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WO2018017998A1 - Recombinant borrelia proteins and methods of use thereof - Google Patents

Recombinant borrelia proteins and methods of use thereof Download PDF

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Publication number
WO2018017998A1
WO2018017998A1 PCT/US2017/043366 US2017043366W WO2018017998A1 WO 2018017998 A1 WO2018017998 A1 WO 2018017998A1 US 2017043366 W US2017043366 W US 2017043366W WO 2018017998 A1 WO2018017998 A1 WO 2018017998A1
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protein
recombinant borrelia
borrelia
ospc
recombinant
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PCT/US2017/043366
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French (fr)
Inventor
Benjamin J. Luft
John F. Bruno
Yun Xu
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The Research Foundation For The State University Of New York
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Publication of WO2018017998A1 publication Critical patent/WO2018017998A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira

Definitions

  • Lyme disease is the most common vector-borne disease in North America and Europe, and its range and incidence are increasing. Human Lyme disease is caused by several members of a group of closely related spirochetes belonging to the Borrelia burgdorferi sensu lato species complex. The spirochete is transmitted to humans via ticks of the genus Ixodes (Steere, A. C, N. Engl. J.Med. 1989; 321 :586-96). It is a progressive multisystem disorder characterized by an initial cutaneous infection that can spread early in infection to secondary sites that include the nervous system, heart and joints (Masuzawa, T. et al, Microbiol. Immunol.
  • the present invention encompasses novel recombinant Borrelia burgdorferi proteins and their use in highly sensitive and specific methods of diagnosing Lyme disease in a subject.
  • the present invention is drawn to recombinant Borrelia fusion protein constructs comprising more than one ⁇ e.g., two or three) single recombinant Borrelia protein antigens, such as those described herein.
  • These constructs are also referred to herein as Borrelia chimeric proteins, or chimeric Borrelia proteins, or Borrelia chimeras, or recombinant Borrelia burgdorferi fusion protein constructs, or recombinant Borrelia fusion protein constructs, or recombinant Borrelia fusion proteins, or recombinant Borrelia proteins, or fusion proteins, or chimeric proteins.
  • Examples of recombinant Borrelia fusion protein constructs are described in Tables 2 and 3 herein.
  • the recombinant Borrelia fusion protein constructs of the present invention can have only one single recombinant Borrelia protein antigen described herein ⁇ e.g., any one of the single recombinant Borrelia protein antigens listed in Table 1, Table 2 or Table 3).
  • the present invention is also drawn to methods of using one or more or all of these recombinant Borrelia fusion protein constructs in assays for assessing test samples for detecting the presence of antibodies to Borrelia burgdorferi in the test sample.
  • the Borrelia fusion protein constructs described herein ⁇ e.g., Tables 2 and 3
  • protein arrays ⁇ e.g., microarrays, Q-Plex Arrays
  • ELISA enzyme-Plex Arrays
  • one or more additional single recombinant Borrelia antigens can be included in such assays ⁇ e.g., microarrays, Q-Plex Array), such as the single recombinant Borrelia antigens listed in Table 1 (see PCT Publication WO 2016/057562, the entire teachings of which are incorporated herein by reference).
  • the microarray and/or Q-Plex Array comprises the 6 recombinant Borrelia fusion protein constructs selected from Table 2.
  • the microarray and/or Q-Plex Array comprises the 5 recombinant Borrelia fusion protein constructs selected from Table 3.
  • the microarray and/or Q-Plex Array comprises the 11 recombinant Borrelia fusion protein constructs of Tables 2 and 3 (e.g., SEQ ID NOS 6-16), and/or subsets thereof.
  • the microarray and/or Q- Plex Array comprises 3 single recombinant Borrelia protein antigens described in Table 2 (e.g., Decorin binding protein A (dbpA), Decorin binding protein B (dbpB) and ABC transporter substrate binding protein (OppA)).
  • the microarray and/or Q-Plex Array comprises recombinant Borrelia fusion protein constructs comprising the three single recombinant Borrelia protein antigens described in Table 2 (e.g., SEQ ID NOS 6-11).
  • the microarray and/or Q-Plex Array comprises 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., Outer surface protein A (OspA), purine-binding chemotaxis protein, VMP-like sequence protein VlsE(Vls), p83/100 antigen, surface lipoprotein P27, Outer surface protein C (OspC) typeB, Outer surface protein C (OspC) typeK and BapA protein).
  • OspA Outer surface protein A
  • VlsE(Vls) VMP-like sequence protein
  • p83/100 antigen e.g., surface lipoprotein P27
  • OspC Outer surface protein C
  • OspC Outer surface protein C
  • BapA protein BapA protein
  • the microarray and/or Q-Plex Array comprises recombinant Borrelia fusion protein constructs comprising the 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., SEQ ID NOS 6-11).
  • a subject such as a mammal, including a human
  • methods of diagnosing Lyme disease in a subject by assessing a test sample obtained from the subject for antibodies reactive with one or more of the recombinant Borrelia
  • burgdorferi fusion protein constructs selected from Tables 2 or 3, or, additionally one or more of the single recombinant Borrelia antigens listed in Table 1, wherein the detection of antibody- antigen reactions (such as antibody-antigen bound complexes, also referred to herein as antibody-antigen reaction products) is indicative of Lyme disease in the individual.
  • antibody- antigen reactions such as antibody-antigen bound complexes, also referred to herein as antibody-antigen reaction products
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise the recombinant Borrelia fusion protein constructs described herein as in Tables 2 and 3, and/or subsets thereof, and also one or more single recombinant Borrelia antigens described in Table 1.
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise the 6 recombinant Borrelia fusion protein constructs selected from Table 2.
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 5 recombinant Borrelia fusion protein constructs selected from Table 3.
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 11 recombinant Borrelia fusion protein constructs of Tables 2 and 3 ⁇ e.g., SEQ ID NOS 6-16), and/or subsets thereof.
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 3 single recombinant Borrelia protein antigens described in Table 2 ⁇ e.g., Decorin binding protein A (dbpA), Decorin binding protein B (dbpB).and ABC transporter substrate binding protein (OppA)).
  • dbpA Decorin binding protein A
  • dbpB Decorin binding protein B
  • OppA ABC transporter substrate binding protein
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q- Plex Arrays comprise recombinant Borrelia fusion protein constructs comprising the 3 single recombinant Borrelia protein antigens described in Table 2 ⁇ e.g., SEQ ID NOS 6-11).
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., Outer surface protein A
  • microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise recombinant Borrelia fusion protein constructs comprising the 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., SEQ ID NOS 6-11).
  • a highly antigenic Borrelia protein an ABC transporter substrate-binding protein (OppA) (Signorino G et.al.,) that is recognized by approximately 50% of all sera samples analyzed including sera from patients with early Lyme disease, was used to create recombinant Borrelia fusion protein constructs.
  • OppA ABC transporter substrate-binding protein
  • OppA along with the library of 48 other highly antigenic single recombinant Borrelia protein antigens shown in Table 1, was used to develop a set of recombinant Borrelia fusion protein constructs as shown in Tables 2 and 3 and a highly sensitive and specific recombinant protein-based assay containing the 11 recombinant Borrelia fusion protein constructs selected from Table 2 and/or 3 and/or subsets thereof, as described herein.
  • the multiplex ELISA array measures multiple proteins simultaneously within a single tissue sample.
  • the Q-PlexTM technology using the Q-Plex Array is a multiplex ELISA platform that can be used to develop a simple yet accurate tool to quantitate a panel of diagnostic proteins in biologic specimens that can be readily implemented into a clinical laboratory setting.
  • Q-Plex Arrays containing proteins encoded by recombinant Borrelia fusion protein constructs (for, example, those described in Tables 2 and 3) were prepared.
  • the assay using Q-Plex technology and the use of Q-Plex Array for improving Lyme disease diagnosis are described in Example 3 of this application.
  • the Q-Plex Array was exposed to sera from individuals previously diagnosed with disseminated Lyme disease. Results indicated that the sera of individuals with Lyme disease reacted with the highly immunogenic recombinant Borrelia fusion protein constructs, shown in Tables 2 and 3. The overall sensitivity and specificity of the assay using the Q-Plex Array are also described in Example 3 of this application.
  • one or more single recombinant Borrelia protein antigens are used.
  • single recombinant Borrelia protein antigens including but not limited to, cell envelope protein antigens, recombinant lipoprotein antigens, extracellular protein antigens, recombinant antigens described herein (for, example, those described in Tables 1, 2 and 3) can be used.
  • a set of two or more ⁇ e.g., three) single recombinant Borrelia protein antigens are used.
  • sets include the set of single recombinant Borrelia protein antigens shown in Table 1, Table 2 and/or Table 3. In certain embodiments, a set of two or more ⁇ e.g., three) single recombinant Borrelia protein antigens are used.
  • Other example sets of single recombinant Borrelia protein antigens include the set of all known and putative cell envelope proteins and /or lipoproteins of B. burgdorferi. Such a set can further include homologs and paralogs of cell envelope lipoproteins and extra-cellular proteins.
  • sets include sets of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or other groups of cell envelope proteins ⁇ e.g., selected from those set forth in Table 2 and/or 3 and additional proteins in Table 1).
  • the set consists essentially of the single recombinant Borrelia protein antigens set forth in Table 1.
  • the set consists essentially of the single recombinant Borrelia protein antigens set forth in Table 2 (for, example, those described under column "gene locus” in Table 2).
  • the set consists essentially of the single recombinant Borrelia protein antigens set forth in Table 3 (for, example, those described under column "gene locus” in Table 3).
  • one or more recombinant Borrelia fusion protein constructs were used.
  • recombinant Borrelia fusion protein constructs including but not limited to, SEQ ID NOS: 6-16 described herein ⁇ e.g., in Tables 2 and 3) can be used.
  • Representative sets include the recombinant Borrelia fusion protein constructs shown in Table 2, and/or Table 3, and subsets thereof. In certain embodiments, only one recombinant Borrelia fusion protein construct was used.
  • two or more ⁇ e.g., three) recombinant Borrelia fusion protein constructs are used.
  • Other example sets of recombinant Borrelia fusion protein constructs include the set of all possible combinations of recombinant Borrelia fusion protein constructs shown in Table 2, and/or Table 3, and subsets thereof.
  • Such a set can further include recombinant Borrelia fusion protein constructs further comprising one or more single recombinant Borrelia protein antigens shown in Table 1.
  • sets include sets of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or other groups of recombinant Borrelia fusion protein constructs ⁇ e.g., selected from those set forth in Table 2 and/or 3 and optionally can include one or more single recombinant Borrelia protein antigens shown in Table 1).
  • the set consists essentially of the recombinant Borrelia fusion protein constructs set forth in Table 2.
  • the set consists essentially of the recombinant Borrelia fusion protein constructs set forth in Table 3.
  • one or more single recombinant Borrelia protein antigens and recombinant Borrelia fusion protein constructs as identified herein are used, and/or combinations of single recombinant Borrelia protein antigens and recombinant Borrelia fusion protein constructs are used ⁇ e.g., combinations of proteins selected from any of Tables 1, 2 and/or 3).
  • Such combinations include sets of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or other groups of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs ⁇ e.g., with one or more selected from Table 1, Table 2 and/or Table 3).
  • a test sample from an individual ⁇ e.g., a human
  • the "test sample” can be a sample of blood, serum, cerebrospinal fluid, or other appropriate biological fluid from the individual.
  • the test sample can be assessed for the presence of antibodies to one or more single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs using routine methods established in the art.
  • the assessment is performed using a microarray of single recombinant Borrelia protein antigens. In another embodiment, the assessment is performed using a microarray of recombinant Borrelia fusion protein constructs. In certain examples, a microarray as described herein, or a protein or set of proteins as described herein, is exposed to the test sample from the individual, and any resultant binding of antibodies (if present in the test sample) to the single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs was determined.
  • the assessment is performed using an ELISA array, such as a multiples ELISA array (e.g., Q-Plex Array) of single recombinant Borrelia protein antigens.
  • the assessment is performed using a Q-Plex Array of recombinant Borrelia fusion protein constructs.
  • a Q-Plex Array as described herein, or a protein or set of proteins as described herein is exposed to the test sample from the individual or multiple samples from the same and/or multiple individuals, and any resultant binding of antibodies (if present in the test sample (s)) to the single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs is determined.
  • antibodies bound to also referred to herein as "reactive with” one or more single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs is indicative of antibodies specific to those proteins of B. burgdorferi.
  • the presence of such antibodies is diagnostic for Lyme disease in the individual from whom the test sample was obtained.
  • the assay methods described herein use protein arrays of B. burgdorferi antigens.
  • the protein array comprises a subset of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B. burgdorferi, such as the set of proteins set forth in Tables 1, 2 or 3.
  • other protein arrays include various subsets of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B.
  • the microarray consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 2, or consists of all of the recombinant Borrelia fusion protein constructs set forth in Table 2.
  • the microarray consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 3, or consists of all of the recombinant Borrelia fusion protein constructs of Table 3.
  • other microarrays include various subsets of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B. burgdorferi, such as subsets of the proteins set forth in Table 1, 2 and 3 (e.g., sets of two or more, four or more, six or more, eight or more, or other combinations of proteins).
  • the Q-Plex Array consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 2, or consists of all of the recombinant Borrelia fusion protein constructs set forth in Table 2.
  • the Q-Plex Array consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 3, or consists of all of the recombinant Borrelia fusion protein constructs of Table 3.
  • other Q-Plex Arrays include various subsets of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B. burgdorferi, such as subsets of the proteins set forth in Table 1, 2 and 3 (e.g., sets of two or more, four or more, six or more, eight or more, or other combinations of proteins).
  • one or more proteins for example, one or more recombinant Borrelia fusion protein constructs of B.
  • burgdorferi such as described herein in Table 2 or Table 3 (e.g., in a microarray and /or Q-Plex Array as described above), are exposed to sera from one or more individuals known to have Lyme disease, and the proteins to which antibodies from the sera bind are then determined.
  • the proteins to which antibodies from the sera bind are then determined.
  • Cy5 intensity/Cy3 intensity ratio of fluorescence as described in the Examples described herein, can be used.
  • the ratio of any proteins greater than the mean ratio of the reactivity of the Lyme sera to a negative control plus three times the standard deviation indicates significant interactions between antibodies present in the Lyme sera and the proteins (e.g., single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs).
  • the proteins identified by such methods are useful in diagnostic tests for Lyme disease (e.g., in the methods described herein), as well as in protein arrays (for, e.g., microarrays, Q-Plex Arrays) as described herein.
  • the present invention is also drawn to diagnostic kits which comprise the proteins described herein (e.g., in a microarray and /or in a Q-Plex Array as described herein).
  • the kit optionally can include reagents for detecting antibody-antigen complexes that are formed between the protein and antibodies that are present in a sample, e.g., a user-supplied host sample.
  • EXAMPLE 1 Discovery of 48 highly antigenic single recombinant Borrelia protein antigens.
  • expressed proteins were purified using IDA resin and printed onto nitrocellulose-coated FAST slides (see, for example, US Serial No. 12/784,584 corresponding to published US Application No. US 20100292096 Aland US Serial No. 12/989,003 corresponding to published US
  • OspC Outer surface protein
  • Arrays were probed with sera from Lyme disease patients, and antibodies were visualized with Cy5-conjugated goat antihuman lgG or Cy5-conjugated goat anti-human lgM to determine antibody isotype (Xu Y et al., 2008).
  • Microarray For protein array ⁇ e.g., microarray), each of the individual 48 single recombinant Borrelia protein antigens ⁇ e.g., in Table 1) were printed onto nitrocellulose-coated FAST glass slides. In addition, recombinant proteins OspB-OspC-Flagellin (B-C-Fia), OspA- p39-p93 (A-39-93) and an OspC dimer comprised of OspC Type B and Type H (OC2/9) from an early study were also printed (Gomes Solecki et al., 2000). Each slide in the arrays also contained 10 immobilized bovine serum albumin (BSA) spots for background determination.
  • BSA bovine serum albumin
  • Proteome chips were probed with serum from patients with untreated early Lyme disease and sick non-Lyme patients using the Fast Pak protein array kit. Briefly, slides were first blocked overnight at 4°C in protein array -blocking buffer before incubation in primary antibody (human sera and mouse anti His-Tag for quantitation) for 2 h. Antibodies were visualized with Cy5- conjugated goat anti-human lgG or Cy 5 -conjugated goat anti-human lgM (to detect bound human antibodies) and Cy 3 -conjugated goat anti mouse lgG (to quantify the amount of recombinant protein in each spot).
  • the slides were stringently washed and then scanned with an Axon GenePix 4200A microarray scanner.
  • the raw data was captured and analyzed with Gene Pix Pro image analysis software.
  • the PMT gain was adjusted to equal 1.0 in all the arrays with power setting at 50%.
  • a global background subtraction method was used to subtract the background from each spot using the average mean intensity value of BSA from each slide (Xu et al., 2008).
  • Table 1 48 highly antigenic single recombinant Borrelia protein antigens.
  • EXAMPLE 2 Recombinant Borrelia fusion protein constructs.
  • Recombinant Borrelia fusion protein constructs From the library of 48 highly antigenic single recombinant Borrelia protein antigens and a representative lipoprotein, OppA, 11 sets of recombinant Borrelia fusion protein constructs each containing at least two of the Borrelia antigens were constructed (Tables 2 and 3). Chimeras of at least two ⁇ e.g., three) single recombinant Borrelia protein antigens in most constructs were chosen to keep the molecular mass of the fusion protein at or less than (below) about 100 kDa to optimize protein yield, which can be significantly affected by protein size.
  • the 5' primer (5'-AATTGGTACCCCAGGATCCCATATG + 15mer ORF specific sequence) (SEQ ID NO: 1) contains Kpnl (underlined), BamHI (bold) and Ndel (italics) recognition sequence.
  • the 3' primer (5'GCGGGATCCGGTACCGTCGAC +15mer ORF specific sequence) (SEQ ID NO:2) contains BamHI, Kpnl and Sail (dashed underline) recognition sequences. GGATCC is bolded in the 5' and 3' primers described above.
  • ten ng of genomic DNA was used as a template in a 50- ⁇ PCR reaction containing two ORF-specific primer pairs (SEQ ID NOS: 1&2).
  • the primer sets were designed to amplify coding regions without a membrane anchoring signal sequence (Dunn et al., 1990). PCR amplification was performed under stringent conditions with Platinum Taq DNA polymerase High Fidelity (Invitrogen) using conditions previously described (Xu et al., 2003). The PCR products were visualized using agarose gel electrophoresis. For quantification, the products were purified (PCR purification kit, Qiagen) and quantified by UV absorbance.
  • the 1 st position fragments were directionally cloned into the unique Ndel and BamHI sites of the T7- based expression vector pET-28 (Novagen).
  • This vector provides an N-terminal poly (His) affinity tag fused to the expressed proteins to aid in purification on nickel-Sepharose columns.
  • Ligation reactions were transformed into E. coli GC5 competent cells and plasmids were purified using Eppendorf Perfectprep Plasmid 96 VAC Direct Bind Kit and verified by sequencing across the inserts. Plasmids containing fragment 1 now served as vectors for all subsequent cloning.
  • vectors were digested with BamHI/XhoIl, and restriction digested amplicons of fragment two and three were ligated simultaneously into the digested vector. Following transformation, plasmids were purified and sequenced.
  • Protein concentration was determined by the measurement of the absorbance shift when Coomassie brilliant blue G-250 reacted with protein (Bio-Rad). Protein purity was checked by SDS-PAGE.
  • EXAMPLE 3 Protein arrays using recombinant Borrelia fusion protein constructs and Diagnostic Test for Lyme Disease.
  • Lyme sera and a number of the control sera were used in previous studies described in Example 1 and the array studies ⁇ e.g., Q-Plex Arrays) described below. All serological assays involving human serum were performed on coded specimens with all investigators blinded to clinical information pertaining to individual samples. No patient identifying information was available to the investigators or technician testing the samples.
  • Q-Plex technology The multiplex assay using Q-Plex technology involves the micro-spotting of individual groups of capture proteins on the bottom of a 96-well plate with each spot being its own 'micro ELISA' assay. Standard ELISA incubation steps including sample incubation, washing, and secondary antibody incubation were employed. The labeling and reporting system used in the Q-Plex Array was chemiluminescent. The binding of secondary antibody was measured via the chemiluminescence produced by streptavidin horseradish peroxidase in the presence of a luminol-based substrate. Chemiluminescent ELISAs have been shown to be more sensitive than colorimetric detection systems. The intensity of
  • Q-Plex Arrays Protein arrays ⁇ e.g., Q-Plex Arrays) comprising the 11 highly antigenic recombinant Borrelia proteins described in Table 2 and 3, were constructed. The sensitivity of Q-Plex Array was tested by screening arrays with sera from patients with early and late Lyme disease using buffers and methods provided by Quansys Biosciences. In addition, specificity was determined using sera from patients with diseases associated with serological responses that are known to produce cross-reactivity in Lyme disease tests. The results of Q- Plex arrays were compared with the results obtained with the standard two-tier assay for the same set of 11 recombinant Borrelia fusion protein constructs.
  • the sensitivity of the Q-Plex Arrays was determined using sera from 97 Lyme disease patients. Of 41 acute-phase sera from patients presenting with erythema migrans, 83% (34/41) were positive with the Q-Plex Arrays while 37% (15/41) were positive by the two- tier test. Testing 40 serum samples from EM patients, convalescent at 3 weeks post-presentation, 90%) (36/40) were positive by the Q-Plex Arrays while 78% (31/40) were positive by the two-tier assay. Assaying 16 late Lyme serum samples, 100 % (16/16) were positive by the Q-Plex Arrays while 94%) (15/16) were positive by two-tier testing. Overall, the sensitivity of the Q-Plex Arrays was 89% (86/97) compared to 63% (60/97) for the two-tier test.
  • results described above demonstrate that Q-Plex Arrays offer superior sensitivity and specificity in detecting antibodies against B. burgdorferi for not only early stages of the disease, but show equivalent or better sensitivity for the late stages of the disease as well.
  • the recombinant Borrelia fusion protein constructs described herein represent strong candidates for the development of diagnostic tests.
  • These variants, as well as variants of the other recombinant Borrelia fusion protein constructs described herein can be used to develop a standardized sensitive and specific single-tier Lyme disease assay of recombinant Borrelia fusion protein constructs that will have a wide range of coverage and specificity against Lyme disease. Furthermore, these assays will have significant potential for the development of a next-generation rapid, single-tier point of care assay.

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Abstract

Novel Borrelia burgdoferi recombinant proteins and methods of assessing a sample for the presence of antibodies to proteins of Borrelia burgdorferi are described, as are methods of diagnosing Lyme disease. Protein arrays comprising Borrelia burgdorferi proteins are also described.

Description

RECOMBINANT BORRELIA PROTEINS AND METHODS OF USE THEREOF
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 62/365,937, filed on July 22, 2016. The entire teachings of the above application are incorporated herein by reference.
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant No. CK000150 from Centers for Disease Control (CDC). The government has certain rights in the invention.
BACKGROUND
[0003] Lyme disease is the most common vector-borne disease in North America and Europe, and its range and incidence are increasing. Human Lyme disease is caused by several members of a group of closely related spirochetes belonging to the Borrelia burgdorferi sensu lato species complex. The spirochete is transmitted to humans via ticks of the genus Ixodes (Steere, A. C, N. Engl. J.Med. 1989; 321 :586-96). It is a progressive multisystem disorder characterized by an initial cutaneous infection that can spread early in infection to secondary sites that include the nervous system, heart and joints (Masuzawa, T. et al, Microbiol. Immunol. 1996; 40:539-45; Stanek, G., Infection 1991; 19:263-7). Accurate, reliable diagnostic assays for Lyme disease are critical to ensure successful treatment and recovery. Improved assays for accurate and reliable diagnosis of Lyme disease are needed to identify patients with Lyme disease.
SUMMARY
[0004] The present invention encompasses novel recombinant Borrelia burgdorferi proteins and their use in highly sensitive and specific methods of diagnosing Lyme disease in a subject. In one embodiment, the present invention is drawn to recombinant Borrelia fusion protein constructs comprising more than one {e.g., two or three) single recombinant Borrelia protein antigens, such as those described herein. These constructs are also referred to herein as Borrelia chimeric proteins, or chimeric Borrelia proteins, or Borrelia chimeras, or recombinant Borrelia burgdorferi fusion protein constructs, or recombinant Borrelia fusion protein constructs, or recombinant Borrelia fusion proteins, or recombinant Borrelia proteins, or fusion proteins, or chimeric proteins. Examples of recombinant Borrelia fusion protein constructs are described in Tables 2 and 3 herein. In other embodiments, the recombinant Borrelia fusion protein constructs of the present invention can have only one single recombinant Borrelia protein antigen described herein {e.g., any one of the single recombinant Borrelia protein antigens listed in Table 1, Table 2 or Table 3).
[0005] The present invention is also drawn to methods of using one or more or all of these recombinant Borrelia fusion protein constructs in assays for assessing test samples for detecting the presence of antibodies to Borrelia burgdorferi in the test sample. For example, the Borrelia fusion protein constructs described herein {e.g., Tables 2 and 3) can be used to prepare protein arrays {e.g., microarrays, Q-Plex Arrays), for use in the detection methods, as well as in ELISA, other immunological assays, or any assay method capable of detecting antibody-antigen binding or antibody-antigen binding complexes. In addition to the use of the Borrelia fusion protein constructs of Tables 2 and 3 in these detection assays, one or more additional single recombinant Borrelia antigens can be included in such assays {e.g., microarrays, Q-Plex Array), such as the single recombinant Borrelia antigens listed in Table 1 (see PCT Publication WO 2016/057562, the entire teachings of which are incorporated herein by reference). In one embodiment of the present invention, the microarray and/or Q-Plex Array comprises the 6 recombinant Borrelia fusion protein constructs selected from Table 2. In another embodiment, the microarray and/or Q-Plex Array comprises the 5 recombinant Borrelia fusion protein constructs selected from Table 3. In another embodiment, the microarray and/or Q-Plex Array comprises the 11 recombinant Borrelia fusion protein constructs of Tables 2 and 3 (e.g., SEQ ID NOS 6-16), and/or subsets thereof. In one embodiment of the present invention, the microarray and/or Q- Plex Array comprises 3 single recombinant Borrelia protein antigens described in Table 2 (e.g., Decorin binding protein A (dbpA), Decorin binding protein B (dbpB) and ABC transporter substrate binding protein (OppA)). In another embodiment of the present invention, the microarray and/or Q-Plex Array comprises recombinant Borrelia fusion protein constructs comprising the three single recombinant Borrelia protein antigens described in Table 2 (e.g., SEQ ID NOS 6-11). In one embodiment of the present invention, the microarray and/or Q-Plex Array comprises 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., Outer surface protein A (OspA), purine-binding chemotaxis protein, VMP-like sequence protein VlsE(Vls), p83/100 antigen, surface lipoprotein P27, Outer surface protein C (OspC) typeB, Outer surface protein C (OspC) typeK and BapA protein). In another embodiment of the present invention, the microarray and/or Q-Plex Array comprises recombinant Borrelia fusion protein constructs comprising the 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., SEQ ID NOS 6-11).
[0006] Further encompassed by the present invention are methods of diagnosing Lyme disease in a subject (such as a mammal, including a human) by assessing a test sample obtained from the subject for antibodies reactive with one or more of the recombinant Borrelia
burgdorferi fusion protein constructs selected from Tables 2 or 3, or, additionally one or more of the single recombinant Borrelia antigens listed in Table 1, wherein the detection of antibody- antigen reactions (such as antibody-antigen bound complexes, also referred to herein as antibody-antigen reaction products) is indicative of Lyme disease in the individual. Such assays to detect antibody-antigen complexes are well-known to those of skill in the art. In a particular embodiment of the present invention, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise the recombinant Borrelia fusion protein constructs described herein as in Tables 2 and 3, and/or subsets thereof, and also one or more single recombinant Borrelia antigens described in Table 1. In one embodiment of the present invention, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise the 6 recombinant Borrelia fusion protein constructs selected from Table 2. In another embodiment, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 5 recombinant Borrelia fusion protein constructs selected from Table 3. In another embodiment, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 11 recombinant Borrelia fusion protein constructs of Tables 2 and 3 {e.g., SEQ ID NOS 6-16), and/or subsets thereof. In one embodiment of the present invention, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 3 single recombinant Borrelia protein antigens described in Table 2 {e.g., Decorin binding protein A (dbpA), Decorin binding protein B (dbpB).and ABC transporter substrate binding protein (OppA)). In another embodiment of the present invention, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q- Plex Arrays comprise recombinant Borrelia fusion protein constructs comprising the 3 single recombinant Borrelia protein antigens described in Table 2 {e.g., SEQ ID NOS 6-11). In one embodiment of the present invention, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., Outer surface protein A
(OspA), purine-binding chemotaxis protein, VMP-like sequence protein VlsE(Vls), p83/100 antigen, surface lipoprotein P27, Outer surface protein C (OspC) typeB, Outer surface protein C (OspC) typeK and BapA protein). In another embodiment of the present invention, microarrays and/or Q-Plex Arrays are used in the detection/diagnostic assays, wherein the microarrays and/or Q-Plex Arrays comprise recombinant Borrelia fusion protein constructs comprising the 8 single recombinant Borrelia protein antigens described in Table 3 (e.g., SEQ ID NOS 6-11).
[0007] As a result of this discovery, highly sensitive and specific methods and microarrays and /or Q-Plex Arrays are now available for the assessment of a test sample for the presence of antibodies to proteins of Borrelia. The presence of such antibodies is diagnostic for Lyme disease. The present invention provides methods to identify potential antigenic candidates for use in diagnosis of Lyme disease.
DETAILED DESCRIPTION OF THE INVENTION
[0008] A description of example embodiments of the invention follows. Accurate diagnosis and treatment of Lyme disease depends on correlating objective clinical abnormalities with serological evidence of exposure to B. burgdorferi. However, serodiagnosis of the disease is particularly difficult due to the high level of genetic heterogeneity of B. burgdorferi isolates, even among those collected from a single location. Furthermore, current serodiagnostic tests for Lyme disease lack sensitivity and specificity for detecting B. burgdorferi infection in the early stages of the disease. In one embodiment of the present invention, a highly antigenic Borrelia protein, an ABC transporter substrate-binding protein (OppA) (Signorino G et.al.,) that is recognized by approximately 50% of all sera samples analyzed including sera from patients with early Lyme disease, was used to create recombinant Borrelia fusion protein constructs. OppA, along with the library of 48 other highly antigenic single recombinant Borrelia protein antigens shown in Table 1, was used to develop a set of recombinant Borrelia fusion protein constructs as shown in Tables 2 and 3 and a highly sensitive and specific recombinant protein-based assay containing the 11 recombinant Borrelia fusion protein constructs selected from Table 2 and/or 3 and/or subsets thereof, as described herein.
[0009] It has been discovered that antibodies to certain cell envelope proteins, lipoproteins and extra-cellular proteins are present in sera of individuals with disseminated Lyme disease. A microarray containing multiple cell envelope proteins, lipoproteins, extracellular proteins and their corresponding homologs was prepared. The microarray was exposed to sera from individuals previously diagnosed with disseminated Lyme disease. Results indicated that the sera of individuals with Lyme disease reacted with single recombinant Borrelia protein antigens, shown in Table 1. In particular, high numbers of the sera from the individuals reacted with a specific subset of those proteins. The sensitivity and specificity of the Lyme disease assay are described in Tables 2 and 3 of the PCT Publication WO 2016/057562, the entire teachings of which are incorporated herein by reference.
[0010] The multiplex ELISA array measures multiple proteins simultaneously within a single tissue sample. The Q-Plex™ technology using the Q-Plex Array (Quansys Bioscience, Logan, UT) is a multiplex ELISA platform that can be used to develop a simple yet accurate tool to quantitate a panel of diagnostic proteins in biologic specimens that can be readily implemented into a clinical laboratory setting. Q-Plex Arrays containing proteins encoded by recombinant Borrelia fusion protein constructs (for, example, those described in Tables 2 and 3) were prepared. The assay using Q-Plex technology and the use of Q-Plex Array for improving Lyme disease diagnosis are described in Example 3 of this application. The Q-Plex Array was exposed to sera from individuals previously diagnosed with disseminated Lyme disease. Results indicated that the sera of individuals with Lyme disease reacted with the highly immunogenic recombinant Borrelia fusion protein constructs, shown in Tables 2 and 3. The overall sensitivity and specificity of the assay using the Q-Plex Array are also described in Example 3 of this application.
[0011] In some embodiments, for example, methods and the assays {e.g., protein arrays including microarrays, Q-Plex Arrays) of the invention, one or more single recombinant Borrelia protein antigens are used. Examples of single recombinant Borrelia protein antigens, including but not limited to, cell envelope protein antigens, recombinant lipoprotein antigens, extracellular protein antigens, recombinant antigens described herein (for, example, those described in Tables 1, 2 and 3) can be used. In some embodiments, a set of two or more {e.g., three) single recombinant Borrelia protein antigens are used. In some examples, sets include the set of single recombinant Borrelia protein antigens shown in Table 1, Table 2 and/or Table 3. In certain embodiments, a set of two or more {e.g., three) single recombinant Borrelia protein antigens are used. Other example sets of single recombinant Borrelia protein antigens include the set of all known and putative cell envelope proteins and /or lipoproteins of B. burgdorferi. Such a set can further include homologs and paralogs of cell envelope lipoproteins and extra-cellular proteins. Other sets include sets of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or other groups of cell envelope proteins {e.g., selected from those set forth in Table 2 and/or 3 and additional proteins in Table 1). In one particular embodiment, the set consists essentially of the single recombinant Borrelia protein antigens set forth in Table 1. In another embodiment, the set consists essentially of the single recombinant Borrelia protein antigens set forth in Table 2 (for, example, those described under column "gene locus" in Table 2). In another embodiment, the set consists essentially of the single recombinant Borrelia protein antigens set forth in Table 3 (for, example, those described under column "gene locus" in Table 3).
[0012] In some embodiments, for example, methods and assays {e.g., protein arrays including microarrays, Q-Plex Arrays) of the invention, one or more recombinant Borrelia fusion protein constructs were used. Examples of recombinant Borrelia fusion protein constructs, including but not limited to, SEQ ID NOS: 6-16 described herein {e.g., in Tables 2 and 3) can be used. Representative sets include the recombinant Borrelia fusion protein constructs shown in Table 2, and/or Table 3, and subsets thereof. In certain embodiments, only one recombinant Borrelia fusion protein construct was used. In other embodiments, two or more {e.g., three) recombinant Borrelia fusion protein constructs are used. Other example sets of recombinant Borrelia fusion protein constructs include the set of all possible combinations of recombinant Borrelia fusion protein constructs shown in Table 2, and/or Table 3, and subsets thereof. Such a set can further include recombinant Borrelia fusion protein constructs further comprising one or more single recombinant Borrelia protein antigens shown in Table 1. Other sets include sets of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or other groups of recombinant Borrelia fusion protein constructs {e.g., selected from those set forth in Table 2 and/or 3 and optionally can include one or more single recombinant Borrelia protein antigens shown in Table 1). In another embodiment, the set consists essentially of the recombinant Borrelia fusion protein constructs set forth in Table 2. In another
embodiment, the set consists essentially of the recombinant Borrelia fusion protein constructs set forth in Table 3.
[0013] In further methods and assays {e.g., protein arrays including microarrays, Q-Plex Arrays) of the invention, one or more single recombinant Borrelia protein antigens and recombinant Borrelia fusion protein constructs as identified herein are used, and/or combinations of single recombinant Borrelia protein antigens and recombinant Borrelia fusion protein constructs are used {e.g., combinations of proteins selected from any of Tables 1, 2 and/or 3). Such combinations include sets of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or other groups of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs {e.g., with one or more selected from Table 1, Table 2 and/or Table 3).
[0014] In one embodiment of the invention, a test sample from an individual {e.g., a human) is assessed for the presence of antibodies to one or more to one or more single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs {e.g., selected from Table 1, Table 2 and/or Table 3) of B. burgdorferi. The "test sample" can be a sample of blood, serum, cerebrospinal fluid, or other appropriate biological fluid from the individual. The test sample can be assessed for the presence of antibodies to one or more single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs using routine methods established in the art. In one r embodiment, the assessment is performed using a microarray of single recombinant Borrelia protein antigens. In another embodiment, the assessment is performed using a microarray of recombinant Borrelia fusion protein constructs. In certain examples, a microarray as described herein, or a protein or set of proteins as described herein, is exposed to the test sample from the individual, and any resultant binding of antibodies (if present in the test sample) to the single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs was determined. In one embodiment, the assessment is performed using an ELISA array, such as a multiples ELISA array (e.g., Q-Plex Array) of single recombinant Borrelia protein antigens. In another embodiment, the assessment is performed using a Q-Plex Array of recombinant Borrelia fusion protein constructs. In certain examples, a Q-Plex Array as described herein, or a protein or set of proteins as described herein, is exposed to the test sample from the individual or multiple samples from the same and/or multiple individuals, and any resultant binding of antibodies (if present in the test sample (s)) to the single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs is determined. The presence of antibodies bound to (also referred to herein as "reactive with") one or more single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs is indicative of antibodies specific to those proteins of B. burgdorferi. The presence of such antibodies is diagnostic for Lyme disease in the individual from whom the test sample was obtained.
[0015] In some embodiments, the assay methods described herein use protein arrays of B. burgdorferi antigens. In one embodiment, the protein array comprises a subset of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B. burgdorferi, such as the set of proteins set forth in Tables 1, 2 or 3. In other embodiments, other protein arrays include various subsets of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B. burgdorferi, such as sets of two or more, four or more, six or more, eight or more, or other groups of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs as set forth in Tables 1, 2 or 3. In one embodiment, the microarray consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 2, or consists of all of the recombinant Borrelia fusion protein constructs set forth in Table 2. In another embodiment, the microarray consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 3, or consists of all of the recombinant Borrelia fusion protein constructs of Table 3. In further embodiments, other microarrays include various subsets of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B. burgdorferi, such as subsets of the proteins set forth in Table 1, 2 and 3 (e.g., sets of two or more, four or more, six or more, eight or more, or other combinations of proteins). In one embodiment, the Q-Plex Array consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 2, or consists of all of the recombinant Borrelia fusion protein constructs set forth in Table 2. In another embodiment, the Q-Plex Array consists essentially of all of the recombinant Borrelia fusion protein constructs set forth in Table 3, or consists of all of the recombinant Borrelia fusion protein constructs of Table 3. In further embodiments, other Q-Plex Arrays include various subsets of single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs of B. burgdorferi, such as subsets of the proteins set forth in Table 1, 2 and 3 (e.g., sets of two or more, four or more, six or more, eight or more, or other combinations of proteins).
[0016] Other embodiments of the present invention encompass methods for the
evaluation/assessment of other or additional proteins of B. burgdorferi as potential candidates for the development of a diagnostic test for Lyme disease. In some embodiments, one or more proteins, for example, one or more recombinant Borrelia fusion protein constructs of B.
burgdorferi, such as described herein in Table 2 or Table 3 (e.g., in a microarray and /or Q-Plex Array as described above), are exposed to sera from one or more individuals known to have Lyme disease, and the proteins to which antibodies from the sera bind are then determined. For example, Cy5 intensity/Cy3 intensity ratio of fluorescence, as described in the Examples described herein, can be used. In one embodiment, the ratio of any proteins greater than the mean ratio of the reactivity of the Lyme sera to a negative control plus three times the standard deviation indicates significant interactions between antibodies present in the Lyme sera and the proteins (e.g., single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs). The proteins identified by such methods (e.g., single recombinant Borrelia protein antigens and/or recombinant Borrelia fusion protein constructs which have or are identified to have significant interactions with the antibodies present in the Lyme sera) are useful in diagnostic tests for Lyme disease (e.g., in the methods described herein), as well as in protein arrays (for, e.g., microarrays, Q-Plex Arrays) as described herein.
[0017] The present invention is also drawn to diagnostic kits which comprise the proteins described herein (e.g., in a microarray and /or in a Q-Plex Array as described herein). The kit optionally can include reagents for detecting antibody-antigen complexes that are formed between the protein and antibodies that are present in a sample, e.g., a user-supplied host sample.
[0018] The specific embodiments of the present invention are described in the following examples, which are not to be interpreted as limiting in any way.
EXEMPLIFICATION
EXAMPLE 1: Discovery of 48 highly antigenic single recombinant Borrelia protein antigens.
[0019] Protein arrays. A total of 416 genes were identified in a B. burgdorferi genome comparison. Of these, 350 (84%) produced a product that was the correct size when PCR was performed, and all 350 were successfully cloned into the T7 expression vector pET28b.
Sequence-confirmed plasmids were expressed using an overnight autoexpression system;
expressed proteins were purified using IDA resin and printed onto nitrocellulose-coated FAST slides (see, for example, US Serial No. 12/784,584 corresponding to published US Application No. US 20100292096 Aland US Serial No. 12/989,003 corresponding to published US
Application No. US20110105355 Al, the teachings of which are incorporated herein). In addition, representatives of several different Outer surface protein (OspC) types were amplified from human isolates. The OspC types included in this study were types A, B, C, D, E, F, G, H, I, J, K and U. This study also included the highly antigenic B31 cell envelope proteins that were identified in an earlier protein array study (Xu Y. et al., 2008). In total, the arrays contained approximately 400 samples including expressed Borrelia proteins, negative controls and blanks. Arrays were probed with sera from Lyme disease patients, and antibodies were visualized with Cy5-conjugated goat antihuman lgG or Cy5-conjugated goat anti-human lgM to determine antibody isotype (Xu Y et al., 2008).
[0020] Microarray. For protein array {e.g., microarray), each of the individual 48 single recombinant Borrelia protein antigens {e.g., in Table 1) were printed onto nitrocellulose-coated FAST glass slides. In addition, recombinant proteins OspB-OspC-Flagellin (B-C-Fia), OspA- p39-p93 (A-39-93) and an OspC dimer comprised of OspC Type B and Type H (OC2/9) from an early study were also printed (Gomes Solecki et al., 2000). Each slide in the arrays also contained 10 immobilized bovine serum albumin (BSA) spots for background determination. Proteome chips were probed with serum from patients with untreated early Lyme disease and sick non-Lyme patients using the Fast Pak protein array kit. Briefly, slides were first blocked overnight at 4°C in protein array -blocking buffer before incubation in primary antibody (human sera and mouse anti His-Tag for quantitation) for 2 h. Antibodies were visualized with Cy5- conjugated goat anti-human lgG or Cy 5 -conjugated goat anti-human lgM (to detect bound human antibodies) and Cy 3 -conjugated goat anti mouse lgG (to quantify the amount of recombinant protein in each spot). After a 2 hr incubation, the slides were stringently washed and then scanned with an Axon GenePix 4200A microarray scanner. The raw data was captured and analyzed with Gene Pix Pro image analysis software. To minimize the variability among samples, the PMT gain was adjusted to equal 1.0 in all the arrays with power setting at 50%. A global background subtraction method was used to subtract the background from each spot using the average mean intensity value of BSA from each slide (Xu et al., 2008).
[0021] Serum samples. For earlier array studies, sera of patients with Lyme disease were obtained from the Centers for Disease Control (CDC). The CDC samples included sera from 31 patients collected upon initial presentation (Samples C =0 days) and at 10 days (Samples D), 20 days (Samples E), 30 days (Samples F), 60 days (Samples G), and 90 days (Samples H) post presentation. Fourteen late Lyme samples (Late) from patients who exhibited late clinical manifestations (Lyme arthritis or neuroborreliosis) obtained from the Lyme Disease Center at Stony Brook University were also analyzed. A total of 115 samples were analyzed that included 21 Sample C, 29 Sample D, 15 Sample E, 8 Sample F, 1 Sample G and 12 Sample H. Normal control sera (n=14) were obtained from healthy donors. The overall sensitivity and specificity of the Lyme disease assay are described in Tables 2 and 3 of the PCT Publication WO
2016/057562, the entire teachings of which are incorporated herein by reference).
[0022] Results. Approximately 140 of the arrayed proteins detectably elicited an antibody response in humans with natural infections using both secondary antibodies (IgG and IgM). Of these 140 antigens, 80 proteins were recognized by at least half of the sera samples from each time point. Importantly, the majority of sera samples recognized at least one antigen from a subset of 48 highly antigenic single recombinant Borrelia protein antigens using lgM and lgG secondary antibodies (Table 1). This high positivity included all sera that were collected when the patients were first seen for their erythema migrans. The Cy5/Cy3 ratios of these 48 single recombinant Borrelia protein antigens were among the highest observed in the arrays, averaging greater than five times the standard deviation above the mean ratio of the reactivity of the Lyme sera to the negative control. In addition, a BLASTx search with highly immunogenic Borrelia proteins against the NCBI non-redundant protein database excluding all recorded Borrelia sequences revealed no significant matches to other organisms. Interestingly, 40 of the 48 single recombinant Borrelia protein antigens are plasmid encoded and the majority of encoded proteins are outer surface proteins, lipoproteins or putative outer membrane proteins. Over 90% of plasmid gene sequences have no significant homologs to non-B. burgdorferi genes. The uniqueness of plasmid sequences suggests that they serve specialized adaptive roles for
Borrelia's survival and propagation in nature and in a wide variety of hosts. Furthermore, the high numbers of outer surface lipoproteins encoded by the plasmids in conjunction with the variability of plasmid sequences supports the view that these sequences are directly involved in parasite-host interactions, and, therefore, are more than likely to be key immunogens (Casjens et al., 2000).
Table 1. 48 highly antigenic single recombinant Borrelia protein antigens.
Figure imgf000012_0001
lipoproteins)
BB H37 hypothetical protein Bbu297 W44 hypothetical protein
BB 116 repetitive antigen A Bbu297 Y03 hypothetical protein
[0023] The overall sensitivity and specificity of the Lyme disease assay are described in Tables 2 and 3 of the PCT Publication WO 2016/057562, the entire teachings of which are incorporated herein by reference). The sensitivity of the assay described in the above-referenced PCT Publication WO 2016/057562 far exceeds that of commercially available Lyme disease assays in patients in the early stages of the disease.
EXAMPLE 2: Recombinant Borrelia fusion protein constructs.
[0024] Overview. A library of recombinant Borrelia fusion protein constructs using the 48 highly antigenic single recombinant Borrelia protein antigens shown in Table 1 was designed for use in developing a recombinant chimera-based diagnostic assay for Lyme disease. Additionally, a representative lipoprotein, OppA (ABC transporter substrate-binding protein), was amplified from human isolates and included in this study. Construction of recombinant Borrelia fusion protein constructs containing genes from several genospecies allows one to generate one protein that confers antigenicity to multiple strains, revealing the great potential and adaptability of this technique. In addition, the use of pure protein preparations as antigens offers greater flexibility in adapting the test to different assay formats. It is reasonable to believe that an assay of recombinant Borrelia fusion protein constructs will present superior sensitivity in detecting antibodies against B. burgdorferi for the early stages of the disease, and equivalent sensitivity for the late stages of the disease, when compared to the currently available whole-cell assays.
[0025] Recombinant Borrelia fusion protein constructs. From the library of 48 highly antigenic single recombinant Borrelia protein antigens and a representative lipoprotein, OppA, 11 sets of recombinant Borrelia fusion protein constructs each containing at least two of the Borrelia antigens were constructed (Tables 2 and 3). Chimeras of at least two {e.g., three) single recombinant Borrelia protein antigens in most constructs were chosen to keep the molecular mass of the fusion protein at or less than (below) about 100 kDa to optimize protein yield, which can be significantly affected by protein size.
[0026] Table 2. Structural Characterization of recombinant Borrelia fusion protein constructs.
Item Protein ID gene locus gene name SEQ ID NOS.
Decorin binding protein A 3
BB_A24-His BB A24
(dbpA)
Figure imgf000014_0001
[0027] Table 3. Structural Characterization of recombinant Borrelia fusion protein constructs.
Figure imgf000014_0002
Figure imgf000015_0001
[0028] Cloning of recombinant fusion proteins. Borrelia genes encoding the components {e.g., single recombinant Borrelia protein antigens) of the 11 recombinant Borrelia fusion protein constructs were amplified using a unique gene-specific primer pair that was specifically designed for chimeric protein construction. The primers were designed from the genomic sequence of B. burgdorferi strains and each contains a novel DNA cassette coding for the recognition sequence for three restriction endonucleases that are in-frame with the gene coding sequence. The 5' primer (5'-AATTGGTACCCCAGGATCCCATATG + 15mer ORF specific sequence) (SEQ ID NO: 1) contains Kpnl (underlined), BamHI (bold) and Ndel (italics) recognition sequence. The 3' primer (5'GCGGGATCCGGTACCGTCGAC +15mer ORF specific sequence) (SEQ ID NO:2) contains BamHI, Kpnl and Sail (dashed underline) recognition sequences. GGATCC is bolded in the 5' and 3' primers described above. For amplification, ten ng of genomic DNA was used as a template in a 50-μΙ PCR reaction containing two ORF-specific primer pairs (SEQ ID NOS: 1&2). To increase the solubility properties of expressed cell envelope proteins, the primer sets were designed to amplify coding regions without a membrane anchoring signal sequence (Dunn et al., 1990). PCR amplification was performed under stringent conditions with Platinum Taq DNA polymerase High Fidelity (Invitrogen) using conditions previously described (Xu et al., 2003). The PCR products were visualized using agarose gel electrophoresis. For quantification, the products were purified (PCR purification kit, Qiagen) and quantified by UV absorbance.
[0029] To create the library of recombinant Borrelia fusion protein constructs comprised of two gene fragments, amplified products were cleaved with Ndel/BamHI (for 1st position fragment), or BamHI/Kpnl (for 2nd position fragment) or Kpnl/Sall (for 3rd position fragment). To create the library of recombinant Borrelia fusion protein constructs comprised of three gene fragments, amplified products were cleaved with Ndel/BamHI (for Γ1 position fragment), or BamHI/Kpnl (for 2nd position fragment) or Kpnl/Sall (for 3rd position fragment). The 1st position fragments were directionally cloned into the unique Ndel and BamHI sites of the T7- based expression vector pET-28 (Novagen). This vector provides an N-terminal poly (His) affinity tag fused to the expressed proteins to aid in purification on nickel-Sepharose columns. Ligation reactions were transformed into E. coli GC5 competent cells and plasmids were purified using Eppendorf Perfectprep Plasmid 96 VAC Direct Bind Kit and verified by sequencing across the inserts. Plasmids containing fragment 1 now served as vectors for all subsequent cloning. For directional cloning of fragments two and three, vectors were digested with BamHI/XhoIl, and restriction digested amplicons of fragment two and three were ligated simultaneously into the digested vector. Following transformation, plasmids were purified and sequenced.
[0030] Protein expression and purification. Purified plasmids were transformed into E. coli BL21/DE3 competent cells for expression. Recombinant Borrelia fusion protein constructs containing an N-terminal poly (His) affinity tag were expressed using the Overnight Express Autoinduction protocol. Induced cells were harvested by centrifugation and resuspended in 8M urea. Aliquots were run on SDS-PAGE. N-terminal poly His-tagged proteins were purified on nickel-Sepharose columns under denaturing conditions using Ni-NTA Spin Kit (Qiagen). The kit is designed for rapid screening and purification of His Tag fusion proteins. Protein concentration was determined by the measurement of the absorbance shift when Coomassie brilliant blue G-250 reacted with protein (Bio-Rad). Protein purity was checked by SDS-PAGE. EXAMPLE 3: Protein arrays using recombinant Borrelia fusion protein constructs and Diagnostic Test for Lyme Disease.
[0031] Overview. Using genomic and proteomic approaches, protein arrays {e.g., microarrays and /or Q-Plex Arrays) were fabricated based on multiple genotypes of Borrelia burgdorferi sensu stricto to identify antigens that may be useful in the development of a highly sensitive and specific single-tier assay that will have a wide range of specificity in detecting Lyme disease. The assay containing 11 recombinant Borrelia fusion protein constructs described above {e.g., SEQ ID NOS: 6-16 in Tables 2 and 3) is very accurate and sensitive. [0032] Serum samples. The antigenicity of the recombinant Borrelia fusion protein constructs was tested using CDC sera samples, as described above in Example 1. For Quansys Array studies, sera from individuals representing all stages of Lyme disease including 41 samples with early localized Lyme disease, 40 with early disseminated infection, and 16 late Lyme disease samples, were used. Late Lyme samples included sera from Lyme arthritis, neurologic Lyme and cardiac Lyme patients. In addition, for specificity studies, 242 sera were obtained from Bioreclamation LLC, Westbury, NY and the CDC from patients with diseases associated with serological responses that are known to produce cross-reactivity in Lyme disease test including syphilis, fibromyalgia, rheumatoid arthritis, mononucleosis, multiple sclerosis, fibromyalgia, psoriasis, osteoarthritis, and EBV. One hundred twenty eight normal control sera from the same sources were obtained from healthy donors from an area of endemicity and healthy donors from non-endemic areas. All of the Lyme sera and a number of the control sera were used in previous studies described in Example 1 and the array studies {e.g., Q-Plex Arrays) described below. All serological assays involving human serum were performed on coded specimens with all investigators blinded to clinical information pertaining to individual samples. No patient identifying information was available to the investigators or technician testing the samples.
[0033] Q-Plex technology. The multiplex assay using Q-Plex technology involves the micro-spotting of individual groups of capture proteins on the bottom of a 96-well plate with each spot being its own 'micro ELISA' assay. Standard ELISA incubation steps including sample incubation, washing, and secondary antibody incubation were employed. The labeling and reporting system used in the Q-Plex Array was chemiluminescent. The binding of secondary antibody was measured via the chemiluminescence produced by streptavidin horseradish peroxidase in the presence of a luminol-based substrate. Chemiluminescent ELISAs have been shown to be more sensitive than colorimetric detection systems. The intensity of
chemiluminescence from each array was measured using the Quansys Q-View chemiluminescent imager. Data were then analyzed using Quansys Q-View software.
[0034] Q-Plex Arrays. Protein arrays {e.g., Q-Plex Arrays) comprising the 11 highly antigenic recombinant Borrelia proteins described in Table 2 and 3, were constructed. The sensitivity of Q-Plex Array was tested by screening arrays with sera from patients with early and late Lyme disease using buffers and methods provided by Quansys Biosciences. In addition, specificity was determined using sera from patients with diseases associated with serological responses that are known to produce cross-reactivity in Lyme disease tests. The results of Q- Plex arrays were compared with the results obtained with the standard two-tier assay for the same set of 11 recombinant Borrelia fusion protein constructs.
[0035] Results. The sensitivity of the Q-Plex Arrays was determined using sera from 97 Lyme disease patients. Of 41 acute-phase sera from patients presenting with erythema migrans, 83% (34/41) were positive with the Q-Plex Arrays while 37% (15/41) were positive by the two- tier test. Testing 40 serum samples from EM patients, convalescent at 3 weeks post-presentation, 90%) (36/40) were positive by the Q-Plex Arrays while 78% (31/40) were positive by the two-tier assay. Assaying 16 late Lyme serum samples, 100 % (16/16) were positive by the Q-Plex Arrays while 94%) (15/16) were positive by two-tier testing. Overall, the sensitivity of the Q-Plex Arrays was 89% (86/97) compared to 63% (60/97) for the two-tier test.
[0036] The specificity of the Q-Plex Arrays was tested using sera from individuals with diseases associated with serological responses that are known to produce cross-reactivity in currently used tests and sera from healthy individuals from both endemic and non-endemic regions. Testing 242 sera from individuals with 11 other disease conditions (sick non-Lyme) resulted in a specificity of 97% with both the Q-Plex Arrays and with two-tier testing.
Specificity was 100% for both the Q-Plex Arrays and the two-tier assay in a population of healthy blood donors from regions of the USA nonendemic for Lyme disease and donors from regions endemic for Lyme disease. Overall, the specificity of the Q-Plex Arrays was 99% (5/370) compared to 99% (5/370) for the two-tier assay.
[0037] Results described above demonstrate that Q-Plex Arrays offer superior sensitivity and specificity in detecting antibodies against B. burgdorferi for not only early stages of the disease, but show equivalent or better sensitivity for the late stages of the disease as well. The recombinant Borrelia fusion protein constructs described herein (e.g., SEQ ID NOS: 6-16 in Tables 2 and 3) represent strong candidates for the development of diagnostic tests. These variants, as well as variants of the other recombinant Borrelia fusion protein constructs described herein, can be used to develop a standardized sensitive and specific single-tier Lyme disease assay of recombinant Borrelia fusion protein constructs that will have a wide range of coverage and specificity against Lyme disease. Furthermore, these assays will have significant potential for the development of a next-generation rapid, single-tier point of care assay.
[0038] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety including the references below. Sequences
BB_A24-His: SEQID NO:3
MGNKTFNNLLKLTILVNLLISCGLTGATKIRLERSAKDITDEIDAIKKDAALKGVNFDAFKDKKT GSGVSENPFILEAKVRATTVAEKFVIAIEEEATKLKETGSSGEFSAMYDLMFEVSKPLQKLGIQ EMTKTVSDAAEENPPTTAQGVLEIAKKMREKLQRVHTKNYCTLKKKENSTFTDEKCKNNVD KLAAALEHHHHHH
BB_A25-His: SEQID N0:4
MGIGLVERTNAALESSSKDLKNKILKIKKEATGKGVLFEAFTGLKTGSKVTSGGLALREAKVQA
IVETGKFLKIIEEEALKLKETGNSGQFLAMFDLMLEVVESLEDVGIIGLKARVLEESKNNPINTA
ERLLAAKAQIENQLKVVKEKQNIENGGEKKNNKSKKKKVDKLAAALEHHHHHH
BB_0329-His: SEQID N0:5
MGNKERKEGVSFKISLGAEPSSLDPQLAEDNVASKMIDTMFRGIVTGDPNTGGNKPGLAKG
WDISSDGTVYTFNLREKITWSDGVAITAEGIRKSYLRILNKETGSKYVEMVKSVIKNGQKYFD
GQVTDSELGIRAIDEKTLEITLESPKPYFIDMLVHQSFIPVPVHVTEKYGQNWTSPENMVTSG
PFKLKERIPNEKYVFEKNNKYYDSNEVELEEITFYTTNDSSTAYKMYENEELDAIFGSIPPDLIKN
LKLRSDYYSSAVNAIYFYAFNTHIKPLDNVKIRKALTLAIDRETLTYKVLDNGTTPTRRATPNFSS
YSYAKSLELFNPEIAKTLLAEAGYPNGNGFPILKLKYNTNEANKKICEFIQNQWKKNLNIDVEL
ENEEWTTYLNTKANGNYEIARAGWIGDYADPLTFLSIFTQGYTQFSSHNYSNPEYNELIKKSD
LELDPIKRQDILRQAEEIIIEKDFPIAPIYIYGNSYLFRNDKWTGWNTNILERFDLSQLKLKNKVD
KLAAALEHHHHHH
Tri-435-His: SEQID NO:6
MGNKTFNNLLKLTILVNLLISCGLTGATKIRLERSAKDITDEIDAIKKDAALKGVNFDAFKDKKT
GSGVSENPFILEAKVRATTVAEKFVIAIEEEATKLKETGSSGEFSAMYDLMFEVSKPLQKLGIQ
EMTKTVSDAAEENPPTTAQGVLEIAKKMREKLQRVHTKNYCTLKKKENSTFTDEKCKNNVD
GTGSHMGNKERKEGVSFKISLGAEPSSLDPQLAEDNVASKMIDTMFRGIVTGDPNTGGNKP
GLAKGWDISSDGTVYTFNLREKITWSDGVAITAEGIRKSYLRILNKETGSKYVEMVKSVIKNG
QKYFDGQVTDSELGIRAIDEKTLEITLESPKPYFIDMLVHQSFIPVPVHVTEKYGQNWTSPEN
MVTSGPFKLKERIPNEKYVFEKNNKYYDSNEVELEEITFYTTNDSSTAYKMYENEELDAIFGSIP
PDLIKNLKLRSDYYSSAVNAIYFYAFNTHIKPLDNVKIRKALTLAIDRETLTYKVLDNGTTPTRRA
TPNFSSYSYAKSLELFNPEIAKTLLAEAGYPNGNGFPILKLKYNTNEANKKICEFIQNQWKKNL
NIDVELENEEWTTYLNTKANGNYEIARAGWIGDYADPLTFLSIFTQGYTQFSSHNYSNPEYN
ELIKKSDLELDPIKRQDILRQAEEIIIEKDFPIAPIYIYGNSYLFRNDKWTGWNTNILERFDLSQLK
LKNKVDGTPGSHMGIGLVERTNAALESSSKDLKNKILKIKKEATGKGVLFEAFTGLKTGSKVTS
GGLALREAKVQAIVETGKFLKIIEEEALKLKETGNSGQFLAMFDLMLEVVESLEDVGIIGLKAR
VLEESKNNPINTAERLLAAKAQIENQLKVVKEKQNIENGGEKKNNKSKKKKVEHHHHHH Tri-453-His: SEQID NO:7
MGNKTFNNLLKLTILVNLLISCGLTGATKIRLERSAKDITDEIDAIKKDAALKGVNFDAFKDKKT
GSGVSENPFILEAKVRATTVAEKFVIAIEEEATKLKETGSSGEFSAMYDLMFEVSKPLQKLGIQ
EMTKTVSDAAEENPPTTAQGVLEIAKKMREKLQRVHTKNYCTLKKKENSTFTDEKCKNNVD
GTGSHMGIGLVERTNAALESSSKDLKNKILKIKKEATGKGVLFEAFTGLKTGSKVTSGGLALRE
AKVQAIVETGKFLKIIEEEALKLKETGNSGQFLAMFDLMLEVVESLEDVGIIGLKARVLEESKN
NPINTAERLLAAKAQIENQLKVVKEKQNIENGGEKKNNKSKKKKVDGTPGSHMGNKERKEG
VSFKISLGAEPSSLDPQLAEDNVASKMIDTMFRGIVTGDPNTGGNKPGLAKGWDISSDGTV
YTFNLREKITWSDGVAITAEGIRKSYLRILNKETGSKYVEMVKSVIKNGQKYFDGQVTDSELGI
RAIDEKTLEITLESPKPYFIDMLVHQSFIPVPVHVTEKYGQNWTSPENMVTSGPFKLKERIPNE
KYVFEKNNKYYDSNEVELEEITFYTTNDSSTAYKMYENEELDAIFGSIPPDLIKNLKLRSDYYSS
AVNAIYFYAFNTHIKPLDNVKIRKALTLAIDRETLTYKVLDNGTTPTRRATPNFSSYSYAKSLELF
NPEIAKTLLAEAGYPNGNGFPILKLKYNTNEANKKICEFIQNQWKKNLNIDVELENEEWTTYL
NTKANGNYEIARAGWIGDYADPLTFLSIFTQGYTQFSSHNYSNPEYNELIKKSDLELDPIKRQD
ILRQAEEIIIEKDFPIAPIYIYGNSYLFRNDKWTGWNTNILERFDLSQLKLKNKVDVEHHHHHH
Tri-543-His: SEQID N0:8
MGIGLVERTNAALESSSKDLKNKILKIKKEATGKGVLFEAFTGLKTGSKVTSGGLALREAKVQA
IVETGKFLKIIEEEALKLKETGNSGQFLAMFDLMLEVVESLEDVGIIGLKARVLEESKNNPINTA
ERLLAAKAQIENQLKVVKEKQNIENGGEKKNNKSKKKKVDGTGSHMGNKTFNNLLKLTILVN
LLISCGLTGATKIRLERSAKDITDEIDAIKKDAALKGVNFDAFKDKKTGSGVSENPFILEAKVRA
TTVAEKFVIAIEEEATKLKETGSSGEFSAMYDLMFEVSKPLQKLGIQEMTKTVSDAAEENPPT
TAQGVLEIAKKMREKLQRVHTKNYCTLKKKENSTFTDEKCKNNVDGTPGSHMGNKERKEG
VSFKISLGAEPSSLDPQLAEDNVASKMIDTMFRGIVTGDPNTGGNKPGLAKGWDISSDGTV
YTFNLREKITWSDGVAITAEGIRKSYLRILNKETGSKYVEMVKSVIKNGQKYFDGQVTDSELGI
RAIDEKTLEITLESPKPYFIDMLVHQSFIPVPVHVTEKYGQNWTSPENMVTSGPFKLKERIPNE
KYVFEKNNKYYDSNEVELEEITFYTTNDSSTAYKMYENEELDAIFGSIPPDLIKNLKLRSDYYSS
AVNAIYFYAFNTHIKPLDNVKIRKALTLAIDRETLTYKVLDNGTTPTRRATPNFSSYSYAKSLELF
NPEIAKTLLAEAGYPNGNGFPILKLKYNTNEANKKICEFIQNQWKKNLNIDVELENEEWTTYL
NTKANGNYEIARAGWIGDYADPLTFLSIFTQGYTQFSSHNYSNPEYNELIKKSDLELDPIKRQD
ILRQAEEIIIEKDFPIAPIYIYGNSYLFRNDKWTGWNTNILERFDLSQLKLKNKVEHHHHHH
Tri-534-His: SEQID N0:9
MGIGLVERTNAALESSSKDLKNKILKIKKEATGKGVLFEAFTGLKTGSKVTSGGLALREAKVQA
IVETGKFLKIIEEEALKLKETGNSGQFLAMFDLMLEVVESLEDVGIIGLKARVLEESKNNPINTA
ERLLAAKAQIENQLKVVKEKQNIENGGEKKNNKSKKKKVDGTGSHMGNKERKEGVSFKISLG
AEPSSLDPQLAEDNVASKMIDTMFRGIVTGDPNTGGNKPGLAKGWDISSDGTVYTFNLREK
ITWSDGVAITAEGIRKSYLRILNKETGSKYVEMVKSVIKNGQKYFDGQVTDSELGIRAIDEKTL
EITLESPKPYFIDMLVHQSFIPVPVHVTEKYGQNWTSPENMVTSGPFKLKERIPNEKYVFEKN
NKYYDSNEVELEEITFYTTNDSSTAYKMYENEELDAIFGSIPPDLIKNLKLRSDYYSSAVNAIYFY AFNTHIKPLDNVKIRKALTLAIDRETLTYKVLDNGTTPTRRATPNFSSYSYAKSLELFNPEIAKTL
LAEAGYPNGNGFPILKLKYNTNEANKKICEFIQNQWKKNLNIDVELENEEWTTYLNTKANGN
YEIARAGWIGDYADPLTFLSIFTQGYTQFSSHNYSNPEYNELIKKSDLELDPIKRQDILRQAEEII
IEKDFPIAPIYIYGNSYLFRNDKWTGWNTNILERFDLSQLKLKNKVDGTPGSHMGNKTFNNLL
KLTILVNLLISCGLTGATKIRLERSAKDITDEIDAIKKDAALKGVNFDAFKDKKTGSGVSENPFIL
EAKVRATTVAEKFVIAIEEEATKLKETGSSGEFSAMYDLMFEVSKPLQKLGIQEMTKTVSDAA
EENPPTTAQGVLEIAKKMREKLQRVHTKNYCTLKKKENSTFTDEKCKNNVEHHHHHH
Tri-345-His: SEQID NO:10
MGNKERKEGVSFKISLGAEPSSLDPQLAEDNVASKMIDTMFRGIVTGDPNTGGNKPGLAKG
WDISSDGTVYTFNLREKITWSDGVAITAEGIRKSYLRILNKETGSKYVEMVKSVIKNGQKYFD
GQVTDSELGIRAIDEKTLEITLESPKPYFIDMLVHQSFIPVPVHVTEKYGQNWTSPENMVTSG
PFKLKERIPNEKYVFEKNNKYYDSNEVELEEITFYTTNDSSTAYKMYENEELDAIFGSIPPDLIKN
LKLRSDYYSSAVNAIYFYAFNTHIKPLDNVKIRKALTLAIDRETLTYKVLDNGTTPTRRATPNFSS
YSYAKSLELFNPEIAKTLLAEAGYPNGNGFPILKLKYNTNEANKKICEFIQNQWKKNLNIDVEL
ENEEWTTYLNTKANGNYEIARAGWIGDYADPLTFLSIFTQGYTQFSSHNYSNPEYNELIKKSD
LELDPIKRQDILRQAEEIIIEKDFPIAPIYIYGNSYLFRNDKWTGWNTNILERFDLSQLKLKNKVD
GTGSHMGNKTFNNLLKLTILVNLLISCGLTGATKIRLERSAKDITDEIDAIKKDAALKGVNFDAF
KDKKTGSGVSENPFILEAKVRATTVAEKFVIAIEEEATKLKETGSSGEFSAMYDLMFEVSKPLQ
KLGIQEMTKTASDAAEENPPTTAQGVLEIAKKMREKLQRVHTKNYCTLKKKENSTFTDEKCK
NNVDGTPGSHMGIGLVERTNAALESSSKDLKNKILKIKKEATGKGVLFEAFTGLKTGSKVTSG
GLALREAKVQAIVETGKFLKIIEEEALKLKETGNSGQFLAMFDLMLEVVESLEDVGIIGLKARVL
EESKNNPINTAERLLAAKAQIENQLKVVKEKQNIENGGEKKNNKSKKKKVEHHHHHH
Tri-354-His: SEQID N0:11
MGNKERKEGVSFKISLGAEPSSLDPQLAEDNVASKMIDTMFRGIVTGDPNTGGNKPGLAKG
WDISSDGTVYTFNLREKITWSDGVAITAEGIRKSYLRILNKETGSKYVEMVKSVIKNGQKYFD
GQVTDSELGIRAIDEKTLEITLESPKPYFIDMLVHQSFIPVPVHVTEKYGQNWTSPENMVTSG
PFKLKERIPNEKYVFEKNNKYYDSNEVELEEITFYTTNDSSTAYKMYENEELDAIFGSIPPDLIKN
LKLRSDYYSSAVNAIYFYAFNTHIKPLDNVKIRKALTLAIDRETLTYKVLDNGTTPTRRATPNFSS
YSYAKSLELFNPEIAKTLLAEAGYPNGNGFPILKLKYNTNEANKKICEFIQNQWKKNLNIDVEL
ENEEWTTYLNTKANGNYEIARAGWIGDYADPLTFLSIFTQGYTQFSSHNYSNPEYNELIKKSD
LELDPIKRQDILRQAEEIIIEKDFPIAPIYIYGNSYLFRNDKWTGWNTNILERFDLSQLKLKNKVD
GTGSHMGIGLVERTNAALESSSKDLKNKILKIKKEATGKGVLFEAFTGLKTGSKVTSGGLALRE
AKVQAIVETGKFLKIIEEEALKLKETGNSGQFLAMFDLMLEVVESLEDVGIIGLKARVLEESKN
NPINTAERLLAAKAQIENQLKVVKEKQNIENGGEKKNNKSKKKKVDGTPGSHMGNKTFNNL
LKLTILVNLLISCGLTGATKIRLERSAKDITDEIDAIKKDAALKGVNFDAFKDKKTGSGVSENPFI
LEAKVRATTVAEKFVIAIEEEATKLKETGSSGEFSAMYDLMFEVSKPLQKLGIQEMTKTVSDA
AEENPPTTAQGVLEIAKKMREKLQRVHTKNYCTLKKKENSTFTDEKCKNNVEHHHHHH A-312-93: SEQID NO:12
MAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVL
EGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRAD
GTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTD
SSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIK
NALKGHPMARKESSKDSRSQLQVAGFKIGKESYGVSIEHIREIIKVPSEGVYAIPNVPEYIIGIYN
LRGSIIPLINLNIKFGVPSISVTEEDMLLTGYLIVKIKNKLLGIFVDRVLKVISFDDSRVQEPPATL
QTLDRKYISGVVKLDEADNLESEYLVLIDIAKIFDKCEFDDIPYKDQYEEAMDEKLLKSKDDKAS
KDGKALDLDRGLNSKASSKEKSKAKEEEITKGKSQKSLGDLNNDENLMMPEDQKLPEVKKLD
SKEEFKPVSEVEKLDKIFKSNNNVGELSPLDKSSYKDIDSKEETVNKDVNLQKTKPQVKDQVT
SLNEDLTTMSIDSSSPVFLEVIDPITNLGTLQLIDLNTGVRLKESTQQGIQRYGIHEREKDLVVIK
MDSGKAKLQILDKLENLKVVSESNFEINKNSSLYVDSKMILVAVRDKDSSNDWRLAKFSPKN
LDEFILSENKIMPFTSFSVRKNFIYLQDEFKSLVILDVNTLKKVKGHHHHHHNM
V-93: SEQID N0:13
MKKNDQIAAAIVLRGMAKDGEFALKNDANNAEKGLKSTVESAVNKTVVDGQKKWAMDE
KLLKSKDDKASKDGKALDLDRGLNSKASSKEKSKAKEEEITKGKSQKSLGDLNNDENLMMPE
DQKLPEVKKLDSKEEFKPVSEVEKLDKIFKSNNNVGELSPLDKSSYKDIDSKEETVNKDVNLQK
TKPQVKDQVTSLNEDLTTMSIDSSSPVFLEVIDPITNLGTLQLIDLNTGVRLKESTQQGIQRYGI
HEREKDLVVIKMDSGKAKLQILDKLENLKVVSESNFEINKNSSLYVDSKMILVAVRDKDSSND
WRLAKFSPKNLDEFILSENKIMPFTSFSVRKNFIYLQDEFKSLVILDVNTLKKVKGHHHHHHN
M
A- V-93: SEQID N0:14
MAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVL
EGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRAD
GTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTD
SSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIK
NALKGHPMAGKVVKVNAAGAAAKGGEETSVNGIASGIKGIVTAAEKAGEEGKLKSEAAGD
G EAN EDAG KLFAKKN DAGG DAKDAE KAAAAVSAVSG KQI LKAI VDAAG KE EKG VVADVKD
AKNPIEAAIGSTGEQNAAAFSHMKKNDQIAAAIVLRGMAKDGEFALKNDANNAEKGLKSTV
ESAVNKTVVDGQKKWAMDEKLLKSKDDKASKDGKALDLDRGLNSKASSKEKSKAKEEEITK
GKSQKSLGDLNNDENLMMPEDQKLPEVKKLDSKEEFKPVSEVEKLDKIFKSNNNVGELSPLD
KSSYKDIDSKEETVNKDVNLQKTKPQVKDQVTSLNEDLTTMSIDSSSPVFLEVIDPITNLGTLQ
LIDLNTGVRLKESTQQGIQRYGIHEREKDLVVIKMDSGKAKLQILDKLENLKVVSESNFEINKN
SSLYVDSKMILVAVRDKDSSNDWRLAKFSPKNLDEFILSENKIMPFTSFSVRKNFIYLQDEFKS
LVILDVNTLKKVKGHHHHHHNM A60-OC2/9-S42: SEQID NO:15
MSINKEQKTKEKTSEKQESEKQNIEKQEPEKQKQNAAKIIPTVSIQTVEIRESNQIPKSIEKYYK
QAYPIQTFTLDFSITREKEFLKPEDKILPTQGKVESLSILINKKLLDFKAPENPKSSTLKNFKEIKNI
ENFFQNQDLLFVLTLKDKNNNNTINIMLNPPNDIQKPKDYILKDLKDTIKKGTGEKYLNPIYRF
QIKNKKDYHSIDYNKVTISEKTIELDLLPHEQVFQMNKNFTKILDTITDLNNLKLVIQKELVVDG
TGSHMACNNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDELAKA
IGKKIKNDGSLDNAANRNEALLAGAATISTLITQKLSKLNGSEGLKEKIAAAKKCSEEFSTKLKD
NHAQLGIQGVTDENAKKAILKANAAGKDKGVEELERLSGSLESLSKAAKEMLANSVKELTSP
VVVESPKKPAMACNNSGKDGNASANSADESVKGPNLTEISKKITESNAVVLAVKEVETLLASI
NQLAKAIGKKIDQNGTLGDAGGQNGALLAGAAAISTVIIEKLSTLKNVEELKEKITKAKDCSEK
FAGKLKNEHASLGKKDATDDDAKKAILKTHGNTDKGAKELKDLSDSVESLVKAAKEMLTNSV
KELTSPVVAESPKKPVDGTPGSHMAEENYTETKRAFSKEDFNLINKRLDNYDFKNEYEKSHVF
SDAPRIRGDLRKIGIKEKSVFLDALEAIEYLIKIKISTDSIFLSEDMIRLIGSYPDSIFNYLIQLNSDKI
DYAEKYGDNARNNFKKDYSEDKANTVKQILKQILADLPKDVEHHHHHH
S42-OC2/9-A60: SEQID N0:16
MAEENYTETKRAFSKEDFNLINKRLDNYDFKNEYEKSHVFSDAPRIRGDLRKIGIKEKSVFLDA
LEAIEYLIKIKISTDSIFLSEDMIRLIGSYPDSIFNYLIQLNSDKIDYAEKYGDNARNNFKKDYSEDK
ANTVKQILKQILADLPKDVDGTGSHMACNNSGKDGNTSANSADESVKGPNLTEISKKITDSN
AVLLAVKEVEALLSSIDELAKAIGKKIKNDGSLDNAANRNEALLAGAATISTLITQKLSKLNGSE
GLKEKIAAAKKCSEEFSTKLKDNHAQLGIQGVTDENAKKAILKANAAGKDKGVEELERLSGSL
ESLSKAAKEMLANSVKELTSPVVVESPKKPAMACNNSGKDGNASANSADESVKGPNLTEISK
KITESNAVVLAVKEVETLLASINQLAKAIGKKIDQNGTLGDAGGQNGALLAGAAAISTVIIEKL
STLKNVEELKEKITKAKDCSEKFAGKLKNEHASLGKKDATDDDAKKAILKTHGNTDKGAKELK
DLSDSVESLVKAAKEMLTNSVKELTSPVVAESPKKPVDGTPGSHMSINKEQKTKEKTSEKQES
EKQNIEKQEPEKQKQNAAKIIPTVSIQTVEIRESNQIPKSIEKYYKQAYPIQTFTLDFSITREKEFL
KPEDKILPTQGKVESLSILISKKLLDFKAPENPKSSTLKNFKEIKNIENFFQNQDLLFVLTLKDKN
NNNTINIMLNPPNDIQKPKDYILKDLKDTIKKGTGEKYLNPIYRFQIKNKKDYHSIDYNKVTISE
KTIELDLLPHEQVFQMNKNFTKILDTITDLNNLKLVIQKELVVEHHHHHH
References
[0039] Casjens, S., Palmer, N., van Vugt, R., Huang, W.G., Stevenson, B., Rosa, P., et al, A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Mol. Microbiol., 2000; 35, 490-516.
[0040] Dunn, J. J., Lade, B.N., Barbour, A.G., Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi: high level expression and purification of a soluble recombinant form of OspA Protein Expr. Purif.1990; 1 : 159-68. [0041] Gomes-Solecki, M.J.C., Dunn, J. J., Luft, B.J., et al., 2000.Recombinant chimeric Borrelia proteins for diagnosis of Lyme disease. J. Clin. Microbial. 2000; 38:2530-2535.
[0042] Xu, Y., Bruno, J.F., Luft, B. J., Profiling the humoral immune response to Borrelia burgdorferi infection with protein microarrays. Microb. Path. 2008; 45: 403-407.
[0043] Xu, Y., Bruno, J.F., Luft, B. J., Detection of Genetic Diversity in Linear Plasm ids 28- 3 and 36 in Borrelia burgdorfesensu stricto isolates by Subtractive Hybridization. Microb. Path. 2003; 35:269-78.
[0044] Signorino G, Arnaboldi PM, Petzke MM, Dattwyler RJ. 2014. Identification of OppA2 linear epitopes as serodiagnostic markers for Lyme disease. Clin Vaccine Immunol 21 :704-711.
[0045] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:
1. A recombinant Borrelia fusion protein construct comprising dbpA, dbpB and OppA.
2. A recombinant Borrelia fusion protein construct selected from a group of constructs selected from Table 2 or Table 3.
3. The recombinant Borrelia fusion protein construct of claim 1 wherein the molecular mass of the recombinant Borrelia fusion protein construct is about 100 kDa or less.
4. The recombinant Borrelia fusion protein construct of claim 2 wherein the molecular mass of the recombinant Borrelia fusion protein construct is about 100 kDa or less.
5. The recombinant Borrelia fusion protein construct of claim 1 wherein the single
recombinant Borrelia protein antigens were amplified by PCR with primers comprising SEQ ID NO: 1 and SEQ ID NO:2.
6. The recombinant Borrelia fusion protein construct of claim 2 wherein the single
recombinant Borrelia protein antigens were amplified by PCR with primers comprising SEQ ID NO: 1 and SEQ ID NO:2.
7. The recombinant Borrelia fusion protein construct of claim 1 further comprising one or more single recombinant Borrelia protein antigens selected from Table 1.
8. The recombinant Borrelia fusion protein construct of claim 2 further comprising one or more single recombinant Borrelia protein antigens selected from Table 1.
9. A vector comprising the recombinant Borrelia fusion protein construct of claim 1.
10. A vector comprising the recombinant Borrelia fusion protein construct of claim 2.
11. A protein array comprising one or more recombinant Borrelia fusion protein constructs wherein the one or more recombinant Borrelia fusion protein constructs comprise single recombinant Borrelia protein antigens selected from a group of dbpA, dbpB,OppA, OspA, purine-binding chemotaxis protein, p83/100 antigen, VMP-like sequence protein VlsE, surface lipoprotein P27, OspC type B, OspC type K, and BapA protein.
12. The protein array of claim 11 wherein the one or more recombinant Borrelia fusion protein constructs comprise one or more single recombinant Borrelia protein antigens selected from the group consisting of dbpA, dbpB and OppA.
13. The protein array of claim 11 wherein the one or more recombinant Borrelia fusion protein constructs comprise one or more single recombinant Borrelia protein antigens selected from the group consisting of purine-binding chemotaxis protein, p83/100 antigen, VMP-like sequence protein VlsE, surface lipoprotein P27, OspC type B, OspC type K, and BapA protein.
14. The protein array of claim 11 further comprising one or more single recombinant
Borrelia protein antigens selected from Table 1.
15. A protein array comprising one or more recombinant Borrelia fusion protein constructs selected from Table 2.
16. A protein array comprising one or more recombinant Borrelia fusion protein constructs selected from Table 3.
17. The protein array of claim 15 further comprising one or more recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC-Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
18. The protein array of claim 16 further comprising one or more recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC-Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
19. A protein array comprising all of the recombinant Borrelia fusion proteins constructs selected from Table 2 and Table 3.
20. The protein array of claim 19 further comprising one or more recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC-Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
21. A method of diagnosing Lyme disease, said method comprising:
A. contacting a test sample obtained from an individual suspected of having Lyme disease with a protein array, said protein array comprising one or more recombinant Borrelia fusion protein constructs wherein the one or more recombinant Borrelia fusion protein constructs comprise single recombinant Borrelia protein antigens selected from a group of dbpA, dbpB,OppA, OspA, purine-binding chemotaxis protein, p83/100 antigen, VMP-like sequence protein VlsE, surface lipoprotein P27, OspC type B, OspC type K, and BapA protein; and
B. detecting the presence of antibodies in the sample reactive to the single recombinant Borrelia protein antigens, wherein detecting the presence of antibodies to one or more single recombinant Borrelia protein antigens indicates the presence of Lyme disease in the individual.
22. The method of claim 21 wherein the protein array consists of all of the single
recombinant Borrelia protein antigens dbpA, dbpB and OppA.
23. The method of claim 21 wherein the protein array comprises two or more of the single recombinant Borrelia protein antigens purine-binding chemotaxis protein, p83/100 antigen, VMP-like sequence protein VlsE, surface lipoprotein P27, OspC type B, OspC type K, and BapA protein.
24. The method of claim 21 wherein the protein array further comprises one or more single recombinant Borrelia protein antigens selected from Table 1.
25. A method of diagnosing Lyme disease, said method comprising:
A. contacting a test sample obtained from an individual suspected of having Lyme disease with a protein array, said protein array comprising one or more recombinant Borrelia fusion protein constructs selected from Table 2; and
B. detecting the presence of antibodies in the sample reactive to the recombinant Borrelia fusion protein constructs, wherein detecting the presence of antibodies to one or more recombinant Borrelia fusion protein constructs indicates the presence of Lyme disease in the individual.
26. A method of diagnosing Lyme disease, said method comprising:
A. contacting a test sample obtained from an individual suspected of having Lyme disease with a protein array, said protein array comprising one or more recombinant Borrelia fusion protein constructs selected from Table 3; and
B. detecting the presence of antibodies in the sample reactive to the recombinant Borrelia fusion protein constructs, wherein detecting the presence of antibodies to one or more recombinant Borrelia fusion protein constructs indicates the presence of Lyme disease in the individual.
27. The method of claim 25 wherein the protein array consists of all of the recombinant Borrelia fusion proteins constructs of Table 2.
28. The method of claim 26 wherein the protein array consists of all of the recombinant Borrelia fusion proteins constructs of Table 3.
29. The method of claim 25 wherein the protein array further comprises one or more
recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC- Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
30. The method of claim 26 wherein the protein array further comprises one or more
recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC- Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
31. A method of diagnosing Lyme disease, said method comprising:
A. contacting a test sample obtained from an individual suspected of having Lyme disease with a protein array, said protein array comprising all of the recombinant Borrelia fusion proteins constructs selected from Table 2 and Table 3; and
B. detecting the presence of antibodies in the sample reactive to the recombinant Borrelia fusion protein constructs, wherein detecting the presence of antibodies to one or more recombinant Borrelia fusion protein constructs indicates the presence of Lyme disease in the individual.
32. The method of claim 31 wherein the protein array further comprises one or more recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC- Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
33. A method for detecting antibodies to Borrelia burgdoferi in a test sample comprising contacting the sample with a protein array of claim 11, under conditions sufficient for the antibodies in the sample to react with one or more single recombinant Borrelia protein antigens and detecting an antibody-antibody reaction.
34. The method of claim 33 wherein the protein array consists of all of the single
recombinant Borrelia protein antigens dbpA, dbpB and OppA.
35. The method of claim 33 wherein the protein array comprises two or more of the single recombinant Borrelia protein antigens selected from the group consisting of purine- binding chemotaxis protein, p83/100 antigen, VMP-like sequence protein VlsE, surface lipoprotein P27, OspC type B, OspC type K, and BapA protein.
36. The method of claim 33 wherein the protein array further comprises one or more single recombinant Borrelia protein antigens selected from Table 1.
37. A method for detecting antibodies to Borrelia burgdoferi in a test sample comprising contacting the sample with a protein array of claim 15, under conditions sufficient for the antibodies in the sample to react with one or more recombinant Borrelia fusion protein constructs and detecting an antibody-antibody reaction.
38. A method for detecting antibodies to Borrelia burgdoferi in a test sample comprising contacting the sample with a protein array of claim 16, under conditions sufficient for the antibodies in the sample to react with one or more recombinant Borrelia fusion protein constructs and detecting an antibody-antibody reaction.
39. The method of claim 37 wherein the protein array consists of all of the recombinant Borrelia fusion proteins constructs of Table 2.
40. The method of claim 38 wherein the protein array consists of all of the recombinant Borrelia fusion proteins constructs of Table 3.
41. The method of claim 37 wherein the protein array further comprises one or more recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC- Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
42. The method of claim 38 wherein the protein array further comprises one or more
recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC- Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
43. A method for detecting antibodies to Borrelia burgdoferi in a test sample comprising contacting the sample with a protein array of claim 19, under conditions sufficient for the antibodies in the sample to react with one or more recombinant Borrelia fusion protein constructs and detecting an antibody-antibody reaction.
44. The method of claim 43 wherein the protein array further comprises one or more
recombinant Borrelia fusion proteins selected from the group consisting of OspB-OspC- Flagellin (B-C-Fla), OspA-p39-p93 (A-39-93), and OspC dimer consisting of OspC Type B and Type H (OC2/9).
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