IgA NEPHROPATHY PREVENTATIVE / THERAPEUTIC AGENT

Abstract

To provide a novel preventative / therapeutic agent which leads to the complete curing and treatment of IgA nephropathy.SOLUTION: This IgA nephropathy preventative / therapeutic agent targets anti-β2 spectrin IgA antibody-induced tonsillar indigenous bacteria.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a prophylactic and therapeutic agent for IgA nephropathy.

Background Art

[0002] IgA nephropathy is the most prevalent primary glomerulonephritis in the world, defined by IgA deposition in the glomerular mesangium region. It often presents with a slow progression of renal dysfunction accompanied by hematuria and proteinuria, and is a disease with a poor prognosis where approximately 40% will progress to end-stage renal failure if left untreated.

[0003] To date, it has been clarified that the IgA deposited in the glomeruli of IgA nephropathy patients is IgA1 with a deficiency of galactose in its hinge region O-linked sugar chain (galactose deficient IgA1: Gd-IgA1). In fact, since Gd-IgA1 is also increased in the sera of IgA nephropathy patients, many basic and clinical studies have been conducted to date aiming at controlling the production of Gd-IgA1 as the first hit for nephritis induction. However, with the worldwide spread of the monoclonal antibody against Gd-IgA1 (KM55; Non-Patent Documents 1 and 2) developed by the present inventors, it has also been found that Gd-IgA1 is present (and sometimes increased) in the sera of healthy individuals. That is, the increase in serum Gd-IgA1 alone cannot fully explain the onset and progression of nephritis, suggesting a latent new pathological condition. That is, it was considered that a further qualitative change in Gd-IgA1 itself is a necessary condition for the progression and chronicity of the pathological condition.

Prior Art Documents

Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] Even now, more than 50 years after its discovery, the detailed etiology and pathogenesis of IgA nephropathy remain unclear, and a radical treatment method based on the pathogenesis has not been established. Therefore, there is no delay for end-stage renal failure patients caused by this disease. Thus, it is essential and urgent to elucidate the pathogenesis leading to radical treatment to prevent the progression to dialysis in this disease. Therefore, an object of the present invention is to provide a new prophylactic and therapeutic agent leading to the radical treatment of IgA nephropathy.

Means for Solving the Problems

[0006] Therefore, as a result of verification regarding the qualitative change of Gd-IgA1, the present inventors found that IgA-type autoantibodies that recognize specific proteins present in mesangial cells are present in the sera of IgA nephropathy model mice (gddY mice) and IgA nephropathy patients. Mass spectrometry using mouse sera identified that the main autoantigen is β2-spectrin, and it was revealed that anti-β2-spectrin IgA antibodies are also frequently present in the sera of IgA nephropathy patients. Furthermore, it was proven that a large number of IgA-positive plasma cells, including anti-β2-spectrin IgA antibody-producing plasma cells, infiltrate the renal tubulointerstitium of gddY mice, and such IgA-positive plasma cells are also present in the renal tubulointerstitium of IgA nephropathy patients. β2-spectrin is a cytoskeleton-forming protein that is ubiquitously expressed in the cytoplasm by nature, but the present inventors found that β2-spectrin is expressed only on the cell surface in renal mesangial cells by flow cytometry analysis of glomerular constituent cells and in vivo implantation experiments of anti-β2-spectrin antibodies. From these facts, it was found that β2-spectrin expressed on the surface of mesangial cells serves as a target autoantigen, causing selective deposition of IgA molecules in the mesangial region in this disease, and it was found that IgA nephropathy can be said to be an autoimmune disease in that it has tissue-specific autoantibodies. In this disease, the deposition of IgA antibodies in the mesangial region of renal glomeruli is considered to form the first hit of the pathological condition. Therefore, the anti-β2 spectrin IgA antibody that selectively deposits in the renal mesangial region can be said to have pathogenicity. As a result of research to elucidate the induction mechanism of this autoantibody, it was found that although IgA antibodies against oral bacteria of gddY were detected in the serum of gddY, IgA antibodies against intestinal bacteria were not detected. Since β2 spectrin IgA antibodies were detected in the serum of gddY, it was suggested that this autoantibody is induced not in the intestinal mucosa of gddY but in the upper respiratory mucosa. Therefore, as a result of verifying whether antibodies that recognize β2 spectrin bind to bacteria present in the oral mucosa of gddY, it became clear that some of the oral bacteria of gddY are recognized by this monoclonal antibody, and it became clear that there is immune cross-reactivity between specific bacteria in the gddY oral cavity and β2 spectrin. That is, it was found that the anti-β2 spectrin IgA antibody is produced by certain commensal bacteria in the oral cavity of gddY mice through the mechanism of molecular mimicry. Furthermore, it was found that the culture supernatant of tonsillar lymphocytes from serum β2 spectrin IgA antibody-positive patients with IgA nephropathy contains anti-β2 spectrin IgA antibodies. In human IgA nephropathy, the tonsils are the responsible mucosa for autoantibody production. As its mechanism, it was found that tonsillar commensal bacteria induce and produce anti-β2 spectrin IgA antibodies. By using an IgA nephropathy vaccine targeting anti-β2 spectrin IgA antibody-inducing tonsillar commensal bacteria or a disinfectant for the bacteria, it was found that radical preventive and therapeutic treatment of IgA nephropathy can be achieved, and the present invention was completed.

[0007] That is, the present invention provides the following inventions [1] to [4]. [1] A prophylactic and therapeutic agent for IgA nephropathy targeting anti-β2 spectrin IgA antibody-inducing tonsillar commensal bacteria. [2] The prophylactic and therapeutic agent for IgA nephropathy according to [1], wherein the prophylactic and therapeutic agent for IgA nephropathy is an IgA nephropathy vaccine or a disinfectant for the tonsillar commensal bacteria. [3] The prophylactic and therapeutic agent according to [2], wherein the vaccine is a vaccine selected from the group consisting of live attenuated vaccines, inactivated vaccines, VLP vaccines, recombinant protein vaccines, mRNA vaccines, DNA vaccines, and viral vector vaccines. [4] The prophylactic and therapeutic agent according to [1] to [3], wherein the tonsil is one or more tonsils selected from the group consisting of palatine tonsils, lingual tonsils, pharyngeal tonsils, and tubal tonsils.

Effects of the Invention

[0008] When the prophylactic and therapeutic agent of the present invention is used, the induction of anti-β2 spectrin IgA antibody in the tonsils can be prevented or suppressed, and as a result, the deposition of anti-β2 spectrin IgA antibody in the mesangial region of the renal glomeruli can be prevented or suppressed, so that the radical prophylactic and therapeutic treatment of IgA nephropathy becomes possible.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

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Modes for Carrying Out the Invention

[0010] One aspect of the present invention is a prophylactic and therapeutic agent for IgA nephropathy that targets anti-β2 spectrin IgA antibody-inducing tonsillar commensal bacteria. Here, the anti-β2 spectrin IgA antibody-inducing tonsillar commensal bacteria are human tonsillar commensal bacteria that induce anti-β2 spectrin IgA antibodies in the tonsillar plasma cells.

[0011] The tonsils include one or more tonsils selected from the palatine tonsils, lingual tonsils, pharyngeal tonsils, and tubal tonsils.

[0012] Examples of human tonsillar commensal bacteria include obligate anaerobic bacteria that are commensal in the human tonsils. For example, bacteria that are sensitive to ampicillin, vancomycin, neomycin, and metronidazole used in the following examples. Specific examples include Actinomyces spp., Streptococcus spp., Streptococcus pyogenes, Staphylococcus spp., and Streptococcus pneumoniae. Among these tonsillar commensal bacteria, anti-β2 spectrin IgA antibody-inducing bacteria are the targets of the vaccine of the present invention.

[0013] Examples of prophylactic and therapeutic agents for IgA nephropathy include vaccines and disinfectants for the above-mentioned human tonsillar commensal bacteria. Examples of vaccines include vaccines selected from attenuated vaccines, inactivated vaccines, VLP vaccines, recombinant protein vaccines, mRNA vaccines, DNA vaccines, and viral vector vaccines. Here, for the live attenuated vaccine, a weakly toxic or attenuated one is used from among the anti-β2 spectrin IgA antibody-inducing tonsillar resident bacteria. For the inactivated vaccine, a vaccine obtained by inactivating the anti-β2 spectrin IgA antibody-inducing tonsillar resident bacteria by formalin treatment or the like is used. For the VLP vaccine and the recombinant protein vaccine, a vaccine produced by artificially synthesizing or recombining a specific protein on the surface of the bacteria is used. For the mRNA vaccine and the DNA vaccine, mRNA or DNA encoding a specific protein on the surface of the bacteria is used. For the viral vector vaccine, a vector obtained by incorporating a gene encoding a specific protein on the surface of the bacteria into a harmless virus can be used. In addition, examples of the disinfectant for the tonsillar resident bacteria include a disinfectant using bacteriophage.

[0014] The vaccine of the present invention prevents the induction of β2 spectrin IgA antibody in the tonsils. Therefore, the vaccine of the present invention is useful as a prophylactic and therapeutic agent for IgA nephropathy. The disinfectant for the tonsillar resident bacteria using bacteriophage is also useful as a prophylactic and therapeutic agent for IgA nephropathy. The prophylactic and therapeutic agent for IgA nephropathy of the present invention may contain the above vaccine or disinfectant, but it is preferably formulated into a pharmaceutical composition in various dosage forms by blending a pharmaceutically acceptable carrier. As the dosage form of such a pharmaceutical composition, injection, preferably subcutaneous injection, intramuscular injection, tonsillar instillation or intravenous drip is preferred. When preparing a preparation such as an injection, water, solubilizing agent, stabilizer and the like can be blended.

Example

[0015] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples at all.

[0016] Example 1 (Materials and Methods) (1) Mice The gddY mice were established by selective mating of early-onset ddY mice for over 20 generations. (2) Human subjects With the informed consent and approval of the Research Ethics Committee of the University of Tokyo Hospital, sera and tonsillar tissues from IgA nephropathy patients and tonsillar tissues from chronic tonsillitis patients were obtained at the University of Tokyo Hospital. (3) Antibiotic administration experiment Ampicillin (1 g / mL), vancomycin (500 mg / L), neomycin (1 g / L), and metronidazole (1 g / L) were mixed into the drinking water of 4-week-old gddY mice and administered for a total of 4 weeks.

[0017] (4) Isolation of mouse mononuclear cells and flow cytometry The perfused kidneys were cut into 2-3 mm pieces, ground with 0.6 mg / mL collagenase D (Roche) and 100 μg / mL DNase (Roche), and then kidney mononuclear cells were isolated by gradient centrifugation using Percoll. After blocking with 2.4G2 (clone name), the single-cell suspension was stained with the following reagents. BD Biosciences: B220 (APC-Cy7), CD138 (PE), IgA (biotin), and streptavidin (BV421). After staining, analysis was performed using a FACS CantoII (BD Biosciences). The data were analyzed using FlowJo (Tree Star). (5) Bacterial flow cytometry The oral cavity of 8-week-old gddY mice was swabbed, and bacteria were cultured in brain heart infusion medium under anaerobic conditions at 37°C for 24 hours. Although a large number of colonies were obtained, 50-100 colonies were collected from each medium, and the cultured bacteria were 2×10 per sample 6Samples were aligned individually, centrifuged at 10000 g to pellet the bacteria, and reacted with a monoclonal antibody recognizing β2 spectrin and a monoclonal antibody recognizing the hapten 4-hydroxy-3-nitrophenylacetyl (NP) as control antibodies at a concentration of 1 μg / mL for 18 hours. After washing with 1% BSA PBS, anti-human-Alexa647 (Jackson) was reacted at 500-fold dilution for 30 minutes. After washing, it was suspended in 100 μL of SYBR TM Green diluted 10000-fold and flow cytometry was performed. After staining, analysis was performed using FACS CantoII (BD Bioscience). The data were analyzed using Flow Jo (Tree Star).

[0018] (6) ELISA assay Recombinant human β2 spectrin was immobilized on a plate at 40 μg / mL (4 °C, 16 hours). Blocking was performed with 3% bovine serum albumin / 0.05%-Tween20 phosphate-buffered saline (1 hour), and the 50-fold diluted serum was reacted for 2 hours. The tonsil culture supernatants were standardized to an IgA concentration of 7 μg / mL in each culture supernatant and reacted for 2 hours. Reaction was performed for 1 hour using an HRP-labeled goat anti-human IgA antibody, and color development was performed using 3,3’,5,5’-tetramethylbenzidine.

[0019] (7) Method for isolating and culturing human tonsil mononuclear cells Tonsil specimens from IgA nephropathy patients and chronic tonsillitis patients were ground with 0.5 mg / mL collagenase D (Roche), and tonsil mononuclear cells were isolated by gradient centrifugation using Percoll. Subsequently, they were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin (GIBCO) for 14 days.

[0020] (Results) (1) Autoantibody-producing IgA plasma cells are induced by bacteria. As described above, IgA-positive plasma cells that produce anti-β2 spectrin IgA antibodies infiltrate the renal tubulointerstitium of gddY mice. Therefore, antibiotics were administered to gddY mice to verify whether the autoreactive IgA-producing plasma cells infiltrating the kidney disappeared. Four-week-old gddY mice were administered ampicillin, vancomycin, neomycin, and metronidazole by mixing them in drinking water. Four weeks after the start of antibiotic treatment, the mice were euthanized, and mononuclear cells infiltrating the renal tubulointerstitium were isolated, and the proportion of IgA-positive plasma cells was analyzed by flow cytometry. As a result, IgA-positive plasma cells disappeared in the antibiotic-treated group (Figs. 1 and 2). From this, it was considered possible that autoreactive IgA-producing plasma cells were induced by specific bacteria present in gddY.

[0021] (2) Immune cross-reactivity between β2 spectrin and gddY oral bacteria The mechanism of autoimmune diseases caused by antibodies against similar structures common to specific bacteria or viruses and autoantigens is called the molecular mimicry mechanism. The present inventor hypothesized that the bacteria present in gddY induced anti-β2 spectrin IgA antibodies by the mechanism of molecular mimicry based on the above results. On the other hand, the present inventor has found that although IgA antibodies against gddY oral bacteria are detected in the serum of gddY, IgA antibodies against intestinal bacteria are not detected. Since β2 spectrin IgA antibodies are detected in the serum of gddY, it was considered that this autoantibody was induced by bacteria present in the oral mucosa rather than the intestinal mucosa of gddY. Therefore, among the recombinant monoclonal antibodies prepared from IgA plasma cells infiltrating the gddY kidney, an antibody that recognizes β2-spectrin was verified by bacterial flow cytometry to determine whether it binds to the bacteria present in the oral mucosa of gddY. As a result, it was revealed that some of the oral bacteria of gddY were recognized by this monoclonal antibody (Figure 3). From these results, it became clear that there is immune cross-reactivity between specific bacteria in the gddY oral cavity and β2-spectrin. That is, it was strongly suggested that anti-β2-spectrin IgA antibodies are produced by certain commensal bacteria in the oral cavity of gddY mice through the mechanism of molecular mimicry.

[0022] (3) Anti-β2-spectrin IgA antibodies are induced and produced in the tonsils of IgA patients. From the results of verification using model mice, it was considered that in IgA nephropathy patients, anti-β2-spectrin IgA antibodies are induced in the upper airway mucosa centered on the tonsils. Therefore, lymphocytes were isolated and cultured from the tonsils of IgA nephropathy patients and chronic tonsillitis patients as a comparison group, and the binding ability to β2-spectrin was evaluated by ELISA using IgA contained in the culture supernatant. As a result, it was revealed that the culture supernatant of tonsil lymphocytes from serum β2IgA antibody-positive patients contained anti-β2-spectrin IgA antibodies (Figure 4). From this, it was suggested that in human IgA nephropathy, the tonsils are the responsible mucosa for autoantibody production, and as its mechanism, tonsil commensal bacteria induce and produce anti-β2-spectrin IgA antibodies.

Industrial Applicability

[0023] Using the vaccine or disinfectant of the present invention, radical preventive and therapeutic treatment of IgA nephropathy can be achieved.

Claims

1. A prophylactic and therapeutic agent for IgA nephropathy targeting IgA antibody - induced tonsillar resident bacteria.

2. The prophylactic and therapeutic agent for IgA nephropathy according to Claim 1, wherein the prophylactic and therapeutic agent for IgA nephropathy is an IgA nephropathy vaccine or an antibacterial agent for the tonsillar resident bacteria.

3. The prophylactic and therapeutic agent according to Claim 2, wherein the vaccine is a vaccine selected from the group consisting of live - attenuated vaccines, inactivated vaccines, VLP vaccines, recombinant protein vaccines, mRNA vaccines, DNA vaccines, and viral vector vaccines.

4. The prophylactic and therapeutic agent according to any one of Claims 1 to 3, wherein the tonsil is one or more tonsils selected from the palatine tonsil, lingual tonsil, pharyngeal tonsil, and tubal tonsil.

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