U.S. patent application number 13/709891 was filed with the patent office on 2013-07-11 for immunoreactive ehrlichia p120/p140 epitopes and uses thereof.
This patent application is currently assigned to RESEARCH DEVELOPMENT FOUNDATION. The applicant listed for this patent is Research Development Foundation. Invention is credited to Tian LUO, Jere W. McBRIDE.
Application Number | 20130177978 13/709891 |
Document ID | / |
Family ID | 42272580 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177978 |
Kind Code |
A1 |
McBRIDE; Jere W. ; et
al. |
July 11, 2013 |
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.
Inventors: |
McBRIDE; Jere W.; (League
City, TX) ; LUO; Tian; (Galveston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Research Development Foundation; |
Carson City |
NV |
US |
|
|
Assignee: |
RESEARCH DEVELOPMENT
FOUNDATION
Carson City
NV
|
Family ID: |
42272580 |
Appl. No.: |
13/709891 |
Filed: |
December 10, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12769352 |
Apr 28, 2010 |
8329189 |
|
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13709891 |
|
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61173345 |
Apr 28, 2009 |
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Current U.S.
Class: |
435/348 ;
435/252.33; 435/254.2; 435/320.1; 435/325; 525/54.2; 536/23.7 |
Current CPC
Class: |
G01N 2469/20 20130101;
C07K 14/29 20130101; C07K 14/195 20130101; G01N 33/56911 20130101;
G01N 2333/29 20130101 |
Class at
Publication: |
435/348 ;
536/23.7; 435/320.1; 435/252.33; 435/254.2; 435/325; 525/54.2 |
International
Class: |
C07K 14/195 20060101
C07K014/195 |
Goverment Interests
[0001] This invention was made with U.S. government support under
grant R01 AI 071145 from the National Institutes of Health. The
government has certain rights in the invention.
Claims
1.-27. (canceled)
28. An isolated nucleic acid segment encoding a peptide 45 amino
acids in length or less, wherein the peptide comprises SEQ ID NO:1
or SEQ ID NO:2, wherein the peptide selectively binds an antibody
that recognizes and binds an Ehrlichia p120 or p140 protein,
provided that if the encoded peptide comprises SEQ ID NO:2, the
encoded peptide is 30 amino acids or less in length.
29. A vector comprising a contiguous sequence consisting of the
nucleic acid segment of claim 28.
30. A host cell comprising the nucleic acid segment of claim
28.
31.-36. (canceled)
37. The isolated nucleic acid segment of claims 28, wherein the
encoded peptide consists of SEQ ID NO:1 or SEQ ID NO:2.
38. The isolated nucleic acid segment of claim 37, wherein the
nucleic acid is immobilized on a surface of a support
substrate.
39. The isolated nucleic acid segment of claim 38, wherein said
support substrate comprises latex, polystyrene, nylon,
nitrocellulose, cellulose, silica, agarose, or magnetic resin.
40. The isolated nucleic acid segment of claim 38, wherein 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.
41. The isolated nucleic acid segment of claim 28, wherein the
segment is comprised in a kit.
42. The isolated nucleic acid segment of claim 28, wherein the
segment is comprised in a pharmaceutical preparation.
Description
[0002] This application is a divisional of U.S. application Ser.
No. 12/769,352, filed Apr. 28, 2010, 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.
BACKGROUND OF THE INVENTION
[0003] I. Field of the Invention
[0004] 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.
[0005] II. Background and Description of Related Art
[0006] 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).
[0007] 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.
[0008] 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; corner 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
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.RTM.
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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] Another aspect of the present invention relates to a vector
comprising a contiguous sequence consisting of the nucleic acid
segment.
[0019] Yet another aspect of the present invention relates to a
host cell comprising the nucleic acid segment.
[0020] 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).
[0021] 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.
[0022] 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."
[0023] 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.
[0024] 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."
[0025] 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.
[0026] 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.
[0027] 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
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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).
[0032] 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).
[0033] 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.
[0034] 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.
[0035] 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]).
[0036] 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.
[0037] 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.
[0038] FIGS. 10A-C. Immunoreactivities of overlapping synthetic
peptides spanning the E. chaffeensis Ank200-N.sub.6, -N.sub.10, and
-C.sub.6 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
N.sub.6-1 were significantly (P<0.05) higher than those of
N.sub.6-2, -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 N.sub.6-1b 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 C.sub.6-4 were significantly (P<0.05)
higher than those of C.sub.6-1, -2, and -3 for all sera, and OD
readings of peptide C6-4b were significantly (P<0.05) higher
than those of C.sub.6-4a 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.
[0039] 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).
[0040] FIGS. 12A-C. Immunoreactivity of overlapping synthetic
peptides spanning E. chaffeensis TRP47-N.sub.4 and synthetic
TRP47-R and TRP47-C peptides as determined by ELISA. (FIG. 12A)
Sequences of three overlapping peptides spanning the TRP47-N.sub.4
fragment and TRP47-R and TRP47-C peptides. (FIG. 12B) TRP47-N.sub.4
peptides reacted with five HME patient sera (nos. 015, 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.
[0041] 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
(R.sub.3+R.sub.4), TRP47 (N.sub.4-1+R+C), TRP120 (R-I.sub.1) and
Ank200 (N.sub.6-1a+N.sub.10-1+C.sub.6-4b) reacted with 31 HME
patient sera (nos. 1.about.31) and an anti-E. chaffeensis dog (no.
2251) serum. (FIG. 13B) An equal mixture of TRP32-R.sub.3,
TRP32-R.sub.4 and TRP120-R-I.sub.1 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
[0042] 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.
[0043] 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
[0044] 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).
[0045] 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.
[0046] 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.
[0047] As used herein, an "p120/p140 immunoreactive peptide" is an
peptide which can selectively bind with an anti-p120 antibody or an
anti-p140 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.
[0048] 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.
[0049] 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
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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
[0054] 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.
[0055] 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.
[0056] Exemplary labels include, but are not limited to, a
particulate label such as colloidal gold, a radioactive isotope
such as astatine.sup.211, .sup.14-carbon, .sup.51chromium,
.sup.36-chlorine, .sup.57cobalt .sup.58cobalt, copper.sup.67,
.sup.152Eu, gallium.sup.67, .sup.3hydrogen, iodine.sup.123,
iodine.sup.125, iodine.sup.131, indium.sup.111, .sup.59iron,
.sup.32phosphorus, rhenium186, rhenium188, .sup.75selenium,
.sup.35sulphur, technicium99, technetium-99m or yttrium.sup.90, 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 6372445, 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
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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).
[0062] 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-3.alpha.-6.alpha.-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.
[0063] 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
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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).
[0068] 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.
[0069] 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,
.beta.-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 .beta.-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.
[0070] 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).
[0071] 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
[0072] 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 (1HA), complement fixation, indirect immunofluorescent 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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
[0086] 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).
[0087] 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.
[0088] 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
(1HA), 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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 (1HA), complement fixation, indirect immunofluorescent 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.RTM. system (Bio-Rad Laboratories, Hercules, Calif.,
USA).
ELISA
[0099] 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.
[0100] 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.).
[0101] 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
[0102] 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.
[0103] Exemplary lateral flow device formats include, but are not
limited to, a dipstick, a card, a chip, a microslide, and a
cassette, and it is widely demonstrated in the art that the choice
of format is largely dependent upon the features of a particular
assay. 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.
[0104] 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.
[0105] 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.
[0106] Typically, a flow-through ELISA may feature a detection
reagent applied to a test strip in a pattern, such as a line, that
can be read visually. As with other lateral flow tests, the absence
of such a pattern 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
[0107] 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.RTM. 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.
[0108] 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
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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, 21.sup.st
Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by
reference).
[0117] 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.
[0118] 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
[0119] 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.
[0120] 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).
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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
[0137] 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
[0138] 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
[0139] Culture and Purification of Ehrlichiae.
[0140] 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.
[0141] Preparation of Ehrlichia Genomic DNA and Antigen.
[0142] 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.).
[0143] PCR Amplification of the Ehrlichia Genes.
[0144] 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).
[0145] 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.degree. C. for 3 min,
30 cycles of 94.degree. C. for 30 s, annealing temperature
(1.degree. C. less than the lowest primer T.sub.m) for 30 s, and
72.degree. C. for the appropriate extension time (1 min/1000 base
pairs) followed by a 72.degree. C. extension for 10 min and a
4.degree. 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 His Select.RTM. 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).
[0146] p120 and p140 Synthetic Peptides.
[0147] 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).
[0148] Antisera.
[0149] 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).
[0150] Gel Electrophoresis and Western Immunoblotting.
[0151] 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.
[0152] ELISA.
[0153] Enzyme-linked immunosorbent assay (ELISA) plates (MaxiSorp;
Nunc, Roskilde, Denmark) were coated (0.5 .mu.g/well; 50 .mu.l)
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 .mu.l Tris-buffered saline
containing 0.2% Tween 20 (TBST). Plates were blocked with 100 .mu.l
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 .mu.l) and incubated at room temperature for 1 h
with gentle agitation. The plates were washed four times, and 50
.mu.l 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 .mu.l; 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 A.sub.650 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.
[0154] Mass Spectrometry.
[0155] Sample solution or protein standard (1 .mu.l) was spotted
directly onto a MALDI target plate and allowed to air dry. Sinapic
acid (Aldrich, Milwaukee, Wis.) matrix solution (1 .mu.l; 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-US-00001 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)
[0156] Sequence Analysis.
[0157] 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).
[0158] Statistics.
[0159] 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.
[0160] Results:
[0161] E. Chaffeensis p120 and E. Canis p140 Composition and
Characteristics.
[0162] 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).
[0163] 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 .about.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.
[0164] Identification of the Native E. Chaffeensis p120 and E.
Canis p140 Proteins.
[0165] Western blotting identified two strongly reactive native
proteins with the molecular mass of .about.95 kDa and .about.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 .about.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-US-00002 TABLE 2 Predicted and observed molecular masses and
amino acid analyses of E. chaffeensis p120 and E. canis p140
proteins. Molecular mass (kDa).sup.a Glutamate Serine Valine
Protein Predicted Observed Mass.sup.c content n (%) content n (%)
content 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)
p120TR.sup.b 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 .sup.aIncluding the fusion tags: all were
thioredoxin (16.3 kDa) except for p120TR and p140TR (GST tag; 28
kDa). .sup.bOnly first two repeats was cloned and expressed, but
the amino acid content values are for the whole repeat region.
.sup.cAs determined by MALDI-TOF mass spectrometry of the
recombinant protein nd = not determined
[0166] Epitope Mapping of E. Chaffeensis p120 and E. Canis p140
with Recombinant Proteins.
[0167] 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.
[0168] 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.
[0169] Peptide Mapping of the Major Immunodeterminants of E.
Chaffeensis p120 and E. canis p140.
[0170] 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 (FIG. 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-I2, 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 (FIG. 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 p12041 (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 (51, 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).
[0171] 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 (FIG. 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).
[0172] Identification of Immunoreactive Regions for Cross Reaction
Between E. Chaffeensis p120 and E. Canis p140.
[0173] 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:
[0174] 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.
[0175] 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.
[0176] 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
.about.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 msp 1a (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.
[0177] 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 continuous epitopes
identified in other Ehrlichial TR proteins, the p120/p140 TR
epitopes could be utilized for species-specific diagnostic
development.
[0178] 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).
[0179] 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
(.about.95 kDa) and E. canis p140 (.about.125 kDa) proteins were
smaller than previously reported masses (.about.120 kDa and
.about.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.
[0180] 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.
TABLE-US-00003 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
[0181] 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.
[0182] 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
[0183] Culture and Purification of E. Chaffeensis.
[0184] 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).
[0185] PCR Amplification of the E. Chaffeensis Genes.
[0186] 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.sub.--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.degree. C. for 3 min, 30 cycles
of 94.degree. C. for 30 s, annealing temperature (1.degree. C. less
than the lowest primer melting temperature [Tm]) for 30 s, and
72.degree. C. for the appropriate extension time (1 min/1,000 bp),
followed by a 72.degree. C. extension for 10 min and a 4.degree. C.
hold.
TABLE-US-00004 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) N.sub.1 CACCATGGCAGATCCAAAACAAG
TACCGCATACAATGGATCTTC 384 (SEQ ID NO: 31) (SEQ ID NO: 32) N.sub.2
CACCCCTTTACCTAAAGGTCAAAG ATCCCTAACACCTTCCC 456 (SEQ ID NO: 33) (SEQ
ID NO: 34) N.sub.3 CACCGCAGTTATTCATGATGAAGAG CAATGGGGATTGATTTC 468
(SEQ ID NO: 35) (SEQ ID NO: 36) N.sub.4 CACCCATGTTATGGTTCAGAACC
ATCATTACCAGCAACAGC 354 (SEQ ID NO: 37) (SEQ ID NO: 38) N.sub.5
CACCATGGCAGATCCAAAACAAG TTGCTGAGAAGGCAAATC 195 (SEQ ID NO: 39) (SEQ
ID NO: 40) N.sub.6 CACCGAAACAGGAGAAACTGTAGAA TACCGCATACAATGGATCTTC
189 (SEQ ID NO: 41) (SEQ ID NO: 42) N.sub.7
CACCGCAGTTATTCATGATGAAGAG AGCTAAATGCAGTAATGTCATTAC 246 (SEQ ID NO:
43) (SEQ ID NO: 44) N.sub.8 CACCGTAATGACATTACTGCATTTAGCT
CAATGGGGATTGATTTC 246 (SEQ ID NO: 45) (SEQ ID NO: 46) N.sub.9
CACCGCAGTTATTCATGATGAAGAG AATTTCTTCTAGATCTGGCTC 123 (SEQ ID NO: 47)
(SEQ ID NO: 48) N.sub.10 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)
C.sub.1 TTTGCTGAAAAGGGTGTAAAAA TCCATGTAGACCATGAACTGC 822 (SEQ ID
NO: 55) (SEQ ID NO: 56) C.sub.2 GCAGTTCATGGTCTACATGGA
TTTGCTCTGGCAAGAACTT 639 (SEQ ID NO: 57) (SEQ ID NO: 58) C.sub.3
GCAGTTCATGGTCTACATGGA CGCTGATGCACCTAGAGA 318 (SEQ ID NO: 59) (SEQ
ID NO: 60) C.sub.4 TCTCTAGGTGCATCAGCG TTTGCTCTGGCAAGAACTT 339 (SEQ
ID NO: 61) (SEQ ID NO: 62) C.sub.5 TCTCTAGGTGCATCAGCG
ACCCTTATCAAATATTCCACT 171 (SEQ ID NO: 63) (SEQ ID NO: 64) C.sub.6
AGTGGAATATTTGATAAGGGT TTTGCTCTGGCAAGAACTT 189 (SEQ ID NO: 65) (SEQ
ID NO: 66) TRP47 N.sub.1 ATGCTTCATTTAACAACAGAA
ATGATAACCACGATCAGGTTC 135 (SEQ ID NO: 67) (SEQ ID NO: 68) N.sub.2
GAACCTGATCGTGGTTATCAT AGGATCAACTAAGAAAGAAGC 135 (SEQ ID NO: 69)
(SEQ ID NO: 70) N.sub.3 GCTTCTTTCTTAGTTGATCCT ATGATCATGTTCATTGTGATG
132 (SEQ ID NO: 71) (SEQ ID NO: 72) N.sub.4 CATCACAATGAACATGATCATG
ATTTCCTTCAAGAACTGGAAC 132 (SEQ ID NO: 73) (SEQ ID NO: 74)
.sup.aLinker sequences for cloning are underlined.
[0187] Expression and Purification of the Recombinant Proteins.
[0188] 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-.beta.-D-thiogalactopyranoside (IPTG) to cultures in log
growth phase incubated for 4 h at 37.degree. 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).
[0189] Synthetic peptides. For E. chaffeensis Ank200, six, four,
and six overlapping peptides corresponding to three regions
(N.sub.6, N.sub.10, and C.sub.6) (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-I.sub.1 [SKVEQEETNPEVLIKDLQDVAS (SEQ ID NO: 1)], TRP47-R
[ASVS EGDAVVNAVSQETPA (SEQ ID NO:75)], TRP32-R.sub.3
[SDLHGSFSVELFDPFKEAVQLGNDLQQSSD (SEQ ID NO:76)], TRP32-R.sub.4
[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-N.sub.6-1 antiserum was generated against the
synthetic keyhole limpet hemocyanin-conjugated peptide
Ank200-N.sub.6-1 by a commercial vendor (Bio-Synthesis).
[0190] Gel Electrophoresis and Western Immunoblotting.
[0191] 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.
[0192] ELISA.
[0193] 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
.mu.g/well; 50 .mu.l) 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.degree. C. The wells were washed
four times with 300 .mu.l 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 .mu.l) and incubated at room temperature for 1 h. The
plates were washed again, and 50 .mu.l 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 A.sub.650, 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.sub.--0170 for TRP32, ECH.sub.--0166 for TRP47, ECH.sub.--0039
for TRP120, ECH.sub.--0684 for Ank200, and Ecaj.sub.--0109 for
TRP36).
Results
[0194] 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).
[0195] Immunoreactivities of the Major E. Chaffeensis Ank200
Fragments.
[0196] 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.
[0197] Major Epitope-Containing Regions in Ank200-N.
[0198] 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).
[0199] Major Epitope-Containing Region in Ank200-C.
[0200] 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).
[0201] Determination of the Major Immunodeterminants of E.
Chaffeensis Ank200 with Synthetic Peptides.
[0202] Synthetic peptides were used to localize the major
epitope(s) in three immunoreactive regions (N.sub.6, N.sub.10, and
C.sub.6) of Ank200, respectively. Four synthetic overlapping
polypeptides (N.sub.6-1,2,3, and 4; FIG. 10A, left panel) covering
the sequence of Ank200-N.sub.6 (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 N.sub.6-2
did not react with two sera, reacted weakly with one and strongly
with two sera; peptide N.sub.6-3 did not react with one sera and
reacted weakly with four sera; peptide N.sub.6-4 did not react with
two sera and reacted weakly with three sera; however, peptide
N.sub.6-1 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-N.sub.6 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, N.sub.6-1 was divided
into two smaller overlapping peptides (N.sub.6-1a and N.sub.6-1b).
By ELISA, peptide N.sub.6-1b did not react with the anti-E.
chaffeensis dog serum and reacted weakly with four patient sera;
however, although peptide N.sub.6-1a 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, N.sub.6-1a (18 aa; ETGETVEEGLYAVPLPKD (SEQ ID NO:80))
contained a major continuous antibody epitope of Ank200 for human,
but longer sequence of peptide N.sub.6-1 (28 aa;
ETGETVEEGLYAVPLPKDQRPTPTQVLE (SEQ ID NO:81)) exhibited the
strongest immunoreactivity and was necessary for full
reconstitution of the major antibody epitope of Ank200.
[0203] To identify the peptide sequence containing the
immunodeterminant in Ank200-N.sub.10 (48 aa) region, four
overlapping peptides (N.sub.10-1, 2, 3 and 4; FIG. 10B, left panel)
covering N.sub.10 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 N.sub.101 and 2 did not react and peptides
N.sub.10-3 and 4 reacted weakly with antibodies in the dog serum
(FIG. 10B, right panel). Since the recombinant Ank200-N.sub.10
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 N.sub.10-2 reacted weakly with two patient sera and
reacted strongly with two patient sera, and peptides N.sub.10-1, 3
and 4 reacted weakly with one patient serum but reacted strongly
with other three patient sera, suggesting that N.sub.10 had two
epitope-containing regions for human, N.sub.10-1 (20 aa;
EPDLEEIVSILKNDKEGISE (SEQ ID NO:82)) and N.sub.10-3 (20 aa;
INEPVQVDIPNNPVREGRNV (SEQ ID NO:83)), and the C-terminal amino
acids (MTLLHLA (SEQ ID NO:84)) of N.sub.10 had no substantial
contribution to the epitope reactivity; moreover, peptide
N.sub.10-1 exhibited substantially stronger immunoreactivity than
did peptides N.sub.10-3 with three patient sera (FIG. 10B, right
panel).
[0204] Four synthetic overlapping peptides (C.sub.6-1, 2, 3 and 4;
FIG. 10C, left panel) covering the sequence of Ank200-C.sub.6 (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 C.sub.6-1, 2 and 3 were only weakly immunoreactive with
one or two patient sera, but peptide C.sub.6-4 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 (C.sub.6-4a and 4b)
representing fragment C.sub.6-4 reacted with anti-E. chaffeensis
dog and patient sera by ELISA. The peptide C.sub.6-4a was not
immunoreactive with all sera, however, peptide C.sub.6-4b 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 C.sub.6-4b (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).
[0205] Identification of TRP47 Antibody Epitopes in the TR Flanking
Terminal Regions.
[0206] 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.
[0207] To locate the epitope in the TRP47-N, four recombinant
overlapping proteins (TRP47-N.sub.1, N.sub.2, N.sub.3, and N.sub.4;
FIG. 11) covering the sequence of whole TRP47-N region were
expressed and reacted with three HME patient sera (nos. O13, O15
and 19) that recognized TRP47-N by Western blotting. The
recombinant N.sub.2 fragment did not react with the patient sera,
the N.sub.1 reacted weakly with one patient serum, the N.sub.3
reacted with two sera (one weakly), while the N.sub.4 fragment
reacted with all three sera strongly, indicating the TRP47-N.sub.4
fragment (44 aa) contained a minor antibody epitope. Three
synthetic overlapping polypeptides (N.sub.4-1, 2, and 3; FIG. 11
and FIG. 12A) covering the sequence of TRP47-N.sub.4 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
N.sub.4-3 was not reactive with any tested serum, N.sub.4-1 was
found to react with five sera (except for no. 19), and N.sub.4-2
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 N.sub.4-1 and 2 fragments contained the
antibody epitope with the TRP47-N region.
[0208] 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. O03 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.
[0209] Evaluation of Synthetic E. Chaffeensis Major
Immunodeterminants for Serologic Diagnosis of HME.
[0210] 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-R.sub.3 (30 aa) and -R.sub.4 (30 aa) peptides were used for
TRP32, an equal mixture of TRP47-N.sub.4-1 (22 aa), -R (19 aa) and
-C (26 aa) peptides was used for TRP47, TRP120-R-I.sub.1 (22 aa)
peptide was used for TRP120, and an equal mixture of
Ank200-N.sub.6-1a (18 aa), N.sub.10-1 (20 aa), and C.sub.6-4b (21
aa) were used for Ank200. In addition, a recombinant TRP120 TR
protein (rTRP120-TR) and an equal mixture of TRP32-R.sub.3,
TRP32-R.sub.4 and TRP120-R-I.sub.1 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.
[0211] 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-I.sub.1 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-US-00005 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 .sup.aSynthetic epitope
peptides of TRP32 (R.sub.3 + R.sub.4), TRP47 (N.sub.4 - 1 + R + C),
TRP120 (R-I.sub.1) and Ank200 (N.sub.6 - 1a + N.sub.10 - 1 +
C.sub.6 - 4b), and an equal mixture of TRP32-R.sub.3, TRP32-R.sub.4
and TRP120-R-I.sub.1 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. .sup.bA sample with a reading
0.1 OD unit above the negative control absorbance was considered
positive.
Discussion
[0212] 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.
[0213] E. chaffeensis and E. canis Ank200 protein orthologs are the
largest Ehrlichial major immunoreactive proteins. They have
identical chromosomal locations, and exhibit .about.50% nucleic
acid identity and .about.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 N.sub.6-1a, N.sub.10-1, N.sub.10-3
and C.sub.6-4b (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-R.sub.4 (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.
[0214] 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.
[0215] 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).
[0216] 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-I.sub.1 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-I.sub.1 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-I.sub.1 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.
[0217] 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.
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Sequence CWU 1
1
123122PRTArtificial SequenceSynthetic peptide 1Ser Lys Val Glu Gln
Glu Glu Thr Asn Pro Glu Val Leu Ile Lys Asp 1 5 10 15 Leu Gln Asp
Val Ala Ser 20 219PRTArtificial SequenceSynthetic peptide 2Ser Lys
Glu Glu Ser Thr Pro Glu Val Lys Ala Glu Asp Leu Gln Pro 1 5 10 15
Ala Val Asp 312PRTArtificial SequenceSynthetic peptide 3Glu Gln Glu
Glu Thr Asn Pro Glu Val Leu Ile Lys 1 5 10 417PRTArtificial
SequenceSynthetic peptide 4Ser Lys Val Glu Gln Glu Glu Thr Asn Pro
Glu Val Leu Ile Lys Asp 1 5 10 15 Leu 516PRTArtificial
SequenceSynthetic peptide 5Glu Gln Glu Glu Thr Asn Pro Glu Val Leu
Ile Lys Asp Leu Gln Asp 1 5 10 15 615PRTArtificial
SequenceSynthetic peptide 6Glu Thr Asn Pro Glu Val Leu Ile Lys Asp
Leu Gln Asp Val Ala 1 5 10 15 719PRTArtificial SequenceSynthetic
peptide 7Ser Ser Ser Glu Val Gly Lys Lys Val Ser Glu Thr Ser Lys
Glu Glu 1 5 10 15 Ser Thr Pro 819PRTArtificial SequenceSynthetic
peptide 8Ser Glu Thr Ser Lys Glu Glu Ser Thr Pro Glu Val Lys Ala
Glu Asp 1 5 10 15 Leu Gln Pro 914PRTArtificial SequenceSynthetic
peptide 9Thr Pro Glu Val Lys Ala Glu Asp Leu Gln Pro Ala Val Asp 1
5 10 1019PRTArtificial SequenceSynthetic peptide 10Thr Pro Glu Val
Lys Ala Glu Asp Leu Gln Pro Ala Val Asp Gly Ser 1 5 10 15 Ile Glu
His 1125PRTArtificial SequenceSynthetic peptide 11Glu His Ser Ser
Ser Glu Val Gly Glu Lys Val Ser Lys Thr Ser Lys 1 5 10 15 Glu Glu
Ser Thr Pro Glu Val Lys Ala 20 25 1210PRTArtificial
SequenceSynthetic peptide 12Ser Lys Val Glu Gln Glu Glu Thr Asn Pro
1 5 10 137PRTArtificial SequenceSynthetic peptide 13Asp Leu Gln Asp
Val Ala Ser 1 5 147PRTArtificial SequenceSynthetic peptide 14Ser
Lys Glu Glu Ser Thr Pro 1 5 158PRTArtificial SequenceSynthetic
peptide 15Glu Asp Leu Gln Pro Ala Val Asp 1 5 1629DNAArtificial
SequenceSynthetic primer 16atggatattg ataatagtaa cataagtac
291727DNAArtificial SequenceSynthetic primer 17tacaatatca
tttactacat tgtgatt 271829DNAArtificial SequenceSynthetic primer
18atggatattg ataatagtaa cataagtac 291921DNAArtificial
SequenceSynthetic primer 19tgtgtcatct tcttgctctt g
212025DNAArtificial SequenceSynthetic primer 20attctagtag
aagatttgcc attag 252127DNAArtificial SequenceSynthetic primer
21tacaatatca tttactacat tgtgatt 272229DNAArtificial
SequenceSynthetic primer 22atggatattg ataacaataa tgtgactac
292327DNAArtificial SequenceSynthetic primer 23tattaaatca
actgtttctt tgttagt 272429DNAArtificial SequenceSynthetic primer
24atggatattg ataacaataa tgtgactac 292522DNAArtificial
SequenceSynthetic primer 25tggatttcct acattgtcat tc
222619DNAArtificial SequenceSynthetic primer 26gaagtacagc ctgttgcag
192727DNAArtificial SequenceSynthetic primer 27tattaaatca
actgtttctt tgttagt 27285PRTArtificial SequenceSynthetic peptide
28Ser Lys Glu Glu Ser 1 5 2922DNAArtificial SequenceSynthetic
primer 29caacaaaatc ctaattcgca ag 223022DNAArtificial
SequenceSynthetic primer 30cgattttata tcattaccag ca
223123DNAArtificial SequenceSynthetic primer 31caccatggca
gatccaaaac aag 233220DNAArtificial SequenceSynthetic primer
32taccgcatac aatggatctt 203324DNAArtificial SequenceSynthetic
primer 33caccccttta cctaaaggtc aaag 243417DNAArtificial
SequenceSynthetic primer 34atccctaaca ccttccc 173525DNAArtificial
SequenceSynthetic primer 35caccgcagtt attcatgatg aagag
253617DNAArtificial SequenceSynthetic primer 36caatggggat tgatttc
173723DNAArtificial SequenceSynthetic primer 37cacccatgtt
atggttcaga acc 233818DNAArtificial SequenceSynthetic primer
38atcattacca gcaacagc 183923DNAArtificial SequenceSynthetic primer
39caccatggca gatccaaaac aag 234018DNAArtificial SequenceSynthetic
primer 40ttgctgagaa ggcaaatc 184125DNAArtificial SequenceSynthetic
primer 41caccgaaaca ggagaaactg tagaa 254221DNAArtificial
SequenceSynthetic primer 42taccgcatac aatggatctt c
214325DNAArtificial SequenceSynthetic primer 43caccgcagtt
attcatgatg aagag 254424DNAArtificial SequenceSynthetic primer
44agctaaatgc agtaatgtca ttac 244528DNAArtificial SequenceSynthetic
primer 45caccgtaatg acattactgc atttagct 284617DNAArtificial
SequenceSynthetic primer 46caatggggat tgatttc 174725DNAArtificial
SequenceSynthetic primer 47caccgcagtt attcatgatg aagag
254821DNAArtificial SequenceSynthetic primer 48aatttcttct
agatctggct c 214925DNAArtificial SequenceSynthetic primer
49caccgagcca gatctagaag aaatt 255024DNAArtificial SequenceSynthetic
primer 50agctaaatgc agtaatgtca ttac 245122DNAArtificial
SequenceSynthetic primer 51tgttcagtta aaggacgtgt tc
225221DNAArtificial SequenceSynthetic primer 52agctaaatgc
agcggtgtat c 215322DNAArtificial SequenceSynthetic primer
53tttgctgaaa agggtgtaaa aa 225428DNAArtificial SequenceSynthetic
primer 54atcttcagat gtaataggag gtagtccc 285522DNAArtificial
SequenceSynthetic primer 55tttgctgaaa agggtgtaaa aa
225621DNAArtificial SequenceSynthetic primer 56tccatgtaga
ccatgaactg c 215721DNAArtificial SequenceSynthetic primer
57gcagttcatg gtctacatgg a 215819DNAArtificial SequenceSynthetic
primer 58tttgctctgg caagaactt 195921DNAArtificial SequenceSynthetic
primer 59gcagttcatg gtctacatgg a 216018DNAArtificial
SequenceSynthetic primer 60cgctgatgca cctagaga 186118DNAArtificial
SequenceSynthetic primer 61tctctaggtg catcagcg 186219DNAArtificial
SequenceSynthetic primer 62tttgctctgg caagaactt 196318DNAArtificial
SequenceSynthetic primer 63tctctaggtg catcagcg 186421DNAArtificial
SequenceSynthetic primer 64acccttatca aatattccac t
216521DNAArtificial SequenceSynthetic primer 65agtggaatat
ttgataaggg t 216619DNAArtificial SequenceSynthetic primer
66tttgctctgg caagaactt 196721DNAArtificial SequenceSynthetic primer
67atgcttcatt taacaacaga a 216821DNAArtificial SequenceSynthetic
primer 68atgataacca cgatcaggtt c 216921DNAArtificial
SequenceSynthetic primer 69gaacctgatc gtggttatca t
217021DNAArtificial SequenceSynthetic primer 70aggatcaact
aagaaagaag c 217121DNAArtificial SequenceSynthetic primer
71gcttctttct tagttgatcc t 217221DNAArtificial SequenceSynthetic
primer 72atgatcatgt tcattgtgat g 217322DNAArtificial
SequenceSynthetic primer 73catcacaatg aacatgatca tg
227421DNAArtificial SequenceSynthetic primer 74atttccttca
agaactggaa c 217515PRTArtificial SequenceSynthetic peptide 75Glu
Gly Asp Ala Val Val Asn Ala Val Ser Gln Glu Thr Pro Ala 1 5 10 15
7630PRTArtificial SequenceSynthetic peptide 76Ser Asp Leu His Gly
Ser Phe Ser Val Glu Leu Phe Asp Pro Phe Lys 1 5 10 15 Glu Ala Val
Gln Leu Gly Asn Asp Leu Gln Gln Ser Ser Asp 20 25 30
7730PRTArtificial SequenceSynthetic peptide 77Ser Asp Ser His Glu
Pro Ser His Leu Glu Leu Pro Ser Leu Ser Glu 1 5 10 15 Glu Val Ile
Gln Leu Glu Ser Asp Leu Gln Gln Ser Ser Asn 20 25 30
7818PRTArtificial SequenceSynthetic peptide 78Thr Glu Asp Ser Val
Ser Ala Pro Ala Thr Glu Asp Ser Val Ser Ala 1 5 10 15 Pro Ala
7912PRTArtificial SequenceSynthetic peptide 79Glu Thr Gly Glu Thr
Val Glu Glu Gly Leu Tyr Ala 1 5 10 8018PRTArtificial
SequenceSynthetic peptide 80Glu Thr Gly Glu Thr Val Glu Glu Gly Leu
Tyr Ala Val Pro Leu Pro 1 5 10 15 Lys Asp 8128PRTArtificial
SequenceSynthetic peptide 81Glu Thr Gly Glu Thr Val Glu Glu Gly Leu
Tyr Ala Val Pro Leu Pro 1 5 10 15 Lys Asp Gln Arg Pro Thr Pro Thr
Gln Val Leu Glu 20 25 8220PRTArtificial SequenceSynthetic peptide
82Glu Pro Asp Leu Glu Glu Ile Val Ser Ile Leu Lys Asn Asp Lys Glu 1
5 10 15 Gly Ile Ser Glu 20 8320PRTArtificial SequenceSynthetic
peptide 83Ile Asn Glu Pro Val Gln Val Asp Ile Pro Asn Asn Pro Val
Arg Glu 1 5 10 15 Gly Arg Asn Val 20 847PRTArtificial
SequenceSynthetic peptide 84Met Thr Leu Leu His Leu Ala 1 5
8513PRTArtificial SequenceSynthetic peptide 85Gln Gly Ala Asp Val
Lys Lys Ser Ser Cys Gln Ser Lys 1 5 10 8621PRTArtificial
SequenceSynthetic peptide 86Gln Ala Val Ser Pro Ser Thr Ser Gln Gly
Ala Asp Val Lys Lys Ser 1 5 10 15 Ser Cys Gln Ser Lys 20
875PRTArtificial SequenceSynthetic peptide 87Ser Cys Gln Ser Lys 1
5 8834PRTArtificial SequenceSynthetic peptide 88Ser Ser Ser Glu Pro
Phe Val Ala Glu Ser Glu Val Ser Lys Val Glu 1 5 10 15 Gln Glu Glu
Thr Asn Pro Glu Val Leu Ile Lys Asp Leu Gln Asp Val 20 25 30 Ala
Ser 8931PRTArtificial SequenceSynthetic peptide 89Ser Ser Ser Glu
Val Gly Glu Lys Val Ser Glu Thr Ser Lys Glu Glu 1 5 10 15 Ser Thr
Pro Glu Val Lys Ala Glu Asp Leu Gln Pro Ala Val Asp 20 25 30
9032PRTArtificial SequenceSynthetic peptide 90Met Asp Ile Asp Asn
Ser Asn Ile Ser Thr Ala Asp Ile Arg Ser Asn 1 5 10 15 Thr Asp Gly
Leu Ile Asp Ile Ile Met Arg Ile Leu Gly Phe Gly Asn 20 25 30
9132PRTArtificial SequenceSynthetic peptide 91Met Asp Ile Asp Asn
Asn Asn Val Thr Thr Ser Ser Thr Gln Asp Lys 1 5 10 15 Ser Gly Asn
Leu Met Glu Val Ile Met Arg Ile Leu Asn Phe Gly Asn 20 25 30
9248PRTArtificial SequenceSynthetic peptide 92Gly Gln Tyr Val Cys
Gly Tyr Glu Met Tyr Met Tyr Gly Phe Gln Asp 1 5 10 15 Val Lys Asp
Leu Leu Gly Gly Leu Leu Ser Asn Val Pro Val Cys Cys 20 25 30 Asn
Val Ser Leu Tyr Phe Met Glu His Asn Tyr Phe Thr Asn His Glu 35 40
45 9348PRTArtificial SequenceSynthetic peptide 93Gly Glu His Val
His Met Tyr Gly Ile Tyr Val Tyr Arg Val Gln Ser 1 5 10 15 Val Lys
Asp Leu Ser Gly Val Phe Asn Ile Asp His Ser Thr Cys Asp 20 25 30
Cys Asn Leu Asp Val Tyr Phe Gly Tyr Asn Ser Phe Thr Asn Lys Glu 35
40 45 9480PRTArtificial SequenceSynthetic peptide 94Ser Ser Ser Glu
Pro Phe Val Ala Glu Ser Glu Val Ser Lys Val Glu 1 5 10 15 Gln Glu
Glu Thr Asn Pro Glu Val Leu Ile Lys Asp Leu Gln Asp Val 20 25 30
Ala Ser His Glu Ser Gly Val Ser Asp Gln Pro Ala Gln Val Val Thr 35
40 45 Glu Arg Glu Ser Glu Ile Glu Ser His Gln Gly Glu Thr Glu Lys
Glu 50 55 60 Ser Gly Ile Thr Glu Ser His Gln Lys Glu Asp Glu Ile
Val Ser Gln 65 70 75 80 9522PRTArtificial SequenceSynthetic peptide
95Ser Ser Ser Glu Pro Phe Val Ala Glu Ser Glu Val Ser Lys Val Glu 1
5 10 15 Gln Glu Glu Thr Asn Pro 20 9616PRTArtificial
SequenceSynthetic peptide 96Glu Thr Asn Pro Glu Val Leu Ile Lys Asp
Leu Gln Asp Val Ala Ser 1 5 10 15 9723PRTArtificial
SequenceSynthetic peptide 97Asp Leu Gln Asp Val Ala Ser His Glu Ser
Gly Val Ser Asp Gln Pro 1 5 10 15 Ala Gln Val Val Thr Glu Arg 20
9822PRTArtificial SequenceSynthetic peptide 98Gln Val Val Thr Glu
Arg Glu Ser Glu Ile Glu Ser His Gln Gly Glu 1 5 10 15 Thr Glu Lys
Glu Ser Gly 20 9921PRTArtificial SequenceSynthetic peptide 99Glu
Thr Glu Lys Glu Ser Gly Ile Thr Glu Ser His Gln Lys Glu Asp 1 5 10
15 Glu Ile Val Ser Gln 20 10036PRTArtificial SequenceSynthetic
peptide 100Ser Ser Ser Glu Val Gly Glu Lys Val Ser Glu Thr Ser Lys
Glu Glu 1 5 10 15 Ser Thr Pro Glu Val Lys Ala Glu Asp Leu Gln Pro
Ala Val Asp Gly 20 25 30 Ser Val Glu His 35 10111PRTArtificial
SequenceSynthetic peptide 101Ser Lys Glu Glu Asn Thr Pro Glu Val
Lys Ala 1 5 10 10263PRTArtificial SequenceSynthetic peptide 102Glu
Thr Gly Glu Thr Val Glu Glu Gly Leu Tyr Ala Val Pro Leu Pro 1 5 10
15 Lys Asp Gln Arg Pro Thr Pro Thr Gln Val Leu Glu Glu Asp Pro Ser
20 25 30 Val Glu Glu Glu Glu Ile Ala Pro Pro Leu Pro Pro Arg Gly
Asp Val 35 40 45 Ala Glu Leu Gln Glu Ala Val Glu Glu Asp Pro Leu
Tyr Ala Val 50 55 60 10316PRTArtificial SequenceSynthetic peptide
103Val Pro Leu Pro Lys Asp Gln Arg Pro Thr Pro Thr Gln Val Leu Glu
1 5 10 15 10419PRTArtificial SequenceSynthetic peptide 104Gln Arg
Pro Thr Pro Thr Gln Val Leu Glu Glu Asp Pro Ser Val Glu 1 5 10 15
Glu Glu Glu 10516PRTArtificial SequenceSynthetic peptide 105Glu Asp
Pro Ser Val Glu Glu Glu Glu Ile Ala Pro Pro Leu Pro Pro 1 5 10 15
10626PRTArtificial SequenceSynthetic peptide 106Ile Ala Pro Pro Leu
Pro Pro Arg Gly Asp Val Ala Glu Leu Gln Glu 1 5 10 15 Ala Val Glu
Glu Asp Pro Leu Tyr Ala Val 20 25 10748PRTArtificial
SequenceSynthetic peptide 107Glu Pro Asp Leu Glu Glu Ile Val Ser
Ile Leu Lys Asn Asp Lys Glu 1 5 10 15 Gly Ile Ser Glu Leu Ile Asn
Glu Pro Val Gln Val Asp Ile Pro Asn 20 25 30 Asn Pro Val Arg Glu
Gly Arg Asn Val Met Thr Leu Leu His Leu Ala 35 40 45
10820PRTArtificial SequenceSynthetic peptide 108Ser Ile Leu Lys
Asn
Asp Lys Glu Gly Ile Ser Glu Leu Ile Asn Glu 1 5 10 15 Pro Val Gln
Val 20 10920PRTArtificial SequenceSynthetic peptide 109Ile Asn Glu
Pro Val Gln Val Asp Ile Pro Asn Asn Pro Val Arg Glu 1 5 10 15 Gly
Arg Asn Val 20 11025PRTArtificial SequenceSynthetic peptide 110Glu
Pro Val Gln Val Asp Ile Pro Asn Asn Pro Val Arg Glu Gly Arg 1 5 10
15 Asn Val Met Thr Leu Leu His Leu Ala 20 25 11163PRTArtificial
SequenceSynthetic peptide 111Ser Gly Ile Phe Asp Lys Gly Glu Gly
Gln His Lys Ala Ser Glu Glu 1 5 10 15 Gln Leu Gln Glu Leu Ser Glu
Glu Ile Thr Asp Ile Val Gln Gly Leu 20 25 30 Pro Pro Ile Thr Ser
Glu Asp Ile Gly Ala Gln Ala Val Ser Pro Ser 35 40 45 Thr Ser Gln
Gly Ala Asp Val Lys Lys Ser Ser Cys Gln Ser Lys 50 55 60
11225PRTArtificial SequenceSynthetic peptide 112Ser Gly Ile Phe Asp
Lys Gly Glu Gly Gln His Lys Ala Ser Glu Glu 1 5 10 15 Gln Leu Gln
Glu Leu Ser Glu Glu Ile 20 25 11326PRTArtificial SequenceSynthetic
peptide 113Ser Glu Glu Gln Leu Gln Glu Leu Ser Glu Glu Ile Thr Asp
Ile Val 1 5 10 15 Gln Gly Leu Pro Pro Ile Thr Ser Glu Asp 20 25
11425PRTArtificial SequenceSynthetic peptide 114Thr Asp Ile Val Gln
Gly Leu Pro Pro Ile Thr Ser Glu Asp Ile Gly 1 5 10 15 Ala Gln Ala
Val Ser Pro Ser Thr Ser 20 25 11525PRTArtificial SequenceSynthetic
peptide 115Asp Ile Gly Ala Gln Ala Val Ser Pro Ser Thr Ser Gln Gly
Ala Asp 1 5 10 15 Val Lys Lys Ser Ser Cys Gln Ser Lys 20 25
11620PRTArtificial SequenceSynthetic peptide 116Asp Ile Gly Ala Gln
Ala Val Ser Pro Ser Thr Ser Gln Gly Ala Asp 1 5 10 15 Val Lys Lys
Ser 20 11721PRTArtificial SequenceSynthetic peptide 117Gln Ala Val
Ser Pro Ser Thr Ser Gln Gly Ala Asp Val Lys Lys Ser 1 5 10 15 Ser
Cys Gln Ser Lys 20 11844PRTArtificial SequenceSynthetic peptide
118His His Asn Glu His Asp His Asp Ala His Gly Arg Gly Ala Ala Ser
1 5 10 15 Ser Val Ala Glu Gly Val Gly Ser Ala Ile Ser Gln Ile Leu
Ser Leu 20 25 30 Ser Asp Ser Ile Val Val Pro Val Leu Glu Gly Asn 35
40 11922PRTArtificial SequenceSynthetic peptide 119His His Asn Glu
His Asp His Asp Ala His Gly Arg Gly Ala Ala Ser 1 5 10 15 Ser Val
Ala Glu Gly Val 20 12022PRTArtificial SequenceSynthetic peptide
120Arg Gly Ala Ala Ser Ser Val Ala Glu Gly Val Gly Ser Ala Ile Ser
1 5 10 15 Gln Ile Leu Ser Leu Ser 20 12122PRTArtificial
SequenceSynthetic peptide 121Gly Ser Ala Ile Ser Gln Ile Leu Ser
Leu Ser Asp Ser Ile Val Val 1 5 10 15 Pro Val Leu Glu Gly Asn 20
12219PRTArtificial SequenceSynthetic peptide 122Ala Ser Val Ser Glu
Gly Asp Ala Val Val Asn Ala Val Ser Gln Glu 1 5 10 15 Thr Pro Ala
12326PRTArtificial SequenceSynthetic peptide 123Thr Gln Pro Gln Ser
Arg Asp Ser Leu Leu Asn Glu Glu Asp Met Ala 1 5 10 15 Ala Gln Phe
Gly Asn Arg Tyr Phe Tyr Phe 20 25
* * * * *
References