Methods for testing autoimmune diseases

Abstract

Provided are a novel biomarker for an autoimmune disease and a method for using the same. A method for examining an autoimmune disease, said examination method comprising (1) a step for detecting a crAss-like phage in an enterovirus-containing sample collected from a subject.

Description

[Technical Field]

[0001] The present invention relates to methods for testing autoimmune diseases and the like. [Background Art]

[0002] Most autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, are difficult to diagnose with a single biomarker, and diagnosis can only be achieved by combining multiple hematological findings, histological findings and clinical symptoms. However, there are many problems to be solved in diagnosis, such as the high invasiveness of histological examination and the high level of expertise required for diagnosis, and the discovery of novel biomarkers is desired.

[0003] The gut microbiota greatly affects human immunity and metabolism, and it has been reported that it may be an etiology for various diseases such as inflammatory bowel disease, colorectal cancer, diabetes, and autism, and is regarded as promising as a biomarker for onset prediction. In recent years, an invention relating to gut microbiota that contributes to the diagnosis of autoimmune diseases has been reported (Patent Document 1).

[0004] As screening for gut microbiota, measurement using 16S rRNA or the like has been carried out so far, but this is a method for quantifying only bacteria, and viruses have not been evaluated. [Prior Art Documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. WO 2016 / 050111 [Summary of the Invention] [Problem to be Solved by the Invention]

[0006] An object of the present invention is to provide a novel biomarker for autoimmune diseases and a method of using the same. Preferably, it is an object to provide a simple testing technique for autoimmune diseases.

[0007] Furthermore, the present invention aims to provide a simpler and more accurate method for metagenomic analysis of the intestinal vir flora. [Means for solving the problem]

[0008] As a result of diligent research, the inventors have found that a method for testing autoimmune diseases, comprising the step of (1) detecting a crAss-like phage in an intestinal virus-containing sample collected from a subject, can solve the above problems. Furthermore, they have found that by assembling shotgun sequencing reads to obtain contigs and comparing them with viral sequences in a database, and performing taxonomic annotation of the contigs, metagenomic analysis of the intestinal virological flora can be performed more simply and accurately. Based on these findings, the inventors have conducted further research and completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Item 1. A method for testing for autoimmune diseases, (1) A step to detect crAss-like phage in an intestinal virus-containing sample collected from a subject, Testing methods, including those mentioned above.

[0010] Item 2. Furthermore, (2) a step of determining that the subject is suffering from an autoimmune disease if the amount or concentration of the crAss-like phage detected in step (1) is less than or equal to a cutoff value. The inspection method described in item 1, including the method described in item 1.

[0011] Item 3. The testing method according to item 1 or 2, wherein the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus.

[0012] Item 4. The testing method described in any of items 1 to 3, wherein the intestinal virus-containing sample is feces.

[0013] Item 5. The testing method described in any of items 1 to 4, wherein the subject is a human.

[0014] Item 6. Diagnostic reagents for autoimmune diseases, including those for detecting crAss-like phage.

[0015] Item 7. A diagnostic kit for autoimmune diseases, including a crAss-like phage detection agent.

[0016] Item 8. A method for screening active ingredients of agents for the prevention or treatment of autoimmune diseases, using the amount or concentration of crAss-like phage in intestinal virus-containing samples collected from animals treated with a test substance as an indicator.

[0017] Item 9. A method for evaluating the potential for inducing or exacerbating autoimmune diseases, using the amount or concentration of crAss-like phage as an indicator, in intestinal virus-containing samples collected from animals treated with a test substance.

[0018] Item 10. A method for metagenomic analysis of the viral flora in a sample containing intestinal viruses, A metagenomic analysis method comprising the step of assembling shotgun sequence reads to obtain contigs, comparing the contigs with viral sequences in a database, and performing taxonomic annotation of the contigs.

[0019] Item 11. The metagenomic analysis method according to Item 10, wherein the contig is a linear or circular contig of 2 kbp or more. [Effects of the Invention]

[0020] According to the present invention, a biomarker for autoimmune diseases can be provided. By utilizing this biomarker, it may be possible to perform tests for autoimmune diseases, screen for active ingredients of agents for the prevention or treatment of autoimmune diseases, and evaluate the inducibility or exacerbation of autoimmune diseases. The testing technique of the present invention can be easily performed as it can utilize samples that can be easily collected.

[0021] Further, according to the present invention, a simpler and more accurate method for metagenomic analysis of the intestinal virome can be provided. Mode for Carrying Out the Invention

[0022] In the present specification, the expressions "comprise" and "contain" include the concepts of "comprise", "contain", "consist essentially of" and "consist only of".

[0023] 1. Testing methods for autoimmune diseases In one aspect, the present invention relates to a method for testing an autoimmune disease, the testing method comprising (1) a step of detecting crAss-like phage in an intestinal virus-containing sample collected from a subject (this may also be referred to as "the autoimmune disease testing method of the present invention" herein). This will be described below.

[0024] 1-1. Process (1) The type of autoimmune disease to be tested is not particularly limited. Autoimmune diseases of all classes, grades and stages according to various classification criteria for autoimmune diseases are subjects of the test. Furthermore, the lesion site is also not particularly limited. From the viewpoint of test accuracy and the like, preferred examples of autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, and the like, and rheumatoid arthritis is particularly more preferred.

[0025] The subject is the target organism of the testing method of the present invention, and the species thereof is not particularly limited. Examples of species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with humans being preferred.

[0026] The condition of the subject is not particularly limited. Examples of the subject include a specimen for which it is unknown whether the subject suffers from an autoimmune disease, a specimen that has already been determined to suffer from an autoimmune disease by another method, a specimen that has already been determined not to suffer from an autoimmune disease by another method, and a specimen from a subject undergoing treatment for an autoimmune disease.

[0027] The intestinal virus-containing sample is not particularly limited as long as it contains intestinal viruses. Examples of intestinal virus-containing samples include intestinal fluid, feces, and other contents of the digestive tract. Of these, feces are preferred from the viewpoint of the burden on the subject. The intestinal virus-containing sample may be used alone or in combination of two or more types. The intestinal virus-containing sample can be collected from the subject by a method known to those skilled in the art.

[0028] The target of detection in step (1) is crAss-like phage (which may be collectively referred to as "target biomarkers" in this specification).

[0029] The target biomarkers are those whose levels are altered in autoimmune diseases, and by using them as indicators, it is possible to differentiate between autoimmune diseases.

[0030] crAss-like phage is a target biomarker that is present in lower amounts in autoimmune disease specimens than in healthy specimens.

[0031] Detection is typically performed by measuring the amount or concentration of the target biomarker. "Concentration" does not necessarily refer to absolute concentration, but may also refer to relative concentration, weight per unit volume, amount per unit of total viral load or total nucleic acid load in the sample, or raw data measured to determine absolute concentration.

[0032] The method for detecting the target biomarker is not particularly limited as long as it can specifically detect some or all of the target biomarker. Specific examples of detection methods include Southern hybridization, Northern hybridization, and DNA microarray methods for detecting genes specific to enteroviruses or mRNA derived from those genes. Metagenomic analysis methods using shotgun sequencing of DNA in the sample (e.g., the method described in Test Example 1 below) can also be used. Furthermore, antibody-based methods for metabolic detection of viral metabolites and proteomics detection of viral proteins can also be used.

[0033] Furthermore, methods for measuring viruses in a sample include, for example, FISH, real-time PCR, and RT-PCR.

[0034] Here, we will explain the RT-PCR method. The RT-PCR method can be used for analysis by, for example, (1) extracting RNA of the target enteric virus from the sample, (2) performing RT-PCR using nucleic acid fragments (primers) that hybridize to the extracted RNA, and (3) detecting the DNA fragment amplified in step (2). By combining the above nucleic acid fragments with template cDNA derived from the sample and performing an amplification reaction, DNA fragments (PCR products) specific to the target enteric virus can be obtained. By observing the PCR product over time and identifying the number of PCR cycles at which a certain amount of DNA is reached, it is possible to quantify the number of target enteric viruses in the sample.

[0035] The time course of amplified PCR products can be observed by labeling the PCR products with an intercalator fluorescent dye such as SYBR(R)GreenI and measuring the fluorescence intensity at each PCR step. Since intercalator dyes have the property of increasing fluorescence intensity when intercalated with double-stranded nucleic acids, it is possible to accurately measure the PCR products generated by the PCR reaction from the cDNA of the target virus, and SYBR(R)GreenI is particularly preferred.

[0036] By identifying the number of PCR cycles (hereinafter referred to as the CT value) at which a certain arbitrarily set fluorescence intensity (DNA amount) is reached, it becomes possible to quantify the target enteric virus in the sample. Alternatively, TaqMan probes or MoleculerBeacons labeled with fluorescent dyes can be used. TaqMan probes and MoleculerBeacons are probes in which a fluorescent dye and a quencher are attached to an oligonucleotide homologous to the internal sequence of the region amplified by PCR, and are used in conjunction with the PCR reaction. Because the interaction between the fluorescent dye and the quencher bound to the probe emits fluorescence corresponding to the PCR amplification reaction, the time course of the amplified PCR product can be observed by measuring the fluorescence intensity at each PCR stage.

[0037] According to the testing method of the present invention, which includes step (1), it is possible to provide the amount and / or concentration of a target biomarker, which is an indicator for detecting autoimmune diseases, thereby assisting in the detection of autoimmune diseases.

[0038] The test results obtained by the testing method of the present invention, including step (1), can be used for determining the effectiveness of treatment, elucidating the pathogenesis of autoimmune diseases, predicting the prognosis of autoimmune diseases, stratifying patients, selecting treatment methods (personalized medicine, treatment response), and so on.

[0039] 1-2. Process (2) In one embodiment, the testing method of the present invention further comprises (2) the step of determining that the subject is suffering from an autoimmune disease if the amount or concentration of the crAss-like phage detected in step (1) is equal to or greater than a cutoff value. It is preferable to include the above. According to the inspection method of the present invention, which includes step 2, it is possible to determine autoimmune diseases.

[0040] The cutoff value can be appropriately set by a person skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc. For example, it can be a value determined on a case-by-case basis based on the amount and / or concentration of the target biomarker in an intestinal virus-containing sample taken from a subject who is not suffering from an autoimmune disease, or a predetermined value. The cutoff value can be, for example, 0.5 to 1.4 times, 0.6 to 1.3 times, 0.7 to 1.2 times, 0.8 to 1.1 times, or 0.9 to 1 times the amount and / or concentration of the target biomarker (mean value, median value, etc., if there are multiple subjects) in an intestinal virus-containing sample taken from a subject who is not suffering from an autoimmune disease.

[0041] In a preferred embodiment of step (2), if the subject is a sample undergoing treatment for an autoimmune disease, the therapeutic effect can be determined by setting the cutoff value to a value based, for example, on the amount and / or concentration of the target biomarker in past samples of the same sample.

[0042] 2. Higher accuracy in the diagnosis of autoimmune diseases If the testing method of the present invention, including step (2), determines that a subject is suffering from an autoimmune disease, the autoimmune disease can be diagnosed with higher accuracy by further combining the testing method of the present invention with a step of applying a diagnosis of autoimmune disease by a physician. Furthermore, since the testing method of the present invention can detect autoimmune diseases more accurately, combining the above step with the testing method of the present invention allows for a more efficient and accurate diagnosis of "suffering from an autoimmune disease."

[0043] 3. Treatment of autoimmune diseases If the testing method of the present invention, including step (2), determines that the subject has an autoimmune disease, the testing method of the present invention can be further modified, or if the subject is diagnosed with an autoimmune disease as described in "2. Higher Accuracy Diagnosis of Autoimmune Diseases" above, the combination of the testing method of the present invention and the step of applying a diagnosis by a physician can be further modified, by performing step (3) of treating the subject who has been determined or diagnosed with an autoimmune disease. Furthermore, since the testing method of the present invention can detect autoimmune diseases more accurately, by combining step (3) with the testing method of the present invention, or with the combination of the testing method of the present invention and the step of applying a diagnosis by a physician, subjects suffering from autoimmune diseases can be treated more efficiently and reliably.

[0044] While there are no particular restrictions on the treatment methods for autoimmune diseases, drug therapy is a typical example. There are no particular restrictions on the types of drugs used in drug therapy, but for example, for the treatment of systemic lupus erythematosus, non-steroidal anti-inflammatory drugs (NSAIDs) and steroids are used, while for the treatment of rheumatoid arthritis, anti-rheumatic drugs, biological agents (biopharmaceuticals), NSAIDs, and steroids (corticosteroids) are used. Drugs can be used individually, in combination of two, or three or more types.

[0045] 4. Diagnostic tests for autoimmune diseases In one aspect, the present invention relates to a diagnostic reagent for autoimmune diseases (which may also be referred to herein as "the diagnostic reagent of the present invention"), comprising a crAss-like phage detection agent (which may also be referred to herein as "the diagnostic reagent of the present invention"). This will be described below.

[0046] The detection agent of the present invention is not particularly limited as long as it can specifically detect the target biomarker. Examples of such detection agents include primers, probes, antibodies, etc., for viral genes that are the target biomarkers, or for their expression products.

[0047] The detection agent of the present invention may be modified, provided that its function is not significantly impaired. Examples of modifications include the addition of labels such as fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, and biotin.

[0048] Suitable fluorescent dyes used in the present invention are those generally used to label nucleotides for the detection and quantification of nucleic acids. Examples include, but are not limited to, HEX (4,7,2',4',5',7'-hexachloro-6-carboxylfluorescein, green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow-green fluorescent dye), rhodamin or its derivatives (e.g., tetramethylrhodamin (TMR)). Any suitable known labeling method can be used to label nucleotides with a fluorescent dye (see Nature Biotechnology, 14, 303-308 (1996)). Commercially available fluorescent labeling kits can also be used (e.g., Amersham Pharmacia's Oligonucleotide ECL 3'-Oligolabeling System).

[0049] The detection agent of the present invention can also be used by immobilizing it on any solid phase. For this reason, the detection agent of the present invention can be provided in the form of a substrate on which the detection agent is immobilized (for example, a microarray chip on which a probe is immobilized).

[0050] The solid phase used for immobilization is not particularly limited as long as it can immobilize polynucleotides, etc., and examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries, or other substrates. The method of immobilizing the detection agent onto the solid phase is not particularly limited. The immobilization method is well known in the art, depending on the type of immobilized probe, for example, using a commercially available spotter (such as one from Amersham) in the case of a microarray [e.g., in situ synthesis of oligonucleotides using photolithographic technology (Affymetrix), inkjet technology (Rosetta Inpharmatics), etc.].

[0051] Primers, probes, etc., are not particularly limited as long as they selectively (specifically) recognize the target biomarker or nucleic acids derived therefrom. Here, "selectively (specifically) recognized" means, for example, that the target biomarker can be specifically detected in the Northern blotting method, or that the target biomarker or nucleic acids (cDNA, etc.) derived therefrom are specifically amplified in the RT-PCR method, but is not limited to these, as long as a person skilled in the art can determine that the detected or amplified product is derived from the target biomarker.

[0052] Specific examples of primers and probes include the polynucleotides listed in (a) below and the polynucleotides listed in (b) below: (a) Polynucleotides having at least 15 consecutive bases in the base sequence of the target biomarker and / or polynucleotides complementary to said polynucleotide, (b) At least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize under stringent conditions to the base sequence of the target biomarker or a base sequence complementary thereto.

[0053] A complementary polynucleotide or complementary base sequence (complementary strand, reverse strand) refers to a polynucleotide or base sequence that is nucleotide-complementary to the full-length polynucleotide sequence consisting of the base sequence of the target biomarker, or to a partial sequence having at least 15 consecutive bases in length (for convenience, these are also referred to here as the "forward strand"). This complementarity is based on base pairings such as A:T and G:C. However, such a complementary strand is not limited to forming a perfectly complementary sequence with the base sequence of the target forward strand; it may also have a complementary relationship sufficient to allow for hybridization under stringent conditions. Here, stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe is bound, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). For example, typical washing conditions after hybridization include conditions of approximately "1×SSC, 0.1%SDS, 37°C". It is preferable that the complementary strand maintains its hybridized state with the target positive strand even after washing under such conditions. While not particularly limited, more stringent hybridization conditions include washing conditions of approximately "0.5×SSC, 0.1%SDS, 42°C", and even more stringent hybridization conditions include washing conditions of approximately "0.1×SSC, 0.1%SDS, 65°C". Specifically, examples of such complementary strands include strands consisting of base sequences that are completely complementary to the base sequence of the target positive strand, and strands consisting of base sequences that have at least 90%, preferably 95%, more preferably 98% or more, identity with the positive strand.

[0054] Primers and probes can be designed, for example, based on the nucleotide sequence of the target biomarker, using various design programs. Specifically, candidate primer or probe sequences obtained by applying a design program to the nucleotide sequence of the target biomarker, or sequences containing at least part of such sequences, can be used as primers or probes.

[0055] The base length of primers, probes, etc., is not particularly limited as long as it has a length of at least 15 consecutive bases, as described above, and can be set appropriately depending on the application. For example, when used as a primer, the base length can be 15 to 35 bases, and when used as a probe, the base length can be 15 to 35 bases.

[0056] The diagnostic reagent of the present invention may contain other detection agents besides the detection agent of the present invention (for example, probes for detecting other nucleic acids, antibodies, etc.). In this case, the diagnostic reagent of the present invention may be a diagnostic reagent that can test for other diseases or conditions in addition to autoimmune diseases. In this case, the detection agent of the present invention is included as a detection agent for testing autoimmune diseases. From this viewpoint, in one embodiment, the diagnostic reagent of the present invention is a diagnostic reagent for autoimmune diseases that includes a detection agent for testing autoimmune diseases comprising the detection agent of the present invention.

[0057] The diagnostic reagent of the present invention may also be in the form of a composition. The composition may optionally contain other components. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.

[0058] The diagnostic reagent of the present invention may be in the form of a kit. In addition to the above-mentioned detection agent or the above-mentioned composition containing the same, the kit may also contain materials that can be used to detect a target biomarker in an intestinal virus-containing sample of a subject. Specific examples of such materials include various reagents (e.g., nucleic acid extraction reagents, buffers, etc.) and equipment (e.g., equipment for the purification and separation of intestinal virus-containing samples).

[0059] 5. Screening methods for active ingredients of agents for the prevention or treatment of autoimmune diseases. In one aspect, the present invention relates to a method for screening active ingredients of agents for the prevention or treatment of autoimmune diseases (sometimes referred to herein as "the active ingredient screening method of the present invention"), using the amount or concentration of crAss-like phage in an intestinal virus-containing sample collected from an animal treated with a test substance as an indicator. This will be described below.

[0060] There are no particular restrictions on the species of animal. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

[0061] The test substance can be a wide range of compounds, both naturally occurring and artificially produced. Furthermore, it is not limited to purified compounds; compositions of various compounds and extracts from plants and animals can also be used. The compounds include not only low-molecular-weight compounds but also high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0062] More specifically, the active ingredient screening method of the present invention includes the step of selecting the test substance as an active ingredient for an autoimmune disease prevention or treatment agent (or a candidate substance for an active ingredient for an autoimmune disease prevention or treatment agent) when the value of the above indicator is higher than the amount or concentration (control value) of the corresponding biomarker in an intestinal virus-containing sample taken from an animal that has not been treated with the test substance.

[0063] A corresponding biomarker refers to the same virus as the target biomarker being used as an indicator.

[0064] "High" means, for example, that the value of the indicator is 2, 5, 10, 20, 50, or 100 times the control value.

[0065] 6. Methods for evaluating the potential for inducing or exacerbating autoimmune diseases. In one aspect, the present invention relates to a method for evaluating the potential for inducing or exacerbating autoimmune diseases (sometimes referred to as "the toxicity evaluation method of the present invention" in this specification) using the amount or concentration of crAss-like phage in an intestinal virus-containing sample collected from an animal treated with a test substance as an indicator. This will be described below.

[0066] More specifically, the toxicity evaluation method of the present invention includes the step of determining that a test substance is capable of inducing or exacerbating autoimmune diseases if the value of the above indicator is lower than the amount or concentration (control value) of the corresponding biomarker in an intestinal virus-containing sample taken from an animal that has not been treated with the test substance.

[0067] A corresponding biomarker refers to the same virus as the target biomarker being used as an indicator.

[0068] "Low" means, for example, that the index value is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the control value.

[0069] 7. Metagenome Analysis Methods In one aspect, the present invention relates to a metagenomic analysis method of the viral flora in an intestinal virus-containing sample, comprising the step of assembling shotgun sequencing reads to obtain contigs, comparing the contigs with viral sequences in a database, and performing taxonomic annotation of the contigs (which may also be referred to as "the metagenomic analysis method of the present invention" in this specification). This will be described below.

[0070] There are no particular restrictions on the method of the shotgun sequence, and any method following or similar to known methods may be employed.

[0071] Shotgun sequencing reads are the determined base sequences of the DNA fragments subjected to shotgun sequencing. The base length of the reads is preferably 50-500 bp, more preferably 80-300 bp, even more preferably 100-250 bp, and even more preferably 120-200 bp.

[0072] A contig is obtained by assembling the reads of a shotgun sequence. That is, the base sequences of each read in the shotgun sequence are compared, and each read is joined together based on the overlapping sequences to generate a longer base sequence (contig). There are no particular restrictions on the assembly method, and a method according to or similar to a known method can be used.

[0073] Conventional methods for analyzing the intestinal virome involved directly comparing shotgun sequence reads with viral sequences in a database for taxonomic annotation. However, this method is impractical due to the cumbersome data processing required and the frequent occurrence of virus misclassification. The metagenomic analysis method of the present invention is characterized by assembling shotgun sequence reads to generate contigs, and then comparing these contigs with viral sequences in a database for taxonomic annotation. This enables simpler and more accurate metagenomic analysis of the intestinal virome.

[0074] The contig is preferably a linear contig with a base length of a certain amount or more, or a circular contig. Here, a linear contig is a base sequence that has a 5' end and a 3' end, and a circular contig is a contig that does not have a 5' end and a 3' end, and the base sequence is circular. A circular contig is generated when the 5' end and 3' end of a linear contig overlap by a certain amount or more (for example, 30 bp or more, preferably 50 bp or more), by superimposing the overlapping portion.

[0075] The base length of the linear contig is preferably 2 kbp or more, more preferably 3 kbp or more, even more preferably 4 kbp or more, and even more preferably 5 kbp or more. There is no particular upper limit to the base length, for example, preferably 10 kbp, more preferably 20 kbp.

[0076] The base length of the circular contig is, for example, 1 kbp or more, preferably 1.5 kbp or more, more preferably 2 kbp or more, even more preferably 3 kbp or more, and even more preferably 5 kbp or more. There is no particular upper limit to the base length, for example 5 kbp, preferably 10 kbp, and more preferably 20 kbp.

[0077] There are no particular restrictions on the methods for comparing contigs with virus sequences in a database, or for taxonomically annotating contigs; known methods or methods similar to them may be adopted.

[0078] Once taxonomic annotation is complete for each contig, that is, once the origin virus of each contig has been identified, the viral composition ratio can be calculated by quantifying each contig. This allows for comparison of viral composition ratios between samples and identification of the types of viruses that have increased or decreased between samples. There are no particular restrictions on the method of quantifying each contig and calculating the composition ratio; known methods or methods similar to them can be employed. [Examples]

[0079] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0080] Example 1: Screening of biomarkers for autoimmune diseases The inventor has constructed a unique pipeline for comprehensive analysis of the vast amount of genomic information (metagenomics) of the microbiome obtained by shotgun sequencing, focusing on three pillars: phylogenetic analysis, genetic analysis, and pathway analysis. In the metagenomic analysis pipeline, fecal samples were first collected from 111 patients with rheumatoid arthritis, 47 patients with systemic lupus erythematosus, and 289 healthy individuals. These samples were lysed using the bead method, and DNA was extracted. Using Hiseq3000, 150 bp paired-end sequencing yielded an average of 6.3 Gb of sequence reads per sample.

[0081] Quality control of sequence reads was performed as follows. A series of quality control steps were performed to maximize the quality of the dataset. The main steps of the quality control process were as follows: (i) trimming of low-quality bases, (ii) identification and masking of human reads, and (iii) removal of duplicate reads. Duplicate reads were marked using PRINSEQ-lite (version 0.20.4, -derep 1). Using Trimmomatic (version 0.39; parameters: ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:8:true LEADING:20 TRAILING:20 SLIDINGWINDOW:3:15 MINLEN:60), raw reads were trimmed, the Illumina adapter was clipped, and low-quality bases at both ends were cut. Reads shorter than 60 bp after trimming were discarded. Next, duplicate reads with the same sequence were removed, leaving only the longest read. Ultimately, the quality-filtered reads were aligned to the human reference genome (hg38) using the default parameters of bowtie2 (version 2.3.5) and BMTagger (version 3.101). Both tools retained only reads where both paired ends were not matched.

[0082] Virus contigs were assembled and identified as follows: Filtered paired-end reads were de novo assembled into contigs using MEGAHIT (version 1.2.9; parameters: -min-contig-len 1500). After assembly, contigs with a 5' and 3' overlap of 50 bp or more were classified as circular contigs. Linear contigs of 5 kbp or more and circular contigs of 1.5 kbp or more were processed with VirSorter (version 1.0.6) and VirFinder (version 1.1). VirSorter was run using both the RefSeqABVir (-db 1) and Viromes (-db 2) databases, and sequences classified as viruses with either the "most confident" prediction (Category 1) or the "most likely" prediction (Category 2) were extracted for further analysis. Furthermore, among the candidate virus sequences identified as "potential" by VirSorter, those with a VirFinder score of 0.7 or higher were extracted for analysis. The remaining contigs were extracted for further analysis if their VirFinder score was 0.9 or higher. To minimize contamination with bacterial sequences, the enrichment of bacterial and viral genes was evaluated, as previously described by Gregory et al. Bacterial single-copy orthologs v4 (BUSCOv4; version 4.1.2) was used to search for single-copy orthologues of 124 bacteria registered in the BUSCO database, and the results were filtered using the HMM score cutoff value provided by BUSCO. Viral gene enrichment was evaluated by performing hmmsearch (version 3.1b2) on the viral contigs against curated viral protein family modules (VPF; https: / / portal.nersc.gov / dna / microbial / prokpubs / EarthVirome_DP / ), with a match of E-value < 0.05 defined as a hit. Furthermore, a threshold of BUSCO hit count / VPF hit count > 0.05 was used to indicate bacterial genome contamination.These procedures yielded a total virus population of 93,254 viruses with an average length of 21.1 kbp.

[0083] Taxonomic annotation was performed as follows: First, viral contigs were classified using the complete viral RefSeq genome (downloaded in June 2020, containing 12,696 genomes; https: / / www.ncbi.nlm.nih.gov / labs / virus / vssi / # / ) as a reference. While the viral genome classification information is based on NCBI classification data, recent papers have modified the family-level classification of crAssphage (NC_024711.1) to crAss-like phage. Viral contig sequences were matched with the reference genome using MegaBlast (ncbi-blast-plus version 2.10.1) with an E-value <10⁻¹⁰, nucleotide identity ≥95%, and contig coverage ≥85%. Viral contigs were assigned to taxonomy at the species level or higher based on the MegaBlast hit with the highest bit score. Contigs that were not assigned to taxonomy by nucleotide-level comparison in the previous step were moved to protein-level comparison for family-level taxonomic annotation. First, open reading frames (ORFs) of viral contigs were predicted using MetaProdigal (version 2.6.3) with the -p meta option. To detect crAss-like phage, the ORFs of viral contigs were compared with the signature genes of crAss-like phage. The protein sequences of the polymerase (UGP_018) and terminase (UGP_092) of crAssphage (NC_024711.1) were matched with the ORFs in the viral contigs using Blastp, with an E-value <10⁻⁵ and an alignment length of 350 or more. Viral contigs that were hit by blastp were assigned family-level taxonomic annotation as crAss-like phage. Subsequently, the remaining contigs that were not classified proceeded to taxonomic annotation through a voting system.ORFs contained in viral contigs were searched for using DIAMOND blastp (v0.9.32.133) against the RefSeq protein database (downloaded in June 2020, containing 420,609 proteins) with an E-value < 10⁻⁵. Taxonomic annotations were assigned to the ORFs based on the DIAMOND blastp hit with the highest bit score. Next, all taxonomic assignments for each contig's ORFs were summarized, and taxonomic information at the family level or higher was assigned based on the majority of the taxonomic assignments among the annotated ORFs. Viral contigs without a majority of taxonomic assignments were classified as unclassified viruses. Additionally, viral contigs with two or fewer annotated ORFs were classified as unclassified viruses.

[0084] The viral abundance was quantified as follows: To calculate the raw abundance of different viral populations in each sample, quality-checked reads from each sample were mapped to viral contigs recovered from the sample using bowtie2 with default parameters. A CoverM filter (version 0.4.0) was used to remove reads mapped to contigs with less than 95% nucleotide identity. Next, the average read depth between viral populations was calculated using CoverM trimmed_mean (parameters: --trim-min 0.05 --trim-max 0.95). The read depth for each viral population was normalized by dividing it by the total sequence length of each sample. The normalized read depths for each viral population were summed for each sample, and the normalized abundance of each clade was calculated at different taxonomic ranks. Finally, outlier samples were detected by principal component analysis (PCA).

[0085] As described above, using a statistical algorithm, we reconstructed the longest possible nucleotide sequences from the metagenomic data of each sample, which consisted of tens of millions of short DNA fragments. By referring to existing viral sequence databases and evaluating homology at the nucleotide level, the presence or absence of viral marker molecules, and homology at the amino acid level, we annotated the reconstructed long nucleotide sequences with viral lineage information. Subsequently, we quantified the abundance of the virus in each sample by quantifying the amount of the annotated nucleotide sequences. As a result, we identified crAss-like phage as a biomarker for rheumatoid arthritis patients and systemic lupus erythematosus.

[0086] Using this biomarker, a regression equation adjusted for age and sex (Disease (rheumatoid arthritis or systemic lupus erythematosus) ~ viral load + age + age) was created. 2 Logistic regression analysis using (+ sex + dataset + total reads) revealed that this biomarker significantly contributes to the diagnosis of rheumatoid arthritis and systemic lupus erythematosus.

[0087] Table 1 shows the effect size, standard error (SE), and p-value (P).

[0088] [Table 1]

[0089] The proportion of crAss-like phage in rheumatoid arthritis was 0.51 times that of healthy individuals, and in systemic lupus erythematosus it was 0.35 times that of healthy individuals.

[0090] When crAss-like phage was used as a biomarker to diagnose rheumatoid arthritis, a receiver operating characteristic (ROC) curve was created, and the AUC was 0.88 (95% confidence interval 0.85~0.92).

[0091] When crAss-like phage was used as a biomarker to diagnose systemic lupus erythematosus, a receiver operating characteristic (ROC) curve was created, and the AUC was 0.76 (95% confidence interval 0.69~0.83).

Claims

1. A method to assist in testing for autoimmune diseases, (1) A step of detecting crAss-like phage in an intestinal virus-containing sample collected from a subject, and (2) A step of determining that the subject is suffering from an autoimmune disease if the amount or concentration of the crAss-like phage detected in step (1) is below a cutoff value, Methods to assist with testing, including those mentioned above.

2. A method to assist in the examination according to claim 1, wherein the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus.

3. The method for assisting the test according to claim 1, wherein the intestinal virus-containing sample is feces.

4. A method for assisting the examination according to any one of claims 1 to 3, wherein the subject is a human being.

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