Species-specific antigenic sequences of tick-borne relapsing fever (TBRF) and their uses

Abstract

The present disclosure provides, in some aspects, antigen-specific amino acid sequences of tick-borne relapsing fever Borrelia spp.

Description

[Technical field]

[0001] Aspects of the present invention provide novel compositions and methods for diagnosing tick-borne relapsing fever (TBRF), which results from infection with various TBRF Borrelia species.

[0002] Sequence Listing This application incorporates by reference a sequence listing, which is an ASCII text file with the filename 0153-2015WO01_SL.txt, a creation date of June 17, 2022, and a file size of 27,349 bytes. [Background technology]

[0003] Borreliosis is caused by two groups of Borrelia, the B. burgdorferi group and the tick-borne relapsing fever (TBRF) group (also referred to herein as RFB (relapsing fever Borrelia)). The B. burgdorferi group was once thought to be the only group that caused Lyme-like symptoms in infected subjects, but it is now known that TBRF Borrelia also cause Lyme-like symptoms. Several Borrelia species cause TBRF, and these are usually associated with specific species of ticks. For example, B. hermsii is transmitted by Ornithodorus hermsi ticks, B. parkerii is transmitted by O. parkeri ticks, and B. turicatae is transmitted by O. turicata ticks. Each tick species has a primary habitat and a primary set of hosts. Typically, hard ticks (Ixodes) transmit Lyme disease pathogens, while soft ticks (Ornithodorus) transmit TBRF pathogens, but there is an expected rule: some RFB pathogens are transmitted only by hard ticks (e.g., B. miyamotoi), and one species, B. recurrentis, is louse-borne.

[0004] The main symptoms of TBRF are high fever (e.g., 103° F), headache, and muscle and joint pain. Symptoms can be recurrent, resulting in a pattern of fever lasting approximately 3 days, followed by 7 days without fever, followed by another 3 days of fever. Without antibiotic treatment, this process can be repeated multiple times.

[0005] Currently, the standard of detection is by identification of TBRF spirochetes in blood smears from subjects exhibiting symptoms consistent with TBRF. After blood is drawn, the sample must be cultured for at least 24 hours to facilitate identification. However, even in the early stages of the disease, when spirochetes are most prevalent, positive identification is only achieved about 70% of the time (see Non-Patent Document 1). Thus, current materials and methods in the field result in delayed diagnoses and provide relatively low levels of sensitivity and specificity. There is a need for new materials and methods suitable for identifying TBRF causative pathogens with reduced assay times and improved sensitivity and specificity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] www.cdc.gov / relapsing-fever / clinicians / index.html Summary of the Invention

[0007] The present invention relates, in part, to compositions and methods that can be used to identify antibodies to infection by one or more TBRF Borrelia species in a sample from a subject suspected of having TBRF. Using the compositions and methods of the present invention, it is possible to identify TBRF Borrelia in a subject sample with greater speed, sensitivity, and specificity than with existing compositions and methods. The antigen-specific amino acid sequences of the present invention can be used in diagnostic and academic assays. Non-limiting examples of suitable assays include immunoblots, ELISA (enzyme-linked immunosorbent assay), and the like. The amino acid sequences of the present invention can be used to detect TBRF Borrelia-specific T cells (e.g., IgXSPOT test; IGeneX, Palo Alto, CA).

[0008] According to certain aspects of the invention, compositions are provided comprising one or more labeled and / or tagged and / or bound amino acid sequences, the one or more labeled and / or tagged and / or bound amino acid sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and variants thereof that retain the immunological binding profile of FIG. 3. In some embodiments, the composition also comprises an amino acid sequence bound to a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride (PVDF), magnetic beads, and agarose. In some embodiments, each of the one or more amino acid sequences is tagged with an antibody having specificity for the amino acid sequence.

[0009] According to one aspect of the present invention, there is provided a method for detecting antibodies resulting from infection with one or more members of the tick-borne relapsing fever (TBRF) Borrelia genus, including B. hermsii, B. turikatae, B. miyamotoi, B. duttonii, and B. parkeri, when present in a sample from a subject suspected of having TBRF, the method comprising: providing a biological sample from a subject suspected of having TBRF; mixing the biological sample with one or more labeled and / or tagged and / or bound amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and variants thereof that retain the immunological binding profile of FIG. 3; and detecting a positive immunobinding reaction indicative of the presence of TBRF-specific antibodies in the sample. In some embodiments, two or more of the labeled and / or tagged and / or bound amino acid sequences are mixed with a biological sample, and the sample is considered positive if at least two of the amino acid sequences are detected. In some embodiments, the labeled and / or tagged and / or bound amino acid sequences are detected with an anti-human IgG or anti-human IgM antibody linked to a detectable moiety. In some embodiments, the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme. In some embodiments, the detectable moiety comprises alkaline phosphatase. In some embodiments, the detectable moiety comprises biotin.

[0010] According to an aspect of the present invention, a vector is provided comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and variants thereof that retain the immunological binding profile of FIG. 3, and a heterologous nucleic acid sequence. In some embodiments, the nucleic acid sequence is operably linked to a promoter sequence. In some embodiments, the vector is a prokaryotic vector. In some embodiments, a cell comprises the vector. In some embodiments, the cell is a bacterial cell. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows micrographs of line immunoblots. TBRF immunoblot strips were tested with rabbit anti-TBRF Borrelia serum samples. Antibodies against TBRF Borrelia species B. turcica, B. miyamotoi, B. hermsii, B. parkeri, and B. tsurikae were detected by TBRF immunoblot strips prepared from recombinant antigens from all five species. This demonstrated that the TBRF immunoblot detected antibodies of the TBRF Borrelia group. N, negative control; TC, B. turcica; MI, B. miyamotoi; BH, B. hermsii; PK, B. parkeri, TT, B. tsurikae; and M, positive control. [Diagram 2]Figure 1 shows micrographs of TBRF immunoblots of representative patient serum samples. Patient serum samples were tested with tick-borne relapsing fever (TBRF) IgM and IgG immunoblots. These results illustrate how detection of antibodies to TBRF Borrelia species identified patients with chronic Lyme disease symptoms that may have been missed by simply testing for Borrelia burgdorferi species antigens. P, positive control; N, negative control; G, TBRF immunoblot IgG; M, TBRF immunoblot IgM; 1, B. hermsii positive IgM; 2, B. tsurukae positive IgG; 3, B. miyamotoi positive IgM; 4, B. tsurushika positive IgG; 5, B. parkeri positive IgM and IgG. [Diagram 3] 1 is a table showing representative results of TBRF immunoblot IgM and IgG testing with SEQ ID NOs: 1-11 on patient serum samples. Pos, positive; I, unknown; Tt, B. tsurikae; Pk, B. parkeri; Mia, B. miyamotoi; Du, B. duttonii; Bh, B. hermsii;

[0012] Sequence description SEQ ID NO:1, B. hermsii Vlp7 MQQPEAGKTGVSGGVNGNLGNSLMELGRSAENAFYAFIELVSDVLGFTAKSDTTKQEVGGYFNSLGAKLGEASNDLEQVAVKAETGVDKSDSSKNPIREAVNEAKEVLGTLKGYVESLGTIGDSNPVGYANNAAGSGTTAADDELRKAFKALQEIVKAATDAGVKALKIGA TTLQANGGADNKEGAKILATSGGNPAAADVAKAAAILSSVSGEEMLSSIVKSGENDAQLAAAADGNTSAISFAKGGSDAHLAGANTPKAAAVAGGIALRSLVKTGKLAAGAADNATGGGKEVQGVGVAAANKLLRAVEDVIKKTVKNVLEKAKEKIDKARGSQEPVSESSK

[0013] SEQ ID NO:2, B. tsurikatae BipA-2H MDNVMSGIDNVIQGAGTFATAAMQGVGTVIDVLQDVGTFVISDIQNMGARMLFGTGENSSVASEGSESVMLSSNDSSEAKDVTVVLSSDVTSKGNDVAVGLSSDETQVIGRLEKYLQSAIKINGRSDSDQSNLESGRKKF FNWLKTSDTNASKRKELVQDLQKVFDLIKEKSSDSTELKHWVQSIVDRIEDKSTIVDIDSDDELNNDKEVDFLIENTLASRDYSGFAVSLLFQSLADTLYDSDNNRDKSEEQIFQDLRKVFSDSSDKSEGVLGFKSKIEATN

[0014] SEQ ID NO:3, B. turikatae BpcA / fHbp MSETSLLNIETNLLNTLDDNQKQALITFKDLLQDKKHLSILEKQQKSILEDLKANQKNYNLQDKLKKTLNSEYDKNQLNKLFDELGNIKTKQFLQQLHIILQSIKDGKPTNFASSNFNNLNQTLEQKKEQALKYIKDKLYTDYYLYINGIQDANYFFERIMSLLE

[0015] SEQ ID NO: 4, B. turikatae P41 / FlaB MRNNSINAANLSKTQEKLSSGRINRASDDAAGMGVAGKINAQIRGLSQASRNTSKAINFIQTTEGNLNEVEKVLVRMKELAVQSGNGTYSDADRGSIQIEIEQLTDEINRIADQAQYNQMHMLSNKSAAQNIKTAEELGMQPAKINTPASLAGSQASWTL RVHVGANQDEAIAVNIYAANVANLFAGEGAQVSPAQEGAQQEGVQAAPAPAAAPAQGGVNSPVNVTTTIDANMSLSKIENAIRMVSDQRANLGAFQNRLESIKASTEYAIENLKSSYAQIQDATMTDEIVASTTNSILTQSAMAMIAQANQVPQYVLSLLR

[0016] SEQ ID NO:5, B. parkeri BipA-1H MDMGSTRDWLTNDDGFVRGTKGFDDSPFRRPDRVDKEVSAGGREIEKAFSRNLGVAGGQRKGTDDVKNGIAGARESGGVLKEAENAGQRDVDDSGEGIKNDVIQNLGSVGVQVAVGSENNGDDSGQEAEKGSQNLGDTGTQRVVSTSDLNSHLGVESKGGMSTNKEGISTNHVTENRNSINSITSTSSGLSTALQIAGTSTRASGYEGEVTTNAQDRSFIDTKTQDSKKQYSDFSDQDIRDKILGNVVGGVV

[0017] SEQ ID NO: 6, B. parkeri BipA-2H MGNVMSGIDNVIQGAGTFATAAMQGVGTVIDVLQDVGTFVISDIQNMGARMFFGTGESSSVASEDSESVMSLSSKASSEAKDTTVGLSSDVTSKGNNVAVGLSSDEIQIIGRLEKYLKSAIKINGRSDSDQSKLESGHKKFFQWLKTSDTNASKRKELVQNLQRVFNLIKEKSSDSTELKKWMQSIVDDIENKSTIIDINSDDKLNNDKEVDFLIEKTLGSSDYSGFAVSLLFQALADTLYDSENSRDKSEEQIFKDLRKVFSDKSEGVLEFKSKIEATN

[0018] SEQ ID NO: 7, B. parkeri GlpQ MCQNEKMSMTNKKSPLTIAHRGASGYLPEHTLESKAFAYALGADYLEQDIVLTKDNVPIIMHDPELDTTTNVAKLFPERARENGRYYSVDFTLDELKSLSLSERFDLETRKPIYPKRFPLNEYNFKIPTLEEEIQFIQGLNKSTGRNVGIYPEIKKPLWHKQQGKDISKIVIEILNKYGYKSKEDKIYLQTFDFDELKRIREELGYQGKLIMLVGENDWNEAPTDYEYIKSQEGMTEVAKYADGIGPWIPQIIIDGKITDLTSLAHKYNMEVHAYTFRIDSLPSYVKDANELLDLLFNQAKIDGLFTDFTDTVVKFIKQ

[0019] SEQ ID NO:8, B. parkeri BpcA / fHbp MSETSLLNTLDNNQKQALITFKDLLQDKNHRSILEKQQKSILKDLEKHQENSNLQDKLKKTLNSEYDKTQLNKLFDELGNIKTKQFLQKLHIMLKSINGTLTSFSSSNFKDSNQTLEQKKEQALQYIKGQLYTDYYLYINGIQDANYFFERIMSVLEI

[0020] SEQ ID NO: 9, B. parkeri P41 / FlaB MRNNSINAANLRKTQEKLSSGRINRASDDAAGMGVAGKINAQIRGLSQASRNTSKAINFIQTTEGNLNEVEKVLVRMKELAVQSGNGTYSDADRGSIQIEIEQLTDEINRIADQAQYNQMHMLSNKSAAQNIKTAEELGMQPAKINTPASLAGAQASWT LRVHVGANQDEAIAVNIYASNVANLFAGEGAQVSPAQEGAQQEGVQAAPAPAAAPAQGGVNSPVNVTTTVDANMSLSKIENAIRMVSDQRANLGAFQNRLESIKASTEYAIENLKSSYAQIKDATMTDEIVASTTNSILTQSAMAMIAQANQVPQYVLSLLR

[0021] SEQ ID NO: 10, B. duttonii GlpQ MENAKINKKSALIIAHRGASGYLPEHTLEAKAYAHALGADYIEQDIVLTKDDIPIVMHDPELDTTTNVAKLFPGRARENGKYYSVDFTLAEIKSLSLSERFDPETQQPIYPNRFPATEYDFKIPTLEEEIKFIQGLNKSTGKNIGIYPEIKKPLWHK QQGKDISKIVIDILNKYGYKSKEDKIYLQTFDFDEIKRIREELGYQGKLIMLVGENDWEEAPTDYEYIKSEEGMAEVAKYADGIGPWIPQIIINGQITGLISLAHKYNMQVHPYTFRIDALPSYVKDPNELLELLFIKAKVDGLFTDFVDISIKFMQ

[0022] SEQ ID NO:11, B. miyamotoi GlpQ MASMTGGQQMGRGSEMGENKKSPLIIAHRGASGYLPEHTLEAKAYAYALGADYLEQDIVLTKDNIPVIMHDPEIDTTTNVAQLFPNRARENGRYYATDFTLTELKSLNLSERFDPENKKPIYPNRFPLNEYNFKIPTLEEEIQFIQGLNKSTGKNVGIYPEIK KPFWHKQQGKDISKIVIEILNKYGYKSKEDKIYLQTFDFDELKRIRKELGYQGKLIMLVGENDWNEAPTDYEYIKSEEGIAEVAKYSDGIGPWIPQIIIDGKITELTNLAHKYNIEVHPYTFRTDALPSYVKNENELLDLFNKAKVDGIFTDFTDTVMNFIKK DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The best known RFB species causing TBRF in the United States (USA) are B. hermsii, B. miyamotoi, B. parkeri, and B. tsurikatae. However, other RFB species causing TBRF continue to be identified. For example, a patient infected with a B. johnsonii-like species, previously found only in bat mites, has been identified in Wisconsin. Furthermore, TBRF has also been reported in Central and South America. B. hispanica, B. persica, and B. miyamotoi are important causes of TBRF in Europe and Asia, while B. hispanica, B. crocidurae, and B. duttonii are important causes of TBRF in Africa. Although most RFB species are transmitted by soft ticks of the genus Ardisia, B. miyamotoi can be transmitted by the same hard ticks of the genus Ixodes that transmit Lyme disease Borrelia (LDB) species. Other RFB species, B. lonestari, the recently described B. turcicia, and B. tachyglossi, are members of a rapidly expanding Borrelia clade associated with reptile (B. turcicia) or echidna (B. tachyglossi) hosts, and are also transmitted by hard ticks. In order to provide timely treatment, clinicians must be able to rapidly and accurately identify whether patients who present with symptoms of tick-borne disease have been exposed to TBRF species.

[0024] Aspects of the invention provide a method for rapid and accurate detection of Borrelia antisera in samples from subjects suspected of having TBRF. Subjects suspected of having TBRF may present with symptoms such as high fever (e.g., 103° F.), headache, and muscle and joint pain. Symptoms are typically recurrent, resulting in a presentation pattern of fever lasting approximately 3 days, followed by 7 days without fever, followed by another 3 days of fever. Without proper antibiotic treatment, this process may be repeated multiple times. Because symptoms of TBRF may resemble, for example, viral flu-like symptoms, accurate diagnosis of TBRF is important in order to provide an effective treatment to the subject. The present invention provides a rapid and simple diagnostic test that detects the presence of antibodies specific to the causative Borrelia species, thereby fulfilling the need for such a test.

[0025] Because TBRF can be caused by multiple RFB species and because the geographic ranges of RFB species can overlap, RFB species testing needs to be comprehensive, i.e., the test needs to be capable of detecting antibodies against multiple species simultaneously. The present invention provides antigenic amino acid sequences specific to various TBRF Borrelia species. The amino acid sequences of the present invention encode antigenic peptides with high specificity and / or sensitivity for the indicated genus. The inclusion of antigenic peptides that cross-react across Borrelia species boundaries is also important for the development of comprehensive serological assays, or other immunologically-based assays, where the goal is to detect infection and not necessarily to identify the specific species causing the infection. For example, the present disclosure includes multi-panel immunoassays, where multiple RFB species can be detected in the context of a single test screen.

[0026] The present invention provides novel compositions and methods for diagnosing infection with TBRF Borrelia species. The invention is based, in part, on the discovery of species-specific amino acid sequences that encode antigenic peptides (which may also be referred to in the art as peptide antigens or antigens), as described herein.

[0027] The present invention, in one aspect, is a composition comprising one or more labeled and / or tagged and / or conjugated amino acid sequences, the one or more labeled and / or tagged and / or conjugated amino acid sequences having at least 90%, 95%, 98%, 99%, 99.5%, or 100% homology with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. Sequences with less than 100% homology may be modified with one or more substitutions, deletions, insertions, or other modifications with reference to the amino acid sequences provided herein. Examples of modifications include, but are not limited to, conservative amino acid substitutions. Conservative amino acid substitutions will result in molecules with similar functional characteristics to those of the molecule in which such modification is made. Conservative amino acid substitutions are substitutions that do not result in significant changes in the activity or conformation of a selected polypeptide or protein. Such substitutions typically involve replacing a selected amino acid residue with a different residue having similar physicochemical properties. For example, the substitution of Glu for Asp is considered a conservative substitution since both are negatively charged amino acids of similar size. Groupings of amino acids according to physicochemical properties are known to those of skill in the art. Each of the following groups contains amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). One skilled in the art can determine whether sequences with less than 100% homology can bind to naturally occurring or non-naturally occurring TBRF-related antibodies, as well as the sensitivity and specificity of the antibodies to modified sequences.One of skill in the art, in light of the teachings herein, will be able to identify sequences with significant homology to SEQ ID NOs: 1-11 of the present invention that give acceptable or equivalent responses in the methods of the present invention without undue experimentation.

[0028] In some embodiments, the invention is a composition comprising one or more labeled and / or tagged and / or conjugated amino acid sequences, the one or more labeled and / or tagged and / or conjugated amino acid sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and variants thereof that retain the immunological binding profile of Figure 3. As used herein, "consisting of," when used as a claim transition phrase referring to an amino acid sequence, refers to an amino acid sequence having 100% homology to the specified amino acid sequence (i.e., SEQ ID NOs:1-11).

[0029] Aspects of the present invention provide antigen-specific amino acid sequences for specific species of TBRF Borrelia. These novel amino acid sequences can be used in assays to identify TBRF specific Borrelia in samples from subjects suspected of having TBRF. Using the amino acid sequences of the present invention, identification of TBRF Borrelia in subject samples is performed with greater speed, sensitivity, and specificity than other current methods. The amino acid sequences of the present invention can be used in diagnostic and academic assays. Non-limiting examples of suitable assays include immunoblots, line immunoblots, ELISA (enzyme-linked immunosorbent assay), and the like. The amino acid sequences of the present invention can be used to detect TBRF Borrelia specific T cells, for example, using the IgXSPOT test (IGeneX, Milpitas, CA).

[0030] In some embodiments, the invention is a composition comprising one or more labeled and / or tagged and / or conjugated amino acid sequences, the one or more labeled and / or tagged and / or conjugated amino acid sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and variants thereof. The term "variant" encompasses any modification(s) of the specified amino acid sequence (i.e., SEQ ID NOs:1-11) that retains the immunological binding profile of FIG. 3. Such modifications can include insertions and deletions (internally or at the N-terminus or C-terminus, or both).

[0031] Nucleic acid sequences, including polynucleotides and oligonucleotides, encoding the amino acid sequences of the present invention and portions thereof can be expressed in cultured cells to provide isolable amounts of peptides that exhibit the biological (e.g., immunological) properties of the antigenic peptides encoded by the amino acid sequences of the present invention. Due to the redundancy of the genetic code, multiple nucleic acid sequences may be suitable for producing the peptide sequences of the present invention. One of skill in the art can identify one or more nucleic acid sequences for producing the peptide sequences of the present invention. The nucleic acid sequences encoding the amino acid sequences of the present invention can be labeled with any suitable label known to those of skill in the art.

[0032] In this regard, the nucleic acid sequence suitable for generating the amino acid sequence of the present invention may be substantially homologous to a naturally occurring sequence. Substantial homology of a nucleic acid sequence, as used herein, means that (a) there is more than 65%, 75%, 85%, 95%, 98%, or 99% homology with a naturally occurring sequence, or (b) the homologous nucleic acid sequence hybridizes to the sequence being compared or its complementary strand under stringent conditions of temperature and salt concentration. Such stringent conditions will generally be temperatures above about 22°C, usually above about 30°C, more usually above about 45°C, and salt concentrations generally below about 1M, usually below about 500mM, preferably below about 200mM. The combination of temperature and salt concentration is more important in the definition of stringency than either temperature or salt concentration alone. Other conditions that affect stringency include the GC content of the sequences being compared, the degree of sequence complementarity, and the length of the sequences involved in hybridization, as well as the nature of the hybridization site. These and other factors that influence stringency include the composition of the buffer(s) used in the lysis mixture. These and other factors that influence stringency are well described in the scientific and patent literature. Those skilled in the art can determine appropriate conditions for identifying the homology of nucleic acid sequences encoding the antigenic peptides of the invention.

[0033] Homologous nucleic acid sequences can be identified based on the nature of nucleotide substitutions in nucleic acid sequences. For example, synonymous nucleotide substitutions, i.e., nucleotide changes in nucleic acid sequences that do not alter the encoded amino acid sequence, are more likely to be tolerated than non-synonymous nucleotide substitutions and therefore more likely to be present in a particular nucleic acid sequence. Those skilled in the art can identify the appropriate number and position of substitutions that can be tolerated in the nucleic acid sequence encoding the amino acid sequence of the present invention without undue experimentation and without adversely affecting the antigenicity of the encoded antigenic peptide.

[0034] Signs and tags One or more amino acid sequences of the present invention can be labeled and / or tagged and / or conjugated. As used herein, a "label" or "tag" is a detectable moiety that can be attached to an amino acid sequence of the present invention. The label or tag can be covalently or non-covalently attached to an amino acid sequence of the present invention. Non-limiting examples of such "tags" include natural and synthetic (i.e., non-naturally occurring) nucleic acid and amino acid sequences (e.g., polyAAA tags), antibodies, and detectable moieties such as labels (discussed elsewhere herein). That is, the definitions of the terms "labeled" and "tagged" may overlap in that a tag can also function as a label in some cases. Furthermore, tags useful in the present invention can be linked to a label.

[0035] The amino acid sequence of the present invention, or any tag attached to the amino acid sequence of the present invention, can be labeled with any suitable label known to those skilled in the art. Such labels can include, but are not limited to, biotin / streptavidin, enzyme complexes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase), fluorescent moieties (e.g., FITC, fluorescein, rhodamine, etc.), biological fluorophores (e.g., green fluorescent protein (GFP), R-phycoerythrin), or other luminescent proteins. Any suitable label known to those skilled in the art can be used in the present invention.

[0036] In some embodiments, the amino acid sequences of the invention can be "bound". A "bound" amino acid sequence is one that has been immobilized in order to allow the amino acid sequence to be used in biological tests such as, for example, immunoassays. In the context of the present invention, a "bound" amino acid sequence is one that is directly or indirectly attached (e.g., covalently or non-covalently attached) to a non-natural surface or material. Additionally or alternatively, a "bound" amino acid sequence of the invention can be directly or indirectly attached to a natural surface or material, none of which is naturally associated with the amino acid sequence. Non-limiting examples of materials to which the amino acid sequences of the invention can be bound include nitrocellulose, nylon, polyvinylidene difluoride (PVDF) plastic, metal, magnetic beads, and agarose (e.g., beads). Linking agents known to those skilled in the art can be used to aid or facilitate the binding of the amino acid sequences of the invention to a surface or material.

[0037] Generation of amino acid sequences In some embodiments, the amino acid sequences of the invention can be non-naturally occurring synthetic sequences, such as sequences produced by recombinant techniques or synthesized by the protein synthesis machinery. Thus, the amino acid sequences of the present invention can be produced by recombinant techniques as described and possible in the literature and generally as referred to in manuals such as, for example, Short Protocols in Molecular Biology, Second Edition, FM Ausubel, Ed., all John Wiley & Sons, NY, edition as of 2008; and Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001, and as is well known to those skilled in the art. In one embodiment, the amino acid sequences of the present invention are produced recombinantly in E. coli.

[0038] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid to which it is operatively linked between different genetic environments. In addition to a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and variants thereof that retain the immunological binding profile of FIG. 3, the vectors of the present invention also contain heterologous nucleic acid sequences. As used herein, heterologous refers to a nucleic acid sequence that does not naturally occur in the organism from which the Markush group sequence is derived. The term "vector" can also refer to a virus or organism that can transport a nucleic acid molecule. One type of vector is a plasmid, a small circular double-stranded extrachromosomal DNA molecule that is physically separate from and capable of self-replicating independently of chromosomal DNA. Some useful vectors are capable of autonomous replication and / or expression of a nucleic acid linked to the vector. A vector capable of directing the expression of an operatively linked nucleic acid is referred to herein as an "expression vector." Other useful vectors include, but are not limited to, bacterial plasmids and bacterial artificial chromosomes (BACs), cosmids, and viruses, such as lentiviruses, retroviruses, adenoviruses, and phages.

[0039] The vectors useful in the methods of the present invention can contain additional sequences, including but not limited to one or more signal sequences and / or promoter sequences, or combinations thereof.Promoters that can be used in the methods and vectors of the present invention include but are not limited to cell-specific promoters or universal promoters.Non-limiting examples of promoters that can be used in the vectors of the present invention include: ubiquitous promoters, such as but not limited to CMV promoter, CAG promoter, CBA promoter, and EF1a promoter.The selection and use of suitable promoters is well known in the art.

[0040] The vectors useful in the methods of the invention can be used to express in cells fusion proteins comprising sequences of the invention. Expression vectors and their preparation and use are well known in the art. In some embodiments of the invention, the nucleic acid sequence of the expression vector encodes a fusion protein comprising an amino acid sequence of the invention. How to prepare and use fusion proteins comprising a polypeptide sequence is well known in the art. In some embodiments, the fusion protein comprising an amino acid sequence of the invention can also include an epitope tag that can be used to purify the fusion protein or can be used in the methods of the invention. Non-limiting examples of epitope tags include FLAG tags, fluorescent tags (including but not limited to green fluorescent protein (GFP)), GST tags, hemagglutinin (HA), polyhistidine (polyHis) tags, Myc tags, MBP tags, or V5 tags. In some embodiments, the fusion protein comprising an amino acid sequence of the invention can also include a detectable label, as described elsewhere herein.

[0041] Subjects and cells As used herein, a subject can be an animal, such as a mammal or a non-mammal. Examples, but not limited to, mammalian subjects include primates (including but not limited to humans), rodents (including but not limited to mice, rats, squirrels, chipmunks, and prairie dogs), lagomorphs, deer, canines (including but not limited to dogs, foxes, coyotes, and wolves), felines (including but not limited to house cats, bobcats, cougars, and other wild cats), bears, horses, cows, sheep, goats, and pigs. Examples, but not limited to, non-mammalian subjects include birds, amphibians, lizards, insects, and arthropods. As used herein, a cell can be a bacterial cell, including but not limited to E. coli, or an animal cell, either mammalian or non-mammalian.

[0042] Assays and Detection Methods The amino acid sequences of the invention can be tagged with antibodies having specificity for any of the amino acid sequences. Specificity for the amino acid sequences, i.e., antibody specificity, is a property of an antibody that allows it to react preferentially with some antigenic determinants and not others. Specificity depends on the chemical composition, physical forces, and molecular structure at the binding site. Sensitivity is how strongly an antibody binds to an antigenic determinant. One skilled in the art can determine the specificity and sensitivity of an antibody for a particular amino acid sequence using standard affinity assays, such as immunoblotting, Ouchterlony assay, titer assay, etc. In another aspect, the invention provides a method for rapid and accurate detection of TBRF Borrelia antisera in a sample from a subject suspected of having TBRF. The method of the present invention for detecting TBRF Borrelia antisera in a sample from a subject suspected of having TBRF can include, for example, providing a biological sample (including but not limited to blood, saliva) obtained from a subject suspected of having TBRF, mixing the biological sample with one or more of the labeled and / or tagged and / or bound amino acid sequences of the present invention, and detecting a positive reaction indicating the presence of Lyme disease antisera in the sample. The antisera can be detected, for example, by immunoblotting, ELISPOT, ELISA, Western blotting, lateral flow assay, or any other suitable immunoassay known to those of skill in the art. These techniques are known to those of skill in the art and can be found in general technical references. Although similar, these techniques each have their own advantages and disadvantages. Other suitable techniques may also be known to those of skill in the art and are incorporated herein.

[0043] To assess the impact of test limitations and to identify levels of exposure to TBRF Borrelia, a modified Western blot technique, the line immunoblot, was developed and employed in the embodiments of the invention described herein. The line immunoblot uses recombinant antigens from common strains and species of the TBRF Borrelia complex to serologically identify and diagnose TBRF in serum from patients suspected of having tick-borne disease. As described elsewhere herein, the serotype makeup of TBRF Borrelia exposure may be more complex than previously recognized, and infection with more than one TBRF Borrelia species may occur.

[0044] Western blotting can involve separating proteins by electrophoresis and then transferring to nitrocellulose or other solid media (e.g., polyvinylidene fluoride or PVDF membranes and nylon membranes), which are described in more detail below. Immunoblotting can also involve manual or mechanical application of proteins to a solid medium. Preferably, proteins are applied in lines or spots and dried, which bind to a solid support medium, e.g., nitrocellulose. Proteins used in immunoblotting can be isolated from biological samples, as is well known to those of skill in the art. The bound protein can be either naturally occurring or recombinantly produced. The bound protein is then contacted with a sample or samples suspected of having antibodies specific for the target protein. Using this technique, known antibodies can be used to identify whether a protein is present in a sample, for example when proteins from lysed cells are separated by electrophoresis and transferred to a solid medium. Western blotting allows for the identification of proteins by size as well as by specificity for a particular antibody.

[0045] Similarly, using a technique called immunoblotting, known proteins can be bound to a solid medium and a sample, such as a sample from a subject suspected of having an infectious disease, can be tested for the presence of a specific antibody in the sample by contacting the bound protein with the sample. The antibody that binds to the target protein is usually referred to as the primary antibody. A secondary antibody is specific for a conserved region of the primary antibody (e.g., a rabbit anti-human IgG antibody can be used to detect a primary human antibody) and is used to detect any bound primary antibody. The secondary antibody is usually labeled with a detectable moiety for visualization. Non-limiting examples of suitable labels include, for example, chromophores such as biotin, radioactive moieties, and enzymes such as alkaline phosphatase. The use of these and other materials for antibody visualization is well known to those skilled in the art.

[0046] The Enzyme-Linked ImmunoSpot (ELISPOT) method can detect human T cells that react to Lyme-specific antigens in vitro. In the ELISPOT assay, the surface of the PVDF membrane of a 96-well microtiter plate is coated with a capture antibody that binds, for example, anti-interferon gamma (IFNγ) or other cytokine-specific antibodies. During the cell incubation and stimulation steps, T cells isolated from whole patient blood are seeded into the wells of the plate together with the sequence(s) described above and are allowed to form a substantial monolayer on the membrane surface of the wells. By stimulating any antigen-specific cells with one or more of the sequences of the invention, they are activated and release IFNγ, which is captured directly on the membrane surface by the immobilized antibody. That is, IFNγ is "captured" in the area immediately surrounding the secreting cell before it diffuses into the culture medium or is degraded by proteases and has a chance to bind to the receptors of bystander cells. A subsequent detection step visualizes the immobilized IFNγ as an immunospot. Immunospots are essentially secretory footprints of activated cells.

[0047] For the specific example of an ELISPOT test, each well of a plate is coated with purified cytokine-specific antibody specific for the cells to be tested or detected. T cells are isolated from a subject (e.g., a subject suspected of having Lyme disease or TBRF), cultured in each well, and stimulated with recombinant antigen of one or more sequences of the invention. Lyme positive patient cells respond to the stimulation by secreting cytokines that are captured by the antibody coated on the well and further detected by ELISA.

[0048] ELISA assays can also be used to detect antigens. ELISA assays allow the quantification of a particular protein in a protein mixture (e.g., a lysate) or can be used to determine whether a peptide is present in a sample. Similarly, ELISA assays can be used to determine whether a specific antibody is present by using a particular antigen as a target. When used in the present invention, the target amino acid sequence(s) is attached to a surface. Then, active antibodies can bind to the antigen, if present in the sample being tested. A second antibody linked to an enzyme is added, and in a final step, a substance containing a substrate for the enzyme is added. The subsequent reaction step produces a detectable signal. This signal is most commonly a color change of the substrate.

[0049] Lateral flow assays are also known by a variety of other names, including lateral flow These include, but are not limited to, lateral flow tests, lateral flow devices, lateral flow immunoassays, lateral flow immunochromatographic assays, and rapid tests. Lateral flow assays are simple, versatile, paper-based platforms that detect and / or quantify the presence of one or more analytes, such as antigens, in a mixture, such as a liquid sample. Lateral flow assays can be qualitative or quantitative. In a lateral flow assay, a sample containing one or more analytes of interest is added to a sample adsorption pad, and the sample is drawn by capillary action through various polymeric test strip zones to which molecules capable of interacting with the analyte(s) are attached. The sample migrates to a complex release pad, which contains molecules complexed with fluorescent, colored, or otherwise detectable particles that specifically bind to the analyte(s) of interest. Finally, the sample, including the bound analyte(s), migrates to the detection zone. Within the porous membrane of the detection zone are biological components, such as antibodies or antigens, which are immobilized in rows and will react with the detectable particles. Lateral flow assays typically have a control line to ensure that the sample is flowing through the strip, and one or more test lines to detect the presence of the analyte(s) of interest. The results can be read by eye or by a machine that can read and interpret the results. Lateral flow assays can be designed as direct assays, as "sandwich" assays (where the presence of a colored line at the test line indicates a positive test), or as competitive assays (where the absence of a colored line indicates a positive test). Direct and competitive assays can be multiplexed.

[0050] In one embodiment of the method of the invention, when two or more of the labeled and / or conjugated amino acid sequences of the invention are mixed with a biological sample, a positive result is indicated if at least two of the amino acid sequences are detected. In another embodiment of the invention, when at least one of the labeled and / or conjugated amino acid sequences of the invention is mixed with a biological sample, a positive result is indicated if at least one of the amino acid sequences is detected.

[0051] In the method of the present invention, any primary antibody bound to a peptide encoded by an amino acid sequence of the present invention can be detected using an anti-human antibody, such as IgG or IgM, used as a secondary antibody conjugated with a detectable moiety. As described elsewhere herein, the detectable moiety can be selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme, or can be other detectable moieties known to those of skill in the art. In one embodiment, the detectable moiety comprises alkaline phosphatase. In another embodiment, the detectable moiety comprises biotin.

[0052] In another aspect of the invention, a method for detecting and distinguishing between various Borrelia species in a sample is provided. The sample can be from a subject suspected of having Lyme disease. The method can include, for example, providing a sample, e.g., a biological sample obtained from a subject suspected of having TBRF, and mixing or contacting the biological sample with one or more of the labeled and / or bound amino acid sequences of the invention. The amino acids can be labeled to confirm their presence if the assay does not give a positive result.

[0053] In some embodiments of the invention, a sample can be considered positive in a TBRF Borrelia multi-species panel assay if at least one amino acid sequence is detected. In some embodiments of the invention, a sample can be considered positive in a TBRF Borrelia multi-species panel assay if at least two amino acid sequences are detected. In some embodiments of the invention, a sample can be considered positive in a TBRF Borrelia multi-species panel assay if at least two amino acid sequences that identify the species are detected. can be considered positive.

[0054] Equivalent Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily contemplate various other means and / or structures for performing the functions and / or results and / or obtaining one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) utilizing the teachings of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. It should therefore be understood that the above-described embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, and / or methods is included within the scope of the present invention, unless such features, systems, articles, materials, and / or methods are mutually inconsistent.

[0055] All definitions herein and as used should be construed to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0056] The indefinite articles "a" and "an," as used herein in the specification and claims, shall be construed to mean "at least one," unless expressly stated to the contrary. The term "and / or," as used herein in the specification and claims, shall be construed to mean "either or both" of the elements so conjoined, i.e., the elements are present jointly in some cases and separately in other cases. Other elements than the elements specifically identified by the "and / or" clause may be optionally present, whether or not related to such specifically identified elements, unless expressly stated to the contrary.

[0057] All references, patents, and patent applications, and publications cited or referred to in this application are hereby incorporated by reference in their entirety. EXAMPLES

[0058] Example 1. TBRF Immunoblot Antigen Comprehensiveness Test The TBRF immunoblot IgG and IgM test, a qualitative immunoblot assay, was designed and performed to detect IgG and IgM antibodies against TBRF Borrelia species in serum samples suspected of having relapsing fever or Lyme-like symptoms. Recombinant TBRF Borrelia antigens were sprayed linearly onto nitrocellulose strips, which were then used in the TBRF immunoblot test. Experiments were performed to determine the specificity of the TBRF immunoblot test.

[0059] method Antigen preparation Hybrid gene constructs or gene parts were cloned into pET vectors and transformed into Escherichia coli as previously described [Liu et al., Healthcare (2018) 6(3) pii: E99; Shah et al., Healthcare (2019) 7:121]. Recombinant target proteins were obtained by expressing the gene products in ELISA (GenScript, Piscataway, NJ) and then isolating the proteins at >90% purity. As previously described [Shah et al., Healthcare (2019) 7:121], recombinant proteins were derived from multiple TBRF Borrelia species (B. hermsii, B. miyamotoi, B. tsurukae, and B. tsurushika) to detect four target antigens, BipA, GlpQ, BpcA / fHbp, and P41 / FlaB.

[0060] Preparation of antigen strips Antigen strips for TBRF immunoblotting were prepared as previously described [Liu et al., Healthcare (2018) 6(3) pii: E99; Shah et al., Healthcare (2019) 7:121]. Purified and control proteins were diluted (7–19 ng protein / line) and sprayed linearly onto nitrocellulose sheets (Cytiva, Marlborough, MA) using a BioDot liquid dispenser (BioDot, Irvine, CA). The sheets were then blocked with 5% nonfat dry milk and sliced ​​into 3 mm wide strips.

[0061] Detection of antibody reactivity Serological immunoblot testing was performed at the IGeneX Reference Laboratory, a Clinical Laboratory Improvement Amendments (CLIA)-certified high-complexity testing facility.

[0062] Reactivity between TBRF Borrelia specific antibodies in test sera and TBRF Borrelia antigens in immunoblots was detected as previously described [Liu et al., Healthcare (2018) 6(3) pii: E99; Shah et al., Healthcare (2019) 7:121]. Strips were labeled and immersed in diluent (100 mM Tris, 0.9% NaCl, 0.1% Tween-20, and 1% nonfat dry milk) in a bath for 5 minutes (min). Either test or control serum was then added to the strips in 10 μL aliquots. Strips were incubated with serum for 1 h at room temperature and washed three times with wash buffer (KPL, Gaithersburg, MD) at room temperature, then the final wash was aspirated. To detect IgG and IgM reactivity, alkaline phosphatase-conjugated goat anti-human IgG at a dilution of 1:10,000 or alkaline phosphatase-conjugated goat anti-human IgM at a dilution of 1:6,000 (KPL, Gaithersburg, MD), respectively, were incubated with the strips for 1 hour and then washed three times. To visualize the antibody / antigen-reactive bands, the strips were reacted with the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP / NBT, KPL, Gaithersburg, MD) and the reaction was terminated by washing with distilled water after the calibration control produced a visible band at 39 kDa. Bands showing lower intensity than that of the calibration control were recorded as negative. Human serum from a patient with confirmed Borrelia infection was used as a positive control and serum from an uninfected individual was used as a negative control. All immunoblot testing of patient samples was performed in parallel with testing of positive and negative control serum samples.

[0063] Scoring immunoblots For TBRF Borrelia immunoblots, detection of either IgG or IgM antibodies against P41 / FlaB (41 kDa) and any two of four antigens, BipA (75 kDa), GlpQ, and BpcA / fHbp (21-23 kDa), provided the best specificity for TBRF species.

[0064] Detection of either IgG or IgM antibodies against two or more proteins within each serotype was considered a positive reaction for that serotype. Optimal detection sensitivity was derived by applying the same criteria for either IgG or IgM antibody reactivity against the four TBRF Borrelia antigens as previously described [Shah et al., Healthcare (2019) 7:121].

[0065] result As shown in Figure 1 , antibodies against TBRF Borrelia species, B. turicica , B. miyamotoi , B. hermsii , B. parkeri , and B. turikatae species were detected by TBRF immunoblot strips prepared from recombinant antigens derived from all five species, indicating that TBRF immunoblots detected TBRF Borrelia group antibodies.

[0066] Example 2. TBRF Immunoblot Clinical Trials of Patients with Suspected Tick-Borne Disease Patients who met the case definition of chronic Lyme disease (CLD) were studied to assess the impact of testing limitations and to identify levels of exposure to TBRF Borrelia. Results revealed that all had been exposed to B. burgdorferi species.

[0067] method The methods used were as described in Example 1 and elsewhere herein.

[0068] Antigen preparation and antigen strip preparation To detect the target antigens BipA, GlpQ, BpcA / fHbp, P41 / FlaB, and Vlp7, additional recombinant proteins were derived from multiple TBRF Borrelia species (B. hermsii, B. miyamotoi, B. tsurikatae, B. parkeri, and B. duttonii) as described elsewhere herein. These recombinant proteins, comprising the amino acid sequences of SEQ ID NOs: 1-11, were included in the TBRF immunoblot strips.

[0069] Clinical Trials A total of 265 patient serum samples were tested by TBRF immunoblot IgM and IgG, including serum samples from 90 patients with CLD symptoms and 212 patients who were TBRF negative.

[0070] Chronic Lyme disease patient cohort (n=90) A cohort of 90 patients with CLD was recruited from a medical facility in San Francisco, CA that specializes in the diagnosis and treatment of tick-borne diseases. The Western Institutional Review Board (WIRB), Puyallup, WA) approved an anonymous retrospective data collection protocol and consent form. Patients of either gender were admitted to the study if they were at least 18 years of age, had a history of musculoskeletal, neuropsychiatric, and / or cardiac symptoms consistent with LD, and provided written informed consent for data collection. Subjects were included in the study if they met the case definition of untreated or previously treated CLD with symptoms lasting more than 6 months. This was as described in detail elsewhere [Cameron et al., Expert Rev Anti Infect Ther. (2014) 12:1103-1135; Stricker et al., Am J Infect Dis (2018) 14:1-44]. Patients were not required to have a documented tick bite or erythema migrans rash to participate in the study, because serological tests were used to detect exposure and not active infection. De-identified patient samples were coded according to the patient's place of residence. Blood was collected and serum was collected and analyzed by independent laboratories (BioReference®, LabCorp®, and AnyLabTestNow®). ), and serum samples were transported to a reference laboratory for immunoblot testing.

[0071] Control patients (n=175) A total of 175 human sera predicted to be negative for B. burgdorferi and TBRF Borrelia species were obtained from the Centers for Disease Control and Prevention (CDC, Atlanta, GA), the College of American Pathologists, the New York State Department of Health, New York Biologics (Southampton, NY, USA), and the IGeneX Reference Laboratory (Milpitas, CA, USA). The IGeneX samples were remnants of sera received for routine testing for tick-borne diseases and would have been discarded if not used in this study. Immunoblot IgM and IgG testing of patient and control sera was performed in a blinded fashion by laboratory technicians in the same format as testing of clinical samples from TBRF patients. The results are shown in Tables 1-2 and Figures 2-3.

[0072] result Immunoblot reactivity of TBRF Borrelia in representative patient serum samples is shown in Figure 2, demonstrating that the TBRF immunoblot was successful in detecting TBRF Borrelia group antibodies in serum samples.

[0073] Results obtained with 175 control sera expected to be negative for B. burgdorferi and RFB resulted in false-positive rates of 2.3% (4 / 175 samples) for B. burgdorferi immunoblots and 2.9% (5 / 175 samples) for RFB immunoblots (Table 1). False-positive tests for RFB were seen in allergy patient sera (one control), multiple sclerosis (one control), viral infection (one control), and syphilis (two controls).

[0074] Using the TBRF immunoblot described in Example 1, which includes SEQ ID NOs: 1-11, testing of patients who met the CLD case definition revealed that all had been exposed to B. burgdorferi species, with 62% having been exposed to TBRF species. In the CLD cohort (Table 2), immunoblot testing revealed that out of 90 subjects suspected of LD, a total of 48 patients (53%) were seropositive for TBRF only, and 8 patients (9%) were co-infected with TBRF Borrelia species and B. burgdorferi species. Figure 2 shows a TBRF immunoblot of a representative patient serum sample. Positive immunoblots were further characterized for the presence of antibodies to one or more RFB species, identifying cases infected with more than one RFB species (Table 2). Immunoblot testing of control sera demonstrated excellent specificity of 97.1% for the TBRF assay (Table 2). As shown in Figure 3, various combinations of SEQ ID NOs: 1-11 were identified in patient serum samples. This demonstrates that comprehensive testing requires antigens from multiple species.

[0075] These results illustrate how detection of antibodies to TBRF Borrelia species identified patients with chronic Lyme disease symptoms that would be missed by testing only for Borrelia burgdorferi antigens. These results also confirm the complexity of the serovar composition of TBRF Borrelia exposures, thereby demonstrating the power and importance of comprehensiveness in TBRF testing, i.e., testing antigens from multiple TBRF species simultaneously.

[0076] [Table 1-1] [Table 1-2]

[0077] [Table 2]

[0078] Considerations of Examples 1 and 2 TBRF are found worldwide and are a significant cause of morbidity and mortality, especially for fever cases of unknown etiology. Advances in molecular testing have led to the widespread identification of Borrelia species in human specimens, thus challenging prevailing ideas regarding the geographic distribution and prevalence of RFB infections. Although RFB is an increasing concern in the Western United States, Central America, and South America, TBRF is not nationally reportable in the United States and no standard case definition exists. In 2011, TBRF was reportable in 12 Western states with 504 cases. Seventy percent of reported cases were contained in three states: California (33%), Washington (25%), and Colorado (11%). In the United States, pathogenic species include B. miyamotoi, B. hermsii, B. ronestalis, and B. moniliforme. , B. parkeri, B. tsurikatae, and B. mazzotii. Most cases in the United States are due to B. hermsii, transmitted by Ornithodoros hermsi ticks, but human infections with B. miyamotoi, B. hermsii, and B. parkeri have been reported in California, where B. coriaceae has been detected in ticks, although no human infections have been confirmed [Fesler et al. Healthcare 8(2): 97-112 (2020)].

[0079] The genetic diversity of Borrelia spirochetes and infectious disease symptoms that are as diverse as the organisms that cause infections make the diagnosis of Borrelia-associated diseases difficult. In summary, exposure to TBRF Borrelia is a cause for concern, and TBRF Borrelia can explain the Lyme disease symptoms in patients who are seronegative for B. burgodorferi sensu lato. As shown herein, some patients can demonstrate dual exposure to both B. burgodorferi and TBRF species, further complicating diagnosis and treatment. Immunoblot testing of RFB species using the methods and compositions of the present invention allows for the detection of a diverse group of TBRF Borrelia serovars, providing a deeper understanding of human exposure to pathogenic Borrelia.

[0080] Equivalent Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily contemplate various other means and / or structures for performing the functions and / or results and / or obtaining one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) utilizing the teachings of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. It should therefore be understood that the above-described embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, and / or methods is included within the scope of the present invention, unless such features, systems, articles, materials, and / or methods are mutually inconsistent.

[0081] All definitions herein and as used should be construed to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0082] The indefinite articles "a" and "an," as used herein in the specification and claims, shall be construed to mean "at least one," unless expressly stated to the contrary. The term "and / or," as used herein in the specification and claims, shall be construed to mean "either or both" of the elements so conjoined, i.e., the elements are present jointly in some cases and separately in other cases. Other elements than the elements specifically identified by the "and / or" clause may be optionally present, whether or not related to such specifically identified elements, unless expressly stated to the contrary.

[0083] All references, patents, and patent applications, and publications cited or referred to in this application are hereby incorporated by reference in their entirety. , which is incorporated herein by reference in its entirety.

Claims

1. One or more labeled and / or tagged and / or conjugated amino acid sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:11, and variants thereof having at least 90% homology to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:11; or two or more labelled and / or tagged and / or conjugated amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, and variants thereof having at least 90% homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, A composition comprising any one of the following:

2. 10. The composition of claim 1, wherein the amino acid sequence is attached to a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride (PVDF), magnetic beads, and agarose.

3. The composition of claim 1 , wherein each of the amino acid sequences is tagged with an anti-human IgG or IgM antibody having specificity for the amino acid sequence.

4. A method for detecting tick-borne relapsing fever (TBRF) antibodies resulting from infection with one or more members of the genus Borrelia, when present in a biological sample from a subject suspected of having TBRF, the method comprising: Providing a biological sample from said subject suspected of having TBRF; mixing the biological sample with the composition of claim 1; and detecting a positive immune binding reaction indicative of the presence of TBRF-specific antibodies in said biological sample; The method comprising:

5. The method described in claim 4, wherein the biological sample is considered TBRF positive if at least two types of amino acid sequences are detected.

6. The method of claim 4, wherein the labeled and / or tagged and / or bound amino acid sequence is detected with an anti-human IgG antibody or an anti-human IgM antibody linked to a detectable moiety.

7. The method of claim 6 , wherein the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme.

8. The method of claim 6 , wherein the detectable moiety comprises alkaline phosphatase.

9. The method of claim 6 , wherein the detectable moiety comprises biotin.

10. The composition described in claim 1, wherein the variant has at least 95% homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

11.

11. The composition described in claim 1, wherein the variant has at least 98% homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

11.

12. The composition described in claim 1, wherein the variant has at least 99% homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

11.

13. The composition described in claim 1, wherein the variant has at least 99.5% homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

11.

14. The composition described in claim 1, wherein the variant has one or more conservative amino acid substitutions.

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