Compositions and methods useful for the detection and treatment of multiple sclerosis and other demyelinating diseases

Abstract

Disclosed herein are methods for detecting one or more microorganisms in a subject having or suspected of having a demyelinating disease, including multiple sclerosis. Also disclosed herein are methods for treating a subject developing or experiencing a demyelination exacerbation based on the detection of a microorganism.

Description

Detailed Description of the Invention

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 785,377, filed Dec. 27, 2018, and U.S. Provisional Application No. 62 / 875,779, filed Jul. 18, 2019. The entire contents of these prior applications are hereby incorporated by reference herein. BACKGROUND OF THE INVENTION

[0002] Multiple sclerosis (MS) is a chronic demyelinating disease of unknown cause that affects approximately 400,000 people's brains and spinal cords in the United States. A number of infectious diseases of the central nervous system (CNS), including distemper (in dogs), measles (SSPE, in humans), JC virus (in humans), and influenza (in humans), can cause demyelination (Atkins, G. et al. Rev. Med. Virol, 291 - 303. (2000)). Microorganisms, particularly viruses, have long been suspected as the causative pathogens of MS based on the epidemiology of the disease, including geographical patterns, sporadic occurrences, and migration studies (Kurtzke, J. Phys. Med. Rehab. Clin. N. Am. 16, 327 - 349 (2005); Kurtzke, J.F. J. Neurovirol. 6, Suppl. 2, S134 - 140 (2000); Meinl, E. Concepts of viral pathogesis of MS. Curr. Opin. Neurology, 12, 303 - 307 (1999); and Murray, J. Infection as a cause of multiple sclerosis. BMJ, 325, 1128 (2002)). SUMMARY OF THE INVENTION

[0003] Disclosed herein is a method for diagnosing a demyelinating disease in a subject, the method comprising: a) obtaining a sample suspected of containing a microorganism-specific antibody from the subject; b) incubating the sample with one or more microorganism capture agents, wherein the one or more microorganism capture agents bind to one or more of the microorganism-specific antibodies and the microorganism-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8; c) detecting the presence of one or more microorganism-specific antibodies in the sample; and d) diagnosing the subject as having a demyelinating disease if one or more microorganism-specific antibodies are present in the sample.

[0004] Disclosed herein is a method for detecting a demyelinating disease in a subject, the method comprising: a) obtaining a sample suspected of containing a microorganism-specific antibody specific for one or more microorganisms selected from the lists of Table 1, Table 2 or Table 8 from the subject; b) contacting the sample with one or more microorganism capture agents under conditions that allow the one or more microorganism capture agents, each specific for one or more microorganism-specific antibodies, and the one or more microorganism-specific antibodies, specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8, to bind to one or more of the microorganism-specific antibodies to produce a mixed biological sample; c) incubating the mixed biological sample under conditions such that the one or more microorganism capture agents specifically bind to at least one of the microorganism-specific antibodies to form a detectable complex; and d) detecting the presence of the detectable complex, thereby detecting the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8, wherein the presence of one or more microorganisms selected from the list of Table 1 in the mixed biological sample detects a demyelinating disease in the subject.

[0005] Disclosed herein is a method for diagnosing and treating a demyelinating disease in a subject, the method comprising: a) obtaining a sample suspected of containing a microorganism-specific antibody from the subject; b) incubating the sample with one or more microbial capture agents, wherein the one or more microbial capture agents bind to one or more of the microorganism-specific antibodies and the microorganism-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8; c) detecting the presence of the one or more microorganism-specific antibodies in the sample; d) diagnosing the subject as having a demyelinating disease if the one or more microorganism-specific antibodies are present in the sample; and e) administering to the subject: i) an antibacterial agent if the one or more detected microorganism-specific antibodies are bacteria selected from the lists of Table 1, Table 2, or Table 8, ii) an antiviral agent if the one or more detected microorganism-specific antibodies are viruses selected from the lists of Table 1, Table 2, or Table 8, iii) an antifungal agent if the one or more detected microorganism-specific antibodies are fungi selected from the lists of Table 1, Table 2, or Table 8, or v) a combination thereof.

[0006] Disclosed herein is a method for detecting and treating a demyelinating disease in a subject, the method comprising: a) obtaining a sample suspected of containing a microorganism-specific antibody specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 from the subject; b) contacting the sample with one or more microbial capture agents under conditions such that each of the one or more microbial capture agents is specific for one or more microorganism-specific antibodies and the one or more microorganism-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8, thereby enabling the one or more microbial capture agents to bind to one or more of the microorganism-specific antibodies to produce a mixed biological sample; c) incubating the mixed biological sample under conditions such that the one or more microbial capture agents specifically bind to at least one of the microorganism-specific antibodies to form a detectable complex; d) detecting the presence of the detectable complex, thereby detecting the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8, wherein the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 in the mixed biological sample detects a demyelinating disease in the subject; and e) administering to the subject: i) an antibacterial agent if the one or more detected microorganisms are bacteria selected from the lists of Table 1, Table 2, or Table 8, ii) an antiviral agent if the one or more detected microorganisms are viruses selected from the lists of Table 1 or Table 2, iii) an antifungal agent if the one or more detected microorganisms are fungi selected from the lists of Table 1, Table 2, or Table 8, or v) a combination thereof.

[0007] Disclosed herein is a method for detecting one or more antibodies in a sample, the method comprising: a) obtaining a sample suspected of containing a microorganism-specific antibody specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 from the subject; b) incubating the sample with one or more microbial capture agents, wherein the one or more microbial capture agents bind to one or more of the microorganism-specific antibodies and the one or more microorganism-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8; and c) detecting the presence of the one or more microorganism-specific antibodies in the sample.

[0008] Disclosed herein is a method for detecting one or more microorganisms in a sample, the method comprising: a) obtaining a sample suspected of containing one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 from a subject; b) incubating the sample with one or more microbial capture agents that bind to one or more of the microorganisms selected from the lists of Table 1, Table 2, or Table 8; and c) detecting the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 in the sample.

[0009] Disclosed herein is a method for treating a patient with multiple sclerosis (MS) or an MS-related disease, the method comprising: a) administering an antibacterial agent to the subject if one or more detected microbial-specific antibodies are microorganisms selected from the lists of Table 1, Table 2, or Table 8; b) administering an antiviral agent to the subject if one or more detected microbial-specific antibodies are viruses selected from the lists of Table 1, Table 2, or Table 8; c) administering an antifungal agent to the subject if one or more detected microbial-specific antibodies are fungi selected from the lists of Table 1, Table 2, or Table 8; or d) administering a combination of a), b), or c) to the subject, wherein the patient is identified as in need of an antibacterial agent, an antiviral agent, an antifungal agent, or a combination thereof by: i) obtaining a sample from the patient suspected of containing microbial-specific antibodies; ii) incubating the sample with one or more microbial capture agents that bind to one or more of the microbial-specific antibodies and wherein the microbial-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8; and iii) detecting the presence of one or more microbial-specific antibodies in the sample.

[0010] The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the invention.

[0011] Further advantages of the present invention will be described in part in the following description, will become apparent in part from the description, or may be learned by practicing the present invention. The advantages of the present invention will be realized and achieved by the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0013] The present disclosure can be more readily understood by reference to the following detailed description of the invention, the drawings, and the examples included herein.

[0014] Before disclosing and describing the methods and compositions, it should be understood that, unless otherwise specified, they are not limited to particular synthesis methods and, unless otherwise specified, are not limited to particular reagents, and thus, may of course vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but exemplary methods and materials are described herein.

[0015] Furthermore, unless otherwise specified, it should be understood that none of the methods described in this specification are intended to be construed as requiring that their steps be performed in a particular order. Thus, if a method claim does not actually recite the order that the steps should follow, or if the steps are not specifically recited in the claim or the specification as being limited to a particular order, no order should be inferred in any way. This holds true for any possible basis of implicit interpretation, including logical issues regarding the arrangement of steps or the flow of operations, the obvious meaning derived from grammatical construction or punctuation, and the number or type of aspects described in this specification.

[0016] All publications mentioned in this specification are hereby incorporated by reference into this specification to disclose and describe the methods and / or materials by which they are cited. The publications discussed in this specification are provided only with respect to their disclosure prior to the filing date of this application. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided in this specification may be different from the actual publication dates and individual verification may be required.

[0017] Definitions As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0018] As used in this specification, the term "or" means any one of a particular list of members and also includes any combination of members of that list.

[0019] A range can be expressed in the present specification as being from "about" or "approximately" one particular value and / or to "about" or "approximately" another particular value. When such a range is expressed, further aspects include from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation, it is understood that the use of the antecedent "about" or "approximately" forms further aspects of that particular value. Further, it will be further understood that each endpoint of the range is significant both in relation to and independent of the other endpoint. It is also understood that there are many values disclosed in the present specification and that each value is also disclosed in the present specification as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0020] As used in the present specification, the terms "any" or "optionally" mean that the event or situation described thereafter may or may not occur, and that the description includes the case where the event or situation occurs and the case where it does not occur.

[0021] As used in the present specification, the term "sample" means a tissue or organ from a subject assayed as described herein; a cell (either a cell within the subject, a cell directly collected from the subject, or a cell derived from a cell maintained in culture or a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cell material (e.g., a polypeptide or nucleic acid). A sample may also be any body fluid or excreted substance (e.g., blood, urine, feces, saliva, tears, bile, cerebrospinal fluid, but not limited thereto) containing cells or cell components. In some aspects, the sample can be taken from the brain, spinal cord, cerebrospinal fluid, or blood.

[0022] As used herein, the term "subject" refers to the target of administration, e.g., a human. Thus, the subject of the disclosed methods can be a vertebrate, e.g., a mammal, fish, bird, reptile, or amphibian. The term "subject" also includes companion animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, flies, etc.). In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. The term does not mean a particular age or sex. Thus, it is intended to encompass subjects of both sexes, adults, children, adolescents, and neonates, as well as fetuses.

[0023] As used herein, the term "patient" refers to a subject suffering from a disease or disorder. The term "patient" includes human subjects and veterinary subjects. In some embodiments of the disclosed methods, the "patient" is diagnosed, for example, as in need of treatment for multiple sclerosis prior to the step of administration. In some embodiments of the disclosed methods, the "patient" is diagnosed, for example, as in need of treatment for a demyelinating disease prior to the step of administration.

[0024] As used herein, the term "normal" refers to an individual, sample, or subject that does not have a demyelinating disease or does not have a high susceptibility to developing a demyelinating disease.

[0025] As used herein, the term "susceptibility" refers to the likelihood that a subject will develop a disease or be clinically diagnosed with a disease. For example, a human subject with a high susceptibility to a demyelinating disease (e.g., multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, or a syndrome consisting of a first episode) can refer to a human subject who is likely to develop a demyelinating disease (e.g., multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, or a syndrome consisting of a first episode) or be clinically diagnosed as such.

[0026] As used herein, the term "comprising" can include the aspects of "consisting of" and "consisting essentially of".

[0027] "Specifically binds" means that an antibody recognizes its cognate antigen, physically interacts therewith (e.g., a microorganism-specific antibody that is specific for one or more microorganisms selected from the lists of Tables 1, 2 or 8), and does not significantly recognize or interact with other antigens; such antibodies may be polyclonal or monoclonal antibodies produced by techniques well known in the art.

[0028] "Probe", "primer", or oligonucleotide means a single-stranded DNA or RNA molecule of a defined sequence that can form base pairs with a second DNA or RNA molecule containing a complementary sequence (the "target"). The stability of the resulting hybrid depends on the degree of base pair formation that occurs. The degree of base pair formation is affected by parameters such as the degree of complementarity between the probe and the target molecule and the stringency of the hybridization conditions. The degree of stringency of hybridization is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to those skilled in the art. Probes or primers specific for a microorganism-specific antibody (a microorganism-specific antibody is specific for one or more microorganisms selected from the lists in Table 1, Table 2, or Table 8) have at least 80% to 90% sequence complementarity, preferably at least 91% to 95% sequence complementarity, more preferably at least 96% to 99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the microorganism-specific antibody with which they hybridize. Probes, primers, and oligonucleotides may be detectably labeled either radioactively or non-radioactively by methods well known to those skilled in the art. Probes, primers, and oligonucleotides are used in methods involving nucleic acid hybridization, such as nucleic acid sequencing, nucleic acid amplification by reverse transcription and / or polymerase chain reaction, single-strand conformational polymorphism (SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, and electrophoretic mobility shift assay (EMSA).

[0029] "Specifically hybridizes" means that a probe, primer, or oligonucleotide recognizes and physically interacts with (i.e., forms base pairs with) a substantially complementary nucleic acid (e.g., a microorganism-specific antibody) under high stringency conditions and does not substantially form base pairs with other nucleic acids.

[0030] "High stringency conditions" means 0.5 M NaHPO at a temperature of 65°C4 、pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V), or at a temperature of 42°C in a buffer containing 48% formamide, 4.8 × SSC, 0.2 M Tris-Cl, pH 7.6, 1 × Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, which means conditions that enable hybridization comparable to that obtained using a DNA probe at least 40 nucleotides in length. Other conditions for high-stringency hybridization, such as PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known to those skilled in the art of molecular biology. (See, for example, F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998). As used herein, the term "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether single-stranded or double-stranded, sense or antisense, that can hybridize to a complementary nucleic acid by Watson-Crick base pairing. The nucleic acids of the present invention can also include nucleotide analogs (e.g., BrdU) and non-phosphodiester nucleoside linkages (e.g., peptide nucleic acid (PNA) or thiophosphate linkages). In particular, the nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0031] As used herein, a "control" is a sample from a normal subject or from non-demyelinated or demyelinated tissue.

[0032] As used herein, "overexpression" means expression that is greater than that detected in normal non-demyelinated tissue. For example, a nucleic acid that is overexpressed may be expressed at about 1 standard deviation above normal, or about 2 standard deviations above normal, or about 3 standard deviations above the normal level of expression. Thus, a nucleic acid that is expressed at about 3 standard deviations above the control level of expression is an overexpressed nucleic acid.

[0033] As used herein, the terms "treat" or "treatment" refer to the medical management of a patient undertaken with the intention of curing, alleviating, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes both active treatment, i.e., treatment specifically directed to the improvement of a disease, pathological condition, or disorder, and causal treatment, i.e., treatment directed to the removal of the cause of the related disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment directed to minimizing or partially or completely suppressing the onset of a related disease, pathological condition, or disorder; and supportive treatment, i.e., treatment employed to assist another specific therapy directed to the improvement of a related disease, pathological condition, or disorder; and supportive treatment, i.e., treatment employed to assist another specific therapy directed to the improvement of a related disease, pathological condition, or disorder. In various aspects, this term encompasses any treatment of a subject, including a mammal (e.g., a human), that (i) prevents the development of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed as having the disease; (ii) suppresses the disease, i.e., arrests the development of the disease; or (iii) alleviates the disease, i.e., causes regression of the disease.

[0034] As used herein, the term "prevent" or "prevention" refers to making something impossible to occur, avoiding, removing, preventing in advance, stopping, or interfering with, particularly by a harmful effect. When "reduce", "suppress", or "prevent" is used herein, it should be understood that the use of the other two terms is also clearly disclosed unless otherwise indicated. For example, "prevent" means minimizing the likelihood that a subject with a high susceptibility to developing a demyelinating disease will develop the demyelinating disease.

[0035] As used herein, the terms "reference", "reference expression", "reference sample", "reference value", "control", "control sample", etc., when used in the context of the expression level of a sample or one or more microorganisms, refer to a reference standard, where the reference is expressed at a certain level among different tissues (i.e., not the same tissue but multiple tissues), is not affected by experimental conditions, and indicates the level in a sample of a given pathological condition (e.g., not suffering from a demyelinating disease). The reference value may be a given reference value or a range of given reference values, and represents disease-free or a disease of a given type or severity.

[0036] As used herein, the term "diagnosed" means having been examined by a person skilled in the art, e.g., a physician, and being known to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, as used herein, "diagnosed with a disorder of a demyelinating disease" means having been examined by a person skilled in the art, e.g., a physician, and being known to have a condition that can be diagnosed or treated by the compounds or compositions disclosed herein.

[0037] As used herein, the term "microorganism" refers to microscopic organisms (i.e., living organisms that are too small to be seen with the naked eye). In general, the term "microorganism" is used to describe a variety of living organisms that can have a variety of sizes and characteristics. For example, microorganisms can refer to bacteria, archaea, fungi, protists, viruses, and microscopic animals.

[0038] As used herein, the term "microorganism capturer" refers to a molecule that can bind to or capture (e.g., directly) at least one microorganism, or that can capture a microorganism-specific antibody that is specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 (e.g., capture or bind to at least one microorganism indirectly).

[0039] Acute tumefactive MS is an acute, tumor-like variant in which some patients with demyelinating disease present large acute lesions, often associated with edema and / or ring enhancement on imaging (Kepes, J. J. Ann. Neurol. 33, 18 - 27, (1993); and Lucchinetti, C. F. et al. Brain, 131, 1759 - 1775, (2008)). This type of inflammatory demyelinating disease is also called pseudotumoral MS, tumefactive sclerosis, diffuse myelolytic sclerosis, and Marburg variant MS. The initial description by Kepes (Kepes, J. J. Ann. Neurol. 33, 18 - 27, (1993)) suggested that some such patients would progress to the development of MS. However, in a more recent and much larger study of 168 patients with biopsy-confirmed CNS inflammatory demyelinating disease, the majority (79%) of such patients subsequently developed clinically definite MS (Lucchinetti, C. F. et al. Brain, 131, 1759 - 1775, (2008)). The syndrome consisting of the first episode (CIS) refers to a single attack that can be compatible with MS, such as optic neuritis. Sixty to eighty percent of patients with CIS and magnetic resonance imaging (MRI) lesions subsequently develop MS, while approximately 20 - 40% have a self-limited course (Frohman, E. M. et al. Neurology, 61, 602 - 611 (2003); Brex, P. A. et al. N. Engl. J. Med. 346, 158 - 164, (2002); and Olek, M. in UpToDate, (ed Francisco Gonzalez-Scarano) (UpToDate, Waltham, MA, 2011)).

[0040] The pathology of multiple sclerosis (MS) has been well summarized by Lucchinetti (Popescu, B.F., Pirko, I. and Lucchinetti, C.F. Continuum (Minneapolis, Minn.), 19, 901-921, (2013): "The pathological features of multiple sclerosis (MS) are multiple foci of myelin loss within the CNS called plaques or lesions... Acute active MS lesions are hypercellular demyelinating plaques with extensive infiltration of macrophages that are uniformly distributed throughout the lesion, forming a classical "sea of macrophages". These macrophages contain myelin remnants, indicating that they have taken up and degraded the remnants of the destroyed myelin sheath (i.e., active demyelination).

[0041] Considering the known infectious causes of demyelination and these factors including the macrophage-predominant pathology of MS plaques, it is contemplated herein that microorganisms within the brain parenchyma may cause the onset of MS or exacerbation of an existing MS disease. Disclosed herein are the results of examining whether the microbial sequence content of primary demyelinated brain samples differs from that in a series of controls. The feasibility of RNA extraction and large-scale sequence analysis from such tissue was demonstrated.

[0042] Disclosed herein is a method that can be used to demonstrate the microbial cause of multiple sclerosis in individual patients. The first step was to obtain RNA sequencing data from MS brain lesions and compare the results to a series of control or reference samples. Next, a list of MS microorganisms or MS microbial families was created using microbial taxa that were significantly overrepresented in the MS group.

[0043] Also disclosed herein are methods that can be used alone or in combination with the methods described herein to demonstrate the microbial cause of multiple sclerosis in individual patients and subsequent treatment targets. For example, disclosed herein are methods that can be used to evaluate that the blood-brain barrier is not impaired. In some embodiments, the methods can be used to detect and / or correct blood-brain barrier leakage that may be associated with multiple sclerosis and other demyelinating diseases. In some embodiments, the methods can be used to determine whether antibodies in the CSF of MS patients directed against some of the MS microbes of the MS microbe family disclosed herein indicate intrathecal antibody synthesis.

[0044] Disclosed herein are a series of microbial reagents that can be used in a series of assays (e.g., serological). Disclosed herein are assays that can be used to detect antibodies in the CSF of MS patients directed against an MS microbe or a portion of an MS microbe family. An exemplary list of MS microbes or MS microbe families is provided in FIGS. 4 and / or 5, similar to Tables 1, 2, and / or 8. Using the results of the methods disclosed herein, antimicrobial therapy can be prescribed for patients with MS and related diseases (e.g., acute disseminated encephalomyelitis or neuromyelitis optica) who are experiencing demyelination (i.e., new onset) or worsening of demyelination (i.e., relapse). For example, a patient with CSF reactivity against Akkermansia mucinophilia may be treated with intravenous meropenem, which is predicted to be active, rather than ceftriaxone, which is predicted to be inactive against this anaerobic bacterium.

[0045] Using the MS microorganisms or MS microorganism families described herein, specific PCR reagents can also be developed to detect one or more of the MS microorganisms or MS microorganism families disclosed herein that cause demyelination. For example, primers can be developed and used to detect the organisms responsible for these MS. CSF samples can be used from patients with demyelination who are in the early stages of the course before specific antibacterial antibodies and oligoclonal bands appear. Specific PCR primers can be developed and used to detect the MS microorganisms or MS microorganism families disclosed herein in brain tissue, spinal cord, CSF, or blood. Multiple primer sets can be multiplexed in a single test. Again, in this case, specific antibacterial treatment can be directed using the detection of microbial DNA (or possibly RNA, although less likely) in the CSF of early demyelination patients (or patients with established MS attacks).

[0046] The methods disclosed herein can be used to detect and treat several related demyelinating disorders including, but not limited to, the presumed underlying cause of MS, and neuromyelitis optica (NMO), acute disseminated encephalomyelitis (ADEM), and the syndrome consisting of the first episode (CIS). The advantage of the methods disclosed herein is that they can help limit or reverse damage to the CNS. For example, patients reactive to CSF against Akkermansia mucinophilia may be treated with intravenous meropenem, which is predicted to be active, rather than ceftriaxone, which is predicted to be inactive against this anaerobic bacterium. Current MS treatments are unreliable and inconsistent when there is no specific factor underlying the treatment choice and no reasonable expectation of success or failure. Current treatments for acute MS attacks include strategies that are broadly immunosuppressive and can have serious side effects (e.g., steroids, rituximab).

[0047] Described herein are methods and microbial reagents that can be used alone or in a series of assays (e.g., serological). Described herein are methods that can be used to detect antibodies in the CSF of MS patients that are directed against some, but not all, of the MS microbes or MS microbial families described herein.

[0048] Described herein are methods that can be used alone or in combination with a series of assays. Described herein are methods that include determining a microbial ELISA index (EI) and comparing the result of the EI to the albumin index of a subject. In some embodiments, the methods can be used to determine whether antibodies in the CSF of MS patients directed against one or more of the MS microbes of the MS microbial families disclosed herein indicate intrathecal antibody synthesis. Further, the methods can be used to identify subjects who may not have been detected or excluded using any of the other detection methods described herein. For example, using the disclosed method of determining a microbial ELISA index (EI) and comparing the result of the EI of a subject to the albumin index of the subject can improve the identification of subjects with demyelinating diseases as compared to determining only the microbial ELISA index (EI).

[0049] Using the assays and methods described herein, antibacterial therapy can be prescribed for patients with MS and related diseases (i.e., ADEM, NMO) who are developing (e.g., newly developing) or experiencing a worsening of demyelination (e.g., relapsing). For example, patients who are reactive to CSF against Akkermansia mucinophilia can be treated with intravenous meropenem, which is predicted to be active, rather than ceftriaxone, which is predicted to be inactive against this anaerobic bacterium.

[0050] Using the MS microorganisms or MS microorganism families described herein, specific PCR reagents can also be developed to detect one or more of the MS microorganisms or MS microorganism families disclosed herein that cause demyelination. Primers can be developed to detect the organisms responsible for MS. The reagents (e.g., primers) can be applied to the CSF of patients with developing demyelination at an early stage of its course, before specific antibacterial antibodies and oligoclonal bands appear. In some embodiments, multiple primer sets are multiplexed in a single assay. Disclosed herein is a method for detecting microbial DNA (or possibly RNA) in the CSF of early demyelinated patients (or patients with established MS attacks) that can be used to direct specific antibacterial therapies.

[0051] Disclosed herein are methods for detecting and treating the putative underlying causes of several related demyelinating disorders, including, but not limited to, MS, and neuromyelitis optica (NMO), acute disseminated encephalomyelitis (ADEM), and clinically isolated syndrome (CIS) consisting of a first episode.

[0052] Disclosed herein is a serological assay for detecting antibodies in the CSF of MS patients directed against one or more of the MS microorganisms or MS microorganism families described herein. Disclosed herein is a method for directing antibacterial therapy in patients with developing (e.g., newly developing) or experiencing a worsening of (e.g., relapsing) demyelination, such as MS and related diseases (e.g., ADEM, NMO). Table 1. Examples of microorganisms. TIFF0007682540000001.tif154168 1 As classified by NCBI taxonomy. Phyla are listed for bacterial and fungal microorganisms but not for viruses. 2As described by MicrobeWiki (https: / / microbewiki.kenyon.edu / index.php / MicrobeWiki), the list of prokaryote names following the nomenclature (LPSN, http: / / www.bacterio.net), UniProt (https: / / www.uniprot.org), and other online information sources. 3 Therapeutic agents were determined using various online information sources such as PubMed articles, microbial websites (such as Microbewiki), and the Sanford Guide. Table 2. Examples of microorganisms. TIFF0007682540000002.tif208159 TIFF0007682540000003.tif73155 MS microorganisms or MS microorganism families derived from large-scale sequencing data. To qualify as an MS microorganism or MS microorganism family at the genus level, at least one specimen from the MS group had significantly increased (q < 0.05) read pair mapping to this taxon compared to a control group with 10 or more MAPQ values (251 separate genera). The MS microorganism genera shown had at least 100 mapped reads among all specimens in the MS group (35 genera).

[0053] Methods Disclosed herein is a method for diagnosing and / or detecting a demyelinating disease in a subject. The methods disclosed herein can be useful for performing microbiological diagnoses. The detection methods described herein can be carried out either directly via PCR amplification of nucleic acids or via direct detection using an antibody or peptide that specifically binds to one or more of the disclosed microorganisms. The detection methods described herein can also be carried out by detecting an antibody specific for one or more of the microorganism-specific antibodies. For example, disclosed herein is a method comprising contacting a sample with one or more microbial capture agents under conditions that allow one or more microbial capture agents to bind to one or more of the microorganism-specific antibodies to produce a mixed biological sample, wherein each of the one or more microbial capture agents is specific for one or more microorganism-specific antibodies and the one or more microorganism-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8.

[0054] Also disclosed herein is a method for treating a subject at risk of developing a demyelinating disease, a method for treating a subject with a demyelinating disease, or a method for treating a subject suspected of having a demyelinating disease. Once the presence of one or more microorganisms or one or more microorganism-specific antibodies is determined, an appropriate treatment can be provided or administered.

[0055] Disclosed herein is a method for diagnosing and / or detecting a demyelinating disease in a subject, wherein the intrathecal production of antibodies can be determined using the method disclosed herein. The method disclosed herein can be useful for performing microbiological diagnosis. The method disclosed herein can also be used to identify subjects with demyelinating diseases not identified in any of the other methods described herein. Using the method disclosed herein, it can be determined that the blood-brain barrier is not impaired. The detection method described herein can compare the microbial ELISA index (EI) of a subject with the albumin index (AI) of the subject. The method disclosed herein can be carried out via ELISA using an antibody that binds or specifically binds to one or more of the disclosed microorganisms. For example, disclosed herein is a method comprising contacting a sample with one or more microbial capture agents under conditions that allow one or more microbial capture agents to bind to one or more of the microbial-specific antibodies to produce a mixed biological sample, wherein each of the one or more microbial capture agents is specific for one or more microbial-specific antibodies, and the one or more microbial-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. EI is a measure of antibodies in a sample (e.g., CSF or cerebrospinal fluid). In some embodiments, the EI value can be compared to a normal AI (e.g., 0-9) indicating an intact blood-brain barrier. Albumin index = CSF albumin (mg / L) / serum albumin (g / L). AI is an index of the integrity of the blood-brain barrier (BBB) adjusted according to the serum albumin concentration. AI increases with BBB dysfunction. For example, the EI of a subject for one or more microorganisms selected from the lists of Table 1, Table 2, and / or Table 8 can be plotted against the AI of the subject. Linear regression analysis can be performed to show the relationship between the EI of each antigen and an intact BBB (as evaluated by the albumin index). A straight line of linear regression or a plot line can be generated to provide the "expected EI" for each bacterial antigen or microorganism examined and can be used to correct for a leaky BBB that may be associated with subjects with MS and other demyelinating diseases.Values above the regression line, particularly values within the normal AI range (e.g., 0 - 9), indicate the possibility of intrathecal antibody synthesis (i.e., the causal relationship of demyelination). In some embodiments, the methods disclosed herein can be used alone or in combination with any of the other methods described herein.

[0056] Methods of diagnosis: Disclosed herein are methods of diagnosing a demyelinating disease in a subject. In some embodiments, the method can include obtaining a biological sample from the subject. In some embodiments, the subject can be suspected of having, or at risk of having, a demyelinating disease. In some embodiments, the method can include detecting the presence of one or more microorganisms in the sample. In some embodiments, the one or more microorganisms in the sample can be selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the presence of the one or more microorganisms can be indirectly detected by detecting the presence of one or more antibodies in the sample. In some embodiments, the one or more antibodies can be specific for the one or more microorganisms in the sample. In some embodiments, the method can include diagnosing the subject as having a demyelinating disease. In some embodiments, the method can include diagnosing the subject as having a demyelinating disease when one or more microorganisms selected from the list of Table 1 or Table 2 are present in the sample.

[0057] In some embodiments, a method of diagnosing a demyelinating disease in a subject can include obtaining a sample from the subject. In some embodiments, the sample can be suspected of containing microorganism-specific antibodies. In some embodiments, the method can include incubating the sample with one or more microorganism capture agents. In some embodiments, the one or more microorganism capture agents can bind to one or more of the microorganism-specific antibodies. In some embodiments, the microorganism-specific antibodies can be specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include detecting the presence of one or more microorganism-specific antibodies in the sample. In some embodiments, the method can include diagnosing the subject as having a demyelinating disease if one or more microorganism-specific antibodies are present in the sample. In some embodiments, the method can further include repeating the steps of incubating the sample with one or more microorganism capture agents and detecting the presence of one or more microorganism-specific antibodies in the sample using different microorganism capture agents. In some embodiments, the various microorganism capture agents can be specific to one or more microorganism-specific antibodies in the sample. In some embodiments, the method can further include detecting the presence of one or more microorganism-specific antibodies specific to one or more of the microorganisms listed in Table 1, Table 2, or Table 8. In some embodiments, the detecting step can include detecting by a microarray. In some embodiments, the detecting step can include detecting by ELISA. In some embodiments, the one or more microorganisms detected can be derived from the genus Akkermansia. In some embodiments, the method of diagnosis can further include detecting the presence of one or more microorganism-specific antibodies in the sample, determining an ELISA index, and comparing the ELISA index to an albumin index. In some embodiments, an EI ratio can be determined. In some embodiments, the EI ratio can be >1.0, indicating that there are more microorganism-specific antibodies to one or more microorganisms in the CSF compared to the serum. In some embodiments, the EI ratio can be <1.0, indicating that there are more microorganism-specific antibodies in the serum than in the CSF.In some embodiments, the method can further include determining an ELISA index for one or more microorganisms, performing a linear regression analysis, and comparing the ELISA index to the AI of the sample by generating a plotted straight line. In some embodiments, the AI can be 9 or less, and the ELISA index can be above the plotted straight line indicating one or more microorganism-specific antibodies in the CSF compared to the serum for one or more microorganisms.

[0058] Disclosed herein is a method for diagnosing and treating a demyelinating disease in a subject. In some embodiments, the method can include obtaining a sample from the subject. In some embodiments, the sample can be suspected of containing a microorganism-specific antibody. In some embodiments, the method can include incubating the sample with one or more microorganism capture agents. In some embodiments, the one or more microorganism capture agents can bind to one or more of the microorganism-specific antibodies. In some embodiments, the microorganism-specific antibody can be specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include detecting the presence of one or more microorganism-specific antibodies in the sample. In some embodiments, the method can include diagnosing the subject as having a demyelinating disease. In some embodiments, the method can include diagnosing the subject as having a demyelinating disease if one or more microorganism-specific antibodies are present in the sample. In some embodiments, the method can include administering an antibacterial agent if the detected one or more microorganism-specific antibodies are bacteria selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include administering an antiviral agent if the detected one or more microorganism-specific antibodies are viruses selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include administering an antifungal agent if the detected one or more microorganism-specific antibodies are fungi selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include administering a combination of an antibacterial agent, an antiviral agent, or an antifungal agent to the subject. In some embodiments, the method for diagnosing and treating a demyelinating disease in a subject can further include detecting the presence of one or more microorganism-specific antibodies in the sample, determining an ELISA index, and comparing the ELISA index to an albumin index. In some embodiments, an EI ratio can be determined. In some embodiments, the EI ratio can be >1.0, indicating that there are more microorganism-specific antibodies against one or more microorganisms in the CSF compared to the serum. In some embodiments, the EI ratio can be <1.0, indicating that there are more microorganism-specific antibodies in the serum than in the CSF.In some embodiments, the method can further include determining an ELISA index for one or more microorganisms, performing a linear regression analysis, and comparing the ELISA index to the subject AI by generating a plotted straight line. In some embodiments, the subject AI can be 9 or less, and the ELISA index can be above the plotted straight line indicating one or more microorganism-specific antibodies in the CSF as compared to the serum for one or more microorganisms.

[0059] Methods of Indirect Detection: Disclosed herein are methods of detecting a demyelinating disease in a subject. In some embodiments, a method of detecting a demyelinating disease in a subject can include obtaining a sample from the subject. In some embodiments, the sample can be suspected of containing a microorganism-specific antibody specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include contacting the sample with one or more microorganism capture agents to produce a mixed biological sample. In some embodiments, each of the one or more microorganism capture agents can be specific to one or more microorganism-specific antibodies. In some embodiments, the one or more microorganism-specific antibodies can be specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include incubating the mixed biological sample under conditions such that the one or more microorganism capture agents can specifically bind to at least one of the microorganism-specific antibodies to form a detectable complex. In some embodiments, the method can include detecting the presence of the detectable complex and thereby detecting the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 in the mixed biological sample can detect a demyelinating disease in the subject. In some embodiments, the method can further include repeating the steps of contacting the sample and incubating the mixed biological sample with various microorganism capture agents. In some embodiments, the various microorganism capture agents can be specific to one or more microorganism-specific antibodies in the sample. In some embodiments, the method can further include detecting the presence of one or more microorganism-specific antibodies specific to one or more of the microorganisms listed in Table 1, Table 2, or Table 8. In some embodiments, the method can further include determining the amount of one or more of the microorganism-specific antibodies in the sample. In some embodiments, the step of detecting can include detecting by a microarray. In some embodiments, the step of detecting can include detecting by ELISA. In some embodiments, the one or more microorganisms detected can be derived from the genus Akkermansia.In some embodiments, the method can further include determining an ELISA index. In some embodiments, the ELISA index can be compared to or plotted against the albumin index. In some embodiments, an EI ratio can be determined. In some embodiments, the EI ratio can be >1.0, indicating that there are more microorganism-specific antibodies against one or more microorganisms in the CSF compared to the serum. In some embodiments, the EI ratio can be <1.0, indicating that there are more microorganism-specific antibodies in the serum than in the CSF. In some embodiments, the method can further include determining an ELISA index for one or more microorganisms, performing a linear regression analysis, and generating a plotted line to compare the ELISA index to the subject's AI. In some embodiments, the subject's AI can be 9 or less, and the ELISA index can be above the plotted line indicating one or more microorganism-specific antibodies in the CSF compared to the serum for one or more microorganisms.

[0060] Disclosed herein is a method for detecting and treating a demyelinating disease in a subject. In some embodiments, the method can include obtaining a sample from the subject. In some embodiments, the sample can be suspected of containing a microbial-specific antibody specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include contacting the sample with one or more microbial capture agents to produce a mixed biological sample. In some embodiments, each of the one or more microbial capture agents can be specific to one or more microbial-specific antibodies. In some embodiments, the one or more microbial-specific antibodies can be specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include incubating the mixed biological sample under conditions such that the one or more microbial capture agents can specifically bind to at least one of the microbial-specific antibodies to form a detectable complex. In some embodiments, the method can include detecting the presence of the detectable complex and thereby detecting the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 in the mixed biological sample can detect a demyelinating disease in the subject. In some embodiments, the method can include administering an antibacterial agent when the one or more detected microbial-specific antibodies are bacteria selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include administering an antiviral agent when the one or more detected microbial-specific antibodies are viruses selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include administering an antifungal agent when the one or more detected microbial-specific antibodies are fungi selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include administering a combination of an antibacterial agent, an antiviral agent, or an antifungal agent to the subject. In some embodiments, the method can include administering an antiparasitic agent. In some embodiments, the method can include administering a combination of an antibacterial agent, an antiviral agent, an antiparasitic agent, or an antifungal agent to the subject.

[0061] In some embodiments, the various microbial capture agents can be specific for one or more microbial-specific antibodies in the sample. In some embodiments, the method can further include detecting the presence of one or more microbial-specific antibodies specific for one or more of the microorganisms listed in Table 1, Table 2, or Table 8. In some embodiments, the method can further include determining the amount of one or more microbial-specific antibodies in the sample. In some embodiments, the detecting step can include detecting by a microarray. In some embodiments, the detecting step can include detecting by ELISA. In some embodiments, one or more of the detected microorganisms can be derived from the genus Akkermansia. In some embodiments, the method can further include detecting the presence of one or more microbial-specific antibodies in the sample, determining an ELISA index, and comparing the ELISA index to an albumin index. In some embodiments, an EI ratio can be determined. In some embodiments, the EI ratio can be >1.0, indicating that there are more microbial-specific antibodies against one or more microorganisms in the CSF compared to the serum. In some embodiments, the EI ratio can be <1.0, indicating that there are more microbial-specific antibodies in the serum than in the CSF. In some embodiments, the method can further include determining an ELISA index for one or more microorganisms, performing a linear regression analysis, and generating a plotted straight line to compare the ELISA index to the subject's AI. In some embodiments, the subject's AI can be 9 or less, and the ELISA index can be above the plotted straight line indicating one or more microbial-specific antibodies in the CSF compared to the serum for one or more microorganisms.

[0062] Also disclosed herein is a method of diagnosing a demyelinating disease in a subject. In some embodiments, the method can include obtaining a sample from the subject. In some embodiments, the subject can be suspected of having, or at risk of having, a demyelinating disease. In some embodiments, the method can include detecting the presence of one or more microorganisms in the sample. In some embodiments, the one or more microorganisms can be selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include diagnosing the subject as having a demyelinating disease if one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 are present in the sample.

[0063] Disclosed herein is a method for detecting one or more antibodies in a sample. In some embodiments, the method can include obtaining a sample from a subject. In some embodiments, the sample can be suspected of containing a microorganism-specific antibody specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include incubating the sample with one or more microorganism capture agents. In some embodiments, the one or more microorganism capture agents can bind to one or more of the microorganism-specific antibodies. In some embodiments, the one or more microorganism-specific antibodies can be specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include detecting the presence of one or more microorganism-specific antibodies in the sample. In some embodiments, the detection of the presence of the one or more microorganism-specific antibodies can indicate a demyelinating disease. In some embodiments, the method can further include detecting the presence of one or more microorganism-specific antibodies specific to one or more of the microorganisms listed in Table 1, Table 2, or Table 8. In some embodiments, the method can further include determining the amount of one or more of the microorganism-specific antibodies in the sample. In some embodiments, the detecting step can include detecting by a microarray. In some embodiments, the detecting step can include detecting by ELISA. In some embodiments, the one or more microorganisms detected can be derived from the genus Akkermansia. In some embodiments, the method can further include determining an ELISA index. In some embodiments, the method can compare the ELISA index to an albumin index. In some embodiments, the method can further include detecting the presence of one or more microorganism-specific antibodies in the sample, determining the ELISA index, and comparing the ELISA index to the albumin index. In some embodiments, an EI ratio can be determined. In some embodiments, the EI ratio can be >1.0, indicating that there are more microorganism-specific antibodies to one or more microorganisms in the CSF compared to the serum. In some embodiments, the EI ratio can be <1.0, indicating that there are more microorganism-specific antibodies in the serum than in the CSF.In some embodiments, the method can further include determining an ELISA index for one or more microorganisms, performing a linear regression analysis, and comparing the ELISA index to the subject AI by generating a plotted straight line. In some embodiments, the subject AI can be 9 or less, and the ELISA index can be above the plotted straight line indicating one or more microorganism-specific antibodies in the CSF as compared to the serum for one or more microorganisms.

[0064] Direct detection method: Disclosed herein is a method for detecting one or more microorganisms in a sample. In some embodiments, the method can include obtaining a sample from a subject. In some embodiments, the sample can be suspected of containing one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include incubating the sample with one or more microbial capture agents. In some embodiments, the one or more microbial capture agents can bind to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can include detecting the presence of one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8 in the sample. In some embodiments, the method can further include detecting the presence of one or more microorganisms listed in Table 1, Table 2, or Table 8. In some embodiments, the method can further include determining one or more amounts of microorganisms in the sample. In some embodiments, detecting the presence of one or more microorganisms indicates a demyelinating disease. In some embodiments, the detecting step can include detecting by a microarray. In some embodiments, the detecting step can include detecting by ELISA. In some embodiments, the one or more microorganisms detected can be derived from the genus Akkermansia. In some embodiments, the method can further include determining an ELISA index. In some embodiments, the method can compare the ELISA index with an albumin index. In some embodiments, the method can further include detecting the presence of one or more microorganism-specific antibodies in the sample, determining an ELISA index, and comparing the ELISA index with the AI of the subject. In some embodiments, an EI ratio can be determined. In some embodiments, the EI ratio can be >1.0, indicating that there are more microorganism-specific antibodies against one or more microorganisms in the CSF compared to the serum. In some embodiments, the EI ratio can be <1.0, indicating that there are more microorganism-specific antibodies in the serum than in the CSF. In some embodiments, the method can further include determining an ELISA index for one or more microorganisms, performing a linear regression analysis, and generating a plotted straight line to compare the ELISA index with the AI of the subject.In some embodiments, the AI of interest can be 9 or less, and the ELISA index can be above the plotted line indicating one or more microbial-specific antibodies in CSF as compared to serum for one or more microorganisms.

[0065] Obtaining tissue samples: The procedures for extracting and collecting samples of the subject's brain or spinal cord tissue, blood, and CSF can be performed by methods known in the art. Frozen tissue specimens can also be used. As described above, tissue samples can be obtained from the subject using a core needle biopsy. The sample can be whole cells or organelles. Cells can be collected by scraping tissue, treating tissue samples to release individual cells, or isolating cells from body fluids. The sample can be fresh tissue, dried tissue, cultured cells or tissue. The sample can be unfixed or fixed. Using the methods described herein, any part of the brain or spinal cord can be obtained and evaluated.

[0066] Disclosed herein is a method for detecting microorganisms in a sample. As will be understood by those skilled in the art, the sample solution can include, but is not limited to, body fluids (including, but not limited to, blood, biopsy tissue, CSF), and can include any number of sources. The sample can be in the range of less than 1 milliliter and can further be in the range of bacterial concentration. Further, the sample can be present in blood, brain tissue, spinal cord tissue, or CSF.

[0067] In any of the methods disclosed herein, the subject can be suspected of having a demyelinating disease. In any of the methods disclosed herein, the subject can be at risk of having a demyelinating disease. In some embodiments, the demyelinating disease can be multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, or a syndrome consisting of the first episode. In some embodiments, the subject can be human. In some embodiments, the sample can be brain tissue, cerebrospinal fluid, spinal cord tissue, or blood.

[0068] In any of the methods disclosed herein, the method can further include administering an antibacterial agent, an antiviral agent, an antifungal agent, or a combination thereof. In some embodiments, the method can include administering an antiparasitic agent. In some embodiments, the method can include administering a combination of an antibacterial agent, an antiviral agent, an antiparasitic agent, or an antifungal agent. In some embodiments, when the one or more microorganism-specific antibodies or microorganisms detected are bacteria selected from the lists in Table 1, Table 2, or Table 8, an antibacterial agent can be administered. In some embodiments, when the one or more microorganism-specific antibodies or microorganisms detected are viruses selected from the lists in Table 1, Table 2, or Table 8, an antiviral agent can be administered. In some embodiments, when the one or more microorganism-specific antibodies or microorganisms detected are fungi selected from the lists in Table 1, Table 2, or Table 8, an antifungal agent can be administered.

[0069] In any of the methods disclosed herein, the method can further include amplifying one or more nucleic acid sequences in the sample. In some embodiments, the one or more nucleic acid sequences correspond to one or more of the microorganisms in Table 1, Table 2, or Table 8. In some embodiments, the one or more nucleic acid sequences can be amplified using a primer pair that can specifically amplify the one or more nucleic acid sequences. In some embodiments, the method can further include determining whether the expression of one or more nucleic acid sequences corresponding to one or more of the microorganisms in Table 1, Table 2, or Table 8 is overexpressed compared to the expression level of the same one or more nucleic acid sequences of the microorganisms in Table 1, Table 2, or Table 8 in a control. In some embodiments, the detecting step can include detecting by a hybridization reaction. In some embodiments, the hybridization reaction can further include hybridizing the sample to one or more primer sets. In some embodiments, the hybridization reaction can be a polymerase chain reaction. In some embodiments, the detecting step can include detecting by expressed sequence tags. In some embodiments, the detecting step can include detecting by localization.

[0070] In some embodiments, the microbial capture agent can be an antibody, peptide, protein, or probe. In some embodiments, one or more of the microorganisms disclosed herein (see, e.g., Tables 1, 2, and 8) can be used to bind to one or more of the CSF-derived antibodies of a subject (e.g., a patient having MS or a patient suspected of having MS). In some embodiments, the bacterial antigen can be sonicated at an OD of 0.5 and can be coated on the bottom of a single-well plate or a multi-well plate. In some embodiments, the bacterial antigen can be derived from Akkermansia, Atopobium, Lactobacillus, Pseudomonas, Bacteroides, and Nitrosospira.

[0071] Any of the methods or assays or tests described herein can further include a positive control, a negative control, or both. Examples of positive controls include, but are not limited to, commercially available CSF formulations (Randox) and anti-Pseudomonas antibodies (ThermoFisher product #PA1-73116). Examples of negative controls include, but are not limited to, PBS and Randox CSF depleted of IgG (devoid of all antibodies).

[0072] In some embodiments, one or more detection methods can be used to demonstrate the binding of specific CSF antibodies to one or more of the microorganisms disclosed herein. In some embodiments, one or more of the microorganisms can be examined (and / or detected) in a subject having or suspected of having MS or another autoimmune disease. In some embodiments, the method of detection can be an indirect method. In some embodiments, the indirect method can be ELISA. In some embodiments, ELISA can be used to examine (and / or detect) two or more microorganisms.

[0073] Methods of treatment: Disclosed herein are methods of treating patients with multiple sclerosis (MS) or MS-related diseases. In some embodiments, the methods can include administering to the patient an antibacterial agent, an antiviral agent, an antifungal agent, an antiparasitic agent, or a combination thereof. In some embodiments, the patient can be identified as in need of an antibacterial agent, an antiviral agent, an antifungal agent, an antiparasitic agent, or a combination thereof. In some embodiments, the patient is identified by obtaining from the patient a sample suspected of containing a microorganism-specific antibody; incubating the sample with one or more microorganism capture agents, wherein the one or more microorganism capture agents bind to one or more of the microorganism-specific antibodies and the microorganism-specific antibodies are specific for one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8; and detecting the presence of the one or more microorganism-specific antibodies in the sample. In some embodiments, the antibacterial agent can be meropenem or ceftriaxone. In some embodiments, the MS-related disease can be neuromyelitis optica, acute disseminated encephalomyelitis, or a syndrome consisting of a first episode. In some embodiments, the detecting step can include detecting by microarray. In some embodiments, the detecting step can include detecting by ELISA. In some embodiments, the one or more microorganisms detected can be derived from the genus Akkermansia. In some embodiments, the sample can be brain tissue, cerebrospinal fluid, spinal cord tissue, or blood.

[0074] In some embodiments, the method can further include detecting the presence of one or more microorganism-specific antibodies specific to one or more microorganisms selected from the lists of Table 1, Table 2, or Table 8. In some embodiments, the method can further include determining one or more amounts of the microorganism-specific antibodies in the sample. In some embodiments, the method can further include amplifying one or more nucleic acid sequences in the sample. In some embodiments, the one or more nucleic acid sequences can correspond to one or more of the microorganisms of Table 1, Table 2, or Table 8. In some embodiments, the one or more nucleic acid sequences can be amplified using a primer pair that can specifically amplify the one or more nucleic acid sequences. In some embodiments, the method can further include determining whether the expression of one or more nucleic acid sequences corresponding to one or more of the microorganisms of Table 1, Table 2, or Table 8 is overexpressed compared to the expression level of the same one or more nucleic acid sequences of the microorganisms of Table 1, Table 2, or Table 8 in a control. In some embodiments, the detecting step can include detecting by a hybridization reaction. In some embodiments, the hybridization reaction can further include hybridizing the sample with one or more primer sets. In some embodiments, the detecting step can include detecting by a polymerase chain reaction.

[0075] Detection methods: The presence, absence, or expression level of one or more microorganisms or microorganism-specific antibodies disclosed herein can be determined directly (e.g., immunoassay, mass spectrometry) or indirectly (e.g., the presence or absence of one or more antibodies specific to one or more microorganisms; determining mRNA expression of proteins or peptides). Examples of mass spectrometry include ionization sources such as EI, CI, MALDI, ESI, and analysis, spectroscopy, isotope ratio mass spectrometry (IRMS), thermal ionization mass spectrometry (TIMS), spark source mass spectrometry, multiple reaction monitoring (MRM), or SRM such as Quad, ion trap, TOF, FT, or combinations thereof, but are not limited thereto. Any of these techniques can be performed in combination with a prefractionation or integration method. Examples of immunoassays include immunoblot, western blot, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay, but are not limited thereto. Immunoassay methods use antibodies for detection, and the measurement of antigen levels is known in the art. Antibodies can be immobilized on solid supports such as sticks, plates, beads, microbeads, or arrays.

[0076] The presence, absence, or expression level of one or more microorganisms or microorganism-specific antibodies disclosed herein can also be determined indirectly by determining the presence or absence of one or more antibodies specific to one or more microorganisms in a tissue sample. Methods of RNA expression include northern blot using a labeled probe that hybridizes to a transcript encoding all or part of a gene, gene-specific primers, polymerase chain reaction (PCR), and amplification of mRNA using reverse transcription polymerase chain reaction (RT-PCR), followed by quantitative detection of gene products by various methods; extraction of RNA from cells (followed by labeling and then used to search for cDNA or oligonucleotides encoding the gene); in situ hybridization; and detection of reporter genes, but are not limited thereto.

[0077] Methods for measuring the expression level of a protein include, but are not limited to, Western blot, immunoblot, ELISA, radioimmunoassay, immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemical analysis, microsite cytometry, microarray, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry. The methods can also include assays based on specific protein properties, including, but not limited to, enzyme activity or interaction with other protein partners. Binding assays can also be used and are well known in the art. For example, using a BIAcore device, the binding constant of a complex between two proteins can be measured. Other assays suitable for measuring or detecting the binding of one protein to another include immunoassays, such as ELISA and radioimmunoassay. In some cases, measurement of binding by observing spectroscopic changes may be used, or the optical properties of the protein may be measured by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). As another method, an immunoassay using a specific antibody can be used to detect the expression of a specific protein in tumor cells.

[0078] The disclosed methods also provide for quantification and / or suitability of responsiveness to a desired treatment based on the identification of one or more microorganisms (or the absence of such identification). Such information can lead to therapeutic decisions.

[0079] Protein arrays: Disclosed herein are arrays of polypeptides or proteins. In some embodiments, the protein array can include probes that include antibodies, aptamers, and other cognate binding ligands specific for components of the microbial panel disclosed herein. Protein arrays and methods for constructing protein arrays are well known to those of skill in the art.

[0080] One type of protein array that can be suitable uses immobilized "capture antibodies". The polypeptides are bound to a solid substrate (e.g., glass) by a treated surface (e.g., aminosilane) or via biotin-streptavidin binding. The array is then incubated with a solution containing probes that can bind to the capture antibodies in a manner that depends on time, buffer components, and recognition specificity. The probes can then be visualized directly if they are pre-labeled, or can be bound to a secondary labeling reagent (such as another antibody). The amount of probe that binds to the capture antibody that is visualized can depend on the labeling method utilized; generally, a CCD imager or laser scanner that uses a filter set appropriate for exciting and detecting the emission of the label can be used. The imager converts the amount of detected photons into an electronic signal (often on an 8-bit or 16-bit scale) that can be analyzed using a commercially available software package.

[0081] The substrate of the array can be organic or inorganic, biological or non-biological, or any combination of these materials. The substrate can be transparent or translucent. Examples of materials suitable for use as the substrate in the array include silicon, silica, quartz, glass, controlled pore glass, carbon, alumina, titanium dioxide, germanium, silicon nitride, zeolite, and gallium arsenide; as well as metals including gold, platinum, aluminum, copper, titanium, and their alloys, but are not limited thereto. Ceramics and polymers can also be used as the substrate. Suitable polymers include polystyrene; poly(tetra)fluoroethylene; (poly)vinylidene difluoride; polycarbonate; polymethyl methacrylate; polyvinyl ethylene; polyethyleneimine; poly(ether ether) ketone; polyoxymethylene (POM); polyvinyl phenol; polylactide; polymethacrylimide (PMI); polyalkene sulfone (PAS); hydroxyethyl polymethacrylate; polydimethylsiloxane; polyacrylamide; polyimide; block copolymer; and Eupergit® (registered trademark), but are not limited thereto. Photoresist, polymerized Langmuir-Blodgett films, and LIGA structures can also serve as the substrate.

[0082] The array can further include a coating that can be formed on or applied to the substrate. The substrate can be modified by a coating using thin film technology based on either physical vapor deposition (PVD) or plasma enhanced chemical vapor deposition (PECVD). Alternatively, the substrate can be directly activated using plasma irradiation. For example, a polymer surface (i.e., polystyrene or polyethylene for exposing polar functional groups such as hydroxyl, carboxylic acid, aldehyde, etc.) can be oxidized using a plasma etching procedure.

[0083] The coating can include a metal film. Examples of metal films include, but are not limited to, aluminum, chromium, titanium, nickel, stainless steel, zinc, lead, iron, magnesium, manganese, cadmium, tungsten, cobalt, and alloys or oxides thereof. In some embodiments, the metal film can be a noble metal film. Examples of noble metals that can be used for the coating include, but are not limited to, gold, platinum, silver, copper, and palladium. In some embodiments, the coating includes gold or a gold alloy. A thin coating of gold can be provided on the surface using electron beam evaporation. In some embodiments, the metal film can have a thickness from about 50 nm to about 500 nm.

[0084] Alternatively, the coating can be silicon, silicon oxide, silicon nitride, silicon hydride, indium tin oxide, magnesium oxide, alumina, glass, a hydroxylated surface, and a polymer.

[0085] The arrays described herein can include a set of specifiable elements. Such elements, such as an array contained within a microtiter plate or an array printed on a plane where each element can be present at a different X and Y coordinate, can be spatially specifiable. Alternatively, elements can be specifiable based on tags, beads, nanoparticles, or physical properties. Microarrays can be prepared according to methods known to those of ordinary skill in the art. As used herein, the term "array" can refer to any biological assay with a plurality of specifiable elements. In some embodiments, the specifiable elements can be polypeptides (e.g., antibodies or fragments thereof) or nucleic acid probes. As used herein, an "element" refers to any probe (based on polypeptides or nucleic acids) that can be bound by a polypeptide fragment or a transcript encoding such a polypeptide that is related to or associated with any of the microorganisms disclosed herein by one or more of the microorganisms disclosed herein. Molecules can be, but are not limited to, proteins, polypeptides, peptides, RNA, DNA, lipids, glycosylated molecules, carbohydrates, polypeptides with phosphorylation modifications, and polypeptides with citrullination modifications, aptamers, oxidized molecules, and other molecules.

[0086] For the elements described herein, "positional specifiability" refers to location, position, tag, cleavable tag or marker, identifier, spectral property, electrophoretic property, or other physical property that enables the identification of the element. An example of positional specifiability, also known as coding, is spatial positional specifiability where the position of a molecule is fixed and its position correlates with uniqueness. This type of spatial array can generally be synthesized or spotted on a planar substrate, for example, to generate a microarray, in which a large number of different molecules are densely arranged in a small area (e.g., containing at least about 400 different sequences per cm2, 1000 sequences per cm 2 or 1000 sequences per cm 2It can be an array of 5000 or more per area). A less dense array (e.g., an ELISA or RIA plate) where each well in the plate contains a separate probe can range from about 96 arrays per plate to up to about 100 arrays per cm 2 including up to the density of a microarray. Other spatial arrays utilize optical fibers, where different probes can be coupled to the fibers and formed into a bundle structure for coupling and analysis. Methods for the manufacture and use of spatial arrays of polypeptides are known in the art.

[0087] An alternative to this type of spatially-coded array is the use of molecular "tags", where the target probe can be linked to a detectable label or tag, which can provide coded information regarding the sequence of the probe. These tags can be cleaved from the element and then detected to identify the element. In some embodiments, a set of probes can be synthesized or attached to a set of coded beads, where each bead can be linked to a separate probe and the beads can be coded in a way that allows identification of the attached probe. In this type of "tag array", flow cytometry can be used to detect binding. For example, microspheres having fluorescent coding can be identified to identify a particular microsphere. The probe can be covalently attached to a "color-coded" object. The labeled target polypeptide can be detected by flow cytometry, and using the coding on the microsphere, the bound probe (e.g., an immunoglobulin, an antigen-binding fragment of an immunoglobulin, or a ligand) can be identified.

[0088] In some embodiments, the array can be an immunoglobulin (e.g., an antibody or an antigen-binding fragment thereof) array. As used herein, an "immunoglobulin array" refers to a set of spatially separated discrete molecular entities that can bind to target polypeptides arranged in a manner that enables the identification of polypeptides contained within a sample. In some embodiments, the array can include one or more of proteins, polypeptides, peptides, RNAs, DNAs, lipids, glycosylated molecules, polypeptides with phosphorylation modifications, and polypeptides with citrullination modifications, aptamers, and other molecules.

[0089] Composition Antibody: Disclosed herein are isolated antibodies, antibody fragments, and antigen-binding fragments thereof that can specifically bind to one or more of the microorganisms or microorganism-specific antibodies disclosed herein. Optionally, the isolated antibody, antibody fragment, or antigen-binding fragment thereof can be a neutralizing antibody.

[0090] As used herein, the term "antibody" is used in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, disclosed herein are also antibody fragments or polymers of those immunoglobulin molecules, and human or humanized forms of immunoglobulin molecules or fragments thereof, as long as they are selected for their ability to interact with the polypeptides disclosed herein. An "antibody fragment" is part of a complete antibody. A complete antibody refers to an antibody having two complete light chains and two complete heavy chains. Antibody fragments lack all or part of one or more chains. Examples of antibody fragments include, but are not limited to, Fab' and fragments of Fab'. A Fab' is composed of a single light chain and a single heavy chain. Fab' and Fab' fragments can be generated by reducing an antibody or antibody fragment having two light chains and two heavy chains. Such antibody fragments are referred to as reduced antibodies. Reduced antibodies have exposed reactive sulfhydryl groups. These sulfhydryl groups can be used for coupling of reactive chemical groups or biomolecules to the antibody fragment. A preferred Fab' fragment is F(ab). The hinge region of an antibody or antibody fragment is the region where the light chain ends and the heavy chain continues.

[0091] Antibody fragments for use in the methods disclosed herein are capable of binding to an antigen (e.g., a microorganism-specific antibody described herein, or one or more of a microorganism). In some embodiments, the antibody fragment can be specific for an antigen. An antibody or antibody fragment is specific for an antigen if it binds to one epitope with significantly higher affinity than to other epitopes. An antigen can be any molecule, compound, composition, or part thereof to which an antibody fragment can bind. For example, the antigen can be a microorganism-specific antibody described herein, or one or more of a microorganism. An analyte can be any molecule, compound, or composition of interest. Antibodies or antibody fragments can be tested for their desired activity using the in vitro assays described herein, or by similar methods.

[0092] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Also disclosed are "chimeric" antibodies in which a portion of the heavy or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies as long as they exhibit the desired antagonistic activity (see, e.g., U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0093] Monoclonal antibodies can be made using any procedure for producing monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using a hybridoma method such as that described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent.

[0094] Monoclonal antibodies may also be produced by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567 (Cabilly et al.). The DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of a mouse antibody). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques as described, for example, in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

[0095] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of the antibody to generate fragments thereof, e.g., Fv, Fab, Fab’ or other antigen-binding portions of the antibody, can be accomplished using routine techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO94 / 29348, published December 22, 1994, and U.S. Patent No. 4,342,566, the contents of which are incorporated herein by reference in their entirety for the teaching of papain digestion of antibodies for preparing monovalent antibodies. Papain digestion of an antibody typically produces two identical antigen-binding fragments, called Fab fragments, each having a single antigen-binding site, and the remaining Fc fragment. Pepsin treatment yields fragments that have two antigen-binding sites and can still cross-link antigens.

[0096] The fragment can include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, regardless of whether it is bound to other sequences, provided that the activity of the antibody or antibody fragment is not significantly changed or impaired as compared to an antibody or antibody fragment whose activity has not been modified. These modifications can provide several additional properties, such as removing / adding disulfide bondable amino acids, increasing its biological lifetime, changing its secretion properties, etc. In any case, the antibody or antibody fragment must have biological activity properties such as specific binding to its cognate antigen. The functional or active region of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein followed by expression and examination of the expressed polypeptide. Such methods will be readily apparent to those of ordinary skill in the art and can include site-directed mutagenesis of the nucleic acid encoding the antibody or antibody fragment (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0097] As used herein, the term "antibody(ies)" can also refer to human antibodies or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and thus can elicit an unwanted immune response when administered to humans. Accordingly, the use of human antibodies or humanized antibodies in methods helps reduce the likelihood that an antibody administered to a human will elicit an unwanted immune response.

[0098] Human antibodies can be prepared using any technique. Examples of techniques for the production of human monoclonal antibodies include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and Boerner et al. (J. Immunol., 147(1): 86 - 95, 1991). Human antibodies (and fragments thereof) can also be generated using phage display libraries (Hoogenboom et al., J. Mol. Biol., 227: 381(1991), Marks et al., J. Mol. Biol., 222: 581(1991)).

[0099] Human antibodies can also be obtained from transgenic animals. For example, transgenic mutant mice that can produce a complete repertoire of human antibodies in response to immunization have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 - 255(1993); Jakobovits et al., Nature, 362: 255, 258(1993); Bruggermann et al., Year in Immunol., 7: 33(1993)). Specifically, homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germline mutant mice results in complete suppression of endogenous antibody production, and successful introduction of such a germline mutant mouse of the human germline antibody gene array results in the production of human antibodies upon antigen loading. Antibodies having the desired activity are selected using the Env - CD4 - coreceptor complex as described herein.

[0100] Optionally, human antibodies can be made from memory B cells using a method for Epstein - Barr virus transformation of human B cells. (See, e.g., Triaggiai et al., An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS, coronavirus, Nat. Med. 2004, Aug;10(8):871 - 5, which is incorporated herein by reference in its entirety for teaching methods of making human monoclonal antibodies from memory B cells). Briefly, memory B cells from subjects who have survived natural infection are isolated in the presence of irradiated monocytes and CpG oligonucleotides that act as polyclonal activators of memory B cells and are immortalized by EBV. The memory B cells are cultured and analyzed for the presence of specific antibodies. Next, EBV - B cells from cultures producing antibodies of the desired specificity are cloned by limiting dilution in the presence of irradiated monocytes and cultured with the addition of CpG2006 to enhance cloning efficiency. After culturing the EBV - B cells, monoclonal antibodies can be isolated. Such methods provide (1) antibodies produced by immortalization of memory B lymphocytes that are stable over a lifetime and can be easily isolated from peripheral blood, and (2) antibodies isolated from immunized natural hosts that have survived natural infection, thus eliminating the need to immunize experimental animals that may show different immune responses because they show different susceptibilities.

[0101] Humanization techniques for antibodies generally involve the use of recombinant DNA techniques to manipulate DNA sequences encoding one or more polypeptide chains of the antibody molecule. Thus, a humanized form of a non - human antibody (or fragment thereof) is a chimeric antibody or antibody chain (or fragment thereof, e.g., Fv, Fab, Fab’, or other antigen - binding portion of the antibody) that contains a portion of the antigen - binding site derived from a non - human (donor) antibody incorporated into the framework of a human (recipient) antibody.

[0102] To generate a humanized antibody, residues derived from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced with residues derived from one or more CDRs of a donor (non-human) antibody molecule known to have the desired antigen binding characteristics (e.g., a particular level of specificity and affinity for a target antigen). Optionally, Fv framework (FR) residues of the human antibody are replaced with the corresponding non-human residues. A humanized antibody may also contain residues not found in either the recipient antibody or the transferred CDR or framework sequences. In general, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. Indeed, a humanized antibody is typically a human antibody in which some CDR residues and perhaps some FR residues are replaced with residues from the analogous sites in a rodent antibody. A humanized antibody generally contains the antibody constant region (Fc), at least a portion of that of a normal human immunoglobulin (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Struct. Biol., 2:593-596 (1992)).

[0103] Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated by using rodent CDRs or sequences of CDRs in place of the corresponding sequences of human antibodies according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)). Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5,939,598 (Kucherlapati et al.), U.S. Patent No. 6,130,364 (Jakobovits et al.), and U.S. Patent No. 6,180,377 (Morgan et al.). The antibodies disclosed herein can also be administered to a subject. There are also nucleic acid approaches for antibody delivery. Broadly neutralizing antibodies and antibody fragments against the polypeptides disclosed herein can also be administered to a subject or to a subject as a nucleic acid preparation (e.g., DNA or RNA) encoding the antibody or antibody fragment so that the subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.

[0104] Kit In some embodiments, kits are disclosed that include one or more probes or primers capable of detecting, amplifying, or measuring the presence or expression of one or more of the microorganisms disclosed herein. Also disclosed are kits that include one or more of the microbial capture agents, microorganisms, or microorganism-specific antibodies disclosed herein.

[0105] Disclosed herein is a solid support comprising one or more primers, probes, polypeptides, or antibodies that can hybridize or bind to one or more of the microorganisms described herein. A solid support is a solid-state substrate or support that can associate molecules such as analytes and analyte-binding molecules. An analyte (e.g., nanoparticle or protein calcification) can be associated with the solid support either directly or indirectly. For example, an analyte can be immobilized directly on the solid support. An analyte capture agent (e.g., a capture compound) can also be immobilized on the solid support.

[0106] As described above, one of ordinary skill in the art can determine the presence or expression level of one or more of the microorganisms or microorganism-specific antibodies (proteins or nucleic acids) disclosed herein in any number of ways. To detect or quantify the level of an RNA product of a biomarker in a sample, arrays such as microarrays, RT-PCR (including quantitative RT-PCR), nuclease protection assays, Northern blot analysis can be used. Thus, in some embodiments, the expression level of one or more of the microorganisms can be determined using an array, microarray, RT-PCR, quantitative RT-PCR, nuclease protection assay, or Northern blot analysis.

[0107] An array is in the form of a solid support. An array detector is also in the form of a solid support to which a plurality of different capture compounds or detection compounds are bound in an array, grid, or other organized pattern.

[0108] Examples of solid substrates for use in a solid support include any solid material to which molecules can bind. Examples of such materials include, but are not limited to, acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene, poly(vinyl acetate), polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, Teflon®, fluorocarbon, nylon, silicone rubber, polyanhydride, polyglycolic acid, polylactic acid, polyorthoester, polypropyl fumerate, collagen, glycosaminoglycan, and polyamino acid. The solid substrate can have any useful form, including thin films, membranes, bottles, dishes, fibers, woven fibers, molded polymers, particles, beads, microparticles, or combinations thereof. The solid substrate and the solid support can be porous or non-porous. An example of a solid substrate is a microtiter dish (e.g., a standard 96-well format). Multi-well slides can also be used. For example, those containing one array per well can be used to enable excellent control of assay reproducibility, improved throughput and sample handling, and ease of automation.

[0109] Different compounds can be used together as a set. The set can be used separately in separate reactions or as a mixture of all or a subset of the compounds immobilized on an array (e.g., microbial capture agents or microbial specific antibodies). The compounds used separately or as a mixture can be physically separated, for example, via association with a solid support or immobilization on a solid support. An array can contain a plurality of compounds immobilized at specified or predefined positions on the array. Each predefined location on the array can generally have one type of component (i.e., all components at that location are the same). Each location can have multiple copies of the component. The spatial separation of different components in the array allows for the separate detection and identification of the polynucleotides or polypeptides disclosed herein.

[0110] The given array need not be a single unit or structure. The set of compounds can be distributed among any number of solid supports. For example, each compound can be immobilized on a separate reaction test tube or vessel, or on separate beads or microparticles. The disclosed methods and various aspects of the use of gene expression panels or arrays or diagnostic devices can be carried out with various components immobilized on a solid support (e.g., various compounds specific for various proteins).

[0111] Some solid supports can have a capture compound such as an antibody attached to a solid-state substrate. Such a capture compound can be specific for calcified nanoparticles or proteins on the calcified nanoparticles. Next, the captured calcified nanoparticles or proteins can be detected by the binding of a second detection compound such as an antibody. The detection compound can be specific for the same protein or a different protein on the calcified nanoparticles.

[0112] Methods for immobilizing nucleic acids, peptides or antibodies (and other proteins) onto substrates in the solid state are well established. Immobilization can be achieved, for example, by attachment to an aminated surface, a carboxylated surface, or a hydroxylated surface using standard immobilization chemistries. Examples of attachment agents include, but are not limited to, cyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin-biotin, photo-crosslinkers, epoxides, maleimides, and N-[γ-maleimidobutyryloxy] succinimide ester (GMBS), and hetero-bifunctional crosslinkers. Antibodies can be bound to the substrate by chemically cross-linking the free amino groups of the antibody to reactive side groups present within the substrate in the solid state. Antibodies can be chemically cross-linked, for example, to substrates containing free amino groups, carboxyl groups, or sulfur groups using glutaraldehyde, carbodiimide, or GMBS, respectively, as cross-linking agents. In this method, an aqueous solution containing the free antibody can be incubated with the substrate in the solid state in the presence of glutaraldehyde or carbodiimide.

[0113] A method for attaching an antibody or other protein to a substrate in the solid state is to functionalize the substrate with an aminosilane or thiolsilane and then activate the functionalized substrate with a homo-bifunctional crosslinker such as bis-sulfo-succinimidyl suberate (BS3) or a hetero-bifunctional crosslinker such as GMBS. For cross-linking with GMBS, the glass substrate can be chemically functionalized by immersion in a solution of mercaptopropyltrimethoxysilane (1% vol / vol in 95% ethanol pH 5.5) for 1 hour, rinsing with 95% ethanol, and heating at 120 °C for 4 hours. Thiol-derivatized slides can be activated by immersion in a 0.5 mg / ml solution of GMBS in 1% dimethylformamide, 99% ethanol at room temperature for 1 hour. The antibody or protein can be added directly to the activated substrate, which can be blocked with a solution containing an agent such as 2% bovine serum albumin and air dried. Other standard immobilization chemistries are known to those skilled in the art.

[0114] Each component immobilized on the solid support (e.g., an antibody) can be placed in different pre-defined regions of the solid support. Each of the different pre-defined regions can be physically separated from each other. The distance between different pre-defined regions of the solid support can be fixed or variable. For example, in an array, each component can be placed at a fixed distance from each other, while components associated with beads will not have a fixed spatial relationship. The use of multiple solid support units (e.g., multiple beads) can result in varying distances.

[0115] Components can associate or immobilize on the solid support at any density. Components can be immobilized on the solid support at densities of over 400 different components per cubic centimeter. An array of components can have any number of components. For example, the array can have at least 1,000 different components immobilized on the solid support, at least 10,000 different components immobilized on the solid support, at least 100,000 different components immobilized on the solid support, or at least 1,000,000 different components immobilized on the solid support.

[0116] Furthermore, the microorganisms described herein can also be used as markers (i.e., biomarkers) for susceptibility to, or the presence or progression of, hypomyelination disorders. The methods and assays described herein can be performed over time to evaluate changes in the levels of the markers. For example, the assays can be performed every 24 - 72 hours for a period of 6 months to 1 year and then as needed. The assays can also be completed before, during, or after a treatment protocol. Overall, the microorganisms or microorganism - specific antibodies disclosed herein can be used to profile the risk or progression of demyelinating diseases in an individual. As used in this context, the terms "differentially expressed" or "differential expression" refer to differences in the expression levels of the disclosed herein biomarkers that can be assayed by measuring the expression levels of the products (e.g., RNA or gene products) of the biomarkers, such as differences in the levels of the expressed messenger RNA transcripts or portions thereof, or the expressed proteins of the biomarker. In some embodiments, this difference is significantly different.

[0117] To improve sensitivity, more than one of the microorganisms disclosed herein can be assayed within a given sample. The various protein, antibody, nucleic acid - specific binding agents provided herein can be combined within a single assay. Further, multiple primers or probes can be used simultaneously. To assist in such assays, specific biomarkers can be useful for the specificity of such tests.

[0118] Generally, disclosed herein are methods for the identification (including diagnosis) of microorganisms (e.g., microorganism) and microbial infections (including polymicrobial infections) in a patient. There are various methods used to identify the various microorganisms within a sample, for example, providing specificity. In some embodiments, a plurality of detection surfaces can be used. In some embodiments, each detection surface can have various specific microbial capture agents. That is, one detection surface may include a microbial capture agent that includes an antibody against a particular microbial species or genus, and another detection surface may include a different microbial capture agent against a different particular species. In some embodiments, a plurality of detection surfaces that are fluidically separated from each other; for example, a plurality of detection modules, for example, detection channels can be used, where one sample can be divided among the detection modules and then subjected to different conditions, for example, different microbial capture agents, for evaluation. As outlined herein, the plurality of different detection surfaces can have non-specific or specific microbial capture agents.

[0119] In some embodiments, the detection surface(s) can rely on non-specific capture of microorganisms or microorganism-specific antibodies, but the detection method can rely on specific binding ligands; for example, microorganism-specific antibodies against specific species or genera of microorganisms can be used with fluorescent labels. In some embodiments, simultaneous detection typically relies on different binding ligands containing different labels, while sequential detection can be performed using different binding ligands with the same label following one or more washing steps. Another aspect of the present invention avoids the use of either specific capture or specific labeling. In some embodiments, the method provides specific identification of microorganisms using spatial separation of microorganisms on a detection surface based on a detectable change or a known change; for example, the ability to detect the division of a single microorganism can include any number of parameters, particularly kinetic parameters including growth rate, assessment of metabolic activity, rate of cell killing by different antibiotics, and the morphology of the microorganism including size, shape, and relationship to sibling organisms (e.g., growth into clusters or chains, two-dimensional growth on a surface, or three-dimensional growth away from a surface), but is not limited thereto, enabling identification based on these kinetic parameters. In addition to assessment of rate, single data point analysis may also be performed (e.g., increase in area associated with individual microorganisms on a surface (e.g., positive growth), stagnant area (no positive growth), or loss of area (e.g., negative growth, apoptosis and / or death).

[0120] In some embodiments, the present invention provides a solid support or kit for detecting a demyelinating disease in a subject. In some embodiments, the subject may be suspected of having a demyelinating disease or at risk of having a demyelinating disease. In some embodiments, the present invention provides a solid support or kit for detecting one or more antibodies in a sample. In some embodiments, detection of the presence of one or more microbe-specific antibodies can indicate a demyelinating disease. In some embodiments, the present invention provides a solid support or kit for detecting one or more microbes in a sample. In some embodiments, the sample can be derived from a subject at risk of developing a demyelinating disease or suspected of having a demyelinating disease. The solid support or kit can include nucleic acid probes, antibodies, microbe-specific antibodies, primers, and / or microbe capture agents that can be useful for determining the presence of one or more microbes selected from the lists of Table 1, Table 2, or Table 8.

[0121] The kit can also include reagents that can be used in various auxiliary substances, such as solvents, washing buffers, etc., so that the kit can be used easily and efficiently.

Examples

[0122] Example 1: Spectrum of microbial sequences and bacterial cell wall antigens in a primary demyelinated brain specimen obtained from a surviving patient

[0123] Abstract: Multiple sclerosis (MS) is an autoimmune disease characterized by multiple lesions in the brain and spinal cord. RNA sequencing was used to identify microbial sequences and to characterize the expression patterns of human genes in 30 human brain biopsy specimens. RNA aligned against known microbial taxa was significantly enriched in 10 out of 12 primary demyelinating (MS) brain specimens compared to a group of 15 epilepsy controls, leading to a list of 29 MS microbes or genera of MS microbial families from 11 different phyla. Most of the MS microbes described herein are anaerobic bacteria. There were some common MS microbes or MS microbial families, but each of the 10 MS samples with significant microbial RNA enrichment had a different set of MS microbes or MS microbial families. The rate of microbial sequencing reads was higher in the MS group (128.8 PPM) compared to controls (77.4 PPM, p = 0.016). Bacterial peptidoglycan was demonstrated in brain tissue sections from some MS subjects. Human gene expression analysis showed increased expression of inflammation-related pathways in the MS group. This data indicates that demyelinating brain lesions are associated with the presence of microbial RNA sequences and bacterial antigens. This suggests that MS is caused by the presence of a diverse set of microbes within the lesions.

[0124] Materials and Methods: Subjects: The characteristics of the subjects under study are shown in Table 3. Twelve brain biopsy samples were selected for sequencing from 11 subjects. Biopsies from 10 of these 11 subjects had lesions indicative of demyelination. Another subject (MS-062) had well-established progressive MS. These 11 subjects are designated as the MS group for clarity and brevity. (One subject, MS-021, was biopsied twice over a 3-month period.) The brain biopsy findings were reviewed by a neuropathologist. The disease courses of the MS subjects ranged from the presentation of a single demyelinating symptom to severe fulminant disease (Marburg disease or tumefactive disease). Clinical information was compiled from electronic records, paper records, and discussions with the treating physicians. Clinical diagnoses and imaging findings were reviewed by an MS neurologist. Three subjects had brain biopsy lesions indicative of some other process called other neurological diseases (OND). The control group consisted of tissue taken from 15 subjects with epilepsy who had had a portion of their brain tissue resected to control seizures. Specimens from the MS and OND subjects were white matter, while specimens from the epilepsy control subjects were primarily from the cerebral cortex (gray and white matter).

[0125] Specimen Preparation, RNA Extraction, and Library Preparation: Brain biopsy blocks were obtained. Multiple procedures were used to prevent exogenous contamination of the samples: a dedicated microtome with a new blade was used for each specimen, the bench area was cleaned, decontamination was performed during specimen processing, the first 50 microns of tissue were discarded, specimens were handled using sterile disposable instruments, and the microtome was disinfected with alcohol and bleach between specimens. Five 10-μm thick sections were taken for RNA extraction.

[0126] RNA extraction from brain biopsies was performed using the Qiagen FFPE RNA kit, which includes DNA-degrading enzymes. RNA from 12 of these MS brain biopsy specimens passed quality control and was sequenced in two separate runs on the Illumina HiSeq 2500 platform. Ten of the 12 sequenced MS samples were from female subjects. One female subject (subject 021) was biopsied twice at 3-month intervals. To enrich samples for microbial sequences, human rRNA was physically depleted from the samples using RiboZero (Illumina Catalog #MRZH116). For sequencing, a non-directional complementary DNA library was constructed and paired-end 125bp sequencing was performed on the HiSeq-2500. Fifteen control epilepsy FFPE samples were processed and sequenced in parallel using the same instrument and procedure. Samples from two cases of chronic encephalitis and one case of hypoxic brain injury were sequenced and used as controls for other neurological diseases (OND). Two blank specimens defined as having no visible tissue (i.e., paraffin) and <10% of the reads of samples mapped to the human genome or transcriptome were sequenced and used in the analysis. 125bp paired-end reads were obtained by RNA-seq.

[0127] Array determination and quality control: Quality control of the 125bp reads of the pairs was performed by the Sickle program. Low-quality pairs were removed and low-quality basecalls at the ends were trimmed (Sickle: Sliding window, adaptive, quality-based trimming tool for FastQ files v.1.33 (2011)). Sickle parameters were set to discard read pairs when either member of the pair was trimmed to a length <40bp. The HQ read pairs of each sample were aligned against several databases using Bowtie2 (settings: -q --k1 --phred33 --local) (Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Meth. 9, 357-359, doi:10.1038 / nmeth.1923, http: / / www.nature.com / nmeth / journal / v9 / n4 / abs / nmeth.1923.html-supplementary-information (2012)). Reads aligned against the human genome and / or human transcriptome (GRCh assembly) were analyzed separately for differences in host gene expression. Next, the remaining non-human reads were aligned against a panmicrobial database curated at the Fischer Lab. The panmicrobial database contains a non-redundant viral database (including complete and partial viral sequences), and the complete genomes of bacteria, archaea, fungi, and protists in GenBank. This 11Gb panmicrobial database contains over 1.3 million sequence records, each identified by a GenBank identifier (gi) representing 10,654 species. Fischer (Fischer, K. F. The Panmicrobial Database, <http: / / pathogenomics.path.utah.edu / sequences / panmicrobial_nrdb.fasta.zip> (2017)). Microbial sequence comparisons were also performed with Bowtie2 (settings: --end-to-end --phred33). Read pairs that aligned to the same microbial sequence (matching pairs) were counted and advanced for analysis.

[0128] Since RNA-seq did not generate exactly the same number of HQ pairs in each sample, the read counts from the samples aligned to each sequence in the pan-microbial database were normalized by dividing by the number of high-quality pair reads (in millions) to obtain pairs per million (PPM). These normalized hit rates (HR) were calculated for each microbial taxon and sample. Taxon PPMs were aggregated when possible and analyzed at the genus and family levels. Data from two sequencing runs (Run 1 and Run 2) were combined and analyzed. Unnatural results unique to Run 2 were observed, with more reads aligning to the phylum Proteobacteria than in Run 1. Taxa affected by this unnatural result were excluded from the HR analysis by comparing the HR of the blank (tissue-free) specimens of Run 2 to the HR of the control samples of Run 1. Next, taxa with an HR in one or both blanks greater than the mean HR of that taxon in the control of sequencing run 1 were excluded. The blanks were separate FFPE brain biopsy specimens from registered subjects without visible tissue.

[0129] Human gene expression: Differential expression levels of known splice variants in the demyelinated sample group and the control sample group were calculated using Bowtie (v2.2.5.0), tophat (v2.0.14), and cuffdiff (v2.2.1) (Trapnell, C. et al. Nat. Biotech. 28, 511-515, (2010); and Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. Biotech. 31, 46-53, (2013). Tophat was run with the "-no-novel-juncs" setting. Cuffdiff was run on the tophat output "accepted_hits.bam" file for each sample. Differentially expressed transcripts were identified between groups with a false discovery rate (FDR) controlled at <0.05. The transcriptome model used was derived from the GRCh37 Ensembl release 75 of May 23, 2014. Pathway enrichment of differentially expressed genes was calculated by Cytoscape and the Reactome Curated pathway database (Fabregat, A. et al. Nucleic Acids Res. 44, D481-487, (2016)).

[0130] Statistical analysis: Demographics and characteristics of the study population were compared using ANOVA and then confirmed with Fisher's exact test for discrete variables (e.g., gender) or the Mann-Whitney non-parametric test (e.g., age, collection year) (Lowry, R. VassarStats: Website for Statistical Computation, <http: / / vassarstats.net / >). The list of microorganisms for each MS brain sample was derived by looking for significant outliers within the dataset. The HR from each taxon for each sample was the transformed log 2It was. The Z-score was calculated for all MS samples using the expected distribution of the control sample with a Z-score of 15. The analysis was performed at the family and genus levels. The Z-score was converted to a p-value using a normal distribution. Since we were interested in microbial sequences that were overrepresented rather than underrepresented in the MS group, a one-sided test was performed. To correct for multiple comparisons, the p-value was converted to a q-value by the false discovery rate control method of Benjamini-Hochberg (Benjamini, Y. & Hochberg, Y. Journal of the Royal Statistical Society, Series B (Methodological) 57, 289-300 (1995)). MS microbes or MS microbial families were defined as those that were significantly (q < 0.05) overrepresented in at least one MS sample compared to a set of 15 controls, and the HR was less than 1.0 PPM. Significance based on the HR was calculated using Python 2.7. Normalization, transformation, and calculation of the Z-score of the HR were performed using numpy and pandas; the p-value and q-value were calculated using scipy.stats (Oliphant, T. E. (2006); McKinney, W. in SciPy 2010; and SciPy: Open source scientific tools for Python (2001)).

[0131] Immunohistochemistry: Five-micron pathological brain tissue sections from the subjects MS-019 and MS-056, epilepsy controls 039 and 040, and brain abscess (positive control) were examined. The tissue sections were treated with antigen demasking (Vector Laboratories, product H3300), followed by 0.5% casein blocking (Sigma). The sections were incubated overnight at 4 degrees with anti-peptidoglycan IgG1 mAb (EMD Millipore product MAB995, Temecula, CA), anti-CD68 (macrophage, ab955, Abcam, Cambridge, MA), anti-lysozyme (macrophage and neutrophil, ab108508, Abcam, Cambridge, MA), or mouse isotype control Ab (IgG1 isotype control, Invitrogen, catalog number MA5-14453). An additional anti-peptidoglycan monoclonal antibody (mAb 2E9) was provided. 2E9 is an IgG3 mAb initially developed in 1994 as a reagent for detecting bacterial antigens from the gut microbiota in splenic macrophages (Kool, J. et al. Journal of Histochemistry & Cytochemistry, 42, 1435-1441, (1994)). This mAb has also been used to show peptidoglycan in brain tissue (Schrijver, I.A. et al. Brain, 124, 1544-1554 (2001); and Branton, W.G. et al. Scientific reports, 6, 37344, (2016)). The sections were developed with biotinylated anti-mouse IgG antibody (BA-2000, Vector, California, USA) and streptavidin-peroxidase (S5512, Sigma, St. Louis, MO, USA). Image analysis was performed with a Zeiss Axioplan microscope.

[0132] Results: Test cohort: The characteristics of the test cohort are shown in Table 3. There were no significant differences between groups in the distribution of age and sex. Brain specimens were collected somewhat later in the control cohort (median 2012) compared to the MS cohort (median 2007, p<0.001). This is due to the abundance of primary demyelinated specimens (relatively rare) compared to surgical controls for epilepsy (more common), and furthermore to the situation where more recent epilepsy control specimens have been registered. The control cohort had significantly more surgical procedures (median 2) one month prior to brain specimen collection compared to the MS cohort (median 1, p=0.005). The higher number of surgical procedures is due to mapping procedures performed in the control prior to resection of epileptogenic foci.

[0133] Characteristics of brain specimens: The brain specimens used in this study were formalin-fixed, paraffin-embedded FFPE. The biopsy sites included a mixture of white and gray matter in the MS cohort and other neurological disease (OND) cohorts. Control biopsies were taken mainly from the cortex (gray matter). Ten out of twelve biopsies from the MS cohort had neuropathological reading data of demyelination consistent with MS. One subject (MS-021) was biopsied twice because the initial specimen had an inadequate diagnosis (i.e., normal brain). Another subject with well-established progressive MS (MS-062) received a biopsy mainly consisting of gray matter showing perivascular inflammation. Three subjects in the OND cohort were initially suspected of having MS based on radiological and clinical findings but were later reclassified based on the neuropathology of their specimens (i.e., not primary demyelination).

[0134] Clinical findings in the MS and OND cohorts: Six out of eleven subjects in the MS cohort had oligoclonal band (OCB) testing recorded. Of these, four were positive and two were negative. One OND subject (OND-003) also had a positive OCB test despite a final diagnosis of acute disseminated encephalomyelitis (ADEM) and encephalitis rather than MS. Eleven subjects in the MS cohort had a disease consistent with multiple sclerosis, and six received disease-modifying therapy (Table 3). Since one subject (MS-21) had two brain biopsies at several-month intervals, there is one less MS subject than MS samples. Table 3. Characteristics of the test population. JPEG0007682540000004.jpg144164 JPEG0007682540000005.jpg171163 JPEG0007682540000006.jpg77162 1 MS refers to the monophasic demyelination group. OND = Other neurological diseases; C = Epilepsy control 2 Age (years) is reported at the time of specimen collection. 3 Site of brain tissue collection as specified in the pathology report. The control sites for epilepsy were mainly derived from the cortex. Laterality is provided if available. 4 Reading data in cases of MS and OND as described by a neuropathologist. Clinical pathology reports are summarized from specimens of these groups not available for review and from controls. FCD = Focal cortical dysplasia 5 Evaluation from medical records and discussions with treating neurologists. RRMS = Relapsing-remitting MS; ADEM = Acute disseminated encephalomyelitis; CVA = Cerebrovascular accident (stroke) 6 ND = Not examined 7 Brain MRI (and / or CT) findings as reviewed by an MS neurologist or as reported in the medical records if not available for review. 8 Disease-modifying therapies (DMTs) provided after diagnostic brain biopsy. Biopsies in the MS group were for establishing a diagnosis, so none of these subjects were receiving disease-modifying therapy at the time of specimen collection. NTZ = Natalizumab; IFNβ = Interferon beta; DMF = Dimethyl fumarate; GA = Glatiramer acetate, NOV = Novantrone, AZP = Azathioprine, RTX = Rituximab

[0135] Sequencing and sequence comparison: Among 32 samples (MS12, control 15, OND3, blank 2), the total amount of sequencing was 1.06 - 2.96×10 8were high-quality read pairs (HQ pairs). There was no significant difference in the number of HQ pairs among the sample groups. Overall, the quality of the sequencing was high, and 2.6% of the original unfiltered read pairs were discarded from the dataset. The remaining HQ pairs were used for sequence comparison with microbial and human databases. Most of the HQ pairs (95.7%) were full-length (125 bp). The average length of the remaining trimmed reads (4.3% of the HQ pairs) was 123.9 bp.

[0136] Table 4 shows the sequence comparison against human and microbial databases. Reads aligned to the human genome were excluded from the microbial analysis. A total of 216,159 aligned paired reads from 30 experimental samples that mapped with high quality and specificity to a single sequence in the pan-microbial database (Table 5). Most of these microbial reads were bacteria. They were mainly mapped to rRNA sequences from the phylum Proteobacteria (50.3%), Actinobacteria (20.3%), Firmicutes (16.2%), Bacteroidetes (4.6%), or other phyla (8.5%). The proportion of microbial reads was higher in the MS group (128.8 PPM) compared to the control (77.4 PPM, p = 0.016).

[0137] Table 4. Summary of sequencing sequence comparison. The total number of mapped microbial read pairs was estimated using single aligned read pairs as reported by Bowtie 2.0 before filtering by mapping quality (MAPQ). TIFF0007682540000007.tif31153 1 Mapping of aligned read pairs to either the human genome / transcriptome or PhiX internal sequencing control 2 Aligned read pairs mapping once to the pan-microbial database ± standard error of the mean (SEM) 3 Value of microbial aligned read pairs divided by HQ read pairs multiplied by 10 6 ± SEM *P < 0.05 compared to the control group Table 5. Taxonomic distribution of sequence comparisons against the pan-microbial database. TIFF0007682540000008.tif52142 1 including bacteria, archaea, fungi, protists, and viruses 2 Taxonomic groups where one or more samples aligned to one or more database sequences (MAPQ value ≥ 10)

[0138] To determine the specific uniqueness of microbial sequences overrepresented in the MS group, additional filtering was performed using the MAPQ measurement criterion, a measure of the specificity of sequence comparison to database sequences (Trapnell, C. et al. Nat. Biotech. 28, 511 - 515, (2010); and Trapnell, C. et al. Nat. Biotech, 31, 46 - 53, (2013)). Reads by humans, reads by PhiX control, and reads with MAPQ less than 10 were excluded from further analysis. The remaining sequence comparisons had a probability of over 90% that the reported mapping was to the single best sequence match in the pan-microbial database. If there was a matching pair, the probability of the correct mapping was over 99%. A significant overrepresentation (q < 0.05) of microbial sequences in at least one of the MS samples was seen in 43 families and 84 genera. The overrepresentation at the family level is shown in Figure 1. The overrepresentation at the genus level is shown in Table 6.

[0139] Table 6. MS microbes or MS microbial families derived from next-generation sequencing data. To qualify as an MS microbe at the genus level, at least one specimen from the MS group had read pair mapping to this taxonomic group that was significantly increased (q < 0.05) compared to the control group with 10 or more MAPQ values (84 separate genera). The MS microbial genera shown had at least 100 mapped reads in all specimens of the MS group (29 genera). TIFF0007682540000009.tif149133 TIFF0007682540000010.tif153129 1 As classified by NCBI taxonomy. The phyla listed are for bacteria and fungal microorganisms, not for viruses. 2 Total number of reads mapped to taxa from the entire MS group (N = 12) or the control group (N = 15). 3 Number of MS specimens (out of 12) in which read mapping to this taxon was significantly increased (q < 0.05) compared to the control group (N = 15). 4 Generally recognized as a human pathogen. 5 As described by MicrobeWiki (https: / / microbewiki.kenyon.edu / index.php / MicrobeWiki), the list of prokaryote names according to the nomenclature (LPSN, http: / / www.bacterio.net), UniProt (https: / / www.uniprot.org), and other online information sources.

[0140] Representative mapping of read - pair alignments for Akkermansia (sample MS - 019) and Pseudomonas phage LUZ24 - like virus (sample MS - 053) is shown in Figure 2. The distribution of RNA abundances observed across these genomes is consistent with expected gene expression. For example, the mapping for the prokaryote (Akkermansia) is concentrated within the rRNA genes and mapping to other bacterial genes is much less. Also, for the LUZ24 - like virus, the observed RNA abundances map to the structural genes, as expected for a replicating bacteriophage.

[0141] MS microbiota: The list of microbiota for each MS brain sample was derived by looking for significant outliers within the dataset (see Methods for details of the analysis). Each sample was compared to a set of controls, and multiple comparisons were adjusted using the false discovery rate (q < 0.05) (Benjamini, Y. & Hochberg, Y. Journal of the Royal Statistical Society. Series B (Methodological) 57, 289 - 300 (1995)). Sequences from 42 microbial families were overrepresented in at least one sample of the MS group. The MS microbiota included one archaeon, 35 bacteria, two fungi, and four viral families (Figure 1).

[0142] Significant overrepresentation (q < 0.05) of microbial sequences in at least one of the MS samples was also seen in 84 genera. The list was filtered to include genera with 100 or more mapped reads among members of the MS group. This led to a more manageable list of 29 MS microbial genera from 11 different phyla (Table 6). The MS microbiota or MS microbial families with the highest number of mapped reads across the set of MS samples included the bacterial genera Nitrosospira, Atopobium, Fusobacterium, and Aggregatibacter, and the fungal genus Ustilago, each with over 1000 mapped reads. The MS microbiota listed in Table 6 included 26 bacterial, two fungal, and one viral genus. The complete list of 84 genera is not shown.

[0143] Another way to rank MS microbes is by the number of specimens that increased significantly more than the control: Bacteroides and Rubrobacter (5 specimens increased); Ustilago, Lactococcus, Capnocytophaga, Thioalkalivibrio, and Aerococcus (4 specimens increased); and Acidothermus and Tolumonas (3 specimens increased). MS microbes or MS microbe families are also listed for each subject (sample). (See Table 3. Since subject MS-021 was biopsied twice, this data is actually specimen-specific.) Ten out of twelve MS subjects had at least one overrepresented microbial genus. The number of microbial candidates observed in the samples ranged from 0 to 28.

[0144] Analysis of MS clusters: Four MS samples 17, 21-2, 55, and 56 showed a similar pattern of accumulation of MS microbes at the family level, as shown on the right side of Figure 1. These samples were compared to the others within the MS group for several technical and clinical parameters. There were no significant differences in total RNA yield or microbial fraction between the "cluster" (N = 4) and "no cluster" (N = 8) MS subgroups. Four cluster samples had prominent macrophages in pathological analysis compared to 6 out of 8 samples without a cluster (p = NS). Similarly, the interval between the onset of neurological symptoms and brain biopsy did not differ between subgroups. As expected, the cluster group had significantly more MS microbes at the genus level (mean 21.0) than the no-cluster group (mean 6.4, p = 0.01).

[0145] Differential gene expression in humans: It was found that 682 genes were differentially expressed between the MS group and the control group (FDR < 0.05). Compared to control samples, MS samples have many overexpressed immune-related genes. Analysis of pathway enrichment shows that five immune-related pathways, one secretory pathway, and two cell surface interaction pathways are significantly enriched for genes overexpressed in MS samples (Table 7). Conversely, control samples with relatively few macrophages show relatively high expression of neurogenes and enrichment of neural pathways.

[0146] Table 7. Human gene expression pathways overexpressed in MS. Differential expression levels of known splice variants in the demyelinated sample group and the control sample group were calculated using Bowtie (v2.2.5.0), tophat (v2.0.14), and cuffdiff (v2.2.1) (Trapnell, C. et al. Nat. Biotech. 28, 511-515, (2010); and Trapnell, C. et al. Nat. Biotech. 31, 46-53, (2013)). Pathway enrichment of differentially expressed genes was calculated by Cytoscape and the Reactome Curated pathway database (Fabregat, A. et al. Nucleic Acids Res. 44, D481-487, (2016)). TIFF0007682540000011.tif140140 TIFF0007682540000012.tif26142

[0147] Immunohistochemical analysis: Unfortunately, not all of the sequenced specimens from the MS group were large enough to allow subsequent immunohistochemical analysis. Four specimens were subjected to staining with an antibacterial (peptidoglycan) monoclonal antibody and a control. Peptidoglycan is a structural cell wall component of both Gram-positive and Gram-negative bacteria. Representative brain tissue sections of this analysis are shown in Fig. 3 along with a control. Signals for peptidoglycan were demonstrated in both the MS specimens and another brain abscess positive control specimen. Some peptidoglycan signals were also observed in some epilepsy control specimens, but the staining patterns were much more limited. A matched IgG1 isotype control supported the specificity of the anti-peptidoglycan signal. Staining with anti-CD68 and anti-lysozyme confirmed the presence of macrophages and neutrophils in the MS-019 specimen. Conventional neuropathology showed the presence of activated macrophages in most of the other MS specimens.

[0148] Investigation: Both RNA sequencing and immunohistochemistry suggest the presence of microorganisms in many MS brain specimens that differ from controls. In the brain biopsy samples in which MS microorganisms were detected, the sequences mapped to a diverse set of bacterial taxa. The samples did not share the same bacterial characteristics, and no single bacterial taxon was found in all samples. This suggests that the MS disease is associated with the presence of various microorganisms within lesions, or that macrophage infiltration provides a route for bacterial transport into lesions. These possibilities are not mutually exclusive, and further studies will be needed to define how microbial RNA and cell wall components reach the brain, and whether the presence of the microorganisms or their components affects disease onset or progression.

[0149] This study has several important limitations. First, the control specimens were taken from tissue affected by epilepsy. Thus, the control specimens were not from completely normal brains. Autopsy samples may be specially selected for normal pathological appearance, but quality analysis of some of these early samples in this study showed that RNA from FFPE autopsy brain samples was of very low quality and too poor to be used for sequencing. Thus, some mismatches between epilepsy controls taken mainly from the cortex (epileptogenic foci) and MS samples containing both cortex and white matter were inevitable. The effect of biopsy site on microbial analysis could not be excluded. Second, many MS patients had received some disease-modifying therapy (DMT in Table 3), while the epilepsy control subjects were treated for their condition with a completely different set of drugs. Since neither MS drugs nor epilepsy drugs are considered antibacterial, this is unlikely, but the effect of some drugs on the microbial composition of the samples could not be excluded. Finally, the initial collection dates of the MS samples compared to controls probably had little or no effect on the results, as no recognizable relationship was found between the collection date and the mapped reads of the microorganisms.

[0150] The data presented here reveal correlations between the presence of taxonomically diverse organisms, mainly bacteria, in MS brain lesions and microbial macromolecules derived from them. This is supported by the unambiguous mapping of sequencing reads to several bacterial genomes (e.g., Atopobium, Fusobacterium, Akkermansia), and by the immunohistochemical detection of bacterial peptidoglycan within the lesions of several subjects. The data reported here do not specifically distinguish between living microorganisms and remnants of microorganisms that can no longer survive (e.g., nucleic acids, peptidoglycan). However, the presence of bacteriophage, LUZ24-like virus, in two MS brain samples (MS-021-2 and MS-053) implies the presence of its host, Pseudomonas aeruginosa, as well. (Pseudomonas itself might have been lost from the final analysis if Gram-negative bacterial sequences were removed by the filter.) Since the LUZ24-like virus is a lytic (or virulent), not a temperate (or lysogenic), bacteriophage, Pseudomonas might also have been actively replicating in these samples at the time of collection (Ceyssens, P.J. et al. Virology, 377, 233-238, (2008)). Although more work will be needed to distinguish replicating bacteria from non-replicating bacteria in MS brain specimens, non-viable bacterial components might be sufficient to stimulate macrophage infiltration and demyelination.

[0151] Microbial RNA was also observed in control specimens, which was also supported by immunohistochemical analysis. The reason for this is not clear, but it might be related to the mapping electrodes placed in the epilepsy controls and the published procedures carried out. These published procedures were necessary to find, map, and remove epileptogenic foci. Although efforts were made to exclude contamination at points along the pipeline, the FFPE specimens themselves are not necessarily completely sterile or free of microbial RNA, and the use of the experimental (MS) study design compared to the control (epilepsy) is required. The microbial reads in the control specimens might also say something about epileptogenic foci within the brain tissue.

[0152] Many of the MS microbiota (enumerated in Table 6) are derived from the genus Anaerobacterium. Many of these may be regarded as commensal bacteria (e.g., Bifidobacterium, Atopobium), and it is currently unknown whether they play a role in MS pathogenesis. However, by sequencing analysis, various anaerobic and non-pathogenic bacterial species have also been observed in brain abscesses (Al Masalma, M. et al. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 54, 202 - 210, (2012)). Since many brain abscess specimens do not grow in culture and the microbiology of these lesions is complex, the microorganisms found within brain abscesses may have some role in the pathological process. This concept of microbiological complexity may also apply to MS.

[0153] Some of the MS group specimens had an accumulation of MS microbiota at the family level, shown as a cluster on the right side of Figure 1. Why these four samples had more MS microbiota than the other MS brain biopsy samples is not clear.

[0154] The Dutch group of Laman was the first to show bacterial antigens, particularly peptidoglycan, by immunohistochemistry in brain tissue from MS donors (Schrijver, I.A. et al. Brain, 124, 1544-1554 (2001)). The authors also demonstrated anti-peptidoglycan antibodies in the CSF of patients with active MS. This group then further hypothesized that peptidoglycan is involved in the development of CNS autoimmunity (Visser, L. et al. Journal of immunology (Baltimore, Md.: 1950) 174, 808-816 (2005)). Since then, the Canadian group led by Chris Power has shown bacterial sequences in brain tissue from patients with various CNS diseases, including one patient with MS (Branton, W.G. et al. PLoS One 8, e54673, (2013)). More recently, this group performed RNA sequencing on six autopsy-derived MS and six control brain samples (Branton, W.G. et al. Scientific reports, 6, 37344, (2016)). Their analysis revealed the predominance of Proteobacteria sequences in progressive MS and control brain samples, and Actinobacteria sequences were predominant in three relapsing-remitting MS brain samples. These results were supported by IHC and gene expression tests and were interpreted as consistent with the disruption of the microbiota within demyelinating lesions characteristic of MS. Although more controls, larger-scale sequencing, different mapping and analysis methods, and formalin-fixed paraffin-embedded specimens from living subjects were used, the findings of Power's group are similar to the reports disclosed herein.

[0155] The accumulation of several bacteria in the feces of MS patients compared to controls has been observed by other groups (Cantarel, B. L. et al. Journal of investigative medicine: the official publication of the American Federation for Clinical Research, 63, 729 - 734, (2015); and Jangi, S. et al. Nature communications, 7, 12015, (2016)). The study by Jangi et al. was also a sequencing study examining the fecal microbiota of MS patients compared to healthy controls. Interestingly, sequences mapped to two genera - Akkermansia and Methanobrevibacter increased in the feces of MS patients. Akkermansia was also identified among MS microbiota in current sequencing studies of brain tissue samples rather than feces (see Table 6).

[0156] Subject MS - 21 was biopsied twice over a period of several months and served as its own unintended control. The first biopsy (MS - 21 - 1) was normal while the second biopsy (MS - 21 - 2) showed clear demyelination. Interestingly, five MS microbiota were detected with relatively few mapped reads (each less than 50) in the first (nearly normal) biopsy. However, the second affected biopsy revealed 28 MS microbiota, and four of these MS microbiota had mapped reads exceeding 100 each. While this data is from a single subject, it supports the hypothesis that demyelinating lesions are associated with a greater number of multiple types of bacteria that are significantly different from controls.

[0157] Human gene expression analysis identified the Toll-like receptor cascade when most significantly overexpressed in brain tissue among MS subjects compared to controls, and the transport and processing of the endosomal TLR pathway. These pathways both include TLR2 and TLR4, which are associated with immunity against bacterial pathogens, and TLR7, which is associated with immunity against ssRNA (viruses). Other groups have shown an association between MS and TLRs (Bustamante, M.F. et al. Ann. Neurol. 70, 634-645, (2011); Hamid, K.M. et al. Iranian journal of allergy, asthma, and immunology, 15, 75-81 (2016); Hossain, M.J., Tanasescu, R. & Gran, B. Oncotarget, 6, 35131-35132, (2015); Nyirenda, M.H. et al. J. Immunol. 194, 5761-5774, (2015); and White, A.T., Light, A.R., Hughen, R.W., Vanhaitsma, T.A. & Light, K.C. Psychosomatic medicine, 74, 46-54, (2012)). Interestingly, while TLR4 expression was lower in PBMCs of MS patients compared to controls, the inventors detected higher expression in the affected brain tissue (Hamid, K.M. et al.; and Iranian journal of allergy, asthma, and immunology, 15, 75-81 (2016); and White, A.T., Light, A.R., Hughen, R.W., Vanhaitsma, T.A. & Light, K.C. Psychosomatic medicine, 74, 46-54, (2012)).

[0158] The source of the microbial sequences and antigens observed in MS lesions is currently unknown. There are at least two possibilities: 1) hematogenous seeding from bacteremia, or 2) the microorganisms were brought in by infiltrating macrophages. If bacteria are actually seeding the MS lesions from bacteremia, it seems strange that this has not been discovered previously. However, many of the MS microorganisms identified in this study are anaerobic or even unculturable, and it is well established that the recovery of microorganisms from brain abscesses (pathological findings different from demyelination) is often difficult. Macrophages may bring bacterial RNA and antigens into these MS lesions as a result of an autoimmune process. While macrophages and neutrophils are usually considered to be on the response side to tissue damage rather than initiators, macrophages can be actively involved in tissue destruction (Dragomir, A.C., Laskin, J.D. & Laskin, D.L. Toxicology and applied pharmacology, 253, 170 - 177, (2011); and Laskin, D.L., Pilaro, A.M. & Ji, S. Toxicology and applied pharmacology, 86, 216 - 226(1986)).

[0159] Several recent mouse studies have shown that a normal gut microbiota affects brain development and behavior via peptidoglycans that cross the blood-brain barrier and interact with the pattern recognition receptor Pglyrp2 (Arentsen, T., Khalid, R., Qian, Y. & Diaz Heijtz, R. Brain, behavior, and immunity, 67, 345-354, (2018); Arentsen, T. et al. Molecular psychiatry, 22, 257-266, (2017); and Diaz Heijtz, R. et al. Proceedings of the National Academy of Sciences of the United States of America, 108, 3047-3052, (2011)). The source of peptidoglycans is thought to be the normal gut microbiota during development. Another group investigating experimental autoimmune encephalomyelitis (EAE, an animal model disease similar to MS) showed that peptidoglycans act via the receptors NOD1, NOD2, and RIP2 and dendritic cells to exacerbate the disease (Shaw, P. J. et al. Immunity, 34, 75-84, (2011)). TLR2 and NOD are also thought to be involved in EAE disease activity in primates (Visser, L. et al. The American journal of pathology, 169, 1671-1685, (2006)). Finally, treatment of EAE mice with oral Lactobacillus paracasei improved the criteria for assessing disease severity (Libbey, J. E. et al. Beneficial microbes, 9, 495-513, (2018)). Whether any of these interesting findings in mice directly apply to human MS is not clear, but they reveal the importance of peptidoglycans and their sensing molecules in the progression of EAE.

[0160] The concept of "immunological scarring," in which infection leads to long-term immune dysfunction, has been proposed (Nathan, C. Science (New York, N.Y.), 350, 161, (2015)). This could help explain the persistent or recurrent dysfunctions that occur in MS, even in the absence of microbial antigen persistence. This study included brain biopsies, which are generally performed early in the disease course for diagnostic purposes.

[0161] Example 2: Spectrum of Microbial Sequences in Brain Specimens

[0162] Table 8 provides a list of MS microbes. Unbiased (large-scale, next-generation) RNA sequencing was performed on 18 formalin-fixed, paraffin-embedded primary demyelinated brain specimens (MS group), 16 epilepsy brain specimens (control group), and 2 blanks (no tissue). Criteria for inclusion in Table 8 included (1) overexpression in the MS group where (a) MS:control ratio ≥ 3.0; and (b) MS:blank ratio > 1.5; and (2) > 1000 total reads summed from members of the MS group (N = 16) (no MAPQ filtering was performed). Table 8. Examples of Microbes. JPEG0007682540000013.jpg156162 JPEG0007682540000014.jpg156163 JPEG0007682540000015.jpg157164 JPEG0007682540000016.jpg104158 *Examined Pseudomonas aeruginosa, the host bacterium of the bacteriophage

[0163] Next, the ELISA index (EI) was averaged for repeated measurements and used to classify serological responses in CSF among enrolled human subjects with demyelinating diseases (DD, MS group), other neurological diseases (OND group), or shunt patients (control group) expected to have normal CSF. The EI value was determined by dividing the experimental OD value by the average OD of three calibrator CSF specimens. Positive and negative controls confirmed the validity of each CSF serological run. Bacterial antigens were selected from previous sequencing data. Indirect ELISA was performed using CSF (undiluted) against whole bacteria that had been sonicated and washed.

[0164] Table 9 shows a summary of serological data for each group collected from CSF serology against six MS microorganisms or microorganism families. Table 9 was organized to show qualitative differences in antibody responses among the MS, OND, and control groups. The data were statistically analyzed and confirmed that the MS group and the OND group were more reactive than the control for most of the antigens examined. The results provided in Table 8 can be used as a panel test to guide the treatment of patients with active MS. Table 9. Serological summary. EI ≤ 1.0 = "-", negative; EI 1.1 - 2.9 = "+ / -", uncertain; EI 3.0 - 4.9 = "+", low positive; EI ≥ 5.0 = "++", high positive. JPEG0007682540000017.jpg210164 JPEG0007682540000018.jpg109164 ND = not performed Negative concordance = same band in CSF and serum

[0165] Microbial ELISA index compared to albumin index in the evaluation of demyelinating diseases. Figure 6 shows the EI compared to the albumin index. These data suggest the possibility of intrathecal synthesis of antibodies against some MS microorganisms or parts of MS microorganism families in some of the subjects. For example, the data generated in this table provide information on which subjects have unexpectedly high antibody levels against a given antigen and explain the leakage of antibodies from serum across the blood-brain barrier. Such tests can be used to guide the treatment of patients with active MS.

[0166] More specifically, FIG. 6 shows the cerebrospinal fluid ELISA index (EI, a measure of antibody) for six MS microbial candidate antigens plotted against the albumin index (AI). The box shows the normal albumin index range (0 - 9) indicating that the blood - brain barrier (BBB) is not compromised. The lines shown are linear regression analyses showing the relationship between the EI for each antigen and the absence of BBB compromise (as evaluated by the albumin index). This line provides the "expected EI" for each bacterial antigen examined and allows correction of the leaky BBB often seen in patients with MS and other demyelinating diseases. Values above the regression line, and especially values within the region surrounded by the normal albumin index, indicate the likelihood of intrathecal antibody synthesis (i.e., causal relationship to demyelination).

[0167] The albumin index of the subject can be calculated as follows: Albumin index = CSF albumin (mg / L) / serum albumin (g / L). The AI is an indicator of the integrity of the BBB adjusted for serum albumin concentration. The AI increases with BBB dysfunction, but there are some limitations. For example, LeVine SM. Albumin and multiple sclerosis. BMC Neurol. 2016, Apr. 12;16:47. doi:10.1186 / s12883 - 016 - 0564 - 9. PMID:27067000; PMCID:PMC48287

[0168] The ELISA index can be calculated / determined as follows. Preparation of Bacterial Antigens: Selection Criteria: Prior to antigen collection, serial dilutions were performed to obtain various ODs for ELISA optimization. Bacterial strains were obtained from ATCC as lyophilized, lyophilized cultures. The cultures were rehydrated using the appropriate medium and incubation conditions specified on the product sheet. The organisms were taken out of the lyophilized state and stock cultures were stored at -80°C at a final concentration of 10% glycerol as soon as they entered the exponential growth phase of proliferation. Broth cultures and plate cultures for the purpose of antigen collection were taken during or immediately after the exponential growth phase. Prior to antigen collection, cells from the broth cultures were washed with PBS and serial dilutions were performed from both the plate cultures and broth cultures to obtain an OD of 1.0 corresponding to approximately 10e8 - 10e9 cells. The plates were read at 600 nm, a common measurement criterion used to estimate the concentration of bacterial cells in liquid, using a Gen5 2.08 ELISA plate reader. The cells were sonicated for 10 seconds at position 4.5 with a continuous cycle time, 40% duty cycle, and Branson Sonifier250. The sonicated organisms were then stored at -80°C for future use

[0169] ELISA Optimization: Flat-bottom 96-well ELISA plates were used and coated with 50 μL / well of the collected antigen (sonicated bacteria), covered, and shaken overnight at 4°C. The plates were washed with phosphate-buffered saline with Tween20 (PBST) (washing was performed with 100 μL / well of the buffer), blocked for 1 hour at room temperature (RT), washed again with PBST, then the appropriate primary antibody was applied and shaken at RT for 2 hours. Following incubation with the primary antibody, the plates were washed with PBST, the appropriate secondary antibody was applied and shaken at RT for 1 hour. Following incubation with the secondary antibody, the plates were washed with PBST and then with 100 μL / well of TMB for 5 minutes. The reaction was stopped with 100 μL / well of 2 normal sulfuric acid (2N HCl) and read at 450 nm using a Gen5 2.08 ELISA plate reader.

[0170] A series of steps were required to optimize the ELISA procedure. 1) Various primary antibacterial antibodies were evaluated for the best positive control consisting of concentrations that gave strong and weak (just above background) positive controls. These antibodies consisted of monoclonal anti - peptidoglycan (mAb995), monoclonal anti - LPS, and polyclonal anti - pseudomonas. 2) In addition to the use of antibacterial antibodies, Randox (Randox Laboratories Ltd, UK), a human CSF control with 10 mg / dl IgG as a positive control, and Randox depleted as a negative control were also used. This depletion was achieved with a HiTrap Protein G HP kit (GE), and the depletion was verified with a Human Total IgG Platinum ELISA kit (Affymetrix eBioscience). Two passes were determined to be sufficient to obtain optimal depletion. 3) Subsequently, various blocking agents were evaluated at each antibody concentration to find out which were the most efficient. These included 5×ELISA / ELISPOT diluent (Invitrogen) (PBS supplemented with bovine serum), blocker BLOTTO (ThermoScientific) in Tris - buffered saline (TBS), blocker casein (ThermoScientific), and starting blocker (ThermoScientific). 4) Along with the optimizations described above, the antigen coating concentration was then adjusted using OD values of approximately 0.5, approximately 1.0, and approximately 2.0. In fact, the plates were then accompanied by appropriate secondary antibodies at the specified dilutions: anti - mouse IgG (Vector PI - 2000, 1:3000), anti - rabbit IgG (Vector PI - 1000, 1:3000), and anti - human (Jackson ImmunoResearch, 1:10,000). These optimization steps were performed for each bacterial organism.

[0171] Throughout the optimization process, rabbit anti-Pseudomonas polyclonal antibody (ThermoFisher Scientific, Rockford, IL) was the optimal primary antibacterial antibody for the organisms examined, with both strong and weak positive concentrations varying. While antibodies specific to the organisms examined could be used, the polyclonal antibody used was sufficient to recognize features common to bacteria and was much more cost-effective. Serial dilutions of the antibody that yielded an OD of approximately 1.0 were used as strong positive controls, and those just above background were used as weak positive controls. Randox, while establishing control of the accuracy of CSF samples, also served as a positive control. Similarly, depleted Randox served as a negative control and was used to calculate the ELISA index (EI).

[0172] ELISA analysis: Performed in duplicate for each CSF and diluted serum sample, and in triplicate for controls. To obtain the EI, the mean OD of each experimental and control sample was obtained and divided by depleted Randox, otherwise known as the inventors' "cut-off calibrator". This was repeated with the serum of each patient to demonstrate reproducibility and to evaluate intrathecal synthesis of IgG. This was achieved by diluting the serum to the same concentration as the CSF and using the ratio of the OD of the diluted serum to the OD of the CSF. All OD values greater than 1 suggest intrathecal synthesis (Halperin et al. 1989).

[0173] Using a method that includes comparing EI to the albumin index, intrathecal antibody synthesis in patients with demyelination (e.g., antibodies against one or more microorganisms disclosed herein, e.g., those listed in Table 8) can be shown and / or determined. Treatment can be prescribed using this method. For example, Subject 3 shows very high EI values against several different MS candidate antigens (e.g., Atopobium, Akkermansia, and Lactobacillus). However, the albumin index of Subject 3 is also very high (about 30), indicating a leaky BBB. Furthermore, the antibody level of Subject 3 against Akkermansia is above the expected level (linear regression line). This data indicates that Subject 3 is producing more antibodies against Akkermansia than can be explained by leakage from the serum alone. Using the disclosed method, intrathecal production of antibodies (i.e., antibodies produced in the brain and spinal cord) that can be used to prove the causal relationship of demyelination and then to prescribe treatment can be evaluated.

[0174] Other methods for determining intrathecal production of antibodies are known. For example, the most direct method is to measure the total IgG concentration in both serum (usually about 1000 mg / dl) and CSF (usually <5), appropriately dilute the serum, and measure the EI of both CSF and serum where the total IgG concentration is the same. This method is called the "direct method" and the ratio of the EI of CSF to the EI of serum is 1.0 or less. A ratio >1.0 indicates intrathecal antibody production.

[0175] A direct comparison of CSF and serum was performed on a portion of demyelinated subjects against five bacterial antigens (Akkermansia, Lactobacillus, Pseudomonas, Atopobium, and Bacteroides). This analysis showed a CSF / serum EI ratio >1.0 for MS Subjects 82 and 83 against a portion of the antigens. The fact that the other EI ratios were <1.0 indicates that there are more antibodies in the serum than in the CSF.

[0176] However, the method of the EI to albumin index of microorganisms identified several other subjects in which the antibody levels in the CSF were higher than expected. These include Subject 10 (Atopobium, Akkermansia, Pseudomonas), Subject 72 (Akkermansia, Odoribacter), Subject 13 (Pseudomonas), and Subject 3 (Akkermansia). This method can be used to identify subjects that might otherwise be missed, using known methods to determine that the BBB is not compromised.

Claims

Claim 1 A method for ex vivo diagnosis of a demyelinating disease in a subject, the method comprising: a) incubating a sample from the subject with one or more capture agents, wherein the one or more capture agents are bound to one or more microbial specific antibodies, and the microbial specific antibodies are specific for one or more microorganisms selected from the group consisting of Akkermansia, Atopobium, Bacteroides, and Lactobacillus; b) detecting the presence of the one or more microbial specific antibodies in the sample; wherein the method indicates that the subject has a demyelinating disease when the one or more microbial specific antibodies are present in the sample. Claim 2 A method for ex vivo detection of a demyelinating disease in a subject, the method comprising: a) contacting a sample from the subject suspected of containing microbial specific antibodies with the one or more capture agents under conditions such that each of the one or more capture agents is specific for one or more microbial specific antibodies, and the one or more microbial specific antibodies are specific for one or more microorganisms selected from the group consisting of Akkermansia, Atopobium, Bacteroides, and Lactobacillus, thereby enabling the one or more capture agents to bind to the one or more microbial specific antibodies to produce a mixed biological sample; b) incubating the mixed biological sample under conditions such that the one or more capture agents specifically bind to at least one of the microbial specific antibodies to form a detectable complex; c) detecting the presence of the detectable complex, thereby detecting the presence of one or more microorganisms selected from the group consisting of Akkermansia, Atopobium, Bacteroides, and Lactobacillus; wherein the presence of one or more microorganisms selected from the group consisting of Akkermansia, Atopobium, Bacteroides, and Lactobacillus in the mixed biological sample indicates that the subject has a demyelinating disease. Claim 3 wherein the subject is suspected of having a demyelinating disease or is at risk of having a demyelinating disease, the method according to claim 1 or 2, wherein the demyelinating disease may be multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, or a syndrome consisting of a first episode. Claim 4 Furthermore, it includes amplifying one or more nucleic acid sequences in the sample, and the one or more nucleic acid sequences correspond to one or more of microorganisms selected from the genus Akamanthia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus. The method according to any one of claims 1 to 3, wherein the one or more nucleic acid sequences are amplified using a primer pair that specifically amplifies the one or more nucleic acid sequences.

5. A composition comprising a drug for use in diagnosing and treating a demyelinating disease in a subject, wherein the use of the drug is a) incubating a sample derived from a subject suspected of containing a microorganism-specific antibody with one or more capture agents, wherein the one or more capture agents bind to one or more of the microorganism-specific antibodies, and the microorganism-specific antibodies are specific to one or more microorganisms selected from the genus Akamanthia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus; b) detecting the presence of the one or more microorganism-specific antibodies in the sample; and c) diagnosing the subject as having a demyelinating disease when the one or more microorganism-specific antibodies are present in the sample; including wherein the drug is an antibacterial agent in which the detected one or more microorganism-specific antibodies are specific to bacteria selected from the genus Akamanthia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus, is the composition.

6. A composition comprising a drug for use in detecting and treating a demyelinating disease in a subject, wherein the use of the drug is a) contacting a sample derived from a subject suspected of containing a microorganism-specific antibody with the one or more capture agents under conditions such that each of the one or more capture agents is specific to one or more microorganism-specific antibodies, and the one or more microorganism-specific antibodies are specific to one or more microorganisms selected from the genus Akamanthia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus, so as to enable the one or more capture agents to bind to one or more of the microorganism-specific antibodies to generate a mixed biological sample; b) incubating the mixed biological sample under conditions such that the one or more capture agents specifically bind to at least one of the microorganism-specific antibodies to form a detectable complex; and c) detecting the presence of the detectable complex, which detects a demyelinating disease in a subject due to the presence of one or more microorganisms selected from the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus in the mixed biological sample, thereby detecting the presence of one or more microorganisms selected from the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus; comprising; wherein the agent is an antibacterial agent specific for bacteria selected from the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus, wherein the detected one or more microorganisms are such that the composition. **Claim 7** The composition according to claim 5 or 6, wherein the use further comprises repeating steps a) and b) with various antibodies specific for the one or more microorganisms in the sample. **Claim 8** The composition according to claim 6, wherein the use further comprises determining the level of the antibody in the sample for each of the microorganisms tested. **Claim 9** The method according to any one of claims 1 to 4, further comprising amplifying one or more nucleic acid sequences in the sample, wherein the one or more nucleic acid sequences correspond to one or more of the microorganisms selected from the list consisting of the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus. **Claim 10** The method according to claim 9, wherein the one or more nucleic acid sequences are amplified using a primer pair that specifically amplifies the one or more nucleic acid sequences. **Claim 11** The method according to claim 10, further comprising determining whether the expression of the one or more nucleic acid sequences corresponding to one or more of the microorganisms in the list consisting of the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus is overexpressed compared to the expression level of the same one or more nucleic acid sequences of the microorganisms in the list consisting of the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus in a control. **Claim 12** The method according to any one of claims 1 to 4, further comprising determining whether the expression of the one or more nucleic acid sequences corresponding to one or more of the microorganisms in the list consisting of the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus is overexpressed compared to the expression level of the same one or more nucleic acid sequences of the microorganisms in the list consisting of the genus Akkermansia, the genus Atopobium, the genus Bacteroides, and the genus Lactobacillus in a control.

13. A composition for use in a method of treating a patient with multiple sclerosis (MS) or an MS-related disease, according to any one of claims 5 to 8, wherein the composition comprises a) an antibacterial agent in which one or more detected microorganism-specific antibodies are specific for bacteria selected from the genera Akkermansia, Atopobium, Bacteroides, Lactobacillus; and i. incubating a sample from a subject suspected of containing a microorganism-specific antibody, together with one or more capture agents, wherein one or more capture agents bind to one or more of the microorganism-specific antibodies and the microorganism-specific antibodies are specific for one or more microorganisms selected from the list consisting of the genera Akkermansia, Atopobium, Bacteroides, Lactobacillus; ii. detecting the presence of the one or more microorganism-specific antibodies in the sample, whereby the patient is identified as in need of an antibacterial agent, the composition.

14. The composition according to claim 13, wherein the antibacterial agent is meropenem or ceftriaxone.

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