JPH0967397A - Anti-jc virus antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new antibody for diagnosing, preventing and treating JC virus infectious diseases, capable of specifically recognizing JC virus by using a peptide fragment having a specific amino acid sequence which is a part of VP-1 protein of JC virus as an antigen. SOLUTION: This anti JC virus antibody is a new anti-JC virus antibody obtained by using a peptide fragment which is a part of VP-1 protein of JC virus and has an amino acid sequence expressed by the formula as an antigen and is useful for diagnosis, prevention and treatment of JC virus infectious diseases and prevention and treatment, etc., of progressive multifocal leukoencephalopat(PML), dementia, etc. The antibody is obtained by synthesizing a peptide fragment having an amino acid sequence expressed by the formula by a solid phase synthesis, etc., injecting the peptide fragment into the subcutis of rat together with Freund' s complete adjuvant to immunize the rat, administering antigen and adjuvant as an additional immune at an interval of 2 weeks, collecting a serum at a point of time at which antibody titre in the serum is raised and separating γ-globulin fraction from the serum.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-JC virus antibody effective for diagnosis, prevention and treatment of JC virus.

[0002]

[Prior art]

BACKGROUND OF THE INVENTION In the past, progressive multifocal leukoencephalopathy (PML) was rarely seen in immunocompromised patients.
Progressive multifocal leukoencephalopathy is an acquired immunodeficiency syndrome (AIDS)
e deficiency syndrome) is increasing. PML is characterized by demyelinating pathology of cranial nerve tissue, and its cause is caused by JC virus (JCV: JC polyomaviru).
s) is believed to be caused by damage to myelin-producing cells, oligodendrocytes. Recently, it has been pointed out that the relationship with JCV is one of the causes of dementia.

[0003] JCV is a human DNA virus and is a member of the genus Polyomavirus of the family Papovaviridae. It causes a lifelong persistent infection with a majority of people being infected during childhood and childhood. Fris
que et al. isolated JCV (Mad-1 strain) from the brain tissue of PML patients and analyzed the DNA sequence of this virus. As a result, BK virus (BKV) and SV
It has been reported that its structure is similar to that of 40 and that it has high homology with both DNA and amino acid sequences (Fri.
Sque, RJ et al., J. Virol., 51 , 458-469, 1984).

Up to now, many methods have been applied and studied to identify JCV existing in brain tissue.
In recent years, PML diagnostic methods have been used for computed tomography (CT: Co).
mputed tomography) and magnetic resonance imaging (MRI: Magne)
Although it has made great progress due to the development of technologies such as tic resonance imaging, JCV in brain tissue can be used for definitive diagnosis of PML.
It is necessary to prove the existence of the above, and in addition to the test of anti-JCV antibody in the blood of the patient, for the confirmation of JCV itself from the brain tissue, DNA analysis, detection of antigen using anti-JCV antibody, immunohistological staining method, etc. Various methods have been tried (EOMaj
Or et al., Clin. Microbiol. Rev., 5 , 49-73, 1992).

Of these diagnostic methods, D by the Southern blot analysis method or the in situ hybridization method is used.
The NA analysis method is considered to be problematic from the viewpoint of reliability and simplicity, such as changes in analysis data due to specificity, sensitivity, frequency of false positives, and the like. On the other hand, the immunohistochemical method is a simple and reliable method, but since the amino acid sequences of the constituent proteins of JCV have a high homology with those of BKV and SV40, the specificity of the antibody used affects its reliability. It becomes an important point.

<Prior Art> Knowles et al.
Mice were immunized with V and an anti-JCV monoclonal antibody was prepared for the first time (WAKnowels et al., J. Med. Viol., 34 , 127).
-131, 1991). Similarly, Bollag et al. Immunize mice with the JCV strain, have reactivity with the large T protein / small t protein of JCV, and have no reactivity with those of BKV and SV40.
Pab2000 created (B. Bollaget al., Virus Res., 25 , 223
-239,1992). Where large T protein and small t protein
Is called a T antigen, and is a generic term for proteins that are gene products of DNA-type oncoviruses such as papovavirus and adenovirus and contribute to cell morphological or oncogenic transformation. Each virus has 2-3 types of T antigens such as large T and small t. Next, Stoner et al. Made rabbit polyclonal antibody against JCV capsid protein,
Immunohistochemical double staining was performed using otein monoclonal antibody and this anti-capsid protein polyclonal antibody, and it was reported to be positive in brain tissue sections of PML patients (GLStoner et al., J. Neuroimmunol., 19 , 223-
236, 1988). As described above, regarding diagnosis using immunological tissue staining, a method using T antigen as an antigen and a method using polyomavirus capsid antigen are disclosed.

[0007]

Although attempts have been made to detect the JCV antigen by immunological tissue staining as described above, 1) JCV and virus can be detected by using a polyclonal antibody or high titer human serum. Cross-react with BKV, which has very similar properties, 2)
In addition, since polyclonal antibodies generally show strong affinity (stainability) because they react with multiple antigenic determinants of the same antigen, monoclonal antibodies specifically react with one antigenic determinant of antigens. Since the affinity is lower than that of polyclonal antibody, it is impossible to obtain the staining property if the specificity is increased in actual immunohistochemical staining, and the specificity is obtained if the staining property is increased. There was a problem that the sex could not be obtained.

Although the antibody prepared by Knowles et al. Strongly reacts with JCV but also weakly with BKV, it is not satisfactory in terms of specificity. Since the antibody created by Bollag et al. Recognizes only part of T antigen, it is possible to detect T antigen subpopulation.
However, it was insufficient for clinical application. Further, these monoclonal antibodies were not sufficient for clinical application in view of the fact that their actual tissue stainability was low as described above. Anti-capsid protein polyclonal antibody created by Stoner et al. Is not only for JCV but also for BKV and SV40.
It reacts with and has no specificity, and cannot be used for detection of JCV.

Further, there are the following problems. Normal,
Biological tissues are mechanically preserved by formalin-fixed paraffin embedding, but conventional antibodies could not stain formalin-fixed paraffin-embedded tissue sections (GLSto
ner et al., Proc.Natl.Acad.Sci.USA, 83 , 2271-2275,198
Because of this, 6) the acetone-fixed tissue was used, but the acetone-fixed tissue was structurally unstable, and the major drawback was that it could not be stained with formalin-fixed paraffin-embedded tissue.
In addition, since the conventionally prepared antibody cultures infected cells and uses the antigen extracted therefrom, it was not possible to eliminate the possibility of cross-reacting with normal brain tissue. As a result of intensive studies on the anti-JCV antibody in order to solve these problems, the present inventors have found an anti-JCV antibody that does not react with BKV or SV40 and has excellent tissue stainability, and completed the present invention.

[0010]

That is, the present invention is based on J
Anti-JC virus antibody obtained by using the peptide fragment of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 which is a part of VP-1 protein of C virus as an antigen, specifically, the VP-1 protein of JC virus. Lys that is part
Ser Ile Ser Ile Ser Asp Thr Phe Glu (SEQ ID NO: 1), L
ys Ser Ile SerIle Ser Asp Thr Phe Glu Ser Asp Ser
Pro Asn Arg Asp Met (SEQ ID NO: 2), or Val His Ser
Asn Gly Gln Ala Thr His Asp Asn Gly Ala Gly Lys P
It is an anti-JC virus antibody obtained by using a peptide fragment of ro ValGln Gly Thr (SEQ ID NO: 3) as an antigen.
JC characterized by not cross-reacting with viruses
SEQ ID NO: 1, which is part of the viral VP-1 protein
Anti-JC virus antibodies obtained by using the described peptide fragment as an antigen, JC virus detection reagents containing these anti-JC virus antibodies, PML preventive / therapeutic agents, dementia preventive / therapeutic agents and JC virus infectious diseases Regarding preventive and therapeutic drugs.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below. As the antigen, a partial peptide of VP-1 protein that constitutes the JCV capsid was used. Because T antigen is an early protein synthesized at the early stage of virus infection (before the initiation of DNA synthesis), an antibody (Pab20
Antibodies prepared using the capsid of the late structural protein that is biosynthesized when the virus is activated and proliferates more accurately than when using the virus (00 etc.) This is because it is considered possible. The selection of this antigen peptide was performed as follows. The VP-1 protein of JCV consists of 354 amino acids, and its amino acid sequence is VP of BKV.
-1 protein has 78% homology. Therefore, in order to prepare JCV-specific antibodies, three regions having low homology with BKV were designated as antigen peptides (see Table 1).

[0012]

[Table 1] [In the table, “•” indicates different amino acid residue portions between JCV and BKV, and “*” indicates the same amino acid residue portion between JCV and BKV. ]

Peptide # 1 (SEQ ID NO: 1) from ⁶⁰ Lys
^{It has} 10 residues of ⁶⁹ Glu (Lys Ser Ile Ser Ile Ser Asp Thr Phe Glu) and has two common amino acids with the corresponding BKV peptide. Peptide # 2 (SEQ ID NO: 2) was converted from ⁶⁰ Lys to ⁷⁷ Met (Lys Ser Ile Ser Ile Ser Asp Thr Phe Glu S
er Asp Ser Pro Asn Arg Asp Met) with 18 residues overlapping with peptide # 1
It has 8 common amino acids compared to the corresponding BKV peptide. Peptide # 3 (SEQ ID NO: 3) from ^{12 1} Val
¹⁴⁰ Thr (Val His Ser Asn Gly Gln Ala Thr His Asp As
n Gly Ala GlyLys Pro Val Gln Gly Thr) with 20 residues and 9 common amino acids with the corresponding BKV peptide. These peptide sequences were also selected by considering the antigenicity predicted from previously reported computer programs (Hopp and Woods, PNAS USA, 78 , 3824, 19).
81; Hopp, Methods in Enzymol., 178 , 571,1989; Chou and
Fasman, Ann Rev Biochem, 47 , 251,1978; Krch'n et al., M
ethods in Enzymol., 178 , 586,1989; Westhof et al., Nat
ure, 311 , 123,1984; Tainer et al., Nature, 312 , 127,198
4; Karplus and Schulz, Naturwissenschaften, 72 , 212,19
85). Furthermore, the synthesis was performed using a MAP system (Maltiple antigen peptide system) developed by Tam JP, which is expected to exhibit high antigenicity (Tam JP, Synthetic Peptide: Approaches to Biologi
cal Problems, p3-18,1989; Tam JP, PNASUSA, 85 , 5409,198
8; Tam et al., J. Exp.Med., 171 , 299,1990; Tam and Lu., P
NAS USA, 86 , 9084, 1989). Each peptide fragment was synthesized by a conventional method using a peptide synthesizer.

The antibody was prepared by a conventional method using the peptides # 1 to # 3 synthesized as described above as antigens. The resulting antibody showed excellent immunological tissue staining in JCV infected cells and brain tissue of PML patients. In particular, the antibody obtained using the above peptide # 1 as an antigen reacted only with JCV and did not cross-react with BKV and SV40. Further, the antibody obtained using peptide # 2, 3 as an antigen reacted not only with JCV but also with BKV, but not with SV40. Therefore, is it possible to infect JCV by combining these antibodies with BKV-specific antibodies?
It is possible to judge whether the infection is KV. Further, unlike the conventional case, the infected cell extract is not used as an antigen but a short polypeptide fragment is used as an antigen, so that it has high specificity with JCV and does not cross-react with normal brain tissue.

The antibody of the present invention is particularly excellent in stainability, and can be used for immunohistochemical assay even in a formalin-fixed paraffin-embedded tissue which is usually used according to the antibody, and can be used for JCV infections such as PML and dementia. A definitive diagnosis of illness etc. is possible. In addition, since a part of the capsid protein is used as an antigen, whole virus can be used for diagnosis, and positive detection can be performed when the virus is activated. Furthermore, by using the antibody of the present invention as a neutralizing antibody, JCV infections such as PML and dementia can be prevented.
It can be treated and is very useful.

When the antibody of the present invention is used as a prophylactic / therapeutic drug, it can be prepared as a lyophilized preparation for preparation at the time of use such as an injection. The dose varies depending on the patient's age and the degree of symptoms, but for example, 1 mg to 10 g / k
g, preferably 10 mg to 1 g / kg, more preferably 50 mg to
Although it is 500 mg / kg, it is not particularly limited to this range and may be added in an amount necessary for exerting a desired pharmacological effect. The dosage form of the prophylactic / therapeutic agent according to the present invention is not particularly limited as long as the object of the present invention can be achieved, but it can be a semi-prepared liquid such as an injection or an infusion, an ointment, a cream or a gel depending on a commonly used method. Solid preparations, solid preparations such as tablets, capsules and suppositories, soft capsules, liposomes and sustained-release preparations can be used. In order to prepare the above-mentioned preparation, in addition to the above-mentioned components, the components generally used as raw materials for pharmaceutical preparations can be blended to obtain a desired dosage form. Further, a preservative, an antioxidant and the like can be added as required.

The antibody of the present invention can be applied to various animals such as humans, mice and rabbits. As long as the object of the present invention can be achieved, a partial structure of the antibody, such as (Fab ') ₂ or Fa, can be used.
Only the b part is acceptable. When the antibody of the present invention is applied to diagnosis, it is usually used, for example, ELISA (enzy
It can be applied to me-linked immunosorbent assay).

[0018]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

Example 1. Preparation of antibody Peptide # 1: designed as described above and manufactured by a conventional method
Lys Ser Ile Ser IleSer Asp Thr Phe Glu (SEQ ID NO: 1), Peptide # 2: Lys Ser Ile Ser Ile SerAsp Thr
Phe Glu Ser Asp Ser Pro Asn Arg Asp Met (SEQ ID NO: 2) and Peptide # 3: Val His Ser Asn Gly Gln Al
a Thr His Asp Asn Gly Ala Gly Lys Pro Val Gln Gly
Antibodies against the respective peptide fragments of Thr (SEQ ID NO: 3) were prepared using two rabbits (Japanese white breed).
First, on day 0, blood for titration of pre-immune serum was sampled, and then 200 μg of peptide was added to rabbits in complete Freund's adjuva (CFA).
nt) and injected subcutaneously. Then 14, 28 and 4
On the second day, 100 μg of the peptide and incomplete Freund's adjuvant (IFA) were subcutaneously injected. Blood samples were obtained containing antibodies to the peptide injected at day 49. Further on the 56th day 10
0 mg of IFA was injected subcutaneously, and 30 to 50 ml of serum containing high titer antibody was obtained from rabbits on day 63. The serum titer assay was carried out by the ELISA method on the peptide fragment used for obtaining the antibody. Antibodies obtained by using peptides # 1, # 2 and # 3 as antigens are JCA, respectively.
b1, JCAb2 and JCAb3.

Embodiment 2 FIG . Immunological staining of tissues with antibodies (experimental method) The specificity of these antibodies is
It was determined by immunological staining of KV-infected cultured cells as well as brain tissue of PML patients. JCV infected IMR32
Cells, BKV infected 293 cells and SV40 infected IMR
32 cells were cultured on a cover glass and fixed with acetone, and then stored at -80 ° C until use. As a control, a paraffin-embedded fragment of formalin-fixed PML patient brain tissue was used. For immunological staining of these cells, LS
The AB kit (Dako) and the high titer serum described above were used as primary antibodies at 50-fold dilution.

(Results) JCV-infected IMR32 cells were subjected to JC
Staining with Abs 1, 2 and 3 stained 3-4% of the cell nuclei. This result supports the previously reported results for these cells. In addition, the cytoplasm of JCV-infected cells was not stained. BKV infection 293
When cells were stained with JCAb 2 and 3, a few% of the cell nuclei were stained, but not JCAb1.
Also, the cytoplasm was not stained in the BKV-infected cells. When SV40-infected IMR32 cells were stained with JCAb1, 2 and 3, no staining was observed with any antibody. The results of the immunological tissue staining with JCAb1, 2, and 3 of the formalin-fixed paraffin-embedded PML patient brain tissue are consistent with the report that JCV is concentrated in a specific part in the PML patient brain tissue. Typical nuclear staining was seen in dendrocytes (oligodendrocytes) and some astrocytes (astrocytes). In addition, as a control, IMR32 cells not infected with virus,
Attempts were made to stain 293 cells, but no staining was observed. The results of the above experiments are summarized in Table 2 below.

[0022]

[Table 2] [In the table, (+) indicates that it was immunologically stained,
Indicates that they were not stained, respectively. In addition, the state of dyeing is shown in a photograph as a drawing substitute. ]

JCAb1 specifically reacted with JCV-infected cells (FIG. 1) and did not cross-react with BKV-infected cells and SV40-infected cells (FIGS. 4 and 7). JCAb2
And 3 did not react with SV40 infected cells, but J
Reacted with CV and BKV infected cells (Fig. 2, 3, 5,
6). Further, all the antibodies of JCAb1 to 3 reacted with PML brain tissue (Figs. 8, 9 and 10) and did not react with uninfected cells as controls (Figs. 11 and 12). In addition, non-infected rabbit serum and JCAb1 did not react.

Embodiment 3 FIG . PML-infected brain group with diluted antibody
Immunological staining of weave (experimental method) Following the method of Example 2, the brain tissue of PML patients was immunologically stained using JCAb1 diluted stepwise. Normal rabbit serum was used as a control. (Results) The results of the above experiments are summarized in Table 3 below.

[0025]

[Table 3] [In the table, * indicates that measurement was performed under a high background, and ND indicates that it was not performed. ]

JCAb1 showed positive cells at a dilution of 1: 5 to 1: 2500. Normal rabbit serum is 1: 5, 1:
It was negative at 2500 times.

Example 4. Immunological Staining of JCV-Infected Cells and BKV-Infected Cells with Diluted Antibody (Experimental Method) In the same manner as in Example 3, JCV-infected cells and BKV-infected cells were immunologically used using JCAb1 which was serially diluted. Stained. Normal rabbit serum was used as a control. (Results) The results of the above experiments are summarized in Table 4 below.

[0028]

[Table 4] [In the table, * indicates that measurement was performed under a high background, and ND indicates that it was not performed. ]

JCAb1 is BK even under high concentration (1: 5)
It showed no cross-reactivity with V. As described above, JCAb1, JCAb2 and JCAb3 according to the present invention react even in a tissue in which formalin-fixed paraffin is embedded and show sufficient immunological tissue stainability for clinical application.
It is useful for definite diagnosis of. In particular, JCAb1 does not react with BKV or SV40, but specifically reacts with JCV, and thus is useful as a JCV detection reagent. Further, since these antibodies specifically react with JCV, they are also useful as anti-JCV antibodies as preventive / diagnostic / therapeutic agents for infectious diseases caused by JCV, such as PML and dementia.

[0030]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Lys Ser Ile Ser Ile Ser Asp Thr Phe Glu 1 5 10

SEQ ID NO: 2 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Ser Ile Ser Ile Ser Asp Thr Phe Glu Ser Asp Ser Pro Asn Arg Asp Met 1 5 10 15

SEQ ID NO: 3 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val His Ser Asn Gly Gln Ala Thr His Asp Asn Gly Ala Gly Lys Pro Val Gln 1 5 10 15 Gly Thr 20

[0033]

[Brief description of drawings]

FIG. 1 is a substitute photograph of a drawing showing the morphology of an organism, specifically, JCV-infected IM immunologically stained with JCAb1.
It is a photograph showing the histological structure of R32 cells.

FIG. 2 is a substitute photograph of a drawing showing the morphology of an organism, specifically, JCV-infected IM immunologically stained with JCAb2.
It is a photograph showing the histological structure of R32 cells.

FIG. 3 is a substitute photograph for a drawing showing the morphology of an organism, specifically, JCV-infected IM immunologically stained with JCAb3.
It is a photograph showing the histological structure of R32 cells.

FIG. 4 is a substitute photograph for a drawing showing the morphology of an organism, specifically, BKV infection 29 immunologically stained with JCAb1.
It is a photograph which shows the histological structure of 3 cells.

[0034]

FIG. 5 is a substitute photograph for a drawing showing the morphology of organisms, specifically, BKV infection 29 immunologically stained with JCAb2.
It is a photograph which shows the histological structure of 3 cells.

FIG. 6 is a substitute photograph for a drawing showing the morphology of organisms, specifically, BKV-infected 29 immunologically stained with JCAb3.
It is a photograph which shows the histological structure of 3 cells.

FIG. 7 is a substitute photograph for a drawing showing the morphology of organisms, specifically, SV40 infection I immunologically stained with JCAb1.
It is a photograph showing the histological structure of MR32 cells.

FIG. 8 is a photograph as a substitute for a drawing showing the morphology of organisms, specifically, a photograph showing the histological structure of PML-infected brain tissue immunologically stained with JCAb1.

[0035]

FIG. 9 is a substitute photograph for a drawing showing the morphology of an organism, specifically, a photograph showing the histological structure of PML-infected brain tissue immunologically stained with JCAb2.

FIG. 10 is a substitute photograph for a drawing showing the morphology of organisms, specifically, a photograph showing the histological structure of PML-infected brain tissue immunologically stained with JCAb3.

FIG. 11 is a substitute photograph for a drawing showing the morphology of an organism, specifically, a non-infected IMR immunologically stained with JCAb1.
It is a photograph showing the histological structure of 32 cells.

FIG. 12 is a substitute photograph for a drawing showing the morphology of an organism, specifically, non-infected 293 immunologically stained with JCAb1.
It is a photograph which shows the histological structure of a cell.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C07K 7/08 C07K 7/08 14/025 ZNA 14/025 ZNA

Claims (9)

[Claims] 1. An anti-JC virus antibody obtained by using the peptide fragment of SEQ ID NO: 1 which is a part of the VP-1 protein of JC virus as an antigen. 2. An anti-JC virus antibody obtained by using the peptide fragment of SEQ ID NO: 2 which is a part of the VP-1 protein of JC virus as an antigen. 3. An anti-JC virus antibody obtained by using the peptide fragment of SEQ ID NO: 3 which is a part of the VP-1 protein of JC virus as an antigen. 4. The anti-JC virus antibody according to claim 1, which does not cross-react with a BK virus antigen. 5. A JC virus detection reagent containing the anti-JC virus antibody according to claim 1. 6. A PML preventive / therapeutic drug comprising the anti-JC virus antibody according to claim 1. 7. A prophylactic / therapeutic drug for dementia, which comprises the anti-JC virus antibody according to claim 1. 8. A preventive / therapeutic agent for JC virus infectious disease, which comprises the anti-JC virus antibody according to claim 1. 9. A medicine comprising the anti-JC virus antibody according to claim 1.