology, for example, they can be produced by introducing an expression vector into host cells into which a polynucleotide encoding BLV in which at least part of the function of the pol gene is lost is incorporated. To lose the function of the pol gene in BLV, all or part of the RT and / or IN encoded by the pol gene may be deleted. Alternatively, the gene may be mutated by substitution, deletion, insertion and / or addition of bases in the pol gene so that normal reverse transcriptase or integrase is not expressed. Or, a foreign gene may be inserted into the RT or IN. Gene deletion or mutation and insertion of foreign genes can be carried out, for example, by known homologous recombination or site-directed mutagenesis.

[0026] The host cells that produce the BLV of the present invention are not particularly limited as long as they are capable of transcribing and translating the expression vector, but examples 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), and mammalian cells are preferred.

[0027] The transformation of host cells can be appropriately selected depending on the type of host cell, and can be carried out by methods such as electroporation, lithium acetate, calcium phosphate, lipofection, and particle gun.

[0028] The cells of the present invention can be used in accordance with the description of the virus of the present invention, in addition to the above.

[0029] <<Virus manufacturing method>> The present invention also provides a method for producing a virus. The method for producing a virus of the present invention includes the step of culturing the cells of the present invention. The method for producing a virus of the present invention may further include the step of recovering the virus produced by the cells of the present invention. The recovered virus may be purified as necessary. The purification method can be appropriately selected from known methods depending on the virus to be produced. In the case of a virus that accumulates in host cells, the host cells may be lysed and then purified. In the case of a virus that is released outside of host cells, for example, the virus can be purified and recovered from the culture supernatant of the host cells. The method for producing a virus of the present invention can also be carried out in accordance with the description of the virus of the present invention, the cells of the present invention, and the method for producing them, in addition to the above.

[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 to BLV (such as the production of specific antibodies and the proliferation of toxic T cells), and promotes the production of neutralizing antibodies against BLV and the production of interferon, etc., in the subject, and can therefore be used for the prevention of BLV infection and the treatment (or improvement of symptoms) of EBL.

[0031] The virus contained in the vaccine of the present invention preferably has at least a gene encoding a structural protein, more preferably at least p24 of the gag gene, and more preferably at least one of gp51 and gp30 of the env gene, and even more preferably at least gp51. Herein, although not bound by the following theory, it is thought that in BLV infection, when gp51 binds to a receptor on the surface of the host (target) cell membrane, the membrane fusion activity of gp30 causes the virus and the host cell membrane to fuse, thereby allowing the BLV core to enter the host cell (Bai L, et al., FASEB Journal, 2019, fj201901528R.).

[0032] In the vaccine of the present invention, the amount of the virus can be appropriately set depending on the target animal to be vaccinated, the method of vaccination, and the form of vaccination. For example, if the target animal is a cow, the amount can be 0.01 mg to 10 mg (preferably 0.5 mg to 5 mg) per animal. Vaccination with the vaccine of the present invention can be performed once, twice, or three or more times depending on the target animal, and if the antibody titer decreases in the target animal, the number of vaccinations may be increased each time.

[0033] The vaccine of the present invention may contain pharmaceutically acceptable carriers, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for osmotic pressure adjustment, buffers, colorants, antioxidants, viscosity modifiers, activators (including apatite carbonate, sodium hydroxide, alum, incomplete / complete Freund's adjuvants, etc. as immunostimulants), or nanoparticles. Examples of pharmaceutically acceptable carriers include water, various salt solutions, alcohols, vegetable oils, and mineral oils.

[0034] Examples of the vaccine of the present invention include injections, liquids, suspensions, emulsions, powders, granules, and capsules. The vaccine may be administered orally, or parenterally (subcutaneous, nasal, intraperitoneal, capsule, intramuscular, or intravenous).

[0035] The vaccine of the present invention can be used for the prevention or treatment of bovine lymphoma. According to another aspect of the present invention, a method for the prevention or treatment of bovine lymphoma is provided, comprising an inoculation step with the vaccine of the present invention.

[0036] In the present invention, the "target" is a non-human animal or non-human mammal, such as cattle (Bos taurus), zebu (Bos indicus), water buffalo (Bubalus bubalis), sheep, goats, pigs, mice, rats, rabbits, cats, and monkeys. Cattle include dairy breeds, meat breeds, dual-purpose dairy and meat breeds, draft breeds, and dual-purpose draft and meat breeds. Specifically, breeds include Japanese Black cattle, Japanese Shorthorn cattle, Holstein, Jersey, and native breeds of various countries. From the viewpoint of preventing or treating bovine infectious lymphoma, the target is preferably cattle, zebu and water buffalo, more preferably cattle and water buffalo, and even more preferably cattle.

[0037] In addition to the above, the vaccine of the present invention can be carried out in accordance with the description of the virus and method for producing the same of the present invention, as well as the cells and method for producing the same of the present invention. [Examples]

[0038] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0039] Example 1: Establishment of a non-replicating BLV-producing cell line The inventors previously constructed a CMVΔU3-pBLV-IF plasmid with high BLV virus production, as disclosed in Japanese Patent Publication No. 2019-24351. However, they constructed the CMVΔU3-pBLV-416 plasmid using a pBLV-416 plasmid cloned as a wild-type strain with even higher virus production as the parent material. The CMVΔU3-pBLV-416 plasmid was prepared in the same manner as the CMVΔU3-pBLV-IF plasmid described in Japanese Patent Publication No. 2019-24351. When the CMVΔU3-pBLV-416 plasmid was introduced into PK15 cells and an attempt was made to establish a cell line with high BLV production, after cell passage and seven cloning cycles, a stable cell line of CMVΔU3-pBLV-416 plasmid-transformed mutant (hereinafter sometimes referred to as the "PK15-BLVΔRT cell line") was successfully created in which the produced virus retained its infectivity but lost its replication and integration capabilities. Analysis of the entire nucleotide sequence of the provirus incorporated into this cell line revealed a deletion in the RT region of the pol gene from 46 bp to 696 bp (see Figure 1).

[0040] Example 2: Study using pBLV-416ΔRT-transformed cells (1) In Example 2, a CMVΔU3-pBLV-416ΔRT plasmid was created by introducing a deletion from 46 bp to 696 bp in the RT region of the pol gene, and the viral protein produced from cells into which this molecular clone was introduced (pBLV-416ΔRT-introduced cells) was analyzed.

[0041] (1) Method A plasmid The CMVΔU3-pBLV-416 plasmid was prepared in the same manner as the CMVΔU3-pBLV-IF plasmid described in Japanese Patent Publication No. 2019-24351. 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.

[0042] I Cell African green monkey kidney cells (COS-1) were used as the cells into which the plasmid was introduced. The COS-1 cells were maintained at 37°C in a CO2 incubator on DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin-glutamine (PSG).

[0043] Transfection Using the reagents and plasmids shown in Tables 3-5, COS-1 cells (5 × 10) were used. 5 Cells were transfected (in a 60mm dish), and the cells and culture supernatant were collected 48 hours after transfection. In this specification, transfected cells may be referred to as "transfected cells."

[0044] [Table 3]

[0045] [Table 4]

[0046] [Table 5]

[0047] Western blot Viral protein expression was confirmed by Western blotting using serum from BLV-infected and BLV-uninfected cattle.

[0048] (2) Results The results are shown in Figure 2. PCR amplification of the pol gene region revealed that the pBLV-416ΔRT plasmid amplified a fragment 650 bp shorter than that of pBLV-416, and that a region corresponding to 46 bp to 696 bp of the RT region of the pol gene was deleted (Figure 2A). Furthermore, Western blotting results showed that in BLV-infected cattle serum, pBLV-416ΔRT-transformed cells did not show any effect on the expression levels of structural proteins such as Gag protein (p24) and Env proteins (gp51, gp30) due to the deletion of the pol gene compared to pBLV-416-transformed cells (Figure 2B left), while no viral proteins were detected in BLV-uninfected cattle serum (Figure 2B right).

[0049] Example 3: Study using pBLV-416ΔRT-transformed cells (2) In Example 3, we investigated the effects of deletion of the pol gene on the intracellular localization and expression levels of viral proteins using pBLV-416ΔRT-transformed cells.

[0050] (1) Method A plasmid A plasmid similar to that used in Example 2(1) was employed.

[0051] I Cell COS-1 cells were used in the same manner as in Example 2(1)(i).

[0052] Western blot Viral proteins were identified by Western blotting using anti-Gag antibodies and anti-Env antibodies.

[0053] E. Immunofluorescence antibody method The intracellular localization of viral proteins was confirmed using immunofluorescence assays with anti-Gag and anti-Env antibodies.

[0054] (2) Results The results are shown in Figures 3 and 4. In pBLV-416ΔRT-transformed cells, no effect on the intracellular localization of Gag protein (p24) and Env protein (gp51) due to the deletion of the pol gene was observed compared to pBLV-416-transformed cells (Figure 3), but increased expression levels of both p24 and gp51 were confirmed (Figures 4A and 4B).

[0055] Example 4: Study using pBLV-416ΔRT-transformed cells (3) In Example 4, the syncytium-forming ability and intercellular infectivity of viruses produced from pBLV-416ΔRT-introduced cells were examined.

[0056] (1) Method A synthium-forming ability The pBLV-416ΔRT plasmid or the pBLV-416 plasmid was introduced into 293T cells along with the pEGFP-N1 plasmid (EGFP expression plasmid), and syncytium formation ability was evaluated. As a negative control, 293T cells introduced with the empty vector pBluescript II KS(-) plasmid along with the EGFP-N1 plasmid were used.

[0057] (i) Evaluation of intercellular (cell-to-cell) infectiousness To evaluate intercellular infectivity, pBLV-416ΔRT plasmid or pBLV-416 plasmid was introduced into COS-1 cells and co-cultured with CC81-GREMG cells (BLV reporter cells). Here, CC81-GREMG cells express EGFP in a BLV tax-dependent manner. When BLV-producing cells and CC81-GREMG cells fuse due to the BLV Env protein, syncytium is formed, and syncytium expressing EGFP is observed, thus allowing evaluation of intercellular infectivity.

[0058] (2) Results The results are shown in Figures 5 and 6. Both pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells were confirmed to form syncytium (Figure 5A). Compared to pBLV-416-transformed cells, pBLV-416-transformed cells were found to have a larger number and size of syncytium (Figure 5B). These results indicate that deletion of the pol gene enhances syncytium formation in pBLV-416ΔRT-transformed cells.

[0059] Furthermore, regarding intercellular infectivity, cells introduced with pBLV-416ΔRT showed a greater number of syncytium molecules formed and larger syncytium sizes compared to cells introduced with pBLV-416 (Figures 6A and B). These results suggest that deletion of the pol gene may enhance intercellular infectivity.

[0060] Example 5: Study using pBLV-416ΔRT-transformed cells (4) In Example 5, the amount of virus produced and the reverse transcriptase activity from cells introduced with pBLV-416ΔRT were analyzed.

[0061] (1) Method A plasmid A plasmid similar to that used in Example 2(1) was employed.

[0062] I Cell COS-1 cells were used in the same manner as in Example 2(1)(i).

[0063] Western blot Viral proteins were detected by Western blotting using anti-Gag antibodies and anti-Env antibodies. E. Virus release amount To confirm the viral production of pBLV-416ΔRT-transformed cells and pBLV-416-transformed cells, the amount of p24 in the culture supernatant was measured by Capture ELISA. The specific procedure was as follows: BLV-positive serum was diluted in carbonate buffer adjusted to pH 9.0 and fixed in a 96-well plate. Next, the culture supernatant of pBLV-416ΔRT-transformed cells or pBLV-416-transformed cells and Tween20 were added to the serum-fixed plate, and p24 was captured. The amount of captured p24 was then measured colorimetrically using anti-BLV p24 antibody (BLV3, VMRD) and HRP-labeled anti-mouse IgG antibody.

[0064] O Reverse transcriptase activity The viruses released into the culture supernatant were recovered by ultracentrifugation, and the reverse transcriptase activity in the viral particles was measured using a reverse transcriptase assay and colorimetric analysis kit (Roche).

[0065] Infectivity of the virus To evaluate the infectivity of the virus, the culture supernatant containing the virus was added to CC81-GREMG cells, and syncytium formation was confirmed. In addition, p24 and gp51 in the virus were quantified by Western blotting using 2 μg of virus.

[0066] (2) Results The results are shown in Figure 7. In pBLV-416ΔRT-introduced cells, no difference in viral release due to pol gene deletion was observed compared to pBLV-416-introduced cells (Figure 7A). Furthermore, the virus released from pBLV-416ΔRT-introduced cells did not show reverse transcriptase activity (Figure 7B) and did not form syncytium (Figure 7C). In addition, Gag protein (p24) and Env protein (gp51) were detected in pBLV-416ΔRT-introduced cells, indicating that pol gene deletion does not affect viral maturation (Figure 7D).

[0067] Example 6: Study using pBLV-IFΔRT-transformed cells In Example 6, a CMVΔU3-pBLV-IFΔRT plasmid was constructed by introducing a deletion from 46 bp to 696 bp in the RT region of the pol gene, and the viral protein produced from cells into which this molecular clone was introduced (pBLV-IFΔRT-introduced cells) was analyzed.

[0068] (1) Method A plasmid The CMVΔU3-pBLV-IF2 plasmid was prepared in accordance with the description in Japanese Patent Publication No. 2019-24351. 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.) (Figure 8).

[0069] I Cell COS-1 cells and 293T cells were used to introduce the plasmid.

[0070] Western blot Viral proteins were identified by Western blotting using anti-Gag antibodies and anti-Env antibodies.

[0071] E syncytium-forming ability The procedure was carried out in the same manner as in Example 4(1)A.

[0072] (2) Results The results are shown in Figure 9. Compared to pBLV-IF2ΔRT-transformed cells (293T cells), the expression levels of Gag protein (p24) and Env protein (gp51) were increased in pBLV-IF2-transformed cells (293T cells) (Figure 9A). In addition, compared to pBLV-IF2-transformed cells, the number of syncytium molecules increased and the syncytium size increased in pBLV-IF2-transformed cells (Figure 9B).

[0073] In pBLV-IF2ΔRT-transformed cells (COS-1 cells), the same amount of Gag protein (p24) as in pBLV-IF2-transformed cells (COS-1 cells) was detected by Capture ELISA, and no effect from pol gene deletion was observed (Figure 9C).

[0074] Example 7: Examination of bovine lymphoma vaccine In Example 7, the effectiveness of a virus produced from pBLV-416ΔRT-introduced cells as a vaccine was investigated by inoculating mice with the virus.

[0075] (1) Method A plasmid A plasmid similar to that used in Example 1(1) was employed.

[0076] I Cell COS-1 cells were used to introduce the plasmid.

[0077] U virus The cell fragments were removed from the culture supernatant of pBLV-416ΔRT-introduced cells or pBLV-416-introduced cells by centrifugation at 3000 rpm for 10 minutes, followed by ultracentrifugation at 141,118 × g, 4°C, for 2 hours. After removing the culture supernatant, the precipitate was resuspended in PBS to purify the virus.

[0078] Inoculation into mice As shown in Figure 10A, 100 μg of the virus was inoculated into mice at weeks 0 and 2, and PBS was administered as a negative control (n=6 in each group). Blood samples were collected from weeks 0 to 6 after inoculation, and the mice were euthanized at week 6, followed by splenectomy. To confirm viral replication in the mice, the BLV gene in blood cells was detected by nested PCR at week 3 after inoculation. Splenocytes and CC81-GREMG cells were co-cultured, and syncytium-forming ability was evaluated. Furthermore, anti-p24 antibodies from week 0 to 6 after inoculation were quantified by ELISA.

[0079] (2) Results The results are shown in Figure 10. In mice inoculated with the virus produced by pBLV-416ΔRT-transformed cells, the BLV gene was not detected (Figure 10B), and no syncytium was observed when spleen cells excised from mice were co-cultured with CC81-GREMG (Figure 10C). On the other hand, in mice inoculated with the virus produced by pBLV-416ΔRT-transformed cells, an increase in anti-p24 antibodies over time was confirmed (Figure 10D). These results suggest that the virus produced by pBLV-416ΔRT-transformed cells may be useful as a non-replicating BLV vaccine.

[0080] Example 8: Study using non-replicating BLV-producing cell lines (1) In Example 8, the entire nucleotide sequence of the provirus incorporated into the PK15-BLVΔRT cell line successfully created in Example 1 was re-analyzed. 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). Although there was a difference of 3 nucleotides in the number of deleted nucleotides in the RT region of the pol gene compared to the analysis results of Example 1, it is thought that further mutations occurred during the passage of the PK15-BLVΔRT cell line after its establishment. In addition, 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.

[0081] Example 9: Study using non-replicating BLV-producing cell lines (2) In Example 9, the expression and localization of viral proteins within cells, as well as the amount of virus released, were investigated using the PK15-BLVΔRT cell line.

[0082] (1) Method A Western blot Viral proteins were identified by Western blotting using anti-Gag antibodies and anti-Env antibodies.

[0083] (i) Immunofluorescence antibody method The intracellular localization of viral proteins was confirmed using immunofluorescence assays with anti-Gag and anti-Env antibodies.

[0084] U. Virus release amount The viral release from the PK15-BLVΔRT cell line was measured by Capture ELISA. The specific procedure was as follows: The cell culture supernatant was centrifuged at 141,118 × g for 2 hours, and the supernatant was removed. The supernatant was then resuspended in 1 / 100th the volume of PBS, the virus was concentrated, and the p24 concentration was measured. Here, the viral p24 concentration was calculated using a standard curve created with His-p24 antigen expressed and purified using E. coli as the standard (Figure 14B).

[0085] (2) Results The results are shown in Figures 12-14. Figure 12 confirms that the PK15-BLVΔRT cell line expresses the viral structural proteins p24 and gp51. Furthermore, compared to FLK-BLV, a positive control of persistently infected BLV cells, no change in the molecular weight of p24 and gp51 due to pol gene deletion was observed. Figure 13 shows that, similar to FLK-BLV, a positive control of persistently infected BLV cells, spot-like accumulation was observed within the cells. This result confirms that pol gene deletion does not affect the intracellular localization of p24. Figure 14A shows that the p24 concentration was 25.1 ± 1.2 μg, confirming that a sufficient amount of virus was released from the PK15-BLVΔRT cell line. These results suggest that the virus produced from the PK15-BLVΔRT cell line may be useful as a non-replicating BLV vaccine.

Claims

1. A bovine lymphoma virus (BLV) lacking the function of the reverse transcriptase of the pol gene, wherein the region of the full length of the sequence between 5'LTR and 3'LTR of the wild-type BLV shown in Sequence ID No. 1, excluding the pol gene, has a sequence that is 90% or more identical to the nucleotide sequence shown in Sequence ID No.

1.

2. The virus according to claim 1, wherein at least a portion of the nucleotide sequence of the reverse transcriptase region (RT) encoded by the pol gene is deleted.

3. The virus according to claim 2, wherein the deletion is a deletion at the 1st to 1000th base from the 5' end of the base sequence of the poll gene shown in Sequence ID No.

2.

4. The virus according to claim 2 or 3, wherein the number of deleted bases in the RT is 50 to 1000.

5. The virus according to claim 1 or 2, which does not replicate in the infected subject.

6. Non-replicating BLV-producing cells comprising a gene for bovine lymphoma virus (BLV) lacking the function of the reverse transcriptase of the pol gene, wherein the virus has a sequence in which the region other than the pol gene of the entire sequence between 5'LTR and 3'LTR of the wild-type BLV shown in SEQ ID NO: 1 has 90% or more identity with the nucleotide sequence shown in SEQ ID NO:

1.

7. A method for producing non-replicating BLVs, comprising the step of culturing the cells described in claim 6.

8. A method for preventing or treating endemic bovine lymphoma (EBL), comprising the step of inoculating a target with the virus described in claim 1 (excluding humans from the target).

9. A method for producing non-replicating BLV-producing cells, comprising the step of introducing the gene of bovine lymphoma virus (BLV) lacking the function of the reverse transcriptase of the pol gene into host cells, wherein the virus has a sequence in which the region other than the pol gene of the entire sequence between 5'LTR and 3'LTR of the wild-type BLV shown in SEQ ID NO: 1 has 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1.

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