# Patent

## Title
Immunoreactive Ehrlichia p120/p140 epitopes and uses thereof

## Abstract

Provided herein are immunoreactive peptides which can selectively bind Ehrlichia-specific anti-p120 or anti-p140 antibodies. Methods and kits utilizing the immunoreactive peptides are also provided. The immunoreactive peptides may be utilized, e.g., for determining whether or not a subject is infected with Ehrlichia chaffeensis or Ehrlichia canis. In certain embodiments, the immunoreactive peptides may be utilized in an ELISA or lateral flow assay.

## Background
This application is a divisional of U.S. application Ser. No. 13/709,891, filed Dec. 10, 2012, now abandoned which is a divisional of U.S. application Ser. No. 12/769,352, filed Apr. 28, 2010, now U.S. Pat. No. 8,329,189 which claims priority to U.S. Application No. 61/173,345 filed on Apr. 28, 2009. The entire text of each of the above referenced disclosures is specifically incorporated herein by reference without disclaimer.


This invention was made with government support under grant R01 AI 071145 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
I. Field of the Invention
The present invention relates generally to the diagnosis and treatment of Ehrlichia infection. In particular, the invention is related to p120/p140 immunoreactive peptides derived from Ehrlichia proteins, and the use of such peptides in the detection of Ehrlichia infection in humans and animals.
II. Background and Description of Related Art
Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted, obligately intracellular bacterium that cause monocytrotropic ehrlichiosis, an emerging life-threatening disease in humans and a mild to severe disease in wild and domestic canids. A number of studies have demonstrated that antibodies play an essential role in immunity against Ehrlichial pathogens (Feng and Walker, 2004; Winslow et al., 2003; Winslow et al., 2000; Yager et al., 2005). However, only a small subset of E. chaffeensis and E. canis proteins react strongly with antibodies in sera from infected humans or dogs, and thus are considered to be major immunoreactive proteins (Chen et al., 1997; Chen et al., 1994; McBride et al., 2003; Rikihisa et al., 1994). Molecularly characterized major immunoreactive proteins of E. chaffeensis and E. canis include four protein ortholog pairs (p200/p200, p120/p140, p47/p36, and VLPT/p19, respectively) (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2003; McBride et al., 2007; McBride et al., 2000; Nethery et al., 2007). Three of these ortholog pairs (p120/p140, p47/p36, and VLPT/p19) have acidic serine-rich tandem repeats (TRs), and continuous species-specific epitopes have been identified in the TRs of p47/p36 and VLPT/p19 (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2007; McBride et al., 2000).
The p120 is differentially expressed by dense-cored E. chaffeensis, and is found on the surface of the organism and free in the morula space; however, the role of this protein in pathobiology or in eliciting a protective immune response is unknown (Popov et al., 2000). E. chaffeensis p120 has two to five nearly identical serine-rich 80-amino acid TRs, and similarly orthologous E. canis p140 contains 12 or 14 nearly identical serine-rich 36-amino acid TRs (Yabsley et al., 2003; Yu et al., 1997; Yu et al., 2000; Zhang et al., 2008). Specific regions of the p120 and p140 proteins are immunoreactive (McBride et al., 2000; Yu et al., 1996; Yu et al., 2000); however, it is presently unclear as to which sequences within the immunoreactive regions may be recognized by a host immune system.
Current methodologies for diagnosing human monocytotropic ehrlichiosis (HME) present significant clinical limitations. Clinical diagnosis of HME is usually confirmed retrospectively by detection of Ehrlichia-specific antibodies in patient sera using an indirect fluorescent-antibody assay (IFA)(Dumler et al., 2007). The limitations of IFA include lack of standardization between laboratories, false positive interpretations due to autoantibodies or antibodies directed at conserved bacterial proteins, and cross-reactive antibodies produced by related organisms (for example, E. canis, E. ewingii, and Anaplasma phagocytophilum) that can make identification of the specific etiologic agent difficult (Carpenter et al., 1999; Chen et al., 1994; Comer et al., 1999; Paddock and Childs, 2003; Unver et al., 2001). Furthermore, IFA requires expensive microscopy equipment and highly skilled technicians to produce the antigen and interpret results. Molecular diagnostic methods such as PCR are useful for specific and sensitive detection of E. chaffeensis prior to development of reactive antibodies (Childs et al., 1999), but PCR is not useful after antibiotic therapy is initiated, and the clinical sensitivity of PCR in the primary care setting has not been unequivocally determined. Therefore, PCR is currently considered only a valuable adjunct to IFA for diagnosis (Walker et al., 2000). HME diagnosis thus presents significant clinical limitations, and Ehrlichiosis continues to be an emerging infectious disease. Clearly, there is a need for new and improved methods for the detection and diagnosis of Ehrlichiosis.
SUMMARY OF THE INVENTION
The present invention overcomes limitations in the prior art by providing compositions and methods for the diagnosis or detection of Ehrlichia infection. The present invention provides, in certain embodiments, p120/p140 immunoreactive peptides derived from Ehrlichia proteins which may be used to identify Ehrlichia-specific antibodies in a sample, diagnose an Ehrlichia infection in a subject, distinguish between infected and immunized subjects, and/or determine whether the Ehrlichia infection in a subject is caused by Ehrlichia chaffeensis or Ehrlichia canis. These immunoreactive peptides may also be included in a vaccine composition or used to induce a protective immune response in a subject against an Ehrlichia infection. The p120/p140 immunoreactive peptides may selectively bind an Ehrlichia-specific antibody, such as antibodies specific for the 120 kD protein of an Ehrlichia chaffeensis or the 140 kD protein of an Ehrlichia canis. One or more of the p120/p140 immunoreactive peptides may be included or used in a diagnostic kit or assay such as, e.g., an enzyme-linked immunosorbent assay (ELISA), a solid phase assay, and/or a lateral flow assay.
Certain aspects of the present invention are based, in part, on the discovery that certain p120 immunoreactive peptides, such as the synthetic TRP120-R-I1 peptide, described herein below, can surprisingly exhibit substantially improved and increased sensitivity for diagnosing ehrlichiosis in humans as compared to other immunoreactive Ehrlichia peptides or even a recombinant Ehrlichia p120 protein. For example, as shown in the below examples, TRP120-R-I1 peptide exhibited a 96.7% specificity for diagnosing HME, whereas p32 immunoreactive peptides, p47 immunoreactive peptides, Ank200 immunoreactive peptides, and recombinant p120 only displayed specificities of 87.1%, 77.4%, 61.3%, and 90.3%, respectively. Further, various p120/p140 immunoreactive peptides of the present invention may be synthesized, e.g., using solid-phase synthesis; without wishing to be bound by any theory, synthetic p120/p140 immunoreactive peptides may provide the advantage of efficient generation in consistently highly pure forms without contaminating E. coli proteins that can result in false positive reactions when utilizing recombinant proteins. The data presented in the below Examples demonstrates that a single synthetic peptide from TRP120 can provide highly sensitive and specific diagnosis of HME infection comparable to the “gold standard” IFA and may be used for standardized specific point-of-care and/or reference laboratory immunodiagnostics for HME.
An aspect of the present invention relates to an isolated peptide 45 amino acids in length or less and comprising the sequence of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, or a sequence having at least 90% identity to SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, wherein the peptide selectively binds an antibody that recognizes and binds an Ehrlichia p120 or p140 protein. In certain embodiments, peptide is from 20 to 30 amino acids in length. The peptide may comprise SEQ ID NO:1 or SEQ ID NO:2. In various embodiments, the peptide consists of SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, the peptide has at least 95% identity to SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10. The peptide may comprise, in certain embodiments, SEQ ID NO: 4, 5, 6, 7, 8, 9, or 10. In various embodiments, the isolated peptide is immobilized on a surface of a support substrate. The support substrate may comprise latex, polystyrene, nylon, nitrocellulose, cellulose, silica, agarose, or magnetic resin. In certain embodiments, the support substrate is a reaction chamber, a well, a membrane, a filter, a paper, an emulsion, a bead, a microbead, a dipstick, a card, a glass slide, a lateral flow apparatus, a microchip, a comb, a silica particle, a magnetic particle, a nanoparticle, or a self-assembling monolayer. The peptide may be comprised in a kit. The peptide may be comprised in a pharmaceutical preparation. In certain embodiments, the peptide is produced via peptide synthesis. In other embodiments, the peptide may be recombinantly produced. The isolated peptide may further comprises a detectable label.
Another aspect of the present invention relates to a method of detecting antibodies that specifically bind an Ehrlichia organism in a test sample, comprising:(a) contacting an isolated p120/p140 immunoreactive peptide (e.g., a peptide 45 amino acids or less in length and comprising the sequence of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, or a sequence having at least 90% identity to SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, wherein the peptide selectively binds an antibody that recognizes and binds an Ehrlichia p120 or p140 protein), with the test sample, under conditions that allow peptide-antibody complexes to form; (b) detecting the peptide-antibody complexes; wherein the detection of the peptide-antibody complexes is an indication that antibodies specific for an Ehrlichia organism are present in the test sample, and wherein the absence of the peptide-antibody complexes is an indication that antibodies specific an Ehrlichia organism are not present in the test sample. The Ehrlichia organism may be an Ehrlichia chaffeensis or an Ehrlichia canis organism. The step of detecting may comprise performing an enzyme-linked immunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a Bio-Plex® suspension array assay, a mass spectrometry assay, or a particulate-based assay. The step of detecting may comprise a lateral flow assay or a an enzyme-linked immunoassay, wherein the enzyme-linked immunoassay is an ELISA.
Yet another aspect of the present invention relates to a method of identifying an Ehrlichia infection in a subject comprising:(a) contacting a sample from the subject with an isolated p120/p140 immunoreactive peptide (e.g., a peptide of 45 amino acids or less in length and comprising the sequence of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, or a sequence having at least 90% identity to SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, wherein the peptide selectively binds an antibody that recognizes and binds an Ehrlichia p120 or p140 protein) under conditions that allow peptide-antibody complexes to form; and (b) detecting the peptide-antibody complexes; wherein the detection of the peptide-antibody complexes is an indication that the subject has an Ehrlichia infection. The step of detecting may comprise performing an enzyme-linked immunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a Bio-Plex suspension array assay, a dipstick test, or a particulate-based assay. In certain embodiments, the subject is a dog or a human. The method may be at least about 90.3%, 91%, 92%, 93%, 94%, 95%, 96%, or about 96.8% sensitive.
Another aspect of the present invention relates to a method of distinguishing between an active Ehrlichia infection and a previous Ehrlichia immunization in a subject, the method comprising:(a) contacting a sample from the subject with at least one isolated p120/p140 immunoreactive peptide that is not a component of an Ehrlichia vaccine; and (b) detecting whether an antibody in the sample specifically binds to the isolated peptide; wherein if an antibody in the sample specifically binds to the isolated peptide, then the subject has an active Ehrlichia infection, and if an antibody does not specifically bind to the isolated peptide, then the subject is either previously immunized with an Ehrlichia vaccine or is not infected with an Ehrlichia organism. The subject may be a dog or a human. The Ehrlichia organism may be an Ehrlichia chaffeensis or an Ehrlichia canis organism.
Yet another aspect of the present invention relates to a method of distinguishing between an Ehrlichia chaffeensis infection and an Ehrlichia canis infection in a subject, the method comprising: (a) contacting a first sample from the subject with an isolated peptide comprising an amino acid sequence having about 95% or more sequence identity with a peptide selected from the group consisting of SEQ ID NOs 1, 4, 5, and 6; (b) contacting a second sample from the subject with an isolated peptide comprising an amino acid sequence having about 95% or more sequence identity with a peptide selected from the group consisting of SEQ ID NOs: 2, 7, 8, 9, and 10; (c) detecting the presence of peptide-antibody complexes in each of the first and second samples; wherein the presence of peptide-antibody complexes in the first sample is an indication that the subject has an Ehrlichia chaffeensis infection, and wherein the presence of peptide-antibody complexes in the second sample is an indication that the subject has an Ehrlichia canis infection. The subject may be a dog.
Another aspect of the present invention relates to an isolated amino acid sequence having about 90% or more sequence identity with SEQ ID NOs 1, 2, 3,4,5,6,7,8,9, or 10; wherein the peptide is from 15 to 40 amino acids in length, and wherein the peptide can selectively bind an Ehrlichia-specific antibody. In certain embodiments, the isolated amino acid has about 95% or more or more sequence identity with SEQ ID NOs 1, 2, 3,4,5,6,7,8,9, or 10.
Yet another aspect of the present invention relates to an isolated nucleic acid segment encoding an isolated peptide, wherein the peptide is 45 amino acids or less in length and comprises the sequence of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, or a sequence having at least 90% identity to SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, wherein the peptide selectively binds an antibody that recognizes and binds an Ehrlichia p120 or p140 protein. The isolated nucleic acid may, in various embodiments, encode an amino acid sequence having about 90% or more, or about 95% or more sequence identity with SEQ ID NOs 1, 2, 4,5,6,7,8,9, or 10; wherein the peptide is from 15 to 40 amino acids in length, and wherein the peptide can selectively bind an Ehrlichia-specific antibody.
Another aspect of the present invention relates to a vector comprising a contiguous sequence consisting of the nucleic acid segment.
Yet another aspect of the present invention relates to a host cell comprising the nucleic acid segment.
Another aspect of the present invention relates to a kit comprising: (a) an isolated p120/p140 immunoreactive peptide (e.g., a peptide 45 amino acids or less in length and comprising the sequence of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, or a sequence having at least 90% identity to SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9 or 10, wherein the peptide selectively binds an antibody that recognizes and binds an Ehrlichia p120 or p140 protein), (b) an anti-dog or anti-human secondary antibody linked to a reporter molecule; and, (c) an appropriate reagent for detection of the reporter molecule. The peptide may be immobilized on a membrane or a microtiter plate. The reporter molecule may be selected from the group consisting of luciferase, horseradish peroxidase, P-galactosidase, and a fluorescent label. The kit may further comprises a dilution buffer for dog or human serum. The kit may comprise a lateral flow immunoassay, a lateral flow immunochromatographic assay, or an enzyme-linked immunosorbent assay (ELISA).
In various embodiments, antibody epitopes of Ehrlichia chaffeensis Ankrin protein 200 and Tandem repeat protein 47 are also provided (e.g., as shown in FIG. 10 and FIG. 12A). These peptides may be used for the diagnosis of Ehrlichia infection. In various embodiments, one or more of these peptides may be included in a vaccine composition or used for vaccination purposes or to induce an immune response against Ehrlichia chaffeensis or Ehrlichia canis. 
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
The use of the term “or” in the claims is used to mean “and/or ” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
Other objects, features and/or advantages of the present invention will become apparent from the following detailed description. It should be understood that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIGS. 1A-1B. (FIG. 1A) Schematic of E. chaffeensis p120 and E. canis p140 proteins showing domains, location of TRs (number of amino acids in parentheses; R=repeat), and recombinant proteins used for epitope mapping. For both p120 and p140, there were two incomplete repeats preceding the first repeat and following the last repeat, respectively, which were homologous to tandem repeats and also shown in gray. The N-terminus (N); C-terminus (C); tandem repeat region (TR); whole protein (W). (FIG. 1B) Schematic of synthetic peptides used to map the tandem repeat epitope of E. chaffeensis p120 and E. canis p140 proteins.
FIG. 2. Alignments of amino acid sequence of homologous regions in tandem repeat unit, N- and C-terminal regions of E. chaffeensis p120 and E. canis p140 proteins. Residues that match the consensus within two distance units are boxed, and gaps are shown by dashes. The major TR epitope of E. chaffeensis p120 (22-mer) and E. canis p140 (19-mer) are identified with a bar.
FIGS. 3A-3B. Identification of native E. chaffeensis p120 and E. canis p140 proteins by Western immunoblot. (FIG. 3A) E. chaffeensis whole-cell lysates (lane 1), supernatants derived from E. chaffeensis-infected cells (lane 2), and E. canis whole-cell lysates (lane 3) reacted with rabbit anti-p120R-I1 antibody. (FIG. 3B) E. canis whole-cell lysates (lane 1), supernatants derived from E. canis-infected cells (lane 2), and E. chaffeensis whole-cell lysates (lane 3) reacted with rabbit anti-p140 peptide antibody. Pre-immunization rabbit serum controls did not recognize Ehrlichia whole-cell lysates. Precision Protein Standard (Bio-Rad).
FIGS. 4A-4B. Immunoreactivity of recombinant proteins of E. chaffeensis p120 and E. canis p140 by Western immunoblot. (FIG. 4A) SDS-PAGE and total protein staining of purified recombinant p120 recombinant fragments (whole protein [W], N-terminus [N], tandem repeats [TR, two repeats], and C-terminus [C]) (left), and corresponding Western immunoblot probed with two anti-E. chaffeensis dog (experimentally infected; 2251 and 2495 [D-2251/Ech and D-2495/Ech]) sera and two HME patient (SC07 and CDC4 [H-SC07/Ech and H-CDC4/Ech]) sera (right). (FIG. 4B) SDS-PAGE and total protein staining of purified recombinant p140 proteins fragments (whole protein [W], N-terminus [N], tandem repeats [TR, fourteen repeats], and C-terminus [C]) (left), and corresponding Western immunoblot probed with three anti-E. canis sera from one experimentally infected dog (2995 [D-2995/Eca]) and two naturally infected dogs (4283 and 2160 [D-4283/Eca and D-2160/Eca]) (right). Human or dog sera did not recognize thioredoxin or GST proteins, and the normal human or dog sera did not recognize these recombinant proteins by Western immunoblot. M, Precision Protein Standard (Bio-Rad).
FIGS. 5A-5E. Immunoreactivity of overlapping synthetic peptides spanning the E. chaffeensis p120 repeat unit by ELISA. (FIG. 5A) Sequence and orientation of all overlapping peptides representing E. chaffeensis p120 repeat unit. (FIG. 5B) E. chaffeensis p120 peptides reacted with the anti-E. chaffeensis dog serum derived from an experimentally infected dog (2251). (FIGS. 5C, 5D, and 5E) E. chaffeensis p120 peptides reacted with three HME patients (3, 18 and 20, respectively) sera. The OD readings represent the means for three wells (±standard deviations), with the OD of the buffer-only wells subtracted. The OD readings of peptide p120R-I1 were significantly higher than those of smaller overlapping peptides (I1-S1, I1-S3 and I1-S4, P<0.05 for all sera; I1-S2, P<0.05 for all patient sera). Normal dog or human serum did not recognize these peptides.
FIGS. 6A-6E. Immunoreactivity of E. canis p140 repeat overlapping synthetic peptides as determined by ELISA. (FIG. 6A) Six overlapping peptides spanning the E. canis p140 repeat unit. (FIGS. 6B, 6C, 6D, and 6E) E. canis p140 peptides reacted with anti-E. canis dog sera obtained from four naturally infected dogs (2160, 6, 10 and 18, respectively). The OD readings represent the means for three wells (±standard deviations), with the OD of the buffer-only wells subtracted. The OD readings of peptide R-4 were significantly higher than those of R-2 with half of the dog sera (10 and 18, P<0.05). The normal dog serum did not recognize these peptides.
FIG. 7. Localization of minor cross-reactive epitopes between E. chaffeensis p120 and E. canis p140 proteins by Western immunoblot. E. chaffeensis p120 and E. canis p140 recombinant proteins (N-terminus [N], tandem repeats [TR], and C-terminus [C]) reacted with anti-E. canis sera (4283 and 2995 [D-4283/Eca and D-2995/Eca]) and anti-E. chaffeensis sera (2251 and CDC3 [D-2251/Ech and H-CDC3/Ech]).
FIG. 8. Immunoreactivities of major antibody epitopes of E. chaffeensis immunodominant proteins with HME patient sera by ELISA. Synthetic epitope peptides of VLPT (R3+R4), p47 (N2C-N+R+C), p120 (R-I1), and the recombinant p120 TR protein (rp120, containing first two tandem repeats of p120) reacted with 10 HME patient sera and an anti-E. chaffeensis dog (no. 2495) serum. The OD readings represent the means for three wells (±standard deviations), with the OD of the negative control wells subtracted. The normal human or dog serum did not recognize these peptides.
FIG. 9. Schematic of E. chaffeensis Ank200 protein, showing domains, predicted isoelectric points (pIs), and the recombinant proteins and synthetic peptides used for epitope mapping. Predicted ankyrin domains are shown in shaded boxes. The recombinant proteins and synthetic peptides are shown in black lines and gray lines, respectively, and solid lines show regions containing an epitope(s), whereas dashed lines show regions which did not react or reacted weakly with anti-E. chaffeensis human and dog sera. The approximate locations of mapped epitopes are designated by arrows.
FIGS. 10A-C. Immunoreactivities of overlapping synthetic peptides spanning the E. chaffeensis Ank200-N6, -N10, and -C6 fragments by ELISA. (FIG. 10A) Ank200-N6 peptides (left) reacted with four HME patient serum samples (no. F3, F5, F13, and F22) and an anti-E. chaffeensis dog serum sample derived from an experimentally infected dog (no. 2251). The OD readings of peptide N6-1 were significantly (P<0.05) higher than those of N6-2, -3, and -4 for the dog serum sample and for most patient sera, and the OD readings of peptide N6-1a were significantly (P<0.05) higher than those of N6-1b for all patient sera. (FIG. 10B) Ank200-N10 peptides (left) reacted with four HME patient serum samples (no. F2, F4, F5, and F21) and the dog serum sample. (FIG. 10C) Ank200-C6 peptides (left) reacted with four HME patient serum samples (no. F2, F4, F15, and SC07) and the dog serum sample. The OD readings of peptide C6-4 were significantly (P<0.05) higher than those of C6-1, -2, and -3 for all sera, and OD readings of peptide C6-4b were significantly (P<0.05) higher than those of C6-4a for all sera. The OD readings represent the mean values for three wells (±standard deviations), with the OD values of the buffer-only wells subtracted. Normal dog or human sera did not recognize these peptides.
FIG. 11. Schematic of TRP47 showing domains, location of TRs (number of amino acids in parentheses), and recombinant proteins and synthetic peptides used for epitope mapping. The recombinant proteins and synthetic peptides are shown in black lines and gray lines, respectively, and solid lines show regions containing epitope(s).
FIGS. 12A-C. Immunoreactivity of overlapping synthetic peptides spanning E. chaffeensis TRP47-N4 and synthetic TRP47-R and TRP47-C peptides as determined by ELISA. (FIG. 12A) Sequences of three overlapping peptides spanning the TRP47-N4 fragment and TRP47-R and TRP47-C peptides. (FIG. 12B) TRP47-N4 peptides reacted with five HME patient sera (nos. O15, 6, 9, 13, 18 and 19) by ELISA. (FIG. 12C) TRP47-R and TRP47-C peptides reacted with seven HME patient sera (nos. O3, O13, 4, 8, 10, 13 and 20) and an anti-E. chaffeensis dog serum (no. 2251) by ELISA. The OD readings represent the means for three wells (±standard deviations), with the OD of the buffer-only wells subtracted. The OD readings of peptide TRP47-R were significantly (P<0.05) higher than those of TRP47-C for all patient sera except for no. O3 and no. 13, for which the OD readings of peptide TRP47-C were significantly (P<0.05) higher than those of TRP47-R. The normal human or dog serum did not recognize TRP47 polypeptides.
FIGS. 13A-B. Immunoreactivity of major antibody epitopes from E. chaffeensis immunoreactive proteins with HME patient sera by ELISA. (FIG. 13A) Synthetic epitope peptides of TRP32 (R3+R4), TRP47 (N4-1+R+C), TRP120 (R-I1) and Ank200 (N6-1a+N10-1+C6-4b) reacted with 31 HME patient sera (nos. 1 ̃31) and an anti-E. chaffeensis dog (no. 2251) serum. (FIG. 13B) An equal mixture of TRP32-R3, TRP32-R4 and TRP120-R-I1 peptides as well as the recombinant TRP120 TR protein (rTRP120-TR, containing first two tandem repeats of TRP120 only) reacted with 31 HME patient (nos. 1-31) sera and an anti-E. chaffeensis dog (no. 2251) serum. The OD readings represent the means for three wells (±standard deviations), with the OD of the negative control (E. canis TRP36-2R peptide) wells subtracted. The cut-off OD (0.1) established for the positive reading is shown by a dotted line. The normal human or dog serum did not recognize these peptides.



DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention is based, in part, on the discovery by the inventors of peptides corresponding to single continuous species-specific major epitopes within each of the E. chaffeensis p120 and E. canis p140 proteins. These immunoreactive peptides may be used for the detection of Ehrlichia infection, for example, by selectively binding Ehrlichia-specific antibodies in a biological sample, such as a blood or serum sample. Alternately, one or more of these peptides may be included in a vaccine formulation to induce a protective immune response in a subject against Ehrlichia. 
Surprisingly, it was observed that various synthetic p120 immunoreactive peptides provided herein can display superior sensitivity and reactivity as compared to other immunoreactive proteins for the diagnosis of the emerging zoonosis human monocytotropic ehrlichiosis (HME) caused by Ehrlichia chaffeensis. As shown in the Examples below, the sensitivity and specificity of synthetic peptides representing immunodeterminants of E. chaffeensis were determined by enzyme-linked immunosorbent assay (ELISA). Thirty-one HME patient sera that had detectable E. chaffeensis antibodies (titers from 64 to 8192) by indirect fluorescent-antibody assay (IFA) were tested. All 31 sera reacted with at least one E. chaffeensis peptide and 30 sera (96.8%) with TRP120 peptide, 27 (87.1%) with TRP32 peptides, 24 (77.4%) with TRP47 peptides, 19 (61.3%) with Ank200 peptides, and 28 (90.3%) with recombinant TRP120-TR protein. A mixture of the two most sensitive peptides from TRP120 and TRP32 did not provide enhanced analytical sensitivity over the TRP120 alone. These results demonstrate that a p120 immunoreactive peptide may be used in a standardized sensitive point-of-care and/or reference laboratory immunodiagnostics for HME. To the inventors knowledge, these are the first studies to compare molecularly-defined major antibody epitopes with IFA for diagnosis of HME.
I. Ehrlichia Immunodominant Proteins and Immunoreactive Peptides Thereof
Most Ehrlichia species, including Ehrlichia chaffeensis and Ehrlichia canis, are obligately intracellular bacteria that exhibit tropism for mononuclear phagocytes (Winslow et al., 2005), interacting with these cells and other components of the immune system through a small subset of their constituent proteins (Collins et al., 2005; Hotopp et al., 2006; Frutos et al., 2006; Mavromatis et al., 2006). Among these host-pathogen interacting proteins are the major immunoreactive proteins which are recognized by antibodies in human and animal hosts (Doyle et al., 2006; McBride et al., 2003; McBride et al., 2000) and include p200, p120, p47 and VLPT in Ehrlichia chaffeensis and their orthologs in Ehrlichia canis, p200, p140, p36, and p19, respectively (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2003; McBride et al., 2007; McBride et al., 2000; Nethery et al., 2007).
E. chaffeensis p120 and E. canis p140 are each major immunoreactive proteins that are differentially expressed and are secreted (Doyle et al., 2006; Popov, et al., 2000) by their respective organisms. Extensive variability in the number and/or sequence of tandem repeats in the E. chaffeensis and E. canis immunoreactive proteins is well documented (Chen et al., 1997; Doyle et al., 2006; Sumner et al., 1999). The p120 protein is a 120 kD protein that contains two to five serine-rich tandem repeats with 80-amino acids each, and the orthologous E. canis p140 is a 140 kD protein that contains twelve to fourteen serine-rich 36-amino acid TRs (Yabsley et al., 2003; Yu et al., 1997; Yu et al., 2000; Zhang et al., 2008). Disclosed herein is the mapping of a single species-specific epitope to each of the Ehrlichia proteins, p120 and p140, and in each protein, the epitope lies within the serine-rich, acidic tandem repeats. Such an epitope may, for example, be comprised in one or more immunoreactive peptides, i.e., p120/p140 immunoreactive peptides, from each of the Ehrlichia proteins and may be bound, identified, or recognized by an Ehrlichia specific antibody.
As used herein, the term “peptide” encompasses amino acid chains comprising less than about 100 amino acids and preferably less than about 50 amino acid residues, wherein the amino acid residues are linked by covalent peptide bonds. As used herein, an “antigenic peptide” is a peptide which, when introduced into a vertebrate, can stimulate the production of antibodies in the vertebrate, i.e., is antigenic, and wherein the antibody can selectively recognize and/or bind the antigenic peptide. An antigenic peptide may comprise an immunoreactive sequence derived from a p120 or p140 Ehrlichia protein, and may comprise additional sequences. The additional sequences may be derived from a native Ehrlichia antigen and may be heterologous, and such sequences may (but need not) be immunogenic.
As used herein, an “p120/p140 immunoreactive peptide” is an peptide which can selectively bind with an anti-p 120 antibody or an anti-p 140 antibody. For example, a p120/p140 immunoreactive peptide may bind one or more antibodies produced by a mammalian host (e.g., a dog or human) which was previously exposed to or infected by Ehrlichia chaffeensis or Ehrlichia canis. Accordingly, a “p120 immunoreactive peptide” refers to a peptide which can selectively bind an anti-p120 antibody, and “p140 immunoreactive peptide” refers to a peptide which can selectively bind an anti-p140 antibody. A p120/p140 immunoreactive peptide may have at least about, or comprise a sequence with at least about, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 disclosed herein. The p120/p140 immunoreactive peptide may be from 10 to 45, 15 to 50, 15 to 45, 15 to 40, 16 to 45, 16 to 40, 18 to 35, or 20 to 30 amino acids in length, or any length or range derivable therein.
In certain embodiments, a p120/p140 peptide may be immunogenic or antigenic. For example, certain p120/p140 peptides may comprise an Ehrlichia antigen which, when introduced into a vertebrate, may stimulate the production of antibodies in the vertebrate which selectively recognize and/or bind a portion of an Ehrlichia p120 or p140 protein. It is envisioned that such peptides could be used to induce some degree of protective immunity.
A p120/p140 immunoreactive peptide may be a recombinant peptide, synthetic peptide, purified peptide, immobilized peptide, detectably labeled peptide, encapsulated peptide, or a vector-expressed peptide. In certain embodiments, a synthetic p120/p140 immunoreactive peptide may be used for diagnostic testing, and synthetic peptides may display certain advantages, such as a decreased risk of bacterial contamination, as compared to recombinantly expressed peptides. In select embodiments, an p120/p140 immunoreactive peptide of the present invention may be comprised in a kit, or may be immobilized onto a surface of a component of the kit. An p120/p140 immunoreactive peptide may also be comprised in a composition, such as, for example, a vaccine composition, which is formulated for administration to a human or canine subject.
Immobilized Immunoreactive Peptides
In certain embodiments, an p120/p140 immunoreactive peptide described herein may be used as diagnostic or prophylactic tools for detection of or immunization against Ehrlichia infection. In particular, p120/p140 immunoreactive peptides disclosed herein may be useful in solution-phase assays, or in assays in which the isolated p120/p140 immunoreactive peptide is immobilized on a surface of a support substrate. Alternatively, an p120/p140 immunoreactive peptide described herein may be comprised in a vaccine formulation to induce a protective immune response in a subject, or an immune response against Ehrlichia chaffeensis or Ehrlichia canis. One or more p120/p140 immunoreactive peptides may be immobilized on a surface by covalent attachment, encapsulation, or adsorption using methods generally known in the art, and may include the use of cross-linkers, capture molecules and such like, to which peptides may be coupled, conjugated, or cross-linked.
A p120/p140 immunoreactive peptide may be immobilized onto a surface of a support or a solid substrate; for example, the p120/p140 immunoreactive peptide may be immobilized directly or indirectly by coupling, cross-linking, adsorption, encapsulation, or by any appropriate method known in the art. By way of non-limiting example, binding of an p120/p140 immunoreactive peptide disclosed herein by adsorption to a well in a microtiter plate or to a membrane may be achieved by contacting the peptide, in a suitable buffer, with the well surface for a suitable amount of time. The contact time can vary with temperature, but is typically between about 1 hour and 1 day when using an amount of peptide ranging from about 50 ng to about 1 mg, and preferably about 500 ng.
In some embodiments, an p120/p140 immunoreactive peptide disclosed herein is covalently attached to a support substrate by first reacting the support with a reagent that will chemically react with both the support and a functional group (i.e., crosslink), such as a hydroxyl or amino group, on the peptide. For example, an p120/p140 immunoreactive peptide may be crosslinked to a surface through an amine or carboxylic group on either end of the peptide, and a peptide may be crosslinked through a group on each end of the peptide (i.e., head-to-tail crosslinked). Such peptomers (i.e., head-to-tail crosslinked or otherwise immobilized peptides) may be used with both diagnostic and therapeutic methods of the present invention.
Numerous support substrates for peptide immobilization are known in the art which may be employed with an p120/p140 immunoreactive peptide disclosed herein, formed from materials such as, for example, latex, polystyrene, nylon, nitrocellulose, cellulose, silica, agarose, inorganic polymers, lipids, proteins, sugars, or magnetic resin. A person of ordinary skill in the art may select the support substrate that is appropriate for a given application. In particular embodiments of the present invention, a support substrate may be a reaction chamber, a microplate well, a membrane, a filter, a paper, an emulsion, a bead, a microbead, a microsphere, a nanocrystal, a nanosphere, a dipstick, a card, a glass slide, a microslide, a lateral flow apparatus, a microchip, a comb, a silica particle, a magnetic particle, a nanoparticle, or a self-assembling monolayer.
Detectably-Labeled Immunoreactive Peptides
A p120/p140 immunoreactive peptide may be conjugated to or attached to detectable label such as, for example, a radioactive isotope, a non-radioactive isotope, a particulate label, a fluorescent label, a chemiluminescent label, a paramagnetic label, an enzyme label or a colorimetric label. The detectably-labelled peptides may be used, e.g., in diagnostic or prophylactic methods and compositions. In certain embodiments, the peptide portion of the detectably labeled p120/p140 immunoreactive peptide may be immobilized on a surface of a support substrate. In other embodiments, the detectable label may be used to immobilize the detectably labeled p120/p140 immunoreactive peptide to the surface of a support substrate.
As used herein, “detectable label” is a compound and/or element that can be detected due to its specific functional properties, and/or chemical characteristics, the use of which allows the peptide to which it is attached be detected, and/or further quantified if desired.
Exemplary labels include, but are not limited to, a particulate label such as colloidal gold, a radioactive isotope such as astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodine123, iodine125, iodine131, indium111, 59iron, 32phosphorus, rhenium186, rhenium188, 75selenium, 35sulphur, technicium99, technetium-99m or yttrium90, a colorimetric label such as dinitrobenzene, dansyl chloride, dabsyl chloride, any of the azo, cyanin or triazine dyes, or chromophores disclosed in U.S. Pat. Nos. 5,470,932, 5,543,504, or 6,372,445, all of which are incorporated herein by reference; a paramagnetic label such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III), a fluorescent label such as Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red, or Lucifer Yellow, an enzyme label such as urease, luciferase, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase, or a chemiluminescent label such as luminol, phthalazinedione, and others disclosed in any of U.S. Pat. Nos. 4,373,932, 4,220,450, 5,470,723, and U.S. Patent Application 2007/0264664, all of which are incorporated herein by reference.
Methods of Producing an Immunoreactive Peptide
Certain p120/p140 immunoreactive peptide of the present invention may be synthesized, e.g., using solid-phase synthesis. Synthetic peptides may provide certain advantages over recombinant proteins; for example, synthetic peptides can be produced consistently in highly pure forms without contaminating E. coli proteins that can result in false positive reactions when utilizing recombinant proteins. In addition, peptides can be produced quickly and efficiently without costly and laborious purification procedures and need for defined expression vectors and hosts.
An isolated p120/p140 immunoreactive peptide disclosed herein may be produced by any appropriate method known in the organic chemistry arts. For example, such peptides may be produced using one of the established solid-phase peptide synthesis techniques, such as those of Merrifield, Carpino, or Atherton [Merrifield 1963; Carpino 1993, Atherton and Sheppard, 1989]. Peptides may be synthesized using equipment for automated peptide synthesis that is widely available from commercial suppliers such as Perkin Elmer (Foster City, Calif.). A p120/p140 immunoreactive peptide of the invention may also be chemically synthesized using solution-phase techniques such as those described in Carpino et al., (2003) or U.S. Patent Application 2009/0005535, both incorporated herein in their entirety by reference. Due to the length of the peptides, in certain embodiments, the peptides may be synthesized, e.g., using solid-phase peptide synthesis (SPPS), t-Boc solid-phase peptide synthesis, or Fmoc solid-phase peptide synthesis.
In alternative embodiments, an isolated p120/p140 immunoreactive peptide may be recombinantly prepared from a nucleic acid encoding the peptide. Such a nucleic acid may be operably linked to an expression vector and used to produce a peptide of the present invention using known methods. By way of nonlimiting example, a p120/p140 immunoreactive peptide may be expressed from a vector and isolated from the growth media of a host cell comprising the vector. Alternatively, the present p120/p140 immunoreactive peptides may be produced in a cell-free system from a nucleic acid encoding the peptide.
An immobilized p120/p140 immunoreactive peptide may be synthesized onto a support substrate, or conjugated, crosslinked, or adsorbed, either directly or indirectly onto a surface of a support substrate.
It is anticipated that virtually any method of peptide immobilization known in the art which would not impact the structure or function of the disclosed peptides may be used to immobilize a p120/p140 immunoreactive peptide. For example, peptide immobilization may be accomplished using a crosslinking or conjugation agent such as methyl-p-hydroxybenzimidate, N-succinimidyl-3-(4-hydroxyphenyl)propionate, using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sSMCC), N-[maleimidocaproyloxy]sulfosuccinimide ester (sEMCS), N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), Bis-diazobenzidine (BDB), or N-acetyl homocysteine thiolactone (NAHT), and others disclosed in any of U.S. Pat. Nos. 5,853,744, 5,891,506, 6,210,708, 6,617,142, 6,875,750, 6,951,765, 7,163,677, and 7,282,194, each incorporated herein by reference. Peptides may be conjugated directly or indirectly to any of the commercially available support substrates having a surface coatings comprising crosslinkers, coupling agents, thiol or hydroxyl derivatizing agents, carboxyl- or amine-reactive groups such as of maleic anhydride (e.g., Pierce Immunotechnology Catalog and Handbook, at A12-A13, 1991).
In some embodiments, peptide of the invention may also be immobilized using metal chelate complexation, employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); EDTA; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Peptides can be immobilized by coupling to other peptides and to condensation groups immobilized on a surface or present in an immobilization buffer such as glutaraldehyde or periodate. Conjugates with fluorescence markers may also prepared in the presence of such agents or by reaction with an isothiocyanate. A peptide may be attached to a surface by conjugation, crosslinking or binding to an affinity binding agent such as biotin, streptavidin, a polysaccharide such as an alginate, a lectin, and the like.
In general, regardless of the method of preparation or immobilization status, the p120/p140 immunoreactive peptides disclosed herein are preferably prepared in a substantially pure form. Preferably, the p120/p140 immunoreactive peptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure.
Nuclieic Acids
In an aspect, the present invention provides a nucleic acid encoding an isolated p120/p140 immunoreactive peptide comprising a sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10. Such a p120/p140 immunoreactive peptide may be from 10 to 45, 15 to 40, 15 to 30, 18 to 35, or 20 to 30 amino acids in length, or any range derivable therein. The term “nucleic acid” is intended to include DNA and RNA and can be either double stranded or single stranded.
Some embodiments of the present invention provide recombinantly produced p120/p140 immunoreactive peptides which can specifically bind Ehrlichia specific antibodies. Accordingly, a nucleic acid encoding a p120/p140 immunoreactive peptide or an antigenic Ehrlichia peptide may be operably linked to an expression vector and the peptide produced in the appropriate expression system using methods well known in the molecular biological arts. A nucleic acid encoding an p120/p140 immunoreactive peptide disclosed herein may be incorporated into any expression vector which ensures good expression of the peptide. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is suitable for transformation of a host cell.
A recombinant expression vector being “suitable for transformation of a host cell”, means that the expression vector contains a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid molecule. The terms, “operatively linked” or “operably linked” are used interchangeably, and are intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
Accordingly, the present invention provides a recombinant expression vector comprising nucleic acid encoding an p120/p140 immunoreactive peptide, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, or viral genes (e.g., see the regulatory sequences described in Goeddel (1990).
Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native protein and/or its flanking regions.
A recombinant expression vector may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant p120/p140 immunoreactive peptide disclosed herein. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of a recombinant expression vector, and in particular, to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.
Recombinant expression vectors can be introduced into host cells to produce a transformant host cell. The term “transformant host cell” is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms “transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel (1991).
A nucleic acid molecule of the invention may also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxy-nucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071).
Biological Functional Equivalents
Preferred p120/p140 immunoreactive peptides or analogs thereof specifically or preferentially bind an Ehrlichia p120 or p140 specific antibody. Determining whether or to what degree a particular p120/p140 immunoreactive peptide or labeled peptide, or an analog thereof, can bind an Ehrlichia p120 or p140 specific antibody can be assessed using an in vitro assay such as, for example, an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immnunofluorescent assay (FA), nephelometry, flow cytometry assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, mass spectrometry assay, particle-based assay, inhibition assay and/or an avidity assay.
An p120/p140 immunoreactive peptide of the present invention may be modified to contain amino acid substitutions, insertions and/or deletions that do not alter their respective interactions with anti-Ehrlichia antibody binding regions. Such a biologically functional equivalent of an p120/p140 immunoreactive peptide derived from an Ehrlichia p120 or p140 protein could be a molecule having like or otherwise desirable characteristics, i.e., binding of Ehrlichia specific antibodies. As a nonlimiting example, certain amino acids may be substituted for other amino acids in an p120/p140 immunoreactive peptide disclosed herein without appreciable loss of interactive capacity, as demonstrated by detectably unchanged antibody binding. It is thus contemplated that an p120/p140 immunoreactive peptide disclosed herein (or a nucleic acid encoding such a peptide) which is modified in sequence and/or structure, but which is unchanged in biological utility or activity remains within the scope of the present invention.
It is also well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while still maintaining an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. Of course, a plurality of distinct peptides with different substitutions may easily be made and used in accordance with the invention.
The skilled artisan is also aware that where certain residues are shown to be particularly important to the biological or structural properties of a peptide, e.g., residues in specific epitopes, such residues may not generally be exchanged. This may be the case in the present invention, as a mutation in an p120/p140 immunoreactive peptide disclosed herein could result in a loss of species-specificity and in turn, reduce the utility of the resulting peptide for use in methods of the present invention. Thus, peptides which are antigenic (i.e., bind anti-Ehrlichia antibodies specifically) and comprise conservative amino acid substitutions are understood to be included in the present invention. Conservative substitutions are least likely to drastically alter the activity of a protein. A “conservative amino acid substitution” refers to replacement of amino acid with a chemically similar amino acid, i.e., replacing nonpolar amino acids with other nonpolar amino acids; substitution of polar amino acids with other polar amino acids, acidic residues with other acidic amino acids, etc.
Amino acid substitutions, such as those which might be employed in modifying an p120/p140 immunoreactive peptide disclosed herein are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
The invention also contemplates isoforms of the p120/p140 immunoreactive peptides disclosed herein. An isoform contains the same number and kinds of amino acids as a peptide of the invention, but the isoform has a different molecular structure. The isoforms contemplated by the present invention are those having the same properties as a peptide of the invention as described herein.
Nonstandard amino acids may be incorporated into proteins by chemical modification of existing amino acids or by de novo synthesis of a peptide disclosed herein. A nonstandard amino acid refers to an amino acid that differs in chemical structure from the twenty standard amino acids encoded by the genetic code.
In select embodiments, the present invention contemplates a chemical derivative of an p120/p140 immunoreactive peptide disclosed herein. “Chemical derivative” refers to a peptide having one or more residues chemically derivatized by reaction of a functional side group, and retaining biological activity and utility. Such derivatized peptides include, for example, those in which free amino groups have been derivatized to form specific salts or derivatized by alkylation and/or acylation, p-toluene sulfonyl groups, carbobenzoxy groups, t-butylocycarbonyl groups, chloroacetyl groups, formyl or acetyl groups among others. Free carboxyl groups may be derivatized to form organic or inorganic salts, methyl and ethyl esters or other types of esters or hydrazides and preferably amides (primary or secondary). Chemical derivatives may include those peptides which comprise one or more naturally occurring amino acids derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for serine; and ornithine may be substituted for lysine.
It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The amino acids described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional properties set forth herein are retained by the protein. In keeping with standard protein nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are known in the art.
Peptidomimetics
In addition to the biological functional equivalents discussed above, the inventors also contemplate that structurally similar compounds may be formulated to mimic the key portions of an p120/p140 immunoreactive peptide of the present invention. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and, hence, also are functional equivalents.
Certain mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
Methods for generating specific structures have been disclosed in the art. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos. 5,446,128; 5,710,245; 5,840,833; and 5,859,184. These structures render the peptide more thermally stable, also increase resistance to proteolytic degradation. Six, seven, eleven, twelve, thirteen and fourteen membered ring structures are disclosed.
Beta II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids. Weisshoff et al. (1999). Also, Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn inducing properties. Methods for generating conformationally restricted beta turns and beta bulges are described, for example, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155.
Beta-turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures. Other types of mimetic turns include reverse and gamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.
II. Ehrlichiosis and Detecting Ehrlichia Infection
Ehrlichiosis in humans generally refers to infections caused by obligate intracellular bacteria in the family Anaplasmataceae, chiefly in the genera Ehrlichia and Anaplasma. The majority of cases of human ehrlichiosis (HE) are caused by 3 distinct species: Ehrlichia chaffeensis, chief among them (Dumler et al., 2007). Ehrlichia infections in animals are also referred to as Ehrlichiosis, along with a variety of diseases caused by a diverse group of pathogens from genuses Ehrlichia, Anaplasma, Neorickettsia, and Cowdria (Dumler et al., 2007). Ehrlichia infections are sustained mostly in monocytes or granulocytes, and studies have demonstrated that antibodies play an essential role in the immune response to Ehrlichia infection (Feng et al., 2004; Winslow et al., 2003; Winslow et al., 2000; Yager et al., 2005).
Accordingly, select embodiments of the present invention provide methods of detecting antibodies that specifically bind an Ehrlichia organism in a sample. Such a method may involve contacting an isolated p120/p140 immunoreactive peptide disclosed herein, with the test sample, under conditions that allow peptide-antibody complexes to form, and detecting the peptide-antibody complexes. In these embodiments, the detection of the peptide-antibody complexes is an indication that antibodies specific for an Ehrlichia organism are present in the test sample, and the absence of the peptide-antibody complexes is an indication that antibodies specific an Ehrlichia organism are not present in the test sample.
In multiple embodiments, the detection of an p120/p140 immunoreactive peptide disclosed herein bound to an Ehrlichia specific antibody (i.e., a peptide-antibody complex) may be accomplished using an enzyme-linked immunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a mass spectrometry assay, latex agglutination, an indirect hemagglutination assay (IHA), complement fixation, an inhibition assay, an avidity assay, a dipstick test, or a particulate-based assay. In preferred embodiments, peptide-antibody complexes described herein are detected using an enzyme-linked immunoassay, a lateral flow assay, or a particle-based assay.
As used herein, a “sample” is any sample that comprises or is suspected to comprise antibodies. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. In some embodiments, the sample is a blood, serum or plasma sample obtained from a subject or patient.
Ehrlichiosis caused by an Ehrlichia chaffeensis infection in humans presents with flu-like symptoms of fever, chills, headache, and muscle aches. In more severe cases, nausea, loss of appetite, weight loss, abdominal pain, cough, diarrhea and change in mental status may also be observed. Ehrlichiosis in humans is potentially fatal.
In dogs, ehrlichiosis is most often caused by either Ehrlichia chaffeensis or Ehrlichia canis bacteria, and progresses in three phases: an acute phase, a subclinical phase, and a chronic phase. The acute phase normally extends weeks after infection and features symptoms similar to those of human ehrlichiosis, i.e., fever, lethargy, loss of appetite, shortness of breath, joint pain and stiffness, and can include more sever symptoms such as anemia, depression, bruising, and enlarged lymph nodes, liver, and spleen. The subclinical phase can persist for years and most often presents no symptoms, although antibodies to Ehrlichia antigens may be detectable. The chronic phase of Ehrlichia infection generally features recurring symptoms of weight loss, anemia, neurological dysfunction, bleeding, ocular inflammation, leg edema, and fever, and presents a blood profile which often leads to a misdiagnosis of leukemia. An Ehrlichia infection that progesses to the chronic stage of disease is often fatal.
The nonspecific symptoms of an Ehrlichia infection and their resemblance to mild and severe influenza symptoms makes diagnosis of Ehrlichiosis difficult in humans and dogs. Diagnosis is further hampered by current laboratory testing procedures for Ehrlichia infection which are not point-of-care tests, i.e., the tests are not available in most hospitals, clinics, and physician or veterinarian offices where a patient can receive treatment.
Accordingly, select embodiments of the present invention provide methods of identifying an Ehrlichia infection in a subject. Such a method may involve contacting a sample from the subject with an isolated p120/p140 immunoreactive peptide disclosed herein under conditions that allow peptide-antibody complexes to form, and detecting the peptide-antibody complexes. In these embodiments, the detection of the peptide-antibody complexes is an indication that the subject has an Ehrlichia infection. The Ehrlichia organism may be an Ehrlichia chaffeensis organism or an Ehrlichia canis organism. In some embodiments, the subject is a human or a dog. As with other methods disclosed herein, the detection step may be accomplished using any appropriate type of assay known in the art, and may be preferrably accomplished using a lateral flow assay or an ELISA.
The terms “subject” and “patient” are used interchangeably herein, and may refer to a mammal, especially a human or a dog. In certain embodiments, a “subject” or “patient” refers to a mammalian Ehrlichia host (i.e., animal infected with an Ehrlichia organism). An Ehrlichia host may be, for example, human or non-human primate, bovine, canine, caprine, cavine, corvine, epine, equine, feline, hircine, lapine, leporine, lupine, murine, ovine, porcine, racine, vulpine, and the like, including livestock, zoological specimens, exotics, as well as companion animals, pets, and any animal under the care of a veterinary practitioner. A subject may be or may not be infected with an Ehrlichia organism, and a subject may be a mammal suspected of being infected with an Ehrlichia organism.
Without wishing to be bound by theory, the p120/p140 immunoreactive peptides disclosed herein each comprise at least a part of a major Ehrlichia epitope that accounts for a species-specific immunogenicity in humans and animals. The term “epitope” is used herein to indicate that portion of an immunogenic substance that is specifically identified, recognized, and bound by, an antibody or cell-surface receptor of a host immune system that has mounted an immune response to the immunogenic substance as determined by any method known in the art. (see, for example, Geysen et al., 1984). Thus, an epitope that is “species-specific” is an epitope that can be used to differentiate one species of the Ehrlichia genus from another Ehrlichia species. By way of non-limiting example, an p120/p140 immunoreactive peptide that has at least 95% identity with SEQ ID NO:1 from Ehrlichia chaffeensis comprises an epitope that may be distinguishable by the immune system of a host mammal from an p120/p140 immunoreactive peptide that has at least 95% identity with SEQ ID NO:2 from Ehrlichia canis. 
Accordingly, an aspect of the present invention provides a method of distinguishing between an Ehrlichia chaffeensis infection and an Ehrlichia canis infection in a subject. Such a method may comprise contacting a first sample from the subject with an isolated p120 immunoreactive peptide (e.g., comprising an amino acid sequence having about 95% or more sequence identity with a peptide selected from the group consisting of SEQ ID NOs 1, 4, 5, and 6); contacting a second sample from the subject with an isolated p140 immunoreactive peptide (e.g., comprising an amino acid sequence having about 95% or more sequence identity with a peptide selected from the group consisting of SEQ ID NOs: 2, 7, 8, 9, and 10); detecting the presence or absence of peptide-antibody complexes in each of the first and second samples. In these embodiments, the presence of peptide-antibody complexes in the first sample is an indication that the subject has an Ehrlichia chaffeensis infection, and the presence of peptide-antibody complexes in the second sample is an indication that the subject has an Ehrlichia canis infection.
Particular embodiments relate to determining whether a subject has been immunized against Ehrlichia or is actively infected with an Ehrlichia organism. In these embodiments, the method comprises contacting a sample from the subject with at least one isolated p120/p140 immunoreactive peptide disclosed herein that is not a component of an Ehrlichia vaccine, and detecting whether an antibody in the sample specifically binds to the isolated p120/p140 immunoreactive peptide. According to the method, if an antibody in the sample specifically binds to the isolated p120/p140 immunoreactive peptide, then the subject has an active Ehrlichia infection, and if an antibody does not specifically bind to the isolated p120/p140 immunoreactive peptide, then the subject is either previously immunized with an Ehrlichia vaccine or is not infected with an Ehrlichia organism. An Ehrlichia organism may be an Ehrlichia chaffeensis organism or an Ehrlichia canis organism.
A p120/p140 immunoreactive peptide may be used to bind an Ehrlichia-specific antibody using a variety of methods or kits. The specific binding between an antibody and an p120/p140 immunoreactive peptide of the present invention may therefore be assessed by any appropriate method known in the art including, but not limited to, an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immnunofluorescent assay (FA), nephelometry, flow cytometry assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, mass spectrometry assay, particle-based assay, inhibition assay and avidity assay. Exemplary methods of detecting the binding of an Ehrlichia-specific antibody to an p120/p140 immunoreactive peptide disclosed herein may include, for example, an ELISA performed in a microplate, a lateral flow test performed using a dipstick or lateral flow device, or a particulate-based suspension array assay performed using the Bio-Plex® system (Bio-Rad Laboratories, Hercules, Calif., USA).
ELISA
In certain embodiments, the detection of an peptide-antibody complex described herein is accomplished using an enzyme linked immunosorbent assay (ELISA). This assay may be performed by first contacting an p120/p140 immunoreactive peptide that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that antibodies specific for the peptide within the sample are allowed to bind to the immobilized peptide. Unbound sample is then removed from the immobilized peptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent.
In some embodiments, the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group or label. Exemplary reporter groups or labels include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of binding agent to reporter group or label may be achieved using standard methods known to those of ordinary skill in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many commercial sources (e.g., Zymed Laboratories, San Francisco, Calif.; and Pierce, Rockford, Ill.).
In an aspect of the present invention, the presence or absence of Ehrlichia specific antibodies may be determined in the sample by comparing the level of a signal detected from a reporter group or label in the sample with the level of a signal that corresponds to a control sample or predetermined cut-off value. In certain embodiments, the cut-off value may be the average mean signal obtained when the immobilized p120/p140 immunoreactive peptide is incubated with samples from an uninfected subject. The cut-off value may be determined using a statistical method or computer program.
Lateral Flow Tests
Lateral flow tests may also be referred to as immunochromatographic strip (ICS) tests or simply strip-tests. In general, a lateral flow test is a form of assay in which the test sample flows laterally along a solid substrate via capillary action, or alternatively, under fluidic control. Such tests are often inexpensive, require a very small amount (e.g., one drop) of sample, and can typically be performed reproducibly with minimal training. The economical simplicity and robustness of many lateral flow assay formats makes these types of tests ideal for identifying an Ehrlichia infection at the point of care, which is particularly important when the subject is, for example, a dog exhibiting detectable antibodies during the treatable acute phase of infection.
Exemplary lateral flow device formats include, but are not limited to, a dipstick, a card, a chip, a microslide, and a cassette, and it is widely deomonstrated in the art that the choice of format is largely dependent upon the features of a particular assay. Accordingly, lateral flow devices are now ubiquitous in human and verinarian medicine and quite varied, providing many options to the ordinarily skilled artisan for detecting a peptide-antibody complex in a sample using a lateral flow assay (See any of U.S. Pat. Nos. 7,344,893, 7,371,582, 6,136,610, and U.S. Patent Applications, 2005/0250141 and 2005/0047972, each incorporated herein by reference.) By way of a nonlimiting example, a sample from a subject suspected of having an Ehrlichia infection is applied to a lateral flow device comprising at least a sample zone and a binding zone. The sample may be a serum sample, and may be drawn laterally from the sample zone to the binding zone which comprises an p120/p140 immunoreactive peptide disclosed herein immobilized to a surface of the lateral flow device. In this example, the binding of the immobilized p120/p140 immunoreactive peptides on the lateral flow device is an indication that Ehrlichia specific antibodies are present in the sample from the subject, indicating an Ehrlichia infection in the subject.
In related embodiments, an ELISA assay as described above may be performed in a rapid flow-through, lateral flow, or strip test format, wherein the antigen is immobilized on a membrane, such as a nitrocellulose membrane. In this flow-through test, Ehrlichia antibodies within the sample bind to the immobilized p120/p140 immunoreactive peptide as the sample passes through the membrane. A detection reagent, such as protein A labeled with gold, a fluorophore, or a chromophore, binds to the peptide-antibody complex as the solution containing the detection reagent flows through the membrane. The detection peptide-antibody complexes bound to detection reagent may then be performed as is appropriate for the detection reagent used.
In an aspect, a flow-through format ELISA may be performed in which one end of the membrane to which p120/p140 immunoreactive peptide is immobilized may be immersed in a solution containing the sample, or the sample may be added to an area (i.e., a sample zone) at one end of the membrane. The sample migrates along the membrane through a region (i.e., a labeling zone) comprising the detection reagent, and flows to the area (i.e., a binding zone) comprising an immobilized p120/p140 immunoreactive peptide disclosed herein. An accumulation of detection reagent at the binding zone indicates the presence of Ehrlichia specific antibodies in the sample.
Typically, a flow-through ELISA may feature a detection reagent applied to to a test strip in a pattern, such as a line, that can be read visually. As with other lateral flow tests, the absence of such a pattern indicates a negative result. It is within the ability of an ordinarily skilled artisan to select an amount of p120/p140 immunoreactive peptide for immobilization on the membrane that can generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in a standard format ELISA. Preferably, the amount of peptide immobilized on the membrane ranges from about 25 ng to about 1 mg.
Particulate-Based Assays
In general, particle-based assays use a capture-binding partner, such as an antibody or an antigen in the case of an immunoassay, coated on the surface of particles, such as microbeads, crystals, chips, or nanoparticles. Particle-based assays may be effectively multi-plexed or modified to assay numerous variables of interest by incorporating fluorescently labeled particles or particles of different sizes in a single assay, each coated or conjugated to one or more labeled capture-binding partners. The use of sensitive detection and amplification technologies with particle-based assay platforms known in the art has resulted in numerous flexible and sensitive assay systems to choose from in performing a method described herein. For example, a multi-plex particle-based assay such as the suspension array Bio-Plex® assay system available from Bio-Rad Laboratories, Inc. (Hercules, Calif.) and Luminex, Inc. (Austin, Tex.) may be useful in identifying Ehrlichia antibodies in a sample.
In an aspect, the present invention contemplates the immobilization of an isolated p120/p140 immunoreactive peptide disclosed herein on a surface of a particle for use in a particle-based immunoassay. As described herein, methods of peptide immobilization onto support surfaces is well known in the art. In a preferred embodiment, a labeled p120/p140 immunoreactive peptide disclosed herein is immobilized onto a surface of a particle and the peptide-particle complex is employed in an ELISA or in a flow cytometry assay according to established protocols.
III. Ehrlichia Vaccine Compositions and Uses Thereof
In select embodiments, it is contemplated that an p120/p140 immunoreactive peptide of the present invention may be comprised in a vaccine composition and administered to a subject to induce a protective immune response in the subject that may substantially prevent or ameliorate infection in the subject by an Ehrlichia organism such as Ehrlichia chaffeensis or Ehrlichia canis. A vaccine composition for pharmaceutical use in a subject may comprises an p120/p140 immunoreactive peptide composition disclosed herein and a pharmaceutically acceptable carrier.
The phrases “pharmaceutical,” “pharmaceutically acceptable,” or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1289-1329, 1990, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the vaccine compositions of the present invention is contemplated.
As used herein, a “protective immune response” refers to a response by the immune system of a mammalian host to an Ehrlichia antigen which results in increased recognition of the antigen and antibody production by the immune system of the mammalian host upon subsequent exposure to an Ehrlichia pathogen. A protective immune response may substantially reduce or prevent symptoms as a result of a subsequent exposure to Ehrlichia chaffeensis or Ehrlichia canis. 
In some embodiments, a vaccine composition of the present invention may comprise an p120/p140 immunoreactive peptide (e.g., having a sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs. 1, 4, 5, 6, or SEQ, ID NOs. 2, 7, 8, 9, and 10). The vaccine composition may comprises at least one p120 immunoreactive peptide (e.g., having a sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs. 1, 4, 5, 6). A vaccine composition comprising a p120 immunoreactive peptide may be used to induce a protective immune response against Ehrlichia chaffeensis. The vaccine composition may comprise least one p140 immunoreactive peptide (e.g., having a sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or more sequence identity to any of SEQ, ID NOs. 2, 7, 8, 9, and 10). A vaccine composition comprising a p140 immunoreactive peptide may be used to induce a protective immune response against Ehrlichia canis. 
A person having ordinary skill in the medical arts will appreciate that the actual dosage amount of a vaccine composition administered to an animal or human patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, vaccine compositions may comprise, for example, at least about 0.1% of an p120/p140 immunoreactive peptide. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. As with many vaccine compositions, frequency of administration, as well as dosage, will vary among members of a population of animals or humans in ways that are predictable by one skilled in the art of immunology. By way of nonlimiting example, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 3 doses may be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals of 3-4 months, and booster vaccinations may be given periodically thereafter.
In some embodiments, a “suitable dose” is an amount of an p120/p140 immunoreactive peptide that, when administered as described above, is capable of raising an immune response in an immunized patient sufficient to protect the subject from an Ehrlichia infection in subsequent exposures to Ehrlichia organisms. In general, the amount of peptide present in a suitable dose (or produced in situ by the nucleic acid in a dose) ranges from about 1 pg to about 500 mg per kg of host, typically from about 10 pg to about 10 mg, preferably from about 100 pg to about 1 mg and more preferably from about 100 pg to about 100 microgram.
A vaccine composition of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. A vaccine composition disclosed herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, and by inhalation, injection, infusion, continuous infusion, lavage, and localized perfusion. A vaccine composition may also be administered to a subject via a catheter, in cremes, in lipid compositions, by ballistic particulate delivery, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference).
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
Of particular interest in an aspect of the present invention is a vaccine composition that may be administered by microstructured transdermal or ballistic particulate delivery. Microstructures as carriers for vaccine formulation are a desirable configuration for vaccine applications and are widely known in the art (Gerstel and Place 1976 (U.S. Pat. No. 3,964,482); Ganderton and McAinsh 1974 (U.S. Pat. No. 3,814,097); U.S. Pat. Nos. 5,797,898, 5,770,219 and 5,783,208, and U.S. Patent Application 2005/0065463). Such a vaccine composition formulated for ballistic particulate delivery may comprise an isolated p120/p140 immunoreactive peptide disclosed herein immobilized on a surface of a support substrate. In these embodiments, a support substrate can include, but is not limited to, a microcapsule, a microparticle, a microsphere, a nanocapsule, a nanoparticle, a nanosphere, or a combination thereof.
Microstructures or ballistic particles that serve as a support substrate for an p120/p140 immunoreactive peptide disclosed herein may be comprised of biodegradable material and non-biodegradable material, and such support substrates may be comprised of synthetic polymers, silica, lipids, carbohydrates, proteins, lectins, ionic agents, crosslinkers, and other microstructure components available in the art. Protocols and reagents for the immobilization of a peptide of the invention to a support substrate composed of such materials are widely available commercially and in the art.
In other embodiments, a vaccine composition comprises an immobilized or encapsulated p120/p140 immunoreactive peptide disclosed herein and a support substrate. In these embodiments, a support substrate can include, but is not limited to, a lipid microsphere, a lipid nanoparticle, an ethosome, a liposome, a niosome, a phospholipid, a sphingosome, a surfactant, a transferosome, an emulsion, or a combination thereof. The formation and use of liposomes and other lipid nano- and microcarrier formulations is generally known to those of ordinary skill in the art, and the use of liposomes, microparticles, nanocapsules and the like have gained widespread use in delivery of therapeutics (e.g., U.S. Pat. No. 5,741,516, specifically incorporated herein in its entirety by reference). Numerous methods of liposome and liposome-like preparations as potential drug carriers, including encapsulation of peptides, have been reviewed (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587, each of which is specifically incorporated in its entirety by reference).
In addition to the methods of delivery described herein, a number of alternative techniques are also contemplated for administering the disclosed vaccine compositions. By way of nonlimiting example, a vaccine composition may be administered by sonophoresis (i.e., ultrasound) which has been used and described in U.S. Pat. No. 5,656,016 for enhancing the rate and efficacy of drug permeation into and through the circulatory system; intraosseous injection (U.S. Pat. No. 5,779,708), or feedback-controlled delivery (U.S. Pat. No. 5,697,899), and each of the patents in this paragraph is specifically incorporated herein in its entirety by reference.
Any of a variety of adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and quil A.
A peptide may be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
Sterile injectable solutions are prepared by incorporating the active peptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
IV. Ehrlichia Detection and Vaccination Kits
Various embodiments of the present invention are concerned with kits for the detection of antibodies in a sample that specifically bind an Ehrlichia organism. The kits may thus be used for the diagnosis or identification of an Ehrlichia infection in a subject. In other embodiments, the invention provides kits for distinguishing between an Ehrlichia chaffeensis infection and an Ehrlichia canis infection in a subject, or for determining whether a subject has been immunized against Ehrlichia or is actively infected with an Ehrlichia organism. In still other embodiments, kits are provided for vaccination of a subject against Ehrlichia chaffeensis infection and an Ehrlichia canis infection.
In select embodiments, a kit of the present invention may be used to perform a method disclosed herein. For example, a kit may be suitable for detecting Ehrlichia antibodies in a sample, for identifying an Ehrlichia infection individual, for distinguishing between an Ehrlichia chaffeensis infection and an Ehrlichia canis infection in a subject, for determining whether a subject has been immunized against Ehrlichia or is actively infected with an Ehrlichia organism, or for vaccinating a subject against an Ehrlichia organism. In these embodiments, one or more p120/p140 immunoreactive peptides (e.g., having about 95% or more sequence identity with any of SEQ ID NOs 1, 2, 3,4,5,6,7,8,9, and 10) may be comprised in the kit. The p120/p140 immunoreactive peptide in the kit may be detectably labeled or immobilized on a surface of a support substrate also comprised in the kit. The p120/p140 immunoreactive peptide(s) may, for example, be provided in the kit in a suitable form, such as sterile, lyophilized, or both.
The support substrate comprised in a kit of the invention may be selected based on the method to be performed. By way of nonlimiting example, a support substrate may be a multi-well plate or microplate, a membrane, a filter, a paper, an emulsion, a bead, a microbead, a microsphere, a nanobead, a nanosphere, a nanoparticle, an ethosome, a liposome, a niosome, a transferosome, a dipstick, a card, a celluloid strip, a glass slide, a microslide, a biosensor, a lateral flow apparatus, a microchip, a comb, a silica particle, a magnetic particle, or a self-assembling monolayer.
As appropriate to the method being performed, a kit may further comprise one or more apparatuses for delivery of a composition to a subject or for otherwise handling a composition of the invention. By way of nonlimiting example, a kit may include an apparatus that is a syringe, an eye dropper, a ballistic particle applicator (e.g., applicators disclosed in U.S. Pat. Nos. 5,797,898, 5,770,219 and 5,783,208, and U.S. Patent Application 2005/0065463), a scoopula, a microslide cover, a test strip holder or cover, and such like.
A detection reagent for labeling a component of the kit may optionally be comprised in a kit for performing a method of the present invention. In particular embodiments, the labeling or detection reagent is selected from a group comprising reagents used commonly in the art and including, without limitation, radioactive elements, enzymes, molecules which absorb light in the UV range, and fluorophores such as fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. In other embodiments, a kit is provided comprising one or more container means and a BST protein agent already labeled with a detection reagent selected from a group comprising a radioactive element, an enzyme, a molecule which absorbs light in the UV range, and a fluorophore.
In particular embodiments, the present invention provides a kit for detecting anti-Ehrlichia antibodies in a sample which may also be used for identification of an Ehrlichia infection in a subject, for distinguishing between an Ehrlichia chaffeensis infection and an Ehrlichia canis infection in a subject, and/or for determining whether a subject has been immunized against Ehrlichia or is actively infected with an Ehrlichia organism. Such a kit may comprise one or more p120/p140 immunoreactive peptides (e.g., having about 95% or more sequence identity with any of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10), and the peptides may be detectably labeled and immobilized to one or more support substrates comprised in the kit.
In some embodiments, a kit comprises an p120/p140 immunoreactive peptide having about 95% or more sequence identity with SEQ ID NO 1 and/or an p120/p140 immunoreactive peptide having about 95% or more sequence identity with SEQ ID NO 2. The peptides may be immobilized to one or more separate lateral flow assay devices, such as a nitrocellulose test strips. In these embodiments, each of the test strips may further comprises a detection reagent, for example, a chromophore-labeled protein A. Such a kit may further comprise one or more containers for sample material, one or more diluents for sample dilution, and one or more control indicator strips for comparison.
When reagents and/or components comprising a kit are provided in a lyophilized form (lyophilisate) or as a dry powder, the lyophilisate or powder can be reconstituted by the addition of a suitable solvent. In particular embodiments, the solvent may be a sterile, pharmaceutically acceptable buffer and/or other diluent. It is envisioned that such a solvent may also be provided as part of a kit.
When the components of a kit are provided in one and/or more liquid solutions, the liquid solution may be, by way of non-limiting example, a sterile, aqueous solution. The compositions may also be formulated into an administrative composition. In this case, the container means may itself be a syringe, pipette, topical applicator or the like, from which the formulation may be applied to an affected area of the body, injected into a subject, and/or applied to or mixed with the other components of the kit.
V. Examples
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the following example represent techniques identified by the applicant to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
Major Species-Specific Antibody Epitopes of the Ehrlichia chaffeensis p120 and Ehrlichia canis p140 Orthologs in Surface-Exposed Tandem Repeat Regions
Here is presented the identification and characterization of the immunodeterminants of the E. chaffeensis p120 and E. canis p140. Major antibody epitope-containing regions of both p120 and p140 were localized to the TR regions, which reacted strongly by Western immunoblot with antibodies in sera from E. chaffeensis-infected dogs/patients and E. canis-infected dogs, respectively. Single continuous species-specific major epitopes within the E. chaffeensis p120 and E. canis p140 TRs were mapped to homologous surface-exposed glutamate/aspartate-rich regions (19 to 22 amino acids). In addition, minor cross-reactive epitopes were localized to homologous N- and C-terminal regions of p120 and p140. Furthermore, although the native and recombinant p120 and p140 proteins exhibited larger-than-predicted molecular masses, posttranslational modifications were not present on abnormally migrating p120 and p140 TR recombinant proteins as determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
Materials and Methods
Culture and purification of Ehrlichiae. E. chaffeensis (Arkansas strain) and E. canis (Jake strain) were propagated and purified by size exclusion chromatography as previously described (McBride et al., 2001; Rikihisa et al., 1992). The fractions containing bacteria were frozen and utilized as antigen and DNA sources.
Preparation of Ehrlichia genomic DNA and antigen. Genomic DNA and antigen were purified from E. chaffeensis (Arkansas strain) and E. canis (Jake strain) as previously described (McBride et al., 1996). Ehrlichia-infected DH82 cell culture supernatants (0.5 ml) were collected five days postinfection without disturbing the cell monolayer and clarified by high speed centrifugation (10,000 g for 5 min) to remove Ehrlichiae. Supernatants were subsequently concentrated 10-fold using Microcon ultra centrifugal filter with a 10-kDa cutoff (Millipore, Billerica, Mass.).
PCR amplification of the Ehrlichia genes. Oligonucleotide primers for the amplification of the E. chaffeensis p120 and E. canis p140 gene fragments were designed manually, or by using PrimerSelect (Lasergene v5.08, DNAStar, Madison, Wis.) according to the sequences in GenBank (accession numbers U49426 and NC—007354, respectively) and synthesized (Sigma-Genosys, Woodlands, Tex.) (Table 1). Gene fragments corresponding to the N-termini (p120N/p140N), the C-termini (p120C/p140C), and the entire open reading frames (p120W/p140W) were amplified by PCR (FIG. 1A). Constructs containing the tandem repeat regions (designated p120TR and p140TR in this report, respectively) were described previously and used in this study (Yu et al., 1996; Yu et al., 2000). The E. chaffeensis p120TR contained only the first two tandem repeats (R1 and R2), whereas the p140TR contained the complete tandem repeat region (14 repeats) of the E. canis p140 (FIG. 1A).
PCR was performed with PCR HotMaster Mix (Eppendorf, Westbury, N.Y.) and the appropriate Ehrlichia genomic DNA as the template. The thermal cycling profile was: 95° C. for 3 min, 30 cycles of 94° C. for 30 s, annealing temperature (1° C. less than the lowest primer Tm) for 30 s, and 72° C. for the appropriate extension time (1 min/1000 base pairs) followed by a 72° C. extension for 10 min and a 4° C. hold.
Expression and purification of the recombinant Ehrlichia p120 and p140 proteins. The amplified PCR products were cloned directly into the pBAD/Thio-TOPO expression vector (Invitrogen, Carlsbad, Calif.) and transformed E. coli TOP10 cells (Invitrogen). The resulting transformants were screened by PCR for correctly oriented inserts, and plasmids from the positive transformants were isolated and sequenced to verify the inserts with an ABI Prism 377XL DNA sequencer (Applied Biosystems, Foster City, Calif.) at the University of Texas Medical Branch Protein Chemistry Core Laboratory. Recombinant protein expression was performed for 4 h after induction with 0.2% arabinose, and proteins were purified under native conditions using HisSelect® columns (Sigma, St. Louis, Mo.). The recombinant TR regions of Ehrlichia p120 and p140 were expressed as glutathione S-transferase (GST) fusion proteins as previously described (Yu et al., 1996; Yu et al., 2000).
p120 and p140 synthetic peptides. For the E. chaffeensis p120, five overlapping peptides corresponding to a single repeat unit (p120R-N, p120R-I1, p120R-I2, p120R-I3, and p120R-C) were commercially synthesized (Bio-Synthesis, Lewisville, Tex.) (FIG. 1B, left panel; see FIG. 5A for sequences). Fine mapping within the p120R-I1 region was performed with four overlapping peptides (p120R-I1-S1, p120R-I1-S2, p120R-I1-S3, and p120R-I1-S4; Bio-Synthesis) (FIG. 1B, left panel; see FIG. 5A for sequences). For p140, six overlapping peptides (p120R-1 to p120R-6) corresponding to the different regions of the E. canis p140R were synthesized (Bio-Synthesis) (FIG. 1B, right panel; see FIG. 6A for sequences). All peptides were supplied as a lyophilized powder and resuspended in molecular biology grade water (1 mg/ml).
Antisera. Two convalescent anti-E. chaffeensis dog sera (nos. 2251 and 2495) and one convalescent anti-E. canis dog sera (no. 2995) were obtained from experimentally infected dogs. Sera from dogs exhibiting clinical signs or hematologic abnormalities consistent with CME were submitted to the Louisiana Veterinary Medical Diagnostic Laboratory from veterinarians statewide and screened by IFA, as described previously (McBride et al., 2001). HME patient sera were kind gifts from Focus Technologies (Cypress, Calif.) and William Nicholson at Centers for Disease Control and Prevention (Atlanta, Ga.). Rabbit anti-p120 and anti-p140 antisera were generated against the synthetic KLH-conjugated peptides located in the epitope-containing region of each respective repeat unit (p120: SKVEQEETNPEVLIKDLQDVAS (SEQ ID NO:1); p140: EHSSSEVGEKVSKTSKEESTPEVKA (SEQ ID NO:11)) by a commercial vendor (Bio-Synthesis).
Gel electrophoresis and Western immunoblotting. Purified E. chaffeensis or E. canis whole-cell lysates or recombinant proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose, and Western immunoblotting performed as previously described (McBride et al., 2003), except that primary dog sera were diluted 1:100, human sera were diluted 1:200, and rabbit antisera were diluted 1:1,000.
ELISA. Enzyme-linked immunosorbent assay (ELISA) plates (MaxiSorp; Nunc, Roskilde, Denmark) were coated (0.5 μg/well; 50 μl) with recombinant proteins or synthetic peptides suspended in phosphate-buffered saline (pH 7.4). Proteins and peptides were absorbed for 1 h at room temperature with gentle agitation, and subsequently washed thrice with 200 μl Tris-buffered saline containing 0.2% Tween 20 (TBST). Plates were blocked with 100 μl 10% equine serum (Sigma) in TBST for 1 h at room temperature with agitation, and washed. Convalescent dog or human sera diluted (1:100 or 1:200, respectively) in 10% equine serum-TBST were added to each well (50 μl) and incubated at room temperature for 1 h with gentle agitation. The plates were washed four times, and 50 μl alkaline phosphatase-labeled goat anti-dog or human IgG (H+L) secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted (1:5,000) in 10% equine serum-TBST was added and incubated for 1 h at room temperature. The plates were washed four times, and substrate (100 μl; BluePhos; Kirkegaard & Perry Laboratories) was added to each well. The plates were incubated in the dark for 30 min with agitation, and color development was determined on a microplate reader (VersaMax; Molecular Devices, Sunnyvale, Calif.) at A650 and data analyzed by SoftmaxPro v4.0 (Molecular Devices). Optical density (OD) readings represent the mean OD for three wells (±standard deviations) after subtracting the OD value of the buffer-only wells. A reading >0.2 OD unit above the negative control absorbance was considered positive for all samples. In addition, a reading 0.2-0.5 OD unit above the control absorbance was considered a weak positive, and a reading >0.5 OD unit above the control absorbance was considered a strong positive.
Mass spectrometry. Sample solution or protein standard (1 μl) was spotted directly onto a MALDI target plate and allowed to air dry. Sinapic acid (Aldrich, Milwaukee, Wis.) matrix solution (1 μl; 50:50 acetonitrile/water) was then applied on the sample spot and allowed to dry. The dried MALDI spot was blown with compressed air (Decon Laboratories, King of Prussia, PA) before inserting into the mass spectrometer. Mass spectrometry was performed using a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (4800 MALDI TOF/TOF Proteomics Analyzer; Applied Biosystems) at the University of Texas Medical Branch Mass Spectrometry Core Laboratory. Data were acquired with the software package including 4000 series explorer (v3.6 RC1; Applied Biosystems). The instrument was operated in positive ion linear mode, mass range as required. 4000 laser shots were acquired and averaged from each sample shot. External calibration was performed using cytochrome C or BSA according to the target molecular weight.







TABLE 1







Oligonucleotide primers for amplification of the E. chaffeensis p120 and E. canis


p140 gene fragments.










Primers
Amplicon size










Fragment
Name
Sequence (5′ to 3′)
(bp)













p120
p120N-F
ATGGATATTGATAATAGTAACATAAGTAC
1,644




(SEQ ID NO: 16)




p120C-R
TACAATATCATTTACTACATTGTGATT





(SEQ ID NO: 17)



 


p120N
p120N-F
ATGGATATTGATAATAGTAACATAAGTAC
162




(SEQ ID NO: 18)




p120N-R
TGTGTCATCTTCTTGCTCTTG





(SEQ ID NO: 19)



 


p120C
p120C-F
ATTCTAGTAGAAGATTTGCCATTAG
444




(SEQ ID NO: 20)




p120C-R
TACAATATCATTTACTACATTGTGATT





(SEQ ID NO: 21)



 


p140
p140N-F
ATGGATATTGATAACAATAATGTGACTAC
2,064




(SEQ ID NO: 22)




p140C-R
TATTAAATCAACTGTTTCTTTGTTAGT





(SEQ ID NO: 23)



 


p140N
p140N-F
ATGGATATTGATAACAATAATGTGACTAC
183




(SEQ ID NO: 24)




p140N-R
TGGATTTCCTACATTGTCATTC





(SEQ ID NO: 25)



 


p140C
p140C-F
GAAGTACAGCCTGTTGCAG
324




(SEQ ID NO: 26)




p140C-R
TATTAAATCAACTGTTTCTTTGTTAGT





(SEQ ID NO: 27)









Sequence analysis. Amino acid sequence alignments of E. chaffeensis p120 and E. canis p140 were performed with MegAlign (Lasergene v5.08; DNAStar). The major epitopes of p120 and p140 were examined for sequence similarity to other proteins by using the protein-protein basic local alignment search tool (BLAST; www.ncbi.nlm.nih.gov/BLAST).
Statistics. Statistical difference between experimental groups were assessed with the two-tailed Student's t-test, and significance was indicated by a P value of <0.05.
Results:
E. chaffeensis p120 and E. canis p140 composition and characteristics. In the E. chaffeensis (Arkansas strain) p120 and E. canis (Jake strain) p140 proteins, glutamate (17.5% in p120; 17.4% in p140), serine (12.2%; 15.8%), and valine (10.8%; 12.9%) were the most frequently occurring amino acids (Table 2). Moreover, in the TRs of p120 and p140, the occurrences of these three (E, S and V) residues were more frequent (22.3%/21.4%; 14.8%/18.5%; and 11.4%/13.3%, respectively). On the contrary, in the N- and C-termini of p120 and p140, the occurrences of these three residues became less frequent, except for the valine content in the C-terminus of p120. Due to the large proportion of glutamate residues, the p120 and p140 proteins were highly acidic (pI 3.8 and 3.9, respectively).
Amino acid sequence similarity within the N-terminus and surface-exposed motif of the repeat region between E. chaffeensis p120 and E. canis p140 has been reported (McBride et al., 2000; Yu et al., 2000), but sequence similarity within the C-terminus and the analysis of specific regions has not been fully explored. The amino acid identity was  ̃50% for the first 32 amino acids of the N-terminus. Similarly, homologous ( ̃39% amino acid identity) regions were identified in the C-terminus of p120 and p140 (FIG. 2). A BLAST search determined no substantial sequence similarity with other known Ehrlichial proteins or proteins from organisms in closely related genera.
Identification of the native E. chaffeensis p120 and E. canis p140 proteins. Western blotting identified two strongly reactive native proteins with the molecular mass of  ̃95 kDa and  ̃75 kDa (both larger than predicted mass of 61 kDa based on the amino acid sequence) and a few less prominent proteins (75-50 kDa) in E. chaffeensis whole-cell lysates and culture supernatants that reacted with monospecific rabbit antiserum against the synthetic p120R-I1 peptide; however, this antiserum did not react with any proteins in E. canis whole-cell lysates (FIG. 3A). Similarly, a native protein with the molecular mass of  ̃125 kDa (larger than predicted mass of 74 kDa) and a few smaller and less prominent proteins in E. canis whole-cell lysates reacted with monospecific rabbit antiserum against the p140TR. Proteins in E. chaffeensis whole-cell lysates did not react with this antiserum (FIG. 3B). Pre-immunization rabbit serum controls did not react with proteins in E. chaffeensis or E. canis whole-cell lysates by Western immunoblot.







TABLE 2







Predicted and observed molecular masses and amino acid analyses of E. chaffeensis


p120 and E. canis p140 proteins.












Molecular mass (kDa)a
Glutamate content
Serine content
Valine content













Protein
Predicted
Observed
Massc
n (%)
n (%)
n (%)










E. chaffeensis p120













p120
77.1
110
nd
96 (17.5)
67 (12.2)
59 (10.8)


p120N
22.3
23
nd
2 (4.0)
4 (8.0)
1 (2.0)


p120TRb
47.0
58
47.1
78 (22.3)
52 (14.8)
40 (11.4)


p120C
33.0
33
nd
16 (10.8)
11 (7.4) 
18 (12.2)


Native p120
60.8
95/75
nd







E. canis p140













p140
89.9
140
nd
120 (17.4) 
109 (15.8) 
89 (12.9)


p140N
21.5
22
nd
4 (6.6)
6 (9.8)
 7 (11.5)


p140TR
85.6
130
85.9
111 (21.4) 
96 (18.5)
69 (13.3)


p140C
28.3
28
nd
5 (4.6)
7 (6.5)
13 (12.0)


Native p140
73.6
125
nd





aIncluding the fusion tags: all were thioredoxin (16.3 kDa) except for p120TR and p140TR (GST tag; 28 kDa).


bOnly first two repeats was cloned and expressed, but the amino acid content values are for the whole repeat region.


cAs determined by MALDI-TOF mass spectrometry of the recombinant protein


nd = not determined






Epitope mapping of E. chaffeensis p120 and E. canis p140 with recombinant proteins. To conclusively determine the major epitope-containing regions of p120 and p140, the recombinant full-length p120 and p140 proteins (p120W/p140W) and fragments corresponding to three distinct domains including the N-terminus (p120N/p140N), tandem repeat region (p120TR/p140TR), and C-terminus (p120C/p140C) were expressed (FIG. 1A). The p120W/p140W and p120TR/p140TR recombinant proteins exhibited molecular masses substantially larger than predicted by their amino acid sequences by SDS-PAGE. In contrast, the recombinant p120N/p140N and p120C/p140C exhibited masses consistent with that predicted by their amino acid sequences. MALDI-TOF mass spectrometry determined that the molecular masses of recombinant p120TR and p140TR proteins were nearly identical to that predicted by the corresponding amino acid sequences (Table 2), and thus the abnormal migration was not associated with posttranslational modifications.
By Western immunoblot, the recombinant p120W and p120TR reacted very strongly with two anti-E. chaffeensis dog sera derived from dogs (nos. 2251 and 2495) experimentally infected with E. chaffeensis and two HME patient (nos. SC07 and CDC4) sera that had detectable E. chaffeensis antibodies by immunofluorescence assay (IFA); however, recombinant fragments of the p120N and p120C did not react, or reacted very weakly with those dog or patient sera, or reacted with only one serum (FIG. 4A). Similarly, the recombinant p140W protein and p140TR reacted very strongly with three anti-E. canis dog sera derived from an experimentally infected dog (no. 2995) and two naturally infected dogs (nos. 2160 and 4283); however, recombinant p140N and p140C did not react or reacted weakly with those dog sera (FIG. 4B). These human or dog sera did not recognize thioredoxin or GST proteins, and the normal human or dog sera did not recognize these recombinant proteins by Western immunoblot.
Peptide mapping of the major immunodeterminants of E. chaffeensis p120 and E. canis p140. To localize the major epitope(s) of E. chaffeensis p120 protein, 5 overlapping peptides (p120R-N, p120R-I1, p120R-I2, p120R-I3 and p120R-C) spanning the TR of p120 (FIGS. 1B [left panel] and 5A) were reacted by ELISA with the anti-E. chaffeensis dog (no. 2251) sera and three HME patient (nos. 3, 18 and 20) sera that demonstrated E. chaffeensis antibodies by immunofluorescence assay (IFA). Four peptides (p120R-N, p120R-12, p120R-I3 and p120R-C) were not immunoreactive, but p120R-I1 (22-mer) located in the N-terminal region of the TR reacted strongly with E. chaffeensis patient sera by ELISA (FIGS. 5B to E). Furthermore, peptides p120R-N and p120R-I2, which contain amino acids (SKVEQEETNP (SEQ ID NO:12) and DLQDVAS (SEQ ID NO:13), respectively) present in the N- and C-termini of the p120R-I1 (22-mer), and the p120-S1 (EQEETNPEVLIK (SEQ ID NO:3)) representing a central overlapping region were not reactive with antibodies individually; however collectively the peptide p120-I1 (SKVEQEETNPEVLIKDLQDVAS (SEQ ID NO:1)) reacted strongly with antibodies in sera, suggesting that 22 amino acids were necessary for full constitution of the p120 TR epitope (FIG. 5A-E). Additional mapping with smaller peptides (p120R-I1-S1, S2, S3 and S4) demonstrated a significant (S1, S3 and S4, P<0.05 for all sera; S2, P<0.05 for all patient sera) contribution by both N-terminal (SKV) or C-terminal (DLQD) amino acids of peptide p120R-I1 and indicated that the continuous epitope was represented by this peptide (FIG. 5A-E).
To identify the peptide sequence containing the immunodeterminant in E. canis p140 protein, six overlapping peptides (designated p140R-1 to p140R-6 from N-terminus to C-terminus) spanning the TR of p140 (FIGS. 1B [right panel] and 6A) were reacted with four anti-E. canis sera from naturally infected dogs (nos. 2160, 6, 10 and 18) (FIG. 6B to E). By ELISA, all overlapping peptides except for peptide p140R-3 (11-mer) reacted with anti-E. canis dog sera. Peptide p140R-4 (19 amino acids; SKEESTPEVKAEDLQPAVD (SEQ ID NO:2)), which was predicted to be surface-exposed and overlapped with the identified E. chaffeensis p120 epitope (see above and FIG. 2), had significantly (P<0.05) stronger immunoreactivity with the majority of sera tested by ELISA. Additional peptide mapping with overlapping peptides (p140-R1) demonstrated that the N-terminal amino acids (SKEESTP (SEQ ID NO:14)) of p140-R4 did react with antibodies and contributed to the epitope as p140-R4 exhibited consistently stronger immunoreactivity than p140R-5, which lacked amino acids SKEES (SEQ ID NO:28) (FIG. 6A-E). Furthermore, peptide p140R-4, which contained additional C-terminal amino acids (EDLQPAVD (SEQ ID NO:15)) compared to p140R-3, exhibited strong immunoreactivity, whereas p140R-3 lacking these amino acids was virtually nonreactive, indicating a dominant contribution associated with these residues (EDLQPAVD (SEQ ID NO:15)) to the epitope. Comparative immunoreactivity between peptides p140R-2 and R-4 indicated that additional C-terminal amino acid residues, AVD, also contributed significantly (P<0.05) to epitope reactivity with half of the dog sera examined (FIG. 6A-E).
Identification of immunoreactive regions for cross reaction between E. chaffeensis p120 and E. canis p140. To examine cross reactions between p120 and p140 and to localize the regions containing cross-reactive epitope(s), the recombinant p120 and p140 proteins corresponding to three distinct domains (N-terminus, TR region and C-terminus) were reacted with the anti-E. canis dog sera and anti-E. chaffeensis dog or patient sera. By Western immunoblot, the recombinant p120TR and p140TR proteins did not react, or reacted weakly with heterologous anti-E. canis sera and anti-E. chaffeensis sera, respectively; however, either recombinant N- or C-terminus of the p120 or p140 proteins did cross react with heterologous sera (FIG. 7).
Discussion:
It is well established that tandem repeat-containing proteins of Ehrlichia spp. are primary targets of the humoral immune response and elicit vigorous, and in many instances, species-specific antibodies (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2000). E. chaffeensis p120 and E. canis p140 protein orthologs are well characterized major immunoreactive proteins strongly recognized by sera from HME patients and E. canis-infected dogs (McBride et al., 2000; Yu et al., 1997; Yu et al., 2000). Although previous studies demonstrated that E. chaffeensis p120 and E. canis p140 proteins reacted with antibodies in dog and/or patient sera (McBride et al., 2001; Yu et al., 1996; Yu et al., 1999; Yu et al., 2000), the immunologic properties of these two proteins were not fully defined, and the extent of the host response directed against them has remained undetermined.
All of the major immunoreactive TR proteins of E. chaffeensis and E. canis that have been characterized, including p120 and p140 orthologs, are highly acidic due to a predominance of glutamate/aspartate, moreover, they also appear to be serine-rich, which usually occurs more frequently within TRs of these proteins (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2003; McBride et al., 2007; McBride et al., 2000). Interestingly, major continuous antibody epitopes of these proteins have been mapped to serine-rich acidic domains (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2007; McBride et al., 2000; Nethery et al., 2007), which indicates a relationship between these domains and the host immune response; however, the specific role of these amino acids in directing the immune response against Ehrlichia is still unknown. The major epitope-containing regions of both E. chaffeensis p120 and E. canis p140 protein orthologs were mapped to the serine-rich tandem repeat units, which is consistent with the location of epitopes in other Ehrlichial TR-containing proteins. The antibody epitopes in p120TR and p140 TR, which exhibited the strongest antibody reactivity with both dog and human sera, were localized to the p120R-I1 (22 amino acids) and p140R-4 (19 amino acids) regions, respectively, which are homologous and predicted to be surface-exposed domains. Therefore, consistent with the location of epitopes mapped in other TR Ehrlichial proteins, the conserved surface-exposed domains of p120 and p140 TRs contained a dominant continuous immunodeterminant.
The length of the E. chaffeensis p120 and E. canis p140 epitopes was similar ( ̃20 amino acids) and consistent in size with that described of other molecularly characterized continuous Ehrlichial epitopes, including those of VLPT/p19, p47/36, and p200 (E. canis) (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2007; Nethery et al., 2007). Although smaller peptides associated with the mapped epitope reacted with antibodies, significantly higher antibody reactivity was observed with peptides consisting of  ̃20 amino acids a finding that is consistent with the epitope length the inventors have mapped on other TR proteins and similar in size to a neutralizing continuous antibody epitope consisting of 15 amino acids recently mapped in the Helicobacter UreB protein (Li et al., 2008). However, a smaller six amino acid continuous epitope has been mapped in Anaplasma marginate msp1a (Allred et al., 1990). Although major continuous epitopes have been mapped on several Ehrlichial TR proteins, one conformational epitope has been mapped in VLPT (Luo et al., 2008), and there may be other discontinuous epitopes associated with these major immunoreactive proteins that were not determined in this study. However, the host response to the continuous epitopes is strong and consistent with the response observed with recombinant folded proteins, suggesting the absence of dominant conformational epitopes.
Unlike other immunoreactive protein orthologs of Ehrlichia, the major epitopes of p120 and p140 exhibit some sequence similarity, raising the possibility that cross-reactive antibodies could be elicited; however, antibodies generated against epitope-containing peptides did not cross react by Western immunoblot, indicating that these epitopes appear to be primarily species-specific, a finding consistent with a previous study using antisera against recombinant p120TR and p140TR (McBride et al., 2000). Hence, the cross reactive immune response elicited by Ehrlichia species does not appear to be directed against the major continuous antibody epitopes identified thus far in E. chaffeensis and E. canis TR proteins, including the p120/p140. However, the inventors did identify that minor cross-reactive epitopes in the N- and C-terminal regions, which is consistent with the fact that substantial sequence similarity occurs in these regions. Therefore, as the inventors have proposed with major continous epitopes identified in other Ehrlichial TR proteins, the p120/p140 TR epitopes could be utilized for species-specific diagnostic development.
The inventors have previously reported that some recombinant Ehrlichial immunoreactive proteins exhibited larger-than-predicted masses similar to their native counterparts by gel electrophoresis (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2007; McBride et al., 2000), which was also observed in this study with both recombinant and native p120 and p140 proteins. The recombinant p120W/p140W and p120TR/p140TR exhibited abnormally large molecular masses, but the recombinant N- and C-terminal regions (p120N/p140N, p120C/p140C) migrated as expected, indicating that the highly acidic serine-rich TR was responsible for the anomalous electrophoretic behavior of these proteins. This abnormal electrophoretic migration was previously associated with detection of carbohydrate based on chemical reactivity, suggesting glycosylation of TRs (McBride et al., 2000).
In this study, the inventors determined by mass spectrometry that the molecular masses of p120TR and p140TR were consistent with those predicted by their amino acid sequences; therefore, the glycosylation is not responsible for the larger-than-predicted masses of the p120 and p140 proteins. It is likely that the high acidity of these proteins, particularly in the TR regions is responsible for the abnormal electrophoretic behavior. This is supported by studies demonstrating that highly acidic proteins exhibit abnormal migration patterns during gel electrophoresis (Garcia-Ortega et al., 2005; Graceffa et al., 1992). Like p120 and p140 proteins, the inventors recently reported that another major immunoreactive protein (VLPT) of E. chaffeensis also exhibited larger-than-predicted mass on gel, but mass spectrometry determined that this protein was not posttranslationally modified (Luo et al., 2008). The molecular masses of the native E. chaffeensis p120 ( ̃95 kDa) and E. canis p140 ( ̃125 kDa) proteins were smaller than previously reported masses ( ̃120 kDa and  ̃140 kDa, respectively) (McBride et al., 2000; Yu et al., 2000). This difference is likely related to differences in SDS-PAGE procedures and accuracy of molecular mass markers. Nevertheless, the native proteins identified from the Ehrlichial lysate by the antibodies against synthetic epitope peptides, and the masses of the recombinant p120 or p140 protein (without fusion tag) were in agreement in this study.
The major immunoreactive proteins of Ehrlichia spp. have been identified and consist of a small subset of proteins. Three of these proteins in E. chaffeensis and E. canis are acidic, serine-rich and contain TRs (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2007; Yu et al., 2000). The host immune response appears to be primarily directed at continuous species-specific epitopes within the TRs, which suggests similar characteristics contribute to immune response stimulation and production of species-specific antibodies directed at these TR epitopes. However, the role of continuous major antibody epitopes within Ehrlichial TR proteins in eliciting a protective immune response is currently undefined. Although protective antibody epitopes have been mapped to an E. chaffeensis major outer membrane protein, p28 (Li et al., 2002), new studies indicate that Ehrlichial TR proteins are secreted and interact with important host cell targets and facilitate pathogen survival (Wakeel et al., 2009). Thus, studies to examine whether host antibody response elicited by continuous epitopes in TR proteins such as the p120/p140 are protective, will provide much needed insight into the protective Ehrlichial antigens and effective immune responses.










SEQUENCES











SEQ ID NO.





I1(22)
SKVEQEETNPEVLIKDLQDVAS
1


 


I1-S1(12)
EQEETNPEVLIK
3


 


I1-S2(17)
SKVEQEETNPEVLIKDL
4


 


I1-S3(16)
EQEETNPEVLIKDLQD
5


 


I1-S4(16)
ETNPEVLIKDLQDVA
6


 


R-1(19)
SSSEVGKKVSETSKEESTP
7


 


R-2(19)
SETSKEESTPEVKAEDLQP
8


 


R-4(19)
SKEESTPEVKAEDLQPAVD
2


 


R-5(14)
TPEVKAEDLQPAVD
9


 


R-6(19)
TPEVKAEDLQPAVDGSIEH
10 









Example 2
p120 Peptides Display Improved Sensitivity of Serodiagnosis of Human Monocytotropic Ehrlichiosis as Compared to the Full-Length p120 Protein or Combinations of Ehrlichia Peptides
The sensitivities and specificities of synthetic peptides representing these and other well-defined major immunodeterminants of E. chaffeensis were determined by enzyme-linked immunosorbent assay (ELISA). Thirty-one human monocytotropic ehrlichiosis (HME) patient serum samples that had detectable E. chaffeensis antibodies (titers from 64 to 8,192) by indirect fluorescent antibody assay (IFA) were tested. All 31 serum samples reacted with at least one E. chaffeensis peptide, 30 (96.8%) with TRP120 peptides, 27 (87.1%) with TRP32 peptides, 24 (77.4%) with TRP47 peptides, 19 (61.3%) with Ank200 peptides, and 28 (90.3%) with recombinant TRP120-TR protein. A mixture of the two most sensitive peptides from TRP120 and TRP32 did not provide enhanced analytical sensitivity compared to that provided by TRP120 alone. These results demonstrate that the TRP120 peptide can be used for standardized sensitive point-of-care and reference laboratory immunodiagnostics for HME. This is the first study to compare analysis of molecularly defined major antibody epitopes with IFA for diagnosis of HME.
Also presented in this example is data mapping the major immunodeterminants of the E. chaffeensis 200-kDa ankyrin protein (Ank200) and the minor immunodeterminants in the N- and C-terminal regions of E. chaffeensis TRP47. Major antibody epitopes of Ank200 were localized to four polypeptide regions (18-mer, 20-mer, 20-mer, and 21-mer, respectively) in terminal acidic domains, which reacted with antibodies in sera from human monocytotropic ehrlichiosis (HME) patients and an E. chaffeensis-infected dog. Two minor epitope-containing regions were identified in the N terminus and the C terminus of TRP47.
Materials and Methods
Culture and purification of E. chaffeensis. E. chaffeensis (Arkansas strain) was propagated in DH82 cells and purified by size exclusion chromatography as previously described (McBride et al., 2001; Rikihisa et al., 1992). The fractions containing bacteria were frozen and utilized for DNA and antigen preparation (McBride et al., 1996).
PCR amplification of the E. chaffeensis genes. Oligonucleotide primers for the amplification of the E. chaffeensis Ank200 and TRP47 gene fragments were designed manually or by using PrimerSelect (Lasergene v5.08; DNAStar, Madison, Wis.) according to the sequences in GenBank (accession numbers YP—507490 and DQ085430, respectively) and synthesized (Sigma-Genosys, Woodlands, Tex.) (Table 3). Gene fragments corresponding to the different regions used for epitope mapping were amplified by PCR (FIG. 1 for Ank200; see FIG. 4A for TRP47). PCR was performed with PCR HotMaster mix (Eppendorf, Westbury, N.Y.) and E. chaffeensis genomic DNA as the template. The thermal cycling profile was as follows: 95° C. for 3 min, 30 cycles of 94° C. for 30 s, annealing temperature (1° C. less than the lowest primer melting temperature [Tm]) for 30 s, and 72° C. for the appropriate extension time (1 min/1,000 bp), followed by a 72° C. extension for 10 min and a 4° C. hold.







TABLE 3







Oligonucleotide primers for amplification of E. chaffeensis


Ank200 and TRP47 gene fragments













Size


Fragment
Forward primer (5′ to 3′)
Reverse primer (5′ to 3′)
(bp)













Ank200





N
CAACAAAATCCTAATTCGCAAG
CGATTTTATATCATTACCAGCA
1,644



(SEQ ID NO: 29)
(SEQ ID NO: 30)



N1
CACCATGGCAGATCCAAAACAAG
TACCGCATACAATGGATCTTC
384



(SEQ ID NO: 31)
(SEQ ID NO: 32)



N2
CACCCCTTTACCTAAAGGTCAAAG
ATCCCTAACACCTTCCC
456



(SEQ ID NO: 33)
(SEQ ID NO: 34)



N3
CACCGCAGTTATTCATGATGAAGAG
CAATGGGGATTGATTTC
468



(SEQ ID NO: 35)
(SEQ ID NO: 36)



N4
CACCCATGTTATGGTTCAGAACC
ATCATTACCAGCAACAGC
354



(SEQ ID NO: 37)
(SEQ ID NO: 38)



N5
CACCATGGCAGATCCAAAACAAG
TTGCTGAGAAGGCAAATC
195



(SEQ ID NO: 39)
(SEQ ID NO: 40)



N6
CACCGAAACAGGAGAAACTGTAGAA
TACCGCATACAATGGATCTTC
189



(SEQ ID NO: 41)
(SEQ ID NO: 42)



N7
CACCGCAGTTATTCATGATGAAGAG
AGCTAAATGCAGTAATGTCATTAC
246



(SEQ ID NO: 43)
(SEQ ID NO: 44)



N8
CACCGTAATGACATTACTGCATTTAGCT
CAATGGGGATTGATTTC
246



(SEQ ID NO: 45)
(SEQ ID NO: 46)



N9
CACCGCAGTTATTCATGATGAAGAG
AATTTCTTCTAGATCTGGCTC
123



(SEQ ID NO: 47)
(SEQ ID NO: 48)



N10
CACCGAGCCAGATCTAGAAGAAATT
AGCTAAATGCAGTAATGTCATTAC
144



(SEQ ID NO: 49)
(SEQ ID NO: 50)



I
TGTTCAGTTAAAGGACGTGTTC
AGCTAAATGCAGCGGTGTATC
1,371



(SEQ ID NO: 51)
(SEQ ID NO: 52)



C
TTTGCTGAAAAGGGTGTAAAAA
ATCTTCAGATGTAATAGGAGGTAGTCCC
1,368



(SEQ ID NO: 53)
(SEQ ID NO: 54)



C1
TTTGCTGAAAAGGGTGTAAAAA
TCCATGTAGACCATGAACTGC
822



(SEQ ID NO: 55)
(SEQ ID NO: 56)



C2
GCAGTTCATGGTCTACATGGA
TTTGCTCTGGCAAGAACTT
639



(SEQ ID NO: 57)
(SEQ ID NO: 58)



C3
GCAGTTCATGGTCTACATGGA
CGCTGATGCACCTAGAGA
318



(SEQ ID NO: 59)
(SEQ ID NO: 60)



C4
TCTCTAGGTGCATCAGCG
TTTGCTCTGGCAAGAACTT
339



(SEQ ID NO: 61)
(SEQ ID NO: 62)



C5
TCTCTAGGTGCATCAGCG
ACCCTTATCAAATATTCCACT
171



(SEQ ID NO: 63)
(SEQ ID NO: 64)



C6
AGTGGAATATTTGATAAGGGT
TTTGCTCTGGCAAGAACTT
189



(SEQ ID NO: 65)
(SEQ ID NO: 66)



 


TRP47





N1
ATGCTTCATTTAACAACAGAA
ATGATAACCACGATCAGGTTC
135



(SEQ ID NO: 67)
(SEQ ID NO: 68)



N2
GAACCTGATCGTGGTTATCAT
AGGATCAACTAAGAAAGAAGC
135



(SEQ ID NO: 69)
(SEQ ID NO: 70)



N3
GCTTCTTTCTTAGTTGATCCT
ATGATCATGTTCATTGTGATG
132



(SEQ ID NO: 71)
(SEQ ID NO: 72)



N4
CATCACAATGAACATGATCATG
ATTTCCTTCAAGAACTGGAAC
132



(SEQ ID NO: 73)
(SEQ ID NO: 74)






aLinker sequences for cloning are underlined.






Expression and purification of the recombinant proteins. The expression of the three largest E. chaffeensis Ank200 fragments (N, I, and C) was performed using the pUni/pRSET-E Echo vector system (Invitrogen, Carlsbad, Calif.). Expression of the recombinant proteins in Escherichia coli BL21(DE3)pLysS (Invitrogen) was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to cultures in log growth phase incubated for 4 h at 37° C. All other Ank200 fragments were expressed by pBAD/Thio-TOPO or pBAD102/D-TOPO vector (Invitrogen). Expression of the recombinant proteins in E. coli TOP10 (Invitrogen) was induced by adding 0.02% arabinose to 4 h cultures. All recombinant proteins were purified under native conditions using His-Select columns (Sigma, St. Louis, Mo.). The expression of the N-terminal region of E. chaffeensis TRP47 (TRP47-N) and the tandem repeat region of E. chaffeensis TRP120 (TRP120-TR; containing first two tandem repeats of TRP120 only) has been previously described (Doyle et al., 2006; Yu et al., 1996).
Synthetic peptides. For E. chaffeensis Ank200, six, four, and six overlapping peptides corresponding to three regions (N6, N10, and C6) (see gray lines for locations in FIG. 1; see FIG. 3A to C, left, for sequences), respectively, were commercially synthesized (Bio-Synthesis, Lewisville, Tex.). For TRP47, the Cterminal peptide and three overlapping peptides corresponding to the N4 region (see FIGS. 4A and 5A) were synthesized (Bio-Synthesis). All other synthetic peptides (TRP120-R-I1 [SKVEQEETNPEVLIKDLQDVAS (SEQ ID NO: 1)], TRP47-R [ASVS EGDAVVNAVSQETPA (SEQ ID NO:75)], TRP32-R3 [SDLHGSFSVELFDPFKEAVQLGNDLQQSSD (SEQ ID NO:76)], TRP32-R4 [SDSHEPSHLELPSLSEEVIQLESDLQQSSN (SEQ ID NO:77)], and E. canis TRP36-2R [TEDSVSAPATEDSVSAPA (SEQ ID NO:78)], which contained two tandem repeat units of TRP36) used in this study have been described previously (Doyle et al., 2006; Luo et al., 2009; Luo et al., 2008). All peptides were supplied as a lyophilized powder and resuspended in molecular biology grade water (1 mg/ml). Antisera. A convalescent-phase anti-E. chaffeensis dog serum sample was obtained from an experimentally infected dog (no. 2251). HME patient serum samples were kind gifts from Focus Technologies (Cypress, Calif.) and the Centers for Disease Control and Prevention (Atlanta, Ga.). Patient serum samples positive for Rickettsia spp. but negative for E. chaffeensis by IFA were kind gifts from Arkansas Public Health Laboratory (Little Rock, Ark.). Rabbit anti-Ank200-N6-1 antiserum was generated against the synthetic keyhole limpet hemocyanin-conjugated peptide Ank200-N6-1 by a commercial vendor (Bio-Synthesis).
Gel electrophoresis and Western immunoblotting. Purified recombinant proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose, and Western immunoblotting was performed as previously described (McBride et al., 2003), except that primary dog sera were diluted 1:100, human sera were diluted 1:200, and rabbit antisera were diluted 1:1,000.
ELISA. For epitope mapping, an ELISA was performed as previously described (Luo et al., 2009). For serologic diagnosis evaluation, an Immobilizer amino plate (Nunc, Roskilde, Denmark) was used to increase the signal-to-noise ratio. Immobilizer amino plates were coated with synthetic peptides or recombinant proteins (0.5 μg/well; 50 μl) suspended in 100 mM sodium carbonate buffer (pH 9.6) and incubated with gentle agitation at room temperature for 1 to 2 h or overnight at 4° C. The wells were washed four times with 300 μl phosphatebuffered saline containing 0.05% (vol/vol) Tween 20 (PBST; pH 7.2) by a plate washer (SkanWasher 400; Molecular Devices, Sunnyvale, Calif.). Dog or human sera diluted (1:100 or 1:200, respectively) in PBST were added to each well (50 μl) and incubated at room temperature for 1 h. The plates were washed again, and 50 μl alkaline phosphatase-labeled goat anti-dog or -human IgG(H+L) secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted (1:5,000) in PBST was added and incubated at room temperature for 1 h. After the addition of substrate (BluePhos; Kirkegaard & Perry Laboratories), plates were incubated in the dark for 30 min, color development was determined on a microplate reader (VersaMax; Molecular Devices, Sunnyvale, Calif.) at A650, and data were analyzed by SoftMax Pro version 4.0 (Molecular Devices). Optical density (OD) readings represent the mean OD value for three wells (±standard deviations) after subtracting the OD value of the negative control wells. All sera negative for E. chaffeensis by IFA had readings of <0.05 OD unit; therefore, a positive sample threshold was set at >0.1 OD unit. In addition, a reading of 0.1 to 0.5 OD unit was considered a weak positive, and a reading of >0.5 OD unit was considered a strong positive. IFA. The anti-E. chaffeensis antibody status in HME patient sera was determined as described previously (McBride et al., 2003). Antigen slides were prepared from DH82 cells infected with E. chaffeensis (Arkansas strain) (McBride et al., 2001). Sera were diluted 2-fold in PBS, starting at 1:64. Statistics. The statistical differences between experimental groups were assessed with the two-tailed Student t test, and significance was indicated by a P value of <0.05. Locus tag numbers of nucleotide sequences. Ehrlichia gene locus tag numbers for the proteins in this study were previously available in the Integrated Microbial Genomes system (img.jgi.doe.gov) (ECH—0170 for TRP32, ECH—0166 for TRP47, ECH—0039 for TRP120, ECH—0684 for Ank200, and Ecaj—0109 for TRP36).
Results
E. chaffeensis Ank200 amino acid composition and domains. The overall Ank200 composition (1,463 amino acids [aa]) was dominated by three hydrophobic amino acids (L, V, and A; 353 aa), three polar amino acids (S, G, and N; 362 aa), and two strongly acidic amino acids (E and D; 198 aa), resulting in a protein with an acidic nature (pI 4.6). Like for E. canis Ank200, three specific domains (N acidic, Ank, and C acidic) were identified, according to amino acid composition and con served motifs (FIG. 9). The distal terminal polypeptides (N acidic, first 390 aa; C acidic, last 267 aa) exhibited a substantially larger proportion of strongly acidic amino acids (D and E; 22.6% in the N-acidic domain and 13.1% in the C-acidic domain) than the internal region (Ank domain, 806 aa [positions 391 to 1196]; 9.3% D and E) of the protein, where ankyrin repeats were located. In contrast, the Ank domain region contained more strongly basic amino acids (K and R; 10.2%) than strongly acidic amino acids. Consequently, the isoelectric points of two terminal domains were acidic (pI 3.6 and 4.7), whereas the internal Ank domain region was slightly basic (pI 8.3) (FIG. 9).
Immunoreactivities of the major E. chaffeensis Ank200 fragments. To determine the major epitope-containing regions of Ank200, the recombinant fragments corresponding to the N terminus (Ank200-N, aa 10 to 557), internal region (Ank200-I, aa 562 to 1018), and C terminus (Ank200-C, aa 984 to 1439), covering 98% of the open reading frame, were expressed (FIG. 9). By Western immunoblotting, the recombinant Ank200-N and Ank200-C (containing the N- and C-acidic domains, respectively) proteins reacted with an HME patient serum sample (no. SC07); however, recombinant protein of the Ank200-I (a majority of the Ank domain) did not react with the patient serum sample. A similar result was obtained by Western blotting probed with the anti-E. chaffeensis dog serum sample derived from a dog (no. 2251) experimentally infected with E. chaffeensis. Thus, the two immunoreactive fragments Ank200-N and Ank200-C were considered to contain antibody epitopes and were investigated further. The anti-E. chaffeensis patient or dog sera did not recognize thioredoxin protein, and the normal human or dog sera did not recognize these recombinant proteins by Western immunoblotting.
Major epitope-containing regions in Ank200-N. The major epitope-containing region(s) in Ank200-N was identified by evaluating the immunoreactivities of four overlapping recombinant proteins (N1 to N4) and of some smaller overlapping recombinant proteins (N5 to N10) (FIG. 1). Western immunoblotting revealed that N1 and N3 fragments were reactive with the patient serum samples, whereas two other fragments (N2 and N4) of Ank200-N were not reactive or only weakly reactive. Western blotting probed with anti-E. chaffeensis dog sera exhibited a similar result. Therefore, smaller overlapping recombinant proteins (N5, N6, N7, and N8) representing N1 and N3 regions were expressed, and two fragments, N6 and N7, were immunoreactive with the patient sera or anti-E. chaffeensis dog sera by Western blotting, while the other two fragments (N5 and N8) were not immunoreactive or were weakly immunoreactive. N7 was further divided into two overlapping polypeptides, N9 and N10, and polypeptide N10 was immunoreactive with the patient sera or anti-E. chaffeensis dog sera by Western blotting, while N9 was not immunoreactive. Thus, the N6 (63-aa) and N10 (48-aa) sections were identified as the major epitope-containing regions of E. chaffeensis Ank200-N, which were located in a highly acidic domain and exhibited high glutamate content (22.2% and 14.6%, respectively) (FIG. 9 and FIGS. 10A and B, left, for sequences).
Major epitope-containing region in Ank200-C. The major epitope(s) in Ank200-C was identified by evaluating the immunoreactivities of six overlapping recombinant proteins Ank200-C was divided into two overlapping fragments (C1 and C2), and Western immunoblotting revealed that the C2 fragment was immunoreactive with a patient serum sample, while C1 was not reactive. Therefore, C2 fragment was further divided into two overlapping polypeptides (C3 and C4), and the C4 fragment was immunoreactive with a patient serum sample by Western blotting, while C3 was not reactive or was only weakly reactive. Smaller overlapping polypeptides (C5 and C6) representing the C4 region were expressed, and the C6 fragment reacted with a patient serum sample by Western blotting, while C5 was not reactive or was weakly reactive. A similar result was obtained by Western blotting probed with an anti-E. chaffeensis dog serum sample. Thus, the C6 (63-aa) section of E. chaffeensis Ank200-C was identified as a major epitope-containing region, which was also located in a highly acidic domain and exhibited a high glutamate content (11.1%) (FIG. 9 and FIG. 10C, left, for the sequence).
Determination of the major immunodeterminants of E. chaffeensis Ank200 with synthetic peptides. Synthetic peptides were used to localize the major epitope(s) in three immunoreactive regions (N6, N10, and C6) of Ank200, respectively. Four synthetic overlapping polypeptides (N6-1, 2, 3, and 4; FIG. 10A, left panel) covering the sequence of Ank200-N6 (63 aa) were generated and reacted by ELISA with an anti-E. chaffeensis dog serum (no. 2251) and four HME patient sera (nos. F3, F5, F13 and F22) that had detectable E. chaffeensis antibodies by IFA. Among five sera, peptide N6-2 did not react with two sera, reacted weakly with one and strongly with two sera; peptide N6-3 did not react with one sera and reacted weakly with four sera; peptide N6-4 did not react with two sera and reacted weakly with three sera; however, peptide N6-1 was found to react strongly with all the anti-E. chaffeensis dog and patient sera, indicating that the N-terminal fragment (28 aa) of the Ank200-N6 region had a significantly (P<0.05 for dog serum and most patient sera) stronger immunoreactivity than other fragments and contained a major antibody epitope (FIG. 10A, right panel). To further determine the amino acid sequence reactive with antibody, N6-1 was divided into two smaller overlapping peptides (N6-1a and N6-1b). By ELISA, peptide N6-1b did not react with the anti-E. chaffeensis dog serum and reacted weakly with four patient sera; however, although peptide N6-1a was also not reactive with antibodies in dog serum, it reacted strongly with all four patient sera, indicating that the N-terminal amino acids (ETGETVEEGLYA (SEQ ID NO:79)) contributed significantly (P<0.05) to epitope reactivity with all patient sera (FIG. 10A, right panel). Therefore, N6-1a (18 aa; ETGETVEEGLYAVPLPKD (SEQ ID NO:80)) contained a major continuous antibody epitope of Ank200 for human, but longer sequence of peptide N6-1 (28 aa; ETGETVEEGLYAVPLPKDQRPTPTQVLE (SEQ ID NO:81)) exhibited the strongest immunoreactivity and was necessary for full reconstitution of the major antibody epitope of Ank200.
To identify the peptide sequence containing the immunodeterminant in Ank200-N10 (48 aa) region, four overlapping peptides (N10-1, 2, 3 and 4; FIG. 10B, left panel) covering N10 region were reacted with an anti-E. chaffeensis dog serum (no. 2251) and four HME patient sera (nos. F2, F4, F5 and F21). By ELISA, peptides N10-1 and 2 did not react and peptides N10-3 and 4 reacted weakly with antibodies in the dog serum (FIG. 10B, right panel). Since the recombinant Ank200-N10 protein reacted strongly with anti-E. chaffeensis dog serum by Western blotting, the data suggested that the sequence longer than above peptides was required to reconstitute the major antibody epitope of Ank200 recognized by antibodies in the dog serum. By ELISA, peptide N10-2 reacted weakly with two patient sera and reacted strongly with two patient sera, and peptides N10-1, 3 and 4 reacted weakly with one patient serum but reacted strongly with other three patient sera, suggesting that N10 had two epitope-containing regions for human, N10-1 (20 aa; EPDLEEIVSILKNDKEGISE (SEQ ID NO:82)) and N10-3 (20 aa; INEPVQVDIPNNPVREGRNV (SEQ ID NO:83)), and the C-terminal amino acids (MTLLHLA (SEQ ID NO:84)) of N10 had no substantial contribution to the epitope reactivity; moreover, peptide N10-1 exhibited substantially stronger immunoreactivity than did peptides N10-3 with three patient sera (FIG. 10B, right panel).
Four synthetic overlapping peptides (C6-1, 2, 3 and 4; FIG. 10C, left panel) covering the sequence of Ank200-C6 (63 aa) were reacted by ELISA with an anti-E. chaffeensis dog serum (no. 2251) and four HME patient sera (nos. F2, F4, F15 and SC07). Peptides C6-1, 2 and 3 were only weakly immunoreactive with one or two patient sera, but peptide C6-4 was found to react with the anti-E. chaffeensis dog serum and react strongly with all patient sera, indicating that the C-terminal fragment (25 aa) of the Ank200 contained a major antibody epitope and the C-terminal sequence (QGADVKKSSCQSK (SEQ ID NO:85), 13 aa) significantly (P<0.05 for all sera) contributed to the epitope reactivity (FIG. 10C). To further determine the amino acid sequence reactive with antibody, two smaller overlapping peptides (C6-4a and 4b) representing fragment C6-4 reacted with anti-E. chaffeensis dog and patient sera by ELISA. The peptide C6-4a was not immunoreactive with all sera, however, peptide C6-4b was found to react with the anti-E. chaffeensis dog serum and react strongly with all patient sera, indicating that the very distal C-terminal fragment C6-4b (21 aa; QAVSPSTSQGADVKKSSCQSK (SEQ ID NO:86)) contained a major continuous antibody epitope of Ank200. Moreover, the very distal C-terminal amino acids (SCQSK (SEQ ID NO:87)) contributed significantly (P<0.05 for all sera) to the epitope immunoreactivity (FIG. 10C).
Identification of TRP47 antibody epitopes in the TR flanking terminal regions. E. chaffeensis TRP47 has N- (157 aa) and short C- (26 aa) termini flanking the TR (19 aa each) region (FIG. 11). In a previous study, it was determined that the TR of TRP47 contained a major antibody epitope (Doyle et al., 2006); however the N- and C-terminal regions were not fully explored. The immunoreactivity of TRP47-N and TRP47-C regions was further investigated using HME patient sera in this report. A large panel of 31 patient sera that had detectable E. chaffeensis antibodies by IFA was used to detect the recombinant TRP47-N protein by Western blot; as a result, 13 of 31 sera reacted with TRP47-N, indicating that the N-terminal region of TRP47 contained a minor antibody epitope.
To locate the epitope in the TRP47-N, four recombinant overlapping proteins (TRP47-N1, N2, N3, and N4; FIG. 11) covering the sequence of whole TRP47-N region were expressed and reacted with three HME patient sera (nos. 013, 015 and 19) that recognized TRP47-N by Western blotting. The recombinant N2 fragment did not react with the patient sera, the N1 reacted weakly with one patient serum, the N3 reacted with two sera (one weakly), while the N4 fragment reacted with all three sera strongly, indicating the TRP47-N4 fragment (44 aa) contained a minor antibody epitope. Three synthetic overlapping polypeptides (N4-1, 2, and 3; FIG. 11 and FIG. 12A) covering the sequence of TRP47-N4 were generated and reacted with six HME patient sera (nos. O15, 6, 9, 13, 18 and 19) that recognized TRP47-N by Western blotting. By ELISA, peptide N4-3 was not reactive with any tested serum, N4-1 was found to react with five sera (except for no. 19), and N4-2 reacted with three sera (nos. 6, 18 and 19), and the reaction with serum no. 19 was very strong (FIG. 12B). Therefore, the assembled sequence (33 aa) of N4-1 and 2 fragments contained the antibody epitope with the TRP47-N region.
Although the TR of TRP47 has previously been reported to react with anti-E. chaffeensis dog serum, its immunoreactivity with the HME patient serum has not been reported. Synthesized TR unit (TRP47-R; 19 aa) and C-terminus (TRP47-C; 26 aa) of TRP47 (FIG. 11 and FIG. 12A) were reacted by ELISA with sera from seven HME patients and one experimentally infected dog. Peptide TRP47-R was recognized by six patient sera and the dog serum; peptide TRP47-C was recognized by three patient sera, but exhibited significantly (P<0.05) stronger reactivity than did TRP47-R with two sera (nos. O3 and 13) (FIG. 12C). Hence, both TRP47-R and TRP47-C exhibited the immunoreactivity with HME patient sera; however, TRP47-R had stronger overall immunoreactivity (P<0.05 for most sera) than TRP47-C. Moreover, TRP47-R exhibited stronger immunoreactivity than did TRP47-C with an anti-E. chaffeensis dog serum.
Evaluation of synthetic E. chaffeensis major immunodeterminants for serologic diagnosis of HME. In order to examine and compare the immunoreactivity of E. chaffeensis major immunoreactive epitopes that have been characterized, a panel of 31 HME patient sera that had detectable E. chaffeensis antibodies by IFA (titer from 64 to 8192) were used to examine and compare the sensitivity of synthetic epitopes from E. chaffeensis TRP32, TRP47, TRP120 and Ank200 with IFA. Epitopes for TRP32, TRP47, and TRP120 mapped in other studies were also included in this evaluation. An equal (w:w) mixture of TRP32-R3 (30 aa) and -R4 (30 aa) peptides were used for TRP32, an equal mixture of TRP47-N4-1 (22 aa), -R (19 aa) and -C (26 aa) peptides was used for TRP47, TRP120-R-I1 (22 aa) peptide was used for TRP120, and an equal mixture of Ank200-N6-1a (18 aa), N10-1 (20 aa), and C6-4b (21 aa) were used for Ank200. In addition, a recombinant TRP120 TR protein (rTRP120-TR) and an equal mixture of TRP32-R3, TRP32-R4 and TRP120-R-I1 peptides were also tested. E. canis TRP36-2R (18 aa) was used as a negative control peptide. Patient sera (n=10) negative for E. chaffeensis antibodies by IFA were also tested.
All 31 HME patient sera reacted with at least one E. chaffeensis peptide and 30 sera (96.8%) reacted with TRP120 peptide, 27 (87.1%) with TRP32 peptides, 24 (77.4%) with TRP47 peptides, 19 (61.3%) with Ank200 peptides (FIG. 13A; Table 4). Only one serum (no. 16) with low IFA titer (1:64) did not reach established positive cutoff with TRP120 peptide, and four sera (nos. 16, 19, 30 and 31) with low IFA titer (three with 1:64 and one with 1:256) did not react with TRP32 peptides. The recombinant TRP120-TR protein was recognized by 28 (90.3%) sera, and a mixture of TRP120 and TRP32 peptides was recognized by only 26 (83.9%) sera and did not provide enhanced sensitivity over the TRP120 alone (FIG. 13B; Table 4). These results suggested that TRP120 is the best candidate for immunodiagnosis of HME, and a single synthetic peptide TRP120-R-I1 from TRP120 repeats exhibited higher sensitivity than the peptide mixture or recombinant TRP120-TR protein did with HME patient sera. Moreover, the peptides were not recognized by patient sera that were positive for Rickettsia spp. but not positive for E. chaffeensis by IFA, indicating that ELISA reactions between synthetic E. chaffeensis immunodeterminants and HME patient sera were specific.







TABLE 4







Analytical sensitivity of synthetic antibody epitopes of E. chaffeensis immunoreactive


proteins for immunodiagnosis of HME by ELISA.














Antigens
TRP32
TRP47
TRP120
Ank200
TRP32 + TRP120
Overall
rTRP120

















No. of patients with
27
24
30
19
26
31
28


detectable antibodies


% of patients with
87.1
77.4
96.8
61.3
83.9
100
90.3


detectable antibodies





aSynthetic epitope peptides of TRP32 (R3 + R4), TRP47 (N4-1 + R + C), TRP120 (R-I1) and Ank200 (N6-1a + N10-1 + C6-4b), and an equal mixture of TRP32-R3, TRP32-R4 and TRP120-R-I1 peptides as well as the rTRP120 (recombinant TRP120-TR protein, containing first two tandem repeats of TRP120 only) reacted with 31 HME patient sera. “Overall” refers to overall number and percentage of patients with detectable antibodies against any tested synthetic peptide.


bA sample with a reading 0.1 OD unit above the negative control absorbance was considered positive.






Discussion

Many of the major immunoreactive proteins of E. chaffeensis and E. canis have been identified and molecularly characterized, and interestingly, most are members of a small group of tandem repeat or ankyrin repeat containing proteins, including TRP32/TRP19, TRP47/TRP36, TRP120/TRP140 and Ank200s (Doyle et al., 2006; McBride et al., 2003; McBride et al., 2007; Sumner et al., 1999; Yu et al., 1997; Yu et al., 2000). Common features among these proteins include serine-rich TRs and an acidic pI (due to a predominance of glutamate/aspartate). Both recombinant and native proteins exhibit electrophoretic masses larger than predicted by amino acid sequence, due to the acidic properties of the proteins and not by the addition of glycans post-translationally (Garcia-Ortega et al., 2005; Luo et al., 2009; Luo et al., 2008). Notably, major continuous antibody epitopes of these proteins have been mapped to acidic domains, which are located in the central TR region in all TRPs or N- and C-terminal regions in E. canis Ank200, indicating Ehrlichial acidic domains, particularly those in TRs, are primary targets of the host humoral immune response (Doyle et al., 2006; Luo et al., 2009; Luo et al., 2008; McBride et al., 2003; McBride et al., 2007; Nethery et al., 2007). The association of these acidic domains with the host immune response is interesting and unique and to the inventor's knowledge, has not been described with respect to any other pathogen; however, the specific role of these domains in Ehrlichial pathobiology or immunity is still unknown.
E. chaffeensis and E. canis Ank200 protein orthologs are the largest Ehrlichial major immunoreactive proteins. They have identical chromosomal locations, and exhibit  ̃50% nucleic acid identity and  ̃32% amino acid identity, and they lack serine-rich TRs present in other Ehrlichial major immunoreactive proteins (McBride et al., 2003). However, they have similar distal N- and C-terminal acidic domains flanking the centralized ankyrin domain containing numerous ankyrin repeats that may mediate protein-protein interactions (Nethery et al., 2007). Like the ankyrin protein AnkA from Anaplasma phagocytophilum (Park et al., 2004), E. chaffeensis Ank200 is also translocated to the nucleus of infected cells, where it interacts with the DNA motif Alu (Zhu et al., 2009). In this study, major epitope-containing regions of E. chaffeensis Ank200 were mapped to the distal N- and C-terminal acidic (pI 3.6 and 4.7) domains, which is consistent with the location of the four epitopes mapped in E. canis Ank200 N- and C-terminal acidic (pI 4 and 4.9) domains (Nethery et al., 2007). The antibody epitopes in E. chaffeensis Ank200, which exhibited the strongest antibody reactivity with both dog and human sera, were localized to four polypeptides N6-1a, N10-1, N10-3 and C6-4b (18-mer, 20-mer, 20-mer, and 21-mer, respectively), with three in the N-terminal domain and only one in the C-terminal domain, demonstrating that the N-terminal domain has multiple epitopes, and thus, is the immunodominant region. The length of the Ank200 epitopes was similar and consistent in size (around 20-mer) with those described of other molecularly characterized continuous Ehrlichial epitopes Doyle et al., 2006; Luo et al., 2009; Luo et al., 2008; McBride et al., 2007; Nethery et al., 2007. However, a smaller six-amino acid epitope has been reported Anaplasma marginate msp1a protein (Allred et al., 1990). One conformational epitope has been mapped in TRP32-R4 (Luo et al., 2008), and there may be other conformational epitopes associated with these major immunoreactive proteins that were not determined, although the host response to the continuous major epitopes in Ehrlichial immunodominant proteins is strong and suggest the absence of dominant conformational epitopes.
A major epitope in the TR region of the TRP47 and corresponding ortholog (TRP36) in E. canis was previously reported (Doyle et al., 2006). However, a comprehensive analysis of the regions flanking the TR was not performed. Hence, in this Example, HME patient sera were used to fully explore these regions and all three regions exhibited the immunoreactivity with patient sera. Two additional epitope-containing regions were identified in the N- and C-termini of TRP47, respectively, but TRP47-TR exhibited the stronger overall immunoreactivity than TRP47-N and -C and was more consistently recognized by antibodies in HME patient sera. Therefore, TRP47 TR appears to be the major antibody epitope and minor epitopes are located in the N- and C-termini. Similarly, minor cross-reactive antibody epitopes have been identified in N- and C-terminal regions of the TRP120 and TRP140 (Luo et al., 2009). Some HME patients only developed antibodies to one or more of the TRP47 minor epitopes and not to the TR epitope. This could be related to diversity in the TR of TRP47, which has been described in Arkansas and Supulpa strains (Doyle et al., 2006; Yu et al., 2007). This is in contrast to other TRPs, such as TRP120 and TRP32, in which the TR epitopes appear to be more conserved (Yabsley et al., 2003; Yu et al., 2007). Therefore, the increased sensitivity attained with a peptide mixture containing all TRP47 epitopes compared to the TR epitope alone, is likely related to antigenic diversity of this protein. Additional characterization of TRP47 variants could provide an explanation for the decreased sensitivity of this protein compared to TRP120 or TRP32 as well as information regarding the kinetics of the antibody response in HME patients.
All of the Ehrlichial major immunoreactive protein orthologs (TRP32/TRP19, TRP47/TRP36, and TRP120/TRP140) identified and characterized recently are antigenically distinct and elicit species-specific antibodies (Doyle et al., 2006; Luo et al., 2009; Luo et al., 2008; McBride et al., 2007). Five major antibody epitopes characterized in E. canis Ank200 are also molecularly distinct (Nethery et al., 2007). Consistent with these findings, the amino acid alignments of the mapped epitopes in Ank200 identified no significant homology with E. canis Ank200 or other proteins from organisms in closely related genera; moreover, antisera against recombinant E. chaffeensis or E. canis Ank200N did not cross-react, indicating that these epitopes appear to be primarily species-specific and could be utilized for species-specific diagnostic development. The inventors have previously reported that minor antibody epitope-containing regions in the N- and C- termini of E. chaffeensis TRP120 and E. canis TRP140 are cross-reactive, further suggesting that cross-reactive antibodies generated between closely related Ehrlichia spp. were directed at some minor epitopes rather than major epitopes (Luo et al., 2009).
Previous studies have concluded that the TRP120 is a sensitive immunodiagnostic antigen for HME (Yu et al., 1999). The data presented in this Example indicates that the TRP120 is the most sensitive immunodiagnostic antigen for HME. It is becoming increasingly evident that all of the major immunoreactive proteins of Ehrlichia spp. have molecularly distinct epitopes, which can be used to serologically identify etiologic agents, a task that has been routinely difficult or impossible to accomplish (Doyle et al., 2006; Luo et al., 2009; Luo et al., 2008; McBride et al., 2007; Nethery et al., 2007). The TRP epitopes are molecularly distinct and therefore, serologic responses specific to E. chaffeensis can be distinguished from those against closely related agents or conserved bacterial proteins using these immunodeterminants. The inventors determined serologically that TRP120-R-I1 is a species-specific epitope, and lack of serologic cross-reactivity with E. canis was related to divergence at the amino acid level (Luo et al., 2009). In addition, the TRP120 has very limited amino acid homology with two A. phagocytophilum repeat-containing proteins, GE100 and GE130; however, the TRP120-R-I1 peptide does not have any amino acid homology with these two proteins (Storey et al., 1998). Compared with TRP32 and TRP47, the TRP120 has less molecular variation among examined E. chaffeensis strains, and this trait is shared with an ortholog, E. canis TRP140 (Yu et al., 2007). However, as observed with other immunoreactive peptides from Ehrlichia, in some cases, but not all, a mixture of TRP120 and TRP32 peptides does not provide enhanced sensitivity over the TRP120 alone, indicating that mixed peptides could compete with each other resulting in decreased sensitivity. To the inventor's knowledge, this is the first study to compare multiple molecularly-defined major antibody epitopes of E. chaffeensis for serodiagnosis of HME in a solid phase assay. The synthetic TRP120-R-I1 peptide exhibited even more sensitive reactivity than the recombinant TRP120-TR with patient sera, indicating that high purity of the immunodeterminant may contribute to enhanced sensitivity of ELISA and could effectively replace recombinant proteins.
* * *
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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## Claims

1. A method of detecting antibodies that specifically bind an Ehrlichia antigenic polypeptide in a test sample of a subject suspected of having an Ehrlichia infection, comprising detecting in said sample the presence of an antibody that immunologically reacts with a polypeptide consisting of SEQ ID NO:1 or SEQ ID NO:2.

2. The method of claim 1, wherein the step of detecting comprises performing an enzyme-linked immunoassay, a radioimmunoassay, an immunoprecipitation, a fluorescence immunoassay, a chemiluminescent assay, an immunoblot assay, a lateral flow assay, a flow cytometry assay, a multi-plex particle-based suspension array assay, a mass spectrometry assay, or a particulate-based assay.

3. The method of claim 2, wherein the step of detecting comprises a lateral flow assay or a an enzyme-linked immunoassay, wherein the enzyme-linked immunoassay is an ELISA.

4. The method of claim 1, wherein the antibody immunologically reacts with a polypeptide consisting of SEQ ID NO:1.

5. The method of claim 1, wherein the antibody immunologically reacts with a polypeptide consisting of SEQ ID NO:2.