WO2016003719A1 - Autoantigens for diagnosis of chronic inflammatory diseases - Google Patents

Abstract

Disclosed herein are methods of diagnosing Lyme arthritis in a subject comprising determining whether the subject is immunologically reactive with one or more of annexin A2, Apolipoprotein B (ApoB), and matrix metalloproteinase 10 (MMP10), wherein immunological reactivity of the subject to one or more of annexin A2, ApoB, and MMP10, as compared to an appropriate control, indicates the subject has Lyme arthritis. Methods of treatment and kits for performing the methods are also disclosed.

Description

AUTOANTIGENS FOR DIAGNOSIS OF CHRONIC INFLAMMATORY DISEASES

RELATED APPLICATIONS

[0001] This Application claims the benefit under 35 U.S. C. § 119(e) of U. S. Provisional Application serial number 62/020,686, filed July 3, 2014, and 62/020,710, filed July 3, 2014, and 62/079,264, filed November 13, 2014, the contents of each of which are incorporated herein by reference in their entirety.

GOVERNMENTAL SUPPORT

[0002] This invention was made with Government support under NIH RO 1 Al-110175, and NIH P41 GM 104603 awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of diagnostics using humoral and cellular autoimmune responses to disease-related autoantigens. In particular, it relates to

autoantigenic biomarkers for forms of inflammatory diseases associated with Lyme disease.

BACKGROUND OF THE INVENTION

[0004] Lyme disease, or borreliosis, is the most common vector-borne infectious disease in North America, Europe and Asia. Lyme disease has major public health and economic effects: the estimated annual cost is approximately $1 billion in the U.S. alone. State health departments reported about 30,000 confirmed cases and 8,500 probable cases of Lyme disease to the Centers for Disease Control and Prevention in 2009, representing a 3.6 percent increase in confirmed cases compared to the previous year.

[0005] Caused by the spirochete bacterium Borrelia, and transmitted to humans through the bite of infected ticks, Lyme disease is a multi-system disorder that is treatable with antibiotics and can affect the nervous system, heart, and in particular the joints. Some patients develop chronic Lyme disease, a condition characterized by persistent musculoskeletal and peripheral nerve pain, fatigue, and memory impairment. In subjects with joint involvement, a small percentage develop proliferative synovitis that persists for months or several years after apparent spirochetal killing with antibiotics, referred to as antibiotic-refractory arthritis. This disease course is hypothesized to result from infection-induced autoimmunity. A novel approach was developed to identify disease-relevant HLA-DR-presented peptides in synovial tissue using discovery-based proteomics and translational research. Using this approach, endothelial cell growth factor (ECGF) was identified as the first autoantigen known to be a target of T and B cell responses in antibiotic-refractory or antibiotic-responsive LA, but not in RA. Further characterization of the repertoire of naturally presented HLA-DR peptides in synovial tissue in patients with RA or LA is still needed.

SUMMARY OF THE INVENTION

[0006] 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 one or more of annexin A2, Apolipoprotein B (ApoB), and matrix metalloproteinase 10 (MMP10) comprising performing an assay for identifying the presence of antibodies that specifically bind one or more of annexin A2, ApoB, or MMP10, or performing an assay for identifying T cells specifically reactive to annexin A2, ApoB, or MMP10, and determining the sample is reactive with the annexin A2, ApoB, and MMP10 if the assay produces positive results compared to an appropriate control.

[0007] In one embodiment, the assay that identifies the presence of the antibodies comprises contacting the sample with annexin A2 protein or a fragment thereof, ApoB protein or a fragment thereof, and/or MMP10 protein or a fragment thereof, under conditions that allow an immunocomplex of the antibody and the annexin A2, ApoB, or MMP10 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 annexin A2, ApoB, and/or MMP10 and wherein the absence of the immunocomplex indicates the biological sample is not immunoreactive with annexin A2, ApoB, and/or MMP10.

[0008] In one embodiment of the invention, the assay is an ELISA, agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, or an

immunoblot assay. In one embodiment, the assay for identifying T cells specifically reactive to annexin A2, ApoB, or MMP10 comprises a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with one or more of annexin A2, ApoB, MMP10 whole protein(s) and/or polypeptide fragment(s), 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 one or more of annexin A2, ApoB, and MMP10 when T cell proliferation or secretion of IFN-γ measured is increased over that of an appropriate control. [0009] In one embodiment of the invention, the assay is a T cell proliferation assay. In one embodiment, the assay is a H-thymdine incorporation assay, CFSE dilution, or an ELISPOT. In one embodiment, the assay is a T cell reactivity assay.

[0010] In one embodiment, the polypeptide fragment is predicted to be presented by HLA-DR molecules associated with chronic inflammatory arthritis. In one embodiment, the polypeptide fragment is selected from the group consisting of GVDEVTIVNILTNRSNAQR (SEQ ID NO: 4), TVILGLLKTPAQYDA (SEQ ID NO: 5), SGDFRKLMVALAKGRRA (SEQ ID NO: 6), DKVLIRIMVSRSEVD (SEQ ID NO: 7), IEGNLIFDPNNYLPK (SEQ ID NO: 8), HDLQLLGKLLLMGARTLQGIP (SEQ ID NO: 9),

VPLLLSEPINIIDALEMRD (SEQ ID NO: 10), EHYHIRVNLVKTIHDLHLFI (SEQ ID NO: 11), HLAFLVLICLPVCSAY (SEQ ID NO: 12), GTNLFLVAAHELGHS (SEQ ID NO: 13) and PTIRKIDAAVSDK (SEQ ID NO: 14).

[0011] In one embodiment, said biological sample is obtained from peripheral blood, synovial fluid, synovial tissue, peripheral blood mononuclear cells (PBMC), or synovial fluid mononuclear cells (SFMC).

[0012] In one embodiment, the subject exhibits symptoms of arthritis or other

automimmune related disease manifestation. In one embodiment, the subject is suspected of having Lyme disease.

[0013] Other aspects of the invention relate to the method of diagnosing Lyme arthritis in a subject comprising determining whether the subject is immunologically reactive with one or more of annexin A2, Apolipoprotein B (ApoB), and matrix metalloproteinase 10 (MMP10), wherein immunological reactivity of the subject to one or more of annexin A2, ApoB, and MMP10, as compared to an appropriate control, indicates the subject has Lyme arthritis.

[0014] In one embodiment, determining is by evaluating a biological sample obtained from the subject for immunological reactivity with the one or more of annexin A2, ApoB, and MMP1. In one embodiment, determining immunological reactivity is by detecting the presence of T cells reactive to one or more of annexin A2, ApoB, and MMP10, comprising the steps a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with one or more of annexin A2, ApoB, MMP10 whole protein or polypeptide fragments, 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 reactive to one or more of annexin A2, ApoB, and MMP10 when T cell proliferation or secretion of IFN-γ is measured as increased over that of an appropriate control. [0015] In one embodiment, determining immunological reactivity comprises determining if the subject has a B-cell response to one or more of annexin A2, ApoB, and MMP10 resulting in the production of autoantibodies that specifically recognize the one or more annexin A2, ApoB, and MMP10, by contacting the sample with annexin A2 protein or a fragment thereof, ApoB protein or a fragment thereof, MMP10 protein or a fragment thereof, under conditions that allow an immunocomplex of the antibody and the annexin A2, ApoB, or MMP10 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 annexin A2, ApoB, and/or MMP10 and wherein the absence of an immunocomplex indicates the subject fails to present a B-cell response to annexin A2, ApoB, and/or MMP10. In one embodiment, the assay is an enzyme-linked immunosorbent assay (ELISA), agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, or an

immunoblot assay.

[0016] In one embodiment, the subject suffers from chronic inflammatory arthritis.

[0017] In one embodiment of the various methods described herein, the method further comprises the step of treating the subject with a non-steroidal anti-inflammatory or disease modifying anti-rheumatic drugs.

[0018] Other aspects of the invention relate to a kit comprising, one or more potential antigen and/or potential epitope of annexin A2, ApoB, and/or MMP10, and reagents for conducting an assay for detecting the presence of an antibody in a sample that binds to the one or more potential antigen and/or potential epitope of the annexin A2, ApoB, and

MMP10.

[0019] In one embodiment, the kit further comprises one or more potential antigens and/or potential epitopes of endothelial cell growth factor (ECGF) and reagents for conducting an assay for detecting the presence of an antibody in a sample that binds to the one or more potential antigen and/or potential epitope of the ECGF.

[0020] In one embodiment, the assay is an enzyme-linked immunosorbent assay

(ELISA).

[0021] In one embodiment, the assay is a western blot.

[0022] In one embodiment, the kit further comprises Borrelia antigens.

[0023] Another aspect of the invention relates to a kit or method disclosed above, for use in identifying a patient with chronic inflammatory arthritis. In one embodiment of the use, the chronic inflammatory arthritis is antibiotic-resistant Lyme arthritis. In one embodiment of the use, the chronic inflammatory arthritis is antibiotic-responsive Lyme arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A-Figure 1C present experimental results that indicate a subset of rheumatoid arthritis and Lyme arthritis patients have T and B cell autoreactivity to the self- protein annexin A2. A) A subset of rheumatoid arthritis and Lyme arthritis patients have T cell autoreactivity to peptides derived from annexin A2. PBMC from RA and Lyme arthritis (LA) patients, and healthy control (HC) subjects were tested for T cell reactivity to the two peptides originally identified from the synovial tissue of the RA patient, plus two additional annexin A2 peptides predicted to be promiscuous HLA-DR binders (promiscuous T cell epitopes). B) A subset of rheumatoid arthritis and Lyme arthritis patients produce

autoantibodies against the self-protein annexin A2. The levels of IgG anti-annexin A2 in the sera of HC subjects, patients with RA, patients with LA, or patients with other arthritides including osteoarthritis (OA), spondylarthropathy (SA) or psoriatic arthritis (PA) were measured by enzyme-linked immunosorbent assay (ELISA). The sera of a subset of both RA and LA patients had autoantibodies that recognized annexin A2. For A) and B), the percentages of positive responses for each patient group are shown below the graph. P values were calculated using an unpaired t-test. Red line indicates the mean of each dataset. C) Another set of results indicating an IgG Antibody Responses to Annexin A2 by patients with various stages of Lyme Disease.

[0025] Figure 2A - Figure 2C present experimental results that indicate apolipoprotein B 100 is a target at T and B cell responses in a subgroup of patients with Lyme disease. A) A subset of Lyme arthritis patients have T cell autoreactivity to peptides derived from apolipoprotein B-100. Peripheral blood mononuclear cells (PBMC) from healthy control (HC) subjects, Lyme disease patients with erythema migrans (EM) [an early disease manifestation] or Lyme arthritis (LA) [a late disease manifestation] were tested for T cell autoreactivity against human full-length recombinant ApoB protein (Millipore). B) A subset of Lyme arthritis patients produce autoantibodies against the self-protein apolipoprotein B 100 . The levels of IgG anti-ApoB in the sera of Lyme disease patients with EM or LA, patients with rheumatoid arthritis (RA) or HC subjects were measured by enzyme-linked immunosorbent assay (ELISA). A significant number of Lyme disease patients with EM or LA express autoantibodies that recognize ApoB. In both A and B, the percentages of positive responses for each patient group are shown below the graph. P values were calculated using an unpaired t-test. The bold line indicates the mean of each dataset. C) Another set of results indicating an IgG Antibody Responses to Apolipoprotein by patients with various stages of Lyme Disease.

[0026] Figure 3 A -Figure 3B present experimental results that indicate Lyme disease patients have T and B cell autoreactivity to matrix metalloproteinase 10. A) A subset of Lyme arthritis patients have T cell autoreactivity to peptides derived from matrix

metalloproteinase 10. Peripheral blood mononuclear cells (PBMC) from healthy control (HC) subjects, Lyme disease patients with erythema migrans (EM) [an early disease manifestation] or Lyme arthritis (LA) [a late disease manifestation] were tested for T cell autoreactivity against human full-length recombinant matrix metalloproteinase 10 (R&D Systems). B) A subset of Lyme arthritis patients produce autoantibodies against the self- protein matrix metalloproteinase 10. The levels of IgG anti-MMPIO in the sera of Lyme disease patients with EM or LA, patients with rheumatoid arthritis (RA) or HC subjects were measured by enzyme-linked immunosorbent assay (ELISA). A significant number of patients with Lyme arthritis express autoantibodies that recognize MMP10. In both figures, the percentages of positive responses for each patient group are shown below the graph. P values were calculated using an unpaired t-test. Bold line indicates the mean of each dataset.

[0027] Figure 4 shows the amino acid sequence of ApoB (SEQ ID NO: 1).

[0028] Figure 5 shows the amino acid sequence of annexin A2 (SEQ ID NO: 2 ).

[0029] Figure 6 shows the amino acid sequence of MMP10 (SEQ ID NO: 3).

DETAILED DESCRIPTION OF THE INVENTION

[0030] Aspects of the invention relate to the discovery of certain peptides in synovial tissue, the target tissue in the various forms of chronic inflammatory arthritis. Aspects of the invention also relate to the discovery that at least a subset of these peptides are HLA-DR presented in the subject and serve as autoantigens, contributing to the development of arthritis (e.g., Rheumatoid Arthritis and Lyme Arthritis). Annexin A2, Apolipoprotein B (ApoB), and Matrix Metalloproteinase 10 (MMP10) were each identified as autoantigenic in patients with Lyme arthritis. The identification of these proteins as autoantigens associated with an inflammatory disorder arising from Lyme disease is novel and unexpected. In addition, annexin A2 was also identified as autoantigenic in patients with Rheumatoid arthritis.

[0031] One aspect of the invention relates to a method for determining immunoreactivity of a subject suspected of having an inflammmatory disorder arising from Lyme disease, to one or more of annexin A2, ApoB, and MMP10. Prior to the findings disclosed herein, Lyme induced autoimmunity to these proteins was not previously known or suspected of playing a role in disease progression or pathology.

[0032] 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. The determination of which method to utilize can be determined by the skilled practitioner.

Immunoreactivity of a subject to the protein 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 autoantigen immunoreactivity. 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.

[0033] In one embodiment, the assay is for the presence of antibodies in the subject that specifically bind to the protein. The assay involves contacting a biological sample obtained from the subject with the full length protein or representative fragments thereof (referred to as the test antigen), 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.

[0034] 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 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).

[0035] In one embodiment, the assay is for identifying the presence of T cells in the biological sample that are specifically reactive to the annexin A2, ApoB or MMP10. This is typically accomplished by exposing the cells in the biological sample to the suspected autoantigen or one or more representative fragments thereof (also referred to as the test antigen), 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.

[0036] 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 H- thymdine incorporation assay, CFSE dilution, or an ELISPOT, and also T cell reactivity assays.

[0037] 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).

[0038] Test antigen for use in the methods herein described include, without limitation, full length annexin 2, ApoB, MMP10, and/or fragments thereof. The entire full length protein or protein fragments can be used in the asays described herein. Combinations of full length protein and protein fragments can also be used, as can combinations of proteins/fragments that represent the different autoantigens (e.g., testing for two or more of annexin 2, ApoB and MMP10 reactivity in the same assay, or side by side in the same patient). The proteins or fragments can be generated and isolated from natural sources or produced recombinantly or synthetically.

[0039] Annexin A2 is a calcium regulated phospholipid binding protein that is expresed abundantly in various human organs. Multiple alternatively spliced transcript variants encoding different isoforms have been found for this gene (NCBI Reference Sequence:

NC 000015.10), shown in Figure 5. Generation in vitro and purification for the detection of anti-annexin A2 antibodies is also described (Tabata et al., J Biosci Bioeng. 2006

Feb; 101(2): 190-7).

[0040] Apolipoprotein B is a component of a lipoprotein and is found in various cells throughout the body. ApoB- 100 functions as a recognition signal for the cellular binding and internalization of LDL particles by the apoB/E receptor. The complete amino acid sequence of ApoB is known (Chen et al, J Biol Chem. 1986 Oct 5;261(28): 12918-21; Law et al, Proc Natl Acad Sci U S A. Nov 1986; 83(21): 8142-8146; ApoB Human Accession:

NM 000384.1 1). Figure 4 shows the complete amino acid sequence that contains the signal peptide (from amino acid 129 - 209), with the mature ApoB 100 protein being amino acid 210 - 13817, and the mature apolipoprotein B48, resulting from a post-transcriptional modification introducing a stop codon, being amino acid 210 - 6665.

[0041] Matrix metalloproteinase 10 degrades proteoglycans and fibronectin in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. It is secreted as inactive proprotein and activated when cleaved by extracellular proteinases. The amino acid sequence of MMP10 is shown in Figure 6

(Genbank Accession: NC OOOOl 1.10). The signal peptide is amino acid 1-17, the propeptide is amino acid 18-98, and the functinoal peptide is amino acid 99-476.

[0042] 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).

[0043] Specific protein fragments can be identified for use in the herein described methods such as those predicted for presentation by HLA-DR. Such protein fragment can be identified by a variety of softward 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

are also available.

[0044] Examples of specific protein fragments of annexin A2 include, without limitation ⁵⁰GVDEVTFVNILTNRSNAQR⁶⁸ (SEQ ID NO: 4), ⁹⁷TVILGLLKTPAQYDA^1U(SEQ ID NO: 5), ¹⁶⁴SGDFR LMVALAKGRRA¹⁸⁰(SEQ ID NO: 6), and

²⁸⁵DKVLIRIMVSRSEVD²⁹⁹(SEQ ID NO: 7). Examples of specific protein fragments of ApoB include, without limitation : ⁶⁵⁵IEGNLIFDPNNYLPK⁶⁶⁹(SEQ ID NO: 8),

7⁹⁰HDLQLLGKLLLMGARTLQGIP⁸¹⁰(SEQ ID NO: 9), ²⁰²⁶VPLLLSEPINIIDALEMRD²⁰⁴⁴ (SEQ ID NO: 10) and ²²¹⁴EHYHIRVNLVKTIHDLHLFI²²³³ (SEQ ID NO: 11). Examples of specific protein fragments of MMP10 include, without limitation,

3HLAFLVLICLPVCSAY¹⁸(SEQ ID NO: 12), ²⁰⁸GTNLFLVAAHELGHS²²²(SEQ ID NO: 13) and ³⁸²PTIRKIDAAVSDK³⁷⁴(SEQ ID NO: 14).

[0045] Reactivity to two or all three of annexin A2, ApoB, or MMP10 can be assayed in a single subject. In one embodiment, reactivity to all three annexin A2, ApoB, and MMP10 are assayed in a subject. In one embodiment, reactivity in a subject to other suspected autoantigens is also performed in conjunction with the herein described assays. One such autoantigen is human endothelial cell growth factor (ECGF). Methods for determination of reactivity of a subject for ECGF for disease diagnosis are described in US Application 2013- 0302329, the contents of which are incorporated herein by reference in their entirety.

[0046] ECGF is available commercially, for example from R&D Systems, Inc.

(Minneapolis, MN). Additionally, ECGF can be obtained from platelets, liver, lung, placenta, spleen, lymph nodes, peripheral lymphocytes, and astrocytes. See Haraguchi et al, 368 Nature 198 (1994); Toi et al, 6 Lancet Oncol. 158 (2005); Akiyama et al, 95 Cane. Sci. 851 (2004). As with the other autoantigens, full length ECGF, fragments of ECGF, or a combination of the two can be used in the herein described assays. In a particular embodiment, the ECGF peptides/epitopes are one or more of the peptides having the following amino acids: LGRFERMLAAQGVDPG (SEQ ID NO: 15);

ADIRGFVAAVVNGSAQGAQI (SEQ ID NO: 16); DKVSLVLAPALAACG (SEQ ID NO: 17 ) ; SKKLVEGLSALWDV (SEQ ID NO: 18); KTLVGVGASLGLRVAAALTAMD (SEQ ID NO: 19 ); LRDLVTTLGGALLWL (SEQ ID NO: 20 ); GTVELVRALPLALVLH (SEQ ID NO: 21); or functionally equivalent portions, fragments, analogs, or derivatives of any of these.

[0047] Identification of immunoreactivity to one or more of these proteins can be used in diagnosis of the respective disease. Immunoreactivity of a subject to one or more of the proteins can determined by a variety of methods available to the skilled practitioner.

[0048] The term "subject" as used herein refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and non-human primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject. A subject as indicated herein can be someone suspected of having or at risk for developing an inflammatory related disease or condition associated with Lyme disease. The subject may have a history indicative of having contracted or being exposed to Lyme disease, such as having been bitten by a deer tick, and/or having experienced the bulls eye rash arising from initial infection, and/or having been diagnosed with Lyme disease (either

symptomatically or having tested positive for the Borrelia spirochete (e.g., B. Burgdorferi ). Where appropraite, a subject as indicated herein can be someone suspected of having or at risk for developing an inflammatory related disease such as rheumatoid arthritis.

[0049] An inflammatory related disease or condition associated with Lyme disease (also referred to herein as a Lyme related disease manifestation) is one that is secondary to systemic infection by the Borrelia spirochete (e.g., Borrelia burgdorferi). Typical conditions known to develop involve inflammation in the joints (e.g., Lyme arthritis), the heart (e.g., Lyme carditis) and the nervous system (e.g., Lyme neuroborreliosis).

[0050] 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), or serum).

[0051] The discoveries presented herein indicate that reactivity to annexin A2, ApoB, and MMP10 can be used as a biomarker for the diagnosis of a condition (e.g., Lyme related autoimmune conditions such as Lyme arthritis, in particular, antibiotic-refractory Lyme arthritis). More specifically, subject immunoreactivity (e.g., autoantigen reactive T cells, and/or anti-autoantigen antibodies) is indicative of the condition (e.g, Lyme arthritis). As such, another aspect of the invention relates to a method of diagnosing the condition in a subject (e.g., Lyme arthritis) by determining whether the subject is immunologically reactive with one or more of annexin A2, ApoB, or MMP10 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 ECGF.

[0052] 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)).

[0053] The methods described herein can also include determination of reactivity to Borrelia antigens. Many such antigens are known in the art, such as p83/100 derived from strain PKo {Borrelia afzelii); p39 (BmpA) and OspC from strains PKa2 (B. burgdorferi sensu stricto), PBi (B. garinii, OspA-type 4), and PKo; p41i (internal flagellin fragment) from PKo and PBi; p58 derived from PBi; Ospl7 from PKo; decorin binding protein A (DbpA) derived from B. garinii strain PBr (OspA-type 3); VlsE from B. burgdorferi sensu stricto strain PKa2; and/or OspC from B. garinii strain 20047. See, e.g., 41 J. Clin. Microbiol. 1299 (2003);

Wilske et al, 188 Med. Micriobiol. Immunol. 139 (1999). It should be noted that the Borrelia antigen(s) can be derived from natural and/or recombinant sources. For example, the test antigen can be included in or 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. In one embodiment, the biological sample can be pre-enriched for antibodies by methods known in the art.

[0054] 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. "Epitope" refers to that portion of any molecule capable of being recognized by, and bound by, an antibody (the corresponding antibody binding region may be referred to as a paratope), and/or eliciting an immune response. In general, 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.

[0055] 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).

[0056] 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.

[0057] 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, gin, 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 Lyme arthritix).

[0058] 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- R A mediated addition of amino acids to proteins such as arginylation, and ubiquitination. [0059] 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.

[0060] 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 antibody 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

125 131 14 35 3

radioactive iodine ( I, I), carbon ( C), sulfur ( S), tritium ( H) 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).

[0061] 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.

[0062] 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).

[0063] 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.

[0064] The herein described methods may further be used to indicate a therapy for the subject who tests positive. The identification of annexin A2, ApoB, or MMP10 for a biomarker in Lyme arthritis, 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 antibiotic-refractory Lyme arthritis is diagnosed, nonsteroidal anti-inflammatory drugs (NSAIDs) or intra-articular steroid; or disease modifying anti-rheumatic drugs (DMARDs), such as hydroxychloroquine or methotrexate, may be prescribed. Additionally or alternatively, anti-TNF therapy (e.g., HUMIRA®

(adalimumab) or ENBREL® (etanercept)) may be beneficial. It is also possible that annexin A2, ApoB, or MMP10 -specific therapy, such as targeting Annexin A2, ApoB, or MMP10 and/or annexin A2, ApoB, or MMP10 -binding antibodies, 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 Lyme related autoimmune condition such as those described herein. 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 Lyme symptoms.

[0065] Another aspect of the invention relates to a kit for identifying a subject with a condition such as chronic inflammatory arthritis, or a Lyme related inflammatory condition (e.g., Lyme arthritis). The kit comprises one or more of annexin A2, ApoB, MMP10, or a portion or fragment thereof, such as a set of synthesized peptides, fragments, or epitopes thereof, and reagents necessary for conducting an assay capable of detecting the presence of immune reactivity of a subject to that test antigen (e.g., an antibody in a sample obtained from said subject that binds to one or more of annexin A2, ApoB, MMP10). 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, ECGF or fragments/epitopes thereof) and/or Borrelia antigens known in the art, such as those described herein. 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.

[0066] 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.

[0067] 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.

[0068] 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%. [0069] 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).

[0070] 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.

[0071] The present invention may be as defined in any one of the following numbered paragraphs.

1. A method for determining whether a biological sample comprising antibodies and/or immunological cells obtained from a subject is immunologically reactive with one or more of annexin A2, Apolipoprotein B (ApoB), and matrix metalloproteinase 10 (MMP10) comprising performing an assay for identifying the presence of antibodies that specifically bind one or more of annexin A2, ApoB, or MMP10, or performing an assay for identifying T cells specifically reactive to annexin A2, ApoB, or MMP10, and determining the sample is reactive with the annexin A2, ApoB, and MMP10 if the assay produces positive results compared to an appropriate control.

2. The method of paragraph 1 , wherein the assay that identifies the presence of the antibodies comprises contacting the sample with annexin A2 protein or a fragment thereof, ApoB protein or a fragment thereof, and/or MMPIO protein or a fragment thereof, under conditions that allow an immunocomplex of the antibody and the annexin A2, ApoB, or MMPIO 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 annexin A2, ApoB, and/or MMPIO and wherein the absence of the immunocomplex indicates the biological sample is not immunoreactive with annexin A2, ApoB, and/or MMPIO.

3. The method of any one of paragraphs 1-2, wherein the assay is an ELISA,

agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, or an immunoblot assay.

4. The method of paragraph 1, wherein the assay for identifying T cells specifically reactive to annexin A2, ApoB, or MMPIO comprises:

a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with one or more of annexin A2, ApoB, MMPIO whole protein(s) and/or polypeptide fragment(s); 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 specifically reactive to one or more of annexin A2, ApoB, and MMPIO when T cell proliferation or secretion of IFN-γ measured is increased over that of an appropriate control.

5. The method of any one of paragraphs 1 or 4, wherein the assay is a T cell proliferation assay.

6. The method of paragraph 5, wherein the assay is a H-thymdine incorporation assay, CFSE dilution, or an ELISPOT.

7. The method of any one of paragraphs 1 or 4, wherein the assay is a T cell reactivity assay.

8. The method of any one of paragraphs 1-7, wherein the polypeptide fragment is predicted to be presented by HLA-DR molecules associated with chronic inflammatory arthritis 9. The method of any one of paragraphs 1-8, wherein the polypeptide fragment is selected from the group consisting of

GVDEVTIVNILTNRSNAQR (SEQ ID NO: 4), TVILGLLKTPAQYDA (SEQ ID NO: 5), S GDFRKLM V AL AKGRRA (SEQ ID NO: 6), DKVLIRIMVSRSEVD (SEQ ID NO: 7), IEGNLIFDPNNYLPK (SEQ ID NO: 8), HDLQLLGKLLLMGARTLQGIP (SEQ ID NO: 9), VPLLLSEPINIIDALEMRD (SEQ ID NO: 10), EHYHIRVNLVKTIHDLHLFI (SEQ ID NO: 11), HLAFLVLICLPVCSAY (SEQ ID NO: 12), GTNLFLVAAHELGHS (SEQ ID NO: 13 ) and PTIRKIDAAVSDK (SEQ ID NO: 14).

10. The method of any one of paragraphs 1-9, wherein said biological sample is obtained from peripheral blood, synovial fluid, synovial tissue, peripheral blood mononuclear cells (PBMC), or synovial fluid mononuclear cells (SFMC).

11. The method of any one of paragraphs 1-10, wherein the subject exhibits symptoms of arthritis or other automimmune related disease manifestation.

12. The method of any one of paragraphs 1-11, wherein the subject is suspected of having Lyme disease.

13. A method of diagnosing Lyme arthritis in a subject comprising determining whether the subject is immunologically reactive with one or more of annexin A2, Apolipoprotein B (ApoB), and matrix metalloproteinase 10 (MMP10), wherein immunological reactivity of the subject to one or more of annexin A2, ApoB, and MMP10, as compared to an appropriate control, indicates the subject has Lyme arthritis.

14. The method of paragraph 13, wherein determining is by evaluating a biological sample obtained from the subject for immunological reactivity with the one or more of annexin A2, ApoB, and MMP10.

15. The method of any one of paragraphs 13 - 14, wherein determining immunological reactivity is by detecting the presence of T cells reactive to one or more of annexin A2, ApoB, and MMP10, comprising the steps:

a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with one or more of annexin A2, ApoB, MMP10 whole protein or polypeptide fragments; 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 one or more of annexin A2, ApoB, and MMPIO when T cell proliferation or secretion of IFN-γ is measured as increased over that of an appropriate control.

16. The method of any one of paragraphs 13-14, wherein determining immunological reactivity comprises determining if the subject has a B-cell response to one or more of annexin A2, ApoB, and MMPIO resulting in the production of autoantibodies that

specifically recognize the one or more annexin A2, ApoB, and MMPIO, by contacting the sample with annexin A2 protein or a fragment thereof, ApoB protein or a fragment thereof, MMPIO protein or a fragment thereof, under conditions that allow an immunocomplex of the antibody and the annexin A2, ApoB, or MMPIO 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 annexin A2, ApoB, and/or MMPIO and wherein the absence of an immunocomplex indicates the subject fails to present a B-cell response to annexin A2, ApoB, and/or MMPIO.

17. The method of paragraph 16, wherein the assay is an enzyme-linked immunosorbent assay (ELISA), agglutination test, direct immunofluorescence assay, indirect

immunofluorescence assay, or an immunoblot assay.

18. The method of any one of paragraphs 1-17 wherein the subject suffers from chronic inflammatory arthritis.

19. The method of any one of paragraphs 1 - 18, further comprising the step of treating the subject with a non-steroidal anti-inflammatory or disease modifying anti-rheumatic drugs.

20. A kit comprising, one or more potential antigen and/or potential epitope of annexin A2, ApoB, and/or MMPIO, and reagents for conducting an assay for detecting the presence of an antibody in a sample that binds to the one or more potential antigen and/or potential epitope of the annexin A2, ApoB, and MMPIO.

21. The kit of paragraph 20, further comprising one or more potential antigens and/or potential epitopes of endothelial cell growth factor (ECGF) and reagents for conducting an assay for detecting the presence of an antibody in a sample that binds to the one or more potential antigen and/or potential epitope of the ECGF.

22. The kit of any one of paragraphs 20-21, wherein the assay is an enzyme-linked immunosorbent assay (ELISA).

23. The kit of any one of paragraphs 20 - 21, wherein the assay is a western blot.

24. The kit of any one of paragraphs 20-23, wherein said kit further comprises

Borrelia antigens.

25. The kit or method of any one of paragraphs 1-24 for use in identifying a patient with chronic inflammatory arthritis.

26. The kit or method of paragraph 25, wherein the chronic inflammatory arthritis is antibiotic-resistant Lyme arthritis.

27. The kit or method of paragraph 25, wherein the chronic inflammatory arthritis is antibiotic-responsive Lyme arthritis.

[0072] The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES

[0073] Antibiotic-refractory Lyme arthritis, defined as persistent synovitis for months to years after spirochetal killing with antibiotic therapy, is hypothesized to result from B.

burgdorferi-induced autoimmunity. Lyme borreliosis, the most common tick-borne disease in the northern hemisphere, results from infection with spirochetes of the Borrelia burgdorferi sensu lato complex. In the U.S., Borrelia burgdorferi sensu stricto (Bb) is the sole cause of the illness, whereas in Europe, Borrelia afzelii (Ba) and Borrelia garinii (Bg) are the primary pathogens. (Steere, 345 N. Engl. J. Med. 115 (2001); Baranton et al, 42 Intl. J. Syst.

Bacteriol. 378 (1992); Canica et al, 25 Scand. J. Infect. Dis. 441 (1993)).

[0074] Infection with each of these species usually begins with a slowly expanding skin lesion, called erythema migrans (EM), which occurs at the site of the tick bite. In the U.S., where Bb is particularly arthritogenic, about 60% of un-treated patients develop intermittent or chronic joint swelling in later months, especially affecting knees (Steere et al., 107 Annals Internal Med. 725 (1987)). These patients can usually be treated successfully with 1 month of oral or intravenous (IV) antibiotics, an outcome called antibiotic-responsive arthritis (Steere et al., 37 Arthritis Rheum. 878 (1994); Dattwyler et al., 117 Wiener klinische Wochenschrift 393 (2005)). In a small percentage of patients, however, proliferative synovitis persists for months or several years after apparent spirochetal killing with >2 months of oral antibiotics, >1 month of intravenous antibiotics, or usually both, referred to as antibiotic-refractory arthritis (Steere & Angelis, 54 Arthritis Rheum. 3079 (2006a)). This disease course is hypothesized to result from infection induced autoimmunity.

[0075] Evidence supporting the hypothesis that Lyme arthritis is an infection induced autoimmunity disease is 3-fold: (1) PCR and culture results of synovectomy specimens have been uniformly negative (Nocton et al, 330 New. Engl. J. Med.229 (1994)); (2) relapse of infection has not been observed with the use of DMARDs after antibiotic therapy (Steere & Angelis, 2006a); and (3) specific HLA-DR alleles are the greatest known genetic risk factor for antibiotic-refractory arthritis, a risk factor commonly associated with autoimmune diseases (Steere et al, 203 J. Experi. Med. 961 (2006b)).

[0076] A number of spirochetal and host risk factors associated with antibiotic-refractory Lyme arthritis. These factors include infection with highly inflammatory B. burgdorferi RST1 (OspC type A) strains; a host Toll-like receptor 1 (TLR1) polymorphism (1805GG) which leads to exceptionally high inflammatory responses; certain HLA-DR molecules, such as the DRB 1 *0401 molecule, that bind an epitope of B. burgdorferi outer- surface protein A (OspA165-173); high levels of inflammatory cytokines and chemokines in joint fluid, particularly CXCL9 and CXCL10, which are chemoattractants for TH1 effector cells; and a dominant TH1 response in joint fluid with persistently low percentages of Treg. The present invention adds to these factors a biomarker, the autoantigen ECGF, which is present and induces autoreactive immune cells in many, but not all, antibiotic-refractory Lyme arthritis patients.

[0077] As with all forms of chronic inflammatory arthritis, including RA, the synovial lesion in in patients with antibiotic-refractory arthritis shows synovial hypertrophy, vascular proliferation, infiltration of mononuclear cells, and intense expression of human leukocyte antigen D-related (HLA-DR) molecules. In antibiotic-refractory Lyme arthritis, the HLA-DR risk alleles include HLA-DRB1 *0101, 0401, 0404, 0405 and 1501/DRB5*0101 (Steere et al, 2006b), similar to those in rheumatoid arthritis (RA) (Deighton et al., 36 Clin. Genet. 178 (1989); Seldin et al, 42 Arthritis Rheum. 1071 (1999)). HLA-DR molecules present peptides, both foreign and self, to CD4+ T cells, which leads to T cell activation and proliferation. With tissue-specific autoimmune diseases, HLA DR molecules in the target tissue, in this case synovial tissue, are thought to present disease-related autoantigenic epitopes, but for the most part these epitopes have not yet been identified.

[0078] Initial attempts to uncover autoantigens in antibiotic-refractory Lyme arthritis were based upon a search for molecular mimicry between an epitope of Bb outer-surface protein A (OspA163 175), which is bound by refractory arthritis-associated HLA-DR molecules, and those in human proteins (Steere et al., 52 Clin. Inf. Dis. S259 (2011)). The first candidate autoantigen identified was LFA-laL332-340 (Gross et al, 281 Sci. 703 (1998)), which has sequence homology with six of the nine core amino acid residues in OspA163-175; and later, MA WD BP280 288 which was shown to have sequence identity with eight of the nine core OspA residues (Drouin et al., 45 Molec. Immunol. 108 (2008)). Only a minority of patients had T cell reactivity with these peptides, however, and none had B-cell responses to these self proteins. Id.; (Steere et al, 48 Arthritis Rheum. 534 (2003)). Others, using recombinant antibody probes derived from B cells in patients' synovial lesions, identified cytokeratin 10 as a candidate autoantigen (Ghosh et al., 117 J. Immunol. 2486 (2006)). This probe cross-reacted with OspA, but only three of fifteen patients with antibiotic-refractory arthritis had slight antibody responses to this self protein. Hence, none of these known candidate autoantigens appeared to explain antibiotic-refractory Lyme arthritis.

[0079] The identification of in vivo HLA-DR-presented peptides in synovial tissue was developed as a novel, unbiased approach that combines discovery-based proteomics with translational research for the identification of immunogenic self antigens. This protocol has steps comprising: (a) a proteomics approach utilizing tandem mass spectrometry (MS/MS) for the identification of HLA-DR-presented peptides in individual subject's synovial tissue; (b) synthesis and testing of all peptides identified in step (a) for reactivity with the same subject's PBMC, thereby asking the subject's own T cells to indicate which HLA-DR self- peptides may be acting as autoantigens; and (c) validation that any immunogenic peptides and their source proteins identified in a single subject in step B also induce T- and B cell reactivity in large numbers of subjects with Lyme arthritis. This method was initially described in Published U.S. Application 2013-0302329, the contents of which are

incorporated herein by reference, where it was used to identify endothelial cell growth factor (ECGF) as the first autoantigen known to be a target of T and B cell responses in antibiotic- refractory or antibiotic-responsive Lyme arthritis (LA), but not in Rheumatoid arthritis (RA). Example 1 - Identification of Annexin A2 as an Autoantigen in Rheumatoid Arthritis and in Lyme Arthritis

[0080] HLA-DR-presented peptides were eluted from synovia, identified by tandem mass spectrometry, synthesized, and tested for reactivity with the matching patient's PBMC.

Immunoreactive peptides or their source proteins were then tested for T cell reactivity by IFN-γ ELISpot assay or for antibody responses by ELISA. All RA patients met the 2010 ACR/EULAPv criteria for RA and the LA patients met the CDC criteria for Lyme disease.

[0081] In one RA patient who lacked positive tests for rheumatoid factor (RF) or anti- citrullinated protein antibodies (ACPA), 1 of 86 non-redundant HLA-DR-presented peptides identified from her synovial tissue induced her PBMC to secrete IFN-γ. The peptide was derived from the protein annexin A2 and others had shown that -10% of RA patients make autoantibodies against this self-protein. We tested serum samples from our cohort of RA patients for anti-annexin A2 autoantibodies, and for comparison, from healthy control subjects or from patients with antibiotic-responsive or antibiotic-refractory LA. In our RA cohort, 24% of 91 patients had antibody responses to annexin A2 that were >3SD above the mean value in healthy control subjects (Figure 1C). Surprisingly, about 20%> of the patients with antibiotic-responsive or antibiotic-refractory LA also had antibody reactivity with this autoantigen. In annexin A2 -positive RA patients, the magnitude of antibody responses to ACPA or RF were less than in annexin A2 -negative patients. Analysis of T and B cell responses in RA and LA patients is expected to show a linkage of T and B cell responses to annexin A2 in both the RA and LA cohorts.

[0082] The results from these experiments indicates that annexin A2, a phospholipid- binding protein that protects damaged endothelial cells, is an autoantigen in a subgroup of patients with RA. Moreover, this protein also serves as an autoantigen in a subgroup of patients with LA. As with reactivity to ECGF, autoantibody responses to annexin A2 in Lyme disease seem to occur as a part of the immune response to the infection, whereas additional factors, such as immune dysregulation, are required for refractory arthritis.

A subset of rheumatoid arthritis and Lyme arthritis patients have T and B cell autoreactivity to the self-protein annexin A2

[0083] Of 116 non-redundant HLA-DR-presented peptides identified from the synovial tissue of a rheumatoid arthritis (RA) patient, 2 peptides derived from annexin A2 were identified and 1 was shown to be immunoreactive when tested using the patient's own peripheral blood mononuclear cells (PBMC). The identification of these peptides is described in Seward et al. (Molecular and Cellular Proteomics. 2011; March, 10(3):M110.002477) and the method used to screen these peptides for immunoreactivity using the patient's own T cells is described in Drouin et al. (Arthritis & Rheumatism. 2013; 65: 186-196).

A. A subset of rheumatoid arthritis and Lyme arthritis patients have T cell autoreactivity to peptides derived from annexin A2

[0084] PBMC from RA and Lyme arthritis (LA) patients, and healthy control (HC) subjects were tested for T cell reactivity to the two peptides originally identified from the synovial tissue of the RA patient, plus two additional annexin A2 peptides predicted to be promiscuous HLA-DR binders (promiscuous T cell epitopes). The sequences of these 4 annexin A2 peptides are as follows with the HLA-DR-presented peptides identified in the patient's synovial tissue sample underlined and the immunoreactive peptide indicated by an * : ⁵⁰GVDEVTIVNILTNRSNAQR⁶⁸ (SEQ ID NO: 4) , ⁹⁷TVILGLLKTPAQYDA¹¹¹ (SEQ ID NO: 5), ¹⁶⁴SGDFR LMVALAKGRRA¹⁸⁰ (SEQ ID NO: 6), ²⁸⁵DKVLIRIMVSRSEVD*²⁹⁹ (SEQ ID NO: 7). The results are presented in Figure 1A.

B. A subset of rheumatoid arthritis and Lyme arthritis patients produce autoantibodies against the self-protein annexin A2

[0085] The levels of IgG anti-annexin A2 in the sera of HC subjects, patients with RA, patients with LA, or patients with other arthritides including osteoarthritis (OA),

spondylarthropathy (SA) or psoriatic arthritis (PA) were measured by enzyme-linked immunosorbent assay (ELISA). The sera of a subset of both RA and LA patients had autoantibodies that recognized annexin A2. Results are presented in Figure IB.

Materials and Methods

[0086] The RA patients' samples were analyzed for annexin A2 immunoreactivity using a human IFN-y/IL17 Double-Color Enzymatic Immunospot kit (Cellular Technology Limited, OH), whereas the LA patients' samples were tested using a human IFN-γ

ELISpot^plus kit (MabTech). The 4 peptides were combined (1 μΜ each) and tested in duplicate wells. As controls, wells were stimulated with either phytohemagglutinin (positive control) or no antigen (negative control). After 5 days, cells were transferred to ELISpot plates coated with IFN-y/IL17 capture antibodies (RA patients) or only with IFN-γ capture antibodies (LA patients), and incubated overnight. Images of wells were captured using ImmunoSpot series 3B analyzer and spots were counted using ImmunoSpot software. For each well, the spot forming units (SFU) per 10⁶ cells was calculated. A positive response was defined as 3 standard deviations (SD) above the mean SFU/10⁶ cells of HC subjects (area above the gray shaded region). Only IFN-γ results are shown.

[0087] ELIS A plates were coated with 1 μg/ml of recombinant human annexin A2 (Novoprotein, Summit, NJ) 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. Then, 100 μΐ of each patient's serum sample (diluted 40-fold) was added in duplicate for 1.5 hours, followed by horseradish-peroxidase (HRP)-conjugated goat anti- human IgG (sc-2453, Santa Cruz Biotech), and then TMB substrate (BD, San Diego, CA). For generation of a standard curve, serial dilutions of rabbit polyclonal antibody against annexin A2 (sc-9061, Santa Cruz Biotech) were included with each assay. The shaded gray area corresponds to 3 SD above the mean of HC subjects.

Example 2 -Apo lipoprotein B100 Is A Target at T and B Cell Responses in a Subgroup of Patients with Lyme Disease

[0088] Using the same approach as in Published U.S. Application 2013-0302329, apolipoprotein B 100 (ApoB) was identified as another novel autoantigen in Lyme disease. HLA-DR presented self-peptides were isolated from ALRA patients' synovia, identified by tandem mass spectrometry, synthesized, and tested for reactivity with the matching patient's PBMC using an IFN-γ ELISpot assay. Immunoreactive peptides

⁶⁵⁵IEGNLIFDPNNYLPK⁶⁶⁹ (SEP ID NO: 8), ⁷⁹⁰HDLQLLGKLLLMGARTLQGIP⁸¹⁰ (SEQ ID NO: 9), ²⁰²⁶ VPLLLS EPINIID ALEMRD²⁰⁴⁴ (SEQ ID NO: 10) and

2²¹⁴EHYHIRVNLVKTIHDLHLFI²²³ (SEQ ID NO: 11) and their full-length source proteins were then tested for T and B cell reactivity using large numbers of patient and control cells and sera. Samples from patients with antibiotic-responsive arthritis were seen prior to antibiotic therapy, when the infection was still active, whereas those from patients with antibiotic-refractory arthritis were collected after antibiotics, during the presumed autoimmune phase of the illness. Antibody responses were quantified by ELISA.

[0089] From the synovial tissue of one ALRA patient, 141 non-redundant HLA-DR- presented self-peptides were identified and tested. One peptide derived from ApoB caused significant secretion of IFN-γ by ELISpot.

[0090] Additional testing of 25 patients showed -10-30% patients with early or late manifestations of Lyme disease had T cell responses to ApoB. To look for linked T and B cell responses, patients' serum samples were also tested for anti-ApoB IgG antibodies. By definition, none of the 55 healthy control subjects had a positive response (defined as >3 SD above the mean value in these subjects) (Figure 2C). In comparison, 5% of patients with EM and 12% each of patients with responsive or refractory arthritis had positive responses for anti-ApoB IgG autoantibodies. Compared with the EM group, the values were significantly higher in both arthritis groups (P<0.0001), particularly in those with responsive arthritis, a group still actively infected (Figure 2C).

A subset of Lyme arthritis have T and B cell autoreactivity to the self-protein apolipoprotein B-100

[0091] Of 217 non-redundant HLA-DR-presented peptides identified from the synovial tissue of a patient with Lyme arthritis (LA), one derived from apolipoprotein B-100 (ApoB) was immunoreactive (⁶⁵⁵IEGNLIFDPNNYLPK⁶⁶⁹ (SEQ ID NO: 8) when tested using the patient's peripheral blood mononuclear cells (PBMC). The method used to identify these peptides is described in Seward et al. (Molecular and Cellular Proteomics. 2011; March, 10(3):M110.002477) and the method used to screen these peptides for immunoreactivity using the patient's own T cells is described in Drouin et al. (Arthritis & Rheumatism. 2013; 65: 186-196).

A. A subset of Lyme arthritis patients have T cell autoreactivity to peptides derived from apolipoprotein B-100

[0092] Peripheral blood mononuclear cells (PBMC) from healthy control (HC) subjects, Lyme disease patients with erythema migrans (EM) [an early disease manifestation] or Lyme arthritis (LA) [a late disease manifestation] were tested for T cell autoreactivity against human full-length recombinant ApoB protein (Millipore). These results are presented in Figure 2A.

B. A subset of Lyme arthritis patients produce autoantibodies against the self-protein apolipoprotein B 100

[0093] The levels of IgG anti-ApoB in the sera of Lyme disease patients with EM or LA, patients with rheumatoid arthritis (RA) or HC subjects were measured by enzyme-linked immunosorbent assay (ELISA). A significant number of Lyme disease patients with EM or LA express autoantibodies that recognize ApoB. The results are presented in Figure 2B. [0094] The results of these experiments indicate that ApoB is a target of T and B cell responses in a subset of patients with Lyme disease. Although the molecular mechanisms are not yet known, B. burgdorferi, an organism with sequences for >100 lipoproteins, including cholesterol, may contribute directly to the development of this autoimmune response. As with reactivity to ECGF, autoantibody responses to ApoB seem to occur as part of the immune response to the infection, whereas additional factors, such as immune dysregulation, are also required for refractory arthritis.

Materials and Methods

[0095] Patient and control samples were analyzed using a human IFN-γ ELISpot^plus kit (MabTech). The ApoB protein (1.3 nM) was tested in duplicate wells. As controls, wells were stimulated with either phytohemagglutinin (positive control) or no antigen (negative control). Due to the fact that the recombinant ApoB was dissolved in a buffer containing sodium deoxycholate (to keep the protein in solution), an equal concentration of sodium deoxycholate (0.16mM) was added to each of the negative control wells. After 5 days, cells were transferred to ELISpot plates coated with IFN-γ antibodies and incubated overnight. Images of wells were captured using ImmunoSpot series 3B analyzer and spots were counted using ImmunoSpot software. For each well, the spot forming units (SFU) per 10⁶ cells was calculated. A positive response was defined as 3 standard deviations (SD) above the mean SFU/10⁶ cells of healthy control subjects (area above the gray shaded region).

[0096] ELISA plates were coated with 0.5 μg/ml recombinant human ApoB (Millipore) overnight at 4°C. For each patient and healthy subject, duplicate apoB coated, apoB uncoated wells, and buffer only were measured. All subsequent steps were performed on a platform shaker set at 200 revolutions per minute at room temperature. The plates were incubated with a 3% bovine serum albumin (BSA; Equitech-Bio, Inc.) in PBS-0.05% Tween 20 (PBST) blocking buffer. Plates were washed with PBST and then incubated with patient serum samples (100 μΐ; diluted 1 :200) or the positive control goat anti-apolipoprotein B-100 antibody (AB742, Millipore). Subsequently, horseradish peroxidase-conjugated goat anti- human IgG (Santa Cruz Biotechnology) or horseradish peroxidase-conjugated donkey anti- goat IgG (Life Technologies) was added, followed by TMB substrate (BD Biosciences). For interplate standardization, the positive control anti-ApoB antibody was included on each plate. A positive B cell response was defined as >3 SD above the mean in 57 healthy subjects (area above the gray shaded region). Example 3- Lyme Disease Patients Have T and B Cell Autoreactivity to Matrix Metalloproteinase 10

[0097] A subset of Lyme arthritis have T and B cell autoreactivity to the self-protein matrix metalloproteinase 10. Of 122 non-redundant HLA-DR-presented peptides identified from the synovial tissue of a patient with Lyme arthritis (LA), one derived from matrix metalloproteinase 10 (MMP10) was immunoreactive (²⁰⁸ GTNLFL V AAHELGHS ²²² (SEQ ID NO: 13)) when tested using the patient's peripheral blood mononuclear cells (PBMC).

Other peptides used in the experiments were ³HL AFLVLICLP VC SAY¹⁸ (SEQ ID NO: 12), and ³⁸²PTIR IDAAVSDK³⁷⁴ (SEQ ID NO: 14). The method used to identify these peptides is described in Seward et al. Molecular and Cellular Proteomics. 2011; March,

10(3):M110.002477 and the method used to screen these peptides for immunoreactivity using the patient's own T cells is described in Drouin et al. Arthritis & Rheumatism. 2013; 65: 186- 196.

A. A subset of Lyme arthritis patients have T cell autoreactivity to peptides derived from matrix metalloproteinase 10

[0098] Peripheral blood mononuclear cells (PBMC) from healthy control (HC) subjects, Lyme disease patients with erythema migrans (EM) [an early disease manifestation] or Lyme arthritis (LA) [a late disease manifestation] were tested for T cell autoreactivity against human full-length recombinant matrix metalloproteinase 10 (R&D Systems). Results are shown in Figure 3A.

B. A subset of Lyme arthritis patients produce autoantibodies against the self-protein matrix metalloproteinase 10

[0099] The levels of IgG anti-MMPIO in the sera of Lyme disease patients with EM or LA, patients with rheumatoid arthritis (RA) or HC subjects were measured by enzyme-linked immunosorbent assay (ELISA). A significant number of patients with Lyme arthritis express autoantibodies that recognize MMP10. Results are shown in Figure 3B.

Materials and Methods

[00100] ELISA plates were coated with 0.5 μg/ml recombinant human MMP10 (R&D Systems)) overnight at 4°C. For each patient and healthy subject, duplicate MMP10 coated, MMP10 uncoated wells, and buffer only were measured. All subsequent steps were performed on a platform shaker set at 200 revolutions per minute at room temperature. The plates were incubated with a 3% bovine serum albumin (BSA; Equitech-Bio, Inc.) in PBS- 0.05% Tween 20 (PBST) blocking buffer. Plates were washed with PBST and then incubated with patient serum samples (100 μΐ; diluted 1 :200) or the positive control anti-MMPIO antibody ( MAB910 R&D Systems). Subsequently, horseradish peroxidase-conjugated goat anti-human IgG (Santa Cruz Biotechnology) or horseradish peroxidase-conjugated donkey anti-goat IgG (Life Technologies) was added, followed by TMB substrate (BD Biosciences). For interplate standardization, the positive control MMP10 antibody was included on each plate. A positive B cell response was defined as >3 SD above the mean of healthy subjects (area above the gray shaded region).

[00101] The patient and control samples were analyzed using a human IFN-γ ELISpot^plus kit (MabTech). The MMP10 protein (85 nM) and tested in duplicate wells. As controls, wells were stimulated with either phytohemagglutinin (positive control) or no antigen (negative control). After 5 days, cells were transferred to ELISpot plates coated with IFN-γ antibodies and incubated overnight. Images of wells were captured using ImmunoSpot series 3B analyzer and spots were counted using ImmunoSpot software. For each well, the spot forming units (SFU) per 10⁶ cells was calculated. A positive response was defined as 3 standard deviations (SD) above the mean SFU/10⁶ cells of healthy control subjects (area above the gray shaded region).

Example 4 -ELISPOT assays can be performed with peptides suspected of being

autoantigens in a subject

[00102] Enzyme-linked immunosorbent spot (ELISPOT) assays are performed using ELISpot^plus for human IFN-γ kits (Mabtech Inc., # 3420-2AW-Plus). Briefly, PBMC collected using Ficoll-Hypaque density centrifugation and stored in liquid nitrogen are thawed quickly and plated in round bottom, 96-well plates (Costar, # 3799) at 2 x 10⁵ per well in 200 μΐ of complete media (RPMI-1640, 2 mM glutamine, 100 units/ml penicillin 100 μg/ml streptomycin, 10 mM HEPES (all from Invitrogen) and 10% human AB serum

(Cellgrow). Peptides are added at a concentration of 1 μΜ in duplicate wells. Positive and negative controls consist of 1%> PHA (Invitrogen, # 10576-015) and no antigen, respectively. After 5 days at 37°C and 5% C0₂, cells are transferred to ELISPOT plates (Mabtech), previously coated with IFN-γ capture antibody, and incubated overnight. All subsequent steps are performed as detailed in the manufacturer's protocol. Images of wells are captured using ImmunoSpot series 3B analyzer and spots counted using ImmunoSpot 5.0 academic software (Cellular Technology Limited).

Example 5 - ELISA assays can be performed with peptides suspected of being autoantigens in a subject.

Serum anti-peptide antibody ELISA :

[00103] EasyWash ELISA plates (CoStar) are coated with 100 μΐ of 0.5 μg/ml carrier free, recombinant human PD-autoantigen dissolved in PBS and incubated overnight at 4°C. All subsequent steps are performed at room temperature with plates on a platform shaker set at 200 rpm. The next day, plates are washed three times with PBST (phosphate buffered saline and 0.05% Tween-20) then incubated with 200 μΐ of blocking buffer (5% nonfat dry milk in PBST) for 1 hr. Afterwards, wells are washed three times with PBST and 100 μΐ of each patient's serum sample diluted 1/100 with blocking buffer is added to individual wells and incubated for 1 hr. As a control, serum from eight healthy subjects is added to each plate to be used for inter-plate standardization. After three more washes with PBST, 100 μΐ goat anti-human IgG conjugated to horseradish peroxidase (KPL # 074-1006) diluted 1 :7500 in blocking buffer is added to each well and incubated for 1 hr. Plates are then washed three times with PBST, followed by three times with PBS and incubated with 100 μΐ of a 1 : 1 mixture of the substrate 3,3',5,5'-tetramethylbenzidine and 0.01%> hydrogen peroxide (TMB substrate reagent kit, # 555214) (BD Biosciences). The reaction is stopped after 3 min with 100 μΐ of 2N sulfuric acid (LabChem Inc., # LC25790-2). Absorbance values (OD₄₅₀) for each well were determined using a microplate reader (Bio-Rad, model 550).

Synovial fluid autoantigen sandwich ELISA :

[00104] EasyWash ELISA plates are coated with 50 μΐ of the capture antibody, goat anti-human PD-autoantigen diluted in PBS (5 μg/ml) and incubated overnight at 4°C. All subsequent steps are performed at room temperature. The next day, plates are washed three times with PBS and incubated with blocking buffer for 30 min. Afterwards, plates are washed three times with PBS and 100 μΐ of each patients' synovial fluid sample diluted 1 : 10 with blocking buffer are added to individual wells and incubated for 2 hr. In order to quantify results, recombinant human PD-autoantigen serially diluted with blocking buffer is also added to each plate to generate a standard curve. After washing the plates three times, wells are filled with 150 μΐ of blocking buffer, gently vortexed and washed again three times with PBS to ensure removal of all unbound proteins. Plates are then incubated with 50 μΐ of the mouse anti-human PD-autoantigen antibody (Santa Cruz, SC-47702) diluted in blocking buffer (5 ng/ml) for 2 hr. Plates are again washed with PBS and 50 μΐ of the detection antibody, goat anti-mouse IgG conjugated to horse radish peroxidase (Santa Cruz, #SC-2005) diluted in blocking buffer (1 : 1000) is added to plates and incubated for 1 hr. After plates are washed three times with PBS, 100 μΐ of TMB was added for ~6 min and then the reaction is stopped with 100 μΐ of 2N sulfuric acid. Plates are read as described above.

Example 6 - Immunoblotting can be performed with peptides suspected of being autoantigens in a subject.

[00105] Autoantigen (e.g, Human recombinant autoantigen) 12 μg is electrophoresed through a 10% mini-PROTEAN TGX gels (Bio-Rad) then transferred to nitrocellulose membranes. All subsequent steps are performed at room temperature with rocking.

Membranes are cut into strips, individually placed into eight channel reservoir liners (Costar, #4878) and incubated for 1 hr in 1.5 ml blocking buffer (5% nonfat dry milk, 0.1% Tween-20 in 20 mM Tris, 500 mM sodium chloride; pH 7.5). Afterwards, strips are washed three times for 1 min intervals with rinse buffer (0.1% Tween-20 in 20 mM Tris, 500 mM sodium chloride; pH 7.5) and each individual strip is incubated for 1 hr with patient's serum diluted 1 : 100 in blocking buffer. Strips are again washed three times with rinse buffer and incubated for 1 hr with goat anti-human IgG antibody conjugated to alkaline phosphatase (KPL, #4751- 1006) diluted 1 :2000 in blocking buffer. Strips are washed three times with rinse buffer and another three times with 20 mM Tris, 500 mM sodium chloride; pH 7.5. Bands are visualized by incubation with NBT/BCIP substrate solution (Roche Diagnostics GmbH, #

11681451001) for 3-5 min after which the strips are washed with copious amounts of water to stop the reaction. Bands are considered positive if darker than the pre-determined positive control sample included in each assay.

Example 7 - Immunohistochemical characterization can be performed with peptides suspected of being autoantigens in a subject.

[00106] Synovial tissue biopsies obtained from antibiotic-refractory Lyme arthritis patients after synovectomies are placed in optimal cutting temperature (Tissue-Tek, Sakura, Japan) and stored in liquid nitrogen. Subsequently, 6-8 μι -ΐΐι^ cryosections are cut and stored at -70°C until use. After an initial review by hematoxylin-eosin of the biopsies;

sections selected are those in which lining, sublining and subsynovium are present. [00107] The presence of the suspected autoantigen is assayed by immunohistochemital staining performing the immunoperoxidase technique. The sections are fixed in cold acetone for 3 min and air dried. Fixed sections are washed in PBS. Endogenous peroxidase is blocked by incubating the sections with 3% hydrogen peroxide in methanol for 10 min. After rinsing with PBS three times, nonspecific reaction is blocked by incubating sections in IX power block solution (Biogenex cat. No. HK085-5K) containing 10% normal donkey serum. The sections are then incubated at 4°C overnight with appropriate dilution (3 μg/ml) of anti-rabbit polyclonal PD-ECGF (Abeam Cat. No. ab75920). Negative controls are done using nonspecific rabbit IgG (Sigma) as the primary antibody at the same IgG concentrations. After 5 min rinses with PBS, the sections are incubated with biotinylated anti-rabbit secondary antibody (Biogenex Cat. No.HK3260709) for 40 min at room temperature, rinsed in PBS, and incubated with peroxidase-streptavidin (Biogenex HK320-UK) for 20 min. After three rinses with PBS, the sections are incubated with diamiobenzidine substrate (Biogenex HK130-5K) for up to 10 min. The sections are washed in distilled water and counterstained with Mayer's hemotoxylin, and glycerol-mounted. Microscopic images are obtained with a Nikon eclipse ME6000 microscope using a Nikon digital camera DXM1200C. The intensity of PD-ECGF staining on each synovial tissue regions (lining, sublining and subsynovium) was graded on an arbitrary scale of 0-3: 0 = no ECGF positive stain cells; 1 = few (-50) positive staining cells; 2 = many (-50 to 100) positive staining cells; and 3= most (>100) positive cells determined by counting a total of five 200X microscopic fields on for each synovial tissue regions (lining, sublining, and subsynovium); to sample a larger area per section every fifth 200X microscopic field are examined. For illustration purposes, whole-slide imaging of five random biopsies with moderate to intense stain in all synovial regions are scanned using with Mirax Viewer Scan (Carl Zeiss), at 20x/0.8 Plan-Apochromat objective and image is prepared using Mirax viewer software.

Claims

What is claimed:

1. A method for determining whether a biological sample comprising antibodies and/or immunological cells obtained from a subject is immunologically reactive with one or more of annexin A2, Apolipoprotein B (ApoB), and matrix metalloproteinase 10 (MMP10) comprising performing an assay for identifying the presence of antibodies that specifically bind one or more of annexin A2, ApoB, or MMP10, or performing an assay for identifying T cells specifically reactive to annexin A2, ApoB, or MMP10, and determining the sample is reactive with the annexin A2, ApoB, and MMP10 if the assay produces positive results compared to an appropriate control.

2. The method of claim 1, wherein the assay that identifies the presence of the antibodies comprises contacting the sample with annexin A2 protein or a fragment thereof, ApoB protein or a fragment thereof, and/or MMP10 protein or a fragment thereof, under conditions that allow an immunocomplex of the antibody and the annexin A2, ApoB, or MMP10 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 annexin A2, ApoB, and/or MMP10 and wherein the absence of the immunocomplex indicates the biological sample is not immunoreactive with annexin A2, ApoB, and/or MMP10.

3. The method of any one of claims 1-2, wherein the assay is an ELISA, agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, or an

immunoblot assay.

4. The method of claim 1, wherein the assay for identifying T cells specifically reactive to annexin A2, ApoB, or MMP10 comprises:

a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with one or more of annexin A2, ApoB, MMP10 whole protein(s) and/or polypeptide fragment(s); 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 specifically reactive to one or more of annexin A2, ApoB, and MMP10 when T cell proliferation or secretion of IFN-γ measured is increased over that of an appropriate control.

5. The method of any one of claims 1 or 4, wherein the assay is a T cell proliferation assay.

6. The method of claim 5, wherein the assay is a H-thymdine incorporation assay, CFSE dilution, or an ELISPOT.

7. The method of any one of claims 1 or 4, wherein the assay is a T cell reactivity assay.

8. The method of any one of claims 1-7, wherein the polypeptide fragment is predicted to be presented by HLA-DR molecules associated with chronic inflammatory arthritis

9. The method of any one of claims 1-8, wherein the polypeptide fragment is selected from the group consisting of

GVDEVTIVNILTNRSNAQR (SEQ ID NO: 4), TVILGLLKTPAQYDA (SEQ ID NO: 5), S GDFRKLM V AL AKGRRA (SEQ ID NO: 6), DKVLIRIMVSRSEVD (SEQ ID NO: 7), IEGNLIFDPNNYLPK (SEQ ID NO: 8), HDLQLLGKLLLMGARTLQGIP (SEQ ID NO: 9), VPLLLSEPINIIDALEMRD (SEQ ID NO: 10), EHYHIRVNLVKTIHDLHLFI (SEQ ID NO: 11), HLAFLVLICLPVCSAY (SEQ ID NO: 12), GTNLFLVAAHELGHS (SEQ ID NO: 13 ) and PTIRKIDAAVSDK (SEQ ID NO: 14).

10. The method of any one of claims 1-9, wherein said biological sample is obtained from peripheral blood, synovial fluid, synovial tissue, peripheral blood mononuclear cells (PBMC), or synovial fluid mononuclear cells (SFMC).

11. The method of any one of claims 1-10, wherein the subject exhibits symptoms of arthritis or other automimmune related disease manifestation.

12. The method of any one of claims 1-11, wherein the subject is suspected of having Lyme disease.

13. A method of diagnosing Lyme arthritis in a subject comprising determining whether the subject is immunologically reactive with one or more of annexin A2, Apolipoprotein B (ApoB), and matrix metalloproteinase 10 (MMP10), wherein immunological reactivity of the subject to one or more of annexin A2, ApoB, and MMP10, as compared to an appropriate control, indicates the subject has Lyme arthritis.

14. The method of claim 13, wherein determining is by evaluating a biological sample obtained from the subject for immunological reactivity with the one or more of annexin A2, ApoB, and MMP10.

15. The method of any one of claims 13 - 14, wherein determining immunological reactivity is by detecting the presence of T cells reactive to one or more of annexin A2, ApoB, and MMP10, comprising the steps:

a) stimulating peripheral blood mononuclear cells (PBMC) of the subject or the synovial fluid mononuclear cells (SFMC) of the subject in vitro with one or more of annexin A2, ApoB, MMP10 whole protein or polypeptide fragments; 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 one or more of annexin A2, ApoB, and MMP10 when T cell proliferation or secretion of IFN-γ is measured as increased over that of an appropriate control.

16. The method of any one of claims 13-14, wherein determining immunological reactivity comprises determining if the subject has a B-cell response to one or more of annexin A2, ApoB, and MMP10 resulting in the production of autoantibodies that specifically recognize the one or more annexin A2, ApoB, and MMP10, by contacting the sample with annexin A2 protein or a fragment thereof, ApoB protein or a fragment thereof, MMP10 protein or a fragment thereof, under conditions that allow an immunocomplex of the antibody and the annexin A2, ApoB, or MMP10 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 annexin A2, ApoB, and/or MMP10 and wherein the absence of an immunocomplex indicates the subject fails to present a B-cell response to annexin A2, ApoB, and/or MMP10.

17. The method of claim 16, wherein the assay is an enzyme-linked immunosorbent assay (ELISA), agglutination test, direct immunofluorescence assay, indirect immunofluorescence assay, or an immunoblot assay.

18. The method of any one of claims 1-17 wherein the subject suffers from chronic inflammatory arthritis.

19. The method of any one of claims 1 - 18, further comprising the step of treating the subject with a non-steroidal anti-inflammatory or disease modifying anti-rheumatic drugs.

20. A kit comprising, one or more potential antigen and/or potential epitope of annexin A2, ApoB, and/or MMP10, and reagents for conducting an assay for detecting the presence of an antibody in a sample that binds to the one or more potential antigen and/or potential epitope of the annexin A2, ApoB, and MMP10.

21. The kit of claim 20, further comprising one or more potential antigens and/or potential epitopes of endothelial cell growth factor (ECGF) and reagents for conducting an assay for detecting the presence of an antibody in a sample that binds to the one or more potential antigen and/or potential epitope of the ECGF.

22. The kit of any one of claims 20-21, wherein the assay is an enzyme-linked

immunosorbent assay (ELISA).

23. The kit of any one of claims 20 - 21, wherein the assay is a western blot.

24. The kit of any one of claims 20-23, wherein said kit further comprises

Borrelia antigens.

25. The kit or method of any one of claims 1-24 for use in identifying a patient with chronic inflammatory arthritis.

26. The kit or method of claim 25, wherein the chronic inflammatory arthritis is antibiotic-resistant Lyme arthritis.

27. The kit or method of claim 25, wherein the chronic inflammatory arthritis is antibiotic-responsive Lyme arthritis.