Bacterial proteins that confer risk of rheumatoid arthritis and related materials and methods

Abstract

Methods for the detection of subjects at risk of developing rheumatoid arthritis, and subjects having early rheumatoid arthritis thus allowing for early intervention. Compositions for inducing immune tolerance in a subject thus treating or slowing progression of RA. Kits useful in the diagnosis of RA.

Description

2848-350-BACTERIAL PROTEINS THAT CONFER RISK OF RHEUMATOID ARTHRITIS AND RELATED MATERIALS AND METHODSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 704,238, filed October 7, 2024, which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Nos. U01 AI101981 and R01 AR075033 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. This XML file, created 06 October 2025, is named 2848-350-PCT.xml and is 2,476,000 bytes in size.BACKGROUND

[0004] Rheumatoid arthritis (RA) is a progressive, systemic autoimmune disease characterized by chronic inflammation of multiple joints with associated symptoms such as fatigue. This inflammation causes joint pain, stiffness, and swelling resulting in loss of joint function due to destruction of bone and cartilage. RA often leads to progressive disability. Patients with RA also have an increased likelihood of developing other systemic complications such as osteoporosis, anemia, cardiovascular disease, and disorders affecting the lungs and skin. The disease shortens the average life expectancy of affected patients by three to seven years. About 0.5-1% of the US adult population is affected by RA and, given the average age of onset is approximately 50, the number of affected individuals is expected to rise. Despite declining rates of disability attributed to RA, about 30% of patients do not achieve a minimally acceptable symptom state.

[0005] Early treatment of RA is associated with decreased joint damage and disability. In addition, studies performed in individuals considered to have pre-RA, defined as clinical risk for RA but not yet exhibiting classifiable disease, have demonstrated only marginally effective prevention of clinical RA onset. Further, identification of individuals at risk for developing RA is a challenging and inexact process. Currently, prediction tools for the development of RA are based on circulating antibodies and genetic factors. These toolscan classify only 30-40% of individuals with RA as high risk of developing RA in the next 2-3 years.

[0006] Proteomic technologies have enabled the high-throughput, multiplex measurement of large panels of antibodies in biological fluids of patients with immune-driven diseases to delineate the natural history of autoantibody formation and epitope spread and gain insight into the pathogenesis of autoimmune diseases such as RA, as well as into host immunity and its shortcomings. Characterization of B- and T-cell reactivity that precedes the onset of clinically apparent disease has the potential to guide disease prevention using either conventional immunosuppression or novel, antigen-specific tolerizing therapies. Additionally, autoantibody profiling in RA has the potential to identify molecular subtypes of a disease, which could allow for prediction of disease outcomes such as severity, tissue damage, and response to therapy. New methods of applying these techniques are needed for identifying individuals at risk for developing RA or in an early stage of RA, and new therapeutic strategies are needed in the pre-RA period to prevent the onset of clinical disease.SUMMARY

[0007] The mucosal origins hypothesis of rheumatoid arthritis (RA) development proposes a central role for mucosal immune responses in which microbes and associated immune dysregulation at mucosal surfaces can drive initially asymptomatic local inflammatory responses that evolve into the systemic pathogenic autoimmunity and clinical findings characteristic of RA (Holers, V.M., et al. 2018 Nat Rev Rheumatol 14; 542-57). As described in PCT / US2021 / 072293 and PCT / US2023 / 069489 (Published as WO 2022 / 099317, WO 2024 / 006983, and US20240011986 , which are incorporated herein, in their entirety, by this reference) using dual-family IgA-IgG isotype clonal plasmablast- derived monoclonal antibodies (PB-mAbs) from individuals in the preclinical phase of RA, the inventors identified a novel species and strain of Subdoligranulum (Subdoligranulum didolesgii strain D8; also referred to as “isolate 7” in this disclosure) demonstrated to be causally involved in the development of human RA in at least a subset of patients and to worsen its disease course and effects on involved tissues.

[0008] As shown herein, the inventors have identified antigens in Subdoligranulum didolesgii strain D8 that activate antigen presenting cells to enhance T cell co-stimulation and are causal of adaptive immunity and development of RA in humans and progression of the RA disease course. Accordingly, the presence and / or abundance of one or more of these causalSubdoligranulum didolesgii strain D8 proteins, in a human subject, such as in a fecal sample or other biological sample disclosed herein, can be used as a diagnostic indicator for RA, and as a predictive indicator for risk of RA onset. Additionally, the detection of Eland T-cell reactivity or co-stimulation, and / or expansion in a biological sample from a subject in contact with one or more of these causal Subdoligranulum didolesgii strain D8 proteins, can be used as a diagnostic indicator for RA, and as a predictive indicator for risk of RA onset.

[0009] The present disclosure is directed to methods for diagnosing a subject with RA and for identifying a subject at risk of developing RA. The methods comprise detecting the presence or absence of one or more of these causal Subdoligranulum didolesgii strain D8 proteins, in a biological sample from a subject. The methods further comprise detecting the presence or absence of B-cell and / or T-cell reactivity to one or more of these causal Subdoligranulum didolesgii strain D8 proteins, in a biological sample from a subject. The methods advantageously permit early therapeutic action that may contribute to the slowing or prevention of tissue damage caused by RA.

[0010] The methods further comprise antigen-specific tolerizing therapies based on the use of one or more of these causal Subdoligranulum didolesgii strain D8 proteins.

[0011] Thus, in one aspect, this disclosure provides ex vivo methods for diagnosing rheumatoid arthritis (RA) in a subject, by obtaining a biological sample from the subject, and detecting the presence or the absence of an antibody that specifically binds a peptide from the Subdoligranulum didolesgii strain D8 in the biological sample. The subject may then be diagnosed with RA, or at risk of developing RA, when an antibody to the peptide from the Subdoligranulum didolesgii strain D8 is detected in the biological sample. In these methods, the peptide may be a cell wall protein from Subdoligranulum didolesgii strain D8 selected from endoglucanase, cellulosome-anchoring protein, and cell surface protein, or a homolog thereof. The peptide may be a peptide fragment comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of SEQ ID NOs: 2-19. In these methods, the antibody may be an IgA and / or IgG class antibody, and the biological sample may be a blood sample, such as whole blood, serum, and / or plasma. In these methods, the antibody may be detected using a labeled secondary antibody, and the antibody may be detected using colorimetry, immunofluorescence, detection of enzymatic activity, chemiluminescence, and / or radioactivity. In these methods, the peptide may belinked to a carrier such as a bead, a test strip, a microtiter plate, a membrane, a lateral flow device, a glass surface, a slide, a microarray, and / or a biochip.

[0012] In another aspect, this disclosure provides ex vivo methods for diagnosing rheumatoid arthritis (RA) in a subject, by contacting a biological sample comprising T cells from the subject with a composition containing a peptide from the Subdoligranulum didolesgii strain D8, and detecting the level of at least one co-stimulatory molecule, cytokine, or chemokine produced by the T cells in the sample. The subject is then diagnosed with RA, or at risk of developing RA, when elevated levels of the co-stimulatory molecule, cytokine, and / or chemokine produced by the T cells in the sample is detected in the biological sample. In these methods, the biological sample may be a peripheral blood sample. In these methods, the peptide may be a cell wall protein selected from endoglucanase, cellulosome-anchoring protein, and cell surface protein, or a homolog thereof, and / or a peptide fragment comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of any one of SEQ ID NOs: 2-19. In these methods, the co-stimulatory molecule, cytokine, and / or chemokine may be related to CD4+ Thl7 T cell activation polarization profile and may include a molecule such as IL-ip, IL-6, TNF, TLR2, CD86, CD40, IL-23, and / or BAFF. In these methods, the cytokine and / or chemokine may be detected by ELISPOT and / or cytometry. In these methods, the co- stimulatory molecule, cytokine, and / or chemokine produced by the T cells in the sample may be assessed relative to a control sample from a subject not diagnosed with RA or not at risk of developing RA. Alternatively or additionally, the level of the co-stimulatory molecule, cytokine, and / or chemokine produced by the T cells in the sample may be assessed relative to a threshold value that is at least two standard deviations above the mean value for controls.[0013| In any of these methods provided in this disclosure, the rheumatoid arthritis diagnosed may be early rheumatoid arthritis.

[0014] Any of these methods provided in this disclosure may further include administering an RA treatment to the subject diagnosed with RA or at risk of developing RA. Such treatments may include administering to the subject any one or more of a non-steroidal inflammatory drug; a corticosteroid; a disease-modifying antirheumatic drug; and / or a biologic response modifier. An exemplary non-steroidal inflammatory drug may include one or more of ibuprofen; naproxen sodium; celecoxib; diclofenac; fenoprofen; flurbiprofen;indomethacin; ketorolac; mefenamic acid; meloxicam; oxaprozin; piroxicam; and sulindac. An exemplary corticosteroid may include one or more of prednisone; bethamethasone; and prednisolone; triamcinolone; methylprednisolone; and / or dexamethasone. An exemplary disease-modifying antirheumatic drug may include one or more of methotrexate; leflunomide; hydroxychloroquine; and / or sulfasalazine. An exemplary biologic response modifier may include abatacept; adalimumab; anakinra; baricitinib; certolizumab; etanercept; golimumab; infliximab; rituximab; sarilumab; tocilizumab; and / or tofacitinib.

[0015] Another aspect of this disclosure provides compositions for inducing immune tolerance in a subject with RA, or at risk of developing RA. These compositions may include a peptide such as endoglucanase, cellulosome-anchoring protein, and / or cell surface protein, or a homolog thereof, and / or a peptide having an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of SEQ ID NOs: 2-19. In these compositions, a carrier may be linked to the peptide. Exemplary carriers may include poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and / or poly(lactic co-glycolic acid) (PLGA).[0016| Another aspect of this disclosure provides methods of treating RA by administering to a subject in need of treatment an effective amount of one of these compositions for inducing immune tolerance in a subject with RA, or at risk of developing RA. In these methods, the composition may be administered intravenously, intramuscularly, subcutaneously, or orally, and the administration may reduce or eliminate the generation of anti-citrullinated protein antibodies, or CD4+ T cell proliferation and / or activity, or B cell proliferation and / or activity, or combinations of these effects in the subject.|0017] Another aspect of this disclosure provides kits for the diagnosis of RA that include a peptide such as endoglucanase, cellulosome-anchoring protein, and cell surface protein, or a homolog thereof; and / or a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of SEQ ID NOs: 2-19. These kits further include a reagent useful for one or both of analysis of antibodies present in a biological sample obtained from a subject; and / or analysis of the activation of T cells in a biological sample obtained from a subject.[0018| The skilled artisan will recognize other benefits of the disclosed methods and materials.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1 A and IB depict amounts of bacterial isolates present in stool samples collected from human subjects. FIG. 1 A is a graph depicting the concentration of S. dido strain D8 (in ng / pl) as assayed by qPCR of stool DNA from a subset of participants detailed in Example 1, Table 1, infra. (P values determined by Kruskall-Wallis test. *P<0.05 across all 3 groups; **P<0.01. NB: The RA group was not powered for statistical significance.) FIG. IB shows the relative abundance of the control reference strain AT. pectinilyticus from stool metagenomics of the same stool samples analyzed in FIG. 1 A (symbols are individual samples with bars as the mean ± SEM. No significant differences as assessed by Kruskall-Wallis).

[0020] FIGS. 2A and 2B show bone marrow derived dendritic cells (BMDCs) from DBA / 1, C56B1 / 6, or TLR2 KO mice cultured with Flt3-L and GM-CSF for 9 days. Gut-BMDCs were plated and stimulated with isolate 1 or strain D8 (MOI 40). LPS (lug / mL) was used as positive control. FIG. 2 A shows relative expression of mRNA harvested at 6 hours calculated by RT-qPCR (2AACT, normalized to media). FIG. 2B shows RNA harvested from the B6 gut-BMDCs at the times indicated on the x-axis. (Data are histograms based on live CD1 lb+ CD1 lc+ monocytes. Data are representative of two independent experiments with N=4 each. Bars represent mean + / - SEM. *,P<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by ANOVA.)

[0021] FIGS. 3 A and 3B depict data characterizing a cell wall protein antigen on S. dido strain D8 targeted by PB-mAbs. FIG. 3 A shows binding capacity of PB-mAb MH91 for cell-wall associated proteins on the surface of strain D8, untreated and digested with proteinase K (black bars are percentage of PB-mAb binding to undigested bacteria and grey bars are binding to the proteinase K digested bacteria). FIG. 3B shows a putative protein antigen was immunoprecipitated from S. dido strain D8 cell wall associated proteins using PB- mAb MH28. The precipitated protein was run on a 10% Native-PAGE gel and silver stained.[00221 FIG. 4 shows assembled genome maps of S. dido strain D8 (inside black circle with kilobase pair positions marked) compared to isolate 1 (light, middle circle), the reference strain M. pectinilyticus (outer darker circle), and mapped transposons (outer broken circle portions). Arrows indicate locations of antigens.DETAILED DESCRIPTION

[0023] In the following sections, various compositions and methods are described to detail various embodiments. Practicing the various embodiments does not require the employment of all the specific details outlined herein, but rather concentrations, times, and other specific details may be modified. In some cases, well known methods or components have not been included in the description.(0024] The inventors have surprisingly found that specific cell wall proteins of the Subdoligranulum didolesgii strain D8 bacterium drive immune responses leading to RA.

[0025] With the present invention, the presence or absence of these bacterial proteins, and a subject’s immune reactions to them can be used to diagnose and predict onset of RA in a subject, including subjects with no clinical RA symptom ology, well in advance of progression to and onset of RA. The ability of the methods of this disclosure to provide early prediction of progression and onset of RA disease is an important added value compared with the standard diagnostic methods currently used in clinical settings, and is very relevant for the planning and treatment of RA that has become a worldwide challenge.Definitions of general terms and expressions

[0026] The term “antigen,” as used herein, refers to a molecule recognized by the products of the adaptative immune response (specifically, recognized by specific T cell receptors (TCR) and B cell receptors (BCR, or antibodies). Antigens include but are not limited to cells, cell extracts, proteins, polypeptides, peptides, lipoproteins, peptidoglycans, polysaccharides, polysaccharide conjugates, peptide and nonpeptide mimics of polysaccharides and other molecules, small molecules, lipids, glyco lipids, carbohydrates, viruses and viral extracts and muticellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule which is recognized by a host's immune system as such including, but not limited to, pathogen-derived antigens, selfantigens, allergens, and toxins.

[0027] The term “biological sample comprising T cells,” as used herein, refers to tissues or bodily fluids removed from a mammalian subject, which contain T cells. In some embodiments, the T cells are isolated from the sample prior to their exposure to the antigen(s) of interest. The term “isolated” with respect to T cells refers to cell population preparation in a form that results in a cell population that has at least 70%, 80%, 90%, 95%, 99%, or 100% T cells. In some embodiments, a desired cell population is isolated from other cellularcomponents, in some instances to specifically exclude other cell types that may interfere with the study of the cells in isolation. However, the “isolated” cell population may incorporate additional cell types that are necessary for cell survival or to achieve the desired results provided by the invention. For example, antigen presenting cells, such as monocytes (macrophages) or dendritic cells, may be present in an “isolated” cell population of T cells or added to a population of isolated T cells for generation of regulatory T cells. In some embodiments, these antigen presenting cells may be activated monocytes or dendritic cells. Cell populations comprising T cells for use in the methods of the invention may be isolated from a biological sample taken from a mammalian subject. The sample may originate from a number of sources, including, but not limited to, peripheral blood, leukapheresis blood product, apheresis blood product, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoid tissue, mucosa associated lymphoid tissue, liver, sites of immunologic lesions (e.g., synovial fluid), and pancreas.[00281 The term “ELISPOT” or “enzyme-linked immunospot” refers to a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. In this assay, cells are cultured on a surface coated with a cytokine-specific capture antibody in the presence or absence of antigenic stimuli. Either a monoclonal or polyclonal antibody specific for the chosen analyte (e.g., cytokine) is usually pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37°C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody, in the immediate vicinity of cells secreting the analyte of interest binds to the secreted analyte. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for the chosen analyte is added to the wells. Following a wash to remove any unbound biotinylated antibody, streptavidin conjugated with an enzyme (e.g., alkaline phosphatase) is added. Unbound enzyme is subsequently removed by washing and an enzyme specific substrate solution (e.g., BCIP / NBT) is added. A colored precipitate (e.g., blue-black in the case of BCIP / NBT) forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual analytesecreting cell. Biotin-streptavidin enzymatic detection can be replaced for fluorescent labelling allowing for direct detection of the secreted analyte by a fluorescence detecting apparatus. The spots can be counted with an automated ELISPOT reader system ormanually, using a stereomicroscope. The ELISPOT assay may be modified to detect cells producing several cytokines by using several cytokine-specific antibodies which are labelled (for instance enzymatic labels like AP or fluorescent labels) for detection and quantification of cells producing one or several of these cytokines. This “boosted ELISPOT”, thus allows detection of a wider set of effector functions than standard, generally monofunctional, ELISPOT assays and provides a higher sensitivity than standard assays because the signals stemming from the individual cytokines produced in a single cell all add up to form a spot.

[0029] The term “flow cytometry” refers to an assay based on the detection of cells secreting one, or simultaneously several, cytokines and detecting the cytokine(s) in the channel of a flow cytometer. The detected signals may be additive across detected cytokines. Simultaneous detection of multiple cytokines thus leads to an increased sensitivity and makes it possible to cover a vastly larger set of effector functions than standard flow cytometry assays.

[0030] The term “CD4+ T cells,” as used herein, refers to T cells presenting the CD4 molecule (Cluster of Differentiation 4 molecule) on their surface. The term refers to T helper cells, which either orchestrate the activation of macrophages and CD8+ T cells (Th-1 cells), the production of antibodies by B cells (Th-2 cells), or which have been shown to play an essential role in autoimmune diseases (examples are Th- 17 cells, Tph cells, Tfh cells). In addition, the term “CD4+ T cells” also refers to other CD4 expressing T cell populations, including but not limited to regulatory CD4+ T cells (Treg), which represent approximately 10% of the total population of CD4+ T cells. Regulatory T cells play an essential role in the dampening of immune responses, in the prevention of autoimmune diseases and in oral tolerance.[0031 [ The term “CD8+ T cells,” as used herein, refers to T cells expressing the CD8 glycoprotein on their surface, wherein the CD8 (Cluster of Differentiation 8 molecule) glycoprotein is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). CD8 binds to a major histocompatibility complex (MHC) molecule but is specific for the class I MHC protein. Exemplary CD8 T cells comprise cytotoxic memory CD8 T cells, regulatory CD8 T cells, cytotoxic effector CD8 T-cells and additional cells identifiable by a skilled person.10032] The term “T cell response”, as used herein, refers to an immune response in which T cells directly or indirectly mediate or otherwise contribute to an immune response in a mammal. The T cell mediated immune response may be associated with cell mediated effects (e.g.,lymphokine mediated effects), and even effects associated with B cells if the B cells are stimulated, for example, by the lymphokines secreted by T cells. For MHC class I restricted CTLs, effector functions may be lysis of peptide-pulsed, peptide-precursor pulsed or naturally peptide-presenting target cells, secretion of cytokines (e.g., IFN-y, TNF-a, IL-2), secretion of effector molecules (e.g., granzymes, perforins), or degranulation. For MHC class Il-restricted T helper cells, effector functions may be peptide induced secretion of cytokines (e.g., IFN-y, TNF-a, IL-4, IL5, IL-10, IL-13, IL-2), or degranulation but in some cases can also include other effector functions, such as cytotoxic activity. T cell responses can also be measured as proliferation of T cells and can be observed in vivo as well as in vitro.

[0033] The abbreviation “PBMC” refers to peripheral blood mononuclear cells, including lymphocytes, monocytes, and macrophages. PBMCs may be isolated using methods known in the art, such as density gradient centrifugation.

[0034] A “subject” means any individual having rheumatoid arthritis (RA), having symptoms of RA, or at risk for developing RA. A subject may be human or non-human, and may include, for example, animals or species used as “model systems” for research purposes, such as a mouse model. The subject may be a human patient having RA or at risk of developing RA.

[0035] As used herein, a subject that is “at-risk of developing RA” is defined as a subject that is a first-degree relative (FDR) of a patient with RA or a subject with preclinical RA. “Preclinical RA” refers to the presence of positive levels of circulating RA-specific autoantibodies, i.e., positive for serum antibodies to citrullinated protein antigens (ACPA+) and / or RF positive (RF+) and / or other antibodies to modified protein antigens (AMP A) and / or biomarkers that define the preclinical state of seronegative RA. Circulating RA-specific autoantibodies refers to serum anti-CCP3 / anti-CCP3.1 positive and / or >2 RF isotypes.

[0036] As used herein, “having RA” is defined as an individual diagnosed using American College of Rheumatology (ACR) 1987 and / or ACR / European League Against Rheumatism (EULAR) 2010 classification criteria; such criteria include exhibiting clinical symptoms and clinical signs and biomarkers of rheumatoid arthritis. As used herein, “early RA” refers to an individual within one year of RA diagnosis.

[0037] The present disclosure provides methods for identifying subjects at risk of developing RA, having pre-RA, having preclinical RA, having clinically suspect arthralgia (CSA), havingearly RA, or having RA. Such methods generally include detecting the presence or absence of the Subdoligranulum proteins of this disclosure in the subject, with the presence of the Subdoligranulum didolesgii strain D8 proteins, and B- and T-cell responsiveness to those proteins, being linked to a risk of developing RA or indicative of pre-RA, early RA, RA, or progression of RA in the subject. Also provided are methods for preventing development and / or progression of RA in a subject, or for treating RA in a subject. Such methods generally involve administering to a subject in need of treatment one or more therapeutic agents.

[0038] As used herein, “treat” in reference to a condition means: (1) to ameliorate or prevent the condition or one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition, (3) to alleviate one or more of the symptoms or effects associated with the condition, and / or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition. The terms “prevent,” “preventing” and the like are to be understood to refer to a method of blocking the onset of disease and / or its attendant symptoms. “Prevent” also encompasses delaying or otherwise impeding the onset of RA disease and / or its attendant symptoms.[00391 As used herein, a “therapeutically effective amount” in reference to an agent means an amount of the agent sufficient to treat the subject’s condition but low enough to avoid serious side effects at a reasonable benefit / risk ratio within the scope of sound medical judgment. The safe and effective amount of an agent will vary with the particular agent chosen (e.g. consider the potency, efficacy, and half-life of the compound); the route of administration chosen; the condition being treated; the severity of the condition being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be determined by the skilled artisan.

[0040] The term “kit”, as used herein, refers to a product containing different reagents useful or necessary for carrying out the method of the invention which is packaged so as to allow their transport and storage. Materials suitable for packaging the components of the kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper and / or envelopes.Methods of diagnosing RA or propensity to RA

[0041] The inventors previously identified a specific bacterial strain in the family Ruminococcus genus Subdoligranulum that is targeted by autoantibodies of individuals at risk for RA and those with classified RA (see, PCT / US2023 / 069489 (WIPO publication No. W02024006983A); and Chriswell, M.E., et al. (2022) Sci Transl Med 14, eabn5166). Cell wall proteins of the Subdoligranulum sp. nov. and, in particular, Subdoligranulum didolesgii strain D8, and immune reactivity to these proteins, are demonstrated herein to be biomarkers of risk of RA or early RA. The strain, designated Subdoligranulum didolesgii strain D8, is maintained in the lab of inventor Kuhn and was deposited with the ATCC Patent Depository under the Budapest Treaty (10801 University Blvd, Manassas, VA 20110), on 9 November 2021, with the ATCC Patent Deposit Designation No. PTA- 126891. A genome contig of S. didolesgii strain D8 is provided in SEQ ID NO: 1.

[0042] With regard to S. didolesgii strain D8, the term “peptide” and / or “protein” as used in accordance with the present disclosure relates to a protein selected from endoglucanase, cellulosome-anchoring protein, and cell surface protein, or a homolog thereof having an amino acid sequence with at least 80% similarity to one of these proteins after optimal alignment, a peptide fragment of these proteins having a T-cell epitope or a chemical analog thereof, including a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or antigenic variants of SEQ ID NOs: 2-19. The peptide variants preferably comprise an amino acid sequence characterized in that the sequence can be derived from any one of SEQ ID NOs: 2-19 by at least one amino acid substitution, addition, and / or deletion. Thus, as used herein, “a peptide of this disclosure” includes one or more of the antigenic cell wall proteins of Subdoligranulum didolesgii strain D8 which the inventors have demonstrated to stimulate human T cells from individuals with RA and to bind ACPA mAbs (see Example 3, infra), as well as bacterial and human homologs, as set forth in Table 1 using the single letter amino acid abbreviations.|0043] Table 1.

[0044] With regard to S. didolesgii strain D8, the term “polynucleotide” as used in accordance with the present disclosure relates to a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1, which encodes a peptide of this disclosure, or a variant thereof. The polynucleotide variants preferably comprise a nucleic acid sequence characterized in that the sequence can be derived from SEQ ID NO: 1 by at least one nucleotide substitution, addition, and / or deletion. Variants also encompass polynucleotides comprising a nucleic acid sequence that is capable of hybridizing to the aforementioned nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6 x sodium chloride / sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 x SSC, 0.1% SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, the temperature and concentration of the buffer.

[0045] Percent identity values are generally calculated over the entire nucleic acid or amino acid sequence region(s). A series of computer programs based on a variety of algorithms are known and available to the skilled artisan for comparing different nucleic acid or amino acid sequences.[00461 The percentage identity between two sequences can be determined using techniques well known to those of skill in the art, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

[0047] In methods of this disclosure, the presence of one or more antibodies that specifically bind to S. didolesgii strain D8 peptides, and / or homolog(s) thereof, and / or fragment(s) thereof in a subject, identifies the subject as being at risk of developing rheumatoid arthritis, such as having preclinical RA, or as having RA, such as early RA.

[0048] In additional methods of this disclosure, the presence of one or more B cells that specifically bind to S. didolesgii strain D8 peptides, and / or homolog(s) thereof, and / or fragment(s) thereof, in a subject, identifies the subject as being at risk of developing rheumatoid arthritis, such as having preclinical RA, or as having RA, such as early RA.

[0049] In additional methods of this disclosure, the presence of one or more T cells that specifically bind to S. didolesgii strain D8 peptides, and / or homolog(s) thereof, and / or fragment(s) thereof, in a subject, identifies the subject as being at risk of developing rheumatoid arthritis, such as having preclinical RA, or as having RA, such as early RA.

[0050] In additional methods of this disclosure, the presence of one or more S. didolesgii strain D8 peptides, and / or homolog(s) thereof, and / or fragment(s) thereof, in a subject, identifies the subject as being at risk of developing rheumatoid arthritis, such as having preclinical RA, or as having RA, such as early RA.[0051 [ In additional methods of this disclosure, the presence of one or more polynucleotides encoding S. didolesgii strain D8 peptides, and / or homolog(s) thereof, and / or fragment(s) thereof, in a subject, identifies the subject as being at risk of developing rheumatoid arthritis, such as having preclinical RA, or as having RA, such as early RA.[0052 [ In a first aspect, this disclosure provides a method for diagnosing RA in a subject by detecting the presence or absence of an antibody to a peptide of this disclosure. The antibody may be an IgG and / or IgA class antibody, in a biological sample from the subject, the presence of the antibody being diagnostic of RA or at risk of developing RA in the subject.

[0053] In these methods, the sample may be a sample of any bodily fluid or tissue from the subject that may comprise an antibody, including, for example, blood, saliva, nasal mucus, or lymph fluid. Preferably, the sample is a blood sample, selected from whole blood, serum, plasma, capillary blood, arterial blood, venous blood, or a mixture thereof. Capillary blood may be in the form of a dried blot spot, which may be collected by ahealth care practitioner or by the subject him- or herself, sent to the lab, followed by extraction of the blood.10054] In these methods, the antibody that binds to a peptide of this disclosure may be detected using a labeled secondary antibody. The term “secondary antibody” in its broadest sense is to be understood to refer to any kind of “binding moiety,” capable of specific binding to an IgA, IgG class antibody or a fragment thereof (such as a constant domain of a particular Ig class). Non-limiting examples of binding moi eties include antibodies derived from any species, for example human, chicken, camel, llama, lamprey, shark, goat, rodent, cow, dog, rabbit, etc., antibody fragments, domains or parts thereof, for example Fab, Fab', F(ab')2, scFab, Fv, scFv, VH, VHH, VL, VLRs, and the like, diabodies, monoclonal antibodies (mAbs), polyclonal antibodies (pAbs), mAbdAbs, phage display-derived binders, affibodies, heteroconjugate antibodies, bispecific antibodies, evibodies, lipocalins, anticalins, affibodies, avimers, maxibodies, heat shock proteins such as GroEL and GroES, trans-bodies, DARPins, aptamers, C-type lectin domains such as tetranectins; human y-crystallin and human ubiquitin-derived binders such as affilins, PDZ domain- derived binders; scorpion toxin and / or Kunitz-type domain binders, fibronectin-derived binders such as adnectins, receptors, ligands, lectins, streptavidin, biotin, including derivatives and / or combinations thereof such as bi- / multi-specific formats formed from two or more of these binding molecules. Various antibody derived and alternative (i.e. non-antibody) binding protein scaffolds including methods of generation thereof are known in the art (e.g. reviewed in Chiu ML et at, Antibodies (Basel), (2019); 8(4): 55; Simeon R. & Chen Z., Protein Cell. (2018);9(1):3- 14; and Chapter 7 - Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007) edited by Stefan Diibel.[00551 Means and methods for developing, screening and identification of suitable binding molecules of various scaffolds, including, without limitation, those described above, toward desired target structures, such as the IgA and / or IgG class antibodies, are well known and routinely employed in the art. Exemplary and routinely-performed methods include, high-throughput (HT) combinatorial library-based display and selection methods, such as phage display, ribosome display, mRNA display, and cell surface display (e.g. yeast display).10056] In a preferred embodiment, the secondary antibody is an immunoglobulin (Ig), preferably IgG raised in a non-human species, wherein the secondary antibody specifically binds immunoglobulins of one or more specific Ig classes or fragments thereof (e.g. a constantdomain of a particular Ig class) of another selected species, preferably human species. An example is a polyclonal antibody raised in goat that specifically recognizes human IgA (z.e., a polyclonal goat anti -human IgA antibody), or human IgA (z.e., a polyclonal goat anti-human IgG antibody). Means and methods for producing (mono- or polyclonal) antibodies capable of specific binding of one or more selected target antigens are well known in the art.

[0057] In these methods, rather than use one type of secondary antibody, a mixture of several different secondary antibodies may be used, wherein the different secondary antibodies either bind to the same or different Ig classes (e.g., a mixture of different antibodies such as polyclonal antibodies binding to IgA, or IgA and IgG, or IgM or wherein the different secondary antibodies bind to different individual target structures such as one secondary antibody specifically binding to IgA, and another specifically binding to IgG).

[0058] In these methods, the antibody binding to a peptide of this disclosure is detected using a labeled secondary antibody, preferably a labeled secondary antibody binding to IgA, or IgG class antibodies. The secondary antibody may be labelled by coupling, preferably physically linking, a detectable substance, such as a radioactive agent or another molecule providing a detectable signal, such as a fluorophore (such as a small organic chemical fluorphore or fluorescent protein), or an enzymatically active label, (such as alkaline phosphatase) whose presence can be assessed and optionally be quantified based on its reactivity with, and / or conversion of, a substrate substance. Various suitable detectable labels are known in the art and some of which are also described herein.

[0059] In these methods, the secondary antibody may be detected using a method selected from colorimetry, immunofluorescence, detection of enzymatic activity, chemiluminescence, and radioactivity.

[0060] In these methods, the subject may be diagnosed with RA or at risk of developing RA when an antibody to a peptide of this disclosure from Subdoligranulum didolesgii strain D8 is detected in the biological sample from the subject. The detection of the presence of the antibody may be assessed relative to a control sample from a subject not diagnosed with RA and / or a control sample from a subject not at risk of RA. Detection may also encompass evaluating a relative abundance of an antibody to a peptide of this disclosure above a threshold. The threshold may be two, or more, standard deviations above the mean value for controls.[00611 These methods of detecting an antibody to a peptide of this disclosure may also be used for monitoring the treatment of a subject for RA and / or monitoring the progression to RA in a subject identified as at risk of developing RA, whereby an increase or decrease in antibodies to a peptide of this disclosure may be indicative of progression of RA or successfully treating RA, respectively, over time.[0O62| In another aspect, this disclosure provides a method for diagnosing RA in a subject by detecting the presence or absence of T cell activation by a peptide of this disclosure. These methods encompass methods of assaying T cell responses specific against a peptide of this disclosure in a subject. These methods include contacting a biological sample comprising T cells from the subject with a composition comprising a peptide of this disclosure, and determining the levels of at least one co-stimulatory molecule, and / or cytokine, and / or chemokine produced by the T cells in the sample. In these methods, the at least one costimulatory molecule, and / or cytokine, and / or chemokine may be detected by ELISPOT and / or flow cytometry. In these methods, a combination of antibodies against a plurality of co-stimulatory molecules, cytokines and / or chemokines may be used and the plurality of co-stimulatory molecules, cytokines, and / or chemokines may be related to a T-cell activation profile identified for activation of T cells responsive to one or more peptides of this disclosure from Subdoligranulum didolesgii strain D8.[00631 In these methods, the peptide may be a cell wall protein from Subdoligranulum didolesgii strain D8 selected from endoglucanase, cellulosome-anchoring protein, and cell surface protein, or a homolog thereof. Alternatively or additionally, the peptide may be a peptide fragment comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or antigenic variants of SEQ ID NOs: 2-19.[0064[ In these methods, the co-stimulatory molecules, cytokines, and / or chemokines may be related to CD4+ Thl7 T cell polarization profile. In a preferred embodiment, the co- stimulatory molecules, cytokines, and / or chemokines may include one or more of IL-1 P, IL-6, TNF, TLR2, CD86, CD40, IL-23, and BAFF.[00651 In these methods, antibodies may be used to detect the co-stimulatory molecules, cytokines, and / or chemokines, and the antibodies may be monoclonal antibodies. In these methods, the biological sample may be a blood sample, preferably a peripheral blood sample prepared for analysis as a peripheral blood cells preparation.

[0066] In these methods, the subject may be diagnosed with RA or at risk of developing RA when T cell activation to a peptide of this disclosure from Subdoligranulum didolesgii strain D8is detected in the biological sample from the subject. The detection of the presence of T cell activation may be assessed relative to a control sample from a subject not diagnosed with RA and / or a control sample from a subject not at risk of RA. Detection may also encompass evaluating a relative level of T cell activation (such as relative level of costimulatory molecules, cytokines, and / or chemokines detected) to a peptide of this disclosure above a threshold value. The threshold value may be two, or more, standard deviations above the mean value for controls.

[0067] These methods of detecting T cell activation may also be used for monitoring the treatment of a subject for RA and / or monitoring the progression to RA in a subject identified as at risk of developing RA, whereby an increase or decrease in T cell activation following contact with a peptide of this disclosure may be indicative of progression of RA or successfully treating RA, respectively, over time.Treatment Methods[0068[ Methods for treating rheumatoid arthritis in a subject, or ameliorating at least one symptom of rheumatoid arthritis in a subject, as known in the art. These methods of treatment may include administering to a subject diagnosed with RA or at risk of developing RA using the methods of this disclosure, a therapeutically effective amount of an agent known for treating RA. These agents include but are not limited to non-steroidal anti-inflammatory drugs, corticosteroids, disease-modifying antirheumatic drugs, and biologic response modifiers.

[0069] Examples of suitable non-steroidal anti-inflammatory drugs include: ibuprofen; naproxen sodium; celecoxib; diclofenac; fenoprofen; flurbiprofen; indomethacin; ketorolac; mefenamic acid; meloxicam; oxaprozin; piroxicam; and sulindac.[0070[ Examples of suitable corticosteroids include: prednisone; bethamethasone; and prednisolone; triamcinolone; methylprednisolone; and dexamethasone.[007.1] Examples of suitable disease-modifying antirheumatic drugs include: methotrexate; leflunomide; hydroxychloroquine; and sulfasalazine.[0072 [ Examples of suitable biologic response modifiers include: abatacept and biosimilars thereof; adalimumab and biosimilars thereof; anakinra and biosimilars thereof; baricitinib; certolizumab and biosimilars thereof; etanercept and biosimilars thereof; golimumab and biosimilars thereof; infliximab and biosimilars thereof; rituximab and biosimilars thereof; sarilumab and biosimilars thereof; tocilizumab and biosimilars thereof; and tofacitinib.[00731 The therapeutic agents may be administered prophylactically or therapeutically to subjects at risk of (or susceptible to) developing RA, those presenting with preclinical RA, or those subjects diagnosed with RA, as diagnosed using the methods of this disclosure. Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of RA, such that RA is prevented or alternatively delayed in its progression of the disease. The goal of prophylactic treatment is to control symptoms and help the subject maintain a normal quality of life. Prevention can occur at primary, secondary, and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

[0074] Administration of the agents can be achieved in several ways using methods known in the art, including oral administration, systemic administration (e.g., enteral or parenteral administration), intradermal delivery, intramuscular delivery, subcutaneous delivery, inhalation, and intranasal delivery.|0075] The agents can be administered to a subject alone, or in conjunction with one or more agents, such as those traditionally utilized in the treatment of RA.[00761 The agents may be administered to a subject as frequently as several times daily, or they may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily determinable by the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated.

[0077] In another aspect of this disclosure, the peptides of this disclosure may be used therapeutically to induce immune tolerance in a subject with RA or at risk of developing RA. Immune tolerance may be defined as the absence of an immune response to a specific antigen in the setting of an otherwise normal immune system. This state of specific immunological tolerance to antigens involves both central and peripheral mechanisms. Central tolerance (negative selection) is a consequence of immature T cells receiving strong intracellular signaling while still resident in the thymus, resulting in clonal deletion of autoreactive cells. Peripheral tolerance occurs when the immune system becomes unreactive to an antigen presented in the periphery, where, in contrast to the thymus, Tcells are assumed to be functionally mature. Peripheral tolerance has been proposed to be the result of various mechanisms, including the development of antigen specific suppressor cells or other means of active tolerance, clonal deletion, and anergy. Autoreactive cells may be physically deleted by the induction of apoptosis after recognition of tolerizing antigen, may become anergic without deletion, or may be functionally inhibited by regulatory cytokines or cells.

[0078] In this aspect, a therapeutic composition that includes any of the peptides of this disclosure is administered to a subject with RA or at risk of developing RA to induce immune tolerance, thereby treating RA. The composition may be administered in an amount effective to reduce the generation of an immune response involved in the development, progression, and / or physical effects of RA. In these therapeutic methods, the composition may be administered to a subject according to protocols shown to reduce the generation of an undesired immune response to citrullinated proteins, such as fibrin, fibrinogen, vimentin, types I and II collagens, and a-enolase, in one or more subjects. The administration of the composition may reduce or eliminate the generation of anti- citrullinated protein antibodies, or CD4+ T cell proliferation and / or activity, or B cell proliferation and / or activity, or combinations of these effects. The administration of the composition may also prevent the development or progression of an undesired immune response altogether or delay the occurrence of an undesired immune response. The composition may cause T cells to undertake early events associated with T-cell activation but not allow T-cells to acquire effector function. For example, administration of compositions of this disclosure may result in T-cells having a quasi-activated phenotype, such as CD86 and / or CD40 upregulation, but without displaying effector function, such as indicated by a lack of IL-ip, IL-6, TNF, or TLR2 synthesis. The administration of compositions of this disclosure may result in T cells having a quasi-activated phenotype without having conversion of naive antigen-specific T-cells to a regulatory phenotype, such as those having CD25+ / Foxp3+phenotypes. The achievement of these effects may be monitored by the methods of this disclosure as well as routine methods of clinical evaluation.

[0079] In these therapeutic compositions, a peptide of this disclosure may be linked to a carrier (e.g., an immune modifying particle). A carrier may include poly(lactic acid) (PLA), poly(glycolic acid) (PGA), or poly(lactic co-glycolic acid) (PLGA) particles. The carrier may be effective to induce antigen-specific tolerance and / or prevent the onset of RAand / or diminish the severity of existing RA disease. The surface of a carrier (e.g., particle) may comprise chemical moieties and / or functional groups that allow attachment (covalently, non-covalently) of antigenic peptides and / or other functional elements to the carrier. A carrier may comprise one or more biological or chemical agents adhered to, adsorbed on, encapsulated within, and / or contained throughout the carrier. A chemical or biological agent, in addition to a peptide of this disclosure, may be encapsulated in and / or contained throughout the carrier. Such additional agents may include, but are not limited to, proteins, nucleic acid molecules, small molecule drugs, lipids, carbohydrates, cells, cell components, and the like. The carrier, or a portion thereof, may be configured for slow release of biological or chemical agents, including a peptide of this disclosure. The slow release may provide release of biologically active agents over a period of at least 30 days (e.g., 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 180 days, etc.). The carrier may comprise a peptide of this disclosure without other non-peptide active agents, such as drugs or immunomodulators.[00801 The carrier may encapsulate a peptide of this disclosure, and / or biological, and / or chemical agents. Encapsulation may better induce immunological tolerance and has several advantages, including a slower cytokine response, and may allow more peptides, biological, and / or chemical agents to be incorporated within the particle. The encapsulation of the peptides, biological, and / or chemical agents in carrier particles may be performed by any method known in the art.

[0081] Thus, this disclosure provides therapeutic methods of inducing antigen-specific tolerance in a subject having RA or at risk of developing RA by administering to the subject an effective amount of a composition comprising a peptide of this disclosure, which may be linked to a carrier, as described above. In these methods, the composition may be administered intravenously, intramuscularly, subcutaneously, or orally.Kits

[0082] This disclosure also provides kits useful in carrying out the methods and treatments of this disclosure. Such kits may comprise components useful in any of the methods described herein, including for example, peptides of this disclosure, which may be linked to a carrier, such as a bead, a test strip, a microtiter plate, a membrane, a lateral flow device, a glass surface, a slide, a microarray, and a biochip; antibodies, including “secondary antibodies” that may be labeled with compounds that enhance their detection; hybridization probes or primers (e.g., labeled probes or primers), reagents for detection oflabeled molecules, means for amplification of nucleic acids, means for analyzing a nucleic acid sequence, antibodies, and instructional materials. For example, a kit may comprise components useful for analysis of antibodies present in a biological sample obtained from a subject, and / or analysis of the activation of T cells in a biological sample obtained from a subject. The kit may comprise components for detecting one or more of the proteins of this disclosure present in a biological sample derived from a subject.

[0083] A kit of this disclosure may include reagents for the detection of co-stimulatory molecule(s), cytokine(s), or chemokine(s) according to the method this disclosure. These reagents may include reagents for detecting one or more cytokines related to a CD4+ Th 17 T cell polarization profile. In some embodiments, the kit comprises antibodies for the detection of one or more compounds selected from the group consisting of IL-ip, IL-6, TNF, TLR2, CD86, CD40, IL-23, and BAFF.

[0084] The components of the kits may be stored in unit or multi-dose containers as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kits may further comprise more containers comprising a pharmaceutically acceptable carrier. They may further include other materials desirable from a commercial and end user standpoint, including, but not limited to, buffers, diluents, filters, needles, syringes, or other active agents. The kits can contain instructions customarily included in commercial packages of diagnostic and therapeutic products that contain information, for example, about the indications, usage, dosage, manufacture, expiration, administration, contraindications or warnings concerning the use of such diagnostic products.EXAMPLES|0085] The materials, methods, and embodiments described herein are further defined in the following Examples. Certain embodiments are defined in the Examples herein. These Examples, while demonstrating certain embodiments, are given by way of illustration only. From the disclosure herein and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.[0086| As described in the inventors’ copending application (PCT / US2021 / 072293, filed 8 November 2021; WO 2022 / 099317, published 12 May 2022), to investigate immune responses to Lachnospiraceae and Ruminococcaceae in RA, primary bacterial isolates from an individual with greater than 40% abundance of these taxa in their feces were obtained. A total of 50 isolates were established, of which seven were identified as Lachnospiraceae / Ruminococcaceae by qPCR (isolates 1-7), and five were confirmed as pure isolates by 16S rRNA sequencing (isolates 1, 3-5, and 7). These five isolates underwent whole genome sequencing. The five isolates were narrowed to two candidates (isolates 1 and 7) for further study due to their differing abilities to induce joint swelling in monocolonized mice. Isolate 1 was designated Subdoligranulum didolesgii (“5. dido") strain H3 and isolate 7 was designated Subdoligranulum didolesgii strain D8. Of these two candidates, S. dido strain D8 (isolate 7), was identified as sufficient to cause rheumatoid arthritis (RA) or to worsen its disease course and effects on involved tissues.EXAMPLE 1: Assessing the abundance of S. dido strain D8 in a human population.[0087| We sought to assess the abundance of S. dido strain D8 in a large human population. Since 2016, we have identified and prospectively followed a cohort of serum anti-citrullinated protein antibody (ACPA)(+) individuals (i.e. participants at-risk for developing RA) who at baseline did not have inflammatory arthritis or RA. This cohort also includes individuals termed ‘ ACPA(+) Early Clinical RA’ (eRA) with their initial study visit occurring less than 12 months from their diagnosis of RA as well as a set of prospectively followed ACPA(-) controls. All participants had extensive clinical metadata, as well as blood and stool collection at baseline and l-2x annually during the approx. 4-years followup. The data and samples collected were banked and available for use and for further study through the NIH P30-supported Center for Mucosal Immunology and Rheumatic Disease Pathogenesis (CMIR). Characteristics of participants in this cohort are summarized in Table 2.10088] Using PCR primers that we have previously designed and shown to specifically detect S. dido strain D8, we profiled the baseline stool sample for most of the individuals listed in Table 1. As shown in FIG. 1A, compared to controls, participants at risk for RA had significantly more S. dido strain D8 abundance, demonstrating that S. dido strain D8 is present in additional individuals beyond the original sample from whom the isolates were obtained. For reference, we examined the abundance of a closely related bacterium, M. pectinilyticus in bacterial metagenomics sequencing data from the same samples and, as shown in FIG. IB, did not find differences between the groups.

[0089] These data allowed us to identify two individuals with the highest abundance of S. dido strain D8 from each group, control, at-risk, and RA. Based on the methodology of Goodman et al. (2011 PNAS 108:6252-57), stool samples from the identified individuals were transferred into an anaerobic Coy chamber for dilutional culturing to obtain single strain isolates. In total, we generated an additional 117 isolates from 6 individuals (2 each RA, at-risk, and control). Of these, 18 were identified as S. dido based on 16S rRNA sequencing and our specific qPCR primers. Intriguingly, we were unable to obtain S. dido isolates from the two controls. Therefore, we confirm that S. dido strain D8 is more abundant in individuals at risk for and with RA compared to controls, and we now show that the impact and prevalence of S. dido in RA is much greater than we originally published, increasing the relevance of this strain.EXAMPLE 2: S. dido strain D8 has unique immune activation properties.

[0090] Bacteria gain access to the host epithelium through utilization of host glycoproteins in the mucus layer based on polysaccharide utilization loci. Additionally, bacterial peptidoglycan content determines its absorption by the host and signaling through pattern recognition receptors. As S. dido is a Gram-positive bacterium, the lipoprotein and peptidoglycan content of Gram-positive cell walls is recognized by mononuclear phagocytes through TLR2 by itself or as a heterodimer with TLR1 or TLR6, as well as NODI and N0D2 signaling, resulting in expression of cytokines like TNF, IL-ip, and IL-6. Therefore, we utilized BMDCs from DBA / 1 and C57BL / 6 mice cultured in conditions to phenocopy gut DC. BMDCs from DBA / 1, C56B1 / 6, or TLR2 KO mice were cultured with 200ng / mL Flt3-L + 4ng / mL GM-CSF for 9 days. Gut-BMDCs were plated at 1.5xl06cells / ml in a 48well plate and stimulated with isolate 1 or strain D8 (MOI 40). LPS (Ipg / mL) was used as positive control. Gut-BMDCs from DBA, B6, and TLR2 KO mice were harvested at 24 hours after stimulation and examined by flow cytometry for the presence of co-stimulatory molecules. When these gut-BMDCs are exposed to oxygen-killed S. dido strain D8 versus isolate 1, they express more RA-relevant cytokines like IL-ip, IL-6, and TNF (FIG. 2A) and TLR2 (FIG. 2B) as well as increased surface co-stimulatory CD86 and CD40 (demonstrated by flow cytometry data). IL-23 and BAFF were also significantly elevated upon exposure to S. dido strain D8 compared to isolate 1, but not IL-10 or TGFp. Because expression of TLR2, but not NODI or N0D2, significantly increased with exposure to S. dido strain D8 compared to isolate 1, we then cultured gut-BMDCs from TLR2-deficient mice and again stimulated with strain D8. In the absence of TLR2, strain D8 no longer stimulated expression of IL-ip, IL-6, TNF, CD86, and CD40 (FIG. 2 A and flow cytometry data), indicating that lipoprotein and peptidoglycan content differences between S. dido strain D8 and isolate 1 enabled antigen-presenting cell functions to enhance pathogenic T cell responses to antigen.EXAMPLE 3: S. dido strain D8 harbors antigens that stimulate human T cells from individuals with RA and bind ACPA mAbs.

[0091] We undertook to identify antigens targeted by human T cells and our autoreactive PB- mAbs used to identify these bacteria. First, to ensure that the putative antigen(s) was protein in nature, we digested S. dido strain D8 with proteinase K (lOOpg / ml), and tested binding of previously generated PB-mAbs that were originally selected for their ability to bind S. dido. This data showed greater than 50% reduction in PB-mAb binding in the Proteinase K digested proteins (FIG. 3 A). We then performed immunoblotting of whole bacterial lysate as well as cell-wall associated peptides. Precipitated protein was run on a 10% Native-PAGE gel and silver stained. FIG. 3B shows that only cell-wall associated protein was targeted and immunoprecipitated. Excised immunoprecipitated bands underwent MADLI-ToF to generate de novo peptide libraries, and three peptides were identified: VATVSPL (SEQ ID NO: 20), PQVNG (SEQ ID NO: 21), and GDRDHMN (SEQ ID NO: 22). These peptides were then cross-referenced with the S. dido strain D8 assembled genome. We conducted further epitope mapping using HLA-DR4 liquid chromatography with tandem mass spectrometry (LC-MS / MS). The peptides presented by the DR4 molecules were eluted, separated, and analyzed by LCMS to directly determine the epitope sequences bound and presented to T cells. Table 3 lists the antigens identifiedby these approaches that had greater than 85% coverage and percent identity with S. dido strain D8 annotated genome (sequences use the single letter amino acid abbreviations).

[0092] Table 3.[0093| These shorter peptides (SEQ ID NOs 20-28, Table 3) are the B cell epitopes from immunoprecipitation with monoclonal antibodies while the longer peptides (SEQ ID NOs 2-19, Tables 1 and 5) are dendritic cell-presented peptides for T cell activation10094] The dissociation constant (Kd) was measured for five PB-mAb clones (MH17, MH28, MH58, MH93, and MH91) and S. dido Strain D8 endoglucanase). The binding affinity for each of these mAb clones is shown in Table 4, which indicates strong(MH91) to moderate (MH17) binding affinity for these PB-mAb clones and S. dido Strain D8 proteins.10095J Table 4.

[0096] We then identified naturally presented HLA-DR peptides derived from S. dido throughHLA peptidomic analysis of isolate pulsed DR0401+ dendritic cells and confirmed their immunogenicity through in vitro T cell assays and T cell cloning. Using the Blast databaseto search for sequence alignment and a custom algorithm for HLA-DRB 1*0401 binding predictions of candidate self-peptides, we identified human proteins which had considerable shared, but not complete, sequence homology with immunogenic peptides derived from the S. dido cell surface protein and cellulose anchoring protein. These mimics included peptides from human glycine decarboxylase, leucyl-cystinyl aminopeptidase, and rapl GTPase-activating protein 2 isoform X9. The identified peptide sequences (using the single letter amino acid abbreviations), their human homologs, and the names of the human proteins are identified in Table 5.[0097| As a second approach to identify T cell relevant peptide antigens, we established BMDCs from B6 mice transgenic for the RA-associated human HLA DR0401 class II molecule and lacking endogenous murine MHC class II. After co-culturing the BMDCs with oxygen-killed S. dido strain D8 overnight, the HLA DR complexes were column-purified and presented peptides were acid eluted. The eluted peptides were analyzed by liquid chromatography mass spectrometry (LCMS) and, surprisingly, overlapped with the antibody-precipitated protein antigens shown in Table 5. We then confirmed that individuals with RA harbor T cells specific for these peptides using peptide loaded tetramers. Importantly, one of the S. dido strain D8 peptides we identified has homology with a human citrullinated analog (Table 5). These data demonstrate S. dido strain D8 harbors cell wall protein antigens that stimulate human T cells isolated from individuals with RA.EXAMPLE 4: S. dido isolates are genetically homogeneous.[0098| Using next generation sequencing, we assembled greater than 90% of the total genomes for our initial S. dido isolates and annotated their predicted genes. The isolates share 96% sequence homology and are approx. 2.9Mbp in size. Alignment of the assembled genomes between strain D8 and isolate 1 does not reveal differentially present genes, indicating that single nucleotide polymorphisms and / or differential gene expression results in the opposing effects of these two isolates on the murine host and human T cells. The reference genome to which we aligned our assembled short-read S. dido genome was Clostridiales bacterium MGYG-HGUT-02424 that has now been updated to Monoglobus pectinilyticus. The genomes of AL pectinilyticus and S. dido are 88% similar with S. dido having additional unique DNA inserts that align closely with Lachnospiraceae and Clostridiales (FIG. 4). These data indicate S. dido is a strain within the AL pectinilyticus species that contains transposons, similar to what was described with P. copri (Nii, T., et al. 2023 Annals of the Rheumatic Diseases 82:621-29), or polymorphisms within specific genes are causal for immunogenicity.[00991 Although embodiments have been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of this disclosure, and would readily be known to the skilled artisan.100100] These and other aspects disclosed herein, including the exemplary diagnostic methods, treatment methods, and uses, will be apparent from the teachings contained herein.

Claims

CLAIMS1. An ex vivo method for diagnosing rheumatoid arthritis (RA) in a subject, the method comprising: a) obtaining a biological sample from the subject; b) detecting presence or absence of an antibody that binds a peptide fromSubdoligranulum didolesgii strain D8 in the biological sample, and c) diagnosing the subject with RA or at risk of developing RA when an antibody that binds the peptide from Subdoligranulum didolesgii strain D8 is detected in the biological sample.

2. The method of claim 1, wherein the peptide is a cell wall protein from Subdoligranulum didolesgii strain D8 selected from endoglucanase, cellulosome- anchoring protein, and cell surface protein, or a homolog thereof.

3. The method of claim 1 or claim 2, wherein the peptide is a peptide fragment comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of SEQ ID NOs: 2-19.

4. The method according to any of claims 1 to 3, wherein the antibody is an IgA and / or IgG class antibody.

5. The method according to any of claims 1 to 4, wherein the biological sample is a blood sample selected from whole blood, serum, and plasma.

6. The method according to any of claims 1 to 5, wherein the antibody is detected using a labeled secondary antibody.

7. The method according to any of claims 1 to 6, wherein the antibody is detected using a method selected from colorimetry, immunofluorescence, detection of enzymatic activity, chemiluminescence, and radioactivity.

8. The method according to any of claims 1 to 7, wherein the peptide is linked to a carrier selected from a bead, a test strip, a microtiter plate, a membrane, a lateral flow device, a glass surface, a slide, a microarray, and a biochip.

9. An ex vivo method for diagnosing rheumatoid arthritis (RA) in a subject, the method comprising: a) contacting a biological sample comprising T cells from a subject with a composition comprising a peptide from Subdoligranulum didolesgii strain D8; b) detecting the level of at least one co-stimulatory molecule, cytokine, or chemokine produced by the T cells in the sample, and c) diagnosing the subject with RA or at risk of developing RA when elevated levels of the at least one co-stimulatory molecule, cytokine, or chemokine produced by the T cells in the sample is detected in the biological sample from the subject.

10. The method of claim 9, wherein the biological sample is a peripheral blood sample.

11. The method according to claim 9 or claim 10, wherein the peptide is a cell wall protein from Subdoligranulum didolesgii strain D8 selected from endoglucanase, cellulosome-anchoring protein, and cell surface protein, or a homolog thereof.

12. The method according to any one of claim 9-11, wherein the peptide is a peptide fragment comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of any one of SEQ ID NOs: 2-19.

13. The method according to any of claims 9-12, wherein the at least one co-stimulatory molecule, cytokine, or chemokine is related to CD4+ Thl7 T cell activation polarization profile.

14. The method according to any of claims 9-13, wherein the at least one co-stimulatory molecule, cytokine, or chemokine includes at least one of IL-ip, IL-6, TNF, TLR2, CD86, CD40, IL-23, and BAFF.

15. The method according to any of claims 9-14, wherein the at least one cytokine and / or chemokine is detected by ELISPOT.

16. The method according to any of claims 9-15, wherein the at least one cytokine and / or chemokine is detected by flow cytometry.

17. The method according to any of claims 9-16, wherein the level of at least one co- stimulatory molecule, cytokine, or chemokine produced by the T cells in the sample is assessed relative to a control sample from a subject not diagnosed with RA or not at risk of developing RA.

18. The method according to any one of claims 9-16, wherein the level of at least one co-stimulatory molecule, cytokine, or chemokine produced by the T cells in the sample is assessed relative to a threshold value that is at least two standard deviations above a mean value for control subjects.

19. The method of any one of the preceding claims, wherein the rheumatoid arthritis is early rheumatoid arthritis.

20. The method of any one of the preceding claims, further comprising administering an RA treatment to the subject diagnosed with RA or at risk of developing RA.

21. The method of claim 20, wherein the RA treatment comprises administering to the subject one or more compounds selected from: a non-steroidal inflammatory drug; a corticosteroid; a disease-modifying antirheumatic drug; and a biologic response modifier.

22. The method of 21, wherein: a. the non-steroidal inflammatory drug is selected from: ibuprofen; naproxen sodium; celecoxib; diclofenac; fenoprofen; flurbiprofen; indomethacin; ketorolac; mefenamic acid; meloxicam; oxaprozin; piroxicam; and sulindac; b. the corticosteroid is selected from: prednisone; bethamethasone; and prednisolone; triamcinolone; methylprednisolone; and dexamethasone; c. the disease-modifying antirheumatic drug is selected from: methotrexate; leflunomide; hydroxychloroquine; and sulfasalazine; and d. the biologic response modifier is selected from: abatacept; adalimumab; anakinra; baricitinib; certolizumab; etanercept; golimumab; infliximab; rituximab; sarilumab; tocilizumab; and tofacitinib.

23. A composition for inducing immune tolerance in a subject with RA or at risk of developing RA comprising a peptide selected from: a) endoglucanase, cellulosome-anchoring protein, and cell surface protein fromSubdoligranulum didolesgii strain D8, or a homolog thereof; and, b) a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of any one of SEQ ID NOs: 2-19.

24. The composition of claim 23, further comprising a carrier linked to the peptide.

25. The composition of claim 24, wherein the carrier comprises at least one of poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic co-glycolic acid) (PLGA).

26. A method of treating RA comprising administering to a subject in need of treatment an effective amount of a composition of any one of claims 23-25.

27. The method of claim 26, wherein the composition is administered intravenously, intramuscularly, subcutaneously, or orally.

28. The method of claim 26 or claim 27, wherein the administration reduces or eliminates the generation of anti-citrullinated protein antibodies, or CD4+ T cell proliferation and / or activity, or B cell proliferation and / or activity, or combinations of these effects in the subject.

29. A kit for the diagnosis of RA comprising at least one peptide selected from: a) endoglucanase, cellulosome-anchoring protein, and cell surface protein fromSubdoligranulum didolesgii strain D8, or a homolog thereof; and, b) a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-19, or an antigenic variant of SEQ ID NOs: 2-19; and, a reagent useful for one or both of: c) analysis of antibodies present in a biological sample obtained from a subject; and, d) analysis of the activation of T cells in a biological sample obtained from a subject.

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