WO2024091264A1 - Protéines recombinantes chimériques et panels de protéines recombinantes pour le diagnostic de la maladie de lyme chez les animaux et les êtres humains - Google Patents

Protéines recombinantes chimériques et panels de protéines recombinantes pour le diagnostic de la maladie de lyme chez les animaux et les êtres humains Download PDF

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WO2024091264A1
WO2024091264A1 PCT/US2022/078580 US2022078580W WO2024091264A1 WO 2024091264 A1 WO2024091264 A1 WO 2024091264A1 US 2022078580 W US2022078580 W US 2022078580W WO 2024091264 A1 WO2024091264 A1 WO 2024091264A1
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variant
seq
amino acids
acid sequence
sequence identity
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Richard MARCONI
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Virginia Commonwealth University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the disclosure generally relates to diagnostics for diseases caused by Borreliella burgdorferi and related species in mammals, such as Lyme disease, also referred to as Lyme Borreliosis.
  • Lyme disease also referred to as Lyme Borreliosis.
  • the invention provides chimeric recombinant proteins comprising Borreliella proteins, or variants thereof, that are highly specific and sensitive when used to detect a i-Borreliella antibodies.
  • Lyme disease is the most common arthropod-bome disease in North America and Europe. It is caused by the spirochetes Borreliella burgdorferi, B. garinii, B. mayonii, B.myiamotoi, B. bissetiae, B. lanei, B. bavariensis, B. afzelii and several other taxonomically related species.
  • the bacterial species that cause Lyme disease were reclassified and assigned a new genus name Borreliella'). We collectively refer to the Borreliella species that cause Lyme disease as the Lyme disease spirochetes.
  • Lyme disease spirochetes Transmission of Lyme disease spirochetes to mammals occurs through the bite of infected Ixodes ticks [Burgdorfer et al, 1982, Benach et al., 1983].
  • Ixodes ticks Infected Ixodes ticks
  • Infection results in a multi-systemic inflammatory disease with early-stage symptoms that may include erythema migrans, low-grade fever, arthralgia, myalgia, and headache [Steere et al., 1977a].
  • Late-stage clinical manifestations can be severe and may include in part, arthritis [Steere et al., 1977a; Eiffert et al., 1998; Steere et al., 2004], carditis [Asch et al., 1994; Nagi et al., 1996 Barthold et al., 1991] and neurological complications [Nachman and Pontrelli, 2003; Coyle and Schutzer 2002].
  • Lyme disease has significant socio-economic costs, manifested by reductions in outdoor recreational and social activities due to concerns about tick exposure.
  • the present disclosure describes an exhaustive analysis of antibody responses that develop during Lyme disease infection and the resulting design, production and testing of “chimeric” recombinant proteins that comprise a plurality of different antigenic Borreliella proteins or variants or peptide segments thereof.
  • 3 or 4 different Borreliella proteins selected from, for example, DbpA, DbpB, BBK19, BBK53, VlsE, BBA73 and BB0238, and/or variants or peptide segments thereof
  • the chimeras including the chimeras referred to herein as HDFL4, DCFL4, and DCFL3a.
  • the chimeras proved to exhibit high specificity and sensitivity in Lyme disease diagnostic assays in humans and animals.
  • chimeric proteins do not yield false positives in vaccinated dogs and can detect infection shortly after exposure to infected Ixodes ticks.
  • studies in humans have demonstrated high sensitivity and specificity using both ELISA and lateral flow (rapid test) based assays.
  • the chimeric proteins are thus useful as diagnostic tools to identify individuals and/or animals that have antibodies to the epitopes in the chimeric proteins.
  • the chimeric proteins can be used to determine whether or not an individual person or animal has been exposed to and/or infected with Lyme disease spirochetes.
  • DbpB (SEQ ID NO: 2), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO: 2, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 2, or a third variant having at least 95% amino acid sequence identity to the first variant; and at least one additional Borreliella protein, or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of the at least one additional Borreliella protein, or a second variant thereof having at least 95% amino acid sequence identity to the at least one additional Borreliella protein, or a third variant having at least 95% amino acid sequence identity to the first variant.
  • the at least one additional Borreliella protein is:
  • BBA73 (SEQ ID NO: 3), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO: 3, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 3, or a third variant having at least 95% amino acid sequence identity to the first variant;
  • BBK53 (SEQ ID NO: 4), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO: 4, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 4, or a third variant having at least 95% amino acid sequence identity to the first variant;
  • BB0238 (SEQ ID NO: 5), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO: 5, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 5, or a third variant having at least 95% amino acid sequence identity to the first variant;
  • DbpA (SEQ ID NO: 13), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO: 13, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 13, or a third variant having at least 95% amino acid sequence identity to the first variant;
  • BBK19 (SEQ ID NO: 15), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of (SEQ ID NO: 15), or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 15, or a third variant having at least 95% amino acid sequence identity to the first variant; or
  • VlsE (SEQ ID NO: 16), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO: 16, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 16, or a third variant having at least 95% amino acid sequence identity to the first variant.
  • the recombinant chimeric polypeptide comprises i) DbpB (SEQ ID NO:2), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of (SEQ ID NO:2), or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO:2, or a third variant having at least 95% amino acid sequence identity to the first variant; ii) BBK53 (SEQ ID NO:4), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO:4, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO:4, or a third variant having at least 95% amino acid sequence identity to the first variant; and iii) BBA73 (SEQ ID NO:3), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids in
  • the recombinant chimeric polypeptide comprises i) DbpB (SEQ ID NO:2), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO:2, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO:2, or a third variant having at least 95% amino acid sequence identity to the first variant; ii) BBK53 (SEQ ID NO:4), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO:4, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO:4, or a third variant having at least 95% amino acid sequence identity to the first variant; iii) BBA73 (SEQ ID NO:3), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are
  • the recombinant chimeric polypeptide comprises i) DbpB (SEQ ID NO:2), ii) BBK53 (SEQ ID NO:4 and iii) BBA73 (SEQ ID NO:3).
  • the recombinant chimeric polypeptide comprises i) DbpB (SEQ ID NO:2), ii) BBK53 (SEQ ID NO:4), iii) BBA73 (SEQ ID NO:3), and iv) BB0238 (SEQ ID NO: 5).
  • the recombinant chimeric polypeptide comprises i) DbpB (SEQ ID NO:2), ii) DbpA (SEQ ID NO: 13), iii) BBK19 (SEQ ID NO: 15), and iv) VlsE (SEQ ID NO: 16).
  • composition comprising at least one recombinant chimeric polypeptide of any of claims 1-8 (i.e., at least one of any of the recombinant polypeptides disclosed herein), and a physiologically acceptable carrier.
  • the disclosure also provides a lyophilized, reconstitutable preparation comprising at least one recombinant chimeric polypeptide of any of claims 1-8 (i.e., at least one of any of the recombinant polypeptides disclosed herein).
  • the disclosure also provides a nucleic acid encoding a recombinant chimeric polypeptide of any of claims 1-8 (i.e., a nucleic acid encoding any of the recombinant polypeptides disclosed herein).
  • the disclosure also provides a vector comprising the nucleic acid of claim 11, (i.e., a vector comprising a nucleic acid encoding any of the recombinant polypeptides disclosed herein).
  • the disclosure also provides a host cell comprising the vector of claim 12 (i.e., a host cell comprising a vector comprising a nucleic acid encoding any of the recombinant polypeptides disclosed herein).
  • the disclosure also provides an Escherichia coli bacterial cell that is genetically engineered to contain and express the nucleic acid of claim 11 (i.e., an Escherichia coli bacterial cell that is genetically engineered to contain and express a nucleic acid encoding any of the recombinant polypeptides disclosed herein).
  • the disclosure also provides method of diagnosing Lyme disease in a subject, comprising i) contacting a biological sample from the mammal with at least one recombinant chimeric polypeptide of any of claims 1-8 i.e., at least one of any of the recombinant polypeptides disclosed herein), and detecting antibody-antigen complexes formed by antibodies in the biological sample and the at least one recombinant chimeric polypeptide, wherein the subject is diagnosed as having Lyme disease when the antibodyantigen complexes are detected.
  • the subject is a human or a non-human mammal.
  • the subject is a non-human mammal and the at least one recombinant chimeric polypeptide is or includes the recombinant chimeric polypeptide of claim 6 and/or claim 7 (i.e., the at least one recombinant chimeric polypeptide is or includes the recombinant chimeric polypeptide comprising i) DbpB (SEQ ID NO:2), ii) BBK53 (SEQ ID NO:4 and iii) BBA73 (SEQ ID NO:3) and/or the recombinant chimeric polypeptide comprises i) DbpB (SEQ ID NO:2), ii) BBK53 (SEQ ID NO:4), iii) BBA73 (SEQ ID NO:3), and iv) BB0238 (SEQ ID NO: 5)).
  • the subject is a human and the at least one recombinant chimeric polypeptide is or includes the recombinant chimeric polypeptide of claim 8 (i.e., the at least one recombinant chimeric polypeptide is or includes the recombinant chimeric polypeptide comprising i) DbpB (SEQ ID N0:2), ii) DbpA (SEQ ID NO: 13), iii) BBK19 (SEQ ID NO: 15), and iv) VlsE (SEQ ID NO: 16)).
  • the disclosure also provides a method of detecting antibodies that specifically bind a i-Borreliella antibodies in a test sample, comprising contacting the test sample with at least one recombinant chimeric polypeptide of claim 1 (i.e., a recombinant chimeric polypeptide comprising: DbpB (SEQ ID NO: 2), or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of SEQ ID NO: 2, or a second variant thereof having at least 95% amino acid sequence identity to SEQ ID NO: 2, or a third variant having at least 95% amino acid sequence identity to the first variant; and at least one additional Borreliella protein, or a first variant thereof that is at least 50 amino acids in length, wherein the at least 50 amino acids are identical to at least 50 contiguous amino acids of the at least one additional Borreliella protein, or a second variant thereof having at least 95% amino acid sequence identity to the at least one additional Borreliella protein
  • the disclosure further provides a device for detecting ni-Borreliella antibodies in a sample comprising: a) a sample application pad for receiving the sample; and b) a membrane substrate comprising a first test line, the first test line comprising at least one immobilized recombinant chimeric polypeptide of any of claims 1-8 (i.e., at least on of any of the recombinant chimeric polypeptides disclosed herein).
  • the device is a lateral flow immunoassay.
  • the membrane substrate further comprises at least one control line.
  • the device further comprises a backing.
  • the device further comprises a wicking pad.
  • Figure 1 Synthesis, generation, and purification of candidate antigens for inclusion in chimeric diagnostic proteins.
  • the genes encoding each protein were codon optimized, synthesized, and ligated into the pET45 expression vector.
  • the proteins were expressed and purified.
  • the SDS-PAGE gel presents an image of the purity of representative proteins (indicated along the top).
  • FIG. 2A and B Analysis of antibody responses to Lyme disease spirochete proteins in wildlife (Canis latrans; Eastern coyotes).
  • A B. burgdorferi cell lysates were immunoblotted and screened with serum collected from Lyme disease-infected eastern coyotes from the commonwealth of Pennsylvania. The results demonstrated that the antibody responses that develop vary among individual animals (as evidenced by the immunoreactive protein profiles).
  • B the coyote sera were used to screen recombinant proteins using a dot-blot format. As can be seen, no single protein was sufficient to confirm infection in all animals.
  • the labels above each panel indicate the geographic sector of Pennsylvania from which the animals and serum samples were collected, Abbreviations are as follows: NW, northwest; NC, north central; NE, northeast; SW, southwest; SC, southcentral, southeast.
  • FIG 3A and B Identification of B. burgdorferi proteins that elicit antibodies by day 12 of infection in mice.
  • A first set of 30 proteins
  • B second set of 30 proteins. Mice were infected and serum recovered from each on days 7, 8, and 12 (left to right in each data set). The proteins listed on the x-axis were screened with the serum samples by ELISA.
  • Figure 4. Identification of proteins that can detect antibodies to Lyme disease spirochetes during early infection. Recombinant proteins (as indicated in the key) were screened by ELISA with serum collected over time from experimentally infected dogs. Approximately 60 different proteins were screened (representative data are shown).
  • Figure 5. Identification of the immunodominant region of candidate diagnostic antigens.
  • BBA24, BBA25, and BBA36 Full- length proteins (minus the leader peptide) and subfragments of each protein (indicated in the schematic), were synthesized, produced, and purified. The proteins were dot-blotted onto nitrocellulose membranes (images on the right) and screened with serum from infected animals. The immunodominant regions of BBA24, BBA25, and BB A36 mapped within residues 75-190, 32-97, and 79-155, respectively. The same mapping strategy was applied to several other proteins. These analyses were central in informing the choice of antigens and specific protein fragments to use in the generation of chimeric diagnostic antigens.
  • FIG. 6 Schematic depiction of representative chimeric diagnostic antigens. The identity of each chimeric is indicated to the left and the proteins, or protein segments, that comprise each are indicated above or within each schematic. The varying segments of the N and C terminal domains of the proteins that comprise REBE and RCBE have been deleted in these constructs, as detailed in the sequences. The schematics are not drawn to scale.
  • DCFL4 does not yield false positives with sera from dogs infected with other pathogens.
  • Recombinant DCFL4, VlsE and FhbB (left, middle and right bars in each data set) were immobilized in ELISA plate wells and screened with serum from dogs verified to be infected with Leptospira (1-5), Anaplasma (6-10), or Ehrlichia (11-15). Positive controls included serum from dogs infected with Lyme disease (LD+) and infection-free dogs (LD-).
  • Figure 8 Analysis of the ability of representative chimeric antigens (recombinant chimeric proteins) to detect antibody responses elicited by infection with Lyme disease spirochetes over time. Mice were infected with B. burgdorferi, blood was collected at the timepoints indicated and used to screen the recombinant chimerics by ELISA. Of those tested, DCFL4 performed the best. Subsequent analyses revealed that HDFL4 and DCFL3a (data not shown) were also highly effective at detecting early antibody responses.
  • FIG 9A and B DCFL4 detects antibodies elicited by infection with Lyme disease spirochetes during early-stage infection and throughout the course of infection.
  • A ELISA results in which the proteins indicated on the X axis were screened with serum collected 21 days (i.e., early) after tick bite from experimentally infected dogs.
  • B ELISA results in which the proteins indicated were screened with serum from infected dogs over longer periods of time. The results demonstrate that DCFL4 detects antibodies during both early and late-stage infection.
  • Figure 10. Demonstration the chimeric recombinant diagnostic antigens do not yield false positive results in dogs vaccinated with commercially available vaccines.
  • Recombinant proteins were spotted onto membranes and then the membranes were screened with sera from vaccinated or control dogs. One set of dot blots was stained with MemCodeTM to verify the presence of the chimerics on the membrane. Representative data are shown for the DCF5A, DCF5B, and DCFL4 chimerics.
  • FhbB is a negative control protein derived from Treponema denticola that should not react with any serum sample.
  • B. burgdorferi OspA is a positive control protein for the detection of antibodies elicited by vaccination (all current vaccines contain OspA).
  • VlsE is a positive control for the detection of antibodies in dogs infected with Lyme disease spirochetes. Trade names for each vaccine are indicated on the left. False positives were not observed in vaccinated dogs with any of the chimeric constructs.
  • FIG. 11 Optimization of ELISA conditions for diagnostic applications.
  • a serum sample from a dog confirmed to be infected with B. burgdorferi was diluted as indicated and then incubated with a chromogenic substrate for increasing lengths of time (as indicated). It can be concluded that about 1:1000 is an optimal dilution for diagnostic assays.
  • An optimal timeframe for incubation with the chromogenic substrate is about 20 minutes (based on convenience and keeping the OD readings from maxing out).
  • FIG. 12 Screening of the Lyme Disease Biobank Test Serum panel.
  • the Lyme Disease Biobank has generated a well-characterized serum panel of human sera that can be used to validate and compare the sensitivity and specificity of diagnostic antigens.
  • a dot blot format was used.
  • the chimerics and control proteins shown to the left were screened with the serum samples.
  • the serum ID numbers assigned by the Biobank are indicated.
  • the two-tier (2TT) scoring results for both IgM and IgG as determined by others are indicated.
  • DCFL4 displayed high sensitivity and specificity. This panel was screened specifically for IgG. DCFL4 proved highly effective in detecting antibodies in human Lyme disease patients with well-characterized disease.
  • test line 106 which comprises immobilized capture molecules (e.g., antibodies specific for binding to complexes that comprise ni-Borreliella antibodies bound to chimeric proteins), control line 107 which lacks capture molecules and sample application pad 103, which receives a sample to be tested.
  • immobilized capture molecules e.g., antibodies specific for binding to complexes that comprise ni-Borreliella antibodies bound to chimeric proteins
  • control line 107 which lacks capture molecules
  • sample application pad 103 which receives a sample to be tested.
  • aspects of the present disclosure are related to diagnosing disease caused by Lyme disease spirochetes. Described herein is an exhaustive analysis of antibody responses that develop during infection with Borreliella species. This was accomplished by generating and individually screening 60 different Lyme disease spirochete proteins to determine if they i) are expressed during mammalian infection and ii) elicit antibody responses during both natural and experimental infection. Sera from humans, dogs, horses, eastern coyotes, foxes (gray and red), pocket gophers, ground hogs, grey squirrels, mice, black vultures, eastern black bears, and raccoons, were screened for antibodies to each of the 60 proteins using single dilution ELISA and immunoblot analyses. While several proteins were identified as being highly antigenic, no single natural protein was able to detect antibodies in all of the infected mammals. Thus, no one single natural protein was identified that would provide the necessary specificity and sensitivity required of a diagnostic assay.
  • chimeric proteins were generated by designing chimeric genes that encode the proteins that had been identified as highly antigenic.
  • the chimeric genes were codon optimized for efficient production in expression systems.
  • the leader peptides or lipidation motifs were omitted to allow for high-level expression.
  • the chimeras which have no parallel or equivalent in nature, comprise non-lipidated amino acid sequences derived from about 3 to about 8 different proteins selected from the 60 that were initially tested.
  • the amino acid sequences are full-length proteins (minus the N-terminal leader sequence and lipidation motif).
  • the amino acid sequences are antigenic peptide segments (e.g., epitopes) derived from the about 3 to about 8 different proteins.
  • epitopes e.g., epitopes
  • Several different variants of each chimeric protein were made in which the ordering of the component proteins or epitopes was varied. These analyses were critical as the optimal ordering of the polypeptides in the chimerics could not be predicted and was not obvious.
  • the chimeric proteins were modified e.g. by the inclusion of non-native tryptophan insertions to allow for accurate quantification.
  • some properties of some proteins or the genes that encode them were modified to make them amenable to mass production, e.g., by identifying variants of the chimeras that could be readily produced at high levels with high purity as soluble proteins in Escherichia coli expression systems.
  • the specificity and sensitivity of the chimeras were tested using ELISA, dot blots, immunoblot and lateral flow assays.
  • the results showed that, in contrast to any of the individual proteins and other prior art Lyme disease diagnostic proteins, the chimeric proteins disclosed herein advantageously exhibited very high specificity and sensitivity toward antibodies elicited during infection with the Lyme disease spirochetes. Significantly, very high specificity and sensitivity was exhibited in both humans and animals infected with Lyme disease spirochetes.
  • recombinant chimeric polypeptides comprising the Lyme disease spirochete proteins DbpB, DbpA, BBK19, BBK53, VlsE, BBA73 and BB0238, typically in a specific order within the chimera, exhibited exceptionally high specificity and sensitivity. Also of high significance, the high specificity and sensitivity was exhibited without generating false positive results.
  • chimeric proteins comprising these proteins are readily produced using in vitro manufacturing processes, such as by using E. coli bacteria that are genetically engineered to encode and express nucleic acids encoding the chimeras.
  • the chimeras can be used in any species of mammal to determine if a mammal is actively infected or was recently infected with Lyme disease spirochetes.
  • the chimeras can be used in young dogs with suspected Lyme disease to differentiate between antibodies resulting from an active infection and antibodies resulting from infection of the mother (maternal antibodies). This is done by testing the serum samples obtained from young dogs at timed intervals, shortly after birth (e.g., 6-15 weeks). Antibodies that are maternal in origin decrease in titer over time whereas persistent and elevated antibody responses indicate an active infection.
  • Antigen- term used historically to designate an entity that is bound by an antibody, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody, while the word “immunogen” is used for the entity that induces antibody production. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen will typically be made according to its intended utility.
  • the terms “antigen”, “antigenic region” “immunogen” and “epitope” may be used interchangeably herein.
  • an antigen, immunogen or epitope is generally a portion of a protein (e.g. a peptide or polypeptide).
  • Recombinant protein sequences that can be added to the N- or C-terminus of a recombinant protein for the purpose of identification or for purifying the recombinant protein for subsequent uses.
  • recombinant protein tags that may be useful in practicing the invention include but are not limited to glutathione-S-transferease (GST), poly-histidine, maltose binding protein (MBP), FLAG, V5, halo, myc, hemaglutinin (HA), S-tag, calmodulin, tag, streptavidin binding protein (SBP), SoftaglTM, Softag3TM, Xpress tag, isopeptag, Spy Tag, biotin carboxyl carrier protein (BCCP), GFP, Nus-tag, strep-tag, thioredoxin tag, TC tag, and Ty tag.
  • GST glutathione-S-transferease
  • MBP maltose binding protein
  • FLAG FLAG
  • V5 halo, myc
  • tags are well-known to those of ordinary skill in the art of recombinant protein production and may or may not be removed before use, using tag specific cleavage protocols.
  • a hexa-histidine tag (His-His-His-His-His-His-His (SEQ ID NO: 74)) is typically removed by incubation of the protein with the enzyme enterokinase which recognizes the following amino acid sequence: AspAspAspAspLys (SEQ ID NO: 75). Cleavage of this motif releases the His tag from the recombinant protein.
  • Epitope a specific chemical domain on an antigen that is recognized by a B-cell receptor, and which can be bound by secreted antibody.
  • the term as used herein is interchangeable with “antigenic determinant”.
  • An epitope may comprise a single, non-interrupted, contiguous chain of amino acids joined together by peptide bonds to form a peptide or polypeptide. Such an epitope can be described by its primary structure, i.e. the linear sequence of amino acids in the peptide chain.
  • Epitope may also refer to conformational epitopes, which are comprised of at least some amino acids that are not part of an uninterrupted, linear sequence of amino acids, but which are brought into proximity to other residues in the epitope by secondary, tertiary and/or quaternary interactions of the protein. Residues in conformational epitopes may be located far from other resides in the epitope with respect to primary sequence but may be spatially located near other residues in the conformational epitope due to protein folding.
  • Panel indicates more than one protein e.g., in an assay.
  • Protein- Generally means a linear sequence of about 100 or more amino acids covalently joined by peptide bonds.
  • Polypeptide- Generally means a linear sequence of about 55 to about 100 amino acids covalently joined by peptide bonds.
  • Peptide- Generally means a linear sequence of about 100 or fewer amino acids covalently joined by peptide bonds.
  • polypeptide and “protein” may be used interchangeably herein. Chimeric or fusion peptide or polypeptide-, a recombinant or synthetic peptide or polypeptide whose primary sequence comprises two or more linear amino acid sequences which do not occur together in a single molecule in nature.
  • the two or more sequences may be, for example, a peptide (e.g. an epitope or antigenic region) and a linker sequence, or two or more peptides (which may be the same or different) which are either contiguous or separated by a linker sequences, etc. Original or native or wild type sequence'.
  • the sequence of a peptide, polypeptide, protein or nucleic acid as found in nature.
  • Recombinant peptide, polypeptide, protein or nucleic acid A peptide, polypeptide, protein or nucleic acid that has been produced and/or manipulated using molecular biology techniques such as cloning, polymerase chain reaction (PCR), etc.
  • Synthetic peptide, polypeptide, protein or nucleic acid A peptide, polypeptide, protein or nucleic acid that has been produced using chemical synthesis procedures.
  • Techniques for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection ), enzymatic reactions, purification, and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology as cited and discussed throughout the present specification.
  • a polypeptide e.g. a first polypeptide
  • a reference polypeptide e.g. a second polypeptide
  • Similarity of two polypeptides can be determined along the entire length of the polypeptides by aligning the residues of the two polypeptides to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • polypeptides may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website.
  • similarity In the comparison of two amino acid sequences, similarity may be referred to by e.g. “percent identity” or “percent similarity.” “Identity” refers to the presence of identical amino acids along the entire length of the polypeptides that are being compared. “Similarity” generally refers to the presence of not only identical amino acids but also the presence of conservative substitutions along the entire length of the polypeptides that are being compared. A conservative substitution for an amino acid may be selected from other members of the class to which the amino acid belongs.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free
  • Specificity as used herein generally refers to the ability of a chimeric protein to discriminate between similar or even dissimilar antibodies, e.g., antibodies to other infectious agents. Specificity measures how often a test correctly gives a negative result when a person does not have the disease. Low specificity can result in false positives, incorrectly identifying non-infected subjects as sick. The higher the specificity, the less chance there will be a false positive result.
  • “Sensitivity” as used herein generally refers to how often a test correctly gives a positive result when a subject has or has had an infection that generated antibodies to an infectious agent. Low sensitivity can result in false negatives, incorrectly identifying infected subjects as not infected. The higher the sensitivity, the less chance there will be a false negative result.
  • compositions of the invention comprise one or more isolated and purified recombinant chimeric proteins/polypeptides.
  • Each chimeric polypeptide comprises a plurality of Lyme disease spirochete proteins.
  • each chimeric polypeptide comprises, for example, from about 2-10 different Borreliella proteins, such as about 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different Borreliella proteins, and/or peptide segments from about 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different Borreliella proteins.
  • each chimeric polypeptide comprises from about 3 to 6 different Borreliella proteins (e.g.
  • Borreliella proteins, and/or peptide segments thereof, that are included in a chimera are selected from, for example: BB0141, BB0147, BB0167, BB0238, BB0283, BB0419, BB0564, BB0603, BB0612, BB0664, BB0733, BBA04, BBA15, BBA16, BBA19, BBA24, BBA25, BBA36, BBA57, BBA60, BBA64, BBA66, BBA68, BBA73, BBA74, BBB07, BBB09, BBB14, BBB19, BBC10, BBE09, BBE16, BBH06, BBI29, BBI36, BBI39, BBJ41, BBK01, BBK19, BBK32
  • BBK50 the leader sequence is underlined
  • NKNTFKQVVQEALKGGIDGFENTASSTCKNS SEQ ID NO:65
  • Exemplary chimeric proteins that comprise at least two, and generally 3 or 4, of these sequences include but are not limited to: i) DCFL4 (SEQ ID NO: 1) which comprises, in order from amino to carboxy terminus, the four proteins: DbpB (SEQ ID NO: 2) (wavy underline); BBA73 (SEQ ID NO: 3) (bold); BBK53 (SEQ ID NO: 4) (no underline) and BB0238 (SEQ ID NO: 5) (italics).
  • DCFL4 SEQ ID NO: 1 which comprises, in order from amino to carboxy terminus, the four proteins: DbpB (SEQ ID NO: 2) (wavy underline); BBA73 (SEQ ID NO: 3) (bold); BBK53 (SEQ ID NO: 4) (no underline) and BB0238 (SEQ ID NO: 5) (italics).
  • DCFL3A (SEQ ID NO: 6) which comprises, in order from amino to carboxy terminus, the three proteins DbpB (SEQ ID NO: 2) (wavy underline), BBA73 (SEQ ID NO: 3) (bold) and BBK53 (SEQ ID NO: 4) (no underline).
  • HDFL4 which comprises, in order from amino to carboxy terminus, the four proteins DbpA (SEQ ID NO: 13) (wavy underline), DbpB, (SEQ ID NO: 2) (bold), BBK19 (SEQ ID NO: 15) (no underline), VlsE (SEQ ID NO: 16) (italics).
  • ETPPAENK SEQ ID NO: 10.
  • DCFL3B the sequence of another chimera, DCFL3B.
  • This chimeric provided low sensitivity for detecting antibody in serum from infected mammals.
  • the genes represented in this construct are also in DCFL4 which has high sensitivity and specificity. It can be concluded that gene order and composition are critical and not obvious.
  • the order of the genes in this construct is: DbpB (SEQ ID NO: 2) (wavy underline), BB0238 (SEQ ID NO: 5) (bold), and BBK53 (SEQ ID NO: 4) (no underline).
  • DbpB-F2 (SEQ ID NO: 69) (wavy underline), BBA73-F5 (SEQ ID NO: 70) (bold), BB0238-F2 (SEQ ID NO: 71) (no underline), and BBK53-F1 (SEQ ID NO: 72) (italics).
  • F stands for “fragment”.
  • an entire native or wild type protein (with or without its leader peptide and/or lipidation motif) is present in a chimera.
  • a portion of the wildtype sequence is used, e.g. a peptide segment or segments of at least about 20-25 contiguous amino acids of the protein that comprise(s) epitopes recognized by mammalian antibodies. “At least about 20-25 contiguous amino acids” refers to about 20, 21, 22, 23, 24, or 25 amino acids.
  • the portion of the wildtype sequence is longer, e.g.
  • amino acids from about 20 to 200 amino acids, such as about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 amino acids.
  • the at least about 25 contiguous amino acids are the amino acids that remain after a deletion of amino acids from the amino terminus of the protein and/or from the carboxy terminus of the protein. In further aspects, the at least about 25 contiguous amino acids are the amino acids that remain after a deletion of amino acids from within the sequence of the protein (internal deletion) but which are joined by a covalent bond. In other aspects, the at least about 50 contiguous amino acids are the amino acids that remain after one or more of these types of deletion, i.e. they remain after a combination of these types of deletions such as one from the amino and/or carboxy terminus and/or one or more internal deletions.
  • the polypeptide e.g. a first polypeptide
  • the polypeptide may be full- length or a peptide segment or peptide segments of the native protein from which it is derived
  • the polypeptide is, independent from the first polypeptide, either full length or a peptide segment or peptide segments of the native protein from which it is derived.
  • the order of the constituent 2-8 proteins and/or variants thereof may be varied.
  • their order within the chimera can be: ABCD, BCDA, CDAB, CABC, AB DC, BACD, etc. with all permutations of these orders permitted, as long as the resulting chimeric polypeptide is capable of the specificity and sensitivity for detecting ni-Borreliella antibodies as described herein.
  • a variant of a wild-type Lyme disease spirochetes protein having at least 50% amino acid sequence identity to i) the amino acid sequence of any individual wildtype Lyme disease spirochete protein disclosed herein or to ii) fragments or segments thereof as disclosed herein or to iii) a complete chimera as disclosed herein.
  • the amino acid sequence of a variant (either a variant of an individual Borreliella protein, segment thereof or a variant of a chimeric polypeptide) has at least about 85 to about 100% amino acid identity along the length of the query protein (e.g. a native sequence, segment thereof or a chimera) i.e.
  • Such protein variants may be included in a chimera, as long as they maintain a suitable, useful level of activity, i.e., as long as they react with ni-Borreliella antibodies in a sample to a degree or level that results in a high degree of specificity and sensitivity described herein.
  • sequences or sequence variants that are present in a chimeric polypeptide are separated from one another by intervening sequences that are more or less neutral in character, i.e. they do not in participate in or contribute to binding to anti- Borreliella antibodies. However, they also do not interfere with antibody binding. If present, such sequences may be e.g., artifacts of recombinant processing procedures that are included for practical reasons (e.g., they arise from restriction sites), or for convenience, e.g. to identify the stop and start sites of a sequence within a chimera, etc.
  • Such sequences may be e.g., from about 2-10 amino acids in length, and are typically known as linker or spacer peptides, many examples of which are known to those of skill in the art. See, for example, Crasto, C. J. and J. A. Feng. 2000. Such linker sequences may or may not be included in identity calculations.
  • chimeric proteins for example sequences that “tag” the protein to facilitate visualization, purification and/ or detection of the protein, the addition of a tryptophan residue to the sequence or the substitution of an amino acid in the sequence by a tryptophan residue, Hexahistidine tags, S-tags, Flagtags, various protein stabilizing motifs, or other N- or C-terminal tags that are added for the purpose of facilitating purification and or detection, etc.
  • tagging sequences may or may not be included in identity calculations.
  • the chimeric proteins may be chemically modified, e.g. by amidation, sulfonylation, lipidation, or other techniques that are known to those of skill in the art.
  • the naturally occurring leader sequences of the native proteins are not included in the chimera. However, chimeras in which one or more leader sequences are included are also encompassed.
  • compositions disclosed herein comprise at least one chimeric recombinant protein as described herein
  • compositions are provided which comprise at least one protein, and typically a plurality of proteins, e.g., a cocktail of proteins.
  • the protein or proteins that are selected for inclusion in the cocktail are typically those identified herein as exhibiting high specificity and sensitivity in Lyme disease diagnostic assays in humans and/or animals.
  • combinations of proteins, or antigenic portions thereof as described herein, that are the same as those included in a recombinant chimera are included in a composition, but they are not covalently linked to each other.
  • one or more selected proteins may be included in a composition together with one or more chimeric proteins.
  • compositions typically include other components as described for chimeric compositions, i.e. a physiologically compatible buffer or carrier (e.g. a phosphate-buffered saline buffer, citrate buffer, a Good’s buffer, etc.) and may further comprise, e.g. a blocking agent (e.g., casein, Tween® reagent, etc.), a surfactant, various additives, and other reagents to increase sensitivity of the assay, as known in the art.
  • a physiologically compatible buffer or carrier e.g. a phosphate-buffered saline buffer, citrate buffer, a Good’s buffer, etc.
  • a blocking agent e.g., casein, Tween® reagent, etc.
  • surfactant e.g., a surfactant, various additives, and other reagents to increase sensitivity of the assay, as known in the art.
  • Polynucleotide encoding the recombinant chimeric polypeptides, or variants thereof, are provided.
  • polynucleotide or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids.
  • Polynucleotides may be single- stranded or double- stranded and either recombinant, synthetic, or isolated.
  • Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
  • pre-mRNA pre-messenger RNA
  • mRNA messenger RNA
  • RNA(+) plus strand RNA
  • RNA(-) minus strand RNA
  • PCR amplified DNA complementary DNA
  • cDNA complementary DNA
  • synthetic DNA or recombinant DNA.
  • polynucleotides may be codon-optimized for gene expression in E. coli or any other suitable expression cell (insect, mammalian, other bacteria).
  • codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide based on e.g.
  • codon biases between two or more organisms or genes or synthetically constructed bias tables variation in the degree of codon bias within an organism, gene, or set of genes, systematic variation of codons including context, variation of codons according to their decoding tRNAs, variation of codons according to GC %, either overall or in one position of the triplet, variation in degree of similarity to a reference sequence, for example, a naturally occurring sequence, variation in the codon frequency cutoff, structural properties of mRNAs transcribed from the DNA sequence, systematic variation of codon sets for each amino acid, and/or isolated removal of spurious translation initiation sites.
  • the polynucleotides encode at least one, and usually only one, of the chimeric polypeptides or a variant thereof as disclosed herein.
  • Various illustrative embodiments of such polynucleotides include but are not limited to those set forth in SEQ ID NOS: 8, 9 and 11 etc., and variants thereof.
  • Exemplary DNA sequence encoding DCFL4 where the DNA sequence encoding the N- terminal tag that is part of the protein to facilitate expression is underlined and may be present or absent or replaced by a different tag in variants of this sequence.
  • ATAACAGCCCGGTTAGCATCCTGCCGACCTAA SEQ ID NO: 9
  • polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
  • sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide over a window of comparison, similar to that of amino acid “identity” discussed herein.
  • a "percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • nucleotides and polypeptides comprising at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% (or even 100%) sequence identity compared to any of the sequences described herein.
  • Sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (including but not limited to GAP, BESTFIT, FASTA, and TFASTA) etc.; or the BLAST family of programs as for example disclosed in the GCG (Genetics Computer Group) product, the Wisconsin Package 11.0 (or later when available), a recognized industry standard for sequence analysis of nucleic acids and protein sequences; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 2012.
  • algorithms including but not limited to GAP, BESTFIT, FASTA, and TFASTA
  • nucleotide sequences that may encode a polypeptide, or fragment of variant thereof, as contemplated herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene or chimeric polypeptide. All such sequences are encompassed herein.
  • nucleic acid cassette or "expression cassette” refers to genetic sequences within a vector which can express an RNA, and subsequently a polypeptide.
  • the nucleic acid cassette contains at least one gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest that encodes at least one chimera as disclosed herein.
  • the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter (e.g.
  • the nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA. Generally, the cassette can be removed and inserted into a plasmid or viral vector as a single unit.
  • a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
  • a desired polypeptide can also be expressed by delivering an mRNA encoding the polypeptide into a cell.
  • vector is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
  • vectors include, but are not limited to plasmids, autonomously replicating sequences, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), various episomal vectors, bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • artificial chromosomes such as yeast artificial chromosome (YAC), various episomal vectors, bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • Exemplary expression vectors include but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST.TM., pLenti6/V5-DEST.TM., and pLenti6.2/V5- GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • a polynucleotide sequence of interest is generally “operably linked” to other elements of the vector so as the intended function of all elements is possible.
  • the phrase refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • Exemplary expression control sequences suitable for use according to the present disclosure include but are not limited to: cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl l promoters from vaccinia virus, short elongation factor 1-alpha (EFla-short) promoter, long elongation factor 1-alpha (EFla-long) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5),
  • the chimeric proteins may be produced by any suitable method, many of which are known to those of skill in the art. For example, they may be chemically synthesized, or produced using recombinant DNA technology (e.g. in bacterial cells, in cell culture (mammalian, yeast or insect cells), in plants or plant cells, or by cell-free prokaryotic or eukaryotic-based expression systems, by other in vitro systems, etc.).
  • the polypeptides are produced using an E. coli recombinant expression system.
  • the disclosure also encompasses bacterial, mammalian, yeast or insect host cells which comprise at least on copy of a nucleotide sequence encoding a recombinant chimeric protein disclosed herein.
  • the host cell is a bacterial cell.
  • the bacterial cell is an E. coli host that has been genetically engineered to contain and express at least one nucleic acid sequence disclosed herein in a manner that results is production of the encoded recombinant chimeric protein.
  • Methods of making the recombinant chimeric proteins are also included.
  • the methods include genetically engineering a host cell to contain and express a nucleic acid encoding at least one recombinant chimeric protein disclosed herein.
  • the host cell is genetically engineered so that the at least one recombinant chimeric protein is produced by the host cell, either within the host cell or excreted into medium in which the host cell is grown.
  • Methods of making recombinant proteins using various types of host cells are known in the art, e.g. by growing/cultivating the host cell in a medium compatible with growth, and then i) harvesting and lysing the host cells to release the proteins or, for excreted proteins ii) harvesting the growth medium.
  • Various known steps of concentrating, purifying and analyzing the harvested proteins are followed to achieve a desired level of purity for use in the diagnostics disclosed herein, e.g. filtration, centrifugation, various column purification methods (e.g. affinity chromatography), and the like. Purity levels and identity may be determined, e.g. by gel electrophoresis, HPLC, sequencing, mass spectrometry, and/or any combination of these.
  • a biological sample from an individual e.g., a human, deer, dog or other mammal susceptible to infection by Borreliella
  • a biological sample is contacted with at least one of the chimeric proteins of the invention.
  • the sample may be contacted with a plurality (e.g., 2 or more) of the chimeric proteins.
  • At least one control protein that is known to not react with antibodies to Borreliella proteins is generally included in the assay.
  • a positive result i.e., binding occurs and antibody-chimeric protein complexes are detected, thus a i-Borreliella antibodies are present
  • a negative result i.e., binding does not occur and antibody-chimeric protein complexes do not form, thus a i-Borreliella antibodies are not present
  • a negative result indicates that the individual is not infected with Borreliella and has not been previously exposed. For the control proteins, a negative result is expected.
  • the assays disclosed herein provide both high specificity and sensitivity with respect to detecting a i-Borreliella antibodies, as described elsewhere herein.
  • the assays are useful for diagnosing a Borreliella infection in a human subject or a non-human mammalian subject.
  • the assays are performed by contacting (mixing, etc.) at least one of the chimeric proteins disclosed herein with a biological sample obtained from a subject who is at risk of having contracted Lyme disease or is suspected of having Lyme disease or is known to have Lyme disease.
  • the test sample is typically blood, plasma, or serum.
  • the assay used to detect antibodies may be any type of immunoassay, examples of which include but are not limited to: an enzyme-linked immunosorbent assay (ELISA) (e.g., a sandwich ELISA, or a competitive ELISA), a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow based assays, a flow cytometry assay, a multiplex immunoassay, a mass spectrometry assay, or a particulate-based assay, immuno staining, latex agglutination, indirect hemagglutination assay (IHA) and indirect immnunofluorescent assay (FA).
  • ELISA enzyme-linked immunosorbent assay
  • IHA indirect hemagglutination assay
  • FA indirect immnunofluorescent assay
  • Exemplary methods of detecting the binding of an anti- Borreliella antibody to an immunoreactive chimeric polypeptide as disclosed herein may include, for example, an ELISA performed in a microplate, a lateral flow test performed using a dipstick or lateral flow device, or a particulate-based suspension array assay, e.g performed using the Bio-Plex® system (Bio-Rad Laboratories, Hercules, Calif., USA).
  • the diagnostic is an immunoblot, an enzyme-linked immunosorbent assay (ELISA) or a lateral flow assay, all three of which were used in the Examples described herein.
  • immunoblots and ELISAs are conducted in a laboratory setting, such as in a commercial or research diagnostic lab, whereas lateral flow assays can be conducted rapidly e.g., at the point of care, such as in a physician’s or veterinarian’s office.
  • the diagnostic aspects of the disclosure are not confined to clinical use or home use, but may also be valuable for use in the laboratory as a research tool, e.g. to identify Lyme disease spirochetes isolated from ticks, screen for the presence of antibodies in sentinel animals such as wildlife, to investigate the geographical distribution of Borreliella species and strains, etc.
  • the diagnostic tools detailed here can also be used for epidemiological analyses.
  • the detection of a chimera-antibody complex described herein is accomplished using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • This assay may be performed by first contacting a chimeric polypeptide that has been immobilized on a solid support, commonly the well of a microtiter plate, with a sample, such that antibodies specific for the chimeric polypeptide within the sample are allowed to bind to the immobilized chimeric polypeptide. Unbound sample is then removed from the immobilized chimeric polypeptide and a detection reagent capable of binding to the immobilized antibody-chimeric polypeptide complex is added.
  • the reagent is capable of detecting IgG and IgG that have bound to the diagnostic antigen. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent.
  • the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated or covalently attached to a reporter group or label.
  • a binding agent such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen
  • reporter groups or labels include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • the conjugation of binding agent to reporter group or label may be achieved using standard methods known to those of ordinary skill in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many commercial sources (e.g., Zymed Laboratories, San Francisco, Calif.; and Pierce, Rockford, Ill.).
  • the presence or absence of Borreliella specific antibodies may be determined in the sample by comparing the level of a signal detected from a reporter group or label in the sample with the level of a signal that corresponds to a control sample or predetermined cut-off value.
  • the cut-off value may be the average mean signal obtained when the immobilized Borreliella immunoreactive chimera is incubated with samples from an uninfected subject. The cut-off value may be determined using a statistical method or computer program.
  • an ELISA assay as described above may be performed in a rapid flow-through, lateral flow, or strip test format, wherein the antigen is immobilized on a membrane, such as a nitrocellulose membrane.
  • a membrane such as a nitrocellulose membrane.
  • a detection reagent such as protein A labeled with gold, a fluorophore, or a chromophore, binds to the chimera- antibody complex as the solution containing the detection reagent flows through the membrane.
  • Chimera-antibody complexes bound to the detection reagent may then be detected, as appropriate for the detection reagent used (e.g., based on the presence or absence of a visibly detectable color or fluorescent label, a nanoparticle, a luminescent rare earth nanoparticle, a luminous nanoparticle, a strontium aluminate nanoparticle (e.g., see Paterson et al., 2014; and Wang et al., 2017, etc.).
  • a visibly detectable color or fluorescent label e.g., based on the presence or absence of a visibly detectable color or fluorescent label, a nanoparticle, a luminescent rare earth nanoparticle, a luminous nanoparticle, a strontium aluminate nanoparticle (e.g., see Paterson et al., 2014; and Wang et al., 2017, etc.).
  • a flow-through format ELISA may be performed in which one end of the membrane to which a Borreliella immunoreactive chimera is immobilized may be immersed in a solution containing the sample, or the sample may be added to an area (i.e., a sample zone) at one end of the membrane.
  • the sample migrates along the membrane through a region (i.e., a labeling zone) comprising the detection reagent, and flows to the area (i.e., a binding zone) comprising the immobilized Borreliella immunoreactive chimera.
  • An accumulation of detection reagent at the binding zone indicates the presence of Borreliella specific antibodies in the sample.
  • a flow-through ELISA may feature a detection reagent applied to a test strip in a pattern, such as a line, that can be read visually. As with other lateral flow tests, the absence of such a pattern typically indicates a negative result. It is within the ability of an ordinarily skilled artisan to select an amount of the Borreliella immunoreactive chimeric polypeptide for immobilization on the membrane that can generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in a standard format ELISA. Preferably, the amount of chimeric polypeptide immobilized on the membrane ranges from about 25 ng to about 1 mg.
  • particle -based assays use a capture-binding partner, such as an antibody or an antigen in the case of an immunoassay, coated on the surface of particles, such as microbeads, crystals, chips, or nanoparticles.
  • Particle-based assays may be effectively multiplexed or modified to assay numerous variables of interest by incorporating fluorescently labeled particles or particles of different sizes in a single assay, each coated or conjugated to one or more labeled capture-binding partners.
  • the use of sensitive detection and amplification technologies with particle-based assay platforms known in the art has resulted in numerous flexible and sensitive assay systems to choose from in performing a method described herein.
  • a multiplex particle-based assay such as the suspension array Bio-Plex® assay system available from Bio-Rad Laboratories, Inc. (Hercules, Calif.) and Luminex, Inc. (Austin, Tex.) may be useful in identifying Borreliella antibodies in a sample.
  • a multiplex particle-based assay such as the suspension array Bio-Plex® assay system available from Bio-Rad Laboratories, Inc. (Hercules, Calif.) and Luminex, Inc. (Austin, Tex.) may be useful in identifying Borreliella antibodies in a sample.
  • the present disclosure contemplates the immobilization of an isolated Borreliella immunoreactive chimeric polypeptide on a surface of a particle for use in a particle-based immunoassay.
  • methods of peptide immobilization onto support surfaces are well-known in the art.
  • a labeled Borreliella immunoreactive chimeric polypeptide disclosed herein is immobilized onto a surface of a particle and the polypeptide-particle complex is employed in an ELISA or in a flow cytometry assay according to established protocols.
  • Lateral flow tests may also be referred to as immunochromatographic strip (ICS) tests or simply strip-tests.
  • ICS immunochromatographic strip
  • a lateral flow test is a form of assay in which the test sample flows laterally along a solid substrate via capillary action, or alternatively, under fluidic control.
  • Such tests are often inexpensive, require a very small amount (e.g., one drop) of sample, and can typically be performed reproducibly with minimal training.
  • the economical simplicity and robustness of many lateral flow assay formats makes these types of tests ideal for identifying a Borreliella infection at the point of care, which can be particularly important when the subject is, for example, a human or pet exhibiting detectable antibodies during a treatable phase of infection.
  • Exemplary lateral flow device formats include, but are not limited to, a dipstick, a card, a chip, a microslide, and a cassette, and it is widely demonstrated in the art that the choice of format is largely dependent upon the features of a particular assay.
  • Lateral flow devices are now ubiquitous in human and veterinarian medicine and quite varied, providing many options to the ordinarily skilled artisan for detecting a polypeptide- antibody complex in a sample. (See any of U.S. Pat. Nos. 7,344,893, 7,371,582, 6,136,610, and U.S. Patent Applications, 2005/0250141 and 2005/0047972, or Koczula et al.
  • a sample from a subject suspected of having an Borreliella infection is applied to a lateral flow device comprising at least a sample zone and a binding zone.
  • the sample may be a serum sample and may be drawn laterally from the sample zone to the binding zone which comprises an Borreliella immunoreactive chimeric polypeptide disclosed herein immobilized to a surface of the lateral flow device.
  • the binding of the immobilized Borreliella immunoreactive chimeric polypeptide on the lateral flow device is an indication that Borre/ze/Zzz-specific antibodies are present in the sample from the subject, indicating a Borreliella infection in the subject.
  • the chimeric proteins as described herein may be used in a device for detecting wa i-Borreliella antibodies, i.e., antibodies raised against one or more Borreliella proteins upon and/or after and/or during infection of a subject with Lyme disease spirochetes.
  • the devices can be used to detect Borreliella infections such as Lyme disease.
  • Figure 13 shows an example of a lateral flow device.
  • Lateral flow device 100 comprises a membrane substrate 101 supported on backing 102, which may be a solid backing.
  • membrane substrate 101 comprises, for example, a woven mesh, a cellulose fiber, glass fiber, mixed glass and cellulose fiber, synthetic fiber, mixed fiber, surface modified plastic (polyester, polypropylene, or polyethylene), graded density polyethersulfone (PES), nitrocellulose, or combinations thereof.
  • the membrane substrate can be of any suitable form, e.g., a strip, circle, etc.
  • the solid backing may comprise any suitable material including, but not limited to, plastic, fiber, and glass. In some instances, the backing comprises an adhesive.
  • Lateral flow device 100 generally further comprise a sample application pad 103 to receive a sample and absorbent pad 104 (e.g., a cellulose filter) to prevent backflow and conjugate release pad 105 which contains e.g., chimeric proteins that are specific to the antibodies to be detected.
  • a sample and absorbent pad 104 e.g., a cellulose filter
  • conjugate release pad 105 which contains e.g., chimeric proteins that are specific to the antibodies to be detected.
  • the chimeric proteins are conjugated to detectable particles, such as colored or fluorescent particles, e.g. colloidal gold or latex microspheres.
  • membrane substrate 101 further comprises at least one test line 106 and at least one control line 107.
  • Test line 106 comprises immobilized capture molecules (e.g. antibodies) specific for binding to complexes that comprise anti- Borreliella antibodies bound to chimeric proteins.
  • Control line 107 lacks capture molecules.
  • test line 106 is placed upstream of at least one control line 107. In other aspects, test line 106 is placed downstream of at least one control line 107.
  • sample application pad 103 a suitable quantity of sample is applied to sample application pad 103.
  • the sample then travels by capillary action across conjugate release pad 105 in the direction of the test lines. If antibodies are present in the sample, they encounter and bind to chimeric proteins in conjugate release pad 105, forming antibody-antigen complexes or conjugates.
  • the complexes or conjugates continue to migrate along the device and reach test line 106 where they are captured and immobilized on the test line. Detection of binding at test line 109 indicates that antibodies are present in the sample. If no binding is detected at test line 109, this indicates that antibodies are not present in the sample.
  • the binding is rendered detectable by any of a variety of methods, e.g., by a chemical reaction that causes a color change at the test line but not at the control line, or by the chimeras having been tagged with a detectable marker, etc.
  • the change may be detectable with the naked eye.
  • results from the device are transferred e.g., wirelessly or through a cable, to a computerized device to process and display information about the change.
  • the result is transmitted to a software program on a computerized device, where the computerized device has a graphical user interface that displays the assay results.
  • the results are transferred to a database.
  • the results from the database are used e.g., for bioinformatics applications.
  • the lateral flow device comprises a buffer which may be referred to as a running or chase buffer.
  • the buffer is a physiologically compatible buffer or carrier (e.g. a phosphate-buffered saline buffer, citrate buffer, a Good’s buffer, etc.) and may further comprise, e.g. a blocking agent (e.g., casein, Tween® reagent, etc.), a surfactant, various additives, and other reagents to increase sensitivity of the assay.
  • the buffer is or comprises a phosphate-based buffer with a divalent cation.
  • lateral flow device 100 further comprises a housing (not shown) that covers at least a portion of the device.
  • the housing may comprise, for example, a sample application port to allow sample application and an optic opening to allow signal visualization.
  • the housing can comprise any suitable material.
  • the housing can comprise a plastic material.
  • the housing comprises an opaque, translucent, or transparent material.
  • a small volume of sample is required to utilize the devices described herein e.g., an volume of from about 1 pL to about lOOOpL, such as from about 5 pL to about 500 pL, or about 10 pL to about 100 pL. In some aspects, about 10, 20, 30, 40, 50, 60, 70, 80 90 or 100 pL of sample is applied to the device.
  • the disclosure provides a method to detect the presence of antibodies to at least one of Borreliella proteins DbpA, DbpB, BBK19, BBK73, BBA53, VlsE and BB0238, or to detect antibodies which react with Borreliella proteins having epitopes in common with or epitopes similar enough to those of DbpA, DbpB, BBK19, BBK73, BBA53, VlsE and BB0238 so as to bind to the chimeras.
  • Devices and systems comprising the devices as described herein can detect Borreliella antibodies in a rapid and reliable manner.
  • the device provides a read out in about 1 minute to about 30 minutes (min). In some aspects, a read out is provided in about 20 minutes.
  • Methods as described herein have a sensitivity of at least about 70% of detecting a i-Borreliella antibodies. In some instances, the methods and systems as described herein are at least about 75%, 80%, 85%. 90%, 95% or more than 95% sensitive in detecting Borreliella antibodies. Using an ELISA format the sensitivity can be even greater than 95%, such as about 96, 97, 98 or 99%. Methods as described herein may have a specificity of at least about 90% for detecting a i-Borreliella antibodies e.g. as compared to other antibodies.
  • the methods and systems as described herein are at least about 90%, 91, 92, 93, 94, 95% or more than 95% specific (such as about 96, 97, 98 or 99%) for detecting anti- Borreliella antibodies.
  • Methods and systems as described herein may have an accuracy of at least about 70% of detecting anti- Borreliella antibodies.
  • the methods and systems as described herein are at least about 75%, 80%, 85%. 90%, 95% or more than 95% accuracy in detecting anti- Borreliella antibodies. In some aspects, no false positives occur.
  • Methods and systems as described herein may have a reliability of at least about 70% of detecting anti- Borreliella antibodies.
  • the methods and systems as described herein are at least about 75%, 80%, 85%. 90%, 95% or more than 95% reliable in detecting the i-Borreliella antibodies
  • Samples that are analyzed as described herein may be from any mammal that is susceptible to infection by Borreliella. Examples include but are not limited to humans, companion animals (dogs, cats), food sources, livestock (cattle, horses, oxen, sheep, pigs, goats) wild-life and zoo animals (mice, deer, rats, raccoons, opossum, coyotes, bears, vultures, gophers, groundhogs, squirrels, etc.).
  • the present disclosure also encompasses methods which include 1) diagnosing the infection in a subject as described herein and then ii) treating the subject who is diagnosed or identified as having Borreliella antibodies by administering a suitable treatment.
  • Subjects diagnosed as having a Borreliella infection are typically treated with antibiotics.
  • antibiotics for example, in dogs treatment usually involves a course of antibiotics which will last for 4 weeks or longer.
  • the antibiotic doxycycline is typically a first-choice option.
  • a short course of oral antibiotics such as doxycycline or amoxicillin, is generally used.
  • Lyme disease is usually treated with three to four weeks of antibiotic therapy.
  • Oral antibiotics are the standard treatment for early-stage Lyme disease. These usually include doxycycline for adults and children older than 8, or amoxicillin or cefuroxime for adults, younger children, and pregnant or breast-feeding women.
  • Intravenous antibiotics may be administered if the disease involves the central nervous system, e.g., typically for 14 to 28 days. Intravenous antibiotics can cause various side effects, including a lower white blood cell count, mild to severe diarrhea, or colonization or infection with other antibiotic -resistant organisms unrelated to Lyme, and other treatment measures may be used to prevent, combat or control such side effects.
  • kits are provided to an administering physician, veterinarian, other health care professional, a patient, or a caregiver.
  • a kit comprises a container which contains a testing device and instructions for using the device.
  • the container contains more than one testing device.
  • the testing devices are individually wrapped.
  • Such a kit may comprise chimeric polypeptides immobilized to one or more separate lateral flow assay devices, such as a nitrocellulose test strips.
  • each of the test strips may further comprise a detection reagent.
  • Such a kit may further comprise one or more containers for sample material, one or more diluents for sample dilution, and one or more control indicator strips for comparison.
  • the assay kit can also include an amount of a chase buffer, e.g., PBS, sufficient to enable proper flow of the tracer reagent on each of the first and second test lines to the control line.
  • the kit may comprise solutions, agents, and chase buffers that may be required to operate the device.
  • reagents and/or components comprising a kit are provided in a lyophilized form (lyophilisate) or as a dry powder.
  • the lyophilisate or powder can be reconstituted by the addition of a suitable solvent.
  • the solvent may be a sterile, pharmaceutically acceptable buffer and/or other diluent. It is envisioned that such a solvent may also be provided as part of a kit.
  • the liquid solution may be, by way of non-limiting example, a sterile, aqueous solution.
  • Additional kit components can include, e.g., an instrument for sample collection, e.g., a sharp instrument for drawing blood, or a swab for collecting mucosal swabs and an instrument for applying the sample to the sample pad, e.g., a dropper.
  • an instrument for sample collection e.g., a sharp instrument for drawing blood, or a swab for collecting mucosal swabs
  • an instrument for applying the sample to the sample pad e.g., a dropper.
  • the kit can optionally also contain one or more other testing devices and diagnostic tools.
  • the kit may also optionally contain therapeutic or other agents.
  • the kit comprises an assisted vision tool to help visually observe the readout such as a light source, a light filter, or a magnifier.
  • the assay kit can further include instructions for use, which can comprise a description of test pattern interpretation, and recommendations for action based on the result obtained.
  • the instructions for use include a cautionary warning based on the result interpretation.
  • a mobile phone application is made available to the user, so that test results is provided to a practitioner and/or epidemiologist.
  • Borreliella isolates were cultivated in standard BSK-II media supplemented with 6% rabbit serum at 34 °C (without gelatin). Growth was monitored using wet-mounts and dark-field microscopy. Cells were harvested from late-log phase cultures by low speed centrifugation.
  • the gene sequences were codon-optimized for expression in Escherichia coli, synthesized (minus the leader sequence) on a fee for service basis, and inserted into the vector pET45b(+) at its BamHI and EagI restriction sites (GenScript). The sequences of the cloned DNA were confirmed by DNA sequence analyses on a fee for service basis.
  • the plasmids were purified and transformed into E. coli BL21(DE3) cells. Protein expression was induced by autoinduction or by the addition of IPTG (1 mM) and the cells were lysed using a high-pressure homogenizer.
  • the His-tagged proteins were purified using nickel affinity chromatography on an AKTATM FPLC platform (Cytvia) or through the use of Ni affinity gravity flow columns ( Figure 1). Some proteins were subjected to a second round of FPLC purification using a cobalt column on the AKTA platform. As detailed below, the chimeric diagnostic antigens that were designed based on the data detailed within were produced and purified in the same manner.
  • Figure 1 presents an SDS-PAGE gel that demonstrates the purity of representative recombinant proteins. SDS-PAGE, immunoblot and dot-blot analyses.
  • ELISA analyses ELISA plate wells were coated with 500 ng of protein (individual recombinant proteins or chimeric recombinant proteins) in bicarbonate buffer overnight at 4 °C. Antisera were used at dilutions indicated in the figures with secondary Antibodies used at a 1:15000 dilution. ABTS (chromogenic substrate) was added, the plates were incubated for 20 min and the absorbance was measured at 405nm. Preimmune serum served as a negative control serum sample and BSA or the FhbB protein served as the immobilized negative control for non-specific antibody binding.
  • Serum samples from B. burgdorferi infected humans, client-owned dogs, horses, laboratory mice, and wildlife have been collected and catalogued over the past decade.
  • Human serum samples were obtained from the CDC, NIH, and the Lyme disease Biobank.
  • Serum samples from wildlife eastern coyotes, red foxes, gray foxes, eastern black bears, pocket gophers, ground hogs, black vultures and squirrels were obtained from the USDA, Pennsylvania Game Commission, Pennsylvania State University and others).
  • the samples from client-owned dogs were tested for antibodies to the panel of recombinant proteins (Tables 1 and 2).
  • mice were infected by needle inoculation (subcutaneous) with 10 4 live B. burgdorferi cells. The spirochetes were administered subcutaneously between the shoulder blades. Serum was harvested from groups of 5 mice on days 0, 7, 8 and 12. The sera were screened against several recombinant proteins (Figure 3) using an ELISA format. Representative data are shown. Select proteins were effective at detecting IgG antibodies as early as day 12 of infection (bottom bar). Proteins that detected specific IgG responses included VlsE, DbpA, DbpB, BBK19, BBA64, BBO383, BBP39, BBN38, BBM27, BBC10, and BBK50. These proteins were selected as candidates for inclusion in chimeric diagnostic antigens as detailed below.
  • the immunodominant regions of BBA24, BB A25 and BBA36 mapped within residues 75-190, 32-97, and 79-155, respectively.
  • the same mapping strategy was applied to several other proteins including but not limited to BBK19, BBA73, BB0238, BBK53, OspC, VlsE and BBG01. These analyses were central in informing the choice of antigens and specific protein fragments to use in the generation of chimeric diagnostic antigens.
  • the protein was immobilized in ELISA wells and then screened with the serum samples indicated in Table 3.
  • DCFL4 Specificity of DCFL4 was further verified using additional serum samples derived from dogs infected with Leptospira, Anaplasma and Ehrlichia ( Figure 7). The results demonstrate the effectiveness of DCFL4 to serve as a diagnostic antigen.
  • DCFL4 The sensitivity of DCFL4 was then compared with several other chimerics. Mice were infected with B. burgdorferi and then sera was collected over time. The chimerics indicated in Figure 8 were used to coat wells of ELISA plates and then were screened with the serum samples. Of the chimerics tested, as shown in Figure 8, DCFL4 displayed the best results. These data indicate that the organization of the constructs is critical and not obvious. Subsequently we test two additional chimerics, DCFL3a and HDFL4. Both yielded strong results that are described below.
  • FIG. 11 presents the results in which serum dilution and substrate exposure time were assessed using a well-characterized serum sample from an infected dog. The optimal serum dilution proved to be 1:1000. Given that serum is not typically limiting, this dilution is appropriate. Chromogenic substrate exposure time need not be absolute but must be consistent and allow for rapid testing. We determined 20 minutes to be ideal as it keeps the absorbance reading in the linear part of the plot.
  • the Lyme Disease Biobank has generated a well-characterized serum panel that can be used to validate and compare the sensitivity and specificity of diagnostic antigens.
  • An initial test serum panel provided by the Biobank was screened against the chimeric proteins using a dot blot format ( Figure 12). The chimerics and control proteins used are shown on the left. The serum ID numbers are indicated along the top (as assigned by the Biobank). Across the top of the image, the two-tier (2TT) scoring results for both IgM and IgG as determined by others are indicated.
  • DCFL4 displayed high sensitivity and specificity. This panel was screened specifically for IgG. DCFL4 proved highly effective in detecting antibody in Lyme disease patients with well-characterized disease.
  • the use of a single protein (DCFL4) offers advantages over the two-tier testing system in terms of speed, cost, sensitivity, and specificity and eliminates the need to test for IgM which can yield false positive results.
  • HDFL4 A highly sensitive diagnostic antigen for Lyme disease infections in humans.
  • the sensitivity of HDFL4 was independently assessed by a third party using a commercially available serum panel (SeraCare; S1-S15) and a proprietary lateral flow test that is in development.
  • the samples are designated as SI -SI 5 and they have been tested by SeraCare for both Lyme specific IgG and IgM antibodies.
  • the test employs a proprietary lateral flow approach. Additional human serum samples (ID numbers are shown) were also tested.
  • the sensitivity and specificity of HDFL4 were compared against a multiplex assay that uses 4 antigens (Agl-Ag4; undisclosed identity). Samples scored as positive are underlined, bolded, and italicized in Table 4 below. The results indicate that HDFL4 detects antibodies with high sensitivity and can be used in place of multiple independent antigens.
  • HDFL4 A highly sensitive diagnostic antigen for Lyme disease infections in humans.

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Abstract

L'invention concerne des protéines recombinantes chimériques pour la détection d'une infection par Borreliella et/ou le diagnostic de la maladie de Lyme. L'invention concerne également des procédés et des dispositifs pour effectuer des dosages immunologiques avec les protéines recombinantes chimériques.
PCT/US2022/078580 2022-10-24 2022-10-24 Protéines recombinantes chimériques et panels de protéines recombinantes pour le diagnostic de la maladie de lyme chez les animaux et les êtres humains WO2024091264A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050147999A1 (en) * 1997-06-20 2005-07-07 Choi Gil H. Lyme disease vaccines
US20080131466A1 (en) * 2006-09-26 2008-06-05 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
US20110117636A1 (en) * 2009-11-18 2011-05-19 Infopia Co., Ltd. Lateral flow immunoassay device with a more rapid and accurate test result
RU2514230C1 (ru) * 2012-12-14 2014-04-27 Федеральное государственное бюджетное учреждение "Научно-исследовательский институт биохимии" Сибирского отделения Российской академии медицинских наук (ФГБУ "НИИ биохимии" СО РАМН) Рекомбинантные химерные полипептиды, несущие эпитопы различных иммунодоминантных белков спирохет комплекса borrelia burgdorferi sensu lato, и способ серодиагностики иксодового клещевого боррелиоза
US20170059566A1 (en) * 2015-08-27 2017-03-02 Quidel Corporation Immunoassay test device with two fluid flow paths for detection and differentiation of two or more analytes
US20180149648A1 (en) * 2014-09-24 2018-05-31 Wellstat Diagnostics, Llc Compositions and methods for the diagnosis of lyme disease
WO2018217897A1 (fr) * 2017-05-23 2018-11-29 David Weiner Méthodes et compositions pour induire une réponse immunitaire

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050147999A1 (en) * 1997-06-20 2005-07-07 Choi Gil H. Lyme disease vaccines
US20080131466A1 (en) * 2006-09-26 2008-06-05 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
US20110117636A1 (en) * 2009-11-18 2011-05-19 Infopia Co., Ltd. Lateral flow immunoassay device with a more rapid and accurate test result
RU2514230C1 (ru) * 2012-12-14 2014-04-27 Федеральное государственное бюджетное учреждение "Научно-исследовательский институт биохимии" Сибирского отделения Российской академии медицинских наук (ФГБУ "НИИ биохимии" СО РАМН) Рекомбинантные химерные полипептиды, несущие эпитопы различных иммунодоминантных белков спирохет комплекса borrelia burgdorferi sensu lato, и способ серодиагностики иксодового клещевого боррелиоза
US20180149648A1 (en) * 2014-09-24 2018-05-31 Wellstat Diagnostics, Llc Compositions and methods for the diagnosis of lyme disease
US20170059566A1 (en) * 2015-08-27 2017-03-02 Quidel Corporation Immunoassay test device with two fluid flow paths for detection and differentiation of two or more analytes
WO2018217897A1 (fr) * 2017-05-23 2018-11-29 David Weiner Méthodes et compositions pour induire une réponse immunitaire

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