Patent Application
20190137491
The present invention relates to the field of diagnostics using humoral and cellular autoimmune responses to gut microbe Prevotella copri (P. copri). In particular it relates to use of P. copri proteins in diagnosis and treatment of Rhuematiod arthritits.
Rheumatoid arthritis (RA), the prototypic autoimmune joint disease, results from a complex interplay between genetic and environmental factors (Firestein, 2003; McInnes and Schett, 2011). Great progress has been made in the identification of genetic factors and inflammatory pathways that influence the disease (McInnes and Schett, 2011; Plenge, 2009), but environmental factors are only now being determined (Catrina et al., 2014). A key hypothesis is that specific organisms in the mouth or gut microbiota, the composition of which is strongly influenced by environmental cues, may shape mucosal and systemic immune responses that affect joints in RA patients (Catrina et al., 2014; Han and Wang, 2013; Longman and Littman, 2015; Scher and Abramson, 2011).
In seminal studies, Littman and colleagues explored the role of the gut microbiota in shaping immune responses in animal models. In mice, segmented filamentous bacteria (SFB) penetrate the mucus layer of the gut and bind epithelial cells where they stimulate Th17-cell responses (Hooper et al., 2012; Ivanov et al., 2009; Ivanov and Honda, 2012). These studies highlight the potential benefits and disadvantageous aspects of Th17 responses. These cells promote barrier protection at the mucosal surface, but they can also contribute to systemic inflammation and autoimmunity.
Although humans do not have SFB, other bacteria may serve a similar function in human autoimmune disease. Using high-through-put sequencing, Scher et al. showed that Prevotella copri in the gut microbiota was over-expanded in stool samples from patients with new-onset RA (NORA) compared with patients with chronic RA (CRA), psoriatic arthritis, or healthy people (Scher et al., 2013). In NORA patients, Prevotella abundance in the gut was at the expense of Bacteroides fragilis, an organism that is important for Treg function (Atarashi et al., 2011; Round et al., 2011). In addition, according to the NCBI database, P. copri has a gene sequence that would code for a peptidylarginine deiminase-related protein (WP_006847349), which could potentially contribute to citrullination of proteins and production of anti-citrullinated protein antibodies (ACPA), a specific autoantibody response in RA (Nishimura et al., 2007; Schellekens et al., 1998; Suzuki et al., 2003).
A recent metagenome-wide analysis of fecal, dental, and salivary samples in RA patients showed similar dysbiosis not only in the gut, but also in the mouth and salivary glands (Zhang et al., 2015). In this study, a trend toward increased abundance of P. copri in stool samples was also seen during the first year of disease. Therefore, two studies have now shown that the gut microbiome in RA patients is distinct from that of healthy individuals. However, it is unclear whether over-expansion of P. copri in stool is simply the result of gut dysbiosis or whether the organism has the potential to regulate immune cell functions at both mucosal and systemic sites, thereby contributing to RA disease pathogenesis.
Aspects of the invention relate to a method for determining whether a biological sample comprising antibodies and/or immunological cells obtained from a subject is immunologically reactive with P. copri Pc-p27 protein or antigen thereof, comprising performing an assay for identifying the presence of antibodies that specifically bind P. copri Pc-p27 protein or antigen thereof or performing an assay for identifying T cells specifically reactive to P. copri Pc-p27 protein or antigen thereof, and determining the sample is reactive with the P. copri Pc-p27 protein or antigen thereof if the assay produces positive results compared to an appropriate control.
In some embodiments, the assay that identifies the presence of the antibodies comprises contacting the sample with P. copri Pc-p27 protein or antigen thereof, under conditions that allow an immunocomplex of the antibody and the P. copri Pc-p27 protein or antigen thereof to form, and detecting the presence or absence of the immunocomplex, wherein the presence of the immunocomplex indicates the biological sample is immunoreactive with the P. copri Pc-p27 protein or antigen thereof and wherein the absence of the immunocomplex indicates the biological sample is not immunoreactive with the P. copri Pc-p27 protein or antigen thereof.
In some embodiments the antibodies are IgG or IgA. In some embodiments the assay is an ELISA, agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, or an immunoblot assay.
In some embodiments, the assay for identifying T cells specifically reactive to P. copri Pc-p27 protein or antigen thereof comprises: (a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with P. copri Pc-p27 protein or antigen thereof; (b) measuring T cell proliferation in vitro or secretion of IFN-γ into cell culture supernatants; and (c) identifying the subject as having T cells specifically reactive to P. copri Pc-p27 protein or antigen thereof, when T cell proliferation or secretion of IFN-γ measured is substantially increased over that of an appropriate control.
In some embodiments of the foregoing aspects the assay for identifying T cells specifically reactive to P. copri Pc-p27 protein or antigen thereof, is a T cell proliferation assay. In some embodiments the assay is a 3H-thymdine incorporation assay, CFSE dilution, or an ELISPOT.
In some embodiments of the foregoing aspects the assay for identifying T cells specifically reactive to P. copri Pc-p27 protein or antigen thereof, is a T cell reactivity assay.
In some embodiments of the foregoing aspects, the biological sample is obtained from peripheral blood, synovial fluid, synovial tissue, peripheral blood mononuclear cells (PBMC), or synovial fluid mononuclear cells (SFMC).
Another aspect of the invention relates to diagnosing Rheumatoid arthritis in a subject comprising determining whether the subject is immunologically reactive with P. copri or a portion thereof, wherein immunological reactivity of the subject to P. copri or a portion thereof as compared to an appropriate control, indicates the subject has Rheumatoid arthritis.
In some embodiments, the determining is by evaluating a biological sample obtained from the subject for immunological reactivity with the P. copri or a portion thereof. In some embodiments, determining immunological reactivity is by detecting a T cell response to P. copri or a portion thereof in the subject by processing a biological sample obtained from the subject.
In some embodiments of the foregoing aspects, detecting a T cell response is by detecting the presence of T cells reactive to P. copri or a portion thereof. In some embodiments, the T cell response is a Th17 response.
In some embodiments, the detecting the presence of T cells comprises: (a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with P. copri or a portion thereof, and (b) measuring T cell proliferation in vitro or secretion of IFN-γ into cell culture supernatants;(c) identifying the subject as having T cells reactive to P. copri or a portion thereof when T cell proliferation or secretion of IFN-γ is measured as significantly increased over that of an appropriate control.
In some embodiments of the foregoing aspects, determining immunological reactivity comprises determining if the subject has a B-cell response to P. copri or a portion thereof resulting in the production of antibodies that specifically recognize the P. copri or a portion thereof, by contacting the sample with the P. copri or a portion thereof, under conditions that allow an immunocomplex of the antibody and the P. copri or a portion thereof to form, and detecting the presence or absence of an immunocomplex, wherein the presence of an immunocomplex indicates the subject presents a B-cell response to the P. copri or a portion thereof and wherein the absence of an immunocomplex indicates the subject fails to present a B-cell response to the P. copri or a portion thereof.
In some embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA), agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, or an immunoblot assay.
In some embodiments the biological sample is obtained from peripheral blood, synovial fluid, synovial tissue, peripheral blood mononuclear cells (PBMC), or synovial fluid mononuclear cells (SFMC).
In some embodiments, the portion of P. copri is a protein or polypeptide fragment(s) of P. copri. In some embodiments the protein is Pc-p27 whole protein or polypeptide fragment(s) thereof. In some embodiments, the Pc-p27 polypeptide fragment is predicted to be presented by HLA-DR molecules associated with chronic inflammatory arthritis.
In some embodiments of the foregoing aspects, the polypeptide fragment is selected from the group consisting of KRIILILTVLLAMLGQVAY (SEQ ID NO: 2), or antigenic portion thereof; DYRGYWTMRYQFDSATVS (SEQ ID NO: 3) or antigenic portion thereof; EKINSLPTSSTGI (SEQ ID NO: 4) or antigenic portion thereof; and combinations thereof.
In some embodiments, the subject exhibits symptoms of arthritis or other autoimmune related disease manifestation. In some embodiments, the subject is suspected of having Rheumatoid arthritis. In some embodiments, the subject is suspected of having or suffers from new onset rheumatoid arthritis or chronic rheumatoid arthritis. In some embodiments, the subject has been further tested for one or more of rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA), Prevotella DNA in their synovial fluid, and one or more HLA-DR alleles. In some embodiments, the subject is further tested for P. copri in a biological sample of the subject.
In some embodiments, the biological sample is synovial fluid or serum, and the subject is tested for the presence of P. copri 16S rDNA.
In some embodiments, the subject tests positive for one or more of (RF), (ACPA), Prevotella DNA in their synovial fluid, and one or more HLA-DR alleles. In some embodiments, the subject tests negative for one or more of (RF), (ACPA), Prevotella DNA in their synovial fluid, and one or more HLA-DR alleles. In some embodiments, the HLA-DR allele is HLA-DRB1*0101 and/or HLA-DRB1*0401.
In some embodiments, the methods of the foregoing aspects further comprises the step of treating the subject who comprises the antibodies and/or immune cells for rheumatoid arthritis. In some embodiments, the treating is by administration of a therapeutically effective amount of one or more of a nonsteroidal anti-inflammatory drug (NSAIDs), a steroid, a disease modifying anti-rheumatic drug (DMARD), an antibiotic, and a biologic, to the subject.
Another aspect of the invention relates to a kit comprising, one or more of P. copri or a portion thereof, and reagents for conducting an assay for detecting the presence of an antibody in a sample that binds to the one or more of P. copri or a portion thereof.
In some embodiments, the kit further comprises reagents for conducting an assay for detecting the presence of one or more cytokines or chemokines. In some embodiments, the cytokines or chemokines comprise one or more of IL-23, IL-22, IL-17E, IL-17F IFN-γ, TNF, IFN-α, MIP-1α, MMP-1β, IL-17F, IL-1β, CXCL9, CXCL10, IL-12.
In some embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the assay is a western blot. In some embodiments, the kit further comprises E. coli and/or B. fragilis whole cells or potential antigens derived from them.
In some embodiments of the present invention, the kit or method of the foregoing aspects are used in identifying a patient with Rheumatoid arthritis. In some embodiments, the Rheumatoid arthritis is new onset rheumatoid arthritis or chronic rheumatoid arthritis.
Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
As used herein, the terms “diagnose” or “diagnosis” or “diagnosing” refers to determining the nature or the identity of a condition or disease or disorder, detecting and/or classifying the disease and/or disorder in a subject. A diagnosis may be accompanied by a determination as to the severity of the disease. The term also encompasses assessing or evaluating the disease status (progression, regression, stabilization, response to treatment, etc.) in a patient known to have the disease). Diagnosis as it relates to the present invention, relates to the diagnosis of chronic inflammatory arthritis e.g., Rheumatoid arthritis. The diagnosis can be differential diagnosis. As used herein, “differential diagnosis” refers to determination of which two or more diseases with similar symptoms (e.g., RA, osteoarthritis, systemic lupus erythematosus, Lyme arthritis, reactive arthritis, psoriatic arthritis, ankylosing spondylitis) is the one from which a patient is suffering from based on an analysis of clinical data such as for example results obtained from methods described herein.
As used herein, the term “biological sample” refers to a sample obtained for evaluation in vitro. The biological sample can be any sample that is expected to contain antibodies and/or immune cells. The sample can be taken from a part of the body that is specifically affected by the disorder, such as taken specifically from a site of inflammation or pathology in the subject (e.g., synnovial fluid, synovial tissue, synovial fluid mononuclear cells (SFMC), spinal fluid, etc.) or can be a more systemic sample (e.g., peripheral blood, peripheral blood mononuclear cells (PBMC), whole blood or whole blood pre-treated with an anticoagulent such as heparin, ethylenediamine tetraacetic acid, plasma or serum). Sample can be pretreated prior to use, such as preparing plasma from blood, diluting viscous liquids, or the like; methods of treatment can also involve separation, filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents.
As used herein, “immunologically reactive” or “immunoreactive” is defined as the capability of the molecule (e.g., P. copri whole cell, whole protein thereof or polypeptide fragments thereof, protein Pc-p27 or one or more polypeptide fragment thereof) of the present invention to induce a specific immune response (e.g., T cell reactivity, B cell response) in appropriate subjects or cells and to bind with specific antibodies. The immunological reactivity can be assayed in a biological sample comprising immunological cells and/or antibodies obtained from a subject by methods described herein.
As used herein “autoantigen/self-antigen” is any substance normally found within a subject which, in an abnormal situation, is no longer recognized as part of the subject itself by the lymphocytes or antibodies of that subject, and is therefore attacked by the immune system as though it were a foreign substance. An autoantigen can be a naturally occurring molecule such as a protein normally produced and used by the subject itself, eliciting an immune response possibly leading to an autoimune disease (e.g. RA) in the subject.
The term “antibody,” as used herein, refers to an immunoglobulin molecule which is able to “specifically recognize” and/or “specifically bind” to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically immunoglobulin molecules. As it relates to the present invention, the antibody can be IgG or IgA antibody.
As used herein, “autoantibody” means an antibody produced by the immune system of a subject that is directed to, and specifically binds to an “autoantigen/self-antigen” or an “antigenic epitope” thereof.
As used herein, the term “epitope” refers to that portion of any molecule capable of being recognized by, and bound by, a T cell or an antibody (the corresponding antibody binding region may be referred to as a paratope), and/or eliciting an immune response. In general, B cell epitopes consist of chemically active surface groupings of molecules, e.g., amino acids, and have specific three-dimensional structural characteristics as well as specific charge characteristics. T cell epitopes are linear groups of amino acids that are presented to the immune system by HLA-DR or DQ molecules on host antigen presenting cells. As used herein, “immunogenic epitope”, as determined by any method known in the art, is defined as a portion of a polypeptide that causes an immune response in a subject. As used herein, “antigenic epitope”, as determined by any methods well known in the art, in that a given antibody or T cell receptor specifically recognizes and specifically binds to a given antigen. It can be, and is defined as a portion of a protein (e.g. Pc-p27). As used herein, “antigenic portion” refers to the portion of Pc-p27 that includes the antigenic epitopes.
“Specifically bind” and/or “specifically recognize” as used herein, refers to the higher affinity of a binding molecule for a target molecule compared to the binding molecule's affinity for non-target molecules. A binding molecule that specifically binds a target molecule does not substantially recognize or bind non-target molecules. e.g., an antibody “specifically binds” and/or “specifically recognize” another molecule, meaning that this interaction is dependent on the presence of the binding specificity of the molecule structure, e.g., an antigenic epitope. For example, an antibody that specifically binds to the antigenic epitope of protein molecules such as Pc-p27 and/or polypeptide fragments thereof, instead of indiscriminately binding to cause non-specific binding and/or background binding. As used herein, “non-specific binding” and “background binding” refers to the interaction that does not depend on the presence of specific structure (e.g., a specific antigenic epitopes).
As used herein, an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.
As used herein, an “appropriate control” refers to one or more biological samples obtained from a subject not afflicted with a disease or disorder that features abnormal level of the molecule and measurement of the molecule therein. An example of an appropriate control can be biological sample from a subject not afflicted from RA. As it relates to the present invention, an appropriate negative control sample would not be positive for T cell reactivity to and/or show presence of antibodies to P. copri or a portion thereof, protein Pc-p27 or one or more polypeptide fragments thereof when tested using the methods described herein.
The terms “disease”, “disorder”, or “condition” are used interchangeably herein, refer to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder can also be related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, or affectation.
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of Rheumatoid arthritis, (e.g. new onset rheumatoid arthritis or chronic rheumatoid arthritis), an associated condition and/or a symptom thereof. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of RA. Treatment is generally “effective” if one or more symptoms or clinical markers (e.g. antibodies to one or more of Pc-p27 or cyclic citrullinated peptide (anti-CCP) and/or rheumatoid factors) are reduced. Alternatively, or in addition, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Accordingly in case of chronic inflammatory arthritis such as RA, “effective treatment” can for example reduce inflammation, swelling and joint pain, bone deformity and/or result in reduction in T or B cell reactivity to P. copri, Pc-p27and/or polypeptides thereof and/or reduction in antibodies to P. copri, Pc-p27and/or polypeptides thereof compred to that observed pre-treatment, as determined by methods described herein. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality. For example, treatment is considered effective if the condition is stabilized. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
As used herein, a “subject”, “patient”, “individual” and like terms are used interchangeably and refers to a vertebrate, preferably a mammal, more preferably a primate, still more preferably a human. Mammals include, without limitation, humans, primates, rodents, wild or domesticated animals, including feral animals, farm animals, sport animals, and pets. Primates include, for example, chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. The terms, “individual,” “patient” and “subject” are used interchangeably herein. In one embodiment the subject is male. In another embodiment, the subject is female.
Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of conditions or disorders associated with RA. Such models are known in the art and are described in (Asquith et al., 2009). In addition, the compositions and methods described herein can be used to diagnose domesticated animals and/or pets.
A subject can be one who has been previously diagnosed with or identified as suffering from or under medical supervision for a chronic inflammatory and/or autoimmune disease (e.g., RA). A subject can be one who is diagnosed and currently being treated for, or seeking treatment, monitoring, adjustment or modification of an existing therapeutic treatment, or is at a risk of developing RA, e.g., due to family history, carrying alleles or genotype associated with RA (e.g. HLA-DRB1*0101 and DRB1*0401, HLA-DRB1*0405 and DRB1*0408). The subject can exhibit one or more symptoms of autoimmune disease (e.g., RA) (e.g. swollen joints, joint pains). The subject may have tested positive with other assays for RA associated factor. Such factors include rheumatoid factor, anti-citrullinated protein antibodies (ACPA), one or more of HLA-DR alleles associated with RA, or other autoantigens. The subject may lack one or more symptoms of autoimmune disease. The subject may be identified as testing negative for other RA associated factors (rheumatoid factor, anti-citrullinated protein antibodies (ACPA), one or more of HLA-DR alleles, and other autoantigens).
As used herein, the terms “protein”, “peptide” and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to each other by bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, “peptide” and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein”, “peptide” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a difference of two standard deviations (2SD) or more.
Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.,” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.,” is synonymous with the term “for example.”
As used in this specification and appended claims, the singular forms “a,” “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, reference to “the method” included one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
In this application and the claims, the use of the singular includes the plural unless specifically stated otherwise. In addition, use of “or” means “and/or” unless stated otherwise. Moreover, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one unit unless specifically stated otherwise.
Aspects of invention relate to the identification of peptides presented by HLA-DR molecules (T cell epitope) in PBMC of RA patients, to be derived from a 27 kD protein (Pc-p27) of the intestinal commensal organism Prevotella copri (P. copri). Aspects of the invention also relate to the findings that P. copri induced immune responses to whole organism, protein Pc-p27 and polypeptide fragments thereof. One subgroup of patients was shown to have IgA antibody responses to Pc-p27 or to P. copri, correlating with Th17 cytokine responses and frequent anti-citrullinated protein antibodies (ACPA). The other subgroup had IgG P. copri antibodies, sometimes with Prevotella DNA in synovial fluid, which was associated with P. copri-specific Th1 responses and infrequent ACPA. P. copri antibody responses were rarely found in patients with other rheumatic diseases for e.g., Lyme disease or in healthy subjects. These findings indicate that P. copri, Pc-p27, and immunogenic polypeptides thereof, can be used to identify immunoreactivity in a subject, with that immunoreactivity being indicative of RA disease.
Test antigen for use in the methods and kits herein described include, without limitation, P. copri or a portion thereof. In some embodiments the portion of P. copri can be protein of P. copri or polypeptide fragments thereof. In some embodiments the protein can be full length Pc-p27 and/or polypeptide fragments thereof.
As used herein the term P. copri refers to the whole intact cell of the gram negative microorganism. In some embodiments of the methods and kits disclosed herein, the whole intact cell of P. copri or a portion thereof is used as a test antigen to determine immunoreactivity of a subject or a sample obtained from a subject. Positive immunoreactivity will indicate the subject has Rheumatoid arthritis. The deposited strain DSM 18205 is a non-limiting representative strain of P. copri which can be used in the assays, methods and kits disclosed herein. A mutant form of the bacterium which retains, at minimum its biological activity of inducing immune reactivity in a subject and or biological sample obtained from a subject as tested by the methods disclosed herein can also be used. In some embodiments the mutant P. copri has at least about 70%, 80%, 90%, or 100% immunological reactivity of the wild-type P. copri. In some embodiments, the P. copri can be one that preferably expresses the protein Pc-p27. The P. copri can be obtained from a commercial vendor or purified from a biological sample or grown as a bacterial culture in vitro under routine laboratory conditions known to one skilled in the art. The P. copri can be a bacterial cell-suspension which is typically heat inactivated or inactivated and fixed using solutions such as formalin.
The term “portion thereof” as used herein is defined as an antigenic component derived from P. copri which retains the ability, at a minimum, to induce immune reactivity in a subject or a biological sample obtained from the subject as determined by methods described herein. In some embodiments, the portion thereof has at least about 70%, 80%, 90%, 100% or more than 100% of the immunological reactivity of whole intact P. copri cell. Non limiting examples of antigenic components derived from P. copri can be cell wall, cell membranes, proteins, lipoproteins, polysaccharides, nucleic acids. The antigenic portion of P. copri of the methods and kits disclosed herein, can be synthetic, recombinant, purified, or contained in a P. copri cell extract. Methods to prepare cell extracts i.e., extracts comprising one or more antigenic portions of P. copri are known in the art, for example can include solubilization of the bacterial cells or cell lysis by sonication. Methods for obtaining portions of P. copri which can be used as test antigens in the methods and kits of the present invention are known to those skilled in the art. In some embodiments, the portion can be P. copri protein or one or more polypeptide fragments of the P. copri protein. In some embodiments the protein can be Pc-p27 or one or more polypeptide fragment thereof.
The term “Pc-p27” as used herein refers to the full length 27-kD protein of P. copri or to a polypeptide fragment or a derivative thereof that retains the ability, at a minimum, to induce immune reactivity in a subject or a biological sample obtained from the subject as determined by methods described herein. The sequence of the full length Pc-p27 is defined by GenBank Accession No. WP_022121928.1, which is incorporated herein by reference in its entirety as SEQ ID NO:1
In some embodiments the “ Pc-p27” of the methods and kits described herein can be a polypeptide fragment including or derived from full length Pc-p27 having the amino acid sequence of SEQ ID NO. 1. The polypeptide fragment refers to fragment of the full length Pc-p27 of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive amino acids of SEQ ID NO:1, that has at least about 70%, 80%, 90%, 100% or more than 100% of the immunological reactivity of Pc-p27 of SEQ ID NO:1. In some embodiments the polypeptide fragment can be signal sequence of Pc-p27. In some embodiments the polypeptide fragment comprises the amino acid sequence; KRIILILTVLLAMLGQVAY (SEQ ID NO: 2), or antigenic portion thereof; DYRGYWTMRYQFDSATVS (SEQ ID NO: 3) or antigenic portion thereof; EKINSLPTSSTGI (SEQ ID NO: 4) or antigenic portion thereof. In some embodiments the polypeptide fragments are predicted to be presented by HLA-DR molecules associated with rheumatoid arthritis. In one embodiment, the Pc-p27, or fragment thereof, is attached to a solid support matrix, such as a plate for ELISAs, such as in the kits described herein.
One aspect of the invention relates to a method for determining immunoreactivity of a biological sample to one or more of P. copri Pc-p27 protein or antigen thereof. Immunoreactivity of a subject to a test antigen (for example protein Pc-p27 or one or more of polypeptide fragments thereof) is determined, for example, by obtaining a biological sample from the subject containing antibodies or immune cells (e.g., T cells), and then assaying that biological sample for test antigen immunoreactivity. A variety of methods are available for determining immunoreactivity to a molecule, a few of which are set forth herein by way of non-limiting example. Immunoreactivity for example can be determined by assaying for B cell reactivity i.e., assaying for presence of antibodies that specifically bind P. copri Pc-p27 protein or antigen thereof. Immunoreactivity can also be determined by performing an assay for identification of T cells specifically reactive to P. copri Pc-p27 protein or antigen thereof. Accordingly in some embodiments, immunoreactivity can be determined by assaying T cell reactivity and/or B cell reactivity. The determination of which method to utilize can be determined by the skilled practitioner. Immunoreactivity to a molecule (e.g., a protein) can be determined using the full length molecule or one or more representative fragments or epitopes thereof.
In one embodiment, the assay is for the presence of antibodies in the subject that specifically bind to P. copri Pc-p27 protein or antigen thereof The assay involves contacting a biological sample obtained from the subject with the full length protein or representative fragments thereof (referred to as the antigen thereof), under conditions that allow an immunocomplex of the antibody and the polypeptide to form, and then assaying for the presence of the immunocomplex. Various methods of detecting the presence of an antibody-antigen complex are available in the art, and are suitable for use in the methods described herein, such as ELISA, agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, and immunoblot assay. In one embodiment, the assay involves a detectable label that is used to facilitate detection of the complex through detection of the label. The label present in the test sample is compared to label present in a positive control and/or absent in a negative control. Appropriate controls can be determined by the skilled practitioner. In one embodiment, a negative control is the same type of biological sample obtained, for example, from a healthy subject. In one embodiment, the antibodies can be IgG or IgA type.
Reactivity that indicates a detection of a significant level of the antibody complex above background (e.g., that in a negative control) is expected to serve as a positive result. In one embodiment, reactivity is indicated by detection of the label. In one embodiment, a determination of reactivity that is ≥3 standard deviations (SD) above the mean of samples of healthy control subjects is considered a positive result. In one embodiment, a result that is at least 2-fold above background (e.g., of the signal of a sample obtained from a healthy control subject) is indicative of positive. In one embodiment, higher levels of detection are used to indicate a positive result (e.g., at least 3-fold, 4-fold, or 5-fold above background).
In one embodiment, the assay is for identifying the presence of T cells in the biological sample that are specifically reactive to the P. copri Pc-p27 protein or antigen thereof This is typically accomplished by exposing the cells in the biological sample to a test antigen or one or more representative fragments thereof, and assaying for a response such as stimulation of proliferation. In one embodiment, peripheral blood mononuclear cells (PBMC) or synovial fluid mononuclear cells (SFMC) are contacted with the test antigen in vitro under conditions conducive to stimulation. The cells are then monitored for a response that indicates stimulation. In one embodiment, T cell proliferation in vitro or cytokine production (e.g., IFN-γ) into the supernatant that indicates stimulation is monitored. Identification of a response (e.g,. T cell proliferation or cytokine secretion) substantially over that of an appropriate control sample indicates stimulation has occurred. The level of one or more cytokines that are indicative of T cell activation can be monitored in the supernatant of the cells as an indication of T cell activation. One such cytokine is IFN-γ. Other cytokines to monitor include, without limitation IL-17, IL-12, and IL-10. The levels of one or more of such cytokines can be monitored. An increase in IFN-γ, IL-17, and/or IL-12, and/or a decrease in IL-10 levels indicates activation. In one embodiment, the T cell reactivity as determined by T cell response is a Th17 and/or Th1 response. Typically a Th17 response can be indicated by an increase in IL-17 and Th1 response can be indicated by an increase in IFN-γ.
Detection of the stimulation indicates that the sample comprises immunological cells that are immunologically reactive to the test antigen, which in turn indicates that the subject from whom the sample was obtained is immunologically reactive to the test antigen. Put another way, a positive assay result indicate that the test antigen is an autoantigen in the subject. Typical assays for use in this method are T cell proliferation assays, such as 3H-thymdine incorporation assay, CFSE dilution, or an ELISPOT, and also T cell reactivity assays.
Reactivity that indicates a detection of a significant level of stimulation above background (e.g., that in a negative control) is expected to serve as a positive result. In one embodiment, a determination of stimulation that is ≥3 standard deviations (SD) above the mean of samples of healthy control subjects is considered a positive result. In one embodiment, a result that is at least 2-fold above background (e.g., of the signal of a sample obtained from a healthy control subject) is indicative of positive. In one embodiment, higher levels of stimulation is used to indicate a positive result (e.g., at least 3-fold, 4-fold, or 5-fold above background).
One aspect of the invention relates to a method of diagnosing rheumatoid arthritis in a subject by determining whether the subject demonstrates immunological reactivity to P. copri or portion thereof, for example proteins such as Pc-p27 or polypeptide fragments thereof. Immunological reactivity of the subject to one or more of P. copri or a portion thereof, e.g., protein Pc-p27 or polypeptide fragments thereof, as compared to an appropriate control, indicates the subject has rheumatoid arthritis.
Immunoreactivity of a subject to the P. copri is determined, for example, by obtaining a biological sample from the subject containing antibodies or immune cells (e.g., T cells), and then assaying that biological sample for immunoreactivity.
Immunoreactivity to a portion thereof (e.g, a protein) can be determined using the full length molecule or one or more representative fragments or epitopes thereof The immunological reactivity demonstrated by the subject can be T cell reactivity or B cell reactivity (e.g., has antibodies that specifically bind to the Pc-p27 or polypeptide fragments thereof. T cell and B cell reactivity of a subject to Pc-p27 or polypeptide fragments thereof can be determined by various methods known in the art. Typically, T cell reactivity is performed with an antigenic fragment of the full length molecule. B-cell reactivity can be determined using a full length protein, however in some embodiment, shorter fragments may be used. Any method known in the art can be used to identify the immunological reactivity.
One aspect of the invention relates to a method for diagnosing rheumatoid arthritis in a subject by identifying the presence of antibodies in the subject that specifically bind to P. copri or portion thereof, (e.g., protein Pc-p27 or polypeptide fragments thereof. The assay involves contacting a biological sample obtained from the subject with P. copri or portion thereof (referred to as the test antigen), under conditions that allow an immunocomplex of the antibody and the test antigen to form, and then assaying for the presence of the immunocomplex. In some embodiments the portion of P. copri can be a full length protein of P. copri or one or more representative polypeptide fragments thereof. In some embodiments, the protein is Pc-p27 or one or more polypeptide fragments thereof. Various methods of detecting the presence of an antibody-antigen complex are available in the art, and are suitable for use in the methods described herein, such as ELISA, agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, radioimmunoassay and immunoblot assay. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein, (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). In one embodiment, the assay involves a detectable label that is used to facilitate detection of the complex through detection of the label. The label present in the test sample is compared to label present in a positive control and/or absent in a negative control. Appropriate controls can be determined by the skilled practitioner. In one embodiment, a negative control is the same type of biological sample obtained, for example, from a healthy subject.
Reactivity that indicates detection of a significant level of the antibody complex above background (e.g., that in a negative control) indicates a positive result. In one embodiment, reactivity is identified by detection of the label. In one embodiment, a determination of reactivity that is ≥3 standard deviations (SD) above the mean of a sample of a healthy control subject is considered a positive result. In one embodiment, a result that is at least 2-fold above background (e.g., of the signal of a sample obtained from a healthy control subject) is indicative of positive. In one embodiment, higher levels of detection are used to indicate a positive result (e.g., at least 3-fold, 4-fold, or 5-fold above background).
One aspect of the invention relates to a method for diagnosing rheumatoid arthritis in a subject by identifying the presence of T cells in the subject that are specifically reactive to P. copri or a portion thereof This is typically accomplished by obtaining a biological sample from the subject that contains PBMC, and exposing the cells in the biological sample to P. copri or one or more portion thereof (also referred to as the test antigen), and assaying for a response to the test antigen such as stimulation of proliferation. In one embodiment, peripheral blood mononuclear cells (PBMC) or synovial fluid mononuclear cells (SFMC) of the subject (e.g., in or obtained from the biological sample) are contacted with the test antigen in vitro under conditions conducive to stimulation. The cells are then monitored for a response that indicates stimulation. In one embodiment, T cell proliferation in vitro or cytokine production (e.g., IFN-γ) into the supernatant that indicates stimulation is monitored. Identification of a response (e.g., T cell proliferation or cytokine secretion) substantially over that of an appropriate control sample indicates stimulation has occurred. The level of one or more cytokines that are indicative of T cell activation can be monitored in the supernatant of the cells as an indication of T cell activation. One such cytokine is IFN-γ.
Other cytokines to monitor include, without limitation IL-17, IL-12, and IL-10. The levels of one or more of such cytokines can be monitored. An increase in IFN-γ, IL-17, and/or IL-12, and/or a decrease in IL-10 levels indicates activation. In some embodiments the portion of P. copri can be a full length protein of P. copri or representative polypeptide fragments thereof. In some embodiments, the protein is Pc-p27 or polypeptide fragments thereof. Detection of the stimulation indicates that the sample comprises immunological cells that are immunologically reactive to the test antigen, which in turn indicates that the subject from whom the sample was obtained is immunologically reactive to the test antigen. Put another way, a positive assay result indicates that the test antigen is an autoantigen in the subject, and that the subject has rheumatoid arthritis. Typical assays for use in this method are T cell proliferation assays, such as 3H-thymdine incorporation assay, CFSE dilution, or an ELISPOT, and also T cell reactivity assays. Methods of determining T cell reactivity are well known in the art and are described in for example U.S. Pat. No. 5,750,356A.
Reactivity that indicates a detection of a significant level of stimulation above background (e.g., that in a negative control) is expected to serve as a positive result. In one embodiment, a determination of stimulation that is ≥3 standard deviations (SD) above the mean of a sample of a healthy control subject is considered a positive result. In one embodiment, a result that is at least 2-fold above background (e.g., of the signal of a sample obtained from a healthy control subject) is indicative of positive. In one embodiment, higher levels of stimulation is used to indicate a positive result (e.g., at least 3-fold, 4-fold, or 5-fold above background).
Test antigen for use in the methods and kits herein described include, without limitation, P. copri or a portion thereof. In some embodiments the portion of P. copri can be protein of P. copri or polypeptide fragments thereof. In some embodiments the protein can be full length Pc-p27 and/or polypeptide fragments thereof. The sequence of the full length Pc-p27 is defined by GenBank Accession No. WP_022121928.1, which is incorporated herein by reference in its entirety as SEQ ID NO:1.
In some embodiments the “Pc-p27” of the methods and kits described herein can be a polypeptide fragment including or derived from full length Pc-p27 having the amino acid sequence of SEQ ID NO. 1. The polypeptide fragment refers to fragment of the full length Pc-p27 of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive amino acids of SEQ ID NO:1, that has at least about 70%, 80%, 90%, 100% or more than 100% of the immunological reactivity of Pc-p27 of SEQ ID NO:1. In some embodiments the polypeptide fragment can be signal sequence of Pc-p27. In some embodiments the polypeptide fragment comprises the amino acid sequence; KRIILILTVLLAMLGQVAY (SEQ ID NO: 2), or antigenic portion thereof; DYRGYWTMRYQFDSATVS (SEQ ID NO: 3) or antigenic portion thereof; EKINSLPTSSTGI (SEQ ID NO: 4) or antigenic portion thereof. In some embodiments the polypeptide fragments are predicted to be presented by HLA-DR molecules associated with rheumatoid arthritis.
The polypeptide and encoding nucleic acid sequences of Pc-p27 are publically available e.g., from the NCBI website. The Pc-p27 or a polypeptide fragment can be a homolog of P. copri Pc-p27 or a polypeptide fragment thereof. In some embodiments, the Pc-p27 polypeptide has an amino acid sequence at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence of SEQ ID NO:1 and has at least about 70%, 80%, 90%, 100% or more than 100% of the immunological reactivity of Pc-p27 of SEQ ID NO:1. In some embodiments, the Pc-p27 polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence homology to amino acid sequence of SEQ ID NO:1 and has at least about 70%, 80%, 90%, 100% or more than 100% of the immunological reactivity of Pc-p27 of SEQ ID NO:1. In some embodiments, the Pc-p27 is a polypeptide fragment of SEQ ID NO:1 of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive amino acids of SEQ ID NO:3, that has at least about 70%, 80%, 90%, 100% or more than 100% of the immunological reactivity of Pc-p27 of SEQ ID NO:1.
Percent (%) amino acid sequence identity for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent (%) amino acid sequence homology for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical or strongly similar amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent homology. Non identities of amino acid sequences include conservative substitutions, deletions or additions that do not affect immunological reactivity of Pc-p27. Strongly similar amino acids can include, for example, conservative substitutions known in the art. Percent identity and/or homology can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software software such as BLAST, Align, ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated.
The immunological reactivity can be determined by T cell reactivity or B cell response to Pc-p27 and one or more of polypeptide fragments thereof by the skilled practitioner by assays known in the art, some of which are described herein. Examples of assays for T cell reactivity include, without limitation, measurement of in vitro T cell proliferation, measurement of in vitro IFN-γ secretion, induced in PBMC or SFMC of the subject in response to Pc-p27 (e.g., using an immunogenic polypeptide fragment). Example of assays for B cell responses include, without limitation, detection and/or measurement of antibodies in a biological sample that specifically recognize and/or specifically bind to Pc-p27. Examples of assays for T cell reactivity include, without limitation, measurement of T cell proliferation and IFN-γ secretion by PBMC or SFMC of the subject by Pc-p27 in vitro.
The entire full length protein Pc-p27or protein fragments can be used in the assays described herein. Combinations of full length protein and protein fragments can also be used, as can combinations of proteins/fragments that represent the different antigens (e.g., testing for one or more of Pc-p27 and other known RA autoantigens reactivity in the same assay, or side by side in the same patient). The appropriate polypeptide or fragment thereof for use in the specific method can be determined by the skilled practitioner. The proteins or fragments can be recombinant, purified, isolated, naturally occurring or synthetically produced. The term “recombinant” when used in reference to a nucleic acid, protein, cell or a vector indicates that the nucleic acid, protein, vector or cell containing them have been modified by introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or a protein, or that the cell is derived from a cell so modified. The term “heterologous” (meaning ‘derived from a different organism’) refers to the fact that often the transferred protein was initially derived from a different cell type or a different species from the recipient. Typically the protein itself is not transferred, but instead the genetic material coding for the protein (often the complementary DNA or cDNA) is added to the recipient cell. Methods of generating and isolating recombinant polypeptides are known to those skilled in the art and can be performed using routine techniques in the field of recombinant genetics and protein expression. For standard recombinant methods, see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989); Deutscher, Methods in Enzymology 182:83-9(1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, NY (1982).
Either the full length protein, or protein fragments derived from the full length protein, can be used to identify immunoreactivity of a subject to the proteins disclosed herein. The specific molecules used in the assays described herein (e.g., full length protein(s), representative fragments thereof, or a combination of the full length and fragments) are referred to as the “test antigen” in the assay descriptions. Which fragments of a full length protein to use in the assay can be determined by the skilled practitioner. Typically, a cocktail of fragments derived from the same protein are used for determination of reactivity of a subject to that protein. Depending upon the length, the number of different protein fragments can be determined and optimized by the skilled practitioner. In one embodiment, 2 or more protein fragments are used. In one embodiment, 3 or more, 4, 5, 6, 7, 8, 9, or 10 or more protein fragments are used. In one embodiment, a protein fragment is at least 8 amino acids in length (e.g., ≥8, ≥9, ≥10, ≥11, ≥12, ≥13, ≥14, ≥15 amino acids).
Specific protein fragments can be identified for use in the herein described methods such as those predicted for presentation by HLA-DR molecules. Such protein fragment can be identified by the methods described in the examples and also by a variety of software programs available to the skilled practitioner (Lin et al. BMC Immunology 2008, 9:8). In one embodiment, TEPITOPE 2000 (Sturniolo et al. 1999 Nature Biotechnology. 17; 555-561) is used. Other freely accessible online programs offered by the Immune Epitope Database and Analysis Resource (http://www.iedb.org/) are also available.
Examples of specific protein fragments of Pc-p27 include, without limitation KRIILILTVLLAMLGQVAY (SEQ ID NO: 2), or antigenic portion thereof; DYRGYWTMRYQFDSATVS (SEQ ID NO: 3) or antigenic portion thereof; EKINSLPTSSTGI (SEQ ID NO: 4) or antigenic portion thereof; and combinations thereof.
In one embodiment, reactivity in a subject to other suspected autoantigens is also performed in combination with the herein described assays. One such autoantigen is cyclic citrullinated peptide (CCP). Several assays for detecting anti-citrullinated protein antibody (ACPAs) are known in the art employing mutated citrullinated Vimentin (MCV-assay), filaggrin-derived peptides (CCP-assay) and viral citrullinated peptides (VCP-assay). Methods for determination of reactivity of a subject for anti-CCP and/or other markers for disease diagnosis are known in the art. Non limiting examples include WO 2005/085858 discloses a method of assessing RA by measuring CCP and serum amyloid A (SAA). WO 2005/064307 and US 2007/0264673 assess RA by measuring CCP and IL-6. WO 2005/029091 and US 2006/094056 provide methods to diagnose, treat, or evaluate inflammatory/autoimmune diseases such as RA by sampling fluids from a human with a suspected diagnosis by detecting CCP. US 2007/0148704 and WO 2007/039280 disclose use of CCP and antibodies as biomarkers in diagnosing RA. WO 2006/008183 discloses various biomarkers for RA.
A subject suspected of having rheumatoid arthritis can be tested by the methods described herein. The subject may exhibit one or more symptoms of rheumatoid arthritis prior to performance of the methods. Rheumatoid arthritis (RA) is an autoimmune disease that causes the body's immune system to attack joint tissues, which leads to inflammation of joint lining. Inflammation of the joints is the primary manifestation of RA. Early symptoms of RA include affected joints being swollen, tender, warm, painful and stiff especially in the morning. The stiffness is shown to last for at least an hour. The pain associated with RA is induced at the site of inflammation. Non limiting examples of other symptoms for RA include presence of rheumatoid nodules in the skin, fatigue, joint deformity and reduction in range of motion of affected joints, dryness of eyes and mouth. Early symptoms associated with RA are also seen in patients with other autoimmune related disease such osteoarthritis, fibromyalgia, lupus. Accordingly, in some embodiments of the present invention, the subject exhibits symptoms of RA and/or other autoimmune related disease manifestation known in the art e.g., Tenosynovitis, osteoporosis, carpal tunnel syndrome.
There is no unique test or feature that is pathognomonic for RA. Rather, the diagnosis is made by recognizing a pattern of signs and symptoms. Classification criteria based on symptoms for identifying subjects suffering to early stages of RA and therefore aiding in disease diagnosis are known in the art and are for example set forth in 1987 American College of Rheumatology (ACR) criteria (Arnett et al; 1987) and recently 2010 critieria by ACR and the European League Against Rheumatism (EULAR) (Aletaha et al; 2010).
The diagnostic methods of RA includes X-ray, MRI and/or ultrasound imaging of the affected joints and blood/serological tests. Non limiting examples of blood/serology tests known in the art to be conducted for diagnosis of RA include measurements of ; Erythrocyte Sedimentation Rate (ESR), Rheumatoid factor (RF), Anti-cyclic Citrullinated Peptide (anti-CCP), Antinuclear Antibody (ANA), Uric Acid, complete blood count: measures the numbers of red and white cells in your blood, C-reactive protein (CRP). In RA, 10 HLA-DR alleles with a shared epitope, including the HLA-DRB1*0101 and 0401 alleles (Sturniolo et al., 1999) convey the greatest risk. Accordingly, in some embodiments, the subject has been or is further tested for one or more of RF, ACPA, and one or more of HLA-DR alleles. Non-limiting examples of HLA-DR alleles that can be tested for include HLA-DRB1*0101, HLA-DRB1*0401.
In some embodiments, identification of immunoreactivity to P. copri or portion thereof described herein can be used in diagnosis of RA. In some embodiments, the methods disclosed herein can be used to identify a patient with RA. In some embodiments, the methods disclosed herein can be used for differential diagnosis of RA. Immunoreactivity of a subject to one or more of the proteins can determined in vitro using suitable biological samples disclosed herein by a variety of methods available to the skilled practitioner.
The discoveries presented herein indicate that reactivity to P. copri or portion thereof can be used as a biomarker for the diagnosis of a condition (e.g., RA and associated symptoms). More specifically, subject immunoreactivity (e.g., antigen reactive T cells, and/or antibody response to antigen) is indicative of the condition. As such, another aspect of the invention relates to a method of diagnosing the condition in a subject (e.g., RA) by determining whether the subject is immunologically reactive with P. copri or portion thereof by the herein described methods. The determination may be made in conjunction with the presence of other such symptoms of the condition present in the subject, for example, arthritic symptoms in a subject, or neurologic symptoms in a subject, or cardiac symptoms in a subject. The determination of immunological reactivity of a subject, as compared to an appropriate control, indicates the subject has, or is likely to develop, the condition. The condition can be further confirmed by the determination of immunological reactivity to additional proteins such as antibodies to citrullinated peptides (ACPA), antinuclear antibody (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and presence of rheumatoid factors and other diagnostic tests known to those skilled in the art and described above. In some embodiments, the subject can test negative for one or more of RF, ACPA, Prevotella DNA in their synovial fluid, and or more HLA-DR alleles.
Various assays can be used to identify antibodies present in a serum sample that bind the test antigen (e.g., ELISA, agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, western blot, an immunoblot assay). For example, the assay could be an immunoblot that carries a recombinant test antigen (full length or peptide fragment(s)).
The methods described herein can also include determination of rheumatoid factor, ACPA and/or ANA. For example, the test antigens disclosed herein can be included in or used in conjunction with an assay such as the Sure-Vue® RF (Fishersci), RFscan™ Card Test Kit (BD Biosciences), Anti-Rheumatoid Factor IgM ELISA Kit (abcam), AVITEX® -RF (Omega diagnostics) Citrullinated Protein Antibodies IgG ELISA Kit (Omega diagnostics) Anti-CCP EIA (Bio-Rad), BioPlex® 2200 Anti-CCP (Bio-Rad), CCPoint® (Eurodiagnostica), DIASTAT® anti-nuclear antibody (ANA) test (Eurodiagnostica), DIASTATR PR3-ANCA® (Eurodiagnostica). The methods described herein can also include determination of one or more of immunological reactivity to filamin-A, immunological reactivity to N-acetylglucosamine-6-sulfatase, such as disclosed in International Patent Publication WO 2016/183310, the contents of which are incorporated herein by reference.
Test-antigen reactive T cells in PBMC and SFMC can be assessed using a number of assays. For example, reactive T cells in PBMC and SFMC can be assessed using tetramer reagents comprising recombinant HLA-DR molecules and test antigen epitopes.
The test antigen may comprise naturally occurring or analog or derivative amino acids, as long as the immunoreactive or immunostimulatory nature of the peptide is retained to sufficient degree to allow T cell activation and/or antibody binding. Thus, some amino acids may be added to or subtracted from the native protein or polypeptide fragments as known in the art. Additionally, some amino acids of the native human protein or polypeptide fragments may be substituted with amino acids that occur in other species, or be substituted as known in the art. Amino acid substitution exchange groups and empirical similarities between amino acid residues, can be found in standard texts such as Schulz et al., Principles of Protein Structure, 14 16 (Springer-Verlag, New York, 1979). There is a limit to how much substitution can be tolerated before the original tertiary structure is lost. Typically, tertiary structure conservation would be lost when the amino acid sequence varies by more than 50%. See, e.g., Chothia & Lesk, Relation between the divergence of sequence & structure in proteins, 5 EMBO J. 823 (1986).
Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., 247 Science 1306 (1990). Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244 Science 1081 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as antibody binding and/or T cell stimulation. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899 (1992); de Vos et al., 255 Science 306 (1992).
The genes and encoded proteins of the autoantigens described herein have been sequenced and are available at numerous sources. Further, the genes are conserved in human, chimpanzee, rat, and zebrafish. Hence, these peptides can include those derived from non-human sources or appropriate sequence information. In an aspect of the invention, the test peptide is predicted to be presented by HLA-DR molecules associated with chronic inflammatory arthritis (e.g., RA). In some embodiments the peptide of the present invention can be presented by one or more HLA-DR disclosed in reference (Sturniolo et al., 1999), the contents of which are incorporated herein in its entirety.
As noted above, generally, amino acid substitutions should be made conservatively; i.e., a substitute amino acid should replace an amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Variants within the scope of this invention may also, or alternatively, contain other modifications, including the deletion or addition of amino acids, that have minimal influence on the stimulatory properties, antibody binding, tertiary structure of the peptide. Thus, for example, conservative substitutions in a protein fragment can be made with the proviso that functional activity is retained to a meaningful degree such that the particular assay (e.g., T cell reactivity or immunoassay) works as intended to provide evidence in diagnosing subjects (e.g., with RA).
Moreover, peptides often contain amino acids other than the twenty “naturally occurring” amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Creighton, PROTEINS—STRUCTURE & MOLECULAR PROPERTIES (2nd ed., W. H. Freeman & Co., New York, 1993). Many detailed reviews are available on this subject, such as by Wold, POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, 1-12 (Johnson, ed., Academic Press, New York, 1983); Seifter et al. 182 Meth. Enzymol. 626 (1990); Rattan et al., 663 Ann. N.Y. Acad. Sci. 48 (1992). Accordingly, the peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code.
Further, “derivatives” of a test antigen contain additional chemical moieties not normally a part of the protein. Covalent modifications of the autoantigens are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents, well-known in the art, is useful for cross-linking the antigen or fragment to a water-insoluble support matrix or to other macromolecular carriers. Derivatives also include radioactively labeled peptides that are labeled, for example, with radioactive iodine (1251, 1311), carbon (14C), sulfur (35S), tritium (3H) or the like; conjugates of peptides with biotin or avidin, with enzymes, such as horseradish peroxidase, alkaline phosphatase, (β-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholine esterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemoluminescent agents (such as acridine esters), or fluorescent agents (such as phycobiliproteins).
Structural analogs of the autoantigens identified herein are provided by known method steps based on the teaching and guidance presented herein. Knowledge of the three-dimensional structures of proteins is crucial in understanding how they function. The three-dimensional structures of hundreds of proteins are currently available in protein structure databases (in contrast to the thousands of known protein sequences in sequence databases). Analysis of these structures shows that they fall into recognizable classes of motifs. It is thus possible to model a three-dimensional structure of a protein based on the protein's homology to a related protein of known structure. Many examples are known where two proteins that have relatively low sequence homology, can have very similar three dimensional structures or motifs.
It is possible to determine the three dimensional structures of proteins of up to about 15 kDa by nuclear magnetic resonance (NMR). The technique only requires a concentrated solution of pure protein. No crystals or isomorphous derivatives are needed. The structures of a number of proteins have been determined by this method. The details of NMR structure determination are well-known in the art. See, e.g., Wuthrich, NMR of Proteins & Nucleic Acids (Wiley, N.Y., 1986); Wuthrich, 243 Science 45 (1989); Clore et al., 24 Crit. Rev. Bioch. Molec. Biol. 479 (1989); Cooke et al., 8 Bioassays 52 (1988).
Thus, according to the present invention, use of NMR spectroscopic data can combined with computer modeling to arrive at structural analogs of at least portions of the autoantigen (peptides or epitopes) based on a structural understanding of the topography. Using this information, one of ordinary skill in the art can achieve structural analogs of the autoantigens such as by rationally-based amino acid substitutions allowing the production of peptides in which the binding affinity or avidity is modulated in accordance with the requirements of the expected diagnostic use of the molecule, for example, the achievement of greater binding specificity or affinity.
The herein described methods may further be used to indicate a therapy for the subject who tests positive. The identification of immunoreactivity to P. copri, portion derived from it e.g., to protein Pc-p27 or polypeptide fragments thereof in RA, as provided herein, is an important addition to the clinician's arsenal in combating chronic inflammatory arthritis, and assists the clinician in choosing the course of therapy. For example, when RA is diagnosed, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, naproxen sodium) or steroids (e.g., corticosteroid medication such as prednisone); or disease modifying anti-rheumatic drugs (DMARDs), such as hydroxychloroquine, sulfasalzine or methotrexate, may be prescribed. DMARDs can be synthetic or biologic in nature. Additionally or alternatively, anti-TNF therapy (e.g., HUMIRA® (adalimumab), ENBREL® (etanercept), may be beneficial. Other non-limiting examples include Orencia® (abatacept), Kineret® (anakinra), certolizumab® (Cimzia), Simponi® (golimumab), Remicade® (infliximab), Actemra® (tocilizumanb) and Xelijanz® (tofacitinib).
It is also contemplated that P. copri—directed therapy, such as targeting P. copri with antibiotics, consumption of probiotic supplements may also be beneficial. In one embodiment, the subject is provided one or more therapies following an indication that they are immunoreactive to an autoantigen. As such, another aspect of the invention relate to a method of treating a subject diagnosed with a RA. The method comprises diagnosing the subject for the disorder by one or more of the herein described methods, and then treating the subject for said condition. Diagnosis may further include assessing the subject for other symptoms such as a neurological condition or heart condition typically seen with secondary RA symptoms. In some embodiments of the present invention, the methods to determine immunoreactivity to autoantigens disclosed herein can be conducted prior to, during and/or after a therapeutic treatment and therefore aid in assessment of effectiveness of the treatment and/or monitor disease progression.
Another aspect of the invention relates to a kit for identifying a subject with a condition such as rheumatoid arthritis. (e.g., citrullinated protein/peptides such as citrullinated filaggrin, fibrinogen, fibronectin, α-enolase, collagen type II, histones, vimentin). The kit comprises one or more of P. copri or a portion thereof as test antigens. In some embodiments the portion of P. copri can be protein of P. copri or a polypeptide fragment thereof. In some embodiments the protein can be Pc-p27 or one or more polypeptide fragments thereof, such as a set of synthesized peptides, fragments, or epitopes thereof. In some embodiments the kit further comprises reagents necessary for conducting an assay capable of detecting the presence of immunoreactivity of a subject to that test antigen (e.g., an antibody in a sample obtained from said subject that binds to Pc-p27 or polypeptide fragment thereof). The kit can be designed for any of the various assays described herein. In one embodiment, the assay in the kit is an enzyme-linked immunosorbent assay (ELISA) or immunoblot, the components for which are well-known in the art. The peptides may be synthesized or obtained from natural or recombinant sources, each of which is well-known in the art. The kit may further include other autoantigens (e.g, citrullinated protein or fragments/epitopes thereof) known in the art, such as those described herein.
In some embodiments, the kit can further comprise reagents for conducting an assay for detecting the presence of one or more cytokines and/or chemokines. Non-limiting examples of cytokines and/or chemokines that can be assayed include one or more of IL-23, IL-22, IL-17E, IL-17F IFN-γ, TNF, IFN-α, MIP-1α, MMP-1β, IL-17F, IL-1β, CXCL9, CXCL10, IL-12. In some embodiments, the kit can further comprise E.coli and/or B. fragilis whole cells or potential antigen derived from it. In some embodiments, the kit can also include components to assay for Prevotella DNA in the biological sample. Methods to assay Prevotella DNA in a biological sample are disclosed herein and are known in the art for example in WO2013056222A1, the teachings of which are incorporated herein in its entirety. The kit may alternately further comprise buffers, enzymes, and/or containers for performing the reactions or analyses. The various reagents within the kit may be provided separately or together as is convenient in a container such as a vial, test tube, flask, bottle or even syringe. The components may be suitably aliquotted for performance of the methods. The kit may further contain one or more positive and/or negative controls. The antigens or other components of the kit may be labeled with a detectable marker. In some embodiments the kit can be used to identify a patient with new onset rheumatoid arthritis or chronic rheumatoid arthritis.
In some embodiments, the subject is further tested for Prevotella DNA in their synovial fluid. Assays for P. copri DNA in a biological sample are disclosed herein and are known in the art for example in WO2013056222A1, the teachings of which are incorporated herein in its entirety. In some embodiments, the subject tests negative for Prevotella DNA. In some embodiments, the subject can test positive for Prevotella DNA. In some embodiments the test antigens can be included in kit for use in differential diagnosis of rheumatoid arthistis and other diseases for example for Lyme disease. Accordingly, the test antigens of the present invention can be used in conjunction with an assay such as the Borrelia ViraStripe® IgG, IgM test kit (Viramed Biotech AG, Planegg, Germany). The Borrelia ViraStripe® is an immunoblot that carries native, purified antigens from Borrelia afzelii (Pko), Borrelia burgdorferi sensu stricto, and recombinant Borrelia antigen VlsE. Alternatively, the test antigen may be included in or used in conjunction with an immunoassay such as the Borrelia B31 ViraBlot® Western blot test kits (Viramed Biotech AG, Planegg, Germany) which identify anti-Borrelia antigen-binding IgG and/or IgM in the serum of suspected Borrelia-infected patients.
Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used to described the present invention, in connection with percentages means ±1%.
In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not (“comprising). In some embodiments, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the invention (“consisting essentially of”). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments, the inventions, compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (“consisting of”).
All patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
The present invention may be as defined in any one of the following numbered paragraphs:
The invention is further illustrated by the following examples, which should not be further construed as further limiting.
An unbiased, discovery-based approach was developed to identify novel, immunogenic T cell epitopes in patients with chronic inflammatory arthritis. With this approach, in vivo HLA-DR presented peptides in patients' synovial tissue, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) are identified by tandem mass spectrometry (LC-MS/MS) (Seward et al., 2011), followed by testing with patients' samples to determine the antigenicity of identified peptides and their source proteins. Using this method, 4 novel autoantigens in Lyme arthritis (Crowley et al., 2015; Crowley et al., 2016; Drouin et al., 2013; Pianta et al., 2015) and 2 new autoantigens in RA (Pianta A, 2015b) that are targets of T and B cell responses in many patients with these diseases were identified.
Recently, this approach was used to search for T cell epitopes of proteins derived from microbes implicated in RA. Reported herein is the identification of an HLA-DR-presented peptide (T cell epitope) derived from a P. copri 27-kD protein (Pc-p27), which stimulated Th1 responses in 42% of RA patients. It was also found that P. copri induced differential immune responses to this protein or the whole organism in a substantial portion of RA patients. Although not mutually exclusive, one type of immune response, a Th17 response, was indicative of a mucosal immunity that may help contain the organism but enhance autoimmunity. The other was indicative of a systemic Th1 immune response, accompanied by spread of Prevotella to joints, but with less frequent ACPA. Taken together, P. copri appears to be an immune-relevant bacterium in RA disease pathogenesis.
RA patients and control subjects. The study, “Autoantigens in RA,” was approved by the Human Investigations Committee at Massachusetts General Hospital (MGH); all subjects gave written informed consent. All RA patients met the American College of Rheumatology/European League Against Rheumatism Collaborative Initiative criteria for that disease (Aletaha et al., 2010). All study patients with RA or other rheumatic diseases were recruited from the Rheumatology Clinic at Massachusetts General Hospital (MGH) or from suburban MGH clinics.
For isolation of HLA-DR-presented peptides, synovial tissue, SFMC or PBMC were obtained from 5 patients with RA. To test implicated peptides and their source proteins for immunoreactivity in larger numbers of patients, the cohort of NORA patients were used from whom systematic clinical information, PBMC and serum samples, and in some cases, SF were available. For comparison, PBMC and serum samples were available from patients with Lyme arthritis. In addition, serum and sometimes SF samples were collected from CRA patients, and serum samples were obtained from patients with other types of arthritis or connective tissue diseases, and from healthy control subjects. HLA-DR typing was performed on blood samples from RA patients at the American Red Cross Laboratory in Dedham, Mass. ACPA and RF determinations were made in the clinical laboratories at MGH.
Enzyme-linked immunospot (ELISpot) T cell assay. A detailed description of the methods for isolation and identification of HLA-DR presented peptides is given in Seward et al., 2011. In initial experiments in this study, one microbial peptide (2KRIILILTVLLAMLGQVAY20)(SEQ ID NO: 6) derived from a 27 kDa P. copri protein (WP_022121928.1) was identified from the PBMC of one RA patient. This P. copri peptide and 2 additional peptides from the same protein, which were predicted to be promiscuous T cell epitopes (52DYRGYWTMRYQFDSATVS69 (SEQ ID NO: 7), 118EKINSLPTSSTGI130 (SEQ ID NO: 8)), were synthesized and HPLC-purified in the Core Proteomics Laboratory at MGH. PBMC from RA patients were then stimulated with the peptides (1 μM) in duplicate along with positive (phytohemagglutinin) and negative (no antigen) controls, and incubated for 5 days in culture in a CO2 incubator at 37° C. Cells were then transferred to Dual Color ELISpot plates coated with IFN-γ/IL17 antibodies (Cellular Technology Limited), and incubated overnight at 37° C. Images of wells were captured using ImmunoSpot series 3B analyzer, and spots were counted using ImmunoSpot software. A positive T cell response was defined as 3 standard deviations (SD) above the mean value of healthy subjects.
ELISA for serum IgG and IgA antibodies to the P. Copri protein (Pc-p27). ELISA plates were coated with 0.25 μg/ml of the P. copri protein Pc-p27 (GenScript USA Inc, Piscataway, N.J.) overnight at 4° C. Subsequent incubations and washes were performed at room temperature. After washing with phosphate buffered saline with 0.05% Tween-20 (PBST), the plates were incubated with blocking buffer (5% nonfat dry milk in PBST) for one hour. Afterwards, 100 μl of each patient's serum sample (diluted 200-fold) was added in duplicate wells for 1.5 hours, followed by horseradish-peroxidase (HRP)-conjugated goat anti-human IgG (sc-2453, Santa Cruz Biotech) or HRP-conjugated goat anti-human IgA (Bio-Rad, Hercules, Calif.) and then TMB substrate (BD, San Diego, Calif.). For inter-plate standardization, 2 control samples were included on each plate.
ELISA for serum IgG and IgA antibodies to gut microbial organisms. The P. copri type strain (DSM 18205) was obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). B. fragilis (ATCC 25285) and E. coli (ATCC 25922) isolates were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). The bacterial cultures were inactivated in 1% formalin for 24 hours and washed twice in PBS and diluted in PBS at a final concentration of 106 cells/ml.
IgG and IgA antibody responses against whole-cell P. copri, B. fragilis or E. coli were determined by ELISA. All analyses were done in duplicate using 100 μl of solution per well. ELISA plates were coated overnight at 4° C. with suspensions of inactivated whole bacterial cells (106 cells/ml). After washing with PBST, the plates were incubated with blocking buffer for one hour. Afterwards, each patient's serum sample (diluted 1:100) was added in duplicate wells for 1.5 hours, followed by horseradish-peroxidase (HRP)-conjugated goat anti-human IgG (sc-2453, Santa Cruz Biotech) or HRP-conjugated goat anti-human IgA (Bio-Rad) and then TMB substrate (BD). For inter-plate standardization, 2 control samples were included on each plate.
Cytokine and chemokine determinations. The levels of 14 cytokines and chemokines associated with innate immune responses (IFN-γ, TNF, MIP-1α, MIP-1β), and Th1 (IFNγ, IL-12, CXCL9, and CXCL10) or Th17 (IL-1β, IL-17A, IL-17E, IL-17F, IL-22, and IL-23) adaptive immune responses were determined in serum or SF samples from RA patients. Samples were diluted 1:5 in PBS and incubated with Heteroblock (Omega Biologicals) at a concentration of 150 μg/ml to limit the possible confounding effects of rheumatoid factor. Protein levels of all 14 inflammatory mediators in serum or SF were assessed in one complete experiment using bead-based Luminex assays (EMD-Millipore) coupled with the Luminex-200 System Analyzer (Luminex, Austin, Tex.). Data were assessed using the xPONENT 3.1 software.
Detection of P. Copri DNA in patients' samples by PCR. DNA was isolated from 200 μl of serum or synovial fluid using the QIAamp DNA mini (QIAGEN), according to manufacturer's protocol. Nested PCR primers were designed to detect DNA for the P. copri ribosomal 16S RNA gene (16S rDNA) using Primer3 software. Target DNA was amplified for outer PCR using the forward primer CCAAGTAGCGTGCAGGATGA (SEQ ID NO: 9) and the reverse primer TTCAAGCCCGGGTAAGGTTC (SEQ ID NO: 10). One μl of the amplified DNA (1:10 in sterile distilled water) was used for the second PCR reaction using the forward primer CGCGGTAATACGGAAGGTCC (SEQ ID NO: 11) and the reverse primer GATACCCGCACTTTCGAGCT (SEQ ID NO: 12). Both reactions were carried out using 2.5 U of HotStarTaq DNA polymerase (QIAGEN), according to manufacturer's protocol. The amplification program included 40 cycles with denaturation 94° C. for 30 s, annealing 59° C. for 30 s, extension 72° C. for 50 s, and a final extension of 10 min. For both outer and nested PCR reactions, one positive control (P. copri DNA derived from the type strain, DSM 18205) and a negative control (sterile distilled water) were included. When enough DNA was available, samples were tested in duplicates. Amplified products (10 μl) were visualized by electrophoresis in a 2% agarose gel. Correct identity of PCR products were validated by direct DNA sequencing carried out in the Center for Computational & Integrative Biology DNA CORE facility at MGH. The sequenced product was aligned with all human and known microbial genomes using Genomic Blast Sequence.
Statistical Analyses. Categorical data were analyzed by Fisher's exact test, and quantitative data were analyzed using unpaired t test with Welch correction. Correlations were sought using Pearson's correlation test. All analyses were performed using GraphPad Prism 6. All P values were two-tailed. P values <0.05 were considered statistically significant.
Using tandem mass spectrometry (LC-MS/MS), HLA-DR-presented peptides in synovial tissue (N=4), SFMC (N=3) or PBMC (N=2) were identified from 5 patients with NORA or chronic RA (CRA). From the 17 HLA-DR-presented peptides identified from the PBMC of one patient (called here RA1), one P. copri sequence was found (
At disease onset, patient RA1, who had 2 copies of the RA “shared epitope” (HLA-DRB1*0401 and 0101), had severe symmetrical polyarthritis affecting large and small joints. During the course of the disease, patient RA1developed a positive test for ACPA, but not RF. Despite treatment with disease modifying anti-rheumatic drugs (DMARDs), patient RA1had recurrent episodes of knee swelling with evidence of destructive changes in cartilage and bone. The HLA-DR-presented peptide derived from P. copri was identified from PBMC obtained during one such episode 7 years after disease onset.
The peptide sequence of 19 amino acids had 100% sequence homology with the signal sequence of a 27-kD protein of P. copri (Pc-p27, WP_022121928.1) (
T Cell Reactivity to P. copri Peptides in NORA Patients
To determine the immunogenicity of HLA-DR-presented peptides of Pc-p27 more broadly, PBMC obtained from the cohort of patients with new-onset RA (NORA) seen prior to DMARD therapy was used, the time when immune responses would be expected to be most robust. When PBMC from 39 NORA patients and from the case patient RA1 (a CRA patient) were stimulated with the signal peptide sequence from Pc-p27 peptide 1, 17 of the 40 patients (42%) secreted levels of IFN-γ that were >3 SD above the mean value of HC (P=0.0002), using an IFN-γ/IL-17 Double-Color ELISpot assay (
To determine whether patients had reactivity with other epitopes of the Pc-p27 protein, TEPITOPE was used to predict 2 additional promiscuous peptides derived from the same protein (Pc-p27 peptides 2 and 3). The 2 peptides together were predicted to be presented by all 25 HLA-DR molecules in the program, and therefore, these peptides were pooled for testing. PBMC from 14 of the 40 patients (35%) secreted levels of IFN-γto peptides 2 and 3 that were >3 SD above the mean value of HC (P=0.0007) or patients with LA (P=0.006) (
Because of the importance of citrullinated proteins in RA pathogenesis (Brink et al., 2013; McInnes and Schett, 2011), peptide 1 was re-synthesized with a citrulline in place of the only arginine in the peptide, which was predicted to be in the—P1 position in the flanking region of the HLA-DR binding pocket. The citrullinated peptide induced an IFN-γ response in 36% of patients compared with 44% who had reactivity with the uncitrullinated peptide (
IgG and IgA Antibody Responses to Pc-p27 and Whole P. copri
Since the role of P. copri-specific CD4+ T cells is to help B cells produce antibodies to the organism, the antibody responses to Pc-p27 in serum samples from 303 individuals was determined. These included samples from 127 patients with new-onset or chronic RA, 28 patients with connective tissue diseases (14 with systemic lupus, 4 with mixed connective tissue disease, 4 with scleroderma, and 6 with Sjogren's syndrome), 28 patients with spondyloarthropathy (15 with psoriatic arthritis, 10 with ankylosing spondylitis, and 3 with reactive arthritis), 70 patients with Lyme arthritis, and 50 healthy control subjects.
Of the 78 NORA patients, 10 (13%) had IgG antibody responses to Pc-p27 that were >3 SD above those in HC (P<0.0001) (
Because the first interactions between P. copri and immune cells would presumably occur in the gut mucosa, the IgA antibody responses to the organism was also determined. About 10% of the patients in both the NORA and CRA groups had IgA antibody responses to Pc-p27 (P<0.0002 and P<0.02, respectively), and the responses was more robust in NORA patients (
When both T and B cell responses were considered together, 3 of the 24 patients who had T cell reactivity with Pc-p27 peptides also had IgG Pc-p27 antibody responses, but none had IgA responses to the protein. In comparison, among 16 patients lacking T cell reactivity to Pc-p27 peptides, only 1 had an IgG antibody response to the protein, but 5 had IgA responses (P=0.05). The frequencies of “shared epitope” alleles in patients with P. copri T or B cell responses was not significantly different than those in patients who lacked these responses.
To confirm and extend these findings, the frequency of IgG and IgA antibody responses to whole P. copri was determined using the same set of 303 serum samples. Six of the 78 NORA patients (8%) and 5 of the 49 CRA patients (10%) had IgG antibody responses to P. copri that were >3 SD above the mean values in healthy controls (HC) (P<0.005 and P<0.0001, respectively) (
When the antibody responses to whole P. copri or recombinant Pc-p27 were combined, 41 (32%) of the 127 RA patients had IgG or IgA antibody reactivity with the organism. With both preparations, antibody responses to P. copri were common in RA patients, both early and late in the disease, yet they were rarely found in patients with other types of arthritis, implying specificity in RA.
Testing for Antibody Responses to other Commensal Bowel Flora
For comparison, antibody responses were determined to two other gut commensal organisms, using whole cell preparations of Bacteroides fragilis and Escherichia coli. In contrast to the findings with P. copri, very few RA patients or those with other types of chronic inflammatory arthritis or connective tissue diseases had IgG or IgA antibody responses to B. fragilis or E. coli that were >3 SD above the mean values in healthy control subjects (
Correlation of P. copri Antibodies with Serum Cytokine Levels and RA Autoantibodies
To link P. copri with inflammatory responses and autoantibody production, IgG and IgA P. copri antibody values were correlated with serum cytokine levels and RA autoantibody frequencies (ACPA and RF) in 120 of the 127 RA patients in whom sufficient serum samples were still available. For this analysis, the values in the 37 NORA and CRA patients who had positive P. copri antibody responses were compared with either Pc-p27 or the whole organism with those in the 83 patients who lacked these antibody responses. The 14 cytokines and chemokines measured were representative of innate, Th1 and Th17 immune responses. These assays were performed with Heteroblock to limit the possible interference by rheumatoid factor.
When the levels of these mediators were compared in patients and healthy controls, two cytokines associated with innate immunity (IFN-α and TNF) and four cytokines and chemokines associated with Th1 immunity (CXCL9, CXCL10, IFN-γ, and IL-12) were significantly higher both in P. copri-positive and P. copri-negative RA patients compared with healthy controls. However, no significant differences were observed between patients who did or did not have P. copri antibodies (
When the magnitude of P. copri IgG or IgA antibody responses were correlated with cytokine levels, there were strong, positive correlations between IgA antibody values and the levels of three innate cytokines (IFN-γ, MIP-1α, and MMP-1β), but no correlations were found with P. copri IgG antibody responses (
Among 14 patients in whom SF samples were available, the innate inflammatory mediators (IFN-γ, TNF, MIP-1α, MIP-1β) and pro-inflammatory Th1 mediators (IFN-γ, IL-12, CXCL9 and CXCL10) were concentrated in SF, whereas the levels of several Th17-associated cytokines (IL-23, IL-22, IL-17E and IL-17F) were significantly greater in serum (
When P. copri IgG or IgA antibody responses were stratified according to RA autoantibodies, only 37% of the patients with IgG P. copri antibody responses had ACPA compared with 74% with IgA P. copri antibodies (P=0.006) and 70% with no P. copri antibodies (P=0.01) (
Detection of P. copri 16S rDNA in Synovial Fluid
Because P. copri IgG antibody responses were indicative of a systemic immune response to the organism and because Th1 cytokines were concentrated in SF, the question of whether P. copri itself may spread to joints was addressed. For this purpose, nested PCR primers were designed in an effort to detect DNA for the 16S ribosomal RNA gene of P. copri (16S rDNA) in patients' samples. Of 18 patients in whom paired serum and SF samples were available, 10 were obtained from NORA patients who were seen prior to DMARD therapy, and 8 were collected from CRA patients who were seen from 3-to-50 years after disease onset. Five of the 18 patients had IgG antibody responses to P. copri; 2 had IgA antibody reactivity with the organism, and 11 did not have P. copri antibody responses.
Of the 5 patients (RA1 to RA5) with IgG P. copri antibodies, 3 had P. copri 16S rDNA detected in SF. Of 2 samples from NORA patient RA2, 16S rDNA was found in one SF sample obtained prior to DMARD therapy and in one collected 2 months after the start of therapy (
All positive results were confirmed by sequencing. Amplicons from patient RA2 had 100% sequence identity with the sequence for P. copri 16S rDNA in the NCBI database, which was obtained from a Japanese isolate. Interestingly, patient RA2 grew up in Korea. Amplicons from patient RA1 and from patient RA5 annealed with P. copri sequences (DSM18205), but they had 86% and 89% sequence homology, respectively (
In this study, using a discovery-based search for HLA-DR-presented peptides derived from P. copri, one spectrum-to-peptide match was identified from 1 of the 2 RA patients in whom sufficient PBMC were available for this type of high-throughput analysis. This peptide sequence was found in the signal domain of a 27-kD protein of P. copri, which was predicted to be a secreted protein. Signal sequences, which are cleaved prior to secretion, can accumulate in transmembrane locations; they are often highly antigenic, and they typically bind many different HLA-DR molecules (Kovjazin et al., 2011). In addition, the secreted portion of the protein may become an immunogenic soluble antigen (Han and Wang, 2013; Yang et al., 2014). It was then found that the signal sequence peptide and two other peptides from the Pc-p27 protein induced Th1 responses in 60% of patients with RA. Although RA SE alleles correlated inversely with P. copri overexpansion in the gut (Scher et al., 2013), the findings showed no significant correlations between these T cell epitopes and SE alleles, consistent with the fact that these epitopes are promiscuous HLA-DR binders.
In addition to T cell reactivity, IgA or IgG antibody responses to P. copri were found, but usually not both, in 32% of NORA patients. Although over-expansion of P. copri in stool samples was previously detected only in NORA patients (Scher et al., 2013), IgG or IgA antibody responses to the organism were found as commonly in CRA patients as in NORA patients, indicating that once initiated, these antibody responses may persist for years. In contrast, immune reactivity with P. copri was rarely detected in patients with other chronic inflammatory arthritides, including those with Lyme arthritis, spondylarthropathies or other connective tissue diseases, implying specificity in RA.
It was recently reported in mice that gut symbiotic gram-negative bacteria may disseminate systemically to other sites and induce IgG antibody responses (Zeng et al., 2016). These homeostatic IgG responses may help later in protection against invasion by pathogenic gram-negative bacteria. High background in E. coli and B. fragilis antibody assays were found, perhaps reflecting positive responses to these bacteria in some individuals. However, the distribution of antibody values to these bacteria was not greater in RA patients than in other comparison groups, whereas P. copri antibody responses were significantly higher in RA patients and correlated with inflammatory cytokine levels. For this reason, these findings could not be explained simply by homeostatic P. copri antibody responses.
The magnitude of IgA P. copri antibody responses in RA patients were found to correlate with serum levels of a range of cytokines and chemokines associated with innate, Th1 and Th17 immune responses, such that the higher the IgA antibody response, the greater the cytokine level. Although Pc-27-specific Th17 cells in PBMC in this subgroup of patients was not found, it is likely that such cells were present in their intestinal mucosa, as shown by the strong correlation of IgA antibody responses with a range of Th17-associated cytokines. Th17 responses are important in containing the organism in the bowel, but they can also promote systemic inflammation and autoimmunity (Ruff and Kriegel, 2015). This idea is indicated here by strong correlations between IgA antibody values and serum levels of innate, Th1, and Th17-associated cytokines along with high frequencies of ACPA, which could react with citrullinated proteins in joints (Romero et al., 2013).
Conversely, the other RA subgroup had IgG P. copri antibody responses, sometimes with Prevotella DNA in joints, which was associated with inflammatory Th1 responses and infrequent ACPA. Although a mucosal IgA response might occur prior to systemic spread of the organism, most of the patients had only IgA or IgG antibody responses, but usually not both. In IgG-positive patients, it was postulated that P. copri, a strict anaerobe, may spread to joints within phagocytic cells. In 2 of 3 patients, Prevotella DNA was detected in SF obtained early in the disease. However, in the remaining patient (RA1), in whom the Pc-p27 HLA-DR-presented peptide was identified in PBMC obtained 7 years after disease onset, P. copri DNA was detected in SF collected 9 years after onset. This indicates that P. copri may sometimes spread from the bowel in repeated waves over a period of years, perhaps explaining the persistence of antibody responses to P. copri in CRA patients. In addition to P. copri, it is possible that other Prevotella species or other yet-to-be-identified commensal gut microbes may have an immune-relevant function in RA.
There are parallels between the potential role P. copri in RA pathogenesis and that of the periodontal pathogen, Porphyromonas gingivalis (Mikuls et al., 2014; Pischon et al., 2008). In patients with periodontitis, the composition of the oral flora shifts in favor of organisms, particularly anaerobes such as P. gingivalis, which thrive in an inflammatory environment (Hajishengallis, 2015). IgG antibody responses to P. gingivalis in RA patients correlate strongly with systemic inflammation and co-existent periodontal disease (Arvikar et al., 2013; Mikuls et al., 2014; Scher and Abramson, 2013). In addition, P. gingivalis has a PPAD enzyme, which catalyzes the post-translational conversion of arginine to peptidylcitrulline (Konig et al., 2015; Rosenstein et al., 2004; Wegner et al., 2010), and this may lead to the generation of neoepitopes and ACPA (Wegner et al., 2010). Furthermore, P. gingivalis may disseminate from the gingiva, presumably via dendritic cells, also leading to infection or inflammation at distant sites (Carrion et al., 2012; Han and Wang, 2013), and DNA from periodontal bacteria has been detected by PCR in SF of a few RA patients with periodontal disease (Martinez-Martinez et al., 2009).
Provided herein is evidence that P. copri is an immune-relevant bacterium in a subgroup of RA patients. Th17 and IgA immune responses may help contain the organism in the bowel, but may enhance systemic inflammation and autoimmunity, whereas P. copri IgG antibodies, sometimes with Prevotella DNA in joints, were associated with P. copri-specific Th1 responses and infrequent ACPA. These new insights are likely to have implications for both the diagnosis and treatment of RA. For example, P. copri IgG antibody responses could provide support for the diagnosis in RA patients who lack ACPA or RF. Regarding therapy, approaches that target P. copri, such as specifically tailored antibiotic regimens or diet alterations or probiotic therapies, could have an adjunctive role to DMARDs in treatment of the disease. Previously, patients with early RA who received tetracycline or its derivatives had significantly better outcomes than placebo-treated patients (O'Dell et al., 1997; Stone et al., 2003). These results are often attributed to the anti-inflammatory effects of tetracyclines, but recent insights about the microbiota indicate an additional explanation. Importantly, insights about specific immune-relevant commensal organisms in the microbiota promise a new era in the understanding and treatment of RA.
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Rheumatoid arthritis (RA) is an HLA class II-associated autoimmune disease, in which arthritogenic T cells drive progressive inflammation and destruction of synovial joints (1). Both genetic and environmental factors are thought to contribute to disease development and progression. The greatest genetic risk factor is HLA-DRB1 susceptibility alleles that share a 5 amino acid sequence in the B1 chain, termed the RA “shared epitope” (SE) (2). HLA-DRB1 SE alleles largely influence the development of seropositive RA, which is defined by positive tests for rheumatoid factor (RF) and/or anti-citrullinated-protein antibodies (ACPA) (3), the latter being the only known specific autoantibodies for this disease (4-6). RF and/or ACPA may develop years before the onset of clinical arthritis (7-9), indicating that autoimmunity may be triggered at sites other than joints in RA patients.
Causative environmental factors are less well characterized. However, T cell epitope mimicry between microbial pathogens and self proteins has been implicated as a possible factor in the induction or exacerbation of autoimmune disease (10-12). In addition, alteration in the oral or gut microbiota may affect mucosal immunity inducing aberrant immune responses affecting joints in RA patients (13, 14). Using high through-put sequencing, Scher et al. showed that Prevotella species, including P. copri, in the gut microbiota were expanded in stool samples from patients with new-onset RA (NORA), indicating that these organisms might have this role in RA pathogenesis (13). Moreover, a recent study in mice showed that gut dysbiosis contributes to arthritis development via activation of autoreactive T cells in the intestine (15). Proposed mechanisms to link infection and autoimmunity include molecular mimicry between T-cell microbial and host epitopes (16); infection-induced alteration and release of sequestered self antigens (11), or non-specific, infection-induced inflammatory responses which function as adjuvants in the induction of pathogenic autoimmunity (17, 18).
Identification of disease-relevant infectious or self antigens has been challenging in any autoimmune disease, but current discovery-based methods offer innovative approaches to this problem.
A novel approach for antigen detection in chronic inflammatory arthritides was developed in which HLA-DR-presented peptides (T cell epitopes) are identified directly from patients' inflamed synovial tissue, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) by tandem mass spectrometry (LC-MS/MS), and tested for immunogenicity using patients' samples (19-24). With this approach, it was recently identified an HLA-DR-presented peptide from a 27-kD protein of P. copri (Pc-p27), which stimulated T and B cell responses in about 40% of RA patients, but not in patients with other rheumatic diseases or in healthy controls (25). Using the same methodology, 2 novel autoantigens were identified, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA), that are targets of T and B cell responses which appear to be specific for RA. Both autoantigens are highly expressed in inflamed synovial tissue; they share homologous T cell epitopes with Prevotella and several other gut microbes, and they are targets of specific T and B cell responses in patients with RA, providing evidence that may link immune responses to microbial peptides from gut commensals and autoimmune responses affecting joints.
Identification of naturally presented HLA-DR-peptides (T cell epitopes). In a recent study (20), HLA-DR-presented peptides were identified in synovial tissue, SFMC, or PBMC from 5 patients with RA using LC-MS/MS, and immunogenicity of the peptides was determined using patients' samples in ELISpot assays. The findings from one patient (referred to herein as RA1) were of particular interest. RA1 had classic, seropositive RA, with severe symmetrical polyarthritis, a positive test for anti-citrullinated protein antibodies (ACPA), and 2 copies of SE alleles (HLA-DRB1*0401 and 0101). In this patient, an immunogenic HLA-DR-presented peptide derived from a Prevotella copri protein (Pc-p27) was identified from her PBMC (25). I was shown that about 40% of RA patients have T and/or B cell responses to Pc-p27 or to the whole Prevotella copri organism (25).
In patient RA1, 2 immunogenic HLA-DR-presented human, self peptides derived from N-acetyl-glucosamine-6-sulfatase (GNS) and filamin A (FLNA) were also identified from RA1synovial tissue, and the same FLNA peptide was also found in her PBMC (20). The HLA-DR-presented peptide derived from GNS was predicted to be promiscuous, binding to 24 of the 25 HLA-DR molecules modeled in the program TEPITOPE (26), and the FLNA-derived peptide was predicted to bind 9 of the 25 HLA-DR molecules. With both peptides, this included binding by HLA-DR molecules encoded by SE alleles *0101, *0401, *0404, and *0405. Neither the GNS nor FLNA protein nor the P. copri protein had previously been noted to be antigens in RA.
T cell reactivity to N-acetylglucosamine-6-sulfatase and filamin A peptides. To determine the immunogenicity of HLA-DR-presented peptides and their source proteins more broadly, cohort was developed of patients with new-onset RA (NORA) seen prior to therapy with disease-modifying anti-rheumatic drugs (DMARDs), the time when immune responses would be expected to be most robust. For comparison, samples from Lyme arthritis (LA) patients and from healthy control subjects were tested. HLA-DR typing showed that 60% of the 40 RA patients had shared epitope (SE) alleles; 50% of the 10 LA patients and 42% of 15 healthy subjects also had SE alleles. Nevertheless, since patients and control subjects had a range of different HLA-DR alleles and since cell numbers are limited in human patients, the initial approach for determining T cell responses in multiple individuals comprises pooling the original peptide with 3 additional peptides from the same protein that are predicted by the program TEPITOPE to be promiscuous HLA-DR binders (26). In addition, because of limited cells, testing of irrelevant, control peptides in these experiments were not included. However, it was previously shown that RA patients do not have reactivity to peptides derived from endothelial cell growth factor (ECGF) or MMP-10 peptides (21, 23). These autoantigens in Lyme arthritis (LA) are irrelevant in RA.
When PBMC from 40 NORA patients were stimulated with the GNS peptides, 14 of the 40 patients (35%) secreted levels of IFN-γ that were >3 SD above the mean value of HC (P=0.006), using an IFN-γ/IL-17 Double-Color ELISpot assay (
B cell reactivity to N-acetylglucosamine-6-sulfatase and filamin A proteins. Since the role of CD4+ T cells would likely be to help B cells to produce autoantibodies to GNS or FLNA, IgG levels to these proteins were examined in serum samples from RA patients and control groups. Since sera (but not PBMC) were also available from patients with chronic RA (CRA), testing was done in 48 NORA patients and in 53 CRA patients. Because the results were similar in both groups, they are presented together here. Of the 101 RA patients, 32 (32%) had IgG antibody responses to GNS that were >3 SD above those in HC (P<0.0001) (
Because autoimmune processes in RA may be triggered at mucosal sites, IgA levels to GNS and FLNA were also tested in serum samples from patients and control subjects. Of the 101 RA patients, 16 (16%) had elevated IgA antibody responses to GNS that were >3 SD above those in HC (P<0.0001) (
When IgG and IgA responses were considered together, 48 of the 101 RA patients (48%) had IgG responses to GNS and/or FLNA; 21 (21%) had IgA responses to 1 or both of the proteins, and 56 (55%) had IgG and/or IgA responses to the proteins. Of the 14 patients who had T cell responses to GNS peptides, 11 (79%) had IgG and/or IgA antibody responses to the GNS protein. Among the 17 patients who had T cell reactivity with the FLNA peptides, 6 (35%) had IgG and/or IgA antibody responses to FLNA protein. Thus, T and B cell concordance was greater with GNS than with FLNA.
Correlation of antibody responses to P. copri, GNS and FLNA. Using these same serum samples (25), IgG and IgA antibody responses were previously tested to two RA-associated bacteria, Prevotella copri, a gut microbe, and Porphyromonas gingivalis, a periodontal pathogen (27). Antibody responses to P. copri were found in 32% of RA patients, but were absent in patients with other connective tissue diseases, spondyloarthropathy, Lyme arthritis, or healthy subjects (25). Therefore, using these data, IgG and IgA antibody responses to these two organisms were correlated with the GNS and FLNA antibody responses determined here.
In RA patients, the levels of anti-GNS IgG and IgA antibodies strongly correlated with P. copri antibody responses (P=0.002 and P<0.0001, respectively) and a similar correlation was found between anti-FLNA IgG and IgA antibody responses and P. copri antibodies (P<0.0001 and P<0.0001,
Testing of citrullinated N-acetylglucosamine-6-sulfatase and filamin A proteins. Because citrullinated autoantigens are thought to play a central role in RA, particularly in those with SE alleles, it was investigated whether autoantibody responses to GNS or FLNA were greater when these proteins were citrullinated. For this purpose, the native proteins were citrullinated in vitro using recombinant human PAD4 enzyme. Using samples from 46 RA patients in whom enough serum still remained, IgG antibody responses were higher to citrullinated GNS compared with responses to the uncitrullinated protein (P=0.005), whereas the responses were negative to both forms of the protein in 15 healthy control subjects (
Utility of GNS and FLNA autoantibodies evaluation in the diagnosis of rheumatoid arthritis. In the patient cohort, 70 (69%) of the 101 new-onset and chronic RA patients were seropositive for ACPA and/or RF, which are standard, commercially available autoantibody determinations for support of the diagnosis in RA patients. Among the 31 patients who did not have a positive test for ACPA and/or RF, 13 had a positive test for IgG and/or IgA GNS autoantibodies and 9 had positive test for IgG and/or IgA FLNA autoantibodies. Taken together, 17 of 31 seronegative patients (55%) had such autoantibodies, 15 of whom could be identified with the IgG test alone. Overall, when autoantibody responses to GNS and FLNA were combined with standard autoantibody determinations, 87 (86%) of the 101 RA patients had a positive test result for support of the diagnosis, and only 14 (14%) lacked a specific marker for RA.
N-acetylglucosamine-6-sulfatase and filamin A protein levels in serum and joints. For a self-protein to become the target of autoimmune responses in RA patients' inflamed joints, one would predict that the protein would be present in high concentrations there. For this purpose, GNS and FLNA protein concentrations in serum samples were measured from the 101 RA patients and in synovial fluid (SF) from 17 patients in whom such samples were available. The levels of GNS were higher in the serum of RA patients compared with those in control groups (P<0.002,
To gain further insight into protein abundance and distribution, synovial tissue from 10 patients, 4 with RA, and 3 each with LA or osteoarthritis (OA), were stained for expression of GNS and FLNA, using immunohistologic methods. GNS showed a fine, reticular pattern in and around endothelial cells in 3 of the 4 RA patients, but not in those with LA or OA (
Sequence homology between T cell epitopes of microbial and self peptides. In an effort to determine whether T cell epitope mimicry may play a role in linking Prevotella reactivity with GNS and FLNA autoimmune responses, the sequence of each of the 2 self-peptides isolated from patient RA1 was used first to search for regions of similarity with any microbial protein using the microbial protein database in the Blastp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). For both self-peptides, Prevotella sp. peptides were among the top sequences producing significant alignment, specifically in areas predicted to be in the HLA-DR binding groove. Therefore, the search for sequence similarity was refined by screening only microbial sequences from Prevotellaceae (taxid:171552). To evaluate sequence homology, self and microbial peptides were aligned using the program Clustal Omega (28). The peptide derived from GNS had 67% sequence homology with a peptide from the Prevotella arylsulfatase protein (WP_062433009) (
For comparison, GNS and FLNA sequences were analyzed for homology with Porphyromonas gingivalis, a periodontal pathogen of interest in RA, using Porphyromonadaceae (taxid:171551) as the reference database in the Blastp search. However, no homology was found between P. gingivalis and GNS or FLNA sequences. P. gingivalis also stimulates antibody responses in the subgroup of RA patients who have periodontal disease (27, 30), but there is little overlap between RA patients with P. gingivalis antibodies and those with P. copri antibodies (25).
Instead, among the Porphyromonadaceae, the GNS epitope had partial sequence similarity with a peptide from the periplasmic protein N-acetylgalactosamine-6-sulfatase of Parabacteroides sp. (WP_046148720) (
T cell responses to homologous microbial and self peptides. To address whether patients had reactivity with these self epitopes and the corresponding microbial epitopes, ELISpot assays were preformed with each of these peptides using PBMC from the 24 NORA patients in whom enough cells remained and in 10 HC. When cells were stimulated with the GNS peptide or each of the 2 corresponding microbial peptides (one derived from Prevotella and the other from Parabacteroides), Th1 cell reactivity was found with all 3 peptides. Of the 24 RA patients, 8 (33%) had T cell reactivity with the GNS peptide, 9 (38%) showed responses to the Prevotella peptide, and 6 (25%) had reactivity with the Parabacteroides peptide that were >3SD above the mean values in HC (
Moreover, when the magnitude of the T cell responses to each self peptide was correlated with that of the corresponding microbial peptides, the responses to the GNS or FLNA peptide strongly correlated with reactivity to each of the two microbial peptides (in each instance, P<0.0001,
Using an in silico prediction method (Immune Epitope Database and Analysis resource, IEDB), the GNS peptide and the corresponding microbial peptides were predicted to bind HLA-DR molecules encoded by “shared epitope” (SE) alleles with significantly higher affinity than non-SE alleles (
Rheumatoid arthritis (RA) is an HLA class-II associated autoimmune disease in which mucosal immunity, often resulting from interaction with oral or gut microbes or from inhaled antigens in the lung, is hypothesized to cause autoimmune phenomena leading to joint inflammation and damage. However, the factors linking mucosal immunity to autoimmunity in joints have been unclear. In this study in which HLA-DR-presented peptides were identified directly from patients' synovial tissue or PBMC, two novel self-antigens, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA), were shown to be targets of T and B cell responses in 52% and 56% of RA patients, respectively. Importantly, the GNS and FLNA HLA-DR-presented T cell epitopes have considerable sequence homology with Prevotella epitopes and with similar epitopes from several related gut commensals belonging to the same order, particularly in areas predicted to be in the HLA-DR binding groove. Moreover, T cell responses to the corresponding microbial and self peptides correlated strongly, indicating that T cell epitope mimicry may provide a potential link between mucosal immunity and immune responses in affected joints. Moreover, GNS and FLNA autoantibodies correlated with P. copri antibody responses. This finding can also be due to cross-reactive Prevotella and host protein B cell epitopes, but the recombinant microbial proteins to test this hypothesis directly is lacking. Alternately, this correlation might simply be a reflection of T cell help resulting from T cell epitope mimicry.
The presence of natural IgM, IgG, or IgA autoantibodies has been reported in the serum of normal control subjects, characterized by broad reactivity to self and microbial antigens (33, 34). However, these findings cannot be explained simply by non-specific physiological responses. First, GNS and FLNA autoantibodies were increased specifically in a large subgroup of RA patients, and not in those with other rheumatic diseases or in HC. Second, these self autoantibodies correlated strongly with P. copri antibodies, but not with Porphyromonas gingivalis antibodies. Third, RA patients, but not healthy subjects, often had both T and B cell responses to these self antigens. For control of the microbiota, the generation of specific, high affinity IgA antibody responses requires T and B cell interactions (35), whereas the “natural” antibody pool consists primarily of low-affinity polyreactive IgA antibodies whose production is independent of T cell help (36-38).
Currently, the only known antibody responses that are specific for RA are directed against citrullinated proteins, particularly to enolase, vimentin, and fibronectin (39). Moreover, there is hyper-citrullination of a range of proteins in the joints of many RA patients (40). The GNS protein appeared to be citrullinated in vivo and antibodies to it correlated with ACPA levels, whereas antibody levels to the FLNA protein, which did not appear to be citrullinated, did not correlate with ACPA levels. Moreover, HLA-DR molecules encoded by RA “shared epitope” alleles bound the GNS peptide and to a lesser degree the FLNA peptide with higher affinity than non-SE alleles, and SE alleles largely influence the generation of ACPA (3).
Both self antigens, which are often highly expressed in RA synovial tissue, represent good autoimmune targets. GNS is an enzyme located in lysosomes and is involved in the degradation and recycling of different molecules, such as glycosaminoglycans (GAGs) (https://ghr.nlm.nih.gov/gene/GNS), a major component of joint cartilage and other soft connective tissues (41). FLNA is an ubiquitous, fundamental protein for building the cell cytoskeleton and for organization of the extracellular matrix (42). In addition, FLNA is required for cell-cell contact in vascular development (43). Consistent with its function, the FLNA protein was especially prominent in the tunica muscularis around blood vessels in RA synovial tissue, and was also faintly expressed in the extracellular matrix. However, it is not yet clear why the GNS protein was seen only in a reticular pattern around blood vessels. Nevertheless, the important point is that both host proteins would be available for HLA-DR presentation in synovial tissue and they represent potential targets for autoimmune responses.
Based on high through-put sequencing of stool samples, gut dysbiosis has been shown in two studies of RA patients (13, 14) and overexpansion of Prevotella sp., particularly P. copri, was reported in one study (13). However, little is known about potential bowel pathology in RA. The major limitation of the study is the lack of specific information about pathology or immune responses in the bowel. The study of NORA patients was originally intended as a study of mouth flora and periodontal disease (30), and therefore, collection of stool samples was not a part of the protocol. Nevertheless, the finding of gut dysbiosis favoring Prevotella (13), the identification of P. copri as an immune-relevant bacterium in RA (25), and the finding here of specific T and B cell responses to Prevotella and corresponding GNS and FLNA epitopes indicate that mucosal immune responses in the gut may be a part of the disease in a sizable subgroup of RA patients. Although DMARDs therapy may resolve dysbiosis (14) and P. copri over-expansion (13), immune reactivity to the organism, once triggered, appeared to persist or even increase in patients with chronic RA who were taking these medications. In contrast, patients with spondyloarthropathy or post-infectious, antibiotic-refractory Lyme arthritis, which may also be treated with DMARDs, did not have reactivity with this organism (25).
Taken together, the studies described herein highlight a mechanism that may link gut and joint inflammation. Thus, dysbiosis or low-grade gut inflammation can compromise the mucosal barrier resulting in leakage of commensal organisms, leading to activation of lymphocytes targeting microbial antigens. Eviendence that T cell epitopes of a related order of gut microbes, particularly Prevotella species, may cross-react with self epitopes of highly expressed proteins in joints, especially in patients with SE alleles is provided. T cells activated by microbial peptides at mucosal surface may then home to inflamed joints and cross-react with homologous self antigens. In addition, as previously reported (25), commensal organisms or their remnants or microbial-activated APCs may occasionally home to joints where they further amplify joint inflammation and re-stimulate self-reactive T cells. Thus, an initial trigger in the gut mucosa activates cross-reactive microbial immune responses which have the potential to shift to an autoimmune phenotype at sites where the homologous self antigens are expressed. Even though proof of cross-reactivity will require the cloning of single T cells, the prominence of homologous epitopes among multiple related families of gut microbes and epitopes from two self proteins in synovial tissue, the greater affinity of HLA-DR molecules encoded by SE alleles in binding these epitopes, and the demonstration that RA patients often have T cell responses to corresponding microbial and self epitopes indicate the potential for these immune responses to have a role in RA pathogenesis. These mechanisms are the entire explanation for the marked inflammatory and proliferative responses in the RA synovial lesion. Rather, they play a role in linking gut and joint immune responses and in amplifying synovial inflammation in a large subgroup of RA patients.
Although knowledge of bowel pathology is not yet clear in RA patients, the identification of immune responses to P. copri, GNS and FLNA has practical implications regarding the diagnosis and treatment of the disease. First, these immune responses appear to be specific for RA. As shown here, the addition of GNS and FLNA to standard autoantibody determinations (ACPA and RF) increased the percentage of seropositive patients from 69% to 86%, improving diagnostic support for some patients with seronegative RA, which currently lack specific markers. Furthermore, inclusion of citrullinated GNS in a test for ACPA increases the sensitivity of that testing. These determinations have increased in importance because earlier treatment with DMARDs may improve long-term outcomes, whereas lack of diagnostic markers for seronegative patients may delay appropriate DMARD treatment, leading to less favorable clinical outcomes (44, 45). Finally, in addition to DMARDs, identification of patients with these antibody responses may allow the testing and development of adjunctive forms of treatment, such as brief, targeted antibiotic regimens or diet alterations. Ultimately, the identification of pathogenic T cell epitopes in synovial tissue, as was done here, may make it possible to engineer blocking peptides, which would limit autoimmune stimulation and ameliorate these presumably disadvantageous autoimmune responses.
Patients and control subjects. The study, “Autoantigens in RA,” was approved by the Human Investigations Committee at Massachusetts General Hospital (MGH) (2008-2014). The 101 study patients met the American College of Rheumatology/European League Against Rheumatism Collaborative Initiative criteria for that disease (46). Of the 101 patients, 49 had new-onset RA (NORA) for <12 months and had not yet been treated with DMARDs. The remaining 52 patients had chronic RA (CRA) of >1 year's duration (usually for many years), often treated with DMARDs. For isolation of HLA-DR-presented peptides, synovial tissue and peripheral blood mononuclear cells (PBMC) were obtained from patient RA1 who was undergoing arthroscopic synovectomy. To test implicated peptides and their source proteins for immunoreactivity in additional patients, PBMC and serum samples were collected from patients with NORA, but only serum samples were available from patients with CRA.
For other comparison groups, serum samples were collected from 28 patients with connective tissue diseases (CTD), including systemic lupus, mixed connective tissue disease, scleroderma, and Sjogren's syndrome, from 28 patients with spondyloarthropathy (SpA), and from 50 patients with Lyme arthritis (LA). Additionally, serum samples and PBMC were collected from 15 healthy hospital personnel who did not have a history of RA or other autoimmune diseases, and serum samples were obtained from 40 healthy blood bank donors. HLA-DR typing was performed on blood samples from all RA or LA patients and from healthy subjects at the American Red Cross Laboratory in Dedham, Mass.. All patients and control subjects gave written informed consent.
Enzyme-linked immunospot (ELISpot) T cell assay. A detailed description of the methods for isolation and identification of HLA-DR presented peptides is given in previous publications (19, 20). In the current study, all non-redundant HLA-DR-presented peptides identified from patient RA1 were synthesized (Mimotopes, Australia) and tested for reactivity in sets of 3 (1 μM of each peptide) with the patient's PBMC using IFN-γ ELISpot assays. In this initial screen, 2 self-peptides derived from GNS and FLNA were implicated; the remaining 138 peptides showed no reactivity. For testing in larger numbers of patients and control subjects who would have a range of HLA-DR genotypes, the original peptides identified from the patient (shown in bold) and 3 additional promiscuous peptides that were predicted to bind >16 HLA-DR molecules were synthesized and HPLC-purified in the MGH Core Facility. The sequences of the GNS peptides were as follows: 47PNVVLLLTDDQDE59 (SEQ ID NO: 13), 222FEPFFMMIATPAPH235 (SEQ ID NO: 14), 451TYACVRTMSALWNLQ465 (SEQ ID NO: 15), 504NYRLMMLQSCSGPTC518 (SEQ ID NO: 16). The sequences of the FLNA peptides were as follows: 73DGLRLIALLEVLSQKK88 (SEQ ID NO: 17), 118SIKLVSIDSKAIVDG132 (SEQ ID NO: 18), 416VEVVIQDPMGQKG428 (SEQ ID NO: 19), 2446NPAEFVVNTSNAGAG2460 (SEQ ID NO: 20).
To assess microbial sequence homology with the GNS peptide 222FEPFFMMIATPAPH235 (SEQ ID NO: 21), the following microbial peptides were tested: 231KKPFFMMVAMNPPH245 (SEQ ID NO: 22), which was derived from the arylsulfatase protein of Prevotella sp. (WP_062433009.1) and 247DVPFFMMWTTPLPH261 (SEQ ID NO: 23), derived from the N-acetylgalactosamine-6-sulfatase of Parabacteroides sp. (WP_046148720.1).
To evaluate the microbial sequence homology for the FLNA peptide 2446NPAEFVVNTSNAGAG2460 (SEQ ID NO: 24), the following microbial peptides were tested: 133QGFVVKTANAGAL146 (SEQ ID NO: 25), which was derived from an uncharacterized protein of Prevotella sp. (WP_028897633.1) and 90ANFMINVSNAGAL103 (SEQ ID NO: 26), derived from an uncharacterized protein of Butyricimonas sp. (WP_065219401.1). The RA patients' PBMC were stimulated with the pool of peptides or single peptides, and analyzed for reactivity with a human IFN-γ/IL17 Double-Color ELISpot kit (Cellular Technology Limited). All peptides (1μM) were tested in duplicate wells, as were positive (phytohemagglutinin) and negative (no antigen) control samples. After 5 days, cells were transferred to ELISpot plates coated with IFN-γ/IL17 antibodies, and incubated overnight. Images of wells were captured using ImmunoSpot series 3B analyzer, and spots were counted using ImmunoSpot software. A positive T cell response was defined as 3 standard deviations (SD) above the mean value of healthy control subjects.
ELISA for serum IgG and IgA anti-GNS and anti-FLNA autoantibodies. ELISA plates were coated with 0.5 μg/ml of recombinant human N-acetylglucosamine-6-sulfatase (Novoprotein, Summit, N.J.) or filamin A (Novusbio, Littleton, Col.) overnight at 4° C. Subsequent incubations and washes were performed at room temperature. After washing with phosphate buffered saline with 0.05% Tween-20 (PBST), the plates were blocked with blocking buffer (5% nonfat dry milk in PBST) for one hour. Afterwards, 100 μl of each patient's serum sample (diluted 50-fold) was added in duplicate wells for 1.5 hours, followed by horseradish-peroxidase (HRP)-conjugated goat anti-human IgG (sc-2453, Santa Cruz Biotech) or HRP-conjugated goat anti-human IgA (Bio-Rad, Hercules, Calif.), and then TMB substrate (BD, San Diego, Calif.). For inter-plate standardization, 2 control samples were included with each assay.
In vitro citrullination assay. Human recombinant N-acetylglucosamine-6-sulfatase (2 μg) or filamin A (2 μg) were incubated with rhPAD-4 (400 ng), generously provided by Maximilian Koenig from the laboratory of Felipe Andrade, in 1 M Tris (pH 7.6) in presence of 200 mM CaCl2. A negative control reaction was performed substituting 200 mM CaCl2 with 200 mM EDTA. Incubation was performed at 3° C. for 3 hours. Protein citrullination was determined by anti-modified citrulline immunoblotting, according to the recommendations of the manufacturer (Millipore).
Quantification of GNS and FLNA protein levels in serum and joint fluid. ELISA plates were coated with 5 μg/ml capture antibody (sc-161669 for GNS, and sc-58764 for FLNA, Santa Cruz Biotech) overnight at 4° C. Plates were then washed with PBS and blocked with 5% milk PBST (blocking buffer) for one hour. Afterwards, serum or joint fluid samples (diluted 1:10 in blocking buffer) were added in duplicate and incubated for two hours at room temperature. Next, detection antibody (5 μg/ml) (SAB1410557 for GNS, Sigma; and sc-28284 for FLNA, Santa Cruz Biotech) was added for 2 hours. The plates were then incubated with goat anti-rabbit IgG-HRP (sc-2030, Santa Cruz Biotech), followed by TMB substrate. For inter-plate standardization, 2 control samples were included on each plate.
Immunohistochemistry. Fresh frozen synovial tissue samples were stained for GNS and FLNA proteins. After blocking, the sections were incubated with rabbit polyclonal antibody against GNS (SAB1410557, Sigma-Aldrich) and with mouse monoclonal antibody against FLNA (sc-17749, Santa Cruz Biotech) at 4° C. overnight. For negative controls, non-specific rabbit or mouse IgG were used. The following day, the sections were incubated with biotinylated anti-rabbit or anti-mouse secondary antibody, peroxidase-streptavidin, and then, diaminobenzidine substrate. The slides were counterstained with Mayer's hemotoxylin and mounted with Permount. Microscopic images were obtained with a Zeiss widefield microscope.
In silico determinations of HLA-DR binding affinity. The affinity of HLA-DR binding of the GNS, FLNA, and related microbial peptides was determined using the T cell epitope—MHC class II molecules binding prediction tool from the Immune Epitope Database and Analysis resource (IEDB). This tool employs different methods to predict MHC Class II epitopes, including a consensus approach which combines NN-align, SMM-align and combinatorial library methods. For the analysis, all HLA-DR alleles found in the RA cohort were analyzed, including 5 shared-epitope alleles and 9 non-SE alleles.
Statistics. Categorical data were analyzed by Fisher's exact test, and quantitative data were analyzed using unpaired t test with Welch correction. Correlations were sought using Spearman correlation test. All analyses were performed using GraphPad Prism 6. All P values were two-tailed. P values<0.05 were considered statistically significant.
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This Application claims the benefit under 35 U.S.C. § 119(e) of U. S. Provisional Application Ser. No. 62/346,666, filed Jun. 7, 2016, the contents of which are incorporated herein by reference in their entirety.
This invention was made with government support under Grant Nos. P41 GM104603, S10 RR020946 and S10 OD010724 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/36198 | 6/6/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62346666 | Jun 2016 | US |
