EP3688010A1 - Dosage sérologique pour la détection d'une exposition à des agents pathogènes portés par les tiques et d'une infection par ceux-ci - Google Patents

Dosage sérologique pour la détection d'une exposition à des agents pathogènes portés par les tiques et d'une infection par ceux-ci

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Publication number
EP3688010A1
EP3688010A1 EP18857606.0A EP18857606A EP3688010A1 EP 3688010 A1 EP3688010 A1 EP 3688010A1 EP 18857606 A EP18857606 A EP 18857606A EP 3688010 A1 EP3688010 A1 EP 3688010A1
Authority
EP
European Patent Office
Prior art keywords
peptide
peptides
amino acid
antibodies
acid sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18857606.0A
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German (de)
English (en)
Other versions
EP3688010A4 (fr
Inventor
Rafal TOKARZ
Walter Ian Lipkin
Nischay MISHRA
Thomas Briese
Adrian CACIULA
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Columbia University in the City of New York
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Columbia University in the City of New York
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Application filed by Columbia University in the City of New York filed Critical Columbia University in the City of New York
Publication of EP3688010A1 publication Critical patent/EP3688010A1/fr
Publication of EP3688010A4 publication Critical patent/EP3688010A4/fr
Pending legal-status Critical Current

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43556—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from ticks

Definitions

  • This invention relates to the field of detection of exposure to tick-borne pathogens using high throughput serology and novel peptide antigens.
  • TBD Tick-borne diseases
  • Adams et al. 2013 9teen bacterial, protozoan and viral agents have been implicated in TBDs.
  • Borrelia Burgdorferi the causative agent for Lyme disease alone accounts for an estimated 300,000 cases of tick-borne disease and the total annual direct medical costs of Lyme disease are estimated to range between $700 million and $1.3 billion (Nelson et al. 2015; Hinkley et al. 2014; Adrion et al. 2015).
  • IFA Indirect immunofluorescent assay
  • TBDs The reported incidence of TBDs has risen continuously over the past three decades in association with geographic expansion of tick populations and the discovery of a wide range of novel tick-borne pathogens (Khatchikian et al. 2015; Kugeler et al. 2015). Nonetheless, the true incidence of TBDs is likely greatly underestimated, as patients with presumed TBDs are rarely tested for the full range of tick-borne pathogens, and only a fraction of positive cases are properly reported (White et al. 2016).
  • New diagnostic assays that can detect infections with the full range of tick-borne pathogens are urgently needed, and in particular there is a need for a sensitive, specific, and inexpensive high throughput serological assay for the detection of tick-borne pathogens including, but not limited to, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, Powassan virus, and, as well as to discriminate between various stages of Lyme disease in order to expand the window of accurate diagnosis.
  • aims will enable new strategies for diagnosing and investigating the epidemiology and pathogenesis of acute TBD and persistent conditions such as Post-treatment Lyme Disease Syndrome (PTLDS).
  • PTLDS Post-treatment Lyme Disease Syndrome
  • the current invention addresses the current limitations in differential diagnosis of TBD through the development of a programmable, multiplex, high-resolution peptide array platform that can detect antibodies to all key pathogens currently implicated in TBD and can be expanded as new pathogens are encountered.
  • the current invention provides compositions, methods, and kits for detecting the exposure to and infection by tick-borne pathogens. Specifically, the current invention allows for the rapid simultaneous and differential serological detection of exposure to, and infection by tick-borne pathogens. In particular, the current invention allows for the rapid simultaneous serological detection of exposure to, and infection by Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus.
  • compositions, methods and kits for detection of exposure to, and infection by Borrelia burgdorferi, as well as Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus comprise specific peptides, isolated and non-isolated, which are strongly reactive with, and specific for each pathogen, i.e., reactive and specific epitopes of antibodies to each pathogen.
  • one embodiment of the present invention are the peptides listed in Table 1, which are reactive with, and specific for antibodies to the tick-borne pathogens (SEQ ID NO: 1;
  • a further embodiment of the present invention are collections or sets of peptides which are strongly reactive with, and specific for each pathogen, i.e., reactive and specific epitopes of antibodies to each pathogen, comprising amino acid sequences shifted one residue across the peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-19 and
  • One embodiment of the current invention is a collection or set of peptides which are strongly reactive with, and specific for all of the following tick-borne pathogens, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Borrelia miyamotoi, Heartland virus, and Powassan virus, comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1-19 and 29-43.
  • a further embodiment of the present invention are peptides listed in Table 6 which are strongly reactive with, and specific for IgG epitopes of Borrelia burgdorferi, as well as collections or sets of peptides comprising amino acid sequences shifted one residue across the peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 4, 7, 8, and 20- 28.
  • a further embodiment of the present invention is a collection or set of peptides comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28. These peptides are particularly useful for diagnosis of acute neuroborreliosis.
  • peptides can comprise peptides that are 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, and up to 12 amino acids in length.
  • the collection or set of peptides used in the present invention can range from 2 peptides to a number in the thousands to tens of thousands to hundreds of thousands. In the some of the Examples over 169,000 unique peptide sequences were used.
  • compositions comprising two or more peptides of the invention.
  • the invention provides nucleic acids comprising a sequence encoding a peptide of the invention.
  • the invention provides vectors comprising such nucleic acids, and host cells comprising such vectors.
  • the vector is a shuttle vector.
  • the vector is an expression vector ⁇ e.g., a bacterial or eukaryotic expression vector).
  • the host cell is a bacterial cell. In other embodiments, the host cell is a eukaryotic cell.
  • a further embodiment of the present invention is a peptide microarray comprising peptides, which are reactive with, and specific for antibodies of the tick-borne pathogens.
  • the peptide microarray comprises: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • the peptide microarray comprises a collection or set of peptides which are strongly reactive with, and specific for all of the following tick- borne pathogens, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus, comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1-19 and 29-43.
  • the peptide array comprises: at least one peptide listed in
  • the peptide array comprises a collection or set of peptides comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28.
  • the invention provides devices.
  • the devices are useful for performing an immunoassay.
  • the device is a lateral flow immunoassay device.
  • the device is an analytical or centrifugal rotor.
  • the device is a tube or a well, e.g., in a plate suitable for an ELISA assay or a microarray.
  • the device is an electrochemical, optical, or opto-electronic sensor.
  • the device comprises at least one peptide of the invention. In other embodiments, the device comprises a collection or set of the peptides of the invention as described herein. In certain embodiments, the peptides are attached to or immobilized upon the device.
  • a further embodiment of the present invention is a method for the serological detection of exposure to and/or infection by one or more of the tick-borne pathogens, comprising the use of a peptide or peptides which are reactive with, and specific for the antibodies to the tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • a further embodiment of the present invention is a method for the simultaneous serological detection of exposure to and/or infection by all of the following tick-borne pathogens, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus, comprising the use of a set or collection of peptides, comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1-19 and 29-43.
  • a further embodiment of the present invention is a method for the serological detection of acute neuroborreliosis, comprising the use of a peptide or peptides which are reactive with, and specific for the antibodies to the tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof.
  • a further embodiment of the present invention is a method for the serological detection of acute neuroborreliosis, comprising the use of a collection or set of peptides comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28.
  • a further embodiment of the present invention is a method for the serological detection of exposure to and/or infection by one or more of the tick-borne pathogens, comprising the use of peptide microarray comprising peptides which are reactive with, and specific for antibodies to the tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • a further embodiment of the present invention is a method for the simultaneous serological detection of exposure to and/or infection by all of the following tick-borne pathogens, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus, comprising the use of peptide microarray comprising a set or collection of peptides, comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1-19 and 29-43.
  • a further embodiment of the present invention is a method for the serological detection of acute neuroborreliosis, comprising the use of peptide microarray comprising a peptide or peptides which are reactive with, and specific for the antibodies to the tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof.
  • a further embodiment of the present invention is a method for the serological detection of acute neuroborreliosis, comprising the use of peptide microarray comprising a collection or set of peptides comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28.
  • the method for the serological detection of exposure to and/or infection by one or more tick-borne pathogens in a sample from a subject comprises: contacting the sample with at least one peptide which is reactive with, and specific for antibodies to the tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof; and detecting the binding between the anti-TBD pathogen antibodies in the sample and the peptide or peptides.
  • the method for the serological detection of exposure to and/or infection by at least one tick-borne pathogen in a sample from a subject comprises: contacting the sample with a peptide microarray comprising peptides which are reactive with, and specific for antibodies to tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof; and detecting the binding between the anti-TBD pathogen antibodies in the sample and the peptide or peptides in the microarray.
  • the method for the diagnosis of acute neuroborreliosis, in a sample from a subject comprises: contacting the sample with at least one peptide which is reactive with, and specific for antibodies to the tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof; and detecting the binding between the anti-TBD pathogen antibodies in the sample and the peptide or peptides.
  • the method for the diagnosis of acute neuroborreliosis in a sample from a subject comprises: contacting the sample with a peptide microarray comprising peptides which are reactive with, and specific for antibodies to tick-borne pathogens comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1 , 2, 4, 7, 8, and 20-28; or at least one collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1 , 2, 4, 7, 8, and 20-28; or combinations thereof; and detecting the binding between the anti-TBD pathogen antibodies in the sample and the peptide or peptides in the microarray.
  • the peptides, and collections and sets of peptides of the invention for the detection of the antibodies to the TBD pathogens can used in any number of immunodetection techniques, which include but are not limited to Western blot, enzyme-linked immunosorbent assay (ELISA), lateral flow, dipstick type of assay or a SNAP test, multiplex antibody detection techniques of various kinds, or any modification of such assays that are suitable for detecting antibodies of interest. Many suitable antibody detection methods are described including, for example, in U.S. Patent No. 9,034,656.
  • the detecting step comprises performing an agglutination assay.
  • the detecting step comprises spinning the sample in an analytical or centrifugal rotor.
  • the detecting step comprises analyzing the sample with an electrochemical sensor, an optical sensor, or an opto-electronic sensor.
  • the detecting step comprises performing a wavelength shift assay.
  • the immunodetection technique is in the form of a programmable peptide array.
  • peptides of the invention are attached to or immobilized on a solid support.
  • the peptides of the invention are attached to a solid support through a metallic nanolayer.
  • the solid support is a bead (e.g., a colloidal particle, metallic nanoparticle or nanoshell, or latex bead), a flow path in a lateral flow immunoassay device (e.g., a porous membrane), a blot (e.g., Western blot, a slot blot, or dot blot), a flow path in an analytical or centrifugal rotor, or a tube or well (e.g., in a plate suitable for an ELISA assay or microarray).
  • the solid support comprises metal, glass, a cellulose-based material (e.g., nitrocellulose), or a polymer (e.g., polystyrene, polyethylene, polypropylene, polyester, nylon, or polysulfone).
  • a cellulose-based material e.g., nitrocellulose
  • a polymer e.g., polystyrene, polyethylene, polypropylene, polyester, nylon, or polysulfone.
  • peptides of the invention are isolated (e.g., synthetic and/or purified) peptides.
  • peptides of the invention are conjugated to a ligand.
  • the peptides are biotinylated.
  • the peptides are conjugated to streptavidin, avidin, or neutravidin.
  • the peptides are conjugated to a carrier protein (e.g., serum albumin, keyhole limpet hemocyanin (KLH), or an immunoglobulin Fc domain).
  • a carrier protein e.g., serum albumin, keyhole limpet hemocyanin (KLH), or an immunoglobulin Fc domain.
  • the patient antibody or antibodies to TBD pathogens can be an IgG or IgM, or other immunoglobulin classes or subtypes.
  • the present invention also includes systems and kits for the serological detection of exposure to and/or infection by agents that cause TBD or for the diagnosis of acute neuroborreliosis.
  • the invention provides for a method for the serological detection of
  • Post-treatment Lyme Disease and other stages of Lyme Disease, using at least one peptide or at least one collection or set of peptides which are reactive with, and specific for antibodies of various stages of Lyme Disease.
  • Figure 1 shows the epitope identification using TBD-Serochip.
  • Figure 1A shows the reactivity data generated for the C6 region of the B. burgdorferi VlsE antigen using patient sera from early acute Lyme disease (LD).
  • Figure IB shows the reactivity data generated for the C6 region of the B. burgdorferi VlsE antigen using patient sera from acute neuroborreliosis.
  • the X-axis in each Figure represents the amino acid span of VlsE (mapped to accession number CAJ41626).
  • the Y-axis in each Figure represents the intensity of the fluorescent signal.
  • the asterisk specifies the location of the 12-mer peptides that make up the major immunoreactive region of the C6 peptide of the VlsE protein of B. burgdorferi, shown in Figure 1A.
  • Figure 2 shows the reproducibility of the peptide assay. This figure shows additional data for the C6 region of the VlsE protein of B. burgdorferi.
  • Figures 2A and 2B represent results from an early acute LD serum tested twice with the same peptide assay.
  • Figure 2C shows the results from the same sample run on a different peptide assay.
  • the Y-axis in Figures 2A-2C represents the intensity of the fluorescence signal, and the X-axis represents a sequence of 9 overlapping peptides within the C6 of the VlsE protein of B. burgdorferi.
  • Figure 2D shows the sequence of the 9 overlapping peptides within the C6 of the VlsE protein of B. burgdorferi.
  • Significant immunoreactive peptides are numbered 5 and 6.
  • Dashed lines in Figures 2A-2C represent the threshold for the MKKDDQIAAAIALRGMA (SEQ ID NO: 1) epitope.
  • Figure 3 shows the identification of the discriminatory epitopes for the diagnosis of babesiosis (infection by Babesia microti).
  • Figure 3 A is a heatmap for SA-1 antigens
  • Figure 3B is a heatmap for BMN 1-17 antigens.
  • the numbers on the Y-axis represent the location of 12-mer peptides positioned along the continuous protein sequence of each antigen.
  • the X-axis are the samples.
  • Bab-1 to Bab- 11 represent B. m croft ' -antibody positive sera
  • LD-1 represents Lyme disease positive serum.
  • Immunoreactivity with the 12-mer peptide is indicated in gray with increasing signal intensity displayed from light to dark. The dark brackets across the map indicates the range of discriminatory peptides from Table 1.
  • FIG. 4 shows the discovery of A. phagocytophilum-specific epitopes on MSP2.
  • a schematic of the MSP2 antigen (accession number WP 044108210) indicating the approximate lengths of the conserved N and C fragments flanking the central variable core is shown on top.
  • the heatmap displays in gray the immunoreactivity of the 176 amino acid N- terminal region, with increasing signal intensity displayed from light to dark.
  • the numbers on the Y-axis represent the location of 12-mer peptides positioned along the contiguous sequence of the N terminal region.
  • the X-axis are the samples.
  • Ana-1 to Ana-7 indicate anaplasmosis-positive sera
  • LD-1 and Ehr-1 indicate Lyme disease and ehlichiosis sera, respectively.
  • the dark brackets indicate the location of the discriminatory epitopes from Table 1.
  • Figure 5 shows the identification of a POWV-specific peptide using the peptide array.
  • the Y-axis represents the intensity of the fluorescence signal for the seven peptides to the right.
  • the bottom six peptides in the Figure are the reactive 12-mer peptides.
  • the X-axis represents the samples.
  • POWV indicate Powassan virus positive samples and LD-NEG corresponds to negative control samples.
  • sample means any substance containing or presumed to contain antibodies, in particular those to a pathogen or agent that can cause a tick-borne disease.
  • the sample can be of natural or synthetic origin and can be obtained by any means known to those of skill in the art.
  • the sample can be a sample of tissue or fluid isolated from a subject including but not limited to, plasma, serum, whole blood, cerebrospinal fluid (CSF), semen, amniotic fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, and tissue. Samples can be research, clinical, or environmental. Sample can also be blood products used to transfuse or treat. Samples can also be synthetic and include but are not limited to in vitro cell culture constituents including but not limited to conditioned medium, recombinant cells, and cell components.
  • the term "subject” means any organism including, without limitation, a mammal such as a fox, a deer, a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate.
  • the subject is a human being, a pet or livestock animal.
  • patient as used in this application means a human subject.
  • tick-borne means arising from a tick, usually from a bite.
  • a "tick-borne disease” or “TBD” is a disease caused by a tick-borne pathogen or agent, which can be a virus or a bacterium.
  • identification means to recognize exposure to a specific pathogen or agent in sample from a subject.
  • peptide includes any sequence of two or more amino acids. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right.
  • amino acid includes the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g., phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, l ,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, alpha-methylalanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butyl
  • the term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., (Ci-C 6 ) alkyl, phenyl or benzyl ester or amide).
  • a conventional amino protecting group e.g., acetyl or benzyloxycarbonyl
  • natural and unnatural amino acids protected at the carboxy terminus e.g., (Ci-C 6 ) alkyl, phenyl or benzyl ester or amide.
  • an "antigen” from antibody-generating or “immunogen” is a substance that prompts the generation of antibodies and can cause an immune response. They may also be used for diagnostic or patient selection or characterization purposes.
  • Antibodies also known as immunoglobulins (Ig) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural units— each with two large heavy chains and two small light chains— to form, for example, monomers with one unit, dimers with two units or pentamers with five units. Antibodies are produced by B cells. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.
  • Antibodies can also neutralize targets directly by, for example, binding to a part of a pathogen that it needs to cause an infection. Production of antibodies is the main function of the humoral immune system.
  • isolated and the like means that the referenced material is free of components found in the natural environment in which the material is normally found. In particular, isolated biological material is free of cellular components. An isolated peptide or protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane- associated protein. An isolated material may be, but need not be, purified.
  • substantially purified refers to a molecule, such as a peptide, that is substantially free of cellular material (proteins, lipids, carbohydrates, nucleic acids), culture medium, chemical precursors, chemicals used in synthesis of the peptide, or combinations thereof.
  • a peptide that is substantially purified has less than about 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of the cellular material, culture medium, other polypeptides, chemical precursors, and/or chemicals used in synthesis of the peptide.
  • a substantially pure molecule, such as a peptide can be at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, by dry weight, the molecule of interest.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation.
  • “about” can mean within 1 or more than 1 standard deviations, per the practice in the art.
  • “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • the term "about” meaning within an acceptable error range for the particular value should be assumed.
  • the current invention enhances the differential diagnosis and management of tick- borne disease by establishing a new serologic assay platform.
  • TBD-Serochip Tick-borne Disease Serochip
  • the TBD-Serochip offers substantial improvement in target selection by employing an extensive range of linear peptides that identifies key specific immunodominant epitopes. These peptides used in the TBD-Serochip are the key to the enhanced diagnostic accuracy, and provide a differential diagnosis not available with currently employed serologic assays for TBDs.
  • peptides and the peptide array-based platform will enable new strategies for investigating the epidemiology and pathogenesis of acute and chronic diseases due to infection and for monitoring humoral responses to vaccines and immunomodulatory drugs. It will also serve as a screening tool for rapid selection of key informative peptides that can be used in established, inexpensive, alternative platforms including lateral flow immunoassays and ELISAs. Such applications will have practical utility for both clinical medicine and public health by enabling retrospective differential diagnosis of an infectious illness (when genetic footprints of the agent may no longer be present), and in facilitating outbreak investigation and surveillance.
  • the array-based assay and peptides of the current invention have several advantages over serologic platforms currently employed for TBD diagnosis.
  • the presence of antibodies to multiple agents can be attributed to concurrent infections as well as asymptomatic, or past infections.
  • the capacity of the assay and peptides of the invention to simultaneously detect both IgG and IgM can be helpful in extrapolating infection status.
  • the peptides of the current invention include a wide range of specific peptides, including the first discriminatory epitopes for anaplasmosis and Powassan disease, as well as new diagnostic targets for Lyme disease and babesiosis. Both IgG and IgM can be detected simultaneously.
  • the assay of the current invention also allows simultaneous detection and confirmation of infection. Whereas the current diagnostic algorithm for diagnosis of Lyme disease requires ELISA followed by confirmatory western blot, the assay of the current invention provides evidence of immunoreactivity to multiple epitopes in a single reaction.
  • the peptides described herein can also readily be adapted to other serologic platforms, such as ELISA, Luminex or lateral flow immunoassays.
  • the assay of the current invention is inherently more flexible than other platforms because new targets can be added to arrays through in situ peptide synthesis.
  • Target protein selection, peptide design, and manufacture of an array or other assay comprising the peptides can be executed in less than 4 weeks, facilitating rapid development of new and specific diagnostic tests for novel TBDs agents.
  • the peptides and assay of the current invention provides a powerful research tool for studies of TBDs as they can be employed to discriminate individual antibody responses in patients with TBD and thus examine the interplay of TBD agents on disease manifestation and progression. It can also be used to assess the impact of genetic diversity of tick-borne pathogens on the host immune response.
  • the array can resolve antibodies to different genotypes of TBD agents, such as OspC types of B. burgdorferi, or analyze the temporal response to the multitude of B. burgdorferi VlsE or A. phagocytophilum MSP2 antigenic variants generated during the course of disease. It can also be utilized to identify potential changes in reactive epitopes in individuals with persistent symptoms following antibiotic treatment.
  • the current invention was developed using the strategy of tiling each antigen in a sliding window of 12-mer peptides that shifted one residue (e.g., residues 1-12, residues 2-13, residues 3-14, etc.).
  • residues 1-12, residues 2-13, residues 3-14, etc. all protein variants were included in the 12-mer design for each antigen.
  • the goal was to obtain peptides that are specific and sensitive for each antigen of the tick-borne pathogens or agents including Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus.
  • Such peptides were obtained. See Example 1 and Table 1.
  • the same tiling strategy can be used to synthesize shorter peptides, (6, 7, 8, 9 or 10 amino acids in length) corresponding to the other regions
  • an additional aspect of the current invention are peptides and peptide array platforms that are diagnostic of PTLDS, and other stages of disease.
  • the present invention includes peptides, isolated and non-isolated, which are strongly reactive with, specific for, and sensitive to, antibodies to tick-borne pathogens including Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus in a sample.
  • tick-borne pathogens including Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus in a sample.
  • These peptides can be used in any type of serological assay or platform to screen for the presence of antibodies to Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus, and to determine if a subject has had an infection by, and/or exposure to, any of these tick-borne pathogens or agents.
  • These peptides can also be used to test for and monitor humoral responses to vaccines and immunomodulatory drugs thus being useful for the development of treatment and preventative agents for any of these tick-borne agents.
  • These peptides can also be used to monitor treatment and management of tick-borne illnesses including both acute TBD as well as persistent conditions such as post-treatment Lyme disease syndrome (PTLDS).
  • PTLDS post-treatment Lyme disease syndrome
  • One embodiment of the present invention is each of the peptides listed in Table 1 which are specifically immunoreactive for the listed tick-borne pathogen or agent. In tests with sera from patients with Lyme disease and other tick-borne diseases, these specific and immunoreactive regions were shown to be useful for the serologic detection of the pathogens. See Examples 2-7. These peptides detect all early and late Lyme disease cases and unlike current methods for diagnosis of Lyme disease that require two consecutive tests (ELISA followed by Western blot), do so in one single step. Table 1 - Peptides of the Invention
  • a further embodiment of the present invention is a collection or set of isolated peptides comprising two or more up to all 34 amino acid sequences listed in Table 1 (SEQ ID NOs 1-19 and 29-43).
  • a further embodiment of the present invention comprises a collection or set of peptides comprising amino acid sequences shifted one residue across any of the peptides listed in Table 1 (SEQ ID NOs: 1-19 and 29-43). For example, see Figures 1 (SEQ ID NOs: 44-49), Figure 2D (SEQ ID NOs: 44-52), and Figure 5 (SEQ ID NOs: 53-59).
  • a further embodiment of the present invention is a collection or set of isolated peptides shifted one residue across all of the peptides listed in Table 1 (SEQ ID NOs: 1-19 and 29-43).
  • a further embodiment of the present invention is each of the isolated peptides listed in Table 6 which are specific for the listed epitopes of Borrelia burgdorferi, and of particular usefulness in the diagnosis of acute neuroborreliosis.
  • a further embodiment of the present invention is a collection or set of isolated peptides comprising two or more up to all 14 amino acid sequences listed in Table 6 (SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28).
  • a further embodiment of the present invention is a collection or set of isolated peptides shifted one residue across at least one of the peptides listed in Table 6 (SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28).
  • a further embodiment of the present invention is a collection or set of isolated peptides shifted one residue across all of the peptides listed in Table 6 (SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28).
  • collections or sets can comprise isolated peptides that are 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, and up to 12 amino acids in length.
  • Peptides of 12 amino acids are preferred based upon work that shows that antibodies bind to linear peptide sequences ranging from 5 to 9 amino acids in length and bind most efficiently when targets are flanked by additional amino acids (Buus et al. 2012). However, peptides containing less than 12 amino acids in length and more than 12 amino acids in length can be used. Peptides 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, up to 25 amino acids in length, up to 30 amino acids in length, up to 35 amino acids in length, up to 40 amino acids in length, and up to 50 amino acids in length can be used.
  • peptides of the invention are produced by synthetic chemistry
  • peptides of the invention are produced biologically.
  • An isolated peptide of the invention can be in water, a buffer, or in a dry form awaiting reconstitution, e.g., as part of a kit.
  • An isolated peptide of the present invention can be in the form of a pharmaceutically acceptable salt.
  • Suitable acids and bases that are capable of forming salts with the peptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.
  • peptides of the invention are modified.
  • the peptides of the invention may be modified by a variety of techniques, such as by denaturation with heat and/or a detergent (e.g., SDS).
  • peptides of the invention may be modified by association with one or more further moieties.
  • the association can be covalent or non- covalent, and can be, for example, via a terminal amino acid linker, such as lysine or cysteine, a chemical coupling agent, or a peptide bond.
  • the additional moiety can be, for example, a ligand, a ligand receptor, a fusion partner, a detectable label, an enzyme, or a substrate that immobilizes the peptide.
  • Peptides of the invention can be conjugated to a ligand, such as biotin (e.g., via a cysteine or lysine residue), a lipid molecule (e.g., via a cysteine residue), or a carrier protein (e.g., serum albumin, immunoglobulin Fc domain, keyhole limpet hemocyanin (KLH) via e.g., a cysteine or lysine residue).
  • ligands such as biotin
  • ligand receptors such as avidin, streptavidin, polymeric streptavidin, or neutravidin.
  • Avidin, streptavidin, polymeric streptavidin, or neutravidin in turn, can be linked to a signaling moiety (e.g., an enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase (ALP), or other moiety that can be visualized, such as a metallic nanoparticle or nanoshell (e.g., colloidal gold) or a fluorescent moiety), or a solid substrate (e.g., nitrocellulose membrane).
  • a signaling moiety e.g., an enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase (ALP), or other moiety that can be visualized, such as a metallic nanoparticle or nanoshell (e.g., colloidal gold) or a fluorescent moiety), or a solid substrate (e.g., nitrocellulose membrane).
  • HRP horse radish peroxidase
  • ALP alkaline phosphatase
  • the peptides of the invention can be fused or linked to a ligand receptor, such as avidin, streptavidin, polymeric streptavidin, or neutravidin, thereby facilitating the association of the peptides with the corresponding ligand, such as biotin and any moiety (e.g. , signaling moiety) or solid substrate attached thereto.
  • a ligand receptor such as avidin, streptavidin, polymeric streptavidin, or neutravidin
  • Peptides of the invention can be fused to a fusion partner (e.g., a peptide or other moiety) that can be used to improve purification, to enhance expression of the peptide in a host cell, to aid in detection, and to stabilize the peptide.
  • a fusion partner e.g., a peptide or other moiety
  • suitable compounds for fusion partners include carrier proteins (e.g., serum albumin, immunoglobulin Fc domain, KLH), and enzymes (e.g., horse radish peroxidase (HRP), beta-galactosidase, glutathione-S- transferase, alkaline phosphatase).
  • HRP horse radish peroxidase
  • beta-galactosidase glutathione-S- transferase
  • alkaline phosphatase alkaline phosphatase
  • peptides of the invention may be modified to include any of a variety of known chemical groups or molecules. Such modifications include, but are not limited to, glycosylation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment to polyethylene glycol (e.g., PEGylation), covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization,
  • Analogues of an amino acid including unnatural amino acids
  • peptides with substituted linkages are also included.
  • Peptides of the invention that consist of any of the sequences discussed herein may be modified by any of the discussed modifications. Such peptides still “comprise” or “consist of the amino acids.
  • the invention provides nucleic acids comprising a sequence encoding a peptide of the invention.
  • Nucleic acids of the invention can be single- or double- stranded.
  • a nucleic acid can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof.
  • the nucleic acids can be purified free of other components, such as proteins, lipids and other polynucleotides.
  • the nucleic acids can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified.
  • the nucleic acids of the invention encode the peptides described herein.
  • nucleic acids encode a peptide having the sequence of SEQ ID NOs: 1-59.
  • Nucleic acids of the invention can comprise other nucleotide sequences, such as sequences coding for linkers, signal sequences, TMR stop transfer sequences, transmembrane domains, or ligands useful in protein purification such as glutathione-S-transferase, histidine tag, and staphylococcal protein A.
  • a nucleic acid of the invention is operably linked when it is positioned adjacent to or close to one or more expression control elements, which direct transcription and/or translation of the polynucleotide.
  • the peptides of the invention can be produced recombinantly following conventional genetic engineering techniques.
  • a nucleic acid encoding the peptide is inserted into a suitable expression system.
  • a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the selected peptide is operably linked to an expression control sequence permitting expression of the peptide.
  • appropriate expression vectors include, e.g., vectors containing bacterial, viral, yeast, fungal, insect or mammalian expression systems. Methods for obtaining and using such expression vectors are well-known.
  • the invention also provides vectors comprising nucleic acids of the invention, and host cells comprising such vectors.
  • the vector is a shuttle vector.
  • the vector is an expression vector ⁇ e.g., a bacterial or eukaryotic expression vector).
  • the host cell is a bacterial cell. In other embodiments, the host cell is a eukaryotic cell.
  • any number of conventional protein assay formats may be designed to utilize the isolated peptides of this invention, and collections and sets of peptides of the invention, for the detection of antibodies to tick-borne pathogens in a subject.
  • This invention is thus not limited by the selection of the particular assay format, and is believed to encompass assay formats that are known to those of skill in the art.
  • the peptides of the present invention can be used in any assay, format or platform for antibody detection including but not limited to ELISA, Luminex, Western blot assays, immunofluorescence assays, and spotted peptide arrays, as well as those platforms that are later developed.
  • the assay comprises: immobilizing the antibody(s) in the sample; adding a peptide of the invention; and detecting the degree of antibody bound to the peptide, e.g., by the peptide being labeled or by adding a labeled substance, such as a labeled binding partner (e.g., streptavidin-HRP or streptavidin-colloidal gold complex) or a labeled antibody which specifically recognizes the peptide.
  • a labeled binding partner e.g., streptavidin-HRP or streptavidin-colloidal gold complex
  • the assay comprises: immobilizing a peptide of the invention; adding the sample containing antibodies; and detecting the amount of antibody bound to the peptide, e.g., by adding another peptide of the invention conjugated, directly or indirectly, to a label (e.g., metallic nanoparticle or metallic nanoshell, fluorescent label, or enzyme (e.g., horseradish peroxidase or alkaline phosphatase)) or by adding a labeled substance, such as a binding partner or a labeled antibody which specifically recognizes the sample antibodies (e.g., anti-human IgG antibodies, or anti-human IgM antibodies).
  • a label e.g., metallic nanoparticle or metallic nanoshell, fluorescent label, or enzyme (e.g., horseradish peroxidase or alkaline phosphatase)
  • a labeled substance such as a binding partner or a labeled antibody which specifically recognizes the sample antibodies (e.g., anti-human IgG antibodies, or
  • the assay comprises: immobilizing a peptide of the invention; adding the sample containing antibodies; and detecting the amount of antibody bound to the peptide, e.g., by adding a first binding partner which specifically recognizes the sample antibodies (e.g., anti-human IgG antibodies, or anti-human IgM antibodies), and further adding a second binding partner, wherein the second binding partner is labeled and recognizes said first binding partner.
  • a first binding partner which specifically recognizes the sample antibodies (e.g., anti-human IgG antibodies, or anti-human IgM antibodies)
  • second binding partner is labeled and recognizes said first binding partner.
  • the assay comprises: reacting the peptide and the sample containing antibodies without any of the reactants being immobilized, and then detecting the amount of complexes of antibody and peptide, e.g., by the peptide being labeled or by adding a labeled substance, such as a labeled binding partner (e.g., streptavidin-HRP or streptavidin- colloidal gold complex) or a labeled antibody which specifically recognizes the peptide.
  • a labeled substance such as a labeled binding partner (e.g., streptavidin-HRP or streptavidin- colloidal gold complex) or a labeled antibody which specifically recognizes the peptide.
  • Immobilization of a peptide of the invention can be either covalent or non-covalent, and the non-covalent immobilization can be non-specific (e.g., non-specific binding to a polystyrene surface in a microtiter well).
  • Specific or semi- specific binding to a solid or semisolid carrier, support or surface can be achieved by the peptide having, associated with it, a moiety which enables its covalent or non-covalent binding to the solid or semi-solid carrier, support or surface.
  • the moiety can have affinity to a component attached to the carrier, support or surface.
  • the moiety may be, for example, a biotin or biotinyl group or an analogue thereof bound to an amino acid group of the peptide, and the component is then avidin, streptavidin, neutravidin, or an analogue thereof.
  • Suitable carriers, supports, and surfaces include, but are not limited to, metallic nanolayers, beads (e.g., magnetic beads, colloidal particles or metallic nanoparticles or nanoshells, such as colloidal gold, or particles or nanoparticles comprising silica, latex, polystyrene, polycarbonate, or PDVF), latex of co-polymers such as styrene-divinyl benzene, hydroxylated styrene-divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy- activated porous magnetic glass, gelatin or polysaccharide particles or other protein particles, red blood cells, mono- or polyclonal antibodies or Fab fragments of such antibodies.
  • metallic nanolayers beads (e.g., magnetic beads, colloidal particles or metallic nanoparticles or nanoshells, such as colloidal gold,
  • the protocols for immunoassays using antigens for detection of specific antibodies are well known in art.
  • a conventional sandwich assay can be used, or a conventional competitive assay format can be used.
  • Solid-phase assay devices include microtiter plates, flow-through assay devices (e.g., lateral flow immunoassay devices), dipsticks, and immunocapillary or immunochromatographic immunoassay devices.
  • the solid or semi-solid surface or carrier is the floor or wall in a microtiter well, a filter surface or membrane (e.g., a nitrocellulose membrane or a PVDF (polyvinylidene fluoride) membrane), a hollow fiber, a beaded chromatographic medium (e.g.
  • an agarose or polyacrylamide gel an agarose or polyacrylamide gel
  • a magnetic bead a fibrous cellulose matrix
  • an HPLC matrix a fibrous cellulose matrix
  • an FPLC matrix a substance having molecules of such a size that the molecules with the peptide bound thereto, when dissolved or dispersed in a liquid phase, can be retained by means of a filter, a substance capable of forming micelles or participating in the formation of micelles allowing a liquid phase to be changed or exchanged without entraining the micelles, a water-soluble polymer, or any other suitable carrier, support or surface.
  • the peptide of the invention is provided with a suitable label which enables detection.
  • suitable labels may be used which are capable, alone or in concert with other compositions or compounds, of providing a detectable signal.
  • Suitable labels include, but are not limited to, enzymes (e.g., HRP, beta-galactosidase, or alkaline phosphatase), fluorescent labels, radioactive labels, colored latex particles, and metal-conjugated labels (e.g., metallic nanolayers, metallic nanoparticle- or metallic nanoshell-conjugated labels).
  • Suitable metallic nanoparticle or metallic nanoshell labels include, but are not limited to, gold particles, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells.
  • Metallic nanolayers suitable for detectable layers include nanolayers comprised of cadmium, zinc, mercury, and noble metals, such as gold, silver, copper, and platinum.
  • Suitable detection methods include, but are not limited to, detection of an agent which is tagged, directly or indirectly, with a colorimetric assay (e.g., for detection of HRP or beta- galactosidase activity), visual inspection using light microscopy, immunofluorescence microscopy, including confocal microscopy, or by flow cytometry (FACS), autoradiography (e.g., for detection of a radioactively labeled agent), electron microscopy, immunostaining, subcellular fractionation, or the like.
  • a radioactive element e.g., a radioactive amino acid
  • a radioactive amino acid is incorporated directly into a peptide chain.
  • a fluorescent label is associated with a peptide via biotin/avidin interaction, association with a fluorescein conjugated antibody, or the like.
  • a detectable specific binding partner for the antibody is added to the mixture.
  • the binding partner can be a detectable secondary antibody or other binding agent (e.g., protein A, protein G, protein L or combinations thereof) which binds to the first antibody.
  • This secondary antibody or other binding agent can be labeled with, for example, a radioactive, enzymatic, fluorescent, luminescent, metallic nanoparticle or metallic nanoshell (e.g. colloidal gold), or other detectable label, such as an avidin/biotin system.
  • the binding partner is a peptide of the invention, which can be conjugated directly or indirectly to an enzyme, such as horseradish peroxidase or alkaline phosphatase or other signaling moiety.
  • the detectable signal is produced by adding a substrate of the enzyme that produces a detectable signal, such as a chromogenic, fluorogenic, or chemiluminescent substrate.
  • the detection procedure comprises visibly inspecting the antibody-peptide complex for a color change or inspecting the antibody- peptide complex for a physical-chemical change.
  • Physical-chemical changes may occur with oxidation reactions or other chemical reactions. They may be detected by eye, using a spectrophotometer, or the like.
  • One assay format is a lateral flow immunoassay format.
  • Antibodies to human or animal immunoglobulins can be labeled with a signal generator or reporter (e.g., colloidal gold) that is dried and placed on a glass fiber pad (sample application pad or conjugate pad).
  • the diagnostic peptide is immobilized on membrane, such as nitrocellulose or a PVDF (polyvinylidene fluoride) membrane.
  • membrane such as nitrocellulose or a PVDF (polyvinylidene fluoride) membrane.
  • antibodies against the diagnostic peptide If antibodies against the diagnostic peptide are present, they bind to the diagnostic peptide striped on the membrane, thereby generating a signal (e.g., a band that can be seen or visualized).
  • a signal e.g., a band that can be seen or visualized.
  • An additional antibody specific to the labeled antibody or a second labeled antibody can be used to produce a control signal.
  • An alternative format for the lateral flow immunoassay comprises the peptides or compositions of the invention being conjugated to a ligand (e.g., biotin) and complexed with labeled ligand receptor (e.g., streptavidin-colloidal gold).
  • the labeled peptide complexes can be placed on the sample application pad or conjugate pad.
  • Anti-human IgG/IgM or anti- animal IgG/IgM antibodies of the invention are immobilized on a membrane, such as nitrocellulose of PVDF, at a test site.
  • the antibodies in the sample are then transported into the next membrane (PVDF or nitrocellulose containing the diagnostic peptide) by capillary action and bind to the immobilized anti-human IgG/IgM or anti-animal IgG/IgM antibodies. If any of the sample antibodies are bound to the labeled peptides of the invention, the label associated with the peptides can be seen or visualized at the test site.
  • PVDF nitrocellulose containing the diagnostic peptide
  • Another assay for the screening of blood products or other physiological or biological fluids is an enzyme linked immunosorbent assay, i.e., an ELISA.
  • an enzyme linked immunosorbent assay i.e., an ELISA.
  • isolated peptides or collection or set of peptides of the invention are adsorbed to the surface of a microtiter well directly or through a capture matrix (e.g., an antibody). Residual, nonspecific protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent).
  • BSA bovine serum albumin
  • NGS heat-inactivated normal goat serum
  • BLOTTO a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent.
  • the well is then incubated with a biological sample suspected of containing specific antibody.
  • the sample can be applied neat, or more often it can be diluted, usually in a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
  • a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
  • the label can be chosen from a variety of enzymes, including horseradish peroxidase (HRP), beta-galactosidase, alkaline phosphatase (ALP), and glucose oxidase.
  • HRP horseradish peroxidase
  • ALP alkaline phosphatase
  • glucose oxidase oxidase
  • Sufficient time is allowed for specific binding to occur again, then the well is washed again to remove unbound conjugate, and a suitable substrate for the enzyme is added. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally (measured at an appropriate wave length).
  • Conditions for performing ELISA assays are well-known in the art.
  • a peptide or a collection or set of peptides of the invention are immobilized on a surface, such as a ninety- six- well ELISA plate A sample is then added and the assay proceeds as above.
  • a peptide or collection or set of peptides of the invention are electro- or dot-blotted onto nitrocellulose paper. Subsequently, a sample, such as a biological fluid is incubated with the blotted antigen, and antibody in the sample is allowed to bind to the antigen(s). The bound antibody can then be detected, e.g., by standard immunoenzymatic methods or by visualization using metallic nanoparticles or nanoshells coupled to secondary antibodies or other antibody binding agents or combinations thereof.
  • Luminex-based systems have been employed that can address up to 100 antigenic targets simultaneously (i.e., 100 individual pathogens, 100 individual antigenic targets for one pathogen, or some variation thereof) (Anderson et al. 2011). Additionally, arrays are established that comprise spotted recombinant proteins expressed in vitro in E. coli, S. cerevesiae, baculoviruses, or cell-free, coupled transcription- translation systems (Vigil et al. 2010).
  • One goal of the present invention is to automate the process of tick-borne disease detection and make it inexpensive, quick and accurate as well as detect exposure per se rather than to rigorously characterize humoral responses to specific pathogens.
  • One assay that meets these requirements is a programmable peptide array.
  • One method to create and validate a programmable array that can measure the humoral immune response to tick-borne pathogens, thus enabling detection of exposure to a tick-borne pathogen (or its gene products in vaccines), comprises the following steps: 1) select peptides using bioinformatic methods; 2) test those peptides printed on arrays for sensitivity and specificity using sera from humans and other animals who have been exposed to antigens of tick-borne pathogens; 3) examine the performance of algorithms typically employed for epitope prediction; 4) use assay results to develop smaller and less comprehensive peptide libraries that can be deployed in smaller and more facile platforms; 5) optimize and validate assay protocols; and 6) develop software to automate assay analysis.
  • the peptides of the present invention were generated.
  • additional specific peptide which are strongly reactive with, and specific for antibodies to tick-borne pathogens, those listed herein as well as others, can be generated and used in all of the assays and methods disclosed herein.
  • the array capacity can be exploited to print multiple arrays per glass slide (configurations of 1, 3, 8, or 12 arrays can be printed).
  • a further embodiment of the present invention is a peptide microarray comprising peptides that are reactive with, and specific for antibodies to tick-borne pathogens.
  • the peptide microarray comprises: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • the peptide array comprises a set or collection of peptides, comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1-19 and 29-43.
  • peptide microarray comprising peptides that are reactive with, and specific for antibodies to Borrelia burgdorferi.
  • the peptide microarray comprises: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof.
  • the peptide array comprises a set or collection of peptides, comprising amino acid sequences shifted one residue across all of the peptides comprising the amino acid sequences of SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28.
  • these peptide microarrays are programmable. Methods and Systems for Serological Detection of Exposure to and/or Infection by Tick- borne Pathogens
  • the present invention includes methods and systems for the detection of exposure to and/or infection by tick-borne pathogens, i.e., antibodies to tick-borne pathogens including, but not limited to, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, Anaplasma phagocytophilum, Ehlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus, in any sample utilizing the various peptides, isolated and non-isolated, and peptide arrays of the present invention.
  • the present invention also includes methods and systems for the diagnosis of acute neuroborreliosis
  • Suitable methods typically include: receiving or obtaining ⁇ e.g., from a patient) a sample of bodily fluid or tissue likely to contain antibodies; contacting ⁇ e.g., incubating or reacting) a sample to be assayed with a peptide or peptides of the invention, under conditions sufficient for the formation of a specific peptide- antibody complex ⁇ e.g., for specific binding of the peptide to the antibody); and assaying the contacted (reacted) sample for the presence of an antibody-peptide reaction ⁇ e.g., determining the amount of an antibody-peptide complex).
  • the presence of an elevated amount of the antibody-peptide complex indicates that the subject was exposed to and infected by a tick-borne pathogen.
  • a peptide, including a modified form thereof, which "binds specifically" to an antibody against a tick-borne pathogen antigen interacts with the antibody, or forms or undergoes a physical association with it, in an amount and for a sufficient time to allow detection of the antibody.
  • the methods and systems of the present invention may be used to detect exposure to antigens of tick-borne pathogens in research and clinical settings.
  • a preferred sample is a biological sample.
  • a biological sample may be obtained from a tissue of a subject or bodily fluid from a subject including but not limited to nasopharyngeal aspirate, blood, cerebrospinal fluid, saliva, serum, plasma, urine, sputum, bronchial lavage, pericardial fluid, or peritoneal fluid, or a solid such as feces.
  • the preferred sample is serum, plasma and cerebrospinal fluid (CSF).
  • the subject may be any animal, particularly a vertebrate and more particularly a mammal, including, without limitation, a cow, dog, human, monkey, mouse, pig, deer, fox, or rat. In one embodiment, the subject is a human.
  • a sample may also be a research, clinical, or environmental sample.
  • Additional applications include, without limitation, detection of the screening of blood products (e.g., screening blood products for infectious agents), biodefense, food safety, environmental contamination, forensics, and genetic-comparability studies.
  • the present invention also provides methods and systems for detecting antibodies in cells, cell culture, cell culture medium and other compositions used for the development of pharmaceutical and therapeutic agents.
  • a sample may also be a research, clinical, or environmental sample, such as cells, cell culture, cell culture medium, and compositions for use as, or the development of pharmaceutical and therapeutic agents.
  • Additional applications include, without limitation, detection of the screening of blood products (e.g., screening blood products for infectious agents), biodefense, food safety, environmental contamination, forensics, and genetic-comparability studies.
  • the present invention also provides methods and systems for detecting viral antibodies in cells, cell culture, cell culture medium and other compositions used for the development of pharmaceutical and therapeutic agents.
  • the subject may have been exposed to antigens of tick-borne pathogens, suspected of having exposure to antigens of tick-borne pathogens or believed not to have had exposure to antigens of tick-borne pathogens.
  • the subject may have been found to be seropositive by tick-borne pathogen ELISA.
  • the subject has been previously diagnosed with a tick-borne disease.
  • the tick-borne disease is Lyme disease.
  • the subject may be a test subject, which has been administered a tick-borne pathogen vaccine or immunomodulatory agent.
  • the systems and methods described herein support the detection and measure of a humoral immune response to tick-borne pathogens.
  • one embodiment of the present invention provides a system for the detection of exposure to and/or infection of tick-borne pathogens, i.e., antibodies to tick-borne pathogens, in any sample.
  • the system includes at least one subsystem wherein the subsystem includes a peptide or peptides which are reactive with, and specific for tick-borne pathogens antibodies, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • the system can also include additional subsystems for the purpose of: preparation of the sample; binding of any tick-borne pathogens antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.
  • a further embodiment of the present invention provides a system for the detection of exposure to and/or infection by tick-borne pathogens, i.e., antibodies to tick-borne pathogens, in any sample.
  • the system includes at least one subsystem wherein the subsystem includes a peptide microarray comprising at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • the system can also include additional subsystems for the purpose of: preparation of the sample; binding of any tick-borne pathogens antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.
  • the present invention provides a method for detecting the exposure to and/or infection by tick-borne pathogens, i.e., antibodies to tick-borne pathogens, in any sample, including the steps of: contacting the sample with a peptide or peptides which are reactive with, and specific for tick-borne pathogens antibodies, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof, under conditions sufficient for any antibodies to tick-borne pathogens in the sample and the peptides to bind; and visualizing and/or quantifying any bound antibody or antibodies to the peptides.
  • the method may optionally include a step for washing any unbound sample.
  • the present invention provides a method for the detecting the exposure to and/or infection by tick-borne pathogens, i.e., antibodies to tick-borne pathogens, in any sample, including the steps of: contacting the sample with a peptide microarray comprising at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29- 43; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof under conditions sufficient for any antibodies to tick-borne pathogens in the sample and the peptides to bind; and visualizing and or quantifying any bound antibody or antibodies to the peptides.
  • the method may optionally include a step for washing any unbound sample.
  • a further embodiment of the present invention provides a system for the diagnosis of acute neuroborreliosis by the detection of exposure to antigens of Borrelia burgdorferi, in any sample.
  • the system includes at least one subsystem wherein the subsystem includes a peptide or peptides which are reactive with, and specific for Borrelia burgdorferi antibodies, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof.
  • the system can also include additional subsystems for the purpose of: preparation of the sample; binding of any Borrelia burgdorferi antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.
  • a further embodiment of the present invention provides a system for the diagnosis of acute neuroborreliosis by the detection of exposure to antigens of Borrelia burgdorferi, in any sample.
  • the system includes at least one subsystem wherein the subsystem includes a peptide microarray comprising at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof.
  • the system can also include additional subsystems for the purpose of: preparation of the sample; binding of any Borrelia burgdorferi antibody in the sample with the peptides(s); washing the unbound sample; and visualization and/or quantification of bound antibody or antibodies.
  • the present invention provides a method for the diagnosis of acute neuroborreliosis by the detection of exposure to antigens of Borrelia burgdorferi, in any sample, including the steps of: contacting the sample with a peptide or peptides which are reactive with, and specific for Borrelia burgdorferi antibodies, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof, under conditions sufficient for any antibodies to Borrelia burgdorferi in the sample and the peptides to bind; and visualizing and/or quantifying any bound antibody or antibodies to the peptides.
  • the method may optionally include a step for washing any unbound sample.
  • the present invention provides a method for the diagnosis of acute neuroborreliosis by the detection of exposure to antigens of Borrelia burgdorferi, in any sample, including the steps of: contacting the sample with a peptide microarray comprising at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof under conditions sufficient for any antibodies to Borrelia burgdorferi in the sample and the peptides to bind; and visualizing and or quantifying any bound antibody or antibodies to the peptides.
  • the method may optionally include a step for washing any unbound sample.
  • the invention also includes reagents and kits for practicing the methods of the invention. These reagents and kits may vary.
  • One reagent of the kit would be a peptide or peptides which are reactive with, and specific for antibodies to tick-borne pathogens, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • the peptides are attached to or immobilized on a solid support. In some embodiments, the peptides are attached to or immobilized on a solid support through a metallic nanolayer ⁇ e.g., cadmium, zinc, mercury, gold, silver, copper, or platinum nanolayer).
  • a metallic nanolayer e.g., cadmium, zinc, mercury, gold, silver, copper, or platinum nanolayer.
  • the solid support is a bead ⁇ e.g., a colloidal particle or a metallic nanoparticle or nanoshell), a flow path in a lateral flow immunoassay device, a flow path in an analytical or centrifugal rotor, a tube or a well ⁇ e.g., in a plate), or a sensor ⁇ e.g., an electrochemical, optical, or opto-electronic sensor).
  • kits of the invention can include a population of beads ⁇ e.g., suitable for an agglutination assay or a lateral flow assay), or a plate ⁇ e.g., a plate suitable for an ELISA assay).
  • the kits comprise a device, such as a lateral flow immunoassay device, an analytical or centrifugal rotor, a Western blot, a dot blot, a slot blot, or an electrochemical, optical, or opto-electronic sensor.
  • One further reagent of the kit would be peptide microarrays comprising a peptide or peptides which are reactive with, and specific for antibodies to tick-borne pathogens, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1-19 and 29-43; or combinations thereof.
  • kits could include a peptide or peptides which are reactive with, and specific for antibodies to Borrelia burgdorferi, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof.
  • One further reagent of this kit would be peptide microarrays comprising a peptide or peptides which are reactive with, and specific for antibodies to Borrelia burgdorferi, comprising: at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or a collection or set of peptides comprising amino acid sequences shifted one residue across at least one peptide comprising any one of the amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 8, and 20-28; or combinations thereof.
  • kits could include reagents for preparing the sample, binding any antibodies in the sample with the peptides, washing any unbound sample, visualizing any bound antibody or antibodies, and quantifying any bound antibody or antibodies.
  • kits can include various diluents and buffers, labeled conjugates or other agents for the detection of specifically bound antigens or antibodies ⁇ e.g., labeling reagents), and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens.
  • the kit comprises an anti-human IgG/IgM antibody conjugated to a detectable label ⁇ e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, colored latex particle, or enzyme) as a labeling reagent.
  • the kit comprises protein A, protein G, protein A/G fusion proteins, protein L, or combinations thereof conjugated to a detectable label ⁇ e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, colored latex particle, or enzyme) as a labeling reagent.
  • the labeling reagents of the kit are a second collection or set of peptides of the invention conjugated to a detectable label (e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, colored latex particle, or enzyme).
  • the second collection or set of peptides can be the same as or different than the collection or set of peptides, which may optionally be attached to or immobilized upon a solid support.
  • kits can easily be determined by one of skill in the art.
  • Such components may include coating reagents, polyclonal or monoclonal capture antibodies specific for a peptide of the invention, or a cocktail of two or more of the antibodies, purified or semi-purified extracts of these antigens as standards, monoclonal antibody detector antibodies, an anti-mouse, anti-dog, anti-cat, anti-chicken, or anti-human antibody conjugated to a detectable label, indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, a sample preparatory cup and buffers or other reagents appropriate for constituting a reaction medium allowing the formation of a peptide- antibody complex.
  • kits provide a convenient, efficient way for a clinical laboratory to detect exposure to, and to diagnose infection by a tick-borne pathogen.
  • the kits further comprise instructions.
  • Example 1 Generation of 12-Mer Peptides from Tick-Borne Agents or Pathogens
  • the array platform employed for the assay can accommodate up to 3 million 12-mer linear peptides.
  • the array slide can be divided into 12 subarrays, each containing approximately 170,000 12-mer peptides. Because peptides are programmed for synthesis in situ, the array composition can be continuously and inexpensively modified based on performance or the need to address new immunological targets.
  • This platform was used to develop the assay of the invention that targeted 8 principal tick-borne pathogens present in the United States, including Anaplasma phagocytophilum (agent of human granulocytic anaplasmosis), Babesia microti (babesiosis), Borrelia burgdorferi (Lyme disease), Borrelia miyamotoi, Ehrlichia chaffeensis (human monocytic ehrlichiosis), Rickettsia rickettsii (Rocky Mountain spotted fever), Heartland virus and Powassan virus.
  • a Long Island tick rhabdovirus a novel virus identified by the inventors in Amblyomma americanum ticks through metagenomic sequencing, was also included (Tokarz et al. 2014). See Table 2
  • 12-mers reflects three considerations: 1) principal and contextual determinants in antibody recognition of linear epitopes; 2) platform constraints; and 3) preliminary data obtained in experiments with spotted peptide arrays.
  • Serum antibodies bind linear peptide sequences ranging from 5 and 9 amino acids (Buus et al. 2012) and bind most efficiently when targets are flanked by additional amino acids.
  • a database of overlapping 12-mer peptides that tiled the proteome of each of these agents was established. For each antigen selected, all available protein sequences from the NCBI protein database were downloaded. Sequences were clustered and used as a scaffold for the design of 12-mer linear peptides that tiled each protein sequence with 11 amino acid overlap in a sliding window pattern. Redundant 12-mers were excluded prior to synthesis. For viral agents, 12-mer peptides were designed that tiled the entire proteome (Table 2). For non-viral agents, antigen selection was more selective, and based on the current understanding of the triggers of humoral immunity for each agent.
  • VMPs variable major proteins
  • the B. microti genome contains one species-specific multi-gene family (bmn) that codes for antigenic surface proteins, which has been shown to be a reliable tool for B. microti detection (Lodes et al. 2000; Masatani et al. 2013; Cornillot et al. 2016; Houghton et al. 2002).
  • bmn species-specific multi-gene family
  • MSPs Anaplasma phagocytophilum major surface proteins
  • the unique peptide sequences (169,205 + random control peptides) were printed on Roche-NimbleGen arrays using manufacturer instructions.
  • This platform consists of a 75 mm x 26 mm array slide divided into 12 subarrays that can accommodate 172,000 peptides per slide allowing a 12 sample query at a cost of less than $200/sample.
  • GPI 12 GPI 12, AMA1 ; Hsp70, DnaK, Bmp32
  • Example 1 To test the utility of peptides and peptide microarrays of Example 1 (the TBD- Serochip), well-characterized sera and cerebrospinal fluid (CSF) containing antibodies to one or more agents of TBDs including A. phagocytophilum, B. burgdorferi, B. microti, E. chaffeensis, and Powassan virus was used (Table 3). To determine the optimal serum dilution, 16 sera at dilutions of 1 :50 and 1 :200 and two sera at dilutions of 1 :20, 1 :50, and 1 :200 were tested.
  • CSF cerebrospinal fluid
  • the 1 :50 serum dilution was determined to be optimal for the TBD-Serochip as the 1 :200 dilution did not achieve the required sensitivity in early disease samples and the 1 :20 dilution generated adverse signal to noise ratio.
  • a 1 :5 dilution was used for CSF.
  • CSF cerebral spinal fluid
  • binding buffer 0.1M Tris-Cl, 1 % alkali soluble casein, 0.05% Tween-20
  • Arrays were washed 3 times for 10 minutes each on Little Dipper (SciGene) with IX TBST (0.05% Tween-20) at room temperature.
  • Secondary antibodies were diluted in binding buffer at concentration of 1 ug/ml. Secondary antibody incubation was done in Plastic Coplin Jar (Fisher Scientific) for 3 hours at room temperature with gentle agitation.
  • the slides were scanned on NimbleGen MS 200 Microarray Scanner (Roche) at 2 ⁇ resolution, with an excitation wavelength of 532 nm for Cy3/IgM and 635 nm for Alexa Flour/IgG respectively.
  • the relative fluorescent unit (RFU) signals for all the probes were extracted from the images using Roche Sequencing Solutions image extraction software.
  • the RFU signals were converted into intensity plots after quantile normalization, background and spatial correction, and deconvolution for redundant peptides. To determine background noise, signal intensity data for each antigen was obtained from 26 control serum and 20 cerebrospinal fluids (CSF) samples found to be negative for all agents tested.
  • CSF cerebrospinal fluids
  • a reactive epitope was identified by a a continuous set of overlapping 12-mer peptides with signal intensities above threshold in samples with known TBDs.
  • the signal threshold was defined for each reactive epitope by calculating the mean plus 3 standard deviations of the signal intensity for the same epitope in the 26 negative control samples.
  • An epitope was considered reactive when the signal intensity for at least two contiguous 12-mer peptides was above threshold.
  • An individual sample was called positive when reactive with at least one specific epitope. Table 3 - Samples Tested in Exampli
  • the most consistently reactive epitope was a 17 amino acid portion of the well- characterized immunodominant C6 region (Bacon et al. 2003; Liang et al. 1999). This epitope was reactive in every IgG positive Lyme serum tested and in 16 out of 27 of early Lyme disease samples.
  • FlaB Although highly reactive in Lyme disease patients, the extensive cross-reactivity of FlaB makes it unsuitable as a diagnostic antigen (Luft et al. 1993).
  • the OspC Nt epitope along with epitopes located within variable OspC regions were useful for detection of IgM in early disease.
  • reactivity to epitopes on other antigens was used for diagnosis, including previously unrecognized epitopes on P100, OppA, Bdr, and paralogs of BBO03, a cp32-encoded lipoprotein.
  • This panel of 13 epitopes was also useful for diagnosis of IgG positive samples (Tables 5 and 6).
  • + indicates signal intensity on the serochip
  • NB# represents individual patient with neuroborreliosis
  • B. microti-antibody positive sera Sixteen B. microti-antibody positive sera were tested and six unique frequently reactive specific epitopes (three within SA-1, five within BMN 1-17, and one on BMN 1-3) were identified (Table 1, Figure 3). Ten of these sera also previously tested positive for B. burgdorferi by the two-tiered testing algorithm and all ten were positive for B. burgdorferi by the TBD-Serochip, displaying the utility of this platform for simultaneous detection of concurrent or past infections with more than one agent. In the remaining 46 Lyme disease positive and 15 Lyme-negative sera, B. microti antibodies were detected in 4 out of 46 Lyme disease sera (9%), and in 1 out of 15 Lyme negative sera. None of these samples were previously tested for babesiosis.
  • MSP2 major surface protein 2
  • P44 major surface protein 2
  • MS3-MS5 major surface protein 2
  • TSGKDIVQFAKAVEIS SEQ ID NO: 18
  • chaffeensis infection a tick-transmitted pathogen that frequently cross-reacts in tests for anaplasmosis, was tested.
  • five of these seven samples had antibodies to another TBD-agent, with three sera positive for B. burgdorferi and two for B. microti.
  • Four out of 56 Lyme disease sera were also reactive to these A. phagocytophilum specific epitopes.
  • Example 6- Utility of Peptides and Assay to Detect Antibodies of TBD Pathogens or Agents using Samples Positive for Powassan Virus
  • BMD B. miyamotoi
  • GlpQ an antigen for serological discrimination between relapsing fever and Lyme borreliosis. /. Clin. Microbiol. 1996 34:2483-92.

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Abstract

La présente invention concerne des compositions, des procédés et des trousses pour la détection de l'exposition à certains virus ou agents pathogènes et une infection par ceux-ci. En particulier, la présente invention permet la détection sérologique différentielle rapide de l'exposition à des virus ou agents pathogènes portés par des tiques et l'infection par ceux-ci au moyen de peptides antigéniques.
EP18857606.0A 2017-09-25 2018-09-25 Dosage sérologique pour la détection d'une exposition à des agents pathogènes portés par les tiques et d'une infection par ceux-ci Pending EP3688010A4 (fr)

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