JP6854462B2 - How to identify animals infected with bovine leukemia virus - Google Patents

Description

The present invention relates to a method for identifying a bovine leukemia virus-infected animal, a pharmaceutical composition such as a bovine leukemia virus vaccine composition, and the like.

Bovine leukemia virus (BLV) is a retrovirus that causes endemic bovine leukemia (EBL), a malignant B lymphoma. About 5% of BLV-infected cattle develop leukemia and die after a long incubation period (5-10 years). There are no effective preventive and curative treatments, and the infection and incidence rates are increasing at home and abroad, and there are concerns about the impact on the livestock industry. The current remedy is to prevent the spread by quarantine or selection. Therefore, there is an urgent need to establish preventive and therapeutic methods using pharmaceutical compositions such as vaccine compositions.

Non-Patent Document 1 describes that an antiserum obtained by immunizing a rabbit with peptides 64 to 73 of the amino acid sequence of gp51, which is an Env protein, inhibits BLV-induced syncytium formation and the like. Has been done. In addition, Non-Patent Document 2 describes that the amino acid sequence at positions 61 to 70 of gp51 was identified as a CD4 epitope.

By the way, in recent years, when livestock are vaccinated, the antibody increases due to the vaccine, so that it is not possible to distinguish them from naturally infected animals. Therefore, for example, vaccination of birds should be carried out under the control of the government after introducing the DIVA (Differntiating infected from vaccinated animals) system by international organizations. It has been recommended (eg, Patent Document 1).

Japanese Patent No. 4084403 (published on February 22, 2008)

I. Callebaut et al., Journal of Virology, Sept. 1993, p. 5321-5327. M. H. Gatei et al., Journal of Virology, Apr. 1993, p. 1796-1802.

Further search for epitopes is needed to efficiently identify BLV-infected animals and to establish prophylactic and therapeutic methods with pharmaceutical compositions such as vaccine compositions. It is also necessary to distinguish between BLV naturally infected animals and BLV pharmaceutical composition inoculated animals.

The present invention has been made in view of the above problems, and an object of the present invention is to include a novel method for identifying a BLV-infected animal, a pharmaceutical composition such as a BLV vaccine composition, and an animal inoculated with the pharmaceutical composition. The purpose is to realize a method for identifying BLV-infected animals.

In order to solve the above problems, the present invention includes the following inventions.

(1) A method for detecting the presence or absence of bovine leukemia virus in an animal, wherein an antibody that recognizes at least a part of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1 is contained in a biological sample collected from the animal. A detection method comprising a detection step of detecting the presence or absence.

(2) A detection step for detecting whether or not an antibody that recognizes at least a part of the amino acid sequence shown in SEQ ID NO: 1 is present in a biological sample collected from a test animal, and a case where the antibody is present. A method for identifying a bovine leukemia virus-infected animal, which comprises a determination step of determining that the test animal is infected with the bovine leukemia virus.

(3) The specific method according to (2) above, wherein in the determination step, it is further determined that the test animal is not infected with bovine leukemia virus in the absence of the antibody.

(4) The antibody that recognizes at least a part of the amino acid sequence shown in SEQ ID NO: 1 is an antibody that recognizes at least a part of the amino acid sequence (GRRRFGARAMVTY) shown in SEQ ID NO: 2 (1) to (3). The method described in any of.

(5) A kit for detecting the presence or absence of bovine leukemia virus in animals, at least one of peptides, proteins, and virus particles containing at least a part of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1. A detection kit containing the virus as an antigen.

(6) At least one of a mutant peptide, a mutant protein, a mutant virus particle, and a nucleic acid encoding these having a mutation in at least one amino acid of a B cell epitope common to bovine leukemia virus-infected animals. Including pharmaceutical compositions.

(7) The pharmaceutical composition according to (6) above, wherein the common B cell epitope is one or more epitopes contained in the amino acid sequence shown in SEQ ID NO: 1.

(8) The epitope having the mutation includes the amino acid sequence having a mutation in at least one of the amino acids 7, 9, 10, 16 and 18 of the amino acid sequence shown in SEQ ID NO: 1 (6) or (7). ). The pharmaceutical composition.

(9) The pharmaceutical composition according to any one of (6) to (8) above, which is a bovine leukemia virus vaccine composition.

(10) A method for identifying a bovine leukemia virus-infected animal from animals inoculated with the pharmaceutical composition according to any one of (6) to (9) above, wherein the animal is infected with bovine leukemia virus. A detection step for detecting whether or not an antibody recognizing the common B cell epitope is present in a biological sample collected from a test animal inoculated with the pharmaceutical composition according to any one, and the antibody. A method for identifying a test animal, which comprises a step of determining that the test animal is infected with bovine leukemia virus when is present.

(11) A detection antibody used in the identification method according to (10) above, which is an antibody that recognizes a B cell epitope common to bovine leukemia virus-infected animals.

The present invention has the effect of being able to provide a novel method for identifying a BLV-infected animal, a pharmaceutical composition such as a BLV vaccine composition, and a method for identifying a BLV-infected animal from animals inoculated with the pharmaceutical composition. Play.

It is a figure which shows the amino acid sequence of the peptide prepared based on BLV-Env (gp51) in an Example. It is a figure which shows the amino acid sequence of the peptide prepared based on BLV-Gag (p15, p24, and p12) in an Example. It is a figure which shows the result of the peptide microarray of gp51 in an Example. It is a figure which shows the result of the peptide microarray of p15 in an Example. It is a figure which shows the result of the peptide microarray of p24 in an Example. It is a figure which shows the result of the peptide microarray of p12 in an Example. It is a figure which shows the result of Elisa of the single alanine substitution of gp51p57 in an Example. It is a figure which shows the evaluation of the expression of the gp51 protein in the Example, the evaluation of the formation of syncytium, and the analysis of the conservativeness. It is a figure which shows the result of pBLV-IF-R9A production of 23CLN cells in an Example.

[1. How to detect the presence or absence of bovine leukemia virus]
The detection method according to the present invention is a method for detecting the presence or absence of bovine leukemia virus in an animal, and at least a part of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1 in a biological sample collected from the animal. Includes a detection step of detecting the presence or absence of an antibody that recognizes.

The amino acid sequence shown in SEQ ID NO: 1 is an amino acid sequence designed based on the amino acid sequence (SEQ ID NO: 4) of gp51 derived from the Env region, which is a coat protein constituting bovine leukemia virus. Specifically, SEQ ID NO: 1 is the 58th to 78th amino acids of SEQ ID NO: 4. The present inventors have found that the amino acid sequence shown in SEQ ID NO: 1 contains a B cell epitope common to BLV-infected animals, as in Examples described later.

Test animals include human and non-human animals. Non-human animals are not particularly limited, but are, for example, mammals such as cows (Bos taurus), zebu (Bos indicus), water buffalo (Bubalus bubalis), sheep, goats, pigs, mice, rats, rabbits, cats, and monkeys. Can be mentioned. Examples of cattle include dairy breeds, beef breeds, dairy meat combined breeds, working breeds, and working meat breeds. Specific examples include, but are not limited to, Japanese Black cattle, Japanese Shorthorn cattle, Holstein, Jersey, and native breeds of each country. The test animals are preferably cows, zebu and water buffalo, more preferably cows and water buffalo, and even more preferably cows. The test animal may be a living animal or a dead animal.

The biological sample is not particularly limited, and examples thereof include samples derived from body fluids such as blood, urine, saliva, milk, and nasal discharge. Preferably, it is derived from blood. Examples of blood-derived samples include whole blood, serum, plasma and the like. The biological sample is preferably whole blood, serum and plasma, more preferably serum and plasma. One embodiment of the method for identifying a bovine leukemia virus-infected animal according to the present invention may include the step of collecting a biological sample from a test animal. In addition, one embodiment of the method for identifying a bovine leukemia virus-infected animal according to the present invention may include a step of pretreating a biological sample collected from a test animal. Pretreatment of the biological sample includes, for example, purification of the collected whole blood to obtain serum, purification of the collected whole blood to obtain plasma, and the like.

In the present specification, the "antibody that recognizes at least a part of the amino acid sequence shown in SEQ ID NO: 1" is an antibody capable of binding to at least a part of the amino acid sequence shown in SEQ ID NO: 1, preferably the amino acid sequence shown in SEQ ID NO: 1. Refers to an antibody that can specifically bind to at least a part of the above, and in one example, it is an antibody that can bind to at least a part of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Another example is an antibody capable of binding to a peptide or protein containing the amino acid sequence shown in SEQ ID NO: 1 and capable of binding to at least a part of the amino acid sequence shown in SEQ ID NO: 1 in the peptide or protein. .. In one embodiment, the antibody is an antibody capable of binding to at least one of the 7, 9, 10, 16 and 18 amino acids of the amino acid sequence shown in SEQ ID NO: 1. In a more preferred embodiment, the antibody is an antibody that recognizes at least a part of the amino acid sequence (PPQGRRRFGARAMVT) shown in SEQ ID NO: 2. In a more preferred embodiment, the antibody is an antibody that recognizes at least a portion of the amino acid sequence (GRRRFGARAMVTY) shown in SEQ ID NO: 3. A further example of this antibody is an antibody capable of binding to at least a portion of the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In yet another example, an antibody capable of binding to a protein containing the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 that binds to at least a portion of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 in this protein. It is a possible antibody.

The detection principle in the detection step is not particularly limited, and examples thereof include a detection method based on an antigen-antibody reaction or the like. The detection step is preferably based on an antigen-antibody reaction, and a peptide, protein, virus particle or the like containing at least a part of the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as “target sequence”) is used as an antigen (hereinafter referred to as “detection”). It is more preferable to be based on an antigen-antibody reaction (referred to as "antigen").

The length of the peptide containing at least a part of the target sequence is not particularly limited, but for example, it is preferably 3 or more amino acid lengths, more preferably 6 or more amino acid lengths, and 13 or more amino acid lengths. It is even more preferable to have an amino acid length of 15 or more. The upper limit of the length is not particularly limited, but the length of the peptide is, for example, preferably 50 or less amino acid length, more preferably 40 or less amino acid length, and 30 or less amino acid length. Is even more preferable. In a peptide containing at least a part of a target sequence, the amino acid sequence of a part other than at least a part of the target sequence is not particularly limited. The peptide containing at least a part of the target sequence may have the same amino acid sequence as any fragment of gp51 shown in SEQ ID NO: 4, for example, 10% or more, 20% or more, 30% or more with SEQ ID NO: 4. , 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more amino acids that are the same as any fragment of a polypeptide having amino acid sequence identity. It may be an array. In addition, an amino acid sequence for fixing to a detection plate or column may be further added.

Proteins that contain at least part of the target sequence are specifically intended to be polypeptides that form tertiary or quaternary structure that contains at least part of the target sequence. In a protein containing at least a part of a target sequence, the amino acid sequence of a part other than at least a part of the target sequence is not particularly limited. The protein containing at least a part of the target sequence may have the same amino acid sequence as the total length of gp51 shown in SEQ ID NO: 4, for example, 10% or more, 20% or more, 30% or more, 40 of SEQ ID NO: 4. It may be a protein consisting of a polypeptide having an amino acid sequence identity of% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. In addition, an amino acid sequence for fixing to a detection plate or column may be further added. It may also form a complex with other proteins.

The viral particle containing at least a part of the target sequence is intended to be a viral particle carrying a peptide or protein containing at least a part of the target sequence. The virus particles containing at least a part of the target sequence may be natural virus particles themselves, inactivated by heating or chemicals, or acclimation or genetic manipulation such as cell passage or animal passage. It may be a virus particle that has been attenuated by. Further, the virus particles may be so-called virus-like particles (VLPs) that do not contain a virus genome. It may also be a viral particle modified to express a peptide or protein containing at least a portion of the target sequence described above. For virus particles containing at least a part of the target sequence, it is desirable that the target sequence is exposed on the surface of the virus particle.

When based on an antigen-antibody reaction, for example, the detection antigen was fixed on a plate, membrane, column, beads, colloidal gold, etc., or placed in a specific region (eg, in a well) and collected from a test animal. The biological sample is brought into contact with the detection antigen, and the antibody in the biological sample is reacted with the detection antigen for detection. The detection method may be any of an optical method, a chemical method, a physical method, and the like.

Preferred antigen-antibody reactions include ELISA, Western blotting, immunoprecipitation, immunoagglutination, radioimmunoassay, immunochromatography, immunofluorescence, magnetic beading, peptide array, immunofluorescence, and colloidal gold. And so on.

A competitive ELISA method using an antibody against a detection antigen (detection antigen-specific antibody) may be used. The competitive ELISA method is a method in which a detection antigen attached to a plate or the like is brought into contact with a biological sample, and then a detection antigen-specific antibody is added, and if at least the target sequence is contained in the biological sample. If an antibody that partially recognizes the antibody (hereinafter referred to as “detection target antibody”) is present, the detection target antibody is covered by the detection target antibody, and the detection antigen-specific antibody added later binds to the detection antigen. I can't. Therefore, for example, if a detection antigen-specific antibody is labeled, it is possible to detect whether or not the detection target antibody is present in the biological sample.

An "antibody against a detection antigen (detection antigen-specific antibody)" includes all classes and subclasses of immunoglobulins, as well as functional fragments of the antibody. The antibody is a concept that includes both native antibodies such as polyclonal antibodies and monoclonal antibodies, and also includes antibodies produced using gene recombination technology and functional fragments of the antibodies. The functional fragment of an antibody refers to an antibody having a region of a part of the above-mentioned antibody and having an antigen-binding ability (synonymous with a binding fragment). Natural antibodies can be derived from any species, including, but not limited to, humans, mice, rats, monkeys, goats, rabbits, camels, llamas, cows, chickens and the like. The antibody produced by using the gene recombination technique is not particularly limited, but is a chimeric antibody such as a humanized antibody or a primated antibody obtained by genetically modifying a natural antibody, a synthetic antibody, a recombinant antibody, and the like. Mutation-introduced antibodies and graft-binding antibodies (eg, antibodies in which other proteins and radioactive labels are conjugated or fused) are mentioned, and as described above, for antibodies already produced using gene recombination technology. Also included are antibodies that have undergone the same modifications as in the case of genetic modification of natural antibodies. Specific examples of the functional fragment of the antibody include F (ab') ₂ , Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv) and dAb (single domain antibody). And so on.

Detection of the above-mentioned antibodies can be performed by forming a complex with a secondary antibody labeled with a reporter molecule capable of attracting a detectable signal. Alternatively, the antibody described above may be pre-labeled with a reporter molecule. The reporter molecule is, for example, an enzyme, a fluorophore-containing molecule, or a radionuclide-containing molecule. Various such reporter molecules are known and may be appropriately selected.

When an enzyme is used as the reporter molecule, for example, peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase or the like can be used as the enzyme. The binding between these enzymes and the antibody can be carried out by a known method using a cross-linking agent such as a maleimide compound. As the substrate, a known substance can be used depending on the type of enzyme used. For example, when peroxidase is used as the enzyme, 3,3', 5,5'-tetramethylbenzicine and the like can be used. When alkaline phosphatase is used as the enzyme, paranitolphenol or the like can be used. As the radionuclide, ¹²⁵ I or ³ H or the like can be used. As the fluorescent dye, fluorolesses isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), or the like can be used.

A peptide probe can also be used instead of the antigen-specific antibody for detection.

At least one of a sample known to contain an antibody to be detected and a sample known not to contain an antibody to be detected may be used as a control.

[2. How to identify animals infected with bovine leukemia virus]
In the method for identifying a bovine leukemia virus-infected animal according to the present invention, whether or not an antibody that recognizes at least a part of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1 is present in a biological sample collected from a test animal. This includes a detection step of detecting the presence of the antibody and a determination step of determining that the test animal is infected with the bovine leukemia virus if the antibody is present.

The description of the detection step in the specific method according to the present invention is described in [1. Method for detecting the presence or absence of bovine leukemia virus] is the same as the description of the detection step. In addition, one embodiment of the specific method according to the present invention may include the step of collecting a biological sample from a test animal. In addition, one embodiment of the specific method according to the present invention may include the step of pretreating a biological sample collected from a test animal.

In the determination step, based on the result of the detection step, if the antibody to be detected is present, it is determined that the test animal is infected with bovine leukemia virus. In the determination step, it may be further determined that the test animal is not infected with bovine leukemia virus in the absence of the antibody to be detected.

After the determination, if the test animal is a non-human animal, measures such as quarantine or selection can be taken for the animal determined to be infected with the bovine leukemia virus.

[3. Detection kit for detecting the presence or absence of bovine leukemia virus]
The present invention is also a kit for detecting the presence or absence of bovine leukemia virus in animals, which comprises at least a part of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1 among peptides, proteins, and virus particles. A detection kit comprising one as an antigen is provided. These antigens more preferably contain at least a part of the amino acid sequence shown in SEQ ID NO: 2, and even more preferably contain at least a part of the amino acid sequence shown in SEQ ID NO: 3.

The detection kit according to the present invention is described in [1. Method for detecting the presence or absence of bovine leukemia virus], and [2. It can be suitably used for the detection step described in [Method for identifying animals infected with bovine leukemia virus].

Descriptions of peptides, proteins, and virus particles (detection antigens) provided as antigens are described in [1. Method for detecting the presence or absence of bovine leukemia virus].

The detection antigen may be bound to the substrate. Examples of the base material include plates, membranes, columns, beads, and colloidal gold. A fluorescent protein, a tag protein, or the like may be immobilized on the base material. The base material is preferably a microwell plate (eg, 96 wells). This is because a large number of samples can be processed in parallel.

The detection kit according to the present invention further comprises a base material, an antigen-specific antibody for detection, a peptide probe, various reagents (secondary antibody, reporter molecule, buffer, etc.), an instrument (pipette, etc.), and detection, if necessary. It may include at least one of the kit's instructions, control samples, and so on. Specific explanations of these are described in [1. Method for detecting the presence or absence of bovine leukemia virus]. In addition, in the instruction manual of the detection kit, for example, [1. The detection method described in [Method for detecting the presence or absence of bovine leukemia virus], or [2. Specific procedure for the detection process described in [Method for identifying animals infected with bovine leukemia virus] is described.

[4. Pharmaceutical composition]
The pharmaceutical composition according to the present invention comprises mutant peptides, mutant proteins, mutant virus particles, and nucleic acids encoding them, which have a mutation in at least one amino acid of a B cell epitope common to animals infected with bovine leukemia virus. Includes at least one of them.

"Common to bovine leukemia virus-infected animals" means that they are substantially common within a certain classification population of bovine leukemia virus-infected animals, for example, the same breed of at least the same species (species). Substantially common among infected animals of breed), or at least substantially common among infected animals of the same species, or at least substantially common among infected animals of the same genus. This may be the case, or at least substantially common among infected animals of the same family, or at least substantially common among infected animals of the same order. .. “Substantially common” means 70% or more, preferably 75% or more, more preferably 80% or more, still more preferably 85% or more, and particularly preferably 90% in a certain classification population of infected animals. It means that the antigen-antibody reaction is detected in the above-mentioned infected animals. The infected animal is not particularly limited, and for example, [1. Method for detecting the presence or absence of bovine leukemia virus], which is exemplified as a test animal. In a more specific example, the species includes bos (Bos taurus), bos indicus and buffalo (Bubalus bubalis), the genus includes Bos and Bubalus, and the family includes bovidae. (Bovidae) and the like, and examples of the genus include the order Bovidae (Artiodactyla) and the like. Specific breeds of cattle include, but are not limited to, Japanese Black cattle, Japanese Shorthorn cattle, Holstein, Jersey, and native cattle of each country.

A "B cell epitope" is a region (consecutive amino acid region including a binding site) or site (a continuous amino acid region including a binding site) or site (a continuous amino acid region including a binding site) or a site (a continuous amino acid region including a binding site) to which an antibody produced by B cells of a bovine leukemia virus-infected animal binds to a wild-type bovine leukemia virus protein or a fragment thereof. Refers to one or more specific amino acids that actually bind. The wild-type amino acid sequence is not limited to one, and includes all naturally found amino acid sequences. In the present invention, the wild-type amino acid sequence is preferably the amino acid sequence found most often among all the amino acid sequences found in nature.

The B cell epitope common to bovine leukemia virus-infected animals is not particularly limited, and examples thereof include one or more epitopes contained in the amino acid sequence shown in SEQ ID NO: 1 (WPPPQGRRRFGARAMVTYDC), and the amino acid shown in SEQ ID NO: 1. The entire sequence can be an epitope. More preferably, the common B cell epitope can be one or more epitopes contained in the amino acid sequence shown in SEQ ID NO: 2 (PPQGRRRFGARAMVT), and the entire amino acid sequence shown in SEQ ID NO: 2 can be an epitope. More preferably, the common B cell epitope can be one or more epitopes contained in the amino acid sequence shown in SEQ ID NO: 3 (GRRRFGARAMVTY), and the entire amino acid sequence shown in SEQ ID NO: 3 can be an epitope.

Mutations in amino acids include substitutions, insertions and deletions of amino acids. In the case of amino acid substitution, the type of amino acid after substitution is not particularly limited as long as it is an amino acid of a different type from the amino acid in the wild type, but the amino acid and properties in the wild type (for example, acidic / neutral / basic, or Amino acids of different sizes (hydrophilic / hydrophobic, etc.) are preferred. If the amino acid is of a different nature or size from the wild-type amino acid, the animal inoculated with the pharmaceutical composition will be recognizable as an antibody produced in a naturally infected animal to the bovine leukemia virus that has the wild-type amino acid. Since different antibodies can be produced, it becomes possible to easily identify a bovine leukemia virus-infected animal (naturally infected animal) from the animals inoculated with the pharmaceutical composition. In addition, in bovine leukemia virus, when there is a variation in the wild-type amino acid of the B cell epitope, it is preferable that the amino acid is mutated to an amino acid in which no variation is found. In one embodiment, the amino acid after substitution is alanine.

The number of mutated amino acids is not particularly limited, but for example, it is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1.

The position of the amino acid mutation is not particularly limited, but is preferably the position where the antibody binds in the wild type. This is because it becomes possible to more easily identify an animal infected with bovine leukemia virus from the animals inoculated with the pharmaceutical composition. In the amino acid sequence shown in SEQ ID NO: 1, for example, the 1st (W), 6th (G), 7th (R), 9th (R), 10th (F), 15th (M), 16th ( It is preferable to have a mutation in at least one of the amino acids at positions V), 17 (T), and 18 (Y), and positions 7 (R), 9 (R), 10 (F), and 16 ( It is more preferable to have a mutation in at least one of the amino acids V) and 18 (Y), and even more preferably to have a mutation in at least one of the 7th (R) and 9th (R) amino acids. It is particularly preferable to have a mutation at the 9th (R) position. Also in SEQ ID NO: 2 and SEQ ID NO: 3, it is preferable to have a mutation at a site corresponding to the above amino acid.

The epitope having the mutation preferably contains an amino acid sequence having a mutation in at least one of the 7, 9, 10, 16 and 18 amino acids of the amino acid sequence shown in SEQ ID NO: 1 (WPPPQGRRRFGARAMVTYDC), and is shown in SEQ ID NO: 1. It is more preferable to include an amino acid sequence having a mutation in at least one of the 7th and 9th amino acids of the amino acid sequence, and further preferably to contain an amino acid sequence having a mutation in the 9th amino acid of the amino acid sequence shown in SEQ ID NO: 1. .. Further, as a preferable example of the epitope having a mutation, an amino acid sequence having a mutation in at least one of the above-mentioned amino acids of the amino acid sequence shown in SEQ ID NO: 1 can be mentioned, and as a more preferable example, SEQ ID NO: 5 to 9 can be used. Examples thereof include those containing the amino acid sequences shown or those consisting of the amino acid sequences shown in SEQ ID NOs: 5-9. A particularly preferable example of the epitope having a mutation is the amino acid sequence shown in SEQ ID NO: 6.

The above-mentioned amino acid substitutions, deletions, and / or additions can be carried out by known methods. Known methods include, for example, a method of biosynthesizing a mutant peptide or protein after introducing a desired mutation into a gene by using a site-specific mutagenesis method such as the Kunkel method. Alternatively, the above-mentioned amino acid substitutions, deletions, and / or additions may be synthesized by a peptide synthesis method.

"Mutant peptide" refers to a peptide that has a mutation in the amino acid of the corresponding peptide (eg, a fragment of a protein) in wild bovine leukemia virus, and is at least one in the B cell epitope common to animals infected with bovine leukemia virus. Has a mutation in the amino acid. The mutant peptide may also have a mutation in an amino acid other than the B cell epitope. The mutant peptide is not particularly limited in length. When the B cell epitope is an epitope contained in the amino acid sequence shown in SEQ ID NO: 1, the peptide length is preferably, for example, 3 or more amino acid lengths, more preferably 6 or more amino acid lengths. It is more preferably 13 or more amino acid lengths, and even more preferably 15 or more amino acid lengths. The upper limit of the length is not particularly limited, but the length of the mutant peptide is preferably, for example, an amino acid length of 50 or less, more preferably 40 or less, and an amino acid length of 30 or less. It is more preferable to have.

"Mutant protein" refers to a protein having a mutation in the amino acid of the corresponding protein in wild-type bovine leukemia virus, and has a mutation in at least one amino acid in a B cell epitope common to animals infected with bovine leukemia virus. The "mutant protein" is specifically intended to be a polypeptide that forms tertiary or quaternary structure. The mutant protein may have, for example, a mutation in a common B cell epitope over the entire length of the wild-type gp51 shown in SEQ ID NO: 4, and may be 10% or more, 20% or more, 30% or more with SEQ ID NO: 4. 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more as a common B cell epitope in a protein consisting of a polypeptide having amino acid sequence identity. It may have a mutation. In addition, the mutant protein may be added with an amino acid sequence for purification. In addition, the mutant protein may form a complex with another protein.

The "mutant virus particle" is intended to be a virus particle carrying the above-mentioned mutant peptide or mutant protein, or carrying a gene encoding the above-mentioned mutant peptide or mutant protein. The mutant virus particles may be virus particles that have been inactivated by heating or a drug, or attenuated by a habituation operation such as cell passage or animal passage or a genetic manipulation. Further, the mutant virus particle may be a so-called virus-like particle (VLP) that does not contain a virus genome. In mutant virus particles, it may be desirable to expose common B cell epitopes on the surface of the virus particles. In addition, when the pharmaceutical composition contains mutant virus particles and the gene encoding the wild-type protein is replaced with the gene encoding the mutant protein, the mutant in the inoculated animal. It is preferable that the mutation does not completely lose the expression of the protein or the function of the mutant protein.

The "nucleic acid encoding these" is a nucleic acid encoding a mutant peptide, a mutant protein, or a mutant virus particle (hereinafter referred to as "mutant nucleic acid"). The mutant nucleic acid may be DNA or RNA. Since the nucleic acid sequence of bovine leukemia virus is known (for example, GenBank accession number: EF600696), those skilled in the art can easily design the sequence of the mutant nucleic acid by referring to a codon table or the like. An example of a nucleic acid sequence encoding an amino acid having the full length of gp51 is as shown in SEQ ID NO: 10, but is not limited thereto. Examples of the nucleic acid encoding the mutant virus particle include a nucleic acid in which a mutation is introduced into an infectious molecular clone of wild-type bovine leukemia virus by genetic manipulation (for example, site-direct mutagenesis).

The pharmaceutical composition is a wild-type peptide, protein, virus corresponding to "a mutant peptide, a mutant protein, a mutant virus particle, and at least one of the nucleic acids encoding these" contained in the pharmaceutical composition. It is desirable that it does not contain particles and the nucleic acids that encode them.

In addition to these, pharmaceutical compositions include pharmaceutically acceptable carriers, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, colorants, antioxidants, and viscosities. It may contain a modifier, an activator, nanoparticles, etc. Pharmaceutically acceptable carriers include, but are not limited to, for example, water, various salt solutions, alcohols, vegetable oils, mineral oils and the like.

The content of the mutant peptide, the mutant protein, the mutant virus particles, and the nucleic acid encoding these in the pharmaceutical composition is not particularly limited and may be set as appropriate.

The form of the pharmaceutical composition is also not particularly limited, and examples thereof include injections, liquids, suspensions, emulsions, powders, granules, and capsules. The administration form of the pharmaceutical composition is not particularly limited, and may be oral administration, or parenteral administration (subcutaneous administration, nasal administration, intraperitoneal administration, membrane administration, intramuscular administration, intravenous administration, etc.). There may be. An example of a pharmaceutical composition according to the present invention is a bovine leukemia virus vaccine composition. Another example of the pharmaceutical composition according to the present invention is a mutant antigen-containing composition. Yet another example of the pharmaceutical composition according to the present invention is a mutant B cell epitope-containing composition.

The pharmaceutical composition according to the present invention can be suitably used for the prevention or treatment of bovine leukemia. Therefore, the present invention also provides a method for preventing or treating bovine leukemia, which comprises a step of inoculating or administering a pharmaceutical composition according to the present invention to a target animal. The present invention also provides a method for preventing bovine leukemia, which comprises a step of inoculating a target animal with the bovine leukemia virus vaccine composition according to the present invention.

[5. How to identify bovine leukemia virus-infected animals from animals inoculated with the pharmaceutical composition]
The identification method according to the present invention is a method for identifying a bovine leukemia virus-infected animal from animals inoculated with the above-mentioned pharmaceutical composition according to the present invention, and is collected from a test animal inoculated with the pharmaceutical composition. A detection step of detecting whether or not an antibody recognizing the common B cell epitope is present in the obtained biological sample, and if the antibody is present, the subject animal is infected with bovine leukemia virus. It includes a determination step of determining that the antibody is present.

The description of the test animal and the biological sample is described in [1. Method for detecting the presence or absence of bovine leukemia virus].

As used herein, the term "antibody that recognizes a common B cell epitope" refers to an antibody that can bind to a common B cell epitope, preferably an antibody that can specifically bind to a common B cell epitope.

The description of the detection process is described in [1. Method for detecting the presence or absence of bovine leukemia virus] is the same as the detection step. However, in the identification method according to the present invention, the "detection antigen" is preferably a peptide, protein, virus particle or the like containing a common B cell epitope, and "a peptide containing the amino acid sequence shown in SEQ ID NO: 1". "Protein, virus particles, etc." is a more preferable example.

The detection step of the identification method according to the present invention is preferably a competitive ELISA method using an antibody (preferably a monoclonal antibody) against a detection antigen. For an example and a concrete explanation of the basic principle of the competitive ELISA method, see [1. Method for detecting the presence or absence of bovine leukemia virus]. The pharmaceutical composition according to the present invention comprises at least one of a mutant peptide, a mutant protein, a mutant virus particle, and a nucleic acid encoding them, which has a mutation in at least one amino acid of a common B cell epitope. Since it is contained, it produces an antibody (antibody against a mutant) whose amino acid is different from that produced when it is naturally infected (antibody against the wild type). Among the animals inoculated with the pharmaceutical composition, the animals not infected with the bovine leukemia virus have only the antibody against the mutant in the body. Therefore, when the detection antigen attached to a plate or the like is brought into contact with the biological sample and then the detection antigen-specific antibody is added, the detection antigen containing a common B cell epitope and the antibody against the mutant are different. Without binding, the detection antigen-specific antibody binds to the detection antigen. On the other hand, among the animals inoculated with the pharmaceutical composition, the animals infected with bovine leukemia virus have not only the antibody against the mutant but also the antibody against the wild type in the body. Therefore, when the detection antigen attached to the plate or the like is brought into contact with the biological sample and then the detection antigen-specific antibody is added, the detection antigen containing the common B cell epitope and the antibody against the wild type bind to each other. However, the detection antigen-specific antibody cannot bind to the detection antigen. Therefore, for example, if a detection antigen-specific antibody is labeled, it is possible to detect whether or not an antibody that recognizes a common B cell epitope is present in a biological sample. By utilizing such a difference in reactivity, a competitive ELISA method can be preferably adopted.

At least one of a sample known to contain an antibody to be detected and a sample known not to contain an antibody to be detected may be used as a control.

In the determination step, based on the result of the detection step, if the antibody to be detected is present, it is determined that the test animal is infected with bovine leukemia virus. In the determination step, it may be further determined that the test animal is not infected with bovine leukemia virus in the absence of the antibody to be detected.

After the determination, if the test animal is a non-human animal, measures such as quarantine or selection can be taken for the animal determined to be infected with the bovine leukemia virus.

In addition, one embodiment of the identification method according to the present invention may include the step of collecting a biological sample from a test animal. In addition, one embodiment of the identification method according to the present invention may include a step of pretreating a biological sample collected from a test animal.

[6. Antibodies for detection]
The detection antibody according to the present invention is a detection antibody used in the identification method according to the present invention, and is an antibody that recognizes a B cell epitope common to animals infected with bovine leukemia virus. The antibody is preferably labeled with a reporter molecule.

The description of "antibodies that recognize B cell epitopes common to animals infected with bovine leukemia virus" is described in [1. Method for detecting the presence or absence of bovine leukemia virus] is the same as the description of "antibody against detection antigen". The antibody is preferably an antibody that does not bind to the mutant peptide, mutant protein, and / or mutant virus particles contained in the pharmaceutical composition.

When the common B cell epitope among the detection antibodies according to the present invention is one or more epitopes contained in the amino acid sequence shown in SEQ ID NO: 1, the specific method according to the present invention and the detection according to the present invention. It can also be used in methods and can be a component of the detection kit according to the present invention.

Examples are shown below, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects can be used for details. Furthermore, the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the claims, and the present invention also relates to an embodiment obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. In addition, all the documents described in the present specification are incorporated by reference.

[Search for common BLV epitopes using peptide microarray]
A candidate peptide consisting of 15 amino acids derived from the amino acid sequence of these proteins to comprehensively search for epitopes on the protein in the Env region of BLV (gp51) and the protein in the Gag region of BLV (p15, p24, and p12). A total of 156 types were synthesized (FIGS. 1 and 2). These are peptides prepared by shifting by 4 amino acids. A peptide microarray (manufactured by JPT Peptide Technologies) in which these were immobilized on a slide glass was prepared. Each type of candidate peptide was fixed at 3 locations. The full-length amino acid sequences of gp51, p15, p24, and p12 are as shown in SEQ ID NOs: 4, 11 to 13, respectively.

For the experiment, four Japanese Black cattle with different susceptibility to BLV infection were prepared. The genotype of the bovine MHC (BoLA) class II DRB3 gene for BLV infection susceptibility was typed using the BoLA-DRB3 PCR-SBT method (see Takeshima SN et al., Tissue Antigens 2011.). Sensitive cow 1 and sensitive cow 2 are alleles of BoLA-DRB3 are DRB3 ^* 1601 / ^* 1601 sensitive cows, and insensitive cow 1 is allele of BoLA-DRB3 is DRB3 ^* 1501 / ^* 2703 neutral cows and are insensitive. Cow 2 is a neutral cow in which the allyl of BoLA-DRB3 is DRB3 ^* 1501 / ^{* 0503.}

First, blood was collected from these four bovine individuals before BLV infection to obtain a serum sample (BLV-negative serum). Subsequently, these four cows were infected with BLV, and after confirming an increase in antibody titer by BLV-ELISA and an increase in proviral load by BLV-CoCoMo-qPCR, blood was collected and a serum sample (BLV-positive serum) was collected. Got

After adding serum to the peptide microarray, bovine antibody bound to BLV-derived peptide was labeled with goat anti-bovine IgG antibody bound to Alexa Fluor 647. This was detected by a microarray reader (GenePix 4000B, manufactured by Molecular Devices), and the intensity of each spot was visually evaluated on a 5-point scale (0 to 4) (FIGS. 3 to 6). For the same individual, candidate peptides for which a BLV-specific reaction was obtained were selected by comparing before and after BLV inoculation. As a result, gp51No16 (PPQGRRRFGARAMVT; SEQ ID NO: 2) was the only candidate peptide whose response increased after BLV infection in all four cows (Fig. 3). On the other hand, none of the candidate peptides derived from p15, p24, and p12 reacted specifically with BLV-infected cattle (FIGS. 4 to 6).

[Establishment of peptide ELISA method using epitope]
Since peptide microarrays are not suitable for searching for epitopes in multiple samples, we attempted to develop a peptide ELISA using a gp51No16 peptide that specifically reacted in four BLV-infected cattle. In order to solidify the peptide on an ELISA plate, the peptide ELISA method using a carrier protein was selected. The carrier protein mcKLH (Mariculture Keyhole Limpet Hemocyanin) was maleimided to Sulfo-SMCC and immobilized on a 96-well flat-bottomed microplate (manufactured by Sumitomo Bakelite Corporation). The maleimide group of this maleimided KLH reacts with cysteine (Cys) containing free sulfhydryl to form a stable thioether bond. Utilizing this reaction, a peptide ELISA method linked to mcKLH was constructed using Cys on the C-terminal side of peptide gp51p57 (WPPPQGRRRFGARAMVTYDC; SEQ ID NO: 1) consisting of 20 amino acids derived from BLV containing the amino acid sequence of gp51No16 peptide. I tried.

The concentration of the carrier protein mcKLH immobilized on the plate was examined and determined to be 0.5 μg / well. Next, the dilution factor of the serum was examined, and a 500-fold dilution having a positive serum absorbance value of more than 1 was selected. Subsequently, when the dilution factor of the secondary antibody was examined, a 625-fold dilution showing an absorbance value of around 1 was selected.

On the other hand, since a high absorbance value was obtained even without the addition of the antigen peptide, it was found that a non-specific reaction was detected. Therefore, the serum was mixed with KLH and mcKLH in advance and cultured at 37 ° C. for 30 minutes. The removal of non-specific antibodies in serum by absorption method, which removes non-specific antibodies, was examined. The concentrations of KLH and mcKLH to be mixed in the serum were examined. It was clear that as the concentration of KLH added to the serum increased, the difference in background absorbance between the negative serum and the positive serum disappeared. Finally, 200 μg / mL, which showed almost the same value for the negative serum and the positive serum, was used as the concentration of KLH used for absorption. Subsequently, the concentration of mcKLH used for absorption was examined. Since a clear absorption effect was obtained as compared with no mcKLH, it was decided to use mcKLH at 2 μg / well. Furthermore, in order to reduce the background, a blocking step using horse serum or porcine serum was examined, blocking with 5% horse serum, and further adding 10% horse serum to the sample.

[Results of identification of common B cell epitopes using 917 BLV-infected cattle]
To attempt to identify a common B cell epitope, gp51p57 (SEQ ID NO: 1) containing gp51No16 identified above was synthesized and reactive using a large number of sample samples (917 samples) with different BoLA-DRB3 alleles. Examined. As a result, gp51p57 had 590 samples (91.2%) of serum samples derived from 647 naturally infected cows with BLV-DRB3 allele having 25 kinds of BoLA-DRB3 allele, and 270 cows with 23 kinds of BoLA-DRB3 allele. A positive reaction was shown for 33 samples (12.2%) of serum samples derived from BLV-negative cattle. From the above results, it was suggested that gp51p57 contains a common B cell epitope.

[Results of identification of common antibody binding site]
The antibody binding site in gp51p57, which contains a common B cell epitope, was identified. To this end, a peptide in which one amino acid in the amino acid sequence of gp51p57 was replaced with alanine was synthesized (Fig. 7, left), and the serum derived from the above-mentioned four BLV experimentally infected cattle (Fig. 7, center) and 71 BLV natural animals were synthesized. An ELISA experiment was performed using serum derived from infected cattle (Fig. 7, right). The wild-type sequence gp51p57 was used as a positive control, and no peptide was used as a negative control. The stimulus index (SI) was calculated using the following formula: SI = (wild-type gp51p57 or mutant gp51p57 OD ₄₅₀ ) / (peptide-free OD ₄₅₀ ). When the SI value was the same as or lower than that in the case where no peptide was added, it was determined that the reactivity was reduced. In experiments with serum samples from 4 BLV experimentally infected cattle and 71 BLV naturally infected cattle, gp51p57 responded to all serum samples, while the single alanine-substituted peptides R7A, R9A, F10A, V16A, and Y18A did not respond to the serum samples examined. From the above results, amino acids that contribute to five antibody bindings: R (7), R (9), F (10), V (16), and Y (18) were identified in the common B cell epitope gp51p57. It was.

[Evaluation of gp51 protein expression and syncytium formation]
In order to utilize the identified antibody binding site as a candidate for the DIVA sequence, it is necessary to confirm that there is no effect on the expression of the gp51 protein and the formation of syncytium. Therefore, a mutant BLV-infectious molecular clone pBLV-IF was constructed in which the amino acid at the identified antibody binding site was replaced with alanine. Specifically, the region containing the env gene of the BLV infectious molecular clone pBLV-IF was cloned into the pBluescript SK-II (-) vector. Using this as a template, single alanine substitution was introduced by site-direct mutagenesis using PrimeSTAR GXL (manufactured by Takara Bio Inc.). The env gene fragment into which this single alanine substitution was introduced was inserted into pBLV-IF using the restriction enzyme sites HindIII and BmgBI.

These mutants pBLV-IF were transfected into 23CLN cells and the expression of the gp51 protein was examined by Western blotting (FIG. 8a). When wild-type pBLV-IF was transfected into 23CLN cells, a gp51 protein with the same molecular weight as FLK-BLV (BLV-infected sheep fetal kidney cells) used as a positive control was detected. On the other hand, when the control vector pSK-II was transfected, BLV Env was not detected. Subsequently, for each of the five mutants pBLV-IF, a band was detected in the transfected cells at the same position as the band of the gp51 protein confirmed by FLK-BLV. From this result, it was clarified that the amino acids that contribute to each of the five identified antibody binding sites have no effect on the expression of the gp51 protein.

In addition, in order to investigate whether the amino acids corresponding to these antibody binding sites are involved in the syncytium-forming ability, which is a function of Env, the mutant pBLV-IF was transfected into 293T cells, and syncytium was obtained after culturing for 36 hours. Observed. As shown in FIG. 8b, the mutant pBLV-IF-transfected cells formed syncytium in the same manner as the wild-type pBLV-IF-transfected cells. From the above results, it was clarified that all the identified antibody binding sites had no effect on the expression of gp51 protein and the formation of syncytium.

[Analysis of conservativeness of common B cell epitopes]
High storage stability is required for use as a DIVA sequence. Therefore, 517 BLV sequences downloaded from the GenBank database were analyzed (c in FIG. 8). No mutations were found in amino acids R (7) and F (10) corresponding to the identified antibody binding sites, indicating that they have extremely high storage stability.

[Preparation of α-DIVA monoclonal antibody]
In the process of preparing a monoclonal antibody for DIVA, some rabbit antisera had reduced reactivity with the single alanine-substituted peptide R9A, and thus R9A was incorporated into a pharmaceutical composition (for example, a vaccine composition). It was considered to be one of the candidates for the DIVA mutant sequence.

[PBLV-IF-R9A production in 23CLN cells]
23CLN cells (pig-derived cells; RIKEN registration number RCB0179) were cultured in D-MEM medium supplemented with 5% FBS and 1x Penicillin-Streptomycin-Glutamine (manufactured by ThermoFisher Scientific). For transfection, 1 × 10 ⁶ 23CLN cells were seeded on 20 10 cmφ dishes and cultured for 1 day.

Transfection was performed by suspending 12 μg of pBLV-IF R9A in 600 μL of Opti-MEM for one dish, adding 48 μL of FugeneHD, allowing it to stand at room temperature for 15 minutes, and adding it to the cells.

The culture supernatant was collected 3 days after transfection, and cell debris was precipitated and removed by centrifugation (3000 rpm, 10 minutes, 4 ° C.). Further, the supernatant was subjected to ultracentrifugal separation (rotor: SW28 beckman, 28000 rpm, 2 hours, 4 ° C.) to precipitate virus particles. After removing the supernatant, it was suspended in PBS (-) and the amount of protein was quantified using the BCA assay.

25 μg of the obtained protein was loaded on a 12.5% acrylamide gel, subjected to SDS-PAGE to separate the protein, and then blotted on a PVDF membrane. Blocking was performed at room temperature for 30 minutes using 5% skim milk. It was then reacted with anti-BLV p24 and anti-BLV gp51 monoclonal antibody (primary antibody) for 1 hour at room temperature. Further, it was reacted with an HRP-labeled anti-mouse IgG antibody (secondary antibody) for 30 minutes at room temperature. Then, after reacting with the detection agent superior best pico at room temperature for 5 minutes, chemiluminescence was detected.

In addition, after performing SDS-PAGE under the same conditions, the gel after electrophoresis was stained with CBB.

Bands of BLV antigens p24 and gp51 were detected in Western blot, confirming that the gp51 antigen containing the R9A mutation was taken up by the pBLV-IF virus particles and released into the culture supernatant.

The present invention can be particularly preferably used in the livestock field where it is necessary to identify an animal infected with bovine leukemia virus and prevent bovine leukemia virus.

Claims (9)

A method for detecting the presence or absence of bovine leukemia virus in animals.
A detection method comprising a detection step of detecting whether or not an antibody that recognizes at least a part of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1 is present in a biological sample collected from the animal. A detection step for detecting whether or not an antibody that recognizes at least a part of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1 is present in a biological sample collected from a test animal, and a detection step.
A method for identifying a bovine leukemia virus-infected animal, which comprises a determination step of determining that the test animal is infected with the bovine leukemia virus when the antibody is present. The specific method according to claim 2, wherein in the determination step, it is further determined that the test animal is not infected with the bovine leukemia virus in the absence of the antibody. The antibody that recognizes at least a part of the amino acid sequence shown in SEQ ID NO: 1 is an antibody that recognizes at least one of the 7, 9, 10, 16 and 18 amino acids of the amino acid sequence shown in SEQ ID NO: 1 (WPPPQGRRRFGARAMVTYDC). The method according to any one of claims 1 to 3. A kit for detecting the presence or absence of bovine leukemia virus in animals.
A detection kit comprising at least one of peptide, protein, and viral particles as an antigen, which comprises at least a portion of the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1. A method for identifying bovine leukemia virus-infected animals from animals inoculated with a pharmaceutical composition.
A detection step for detecting whether or not an antibody that recognizes a B cell epitope common to bovine leukemia virus-infected animals is present in a biological sample collected from a test animal inoculated with the pharmaceutical composition.
In the presence of the antibody, it includes a determination step of determining that the test animal is infected with bovine leukemia virus.
The pharmaceutical composition has a mutation in at least one amino acid of said common B-cell epitope comprises the variant peptide, mutant proteins, mutant virus particles, and at least one of the nucleic acids encoding these,
The identification method, wherein the common B cell epitope is one or more epitopes contained in the amino acid sequence (WPPPQGRRRFGARAMVTYDC) shown in SEQ ID NO: 1. The pharmaceutical composition comprises an epitope in which the mutation-carrying epitope has a mutation in at least one of the 7, 9, 10, 16 and 18 amino acids of the amino acid sequence shown in SEQ ID NO: 1 (WPPPQGRRRFGARAMVTYDC). Item 6. The identification method according to Item 6. The identification method according to claim 6 or 7, wherein the pharmaceutical composition is a bovine leukemia virus vaccine composition. A detection antibody used in the identification method according to any one of claims 6 to 8.
An antibody for detection, which is an antibody that recognizes a B cell epitope common to animals infected with bovine leukemia virus.

Images