Method for assisting diagnosis of secondary progressive multiple sclerosis, and method for screening for therapeutic agent for multiple sclerosis

Abstract

The present invention relates to a method for assisting the diagnosis of secondary progressive multiple sclerosis, the method including: a step in which the relative abundance of Tyzzerella nexilis in a fecal sample collected from a subject is measured; and a step in which, when the relative abundance of Tyzzerella nexilis is larger than that in a fecal sample collected from a normal person, it is determined that the subject has been affected with or is potentially affected with secondary progressive multiple sclerosis. The Tyzzerella nexilis is a bacterium that carries genome DNA having a 97% or higher average nucleotide identity with the nucleotide sequence for the genome DNA represented by SEQ ID NO: 1.

Description

Method for assisting diagnosis of secondary progressive multiple sclerosis and method for screening therapeutic agents for multiple sclerosis

[0001] The present invention relates to a method for assisting in the diagnosis of secondary progressive multiple sclerosis and a method for screening for therapeutic agents for multiple sclerosis.

[0002] Multiple sclerosis (MS) is an autoimmune disease that causes neuroinflammation and presents a variety of symptoms, including vision loss, motor paralysis, sensory impairment, and higher brain dysfunction. Many MS patients develop relapsing-remitting MS (RRMS), in which symptoms repeatedly worsen and improve. However, approximately 30% of patients progress to secondary progressive MS (SPMS), in which symptoms accumulate without improvement. While numerous disease-modifying drugs for MS have become available in recent years, their therapeutic efficacy for SPMS is limited, and elucidating its pathogenesis and developing new treatments remain major challenges.

[0003] Numerous research results indicating that dysbiosis of the resident bacterial flora plays an important role in the pathology of MS have been reported worldwide, and characteristics of the resident bacterial flora of MS patients based on 16S ribosomal RNA (16S rRNA) gene analysis and metagenomic analysis have been reported (e.g., Patent Documents 1 and 2, and Non-Patent Documents 1 and 2).

[0004] International Publication No. 2017 / 034031 International Publication No. 2021 / 039932

[0005] Miyake S, et al., “Dysbiosis in the Gut Microbiota of Patients with Multiple Sclerosis, with a Striking Depletion of Species Belonging to Clostridia XIVa and IVC lusters.” PLoS One. 2015 Sep 14;10(9):e0137429.Takewaki D, et al., “Alterations of the gut ecological and functional microenvironment in different stages of multiple sclerosis.” Proc Natl Acad Sci U S A. 2020 Sep 8;117(36):22402-22412.Kim, S. W. et al. “Robustness of gut microbiota of healthy adults in response to probiotic intervention revealed by high-throughput pyrosequencing.” DNA research: an international journal for rapid publication of reports on genes and genomes 20, 241-253, doi:10.1093 / dnares / dst006 (2013).Furusawa Y, et al., “Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells.” Nature. 2013 Dec 19;504(7480):446-50.

[0006] Intestinal bacteria that may play an important role in the progression of MS pathology have not been fully analyzed to date. Therefore, an object of the present invention is to identify intestinal bacteria that may play an important role in the progression of MS pathology, thereby providing a method for assisting in the diagnosis of SPMS. Another object of the present invention is to provide a method for screening therapeutic agents for multiple sclerosis.

[0007] The present inventors have identified Tyzzerella nexilis (hereinafter also referred to as "T. nexilis") as a gut bacterial species whose relative abundance is specifically increased in the intestines of SPMS patients and significantly correlates with the EDSS clinical score, which indicates the degree of neurological impairment. Furthermore, we isolated and cultured T. nexilis from fecal samples from two MS patients (one with RRMS and one with SPMS) that contained high levels of T. nexilis. By performing long-read sequencing, we were able to obtain the full-length genomes of each strain. Analysis of these full-length genomes revealed that the two strains were novel T. nexilis strains. Furthermore, when the two novel strains were colonized in a germ-free disease model mouse, they were found to exacerbate neuroinflammation in the model mouse, suggesting that these two strains likely play an important role in the progression of MS. The present invention is based on these findings.

[0008] That is, the present invention relates to, for example, the following inventions: [1] A method for assisting in the diagnosis of secondary progressive multiple sclerosis, comprising the steps of measuring the relative abundance of Tyzzerella nexilis in a fecal sample collected from a subject, and determining that the subject has or is likely to have secondary progressive multiple sclerosis if the relative abundance of Tyzzerella nexilis is higher than the relative abundance of Tyzzerella nexilis in a fecal sample collected from a healthy control, wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of genomic DNA shown in SEQ ID NO: 1. [2] A method for assisting in the evaluation of the therapeutic effect of a patient with secondary progressive multiple sclerosis who has received treatment, comprising the steps of: measuring the relative abundance of Tyzzerella nexilis in a fecal sample collected from the patient before treatment and in a fecal sample collected from the patient after treatment; and determining that the condition of secondary progressive multiple sclerosis in the patient has improved, or is likely to have improved, by the treatment if the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient after treatment is lower than the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient before treatment, wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1. [3] A method for screening for a therapeutic agent for multiple sclerosis, comprising the steps of contacting a test substance with Tyzzerella nexilis and evaluating whether or not the Tyzzerella nexilis has been inactivated by the contacting step. [4] The screening method according to [3], wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1 or 2.[5] The screening method according to [3] or [4], wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1. [6] The screening method according to [5], wherein the multiple sclerosis is secondary progressive multiple sclerosis. [7] The screening method according to [5] or [6], wherein the inactivation reduces the adhesive ability of the Tyzzerella nexilis to colon epithelial cells and / or reduces the function of flagella possessed by the Tyzzerella nexilis. [8] The screening method according to any of [3] to [7], wherein the therapeutic agent for multiple sclerosis suppresses neuroinflammation caused by the multiple sclerosis. [9] A preventive or therapeutic agent for secondary progressive multiple sclerosis, comprising an endolysin derived from Tyzzerella nexilis, wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1.

[10] The preventive or therapeutic agent according to [9], wherein the endolysin is a protein consisting of an amino acid sequence with 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 10 and has the activity of degrading peptidoglycan that constitutes the cell wall of the Tyzzerella nexilis.

[11] A method for preventing or treating secondary progressive multiple sclerosis, comprising administering an endolysin derived from Tyzzerella nexilis to a subject in need thereof, wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1.

[12] The method for preventing or treating secondary progressive multiple sclerosis according to

[11] , wherein the endolysin is a protein consisting of an amino acid sequence with 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 10 and has the activity of degrading peptidoglycan that constitutes the cell wall of the Tyzzerella nexilis.

[13] Tyzzerella nexilis having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1.

[14] Tyzzerella nexilis having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 2.

[0009] According to the present invention, it is possible to provide a method for assisting in the diagnosis of SPMS. According to the present invention, it is also possible to provide a method for screening therapeutic agents for multiple sclerosis.

[0010] Figure 1 shows a comparison of the gut microbiota composition between the RRMS and SPMS groups. Figure 2 shows changes in the global microbiome in progressive MS. Figure 3 shows the relative abundance of T. nexilis A MAG strain (A) and T. nexilis B MAG strain (B) in the healthy control (HC), RRMS, and SPMS groups. Figure 4 shows the structure of the four T. nexilis closed circular genomes, and the average nucleotide identity (ANI) between T. nexilis_strain A MAG or T. nexilis_strain B MAG and the resulting T. nexilis genomes A1, A2, B1, and B2. The A1 and B1 strains are strains whose full-length genomes were obtained by long-read sequencing after isolation and culture from RRMS and SPMS patients, while the A2 and B2 strains are high-quality genomes obtained by long-read metagenomics targeting fecal samples. Both strains have circular closed genomes. This figure shows the phylogenetic tree of the T. nexilis genome. This figure shows a graph showing the EAE clinical scores of germ-free (GF) mice or GF mice inoculated with the T. nexilis A1 or B1 strain. Figure 7 shows the CD4 expression levels in the small intestinal lamina propria (SILP) (A), large intestinal lamina propria (LILP) (B), and central nervous system (CNS) (C). ＋1 is a graph showing the proportion of TH17 cells in cells. 2 is a graph showing the ratio of the number of KO in each pathway of the B1 strain to the number of KEGG orthology (KO) in each pathway of the A1 strain. 3 is a graph showing the IL-6 concentration in the culture supernatant. 4 is an image showing a representative scanning electron microscope image of the colon surface (epithelial cells) of a mouse monocolonized with the A1 or B1 strain. 5 is a graph showing the gene expression level of SAA1 in colon epithelial cells of a mouse colonized with the A1 or B1 strain. 6 is an image showing the results of culturing the A1 strain, the B1 strain, or Escherichia coli ATCC10798 in an endolysin-containing medium or an endolysin-free medium for 24 hours.

[0011] The embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0012] Multiple sclerosis is an autoimmune disease caused by a reaction of the patient's own immune system against normal cells and tissues. Some patients with multiple sclerosis have optic neuritis and myelitis as their primary symptoms, and are sometimes classified as neuromyelitis optica-like disorder (NMOSD), a disease independent of multiple sclerosis. NMOSD is characterized by the presence of anti-aquaporin 4 (AQP4) antibodies and long spinal cord lesions.

[0013] Multiple sclerosis is classified into two types: relapsing-remitting MS (RRMS), which is characterized by repeated acute exacerbations and remissions, and progressive MS. Progressive MS is classified into primary progressive MS (PPMS) and secondary progressive MS (SPMS), which develops into a progressive form after a period of RRMS symptoms.

[0014] [Method for assisting in the diagnosis of SPMS] The method for assisting in the diagnosis of SPMS according to this embodiment includes a step of measuring the relative abundance of Tyzzerella nexilis in a fecal sample collected from a subject (measurement step A), and a step of determining that the subject has or is likely to have SPMS if the relative abundance of Tyzzerella nexilis is higher than the relative abundance of Tyzzerella nexilis in a fecal sample collected from a healthy control (determination step A). ​​Here, Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity (ANI) of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1.

[0015] As shown in the Examples below, a T. nexilis strain (hereinafter also referred to as "B1 strain") having genomic DNA consisting of the base sequence set forth in SEQ ID NO: 1 was identified as a bacterium whose relative abundance is specifically and significantly increased in SPMS patients compared to healthy controls or RRMS patients. Therefore, by using the relative abundance of bacteria having genomic DNA with an average nucleotide identity (ANI) of 97% or more to the base sequence of the genomic DNA set forth in SEQ ID NO: 1 contained in a fecal sample as an index, it becomes possible to determine the presence or absence of SPMS, or the possibility of SPMS, and to determine the effectiveness of a treatment for SPMS. Note that the B1 strain has a genome structure significantly different from that of previously known T. nexilis strains and is considered to be a novel strain discovered by the present inventors. More specifically, T. The genome size of the type strain of T. nexilis (T. nexilis_DSM1787) is 3,648,318 bp, while the genome size of the B1 strain is 4,290,097 bp. The number of genes in the type strain of T. nexilis is 3,457, while that in the B1 strain is 4,190.

[0016] As used herein, "average nucleotide identity (ANI)" is calculated by fragmenting the genome sequence of a control strain into approximately 1,000 bp fragments on a computer, searching for homology of each fragment with the genome sequence of a comparison strain, and averaging the homology values. An ANI of 100% indicates the same strain; an ANI of 97% or more but less than 100% indicates a closely related strain; and an ANI of less than 97% indicates a different strain (excluding closely related strains). Therefore, bacteria having genomic DNA with an average nucleotide identity (ANI) of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1 are the B1 strain and its closely related strains, and these are collectively referred to herein as "B1 strain, etc."

[0017] (Measurement Step A) In the present specification, the term "subject" is not particularly limited as long as it refers to a human subject in need of a diagnosis of SPMS. Such a subject may be, for example, an RRMS patient, particularly a patient who has been diagnosed with RRMS and whose neurological disorder has been slowly accumulating without improvement for a period of 3 to 6 months, 4 to 6 months, or 5 to 6 months.

[0018] The fecal sample collected from a subject and used in the method for assisting diagnosis according to this embodiment may be any sample that can analyze the composition of the subject's intestinal microbiota and measure the relative abundance of T. nexilis. The fecal sample may be feces excreted from the subject's anus, or feces collected from the subject's intestine (particularly the large intestine) before excretion.

[0019] As used herein, the relative abundance of the B1 strain refers to the proportion of the B1 strain in the entire bacterial flora. The relative abundance of the B1 strain can be determined, for example, from the total number of bacteria constituting the bacterial flora and the number of B1 strain bacteria contained in the bacterial flora. More specifically, for example, genes (e.g., 16S rRNA genes) and / or whole genomes having a base sequence common to bacteria contained in the bacterial flora and a base sequence characteristic of the B1 strain are comprehensively decoded, and the total number of decoded genes and the total number of genes attributed to the B1 strain are used as the total number of bacteria constituting the bacterial flora and the number of B1 strains, respectively, to determine the relative abundance of the B1 strain. The relative abundance of closely related strains of the B1 strain can also be determined in the same manner as for determining the relative abundance of the B1 strain.

[0020] From the viewpoint of further improving the accuracy of the judgment in the judgment step A described below, the ANI may be 98% or more, or 99% or more, preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, or 99.5% or more, more preferably 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, and even more preferably 100%.

[0021] (Determination Step A) In this specification, the relative abundance of the B1 strain in healthy controls may be a relative abundance (database value) obtained by a prior measurement, or a relative abundance (measured value) obtained by measurement in parallel with the subject. The relative abundance of the B1 strain in healthy controls may be the average value of multiple healthy controls. In the case of a measured value, it is preferably the average value of 2 or more, 5 or more, or 10 or more healthy controls, and a more precise database value is the average value of 20 or more, 30 or more, or 50 or more healthy controls. Measurement of the relative abundance of the B1 strain in healthy controls can be performed in the same manner as measurement in subjects. The same applies to the relative abundance of strains closely related to the B1 strain.

[0022] As demonstrated in the Examples, the relative abundance of the B1 strain in fecal samples is specifically and significantly increased in SPMS patients compared to healthy controls. Therefore, in determination step A, if the relative abundance of the B1 strain in a fecal sample collected from a subject is greater than the relative abundance of the B1 strain in a fecal sample collected from a healthy control, the subject can be determined to have or be likely to have SPMS. Conversely, if the relative abundance of the B1 strain in a fecal sample collected from a subject is less than the relative abundance of the B1 strain in a fecal sample collected from a healthy control, the subject can be determined to not have or be unlikely to have SPMS. Even when the relative abundance of closely related strains of the B1 strain is measured, a determination similar to that obtained when the relative abundance of the B1 strain itself is measured is possible.

[0023] "Higher relative abundance" or "lower relative abundance" can be determined by analyzing the relative abundance of the B1 strain in healthy controls and the relative abundance of the B1 strain in a subject by statistical analysis (e.g., Wilcoxon rank sum test, Welch's t-test, etc.) to determine whether there is a significant difference. If there is a significant difference, the accuracy of the determination is high. It can also be determined by the difference in relative abundance (e.g., difference = relative abundance of the B1 strain in a subject - relative abundance of the B1 strain in healthy controls) or the percentage difference (e.g., percentage difference = (relative abundance of the B1 strain in a subject - relative abundance of the B1 strain in healthy controls) / relative abundance of the B1 strain in healthy controls x 100 (%)). If the difference in relative abundance or the percentage difference is a positive value, it can be determined that the subject is suffering from or may be suffering from SPMS. Furthermore, the greater the difference or percentage difference in relative abundance, the higher the accuracy of the determination. Even when the relative abundance of a strain closely related to the B1 strain is measured, it is possible to analyze whether the relative abundance is "higher" or "lower" in the same way as when the relative abundance of the B1 strain is measured.

[0024] The method for assisting in the diagnosis of SPMS according to this embodiment can also be regarded as a data collection method for determining whether a subject is suffering from or is likely to suffer from SPMS, comprising the measurement step A described above.

[0025] [Method for assisting evaluation of therapeutic effect] The method for assisting evaluation of therapeutic effect of a treated SPMS patient according to this embodiment includes a step of measuring the relative abundance of Tyzzerella nexilis in a fecal sample collected from the patient before treatment and a fecal sample collected from the patient after treatment (measurement step B), and a step of determining that the SPMS condition of the patient has improved or is likely to have improved by the treatment, if the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient after treatment is lower than the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient before treatment (determination step B). Here, the Tyzzerella nexilis is a bacterium (such as strain B1) having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA set forth in SEQ ID NO: 1.

[0026] (Measurement Step B) In this specification, the term "patient" is not particularly limited as long as it is a human subject who is suffering from or has suffered from SPMS. Other aspects are the same as those in the above-mentioned measurement step A.

[0027] (Determination Step B) In this specification, analysis of the relative abundance of the B1 strain in a fecal sample from a patient before treatment may be performed in advance, or may be performed approximately simultaneously with analysis of the relative abundance of the B1 strain in a fecal sample from a patient after treatment. The terms "pre-treatment" and "post-treatment" as used herein include, for example, before and after a treatment (e.g., a third dose) administered during a period of continuous treatment (e.g., regular medication). That is, "pre-treatment" refers to the beginning of a treatment period, and "post-treatment" refers to the end of the treatment period. The length of the treatment period is not particularly limited and may be, for example, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, etc. Continuous treatment, such as medication, may be administered throughout the entire treatment period, or treatment may be administered only for a portion of the treatment period. Analysis of the relative abundance of closely related strains of the B1 strain in a fecal sample from a patient before treatment is similar to analysis of the relative abundance of the B1 strain in a fecal sample from a patient before treatment.

[0028] As described above, the relative abundance of the B1 strain in fecal samples is specifically and significantly increased in SPMS patients compared to healthy controls. Therefore, in determination step B, if the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient after treatment is lower than the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient before treatment, it can be determined that the patient's SPMS condition has improved or is likely to have improved by treatment. Conversely, if the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient after treatment is lower than the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient before treatment, it can be determined that the patient's SPMS condition has not improved or is likely to have not improved by treatment. Even when the relative abundance of a strain closely related to the B1 strain is measured, the same determination as when the relative abundance of the B1 strain is measured is possible.

[0029] The method for assisting in the evaluation of the therapeutic effect of SPMS patients who have received treatment according to this embodiment can also be regarded as a data collection method for determining the therapeutic effect of SPMS, which includes the measurement step B described above.

[0030] [Screening Method] The screening method for a therapeutic agent for multiple sclerosis according to this embodiment (hereinafter also simply referred to as "screening method") comprises a step of contacting a test substance with Tyzzerella nexilis (contacting step), and a step of evaluating whether or not Tyzzerella nexilis has been inactivated by the contacting step (evaluating step).

[0031] As described above, multiple sclerosis causes neuroinflammation and exhibits various symptoms. As described in detail in the Examples below, neuroinflammation was exacerbated in multiple sclerosis model mice colonized with two strains of T. nexilis with significantly different genome structures, i.e., T. nexilis (B1 strain and A1 strain) having the genomic DNA base sequences shown in SEQ ID NOs: 1 and 2. This suggests that T. nexilis exacerbates multiple sclerosis. Therefore, if a substance capable of inactivating T. nexilis can be found, it could serve as a therapeutic agent for multiple sclerosis. The therapeutic agent may particularly suppress neuroinflammation caused by multiple sclerosis.

[0032] The therapeutic agent to be screened may be any therapeutic agent for multiple sclerosis, and the multiple sclerosis may be relapsing-remitting MS (RRMS) or progressive MS, and the progressive MS may be primary progressive MS (PPMS) or secondary progressive MS (SPMS).

[0033] (Contacting Step) In the contacting step, the test substance is brought into contact with T. nexilis to allow the test substance to act on T. nexilis. The contacting step may be, for example, mixing a predetermined amount of the test substance with a culture medium for T. nexilis, or adding an aqueous medium containing a predetermined amount of the test substance to T. nexilis.

[0034] Culture media for T. nexilis may be any commonly used medium, such as EGe medium, YCFA medium, or BHI medium. Examples of aqueous media include deionized water, distilled water, Tris-HCl buffer, and buffers such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0035] In the contact step, the contact time between the test substance and T. nexilis may be, for example, 30 minutes to 24 hours. Furthermore, in the contact step, the temperature at which the test substance is brought into contact with T. nexilis (contact temperature) may be, for example, the culture temperature of T. nexilis, which may be 4°C to 40°C.

[0036] The test substance is not particularly limited as long as it is a substance that is expected to be a therapeutic agent for multiple sclerosis. It may be randomly selected from known compounds, or may be selected from compounds such as fungicides.

[0037] The T. nexilis used in the screening method according to this embodiment is not particularly limited, and examples thereof include the type strain of T. nexilis (T. nexilis_DSM1787), bacteria having genomic DNA with an ANI of 97% or more relative to the base sequence of the genomic DNA shown in SEQ ID NO: 1 or 2 (referred to herein as the B1 strain etc. and the A1 strain etc., respectively), T. nexilis_MSK.23.83_GCA_013300925.1 (Genbank accession number: GCA_013300925.1), T. nexilis_MSK.23.13_GCA_020553265.1 (Genbank accession number: GCA_020553265.1), and T. nexilis_MSK. 23.79_GCA_020553215.1 (Genbank accession number: GCA_020553215.1), T. nexilis_MSK. 23.22_GCA_013300945.1 (Genbank accession number: GCA_013300945.1), T. nexilis_AM23-10LB_GCA_003470975.1 (Genbank accession number: GCA_003470975.1), T. nexilis_GCA_019012985.1 (Genbank accession number: GCA_019012985.1), T. nexilis_GCA_018376305.1 (Genbank accession number: GCA_018376305.1), T. nexilis_GCA_017850395.1 (Genbank accession number: GCA_017850395.1), T. nexilis_GCA_003463185.1 (Genbank accession number: GCA_003463185.1), T. nexilis_GCA_008680635.1 (Genbank accession number: GCA_008680635.1), T. nexilis_GCA_902167955.1 (Genbank accession number: GCA_902167955.1), T. nexilis_GCA_019012585.1 (Genbank accession number: GCA_019012585.1), etc.Preferably, the bacteria have genomic DNA with an ANI of 97% or more relative to the base sequence of the genomic DNA shown in SEQ ID NO: 1 or 2 (B1 strain, etc. or A1 strain, etc.), and more preferably, the bacteria have genomic DNA with an ANI of 97% or more relative to the base sequence of the genomic DNA shown in SEQ ID NO: 1. As with the B1 strain, the A1 strain and its closely related strains are collectively referred to as "A1 strain, etc."

[0038] The bacterium (A1 strain) having the genomic DNA shown in SEQ ID NO: 2 has a genome structure different from that of previously known T. nexilis strains and is believed to be a novel strain discovered by the present inventors. More specifically, the genome size of the T. nexilis type strain (T. nexilis_DSM1787) is 3,648,318 bp, while the genome size of the A1 strain is 3,686,041 bp, and the number of genes of the T. nexilis type strain is 3,457, while that of the A1 strain is 3,567.

[0039] When T. nexilis is a bacterium having genomic DNA with an ANI of 97% or more relative to the base sequence of the genomic DNA shown in SEQ ID NO: 1 or 2, the ANI may be 98% or more, or 99% or more, preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, or 99.5% or more, more preferably 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, and even more preferably 100%.

[0040] The contact step may be carried out in the presence of bacteria other than T. nexilis. The bacteria other than T. nexilis are preferably enterobacteria. Examples of enterobacteria include bacteria belonging to the genus Ackermansia, Streptococcus, Clostridium, Lactobacillus, Ruminococcus, Bacteroides, Eubacterium, Megamonas, and the family Lachnospiraceae.

[0041] (Evaluation Step) In the evaluation step, whether T. nexilis has been inactivated by the contact step is evaluated. Here, "inactivation" refers to the loss of at least a portion of the pathogenicity of T. nexilis, and may mean that the contribution of T. nexilis to the aggravation of multiple sclerosis is reduced. Inactivation also includes, for example, killing or inhibiting proliferation. Although bacteria other than T. nexilis may also be inactivated, it is preferable that T. nexilis is specifically inactivated.

[0042] Bacteria having genomic DNA with an ANI of 97% or more relative to the base sequence of the genomic DNA shown in SEQ ID NO: 1 have flagella, which, unlike other T. nexilis strains, promote adhesion to colonic epithelial cells. Therefore, when T. nexilis is a bacterium of the above strain, inactivation may involve reducing the ability of Tyzzerella nexilis to adhere to colonic epithelial cells and / or reducing the function of the flagella of the Tyzzerella nexilis. This is considered to result in specific inactivation of the strain.

[0043] The evaluation step may be performed in vivo or in vitro. In the case of in vivo, for example, T. nexilis is recovered after the contact step, and then allowed to settle in a germ-free mouse model of multiple sclerosis, and the neuropathic score, the abundance or function of immune cells, or the like, can be evaluated. In particular, when the inactivation is to reduce the adhesive ability of T. nexilis to colonic epithelial cells, it can be performed using the method described in the Examples below. For example, it can be performed by recovering T. nexilis after the contact step, allowing it to settle in a germ-free mouse model of multiple sclerosis, collecting colonic epithelial cells from the mouse, and observing them under a microscope. Furthermore, when the inactivation is to reduce the function of the flagella of T. nexilis, it can be performed, for example, by analyzing the expression level of the flagellar gene.

[0044] In the case of in vitro testing, for example, a colon epithelial cell line or an artificial organ mimicking colon tissue can be used. The colon epithelial cells used are not particularly limited. After the contact step, T. nexilis is collected and placed in a colon epithelial cell line or an artificial organ mimicking colon tissue. After a predetermined time (e.g., 24 hours), the colon epithelial cells or the colon epithelial cells in the artificial organ can be observed under a microscope or the gene expression of the collected intestinal epithelial cells can be analyzed for evaluation.

[0045] [Preventive or Therapeutic Drug] The preventive or therapeutic drug for secondary progressive multiple sclerosis according to this embodiment contains an endolysin derived from Tyzzerella nexilis, where Tyzzerella nexilis is a bacterium (such as the B1 strain) having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA set forth in SEQ ID NO: 1.

[0046] As used herein, "prevention" refers to preventing, reducing the risk of, or delaying the onset of a disease or symptom in an individual who has not yet contracted or developed the disease, and "treatment" refers to curing, suppressing, or ameliorating a disease or symptom in an individual who has already contracted or developed the disease.

[0047] As will be understood from the examples described below, endolysin derived from the B1 strain, etc., can specifically inhibit the proliferation of the B1 strain, etc. Furthermore, as demonstrated in the examples, the relative abundance of the B1 strain in fecal samples of SPMS patients is specifically and significantly increased compared to healthy controls. Because endolysin derived from the B1 strain, etc., can inhibit the proliferation of the B1 strain, etc., and reduce their relative abundance, it can serve as an active ingredient in a drug for preventing or treating SPMS.

[0048] Endolysins are enzymes derived from bacteriophages and are used by bacteriophages to degrade peptidoglycan, which constitutes the cell wall of host bacteria, causing bacteriolysis, and are also called lytic enzymes. "Endolysins derived from Tyzzerella nexilis" refer to endolysins expressed from endolysin gene sequences contained in the genomic DNA of Tyzzerella nexilis. As described above, endolysins are derived from bacteriophages, but the endolysin gene derived from Tyzzerella nexilis is one in which the bacteriophage DNA has been incorporated into the genomic DNA of the bacterium, or one that has been taken up from another bacterium or bacteriophage by horizontal transfer.

[0049] The endolysin derived from Tyzzerella nexilis may be, for example, a protein consisting of an amino acid sequence that has 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 10. Furthermore, the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0050] The amino acid sequence shown in SEQ ID NO: 10 is one of the endolysins present in the genome of the B1 strain, and is expressed, for example, from the base sequence shown in SEQ ID NO: 11.

[0051] The prophylactic or therapeutic agent according to this embodiment may contain only the active ingredient, or may further contain a pharmaceutically acceptable carrier (excipient, binder, disintegrant, filler, emulsifier, flow additive, etc.) or additive (isotonicity agent, lubricant, flavoring agent, solubilizer, suspending agent, diluent, surfactant, stabilizer, absorption enhancer, bulking agent, pH adjuster, humectant, adsorbent, disintegration inhibitor, coating agent, colorant, preservative, antioxidant, fragrance, flavoring agent, sweetener, buffer, soothing agent, etc.).

[0052] The dosage form of the prophylactic or therapeutic drug according to this embodiment may be selected appropriately depending on the administration method and prescription conditions. Examples of dosage forms include tablets, pills, granules, powders, capsules, drops, sublingual tablets, lozenges, and liquids. Furthermore, from the viewpoint of efficiently delivering the active ingredient to the large intestine, the preparation may be enteric coated. Known enteric coatings can be used without particular limitation.

[0053] The prophylactic or therapeutic agent according to this embodiment may be administered either orally or parenterally. In the case of parenteral administration, the agent may be administered directly into the intestinal tract.

[0054] The dose of the prophylactic or therapeutic drug according to this embodiment can be appropriately determined depending on the sex, age, weight, and symptoms of the subject, as well as the administration method of the prophylactic or therapeutic drug, etc. The drug may be administered in multiple divided doses.

[0055] [Preventive or therapeutic method] The preventive or therapeutic method according to this embodiment comprises administering an endolysin derived from Tyzzerella nexilis to a subject in need thereof. Here, the Tyzzerella nexilis is a bacterium (such as the B1 strain) having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1.

[0056] The endolysin derived from Tyzzerella nexilis is as described above, and the administration method, dosage, etc. of the endolysin are also as described above.

[0057] [Tyzerella nexilis] According to one embodiment, Tyzerella nexilis has genomic DNA that has an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1. According to another embodiment, Tyzerella nexilis has genomic DNA that has an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 2. The genomic DNA that has an average nucleotide identity of 97% or more to the base sequence of the genomic DNA may be naturally occurring genomic DNA, or may be genomic DNA that has been artificially modified to have a sequence different from that of naturally occurring genomic DNA.

[0058] As described above, these Tyzerella nexilis strains are novel bacteria whose genome structures are significantly different from those of conventionally known T. nexilis strains (type strain T. nexilis_DSM1787).

[0059] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0060] [Test Example 1: Identification of Gut Bacteria Associated with MS Progression] The composition of the gut microbiota of MS patients was analyzed using short-read metagenomic data and marker gene-based operational taxonomic units (mOTUs). To identify bacteria characteristic of SPMS patients, MaAsLin2 (Microbiome Multivariable Associations with Linear Models) was used. To confirm whether T. nexilis (id 03689) is ubiquitously elevated in the gut of progressive MS patients worldwide, mOTU analysis was also performed using publicly available large-scale metagenomic data provided by the International MS Microbiome Study (iMSMS) Consortium.

[0061] (1. Subjects) The subjects included 29 healthy controls (HCs), 62 RRMS patients, and 15 SPMS patients. All MS patients included in this study met McDonald's diagnostic criteria. Of the 77 MS patients, RRMS was defined based on a relapsing-remitting clinical course, whereas SPMS was diagnosed based on a clinical course of persistent worsening of neurological impairment without improvement. No patient had a relapse at the time of sample collection. The clinical severity of the patients was assessed by the EDSS score, a measure of neurological impairment based on the clinical evaluation of MS. Patients who had an infection and were treated with antibiotics during the collection of stool samples were excluded. Twelve RRMS patients and two SPMS patients had not received immunotherapy within at least two months prior to collection of stool samples.

[0062] (2. Preparation of DNA from Feces) Freshly collected fecal samples were transported to the laboratory at 4°C in disposable plastic bags containing oxygen absorbers and carbon dioxide generators to ensure the survival of oxygen-sensitive anaerobic bacteria, according to a previously published literature method (Non-Patent Document 3). In the laboratory, the fecal samples were suspended in phosphate-buffered saline containing 20% ​​glycerol, immediately frozen using liquid nitrogen, and stored at -80°C until use. Bacterial DNA was isolated and purified from the fecal samples by enzymatic lysis.

[0063] (3. Short-Read Metagenomic Analysis) A metagenomic shotgun library (insert size: 350 base pairs) was prepared from metagenomic DNA using the ThruPLEX DNA-Seq kit (Takara Bio) according to the manufacturer's protocol. The prepared metagenomic shotgun library was quantified by qPCR and then sequenced using a NovaSeq 6000 sequencing system (Illumina) in 150-bp paired-end mode. Quality filtering of the metagenomic sequences was performed using fastp (version 0.20.0) and ParDRe (version 2.1.5) to remove low-quality reads (meanQV < 20 and < 50 bp) and duplicate reads, respectively. PhiX and human reads were filtered out by mapping quality-filtered reads to phiX and the human genome (hg38) using the short read mapping option in minimap2 (version 2.13-r850). Taxonomic profiles were obtained using mOTUProfiler (version 3.0.1) with 10 million reads per sample. Metagenome assembly was performed using metaSPAdes (v3.14.0) with default parameters. Metagenome-assembled genomes (MAGs) were constructed using MetaBAT2 (version 2.11.2) with default parameters. MAG quality was assessed using CheckM (v1.1.3) and GUNC (v1.0.5). High-quality (HQ) MAGs with estimated completeness ≥ 90%, contamination ≤ 5%, and distortion heterogeneity ≤ 0.5% were selected and passed through GUNC filtering. Representative HQ MAGs were selected by dereplication of MAGs using dRep (v3.2.2) with 99% identity and 90% coverage. The composition of HQ MAGs was calculated using CoverM (0.6.1) (https: / / github.com / wwood / CoverM). The MAG from the RRMS patient was designated T. nexilis_strain A MAG, and the MAG from the SPMS patient was designated T. nexilis_strain B MAG.

[0064] 4. Confirmation of Bacterial Signatures Using the iMSMS Cohort As a validation cohort, we obtained publicly available metagenomic data (ERP115476) and clinical information from the iMSMS cohort. We analyzed 1,139 samples (435 RRMS samples, 133 progressive MS samples, and 571 healthy control samples) pooled based on sample ID and with a metagenomic sequencing depth of over 1 million reads. Taxonomic profiles were performed using mOTU Profiler (v3.0.1) and compared with our own data.

[0065] (5. Results) The results are shown in Figures 1 to 3. Figure 1 shows a comparison of the gut microbiota composition between the RRMS and SPMS groups. Figure 2 shows changes in the overall microbiome in progressive MS. mOTUs increased or decreased (p<0.05 and |coefficient|>1) in SPMS are shown in the left column. Differences in mOTUs increased or decreased in SPMS between progressive MS and HC groups in each country using the validation cohort of the International MS Microbiome Study (iMSMS) are shown. The coefficient values ​​based on MaAsLin2 for the comparison between progressive MS patients and healthy controls are shown from lowest (lower) to highest (upper), as shown on the right-hand scale (upper) of Figure 2. The coefficient values ​​based on MaAsLin2 for the correlation between the relative abundance of each mOTU and EDSS score are also shown from lowest (lower) to highest (upper), as shown on the shaded scale (lower) on the right-hand scale (lower) of Figure 2. 3 shows the relative abundance of T. nexilis A MAG strain and T. nexilis B MAG strain in the HC, RRMS, and SPMS groups.

[0066] In a preliminary comparison between 62 RRMS patients and 15 SPMS patients, 38 mOTUs were significantly increased or decreased in the SPMS group. Among these, T. nexilis (id 03689) was the most abundant species in the SPMS group based on the coefficient values ​​of MaAsLin2 (Figure 1). Furthermore, in a comparison between 15 SPMS patients and 29 healthy controls (HCs), 40 mOTUs were significantly increased or decreased in the SPMS group (|coefficient| > 1 and p < 0.05, as shown in Figure 2). Among these, T. nexilis (id 03689) was the most abundant species in the SPMS group (Figure 2).

[0067] Using publicly available metagenomic data provided by the International MS Microbiome Study (iMSMS) Consortium, mOTU analysis revealed significantly higher levels of T. nexilis in patients with progressive MS compared with those with HC in three Western countries (the United States, the United Kingdom, and Argentina) (Fig. 2). Correlation analysis between gut microbial burden and EDSS score, a clinically based measure of neurological impairment, revealed that higher levels of T. nexilis were positively correlated with EDSS score (Fig. 2).

[0068] The relative amounts of T. nexilis_strain A MAG and T. nexilis_strain B MAG were evaluated among the HC, RRMS, and SPMS groups. In particular, the relative amount of T. nexilis_strain B MAG was significantly higher in the SPMS group compared to the HC and RRMS groups, but the relative amount of T. nexilis_strain A MAG did not differ significantly among the three groups (Figure 3).

[0069] These results suggest that T. nexilis specifically increases in SPMS patients. Therefore, measuring the relative abundance of T. nexilis in fecal samples may enable us to determine whether or not a patient has SPMS, or whether they may be susceptible to it, and to assess the effectiveness of SPMS treatment.

[0070] [Test Example 2: Discovery of a New T. nexilis Strain] To analyze detailed genomic information at high resolution, four MS patients (two RRMS patients and two SPMS patients) with high abundances of T. nexilis were selected to attempt to obtain high-quality T. nexilis genomes. Two T. nexilis strains were successfully isolated from fecal samples of one RRMS patient and one SPMS patient, and closed circular genomes were obtained by de novo assembly using PacBio HiFi reads. Furthermore, long-read metagenomic analysis was performed on fecal samples from two other RRMS and SPMS patients to obtain closed circular genomes of T. nexilis. The four obtained closed circular genomes of T. nexilis were identified as T. nexilis_strain A MAG and T. nexilis_strain A MAG. The four closed circular genomes obtained, T. nexilis_strain A MAG, T. nexilis_strain B MAG, and the publicly available genomes of 13 T. nexilis strains, were used to construct a phylogenetic tree of T. nexilis.

[0071] (1. Isolation of T. nexilis from Fecal Samples) Fecal samples were homogenized, diluted with PBS containing 20% ​​glycerol, and then stored at -80°C for bacterial isolation. To isolate T. nexilis from RRMS and SPMS patients, frozen fecal dilutions were plated onto EGe agar plates and incubated anaerobically at 37°C for 2 to 4 days. 16S rRNA genes from individual colonies were amplified using the 27F and 1492R primers shown in Table 1. Subsequently, Sanger sequencing was performed using the 27F and 519R primers to identify colonies corresponding to T. nexilis strains from each patient. These bacteria were cultured in GAM medium and stored in 20% glycerol at -80°C.

[0072]

[0073] 2. Long-Read Sequencing. SMRTbell libraries were constructed from 5 μg of fecal metagenomic DNA using Metagenomic Pacific Biosciences (PacBio) according to the manufacturer's protocol (Part No. 101-853-100 Version 05). Bacterial DNA was also obtained from the culture medium of isolated T. nexilis strains by enzymatic lysis. For PacBio Sequel II multiplexed sequencing, SMRTbell libraries were constructed from 1 μg of DNA according to the manufacturer's protocol (Part No. 101-696-100 Version 02). HiFi reads were generated using Circular Consensus Sequencing (CCS) with the parameters --min-passes 3 and --min-rq 0.95.

[0074] (3. Long-Read Assembly of Isolated Genomes) HiFi reads were assembled using hiCanu with the following options: minReadLength = 5000, minOverlapLength = 5000, genomeSize = 4m, and -pacbio-hifi. Circular contigs were detected using minimap2 by terminal direct repeat sequences with ≥95% identity and an alignment length of ≥1,000 base pairs. The genome quality of the circular contigs was assessed using CheckM and GUNC. High-quality genomes with completeness ≥95% and contamination ≤5% were selected and passed through GUNC filtering. The lack of mobile genetic elements (MGEs) in short-read MAGs compared to high-quality genomes obtained with long reads was visualized using Gview. Complete genomes were visualized using Proksee. The genome obtained from the T. nexilis strain isolated from the fecal sample of an RRMS patient was designated A1, and the genome obtained from the T. nexilis strain isolated from the fecal sample of an SPMS patient was designated B1. The T. nexilis strains containing these genomes were designated T. nexilis_A1 and T. nexilis_B1, respectively.

[0075] (4. Long-Read Metagenome Assembly) HiFi reads were assembled using hiCanu with the following options: minReadLength=1500, minOverlapLength=1500, genomeSize=100m, maxInputCoverage=100000, minInputCoverage=1, stopOnLowCoverage=1, and -pacbio-hifi. Circular contigs were detected using minimap2 by terminal direct repeat sequences with ≥95% identity and an alignment length of ≥1,000 base pairs. The genomic quality of the circular contigs was assessed using CheckM and GUNC. High-quality (HQ) contigs were selected based on completeness >90% and contamination <5% and passed through GUNC filtering. HQ circular contigs of T. nexilis were determined by aligning the ORFs of the HQ circular contigs with 20 bacterial marker genes in the T. nexilis mOTU (ref_mOTU_v3_03689). Contigs with an average nucleotide similarity of >96.5% between the marker genes were identified as HQ circular contigs of T. nexilis. Using this method, a closed circular genome of T. nexilis was constructed from a fecal sample of a SPMS patient. This genome was designated B2, and the T. nexilis strain containing this genome was designated T. nexilis_B2.

[0076] In the assembly of the T. nexilis genome from the fecal samples of RRMS patients, we were unable to identify T. nexilis HQ contigs from the circular contigs. Therefore, short reads were mapped to long read contigs and binned using MetaBAT2. HQ bins were identified using the same criteria as for the circular contigs, and T. nexilis HQ bins with an average nucleotide similarity of >96.5% for the 20 bacterial marker genes in ref_mOTU_v3_03689 were identified. Here, according to the phylogenetic tree of 120 bacterial marker genes determined by GTDB-tk, the HQ bin of this T. nexilis strain was clustered in cluster A (the cluster to which T. nexilis_strain A MAG belongs). Therefore, reference-guided assembly was performed to identify T. nexilis from the fecal samples of RRMS patients. A closed circular genome of T. nexilis was constructed. The HQ bin of T. nexilis was combined with the complete genome of a T. nexilis strain isolated from an RRMS patient, and metagenomic HiFi reads were mapped to the combined genome with greater than 99% identity and greater than 80% coverage. The mapped reads were assembled using hifiasm_meta with default parameters to construct a closed circular genome of T. nexilis from a fecal sample of an RRMS patient. This genome was designated A2, and the T. nexilis strain containing this genome was designated T. nexilis_A2 strain.

[0077] (5. Comparison of T. nexilis Genomes) ORFs for each T. nexilis genome were predicted using prokka with default parameters (v1.13.3). ORFs for the T. nexilis genome were aligned to the eggNOG database (v5.0.2) using eggNOG mapper (ultrasensitive mode) (v2.1.5) with an e-value < 1e-10, and the assigned K numbers were mapped to the KEGG pathway using KEGG mapper. These ORFs were aligned to the Comprehensive Antibiotic Resistance Database (CARD) (v3.2.6) and the Virulence Factor Database (VFDB) (download date: 2022 / 01 / 14) using DIAMOND blastp (sensitive mode) with an e-value <1e-100 to annotate antibiotic resistance and virulence genes, respectively. Integrative conjugation element (ICE)-associated genes were also annotated by aligning the ORFs with the IceBerg database (last updated: May 2, 2018) using DIAMOND blastp (sensitive mode) with >50% identity and coverage. Insertion sequences were predicted using ISEscan (v1.7.2.3) with default parameters. ICE signature genes were determined using ICEscreen (v1.2.0) with default parameters. The number of complete ICEs containing all ICE characteristic genes determined by ICEscreen was counted in each genome. Defense systems were predicted using defense finder (v1.2.2) with default parameters. Publicly available T. nexilis genomes were obtained from the RefSeq database, and genomes meeting completeness ≥ 90% and contamination ≤ 5% (CheckM) were used for comparison. Additionally, the T. nexilis reference strain (T. nexilis_DSM1787) was cultivated to obtain a complete genome for comparison. Multiple alignment of 120 bacterial marker genes of the T. nexilis genome was performed using GDTB-tk with default parameters.

[0078] Phylogenetic trees were constructed using maximum likelihood estimation in MEGA X60. Dot plots for genome structure comparison were generated using D-Genies with minimap2 aligner (v2.24). To compare the number of MGEs in the T. nexilis genome, which belongs to the Firmicutes phylum, a representative and complete genome was downloaded from the GenBank database (Release 255).

[0079] (6. Results) The results are shown in Figures 4 and 5. Figure 4 shows the structure of the four T. nexilis closed circular genomes and shows the average nucleotide identity (ANI) between T. nexilis_strain A MAG (or B MAG) and the obtained T. nexilis genomes A1, A2, B1, and B2. Figure 5 shows a phylogenetic tree of the T. nexilis genome.

[0080] Based on the phylogenetic tree of short-read MAGs, five closed circular genomes, and 13 publicly available genomes, we found two distinct clusters of T. nexilis. One cluster was the T. nexilis_strain A MAG, and the other was the T. nexilis_strain B MAG (Figure 5). Furthermore, among the five closed circular genomes obtained in this study, the genome of the T. nexilis type strain (T. nexilis_DSM1787) and two genomes, A1 and A2, derived from RRMS patients, clustered into the A MAG lineage (Figure 5). Furthermore, A1 shared 98.6% ANI with A MAG, and A2 shared 99.8% ANI with A MAG (Figure 4). On the other hand, the two genomes, B1 and B2, derived from SPMS patients, clustered into the B MAG lineage (Figure 5). Furthermore, B1 shared 100% ANI with B MAG, and B2 shared 99.6% ANI with B MAG (Figure 4). These results suggest that the T. nexilis_A1 strain (hereinafter also referred to as "A1 strain") and T. nexilis_B1 strain (hereinafter also referred to as "B1 strain"), which were successfully isolated from RRMS and SPMS patients, have distinct genomes compared to previously known T. nexilis strains and are considered to be novel strains. In particular, the B1 strain had a significantly different genome size compared to A1, and its genome structure was clearly distinct, as it was clustered into a different lineage from publicly available genomes (Figure 4).

[0081] Test Example 3: Evaluation of the effect of T. nexilis strains on the degree of neurological damage. An in vivo bacterial transfer experiment was conducted using two isolated T. nexilis strains (A1 and B1). Experimental autoimmune encephalomyelitis (EAE) germ-free mice, a mouse model of multiple sclerosis, were administered culture media containing the A1 or B1 strain, and the EAE clinical score was evaluated.

[0082] (1. EAE Induction) Germ-free C57BL / 6 mice were purchased from Claire and housed in a pathogen-free facility. To induce EAE, germ-free C57BL / 6 female mice (8-10 weeks old) were injected with 200 μg of myelin oligodendrocyte glycoprotein (MOG) peptide fragment 35-55 (MOG) emulsified in incomplete Freud's adjuvant (Difco). ３５－５５ Mice were immunized subcutaneously at two sites on the back with 1000 mg of pertussis toxin (PTX). On days 0 and 2 after immunization, mice were intraperitoneally injected with 400 ng of pertussis toxin (PTX). EAE was clinically scored daily (0: no clinical signs, 0.5: tail weakness, 1: partial tail paralysis, 1.5: severe tail paralysis, 2: flaccid tail, 2.5: flaccid tail and hind limb weakness, 3: partial hind limb paralysis, 4: total hind limb paralysis, 5: hind limb and forelimb paralysis).

[0083] (2. Administration of A1 and B1 strains to mice) Five to seven-week-old germ-free female C57BL / 6 mice were administered the A1 or B1 strain medium in a MOG. ３５－５５ These gnotobiotic mice were housed in a vinyl isolator for 2 to 3 weeks before use in the experiment.

[0084] (3. Results) The results are shown in Figure 6. Figure 6 is a graph showing the EAE clinical scores of germ-free (GF) mice or GF mice inoculated with the T. nexilis A1 or B1 strain. Mice colonized with the A1 or B1 strain had increased EAE clinical scores compared to germ-free mice (Figure 3B). These results suggest that T. nexilis exacerbates the severity of multiple sclerosis.

[0085] Test Example 4: Evaluation of the effect of T. nexilis strains on the amount of TH17 cells Next, in mice colonized with the A1 and B1 strains, the CD4 ＋ The proportion of T helper 17 (TH17, IL-17A-producing) cells among T cells was measured by flow cytometry.

[0086] (1. Preparation of Lymphocytes) Isolation of colonic or small intestinal lamina propria lymphocytes was performed according to a previously published method (Non-Patent Document 4). More specifically, the colon or small intestine was incubated in Hank's Balanced Salt Solution (HBSS) containing 2% fetal bovine serum (FBS), 1 mM dithiothreitol, and 20 mM EDTA at 37°C for 30 minutes with agitation, and the epithelial layer was then removed by vigorous shaking in the HBSS. The remaining tissue was minced and incubated in RPMI 1640 containing 2% FBS, 400 U / ml collagenase D (Roche), 0.25 U / ml dispase (BD Biosciences), and 0.1 mg / ml DNase I (Wako) at 37°C for 30 minutes with agitation. The digested tissue was suspended in 37% Percoll (GE Healthcare), layered on 70% Percoll® (GE Healthcare), and then centrifuged at 800 × g for 20 minutes. Lymphocytes at the interface were then collected. To isolate lymphocytes from the CNS, tissue from mice was minced and incubated with agitation for 45 minutes at 37°C in RPMI 1640 containing 2% FBS, 1.33 mg / ml collagenase H (Roche), and 20 μg / ml DNase I (Wako). The digested tissue was suspended in 37% Percoll (GE Healthcare), layered on 70% Percoll (GE Healthcare), and then centrifuged at 800 × g for 20 minutes. Lymphocytes were collected from the 37% / 40% Percoll interphase.

[0087] (2. Flow Cytometry) For intracellular cytokine staining, lymphocytes were stimulated with 25 ng / ml phorbol myristate acetate (PMA) and 1 μg ionomycin (Sigma) in the presence of GoldiStop (BD Biosciences) for 4 hours at 37°C. Dead cells were stained with Zombie Aqua dye (BioLegend). After blocking Fc receptors with anti-CD16 / 32 antibody (BD Biosciences), cells were stained with anti-CD4 antibody (eBioscience) and anti-IL-17A antibody (BioLegend). All data were collected on a FACS Canto II cytometer (BD Biosciences) and analyzed using Flowjo (v.10.8) (Tree Star).

[0088] (3. Results) The results are shown in Figure 7. Figure 7 shows the CD4 ＋ Graph showing the percentage of TH17 cells in CD4+ cells in the SILP, LILP, and CNS. ＋ The proportion of TH17 cells among T cells was higher in mice colonized with the A1 or B1 strain than in GF mice. ＋ The proportion of TH17 cells among T cells was significantly higher in mice colonized with the B1 strain than in mice colonized with the A1 strain.

[0089] The results of Test Examples 3 and 4 showed that neuroinflammation worsened in multiple sclerosis model mice colonized with two strains of T. nexilis that differ significantly in genome structure. This suggests that if a substance that inactivates T. nexilis can be discovered, the substance may be considered useful as a therapeutic agent for multiple sclerosis.

[0090] Test Example 5: Comparison of genes possessed by T. nexilis strains As performed in "5. Comparison of T. nexilis genomes" in Test Example 2, the A1 and B1 ORFs were annotated based on the Kyoto Encyclopedia genome and gene (KEGG) database. Then, the differences in the number of KEGG orthologies (KO) in the KEGG pathway were compared.

[0091] The results are shown in Figure 8. Figure 8 is a graph showing the ratio of the number of KOs in each pathway in the B1 strain to the number of KOs in each pathway in the A1 strain. The B1 strain had a particularly high number of KOs in the "Flagellar assembly" category compared to the A1 strain, suggesting that the B1 strain has a higher content of genes involved in the flagella formation pathway.

[0092] Test Example 6: Evaluation of the effect of T. nexilis strains on IL-6 production Bacterial flagella are known to be selective agonists of Toll-like receptor 5 (TLR5), and when flagella are recognized by TLR5, the production of, for example, inflammatory cytokines such as IL-6 is promoted. As shown in Test Example 5, the B1 strain was thought to contain many genes involved in flagellum formation, so a co-culture experiment was conducted between the A1 and B1 strains and lamina propria dendritic cells, which are TLR5 reporter cells, to evaluate IL-6 release.

[0093] (1. Co-culture with lamina propria dendritic cells) CD11c selected by FACS ｈｉｇｈ CD11b ｈｉｇｈ Lamina propria dendritic cells (5 x 10 ３ Cells were cultured in the presence or absence of a TLR5 antagonist (1 μM) in a medium containing bacteria (A1 or B1 strain) that had been heat-inactivated at 60°C for 30 minutes. After 18 hours of incubation, the IL-6 concentration in the culture supernatant was measured by ELISA.

[0094] (2. Results) The results are shown in Figure 9. Figure 9 is a graph showing the IL-6 concentration in the culture supernatant. ｈｉｇｈ CD11b ｈｉｇｈWhen lamina propria dendritic cells (hereinafter also referred to as "dendritic cells") were cocultured with the A1 strain, IL-6 was barely detectable in the culture supernatant, and was unchanged from when dendritic cells were cultured alone (compare the first and second panels from the left in Figure 9). In contrast, when dendritic cells were cocultured with the B1 strain, the IL-6 concentration in the culture supernatant was significantly higher than the IL-6 concentrations in the culture supernatants obtained when dendritic cells were cocultured with the A1 strain and when dendritic cells were cultured alone (compare the first and second and third panels from the left in Figure 9). Furthermore, the IL-6 concentration in the culture supernatant obtained when dendritic cells were cocultured with the B1 strain in the presence of a TRL5 antagonist was reduced compared to the IL-6 concentration in the culture supernatant obtained when dendritic cells were cocultured with the B1 strain in the absence of a TRL5 antagonist (compare the third and fourth panels from the left in Figure 9).

[0095] The B1 strain was confirmed to have the ability to stimulate TLR5, and this result suggested that the B1 strain had flagella.

[0096] Test Example 7: Evaluation of Adhesion of T. nexilis Strains to Intestinal Epithelial Cells This test example focused on another functional aspect of flagella: promoting adhesion to intestinal epithelial cells. To examine bacterial adhesion to intestinal epithelial cells, mice were generated that were monocolonized with the A1 or B1 strain. The colons were then harvested from the mice, the colonic contents were washed, and the colonic surface was observed under a scanning electron microscope.

[0097] (1. Isolation of colonic epithelial cells) Colons collected from mice colonized with the A1 or B1 strain were incised longitudinally and thoroughly washed with PBS. The tissue was incubated with 5 mM EDTA in HBSS at 37°C for 30 minutes with shaking to promote dissociation of epithelial cells from the lamina propria. Epithelial cells were collected by centrifugation, added with RNAprotect Cell Reagent (QIAGEN), and stored at -80°C.

[0098] (2. Results) The results are shown in Figure 10. Figure 10 is an image showing a representative scanning electron microscope image of the colon surface (epithelial cells) of a mouse solely colonized with the A1 or B1 strain. No bacterial adhesion was observed on the colon surface of the mouse solely colonized with the A1 strain. On the other hand, many bacteria were observed adhering to the colon surface of the mouse solely colonized with the B1 strain. It is believed that the bacteria adhering to the colon surface of the mouse solely colonized with the B1 strain were the B1 strain, suggesting that the presence of flagella promotes adhesion of the B1 strain to intestinal epithelial cells.

[0099] Test Example 8: Evaluation of the effect of T. nexilis strains on serum amyloid A1 expression levels. Several studies have shown that serum amyloid A (SAA), secreted from intestinal epithelial cells upon bacterial adhesion, promotes differentiation into TH17 cells. Therefore, this test example focused specifically on serum amyloid A (SAA). Mice monocolonized with the A1 or B1 strain were generated, and the expression levels of SAA in colonic epithelial cells were examined by quantitative PCR (qPCR).

[0100] (1. qPCR) Total RNA was extracted from colonic epithelial cells using an RNeasy Mini Kit (Qiagen), and cDNA was obtained using PrimeScript™ RT Master Mix (TaKaRa). qPCR was performed using SYBR Premix Ex Taq (TaKaRa) with a Thermal Cycler Dice Real Time System (TaKaRa). The primers used were those listed in Table 2 below.

[0101]

[0102] (2. Results) The results are shown in Figure 11. Figure 11 is a graph showing the gene expression levels of SAA1 in colonic epithelial cells of mice colonized with the A1 or B1 strain. SAA1 expression was increased in colonic epithelial cells of mice colonized with the A1 or B1 strain compared to germ-free mice. In particular, the expression level of SAA1 was significantly increased in colonic epithelial cells of mice colonized with the B1 strain compared to colonic epithelial cells of mice colonized with the A1 strain.

[0103] [Mechanism of Action of B1 Strain Aggravating Neuroinflammation in the Large Intestine] Based on the results of Test Examples 3 to 8, it is believed that the B1 strain aggravates neuroinflammation by possessing flagella. In particular, the large intestine has two layers, an outer mucus layer and an inner mucus layer, and colonic epithelial cells are in contact with the inner mucus layer on the side opposite the outer mucus layer. While enterobacteria generally reside in the outer mucus layer, the B1 strain possesses flagella, allowing it to swim through the inner mucus layer and promoting adhesion to colonic epithelial cells. This promotes SAA1 production. Furthermore, the flagella possessed by the B1 strain stimulate TLR5, promoting IL-6 production. It has already been reported that increased production of SAA1 and IL-6 promotes the differentiation of Th17 cells, and the amount of Th17 cells was actually increased by the B1 strain. These factors are thought to be one of the factors that contribute to the exacerbation of neuroinflammation.

[0104] Based on the mechanism of action described above, it was suggested that if a substance could be discovered that could reduce the adhesive ability of the B1 strain to colonic epithelial cells and / or reduce the function of the flagella of the B1 strain, it would be possible to specifically inactivate the B1 strain and determine that this substance would be useful as a therapeutic agent for multiple sclerosis.

[0105] Test Example 9: In Vitro Evaluation of the Bacteriolytic Activity of Endolysin The B1 strain, isolated and cultured from the feces of a patient with secondary progressive multiple sclerosis (SPMS), is a previously unidentified new strain. Further analysis of this strain revealed that it contains an overwhelmingly larger number of dynamic genetic elements (e.g., prophage and insertion sequences) in its bacterial genome than other indigenous gut bacteria. Aiming to develop a new treatment or drug for SPMS by selectively eliminating the B1 strain, we focused on the dynamic genetic elements contained in the genome of the B1 strain, particularly endolysin derived from the B1 strain. As part of the screening process, the bacteriolytic activity of this endolysin was evaluated in vitro.

[0106] (1. Cloning of endolysin gene and purification of endolysin) We commissioned FUJIFUILM to synthesize one endolysin gene present in the genome of the B1 strain, construct an expression vector, and purify the protein expressed from the expression vector.

[0107] Specifically, based on the sequence information of the endolysin gene derived from the B1 strain (one of six endolysin genes present in the genome of the B1 strain), a His tag and a SUMO tag were added in tandem in this order to the N-terminus of the endolysin protein, and the gene was synthesized after further codon optimization for E. coli (SEQ ID NO: 10).The synthesized gene was then inserted into the NcoI-XhoI site of pET28a(+) to construct an expression vector (SEQ ID NO: 12).

[0108] BL21 (DE3) Competent Cells (Novagen) were transformed to produce a recombinant. The resulting recombinant was pre-cultured (37°C, 18 hours) in 20 mL of LB medium (20 g / L LB Broth Base (Invitrogen), 50 μg / mL kanamycin). The pre-culture solution was inoculated into 1.2 L of LB medium at 1% concentration, and main culture was performed (37°C, 3 hours, 120 rpm). Subsequently, the target protein was induced with IPTG at a final concentration of 1 mM. The induction conditions were 16°C, 18 hours. After completion of the culture, the expressing cells (bacterial cells) were collected by centrifugation.

[0109] The collected bacterial cells were suspended in 120 mL of binding buffer and then disrupted by ultrasound to obtain a lysate. The resulting lysate was centrifuged, and the supernatant was sterilized by filtration through a 0.2 μm filter to obtain a sample. The sample was applied to 5 mL of Ni-NTACartridge (FFwako) that had been pre-equilibrated with binding buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.6). The flow-through fraction was collected, and the column was washed with wash buffer (20 mM sodium phosphate, 150 mM sodium chloride, 25 mM imidazole, pH 7.6). The endolysin protein was purified by elution with elution buffer (20 mM sodium phosphate, 500 mM sodium chloride, 500 mM imidazole, pH 7.6).

[0110] (2. In vitro evaluation of the bacteriolytic activity of endolysin) In addition to the A1 and B1 strains, Escherichia coli ATCC10798 (E. coli ATCC10798) was used as a control. E. coli ATCC10798 was obtained from the Microbial Materials Development Laboratory, BioResource Center, RIKEN, National Research and Development Agency. YCFA liquid medium, which had previously been confirmed to be capable of growing all of the bacteria, was used as the liquid medium. For the endolysin-administered group, 150 μl of YCFA liquid medium and 50 μl of a solution containing the endolysin protein purified in 1 above (final concentration: 20 μg / ml) were added to a 96-well round-bottom plate, and one of the three bacteria was then added. For the control group (endolysin-untreated group), 150 μl of YCFA liquid medium and 50 μl of PBS were added to a 96-well round-bottom plate, and one of the three bacteria was added. All groups were incubated in an anaerobic environment (80% N) in an anaerobic chamber. ２ , 10% H ２ , 10% CO ２ ) and after 24 hours, the growth inhibitory effect of endolysin on the target bacteria was evaluated based on the presence or absence of bacterial precipitate after growth.

[0111] (3. Results) The results are shown in Figure 12. Figure 12 is an image showing the results of culturing the A1 strain, the B1 strain, and E. coli ATCC10798 in an endolysin-containing medium or an endolysin-free medium for 24 hours. As shown in Figure 12, the growth of the control groups of each bacterium was not inhibited even after 24 hours of culture. Furthermore, the growth of the endolysin-administered groups of the A1 strain and E. coli ATCC10798 was not inhibited even after 24 hours of culture. On the other hand, the growth of the endolysin-administered group of the B1 strain was inhibited after 24 hours of culture. In other words, endolysin derived from the B1 strain specifically inhibited the growth of the B1 strain.

[0112] Furthermore, as can be seen from the results of Test Example 9, it is possible to screen for therapeutic agents for multiple sclerosis by contacting a test substance with Tyzzerella nexilis and evaluating whether or not the Tyzzerella nexilis is inactivated by the contact.

Claims

1. A method for assisting in the diagnosis of secondary progressive multiple sclerosis, comprising the steps of: measuring the relative abundance of Tyzzerella nexilis in a fecal sample collected from a subject; and determining that the subject has or may have secondary progressive multiple sclerosis if the relative abundance of Tyzzerella nexilis is greater than the relative abundance of Tyzzerella nexilis in a fecal sample collected from a healthy control, wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO:

1.

2. A method for assisting in the evaluation of the therapeutic effect of a patient with secondary progressive multiple sclerosis who has received treatment, comprising the steps of: measuring the relative abundance of Tyzzerella nexilis in a fecal sample collected from the patient before treatment and in a fecal sample collected from the patient after treatment; and determining that the condition of secondary progressive multiple sclerosis of the patient has been improved or is likely to have been improved by the treatment if the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient after treatment is lower than the relative abundance of Tyzzerella nexilis in the fecal sample collected from the patient before treatment, wherein the Tyzzerella nexilis is a bacterium having genomic DNA having an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO:

1.

3. A method for screening a therapeutic agent for multiple sclerosis, comprising the steps of contacting a test substance with Tyzzerella nexilis, and evaluating whether or not the Tyzzerella nexilis is inactivated by the contacting step.

4. The screening method according to claim 3, wherein the Tyzzerella nexilis is a bacterium having genomic DNA having an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO: 1 or 2.

5. The screening method according to claim 3, wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO:

1.

6. The screening method according to claim 5, wherein the multiple sclerosis is secondary progressive multiple sclerosis.

7. The screening method according to claim 5, wherein the inactivation comprises reducing the adhesion ability of the Tyzzerella nexilis to colon epithelial cells and / or reducing the function of flagella possessed by the Tyzzerella nexilis.

8. A screening method according to any one of claims 3 to 7, wherein the therapeutic drug for multiple sclerosis suppresses neuroinflammation caused by multiple sclerosis.

9. A preventive or therapeutic agent for secondary progressive multiple sclerosis, comprising an endolysin derived from Tyzzerella nexilis, wherein the Tyzzerella nexilis is a bacterium having genomic DNA with an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO:1, and the endolysin has activity of degrading peptidoglycan that constitutes the cell wall of the Tyzzerella nexilis.

10. The preventive or therapeutic drug according to claim 9, wherein the endolysin is a protein consisting of an amino acid sequence having 90% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 10 and having the activity of degrading peptidoglycan that constitutes the cell wall of Tyzzerella nexilis.

11. Tyzzerella nexilis, having genomic DNA having an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO:

1.

12. Tyzzerella nexilis, having genomic DNA having an average nucleotide identity of 97% or more to the base sequence of the genomic DNA shown in SEQ ID NO:2.

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