Compositions and methods for diagnosing lyme disease and for predicting elimination of lyme disease spirochetes after treatment

Abstract

Compositions and methods are provided for the detection, diagnosis and prognosis of Lyme disease (LD), including methods for confirming Borrelia spp. infection by contacting a whole blood sample from a subject suspected of having LD with a plurality of synthetic peptides comprising T cell epitope-containing regions derived from Borrelia proteins expressed at different stages of Lyme disease in vitro, and indirectly detecting LD-specific activated T cells by assaying for the production of indicators of T cell immune response (e.g., interferon-gamma) produced in response to stimulation by the peptides. Also disclosed are methods for predicting the elimination of LD spirochetes in a LD patient who has undergone treatment for LD by contacting a whole blood sample from the subject with peptides comprising specific T cell epitope regions of Borrelia proteins expressed at different stages of Lyme disease, and confirming the absence of Borrelia-specific activated T cells in the sample by the absence of detectable indicators of T cell immune response (e.g., interferon-gamma).

Description

[0001] SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and therefore is incorporated by reference herein. The text file contains 12.3 KB and is named 770025_466WO_SEQUENCE_LISTING.txt. The text file was created on September 14, 2016, and is electronically submitted by EFS-Web. BACKGROUND TECHNICAL FIELD

[0004] The present disclosure relates generally to compositions and methods for diagnosing Lyme disease and for assessing the efficacy of treatment of Lyme disease. More specifically, peptides comprising multiple T cell epitopes derived from different Borrelia polypeptide antigens expressed at different stages of Lyme disease are provided for use in a sensitive secondary in vitro immune response assay for Borrelia-specific T cell reactivity.

[0005] Description of the Related Art

[0006] Lyme disease is a tick-borne infectious disease caused by pathogenic spirochetes of the genus Borrelia, including B. burgdorferi, B. afzelii, B. garinii, and other Borrelia spp. The infectious microorganisms are transmitted from the tick saliva into the bite wound, and the "early localized" stage, the first of three defined clinical stages of the disease, usually occurs within two weeks of the tick bite. The early localized stage is often, but not always, accompanied by erythema migrans (EM), a red macular skin lesion at the site of the tick bite. As the infectious Borrelia bacteria disseminate systemically through the bloodstream and / or lymphatic circulation, the disease progresses to the second stage, the "early disseminated" stage, and can be accompanied by one or more of other skin lesions, fatigue, myalgias, arthralgias, neurological and cardiac symptoms. If untreated, the disease can further progress to "late stage" Lyme disease, characterized by arthritis, encephalomyelitis and / or peripheral neuropathy, and possibly other symptoms, including chronic Lyme disease. About 25,000 to 30,000 cases of Lyme disease are reported annually in the United States, and the CDC estimates the actual incidence to be ten times that amount due to incomplete reporting; other cases have been reported in Europe and Asia (Wright et al., 2012 Am. Fam. Physician 85:1086; Shapiro, 2014 N. Engl. J. Med. 370:1724).

[0007] Changes in the profile of expression of antigenic proteins by Borrelia spp. accompany the development of Lyme disease and are thought to underlie the immune invasion mechanism that can lead to chronic Lyme disease (e.g., Drecktrah et al., 2013 PLoS One 8(7): e68799; Schmit et al., 2011 Front. Microbiol. 2: 141; Salo et al., 2011 J. Infect. Dis. 204: 65; Liang et al., 2004 Infect. Immun. 72: 5759; Aberer, 2007 J. Dtsch. Dermatol. Ges. 5(5): 406).

[0008] Effective treatment of Lyme disease (LD) depends on accurate detection of early infection by Borrelia spp. In some cases, the presence of characteristic erythema migrans (EM) skin lesions is sufficient to make an accurate diagnosis when the disease is still in the early localized stage (e.g., Nadelman et al., 1996 Am J Med 100: 502-508). However, a significant number of patients do not develop EM lesions, or the rash is not detected or misidentified. In these cases, traditional serological tests that detect anti-Borrelia IgM and / or IgG antibody responses are the most widely used procedures to confirm the disease (e.g., Dattwyler et al., 2010 Clin Infect Dis 50: 521-522; Johnson et al., 1996 J. Infect. Dis. 174: 346).

[0009] There is currently no laboratory test that reliably predicts successful treatment of LD after administration of a standard course of therapy (e.g., an antibiotic regimen). Circulating antibodies elicited by Borrelia infection can persist for years, even after successful antibiotic treatment that clears the infection. Thus, tests for Borrelia-specific antibodies have limited effectiveness, e.g., because Borrelia spp. spirochetes can colonize a variety of mammalian tissues and cause a variety of non-specific clinical abnormalities, such as joint and / or muscle aches or general malaise. These symptoms can be difficult to distinguish from similar clinical manifestations due to persistent Borrelia infection, leading to considerable confusion and increased healthcare costs. Thus, it would be extremely useful to be able to clinically confirm the effective elimination of pathogenic Borrelia spp. spirochetes after treatment of LD.

[0010] WO 2013 / 116668 describes peptide antigens recognized by antibodies produced in response to Borrelia spp. However, anti-Borrelia antibodies are not typically produced at detectable levels for several weeks after infection, thus missing many early cases of LD. It is also noted that antibody levels produced in response to Borrelia burgdorferi infection can remain elevated for years after infection, even after successful treatment (e.g. antibiotic treatment) that eliminates the Borrelia spirochete. Thus, determination of an anti-Borrelia antibody response is poorly sensitive and unreliable as a diagnostic test for pathogenic Borrelia spp., and is of little use as a post-treatment prognostic test for Borrelia infection.

[0011] Borrelia burgdorferi, developed a vigorous and long-lasting T cell response, which was evidenced by inflammatory cytokines present during the early local stage of the disease, accompanied by EM. This T cell response preceded any detectable antibody response. For example, T cells activated in response to Lyme disease spirochetes reliably produced interferon-γ (Ekerfelt et al., 1999 Clin Exp. Immunol. 115:498; Sikand et al., 1999 Clin. Diagnost. Lab. Immunol. 6:445). Activated T cells occurred at the same incidence in two patient populations: (i) those who were clinically asymptomatic for LD but had detectable circulating anti-Borrelia antibodies in their bodies, and (ii) patients who showed characteristic clinical signs of Borrelia infection (i.e., LD symptoms) (Ekerfelt et al., 1999). WO 2012 / 039614 describes in vitro T cell cytokine responses elicited by peripheral blood leukocytes from individuals infected with Borrelia spirochetes following incubation with whole, fixed or crudely fractionated Borrelia cells. However, in WO 2012 / 039614 there is no description of identifying at which specific stage of Borrelia infection (e.g., early local, early disseminated, late disseminated) the Borrelia pathogen can be recognized by T cells, nor is there a description of which specific Borrelia antigens are capable of eliciting such responses, or a description of the structure of any Borrelia epitopes recognized by T cells. Thus, the significance and specificity of the T cell immune response in early LD remains unclear, and in particular, the pathogenesis of LD has not been identified with respect to specific Borrelia spp. antigens recognized by T cells, and Borrelia-specific T cell epitopes remain unknown.

[0012] It is clear that there remains a need for improved diagnostic and prognostic assessments for Lyme disease, including earlier and more sensitive detection of LD, as well as the ability to monitor the therapeutic efficacy of treatments administered for LD. The presently disclosed embodiments of the invention address these needs, and provide other related benefits as well. SUMMARY

[0013] According to certain embodiments disclosed herein of the present application, there is provided a composition for the diagnosis or prognosis of Lyme disease selected from a first composition and a second composition: (I) the first composition comprises: (a) 1, 2, 3, 4, or 5 isolated FlaB peptides each comprising a B. burgdorferi T-cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequence set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides each comprising a B. burgdorferi T-cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequence set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides each comprising a B. burgdorferi T-cell epitope and selected from the group consisting of p66 peptides having the amino acid sequence set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 19-31; and (d) 1 or 2 isolated OspC peptides each comprising a B. burgdorferi T-cell epitope and selected from the group consisting of OspC peptides having the amino acid sequence set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 32-33, wherein the composition is capable of eliciting a secondary in vitro immune response by T-cells following contact with whole blood obtained from a subject infected with a B. burgdorferi species associated with Lyme disease; and

[0014] (II) the second composition comprises: (a) 5 isolated FlaB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 13 isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 13 isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides having the amino acid sequences set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 32-33.

[0015] In certain other embodiments, at least one of: (a) the composition comprises at least about 1 nanogram and no more than about 100 nanograms of each peptide, (b) the composition comprises at least about 100, 200, 300, or 400 nanograms and no more than about 500 nanograms of each peptide, (c) the composition comprises at least about 500, 600, 700, 800, or 900 nanograms and no more than about 1000 nanograms of each peptide, (d) the composition comprises at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 micrograms and no more than about 2 micrograms of each peptide, or (e) the composition comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9 micrograms and no more than about 10 micrograms of each peptide.

[0016] In another embodiment of the application, a method is provided for detecting Lyme disease in a subject, or for monitoring the efficacy of a treatment for Lyme disease in a subject, comprising: (A) contacting in vitro: (i) a first biological sample obtained at a first time point from a subject known to have Lyme disease or suspected to be at risk of having Lyme disease, wherein the biological sample comprises T cells and antigen presenting cells, and (ii) a peptide composition for the diagnosis or prognosis of Lyme disease, to obtain a first test incubation mixture; (B) incubating the first test incubation mixture under conditions and for a time sufficient for the T cells to specifically recognize a Borrelia T cell epitope present in the peptide composition to stimulate production of a T cell immune response indicator; and (C) detecting a first level of the T cell immune response indicator in the first test incubation mixture, wherein an increase in the first level of the T cell immune response indicator detected in (C) relative to a first control level of the T cell immune response indicator obtained by incubating the first biological sample in a first control incubation in the absence of the peptide composition for the diagnosis or prognosis of Lyme disease indicates the presence of a Borrelia infection in the subject, and wherein the peptide composition for the diagnosis or prognosis of Lyme disease comprises: (a) 1, 2, 3, 4, or 5 isolated FlaB peptides each comprising a Borrelia T cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides each comprising a Borrelia T cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides each comprising a Borrelia T cell epitope and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31.and (d) 1 or 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from an OspC peptide having an amino acid sequence as set forth in SEQ ID NO: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 32-33, thereby detecting Lyme disease in the subject, or monitoring efficacy of a treatment for Lyme disease in the subject.

[0017] In certain other embodiments, the first time point is prior to administering to the subject a treatment for Lyme disease.

[0018] In certain related embodiments, the peptide composition for the diagnosis or prognosis of Lyme disease comprises: (a) 5 isolated FlaB peptides each comprising a B. burgdorferi T cell epitope and selected from a FlaB peptide having an amino acid sequence as set forth in SEQ ID NO: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1-5; (b) 13 isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from a DbpB peptide having an amino acid sequence as set forth in SEQ ID NO: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 6-18; (c) 13 isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from a p66 peptide having an amino acid sequence as set forth in SEQ ID NO: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 19-31; and (d) 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from an OspC peptide having an amino acid sequence as set forth in SEQ ID NO: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 32-33.

[0019] In certain other embodiments of the above methods, the methods further comprise: (D) contacting, in vitro, (i) a second biological sample obtained from the subject at a second time point after the first time point and after administration of a treatment for Lyme disease to the subject, wherein the second biological sample comprises T cells and antigen presenting cells, and (ii) the peptide composition for the diagnosis or prognosis of Lyme disease, to obtain a second test incubation mixture; (E) incubating the second test incubation mixture under conditions and for a time sufficient for the T cells to specifically recognize the Borrelia T-cell epitopes present in the peptide composition to stimulate production of a T-cell immune response indicator; and (F) detecting a second level of the T-cell immune response indicator in the second test incubation mixture, wherein an increase in the second level of the T-cell immune response indicator detected in (F) relative to a second control level of the T-cell immune response indicator obtained by incubating the second biological sample in a second control incubation in the absence of the peptide composition for the diagnosis or prognosis of Lyme disease indicates the presence of a Borrelia infection in the subject, and wherein a decrease in the second level of the T-cell immune response indicator detected in (F) relative to the first level of the T-cell immune response indicator detected in (C) indicates efficacy of the treatment for Lyme disease.

[0020] In certain other embodiments, the peptide composition for the diagnosis or prognosis of Lyme disease comprises: (a) 5 isolated FlaB peptides each comprising a Borrelia T-cell epitope and selected from FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 13 isolated DbpB peptides each comprising a Borrelia T-cell epitope and selected from DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 13 isolated p66 peptides each comprising a Borrelia T-cell epitope and selected from p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 2 isolated OspC peptides each comprising a Borrelia T-cell epitope and selected from OspC peptides having the amino acid sequences set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 32-33.

[0021] In certain embodiments of any of the above methods, the Lyme disease comprises infection with at least one pathogenic Borrelia species, and in certain other embodiments, the pathogenic Borrelia species is pathogenic to humans. In certain other embodiments, the Borrelia species pathogenic to humans is selected from the group consisting of Borrelia burgdorferi, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia spielmanii, Borrelia bissettii, Borrelia lusitaniae, and Borrelia bavariensis.

[0022] In certain embodiments of any of the above methods, the treatment for Lyme disease comprises administering to the subject an antibiotic. In certain other embodiments, the antibiotic is selected from the group consisting of tetracyclines; oxytetracycline, tetracycline, multicycline, or minocycline; penicillins; amoxicillin or penicillin; cephalosporins; cefaclor, cefoperazone, cefminox, cefotaxime, cefotetan, cefmetazole, cefoxitin, cefuroxime axetil, cefuroxime acetyl, carbendazim, or ceftriaxone sodium; macrolides; azithromycin, clarithromycin, or erythromycin.

[0023] In certain embodiments of any of the above methods, the biological sample comprises at least one of whole blood, cerebrospinal fluid, or synovial fluid. In certain embodiments of any of the above methods, the biological sample comprises at least one of (a) whole blood, (b) a cellular component of whole blood, (c) isolated peripheral blood leukocytes, or (d) isolated peripheral blood mononuclear cells.

[0024] In certain embodiments of any of the above methods, the T cell immune response indicator is interferon-gamma (IFN-γ). In certain other embodiments, the IFN-γ is soluble IFN-γ released by T cells. In certain embodiments of any of the above methods, the T cell immune response indicator comprises at least one of T cell proliferation and T cell cytokine expression. In certain other embodiments, the T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ. In certain embodiments, T cell cytokine expression is detected by soluble T cell cytokine released by T cells. In certain other embodiments, the T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ. In certain embodiments, the T cell cytokine is detected by assay binding agent detectable specific binding to T cell cytokine. In certain other embodiments, the binding agent comprises at least one antibody that specifically binds to T cell cytokine. In certain other embodiments, the at least one antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody. In certain embodiments, the at least one antibody is immobilized on a solid phase.

[0025] In certain embodiments of any of the above methods, the Lyme disease comprises infection by at least one pathogenic Borrelia species. In certain embodiments, the pathogenic Borrelia species is pathogenic to humans. In certain other embodiments, the Borrelia species pathogenic to humans is selected from the group consisting of Borrelia burgdorferi, Borrelia burgdorferi sensu stricto, Borrelia azfelii, Borrelia garinii, Borrelia valaisiana, Borrelia spielmanii, Borrelia bissettii, Borrelia lusitaniae, and Borrelia bavariensis.

[0026] In certain other embodiments of the above methods (e.g., those comprising steps (D)-(F) with respect to a second biological sample), the treatment for Lyme disease comprises administering to the subject an antibiotic. In certain other embodiments, the antibiotic is selected from the group consisting of: tetracyclines; oxytetracycline, tetracycline, multicycline, or minocycline; penicillins; amoxicillin or penicillin G; cephalosporins; cefaclor, cefoperazone, cefminox, cefotaxime, cefotetan, cefmetazole, cefoxitin, cefuroxime axetil, cefuroxime acetyl, carbendazim, or ceftriaxone sodium; macrolides; azithromycin, clarithromycin, or erythromycin. In certain embodiments, the biological sample comprises at least one of whole blood, cerebrospinal fluid, or synovial fluid. In certain embodiments, the biological sample comprises at least one of: (a) whole blood, (b) a cellular fraction of whole blood, (c) isolated peripheral blood leukocytes, or (d) isolated peripheral blood mononuclear cells. In certain embodiments, the T cell immune response indicator is interferon-gamma (IFN-γ), which in certain other embodiments is soluble IFN-γ released by T cells. In certain embodiments, the T cell immune response indicator comprises at least one of T cell proliferation and expression of a T cell cytokine, which in certain other embodiments is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ. In certain embodiments, expression of a T cell cytokine is detected by soluble T cell cytokine released by T cells. In certain embodiments, the T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ. In certain other embodiments, the T cell cytokine is detected by assay for detectable specific binding of an assay binding agent to the T cell cytokine. In certain other embodiments, the binding agent comprises at least one antibody that specifically binds to the T cell cytokine. In certain other embodiments, the at least one antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody. In certain other embodiments, the at least one antibody is immobilized on a solid phase. In certain other embodiments, the method comprises repeating steps (D), (E), and (F) at a plurality of second time points, which are different from each other and later than the first time point, and after administering the treatment for Lyme disease to the subject.In certain other embodiments, the plurality of second time points comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 time points.

[0027] In another embodiment, a composition is provided selected from a first nucleic acid composition and a second nucleic acid composition: (I) the first nucleic acid composition comprising one or more isolated nucleic acid molecules encoding: (a) 1, 2, 3, 4, or 5 isolated FlaB peptides each comprising a Borrelia T-cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides each comprising a Borrelia T-cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides each comprising a Borrelia T-cell epitope and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 1 or 2 isolated OspC peptides each comprising a Borrelia T-cell epitope and selected from the group consisting of OspC peptides having the amino acid sequences set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 32-33, wherein the FlaB, DbpB, p66, and OspC peptides are capable of eliciting a secondary in vitro immune response by T-cells following contact with whole blood obtained from a subject infected with a Borrelia species associated with Lyme disease; and

[0028] (II) a second nucleic acid composition comprising one or more isolated nucleic acid molecules encoding: (a) five isolated FlaB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) thirteen isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) thirteen isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) two isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides having the amino acid sequences set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 32-33, wherein the FlaB, DbpB, p66 and OspC peptides are capable of eliciting a secondary in vitro immune response by T cells following contact with whole blood obtained from a subject infected with a B. burgdorferi species associated with Lyme disease. In another embodiment, a vector composition is provided comprising one or more nucleic acid vectors comprising the first nucleic acid composition described above and / or the second nucleic acid composition described above. In another embodiment, a host cell is provided comprising the vector composition described above.

[0029] These and other aspects and embodiments of the present application will become apparent after a reading of the following detailed description of the embodiments when taken in conjunction with the accompanying drawings. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification are hereby incorporated by reference in their entirety. Aspects and embodiments of the present application can be modified, if necessary, to employ the concepts of the various patents, applications and publications to provide yet further embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1The display employs IFN-gamma detection following secondary in vitro T cell activation with a pool of peptides (SEQ ID NO: 1-33) in blood samples taken prior to treatment and about 60 days post-treatment. DETAILED DESCRIPTION

[0031] The presently disclosed embodiments of the invention relate to an artificial composition that surprisingly allows for sensitive diagnosis and prognosis of Lyme disease (LD) and comprises a combination of non-naturally occurring defined peptides. Thus, as described herein, a composition is provided that comprises a combination of peptides including one or more peptides of a peptide region from each of the presently disclosed peptides of flagellin (FlaB) [SEQ ID NO: 1-5], decorin binding protein B (DbpB) [SEQ ID NO: 6-18], common antigen (p66) [SEQ ID NO: 19-31], and outer surface protein C (OspC) [SEQ ID NO: 32-33], or variant peptides thereof. Each of the peptides comprises a Borrelia spp. specific T cell epitope as first disclosed herein. Related methods are also provided, including methods for diagnosing Lyme disease in a subject, and methods for monitoring clearance of pathogenic Borrelia spp. microorganisms following treatment of a subject.

[0032] The combination of peptides described herein is not naturally occurring, as FlaB, DbpB, p66, and OspC are known to be expressed at different stages of the Lyme disease process (commonly referred to as early localized, early disseminated, and late disseminated stages), and thus would not be encountered by the immune system of an infected host in tandem in a natural setting. For example, OspC is expressed by Borrelia bacteria during the early localized stage of infection, DbpB and p66 are expressed following dissemination of Borrelia infection, and FlaB is expressed during the late disseminated and early localized stages. The presently disclosed peptide cocktail can also comprise artificial peptides that are different from naturally processed Borrelia spp. antigen fragments displayed by antigen presenting cells in vivo. Furthermore, the presently disclosed FlaB, DbpB, p66, and OspC peptides do not all naturally occur in a single pathogenic Borrelia species, and thus are not expected to have been encountered by the immune system of a single Lyme disease patient. Thus, the present embodiments surprisingly allow for early detection of Lyme disease in a multitude of subjects having or suspected of having Lyme disease, which can be achieved by detecting secondary in vitro responses of T cells from the subject to the composition.

[0033] As described below, for example, even though the T cells of certain samples did not respond to peptides from all four of FlaB, DbpB, p66, and OspC, all samples comprising T cells from LD subjects responded to at least one peptide of at least one of the polypeptides described herein comprising FlaB, DbpB, p66, and OspC Borrelia T cell epitopes (see Tables 1 and 2). Moreover, all samples comprising T cells from LD subjects responded to a peptide cocktail comprising at least one peptide from at least one of all four of FlaB, DbpB, p66, and OspC. Thus, according to non-limiting theory, the current embodiments allow for rapid and sensitive detection of Borrelia spp. specific T cells in a sample, regardless of which specific Borrelia spp. pathogen the subject is infected with, regardless of which stage of disease progression the T cells are obtained from (early localized, early disseminated, or late disseminated). The current embodiments also allow for detection of Borrelia spp. specific T cells in a sample by providing a combination of peptides comprising Borrelia T cell epitopes that include representative epitopes present at all stages of Lyme disease progression, even when the subject is unknown for having LD. The presently disclosed embodiments can be used to detect Lyme disease that can result from infection with any of a number of pathogenic Borrelia species, such as Borrelia species that are pathogenic to humans, including B. burgdorferi, B. burgdorferi s.s., B. afzelii, B. garinii, B. valaisiana, B. spielmanii, B. bissettii, B. lusitaniae, and B. bavariensis. (See, e.g., (Wright et al., 2012 Am. Fam. Physician 85:1086; Shapiro, 2014 N. Engl. J. Med. 370:1724; Schutzer et al., 2012 J. Bacteriol. 194(2):545).

[0034] As described herein, the compositions, comprising combinations of at least one of the peptides disclosed herein containing a T cell epitope of Borrelia spp. from each of FlaB, DbpB, p66, and OspC (e.g., SEQ ID NOS: 1-33 shown in Table 2), have properties that are significantly different from any individual constituent peptide provided herein or any subset of peptides provided herein that are co-expressed (excluding the properties of at least each of the FlaB [SEQ ID NOS: 1-5], DbpB [SEQ ID NOS: 6-18], p66 [SEQ ID NOS: 19-31], and OspC [SEQ ID NOS: 32-33] peptides). Surprisingly, the compositions elicit a more robust secondary in vitro immune response of T cells in subjects affected by LD, including early stage LD, than any individual constituent peptide (or incomplete subset lacking at least one of the FlaB, DbpB, p66, or OspC peptides), thereby providing unprecedented sensitivity of detection of LD, including early detection. Thus, the current embodiments can provide significantly more than can be achieved with any naturally occurring subset of Borrelia spp. polypeptides by facilitating earlier detection of LD than can be achieved by measuring secondary in vitro T cell responses to any individual constituent peptide described herein (or to incomplete subsets of peptides comprising only peptides derived from co-expressed Borrelia spp. polypeptides but lacking at least one of the FlaB, DbpB, p66, or OspC peptides).

[0035] Thus, the presently disclosed compositions comprise combinations of Borrelia spp. FlaB, DbpB, p66, and OspC peptides or variants thereof containing Borrelia-specific T cell epitopes that are not naturally occurring, and in certain embodiments, the presently disclosed compositions comprise Borrelia spp. FlaB, DbpB, p66, and OspC peptides or variants thereof in non-naturally occurring amounts relative to one another, thereby (and further according to non-limiting theory) achieving certain benefits described herein.

[0036] Accordingly, disclosed herein is one of the ability to replace any of the previously known methods for confirming the presence of Lyme disease (LD) in a subject having or suspected of having LD by providing a test that detects an indicator of an immune response (e.g., interferon-γ) produced by T cells that have been activated by contacting specific regions in individual Borrelia burgdorferi proteins. Specific T cell reactive epitopes within individual Borrelia spp. protein antigens FlaB (GenBank Accession No. ACI49679.1), DbpB (GenBank Accession No. AAC70029.1), p66 (GenBank Accession No. AAC66949.1), and OspC (GenBank Accession No. ABQ42983.1) are described herein and, according to non-limiting theory, are believed to play a role in the compositions and methods described herein as a result of T cell recognition of linear antigenic peptide epitopes.

[0037] It is believed that the LD test based on this strategy can provide at least two important benefits: first, activated T cells proliferate more rapidly than antibody-producing B cells, and thus the test can provide a sensitivity that is superior to any antibody detection type test performed during the early stages of pathogenic Borrelia infection.

[0038] Second, the level of antibody response to B. burgdorferi typically increases over a period of months to years regardless of treatment success, while the level of T cell activity typically subsides, often showing T cell regulation, following clearance of the inciting stimulus (e.g., infectious agent). Thus, the presently disclosed prognostic methods can provide a superior assessment of whether a course of treatment for LD has reduced (e.g., in a statistically significant manner relative to a suitable control) or eliminated pathogenic Borrelia infection. These benefits are believed to arise because T cells typically remain activated only for a short period of time following Borrelia infection resolution. Thus, the absence of elevated levels of T cell immune response indicators (e.g., interferon-γ release by activated T cells) in response to secondary in vitro challenge by the compositions described herein from T cells present in a sample obtained from an LD subject following treatment for LD can also provide an accurate prediction of successful treatment.

[0039] As described herein, specific peptides comprising Borrelia spp. (e.g., B. garinii, B. burgdorferi, B. burgdorferi B31) proteins of clinical importance contained in a polypeptide cocktail are evaluated for their ability to induce production of interferon-gamma (an exemplary T cell immune response indicator) by activated T cells obtained from LD patients in a secondary in vitro immune response. As a proof of concept, the ability of the cocktail comprising peptides having Borrelia spp. specific T cell epitopes to induce Lyme disease specific activated T cells to produce interferon-gamma is evaluated, particularly in blood samples from patients in the early stages of B. burgdorferi infection. The level of interferon-gamma produced by T cells taken from LD patients who are about to undergo antibiotic treatment is compared to the level of interferon-gamma produced by T cells taken from LD patients after about 60 days of effective treatment with antibiotics.

[0040] Peptides comprising Borrelia T cell epitopes.

[0041] In certain embodiments, the present disclosure provides a composition for the diagnosis or prognosis of Lyme disease selected from the first composition and the second composition:

[0042] (I) a first composition comprising: (a) 1, 2, 3, 4, or 5 isolated FlaB peptides each comprising a Borellia T-cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides each comprising a Borellia T-cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides each comprising a Borellia T-cell epitope and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 1 or 2 isolated OspC peptides each comprising a Borellia T-cell epitope and selected from the group consisting of OspC peptides having the amino acid sequences set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 32-33, wherein the composition, upon contact with whole blood obtained from a subject infected with a Borellia species associated with Lyme disease, is capable of eliciting a secondary in vitro immune response by T-cells; and

[0043] (II) a second composition comprising: (a) 5 isolated FlaB peptides each comprising a Borellia T-cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 13 isolated DbpB peptides each comprising a Borellia T-cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 13 isolated p66 peptides each comprising a Borellia T-cell epitope and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 2 isolated OspC peptides each comprising a Borellia T-cell epitope and selected from the group consisting of OspC peptides having the amino acid sequences set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 32-33.

[0044] Borrelia spp. FlaB, DbpB, p66 or OspC peptides comprising a Borrelia T-cell epitope, which are used in certain embodiments contemplated herein, can comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1-5 for a FlaB peptide, an amino acid sequence set forth in any one of SEQ ID NOs: 6-18 for a DbpB peptide, an amino acid sequence set forth in any one of SEQ ID NOs: 19-31 for a p66 peptide, and, an amino acid sequence set forth in any one of SEQ ID NOs: 32-33 for an OspC peptide, and, in certain other embodiments, can comprise a Borrelia T-cell epitope comprising peptide variant comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to at least one of the peptides set forth in SEQ ID NOs: 1-33, and which is capable of being specifically recognized by a T-cell reactive to at least one pathogenic Borrelia species, preferably a Borrelia species pathogenic to humans, which can include but are not necessarily limited to: B. burgdorferi, B. burgdorferi s.s., B. afzelii, B. garinii, B. valaisiana, B. spielmanii, B. bissettii, B. luciae, and B. bavariensis.

[0045] Borrelia T-cell epitope-containing peptides of SEQ ID NOs: 1-33 are shown in Table 1.

[0046] Table 1. Peptides containing Borrelia T cell epitopes:

[0047] Borrelia spp. specific regions in the FlaB, DbpB, p66 and OspC proteins Table 2.

[0048]

[0049]

[0050]

[0051] A Borrelia T cell epitope-containing peptide variant of a Borrelia T cell epitope-containing peptide (e.g., any of the peptides set forth in SEQ ID NO: 1-33) can comprise one or more amino acid substitutions, additions, deletions, and / or insertions relative to the peptide sequence set forth in SEQ ID NO: 1-33 (e.g., wild-type or dominant or naturally occurring allelic forms) of a natural Borrelia T cell epitope-containing peptide. Relative to the corresponding portion of a region of a polypeptide sequence containing a natural Borrelia T cell epitope, e.g., the regions (Table 2) first shown herein that contain T cell epitopes from the Borrelia polypeptide antigens B. burgdorferi FlaB (GenBank Accession No. ACI49679.1), B. burgdorferi DbpB (GenBank Accession No. AAC70029.1), B. burgdorferi p66 (GenBank Accession No. AAC66949.1), and B. burgdorferi OspC (GenBank Accession No. ABQ42983.1), a variant preferably exhibits at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity, and more preferably, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. The percent (%) identity can be readily determined by comparing the sequence of the peptide variant to the corresponding portion of the full-length polypeptide, wherein the corresponding portion can be readily identified according to established methods (e.g., by aligning sequence regions that show a high degree of sequence identity or sequence homology), optionally allowing for short sequence gaps as they can result from insertions or deletions, or allowing for conservative substitutions, or allowing for short regions of mismatches, etc. Some techniques for sequence comparison include the use of computational algorithms well known to those of ordinary skill in the art, e.g., the Align or BLAST algorithms (Altschul, J. Mol. Biol. 219:555-565, 1991; Henikoff and Henikoff, PNAS USA 89: 10915-10919, 1992), which are available from the NCBI website (see [online] Internet site: <URL:http: / / www / ncbi.nlm.nih.gov / cgi-bin / BLAST). Default or custom parameters can be employed.

[0052] Furthermore, computer methods available in the art enable one skilled in the art to predict the three-dimensional structure of a protein or peptide to determine functional variants of a particular polypeptide. For example, variants can be identified in which all or a portion of the three-dimensional structure is not materially altered by one or more modifications, substitutions, additions, deletions, and / or insertions. (See, e.g., Seemayer et al., 2014 Bioinformat. 30:3128; Raman et al., 2010 Science Express February 4, 2010 10.1126 / science.1183649; Gribenko et al., 2009 Proc. Nat. Acad. Sci. USA 106:2601; Bradley et al., Science 309:1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Baker, 2014 Biochem. Soc. Trans. 42:225; Correia et al., 2014 Nature 507:201; King et al., 2014 Proc. Nat. Acad. Sci. USA 111:8577; Roche et al., 2012 PLoS One 7(5):e38219; Zhang et al., 2013 Meths. Enzymol. 523:21; Khoury et al., 2014 Trends Biotechnol. 32:99; O'Meara et al., 2015 J. Chem. Theory Comput. 11:609; Park et al., 2015 Structure 23:1123; Bale et al., 2015 Protein Sci. doi:10.1002 / pro.2748 Epub PMID 26174163; Park et al., 2015 Proteins doi:10.1002 / prot.24862 Epub PMID 26205421; Lin et al., 2015 Proc. Nat. Acad. Sci. USA pii:201509508 Epub PMID 26396255). From this, one skilled in the art can readily determine whether a particular peptide variant containing a B. burgdorferi T cell epitope or functional fragment thereof retains sufficient epitope structure to be specifically recognized by T cells reactive against at least one pathogenic B. burgdorferi species.

[0053] The Borrelia T cell epitope-containing peptides can have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids, and in certain embodiments, the length can generally be no more than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 amino acids. In certain preferred embodiments, the Borrelia T cell epitope-containing peptides can be peptides of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous amino acids in length.

[0054] It is understood that a T cell epitope refers to a structural region of an antigen that can be specifically recognized by a T cell receptor ("T cell receptor") specific for the antigen, typically in the context of the epitope being presented to the T cell receptor by an appropriate major histocompatibility complex (MHC) class I or class II molecule. T cell epitope-containing peptides of about 8-13 amino acids in length are typically presented to CD8 T cell receptors by class I MHC molecules; T cell epitope-containing peptides of about 15-25 amino acids in length are typically presented to CD4 T cell receptors by class II MHC molecules. T cell receptors are not absolutely specific for their specificity, but are rather considered to be promiscuous; that is, a given T cell receptor can be able to specifically recognize a particular T cell epitope structure as well as a range of closely related epitope structures. Specific recognition of a correctly presented T cell epitope by a T cell can be detected in the form of a stimulation of the production of a T cell immune response indicator (such as those described herein, e.g., cytokine release by the T cell, e.g., IFN-γ release). Thus, a Borrelia T cell epitope-containing peptide can refer to a peptide antigen that is capable of stimulating a T cell activated (e.g., activated) to recognize a Borrelia spp. antigen in a secondary in vitro immune response, including where the Borrelia T cell epitope-containing peptide is different from the Borrelia spp. antigen that activated the T cell in vivo.

[0055] The methods provided herein for designing, producing, and testing peptides containing a Borrelia T cell epitope and functional fragments of variants thereof can be adapted with minor modifications based on knowledge in the art, for example, using conventional virology, immunology, microbiology, molecular biology, and recombinant DNA techniques methods, which are explained fully in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Vols. I and II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

[0056] The terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to an amino acid polymer without regard to length. The terms do not exclude modification such as myristylation, sulfation, glycosylation, phosphorylation, formylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" refers to one or more chains of amino acids, wherein each chain contains amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein can comprise multiple chains covalently and / or non-covalently linked together, having a sequence of amino acids of a naturally occurring protein, i.e., a protein produced by a naturally occurring and in particular a non-recombinant cell, or such a protein produced by a genetically engineered or recombinant cell, which comprises a molecule having the sequence of amino acids of the naturally occurring protein, or a molecule having one or more deletions, additions and / or substitutions of amino acids compared to the natural sequence. Thus, a "polypeptide" or "protein" can comprise one (referred to as a "monomer") or more (referred to as a "multimer") chains of amino acids. The terms "peptide," "polypeptide," and "protein" specifically encompass a peptide of the present disclosure, or a sequence having one or more deletions, additions and / or substitutions of amino acids compared to a peptide described herein.

[0057] The term "isolated" means that a substance is removed from its original environment (e.g., the natural environment if it is naturally produced). For example, a naturally produced polypeptide or nucleic acid present in a living animal is not isolated, but the same polypeptide or nucleic acid separated from some or all of the coexisting materials of the natural system is isolated. The nucleic acid can be part of a vector and / or the nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate), and still be isolated in that the vector or composition is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer), as well as intervening sequences between individual coding segments (exons).

[0058] The terms "isolated protein," "isolated polypeptide," and "isolated peptide" refer herein to a subject protein, peptide, or polypeptide that is (1) free of at least some of the other proteins, peptides, or polypeptides with which it is found in nature, (2) substantially free of other proteins, peptides, or polypeptides of the same origin (e.g., from the same species), (3) expressed by a cell from a different species, (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances normally associated with it in nature, (5) not associated (by covalent or noncovalent interaction) with parts of proteins or polypeptides with which it can be associated in nature with the isolated protein, isolated peptide, or isolated polypeptide, (6) operatively associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. The isolated protein, peptide, or polypeptide can be encoded by genomic DNA, cDNA, mRNA, or other RNA, or can have a synthetic origin according to many well-known chemical techniques for artificial peptide and protein synthesis, or any combination thereof. In certain embodiments, the isolated protein, peptide, or polypeptide is substantially free of proteins or polypeptides or other contaminants present in its natural environment that can interfere with its use (therapeutic use, diagnostic use, prophylactic use, research use, etc.).

[0059] A "peptide fragment" or "polypeptide fragment" refers to a peptide or polypeptide, which can be in monomeric form or multimeric form, that has an amino-terminal deletion, a carboxy-terminal deletion, and / or internal deletions or substitutions of a naturally occurring or recombinantly produced polypeptide. "Contiguous amino acids" as used herein refers to covalently linked amino acids that correspond to an uninterrupted linear portion of a disclosed amino acid sequence. In certain embodiments, a polypeptide fragment can comprise an amino acid chain of at least 5 to about 100 amino acids in length. It is understood that in certain embodiments, the fragment length is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0060] Certain preferred embodiments contemplate the total artificial chemical synthesis of the peptides described herein (e.g., the peptides comprising a B. burgdorferi T cell epitope), using any of a variety of well-established methods, such as those described in Amino Acid and Peptide Synthesis (Jones, J., 2002 New York, Oxford Univ. Press USA), Ramakers et al. (2014 Chem. Soc. Rev. 43:2743), Verzele et al. (2013 Chembiochem. 14:1032), Chandrudu et al. (2013 Molecules 18:4373), and / or For example, manual or, preferably, automated solid-phase peptide synthesis, based on the Merrifield method or other solid-phase peptide synthesis techniques and subsequent improvements (e.g., Merrifield, 1963 J. Am. Chem. Soc. 85:2149; Mitchell et al., 1978 J. Org. Chem. 43:2485; Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1st ed.). Boca Raton: CRC Press; Nilsson et al., 2005 Annu. Rev. Biophys. Biomol. Struct. 34; Schnolzer et al., Int. J. Peptide Res. Therap. 13(1-2):31; Li et al., 2013 Molecules 18:9797), is a routine technique in the art of peptide synthesis and can be used to chemically synthesize the peptides described herein.

[0061] A polypeptide or peptide can comprise a signal (or leader) sequence at the N-terminus of the protein, which directs the transfer of the protein in a co-translational or post-translational manner. The polypeptide or peptide can also be fused or coupled in-frame to a linker or other sequence to facilitate synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide or peptide to a solid support. Fusion domain polypeptides can be linked to the N- and / or C-terminus of the polypeptide or peptide, and can include (by way of non-limiting example): immunoglobulin-derived sequences, such as Ig constant region sequences or portions thereof, affinity tags such as His tags (e.g., hexa- or other poly-histidine), FLAG TMor myc or other peptide affinity tags, detectable polypeptide moieties such as green fluorescent protein (GFP) or variants thereof (e.g., yellow fluorescent protein (YFP), blue fluorescent protein (BFP), other luminescent proteins or derivatives thereof, etc.), or other detectable polypeptide fusion domains, enzymes or portions thereof such as glutathione-S-transferase (GST) or other known enzyme detection and / or reporter fusion domains, etc., as are well known to those of skill in the art.

[0062] Systems for recombinant expression of peptides, polypeptides and proteins are known in the art and can be used in certain embodiments to produce the peptides described herein. For example, certain bacterial expression systems such as the E. coli recombinant protein expression system produce polypeptide products with an N-terminal formylated methionine. In some cases, recombinantly produced peptides can thus include an N-terminal methionine residue (which can be an unmodified methionine or a formyl-methionine or another methionine analog, variant, mimetic or derivative as provided herein), sometimes referred to as a starter methionine, immediately preceding the desired peptide sequence (e.g., a Borrelia T-cell epitope-containing peptide). Accordingly, embodiments are also contemplated in which one or more of the Borrelia T-cell epitope-containing peptides described herein are produced in a form that includes an N-terminal methionine (e.g., methionine or N-formylmethionine) and can be recombinantly expressed in host cells that also express methionine aminopeptidase (MAP) enzymes that are capable of cleaving the N-terminal methionine to remove it from the initial polypeptide product. See, e.g., Natarajan et al., 2011 PLoS ONE 6(5):e20176; Shen et al., 1993 Proc. Natl. Acad. Sci. USA 90:8108; Shen et al., 1997 Prot. Eng. 10:1085. Alternatively, MAP enzymes can be recombinantly produced (e.g., Tsunasawa et al., 1997 J. Biochem. 122:843; Bradshaw et al., 1998 Trends Bioch. Sci. 23:263; Ben-Bassat et al., 1987 J. Bacteriol. 169:751) or obtained commercially (Sigma-Aldrich, St. Louis, MO, e.g., catalog no. M6435) and used to remove N-terminal methionines from existing peptides after synthesis.

[0063] According to certain preferred embodiments, the Borrelia T cell epitope-containing peptides can comprise a peptide, polypeptide, or peptidomimetic that includes or shares close sequence identity or structural features with a polypeptide comprising at least 5 and no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 1-33, wherein the peptide in which the Borrelia T cell epitope is present is capable of being specifically recognized by T cells reactive against at least one pathogenic Borrelia species, preferably, a Borrelia species pathogenic to humans, which can include, but is not necessarily limited to, B. burgdorferi, B. burgdorferi s.s., B. afzelii, B. garinii, B. valaisiana, B. spielmanii, B. bissettii, B. lusitaniae, and B. bavariensis. In addition to the Borrelia T cell epitope-containing proteins and peptides disclosed herein for the first time, assay methods for determining the T cell reactivity are described herein and are generally known in the art.

[0064] As generally referred to in the art and as used herein, sequence identity and sequence homology are used interchangeably and generally refer to the percentage of nucleotides or amino acid residues in a candidate sequence that are, respectively, identical with the nucleotides or amino acid residues in a reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and optionally not considering any conservative substitutions as part of sequence identity. In certain embodiments, a variant of a peptide (e.g., a Borrelia spp. T cell epitope-containing peptide disclosed herein (e.g., a peptide according to one of SEQ ID NOs: 1-33) shares at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, or 94%, or at least about 95%, 96%, 97%, 98%, or 99% of the amino acid residues (or nucleotides in a polynucleotide encoding the peptide) of the sequence of the peptide set forth in any one of SEQ ID NOs: 1-33. The sequence identity can be determined according to well-known sequence analysis algorithms (as described above), and include those available from the University of Wisconsin Genetics Computer Group (Madison, Wisconsin), e.g., FASTA, Gap, Bestfit, BLAST, etc.

[0065] "Naturally or non-naturally occurring amino acids" include any of the common naturally occurring amino acids as a constituent part of the biosynthesis of peptides, polypeptides and proteins (e.g., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y)), and also include modified, derivatized, enantiomeric, rare and / or unusual amino acids, naturally occurring or synthetic, such as, for example, N-formylmethionine, hydroxyproline, hydroxylysine, desmosine, isodesmosine, ε-N-methyl lysine, ε-N-trimethyl lysine, methylhistidine, dehydrotyrosine, dehydroalanine, alpha-aminobutyric acid, beta-alanine, gamma-aminobutyric acid, homocysteine, homoserine, citrulline, ornithine, and other amino acids isolatable from natural sources and / or chemically synthesizable, such as, for example, those appearing in the Protein, Peptide and Amino Acid Sourcebook (White, J. S. and White, D. C., 2002 Humana Press, Totowa, NJ) or Amino Acid and Peptide Synthesis (Jones, J., 2002 Oxford Univ. Press USA, New York) or Unnatural Amino Acids, ChemFiles Vol. 1, No. 5 (2001 Fluka Chemie GmbH; Sigma-Aldrich, St. Louis, MO) or Unnatural Amino Acids II, ChemFiles Vol. 2, No. 4 (2002 Fluka Chemie GmbH; Sigma-Aldrich, St. Louis, MO).Additional descriptions of natural and / or unnatural amino acids can be found, for example, in Kotha, 2003 Acc. Chem. Res. 36:342; Maruoka et al., 2004 Proc. Nat. Acad. Sci. USA 101 :5824; Lundquist et al., 2001 Org. Lett. 3:781; Tang et al., 2002 J. Org. Chem. 67:7819; Rothman et al., 2003 J. Org. Chem. 68:6795; Krebs et al., 2004 Chemistry 10:544; Goodman et al., 2001 Biopolymers 60:229; Sabat et al., 2000 Org. Lett. 2:1089; Fu et al., 2001 J. Org. Chem. 66:7118; and Hruby et al., 1994 Meths. Mol. Biol. 35:249. Standard three letter abbreviations and one letter symbols are used herein to designate natural and unnatural amino acids.

[0066] Other non-natural amino acids or amino acid analogs are known in the art, including but not limited to, non-natural L or D derivatives (e.g., D-amino acids present in peptides and / or peptidomimetics (such as those described above and elsewhere herein)), fluorescently labeled amino acids, and specific examples include O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, 3-idio-tyrosine, O-propargyl-tyrosine, homoglutamine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, 3-nitro-L-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-acetyl-L-phenylalanine, m-acetyl-L-phenylalanine, selenomethionine, telluromethionine, selenocysteine, acetylene phenylalanine, O-allyl-L-tyrosine, O-(2-propynyl)-L-tyrosine, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutyryl)-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, homopropargylglycine, azidohomoalanine, p-iodo-phenylalanine, p-bromo-L-phenylalanine, dihydroxy-phenylalanine, dihydroxy-L-phenylalanine, p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine, trifluoro leucine, norleucine (“Nle”), D-norleucine (“dNle” or “D-Nle”), 5-fluoro-tryptophan, p-halo-phenylalanine, homo-phenylalanine (“homo-Phe”), seleno-methionine, ethionyl tyrosine, S-nitroso-homo cysteine, thia-proline, 3-thienyl-alanine, homo-allyl-glycine, trifluoro isoleucine, trans and cis-2-amino-4-butenoic acid, 2-butynyl-glycine, allyl-glycine, p-azido-phenylalanine, p-cyano-phenylalanine, p-ethynyl-phenylalanine, hexafluoro leucine, 1,2,4-triazole-3-alanine, 2-fluoro-histidine, L-methylhistidine, 3-methyl-L-histidine, β-2-thienyl-L-alanine, β-(2-thiazolyl)-DL-alanine, homopropargylglycine (HPG) and azidohomoalanine (AHA), and the like.

[0067] In certain embodiments, there can be naturally or non-naturally occurring amino acids that contain aromatic side chains, as in phenylalanine or tryptophan or analogs thereof, including other naturally or non-naturally occurring amino acids that one of skill in the art would readily recognize as containing a structure in which an aromatic ring system is present, typically in the form of an aromatic monocyclic or polycyclic hydrocarbon ring system, composed of only carbon and hydrogen, and containing 6-19 carbon atoms, in which the ring system can be partially or fully saturated, and which can be present as a group, including but not limited to, groups such as fluorenyl, phenyl, and naphthyl.

[0068] In certain embodiments, there can be naturally or non-naturally occurring amino acids that contain hydrophobic side chains, as in, for example, alanine, valine, isoleucine, leucine, proline, phenylalanine, tryptophan, or methionine or analogs thereof, including other naturally or non-naturally occurring amino acids that one of skill in the art would readily recognize based on structures in which a hydrophobic side chain (e.g., a hydrophobic side chain that is typically non-polar in a physiological environment) is present. In certain embodiments, there can be naturally or non-naturally occurring amino acids that contain basic side chains, as in, for example, lysine, arginine, or histidine or analogs thereof, including other naturally or non-naturally occurring amino acids that one of skill in the art would readily recognize based on structures in which a basic side chain (e.g., a side chain that is typically polar and positively charged in a physiological environment) is present.

[0069] The peptides disclosed herein can include, in certain embodiments, L- and / or D-amino acids, as long as the biological activity of the peptide is preserved (e.g., a spirochete T cell epitope-containing peptide can be recognized by T cells from a subject infected with a (Lyme disease-associated) spirochete species in a secondary in vitro immune response, as shown by stimulation of production of T cell immune response indicators, as described herein). In certain embodiments, the peptides can also contain any of a variety of known natural and artificial post-translational or post-synthetic covalent chemical modifications, which can be achieved by reactions that can include glycosylation (e.g., addition of N-linked oligosaccharides at asparagine residues, O-linked oligosaccharides at serine or threonine residues, glycation, etc.), fatty acylation, acetylation, formylation, pegylation, and phosphorylation. The peptides disclosed herein can also include analogs, alleles, and allelic variants, which can contain amino acid deletions of one or more amino acids, or additions or substitutions by other naturally occurring amino acid residues or non-natural amino acid residues.

[0070] Peptide and non-peptide analogs can be referred to as peptidomimetics or peptidomimetic peptides, and are known in the pharmaceutical arts (Fauchere, J. Adv. Drug Res. 15:29 (1986); Evans et al. J. Med. Chem. 30:1229 (1987)). These compounds can contain one or more non-natural amino acid residues, one or more chemically modified moieties (e.g., glycosylation, pegylation, fluorescence, radioactivity, or other moieties), and / or one or more non-natural peptide bonds (e.g., a reduced peptide bond: --CH2-NH2--). Peptidomimetics can be developed by a variety of methods, including by computer molecular modeling, random or site-directed mutagenesis, PCR-based strategies, chemical mutagenesis, and the like.

[0071] As noted above, certain embodiments also relate to peptidomimetics, or "artificial" polypeptides. Such polypeptides can contain one or more amino acid insertions, deletions, or substitutions, one or more altered or artificial peptide bonds, one or more chemical moieties (e.g., polyethylene glycol, glycosylations, detectable labels, or other moieties), and / or one or more non-natural amino acids. The synthesis of peptidomimetics is well known in the art, and can include altering a naturally-occurring protein or polypeptide by chemical mutagenesis, single or multiple site-directed mutagenesis, PCR shuffling, application of altered aminoacyl tRNA or aminoacyl tRNA synthetase molecules, application of "stop" codons such as amber suppressor genes, application of four-base pair or five-base pair codons, or other means.

[0072] Table 2 shows the Borrelia source, characteristics of the source protein, and the amino acid sequence region from which the Borrelia T-cell epitope-containing peptides (shown in Table 3) of SEQ ID NOs: 1-33 were generated or derived.

[0073] Amino acid sequences of Borrelia specific peptide regions in the FlaB, DbpB, p66 and OspC proteins

[0074] Table 3.

[0075]

[0076]

[0077]

[0078] Synthetic peptides prepared from Borrelia specific regions in the FlaB, DbpB, p66 and OspC proteins

[0079] Selection and preparation of Lyme disease specific protein peptides

[0080]

[0081]

[0082]

[0083] Any combination of the peptides of Table 3 can be used in certain embodiments of the presently contemplated compositions disclosed herein, particularly in the preferred embodiments, where at least one peptide from each of the four disclosed Borrelia polypeptide regions, FlaB, DbpB, p66, and OspC: FlaB [SEQ ID NO: 1-5], DbpB [SEQ ID NO: 6-18], p66 [SEQ ID NO: 19-31], and OspC [SEQ ID NO: 32-33] is present. The amounts of the several peptides relative to one another, as well as the absolute amounts of one or more of the peptides, can vary depending on the assay design and specific technique in which the compositions are employed, as will be familiar to those of skill in the art in light of the particular immunochemical, immunological, and / or biochemical methods. It is intended to be illustrative, not limiting, that in certain embodiments the compositions can comprise at least about 1 nanogram of each peptide and no more than about 100 nanograms of each peptide; in certain embodiments the compositions can comprise at least about 100, 200, 300, or 400 nanograms and no more than about 500 nanograms of each peptide; in certain embodiments the compositions can comprise at least about 500, 600, 700, 800, or 900 nanograms and no more than about 1000 nanograms of each peptide; in certain embodiments the compositions can comprise at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 micrograms and no more than about 2 micrograms of each peptide, while in certain embodiments the compositions can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9 micrograms and no more than about 10 micrograms of each peptide.

[0084] Methods for diagnosis or prognosis of Lyme disease

[0085] As disclosed herein, compositions and methods are provided in certain embodiments for detecting Lyme disease in a subject, or for monitoring the efficacy of a treatment for Lyme disease in a subject, based in part on the discovery that a peptide cocktail comprising at least one Borrelia-specific T cell epitope-containing peptide from each of the FlaB, DbpB, p66, and OspC protein regions of Borrelia (e.g., Tables 2 and 3) identified herein is capable of providing such a composition with which T cells from substantially all LD patients will react in a secondary in vitro immune response, producing a T cell immune response indicator (e.g., interferon-γ release) as provided herein.

[0086] Accordingly, these and related embodiments allow for the detection of LD in a subject at an earlier point in the disease process than the time at which borrelia-specific antibodies can be detected in the subject. Moreover, and as mentioned above, the present disclosure allows for the assessment of the efficacy of treatment against LD based on a decrease in detectable borrelia-specific T cell reactivity by circulating T cells obtained from a subject who has undergone successful LD treatment to eradicate borrelia spirochetes, as opposed to assessment of detectable borrelia-specific antibodies, which can remain at high levels in circulation for months or even years after successful LD treatment, thus masking the effects of successful eradication of borrelia spirochetes.

[0087] Accordingly, in certain embodiments, a method is provided for detecting Lyme disease in a subject, or for monitoring the efficacy of treatment against Lyme disease in a subject, comprising: (A) contacting in vitro (i) a first biological sample obtained at a first point in time from a subject known to have Lyme disease or suspected to be at risk of having Lyme disease, wherein the biological sample comprises T cells and antigen presenting cells, and (ii) a peptide composition for the diagnosis or prognosis of Lyme disease, to obtain a first test incubation mixture; (B) incubating the first test incubation mixture under conditions and for a time sufficient to allow the T cells to specifically recognize a borrelia T cell epitope present in the peptide composition to stimulate production of a T cell immune response indicator; and (C) detecting a first level of the T cell immune response indicator in the first test incubation mixture, wherein an increase in the first level of the T cell immune response indicator detected in (C) relative to a first control level of the T cell immune response indicator obtained by incubating the first biological sample in a first control incubation in the absence of the peptide composition for the diagnosis or prognosis of Lyme disease, indicates the presence of a borrelia infection in the subject, and wherein the peptide composition for the diagnosis or prognosis of Lyme disease comprises a peptide admixture comprising at least one borrelia-specific T cell epitope-containing peptide from each of the borrelia FlaB, DbpB, p66, and OspC protein regions identified herein (e.g., Tables 2 and 3), for example:

[0088] (a) 1, 2, 3, 4, or 5 isolated FlaB peptides, each comprising a B. burgdorferi T cell epitope, and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides, each comprising a B. burgdorferi T cell epitope, and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides, each comprising a B. burgdorferi T cell epitope, and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 1 or 2 isolated OspC peptides, each comprising a B. burgdorferi T cell epitope, and selected from the group consisting of OspC peptides having the amino acid sequences set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 32-33, thereby detecting Lyme disease in the subject, or monitoring efficacy of a treatment for Lyme disease in the subject.

[0089] In certain preferred embodiments, the first time point is prior to administering to the subject a treatment for Lyme disease.

[0090] In certain other embodiments, the method further comprises: (D) contacting in vitro: (i) a second biological sample, obtained from the subject at a second time point after the first time point and after administration of the treatment for Lyme disease to the subject, wherein the second biological sample comprises T cells and antigen presenting cells, and (ii) the peptide composition for the diagnosis or prognosis of Lyme disease, to obtain a second test incubation mixture; (E) incubating the second test incubation mixture under conditions and for a time sufficient for the T cells to specifically recognize the Borrelia T-cell epitopes present in the peptide composition to stimulate production of a T-cell immune response indicator; and (F) detecting a second level of the T-cell immune response indicator in the second test incubation mixture, wherein an increase in the second level of the T-cell immune response indicator detected in (F) relative to a second control level of the T-cell immune response indicator obtained by incubating the second biological sample in a second control incubation in the absence of the peptide composition for the diagnosis or prognosis of Lyme disease indicates the presence of a Borrelia infection in the subject, and wherein a decrease in the second level of the T-cell immune response indicator detected in (F) relative to the first level of the T-cell immune response indicator detected in (C) indicates efficacy of the treatment for Lyme disease.

[0091] In these and certain related embodiments, it is thus appreciated that the first time point is prior to administration of a given treatment for Lyme disease to the subject, while the second or subsequent time point can be after administration of the given treatment for LD to the subject, such that efficacy of the treatment can be determined as indicated, for example, by a decrease in the second level of the T-cell immune response indicator detected (e.g., a statistically significant decrease relative to a suitable control). By way of non-limiting theory, this result would be expected at the second or subsequent time point when the second biological sample comprises a low level of Borrelia-specific T-cell reactivity as a result of negative regulation and / or the absence of T-cell reactivity in the second sample due to substantial clearance of the Borrelia infection in the subject following the treatment for LD.

[0092] Thus, it will be appreciated that the severity of the Borrelia infection associated with LD can be monitored at multiple different time stages, e.g., over a time course between one or more second time points, to assess the disease progression reflected by the levels of T cell immune response indicators as apparent indicators of Borrelia bacterial load in the subject, and also to assess the efficacy of one or more treatments for LD. In certain instances, it can be desirable to repeat testing of multiple biological samples sequentially obtained from a subject at second and subsequent time points to monitor Borrelia-specific T cell activity in the subject. Thus, in certain embodiments, if it is desirable to monitor LD in a subject over a prolonged period of time, e.g., over multiple second time points, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more second time points, which can be separated from each other by varying intervals, which can be intervals of days, weeks, months, or years, the contacting, incubating, and detecting steps described herein can be repeated any number of times.

[0093] Thus, certain embodiments described herein contemplate compositions and / or methods relating to one or more antibiotic compounds known in the art to be suitable for use in treating Lyme disease (e.g., antibiotics based on having effective activity against pathogenic Borrelia species, such as any of the Borrelia species mentioned herein, particularly against human pathogenic Borrelia). Those skilled in the medical arts will appreciate that the terms "treat" and "treatment" refer to medical treatment of a disease, disorder, or condition in a subject (i.e., a patient, host, which can be a human or non-human animal) (see, e.g., Stedman's Medical Dictionary).

[0094] Antibiotics are known in the art and generally include a drug prepared from a species of microorganism that produces a compound capable of killing another species of microorganism, or a synthetic product having an equivalent or similar chemical structure and mechanism of action, e.g., a drug that destroys microorganisms in a living organism or on a body surface, including such drugs that are sometimes applied topically.

[0095] Suitable antibiotics known in the art for treating Lyme disease can include, but are not necessarily limited to, one or more of the following: tetracyclines; oxytetracycline, tetracycline, multicycline, or minocycline; penicillins; amoxicillin or penicillin; cephalosporins; cefaclor, cefoperazone, cefminox, cefotaxime, cefotetan, cefmetazole, cefoxitin, cefuroxime axetil, cefuroxime acetyl, hymexazol, or ceftriaxone sodium; macrolides; azithromycin, clarithromycin, or erythromycin (see, e.g., Cameron et al., 2014 Expert Rev Anti Infect Ther. 12(9): 1103-1135; Shapiro, 2014 N. Engl. J. Med. 370(18): 1724-31; Wright et al., 2012 Am. Fam. Physician 85: 1086.). An overview of these and other clinically useful antibiotics is available and known to those familiar with the art (e.g., Washington University School of Medicine, The Washington Manual of Medical Therapeutics (32ndedition), 2007 Lippincott, Williams and Wilkins, Philadelphia, PA; Hauser, AL, Antibiotic Basics for Clinicians, 2007 Lippincott, Williams and Wilkins, Philadelphia, PA).

[0096] A biological sample for use in the methods described herein can be obtained from a subject for determining the presence and level of a T cell immune response indicator described herein. Suitable biological samples can include, for example, a whole blood sample, or a cerebrospinal fluid sample, or a synovial fluid sample, which can be obtained from a subject (e.g., a human or animal subject, and in preferred embodiments, a human) having or at risk of having Lyme disease, e.g., a patient with a persistent tick bite wound or a patient with clinically migrating erythema or other case in which one or more risk factors for Lyme disease can be present, which can be identified by one of skill in the art (see, e.g., Shapiro, 2014 N. Engl. J. Med. 370: 1724; Wright et al., 2012 Am. Fam. Physician 85: 1086). In certain embodiments, the biological sample can comprise at least one of: whole blood (optionally with an anticoagulant), or a cellular component of whole blood, or isolated peripheral blood leukocytes, or isolated peripheral blood mononuclear cells. A biological sample can be obtained from a subject at a first time point prior to administering an LD treatment to the subject, which can have diagnostic utility and / or can serve as a control for establishing baseline (i.e., pre-treatment) data; other biological samples can be obtained from the subject at one or more second time points after administering an LD treatment to the subject.

[0097] Detection of T cell immune response indicators

[0098] Antigen-specific T cell responses in vitro are typically determined by comparing the observed T cell responses based on any of a variety of described measurable parameters of T cell functionality (e.g., proliferation, cytokine expression, cytokine biosynthesis, cytokine release, altered cell surface marker phenotype, etc.) between T cells that have been contacted with an antigen of interest (e.g., an antigen for priming or activating T cells when presented by immunocompatible antigen presenting cells) and T cells from the same source population (i.e., T cells that have been contacted with a structurally different or unrelated control antigen). A statistically significant greater response to the antigen of interest than to the control antigen indicates antigen specificity.

[0099] Secondary in vitro Borrelia-specific immune response levels can be determined by any of a variety of immunological methods described herein and / or routinely practiced in the art. Secondary in vitro Borrelia-specific immune response levels can be determined at one or more time points, including before and after administering any one or more of the treatments for Lyme disease described herein (e.g., one or more antibiotics) to a subject providing a biological sample comprising T cells and antigen presenting cells (e.g., a subject known to have or suspected of having Lyme disease).

[0100] As described in the Examples, the presently disclosed compositions for diagnosis or prognosis of a condition comprising a peptide containing a Borrelia T-cell epitope (e.g., one or more of the FlaB, DbpB, p66, and OspC peptides of SED ID NOS: 1-33 or variants thereof, as described herein) were shown to be capable of stimulating the production of detectable indicators of T-cell immune response in in vitro test incubations employing a biological sample comprising T-cells and antigen presenting cells from a subject having an early stage infection with a Borrelia species associated with Lyme disease. The level of the detected indicator of T-cell immune response was increased relative to a control level of the indicator obtained by incubating the biological sample in a control incubation identical to the test incubation except that the Borrelia peptide composition was omitted.

[0101] Thus, in certain embodiments, an elevated level of an indicator of T-cell immune response provided herein relative to the level of the indicator detected under suitable control conditions (e.g., in the absence of the Borrelia peptide composition) can be in the form of a statistically significant increase in the level of the indicator that can be detected after incubating T-cells and antigen presenting cells with a peptide composition containing a Borrelia T-cell epitope (FlaB, DbpB, p66, OspC) as described herein under conditions and for a time sufficient to allow the T-cells to recognize the particular antigen.

[0102] By way of illustration and not limitation, in preferred embodiments, the indicator of a T cell immune response produced by stimulating T cells with a Borrelia T cell epitope-containing peptide composition is at least one T cell cytokine induced, expressed and / or released by T cells following in vitro incubation. Preferably, the T cell cytokine is selected from the group consisting of IL-1a, IL-1b, IL-2, IL-10, IL-12, IL-17, TNF-a, TNF-b, and IFN-g. In certain preferred embodiments, the release of IFN-g is the indicator of a T cell immune response produced by stimulating T cells with a Borrelia T cell epitope-containing peptide composition. However, contemplated embodiments are not intended to be so limited, and thus can include any of a number of methods for assessing the ability of a biological sample provided herein to mount a secondary in vitro antigen-specific response to a Borrelia T cell epitope-containing peptide composition described herein. Various assay configurations and techniques are known in the art (e.g., Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009)), and can be adapted to the methods herein based on the present disclosure. Thus, the level of a cytokine can be determined according to methods described herein and practiced in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining, and / or flow cytometry.

[0103] In preferred embodiments, the indicator of a T cell immune response is IFN-g released by T cells of the biological sample in the step of incubating the sample with a Borrelia T cell epitope-containing peptide composition described herein, and the level of released IFN-g is determined by immunochemistry, employing any of a variety of in vitro techniques whereby IFN-g is detected by determining the detectable specific binding of a binding agent to the T cell cytokine (i.e., IFN-g). Thus, the binding agent can comprise at least one antibody that specifically binds to the cytokine (e.g., IFN-g), which can comprise at least one monoclonal antibody or it can comprise a polyclonal antibody. According to certain embodiments, the indicator of a T cell immune response is a cytokine released by T cells and is detected by binding to an antibody immobilized on a solid phase.

[0104] An exemplary immunoassay for IFN-g is The assay (available from Cellestis, Ltd., Carnegie Victoria, Australia) has been established as the gold standard for IFN-γ assays and is widely used for testing for tuberculosis (e.g., Pai et al., 2004 Lancet Infect Dis 4:761), and thus quantitative detection of IFN-γ can be adapted to the use of the spirochete antigen described herein (as an alternative to tuberculosis antigen). (See also, e.g., Yun et al., 2014 J. Clin. Microbiol. 52:90; Belknap et al., 2014 Clin. Lab. Med. 34:337; Ferguson et al., 2015 Transplantation 99:1084; Ruan et al., 2014 Clin. Rheumatol. Epub PMID 25376466).

[0105] If the antibody reacts with the antigen at a detectable level, preferably at a level greater than or equal to about 10... 4 M -1 , or greater than or equal to 10 5 M -1 , greater than or equal to approximately 10 6 M -1 , greater than or equal to approximately 10 7 M -1 , or greater than or equal to 10 8 M -1 Affinity constant (K) a When an antibody reacts with an antigen, the binding partner or antibody is referred to as "immune-specific," "specific for," or "specifically binds" to the antigen of interest (e.g., a cytokine to be tested as an indicator of a detectable T-cell immune response, such as IFN-γ). The affinity of an antibody for its associated antigen is also generally expressed as a dissociation constant K. D Furthermore, if the antibody is less than or equal to 10 -4 M, less than or equal to approximately 10 -5 M, less than or equal to approximately 10 -6 M, less than or equal to 10 -7 M, or less than or equal to 10 - 8 M of K D When it binds to an antigen of interest, it binds specifically to that antigen of interest.

[0106] Combining chaperones or antibodies can be achieved using conventional techniques such as those described by Scatchard et al. (Ann. NYAcad. Sci. USA 51:660 (1949)) as well as through surface plasmon resonance (SPR; BIAcore). TMBiacore (Biosensor, Piscataway, NJ) is readily determined. For surface plasmon resonance, target molecules are immobilized on a solid phase and contacted with a binding partner (or ligand) in a flow phase that is run along a flow cell. If the ligand binds to the immobilized target, a local refractive index change occurs, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in reflected light intensity. The rate of change of the SPR signal can be analyzed to yield apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values gives the apparent equilibrium constant (affinity) (see, e.g., Wolff et al., Cancer Res. 53:2560-2565 (1993)).

[0107] As used herein, the term "polyclonal antibody" refers to antibodies obtained from a population of antigen-specific antibodies that recognize more than one epitope of a particular antigen. An "antigen" or "immunogen" refers to a peptide, lipid, polysaccharide, or polynucleotide that is recognized by the adaptive immune system. An antigen can be a self or non-self molecule. Examples of antigens include, but are not limited to, bacterial cell wall components, pollen, and rh factors. An antigenic region that is specifically recognized by a specific antibody or specific T cell receptor is an "epitope" or "antigenic determinant." A single antigen can have multiple epitopes.

[0108] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be made using recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567) in bacterial, eukaryotic animal or plant cells. "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991), for example.

[0109] Nucleic acids and polynucleotides

[0110] The Borrelia T-cell epitope-containing polypeptides and peptides, as well as the encoding nucleic acid molecules and vectors provided herein, can be isolated and / or purified, e.g., from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acids, free or substantially free of nucleic acids or genes of different origin than the sequence encoding the polypeptide having the desired function. The nucleic acids can comprise DNA or RNA and can be wholly or partially synthetic. Reference to a nucleotide sequence as set forth herein includes both DNA molecules having the set forth sequence, as well as RNA molecules having the set forth sequence, wherein U is substituted for T, unless otherwise indicated.

[0111] Accordingly, the present application also provides, in certain embodiments, isolated nucleic acids encoding any of the Borrelia T-cell epitope-containing peptides having the amino acid sequences set forth in SEQ ID NOs: 1-33.

[0112] The term "operably linked" means that the components to which the term applies are in a relationship permitting them to carry out their inherent functions under suitable conditions. For example, a transcription control sequence "operably linked" to a protein coding sequence is ligated to it in a manner that enables expression of the protein coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0113] The term "control sequence" as used herein refers to a polynucleotide sequence that can affect the expression, processing or cellular location of a coding sequence to which it is linked or operably linked. The nature of such control sequences can depend on the host organism. In particular embodiments, transcription control sequences for prokaryotes can include promoters, ribosome binding sites and transcription termination sequences. In other particular embodiments, transcription control sequences for eukaryotes can include promoters comprising one or more recognition sites for transcription factors, transcription enhancer sequences, transcription termination sequences and polyadenylation sequences. In certain embodiments, "control sequences" can include leader sequences and / or fusion partner sequences.

[0114] The term "polynucleotide" as described herein refers to a single- or double- stranded nucleic acid polymer. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides and deoxyribonucleotides or modified forms of either type of nucleotide. These modifications can include base modifications such as bromouridine, sugar modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate and phosphoramidate. The term "polynucleotide" specifically includes DNA in both single- and double-stranded forms.

[0115] The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroanilide, phosphoramidate, etc. See, e.g., LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc, 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed.), Oxford University Press, Oxford, England; Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are incorporated herein by reference for any purpose. Oligonucleotides can include detectable labels to enable detection of the oligonucleotide or its hybridization.

[0116] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing if introns are present.

[0117] It will be understood by those skilled in the art that polynucleotides can include genomic sequences, extragenomic and plasmid encoded sequences, and smaller engineered genetic segments that express proteins, polypeptides, peptides, etc., or can be adapted to express proteins, polypeptides, peptides, etc. Such segments can be naturally isolated or modified by the skilled artisan by synthetic means.

[0118] Those skilled in the art will recognize that polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules can include HnRNA molecules, which contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences can or can not be present within the polynucleotides according to the present disclosure, and polynucleotides can or can not be linked to other molecules and / or support materials. Polynucleotides can comprise native sequences or can comprise sequences encoding variants or derivatives of such sequences.

[0119] Accordingly, the present disclosure also provides, in accordance with these and related embodiments, polynucleotides encoding the Borrelia T-cell epitope-containing peptides described herein. In certain embodiments, polynucleotides are provided that comprise some or all of the polynucleotide sequences encoding the peptides as described herein, and complements of such polynucleotides.

[0120] In other related embodiments, polynucleotide variants can be substantially identical to polynucleotide sequences encoding the Borrelia T-cell epitope-containing peptides described herein. For example, a polynucleotide can be one that comprises at least 80% sequence identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, compared to a reference polynucleotide sequence, e.g., a sequence encoding a Borrelia T-cell epitope-containing peptide described herein (e.g., a peptide having one of SEQ ID NOs: 1-33 as its amino acid sequence), using methods described herein (e.g., BLAST analysis, using standard parameters, as described below). Those skilled in the art will recognize that these values can be adjusted appropriately by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like, to determine the corresponding identity of the polypeptides or peptides encoded by two nucleotide sequences.

[0121] In another embodiment, there is provided a polynucleotide capable of hybridizing under moderately to highly stringent conditions to a polynucleotide sequence encoding a Borrelia T cell epitope-containing peptide provided herein, or a fragment thereof, or a complement thereof. Hybridization techniques are well known in the art of molecular biology. For illustrative purposes, suitable moderately stringent conditions for testing the hybridization of a polynucleotide provided herein to other polynucleotides include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-60°C in 5X SSC; and then washing in 0.1% SDS, 2X, 0.5X, and 0.2X SSC, for 20 minutes each at 65°C. Those of skill in the art will appreciate that the stringency of hybridization can be readily manipulated, e.g., by altering the salt content and / or temperature of the hybridization solution. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, except that the temperature of hybridization is increased, e.g., to 60°C-65°C or 65°C-70°C.

[0122] As also described elsewhere herein, the three-dimensional structure of a representative Borrelia T cell epitope-containing peptide (e.g., SEQ ID NO: 1-33 or a variant thereof, as provided herein) can be determined by routine methods, such that virtual modeling of substitution, addition, deletion, or insertion of one or more amino acids with selected natural or unnatural amino acids can be performed to determine whether the derived structural variant retains the spatial packing properties of the presently disclosed class. Various computer programs are known to those of skill in the art for determining appropriate amino acid substitutions within a peptide (or suitable polynucleotides encoding the amino acid sequence) such that, for example, Borrelia-specific T cell recognition is maintained.

[0123] Regardless of the length of the coding sequence itself, the polynucleotides described herein, or fragments thereof, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary greatly. It is therefore contemplated that virtually any length of nucleic acid fragment can be used, with the overall length preferably limited by the ease of preparation and the intended recombinant DNA protocol for producing the presently disclosed Borrelia T cell epitope-containing peptides. For example, it is contemplated that illustrative polynucleotide segments having an overall length of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and the like base pairs (including all intermediate lengths) are useful.

[0124] When comparing polynucleotide sequences, two sequences are said to be "identical" if the sequence of nucleotides in the two sequences are the same after maximal correspondence alignment, as described below. Comparison between two sequences is typically performed by comparing sequences over a comparison window, to identify and compare local regions of sequence similarity. A "comparison window" as used herein, is a segment of at least about 20 contiguous positions, usually 30 to about 75 contiguous positions, 40 to about 50 contiguous positions, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can comprise additions or deletions, i.e., gaps, of about 20% of the total number of positions in the comparison window, usually about 5 to about 50% of the number of positions in the comparison window, more usually about 10 to about 30% of the number of positions in the comparison window.

[0125] Optimal alignment of sequences for comparison can be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC vol. 5, suppl. 3, pp. 345- 358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., CABIOS 5: 151-153 (1989); Myers, E.W. and Muller W., CABIOS 4: 11-17 (1988); Robinson, E.D., Comb. Theor 11: 105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4: 406-425 (1987); Sneath, P.H.A. and Sokal, R.R., Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA (1973); Wilbur, W.J. and Lipman, D.J., Proc. Natl. Acad., Sci. USA 80: 726-730 (1983).

[0126] Alternatively, optimal sequence alignments can be performed to compare, for example, by the local homology algorithm of Smith and Waterman, Add. APL Math 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988); by computer implementation of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by visual inspection.

[0127] One preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, e.g., with the parameters described herein, to determine percent sequence identity between two or more polynucleotides or between two or more polypeptides. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0) are used to calculate the cumulative score. Extension of the word matches in each direction are terminated when: the cumulative score falls off by a quantity X from its maximum value; the sum of the positive- and negative-scoring residues in a window of length W positions falls off by a quantity Y from its maximum value; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the comparison. The default values for the BLASTN program (for amino acid sequences) are: wordlength (W) 11, expectation value (E) 10, and BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) comparison matrix, (B) 50, expectation value (E) 10, M=5, N=-4, and comparisons between two strands.

[0128] In certain embodiments, the "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e. gaps) of 20% or less, usually 5 to 15%, or 10 to 12%, as compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percent sequence identity is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the result by 100 to yield the percent sequence identity.

[0129] It will be understood by one of ordinary skill in the art that, as a result of the degenerative nature of the genetic code, numerous nucleotide sequences encoding the Borrelia T cell epitope-containing peptides as described herein exist. Some of these polynucleotides have minimal sequence identity with the nucleotide sequence of the natural or original polynucleotide sequence encoding the Borrelia T cell epitope-containing peptides described herein. Nevertheless, polynucleotides which vary due to the degeneracy of the code are expressly encompassed by the disclosure. In certain embodiments, sequences which are codon-optimized for mammalian expression are specifically contemplated.

[0130] Thus, in another embodiment of the application, mutagenic methods such as site-specific mutagenesis can be employed to prepare variants and / or derivatives of the Borrelia T cell epitope-containing peptides described herein. By this method, specific modifications can be made to the polypeptide sequence by mutagenizing the underlying polynucleotide encoding them. These techniques provide a direct means of making and testing sequence variants, for example, by introducing one or more nucleotide sequence changes into a polynucleotide, introducing one or more of the foregoing considerations.

[0131] Site-directed mutagenesis allows the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex across the deletion junction points flanking the mutation, thereby generating the mutant. Mutations can be used in selected polynucleotide sequences to improve, alter, reduce, modify, or otherwise change the properties of the polynucleotide itself, and / or to change the properties, activities, composition, stability, or primary sequence of the encoded polypeptide.

[0132] According to certain related embodiments, a recombinant host cell is provided, comprising one or more constructs as described herein; a nucleic acid encoding a Borrelia T cell epitope-containing peptide or variant thereof; and, a method for producing the encoded product, said method comprising expression from its encoding nucleic acid. Expression is readily achieved by culturing the recombinant host cell containing the nucleic acid under suitable conditions. Following production by expression, the Borrelia T cell epitope-containing peptide can be isolated and / or purified using any suitable technique, and then used as desired.

[0133] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines useful in the expression of heterologous polypeptides in the art include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A commonly used preferred bacterial host is E. coli. Recombinant expression of peptides in prokaryotic cells such as E. coli has been well established in the art, as has expression in cultured eukaryotic cells.

[0134] Suitable vectors containing appropriate regulatory sequences can be selected or constructed, including promoters, terminators, polyadenylation sequences, enhancers, marker genes, and other sequences as appropriate. The vector can be a plasmid, virus, or phage, as appropriate. See, e.g., Molecular Cloning: A Laboratory Manual, 4th Ed., Green and Sambrook, 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, for more detailed information. Many known techniques and protocols for nucleic acid manipulation, such as those in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, New York, 2015, or subsequent updates.

[0135] The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more of the leptospiral T cell epitope-containing peptides described herein has been or can be introduced, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding any of the leptospiral T cell epitope-containing peptides described herein. The term includes the progeny of the parent cell, whether or not the progeny is identical to the original parent cell in morphology or in genetic constitution, so long as the selected gene is present. Thus, a method is also contemplated that includes introducing the nucleic acid into a host cell. Such introduction can be performed using any suitable technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia virus, or, in the case of insect cells, baculovirus. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophage. The nucleic acid can be expressed after introduction, for example, by culturing the host cell under conditions in which the gene is expressed. In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques.

[0136] In certain embodiments, the present application also provides a method that includes employing a construct as described above in an expression system to express a particular polypeptide, such as a leptospiral T cell epitope-containing peptide described herein. The term "transduction" is used to refer to the transfer of genes from one bacterium to another, usually by a bacteriophage. "Transduction" also refers to the acquisition and transfer of eukaryotic sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are known in the art and are disclosed herein. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, New York, 2015, or subsequent updated editions. The techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0137] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell and the cell has been transformed when it has been modified by the introduction of new DNA. For example, a cell is transformed from the time that it is genetically modified from its native state. Following transfection or transduction, the transforming DNA can become recombined with the cell's chromosome(s) by a physical integration, or it can exist as an extra-chromosomal element, replicating episomally, or it can replicate independently as a plasmid. A cell is considered stably transformed when the DNA is replicated with the cell division, and is passed from mother cell to daughter cell. The term "naturally occurring" or "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, etc., refers to materials that are found in nature and have not been processed by human intervention. Similarly, "non-naturally occurring" or "non-native" as used herein refers to materials that do not exist in nature or have been structurally modified or synthesized by humans.

[0138] It is to be understood that, unless otherwise specified, the practice of several embodiments of the present application will employ, unless otherwise specified, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, and many of the parts thereof are described below for illustrative purposes. Such techniques are fully described in the literature.See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, New York, 2015; Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Ed. 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volumes I and II (D. Glover ed.) ; Oligonucleotide Synthesis (N. Gait ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins eds., 1985); Transcription and Translation (B. Hames & S. Higgins eds., 1984); Animal Cell Culture (R. Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); and other like references.

[0139] Every embodiment described in this specification, unless otherwise indicated, is applicable to each other embodiment so far as is necessary.

[0140] Standard techniques can be used for biochemical and immunochemical and immunological assays, recombinant DNA, oligonucleotide synthesis, microbial and mammalian cell and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures can be generally performed according to conventional methods in the art and as described in various general and more specific references that are cited throughout this specification and that are well known in the art. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (John Wiley & Sons, Inc., July 2008 New Edition); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, Volumes I and II (IRL Press, Oxford University Press, USA, 1985); Current Protocols in Immunology John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober, eds., 2001, John Wiley & Sons, Inc., New York, NY); Real-Time PCR: Current Technology and Applications, Julie Logan, Kirstin Edwards, and Nick Saunders, eds., 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames and S. Higgins, eds., 1985); Transcription and Translation (B. Hames and S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, ed., 3rded., 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology Vols. 154 and 155 (Wu et al., eds., 1987, Academic Press); Methods In Enzymology Vol. 217 (Wu et al., eds., 1993, Academic Press); Methods In Enzymology Vol. 217 (Wu et al., eds., 1993, Academic Press); Methods In Enzymology Vol. 272 (Wu et al., eds., 1996, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304 (Wu et al., eds., 1999, Academic Press); Methods In Enzymology Vol. 304Calos, ed., 1987, Cold Spring Harbor Laboratory Press); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, ed., 2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kurstad Turksen, ed., 2006); Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell, eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug and Craig T.Jordan, 2001); Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting, ed., 2008); Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner, ed., 2008).

[0141] Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical formulation, manufacture, and delivery, and treatment of patients.

[0142] Unless otherwise indicated, the word "comprising" is used in the description and claims of this patent application to mean that the listed steps or options need not be exhaustive. The word "consisting" is used in the description and claims of this patent application to mean that the listed steps or options are to be exhaustive. The word "comprising" is used in the description and claims of this patent application to mean that the listed steps or options need not be exhaustive. The word "consisting essentially of is used in the description and claims of this patent application to mean that the listed steps or options need not be exhaustive, but that other steps or options not recited are optional and do not materially affect the basic and novel characteristics of the claimed application. Thus, the phrase "consisting essentially of indicates that the enumerated elements need not be exhaustive, but that other elements not listed are optional and do not materially affect the basic and novel characteristics of the claimed application.

[0143] As used in this specification and claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the term "about" or "approximately," when used in a particular embodiment, indicates that the value is within 5%, 6%, 7%, 8%, or 9% of the stated value. In other embodiments, the term "about" or "approximately," when used in a range of values, indicates that the range is within 10%, 11%, 12%, 13%, or 14% of the stated range. In other embodiments, the term "about" or "approximately," when used in a range of values, indicates that the range is within 15%, 16%, 17%, 18%, 19%, or 20% of the stated range.

[0144] Reference in the specification to "one embodiment" or "an embodiment" or "one aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. The appearance of the phrase "in one embodiment" or "in an embodiment" in various places in the specification are not necessarily all referring to the same embodiment. Also, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0145] EMBODIMENT

[0146] Example 1. Lyme Disease Detection and Treatment Efficacy Monitoring by Secondary in vitro T Cell Response to Artificial Peptide Compositions

[0147] This example describes the evaluation of the ability of specific peptides (derived from regions of individual B. burgdorferi proteins and contained in a multiplexed peptide cocktail) to induce interferon-γ production in vitro by activated T cells obtained from LD patients. Specifically, the ability of a cocktail containing B. burgdorferi specific peptides to induce interferon-γ release in vitro by Lyme disease specific activated T cells in blood samples obtained from patients with very early B. burgdorferi infection was tested. The level of interferon-γ production in vitro was also determined for T cells collected from an LD patient at a first time point just prior to antibiotic treatment and at a second time point approximately 60 days after effective treatment of the patient with antibiotics.

[0148] MATERIALS AND METHODS

[0149] Borrelia The flagellin protein (FlaB), the core protein polysaccharide binding protein (DbpB), the common antigen (p66), and the outer surface protein C (OspC) are expressed in large amounts on the surface of B. burgdorferi at different times during mammalian infection. Specifically, FlaB is the major filamentous protein of the flagella, which is expressed predominantly during the late disseminated and early localized stages of infection. DbpB is an adhesin that is upregulated during and / or shortly after a tick bite and appears during the early disseminated stage of infection, while p66 is an integrin binding protein that facilitates bacterial adhesion to immune cells and is expressed after infection dissemination. In addition, OspC expression appears during the early localized stage and is believed to be required for establishment of mammalian infection, while OspC specific antibodies are the dominant antibodies during early human LD. B. burgdorferi FlaB, DbpB, p66, and OspC have each been reliably used to detect antibodies produced in response to B. burgdorferi infection.

[0150] A cocktail of peptides comprising peptide regions specific for B. burgdorferi selected from a range of individual B. burgdorferi proteins (Tables 2-3) was evaluated for its ability to activate T cells in peripheral blood samples obtained from LD patients at short-term post-Lyme spirochete infection. To increase the specificity of the peptide composition for potential stimulation of LD-associated T cells, the complete protein sequences were first analyzed by BLAST algorithm to identify suitable B. burgdorferi specific regions (Table 4). Synthetic peptides were then synthesized from specific regions in each protein; each peptide was about 15-25 amino acids in length with an overlap of 10-15 amino terminal ends (Table 3).

[0151] Patient recruitment Patients suspected of having early Lyme disease, characterized by exposure to ticks and subsequent development of skin lesions with features consistent with erythema migrans (EM), were then recruited to participate in a proof-of-concept study. Informed consent was obtained from each volunteer, and each subject had a presumptive diagnosis of Lyme disease that required treatment with doxycycline 100 mg twice daily for a minimum of 10 days, except for one subject (#6) who was treated for only one day.

[0152] Table 4. Amino acid sequences of B. burgdorferi specific regions in FlaB, BbpB, p66 and OspC.

[0153] FlaB, flagellin, partial [Borrelia burgdorferi], GenBank: ACI49679.1

[0154] NVRTAEELGM QPAKINTPAS LSGSQASWTL RVHVGANQDE (51-90)

[0155] [SEQ ID NO: 34]

[0156] QDEAIAVNIYAANVANLFSG (88-107) [SEQ ID NO: 35]

[0157] SLAKIENAIRMISDQRANL (156-174) [SEQ ID NO: 36]

[0158] DbpB (decorin, B. burgdorferi), partial [Borrelia burgdorferi], GenBank: AAC70029.1

[0159] SIVMVLFFDL LVACSIGLVE RTNAALESSS KDLKNKILKI KKEATGKGVL FEAFTGLKTG

[0160] SKVTSGGLAL REAKVQAIVE TGKFLKIIEE EALKLKETGN SGQFLAMFDL MLEVVESLED

[0161] VGIIGLKARV LEESKNNPIN TAERLLAAKA QIENQLKVVK EKQNIENGGE KKNNKSKKKK (1-180)

[0162] [SEQ ID NO: 37]

[0163] p66, complete outer membrane protein p66 [Borrelia burgdorferi B31], GenBank: AAC66949.1

[0164] FGLSGAYGNE TFNNSSITYS LKDKSVVGND LLSPTLSNSA ILASFGAKYK LGLTKINDKN

[0165] TYLILQMGTD FGIDPFASDF SIFGHISKAA NFKKETPSDP NKKAEIFDPN GNALNFSKNT

[0166] ELGIAFSTGA SIGFAWNKDT GEKESWAIKG (251-400)

[0167] [SEQ ID NO: 38]

[0168] OspC, outer surface protein C [Borrelia burgdorferi], GenBank: ABQ42983.1

[0169] ATKAIGKKIQQNGGLAVEAGH (55-75) [SEQ ID NO: 39]

[0170] KEMLANSVKELTSPI (171-185) [SEQ ID NO: 40]

[0171] Collection and processing of blood samples.Blood samples were collected in green-top (lithium-heparin) tubes and processed promptly at the pre-treatment (first symptoms) and post-treatment 60-day visit. One tube was centrifuged to separate the plasma, which was removed and stored at -80°C until testing. A separate 1-ml volume of blood was immediately transferred to a sterile pyrogen-free tube (Cytos Biotech Pty Ltd, Carnegie, Victoria, Australia) pre-loaded with a 10-μl volume of a pool of 33 Borrelia burgdorferi peptides [SEQ ID NO: 1-33] (1 μg of each peptide) as shown in Table 3. In addition, a separate 1-ml volume of blood was added to separate tubes containing no peptide (nil) or pre-loaded with mitogen controls. The contents of each tube were then mixed thoroughly and incubated at 37°C for 18-24 hours.

[0172] Detection of interferon-γ. After incubation, the blood plasma was harvested after centrifugation at 3000 x g for 15 minutes to remove cells, and the levels of interferon-γ in the plasma were quantified by immunochemistry using the TB Gold ELISA kit (Cytos Biotech Pty Ltd) according to the manufacturer's instructions. The assay was established to test blood samples from patients with tuberculosis (TB) to confirm Mycobacterium tuberculosis infection by IFN-γ produced by T cells that have been activated in vitro twice after they have been stimulated by exposure to Mycobacterium spp. peptides. Herein, the assay is referred to as the "TB assay". The platform was also adapted to detect IFN-γ produced by T cells activated in response to Borrelia burgdorferi, rather than Mycobacterium tuberculosis infection. Prior to evaluating plasma samples, significant reactivity cut-off values were first determined by testing sera from eight volunteer subjects with no prior history of Lyme disease or suspected Lyme disease. Blood was collected by venepuncture into clot activator tubes, and the sera were removed and tested immediately. The mean value of the resulting values was then calculated, and OD values above the mean > two standard deviations (> 0.452) were considered significant (" a ").

[0173] Serodiagnosis of LD confirmationPaired (pre- and post-treatment, from the same subject) plasma samples were also tested for anti-C6 antibodies and Western blot reactivity. Anti-C6 antibodies were detected by ELISA as previously described (Jobe et al., 2008. Significantly improved accuracy of diagnosis of early Lyme disease by peptide enzyme-linked immunosorbent assay based on the borrelial antibody epitope of Borrelia burgdorferi OspC. Clin Vacc Immunol 15:981-985). Prior to evaluation, a significant reactivity cutoff was determined by testing 15 well-characterized sera from volunteer subjects with no prior history or suspicion of LD. Blood was collected by venipuncture into clot activator tubes, and serum was removed and immediately tested. The mean of the resulting values was then calculated, and optical density (OD) values above the mean > two standard deviations (IgM > 0.534, IgG > 0.140) were considered significant (" a "). Appropriate positive and normal control sera were also included in separate wells on the ELISA plate.

[0174] Western immunoblot reactivity of serum immunoglobulins to electrophoretically separated B. burgdorferi antigens (Mogilyanski et al., 2004 Clin Diag Lab Immunol 11:924) was tested using a commercially available kit (Marblot TM , Trinity Biotech, Bray, County Wicklow, Ireland) according to the manufacturer's recommended method. The Centers for Disease Control (CDC, Atlanta, GA) recommended criteria were used to determine positivity (IgM - detection of at least two of 23, 39, or 41 bands, IgG - detection of at least five of 18, 23, 28, 30, 39, 41, 45, 58, 66, or 93 bands).

[0175] Results

[0176] Study group list.A total of 21 patients (18 years of age or older) completed the study. Each had been exposed to an interrogative deer tick (Ixodes scapularis) or had a documented tick bite within 30 days prior to the detection of a putative single or multiple erythema migrans lesions, and each received a tetracycline treatment of at least 10 days, except for subject #6 who received antibiotic treatment (doxycycline) for only one day, a treatment regimen that is almost universally successful in eradicating spirochetes at the early stages of disease (Kowalski et al., 2010 Clin Infect Dis 50:512-520). As further support for effective treatment, each subject was interrogated at the time of providing convalescent blood samples and none complained of persistent abnormalities.

[0177] Serodiagnosis of LD confirmation . Initial evaluation of acute and convalescent sera from each subject for anti-C6 antibodies or Western blot reactivity for serodiagnostic confirmation, each participant was infected with B. burgdorferi. Acute sera from 10 (48%) subjects contained significant levels of IgM anti-C6 antibodies and two (10%) other subjects seroconverted at convalescence (Table 5). In addition, acute sera from 12 (57%) subjects contained IgG anti-C6 antibodies, and eight of the patients who produced IgG antibodies also produced IgM antibodies. Furthermore, significant levels of IgG antibodies were detected in convalescent sera from four (19%) other subjects. The overall results provided serodiagnostic confirmation of Lyme disease in 17 (81%) of the 21 enrollees.

[0178] In contrast, acute sera from only two (10%) subjects produced sufficient IgM antibody reactivity to meet the Centers for Disease Control (CDC) criteria for confirmation of B. burgdorferi infection (Table 6). Furthermore, 60 days later, only in four other subjects did the response increase (more reactive IgM and IgG bands) to confirmatory levels. Western blots provided serodiagnostic confirmation of Lyme disease in only 6 (29%) of the 21 enrollees. In addition, Western blots from subjects who failed to develop anti-C6 antibodies were also negative. The combined results from the C6 test and Western blots failed to provide serodiagnostic confirmation of LD in four (19%) of the study subjects. More significantly, the levels of antibodies detected by each test procedure often increased at convalescent, post-treatment sera, even in cases where the LD spirochetes were almost certainly eradicated by antibiotic treatment. Thus, the C6 test or Western blots cannot be relied upon to provide an accurate prediction of successful treatment.

[0179] Table 5. Serodiagnostic confirmation of early Lyme disease by detection of anti-C6 antibodies

[0180]

[0181]

[0182] Table 6. Confirmation of serodiagnosis of early Lyme disease by Western blot.

[0183]

[0184]

[0185] Detection of interferon-γ after T cell stimulation by Borrelia peptide pools. IFN-γ levels were determined by immunoassay of IFN-γ released by peripheral blood T cells following in vitro stimulation with pools of 33 B. burgdorferi peptides shown in Table 2, collected during the acute and convalescent phases of disease in 17 subjects confirmed to be infected with LD by serodiagnosis (supra). Significant levels of IFN-γ were detected in the test incubation mixtures of 10 (59%) of the subjects who had provided acute sera (determined by antibody testing) (Table 7, Acute, "+"). Furthermore, no IFN-γ response was detected in any of the other subjects following convalescence (Table 7, Convalescent), and the levels of IFN-γ in the initial 10 positive patients either decreased significantly (n=4, Table 7, Convalescent, "+") or the IFN-γ response was no longer detectable (n=6), unlike the C6 and Western immunoblot tests for antibodies in the samples.

[0186] The possibility that detectable activated (based on IFN-γ release in response to B. burgdorferi peptide pools) T cells were not present in blood samples collected immediately following antibiotic treatment, coincident with the elimination of spirochetes following treatment for LD, was then explored. To address this question, the average IFN-γ reactivity detected immediately prior to antibiotic treatment (see Table 7, "IFN-γ pre") was compared to the average IFN-γ reactivity detected following treatment (see Table 7, "IFN-γ post"). It was observed that the average reactivity decreased significantly (p value = 0.0002), coincident with the administration of treatment and the clinical resolution of symptoms. These results provide strong evidence that quantification of IFN-γ production by T cells in secondary in vitro responses to pools of B. burgdorferi peptides disclosed herein is advantageous for confirming the disease during the early stages of LD. Furthermore, the rapid decrease in activated T cells, coincident with the successful elimination of spirochetes by antibiotic treatment, was confirmed by the decrease in levels of detectable IFN-γ in test incubation mixtures produced in vitro by T cells collected following antibiotic treatment in response to B. burgdorferi peptide pools described herein. Figure 1 Figure 1

[0187] Table 7. IFN-γ detection in LD patients confirmed following T cell activation with peptide pools.

[0188] ​​

[0189]

[0190] The various embodiments described can also be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ various patents, applications, and publications to provide yet further embodiments.

[0191] Embodiments of the present application include, but are not limited to:

[0192] 1. A composition for the diagnosis or prognosis of Lyme disease selected from a first composition and a second composition:

[0193] (I) the first composition comprising:

[0194] (a) 1, 2, 3, 4, or 5 isolated FlaB peptides each comprising a Borrelia T-cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequence set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1-5;

[0195] (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides each comprising a Borrelia T-cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequence set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 6-18;

[0196] (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides each comprising a Borrelia T-cell epitope and selected from the group consisting of p66 peptides having the amino acid sequence set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 19-31; and

[0197] (d) 1 or 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides having the amino acid sequence set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 32-33,

[0198] wherein the composition, upon contact with whole blood obtained from a subject infected with a B. burgdorferi species associated with Lyme disease, is capable of eliciting a secondary in vitro immune response by T cells; and

[0199] (II) a second composition comprising:

[0200] (a) 5 isolated FlaB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequence set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1-5;

[0201] (b) 13 isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequence set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 6-18;

[0202] (c) 13 isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of p66 peptides having the amino acid sequence set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 19-31; and

[0203] (d) 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides having the amino acid sequence set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 32-33.

[0204] 2. The composition of embodiment 1, wherein at least one:

[0205] (a) the composition comprises at least about 1 nanogram of each peptide and no more than about 100 nanograms of each peptide,

[0206] (b) the composition comprises at least about 100, 200, 300, or 400 nanograms and no more than about 500 nanograms of each peptide,

[0207] (c) the composition comprises at least about 500, 600, 700, 800, or 900 ng and no more than about 1000 ng of each peptide,

[0208] (d) the composition comprises at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 μg and no more than about 2 μg of each peptide, or

[0209] (e) the composition comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9 μg and no more than about 10 μg of each peptide.

[0210] 3. A method for detecting Lyme disease in a subject, or for monitoring the efficacy of a treatment for Lyme disease in a subject, comprising:

[0211] (A) contacting, in vitro, (i) a first biological sample obtained at a first time point from a subject known to have Lyme disease or suspected to be at risk of developing Lyme disease, wherein the biological sample comprises T cells and antigen presenting cells, and (ii) a peptide composition for the diagnosis or prognosis of Lyme disease, to obtain a first test incubation mixture;

[0212] (B) incubating the first test incubation mixture under conditions and for a time sufficient for the T cells to specifically recognize a Borrelia T cell epitope present in the peptide composition to stimulate the production of a T cell immune response indicator; and

[0213] (C) detecting a first level of the T cell immune response indicator in the first test incubation mixture,

[0214] wherein if the first level of the T cell immune response indicator detected in (C) is increased relative to a first control level of the T cell immune response indicator obtained by incubating the first biological sample in a first control incubation in the absence of the peptide composition for the diagnosis or prognosis of Lyme disease, then it is indicative of the presence of a Borrelia infection in the subject, and

[0215] wherein the peptide composition for the diagnosis or prognosis of Lyme disease comprises:

[0216] (a) 1, 2, 3, 4, or 5 isolated FlaB peptides each comprising a Borrelia T cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequence set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1-5;

[0217] (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or 13 isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequence set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 6-18;

[0218] (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or 13 isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of p66 peptides having the amino acid sequence set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 19-31 ; and

[0219] (d) 1 or 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides having the amino acid sequence set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 32-33,

[0220] thereby detecting Lyme disease in the subject, or monitoring efficacy of a treatment for Lyme disease in the subject.

[0221] 4. The method of embodiment 3, wherein the first time point is prior to administering a treatment for Lyme disease to the subject.

[0222] 5. The method of embodiment 3 or embodiment 4, wherein the peptide composition for the diagnosis or prognosis of Lyme disease comprises:

[0223] (a) 5 isolated FlaB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequence set forth in SEQ ID NOs: 1 -5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 -5;

[0224] (b) 13 isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequence set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 6-18;

[0225] (c) 13 isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of p66 peptides having an amino acid sequence as set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 19-31; and

[0226] (d) 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides having an amino acid sequence as set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 32-33.

[0227] 6. The method of embodiment 3 or 4, further comprising:

[0228] (D) contacting in vitro (i) a second biological sample obtained from the subject at a second time point after the first time point and after administration of a treatment for Lyme disease to the subject, wherein the second biological sample comprises T cells and antigen presenting cells, and (ii) the peptide composition for the diagnosis or prognosis of Lyme disease, to obtain a second test incubation mixture;

[0229] (E) incubating the second test incubation mixture under conditions and for a time sufficient to allow the T cells to specifically recognize the B. burgdorferi T cell epitopes present in the peptide composition to stimulate production of a T cell immune response indicator; and

[0230] (F) detecting a second level of the T cell immune response indicator in the second test incubation mixture,

[0231] wherein if the second level of the T cell immune response indicator detected in (F) is increased relative to a second control level of the T cell immune response indicator obtained by incubating the second biological sample in a second control incubation in the absence of the peptide composition for the diagnosis or prognosis of Lyme disease, it is indicative of the presence of a B. burgdorferi infection in the subject, and

[0232] wherein if the second level of the T cell immune response indicator detected in (F) is decreased relative to the first level of the T cell immune response indicator detected in (C), it is indicative of the efficacy of the treatment for Lyme disease.

[0233] 7. The method of embodiment 6, wherein the peptide composition for the diagnosis or prognosis of Lyme disease comprises:

[0234] (a) 5 isolated FlaB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of FlaB peptides having the amino acid sequence set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1-5;

[0235] (b) 13 isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of DbpB peptides having the amino acid sequence set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 6-18;

[0236] (c) 13 isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of p66 peptides having the amino acid sequence set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 19-31; and

[0237] (d) 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides having the amino acid sequence set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 32-33.

[0238] 8. The method of embodiment 3, wherein the Lyme disease comprises an infection by at least one pathogenic B. burgdorferi species.

[0239] 9. The method of embodiment 8, wherein the pathogenic B. burgdorferi species is pathogenic to humans.

[0240] 10. The method of embodiment 9, wherein the B. burgdorferi species pathogenic to humans is selected from the group consisting of Borrelia burgdorferi, Borrelia burgdorferi sensu stricto, Borrelia azfelii, Borrelia garinii, Borrelia valaisiana, Borrelia spielmanii, Borrelia bissettii, Borrelia lusitaniae, and Borrelia bavariensis.

[0241] 11. The method of embodiment 3, wherein the treatment for Lyme disease comprises administering an antibiotic to the subject.

[0242] 12. The method of embodiment 11, wherein the antibiotic is selected from the group consisting of: tetracyclines; oxytetracycline, tetracycline, chlortetracycline, or minocycline; penicillins; amoxicillin or penicillin; cephalosporins; cefaclor, cefoperazone, cefminox, cefotaxime, cefotetan, cefmetazole, cefoxitin, cefuroxime axetil, acetylcefuroxime, carbendazim, or ceftriaxone sodium; macrolides; azithromycin, clarithromycin, or erythromycin.

[0243] 13. The method of embodiment 3, wherein the biological sample comprises at least one of whole blood, cerebrospinal fluid, or synovial fluid.

[0244] 14. The method of embodiment 3, wherein the biological sample comprises at least one of (a) whole blood, (b) a cellular component of whole blood, (c) isolated peripheral blood leukocytes, or (d) isolated peripheral blood mononuclear cells.

[0245] 15. The method of embodiment 3, wherein the T cell immune response indicator is interferon-gamma (IFN-γ).

[0246] 16. The method of embodiment 15, wherein the IFN-γ is soluble IFN-γ released by the T cells.

[0247] 17. The method of embodiment 3, wherein the T cell immune response indicator comprises at least one of T cell proliferation and T cell cytokine expression.

[0248] 18. The method of embodiment 17, wherein the T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

[0249] 19. The method of embodiment 17, wherein the T cell cytokine expression is detected as soluble T cell cytokine released by the T cells.

[0250] 20. The method of embodiment 19, wherein the T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

[0251] 21. The method of embodiment 20, wherein the T cell cytokine is detected by assaying for detectable specific binding of a binding agent to the T cell cytokine.

[0252] 22. The method of embodiment 21, wherein the binding agent comprises at least one antibody that specifically binds to the T cell cytokine.

[0253] 23. The method of embodiment 22, wherein the at least one antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody.

[0254] 24. The method of embodiment 22, wherein the at least one antibody is immobilized on a solid phase.

[0255] 25. The method of embodiment 6, wherein the Lyme disease comprises an infection by at least one pathogenic Borrelia species.

[0256] 26. The method of embodiment 25, wherein the pathogenic Borrelia species is pathogenic to humans.

[0257] 27. The method of embodiment 26, wherein the Borrelia species pathogenic to humans is selected from the group consisting of Borrelia burgdorferi, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia spielmanii, Borrelia bissettii, Borrelia lusitaniae, and Borrelia bavariensis.

[0258] 28. The method of embodiment 6, wherein the treatment for Lyme disease comprises administering to the subject an antibiotic.

[0259] 29. The method of embodiment 28, wherein the antibiotic is selected from the group consisting of tetracyclines; oxytetracycline, tetracycline, multicitracycline, or minocycline; penicillins; amoxicillin or penicillin; cephalosporins; cefaclor, cefoperazone, cefminox, cefotaxime, cefotetan, cefmetazole, cefoxitin, cefuroxime axetil, acetylcefuroxime, carbendazim, or ceftriaxone sodium; macrolides; azithromycin, clarithromycin, or erythromycin.

[0260] 30. The method of embodiment 6, wherein the biological sample comprises at least one of whole blood, cerebrospinal fluid, or synovial fluid.

[0261] 31. The method of embodiment 6, wherein the biological sample comprises at least one of (a) whole blood, (b) a cellular fraction of whole blood, (c) isolated peripheral blood leukocytes, or (d) isolated peripheral blood mononuclear cells.

[0262] 32. The method of embodiment 6, wherein the T cell immune response indicator is interferon-γ (IFN-γ).

[0263] 33. The method of embodiment 32, wherein the IFN-γ is soluble IFN-γ released by the T cells.

[0264] 34. The method of embodiment 6, wherein the T cell immune response indicator comprises at least one of T cell proliferation and T cell cytokine expression.

[0265] 35. The method of embodiment 34, wherein the T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

[0266] 36. The method of embodiment 34, wherein the T cell cytokine expression is detected as soluble T cell cytokine released by the T cells.

[0267] 37. The method of embodiment 36, wherein the T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

[0268] 38. The method of embodiment 37, wherein the T cell cytokine is detected by assaying a binding agent for detectable specific binding to the T cell cytokine.

[0269] 39. The method of embodiment 38, wherein the binding agent comprises at least one antibody that specifically binds to the T cell cytokine.

[0270] 40. The method of embodiment 39, wherein the at least one antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody.

[0271] 41. The method of embodiment 39, wherein the at least one antibody is immobilized on a solid phase.

[0272] 42. The method of embodiment 6, comprising repeating steps (D), (E), and (F) at a plurality of second time points, which are different from each other and later than the first time point, and after administration of the treatment for Lyme disease to the subject.

[0273] 43. The method of embodiment 42, wherein the plurality of second time points comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 time points.

[0274] 44. A composition selected from the group consisting of a first nucleic acid composition and a second nucleic acid composition:

[0275] (I) a first nucleic acid composition comprising one or more isolated nucleic acid molecules encoding:

[0276] (a) 1, 2, 3, 4, or 5 isolated FlaB peptides, each comprising a Borrelia T-cell epitope, and selected from the group consisting of FlaB peptides having the amino acid sequences set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-5;

[0277] (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides, each comprising a Borrelia T-cell epitope, and selected from the group consisting of DbpB peptides having the amino acid sequences set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 6-18;

[0278] (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides, each comprising a Borrelia T-cell epitope, and selected from the group consisting of p66 peptides having the amino acid sequences set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOs: 19-31; and

[0279] (d) 1 or 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from OspC peptides having an amino acid sequence as set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 32-33,

[0280] wherein the FlaB, DbpB, p66, and OspC peptides are capable of eliciting a secondary in vitro immune response by T cells after contact with whole blood obtained from a subject infected with a B. burgdorferi species associated with Lyme disease.

[0281] (II) a second nucleic acid composition comprising one or more isolated nucleic acid molecules encoding:

[0282] (a) 5 isolated FlaB peptides each comprising a B. burgdorferi T cell epitope and selected from FlaB peptides having an amino acid sequence as set forth in SEQ ID NOs: 1-5, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 1-5;

[0283] (b) 13 isolated DbpB peptides each comprising a B. burgdorferi T cell epitope and selected from DbpB peptides having an amino acid sequence as set forth in SEQ ID NOs: 6-18, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 6-18;

[0284] (c) 13 isolated p66 peptides each comprising a B. burgdorferi T cell epitope and selected from p66 peptides having an amino acid sequence as set forth in SEQ ID NOs: 19-31, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 19-31; and

[0285] (d) 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from OspC peptides having an amino acid sequence as set forth in SEQ ID NOs: 32-33, or one or more variants thereof having at least 80% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 32-33,

[0286] wherein the FlaB, DbpB, p66, and OspC peptides are capable of eliciting a secondary in vitro immune response by T cells after contact with whole blood obtained from a subject infected with a B. burgdorferi species associated with Lyme disease.

[0287] 45. A vector composition comprising one or more nucleic acid vectors comprising the composition of embodiment 44.

[0288] 46. A host cell comprising the vector composition of embodiment 45.

[0289] These and other changes can be made to the embodiments in light of the above -detailed description. In general, the terms used in the claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments that would be resolved to the same scope of the claims. Accordingly, the claims are not limited to the disclosure. SEQUENCE LIST <110> Qiagen Sciences LLC Biopeptides Corp. Gundersen Lutheran Medical Foundation, Inc. <120> Compositions and methods for diagnosing Lyme disease and for predicting elimination of Lyme disease spirochetes after treatment <130> 770025.466WO <140> PCT <141> 2016-09-15 <150> US 62 / 233,192 <151> 2015-09-25 <160> 40 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 25 <212> PRT <213> Borrelia sp. <400> 1 Asn Val Arg Thr Ala Glu Glu Leu Gly Met Gln Pro Ala Lys Ile Asn 1 5 10 15 Thr Pro Ala Ser Leu Ser Gly Ser Gln 20 25 <210> 2 <211> 20 <212> PRT <213> Borrelia sp. <400> 2 Asn Thr Pro Ala Ser Leu Ser Gly Ser Gln Ala Ser Trp Thr Leu Arg 1 5 10 15 Val His Val Gly 20 <210> 3 <211> 20 <212> PRT <213> Borrelia sp. <400> 3 Leu Ser Gly Ser Gln Ala Ser Trp Thr Leu Arg Val His Val Gly Ala 1 5 10 15 Asn Gln Asp Glu 20 <210> 4 <211> 20 <212> PRT <213> Borrelia sp. <400> 4 Gln Asp Glu Ala Ile Ala Val Asn Ile Tyr Ala Ala Asn Val Ala Asn 1 5 10 15 Leu Phe Ser Gly 20 <210> 5 <211> 19 <212> PRT <213> Borrelia sp. <400> 5 Ser Leu Ala Lys Ile Glu Asn Ala Ile Arg Met Ile Ser Asp Gln Arg 1 5 10 15 Ala Asn Leu <210> 6 <211> 20 <212> PRT <213> Borrelia sp. <400> 6 Ser Ile Val Met Val Leu Phe Phe Asp Leu Leu Val Ala Cys Ser Ile 1 5 10 15 Gly Leu Val Glu 20 <210> 7 <211> 20 <212> PRT <213> Borrelia sp. <400> 7 Leu Phe Phe Asp Leu Leu Val Ala Cys Ser Ile Gly Leu Val Glu Arg 1 5 10 15 Thr Asn Ala Ala 20 <210> 8 <211> 25 <212> PRT <213> Borrelia sp. <400> 8 Ile Gly Leu Val Glu Arg Thr Asn Ala Ala Leu Glu Ser Ser Ser Lys 1 5 10 15 Asp Leu Lys Asn Lys Ile Leu Lys Ile 20 25 <210> 9 <211> 25 <212> PRT <213> Borrelia sp. <400> 9 Lys Asp Leu Lys Asn Lys Ile Leu Lys Ile Lys Lys Glu Ala Thr Gly 1 5 10 15 Lys Gly Val Leu Phe Glu Ala Phe Thr 20 25 <210> 10 <211> 25 <212> PRT <213> Borrelia sp. <400> 10 Gly Lys Gly Val Leu Phe Glu Ala Phe Thr Gly Leu Lys Thr Gly Ser 1 5 10 15 Lys Val Thr Ser Gly Gly Leu Ala Leu 20 25 <210> 11 <211> 25 <212> PRT <213> Borrelia sp. <400> 11 Ser Lys Val Thr Ser Gly Gly Leu Ala Leu Arg Glu Ala Lys Val Gln 1 5 10 15 Ala Ile Val Glu Thr Gly Lys Phe Leu 20 25 <210> 12 <211> 25 <212> PRT <213> Borrelia sp. <400> 12 Gln Ala Ile Val Glu Thr Gly Lys Phe Leu Lys Ile Ile Glu Glu Glu 1 5 10 15 Ala Leu Lys Leu Lys Glu Thr Gly Asn 20 25 <210> 13 <211> 25 <212> PRT <213> Borrelia sp. <400> 13 Glu Ala Leu Lys Leu Lys Glu Thr Gly Asn Ser Gly Gln Phe Leu Ala 1 5 10 15 Met Phe Asp Leu Met Leu Glu Val Val 20 25 <210> 14 <211> 25 <212> PRT <213> Borrelia sp. <400> 14 Ala Met Phe Asp Leu Met Leu Glu Val Val Glu Ser Leu Glu Asp Val 1 5 10 15 Gly Ile Ile Gly Leu Lys Ala Arg Val 20 25 <210> 15 <211> 25 <212> PRT <213> Borrelia sp. <400> 15 Val Gly Ile Ile Gly Leu Lys Ala Arg Val Leu Glu Glu Ser Lys Asn 1 5 10 15 Asn Pro Ile Asn Thr Ala Glu Arg Leu 20 25 <210> 16 <211> 25 <212> PRT <213> Borrelia sp. <400> 16 Asn Asn Pro Ile Asn Thr Ala Glu Arg Leu Leu Ala Ala Lys Ala Gln 1 5 10 15 Ile Glu Asn Gln Leu Lys Val Val Lys 20 25 <210> 17 <211> 25 <212> PRT <213> Borrelia sp. <400> 17 Gln Ile Glu Asn Gln Leu Lys Val Val Lys Glu Lys Gln Asn Ile Glu 1 5 10 15 Asn Gly Gly Glu Lys Lys Asn Asn Lys 20 25 <210> 18 <211> 25 <212> PRT <213> Borrelia sp. <400> 18 Leu Lys Val Val Lys Glu Lys Gln Asn Ile Glu Asn Gly Gly Glu Lys 1 5 10 15 Lys Asn Asn Lys Ser Lys Lys Lys Lys 20 25 <210> 19 <211> 25 <212> PRT <213> Borrelia sp. <400> 19 Phe Gly Leu Ser Gly Ala Tyr Gly Asn Glu Thr Phe Asn Asn Ser Ser 1 5 10 15 Ile Thr Tyr Ser Leu Lys Asp Lys Ser 20 25 <210> 20 <211> 25 <212> PRT <213> Borrelia sp. <400> 20 Ser Ile Thr Tyr Ser Leu Lys Asp Lys Ser Val Val Gly Asn Asp Leu 1 5 10 15 Leu Ser Pro Thr Leu Ser Asn Ser Ala 20 25 <210> twenty one <211> 25 <212> PRT <213> Borrelia sp. <400> twenty one Leu Leu Ser Pro Thr Leu Ser Asn Ser Ala Ile Leu Ala Ser Phe Gly 1 5 10 15 Ala Lys Tyr Lys Leu Gly Leu Thr Lys 20 25 <210> twenty two <211> 20 <212> PRT <213> Borrelia sp. <400> twenty two Gly Ala Lys Tyr Lys Leu Gly Leu Thr Lys Ile Asn Asp Lys Asn Thr 1 5 10 15 Tyr Leu Ile Leu 20 <210> twenty three <211> 20 <212> PRT <213> Borrelia sp. <400> twenty three Leu Gly Leu Thr Lys Ile Asn Asp Lys Asn Thr Tyr Leu Ile Leu Gln 1 5 10 15 Met Gly Thr Asp 20 <210> 24 <211> 20 <212> PRT <213> Borrelia sp. <400> 24 Thr Tyr Leu Ile Leu Gin Met Gly Thr Asp Phe Gly Ile Asp Pro Phe 1 5 10 15 Ala Ser Asp Phe 20 <210> 25 <211> 20 <212> PRT <213> Borrelia sp. <400> 25 Gln Met Gly Thr Asp Phe Gly Ile Asp Pro Phe Ala Ser Asp Phe Ser 1 5 10 15 Ile Phe Gly His 20 <210> 26 <211> 25 <212> PRT <213> Borrelia sp. <400> 26 Phe Ala Ser Asp Phe Ser Ile Phe Gly His Ile Ser Lys Ala Ala Asn 1 5 10 15 Phe Lys Lys Glu Thr Pro Ser Asp Pro 20 25 <210> 27 <211> 25 <212> PRT <213> Borrelia sp. <400> 27 Asn Phe Lys Lys Glu Thr Pro Ser Asp Pro Asn Lys Lys Ala Glu Ile 1 5 10 15 Phe Asp Pro Asn Gly Asn Ala Leu Asn 20 25 <210> 28 <211> 25 <212> PRT <213> Borrelia sp. <400> 28 Ile Phe Asp Pro Asn Gly Asn Ala Leu Asn Phe Ser Lys Asn Thr Glu 1 5 10 15 Leu Gly Ile Ala Phe Ser Thr Gly Ala 20 25 <210> 29 <211> 20 <212> PRT <213> Borrelia sp. <400> 29 Glu Leu Gly Ile Ala Phe Ser Thr Gly Ala Ser Ile Gly Phe Ala Trp 1 5 10 15 Asn Lys Asp Thr 20 <210> 30 <211> 20 <212> PRT <213> Borrelia sp. <400> 30 Phe Ser Thr Gly Ala Ser Ile Gly Phe Ala Trp Asn Lys Asp Thr Gly 1 5 10 15 Glu Lys Glu Ser 20 <210> 31 <211> 25 <212> PRT <213> Borrelia sp. <400> 31 Phe Ser Thr Gly Ala Ser lie Gly Phe Ala Trp Asn Lys Asp Thr Gly 1 5 10 15 Glu Lys Glu Ser Trp Ala lie Lys Gly 20 25 <210> 32 <211> 15 <212> PRT <213> Borrelia sp. <400> 32 Lys Glu Met Leu Ala Asn Ser Val Lys Glu Leu Thr Ser Pro lie 1 5 10 15 <210> 33 <211> 21 <212> PRT <213> Borrelia sp. <400> 33 Ala Thr Lys Ala lie Gly Lys Lys lie Gin Gin Asn Gly Gly Leu Ala 1 5 10 15 Val Glu Ala Gly His 20 <210> 34 <211> 40 <212> PRT <213> Borrelia burgdorferi <400> 34 Asn Val Arg Thr Ala Glu Glu Leu Gly Met Gin Pro Ala Lys lie Asn 1 5 10 15 Thr Pro Ala Ser Leu Ser Gly Ser Gin Ala Ser Trp Thr Leu Arg Val 20 25 30 His Val Gly Ala Asn Gin Asp Glu 35 40 <210> 35 <211> 20 <212> PRT <213> Borrelia burgdorferi <400> 35 Gln Asp Glu Ala lie Ala Val Asn lie Tyr Ala Ala Asn Val Ala Asn 1 5 10 15 Leu Phe Ser Gly 20 <210> 36 <211> 19 <212> PRT <213> Borrelia burgdorferi <400> 36 Ser Leu Ala Lys lie Glu Asn Ala lie Arg Met lie Ser Asp Gin Arg 1 5 10 15 Ala Asn Leu <210> 37 <211> 180 <212> PRT <213> Borrelia burgdorferi <400> 37 Ser lie Val Met Val Leu Phe Phe Asp Leu Leu Val Ala Cys Ser lie 1 5 10 15 Gly Leu Val Glu Arg Thr Asn Ala Ala Leu Glu Ser Ser Ser Lys Asp 20 25 30 Leu Lys Asn Lys lie Leu Lys lie Lys Lys Glu Ala Thr Gly Lys Gly 35 40 45 Val Leu Phe Glu Ala Phe Thr Gly Leu Lys Thr Gly Ser Lys Val Thr 50 55 60 Ser Gly Gly Leu Ala Leu Arg Glu Ala Lys Val Gin Ala lie Val Glu 65 70 75 80 Thr Gly Lys Phe Leu Lys lie lie Glu Glu Glu Ala Leu Lys Leu Lys 85 90 95 Glu Thr Gly Asn Ser Gly Gin Phe Leu Ala Met Phe Asp Leu Met Leu 100 105 110 Glu Val Val Glu Ser Leu Glu Asp Val Gly lie lie Gly Leu Lys Ala 115 120 125 Arg Val Leu Glu Glu Ser Lys Asn Asn Pro lie Asn Thr Ala Glu Arg 130 135 140 Leu Leu Ala Ala Lys Ala Gin lie Glu Asn Gin Leu Lys Val Val Lys 145 150 155 160 Glu Lys Gin Asn lie Glu Asn Gly Gly Glu Lys Lys Asn Asn Lys Ser 165 170 175 Lys Lys Lys Lys 180 <210> 38 <211> 150 <212> PRT <213> Borrelia burgdorferi <400> 38 Phe Gly Leu Ser Gly Ala Tyr Gly Asn Glu Thr Phe Asn Asn Ser Ser 1 5 10 15 Ile Thr Tyr Ser Leu Lys Asp Lys Ser Val Val Gly Asn Asp Leu Leu 20 25 30 Ser Pro Thr Leu Ser Asn Ser Ala Ile Leu Ala Ser Phe Gly Ala Lys 35 40 45 Tyr Lys Leu Gly Leu Thr Lys Ile Asn Asp Lys Asn Thr Tyr Leu Ile 50 55 60 Leu Gln Met Gly Thr Asp Phe Gly Ile Asp Pro Phe Ala Ser Asp Phe 65 70 75 80 Ser Ile Phe Gly His Ile Ser Lys Ala Ala Asn Phe Lys Lys Glu Thr 85 90 95 Pro Ser Asp Pro Asn Lys Lys Ala Glu Ile Phe Asp Pro Asn Gly Asn 100 105 110 Ala Leu Asn Phe Ser Lys Asn Thr Glu Leu Gly Ile Ala Phe Ser Thr 115 120 125 Gly Ala Ser Ile Gly Phe Ala Trp Asn Lys Asp Thr Gly Glu Lys Glu 130 135 140 Ser Trp Ala Ile Lys Gly 145 150 <210> 39 <211> 21 <212> PRT <213> Borrelia burgdorferi <400> 39 Ala Thr Lys Ala lie Gly Lys Lys lie Gin Gin Asn Gly Gly Leu Ala 1 5 10 15 Val Glu Ala Gly His 20 <210> 40 <211> 15 <212> PRT <213> Borrelia burgdorferi <400> 40 Lys Glu Met Leu Ala Asn Ser Val Lys Glu Leu Thr Ser Pro lie 1 5 10 15

Claims

1. A composition for the diagnosis or prognosis of Lyme disease, comprising: (a) 1, 2, 3, 4, or 5 isolated FlaB peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 1 or 2 isolated OspC peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 32-33; wherein said composition being capable of eliciting a secondary in vitro immune response by T cells after contact with whole blood obtained from a subject infected with a Borrelia species associated with Lyme disease.

2. The composition of claim 1, comprising at least 1 nanogram of each peptide and no more than 100 nanograms of each peptide.

3. The composition of claim 1, comprising at least 100 nanograms and no more than 500 nanograms of each peptide.

4. The composition of claim 1, comprising at least 200 nanograms and no more than 500 nanograms of each peptide.

5. The composition of claim 1, comprising at least 300 nanograms and no more than 500 nanograms of each peptide.

6. The composition of claim 1, comprising at least 400 nanograms and no more than 500 nanograms of each peptide.

7. The composition of claim 1, comprising at least 500 nanograms and no more than 1000 nanograms of each peptide.

8. The composition of claim 1, comprising at least 600 nanograms and no more than 1000 nanograms of each peptide.

9. The composition of claim 1, comprising at least 700 nanograms and no more than 1000 nanograms of each peptide.

10. The composition of claim 1, comprising at least 800 nanograms and no more than 1000 nanograms of each peptide.

11. The composition of claim 1, comprising at least 900 nanograms and no more than 1000 nanograms of each peptide.

12. The composition of claim 1, comprising at least 1.0 microgram and no more than 2 micrograms of each peptide.

13. The composition of claim 1, comprising at least 1.1 microgram and no more than 2 micrograms of each peptide.

14. The composition of claim 1, comprising at least 1.2 microgram and no more than 2 micrograms of each peptide.

15. The composition of claim 1, comprising at least 1.3 microgram and no more than 2 micrograms of each peptide.

16. The composition of claim 1, comprising at least 1.4 micrograms and no more than 2 micrograms of each peptide.

17. The composition of claim 1, comprising at least 1.5 micrograms and no more than 2 micrograms of each peptide.

18. The composition of claim 1, comprising at least 1.6 micrograms and no more than 2 micrograms of each peptide.

19. The composition of claim 1, comprising at least 1.7 micrograms and no more than 2 micrograms of each peptide.

20. The composition of claim 1, comprising at least 1.8 micrograms and no more than 2 micrograms of each peptide.

21. The composition of claim 1, comprising at least 1.9 micrograms and no more than 2 micrograms of each peptide.

22. The composition of claim 1, comprising at least 1 microgram and no more than 10 micrograms of each peptide.

23. The composition of claim 1, comprising at least 2 micrograms and no more than 10 micrograms of each peptide.

24. The composition of claim 1, comprising at least 3 micrograms and no more than 10 micrograms of each peptide.

25. The composition of claim 1, comprising at least 4 micrograms and no more than 10 micrograms of each peptide.

26. The composition of claim 1, comprising at least 5 micrograms and no more than 10 micrograms of each peptide.

27. The composition of claim 1, comprising at least 6 micrograms and no more than 10 micrograms of each peptide.

28. The composition of claim 1, comprising at least 7 micrograms and no more than 10 micrograms of each peptide.

29. The composition of claim 1, comprising at least 8 micrograms and no more than 10 micrograms of each peptide.

30. The composition of claim 1, comprising at least 9 micrograms and no more than 10 micrograms of each peptide.

31. Use of a peptide composition for the manufacture of a product for detecting Lyme disease in a subject or for monitoring the efficacy of a treatment against Lyme disease in a subject, wherein, The peptide composition comprises: (a) 1, 2, 3, 4, or 5 isolated FlaB peptides, each comprising a Borrelia T-cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides, each comprising a Borrelia T-cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides, each comprising a Borrelia T-cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 1 or 2 isolated OspC peptides, each comprising a Borrelia T-cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 32-33; the detecting or monitoring comprises: (A) contacting in vitro (i) a first biological sample, which is obtained at a first time point from a subject known to have Lyme disease or suspected to be at risk of having Lyme disease, wherein the biological sample comprises T cells and antigen presenting cells, and (ii) the peptide composition, to obtain a first test incubation mixture; (B) incubating the first test incubation mixture under conditions and for a time sufficient to allow the T cells to specifically recognize the Borrelia T cell epitopes present in the peptide composition to stimulate production of a T cell immune response indicator; and (C) detecting a first level of the T cell immune response indicator in the first test incubation mixture, wherein if the first level of the T cell immune response indicator detected in (C) is increased relative to a first control level of the T cell immune response indicator, which is obtained by incubating the first biological sample in a first control incubation in the absence of the peptide composition, then the presence of a Borrelia infection in the subject is indicated.

32. The use of claim 31, wherein, The first time point is prior to administering a treatment for Lyme disease to the subject.

33. The use of claim 31 or claim 32, wherein, The peptide composition for the diagnosis or prognosis of Lyme disease comprises: (a) 5 isolated FlaB peptides each comprising a Borrelia T cell epitope and selected from the group consisting of FlaB peptides of the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 13 isolated DbpB peptides each comprising a Borrelia T cell epitope and selected from the group consisting of DbpB peptides of the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 13 isolated p66 peptides each comprising a Borrelia T cell epitope and selected from the group consisting of p66 peptides of the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 2 isolated OspC peptides each comprising a Borrelia T cell epitope and selected from the group consisting of OspC peptides of the amino acid sequences set forth in SEQ ID NOs: 32-33.

34. The use of claim 31 or 32, further comprising: (D) contacting in vitro (i) a second biological sample, which is obtained at a second time point after the first time point and after administering the treatment for Lyme disease to the subject, wherein the second biological sample comprises T cells and antigen presenting cells, and (ii) the peptide composition for the diagnosis or prognosis of Lyme disease, to obtain a second test incubation mixture; (E) incubating the second test incubation mixture under conditions and for a time sufficient to allow the T cells to specifically recognize the Borrelia T cell epitopes present in the peptide composition to stimulate production of a T cell immune response indicator; and (F) detecting a second level of the T cell immune response indicator in the second test incubation mixture, ​ wherein if the second level of the T cell immune response indicator detected in (F) is increased relative to a second control level of the T cell immune response indicator, the second control level obtained by incubating a second biological sample in a second control incubation in the absence of the peptide composition for the diagnosis or prognosis of Lyme disease, then the presence of a Borrelia infection in the subject is indicated, and wherein if the second level of the T cell immune response indicator detected in (F) is decreased relative to the first level of the T cell immune response indicator detected in (C), then the efficacy of a treatment for Lyme disease is indicated.

35. The use of claim 34, wherein, The peptide composition for the diagnosis or prognosis of Lyme disease comprises: (a) 5 isolated FlaB peptides each comprising a Borrelia T cell epitope and selected from the group consisting of FlaB peptides of the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 13 isolated DbpB peptides each comprising a Borrelia T cell epitope and selected from the group consisting of DbpB peptides of the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 13 isolated p66 peptides each comprising a Borrelia T cell epitope and selected from the group consisting of p66 peptides of the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 2 isolated OspC peptides each comprising a Borrelia T cell epitope and selected from the group consisting of OspC peptides of the amino acid sequences set forth in SEQ ID NOs: 32-33.

36. The use of claim 31, wherein, Lyme disease comprises an infection by at least one pathogenic Borrelia species.

37. The use of claim 36, wherein, The pathogenic Borrelia species is pathogenic to humans.

38. The use of claim 37, wherein, The Borrelia species pathogenic to humans is selected from the group consisting of: Borrelia burgdorferi, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia spielmanii, Borrelia bissettii, Borrelia lusitaniae, and Borrelia bavariensis.

39. The use of claim 31, wherein, The treatment for Lyme disease comprises administering to the subject an antibiotic.

40. The use of claim 39, wherein, The antibiotic is selected from the group consisting of: tetracyclines, penicillins, cephalosporins, and macrolides.

41. The use of claim 39, wherein, The antibiotic is selected from the group consisting of: oxytetracycline, tetracycline, polytrimycin, minocycline, amoxicillin, penicillin, cefaclor, cefoperazone, cefminox, cefotaxime, cefotetan, cefmetazole, cefoxitin, cefuroxime axetil, acetylcefuroxime, enilconazole, ceftriaxone, azithromycin, clarithromycin, and erythromycin.

42. The use of claim 31, wherein, The biological sample comprises at least one of whole blood, cerebrospinal fluid, or synovial fluid.

43. The use of claim 31, wherein, The biological sample comprises at least one of (a) whole blood and (b) a cellular component of whole blood.

44. The use of claim 31, wherein, The biological sample comprises isolated peripheral blood leukocytes.

45. The use of claim 31, wherein, The biological sample comprises isolated peripheral blood mononuclear cells.

46. The use of claim 31, wherein, The T cell immune response indicator is IFN-γ.

47. The use of claim 46, wherein the IFN-γ is soluble IFN-γ released by the T cells.

48. The use of claim 31, wherein, The T cell immune response indicator comprises at least one of T cell proliferation and T cell cytokine expression.

49. The use of claim 48, wherein, The T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

50. The use of claim 48, wherein, The T cell cytokine expression is detected as soluble T cell cytokine released by the T cells.

51. The use of claim 50, wherein, The T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

52. The use of claim 51, wherein, The T cell cytokine is detected by assaying a binding agent for detectable specific binding to the T cell cytokine.

53. The use of claim 52, wherein, The binding agent comprises at least one antibody that specifically binds to the T cell cytokine.

54. The use of claim 53, wherein, The at least one antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody.

55. The use of claim 53, wherein, The at least one antibody is immobilized on a solid phase.

56. The use of claim 34, wherein, Lyme disease comprises an infection by at least one pathogenic Borrelia species.

57. The use of claim 56, wherein, The pathogenic Borrelia species is pathogenic to humans.

58. The use of claim 57, wherein, The Borrelia species pathogenic to humans is selected from the group consisting of Borrelia burgdorferi, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia spielmanii, Borrelia bissettii, Borrelia lusitaniae, and Borrelia bavariensis.

59. The use of claim 34, wherein, The treatment for Lyme disease comprises administering to the subject an antibiotic.

60. The use of claim 59, wherein, The antibiotic is selected from the group consisting of tetracyclines, penicillins, cephalosporins, and macrolides.

61. The use of claim 59, wherein, The antibiotic is selected from the group consisting of oxytetracycline, tetracycline, polytrimycin, minocycline, amoxicillin, penicillin, cefaclor, cefoperazone, cefminox, cefotaxime, cefotetan, cefmetazole, cefoxitin, cefuroxime axetil, acetylcefuroxime, enilconazole, ceftriaxone, azithromycin, clarithromycin, and erythromycin.

62. The use of claim 34, wherein, The biological sample comprises at least one of whole blood, cerebrospinal fluid, or synovial fluid.

63. The use of claim 34, wherein, The biological sample comprises at least one of: (a) whole blood and (b) a cellular component of whole blood.

64. The use of claim 34, wherein, The biological sample comprises isolated peripheral blood leukocytes.

65. The use of claim 34, wherein, The biological sample comprises isolated peripheral blood mononuclear cells.

66. The use of claim 34, wherein, The T cell immune response indicator is IFN-γ.

67. The use of claim 66, wherein the IFN-γ is soluble IFN-γ released by the T cells.

68. The use of claim 34, wherein, The T cell immune response indicator comprises at least one of: T cell proliferation and T cell cytokine expression.

69. The use of claim 68, wherein, The T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

70. The use of claim 68, wherein, The T cell cytokine expression is detected as soluble T cell cytokine released by the T cells.

71. The use of claim 70, wherein, The T cell cytokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-10, IL-12, IL-17, TNF-α, TNF-β, and IFN-γ.

72. The use of claim 71, wherein, The T cell cytokine is detected by assaying a binding agent for detectable specific binding to the T cell cytokine.

73. The use of claim 72, wherein, The binding agent comprises at least one antibody that specifically binds to the T cell cytokine.

74. The use of claim 73, wherein, The at least one antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody.

75. The use of claim 73, wherein, The at least one antibody is immobilized on a solid phase.

76. The use of claim 34, comprising repeating steps (D), (E), and (F) at a plurality of second time points, which are different from each other and later than the first time point, and after administration of the treatment for Lyme disease to the subject.

77. The use of claim 76, wherein, The plurality of second time points comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 time points.

78. A nucleic acid composition comprising isolated nucleic acid molecules encoding one or more of: (a) 1, 2, 3, 4, or 5 isolated FlaB peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1-5; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated DbpB peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 6-18; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p66 peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 19-31; and (d) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 isolated p39 peptides, each comprising a Borrelia T cell epitope and selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 32-44. ​ ​ ​ (d) 1 or 2 isolated OspC peptides each comprising a B. burgdorferi T cell epitope and selected from the group consisting of OspC peptides of the amino acid sequences set forth in SEQ ID NOs: 32-33; wherein, said FlaB, DbpB, p66 and OspC peptides are capable of eliciting a secondary in vitro immune response by T cells after contact with whole blood obtained from a subject infected with a B. burgdorferi species associated with Lyme disease.

79. A vector composition comprising one or more nucleic acid vectors comprising the composition of claim 78.

80. A host cell comprising the vector composition of claim 79.

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